Assays and related methods, kits and devices

Abstract

The present disclosure relates to correlating the ratio of the determined level of fetal globulin A fragment to the determined level of urine creatinine to the likelihood of graft function deterioration in a recipient.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 441,751, filed January 27, 2023, entitled “ASSAY AND RELATED METHODS, KITS AND DEVICES”, and U.S. Provisional Patent Application No. 63 / 532,655, filed August 14, 2023, entitled “ASSAY AND RELATED METHODS, KITS AND DEVICES”, each of which is incorporated herein by reference in its entirety. Background Technology

[0003] End-stage renal disease (ESKD) is a leading cause of morbidity and mortality and a major global health burden. To date, kidney transplantation remains the preferred treatment for ESKD because it offers better quality of life and is cost-effective compared to other kidney replacement therapies. Summary of the Invention

[0004] This summary is provided to introduce, in a simplified form, some concepts that will be further described below in the detailed description. This summary is not intended to identify key or essential aspects of the claimed subject matter.

[0005] All features of the exemplary embodiments described in this disclosure, which are not mutually exclusive, can be combined with each other. Elements of one embodiment may be used in other embodiments without further mention. Other aspects and features of the invention will become apparent to those skilled in the art upon review of the following description of specific embodiments in conjunction with any of the accompanying drawings.

[0006] The present disclosure can relate to a method of preparing an assay, the method can comprise providing a first solution comprising a urine sample from a recipient of a kidney transplant after the kidney transplant and a first reagent that interacts with a post-translationally modified fetuin-A fragment in a urine fragment to determine a level of the fetuin-A fragment in the urine sample; providing a second solution comprising the urine sample and a second reagent that interacts with urine creatinine to determine a level of the urine creatinine in the urine sample; in response to the determined level of the fetuin-A fragment and the determined level of the urine creatinine, correlating a ratio of the determined level of the fetuin-A fragment to the determined level of the urine creatinine with a likelihood of a deterioration of graft function in the recipient, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 20 ng / mg, indicates a higher likelihood of a deterioration of graft function in a time period of about ten years or less, and wherein the first solution and the second solution can be the same solution or different solutions from each other.

[0007] In some embodiments, a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to avoid a deterioration of graft function.

[0008] In some embodiments, the higher likelihood can be higher than a lower likelihood of a deterioration of graft function in a time period of about ten years or less in a second subject having a lower ratio of the determined level of the fetuin-A fragment to the level of urine creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0009] In some embodiments, the deterioration of graft function can be indicated by a doubling of serum creatinine, a need for a second kidney transplant, or a need for dialysis.

[0010] In some embodiments, the deterioration of graft function can be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0011] In some embodiments, the interaction with the fetuin-A fragment can comprise binding to the fetuin-A fragment.

[0012] In some embodiments, the interaction with the urine creatinine can comprise binding to the urine creatinine.

[0013] In some embodiments, at least one of the first reagent and the second reagent can comprise an antibody.

[0014] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 27 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.3 ng / mg.

[0015] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 75 ng / mg.

[0016] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 95 ng / mg.

[0017] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 13 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 12 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 11 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 10 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0018] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.2 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.1 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 6 ng / mg.

[0019] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.6 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.5 ng / mg.

[0020] In some embodiments, the higher likelihood of graft function worsening can be about 5% or more. In some embodiments, the higher likelihood of graft function worsening can be about 8% or more. In some embodiments, the higher likelihood of graft function worsening can be about 10% or more. In some embodiments, the higher likelihood of graft function worsening can be about 15% or more. In some embodiments, the higher likelihood of graft function worsening can be about 16% or more.

[0021] In some embodiments, the higher likelihood of graft function worsening can be about 20% or more. In some embodiments, the higher likelihood of graft function worsening can be about 22% or more. In some embodiments, the higher likelihood of graft function worsening can be about 25% or more. In some embodiments, the higher likelihood of graft function worsening can be about 30% or more. In some embodiments, the higher likelihood of graft function worsening can be about 32% or more. In some embodiments, the higher likelihood of graft function worsening can be about 35% or more. In some embodiments, the higher likelihood of graft function worsening can be about 36% or more. In some embodiments, the higher likelihood of graft function worsening can be about 40% or more. In some embodiments, the higher likelihood of graft function worsening can be about 45% or more. In some embodiments, the higher likelihood of graft function worsening can be about 50% or more.

[0022] In some embodiments, the higher likelihood of graft function worsening can be about 5% to about 50%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 35%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 20%. In some embodiments, the higher likelihood of graft function worsening can be about 15% to about 25%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 30% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 35% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 40% to about 50%.

[0023] In some embodiments, the graft function worsening occurs after 2 years. In some embodiments, the graft function worsening occurs after 3 years. In some embodiments, the graft function worsening occurs after 4 years. In some embodiments, the graft function worsening occurs after 5 years. In some embodiments, the graft function worsening occurs after 6 years. In some embodiments, the graft function worsening occurs after 7 years. In some embodiments, the graft function worsening occurs after 8 years. In some embodiments, the graft function worsening occurs after 9 years. In some embodiments, the graft function worsening occurs within 7 years. In some embodiments, the graft function worsening occurs within 8 years. In some embodiments, the graft function worsening occurs within 9 years.

[0024] In some embodiments, the method can further comprise assessing the probability of graft function worsening based on the ratio and at least one other marker.

[0025] In some embodiments, the at least one other marker can comprise age, gender, time post-transplant at inclusion, urinary albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0026] In some embodiments, the assessed probability can exhibit an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 80 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 85 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 90 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 95 (%).

[0027] The present disclosure can relate to an assay kit for determining the likelihood of progressive decline in kidney function, wherein the assay kit can comprise a first solution comprising a first reagent that interacts with a post-translationally modified fetuin-A fragment in a urine fragment to indicate a level of the fetuin A fragment in a urine sample from a recipient of a kidney transplant; a second solution comprising a second reagent that interacts with urine creatinine to indicate a level of the urine creatinine in the urine sample; a device for assaying the level of the fetuin A fragment and the level of the urine creatinine in the urine sample to determine a ratio of the assayed level of the fetuin A fragment to the assayed level of the urine creatinine, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg 20 ng / mg indicates a higher likelihood of graft function deterioration within a period of about ten years or less, and wherein the first solution and the second solution can be the same solution or different solutions from each other.

[0028] In some embodiments, a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg triggers a medical intervention to avoid graft function deterioration.

[0029] In some embodiments, the higher likelihood can be higher than a lower likelihood of graft function deterioration within a period of about ten years or less in a second subject having a lower ratio of the assayed level of the fetuin A fragment to the level of urine creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0030] In some embodiments, graft function deterioration can be indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

[0031] In some embodiments, the worsening of graft function can be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0032] In some embodiments, the interaction with fetuin-A fragment can comprise binding to fetuin-A fragment.

[0033] In some embodiments, the interaction with urine creatinine can comprise binding to urine creatinine.

[0034] In some embodiments, at least one of the first agent and the second agent can comprise an antibody.

[0035] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 27 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.3 ng / mg.

[0036] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 75 ng / mg.

[0037] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 95 ng / mg.

[0038] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 13 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 12 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 11 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 10 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0039] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.2 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.1 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 6 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.6 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.5 ng / mg.

[0040] In some embodiments, the higher likelihood of graft function worsening can be about 5% or more. In some embodiments, the higher likelihood of graft function worsening can be about 8% or more. In some embodiments, the higher likelihood of graft function worsening can be about 10% or more. In some embodiments, the higher likelihood of graft function worsening can be about 15% or more. In some embodiments, the higher likelihood of graft function worsening can be about 16% or more. In some embodiments, the higher likelihood of graft function worsening can be about 20% or more. In some embodiments, the higher likelihood of graft function worsening can be about 22% or more. In some embodiments, the higher likelihood of graft function worsening can be about 25% or more. In some embodiments, the higher likelihood of graft function worsening can be about 30% or more. In some embodiments, the higher likelihood of graft function worsening can be about 32% or more. In some embodiments, the higher likelihood of graft function worsening can be about 35% or more. In some embodiments, the higher likelihood of graft function worsening can be about 36% or more. In some embodiments, the higher likelihood of graft function worsening can be about 40% or more. In some embodiments, the higher likelihood of graft function worsening can be about 45% or more. In some embodiments, the higher likelihood of graft function worsening can be about 50% or more.

[0041] In some embodiments, the higher likelihood of graft function worsening can be about 5% to about 50%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 35%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 20%. In some embodiments, the higher likelihood of graft function worsening can be about 15% to about 25%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 30% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 35% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 40% to about 50%.

[0042] In some embodiments, the worsening of graft function occurs after 2 years. In some embodiments, the worsening of graft function occurs after 3 years. In some embodiments, the worsening of graft function occurs after 4 years. In some embodiments, the worsening of graft function occurs after 5 years. In some embodiments, the worsening of graft function occurs after 6 years. In some embodiments, the worsening of graft function occurs after 7 years. In some embodiments, the worsening of graft function occurs after 8 years. In some embodiments, the worsening of graft function occurs after 9 years. In some embodiments, the worsening of graft function occurs within 7 years. In some embodiments, the worsening of graft function occurs within 8 years. In some embodiments, the worsening of graft function occurs within 9 years.

[0043] In some embodiments, the device can be used to determine the level of fetuin-A fragment and the level of urine creatinine to assess the probability of the worsening of graft function based on the ratio and at least one other marker.

[0044] In some embodiments, the at least one other marker can include age, gender, time post-transplant at inclusion, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0045] In some embodiments, the assessed probability can show an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%). In some embodiments, the assessed probability can show an AUC of at least about 80 (%). In some embodiments, the assessed probability can show an AUC of at least about 85 (%). In some embodiments, the assessed probability can show an AUC of at least about 90 (%). In some embodiments, the assessed probability can show an AUC of at least about 95 (%).

[0046] The present disclosure can relate to a method of correlating likelihood of graft function deterioration, wherein the method can comprise determining a level of a first biomarker and a second biomarker in a urine sample from a recipient of a kidney transplant after the kidney transplant, wherein the first biomarker can be a fragment of a fragment of a post-translationally modified fetuin-A fragment in a urine fragment, wherein the second biomarker can be urine creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio to a likelihood of graft function deterioration in the recipient over a period of about ten years, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 20 ng / mg, indicates a higher likelihood of graft function deterioration over a period of about ten years or less.

[0047] In some embodiments, a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to avoid graft function deterioration.

[0048] In some embodiments, the higher likelihood can be higher than a lower likelihood of graft function deterioration in a second subject over a period of about ten years or less, the second subject having a lower ratio of a determined level of fetuin-A fragment to a level of urine creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0049] In some embodiments, graft function deterioration can be indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

[0050] In some embodiments, graft function deterioration can be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0051] In some embodiments, the interaction with the fetuin-A fragment can comprise binding to the fetuin-A fragment.

[0052] In some embodiments, the interaction with the urine creatinine can comprise binding to the urine creatinine.

[0053] In some embodiments, at least one of the first reagent and the second reagent can comprise an antibody.

[0054] In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 25 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 25.5 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 26 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 27 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 27.5 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 28 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 28.5 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 28.7 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 28.8 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 28.9 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 29.0 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 29.1 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 29.2 ng / mg. In some embodiments, a ratio higher than about 20 ng / mg can be higher than about 29.3 ng / mg.

[0055] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 75 ng / mg.

[0056] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 95 ng / mg.

[0057] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 13 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 12 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 11 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 10 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0058] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.2 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.1 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 6 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.6 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.5 ng / mg.

[0059] In some embodiments, the higher likelihood of graft function worsening can be about 5% or more. In some embodiments, the higher likelihood of graft function worsening can be about 8% or more. In some embodiments, the higher likelihood of graft function worsening can be about 10% or more. In some embodiments, the higher likelihood of graft function worsening can be about 15% or more. In some embodiments, the higher likelihood of graft function worsening can be about 16% or more. In some embodiments, the higher likelihood of graft function worsening can be about 20% or more. In some embodiments, the higher likelihood of graft function worsening can be about 22% or more. In some embodiments, the higher likelihood of graft function worsening can be about 25% or more. In some embodiments, the higher likelihood of graft function worsening can be about 30% or more. In some embodiments, the higher likelihood of graft function worsening can be about 32% or more. In some embodiments, the higher likelihood of graft function worsening can be about 35% or more. In some embodiments, the higher likelihood of graft function worsening can be about 36% or more. In some embodiments, the higher likelihood of graft function worsening can be about 40% or more. In some embodiments, the higher likelihood of graft function worsening can be about 45% or more. In some embodiments, the higher likelihood of graft function worsening can be about 50% or more.

[0060] In some embodiments, the higher likelihood of graft function worsening can be about 5% to about 50%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 35%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 20%. In some embodiments, the higher likelihood of graft function worsening can be about 15% to about 25%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 30% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 35% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 40% to about 50%.

[0061] In some embodiments, the worsening of graft function occurs after 2 years. In some embodiments, the worsening of graft function occurs after 3 years. In some embodiments, the worsening of graft function occurs after 4 years. In some embodiments, the worsening of graft function occurs after 5 years. In some embodiments, the worsening of graft function occurs after 6 years. In some embodiments, the worsening of graft function occurs after 7 years. In some embodiments, the worsening of graft function occurs after 8 years. In some embodiments, the worsening of graft function occurs after 9 years. In some embodiments, the worsening of graft function occurs within 7 years. In some embodiments, the worsening of graft function occurs within 8 years. In some embodiments, the worsening of graft function occurs within 9 years.

[0062] In some embodiments, the method can further comprise assessing the probability of the worsening of graft function based on the ratio and at least one other marker.

[0063] In some embodiments, the at least one other marker can comprise age, gender, time post-transplant at inclusion, urinary albumin to creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0064] In some embodiments, the assessed probability can show an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%). In some embodiments, the assessed probability can show an AUC of at least about 80 (%). In some embodiments, the assessed probability can show an AUC of at least about 85 (%). In some embodiments, the assessed probability can show an AUC of at least about 90 (%). In some embodiments, the assessed probability can show an AUC of at least about 95 (%). BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 Kaplan-Meier survival curves of kidney transplant recipients (KTRs) with death censored graft loss are shown in the main cohort, according to non-limiting embodiments.

[0066] Figure 2A Kaplan-Meier curves of KTRs with (a) non-proteinuria according to uPTM-FetA / UCr risk category in the main cohort, according to non-limiting embodiments, are shown.

[0067] Figure 2B Kaplan-Meier curves of KTRs with (b) proteinuria according to uPTM-FetA / UCr risk category in the main cohort, according to non-limiting embodiments, are shown.

[0068] Figure 3 Kaplan-Meier survival curve of KTRs with death censored graft loss according to non-limiting embodiments.

[0069] Figure 4A Kaplan-Meier curve of subjects with (a) non-proteinuria according to uPTM-FetA / uCr risk category in the validation set according to non-limiting embodiments.

[0070] Figure 4B Kaplan-Meier curve of subjects with (b) proteinuria according to uPTM-FetA / uCr risk category in the validation set according to non-limiting embodiments.

[0071] Figure 5 Kaplan-Meier survival curve of 635 KTRs with death censored graft loss according to non-limiting embodiments.

[0072] Figure 6A Kaplan-Meier curve of KTRs with (a) non-proteinuria according to uPTM-FetA / uCr risk category in the 24-hour collection according to non-limiting embodiments.

[0073] Figure 6B Kaplan-Meier curve of KTRs with (b) proteinuria according to uPTM-FetA / uCr risk category in the 24-hour collection according to non-limiting embodiments.

[0074] Figure 7 Receiver operating characteristic (ROC) curve for uPTM-FetA / uCr and corresponding Kaplan-Meier curve for the marker combination model of KTRs with graft loss within 2 years using random urine according to non-limiting embodiments.

[0075] Figure 8 Receiver operating characteristic (ROC) curve for the combination of uPTM-FetA / uCr, age, sex, time, and UACR and corresponding Kaplan-Meier curve for the marker combination model of KTRs with graft loss within 2 years using random urine according to non-limiting embodiments.

[0076] Figure 9 Receiver operating characteristic (ROC) curve for uPTM-FetA / uCr, age, sex, time, UACR, and eGFR and corresponding Kaplan-Meier curve for the marker combination model of KTRs with graft loss within 2 years using random urine according to non-limiting embodiments.

[0077] Figure 10Receiver Operating Characteristic (ROC) curves for uPTM-FetA / uCr, and corresponding Kaplan-Meier curves for a marker combination model with graft loss within 2 years using random urine for KTRs are shown according to non-limiting embodiments.

[0078] Figure 11 Receiver Operating Characteristic (ROC) curves for uPTM-FetA / uCr, and corresponding Kaplan-Meier curves for a marker combination model with graft loss within 5 years using random urine for KTRs are shown according to non-limiting embodiments.

[0079] Figure 12 Receiver Operating Characteristic (ROC) curves for a combination of uPTM-FetA / uCr, age, sex, time, and UACR, and corresponding Kaplan-Meier curves for a marker combination model with graft loss within 5 years using random urine for KTRs are shown according to non-limiting embodiments.

[0080] Figure 13 Receiver Operating Characteristic (ROC) curves for uPTM-FetA / uCr, age, sex, time, UACR, and eGFR, and corresponding Kaplan-Meier curves for a marker combination model with graft loss within 5 years using random urine for KTRs are shown according to non-limiting embodiments.

[0081] Figure 14 Receiver Operating Characteristic (ROC) curves for uPTM-FetA / uCr, age, sex, time, UACR, eGFR, and presence of human leukocyte antigen class II antibody (HLA II), and corresponding Kaplan-Meier curves for a marker combination model with graft loss within 5 years using random urine for KTRs are shown according to non-limiting embodiments.

[0082] Figure 15 A flow diagram of study population selection according to non-limiting embodiments is shown.

[0083] Figure 16 A scatter plot and visual presentation of the correlation of 24-hour uPTM-FetA excretion with serum creatine levels according to non-limiting embodiments is shown.

[0084] Figure 17 A scatter plot and visual presentation of the correlation of 24-hour uPTM-FetA excretion with eGFR according to non-limiting embodiments is shown.

[0085] Figure 18A scatter plot and visual presentation showing the correlation of 24 hour uPTM-FetA excretion with 24 hour albumin excretion, according to non-limiting embodiments.

[0086] Figure 19 A scatter plot and visual presentation showing the correlation of 24 hour uPTM-FetA excretion with 24 hour urine protein excretion, according to non-limiting embodiments.

[0087] Figure 20 A Kaplan-Meier curve showing death censored graft loss above and below the median 24 hour urine post-translationally modified Fetuin-A excretion, according to non-limiting embodiments.

[0088] Figure 21 A Kaplan-Meier curve showing death censored graft loss above and below the median 24 hour urine post-translationally modified Fetuin-A excretion, according to non-limiting embodiments.

[0089] Figure 22 A Kaplan-Meier curve showing all-cause mortality above and below the median 24 hour urine post-translationally modified Fetuin-A excretion, according to non-limiting embodiments.

[0090] Figure 23 A flowchart showing study participant selection, according to non-limiting embodiments;

[0091] Figure 24 A Kaplan-Meier analysis showing death censored graft function worsening survival per tertile of 24 hour uC-FetA excretion, according to non-limiting embodiments; and

[0092] Figure 25 A forest plot showing the association of uC-FetA with death censored graft function worsening in subgroups, according to non-limiting embodiments.

[0093] Figure 26 A Kaplan-Meier analysis showing patient survival per tertile of 24 hour uC-FetA excretion, according to non-limiting embodiments.

[0094] Figure 27 A calibration curve for the human uPTM3-DKD ELISA, according to non-limiting embodiments (calibration range is 7.813-500 ng / mL) is shown. DETAILED DESCRIPTION

[0095] The present disclosure is based on the surprising finding that urinary proteins and fragments thereof (post-translationally modified fetuin-A fragments in urine (uPTM-FetA)) are differentially present in kidney transplant recipients (KTRs) with a higher likelihood of having a worsening of graft function. Thus, these protein molecules are useful markers for diagnosing a worsening of graft function at different stages, including early stages. Thus, uPTM-FetA has been identified as a biomarker of acute kidney injury, and in its extension, as a biomarker for early detection of a decline in kidney function.

[0096] To date, kidney transplantation remains the most preferred treatment for ESKD as it offers better quality of life and is most cost-effective compared to other renal replacement therapy modalities. However, despite this success, kidney transplant recipients (KTRs) can still be at risk of loss of graft function and of morbidity and premature death, which is largely attributed to a worsening of kidney graft function. From a patient perspective, survival of the kidney graft is considered more important than life itself and patients would rather die than undergo dialysis again. Therefore, greater efforts should be made to detect loss of graft function as early as possible so that appropriate treatment can be given and graft failure can be prevented. In the current clinical setting, routine evaluation of graft function is based on serum creatinine and proteinuria. In case of a significant increase in serum creatinine or presence of proteinuria, a kidney biopsy is considered. Unfortunately, these evaluations are not perfect, especially with respect to prognostic prediction of the graft. Therefore, there is a need for biomarkers that can be used to identify KTRs at risk of a worsening of graft function and subsequent graft failure, such as alternative non-invasive markers that can independently identify patients at risk of a worsening of graft function and subsequent graft failure.

[0097] As used herein, the term "medical intervention" with respect to a worsening of kidney graft function in a KTR means a treatment performed on the recipient that attempts to stabilize, slow, delay, or inhibit the worsening, which can result in a reduced rate of worsening in the recipient. Various types of medical interventions can be implemented. Examples of medical interventions include a guided diet, a restricted diet, restriction of sodium and other ion intake, reduction or avoidance of alcohol consumption, reduction or avoidance of smoking or consumption of related products, taking various medications, weight control, kidney transplantation or retransplantation, dialysis, other medically identifiable treatments, and any combination thereof.

[0098] As used herein, the term "average fetuin-A fragment excretion" means the amount of fetuin-A fragments (such as fetuin-A containing urine link peptide) excreted into urine averaged over a predetermined period of time. For example, average fetuin-A fragment excretion can be averaged over 2 to 48 hours or longer or shorter.

[0099] Accordingly, the present disclosure relates to diagnostic methods using biomarkers, which can be proteins or fragments of proteins that can be considered proteins. Urine samples, serum samples, or both urine and serum samples can be collected from a subject, and urine levels, serum levels, or both levels of biomarkers can be determined by various methods, such as mass spectrometry and immunoassays.

[0100] When a biomarker contains a single protein molecule, its level in a subject can be compared to a reference point to determine a corresponding indication or likelihood of a target condition. A reference point representing a level of the same biomarker, such as a level of the biomarker in a normal subject without substantial worsening of kidney transplant function in a KTR, can be determined based on representative levels of the biomarker in a KTR group. For example, it can be a midpoint between the average levels of the two groups. A biomarker level above the reference point indicates the target condition.

[0101] There is a clinical need for new markers to predict a decline in kidney function in ESKD or KTR patients. A post-translationally modified fetuin-A fragment in urine (uPTM-FetA) can be a biomarker indicating the likelihood of a decline in kidney function to kidney end-point and can predict a decline in kidney function in ESKD or KTR patients.

[0102] Fetuin-A (also known as alpha-2-Heremans-Schmid glycoprotein) is a heterodimeric glycoprotein with a 367 amino acid sequence that undergoes various post-translational modifications before being secreted outside of the producing cell, containing 18 amino acids of a signal peptide, 282 amino acids of an A chain, a 40 amino acid linker chain, and 27 amino acids of a B chain. Fetuin-A, such as post-translationally modified fetuin-A fragments in urine (uPTM-FetA), which can include post-translationally modified fetuin-A fragments in urine (uPTM-FetA) (also known as alpha-2-Heremans-Schmid glycoprotein), has characteristics that suggest it can serve as such a surrogate, independent, non-invasive marker. Fetuin-A is elevated in urine of patients with acute kidney injury (AKI) and recent evidence suggests that it is also related to (the extent of) interstitial fibrosis / tubular atrophy (IFTA). The incidence of AKI and the presence of IFTA after kidney transplantation are known to be associated with adverse long-term graft outcomes. Urinary fetuin-A is also elevated in patients with chronic kidney disease, such as autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis. Furthermore, urinary fetuin-A has previously been shown to be associated with a decline in kidney function in patients with chronic kidney disease, including diabetic nephropathy, and this protein has been proposed for use as a biomarker for early detection of these diseases. However, no studies have been conducted to measure urinary fetuin-A in KTR populations and investigate a plausible association with graft outcomes.

[0103] In some embodiments, post-translationally modified fetuin-A fragments in urine (uPTM-FetA) can be used as a biomarker for acute kidney injury, and in its extension, as a biomarker for early detection of reduced kidney function. In some embodiments, uPTM-FetA proteins can serve as a marker for graft function deterioration in KTRs. For example, uPTM-FetA levels in stable out-patient KTRs can be used to determine the likelihood of a higher probability of graft function deterioration over time, such as about ten years or less. In some embodiments, the primary endpoint can be death censored graft function deterioration, i.e., the need for retransplantation or (re)start of dialysis, and the secondary endpoint is all-cause mortality.

[0104] Due to the potential pathophysiological role of urinary fetuin-A in the occurrence of reduced kidney function, the association of urinary fetuin-A or fragments thereof with graft function deterioration can be used to determine the likelihood of graft function deterioration over time.

[0105] Accordingly, in some embodiments, a method of making, for example, a determination for the likelihood of graft function deterioration in a recipient of a kidney transplant can comprise providing a first solution comprising a urine sample from the recipient of the kidney transplant after the kidney transplant and a first reagent that interacts with post-translationally modified fetuin-A fragments in urine fragments to determine a level of the fetuin-A fragments in the urine sample; providing a second solution comprising the urine sample and a second reagent that interacts with urinary creatinine to determine a level of the urinary creatinine in the urine sample; correlating, in response to the determined level of the fetuin-A fragments and the determined level of the urinary creatinine, a ratio of the determined level of the fetuin-A fragments to the determined level of the urinary creatinine to a likelihood of graft function deterioration in the recipient, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 20 ng / mg, indicates a higher likelihood of graft function deterioration over a period of about ten years or less, wherein the first solution and the second solution are the same solution or different solutions from each other.

[0106] Further, in some embodiments, an assay kit for determining the likelihood of progressive decline in kidney function can comprise a first solution comprising a first reagent that interacts with a post-translationally modified fetuin-A fragment in a urine fragment to indicate a level of the fetuin A fragment in a urine sample from a recipient of a kidney transplant after the kidney transplant; a second solution comprising a second reagent that interacts with urine creatinine to indicate a level of the urine creatinine in the urine sample; a device for assaying the level of the fetuin A fragment and the level of the urine creatinine in the urine sample to determine a ratio of the assayed level of the fetuin A fragment to the assayed level of the urine creatinine, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg indicates a higher likelihood of graft function deterioration within a period of about ten years or less, wherein the first solution and the second solution are the same solution or different solutions from each other.

[0107] Further, in some embodiments, a method of correlating the likelihood of graft function deterioration can comprise assaying a level of a first biomarker and a second biomarker in a urine sample from a recipient of a kidney transplant after the kidney transplant, wherein the first biomarker can be a fragment of a post-translationally modified fetuin-A fragment in a urine fragment, wherein the second biomarker can be urine creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio to a likelihood of graft function deterioration within a period of about ten years for the recipient, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0108] In some embodiments, a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg triggers a medical intervention to avoid graft function deterioration.

[0109] In some embodiments, the higher likelihood can be higher than a lower likelihood of graft function worsening in a second subject having a lower ratio of a level of fetuin-A fragment to a level of urine creatinine determined to be lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg over a period of about ten years or less.

[0110] In some embodiments, the graft function worsening can be indicated by doubling of serum creatinine, need for re- kidney transplantation, or need for dialysis.

[0111] In some embodiments, the graft function worsening can be indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0112] In some embodiments, the interaction with fetuin-A fragment can comprise binding to fetuin-A fragment.

[0113] In some embodiments, the interaction with urine creatinine can comprise binding to urine creatinine.

[0114] In some embodiments, at least one of the first agent and the second agent can comprise an antibody.

[0115] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 25 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 25.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 26 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 27 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 27.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.5 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.7 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.8 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 28.9 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.0 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.1 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.2 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 29.3 ng / mg.

[0116] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 30 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 34 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 35 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 37 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 38 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 40 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 50 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 60 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 65 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 70 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 71 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 72 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 73 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 75 ng / mg.

[0117] In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 80 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 85 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 91 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 92 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 93 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 94 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 90 ng / mg. In some embodiments, the ratio higher than about 20 ng / mg can be higher than about 95 ng / mg.

[0118] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 13 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 12 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 11 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 10 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8.2 ng / mg.

[0119] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.3 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.2 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7.1 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 6 ng / mg.

[0120] In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 5 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 4 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.9 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.8 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.7 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.6 ng / mg. In some embodiments, the second ratio of less than about 14 ng / mg can be less than about 3.5 ng / mg.

[0121] In some embodiments, the higher likelihood of graft function worsening can be about 5% or more. In some embodiments, the higher likelihood of graft function worsening can be about 8% or more. In some embodiments, the higher likelihood of graft function worsening can be about 10% or more. In some embodiments, the higher likelihood of graft function worsening can be about 15% or more. In some embodiments, the higher likelihood of graft function worsening can be about 16% or more.

[0122] In some embodiments, the higher likelihood of graft function worsening can be about 20% or more. In some embodiments, the higher likelihood of graft function worsening can be about 22% or more. In some embodiments, the higher likelihood of graft function worsening can be about 25% or more. In some embodiments, the higher likelihood of graft function worsening can be about 30% or more. In some embodiments, the higher likelihood of graft function worsening can be about 32% or more. In some embodiments, the higher likelihood of graft function worsening can be about 35% or more. In some embodiments, the higher likelihood of graft function worsening can be about 36% or more. In some embodiments, the higher likelihood of graft function worsening can be about 40% or more. In some embodiments, the higher likelihood of graft function worsening can be about 45% or more. In some embodiments, the higher likelihood of graft function worsening can be about 50% or more.

[0123] In some embodiments, the higher likelihood of graft function worsening can be about 5% to about 50%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 35%. In some embodiments, the higher likelihood of graft function worsening can be about 25% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 10% to about 20%. In some embodiments, the higher likelihood of graft function worsening can be about 15% to about 25%. In some embodiments, the higher likelihood of graft function worsening can be about 20% to about 30%. In some embodiments, the higher likelihood of graft function worsening can be about 30% to about 40%. In some embodiments, the higher likelihood of graft function worsening can be about 35% to about 45%. In some embodiments, the higher likelihood of graft function worsening can be about 40% to about 50%.

[0124] In some embodiments, the graft function worsening occurs after 2 years. In some embodiments, the graft function worsening occurs after 3 years. In some embodiments, the graft function worsening occurs after 4 years. In some embodiments, the graft function worsening occurs after 5 years. In some embodiments, the graft function worsening occurs after 6 years. In some embodiments, the graft function worsening occurs after 7 years. In some embodiments, the graft function worsening occurs after 8 years. In some embodiments, the graft function worsening occurs after 9 years. In some embodiments, the graft function worsening occurs within 7 years. In some embodiments, the graft function worsening occurs within 8 years. In some embodiments, the graft function worsening occurs within 9 years.

[0125] In some embodiments, the method can further comprise assessing the probability of graft function worsening based on the ratio and at least one other marker.

[0126] In some embodiments, the at least one other marker can comprise age, gender, time post-transplant at inclusion, urinary albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0127] In some embodiments, the assessed probability can exhibit an area under the receiver operator characteristic (ROC) curve (AUC) of at least about 70 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 80 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 85 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 90 (%). In some embodiments, the assessed probability can exhibit an AUC of at least about 95 (%).

[0128] In some embodiments, the interaction with fetuin-A fragment comprises binding to fetuin-A. In some embodiments, the agent comprises an antibody.

[0129] uPTM-FetA excretion is associated with kidney function and also with markers of proximal tubular injury and increased 24-hour urinary protein excretion. Importantly, uPTM-FetA excretion is independently associated with graft function deterioration in stable KTRs, particularly in patients with relatively good kidney function.

[0130] In some embodiments, in KTRs, shorter time post-transplant, live donor, older donor age, use of proliferation inhibitors, and lower AST levels can be independently associated with higher uPTM-FetA excretion. In some embodiments, urinary L-FABP excretion, a marker of proximal tubular injury, can also be independently associated with uPTM-FetA excretion. In some embodiments, increased uPTM-FetA excretion can be associated with an increased risk of graft function deterioration even after adjustment for potential confounders. In healthy adults, the kidney does not express fetuin-A. However, upon injury, proximal tubular epithelial cells (PTECs) are able to express and release fetuin-A to the luminal side of the tubule. In cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary fetuin-A can be predominantly present in the urinary exosome fraction, but not from non-exosomal fractions. Upon hypoxia-inducible transcription factor stimulation, PTECs can locally produce fetuin-A under hypoxic conditions. The presence of fetuin-A in the proximal tubule can help protect the kidney from hypoxia-induced kidney inflammation by preventing macrophages from turning into pro-inflammatory macrophages M1 and protect the kidney from hypoxia-induced fibrosis by antagonizing TGF-b signaling.

[0131] The human precursor protein of fetuin-A consists of three parts, the A chain, the connecting peptide and the B chain, with lengths of 321, 40 and 27 amino acids, respectively. The connecting peptide of the precursor is removed by post-translational modification, restricted proteolysis, after which only the A and B chain form the active fetuin-A protein. The monoclonal antibody in the ELISA kit can detect fetuin-A containing the connecting peptide. Therefore, the uPTM fetuin-A detected by this ELISA kit can be inactive fetuin-A, without a kidney function protective effect, and elevated uPTM-FetA is associated with an increased risk of eGFR decline in patients. In addition to local production, fetuin-A in urine can also originate from circulation. Fetuin-A is a negatively charged molecule with a molecular weight of approximately 60 kilodaltons, similar to that of albumin. Under physiological conditions, a selective amount of albumin can pass the glomerular filtration barrier (GFB), where it will be reabsorbed in the renal tubular compartment by proximal tubular epithelial cells (PTEC). As fetuin-A and albumin have similar properties, an equivalent handling by the kidney can be expected. Fetuin-A in healthy rats is reabsorbed in the proximal tubule, mainly in the SI segment, to a lesser extent in the S2 segment, by megalin-mediated endocytosis. In case of cumulative damage to the glomerulus, the GFB will be compromised and excess amounts of different types of plasma proteins will leak into the tubular compartment. This leads to an increased amount of filtered proteins that need to be reabsorbed by the proximal tubule. As the reabsorptive capacity of PTEC is not unlimited, once this mechanism is saturated, proteins from circulation will be present in the urine. In addition to this, filtered proteins can cause damage in the proximal tubule by various mechanisms, i.e. by obstructing the lumen with protein casts, by energy depletion and lysosomal rupture due to overload of the amount of proteins that need to be reabsorbed by PTEC, by triggering PTEC to release pro-inflammatory and pro-fibrotic cytokines, by causing intracellular accumulation of lipidized proteins that will trigger apoptosis of the renal tubule, and by activating the complement system to cause direct damage to PTEC. When this happens, the capacity of PTEC to locally produce fetuin-A and reabsorb filtered fetuin-A can be compromised. As uPTM-FetA excretion is significantly associated with an increased risk of graft dysfunction, the origin of uPTM-FetA can be from local production by PTEC, rather than from a compromised reabsorption process, even after adjustment for potential confounders including urinary protein excretion. In a sensitivity analysis, uPTM-FetA can not be associated with graft dysfunction in patients with eGFR below 30 ml / min / 1.73m2. Fetuin-A from circulation can also end up in the urine when there is cumulative damage in the kidney. Therefore, uPTM-FetA measured in this patient subgroup can consist of locally produced fetuin-A and circulating fetuin-A. Fetuin-A in circulation is not associated with kidney function.

[0132] Both acute and chronic kidney injury are closely associated with the occurrence of hypoxia. Among all structures within the kidney organ, PTECs are the most sensitive and vulnerable because these cells are highly active and have a high oxygen demand. Since hypoxia plays an important role in the progression of kidney disease and can exist at a relatively early stage of kidney injury, even before structural damage occurs, early identification of this condition can be beneficial in preventing further deterioration. In cisplatin-induced and ischemia / reperfusion-induced AKI, urinary fetuin-A excretion increases before serum creatinine spikes. Moreover, fetuin-A deposition in PTECs increases before morphologically present structural damage of the renal tubules. In patients with type 2 diabetes, urinary fetuin-A can show progression of worsening kidney function earlier than albuminuria. Since one of the most important requirements for a biomarker is to reflect the underlying pathophysiology of the disease, in some embodiments, measurement of fetuin-A in urine can provide an additional benefit as a biomarker over the currently used parameters for early detection of graft injury in KTRs, such as serum creatinine or proteinuria.

[0133] In some embodiments, correlating fetuin-A with at least one other biomarker associated with the kidney can be an indicator of kidney function or its deterioration. For example, correlating fetuin-A with creatinine can be such an indicator. Kidney function can be judged by the level of filtration that occurs. Filtration is accomplished by tiny blood vessels in the kidneys called "glomeruli." Thus, overall kidney function is referred to as "glomerular filtration rate" (GFR), measured in volume of blood filtered per minute (milliliters per minute). GFR can vary with other variables, such as age and gender. GFR can be estimated (eGFR) using a formula that takes into account your age, gender, and a blood test called creatinine. Kidney failure is most commonly seen when creatinine levels are high, indicating a decrease in kidney function. Creatinine is a molecule produced by your muscles. Normal kidneys remove creatinine from the blood and excrete it through urine. More creatinine in the blood indicates that the kidneys are not cleaning the blood as they should. Thus, correlating fetuin-A with GFR or creatinine can be a relatively reliable indicator of deterioration of kidney function in KTRs.

[0134] Assay

[0135] In some embodiments, various methods, reagents, devices, and kits can be used to measure the level of fetuin-A or fetuin-A based fragments in various media, such as urine, serum, and other bodily fluids. In some embodiments, the level of fetuin-A can be measured using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA can be used to quantitatively measure fetuin-A with specific post-translational modifications (PTMs) in human urine and should be performed by a certified medical professional, such as a medical technician, in a qualified clinical laboratory. For example, a calibrator or unknown urine sample is mixed with an antibody with binding affinity for a portion of fetuin-A, such as an anti-unique PTM fetuin-A monoclonal antibody (mAb), and then incubated in a microplate previously bound with fetuin-A or a segment thereof, such as unique PTM fetuin-A. The monoclonal antibody recognizes fetuin-A in the calibrator or unknown sample under competition in the microplate well. After incubation, in some embodiments, a signal generating molecule, such as a signal molecule-labeled antibody, can be used. In some embodiments, a horseradish peroxidase (HRP)-conjugated secondary antibody is added and then incubated with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison to a predetermined unique PTM fetuin-A calibration curve.

[0136] In some embodiments, a method of correlating likelihood of graft function deterioration in a recipient of a kidney transplant can include obtaining a solution comprising a urine sample from the recipient of the kidney transplant after the kidney transplant and a reagent that interacts with a fetuin A fragment containing a urine-linking peptide; measuring average fetuin A fragment excretion into the urine from the solution; correlating the measured average fetuin A fragment excretion with likelihood of graft function deterioration in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0137] In some embodiments, a method of preparing an assay can include providing a solution comprising a urine sample from a recipient of a kidney transplant after the kidney transplant and a reagent that interacts with a fetuin A fragment containing a urine-linking peptide; measuring average fetuin A fragment excretion into the urine from the solution; correlating the measured average fetuin A fragment excretion with likelihood of graft function deterioration in the recipient, wherein a measured average fetuin A fragment excretion higher than about 0.8 micrograms per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0138] In some embodiments, an assay kit for determining a likelihood of graft function deterioration of a transplanted kidney can include a solution comprising a reagent at a concentration that interacts with an amount of fetuin-A fragment containing a urine-linked peptide in a urine sample from a recipient of a kidney transplant. A device can be provided to measure average fetuin-A fragment excretion into the urine from the solution to correlate the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0139] In some embodiments, a method of correlating a likelihood of graft function deterioration in a recipient of a kidney transplant can include obtaining a solution comprising a urine sample from the recipient of the kidney transplant after the kidney transplant and a reagent that interacts with fetuin-A fragment containing a urine-linked peptide; measuring average fetuin-A fragment excretion into the urine from the solution; correlating the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0140] In some embodiments, a method of preparing an assay can include providing a solution comprising a urine sample from a recipient of a kidney transplant after the kidney transplant and a reagent that interacts with fetuin-A fragment containing a urine-linked peptide; measuring average fetuin-A fragment excretion into the urine from the solution; correlating the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0141] In some embodiments, an assay kit for determining a likelihood of graft function deterioration of a transplanted kidney can include a solution comprising a reagent at a concentration that interacts with an amount of fetuin-A fragment containing a urine-linked peptide in a urine sample from a recipient of a kidney transplant. A device can be provided to measure average fetuin-A fragment excretion into the urine from the solution to correlate the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0142] The present disclosure relates to diagnostic methods using biomarkers, which can be proteins or fragments of proteins that can be considered proteins. Urine samples, serum samples, or both urine and serum samples can be collected from a subject, and urine levels, serum levels, or both levels of biomarkers can be determined by various methods, such as mass spectrometry and immunoassays.

[0143] When a biomarker contains a single protein molecule, its level in a subject can be compared to a reference point to determine a corresponding indication or likelihood of a target condition. A reference point representing a level of the same biomarker, such as a level of the biomarker in normal subjects without substantial worsening of kidney transplant function in KTRs, can be determined based on representative levels of the biomarker in a group of KTRs. For example, it can be a midpoint between the average levels of the two groups. A biomarker level above the reference point indicates the target condition.

[0144] Fetuin-A, such as fetuin-A containing the urine-linking peptide (uC-FetA) (also known as alpha-2-Heremans-Schmid glycoprotein), has characteristics that suggest it can serve as such an alternative, independent, non-invasive marker. Fetuin-A is elevated in the urine of patients with acute kidney injury (AKI), and recent evidence suggests that it is also related to (the extent of) interstitial fibrosis / tubular atrophy (IFTA). The incidence of AKI and the presence of IFTA after kidney transplantation are known to be associated with adverse long-term graft outcomes. Urinary fetuin-A is also elevated in patients with chronic kidney disease, such as autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis. In addition, urinary fetuin-A has previously been shown to be associated with a decline in kidney function in patients with chronic kidney disease, including diabetic nephropathy, and this protein has been proposed for use as a biomarker for early detection of these diseases. However, no studies have been conducted to measure urinary fetuin-A in a KTR population and investigate a plausible association with graft outcomes.

[0145] In some embodiments, fetuin-A containing the urine-linking peptide (uC-FetA) can be used as a biomarker for acute kidney injury, and in its extension, as a biomarker for early detection of a decline in kidney function. In some embodiments, the uC-FetA protein can serve as a marker of worsening graft function in KTRs. For example, uC-FetA levels in stable ambulatory KTRs can be used to determine the likelihood of a higher probability of worsening graft function over time, such as about ten years or less. In some embodiments, the primary endpoint can be death censored graft function, i.e., the need for retransplantation or (re)start of dialysis, and the secondary endpoint is all-cause mortality.

[0146] Because of the potential pathophysiological role of fetuin-A in the development of reduced kidney function, the association of fetuin-A or fragments thereof with graft function deterioration can be used to determine the likelihood of graft function deterioration over time.

[0147] Thus, in some embodiments, a method of associating a likelihood of graft function deterioration in a recipient of a kidney transplant can comprise: obtaining a solution comprising a urine sample from the recipient of the kidney transplant after the kidney transplant and a reagent that interacts with a fetuin-A fragment containing a urine-linked peptide; measuring from the solution an average fetuin-A fragment excretion into the urine; correlating the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0148] In some embodiments, a method of preparing an assay can comprise providing a solution comprising a urine sample from a recipient of a kidney transplant after the kidney transplant and a reagent that interacts with a fetuin-A fragment containing a urine-linked peptide; measuring from the solution an average fetuin-A fragment excretion into the urine; correlating the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0149] In some embodiments, an assay kit for determining a likelihood of graft function deterioration of a transplanted kidney can comprise a solution comprising a reagent at a concentration that interacts with an amount of a fetuin-A fragment containing a urine-linked peptide in a urine sample from a recipient of a kidney transplant. A device such as an ELISA reader can be provided to measure from the solution an average fetuin-A fragment excretion into the urine to correlate the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient, wherein a measured average fetuin-A fragment excretion higher than about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0150] In some embodiments, an excretion higher than about 0.8 pg / h can trigger a medical intervention to inhibit graft function deterioration.

[0151] In some embodiments, graft function deterioration can be indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

[0152] In some embodiments, the recipient can have been instructed to have an eGFR of about 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater at a time before and after the urine sample is collected. 2 ) or greater at a time before and after the urine sample is collected.

[0153] In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 2 hours. In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 4 hours. In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 6 hours. In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 12 hours. In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 24 hours. In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 36 hours. In some embodiments, the average fetuin-A fragment excretion into urine can be averaged over no less than about 48 hours.

[0154] In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 1 week after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 2 weeks after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 1 month after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant. In some embodiments, the urine sample can be collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0155] In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about nine years or less. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about seven years or less. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about five years or less. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about three years or less. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about one year or less.

[0156] In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 0.9 pg / h. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 1 pg / h. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 2 pg / h. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 2.1 pg / h. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 2.2 pg / h. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 2.3 pg / h. In some embodiments, an average fetuin-A fragment excretion of greater than about 0.8 pg / h can be greater than about 2.5 pg / h.

[0157] In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 2 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.5 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.59 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.6 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.7 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.78 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.79 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 1.8 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 2.1 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 2.15 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 2.18 or greater. In some embodiments, the risk ratio for average Fetuin A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin A fragment excretion corresponding to about 0.9 pg / h or less can be about 2.2 or greater.In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2.22 or greater.

[0158] In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 1.5 to about 2.5. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 1.6 to about 2.3. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 1.59 to about 2.22.

[0159] In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2.2 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2.26 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2.5 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2.7 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 3 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 3.5 or more. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 3.6 or more.

[0160] In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 3.7 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 3.8 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 4 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 4.5 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 4.7 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 4.8 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 4.9 or greater. In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 5 or greater.

[0161] In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2 to about 5.

[0162] In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2 to about 4.5.

[0163] In some embodiments, the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less can be about 2.5 to about 4.5.

[0164] In some embodiments, the interaction with Fetuin-A fragment comprises binding to Fetuin-A.

[0165] In some embodiments, the agent comprises an antibody.

[0166] uC-FetA excretion is associated with kidney function and also with increased 24-hour urinary protein excretion and markers of proximal tubular injury. Importantly, uC-FetA excretion is independently associated with graft function deterioration in stable KTRs, particularly in patients with relatively good kidney function.

[0167] In some embodiments, shorter time post-transplant, live donor, older donor age, use of proliferation inhibitors, and lower AST levels can be independently associated with higher 24-hour uC-FetA excretion in KTRs. In some embodiments, 24-hour urinary L-FABP excretion, a marker of proximal tubular injury, can also be independently associated with 24-hour uC-FetA excretion. In some embodiments, increased uC-FetA excretion can be associated with an increased risk of graft function deterioration even after adjustment for potential confounders. In healthy adults, the kidney does not express fetuin-A. However, upon injury, proximal tubular epithelial cells (PTECs) are able to express fetuin-A and release it to the luminal side of the tubule. In cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary fetuin-A can be predominantly present in the urinary exosome fraction, but not from non-exosomal fractions. Upon hypoxia-inducible transcription factor stimulation, PTECs can locally produce fetuin-A under hypoxic conditions. The presence of fetuin-A in the proximal tubule can help protect the kidney from hypoxia-induced kidney inflammation by preventing macrophages from transforming into proinflammatory macrophages M1 and protect the kidney from hypoxia-induced fibrosis by antagonizing TGF-beta signaling.

[0168] The human precursor protein of fetuin-A consists of three parts, the A chain, the connecting peptide and the B chain, with a length of 321, 40 and 27 amino acids, respectively. The connecting peptide of the precursor is removed by post-translational modification, restricted proteolysis, after which only the A and B chain form the active fetuin-A protein. The monoclonal antibody in the ELISA kit can detect fetuin-A containing the connecting peptide. Therefore, the uC-FetA detected by this ELISA kit can be inactive fetuin-A, without a renal function protective effect, and elevated uC-FetA is associated with the risk of eGFR decline in patients. In addition to local production, fetuin-A in urine can also originate from circulation. Fetuin-A is a negatively charged molecule with a molecular weight of approximately 60 kilodaltons, similar to that of albumin. Under physiological conditions, a selective amount of albumin can pass the glomerular filtration barrier (GFB), where it will be reabsorbed in the proximal tubular compartment by proximal tubular epithelial cells (PTEC). Since fetuin-A and albumin have similar properties, an equivalent handling by the kidney can be expected. Fetuin-A in healthy rats is reabsorbed in the proximal tubule, mainly in the SI segment, to a lesser extent in the S2 segment, by megalin-mediated endocytosis. In case of cumulative damage in the glomerulus, the GFB will be disrupted and excess of different types of plasma proteins will leak into the tubular compartment. This leads to an increase in the amount of filtered proteins that need to be reabsorbed by the proximal tubule. Since the reabsorptive capacity of PTEC is not unlimited, once this mechanism is saturated, proteins from circulation will be present in the urine. In addition to this, filtered proteins can cause damage in the proximal tubule by various mechanisms, i.e. by obstructing the lumen with protein casts, by energy depletion and lysosomal rupture due to overload of the amount of proteins that need to be reabsorbed by PTEC, by triggering PTEC to release pro-inflammatory and pro-fibrotic cytokines, by causing intracellular accumulation of lipidized proteins that will trigger apoptosis of the tubule, and by activating the complement system to cause direct damage to PTEC. When this happens, the capacity of PTEC to locally produce fetuin-A and reabsorb filtered fetuin-A can be impaired. Since 24-hour uC-FetA excretion is significantly associated with an increased risk of graft dysfunction, the origin of uC-FetA can be from local production by PTEC, rather than from impaired reabsorption processes, even after adjustment for potential confounders including 24-hour urinary protein excretion. In a sensitivity analysis, uC-FetA can not be associated with graft dysfunction in patients with eGFR below 30 ml / min / 1.73m2. Fetuin-A from circulation can also end up in the urine when there is cumulative damage in the kidney. Therefore, uC-FetA measured in this patient subgroup can consist of locally produced fetuin-A and circulating fetuin-A. Fetuin-A in circulation is not associated with renal function.

[0169] Both acute and chronic kidney injury are closely associated with the occurrence of hypoxia. Among all structures within the kidney organ, PTECs are the most sensitive and vulnerable because these cells are highly active and have a high oxygen demand. Since hypoxia plays an important role in the progression of kidney disease and can exist at a relatively early stage of kidney injury, even before structural damage occurs, early identification of this condition can be beneficial in preventing further deterioration. In cisplatin-induced and ischemia / reperfusion-induced AKI, urinary fetuin-A excretion increases before serum creatinine spikes. Furthermore, fetuin-A deposition in PTECs increases before morphologically present structural damage of the renal tubules. In patients with type 2 diabetes, urinary fetuin-A can show progression of worsening kidney function earlier than albuminuria. Since one of the most important requirements for a biomarker is to reflect the underlying pathophysiology of the disease, in some embodiments, measurement of fetuin-A in urine can provide an additional benefit as a biomarker over the currently used parameters for early detection of graft injury in KTRs, such as serum creatinine or proteinuria.

[0170] Assay

[0171] In some embodiments, various methods, reagents, devices, and kits can be used to measure the level of fetuin-A or fragments based on fetuin-A in various media, such as urine, serum, and other bodily fluids. In some embodiments, the level of fetuin-A can be measured using an enzyme-linked immunosorbent assay (ELISA). For example, an ELISA can be used to quantitatively measure fetuin-A with specific post-translational modifications (PTMs) in human urine and should be performed by a certified medical professional, such as a medical technician, in a qualified clinical laboratory. For example, a calibrator or unknown urine sample is mixed with an antibody that has binding affinity for a portion of fetuin-A, such as an anti-unique PTM fetuin-A monoclonal antibody (mAb), and then incubated in a microplate that has been pre-bound with fetuin-A or a segment thereof, such as a unique PTM fetuin-A. The monoclonal antibody recognizes fetuin-A in the calibrator or unknown sample under competition in the microplate well. After incubation, in some embodiments, a signal generating molecule, such as a signal molecule-labeled antibody, can be used. In some embodiments, a horseradish peroxidase (HRP)-conjugated secondary antibody is added and then incubated with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison to a predetermined unique PTM fetuin-A calibration curve.

[0172] First Set of Non-limiting Embodiments

[0173] The present disclosure is also described by the following first non-limiting set of embodiments. However, the use of these and other embodiments anywhere in the specification only is illustrative. Accordinly, the disclosure is not limited to any particular preferred embodiment or aspect described herein. Rather, the disclosure is capable of modification and adaptation in various ways while still coming within the spirit or scope of the disclosure.

[0174] 1. A method of preparing an assay, the method comprising:

[0175] providing a first solution comprising a urine sample from a recipient of a kidney transplant after the kidney transplant and a first reagent that interacts with a post-translationally modified fetuin-A fragment in a urine fragment to assay a level of the fetuin-A fragment in the urine sample;

[0176] providing a second solution comprising the urine sample and a second reagent that interacts with urine creatinine to assay a level of the urine creatinine in the urine sample;

[0177] in response to the assayed level of the fetuin-A fragment and the assayed level of the urine creatinine, correlating a ratio of the assayed level of the fetuin-A fragment to the assayed level of the urine creatinine to a likelihood of a worsening of graft function in the recipient,

[0178] wherein the first solution and the second solution are the same solution or different solutions from each other.

[0179] 2. The method of embodiment 1, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg triggers a medical intervention to avoid the worsening of graft function.

[0180] 3. The method of any of the preceding embodiments, wherein the higher likelihood is higher than a likelihood of a worsening of graft function in a comparable recipient of a kidney transplant having a ratio of the assayed level of the fetuin-A fragment to the level of the urine creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg in a time period of about ten years or less.

[0181] 4. The method of any of the preceding embodiments, wherein the worsening of graft function is indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

[0182] 5. The method of any of the preceding embodiments, wherein the worsening of graft function is indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0183] 6. The method of any of the preceding embodiments, wherein the interaction with the fetuin-A fragment comprises binding to the fetuin-A fragment.

[0184] 7. The method of any of the preceding embodiments, wherein the interaction with the urine creatinine comprises binding to the urine creatinine.

[0185] 8. The method of any of the preceding embodiments, wherein at least one of the first agent and the second agent comprises an antibody.

[0186] 9. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 25 ng / mg.

[0187] 10. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 25.5 ng / mg.

[0188] 11. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 26 ng / mg.

[0189] 12. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 27 ng / mg.

[0190] 13. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 27.5 ng / mg.

[0191] 14. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 28 ng / mg.

[0192] 15. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 28.5 ng / mg.

[0193] 16. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 28.7 ng / mg.

[0194] 17. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 28.8 ng / mg.

[0195] 18. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 28.9 ng / mg.

[0196] 19. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 29.0 ng / mg.

[0197] 20. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 29.1 ng / mg.

[0198] 21. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 29.2 ng / mg.

[0199] 22. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 29.3 ng / mg.

[0200] 23. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 30 ng / mg.

[0201] 24. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 34 ng / mg.

[0202] 25. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 35 ng / mg.

[0203] 26. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 37 ng / mg.

[0204] 27. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 38 ng / mg.

[0205] 28. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 40 ng / mg.

[0206] 29. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 50 ng / mg.

[0207] 30. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 60 ng / mg.

[0208] 31. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 65 ng / mg.

[0209] 32. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 70 ng / mg.

[0210] 33. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 71 ng / mg.

[0211] 34. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 72 ng / mg.

[0212] 35. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 73 ng / mg.

[0213] 36. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 75 ng / mg.

[0214] 37. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 80 ng / mg.

[0215] 38. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 85 ng / mg.

[0216] 39. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 90 ng / mg.

[0217] 40. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 91 ng / mg.

[0218] 41. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 92 ng / mg.

[0219] 42. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 93 ng / mg.

[0220] 43. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 94 ng / mg.

[0221] 44. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 90 ng / mg.

[0222] 45. The method of any of the preceding embodiments, wherein the ratio above about 20 ng / mg is above about 95 ng / mg.

[0223] 46. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 13 ng / mg.

[0224] 47. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 12 ng / mg.

[0225] 48. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 11 ng / mg.

[0226] 49. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 10 ng / mg.

[0227] 50. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 9 ng / mg.

[0228] 51. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 8.8 ng / mg.

[0229] 52. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 8.5 ng / mg.

[0230] 53. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 8.4 ng / mg.

[0231] 54. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 8.2 ng / mg.

[0232] 55. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 8 ng / mg.

[0233] 56. The method of any of the preceding embodiments, wherein the second ratio below about 14 ng / mg is below about 7.5 ng / mg.

[0234] 57. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 7.4 ng / mg.

[0235] 58. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 3.3 ng / mg.

[0236] 59. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 7.3 ng / mg.

[0237] 60. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 7.2 ng / mg.

[0238] 61. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 7.1 ng / mg.

[0239] 62. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 7 ng / mg.

[0240] 63. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 6 ng / mg.

[0241] 64. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 5 ng / mg.

[0242] 65. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 4 ng / mg.

[0243] 66. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 3.9 ng / mg.

[0244] 67. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 3.8 ng / mg.

[0245] 68. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 3.7 ng / mg.

[0246] 69. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 3.6 ng / mg.

[0247] 70. The method of any of the preceding embodiments, wherein the second ratio of less than about 14 ng / mg is less than about 3.5 ng / mg.

[0248] 71. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 5% or more.

[0249] 72. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 8% or more.

[0250] 73. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 10% or more.

[0251] 74. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 15% or more.

[0252] 75. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 16% or more.

[0253] 76. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 20% or more.

[0254] 77. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 22% or more.

[0255] 78. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 25% or more.

[0256] 79. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 30% or more.

[0257] 80. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 32% or more.

[0258] 81. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 35% or more.

[0259] 82. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 36% or more.

[0260] 83. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 40% or more.

[0261] 84. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 45% or more.

[0262] 85. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 50% or more.

[0263] 86. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 5% to about 50%.

[0264] 87. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 10% to about 45%.

[0265] 88. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 20% to about 40%.

[0266] 89. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 25% to about 35%.

[0267] 90. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 25% to about 30%.

[0268] 91. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 10% to about 20%.

[0269] 92. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 15% to about 25%.

[0270] 93. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 20% to about 30%.

[0271] 94. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 30% to about 40%.

[0272] 95. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 35% to about 45%.

[0273] 96. The method of any of the preceding embodiments, wherein the higher likelihood of worsening graft function is about 40% to about 50%.

[0274] 97. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 2 years.

[0275] 98. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 3 years.

[0276] 99. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 4 years.

[0277] 100. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 5 years.

[0278] 101. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 6 years.

[0279] 102. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 7 years.

[0280] 103. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 8 years.

[0281] 104. The method of any of the preceding embodiments, wherein the worsening graft function occurs after 9 years.

[0282] 105. The method of any of the preceding embodiments, wherein the worsening graft function occurs within 7 years.

[0283] 106. The method of any of the preceding embodiments, wherein the worsening graft function occurs within 8 years.

[0284] 107. The method of any of the preceding embodiments, wherein the worsening graft function occurs within 9 years.

[0285] 108. The method of any of the preceding embodiments, further comprising assessing the probability of worsening graft function based on the ratio and at least one other marker.

[0286] 109. The method of embodiment 108, wherein the at least one other marker comprises age, sex, time post-transplant at inclusion, urinary albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0287] 110. The method of embodiment 109, wherein the probability assessed exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%).

[0288] 111. The method of embodiment 110, wherein the probability assessed exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 80 (%).

[0289] 112. The method of embodiment 110, wherein the probability assessed exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 85 (%).

[0290] 113. The method of embodiment 110, wherein the probability assessed exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 90 (%).

[0291] 114. The method of embodiment 110, wherein the probability assessed exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 95 (%).

[0292] 115. An assay kit for determining the likelihood of progressive decline in kidney function, the assay kit comprising:

[0293] a first solution comprising a first reagent that interacts with a post-translationally modified fetuin-A fragment in a urine fragment to indicate a level of the fetuin-A fragment in a urine sample from a recipient of a kidney transplant after the kidney transplant;

[0294] a second solution comprising a second reagent that interacts with urine creatinine to indicate a level of the urine creatinine in the urine sample;

[0295] a device for assaying the level of the fetuin-A fragment and the level of the urine creatinine in the urine sample to determine a ratio of the assayed level of the fetuin-A fragment to the assayed level of the urine creatinine,

[0296] wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, indicates a higher likelihood of graft function worsening in a period of about ten years or less, and

[0297] wherein the first solution and the second solution are the same solution or different solutions from each other.

[0298] 116. The assay kit of embodiment 115, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg triggers a medical intervention to avoid worsening of graft function.

[0299] 117. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood is higher than a likelihood of graft function worsening within a period of about ten years or less in a comparative recipient of a kidney transplant having a lower ratio of the determined level of the Fetuin-A fragment to the level of the urinary creatinine lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0300] 118. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function is indicated by doubling of serum creatinine, need for re- kidney transplantation, or need for dialysis.

[0301] 119. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function is indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0302] 120. The assay kit of any of the preceding embodiments following 115, wherein the interaction with the Fetuin-A fragment comprises binding to the Fetuin-A fragment.

[0303] 121. The assay kit of any of the preceding embodiments following 115, wherein the interaction with the urinary creatinine comprises binding to the urinary creatinine.

[0304] 122. The assay kit of any of the preceding embodiments following 115, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0305] 123. The assay kit of any of the preceding embodiments following 115, wherein the ratio higher than about 20 ng / mg is higher than about 25 ng / mg.

[0306] 124. The assay kit of any of the preceding embodiments following 115, wherein the ratio higher than about 20 ng / mg is higher than about 25.5 ng / mg.

[0307] 125. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 26 ng / mg.

[0308] 126. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 27 ng / mg.

[0309] 127. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 27.5 ng / mg.

[0310] 128. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 28 ng / mg.

[0311] 129. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 28.5 ng / mg.

[0312] 130. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 28.7 ng / mg.

[0313] 131. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 28.8 ng / mg.

[0314] 132. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 28.9 ng / mg.

[0315] 133. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 29.0 ng / mg.

[0316] 134. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 29.1 ng / mg.

[0317] 135. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 29.2 ng / mg.

[0318] 136. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 29.3 ng / mg.

[0319] 137. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 30 ng / mg.

[0320] 138. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 34 ng / mg.

[0321] 139. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 35 ng / mg.

[0322] 140. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 37 ng / mg.

[0323] 141. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 38 ng / mg.

[0324] 142. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 40 ng / mg.

[0325] 143. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 50 ng / mg.

[0326] 144. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 60 ng / mg.

[0327] 145. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 65 ng / mg.

[0328] 146. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 70 ng / mg.

[0329] 147. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 71 ng / mg.

[0330] 148. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 72 ng / mg.

[0331] 149. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 73 ng / mg.

[0332] 150. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 75 ng / mg.

[0333] 151. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 80 ng / mg.

[0334] 152. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 85 ng / mg.

[0335] 153. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 90 ng / mg.

[0336] 154. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 91 ng / mg.

[0337] 155. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 92 ng / mg.

[0338] 156. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 93 ng / mg.

[0339] 157. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 94 ng / mg.

[0340] 158. The assay kit of any of the preceding embodiments following 115, wherein the ratio above about 20 ng / mg is above about 90 ng / mg.

[0341] 159. The assay kit of any of the preceding embodiments following 115, wherein a ratio above about 20 ng / mg is above about 95 ng / mg.

[0342] 160. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 13 ng / mg.

[0343] 161. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 12 ng / mg.

[0344] 162. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 11 ng / mg.

[0345] 163. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 10 ng / mg.

[0346] 164. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 9 ng / mg.

[0347] 165. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 8.8 ng / mg.

[0348] 166. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 8.5 ng / mg.

[0349] 167. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 8.4 ng / mg.

[0350] 168. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 8.2 ng / mg.

[0351] 169. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 8 ng / mg.

[0352] 170. The assay kit of any of the preceding embodiments following 115, wherein a second ratio below about 14 ng / mg is below about 7.5 ng / mg.

[0353] 171. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 7.4 ng / mg.

[0354] 172. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 3.3 ng / mg.

[0355] 173. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 7.3 ng / mg.

[0356] 174. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 7.2 ng / mg.

[0357] 175. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 7.1 ng / mg.

[0358] 176. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 7 ng / mg.

[0359] 177. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 6 ng / mg.

[0360] 178. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 5 ng / mg.

[0361] 179. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 4 ng / mg.

[0362] 180. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 3.9 ng / mg.

[0363] 181. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 3.8 ng / mg.

[0364] 182. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 3.7 ng / mg.

[0365] 183. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 3.6 ng / mg.

[0366] 184. The assay kit of any of the preceding embodiments following 115, wherein the second ratio of less than about 14 ng / mg is less than about 3.5 ng / mg.

[0367] 185. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 5% or more.

[0368] 186. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 8% or more.

[0369] 187. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 10% or more.

[0370] 188. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 15% or more.

[0371] 189. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 16% or more.

[0372] 190. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 20% or more.

[0373] 191. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 22% or more.

[0374] 192. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 25% or more.

[0375] 193. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of graft function deterioration is about 30% or more.

[0376] 194. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 32% or more.

[0377] 195. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 35% or more.

[0378] 196. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 36% or more.

[0379] 197. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 40% or more.

[0380] 198. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 45% or more.

[0381] 199. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 50% or more.

[0382] 200. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is from about 5% to about 50%.

[0383] 201. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is from about 10% to about 45%.

[0384] 202. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is from about 20% to about 40%.

[0385] 203. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is from about 25% to about 35%.

[0386] 204. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is from about 25% to about 30%.

[0387] 205. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 10% or up to about 20%.

[0388] 206. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 15% or up to about 25%.

[0389] 207. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 20% or up to about 30%.

[0390] 208. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 30% or up to about 40%.

[0391] 209. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 35% or up to about 45%.

[0392] 210. The assay kit of any of the preceding embodiments following 115, wherein the higher likelihood of worsening graft function is about 40% or up to about 50%.

[0393] 211. The assay kit of any of the preceding embodiments following 115, wherein the worsening graft function occurs after 2 years.

[0394] 212. The assay kit of any of the preceding embodiments following 115, wherein the worsening graft function occurs after 3 years.

[0395] 213. The assay kit of any of the preceding embodiments following 115, wherein the worsening graft function occurs after 4 years.

[0396] 214. The assay kit of any of the preceding embodiments following 115, wherein the worsening graft function occurs after 5 years.

[0397] 215. The assay kit of any of the preceding embodiments following 115, wherein the worsening graft function occurs after 6 years.

[0398] 216. The assay kit of any of the preceding embodiments following 115, wherein the worsening graft function occurs after 7 years.

[0399] 217. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function occurs after 8 years.

[0400] 218. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function occurs after 9 years.

[0401] 219. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function occurs within 7 years.

[0402] 220. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function occurs within 8 years.

[0403] 221. The assay kit of any of the preceding embodiments following 115, wherein the worsening of graft function occurs within 9 years.

[0404] 222. The assay kit of any of the preceding embodiments following 115, wherein the device is used to assay the level of fetuin-A fragment and the level of urine creatinine to assess the probability of the worsening of graft function based on the ratio and at least one other marker.

[0405] 223. The assay kit of embodiment 222, wherein the at least one other marker comprises age, gender, time post-transplant at inclusion, urine albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0406] 224. The assay kit of embodiment 223, wherein the assessed probability displays an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%).

[0407] 225. The assay kit of embodiment 224, wherein the assessed probability displays the AUC of at least about 80 (%).

[0408] 226. The assay kit of embodiment 224, wherein the assessed probability displays the AUC of at least about 85 (%).

[0409] 227. The assay kit of embodiment 224, wherein the assessed probability displays the AUC of at least about 90 (%).

[0410] 228. The assay kit of embodiment 224, wherein the assessed probability displays the AUC of at least about 95 (%).

[0411] 229. A method of associating a likelihood of worsening graft function, the method comprising:

[0412] determining levels of a first biomarker and a second biomarker in a urine sample from a recipient of a kidney transplant after the kidney transplant, wherein the first biomarker is a fragment of a fragment of post-translationally modified fetuin-A in a urine fragment, wherein the second biomarker is urine creatinine;

[0413] determining a ratio of the level of the first biomarker to the level of the second biomarker;

[0414] associating the ratio with a likelihood of worsening graft function in the recipient over a period of about ten years,

[0415] wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, higher than about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, indicates a higher likelihood of worsening graft function over a period of about ten years or less.

[0416] 230. The method of embodiment 229, wherein a ratio higher than about 20 ng / mg, higher than about 21 ng / mg, higher than about 22 ng / mg, about 23 ng / mg, more preferably higher than about 23.5 ng / mg, more preferably higher than about 24 ng / mg, triggers a medical intervention to avoid the worsening graft function.

[0417] 231. The method of any of the preceding embodiments following 229, wherein the higher likelihood is higher than a lower likelihood of worsening graft function over a period of about ten years or less in a comparative recipient of a kidney transplant having a ratio of the level of the fetuin-A fragment to the level of the urine creatinine determined lower than about 16 ng / mg, lower than about 15.5 ng / mg, lower than about 15 ng / mg, lower than about 14.5 ng / mg, lower than about 14 ng / mg.

[0418] 232. The method of any of the preceding embodiments following 229, wherein the worsening graft function is indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

[0419] 233. The method of any of the preceding embodiments following 229, wherein the worsening graft function is indicated by a decrease in estimated glomerular filtration rate (eGFR).

[0420] 234. The method of any of the preceding embodiments following 229, wherein the interaction with the fetuin A fragment comprises binding to the fetuin A fragment.

[0421] 235. The method of any of the preceding embodiments following 229, wherein the interaction with the urine creatinine comprises binding to the urine creatinine.

[0422] 236. The method of any of the preceding embodiments following 229, wherein at least one of the first agent and the second agent comprises an antibody.

[0423] 237. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 25 ng / mg.

[0424] 238. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 25.5 ng / mg.

[0425] 239. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 26 ng / mg.

[0426] 240. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 27 ng / mg.

[0427] 241. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 27.5 ng / mg.

[0428] 242. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 28 ng / mg.

[0429] 243. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 28.5 ng / mg.

[0430] 244. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 28.7 ng / mg.

[0431] 245. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 28.8 ng / mg.

[0432] 246. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 28.9 ng / mg.

[0433] 247. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 29.0 ng / mg.

[0434] 248. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 29.1 ng / mg.

[0435] 249. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 29.2 ng / mg.

[0436] 250. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 29.3 ng / mg.

[0437] 251. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 30 ng / mg.

[0438] 252. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 34 ng / mg.

[0439] 253. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 35 ng / mg.

[0440] 254. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 37 ng / mg.

[0441] 255. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 38 ng / mg.

[0442] 256. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 40 ng / mg.

[0443] 257. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 50 ng / mg.

[0444] 258. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 60 ng / mg.

[0445] 259. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 65 ng / mg.

[0446] 260. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 70 ng / mg.

[0447] 261. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 71 ng / mg.

[0448] 262. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 72 ng / mg.

[0449] 263. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 73 ng / mg.

[0450] 264. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 75 ng / mg.

[0451] 265. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 80 ng / mg.

[0452] 266. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 85 ng / mg.

[0453] 267. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 90 ng / mg.

[0454] 268. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 91 ng / mg.

[0455] 269. The method of any of the preceding embodiments following 229, wherein the ratio above about 20 ng / mg is above about 92 ng / mg.

[0456] 270. The method of any of the preceding embodiments after 229, wherein the ratio above about 20 ng / mg is above about 93 ng / mg.

[0457] 271. The method of any of the preceding embodiments after 229, wherein the ratio above about 20 ng / mg is above about 94 ng / mg.

[0458] 272. The method of any of the preceding embodiments after 229, wherein the ratio above about 20 ng / mg is above about 90 ng / mg.

[0459] 273. The method of any of the preceding embodiments after 229, wherein the ratio above about 20 ng / mg is above about 95 ng / mg.

[0460] 274. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 13 ng / mg.

[0461] 275. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 12 ng / mg.

[0462] 276. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 11 ng / mg.

[0463] 277. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 10 ng / mg.

[0464] 278. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 9 ng / mg.

[0465] 279. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 8.8 ng / mg.

[0466] 280. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 8.5 ng / mg.

[0467] 281. The method of any of the preceding embodiments after 229, wherein the second ratio below about 14 ng / mg is below about 8.4 ng / mg.

[0468] 282. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 8.2 ng / mg.

[0469] 283. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 8 ng / mg.

[0470] 284. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 7.5 ng / mg.

[0471] 285. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 7.4 ng / mg.

[0472] 286. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 3.3 ng / mg.

[0473] 287. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 7.3 ng / mg.

[0474] 288. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 7.2 ng / mg.

[0475] 289. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 7.1 ng / mg.

[0476] 290. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 7 ng / mg.

[0477] 291. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 6 ng / mg.

[0478] 292. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 5 ng / mg.

[0479] 293. The method of any of the preceding embodiments after 229, wherein the second ratio of less than about 14 ng / mg is less than about 4 ng / mg.

[0480] 294. The method of any of the preceding embodiments following 229, wherein the second ratio of less than about 14 ng / mg is less than about 3.9 ng / mg.

[0481] 295. The method of any of the preceding embodiments following 229, wherein the second ratio of less than about 14 ng / mg is less than about 3.8 ng / mg.

[0482] 296. The method of any of the preceding embodiments following 229, wherein the second ratio of less than about 14 ng / mg is less than about 3.7 ng / mg.

[0483] 297. The method of any of the preceding embodiments following 229, wherein the second ratio of less than about 14 ng / mg is less than about 3.6 ng / mg.

[0484] 298. The method of any of the preceding embodiments following 229, wherein the second ratio of less than about 14 ng / mg is less than about 3.5 ng / mg.

[0485] 299. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 5% or more.

[0486] 300. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 8% or more.

[0487] 301. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 10% or more.

[0488] 302. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 15% or more.

[0489] 303. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 16% or more.

[0490] 304. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 20% or more.

[0491] 305. The method of any of the preceding embodiments following 229, wherein the higher likelihood of graft function deterioration is about 22% or more.

[0492] 306. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 25% or more.

[0493] 307. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 30% or more.

[0494] 308. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 32% or more.

[0495] 309. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 35% or more.

[0496] 310. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 36% or more.

[0497] 311. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 40% or more.

[0498] 312. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 45% or more.

[0499] 313. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 50% or more.

[0500] 314. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is from about 5% to about 50%.

[0501] 315. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is from about 10% to about 45%.

[0502] 316. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is from about 20% to about 40%.

[0503] 317. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is from about 25% to about 35%.

[0504] 318. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 25% or up to about 30%.

[0505] 319. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 10% or up to about 20%.

[0506] 320. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 15% or up to about 25%.

[0507] 321. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 20% or up to about 30%.

[0508] 322. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 30% or up to about 40%.

[0509] 323. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 35% or up to about 45%.

[0510] 324. The method of any of the foregoing embodiments following 229, wherein the higher likelihood of worsening graft function is about 40% or up to about 50%.

[0511] 325. The method of any of the foregoing embodiments following 229, wherein the worsening graft function occurs after 2 years.

[0512] 326. The method of any of the foregoing embodiments following 229, wherein the worsening graft function occurs after 3 years.

[0513] 327. The method of any of the foregoing embodiments following 229, wherein the worsening graft function occurs after 4 years.

[0514] 328. The method of any of the foregoing embodiments following 229, wherein the worsening graft function occurs after 5 years.

[0515] 329. The method of any of the foregoing embodiments following 229, wherein the worsening graft function occurs after 6 years.

[0516] 330. The method of any of the foregoing embodiments following 229, wherein the worsening graft function occurs after 7 years.

[0517] 331. The method of any of the preceding embodiments after 229, wherein the graft function deterioration occurs after 8 years.

[0518] 332. The method of any of the preceding embodiments after 229, wherein the graft function deterioration occurs after 9 years.

[0519] 333. The method of any of the preceding embodiments after 229, wherein the graft function deterioration occurs within 7 years.

[0520] 334. The method of any of the preceding embodiments after 229, wherein the graft function deterioration occurs within 8 years.

[0521] 335. The method of any of the preceding embodiments after 229, wherein the graft function deterioration occurs within 9 years.

[0522] 336. The method of any of the preceding embodiments after 229, further comprising assessing the probability of the graft function deterioration based on the ratio and at least one other marker.

[0523] 337. The method of embodiment 336, wherein the at least one other marker comprises age, sex, time post-transplant at inclusion, urinary albumin-to-creatinine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

[0524] 338. The method of embodiment 337, wherein the assessed probability exhibits an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%).

[0525] 339. The method of embodiment 338, wherein the assessed probability exhibits the AUC of at least about 80 (%).

[0526] 340. The method of embodiment 338, wherein the assessed probability exhibits the AUC of at least about 85 (%).

[0527] 341. The method of embodiment 338, wherein the assessed probability exhibits the AUC of at least about 90 (%).

[0528] 342. The method of embodiment 338, wherein the assessed probability exhibits the AUC of at least about 95 (%).

[0529] Second Non-Limiting Set of Embodiments

[0530] The present disclosure is also described by the following second, non-limiting set of embodiments. However, the use of these and other embodiments anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure. Likewise, the disclosure is not limited to any particular preferred embodiments or aspects described herein. Indeed, modifications and variations such as would be apparent to those of ordinary skill in the art can be made in the described embodiments, and such variations fall within the scope of the present disclosure.

[0531] 1. A method of preparing an assay, the method comprising:

[0532] providing a solution comprising a urine sample from a recipient of a kidney transplant after the kidney transplant and a reagent that interacts with fetuin A fragments with a urine-linked peptide;

[0533] measuring average fetuin A fragment excretion into the urine from the solution;

[0534] correlating the measured average fetuin A fragment excretion with a likelihood of graft function deterioration in the recipient,

[0535] wherein a measured average fetuin A fragment excretion above about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a time period of about ten years or less.

[0536] 2. The method of embodiment 1, wherein the excretion above about 0.8 pg / h triggers a medical intervention to inhibit the graft function deterioration.

[0537] 3. The method of any one of embodiments 1-2, wherein the graft function deterioration is indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

[0538] 4. The method of any one of embodiments 1-3, wherein the recipient has been indicated to have an eGFR of about 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 ) or higher at a time before and after the urine sample is collected.

[0539] 5. The method of any one of embodiments 1-4, wherein the average fetuin A fragment excretion into urine is averaged over no less than about 2 hours.

[0540] 6. The method of any one of embodiments 1-4, wherein the average fetuin A fragment excretion into urine is averaged over no less than about 4 hours.

[0541] 7. The method of any one of embodiments 1-4, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 6 hours.

[0542] 8. The method of any one of embodiments 1-4, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 12 hours.

[0543] 9. The method of any one of embodiments 1-4, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 24 hours.

[0544] 10. The method of any one of embodiments 1-4, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 36 hours.

[0545] 11. The method of any one of embodiments 1-4, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 48 hours.

[0546] 12. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant.

[0547] 13. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant.

[0548] 14. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 week after the kidney transplant.

[0549] 15. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 weeks after the kidney transplant.

[0550] 16. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 month after the kidney transplant.

[0551] 17. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant.

[0552] 18. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant.

[0553] 19. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant.

[0554] 20. The method of any one of embodiments 1-11, wherein the urine sample is collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0555] 21. The method of any one of embodiments 1-20, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney within a time period of about nine years or less.

[0556] 22. The method of any one of embodiments 1-20, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney within a time period of about seven years or less.

[0557] 23. The method of any one of embodiments 1-20, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney within a time period of about five years or less.

[0558] 24. The method of any one of embodiments 1-20, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney within a time period of about three years or less.

[0559] 25. The method of any one of embodiments 1-20, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h indicates a higher likelihood of graft function deterioration of the transplanted kidney within a time period of about one year or less.

[0560] 26. The method of any one of embodiments 1-25, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h is above about 0.9 pg / h.

[0561] 27. The method of any one of embodiments 1-25, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h is above about 1 pg / h.

[0562] 28. The method of any one of embodiments 1-25, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2 pg / h.

[0563] 29. The method of any one of embodiments 1-25, wherein an average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.1 pg / h.

[0564] 30. The method of any of embodiments 1-25, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.2 pg / h.

[0565] 31. The method of any of embodiments 1-25, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.3 pg / h.

[0566] 32. The method of any of embodiments 1-25, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.5 pg / h.

[0567] 33. The method of any of embodiments 1-32, wherein the hazard ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 or greater.

[0568] 34. The method of any of embodiments 1-32, wherein the hazard ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.5 or greater.

[0569] 35. The method of any of embodiments 1-32, wherein the hazard ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.59 or greater.

[0570] 36. The method of any of embodiments 1-32, wherein the hazard ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.6 or greater.

[0571] 37. The method of any of embodiments 1-32, wherein the hazard ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.7 or greater.

[0572] 38. The method of any of embodiments 1-32, wherein the hazard ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.78 or greater.

[0573] 39. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.79 or greater.

[0574] 40. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.8 or greater.

[0575] 41. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.1 or greater.

[0576] 42. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.15 or greater.

[0577] 43. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.18 or greater.

[0578] 44. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.2 or greater.

[0579] 45. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.22 or greater.

[0580] 46. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.5 to about 2.5.

[0581] 47. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.6 to about 2.3.

[0582] 48. The method of any of embodiments 1-32, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.59 to about 2.22.

[0583] 49. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2 or greater.

[0584] 50. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.2 or greater.

[0585] 51. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.26 or greater.

[0586] 52. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.5 or greater.

[0587] 53. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.7 or greater.

[0588] 54. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3 or greater.

[0589] 55. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.5 or more.

[0590] 56. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.6 or more.

[0591] 57. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.7 or more.

[0592] 58. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.7 or more.

[0593] 59. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4 or more.

[0594] 60. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4.5 or more.

[0595] 61. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4.7 or more.

[0596] 62. The method of any of embodiments 1-48, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4.8 or more.

[0597] 63. The method of any of embodiments 1-48, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4.9 or more.

[0598] 64. The method of any of embodiments 1-48, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 5 or more.

[0599] 65. The method of any of embodiments 1-48, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is from about 2 to about 5.

[0600] 66. The method of any of embodiments 1-48, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is from about 2 to about 5.

[0601] 67. The method of any of embodiments 1-48, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is from about 2.5 to about 5.

[0602] 68. The method of any of embodiments 1-48, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is from about 3.5 to about 5.

[0603] 69. The method of any of embodiments 1-68, wherein the interaction with the Fetuin-A fragment comprises binding to the Fetuin-A.

[0604] 70. The method of any of embodiments 1-68, wherein the agent comprises an antibody.

[0605] 71. An assay kit for determining the likelihood of a worsening of graft function of a transplanted kidney, the assay kit comprising:

[0606] a solution comprising an agent at a concentration that interacts with an amount of Fetuin-A fragment comprising a urinary connecting peptide in a urine sample from a recipient of a kidney transplant;

[0607] an apparatus for measuring average fetuin-A fragment excretion into urine from the solution to correlate the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient,

[0608] wherein a measured average fetuin-A fragment excretion above about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a period of about ten years or less.

[0609] 72. The assay kit of embodiment 71, wherein the excretion above about 0.8 pg / h triggers a medical intervention to inhibit the graft function deterioration.

[0610] 73. The assay kit of any one of embodiments 71-72, wherein the graft function deterioration is indicated by doubling of serum creatinine, need for re- kidney transplantation, or need for dialysis.

[0611] 74. The assay kit of any one of embodiments 71-73, wherein the recipient has an eGFR of about 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 ) or higher at a time before and after the urine sample is collected.

[0612] 75. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 2 hours.

[0613] 76. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 4 hours.

[0614] 77. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 6 hours.

[0615] 78. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 12 hours.

[0616] 79. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 24 hours.

[0617] 80. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 36 hours.

[0618] 81. The assay kit of any one of embodiments 71-74, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 48 hours.

[0619] 82. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant.

[0620] 83. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant.

[0621] 84. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 week after the kidney transplant.

[0622] 85. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 weeks after the kidney transplant.

[0623] 86. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 month after the kidney transplant.

[0624] 87. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant.

[0625] 88. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant.

[0626] 89. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant.

[0627] 90. The assay kit of any one of embodiments 71-81, wherein the urine sample is collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0628] 91. The assay kit of any one of embodiments 71-90, wherein a fetuin-A fragment excretion higher than about 0.8 pg / h indicates a higher likelihood of a graft dysfunction of the transplanted kidney over a period of about nine years or less.

[0629] 92. The assay kit of any of embodiments 71-90, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a period of about seven years or less.

[0630] 93. The assay kit of any of embodiments 71-90, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a period of about five years or less.

[0631] 94. The assay kit of any of embodiments 71-90, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a period of about three years or less.

[0632] 95. The assay kit of any of embodiments 71-90, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a period of about one year or less.

[0633] 96. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 0.9 pg / h.

[0634] 97. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 1 pg / h.

[0635] 98. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2 pg / h.

[0636] 99. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.1 pg / h.

[0637] 100. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.2 pg / h.

[0638] 101. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.3 pg / h.

[0639] 102. The assay kit of any of embodiments 71-95, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.5 pg / h.

[0640] 103. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 or greater.

[0641] 104. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.5 or greater.

[0642] 105. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.59 or greater.

[0643] 106. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.6 or greater.

[0644] 107. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.7 or greater.

[0645] 108. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.78 or greater.

[0646] 109. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.79 or greater.

[0647] 110. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.8 or greater.

[0648] 111. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.1 or greater.

[0649] 112. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.15 or greater.

[0650] 113. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.18 or greater.

[0651] 114. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.2 or greater.

[0652] 115. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.22 or greater.

[0653] 116. The assay kit of any of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.5 to about 2.5.

[0654] 117. The assay kit of any one of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.6 to about 2.3.

[0655] 118. The assay kit of any one of embodiments 71-102, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 0.9 pg / h to about 2.4 pg / h and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.59 to about 2.22.

[0656] 119. The assay kit of any one of embodiments 71-117, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 or greater.

[0657] 120. The assay kit of any one of embodiments 71-117, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.2 or greater.

[0658] 121. The assay kit of any one of embodiments 71-117, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.26 or greater.

[0659] 122. The assay kit of any one of embodiments 71-117, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.5 or greater.

[0660] 123. The assay kit of any one of embodiments 71-117, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.7 or greater.

[0661] 124. The assay kit of any one of embodiments 71-117, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3 or greater.

[0662] 125. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3.5 or more.

[0663] 126. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3.6 or more.

[0664] 127. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3.7 or more.

[0665] 128. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3.7 or more.

[0666] 129. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4 or more.

[0667] 130. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4.5 or more.

[0668] 131. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4.7 or more.

[0669] 132. The assay kit of any of embodiments 71-117, wherein the hazard ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4.8 or more.

[0670] 133. The assay kit of any of embodiments 71-117, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4.9 or more.

[0671] 134. The assay kit of any of embodiments 71-117, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 5 or more.

[0672] 135. The assay kit of any of embodiments 71-117, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 to about 5.

[0673] 136. The assay kit of any of embodiments 71-117, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 to about 5.

[0674] 137. The assay kit of any of embodiments 71-117, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.5 to about 5.

[0675] 138. The assay kit of any of embodiments 71-117, wherein the odds ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3.5 to about 5.

[0676] 139. The assay kit of any of embodiments 71-138, wherein the interaction with the Fetuin-A fragment comprises binding to the Fetuin-A.

[0677] 140. The assay kit of any of embodiments 71-138, wherein the reagent comprises an antibody.

[0678] 141. A method of correlating the likelihood of a worsening of graft function in a recipient of a kidney transplant, the method comprising:

[0679] obtaining a solution comprising a urine sample from the recipient of the kidney transplant after the kidney transplant and a reagent that interacts with a Fetuin-A fragment containing a urine-linked peptide.

[0680] measuring average fetuin-A fragment excretion in the urine from the solution;

[0681] associating the measured average fetuin-A fragment excretion with a likelihood of graft function deterioration in the recipient,

[0682] wherein a measured average fetuin-A fragment excretion above about 0.8 microgram per hour (pg / h) indicates a higher likelihood of graft function deterioration within a time period of about ten years or less.

[0683] 142. The method of embodiment 141, wherein the excretion above about 0.8 pg / h triggers a medical intervention to inhibit the graft function deterioration.

[0684] 143. The method of any one of embodiments 141-142, wherein the graft function deterioration is indicated by doubling of serum creatinine, need for re- kidney transplantation, or need for dialysis.

[0685] 144. The method of any one of embodiments 141-143, wherein the recipient has been indicated to have an eGFR of about 25 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or higher at a time before and after the urine sample is collected. 2 ) or higher at a time before and after the urine sample is collected.

[0686] 145. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over not less than about 2 hours.

[0687] 146. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over not less than about 4 hours.

[0688] 147. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over not less than about 6 hours.

[0689] 148. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over not less than about 12 hours.

[0690] 149. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over not less than about 24 hours.

[0691] 150. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 36 hours.

[0692] 151. The method of any one of embodiments 141-144, wherein the average fetuin-A fragment excretion into urine is averaged over no less than about 48 hours.

[0693] 152. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 day after the kidney transplant.

[0694] 153. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 days after the kidney transplant.

[0695] 154. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 week after the kidney transplant.

[0696] 155. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 2 weeks after the kidney transplant.

[0697] 156. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 1 month after the kidney transplant.

[0698] 157. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 3 months after the kidney transplant.

[0699] 158. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 6 months after the kidney transplant.

[0700] 159. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 12 months after the kidney transplant.

[0701] 160. The method of any one of embodiments 141-151, wherein the urine sample is collected from the recipient of the kidney transplant at least about 24 months after the kidney transplant.

[0702] 161. The method of any of embodiments 141-160, wherein average fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about nine years or less.

[0703] 162. The method of any of embodiments 141-160, wherein average fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about seven years or less.

[0704] 163. The method of any of embodiments 141-160, wherein average fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about five years or less.

[0705] 164. The method of any of embodiments 141-160, wherein average fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about three years or less.

[0706] 165. The method of any of embodiments 141-160, wherein average fetuin-A fragment excretion above about 0.8 pg / h is indicative of a higher likelihood of graft function deterioration of the transplanted kidney in a time period of about one year or less.

[0707] 166. The method of any of embodiments 141-165, wherein average fetuin-A fragment excretion above about 0.8 pg / h is above about 0.9 pg / h.

[0708] 167. The method of any of embodiments 141-165, wherein average fetuin-A fragment excretion above about 0.8 pg / h is above about 1 pg / h.

[0709] 168. The method of any of embodiments 141-165, wherein average fetuin-A fragment excretion above about 0.8 pg / h is above about 2 pg / h.

[0710] 169. The method of any of embodiments 141-165, wherein average fetuin-A fragment excretion above about 0.8 pg / h is above about 2.1 pg / h.

[0711] 170. The method of any of embodiments 141-165, wherein average fetuin-A fragment excretion above about 0.8 pg / h is above about 2.2 pg / h.

[0712] 171. The method of any of embodiments 141-165, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.3 pg / h.

[0713] 172. The method of any of embodiments 141-165, wherein average Fetuin-A fragment excretion above about 0.8 pg / h is above about 2.5 pg / h.

[0714] 173. The method of any of embodiments 141-172, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 or greater.

[0715] 174. The method of any of embodiments 141-172, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.5 or greater.

[0716] 175. The method of any of embodiments 141-172, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.59 or greater.

[0717] 176. The method of any of embodiments 141-172, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.6 or greater.

[0718] 177. The method of any of embodiments 141-172, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.7 or greater.

[0719] 178. The method of any of embodiments 141-172, wherein the risk ratio corresponding to average Fetuin-A fragment excretion from about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 1.78 or greater.

[0720] 179. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.79 or greater.

[0721] 180. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.8 or greater.

[0722] 181. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.1 or greater.

[0723] 182. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.15 or greater.

[0724] 183. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.18 or greater.

[0725] 184. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.2 or greater.

[0726] 185. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.22 or greater.

[0727] 186. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.5 to about 2.5.

[0728] 187. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.6 to about 2.3.

[0729] 188. The method of any of embodiments 141-172, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 0.9 pg / h to about 2.4 pg / h and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 1.59 to about 2.22.

[0730] 189. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2 or greater.

[0731] 190. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.2 or greater.

[0732] 191. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.26 or greater.

[0733] 192. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.5 or greater.

[0734] 193. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 2.7 or greater.

[0735] 194. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3 or greater.

[0736] 195. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.5 or more.

[0737] 196. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.6 or more.

[0738] 197. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.7 or more.

[0739] 198. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 3.7 or more.

[0740] 199. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4 or more.

[0741] 200. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4.5 or more.

[0742] 201. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4.7 or more.

[0743] 202. The method of any of embodiments 141-188, wherein the hazard ratio for average Fetuin-A fragment excretion corresponding to about 2.4 pg / h or more and average Fetuin-A fragment excretion corresponding to about 0.9 pg / h or less is about 4.8 or more.

[0744] 203. The method of any of embodiments 141-188, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 4.9 or greater.

[0745] 204. The method of any of embodiments 141-188, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 5 or greater.

[0746] 205. The method of any of embodiments 141-188, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 to about 5.

[0747] 206. The method of any of embodiments 141-188, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2 to about 5.

[0748] 207. The method of any of embodiments 141-188, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 2.5 to about 5.

[0749] 208. The method of any of embodiments 141-188, wherein the risk ratio corresponding to an average Fetuin-A fragment excretion of about 2.4 pg / h or more and to an average Fetuin-A fragment excretion of about 0.9 pg / h or less is about 3.5 to about 5.

[0750] 209. The method of any of embodiments 141-208, wherein the interaction with the Fetuin-A fragment comprises binding to the Fetuin-A.

[0751] 210. The method of any of embodiments 141-208, wherein the agent comprises an antibody.

[0752] Non-limiting Examples

[0753] The following examples are provided to illustrate selected embodiments. They should not be considered to limit the scope of the application, but merely an illustration of the same. Thus, while the examples provided below are illustrated with specific medical devices or active agents, they are applicable to the range of medical devices and active agents described herein.

[0754] Example 1-1

[0755] The aim of the clinical performance study shown in Examples 1-5 was to evaluate the predictive performance of the human uPTM3-DKD ELISA test for graft loss in kidney transplant recipients (KTR).

[0756] Design and study population

[0757] We used data from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT03272841)2. All adult KTRs who visited the outpatient clinic at the University Medical Center Groningen (UMCG) between November 2008 and March 2011, had normal graft function at least 1 year after transplantation, did not have any drug or alcohol addiction or systemic disease, were invited to participate in the cohort. During the recruitment period, 817 KTRs were invited, of whom 707 (87%) agreed to participate and gave written informed consent.

[0758] Example 1-2

[0759] Determined clinical performance parameters

[0760] The hazard ratio and 95% confidence interval calculated based on Cox proportional hazards regression analysis will be used to evaluate the ability of uPTM-FetA in assessing the risk of kidney graft loss, defined as the need for retransplantation or resuming dialysis.

[0761] Statistical data and sample size calculation

[0762] The required sample size was estimated based on the following assumptions:

[0763] 1. The hazard ratio (Q) of the high-risk group relative to the low-risk group is expected to be a value greater than 1, and the study set a hazard ratio of 2.5;

[0764] 2. According to literature statistics, about 20% of KTRs will develop a decrease in graft function6. Therefore, the study estimated the incidence to be 20% (pE=0.20);

[0765] 3. The proportion of subjects in the low-risk group and the high-risk group is 3:1 (pL=0.75; pH=0.25);

[0766] 4. The Type I error (a) is 0.05;

[0767] 5. The power (1 - b) is 80%;

[0768] The sample size was calculated as 7 ((z1-a / 2 + z1-β) / (ln θ - ln(1)))2 / (pLpHpE). According to the above assumptions, 249 patients are needed.

[0769] Example 1-3

[0770] Data and results management

[0771] The Excel file containing the series of clinical records of KTRs in the TransplantLines Food and Nutrition Biobank and cohort study will be manually downloaded by the principal investigator and saved in a specific folder on a computer with encrypted disk and other privacy and security measures, in compliance with GDPR. This folder will only be accessible to the PI and the statistician. Further details on data storage, backup, selection and preservation are described below. Urine samples will be transferred to BPM and then stored at -80°C following the sample management procedures of BPM and will be taken out until use. After testing, the test results will be recorded in the Excel file and saved by the BPM test laboratory personnel in the specific folder as previously described.

[0772] Data and results analysis

[0773] The focus of this study seems to be the determination of the optimal threshold for using uPTM-FetA / UCr to differentiate between high-risk and low-risk subjects for kidney graft loss in KTRs. The primary endpoint of this study is death-censored graft loss, defined as the need for retransplantation or resuming dialysis. The study design involves the division of KTRs with random urine samples into two subsets: a main set, which will consist of 50% of KTRs and will be used to determine the clinical cut-off value using the Youden index8; and a validation set, consisting of the remaining 50% of KTRs and will be used to validate the cut-off value.

[0774] In the routine practice of urine tests, random urine is preferred over 24-hour urine samples, especially in cases where 24-hour urine samples are difficult to obtain or unavailable. However, 24-hour urine analysis is still commonly used for metabolic evaluation of several types of kidney disorders. In this study, to validate the generalizability of the predictive ability of the kit for different types of urine samples, we will define KTRs with uPTM-FetA and urine creatinine measured from 24-hour urine samples as the "24-hour set" and perform cut-off validation analysis.

[0775] After determining the cut-off value in the main set, KTRs will be divided into two groups based on their uPTM-FetA / UCr levels: low risk (uPTM-FetA / UCr < cut-off value) and high risk (uPTM-FetA / UCr > cut-off value). Cox proportional hazards model and Kaplan-Meier plots / log-rank test will be used to assess hazard ratios and compare survival functions of the two groups, and Cox proportional hazards model and Kaplan-Meier plots / log-rank test will be used to assess the predictive ability of uPTM-FetA / UCr in kidney graft loss in both sets.

[0776] Proteinuria is commonly used as a prognostic marker after kidney transplantation9,10. It is generally accepted that proteinuria above 0.5 gm / day one year after transplantation is predictive of the development of chronic allograft nephropathy and poor graft outcome11-13. To demonstrate the ability of uPTM-FetA / UCr to provide additional explanatory information in clinical use, we performed comparative analyses to evaluate the event rate of kidney graft loss compared to uPTM-FetA / UCr (above and below the clinical cut-off value) and proteinuria (above and below 0.5 g / day) after adjustment for baseline risk factors for age and gender by using Cox regression models.

[0777] Example 1-4

[0778] Results

[0779] Out of 707 KTRs, 173 patients did not have sufficient amount of random urine and 72 patients did not have sufficient amount of 24-hour urine available for uPTM-FetA measurement, respectively. Therefore, we excluded these patients and 534 KTRs were used for random urine-based analysis and 635 KTRs were used for 24-hour urine sample-based analysis.

[0780] The 534 KTRs with random urine samples were divided into two sets: the main set included 267 (50%) KTRs and the validation set included 267 (50%) KTRs. The 635 KTRs with uPTM-FetA and urine creatinine measured from 24-hour urine samples were defined as the 24-hour set.

[0781] Results of the main set

[0782] The mean age of the 267 KTRs was 53.93 years, 116 (43%) were female, eGFR was 51.69 ml / min / 1.73m 2and the median baseline protein excretion was 0.21 g / d. Among the 267 KTRs, 35 (13%) experienced kidney graft loss during follow-up, and the median time to event was 6.04 years (IQR, 4.62-6.40 years). The clinical cutoff of uPTM-FetA / UCr obtained by the Youden index method was 29.1 ng / mg (corresponding to the IVD103 cutoff of 1.464). According to this clinical cutoff, the subjects were divided into a low-risk group (uPTM-FetA / UCr < 29.1 ng / mg) and a high-risk group (uPTM-FetA / UCr > 29.1 ng / mg) (Table 1). As can be seen from Table 1, there was a significant difference in the frequency of kidney graft loss between the low-risk group and the high-risk group (8.1% and 31%, p-value < 0.001), and the time to event was significantly shorter in the high-risk group than in the low-risk group (4.64 [2.82, 6.15] vs. 6.08 [5.65, 6.46], p-value < 0.001).

[0783] Table 1. Summary of the 267 KTRs in the main cohort

[0784]

[0785]

[0786] 1 Mean ± SD; n (%); median [IQR]

[0787] 2 Welch two-sample t-test; Pearson’s chi-squared test; Kruskal-Wallis rank-sum test

[0788] Table 2. Cumulative probability of kidney graft loss events at different time points in KTRs in the main cohort (with 95% confidence intervals).

[0789]

[0790] Table 3. Cox proportional hazards regression model for the main cohort

[0791]

[0792]

[0793] Note: The reference category for uPTM-FetA / UCr is low level uPTM-FetA / UCr < 29.1 ng / mg; the reference group for gender is male; age and eGFR are included as continuous variables.

[0794] Figure 6 shows Kaplan-Meier survival curves for KTRs with death censored kidney graft loss in the main cohort in some embodiments.Figure 1 Kaplan-Meier estimates showed that KTRs in the high-risk group had a significantly higher risk of events compared to KTRs in the low-risk group, log-rank test p-value < 0.0001. Cumulative probabilities of primary composite events at time points 2-5 years after baseline according to baseline uPTM-FetA / UCr risk categories are also shown in Table 2.

[0795] Further investigation using univariate Cox proportional hazards models showed that the high-risk group had a significantly higher risk of graft loss compared to the low-risk group, hazard ratio 4.98 (95% CI: 2.56-9.69, p-value < 0.001). Thus, uPTM-FetA / UCr was a significant predictor of increased risk of kidney graft loss (Table 3). Multivariate Cox proportional hazards models adjusted for age, sex, and eGFR are also shown in Table 3. uPTM-FetA / UCr was attenuated in the multivariate analysis, but remained a highly significant and independent predictor of risk of kidney graft loss, hazard ratio 2.88 (95% CI: 1.42-5.84, p-value = 0.003). The reduced risk can reflect that uPTM-FetA / UCr can be related to clinical factors, but clearly provides additional information.

[0796] In addition, a comparative analysis of uPTM-FetA / UCr, proteinuria, and event rates using urine samples from the main cohort was performed in order to provide additional explanatory information for the clinical use of uPTM-FetA / UCr (Table 4). This analysis evaluated the event rates of the primary endpoint compared to uPTM-FetA / UCr (above and below the cut-off of 29.1 ng / mg) and proteinuria (total protein excretion above and below 0.5 g / day). The hazard ratios for the second, third, and fourth categories were 3.01 (95% CI: 0.79-11.5, p-value = 0.107), 8.82 (95% CI: 3.39-22.9, p-value < 0.001), and 18.5 (95% CI: 7.77-44.2, p-value < 0.001), respectively, compared to the reference group of uPTM-FetA / UCr < 29.1 ng / mg and no proteinuria.

[0797] Figure 2A Kaplan-Meier curves showing KTRs with non-proteinuria in the main cohort according to non-limiting embodiments. Figure 2B Kaplan-Meier curves showing KTRs with proteinuria in the main cohort according to uPTM-FetA / UCr risk categories according to non-limiting embodiments. Reference Figure 2A and 2B , Figure 2A and 7Kaplan-Meier curves in BN showed that KTRs with uPTM-FetA / UCr > 29.1 ng / mg had a slightly significant risk of events compared to the low uPTM-FetA / UCr group for both non-proteinuric and proteinuric, with log-rank test p-values of 0.12 and 0.074, respectively. This suggests that uPTM-FetA / UCr can provide additional information to the risk of kidney graft loss assessment for proteinuria.

[0798] Table 4. Risk ratios of kidney graft loss according to uPTM-FetA / UCr and proteinuria categories in the main cohort of 267 KTRs.

[0799]

[0800] 1 HR = hazard ratio, CI = confidence interval

[0801] *Adjusted for age and sex

[0802] Results in the validation set

[0803] The mean age of the 267 KTRs was 52.32 years, 111 (42%) were female, the mean baseline eGFR was 51.95 ml / min / 1.73 m2, and the median baseline protein excretion was 0.18 g / d. Among the 267 KTRs, 29 (11%) experienced kidney graft loss during follow-up, and the median time to event was 5.06 years (IQR: 4.47-5.36 years). According to the clinical cut-off value of 29.1 ng / mg derived from the main cohort, KTRs were divided into a low-risk group (uPTM-FetA / UCr < 29.1 ng / mg) and a high-risk group (uPTM-FetA / UCr > 29.1 ng / mg) (Table 5). As can be seen from Table 5, there was a significant difference in the frequency of kidney graft loss between the low-risk and high-risk groups (7.6% and 29%, respectively, p-value < 0.001), and the time to event was significantly shorter in the high-risk group than in the low-risk group (4.98 [2.34, 5.34] vs. 5.06 [4.55, 5.36], p-value = 0.100).

[0804] Table 5. Summary of 267 KTRs in the validation set

[0805]

[0806] 1 Mean ± SD; n (%); median [IQR]

[0807] 2 Welch two-sample t-test; Pearson’s chi-squared test; Kruskal-Wallis rank-sum test

[0808] Figure 3 Kaplan-Meier survival curves for KTRs with death censored graft loss in the validation set are shown according to non-limiting embodiments. Referring to FIG. 6, Kaplan-Meier estimates indicate that KTRs in the high risk group have a significantly higher risk of an event relative to KTRs in the low risk group, log-rank test p-value < 0.0001. Cumulative probabilities of events in the validation set at time points 2-5 years after baseline according to baseline uPTM-FetA / UCr risk category are also shown in Table 6. Figure 3

[0809] Table 6. Cumulative probabilities of kidney transplant graft loss events at different time points for subjects in the validation set (with 95% confidence intervals).

[0810]

[0811] Table 7. Cox proportional hazards regression model for the validation set

[0812]

[0813] Note: The reference category for uPTM-FetA / UCr is low level uPTM-FetA / UCr (<29.1 ng / mg); the reference group for gender is male; age and eGFR are included as continuous variables.

[0814] Further investigation using a univariate Cox proportional hazards model indicated that the high risk group had a significantly higher risk of graft loss compared to the low risk group, hazard ratio of 4.51 (95% CI: 2.15-9.45, p-value < 0.001). Thus, uPTM-FetA / UCr is a significant predictor of increased risk of kidney graft loss (Table 7). A multivariate Cox proportional hazards model adjusted for age, gender, and eGFR is also shown in Table 7. uPTM-FetA / UCr was attenuated in the multivariate analysis, but remained a significant and independent predictor of risk of kidney graft loss, hazard ratio of 2.36 (95% CI: 1.09-5.15, p-value = 0.030). The reduced risk can reflect that uPTM-FetA / UCr can be related to clinical factors, but clearly provides additional information.

[0815] ​In addition, comparative analysis of uPTM-FetA / UCr, proteinuria and event rates using urine samples from the main set was performed in order to provide additional explanatory information for the clinical use of uPTM-FetA / UCr (Table 8). This analysis evaluated the event rates of the primary endpoint compared to uPTM-FetA / UCr (above and below the cut-off 29.1 ng / mg) and proteinuria (total protein excretion above and below 0.5 g / d). The hazard ratios for the second, third and fourth categories were 2.93 (95% CI: 0.93-9.23, p-value = 0.066), 3.46 (95% CI: 1.19-10.1, p-value = 0.023) and 13.1 (95% CI: 5.08-33.5, p-value < 0.001), respectively, compared to the reference group of uPTM-FetA / UCr < 29.1 ng / mg and no proteinuria.

[0816] Figure 4A Kaplan-Meier curves of subjects in the validation set with non-proteinuria according to uPTM-FetA / UCr risk categories are shown according to non-limiting embodiments. Figure 4B Kaplan-Meier curves of subjects in the validation set with proteinuria according to uPTM-FetA / UCr risk categories are shown according to non-limiting embodiments. Reference Figure 4A and 4B Kaplan-Meier curves also showed that for both non-proteinuria and proteinuria, patients with uPTM-FetA / UCr > 29.1 ng / mg had a significant event risk relative to the low uPTM-FetA / UCr group with log-rank test p-values of 0.061 and 0.015, respectively. This indicates that uPTM-FetA / UCr can provide additional information for the risk assessment of kidney graft loss by proteinuria.

[0817] The analysis performed by the validation set provided a positive validation confirming the utility of the clinical cut-off of uPTM-FetA / UCr with good predictive power for KTRs at risk of graft loss.

[0818] Table 8. Hazard ratios of kidney graft loss according to uPTM-FetA / UCr and proteinuria categories in 267 KTRs in the validation set.

[0819]

[0820] 1 HR = hazard ratio, CI = confidence interval

[0821] *Adjusted for age and sex

[0822] Results for 24-hour collections

[0823] The mean age of the 635 KTRs was 53 years, 268 (42%) were female, the mean baseline eGFR was 52.31 ml / min / 1.73 m2, and the median baseline protein excretion was 0.20 g / d. Of the 635 KTRs, 75 (12%) experienced kidney graft loss during follow-up, and the median time to event was 5.32 years (IQR: 4.50-6.03 years). According to the clinical cutoff of 29.1 ng / mg derived from the main set, the subjects were divided into a low-risk group (uPTM-FetA / UCr < 29.1 ng / mg) and a high-risk group (uPTM-FetA / UCr > 29.1 ng / mg) (Table 9). As can be seen from Table 9, there was a significant difference in the frequency of kidney graft loss between the low-risk group and the high-risk group (4.8% and 19%, p-value < 0.001), and the time to event was significantly shorter in the high-risk group than in the low-risk group (5.10 [3.71, 5.80] vs. 5.44 [4.93, 6.26], p-value < 0.001).

[0824] Table 9. Summary of the 635 KTRs in the 24-hour set

[0825]

[0826]

[0827] 1 Mean ± SD; n (%); Median [IQR]

[0828] 2 Welch two-sample t-test; Pearson’s chi-squared test; Kruskal-Wallis rank-sum test

[0829] Figure 5 The Kaplan-Meier survival curves for the 635 KTRs with death censored kidney graft loss are shown according to non-limiting embodiments. Reference Figure 5 The Kaplan-Meier estimates indicate that KTRs in the high-risk group have a significantly higher risk of events relative to KTRs in the low-risk group, log-rank test p-value < 0.0001. The cumulative probabilities of events in the 24-hour set at time points 2-5 years after baseline according to baseline uPTM-FetA / UCr risk category are also shown in Table 10.

[0830] Table 10. Cumulative probabilities of kidney graft loss events (with 95% confidence intervals) at different time points for the 635 KTRs.

[0831]

[0832] Table 11. Cox proportional hazards regression model for the 24-hour set

[0833]

[0834] Note: Reference category for uPTM-FetA / UCr is low level uPTM-FetA / UCr (<29.1 ng / mg); reference group for gender is male; age and eGFR are included as continuous variables.

[0835] Further investigation using univariate Cox proportional hazards model showed that the high risk group had a significantly higher risk of graft loss compared to the low risk group with a hazard ratio of 4.71 (95% CI: 2.71-8.19, p-value <0.001). Thus, uPTM-FetA / UCr is a significant predictor of increased risk of kidney graft loss (Table 11). Multivariate Cox proportional hazards model adjusted for age, gender and eGFR is also shown in Table 11. uPTM-FetA / UCr was attenuated in the multivariate analysis, but remained a highly significant and independent predictor of risk of kidney graft loss with a hazard ratio of 2.80 (95% CI: 1.58-4.94, p-value <0.001). The reduced risk can reflect that uPTM-FetA / UCr can be related to clinical factors, but clearly provides additional information.

[0836] In addition, a comparative analysis of uPTM-FetA / UCr, proteinuria and event rates using urine samples from the 24-hour collection was performed in order to provide additional explanatory information for the clinical use of uPTM-FetA / UCr (Table 12). This analysis evaluated the event rates of the primary endpoint compared to uPTM-FetA / UCr (above and below the cut-off of 29.1 ng / mg) and proteinuria (total protein excretion above and below 0.5 g / d). The hazard ratios for the second, third and fourth categories were 3.08 (95% CI: 1.48-6.40, p-value = 0.003), 6.10 (95% CI: 2.10-17.7, p-value <0.001) and 15.5 (95% CI: 7.90-30.5, p-value <0.001), respectively, compared to the reference group of uPTM-FetA / UCr <29.1 ng / mg and no proteinuria.

[0837] Figure 6A Kaplan-Meier curves showing KTRs with (a) no proteinuria according to uPTM-FetA / UCr risk categories in the 24-hour collection according to non-limiting embodiments. Figure 6B Kaplan-Meier curves showing KTRs with (b) proteinuria according to uPTM-FetA / UCr risk categories in the 24-hour collection according to non-limiting embodiments. Reference Figure 6A and 6B , Figure 6A and6B The Kaplan-Meier curves in Figure 8 also show that KTRs with uPTM-FetA / uCr > 29.1 ng / mg had a significantly higher event risk relative to the low uPTM-FetA / uCr group for both non-proteinuric and proteinuric patients with log-rank test p-values of 0.013 and 0.031, respectively. This indicates that uPTM-FetA / uCr can provide additional information for the risk assessment of kidney graft loss in proteinuric patients.

[0838] Table 12. Hazard ratios for kidney graft loss according to uPTM-FetA / uCr and proteinuria categories in the 24-hour cohort of 635 KTRs.

[0839]

[0840] 1 HR = hazard ratio, CI = confidence interval

[0841] *adjusted for age and sex

[0842] Conclusions

[0843] The results of this study demonstrate that uPTM-FetA / uCr can effectively predict the risk of graft loss in KTRs independent of age, sex, and eGFR. The additional prognostic value of uPTM-FetA / uCr on top of proteinuria is evident. The clinical validity of the uPTM-FetA / uCr clinical cut-off (= 29.1 ng / mg) was verified in an independent validation cohort and also in the 24-hour cohort using uPTM-FetA and urine creatinine measured on 24-hour urine samples.

[0844] These results indicate that uPTM-FetA / uCr can effectively predict the actual decrease in eGFR. Patients with high risk can be treated or related adjustments can be made early to achieve a preventive effect, which has considerable clinical benefits and would be a boon for KTRs.

[0845] Example 1-5

[0846] Additional marker combination models were tried and analyzed to be included in the kidney transplant follow-up. In total, the following 4 models were tried and analyzed for predicting graft loss in KTRs:

[0847] • Model 1 : uPTM-FetA / uCr only:

[0848] • Model 2: adjusted for age, sex, and post-transplant time at inclusion (log2) and UACR (urinary albumin to creatinine ratio)

[0849] • Model 3 was further adjusted for eGFR (estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine and cystatin C).

[0850] • Model 4 was further adjusted for the presence of human leukocyte antigens

[0851] Table 13. Logistic regression for graft loss within 2 years (random urine)

[0852]

[0853] Figure 7 Receiver operating characteristic (ROC) curves for uPTM-FetA / uCr and corresponding Kaplan-Meier curves for the marker combination model of KTRs with graft loss within 2 years using random urine are shown, according to non-limiting embodiments.

[0854] Figure 8 Receiver operating characteristic (ROC) curves for the combination of uPTM-FetA / uCr, age, sex, time post-transplant at inclusion (log2), and UACR and corresponding Kaplan-Meier curves for the marker combination model of KTRs with graft loss within 2 years using random urine are shown, according to non-limiting embodiments.

[0855] Figure 9 Receiver operating characteristic (ROC) curves for uPTM-FetA / uCr, age, sex, time post-transplant at inclusion (log2), UACR, and eGFR and corresponding Kaplan-Meier curves for the marker combination model of KTRs with graft loss within 2 years using random urine are shown, according to non-limiting embodiments.

[0856] Figure 10 Receiver operating characteristic (ROC) curves for uPTM-FetA / uCr, age, sex, time post-transplant at inclusion (log2), UACR, eGFR, and the presence of human leukocyte antigen class II antibodies (HLA II) and corresponding Kaplan-Meier curves for the marker combination model of KTRs with graft loss within 2 years using random urine are shown, according to non-limiting embodiments.

[0857] Referring to Table 13 and Figures 7-10 Combination with additional markers resulted in an increased AUC value for KTRs with graft loss within 2 years.

[0858] Table 14. Logistic regression for graft loss within 5 years (random urine)

[0859]

[0860] Figure 11 Receiver Operating Characteristic (ROC) curves and corresponding Kaplan-Meier curves for a marker combination model using random urine for KTRs with graft loss within 5 years are shown in relation to uPTM-FetA / uCr according to non-limiting embodiments.

[0861] Figure 12 Receiver Operating Characteristic (ROC) curves and corresponding Kaplan-Meier curves for a marker combination model using random urine for KTRs with graft loss within 5 years are shown in relation to the combination of uPTM-FetA / uCr, age, gender, time post-transplant at inclusion (log2), and UACR according to non-limiting embodiments.

[0862] Figure 13 Receiver Operating Characteristic (ROC) curves and corresponding Kaplan-Meier curves for a marker combination model using random urine for KTRs with graft loss within 5 years are shown in relation to uPTM-FetA / uCr, age, gender, time post-transplant at inclusion (log2), UACR, and eGFR according to non-limiting embodiments.

[0863] Figure 14 Receiver Operating Characteristic (ROC) curves and corresponding Kaplan-Meier curves for a marker combination model using random urine for KTRs with graft loss within 5 years are shown in relation to uPTM-FetA / uCr, age, gender, time post-transplant at inclusion (log2), UACR, eGFR, and presence of human leukocyte antigen class II antibodies (HLA II) according to non-limiting embodiments.

[0864] The addition of additional markers resulted in an increase in the AUC value for KTRs with graft loss within 5 years with reference to Table 14 and Figures 11-14

[0865] Purpose

[0866] The Human uPTM3-DKD ELISA Kit is a colorimetric immunoassay for the quantification of a unique fetuin-A with specific post-translational modifications in urine (hereinafter E103). This product is a non-automated IVD for prognostic use and should be performed by certified medical professionals, such as medical technicians, in a qualified clinical laboratory. Prior to application in the clinic, the concentration of E103 should be corrected by urine creatinine. The Human uPTM3-DKD ELISA Kit will be used in conjunction with clinical evaluation to help assess the renal function prognosis of kidney transplant recipients.

[0867] Technical and functional features

[0868] ​The human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, the calibrators or unknown urine samples are mixed with an anti-unique PTM Fetuin-A monoclonal antibody (mAb) and then incubated in microplate wells pre-coated with unique PTM Fetuin-A. The monoclonal antibody recognizes the unique PTM Fetuin-A in the calibrators or unknown samples under competition in the microplate wells. After incubation, a horseradish peroxidase (HRP)-conjugated secondary antibody is added followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nm wavelength and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve.

[0869] Identified

[0870] Table 15. Identified

[0871]

[0872] Example 2-1

[0873] The publication, Firas F. Alkaff et al., "Urinary Post-Translationally Modified Fetuin-A Protein Is Associated with Increased Risk of Graft Failure in Kidney Transplant Recipients," published in Am J Nephrol 2023, available online at https: / / doi.org / 10.1159 / 000534829, is incorporated herein by reference in its entirety. The purpose of the clinical performance studies shown in Examples 2-1 to 2-5 was to evaluate the predictive performance of the human uPTM3-DKD ELISA test for graft failure in kidney transplant recipients (KTRs). In Examples 2-1 to 2-5, KT data were derived from 24-hour urine.

[0874] Design and Study Population

[0875] In this prospective cohort study, we used data from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT02811835). All adult KTRs who visited the outpatient clinic of the University Medical Center Groningen (UMCG) between November 2008 and March 2011, had a functioning graft at least 1 year after transplantation, did not have any drug or alcohol addiction or systemic disease, were invited to participate in the cohort. During the recruitment period, 817 KTRs were invited, of whom 707 (87%) agreed to participate and gave written informed consent. For the current study, participants with missing 24-hour uPTM-FetA excretion measurements were excluded from the analysis. The study was conducted in accordance with the Declaration of Helsinki and Istanbul, and the study protocol was approved by the UMCG Institutional Review Board (METc 2008 / 186).

[0876] The primary endpoint of the study was graft loss, defined as the need for retransplantation or resuming dialysis. Secondary endpoints were graft dysfunction (graft loss or doubling of serum creatinine) and all-cause mortality. For the graft loss and graft dysfunction endpoints, KTRs with a functioning graft at death were censored at the time of death. Endpoints were recorded until September 2015. No KTRs were lost to follow-up through the UMCG outpatient clinic’s continuous monitoring system.

[0877] Example 2-2

[0878] Clinical parameters

[0879] All baseline measurements were performed during the morning outpatient clinic visit. Blood pressure was measured twice, with an interval of 15 minutes, after which the results were averaged. Measurements were performed using a semi-automatic device (Dinamap 1846, Critikon, Tampa, USA). Body weight and height were measured with the participant wearing indoor clothing and without shoes. Body mass index was calculated as body weight (kg) divided by the square of height (m 2 ), and body surface area (m 2 ) was estimated using the DuBois and DuBois formula. Diabetes was defined according to American Diabetes Association criteria. Estimated glomerular filtration rate (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula based on creatinine. Primary CMV infection was defined as a CMV infection occurring in a previously seronegative KTR before transplantation of a kidney from a seropositive donor. Secondary CMV infection was defined as a CMV infection occurring in a KTR who was seropositive before transplantation. Relevant donor, recipient, and transplantation information was extracted from medical records as previously described.

[0880] Example 2-3

[0881] Laboratory methods and uPTM-FetA measurement

[0882] Blood samples were drawn in the morning at the outpatient clinic after overnight fasting (approximately 8-12 hours) which included no medication intake. For urine collection, all participants were instructed to collect their 24-hour urine during the day before their visit.

[0883] uPTM-FetA was measured using a novel human uPTM3-DKD ELISA kit (CE IVD marked, Bio Preventive Medicine Corp., Hsinchu, Taiwan, China; trade name: DNlite-IVD103). This assay detects post-translationally modified fetuin-A containing a linking peptide. Total urinary protein excretion was determined using the biuret reaction (MEGAAU 150, Merck Diagnostica, Darmstadt, Germany). Other biochemical parameters including serum creatinine and high-sensitivity C-reactive protein (hs-CRP) were measured using routine laboratory methods.

[0884] Statistical analysis

[0885] All data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R version 4.0.5 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, a p-value < 0.05 was considered statistically significant. The distribution of continuous variables was assessed by visual inspection of histograms and quantile-quantile plots. Normally distributed variables were expressed as mean ± standard deviation, skewed variables were expressed as median (interquartile range), and categorical variables were expressed as frequency (effective percentage). Univariate linear regression analysis was performed to assess the association between 24-hour uPTM-FetA excretion and clinical and biochemical parameters. After univariate analysis, we adjusted 24-hour uPTM-FetA excretion for gender, serum creatinine, and 24-hour urinary protein excretion ≥ 0.5 g / 24 hours. During the linear regression analysis, skewed variables were log2-transformed to meet the assumptions of linear regression. If log2-transformation did not meet the assumptions, variables were reclassified as categorical variables according to median or acceptable cut-off levels.

[0886] The Kaplan-Meier curves were used to visualize the difference in graft and patient survival rates between subgroups of KTRs according to median level 24-hour uPTM-FetA excretion (< 34 pg / 24 hours versus > 34 pg / 24 hours) and the significance of the difference between subgroups was calculated using the log-rank test. Cox proportional hazards regression analysis was used to assess the association of 24-hour uPTM-FetA excretion with graft loss, graft function deterioration, and all-cause mortality, with adjustment for potential confounders. Variables associated with 24-hour uPTM-FetA excretion in unadjusted linear regression analysis were considered potential confounders. In Model 1, 24-hour uPTM-FetA excretion was adjusted for age, sex, and time post-transplantation at inclusion (log2). In Model 2, we additionally adjusted for eGFR. In Model 3, we additionally adjusted for 24-hour urine protein excretion (log2). In Model 4, we additionally adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of proliferation inhibitors. In Model 5, we additionally adjusted for donor characteristics (donor type, donor age, and donor sex). In the full model (Model 6), we additionally adjusted for hs-CRP (log2).

[0887] Schoenfeld residuals were performed as a function of time to assess the proportional hazards assumption in the final Cox regression models for each endpoint. The proportional hazards assumption was violated for graft loss and graft function deterioration (p = 0.004 and p = 0.005, respectively), but not for all-cause mortality (p = 0.073). Therefore, Cox models with time-dependent covariates were used to calculate hazard ratios (HRs) for graft loss and graft function deterioration over time. HRs were expressed as per doubling of 24-hour uPTM-FetA excretion, with 95% confidence intervals (95% CIs).

[0888] For the primary endpoint, we assessed potential interactions of age, sex, eGFR, and 24-hour urine protein excretion with 24-hour uPTM-FetA excretion by adding interaction terms to the full model. For the sensitivity analysis, we re-evaluated the association of 24-hour uPTM-FetA excretion with graft loss after exclusion of outliers. Outliers in this study were defined as values deviating more than two standard deviations from the mean of log2 24-hour uPTM-FetA excretion. In addition, we used uPTM-FetA concentration and uPTM-FetA concentration indexed to creatinine (uPTM-FetA / creatinine ratio) instead of 24-hour uPTM-FetA excretion.

[0889] For all cross-sectional analyses, the original non-imputed dataset was used, and variables with >20 missing values (3.16%) were reported in the table footnotes. For all prospective analyses, multiple imputation using chained equations (MICE) was performed using the R package “mice” (number of multiple imputations = 10) to account for missing data in variables other than 24-hour uPTM-FetA excretion.

[0890] Example 2-4

[0891] Results

[0892] Figure 15 A flowchart showing the study population selection according to non-limiting embodiments. Referring to Figure 15 , the study included a total of 627 KTRs. The mean age was 53 ± 13 years, 42% were female, the median time post-transplant was 5.3 (1.9-12.2) years, and the mean eGFR was 52 ± 20 mL / min / 1.73 m 2 . The median 24-hour uPTM-FetA excretion was 34 (17-74) pg / 24 hours. The median 24-hour urine protein excretion was 0.21 (0.01-0.34) g / 24 hours, with 504 (81%) having a urine protein excretion <0.5 g / 24 hours and only 8 (1%) having a urine protein excretion >3 g / 24 hours. More detailed baseline characteristics of the study population are shown in Table 2-1. Referring to Table 2-1, normally distributed variables are presented as mean ± standard deviation, skewed variables are presented as median (interquartile range), and categorical variables are presented as number (percent valid). Smoking status was missing for 39 patients, alcohol consumption status was missing for 66 patients, CMV infection status was missing for 52 patients, hemoglobin A1C was missing for 21 patients, and hs-CRP levels were missing for 32 patients. All other variables had missing values in <20 patients. eGFR, estimated glomerular filtration rate calculated using the CKD-EPI formula based on creatinine; HLA, human leukocyte antigen; hs-CRP, high-sensitivity C-reactive protein; mTOR, mammalian target of rapamycin; Std. b, standardized b coefficient; uPTM-FetA, urinary post-translational modified FetA; BMI, body mass index. Variables were log2-transformed to meet assumptions in linear regression analysis.

[0893] Table 2-1. Baseline characteristics and linear regression analysis of 24-hour uPTM-FetA excretion

[0894]

[0895] Figure 16Scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with serum creatinine levels according to non-limiting embodiments. Figure 17 Scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with eGFR according to non-limiting embodiments. Figure 18 Scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with 24h albumin excretion according to non-limiting embodiments. Figure 19 Scatter plot and visual presentation of the correlation of 24h uPTM-FetA excretion with 24h urinary protein excretion according to non-limiting embodiments. Reference Figures 16-19 24h uPTM-FetA excretion was negatively correlated with eGFR and positively correlated with plasma creatinine concentration, urinary albumin and 24h urinary protein excretion.

[0896] Cross-sectional association of 24h uPTM-FetA excretion with clinical and biochemical parameters

[0897] In univariate linear regression analysis, 24h uPTM-FetA excretion was most strongly associated with 24h urinary protein excretion > 0.5 g / 24h (standardized beta coefficient [Std. β] 0.94, p < 0.001). In addition, 24h uPTM-FetA excretion was also significantly associated with 24h urinary albumin excretion (Std. β 0.45, p < 0.001), serum creatinine (Std. β 0.34, p < 0.001) and eGFR (Std. β -0.27, p < 0.001). Furthermore, female gender was also significantly associated with higher 24h uPTM-FetA excretion (Std. β -0.22, p = 0.006). Older donor age, living donor, use of proliferation inhibitors and 24h urinary albumin excretion were significantly associated with higher 24h uPTM-FetA excretion, independent of gender, serum creatinine and 24h urinary protein excretion > 0.5 g / 24h. In contrast, longer duration after transplantation and congenital disease as primary renal disease were negatively associated with 24h uPTM-FetA excretion.

[0898] Prospective analysis of the association of 24h uPTM-FetA excretion with graft loss

[0899] Table 2-2 is a prospective analysis of the association of 24-hour uPTM-FetA excretion with death-censored graft failure in 627 KTRs. With reference to Table 2-2, Cox proportional hazards regression analysis was performed to assess the association of 24-hour uPTM-FetA excretion with the risk of death-censored graft failure (i.e., need for retransplantation or (re)start of dialysis). Model 1 was adjusted for age, sex, and time post-transplantation at inclusion (log2). Model 2 was further adjusted for eGFR according to the CKD-EPI formula based on creatinine. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A. Figure 20 The Kaplan-Meier curves showing death-censored graft failure above and below the median of 24-hour urinary post-translationally modified fetuin-A excretion according to non-limiting embodiments are shown. With reference to Table 2-2 and Figure 20During a median follow-up of 5.3 (4.5-6.0) years from the baseline measurement, 73 (12%) KTR developed graft loss. The most common causes of graft loss were chronic graft dysfunction (77%) and recurrence of primary nephropathy (10%). Other causes of graft loss included vascular problems and infections. Among KTR with 24-hour uPTM-FetA excretion below and above the median at baseline, the rates of graft loss were 4% and 19%, respectively (p log rank < 0.001). The association of 24-hour uPTM-FetA excretion with the risk of graft loss was not constant over time (p schoenfeld residual = 0.004). In unadjusted models, 24-hour uPTM-FetA excretion was associated with graft loss within 1 year or between 1 and 3 years from the baseline measurement; however, the association disappeared after adjustment for confounders (HR 0.99, 95% CI: 0.77-1.27 and 0.96, 95% CI: 0.78-1.19, respectively). In contrast, 24-hour uPTM-FetA excretion was associated with an increased risk of graft loss within 3 years from the baseline measurement (HR 1.85, 95% CI: 1.55-2.22), and the association remained significant even after adjustment for potential confounders (HR 1.31, 95% CI: 1.06-1.61) (Table 2). There was no interaction between 24-hour uPTM-FetA excretion and sex, age, eGFR, or 24-hour urinary protein excretion for the association of 24-hour uPTM-FetA excretion with graft loss (all p interactions > 0.05).

[0900] Table 2-2. Prospective analysis of the association of 24-hour uPTM-FetA excretion with death-censored graft loss in 627 KTR.

[0901]

[0902]

[0903] Sensitivity analysis

[0904] We identified 34 KTRs (5.4%) with outliers (11 KTRs with 24-hour uPTM-FetA excretion <3.33 μg / 24 hours and 23 KTRs with uPTM-FetA excretion >408 μg / 24 hours). Tables 2-3 indicate prospective analyses of the association between 24-hour uPTM-FetA excretion and graft failure in death according to some implementation schemes, where outliers of 24-hour uPTM-FetA excretion were excluded. Referring to Table 2-3, outliers were defined as ±2 standard deviations from the log2-transformed mean of 24-hour uPTM-FetA excretion (<3.33 μg / 24 hours and >408 μg / 24 hours). Based on this, 34 KTRs were excluded, leaving 593 KTRs for Cox proportional hazards regression analysis. Model 1 was adjusted for age, sex, and post-transplant time (log2) at inclusion. Model 2 was further adjusted for the estimated glomerular filtration rate based on the creatinine-based CKD-EPI formula. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for clinical characteristics of KTR (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translational modified fetoglobulin-A. After excluding outliers, 24-hour uPTM-FetA excretion remained independently associated only with the risk of graft failure 3 years from baseline (HR 1.77, 95% CI: 1.25–2.49).

[0905] Table 2-3. Prospective analysis of the association between 24-hour uPTM-FetA excretion and graft failure in dead grafts, excluding outliers in 24-hour uPTM-FetA excretion.

[0906]

[0907]

[0908] In other sensitivity analyses, we used uPTM-FetA concentration and uPTM-FetA / creatinine ratio instead of 24-hour uPTM-FetA excretion. Table 2-4 is a prospective analysis of the association of uPTM-FetA concentration with graft loss in 627 kidney transplant recipients. With reference to Table 2-4, Cox proportional hazards regression analysis was performed to assess the association of uPTM-FetA concentration with the risk of death-censored graft loss. Model 1 was adjusted for age, sex, and time post-transplant at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A.

[0909] Table 2-4. Prospective analysis of the association of uPTM-FetA concentration with graft loss in 627 kidney transplant recipients.

[0910]

[0911] Table 2-5 indicates a prospective analysis of the association of uPTM-FetA / creatinine ratio with graft loss in 627 kidney transplant recipients according to some embodiments. With reference to Table 2-5, Cox proportional hazards regression analysis was performed to assess the association of uPTM-FetA / urinary creatinine ratio with the risk of death-censored graft loss. Model 1 was adjusted for age, sex, and time post-transplant at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A.

[0912] Table 2-5. Prospective analysis of the association of uPTM-FetA / creatinine ratio with graft loss in 627 kidney transplant recipients.

[0913]

[0914] Referring to Tables 2-3 and 2-4, similarly, uPTM-FetA concentration and uPTM-FetA / creatinine ratio were also significantly associated with graft failure only after 3 years from baseline measurement, even after adjustment for potential confounders (HR 1.35, 95% CI: 1.10-1.65 and HR 1.36, 95% CI: 1.10-1.67, respectively)

[0915] Secondary analysis of the association between 24-hour uPTM-FetA and graft function deterioration and all-cause mortality

[0916] During a median follow-up of 5.3 (4.0-6.0) years from baseline measurement, 121 (19%) KTR developed graft function deterioration. Figure 21 Kaplan-Meier curves showing death-censored graft function deterioration above and below the median 24-hour urinary post-translationally modified fetuin-A excretion according to non-limiting embodiments. Table 2-6 represents supplementary table 5. Prospective analysis of the association between 24-hour uPTM-FetA excretion and death-censored graft function deterioration in 627 kidney transplant recipients. Referring to Table 2-6, Cox proportional hazards regression analysis was performed to assess the association between 24-hour uPTM-FetA excretion and the risk of graft function deterioration. Model 1 was adjusted for age, sex, and time post-transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary post-translationally modified fetuin-A. Referring to Table 2-6 and Figure 21Among KTRs with 24-hour uPTM-FetA excretion below and above the median at baseline, the rates of graft dysfunction were 9% and 30%, respectively (plog rank < 0.001). The association of 24-hour uPTM-FetA excretion with the risk of graft loss was not constant over time (pschoenfeld residual = 0.005). 24-hour uPTM-FetA excretion was significantly associated with an increased risk of graft dysfunction only after 3 years from the baseline measurement (HR 1.64, 95% CI: 1.41-1.92), and the association remained significant even after adjustment for potential confounders (HR 1.39, 95% CI: 1.17-1.65).

[0917] Table 2-6. Prospective analysis of the association of 24-hour uPTM-FetA excretion with death-censored graft dysfunction in 627 kidney transplant recipients.

[0918]

[0919] During a median follow-up of 5.4 (4.9-6.1) years from the baseline measurement, 132 (21%) KTRs died. Figure 22 A Kaplan-Meier curve showing all-cause mortality above and below the median of 24-hour urinary translationally modified fetuin-A excretion is shown in accordance with non-limiting embodiments. There was no significant difference in mortality between KTRs with 24-hour uPTM-FetA excretion below (19%) and above (23%) the median level (plog rank = 0.2). Table 2-7 indicates a prospective analysis of the association of 24-hour uPTM-FetA excretion with all-cause mortality in 627 kidney transplant recipients in accordance with non-limiting embodiments. Reference is made to Table 2-7. Cox proportional hazards regression analysis was performed to assess the association of 24-hour uPTM-FetA excretion with the risk of all-cause death. Model 1 was adjusted for age, sex, and time post-transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for KTR clinical characteristics (systolic blood pressure, body surface area, smoking status, and CMV infection status) and the use of proliferation inhibitors. Model 5 was further adjusted for donor characteristics (donor type, donor age, and donor sex). Model 6 was further adjusted for serum high-sensitivity C-reactive protein (log2). 95% CI, 95% confidence interval; HR, hazard ratio; uPTM-FetA, urinary translationally modified fetuin-A. Unadjusted and adjusted Cox regression models showed no prospective association between 24-hour uPTM-FetA excretion and all-cause mortality.

[0920] Table 2-7. Prospective analysis of the association of 24-hour uPTM-FetA excretion with all-cause mortality in 627 kidney transplant recipients.

[0921]

[0922]

[0923] Discussion

[0924] In this cohort of 627 KTRs, the association of 24-hour uPTM-FetA excretion with 24-hour urine protein excretion >0.5 g / 24h was strongest. In addition, shorter time post-transplant, living donor, older donor age, congenital disease as primary renal disease, use of proliferation inhibitors, and 24-hour urine albumin excretion were independently associated with higher 24-hour uPTM-FetA excretion. In the prospective analysis, 24-hour uPTM-FetA excretion was independently associated with an increased risk of graft loss. However, the risk was not constant over time, and the increased risk was only observed 3 years or more after the baseline measurement. In several sensitivity analyses, the association was robust. Similarly, 24-hour uPTM-FetA excretion was only associated with graft function deterioration 3 years after the baseline measurement. In contrast, 24-hour uPTM-FetA excretion was not associated with an increased risk of mortality.

[0925] Human Fetuin-A in its single chain precursor form comprises, in addition to the signal peptide, a chain A, a connecting peptide, and a chain B. Subsequently, the precursor protein is post-translationally modified by glycosylation and proteolysis. During the proteolytic processing, the connecting peptide region is removed from the precursor protein, and the single chain form is converted into the mature double chain form connected by disulfide bonds. Under certain conditions where proteolysis does not occur, the single chain Fetuin-A instead undergoes phosphorylation, mainly at serine residues in the connecting peptide region. The ELISA kit used in this study specifically detects the later post-translationally modified form of Fetuin-A.

[0926] In healthy adults, the kidney does not express fetuin-A. However, proximal tubular epithelial cells (PTECs) gain the ability to produce and release fetuin-A into the luminal side of the tubule upon injury. Zhou et al. showed that in cisplatin-induced and ischemia / reperfusion-induced AKI rat models, fetuin-A was predominantly present in the urinary exosomal fraction but not in the non-exosomal fraction, suggesting that fetuin-A is produced by PTECs. Recently, Rudloff et al. also showed that PTECs can locally produce fetuin-A under hypoxic conditions upon stimulation by hypoxia-inducible transcription factors. It is hypothesized that the presence of fetuin-A in the proximal tubule helps to protect the kidney from hypoxia-induced renal inflammation by preventing macrophages from transforming into pro-inflammatory macrophages Ml and from hypoxia-induced fibrosis by antagonizing transforming growth factor-beta signaling.

[0927] Both acute and chronic kidney injury are closely related to the occurrence of hypoxia. In the context of kidney transplantation, the kidney is exposed to various conditions that will enhance hypoxia and hypoxia injury, such as ischemia and reperfusion during organ donation and postoperative vascular complications. Of all structures within the kidney, PTECs are the most sensitive and vulnerable, as these cells are highly active and have a high oxygen demand. Since hypoxia plays an important role in the progression of kidney disease and can be present in relatively early stages of kidney injury, even before structural damage occurs, early identification of this condition can be beneficial to prevent further deterioration. In cisplatin-induced and ischemia / reperfusion-induced AKI, urinary fetuin-A excretion increased before the serum creatinine surge and before morphological damage was present. In the current study, we found that uPTM-FetA was significantly associated with an increased risk of graft loss and graft function deterioration 3 years from baseline measurement. Since one of the most important requirements for a biomarker is to reflect the underlying pathophysiology of the disease, the measurement of uPTM-FetA in urine can provide an additional benefit over the currently used parameters, such as serum creatinine or proteinuria, as a biomarker for early detection of graft injury in KTRs.

[0928] This study has three important limitations that need to be mentioned. First, this study was performed in a single center in the Netherlands, with an over-representation of Caucasian population. Second, since this is an observational study, although we have adjusted for many potential confounders, residual confounding can still be present and the nature of this study does not allow to draw definite conclusions on causality. Third, we did not adjust the p-values for multiple testing, as this was an exploratory study. Nonetheless, this is the first study to evaluate the prospective association of urinary fetuin-A with long-term outcomes in the context of kidney transplantation.

[0929] In conclusion, 24-hour uPTM-FetA excretion was significantly associated with lower kidney function and higher 24-hour urinary albumin and protein excretion. Prospectively, 24-hour uPTM-FetA excretion was independently associated with an increased risk of graft loss and graft function deterioration more than 3 years after measurement. Our study results suggest that uPTM-FetA can be used as a clinical marker to allow early detection of graft loss in KTRs. Further studies in larger and more heterogeneous KTR populations are needed to confirm our study results.

[0930] Example 3-1

[0931] Design and study population

[0932] In this prospective cohort study, we used data from the TransplantLines Food and Nutrition Biobank and Cohort Study (NCT03272841). All adult KTRs who visited the outpatient clinic of the University Medical Center Groningen (UMCG) between November 2008 and March 2011, had normal graft function at least 1 year after transplantation, did not have any drug or alcohol addiction or systemic disease, were invited to participate in the cohort (18). During the recruitment period, 817 KTRs were invited, of whom 707 (87%) agreed to participate and gave written informed consent. For the current study, participants for whom no 24-hour uC-FetA excretion measurements were available were excluded from the analysis. The study was performed in accordance with the Declaration of Helsinki and the study protocol was approved by the Institutional Review Board (METc 2008 / 186).

[0933] The primary endpoint of the study was death-censored graft function deterioration (defined as doubling of serum creatinine or graft loss, i.e. the need for retransplantation or (re)start of dialysis). The secondary endpoint was all-cause mortality. Endpoints were recorded until September 2015. No participants were lost to follow-up.

[0934] Example 3-2

[0935] Clinical parameters

[0936] All measurements were performed during the morning outpatient clinic visit. Blood pressure and heart rate were measured three times with 15 minutes interval in between and the results were averaged. Measurements were performed using a semi-automatic device (Dinamap 1846, Critikon, Tampa, USA). Body weight and height were measured with the participant wearing indoor clothing and without shoes. Body mass index (BMI) was calculated as body weight (kg) divided by the square of height (m) (kg / m2). Blood samples were collected in tubes containing ethylenediaminetetraacetic acid (EDTA) and were centrifuged at 3000 rpm for 10 minutes within 1 hour after collection. Serum was stored at -80°C until analysis. 2), and body surface area (BSA) (square meters) (m2) was estimated using the DuBois and DuBois formula. 2 Diabetes was defined according to American Diabetes Association criteria. Estimated glomerular filtration (eGFR) was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula based on creatinine and cystatin C. Relevant donor, recipient, and transplant information were extracted from medical records as previously described.

[0937] Example 3-3

[0938] Laboratory methods and uC-FetA measurement

[0939] Blood was drawn in the morning at the outpatient clinic after overnight fasting (approximately 8 to 12 hours) which included no medication intake. For urine collection, all participants were instructed to collect their 24-hour urine during the day before their visit.

[0940] Cystatin C concentration in EDTA plasma was measured using a validated particle-enhanced immunoturbidimetric method (Gentian, Moss, Norway). A nanoparticle-based test was employed to measure the Calcification propensity test to assess the calciprotein particle maturation time (T50). Total urinary protein excretion was determined using the biuret reaction (MEGAAU 150, Merck Diagnostica, Darmstadt, Germany). Urinary liver-type fatty acid-binding protein (L-FABP) was measured using the human uL-FABP ELISA assay kit (CMIC holdings Co., Tokyo, Japan). Other biochemical parameters, including creatinine, high-sensitivity C-reactive protein (hs-CRP), and lipid profile were measured using routine laboratory methods. uC-FetA was measured using the novel human uPTM3-DKD ELISA kit (manufacturer: BioPreventive Medicine Corp., trade name: DNlite-IVD103).

[0941] Statistical analysis All data were analyzed using SPSS version 28.0 (IBM Corp., Armonk, NY, USA) and R version 4.2.1 (R Foundation for Statistical Computing, Vienna, Austria). For all analyses, a p-value < 0.05 was considered significant. Baseline characteristics were presented as mean ± standard deviation, median [interquartile range] and frequency (percentage) for normally distributed, skewed and nominal data, respectively. Visual assessment of Q-Q plots and histograms was performed to determine whether data were normally distributed or skewed. Univariate linear regression analysis was performed to assess the association between uC-FetA and clinical and biochemical parameters. After univariate analysis, we adjusted uC-FetA for gender, serum creatinine and 24-hour urinary protein excretion. Skewed data were log2-transformed to meet the assumption of linear regression during the linear regression analysis.

[0942] The association of uC-FetA with death-censored graft dysfunction and all-cause mortality was assessed using Kaplan-Meier curves, and the significance of differences between tertiles was calculated using the log-rank test. To further assess the association of uC-FetA with outcomes, Cox proportional hazards regression analysis was performed and adjusted for potential confounders. In model 1, 24-hour uC-FetA excretion was adjusted for age, gender and time post-transplantation at inclusion. In model 2, we additionally adjusted for eGFR according to the CKD-EPI formula based on creatinine and cystatin C. In model 3, we additionally adjusted for 24-hour urinary protein excretion. In model 4, we additionally adjusted for the presence of human leukocyte antigen class II antibodies, type of donor and donor age. In model 5, we additionally adjusted for hs-CRP. In the final model (model 6), we additionally adjusted for the use of proliferative inhibitors. Schoenfeld residuals were visually inspected and tested, and the final model did not violate the assumption of proportional hazards (p = 0.115 for death-censored graft dysfunction and p = 0.580 for all-cause mortality). We further assessed potential interactions of age, gender, eGFR and 24-hour urinary protein excretion with 24-hour uC-FetA excretion by adding interaction terms to the final model. Hazard ratios were expressed as per doubling of 24-hour uC-FetA with 95% confidence intervals (95% CI).

[0943] Since we identified an interaction between uC-FetA and eGFR with graft function worsening as an outcome of death, we repeated the final model of Cox regression analysis in a subgroup. We used the median eGFR value of patients with graft function worsening (eGFR = 30 mL / min / 1.73 m2) as a cutoff to divide patients into two groups. For all cross-sectional analyses, the original non-imputed dataset was used, with variables having >20 missing values (3.16%) reported in the table footnotes. For all prospective analyses, multiple imputation using the complete case specification was performed using the R package “mice” (number of multiple imputations = 10) to account for missing data in variables other than data on 24-hour uC-FetA excretion.

[0944] Example 3-4

[0945] Results

[0946] Baseline characteristics

[0947] Figure 243 shows a flowchart of study participant selection according to non-limiting embodiments. Referring to Figure 243, the present study included a total of 632 KTRs. The mean age was 53 ± 13 years, 42% were female, the median time post-transplant was 5.3 [1.7 to 12.2] years, and the mean eGFR was 45.3 ± 18.7 mL / min / 1.73. The median 24-hour uC-FetA excretion was 33.9 [17.2-74] pg / 24 hours. The median 24-hour urine excretion was 0.21 [0.2-0.34] g / 24 hours, with 511 (81%) having a urine protein excretion of <0.5 g / 24 hours, and 8 (1.26%) having a urine protein excretion of >3 g / 24 hours. More detailed baseline characteristics of the study population are shown in Table 1.

[0948] Table 1. Study population characteristics and linear regression analysis of 24-hour uC-FetA excretion at baseline

[0949]

[0950]

[0951] Normally distributed data are presented as mean ± standard deviation, skewed data as median [interquartile range], and categorical data as number (percentage valid). Variables were log2-transformed to meet assumptions in linear regression analysis. Diabetes was defined according to American Diabetes Association criteria. Smoking status was missing for 39 patients, alcohol consumption for 66 patients, HbAlC for 22 patients, hs-CRP levels for 33 patients, creatinine clearance for 26 patients, and urinary liver-type fatty acid binding protein for 61 patients. All other variables had missing values in < 20 patients. Abbreviations: eGFR, estimated glomerular filtration rate calculated using the CKD-EPI formula based on creatinine and cystatin C; HLA, human leukocyte antigen; hs-CRP, high-sensitivity C-reactive protein; L-FABP, liver-type fatty acid binding protein; mTOR, mammalian target of rapamycin; uC-FetA, fetuin-A containing urinary connecting peptide. Cross-sectional association univariate linear regression analysis of 24-hour uC-FetA excretion with clinical and biochemical parameters showed that male gender was significantly associated with higher 24-hour uC-FetA excretion (St. β = 0.11). Older donor age, living donor, and use of proliferation inhibitors were significantly associated with higher uC-FetA excretion, independent of gender, serum creatinine, and 24-hour urinary protein excretion. In contrast, longer duration after transplantation and higher circulating aspartate aminotransferase (AST) levels were negatively associated with uC-FetA excretion.

[0952] The association of 24-hour uC-FetA excretion with 24-hour urinary L-FABP excretion (St. β = 0.50) and 24-hour urinary protein excretion (St. β = 0.44) was strongest in univariate linear regression analysis. uC-FetA excretion was also positively associated with plasma NGAL and plasma fibroblast growth factor 23 (FGF23) (St. β = 0.24 and St. β = 0.19, respectively) and negatively associated with eGFR and T50 (St. β = -0.29 and St. β = -0.17, respectively). After adjustment for gender, serum creatinine, and 24-hour urinary protein excretion, urinary L-FABP remained significantly associated with uC-FetA excretion (Std β = 0.32), whereas plasma NGAL, plasma FGF23, and T50 lost their association (Table 1). Prospective analysis of the association of 24-hour uC-FetA excretion with death-censored graft dysfunction. During a median follow-up of 5.3 [4.0-6.0] years after baseline measurement, 121 (19%) KTR developed graft dysfunction. The rate of graft dysfunction was 8%, 16.6%, and 32.9% in KTR with baseline 24 uC-FetA excretion in the lowest, intermediate, and highest tertile, respectively (plog rank < 0.001) Figure 24). In Cox regression analysis, KTRs with 24-hour uC-FetA excretion in the 2nd and 3rd tertile had a higher risk of graft dysfunction compared to KTRs with 24-hour uC-FetA excretion in the lowest tertile (HR 2.18, 95% CI: 1.22-3.90, and HR 4.79, 95% CI: 2.82-8.14, respectively). The association of the 3rd tertile of 24-hour uC-FetA excretion remained significant after adjustment for potential confounders (HR 2.68; 95% CI, 1.49-4.82). Similarly, 24-hour uC-FetA excretion was also associated with a persistently higher risk of graft dysfunction even after adjustment for potential confounders (HR per doubling 1.19, 95% CI: 1.06-1.33) (Table 2).

[0953] Table 2. Prospective analysis of the association of 24-hour uC-FetA excretion with death-censored graft dysfunction in 632 kidney transplant recipients.

[0954]

[0955] Cox proportional hazards regression analysis was performed to assess the association of 24-hour uC-FetA excretion with the risk of death-censored graft dysfunction (doubling of serum creatinine or graft loss, i.e. need for retransplantation or (re)start of dialysis). Model 1 was adjusted for age, sex, and time post-transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine and cystatin C. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for the presence of human leukocyte antigen class II antibodies, type of donor, and donor age. Model 5 was further adjusted for serum high-sensitivity C-reactive protein (log2). Model 6 was further adjusted for use of proliferation inhibitors. CI, confidence interval; HR, hazard ratio, uC-FetA, urine connecting peptide fetuin-A.

[0956] Example 3-5

[0957] Sensitivity analysis

[0958] For the association of 24-hour uC-FetA excretion with graft function deterioration, there was no interaction between 24-hour uC-FetA excretion and gender, age, or 24-hour urinary protein excretion (pinteraction=0.07, 0.913, and 0.202, respectively). However, a significant interaction with eGFR was present (pinteraction=0.01). Sensitivity analysis in subgroups was performed for the final model (model 6). Analysis showed that 24-hour uC-FetA excretion was only significantly associated with graft function deterioration in patients with eGFR≥30 mL / min / 1.73 m2(HR 1.19, 95% CI 1.01-1.41; p=0.040), but not in patients with eGFR<30 mL / min / 1.73 m2(HR 1.02, 95% CI 0.84-1.23, p=0.858), as shown in Figure 25 . Figure 25 Forest plot showing the association of uC-FetA with death-censored graft function deterioration in subgroups, according to non-limiting embodiments. Reference is made to Figure 25 , Forest plot of the association of uC-FetA with death-censored graft function deterioration in subgroups. The model was adjusted for the complete model of age, gender, log2 time on transplant at inclusion, eGFR, log2 24-hour urinary protein excretion, presence of human leukocyte antigen class II antibodies, type of donor, donor age, log2 serum high-sensitivity C-reactive protein, and use of immunosuppressants.

[0959] Prospective analysis of the association between uC-FetA and all-cause mortality

[0960] In a secondary analysis, we evaluated the association of uC-FetA with all-cause mortality. During a median follow-up of 5.4 [4.9 to 6.1] years after the baseline measurement, 133 (21%) KTRs died. There was no significant difference in mortality between tertiles of uC-FetA (18.5% in the first tertile, 20.4% in the second tertile, and 24.3% in the third tertile; plog-rank=0.282) Figure 26 ). Unadjusted and adjusted Cox regression models also showed no prospective association between 24-hour uC-FetA excretion and all-cause mortality (Table 3).

[0961] Table 36 Prospective analysis of the association of 24-hour uC-FetA excretion with all-cause mortality in 632 kidney transplant recipients

[0962]

[0963] Cox proportional hazards regression analysis was performed to assess the association of 24-hour uC-FetA excretion with the risk of all-cause mortality. Model 1 was adjusted for age, sex, and time post-transplantation at inclusion (log2). Model 2 was further adjusted for estimated glomerular filtration rate according to the CKD-EPI formula based on creatinine and cystatin C. Model 3 was further adjusted for 24-hour urinary protein excretion (log2). Model 4 was further adjusted for the presence of human leukocyte antigen class II antibodies, type of donor, and donor age. Model 5 was further adjusted for serum high-sensitivity C-reactive protein (log2). Model 6 was further adjusted for the use of proliferation inhibitors. CI, confidence interval; HR, hazard ratio, uC-FetA, uC-peptide-containing FetA.

[0964] While preferred embodiments of the application have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the application. It should be understood that various alternatives to the embodiments of the application described herein can be employed in practicing the application. The following claims are intended to define the scope of the application and are accordingly not to be limited by the steps recited in the claims.

[0965] Example 3-6

[0966] The human uPTM3-DKD ELISA is a colorimetric immunoassay intended for the quantitative measurement of unique Fetuin-A with specific post-translational modification (PTM) in human urine and should be performed by certified medical professionals such as medical technicians in a qualified clinical laboratory.

[0967] Principle of the test

[0968] The human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrators or unknown urine samples are mixed with an anti-unique PTM Fetuin-A monoclonal antibody (mAb) and then incubated in microplates previously bound with unique PTM Fetuin-A. The monoclonal antibody recognizes unique PTM Fetuin-A in calibrators or unknown samples under competition in the microplate wells. After incubation, a horseradish peroxidase (HRP)-conjugated secondary antibody is added and then incubated with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve.

[0969] Intended use

[0970] The Human uPTM3-DKD ELISA is a colorimetric immunoassay intended for the quantitative measurement of unique Fetuin-A with specific post-translational modifications (PTMs) in human urine and should be performed by certified medical professionals such as medical technicians in a qualified clinical laboratory. This product is for in vitro diagnostic use as an aid in the assessment of the risk of renal complications in diabetic patients. In addition, the uPTM3-DKD assay can also be applied to accurately distinguish the progressive decline of renal function in type 2 diabetic patients with microalbuminuria.

[0971] Contents

[0972] Sufficient for 96 assays

[0973]

[0974] Storage and stability

[0975]

[0976] * Do not expose reagents to sunlight, heat and moisture during storage and use.

[0977] * Reagents are stable within the shelf life indicated on the vial.

[0978] * Once opened, diluent, 10X wash buffer, TMB substrate and stop solution are stable for 4 weeks at 4°C and coated microplates, 1000X mAb anti-unique PTM Fetuin-A (E103) and 4000X HRP conjugate are stable for 4 weeks at -20°C.

[0979] Reagent preparation

[0980] 1. Human unique PTM Fetuin-A (E103) calibrators

[0981] Reconstitute the E103 calibrators with 0.2 mL of distilled or deionized water, let stand at room temperature for 10 minutes until completely dissolved and mix gently. The reconstituted E103 calibrators are now at a concentration of 5 pg / mL. Dilute the 5 pg / mL E103 calibrators 5-fold with diluent, then mix gently to obtain 1 pg / mL E103 calibrators. The procedure for generating serial dilutions of all calibrators for establishing the E103 calibration curve is shown in the table below and all calibrators must be prepared immediately before use and mixed thoroughly.

[0982]

[0983] 2. 1X Wash Buffer

[0984] Bring the 10X wash buffer to room temperature until all salt crystals are dissolved before use. Calculate the amount of 1X wash buffer needed for each assay. For each microplate, mix 50 mL of 10X wash buffer with 450 mL of distilled or deionized water. Mix evenly but gently.

[0985] 3. 1X mAb Anti-Unique PTM Fetuin-A (E103)

[0986] Calculate the amount of 1X mAb Anti-Unique PTM Fetuin-A (E103) needed for each assay and mix the 1000X mAb Anti-Unique PTM Fetuin-A (E103) with diluent according to the amount needed. For each microplate, mix 8 μL of 1000X mAb Anti-Unique PTM Fetuin-A (E103) with 8 mL of diluent. Mix evenly but gently.

[0987] 4. 1X HRP Conjugate

[0988] Calculate the amount of 1X HRP Conjugate needed for each assay and mix the 4000X HRP Conjugate with diluent according to the amount needed. For each microplate, mix 3 μL of 4000X HRP Conjugate with 12 mL of diluent. Mix evenly but gently.

[0989] Materials Needed but Not Provided

[0990] • Precision single micropipettes (1-10 μL, 20-100 μL, 100-200 μL, 200-1000 μL) and multichannel pipettes (100 μL)

[0991] • Microcentrifuge tubes and disposable tips

[0992] • Graduated cylinder 500 mL

[0993] • Vortex mixer and microcentrifuge

[0994] • Orbital shaker

[0995] • Plastic containers for reagent preparation

[0996] • Microplate reader capable of endpoint measurement at 450 ± 10 nm

[0997] • Distilled or deionized water

[0998] • Adhesive plate seal

[0999] • Reagent reservoirs

[1000] Warnings and Precautions

[1001] • This test is for professional in vitro diagnostic use only.

[1002] • Do not use expired reagents.

[1003] • Improper temperature exposure during all storage and assay procedures can adversely affect results.

[1004] • Do not reuse microwell plate wells.

[1005] • Wear protective gloves during all assay procedures.

[1006] • Due to the light sensitivity and skin irritating properties of the TMB substrate, avoid direct light exposure and skin contact during all storage and assay procedures.

[1007] • Avoid skin contact with the stop solution containing 0.5N sulfuric acid. It can cause skin irritation and burns.

[1008] • Consider all clinical specimens to be potentially infectious.

[1009] • Dispose of all waste materials in accordance with local requirements and current Good Laboratory Practice regulations.

[1010] Specimen Collection and Handling

[1011] • Morning urine samples must be collected in a clean and dry container. Avoid cross contamination.

[1012] • No additives or preservatives are required to ensure the integrity of the urine sample.

[1013] • Store urine samples at -20°C until use. Avoid repeated freeze-thawing of urine samples.

[1014] • Prior to performing the assay, bring the urine sample to room temperature. Centrifuge the urine sample at 1,000 ± 20 x g for 5 minutes. Harvest the supernatant and assay immediately.

[1015] • If necessary, dilute the sample using diluent.

[1016] Assay Procedure

[1017] Prepare sufficient microwell plate modules for all calibrators and urine samples and secure them in the holder. Bring all reagents to room temperature prior to use.

[1018] 1. Mix calibrators 1-8 and each centrifuged urine sample with IX mAb Anti-E103 in a 1:1 ratio in a microcentrifuge tube (for a triplicate test, it is recommended to mix 180 μL of each calibrator / sample with 180 μL of IX mAb Anti-E103). Incubate on an orbital shaker at 25°C and 200 rpm for 2 hours.

[1019] 2. Transfer 100 pL of each incubation mixture to the designated wells of the E103 coated microplate and incubate on an orbital shaker at 25°C and 200 rpm for 1.5 hours. Keep the microplate covered and horizontal during all incubations.

[1020] 3. After incubation, discard the contents of the wells.

[1021] 4. Wash each well with 300-400 pL of IX wash buffer. Discard the contents and tap the wells on absorbent paper to remove residual liquid. Wash a total of 4 times.

[1022] 5. Add 100 pL of IX HRP conjugate to each well. Incubate the microplate on an orbital shaker at 25°C and 200 rpm for 60 minutes.

[1023] 6. After incubation, discard the contents of the wells and wash the wells as described in step 4.

[1024] 7. Add 100 pL of TMB substrate to each well. Incubate at room temperature for 30 minutes in the dark.

[1025] 8. Add 100 pL of stop solution to each well. Mix by brief shaking until the mixture becomes homogeneous.

[1026] 9. Measure the absorbance at 450 ± 10 nm within 30 minutes and calculate the results.

[1027] Calculation of results

[1028] 1. Use a 5-parameter or 4-parameter logistic curve fit to establish the calibration curve. The concentration of unique PTM Fetuin-A in patient urine samples can be calculated by interpolation from the calibration curve. The range of this assay is 7.813 - 500 ng / mL.

[1029] 2. Calculate the unique PTM Fetuin-A amount using urine creatinine correction (ng / mg Cr).

[1030] Performance characteristics

[1031] Precision

[1032] The precision of the human uPTM3-DKD ELISA was evaluated in a 5-day study. Three human urine samples from known diabetic patients and five spiked human urine samples were tested in duplicate per day in triplicate. Intra-batch precision: The coefficient of variation (CV) for each of the eight samples was calculated from the results of 3 determinations in each run. Inter-batch precision: The coefficient of variation (CV) for each of the eight samples was calculated from the results of 3 determinations in 10 different runs.

[1033] The precision results are summarized in the table below:

[1034]

[1035] Measurement range

[1036] 7.813-500 ng / mL

[1037] Linearity

[1038] The assay was evaluated and found to be linear from 1.769 to 500 ng / mL (R2= 0.99). 2

[1039] Limit of detection

[1040] Limit of blank (LoB) = 0.873 ng / mL

[1041] Limit of detection (LoD) = 4.510 ng / mL

[1042] Limit of quantitation (LoQ) = 18.983 ng / mL

[1043] Interfering substances

[1044]

[1045] For example, Figure 27 A calibration curve for the human uPTM3-DKD ELISA is shown according to non-limiting embodiments (the calibration range is 7.813-500 ng / mL).

[1046] Discussion

[1047] In this cohort of 632 stable KTRs, shorter time post-transplant, living donor, older donor age, use of proliferation inhibitors, and lower AST levels were independently associated with higher 24-hour uC-FetA excretion. In addition, 24-hour urine L-FABP excretion, a marker of proximal tubular injury, was also independently associated with 24-hour uC-FetA excretion.

[1048] ​In the prospective analysis, increased uC-FetA excretion was also strongly associated with an increased risk of graft function deterioration, even after adjustment for potential confounders. However, 24-hour uC-FetA excretion was not associated with an increased risk of death. In healthy adults, the kidney can not express Fetuin-A. However, upon injury, proximal tubular epithelial cells (PTEC) are able to express Fetuin-A and release it to the luminal side of the renal tubule. Zhou et al. (2006) showed that in cisplatin-induced and ischemia / reperfusion-induced AKI rat models, urinary Fetuin-A was mainly present in the urinary exosome fraction, but not from the non-exosomal fraction. Recently, Rudloff et al. (2021) also showed that upon hypoxia-inducible transcription factor stimulation, PTEC can locally produce Fetuin-A under hypoxic conditions. It is hypothesized that the presence of Fetuin-A in the proximal tubule can help protect the kidney from hypoxia-induced renal inflammation by preventing macrophages from transforming into pro-inflammatory macrophages M1 and protect the kidney from hypoxia-induced fibrosis by antagonizing TGF-beta signaling. The human precursor protein of Fetuin A consists of three parts, the A chain, the connecting peptide, and the B chain, with lengths of 321, 40, and 27 amino acids, respectively. The connecting peptide of the precursor is removed by post-translational modification, restricted proteolysis, after which only the A and B chains form the active Fetuin-A protein. The monoclonal antibody in the ELISA kit used in the current study (human uPTM3-DKD ELISA kit, BPM Corp.) can detect Fetuin-A containing the connecting peptide. Therefore, the uC-Fet detected by this ELISA kit can be inactive Fetuin-A, without kidney function-protecting effects, and elevated uC-FetA is associated with a risk of eGFR decline in patients.

[1049] In addition to local production, Fetuin-A in urine can also originate from circulation. Fetuin-A is a negatively charged molecule with a molecular weight of approximately 60 kilodaltons, similar to that of albumin. Under physiological conditions, a selective amount of albumin can pass the glomerular filtration barrier (GFB), where it will be reabsorbed by proximal tubular epithelial cells (PTEC) in the tubular compartment. As Fetuin-A and albumin have similar properties, an equivalent handling by the kidney can be expected. It has been shown in animal models that Fetuin-A is reabsorbed in the proximal tubule in healthy rats by megalin-mediated endocytosis, mainly in the S1 segment, with a lower degree of reabsorption in the S2 segment.

[1050] In the case of cumulative damage, the GFB will be destroyed and excess amounts of different types of plasma proteins will leak into the tubular compartment. This leads to an increased amount of filtered proteins that need to be reabsorbed by the proximal tubule. Since the reabsorptive capacity of PTECs is not unlimited, once this mechanism is saturated, proteins from the circulation will be present in the urine. In addition to this, filtered proteins can cause damage in the proximal tubule through various mechanisms, i.e. by obstructing the lumen with protein casts, by energy depletion and lysosomal rupture due to overload of PTECs with large amounts of proteins that need to be reabsorbed, by triggering PTECs to release pro-inflammatory and pro-fibrotic cytokines, by causing intracellular accumulation of lipidated proteins that will trigger apoptosis of the tubule, and by activating the complement system to cause direct damage to PTECs. When this happens, the ability of PTECs to locally produce fetuin-A and to reabsorb filtered fetuin-A will be impaired. Since 24h uC-FetA excretion in this study was still significantly associated with an increased risk of graft dysfunction, the source of uC-FetA can even after adju...

Claims

1. An assay kit for determining the likelihood of progressive decline in kidney function, the assay kit comprising: a first solution comprising a first reagent that interacts with a post-translationally modified fetuin-A fragment in a urine fragment to indicate a level of the fetuin A fragment in a urine sample from a recipient of a kidney transplant; a second solution comprising a second reagent that interacts with urine creatinine to indicate a level of the urine creatinine in the urine sample; a device for assaying the level of the fetuin A fragment and the level of the urine creatinine in the urine sample to determine a ratio of the assayed level of the fetuin A fragment to the assayed level of the urine creatinine, wherein a ratio higher than about 20 ng / mg indicates a higher likelihood of graft function deterioration in a period of about ten years or less, and wherein the first solution and the second solution are the same solution as one another or different solutions.

2. The assay kit of claim 1, wherein a ratio higher than about 20 ng / mg indicates a need for medical intervention to avoid the graft function deterioration.

3. The assay kit of claim 1, wherein the graft function deterioration is indicated by doubling of serum creatinine, need for a second kidney transplant, or need for dialysis.

4. The assay kit of claim 1, wherein the graft function deterioration is indicated by a decrease in estimated glomerular filtration rate (eGFR).

5. The assay kit of claim 1, wherein the interaction with the fetuin A fragment comprises binding to the fetuin A fragment.

6. The assay kit of claim 1, wherein the interaction with the urine creatinine comprises binding to the urine creatinine.

7. The assay kit of claim 1, wherein at least one of the first reagent and the second reagent comprises an antibody.

8. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 25 ng / mg.

9. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 30 ng / mg.

10. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 35 ng / mg.

11. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 40 ng / mg.

12. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 50 ng / mg.

13. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 70 ng / mg.

14. The assay kit of claim 1, wherein the ratio higher than about 20 ng / mg is higher than about 90 ng / mg.

15. The assay kit of claim 1, wherein the higher likelihood is higher than a likelihood of graft function deterioration in a comparative recipient of a kidney transplant having a ratio of a level of the Fetuin A fragment to a level of the urinary creatinine of less than about 14 ng / mg over a period of about ten years or less.

16. The assay kit of claim 15, wherein the second ratio of less than about 14 ng / mg is less than about 13 ng / mg.

17. The assay kit of claim 15, wherein the second ratio of less than about 14 ng / mg is less than about 9 ng / mg.

18. The assay kit of claim 15, wherein the second ratio of less than about 14 ng / mg is less than about 7.5 ng / mg.

19. The assay kit of claim 15, wherein the second ratio of less than about 14 ng / mg is less than about 7 ng / mg.

20. The assay kit of claim 15, wherein the second ratio of less than about 14 ng / mg is less than about 4 ng / mg.

21. The assay kit of claim 1, wherein the higher likelihood of graft function deterioration is about 5% or more.

22. The assay kit of claim 1, wherein the higher likelihood of graft function deterioration is about 10% or more.

23. The assay kit of claim 1, wherein the higher likelihood of graft function deterioration is about 30% or more.

24. The assay kit of claim 1, wherein the graft function deterioration occurs after 2 years.

25. The assay kit of claim 1, wherein the graft function deterioration occurs after 5 years.

26. The assay kit of claim 1, wherein the device is used to assay a level of the Fetuin A fragment and a level of the urinary creatinine to assess a probability of the graft function deterioration based on the ratio and at least one supplemental marker.

27. The assay kit of claim 26, wherein the at least one supplemental marker comprises age, gender, time post-transplant at inclusion, urinary albumin to creatine ratio (UACR), estimated glomerular filtration rate (eGFR) according to CKD-EPI formula based on creatinine and Cystatin C, presence of human leukocyte antigen, or any combination thereof.

28. The assay kit of claim 27, wherein the assessed probability shows an area under the receiver operating characteristic (ROC) curve (AUC) of at least about 70 (%).

29. The assay kit of claim 28, wherein the assessed probability shows the AUC of at least about 90 (%).

30. The assay kit of claim 28, wherein the assessed probability shows the AUC of at least about 95 (%).

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