Assays and Related Methods, Kits and Devices

Abstract

The present disclosure relates to correlating the ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine with the likelihood of progressive decline in renal function over time in a subject, leading to a renal endpoint.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 441,745, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," filed January 27, 2023, and U.S. Provisional Patent Application No. 63 / 532,636, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," filed August 14, 2023, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Diabetic nephropathy (DN), also known as diabetic kidney disease (DKD), is a progressive kidney disease associated with long-term diabetes mellitus. Over time, poorly controlled diabetes can cause damage to the vascular clusters in the kidneys that filter waste products from the blood. This can lead to kidney damage, which can cause abnormalities in fluid filtration and increased urinary albumin excretion, ultimately leading to kidney failure. Summary of the Invention

[0003] This Summary is provided to introduce a selection of concepts in a simplified form that are more fully described below in the Detailed Description. This Summary is not intended to identify key or essential aspects of the claimed subject matter.

[0004] All features of the exemplary embodiments described in this disclosure that are not mutually exclusive may be combined with each other. Elements of one embodiment may be utilized in other embodiments without further recitation. Other aspects and features of the present invention will become apparent to those skilled in the art upon review of the following description of specific embodiments in conjunction with any accompanying drawings.

[0005] In some embodiments, the present disclosure relates to a method of preparing an assay, the method comprising: providing a first solution comprising a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent that interacts with urinary post-translationally modified fetuin A fragment (uPTM-FetA) to measure a level of fetuin A fragment in the urine sample; providing a second solution comprising the urine sample and a second reagent that interacts with urinary creatinine to measure a level of urinary creatinine in the urine sample; and measuring the measured level of urinary creatinine in response to the measured level of fetuin A fragment and the measured level of urinary creatinine. and correlating a ratio of measured levels of fetuin-A fragments relative to the likelihood of progressive renal decline to a renal endpoint in the subject over a period of about 10 years, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive renal decline to a renal endpoint, and wherein the first solution and the second solution are the same or different solutions.

[0006] In some embodiments, the present disclosure relates to a method, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall progressive decline in renal function.

[0007] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is greater than a lower likelihood of progressive renal function decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg.

[0008] In some embodiments, the disclosure relates to a method, wherein the higher likelihood is less than the likelihood of progressive renal function decline over a period of about 10 years in a third subject having a ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine lower than about 29.0 ng / mg.

[0009] In some embodiments, the present disclosure relates to a method, wherein a ratio value greater than 7.5 ng / mg indicates a higher likelihood of progressive renal failure.

[0010] In some embodiments, the present disclosure relates to a method, the method further comprising assessing the probability of occurrence of progressive renal decline to a renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0011] In some embodiments, the present disclosure relates to a method, wherein at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level, respectively.

[0012] In some embodiments, the present disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline to a renal endpoint is based on formula (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg)……Formula (1) where I(E103 / UCr>7.53ng / mg)=1 if E103 / UCr>7.53ng / mg, or I(E103 / UCr>7.53ng / mg)=0 if E103 / UCr is 7.53ng / mg or less, y is a first probabilistic value that serves as the basis for assessing the probability of occurrence of progressive renal decline leading to a renal endpoint, and a0, a1, a2, a3, a4, a5, and a6 are assigned coefficients.

[0013] In some embodiments, the disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (2): p=exp(y) / (1+exp(y)) Equation (2) where p is a second probabilistic value that serves as the basis for assessing the probability of cancer occurrence.

[0014] In some embodiments, the present disclosure relates to a method, wherein a0 has a value of about −10.0 to about 0.5, a1 has a value of about −0.0 to about 0.1, a2 has a value of about −0.3 to about 1.2, a3 has a value of about −0.1 to about 0.0, a4 has a value of about 0.1 to about 0.7, a5 has a value of about −0.3 to about 0.3, and a6 has a value of about 1.2 to about 3.7.

[0015] In some embodiments, the disclosure relates to a method, the method further comprising determining that the patient who provided the urine is at elevated risk of progressive renal decline leading to a renal endpoint in response to the second probabilistic value exceeding the first threshold.

[0016] In some embodiments, the disclosure relates to a method, the method further comprising determining, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold, that the patient who donated the urine is at a higher than increased risk of progressive renal decline leading to a renal endpoint.

[0017] In some embodiments, the present disclosure relates to a method, wherein the first threshold is about 0.1.

[0018] In some embodiments, the present disclosure relates to a method, wherein the first threshold is about 0.11.

[0019] In some embodiments, the present disclosure relates to a method, wherein the second threshold is about 0.25.

[0020] In some embodiments, the present disclosure relates to a method, wherein the second threshold is about 0.3.

[0021] In some embodiments, the present disclosure relates to a method, wherein the second threshold is about 0.27.

[0022] In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0023] In some embodiments, the present disclosure relates to a method, wherein the subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater.

[0024] In some embodiments, the present disclosure relates to a method, wherein the second subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2).

[0025] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0026] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collecting the urine sample.

[0027] In some embodiments, the disclosure relates to a method, wherein the subject or second subject was diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0028] In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg. In some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg, hi some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is between about 7.5 ng / mg and about 29 ng / mg. In some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg, hi some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0029] In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0030] In some embodiments, the present disclosure relates to a method, wherein the second ratio lower than about 7.5 ng / mg is lower than about 6 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4.5 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.5 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.3 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio lower than about 7.5 ng / mg is lower than about 1 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.5 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.2 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.1 ng / mg.

[0031] In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 10% or greater. In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 15% or greater. In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 20% or greater. In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 25% or greater. In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 30% or greater. In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 35% or greater. In some embodiments, the disclosure relates to a method, wherein the higher likelihood of progressive renal function decline to a renal endpoint is about 40% or greater. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 45% or greater. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 50% or greater.

[0032] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 10% to about 50%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 15% to about 45%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 20% to about 40%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 25% to about 35%.

[0033] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 25% to about 30%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 10% to about 20%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 15% to about 25%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 20% to about 30%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 30% to about 40%.

[0034] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 35% to about 45%. In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 40% to about 50%.

[0035] In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 4 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 5 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 6 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 7 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 8 years.

[0036] In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 9 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 7 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 8 years. In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 9 years.

[0037] In some embodiments, the disclosure relates to a method, wherein interacting with fetuin A fragment comprises binding to the fetuin A fragment. In some embodiments, the disclosure relates to a method, wherein interacting with urinary creatinine comprises binding to urinary creatinine. In some embodiments, the disclosure relates to a method, wherein at least one of the first reagent and the second reagent comprises an antibody. In some embodiments, the disclosure relates to an assay kit for determining the likelihood of progressive renal function decline, the assay kit comprising: a first solution comprising a first reagent that interacts with fetuin A fragment to indicate a level of urinary post-translationally modified fetuin A fragment (uPTM-FetA) in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution comprising a second reagent that interacts with urinary creatinine to indicate a level of urinary creatinine in the urine sample; and a second solution comprising a second reagent that interacts with urinary creatinine to determine a ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine. and a device for measuring the level of fetuin A fragment and the level of urinary creatinine in the urine sample for determining whether or not a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive decline in renal function to a renal endpoint over a period of about 10 years in the subject, and wherein the first solution and the second solution are the same or different solutions.

[0038] In some embodiments, the present disclosure relates to an assay kit, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall progressive decline in kidney function.

[0039] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood is greater than the lower likelihood of progressive renal function decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg.

[0040] In some embodiments, the disclosure relates to an assay kit, wherein the higher likelihood is less than the likelihood of progressive renal decline over a period of about 10 years in a third subject having a ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine lower than about 29.0 ng / mg.

[0041] In some embodiments, the present disclosure relates to an assay kit, wherein a ratio value greater than 7.5 ng / mg indicates a higher likelihood of progressive renal failure.

[0042] In some embodiments, the present disclosure relates to an assay kit, the assay kit further comprising assessing the probability of occurrence of progressive renal decline to a renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0043] In some embodiments, the present disclosure relates to an assay kit, wherein at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level, respectively.

[0044] In some embodiments, the present disclosure relates to an assay kit, wherein assessing the probability of occurrence of progressive renal decline to a renal endpoint is based on formula (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg)……Formula (1) where I(E103 / UCr>7.53ng / mg)=1 if E103 / UCr>7.53ng / mg, or I(E103 / UCr>7.53ng / mg)=0 if E103 / UCr is 7.53ng / mg or less, y is a first probabilistic value that serves as the basis for assessing the probability of occurrence of progressive renal decline leading to a renal endpoint, and a0, a1, a2, a3, a4, a5, and a6 are assigned coefficients.

[0045] In some embodiments, the present disclosure relates to an assay kit, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on formula (2): p=exp(y) / (1+exp(y))……Equation (2) where p is a second probabilistic value that serves as the basis for assessing the probability of cancer occurrence.

[0046] In some embodiments, the present disclosure relates to an assay kit, wherein the value of a0 is about −10.0 to about 0.5, the value of a1 is about −0.0 to about 0.1, the value of a2 is about −0.3 to about 1.2, the value of a3 is about −0.1 to about 0.0, the value of a4 is about 0.1 to about 0.7, the value of a5 is about −0.3 to about 0.3, and the value of a6 is about 1.2 to about 3.7.

[0047] In some embodiments, the disclosure relates to an assay kit, wherein the method further includes determining that the patient providing the urine is at elevated risk of progressive renal decline leading to a renal endpoint in response to the second probabilistic value exceeding the first threshold.

[0048] In some embodiments, the disclosure relates to an assay kit, wherein the method further includes determining, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold, that the patient providing the urine is at a higher than increased risk of progressive renal decline leading to a renal endpoint.

[0049] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0050] In some embodiments, the present disclosure relates to an assay kit, wherein a subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater.

[0051] In some embodiments, the present disclosure relates to an assay kit, wherein a second subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2).

[0052] In some embodiments, the present disclosure relates to an assay kit, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0053] In some embodiments, the present disclosure relates to an assay kit, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0054] In some embodiments, the present disclosure relates to an assay kit, wherein the subject or second subject has been diagnosed with type 2 diabetes mellitus about 7 to about 20 years prior to collection of the urine sample.

[0055] In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0056] In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0057] In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is between about 7.5 ng / mg and about 29 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg.

[0058] In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0059] In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg. In some embodiments, the disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0060] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio lower than about 7.5 ng / mg is lower than about 6 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio lower than about 7.5 ng / mg is lower than about 5 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.5 ng / mg.

[0061] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 3.3 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 1 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 0.5 ng / mg.

[0062] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 0.2 ng / mg.

[0063] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 0.1 ng / mg.

[0064] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 10% or greater.

[0065] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 15% or greater.

[0066] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% or greater.

[0067] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 25% or greater.

[0068] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 30% or greater.

[0069] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 35% or greater.

[0070] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 40% or greater.

[0071] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 45% or greater.

[0072] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 50% or greater.

[0073] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 10% and about 50%.

[0074] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 15% and about 45%.

[0075] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 40%.

[0076] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 35%.

[0077] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 30%.

[0078] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 10% to about 20%.

[0079] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 15% to about 25%.

[0080] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 30%.

[0081] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 30% to about 40%.

[0082] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 35% to about 45%.

[0083] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 40% to about 50%.

[0084] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after four years.

[0085] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 5 years.

[0086] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 6 years.

[0087] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 7 years.

[0088] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 8 years.

[0089] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 9 years.

[0090] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is expected to lead to a renal endpoint within seven years.

[0091] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is expected to lead to a renal endpoint within 8 years.

[0092] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is expected to lead to a renal endpoint within 9 years.

[0093] In some embodiments, the present disclosure relates to an assay kit, wherein interacting with a fetuin A fragment comprises binding to a fetuin A fragment.

[0094] In some embodiments, the present disclosure relates to an assay kit, wherein interacting with urinary creatinine includes binding to urinary creatinine.

[0095] In some embodiments, the present disclosure relates to an assay kit, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0096] In some embodiments, the present disclosure relates to a method of correlating the likelihood of progressive renal function decline, the method comprising measuring the levels of a first biomarker and a second biomarker in a urine sample from a subject diagnosed with type 2 diabetes mellitus, wherein the first biomarker is a urinary post-translationally modified fragment of fetuin A (uPTM-FetA) and the second biomarker is urinary creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; and correlating the ratio with the likelihood of progressive renal function decline to a renal endpoint in the subject over a period of about 10 years, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive renal function decline to a renal endpoint.

[0097] In some embodiments, the present disclosure relates to a method, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall progressive decline in renal function.

[0098] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is greater than a lower likelihood of progressive renal function decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg.

[0099] In some embodiments, the disclosure relates to a method, wherein the higher likelihood is less than the likelihood of progressive renal function decline over a period of about 10 years in a third subject having a ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine lower than about 29.0 ng / mg.

[0100] In some embodiments, the present disclosure relates to a method, wherein a ratio value greater than 7.5 ng / mg indicates a higher likelihood of progressive renal failure.

[0101] In some embodiments, the present disclosure relates to a method, the method further comprising assessing the probability of occurrence of progressive renal decline to a renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0102] In some embodiments, the present disclosure relates to a method, wherein at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level, respectively.

[0103] In some embodiments, the disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg)...Equation (1), where I(E103 / UCr>7.53ng / mg)=1 if E103 / UCr>7.53ng / mg, or I(E103 / UCr>7.53ng / mg)=0 if E103 / UCr is less than or equal to 7.53ng / mg, and y is a first probabilistic value underlying assessing the probability of occurrence of progressive renal decline leading to a renal endpoint, and a0, a1, a2, a3, a4, a5, and a6 are assigned coefficients.

[0104] In some embodiments, the present disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (2): p=exp(y) / (1+exp(y)) Equation (2), where p is a second probabilistic value based on which the probability of occurrence of cancer is assessed.

[0105] In some embodiments, the present disclosure relates to a method, wherein a0 has a value of about −10.0 to about 0.5, a1 has a value of about −0.0 to about 0.1, a2 has a value of about −0.3 to about 1.2, a3 has a value of about −0.1 to about 0.0, a4 has a value of about 0.1 to about 0.7, a5 has a value of about −0.3 to about 0.3, and a6 has a value of about 1.2 to about 3.7.

[0106] In some embodiments, the disclosure relates to a method, the method further comprising determining that the patient who provided the urine is at elevated risk of progressive renal decline leading to a renal endpoint in response to the second probabilistic value exceeding the first threshold.

[0107] In some embodiments, the disclosure relates to a method, the method further comprising determining, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold, that the patient who donated the urine is at a higher than increased risk of progressive renal decline leading to a renal endpoint.

[0108] In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0109] In some embodiments, the present disclosure relates to a method, wherein the subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater.

[0110] In some embodiments, the present disclosure relates to a method, wherein the second subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2).

[0111] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0112] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collecting the urine sample.

[0113] In some embodiments, the disclosure relates to a method, wherein the subject or second subject was diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0114] In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg. In some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg. In some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is between about 7.5 ng / mg and about 29 ng / mg. In some embodiments, the disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg.

[0115] In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg. In some embodiments, the disclosure relates to a method wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0116] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 6 ng / mg.

[0117] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 4.5 ng / mg.

[0118] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 4 ng / mg.

[0119] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 3.5 ng / mg.

[0120] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 3.3 ng / mg.

[0121] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 1 ng / mg.

[0122] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.5 ng / mg.

[0123] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.2 ng / mg.

[0124] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.1 ng / mg.

[0125] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 10% or greater.

[0126] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 15% or greater.

[0127] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 20% or greater.

[0128] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 25% or greater.

[0129] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 30% or greater.

[0130] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 35% or greater.

[0131] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 40% or greater.

[0132] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 45% or greater.

[0133] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 50% or greater.

[0134] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is between about 10% and about 50%.

[0135] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is between about 15% and about 45%.

[0136] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 20% and about 40%.

[0137] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 25% to about 35%.

[0138] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 30%.

[0139] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 10% to about 20%.

[0140] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 15% to about 25%.

[0141] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 30%.

[0142] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 30% to about 40%.

[0143] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 35% to about 45%.

[0144] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 40% to about 50%.

[0145] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after four years.

[0146] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 5 years.

[0147] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 6 years.

[0148] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 7 years.

[0149] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 8 years.

[0150] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 9 years.

[0151] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within seven years.

[0152] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 8 years.

[0153] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 9 years.

[0154] In some embodiments, the present disclosure relates to a method, wherein interacting with a fetuin A fragment comprises binding to the fetuin A fragment.

[0155] In some embodiments, the present disclosure relates to a method, wherein interacting with urinary creatinine includes binding to urinary creatinine.

[0156] In some embodiments, the present disclosure relates to a method, wherein at least one of the first reagent and the second reagent comprises an antibody. [Brief explanation of the drawings]

[0157] [Figure 1A]Illustrated are Kaplan-Meier curves with endpoints of 30% decline in eGFR or eGFR<15 for non-CKD NTUH patients classified as high risk and low risk by IVD103 according to non-limiting embodiments. [Figure 1B] Illustrated are Kaplan-Meier curves with an endpoint of 30% decline in eGFR or eGFR<15 for patients in the DIALECT study classified as high risk and low risk by IVD103 according to non-limiting embodiments. [Figure 2] 1 illustrates a standard curve for the human uPTM3-DKD ELISA (calibration range 7.813-500 ng / mL), according to a non-limiting embodiment. [Figure 3] 1 illustrates Kaplan-Meier curves for 294 subjects from the NTUH cohort of Examples 5-7, according to non-limiting embodiments. [Figure 4A] 1 illustrates Kaplan-Meier curves by uPTM-FetA / UCr risk category for subjects with UCr<30 mg / g in the NTUH cohort in Examples 5-7, according to non-limiting embodiments. [Figure 4B] 1 illustrates Kaplan-Meier curves by uPTM-FetA / UCr risk category for subjects with UCr≧30 mg / g from the NTUH cohort in Examples 5-7, according to non-limiting embodiments. [Figure 5A] 1 illustrates Kaplan-Meier curves for the NTUH cohort, showing that adding uPTM-FetA / UCr to a predictive model according to non-limiting embodiments improves the ability to predict worsening renal function. [Figure 5B] 1 illustrates Kaplan-Meier curves for the DIALECT cohort, showing that adding uPTM-FetA / UCr to the predictive model according to non-limiting embodiments improves the ability to predict worsening renal function. [Figure 6]1 illustrates an ROC curve, showing that adding uPTM-FetA / UCr to a predictive model according to a non-limiting embodiment improves the ability to predict worsening renal function. [Figure 7] 1 illustrates a Kaplan-Meier curve indicating survival probability in years over time for a uPTM-FetA / UCr cutoff value of 1, according to a non-limiting embodiment. [Figure 8] 1 illustrates a Kaplan-Meier curve indicating event-free probability over time in years for uPTM-FetA / UCr cutoff 2 values, according to a non-limiting embodiment. [Figure 9] 1 illustrates a Kaplan-Meier curve showing progression-free probability over time in years for two cutoff values ​​of the incidence score, according to a non-limiting embodiment. [Figure 10] 1 illustrates a cumulative incidence plot against incidence score, according to a non-limiting embodiment. [Figure 11] 1 illustrates Kaplan-Meier curves for the DIALECT cohort showing progression-free probability over time in years for two cutoff values ​​of the incidence score, according to non-limiting embodiments. [Figure 12] 1 illustrates a cumulative incidence plot against incidence score for the DIALECT cohort, according to a non-limiting embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0158] The present invention is based on the unexpected discovery that urinary and serum proteins and their fragments, either alone or in combination, are differentially expressed in patients with diabetic kidney disease (DKD) compared with subjects without DKD. Therefore, these protein molecules are useful markers for diagnosing early DKD. It is of great importance to identify reliable biomarkers useful for diagnosing early DKD. DKD or DN is a kidney disorder associated with diabetes.

[0159] Due to the increasing incidence and prevalence of type 2 diabetes mellitus (T2DM) worldwide, diabetic kidney disease (DKD) has become one of the most common causes of chronic kidney disease (CKD) and end-stage kidney disease (ESKD). DKD occurs in up to 40% of T2DM patients, and its progression can be slowed or prevented by intervention. Therefore, timely identification of patients at high risk of renal function decline is of utmost importance.

[0160] In some embodiments, DKD can have five stages of progression:

[0161] Stage 1: characterized by diabetes mellitus with normal GFR and normoalbuminuria (e.g., ACR<30 mg / g);

[0162] Stage 2: Glomerular hyperfiltration (120 mL / min / 1.73 m 2 characterized by enlarged kidneys with normal GFR and normal albuminuria (e.g., ACR<30 mg / g),

[0163] Stage 3: characterized by microalbuminuria,

[0164] Stage 4: characterized by overt albuminuria and progressive decline in GFR, and

[0165] Stage 5: 15 mL / min / 1.73 m 2 Generally, stages 1-3 are considered early, and stages 4 and 5 are considered late.

[0166] In other words, DKD may not show symptoms in its early stages. Thus, detecting the onset of the disease can be difficult. In fact, current DKD diagnosis relies on the onset of microalbuminuria, which occurs when the kidneys are already damaged. The lack of early diagnostic tests hinders effective treatment of early DKD.

[0167] To assess the risk of progression to CKD, a commonly used marker in current clinical practice is the creatinine-based estimated glomerular filtration rate (eGFR). Unfortunately, eGFR is burdened by confounding from muscle mass, and its intra-individual trajectory is highly variable, preventing eGFR from reliably predicting the progression of kidney disease, especially in early DKD. Albuminuria is another commonly used biomarker to assess the risk of DKD. However, up to 40% of T2DM patients with worsening renal function remain normal in albuminuria. Therefore, the use of albuminuria in predicting DKD progression is limited by its low sensitivity.

[0168] As a result, eGFR and albuminuria are considered insufficient to predict an individual's renal function decline. Furthermore, these assessment scales may not be appropriately used to evaluate the response to renal-protective treatment in DKD. The limited capabilities of these and other existing markers of renal function decline are explained by the fact that they are "late" markers that indicate renal injury / dysfunction when damage has already occurred. Therefore, there is a need for biomarkers that accurately represent the underlying molecular mechanisms involved in disease progression, ideally ones that could serve as therapeutic targets. However, such biomarkers that have proven to have additional predictive value for DKD are currently lacking.

[0169] As used herein, the term "renal endpoint" refers to the degree of deterioration of renal function that requires renal replacement therapy, such as kidney transplantation, initiation of peritoneal dialysis, or hemodialysis. Deterioration of renal function can be indicated, for example, by estimated glomerular filtration rate (eGFR), such as eGFR calculated by the Chronic Kidney Disease Epidemiology Collaboration formula. For example, an eGFR of <15 mL / min / 1.73 m 2 Attainment of 0.05 is considered an indicator of the renal endpoint.

[0170] As used herein, the term "medical intervention" for declining kidney function refers to treatment of a recipient in an attempt to stabilize, slow, delay, or arrest deterioration, thereby reducing the rate of deterioration in the recipient. Various types of medical interventions can be implemented. Examples of medical interventions include dietary advice, dietary restrictions, limiting sodium and other ion intake, reducing or avoiding alcohol consumption, reducing or avoiding tobacco smoking or consumption of related products, taking various medications, weight management, kidney transplantation or retransplantation, dialysis, other medically acceptable treatments, and any combination thereof.

[0171] Thus, the present disclosure relates to diagnostic methods using biomarkers, which can be proteins that can be considered proteins or fragments of those proteins. A urine sample, a serum sample, or both urine and serum samples can be collected from a subject, and the urine level, serum level, or both levels of the biomarker can be measured via various methods (e.g., mass spectrometry and immunoassay).

[0172] When a biomarker contains a single protein molecule, its level in a subject can be compared to a reference point to determine the corresponding sign or likelihood of a target disease state. A reference point (e.g., a single reference point or multiple reference points) representing the level of the same biomarker, such as the level of a biomarker in subjects without DKD, can be determined based on representative levels of the biomarker in a group of DKD patients and subjects without DKD. For example, the reference point can be the midpoint between the average levels of two groups of DKD patients and subjects without DKD. For example, the reference point can be multiple points, an average point, or any point between multiple groups of DKD patients (e.g., groups of patients at different stages of DKD progression) and a group of subjects without DKD. In some embodiments, a biomarker level higher than the reference point indicates a target disease state such as DKD. In some embodiments, biomarker levels at multiple points indicate different stages or progression of a target disease state such as DKD.

[0173] There is a great clinical need for novel markers to predict renal function decline in patients with type 2 diabetes mellitus (T2DM).

[0174] Urinary post-translationally modified fetuin A fragments (uPTM-FetA), which may include urinary binding peptide-containing fetuin A fragments (uC-FetA), may be a biomarker indicating the possibility of progressive renal function decline leading to renal endpoints and may predict renal function decline in T2DM patients.

[0175] Fetuin A, encoded by the AHSG gene, is a hepatokine associated with insulin resistance and an independent risk factor for T2DM. As a hepatic secretory protein, fetuin A is known to bind to insulin receptors in muscle and adipose tissue, inhibiting insulin action. In insulin-resistant states, insulin signaling in renal arteriolar endothelial cells shifts from the PI3K axis to the MAPK axis, resulting in renal vasoconstriction. Fetuin A also stimulates the secretion of proinflammatory cytokines by perivascular adipose tissue. Therefore, fetuin A may impair renal function by affecting renal sinus fat located around the renal artery. In addition to the renal vasculature, insulin resistance directly impacts podocyte viability and tubular function.

[0176] Fetuin A or fetuin A-based fragments, such as urinary post-translationally modified fetuin A fragments (uPTM-FetA) (also known as alpha-2-Heremans-Schmid glycoprotein), possess or may possess properties suggesting that they may serve as such an alternative, independent, noninvasive marker. Fetuin A is elevated in the urine of patients with acute kidney injury (AKI), and recent evidence suggests that fetuin A is also associated with the degree of interstitial fibrosis / tubular atrophy (IFTA). Urinary fetuin A has also been elevated in patients with chronic kidney diseases such as autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis. Subsequently, urinary fetuin A has previously been shown to be associated with decreased renal function in patients with chronic kidney disease, including diabetic nephropathy, and this protein has been proposed as a biomarker for the early detection of these diseases.

[0177] Thus, fetuin A or its fragments may be causally involved markers and may have additional predictive value in the development and progression of DKD. This is supported by the results of large-scale urinary proteomic profiling among participants in the Taiwan Renal Biomarker Study, in which urinary concentrations of post-translationally modified binding peptide-containing fetuin A fragments (uPTM-FetA) were identified as a candidate marker for DKD in patients with T2DM. This study supports the identification of the uPTM-FetA marker as an early marker of worsening renal function in two large, independent, bi-ethnic, prospective cohort studies in patients with T2DM. This disclosure supports the independent association of uPTM-FetA with declining renal function in patients with type 2 diabetes, highlighting the potential clinical use of uPTM-FetA as a prognostic marker for kidney disease in patients with T2DM.

[0178] In some embodiments, the present disclosure relates to a method of preparing an assay, the method comprising: providing a first solution comprising a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent that interacts with urinary post-translationally modified fetuin A fragment (uPTM-FetA) to measure a level of fetuin A fragment in the urine sample; providing a second solution comprising the urine sample and a second reagent that interacts with urinary creatinine to measure a level of urinary creatinine in the urine sample; and measuring the measured level of urinary creatinine in response to the measured level of fetuin A fragment and the measured level of urinary creatinine. and correlating a ratio of measured levels of fetuin-A fragments relative to the likelihood of progressive renal decline to a renal endpoint in the subject over a period of about 10 years, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive renal decline to a renal endpoint, and wherein the first solution and the second solution are the same or different solutions.

[0179] In some embodiments, the present disclosure relates to a method, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall progressive decline in renal function.

[0180] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is greater than a lower likelihood of progressive renal function decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg.

[0181] In some embodiments, the disclosure relates to a method, wherein the higher likelihood is less than the likelihood of progressive renal function decline over a period of about 10 years in a third subject having a ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine lower than about 29.0 ng / mg.

[0182] In some embodiments, the present disclosure relates to a method, wherein a ratio value greater than 7.5 ng / mg indicates a higher likelihood of progressive renal failure.

[0183] In some embodiments, the present disclosure relates to a method, the method further comprising assessing the probability of occurrence of progressive renal decline to a renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0184] In some embodiments, the present disclosure relates to a method, wherein at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level, respectively.

[0185] In some embodiments, the disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg)...Equation (1), where I(E103 / UCr>7.53ng / mg)=1 if E103 / UCr>7.53ng / mg, or I(E103 / UCr>7.53ng / mg)=0 if E103 / UCr is less than or equal to 7.53ng / mg, and y is a first probabilistic value underlying assessing the probability of occurrence of progressive renal decline leading to a renal endpoint, and a0, a1, a2, a3, a4, a5, and a6 are assigned coefficients.

[0186] In some embodiments, the present disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (2): p=exp(y) / (1+exp(y)) Equation (2), where p is a second probabilistic value based on which the probability of occurrence of cancer is assessed.

[0187] In some embodiments, the present disclosure relates to a method, wherein a0 has a value of about −10.0 to about 0.5, a1 has a value of about −0.0 to about 0.1, a2 has a value of about −0.3 to about 1.2, a3 has a value of about −0.1 to about 0.0, a4 has a value of about 0.1 to about 0.7, a5 has a value of about −0.3 to about 0.3, and a6 has a value of about 1.2 to about 3.7.

[0188] In some embodiments, the disclosure relates to a method, the method further comprising determining that the patient who provided the urine is at elevated risk of progressive renal decline leading to a renal endpoint in response to the second probabilistic value exceeding the first threshold.

[0189] In some embodiments, the disclosure relates to a method, the method further comprising determining, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold, that the patient who donated the urine is at a higher than increased risk of progressive renal decline leading to a renal endpoint.

[0190] In some embodiments, the present disclosure relates to a method, wherein the first threshold is about 0.1.

[0191] In some embodiments, the present disclosure relates to a method, wherein the first threshold is about 0.11.

[0192] In some embodiments, the present disclosure relates to a method, wherein the second threshold is about 0.25.

[0193] In some embodiments, the present disclosure relates to a method, wherein the second threshold is about 0.3.

[0194] In some embodiments, the present disclosure relates to a method, wherein the second threshold is about 0.27.

[0195] In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0196] In some embodiments, the present disclosure relates to a method, wherein the subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater.

[0197] In some embodiments, the present disclosure relates to a method, wherein the second subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2).

[0198] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0199] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collecting the urine sample.

[0200] In some embodiments, the disclosure relates to a method, wherein the subject or second subject was diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0201] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg.

[0202] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg.

[0203] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0204] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg.

[0205] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg.

[0206] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg.

[0207] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0208] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg.

[0209] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 25 ng / mg.

[0210] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 30 ng / mg.

[0211] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0212] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg.

[0213] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0214] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg.

[0215] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg.

[0216] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0217] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 6 ng / mg.

[0218] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 4.5 ng / mg.

[0219] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 4 ng / mg.

[0220] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 3.5 ng / mg.

[0221] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 3.3 ng / mg.

[0222] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 1 ng / mg.

[0223] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.5 ng / mg.

[0224] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.2 ng / mg.

[0225] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.1 ng / mg.

[0226] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 10% or greater.

[0227] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 15% or greater.

[0228] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 20% or greater.

[0229] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 25% or greater.

[0230] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 30% or greater.

[0231] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 35% or greater.

[0232] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 40% or greater.

[0233] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 45% or greater.

[0234] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 50% or greater.

[0235] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is between about 10% and about 50%.

[0236] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is between about 15% and about 45%.

[0237] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 20% and about 40%.

[0238] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 25% to about 35%.

[0239] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 30%.

[0240] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 10% to about 20%.

[0241] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 15% to about 25%.

[0242] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 30%.

[0243] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 30% to about 40%.

[0244] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 35% to about 45%.

[0245] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 40% to about 50%.

[0246] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after four years.

[0247] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 5 years.

[0248] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 6 years.

[0249] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 7 years.

[0250] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 8 years.

[0251] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 9 years.

[0252] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within seven years.

[0253] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 8 years.

[0254] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 9 years.

[0255] In some embodiments, the present disclosure relates to a method, wherein interacting with a fetuin A fragment comprises binding to the fetuin A fragment.

[0256] In some embodiments, the present disclosure relates to a method, wherein interacting with urinary creatinine includes binding to urinary creatinine.

[0257] In some embodiments, the present disclosure relates to a method, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0258] In some embodiments, the present disclosure relates to an assay kit for determining the likelihood of progressive kidney function decline, the assay kit comprising: a first solution comprising a first reagent that interacts with fetuin A fragment to indicate the level of urinary post-translationally modified fetuin A fragment (uPTM-FetA) in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution comprising a second reagent that interacts with urinary creatinine to indicate the level of urinary creatinine in the urine sample; and a second reagent that interacts with urinary creatinine to determine the ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine. and a device for measuring the level of fetuin A fragment and the level of urinary creatinine in the urine sample for determining whether or not a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive decline in renal function to a renal endpoint over a period of about 10 years in the subject, and wherein the first solution and the second solution are the same or different solutions.

[0259] In some embodiments, the present disclosure relates to an assay kit, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall progressive decline in kidney function.

[0260] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood is greater than the lower likelihood of progressive renal function decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg.

[0261] In some embodiments, the disclosure relates to an assay kit, wherein the higher likelihood is less than the likelihood of progressive renal decline over a period of about 10 years in a third subject having a ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine lower than about 29.0 ng / mg.

[0262] In some embodiments, the present disclosure relates to an assay kit, wherein a ratio value greater than 7.5 ng / mg indicates a higher likelihood of progressive renal failure.

[0263] In some embodiments, the present disclosure relates to an assay kit, the assay kit further comprising assessing the probability of occurrence of progressive renal decline to a renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0264] In some embodiments, the present disclosure relates to an assay kit, wherein at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level, respectively.

[0265] In some embodiments, the disclosure relates to an assay kit, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg)...Equation (1), where I(E103 / UCr>7.53ng / mg)=1 if E103 / UCr>7.53ng / mg, or I(E103 / UCr>7.53ng / mg)=0 if E103 / UCr is less than or equal to 7.53ng / mg, and y is a first probabilistic value underlying assessing the probability of occurrence of progressive renal decline leading to a renal endpoint, and a0, a1, a2, a3, a4, a5, and a6 are assigned coefficients.

[0266] In some embodiments, the present disclosure relates to an assay kit, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (2): p=exp(y) / (1+exp(y)) Equation (2), where p is a second probabilistic value based on which the probability of occurrence of cancer is assessed.

[0267] In some embodiments, the present disclosure relates to an assay kit, wherein the value of a0 is about −10.0 to about 0.5, the value of a1 is about −0.0 to about 0.1, the value of a2 is about −0.3 to about 1.2, the value of a3 is about −0.1 to about 0.0, the value of a4 is about 0.1 to about 0.7, the value of a5 is about −0.3 to about 0.3, and the value of a6 is about 1.2 to about 3.7.

[0268] In some embodiments, the disclosure relates to an assay kit, wherein the method further includes determining that the patient providing the urine is at elevated risk of progressive renal decline leading to a renal endpoint in response to the second probabilistic value exceeding the first threshold.

[0269] In some embodiments, the disclosure relates to an assay kit, wherein the method further includes determining, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold, that the patient providing the urine is at a higher than increased risk of progressive renal decline leading to a renal endpoint.

[0270] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0271] In some embodiments, the present disclosure relates to an assay kit, wherein a subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater.

[0272] In some embodiments, the present disclosure relates to an assay kit, wherein a second subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2).

[0273] In some embodiments, the present disclosure relates to an assay kit, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0274] In some embodiments, the present disclosure relates to an assay kit, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0275] In some embodiments, the present disclosure relates to an assay kit, wherein the subject or second subject has been diagnosed with type 2 diabetes mellitus about 7 to about 20 years prior to collection of the urine sample.

[0276] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg.

[0277] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg.

[0278] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0279] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg.

[0280] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg.

[0281] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg.

[0282] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0283] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg.

[0284] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 25 ng / mg.

[0285] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 30 ng / mg.

[0286] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0287] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg.

[0288] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0289] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg.

[0290] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg.

[0291] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0292] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 6 ng / mg.

[0293] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 5 ng / mg.

[0294] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 4 ng / mg.

[0295] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 3.5 ng / mg.

[0296] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 3.3 ng / mg.

[0297] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 1 ng / mg.

[0298] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 0.5 ng / mg.

[0299] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 0.2 ng / mg.

[0300] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio less than about 7.5 ng / mg is less than about 0.1 ng / mg.

[0301] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 10% or greater.

[0302] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 15% or greater.

[0303] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% or greater.

[0304] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 25% or greater.

[0305] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 30% or greater.

[0306] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 35% or greater.

[0307] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 40% or greater.

[0308] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 45% or greater.

[0309] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 50% or greater.

[0310] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 10% and about 50%.

[0311] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 15% and about 45%.

[0312] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 40%.

[0313] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 35%.

[0314] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 30%.

[0315] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 10% to about 20%.

[0316] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 15% to about 25%.

[0317] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 30%.

[0318] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 30% to about 40%.

[0319] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of progressive renal decline leading to a renal endpoint is about 35% to about 45%.

[0320] In some embodiments, the present disclosure relates to an assay kit, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 40% to about 50%.

[0321] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after four years.

[0322] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 5 years.

[0323] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 6 years.

[0324] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 7 years.

[0325] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 8 years.

[0326] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is predicted to lead to a renal endpoint after 9 years.

[0327] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is expected to lead to a renal endpoint within seven years.

[0328] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is expected to lead to a renal endpoint within 8 years.

[0329] In some embodiments, the present disclosure relates to an assay kit, wherein progressive renal decline is expected to lead to a renal endpoint within 9 years.

[0330] In some embodiments, the present disclosure relates to an assay kit, wherein interacting with a fetuin A fragment comprises binding to a fetuin A fragment.

[0331] In some embodiments, the present disclosure relates to an assay kit, wherein interacting with urinary creatinine includes binding to urinary creatinine.

[0332] In some embodiments, the present disclosure relates to an assay kit, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0333] In some embodiments, the present disclosure relates to a method of correlating the likelihood of progressive renal function decline, the method comprising measuring the levels of a first biomarker and a second biomarker in a urine sample from a subject diagnosed with type 2 diabetes mellitus, wherein the first biomarker is a urinary post-translationally modified fragment of fetuin A (uPTM-FetA) and the second biomarker is urinary creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; and correlating the ratio with the likelihood of progressive renal function decline to a renal endpoint in the subject over a period of about 10 years, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive renal function decline to a renal endpoint.

[0334] In some embodiments, the present disclosure relates to a method, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall progressive decline in renal function.

[0335] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is greater than a lower likelihood of progressive renal function decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg.

[0336] In some embodiments, the disclosure relates to a method, wherein the higher likelihood is less than the likelihood of progressive renal function decline over a period of about 10 years in a third subject having a ratio of measured levels of fetuin A fragment to measured levels of urinary creatinine lower than about 29.0 ng / mg.

[0337] In some embodiments, the present disclosure relates to a method, wherein a ratio value greater than 7.5 ng / mg indicates a higher likelihood of progressive renal failure.

[0338] In some embodiments, the present disclosure relates to a method, the method further comprising assessing the probability of occurrence of progressive renal decline to a renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0339] In some embodiments, the present disclosure relates to a method, wherein at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, age, sex, eGFR, UACR, and HbA1c level, respectively.

[0340] In some embodiments, the disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg)...Equation (1), where I(E103 / UCr>7.53ng / mg)=1 if E103 / UCr>7.53ng / mg, or I(E103 / UCr>7.53ng / mg)=0 if E103 / UCr is less than or equal to 7.53ng / mg, and y is a first probabilistic value underlying assessing the probability of occurrence of progressive renal decline leading to a renal endpoint, and a0, a1, a2, a3, a4, a5, and a6 are assigned coefficients.

[0341] In some embodiments, the present disclosure relates to a method, wherein assessing the probability of occurrence of progressive renal decline leading to a renal endpoint is based on Equation (2): p=exp(y) / (1+exp(y)) Equation (2), where p is a second probabilistic value based on which the probability of occurrence of cancer is assessed.

[0342] In some embodiments, the present disclosure relates to a method, wherein a0 has a value of about −10.0 to about 0.5, a1 has a value of about −0.0 to about 0.1, a2 has a value of about −0.3 to about 1.2, a3 has a value of about −0.1 to about 0.0, a4 has a value of about 0.1 to about 0.7, a5 has a value of about −0.3 to about 0.3, and a6 has a value of about 1.2 to about 3.7.

[0343] In some embodiments, the disclosure relates to a method, the method further comprising determining that the patient who provided the urine is at elevated risk of progressive renal decline leading to a renal endpoint in response to the second probabilistic value exceeding the first threshold.

[0344] In some embodiments, the disclosure relates to a method, the method further comprising determining, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold, that the patient who donated the urine is at a higher than increased risk of progressive renal decline leading to a renal endpoint.

[0345] In some embodiments, the disclosure relates to a method, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0346] In some embodiments, the present disclosure relates to a method, wherein the subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2) or greater.

[0347] In some embodiments, the present disclosure relates to a method, wherein the second subject's eGFR is indicated to be about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m2).

[0348] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0349] In some embodiments, the present disclosure relates to a method, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collecting the urine sample.

[0350] In some embodiments, the disclosure relates to a method, wherein the subject or second subject was diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0351] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg.

[0352] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg.

[0353] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0354] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg.

[0355] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg.

[0356] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg.

[0357] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0358] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg.

[0359] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 25 ng / mg.

[0360] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 30 ng / mg.

[0361] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0362] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg.

[0363] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0364] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg.

[0365] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg.

[0366] In some embodiments, the present disclosure relates to a method, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0367] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 6 ng / mg.

[0368] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 4.5 ng / mg.

[0369] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 4 ng / mg.

[0370] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 3.5 ng / mg.

[0371] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 3.3 ng / mg.

[0372] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 1 ng / mg.

[0373] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.5 ng / mg.

[0374] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.2 ng / mg.

[0375] In some embodiments, the present disclosure relates to a method, wherein the second ratio less than about 7.5 ng / mg is less than about 0.1 ng / mg.

[0376] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 10% or greater.

[0377] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 15% or greater.

[0378] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 20% or greater.

[0379] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 25% or greater.

[0380] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 30% or greater.

[0381] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 35% or greater.

[0382] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 40% or greater.

[0383] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 45% or greater.

[0384] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal function decline to a renal endpoint is about 50% or greater.

[0385] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is between about 10% and about 50%.

[0386] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is between about 15% and about 45%.

[0387] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is between about 20% and about 40%.

[0388] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 25% to about 35%.

[0389] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 25% to about 30%.

[0390] In some embodiments, the disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 10% to about 20%.

[0391] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 15% to about 25%.

[0392] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 20% to about 30%.

[0393] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline leading to a renal endpoint is about 30% to about 40%.

[0394] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 35% to about 45%.

[0395] In some embodiments, the present disclosure relates to a method, wherein the increased likelihood of progressive renal decline to a renal endpoint is about 40% to about 50%.

[0396] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after four years.

[0397] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 5 years.

[0398] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 6 years.

[0399] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 7 years.

[0400] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 8 years.

[0401] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint after 9 years.

[0402] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within seven years.

[0403] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 8 years.

[0404] In some embodiments, the present disclosure relates to a method, wherein the progressive decline in renal function is expected to lead to a renal endpoint within 9 years.

[0405] In some embodiments, the present disclosure relates to a method, wherein interacting with a fetuin A fragment comprises binding to the fetuin A fragment.

[0406] In some embodiments, the present disclosure relates to a method, wherein interacting with urinary creatinine includes binding to urinary creatinine.

[0407] In some embodiments, the present disclosure relates to a method, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0408] In some embodiments, the method of preparing the assay includes providing a first solution comprising a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent that interacts with urinary post-translationally modified fetuin A fragment (uPTM-FetA) to measure the level of fetuin A fragment in the urine sample; providing a second solution comprising the urine sample and a second reagent that interacts with urinary creatinine to measure the level of urinary creatinine in the urine sample; and determining a ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine in response to the measured level of fetuin A fragment and the measured level of urinary creatinine. and correlating the ratio with a likelihood of progressive decline in renal function to a renal endpoint in the subject over a period of about 10 years, wherein a ratio greater than about 3.3 ng / mg, more preferably greater than about 3.5 ng / mg, more preferably greater than about 4 ng / mg, more preferably greater than about 5 ng / mg, more preferably greater than about 6 ng / mg, more preferably greater than about 7 ng / mg, more preferably greater than about 7.5 ng / mg indicates a higher likelihood of progressive decline in renal function to a renal endpoint, and wherein the first solution and the second solution are the same or different solutions.

[0409] In some embodiments, an assay kit for determining likelihood of progressive kidney function decline comprises a first solution comprising a first reagent that interacts with fetuin A fragment to indicate a level of urinary post-translationally modified fetuin A fragment (uPTM-FetA) in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution comprising a second reagent that interacts with urinary creatinine to indicate a level of urinary creatinine in the urine sample; and a second reagent that interacts with fetuin A fragment in the urine sample to determine a ratio of a measured level of fetuin A fragment to a measured level of urinary creatinine. The device may include a device for measuring the level of a urinary creatinine fragment and a measured level of urinary creatinine, wherein the ratio is greater than about 3.3 ng / mg, more preferably greater than about 3.5 ng / mg, more preferably greater than about 4 ng / mg, more preferably greater than about 5 ng / mg, more preferably greater than about 6 ng / mg, more preferably greater than about 6.5 ng / mg, more preferably greater than about 7 ng / mg, more preferably greater than about 7.2 ng / mg, more preferably greater than about 7.3 ng / mg, more preferably greater than about 7.4 ng / mg, more preferably greater than about 7.45 ng / mg.

[0410] In some embodiments, the ratio may be greater than about 7.5 ng / mg, more preferably greater than about 7.51 ng / mg, more preferably greater than about 7.52 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably 9 ng / mg, more preferably 10 ng / mg, more preferably 12 ng / mg, more preferably 14 ng / mg, more preferably 15 ng / mg, more preferably 20 ng / mg, more preferably 24 ng / mg, more preferably 25 ng / mg, more preferably 30 ng / mg, more preferably 40 ng / mg, more preferably 45 ng / mg, more preferably 47 ng / mg, more preferably 48 ng / mg, more preferably 49 ng / mg, more preferably 50 ng / mg, indicating a greater likelihood of progressive decline in renal function leading to a renal endpoint over a period of about 10 years in the subject, and wherein the first solution and the second solution are the same or different solutions.

[0411] In some embodiments, a method of correlating the likelihood of progressive renal decline can include measuring the levels of a first biomarker and a second biomarker in a urine sample from a subject diagnosed with type 2 diabetes mellitus, where the first biomarker can be a fragment of urinary post-translationally modified fetuin A fragment (uPTM-FetA) and the second biomarker can be urinary creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; and correlating the ratio with the likelihood of progressive renal decline to a renal endpoint in the subject over a period of about 10 years, where the ratio is greater than about 7.5 ng / mg, more preferably greater than about 7. A level greater than 51 ng / mg, more preferably greater than about 7.52 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably 9 ng / mg, more preferably 10 ng / mg, more preferably 12 ng / mg, more preferably 14 ng / mg, more preferably 15 ng / mg, more preferably 20 ng / mg, more preferably 24 ng / mg, more preferably 25 ng / mg, more preferably 30 ng / mg, more preferably 40 ng / mg, more preferably 45 ng / mg, more preferably 47 ng / mg, more preferably 48 ng / mg, more preferably 49 ng / mg, more preferably 50 ng / mg indicates a higher likelihood of progressive decline in renal function leading to a renal endpoint.

[0412] In some embodiments, a ratio greater than about 7.5 may trigger medical intervention to forestall progressive decline in renal function.

[0413] In some embodiments, a higher likelihood indicates a ratio of the measured level of fetuin-A fragment to the measured level of urinary creatinine is less than about 7.53 ng / mg, less than about 7.5 ng / mg, less than about 7 ng / mg, less than about 6 ng / mg, less than about 5.95 ng / mg, less than about 5.5 ng / mg, less than about 5 ng / mg, less than about 4.5 ng / mg, less than about 4.4 ng / mg, less than about 4.38 ng / mg, less than about 4.3 ng / mg, less than about 4.2 ng / mg, less than about 4.1 ng / mg The likelihood of progressive renal function decline over a period of about 10 years in a second subject having a ratio of less than about 4.0 ng / mg, less than about 3.95 ng / mg, less than about 3.9 ng / mg, less than about 3.5 ng / mg, less than about 3.4 ng / mg, less than about 3.3 ng / mg, less than about 3 ng / mg, less than about 2.5 ng / mg, less than about 2 ng / mg, less than about 1 ng / mg, less than about 0.5 ng / mg, or less than about 0.1 ng / mg.

[0414] In some embodiments, the progressive decline in renal function can be a decline in estimated glomerular filtration rate (eGFR).

[0415] In some embodiments, the subject's eGFR is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 ) or higher.

[0416] In some embodiments, the second subject's eGFR is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 ) has been shown to be

[0417] In some embodiments, the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for the 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0418] In some embodiments, the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or both before and after the time of collection of the urine sample.

[0419] In some embodiments, the subject or second subject has been diagnosed with type 2 diabetes mellitus about 7 to about 20 years prior to collection of the urine sample.

[0420] In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7 ng / mg. In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7.1 ng / mg. In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7.2 ng / mg. In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7.3 ng / mg. In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7.4 ng / mg. In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7.45 ng / mg. In some embodiments, a ratio greater than about 6 ng / mg can be greater than about 7.5 ng / mg.

[0421] In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 7.51 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 7.52 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 7.53 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 7.55 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 7.6 ng / mg.

[0422] In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 9 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 10 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 12 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 14 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 15 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 20 ng / mg. In some embodiments, a ratio greater than about 7.5 ng / mg can be greater than about 24 ng / mg.

[0423] In some embodiments, the higher likelihood may be greater than a lower likelihood of progressive renal decline over a period of about 10 years in a second subject having a lower ratio of measured levels of fetuin-A fragment to measured levels of urinary creatinine of less than about 7.5 ng / mg (such as 7.53 ng / mg).

[0424] In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 7 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be 6 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 5.95 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 5.5 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 5 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 4.5 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 4.4 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 4.38 ng / mg. In some embodiments, the second ratio less than about 7.5 ng / mg can be less than about 4.3 ng / mg. In some embodiments, the second ratio less than about 7.5 ng / mg can be less than about 4.2 ng / mg. In some embodiments, the second ratio less than about 7.5 ng / mg can be less than about 4.1 ng / mg.

[0425] In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 4.0 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 3.95 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 3.9 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 3.5 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 3.4 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 3.3 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 3 ng / mg. In some embodiments, the second ratio lower than about 7.5 ng / mg can be lower than about 2.5 ng / mg. In some embodiments, the second ratio less than about 7.5 ng / mg can be less than about 2 ng / mg, hi some embodiments, the second ratio less than about 7.5 ng / mg can be less than about 1 ng / mg, less than about 0.5 ng / mg, or less than about 0.1 ng / mg.

[0426] In some embodiments, the greater likelihood of progressive renal function decline to a renal endpoint may be about 5% or greater, hi some embodiments, the greater likelihood of progressive renal function decline to a renal endpoint may be about 10% or greater.

[0427] In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 11% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 12% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 13% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 15% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 16% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 18% or greater.

[0428] In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 20% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 22% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 24% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 25% or greater.

[0429] In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 30% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 33% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 35% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 38% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 39% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 40% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 45% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 46% or greater. In some embodiments, the higher likelihood of progressive renal function decline to a renal endpoint may be about 48% or greater. In some embodiments, the increased likelihood of progressive renal decline to a renal endpoint may be about 50% or greater. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 10% to about 50%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 10% to about 40%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 11% to about 39%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 5% to about 20%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 6% to about 16%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 10% to about 25%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 12% to about 24%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 15% to about 45%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 20% to about 40%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 25% to about 35%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 25% to about 40%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 25% to about 30%.

[0430] In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 10% to about 20%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 15% to about 25%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 20% to about 30%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 30% to about 50%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 30% to about 46%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 35% to about 45%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 30% to about 40%. In some embodiments, the greater likelihood of progressive renal function decline leading to a renal endpoint may be about 30% to about 45%. In some embodiments, the higher likelihood of progressive renal decline leading to a renal endpoint may be about 40% to about 50%.

[0431] In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 1 year. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 2 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 3 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 4 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 5 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 6 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 7 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 8 years. In some embodiments, the progressive renal function decline may be expected to reach a renal endpoint after about 9 years.

[0432] In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 1 year. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 2 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 3 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 4 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 5 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 6 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 7 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 8 years. In some embodiments, the progressive renal function decline may be expected to lead to a renal endpoint within about 9 years.

[0433] In some embodiments, the interaction with fetuin A fragment comprises binding to fetuin A fragment. In some embodiments, the interaction with urinary creatinine comprises binding to urinary creatinine. In some embodiments, at least one of the first reagent and the second reagent comprises an antibody.

[0434] As supported by the present disclosure, uPTM-FetA may be associated with the risk of eGFR decline in type 2 diabetes patients, which may be independent of or substantially less dependent on age, sex, eGFR, UACR, baseline HbA1c, multiple other potential confounding factors, or any combination thereof. These findings have been validated in a second, larger cohort of type 2 diabetes patients, highlighting the potential clinical use of uPTM-Fet-A as a predictive marker for DKD. Human fetuin-A precursor protein contains three parts: the A chain, the connecting peptide, and the B chain, which are 321, 40, and 27 amino acids in length, respectively. The precursor connecting peptide is removed by limited proteolysis, a post-translational modification, after which only the A and B chains form active fetuin-A protein. To detect fetuin-A containing the connecting peptide, a monoclonal antibody in an ELISA kit (e.g., Human uPTM3-DKD ELISA Kit, BPM Corp.) can be used. This disclosure supports the association of elevated uPTM-FetA with progressive renal function decline in patients with type 2 diabetes. In some embodiments, uPTM-FetA concentrations may not be substantially related to serum c-FetA concentrations among patients (internal data from the NTUH cohort, data not shown). In some embodiments, discrepancies between circulating and urinary fetuin A concentrations may be observed. Ostensibly, uPTM-FetA appears to accumulate specifically in the urine of patients with progressive renal function decline. This may be due to impaired tubular reabsorption of the molecule rather than increased filtration or secretion. uPTM-FetA may be a predictive marker of progressive kidney disease. uPTM-FetA may have direct pathogenic effects.

[0435] In type 2 diabetes patients without CKD, uPTM-FetA was analyzed against UACR using multivariate-adjusted Cox regression. This suggests that uPTM-FetA may be a better predictor of declining renal function than UACR. Furthermore, the use of antihypertensive drugs, such as angiotensin receptor blockers and angiotensin-converting enzyme inhibitors, frequently prescribed in type 2 diabetes patients, reduces albuminuria, limiting the utility of UACR as a predictive marker for DKD. Potentially, uPTM-FetA may not have this drawback.

[0436] Previous studies have used clinical parameters, with or without the assistance of machine learning, to establish DKD prediction models for patients with type 2 diabetes, but the follow-up period was quite short. Because of the high intra-individual variability of eGFR among patients with early DKD, assessment of eGFR within only a 5-year period is considered insufficient to assess the deterioration of renal function in patients with type 2 diabetes. Given the fact that global renal hyperfiltration associated with increasing eGFR is observed in a significant proportion of this population and that hyperfiltration can mask the development of early-stage kidney disease, long-term follow-up of renal function in patients with type 2 diabetes is essential. This study has several strengths. It included the use of two large, well-characterized cohorts, allowing extensive adjustment for potential confounders. The long follow-up period in the NTUH cohort allowed for a proper assessment of the association between renal outcomes and uPTM-FetA, preventing underestimation due to the issue of renal hyperfiltration in early DKD. Finally, the use of two independent, bi-ethnic cohorts, including patients from different geographic areas, is encouraging regarding the external validity of these findings to other type 2 diabetes populations worldwide.

[0437] Thus, the present disclosure demonstrates that uPTM-FetA is strongly associated with worsening renal function, independent of albuminuria, eGFR, age, sex, and multiple other risk factors, in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes. The additive prognostic value of uPTM-FetA over albuminuria and other markers is particularly evident in patients without CKD at baseline. These findings were validated in a second large, independent cohort, providing promise regarding the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0438] Assay

[0439] In some embodiments, various methods, reagents, devices, and kits can be used to measure levels of fetuin-A or fragments thereof in various media, such as urine, serum, and other body fluids.

[0440] In some embodiments, enzyme-linked immunosorbent assay (ELISA) can be used to measure the level of fetuin A. For example, ELISA can be used to quantitatively measure specific post-translational modification (PTM) fetuin A in human urine and should be performed in a qualified clinical laboratory by a licensed medical professional, such as a clinical laboratory technician. For example, a calibrator or unknown urine sample is mixed with an antibody (such as an anti-specific PTM fetuin A monoclonal antibody (mAb)) that has binding affinity for a portion of fetuin A, and then incubated in a microplate to which fetuin A or a segment thereof (such as a specific PTM fetuin A) has been pre-bound. Under competition in the microplate wells, the monoclonal antibody recognizes fetuin A in the calibrator or unknown sample. After incubation, in some embodiments, a signal-generating molecule, such as an antibody labeled with the signal molecule, can be used. In some embodiments, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate, and their relative reactivities are determined by absorbance measurements at 450 nanometers (nm) and plotted against a predetermined specific PTM fetuin A standard curve.

[0441] Non-limiting embodiments The present disclosure is also described in terms of the following non-limiting embodiments. However, the use of these embodiments and other embodiments anywhere in this specification is merely illustrative and in no way limits the scope and meaning of the present disclosure. Likewise, the present disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed, upon reading this specification, modifications and variations may become apparent to those skilled in the art, and such variations may be made without departing from the spirit or scope of the present disclosure.

[0442] 1. A method of preparing an assay, comprising: providing a first solution comprising a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent that interacts with post-translationally modified fetuin A fragment (uPTM-FetA) in urine, and measuring the level of said fetuin A fragment in said urine sample; providing a second solution comprising the urine sample and a second reagent that interacts with urinary creatinine to measure the level of urinary creatinine in the urine sample; correlating a ratio of the measured level of fetuin-A fragment to the measured level of urinary creatinine to a likelihood of progressive renal decline to a renal endpoint in the subject over a period of about 10 years in response to the measured level of fetuin-A fragment and the measured level of urinary creatinine; wherein said ratio being greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of said progressive decline in renal function leading to said renal endpoint; The method, wherein the first solution and the second solution are the same or different solutions.

[0443] 2. The method of embodiment 1, wherein the ratio being greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggers medical intervention to forestall the progressive decline in renal function.

[0444] 3. The method of any of embodiments 1-2, wherein the higher likelihood is greater than the lower likelihood of progressive renal function decline over the approximately 10-year period in a second subject having a lower ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine of less than about 7.5 ng / mg.

[0445] 4. The method of any one of embodiments 1-3, wherein the higher likelihood is lower than the likelihood of progressive renal decline over the approximately 10-year period in a third subject having a higher ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine of less than about 29.0 ng / mg.

[0446] 5. The method described in embodiments 1-4, wherein a ratio value higher than 7.5 ng / mg indicates that the progressive renal failure is at a higher probability level.

[0447] 6. The method described in any one of embodiments 1 to 5, further comprising assessing the probability of the occurrence of the progressive decline in renal function leading to the renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0448] 7. The method of embodiment 6, wherein at least one of the ratio, the age, the sex, the eGFR, the UACR, and the HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, the age, the sex, the eGFR, the UACR, and the HbA1c level, respectively.

[0449] 8. The step of assessing the probability of occurrence of the progressive renal decline leading to the renal endpoint is based on formula (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg) ...Formula (1) During the ceremony, If E103 / UCr>7.53ng / mg, then I(E103 / UCr>7.53ng / mg)=1, or if E103 / UCr is 7.53ng / mg or less, then I(E103 / UCr>7.53ng / mg)=0; y is a first probabilistic value based on which the probability of occurrence of the progressive renal decline leading to the renal endpoint is assessed; 7. The method of embodiment 6, wherein a0, a1, a2, a3, a4, a5, and a6 are the assigned coefficients.

[0450] 9. The step of assessing the probability of occurrence of the progressive renal decline leading to the renal endpoint is based on Equation (2): p=exp(y) / (1+exp(y))……Equation (2) 9. The method of embodiment 8, wherein p is a second probabilistic value based on which said probability of occurrence of cancer is assessed.

[0451] 10. The value of a0 is approximately -10.0 to approximately 0.5. The value of a1 is about -0.0 to about 0.1, The value of a2 is about -0.3 to about 1.2, The value of a3 is about -0.1 to about 0.0, The value of a4 is about 0.1 to about 0.7, The value of a5 is about -0.3 to about 0.3, 25. The method of any one of claims 20-24, wherein the value of a6 is from about 1.2 to about 3.7.

[0452] 11. The method described in embodiments 9-10, wherein the method further comprises determining an increased risk of the progressive decline in renal function leading to the renal endpoint in the patient who provided the urine in response to the second probabilistic value exceeding a first threshold.

[0453] 12. The method of embodiment 11, wherein the method further comprises determining a higher elevated risk in the patient who provided the urine, the higher elevated risk, of progressive renal function decline leading to the renal endpoint, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold.

[0454] 13. The method of embodiments 11-12, wherein the first threshold is approximately 0.1.

[0455] 14. The method of embodiment 11-12, wherein the first threshold is approximately 0.11.

[0456] 15. The method of embodiment 12, wherein the second threshold is about 0.25.

[0457] 16. The method of embodiment 12, wherein the second threshold is approximately 0.3.

[0458] 17. The method of embodiment 12, wherein the second threshold is approximately 0.27.

[0459] 18. A method described in any one of embodiments 1-3, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0460] 19. The subject's eGFR is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 19. The method of embodiment 18, wherein the .alpha.-methyl-.beta ...

[0461] 20. The eGFR of the second subject is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 20. The method of embodiment 19, wherein:

[0462] 21. The method of any one of embodiments 1-20, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be lower than about 30 milligrams per gram (mg / g) for the 12 months prior to collection of the urine sample, or both before and after the time of collection of the urine sample.

[0463] 22. The method of any one of embodiments 1-21, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be lower than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or before and after the time of collecting the urine sample.

[0464] 23. The method of any of embodiments 1-22, wherein the subject or the second subject has been diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0465] 24. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg.

[0466] 25. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg.

[0467] 26. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0468] 27. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg.

[0469] 28. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg.

[0470] 29. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg.

[0471] 30. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0472] 31. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg.

[0473] 32. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is between about 7.5 ng / mg and about 29 ng / mg.

[0474] 33. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg.

[0475] 34. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0476] 35. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg.

[0477] 36. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0478] 37. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg.

[0479] 38. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg.

[0480] 39. The method of any one of embodiments 1-23, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0481] 40. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 6 ng / mg.

[0482] 41. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4.5 ng / mg.

[0483] 42. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4 ng / mg.

[0484] 43. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.5 ng / mg.

[0485] 44. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.3 ng / mg.

[0486] 45. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 1 ng / mg.

[0487] 46. ​​The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.5 ng / mg.

[0488] 47. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.2 ng / mg.

[0489] 48. The method of any one of embodiments 1-39, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

[0490] 49. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 10% or greater.

[0491] 50. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 15% or greater.

[0492] 51. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 20% or greater.

[0493] 52. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 25% or greater.

[0494] 53. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 30% or greater.

[0495] 54. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 35% or greater.

[0496] 55. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 40% or greater.

[0497] 56. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 45% or greater.

[0498] 57. The method of any of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 50% or greater.

[0499] 58. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 10% and about 50%.

[0500] 59. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 15% and about 45%.

[0501] 60. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 20% and about 40%.

[0502] 61. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 25% and about 35%.

[0503] 62. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 25% to about 30%.

[0504] 63. A method according to any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 10% and about 20%.

[0505] 64. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 15% and about 25%.

[0506] 65. A method according to any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 20% and about 30%.

[0507] 66. A method according to any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 30% and about 40%.

[0508] 67. A method according to any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 35% and about 45%.

[0509] 68. The method of any one of embodiments 1-48, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 40% to about 50%.

[0510] 69. The method of any one of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 4 years.

[0511] 70. The method of any of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 5 years.

[0512] 71. The method of any of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 6 years.

[0513] 72. The method of any one of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 7 years.

[0514] 73. The method of any one of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 8 years.

[0515] 74. The method of any one of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 9 years.

[0516] 75. The method of any of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 7 years.

[0517] 76. The method of any of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 8 years.

[0518] 77. The method of any of embodiments 1-68, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 9 years.

[0519] 78. The method of any one of embodiments 1-77, wherein said interaction with said fetuin A fragment comprises binding to said fetuin A fragment.

[0520] 79. The method of any one of embodiments 1-78, wherein the interaction with urinary creatinine includes binding to the urinary creatinine.

[0521] 80. The method of any one of embodiments 1-79, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0522] 81. An assay kit for determining the likelihood of progressive renal function decline, comprising: a first solution comprising a first reagent that interacts with a urinary post-translationally modified fetuin A fragment (uPTM-FetA) for indicating the level of said fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution comprising a second reagent that interacts with urinary creatinine to indicate the level of urinary creatinine in the urine sample; a device for measuring the level of fetuin A fragment and the level of urinary creatinine in the urine sample to determine a ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine; wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of progressive decline in renal function leading to a renal endpoint in the subject over a period of about 10 years; The assay kit, wherein the first solution and the second solution are the same or different from each other.

[0523] 82. The assay kit of embodiment 81, wherein the ratio being higher than about 7.5 ng / mg, more preferably higher than about 7.53 ng / mg, more preferably higher than about 8 ng / mg, more preferably higher than about 8.5 ng / mg, more preferably higher than about 9 ng / mg, triggers medical intervention to forestall the progressive decline in renal function.

[0524] 83. An assay kit described in any of embodiments 81-82, wherein the higher likelihood is higher than the lower likelihood of progressive renal function decline over the approximately 10-year period in a second subject having a lower ratio of the measured level of the fetuin A fragment to the measured level of the urinary creatinine, that is, less than about 7.5 ng / mg.

[0525] 84. An assay kit described in any of embodiments 81-83, wherein the higher likelihood is lower than the likelihood of progressive decline in renal function over the approximately 10-year period in a third subject having a higher ratio of the measured level of the fetuin A fragment to the measured level of the urinary creatinine, that is lower than about 29.0 ng / mg.

[0526] 85. An assay kit described in embodiments 81-84, wherein a ratio value higher than 7.5 ng / mg indicates that the progressive renal failure is at a higher probability level.

[0527] 86. The assay kit of any one of embodiments 81-85, further comprising assessing the probability of the occurrence of the progressive decline in renal function leading to the renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0528] 87. The assay kit of embodiment 86, wherein at least one of the ratio, the age, the sex, the eGFR, the UACR, and the HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, the age, the sex, the eGFR, the UACR, and the HbA1c level.

[0529] 88. The step of assessing the probability of the progressive decline in renal function leading to the renal endpoint is based on formula (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg) ...Formula (1) During the ceremony, If E103 / UCr>7.53ng / mg, then I(E103 / UCr>7.53ng / mg)=1, or if E103 / UCr is 7.53ng / mg or less, then I(E103 / UCr>7.53ng / mg)=0; y is a first probabilistic value based on which the probability of occurrence of the progressive renal decline leading to the renal endpoint is assessed; 87. The assay kit of embodiment 86, wherein a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients.

[0530] 89. The step of assessing the probability of occurrence of the progressive decline in renal function leading to the renal endpoint is based on formula (2): p=exp(y) / (1+exp(y))……Equation (2) 89. The assay kit of embodiment 88, wherein p is a second probabilistic value based on which the probability of occurrence of cancer is assessed.

[0531] 90. The value of a0 is approximately -10.0 to approximately 0.5. The value of a1 is about -0.0 to about 0.1, The value of a2 is about -0.3 to about 1.2, The value of a3 is about -0.1 to about 0.0, The value of a4 is about 0.1 to about 0.7, The value of a5 is about -0.3 to about 0.3, The assay kit according to any one of embodiments 20 to 24, wherein the value of a6 is about 1.2 to about 3.7.

[0532] 91. The assay kit described in embodiments 89-90, wherein the method further comprises determining an increased risk of the progressive decline in renal function leading to the renal endpoint in the patient who provided the urine in response to the second probabilistic value exceeding a first threshold.

[0533] 92. The assay kit of embodiment 91, wherein the method further comprises determining a higher elevated risk in the patient who provided the urine, the higher elevated risk being greater than the elevated risk of the progressive decline in renal function leading to the renal endpoint, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold.

[0534] 93. An assay kit described in any of embodiments 81-83, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0535] 94. The subject's eGFR is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 94. The assay kit of embodiment 93, wherein the assay kit is indicated to have a nucleotide sequence of 0.1 or more.

[0536] 95. The eGFR of the second subject is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 95. The assay kit of embodiment 94, wherein the assay kit is indicated to be:

[0537] 96. An assay kit described in any of embodiments 81-95, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be lower than about 30 milligrams per gram (mg / g) for the 12 months prior to collecting the urine sample, or both before and after the time of collecting the urine sample.

[0538] 97. An assay kit described in any of embodiments 81-96, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) during the 12 months prior to collecting the urine sample, or before and after the time of collecting the urine sample.

[0539] 98. An assay kit described in any of embodiments 81-97, wherein the subject or the second subject has been diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0540] 99. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg.

[0541] 100. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg.

[0542] 101. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0543] 102. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg.

[0544] 103. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg.

[0545] 104. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg.

[0546] 105. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0547] 106. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg.

[0548] 107. An assay kit described in any of embodiments 81-98, wherein the ratio higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg.

[0549] 108. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg.

[0550] 109. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0551] 110. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg.

[0552] 111. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0553] 112. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg.

[0554] 113. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg.

[0555] 114. An assay kit described in any of embodiments 81-98, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0556] 115. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 6 ng / mg.

[0557] 116. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 5 ng / mg.

[0558] 117. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4 ng / mg.

[0559] 118. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.5 ng / mg.

[0560] 119. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.3 ng / mg.

[0561] 120. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 1 ng / mg.

[0562] 121. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.5 ng / mg.

[0563] 122. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.2 ng / mg.

[0564] 123. An assay kit described in any of embodiments 81-114, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

[0565] 124. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 10% or more.

[0566] 125. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 15% or more.

[0567] 126. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 20% or more.

[0568] 127. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 25% or more.

[0569] 128. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 30% or more.

[0570] 129. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 35% or greater.

[0571] 130. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 40% or more.

[0572] 131. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 45% or greater.

[0573] 132. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 50% or more.

[0574] 133. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 10% and about 50%.

[0575] 134. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 15% and about 45%.

[0576] 135. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 20% and about 40%.

[0577] 136. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 25% and about 35%.

[0578] 137. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 25% to about 30%.

[0579] 138. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 10% and about 20%.

[0580] 139. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 15% and about 25%.

[0581] 140. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 20% and about 30%.

[0582] 141. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 30% and about 40%.

[0583] 142. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 35% and about 45%.

[0584] 143. An assay kit described in any of embodiments 81-123, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is between about 40% and about 50%.

[0585] 144. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 4 years.

[0586] 145. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 5 years.

[0587] 146. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 6 years.

[0588] 147. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 7 years.

[0589] 148. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 8 years.

[0590] 149. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 9 years.

[0591] 150. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 7 years.

[0592] 151. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 8 years.

[0593] 152. An assay kit described in any of embodiments 81-143, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 9 years.

[0594] 153. The assay kit of any of embodiments 81-152, wherein the interaction with the fetuin A fragment comprises binding to the fetuin A fragment.

[0595] 154. An assay kit described in any of embodiments 81-153, wherein the interaction with urinary creatinine includes binding to the urinary creatinine.

[0596] 155. An assay kit described in any of embodiments 81-154, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0597] 156. Measuring the levels of a first biomarker and a second biomarker in a urine sample from a subject diagnosed with type 2 diabetes mellitus, wherein the first biomarker is a urinary post-translationally modified fragment of fetuin A (uPTM-FetA) and the second biomarker is urinary creatinine; determining a ratio of the level of the first biomarker to the level of the second biomarker; 10. A method of correlating likelihood of progressive renal function decline, comprising correlating said ratio with likelihood of progressive renal function decline to a renal endpoint in said subject over a period of about 10 years, The method, wherein a ratio greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg indicates a higher likelihood of the progressive decline in renal function leading to the renal endpoint.

[0598] 157. The method described in embodiment 156, wherein the ratio is greater than about 7.5 ng / mg, more preferably greater than about 7.53 ng / mg, more preferably greater than about 8 ng / mg, more preferably greater than about 8.5 ng / mg, more preferably greater than about 9 ng / mg, triggering medical intervention to forestall the progressive decline in renal function.

[0599] 158. A method described in any of embodiments 156-157, wherein the higher likelihood is higher than the lower likelihood of progressive renal function decline over the approximately 10-year period in a second subject having a lower ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine of less than about 7.5 ng / mg.

[0600] 159. The method described in any of embodiments 156-158, wherein the higher likelihood is lower than the likelihood of progressive decline in renal function over the approximately 10-year period in a third subject having a higher ratio of the measured level of fetuin A fragment to the measured level of urinary creatinine of less than about 29.0 ng / mg.

[0601] 160. The method of embodiments 156-159, wherein a ratio value higher than 7.5 ng / mg indicates that the progressive renal failure is at a higher probability level.

[0602] 161. The method described in embodiments 156-160, further comprising assessing the probability of occurrence of the progressive decline in renal function leading to the renal endpoint based on correlating the ratio with at least one of age, sex, eGFR, UACR, and HbA1c level.

[0603] 162. The method described in embodiment 161, wherein at least one of the ratio, the age, the gender, the eGFR, the UACR, and the HbA1c level is adjusted based on at least one respective coefficient corresponding to at least one of the ratio, the age, the gender, the eGFR, the UACR, and the HbA1c level, respectively.

[0604] 163. The step of assessing the probability of occurrence of the progressive decline in renal function leading to the renal endpoint is based on formula (1): y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg) ...Formula (1) During the ceremony, If E103 / UCr>7.53ng / mg, then I(E103 / UCr>7.53ng / mg)=1, or if E103 / UCr is 7.53ng / mg or less, then I(E103 / UCr>7.53ng / mg)=0; y is a first probabilistic value based on which the probability of occurrence of the progressive renal decline leading to the renal endpoint is assessed; The method of embodiment 161, wherein a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients.

[0605] 164. The step of assessing the probability of occurrence of the progressive decline in renal function leading to the renal endpoint is based on formula (2): p=exp(y) / (1+exp(y))……Equation (2) 164. The method of embodiment 163, wherein p is a second probabilistic value based on which the probability of occurrence of cancer is assessed.

[0606] 165. The value of a0 is approximately -10.0 to approximately 0.5. The value of a1 is about -0.0 to about 0.1, The value of a2 is about -0.3 to about 1.2, The value of a3 is about -0.1 to about 0.0, The value of a4 is about 0.1 to about 0.7, The value of a5 is about -0.3 to about 0.3, 25. The method of any one of claims 20-24, wherein the value of a6 is from about 1.2 to about 3.7.

[0607] 166. The method described in embodiments 164-165, wherein the method further comprises determining an increased risk of the progressive decline in renal function leading to the renal endpoint in the patient who provided the urine in response to the second probabilistic value exceeding a first threshold.

[0608] 167. The method of embodiment 166, wherein the method further comprises determining a higher elevated risk in the patient who provided the urine, the higher elevated risk being greater than the elevated risk of the progressive decline in renal function leading to the renal endpoint, in response to the second probabilistic value exceeding a second threshold that is higher than the first threshold.

[0609] 168. The method of any of embodiments 156-158, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

[0610] 169. The subject's eGFR is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 169. The method of embodiment 168, wherein the method is indicated to have a .times. ...

[0611] 170. The eGFR of the second subject is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m 2 169. The method of claim 169, wherein:

[0612] 171. A method described in any of embodiments 156-170, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be lower than about 30 milligrams per gram (mg / g) for the 12 months prior to collecting the urine sample, or both before and after the time of collecting the urine sample.

[0613] 172. The method of any of embodiments 156-171, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be lower than about 30 milligrams per gram (mg / g) during the 12 months prior to collection of the urine sample, or before and after the time of collection of the urine sample.

[0614] 173. The method of any of embodiments 156-172, wherein the subject or the second subject has been diagnosed with type 2 diabetes mellitus about 7 years to about 20 years prior to collection of the urine sample.

[0615] 174. The method of any of embodiments 156-173, wherein the ratio greater than about 7.5 ng / mg is greater than about 9.5 ng / mg.

[0616] 175. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 10 ng / mg.

[0617] 176. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 11 ng / mg.

[0618] 177. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 12 ng / mg.

[0619] 178. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 14 ng / mg.

[0620] 179. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 15 ng / mg.

[0621] 180. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 20 ng / mg.

[0622] 181. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 24 ng / mg.

[0623] 182. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg.

[0624] 183. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg.

[0625] 184. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 40 ng / mg.

[0626] 185. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 45 ng / mg.

[0627] 186. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 47 ng / mg.

[0628] 187. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 48 ng / mg.

[0629] 188. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 49 ng / mg.

[0630] 189. The method of any of embodiments 156-174, wherein the ratio greater than about 7.5 ng / mg is greater than about 50 ng / mg.

[0631] 190. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 6 ng / mg.

[0632] 191. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4.5 ng / mg.

[0633] 192. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 4 ng / mg.

[0634] 193. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.5 ng / mg.

[0635] 194. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 3.3 ng / mg.

[0636] 195. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 1 ng / mg.

[0637] 196. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.5 ng / mg.

[0638] 197. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.2 ng / mg.

[0639] 198. The method of any of embodiments 156-189, wherein the second ratio lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

[0640] 199. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 10% or greater.

[0641] 200. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 15% or greater.

[0642] 201. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 20% or greater.

[0643] 202. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 25% or greater.

[0644] 203. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 30% or greater.

[0645] 204. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 35% or greater.

[0646] 205. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 40% or greater.

[0647] 206. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 45% or greater.

[0648] 207. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 50% or greater.

[0649] 208. A method according to any of embodiments 156-198, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 10% and about 50%.

[0650] 209. A method according to any of embodiments 156-198, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 15% and about 45%.

[0651] 210. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 20% and about 40%.

[0652] 211. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 25% to about 35%.

[0653] 212. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 25% to about 30%.

[0654] 213. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 10% to about 20%.

[0655] 214. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 15% to about 25%.

[0656] 215. A method according to any of embodiments 156-198, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 20% and about 30%.

[0657] 216. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 30% to about 40%.

[0658] 217. The method of any of embodiments 156-198, wherein the higher likelihood of progressive renal function decline leading to the renal endpoint is about 35% to about 45%.

[0659] 218. A method according to any of embodiments 156-198, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 40% and about 50%.

[0660] 219. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 4 years.

[0661] 220. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 5 years.

[0662] 221. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 6 years.

[0663] 222. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 7 years.

[0664] 223. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 8 years.

[0665] 224. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint after 9 years.

[0666] 225. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 7 years.

[0667] 226. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 8 years.

[0668] 227. The method of any of embodiments 156-218, wherein the progressive decline in renal function is expected to lead to the renal endpoint within 9 years.

[0669] 228. The method of any of embodiments 156-227, wherein said interaction with said fetuin A fragment comprises binding to said fetuin A fragment.

[0670] 229. The method of any of embodiments 156-228, wherein the interaction with urinary creatinine includes binding to the urinary creatinine.

[0671] 230. The method of any of embodiments 156-229, wherein at least one of the first reagent and the second reagent comprises an antibody.

[0672] Non-limiting Examples The following examples are provided to illustrate selected embodiments. They should not be considered as limiting the scope of the invention, but merely as being exemplary and representative thereof. Thus, the examples provided below, while exemplified with particular medical devices or active agents, are applicable to the range of medical devices and active agents described herein.

[0673] Example 1 Study design

[0674] For this study, we included patients from two independent, non-overlapping prospective cohort studies involving type 2 diabetes patients from different geographic areas. First, all analyses were performed in the NTUH cohort, which was initiated at the National Taiwan University Hospital (NTUH) in Taiwan. These findings were then independently replicated in the Dutch DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort.

[0675] Supplementary Table 1 indicates the cohort characteristics and inclusion and exclusion criteria for Examples 1-3. [Table 1]

[0676] uPTM-FetA measurement

[0677] At baseline, spot urine samples (NTUH) or 24-hour urine samples (DIALECT) were collected. These samples were stored at -80°C until measurement. uPTM-FetA was measured using a human uPTM3-DKD ELISA kit (CE-IVD, manufacturer: Bio Preventive Medicine Corp., trade name: DNlite-IVD103). Samples with uPTM-FetA concentrations below the detection limit were set to the lower detection limit. uPTM-FetA concentrations were divided by urinary creatinine and log10 transformed to obtain IVD103, which was used in all analyses.

[0678] Renal endpoint definition

[0679] Renal function was assessed using estimated glomerular filtration rate (eGFR) calculated by the Chronic Kidney Disease Epidemiology Collaboration formula. For both cohorts, the primary outcome of worsening renal function was a ≥30% decline in eGFR from baseline and an eGFR <15 mL / min / 1.73 m. 2Renal endpoints were defined as reaching ≥ 100% or the need for renal replacement therapy (i.e., kidney transplantation, peritoneal dialysis, or initiation of hemodialysis). Follow-up was censored at the end of follow-up or the date on which the renal endpoint was met.

[0680] Covariates

[0681] At baseline, clinical, anthropometric, and demographic data were extracted via electronic patient records and / or questionnaires. The severity of albuminuria was determined based on the urinary albumin / creatinine ratio (UACR). All other laboratory measurements were performed using routine laboratory methods.

[0682] statistical analysis

[0683] Baseline characteristics were presented as mean ± standard deviation (SD), median [interquartile range (IQR)], or frequency (percentage), depending on the data distribution. We performed receiver operating characteristic (ROC) analysis and used the Youden index to determine the optimal sensitivity and specificity of the IVD103 cutoff value. According to the established cutoff, patients were classified into a low-risk group (IVD103 < optimal cutoff) or a high-risk group (IVD103 ≥ optimal cutoff). The statistical significance of differences between groups was assessed using Student's t-test, Mann-Whitney U test, or Fisher's exact test, depending on the data distribution.

[0684] To visualize the occurrence of renal endpoints during follow-up between groups, Kaplan-Meier curves were presented, and the log-rank test was used to assess the statistical significance of differences between groups. The association between IVD103 and the risk of eGFR decline was analyzed using Cox proportional hazards regression analysis. Covariates with non-normal distributions were transformed into natural logarithms before analysis. Hazard ratios and 95% confidence intervals were presented for each relative increase of 1 standard deviation (for continuous risk factors) or for each change compared to the reference group (for categorical risk factors). In Model 1, we performed a multivariate-adjusted analysis using traditional risk factors (age, sex, eGFR, UACR, and HbA1c). We then calculated additional models with additional adjustments to Model 1, including lipid-related parameters (i.e., total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and lipid-lowering drug use (Model 2)), blood pressure-related parameters (systolic blood pressure, diabetic blood pressure, and antihypertensive drug use (Model 3)), other clinical parameters (body mass index, smoking status (Model 4)), and a combination of all the above factors (Model 5).

[0685] All data were analyzed using R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria). For all statistical analyses, a p-value <0.05 was considered significant.

[0686] Example 2 NTUH Cohort (NTUH)

[0687] This prospective cohort study (Project Name: Validation of Early Diagnostic Biomarkers for Diabetic Nephropathy and its Extension, Registered Project Numbers: 201107004RC and 201705062RIPD) was conducted in patients with type 2 diabetes at National Taiwan University Hospital (NTUH), Taiwan, from November 2011 to February 2020. The study included adult patients with type 2 diabetes according to the American Diabetes Association criteria. Given the predefined hypothesis of uPTM-FetA as an early marker of diabetic kidney disease (CKD), we aimed to verify whether the prospective association between uPTM-FetA and renal endpoints would be observed in patients without CKD at baseline. Therefore, the NTUH cohort of the NTUH study included patients with an eGFR >60 ml / min / 1.73 m at baseline. 2 Only individuals with a ≥ 100% CI of 0.01 to 0.01% and a UACR < 30 mg / g were included. A detailed summary of the inclusion and exclusion criteria is shown in Supplementary Table 1. The study was approved by the NTUH Institutional Review Board (IRB number: 1063703310), and was conducted in accordance with the Good Clinical Practice guidelines and the WMA Declaration of Helsinki. All subjects provided written informed consent before participating in the study.

[0688] result Baseline characteristics

[0689] NTUH cohort

[0690] Of all participants enrolled in the NTUH cohort, 223 were ultimately included in this study. The mean age was 60.38 ± 9.66 years, 125 (56%) were men, and the eGFR was 90.00 ± 13.87 ml / min / 1.73 m. 2 The median UACR was 5.87 [3.49–11.59] mg / g (Table 1).

[0691] In Table 1, normally distributed data are presented as mean ± standard deviation, skewed data as median [interquartile range], and categorical data as number (percentage). Undetectable u-PTM-FetA was set to the lowest available value. Abbreviations: eGFR (estimated glomerular filtration rate calculated using the creatinine-based CKD-EPI equation), HbA1c (glycosylated hemoglobin), HDL (high-density lipoprotein cholesterol), LDL (low-density lipoprotein cholesterol), SBP (systolic blood pressure), DBP (diastolic blood pressure), BMI (body mass index). [Table 2]

[0692] Determination of cutoffs and association with renal endpoints - NTUH cohort

[0693] During a median follow-up of 9.25 years (range, 7.52–9.66 years), 59 participants (26%) in the NTUH cohort experienced a renal endpoint. The optimal IVD103 cutoff for risk stratification of renal endpoints was 0.644, based on the Youden index. Population characteristics for the IVD103 stratification groups are presented in Table 1. Figure 1A illustrates Kaplan-Meier curves for non-CKD NTUH patients classified as high-risk and low-risk by IVD103, with an eGFR decline of 30% or eGFR <15 as the endpoint. In the high-risk IVD103 group, 36% experienced a renal endpoint, which was significantly higher than the 19% observed in the low-risk group (P = 0.0054, Figure 1A).

[0694] The association between high-risk IVD103 groups and renal endpoints was also observed in univariate Cox regression analysis (HR: 2.06, 95% CI: 1.23-3.47, P = 0.006) and became even more pronounced in the fully adjusted model 5 (HR: 2.96, 95% CI: 1.64-5.34, P < 0.001), whereas UACR was not significantly associated with renal endpoints (Table 2).

[0695] In Table 2, Cox proportional hazards models were performed to evaluate the association between IVD103 and the risk of worsening renal function (eGFR ≥ 30% from baseline, eGFR < 15 mL / min / 1.73 m2, or need for renal replacement therapy). Model 1 was adjusted for age, sex, eGFR, UACR, and HbA1c. Model 2 was additionally adjusted for total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering medications. Model 3 was additionally adjusted for systolic blood pressure, diabetic blood pressure, and use of angiotensin II receptor blockers (ARBs) or angiotensin-converting enzyme inhibitors (ACEi). Model 4 was additionally adjusted for body mass index and smoking status. Model 5 was adjusted for the above factors. HR = hazard ratio; CI = confidence interval. [Table 3]

[0696] Example 3 DIALECT (validation) cohort (DIALECT)

[0697] To validate the optimal cutoff for assessing the risk of kidney function loss, we used data from the DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort study, described in detail in this disclosure. Briefly, DIALECT is a prospective cohort study of type 2 diabetes patients conducted at Ziekenhuisgroep Twente Hospital, located in Almelo and Hengelo, the Netherlands. Adult patients with type 2 diabetes treated at the outpatient clinic of this secondary care hospital were included. Patients were recruited between March 2009 and May 2019. The study was conducted in accordance with the Good Clinical Practice guidelines and the WMA Declaration of Helsinki, and all subjects provided written informed consent before participating in the study. The study was approved by the local institutional review board (METC registration numbers NL57219.044.16 and 1009.68020) and registered in the Netherlands Trial Register (NTR trial code 5855).

[0698] result Baseline characteristics

[0699] DIALECT cohort

[0700] Overall, 621 participants from the DIALECT cohort were eligible for inclusion in this study. The mean age was 64.11 ± 9.97 years, and 381 (61%) were men.

[0701] eGFR was 75.90 ± 24.49 ml / min / 1.73 m 2 The median UACR was 8.85 [3.54–48.67] mg / g (Table 3).

[0702] In Table 3, normally distributed data are presented as mean ± standard deviation, skewed data as median [interquartile range], and categorical data as number (percentage). Undetectable u-PTM-FetA was set to the lowest available value. Abbreviations: eGFR (estimated glomerular filtration rate calculated using the creatinine-based CKD-EPI equation), HbA1c (glycosylated hemoglobin), HDL (high-density lipoprotein cholesterol), LDL (low-density lipoprotein cholesterol), SBP (systolic blood pressure), DBP (diastolic blood pressure), BMI (body mass index). [Table 4]

[0703] Validation Study Results - DIALECT Cohort

[0704] In the DIALECT cohort, 277 participants (45%) experienced a renal endpoint during a median follow-up of 4.16 [2.53, 6.41] years. Figure 1B illustrates Kaplan-Meier curves with an endpoint of 30% decline in eGFR or eGFR<15, where Figure 1B is for patients in the DIALECT study classified as high-risk and low-risk by IVD103, according to a non-limiting embodiment.

[0705] In the high-risk IVD103 group, 46% experienced a renal endpoint compared with 42% observed in the low-risk group (P=0.0039, Figure 1B).

[0706] DIALECT (validation) cohort analysis

[0707] Similar to the primary analysis, patients in the DIALECT validation cohort (DIALECT) were stratified into high- and low-risk groups based on the IVD103 cutoff obtained from the NTUH cohort study. Survival analyses, including Cox proportional hazards regression models, described for the primary analysis were repeated in this DIALECT validation cohort with the same renal endpoints as outcome variables.

[0708] Analysis of Examples 1-3 In this study, uPTM-FetA was associated with the risk of eGFR decline in patients with type 2 diabetes, independent of age, sex, eGFR, UACR, baseline HbA1c, and several other potential confounders. These findings, which have been validated in a second, larger cohort of patients with type 2 diabetes, highlight the potential clinical use of uPTM-Fet-A as a predictive marker for DKD.

[0709] The human fetuin-A precursor protein consists of three parts: the A chain, the connecting peptide, and the B chain, which are 321, 40, and 27 amino acids long, respectively. The connecting peptide of the precursor is removed by limited proteolysis, a post-translational modification, after which only the A and B chains form the active fetuin-A protein. The monoclonal antibody in the ELISA kit used in this study (Human uPTM3-DKD ELISA kit, BPM Corp.) detects only the connecting peptide-containing fetuin-A.

[0710] We found that elevated uPTM-FetA levels are associated with progressive renal function decline in patients with type 2 diabetes. These findings are generally consistent with previous studies demonstrating an association between urinary fetuin A levels and albuminuria and poorer renal function in type 2 diabetes patients with DKD. In some embodiments, uPTM-FetA levels are not related to serum c-FetA levels among patients (internal data from the NTUH cohort, data not shown). Other studies have also observed discrepancies between circulating and urinary fetuin A levels. uPTM-FetA specifically accumulates in the urine of patients with progressive renal function decline. This may be due to impaired tubular reabsorption of the molecule rather than increased filtration or secretion. In some embodiments, uPTM-FetA may be a predictive marker of progressive kidney disease. In some embodiments, uPTM-FetA may have direct pathogenic effects that need to be further investigated in future preclinical and interventional studies. These results show that uPTM-FetA was compared with UACR in multivariate-adjusted Cox regression in patients with type 2 diabetes without CKD. This suggests that uPTM-FetA may be more suitable for predicting renal function decline than UACR. Furthermore, the use of antihypertensive drugs, such as angiotensin receptor blockers and angiotensin-converting enzyme inhibitors, frequently prescribed in patients with type 2 diabetes, reduces albuminuria, limiting the utility of UACR as a predictive marker for DKD. uPTM-FetA may not have this drawback.

[0711] Although there have been studies using clinical parameters with or without the assistance of machine learning to establish DKD prediction models for patients with type 2 diabetes, the follow-up period was quite short. Because of the high intra-individual variability of eGFR among patients with early DKD, assessment of eGFR within a period of only 5 years is considered insufficient to assess the deterioration of renal function in patients with type 2 diabetes. Given the fact that global renal hyperfiltration with increasing eGFR is observed in a significant proportion of this population and that hyperfiltration may mask the development of early-stage kidney disease, long-term follow-up of renal function in patients with type 2 diabetes is essential.

[0712] This study has several strengths. It included the use of two large, well-characterized cohorts, allowing extensive adjustment for potential confounders. The long follow-up period in the NTUH cohort allowed for a proper assessment of the association between renal outcomes and uPTM-FetA, which is not underestimated by the issue of renal hyperfiltration in early DKD. Finally, the use of two independent, bi-ethnic cohorts, including patients from different geographic areas, is encouraging regarding the external validity of these findings to other type 2 diabetes populations worldwide.

[0713] In conclusion, this study demonstrates that uPTM-FetA is strongly associated with worsening renal function, independent of albuminuria, eGFR, age, sex, and multiple other risk factors, in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes. The additive prognostic value of uPTM-FetA over albuminuria and other markers is particularly clear in patients without CKD at baseline. These findings, validated in a second large, independent cohort, offer promise regarding the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0714] Example 4 The human uPTM3-DKD ELISA is a colorimetric immunoassay designed to quantitatively measure the intrinsic specific post-translational modification (PTM) fetuin A in human urine and must be performed in a competent clinical laboratory by licensed medical personnel.

[0715] Test Principles

[0716] The human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrator or unknown urine samples are mixed with an anti-specific PTM fetuin A monoclonal antibody (mAb) and then incubated in a microplate pre-bound with specific PTM fetuin A. Under competition in the microplate wells, the monoclonal antibody recognizes the specific PTM fetuin A in the calibrator or unknown sample. After incubation, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivities are determined by absorbance measurements at 450 nanometers (nm) and plotted by comparison with a predetermined specific PTM fetuin A standard curve.

[0717] Example 5 Study design

[0718] For the present study (Examples 5 to 7), we included patients from two independent, non-overlapping prospective cohort studies involving type 2 diabetes patients from different geographic areas. All analyses were first performed in the NTUH cohort, which was initiated at the National Taiwan University Hospital (NTUH) in Taiwan. These findings were then independently replicated in the Dutch DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort.

[0719] For this study, the settings indicated in Supplementary Table 2 below were used. [Table 5]

[0720] The Human uPTM3-DKD ELISA Kit is a colorimetric immunoassay for quantifying the unique specific post-translational modification of fetuin A (hereafter referred to as E103) in urine. This product is a non-automated IVD for use in prognostic prediction and must be performed in a competent clinical laboratory by a licensed medical professional such as a clinical laboratory technician. E103 concentrations should be corrected for urinary creatinine before clinical application. The Human uPTM3-DKD ELISA Kit is intended to be used in conjunction with clinical evaluation as an aid in assessing the prognosis of renal function in type 2 diabetes.

[0721] The human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrator or unknown urine samples are mixed with an anti-specific PTM fetuin A monoclonal antibody (mAb) and then incubated in a microplate pre-coated with specific PTM fetuin A. Under competition in the microplate wells, the monoclonal antibody recognizes the specific PTM fetuin A in the calibrator or unknown sample. After incubation, a horseradish peroxide (HRP)-conjugated secondary antibody is added, followed by incubation with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Their relative reactivities are determined by absorbance measurement at 450 nm and plotted by comparison with a predetermined specific PTM fetuin A standard curve.

[0722] Defined clinical performance parameters

[0723] Using hazard ratios and 95% confidence intervals calculated based on Cox proportional hazards regression analysis, E103 was associated with a ≥30% decline from baseline in estimated glomerular filtration rate (eGFR), an eGFR <15 mL / min / 1.73 m 2 or to assess the association with worsening renal function accompanied by the need for renal replacement therapy.

[0724] Statistics & Sample Size Calculations

[0725] The required sample size was estimated based on the following hypotheses: The hazard ratio (θ) for the high-risk group compared to the low-risk group is expected to be greater than 1, and in this study we set the hazard ratio at 2.0. According to literature statistics, approximately 25% to 40% of type 2 diabetes patients develop diabetic nephropathy. Therefore, in this study, we set the incidence of renal function deterioration at 25% (p < 0.01). E =0.25) The proportion of subjects in the low-risk and high-risk groups was the same (p L =p H =0.5), The type I error (α) is 0.05. The power (1-β) is 80%. The number of samples is ((z 1-α / 2 +z 1-β ) / (lnθ-ln(1)))2 / (p L p H p E ) Under the above hypothesis, 261 patients are required.

[0726] Study population

[0727] We used data from the National Taiwan University Hospital (NTUH) cohort in Taiwan and the DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort in the Netherlands. The NTUH cohort (project name: Validation of Early Diagnostic Biomarkers for Diabetic Nephropathy and its extension, registered project numbers: 201107004RC and 201705062RIPD) was conducted on patients with type 2 diabetes at NTUH from November 2011 to February 2020. Adult patients with type 2 diabetes according to the American Diabetes Association criteria were included in this study. Exclusion criteria included a diagnosis of type 1 diabetes mellitus, pregnancy, preexisting malignancy, acute or chronic infection, other primary or secondary renal disease, and illicit drug use. DIALECT was a prospective cohort study of type 2 diabetes patients conducted at Ziekenhuisgroep Twente Hospital, located in Almelo and Hengelo, the Netherlands. Adult patients with type 2 diabetes treated at the outpatient clinic of this secondary care hospital were included. Patients were recruited between March 2009 and May 2019. The study was conducted in accordance with the Good Clinical Practice guidelines and the WMA Declaration of Helsinki, and all subjects provided written informed consent before participating in the study.

[0728] NTUH Cohort (NTUH)

[0729] This prospective cohort study (project name: Validation of Early Diagnostic Biomarkers for Diabetic Nephropathy and its extension, registered project numbers with institutional review board approval: 201107004RC and 201705062RIPD) was conducted in patients with type 2 diabetes at National Taiwan University Hospital (NTUH), Taiwan, from November 2011 to February 2018. A detailed summary of the inclusion and exclusion criteria is shown in Supplementary Table 3. [Table 6]

[0730] This study was conducted in accordance with the Good Clinical Practice guidelines and the WMA Declaration of Helsinki. All subjects provided written informed consent before participating in the study.

[0731] DIALECT (validation) cohort (DIALECT)

[0732] To validate the optimal cutoff for assessing the risk of kidney function loss, we used data from the DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort study, described in detail elsewhere (16). Briefly, DIALECT is a prospective cohort study of type 2 diabetes patients conducted at Ziekenhuisgroep Twente Hospital, located in Almelo and Hengelo, the Netherlands. Adult patients with type 2 diabetes treated at the outpatient clinic of this secondary care hospital were recruited between March 2009 and May 2019 (Supplementary Table 3). The study was conducted in accordance with the Good Clinical Practice guidelines and the WMA Declaration of Helsinki, and all subjects provided written informed consent before participating in the study. The study was approved by the local institutional review board (METC registration numbers NL57219.044.16 and 1009.68020) and registered in The Netherlands Trial Register (NTR trial code 5855).

[0733] Test Procedure

[0734] uPTM-FetA measurement

[0735] At baseline, spot urine samples (NTUH) or 24-hour urine samples (DIALECT) were collected. These samples were stored at -80°C until measurement. uPTM-FetA was measured using a human uPTM3-DKD ELISA kit (CE-IVD, manufacturer: BioPreventive Medicine Corp., trade name: DNlite-IVD103). Samples with uPTM-FetA concentrations below the detection limit were set to the lower detection limit. For normalization, uPTM-FetA concentrations were divided by urinary creatinine (UCr) to obtain uPTM-FetA / UCr.

[0736] 1. Prepare the microplate modules required for all calibrators and urine samples and secure them in the holder. Allow all reagents to come to room temperature before use.

[0737] 2. Add 50 μL of Calibrators 1-8 and urine samples to each well of the E103-coated microplate.

[0738] 3. Add 50 μL of 1× mAb anti-E103 to each well of the E103-coated microplate.

[0739] 4. Incubate for 2 hours at 25°C and 200 rpm on an orbital shaker.

[0740] 5. After incubation, discard the contents of the wells.

[0741] 6. Wash each well with 300-400 μL of 1X Wash Buffer. Discard the contents and tap the well vigorously on absorbent paper to remove any residual liquid. Wash a total of four times.

[0742] 7. Add 100 μL of 1× HRP conjugate into each well. Incubate the microplate on an orbital shaker at 25° C. and 200 rpm for 30 minutes.

[0743] 8. After incubation, discard the contents of the wells and wash the wells as described in steps 4 and 5.

[0744] 9. Add 100 μL of TMB substrate to each well. Incubate in the dark at room temperature for 30 minutes.

[0745] 10. Add 100 μL of Stop Solution into each well. Mix by shaking briefly until the mixture is homogenous.

[0746] 11. Measure the absorbance at 450±10 nm within 30 minutes and calculate the results.

[0747] Renal endpoint definition

[0748] Renal function was assessed using estimated glomerular filtration rate (eGFR) calculated by the Chronic Kidney Disease Epidemiology Collaboration formula. For both cohorts, the primary outcome of worsening renal function was a ≥30% decline in eGFR from baseline and an eGFR <15 mL / min / 1.73 m. 2 Renal endpoints were defined as reaching ≥ 100% or the need for renal replacement therapy (i.e., kidney transplantation, peritoneal dialysis, or initiation of hemodialysis). Follow-up was censored at the end of follow-up or the date on which the renal endpoint was met.

[0749] Covariates

[0750] At baseline, clinical, anthropometric, and demographic data were extracted via electronic patient records and / or questionnaires. The severity of albuminuria was determined based on the urinary albumin / creatinine ratio (UACR). All other laboratory measurements were performed using routine laboratory methods.

[0751] statistical analysis

[0752] Baseline characteristics were presented as mean ± standard deviation (SD), median [interquartile range (IQR)], or frequency (percentage), depending on the data distribution. We performed receiver operating characteristic (ROC) analysis and used the Youden index to determine the optimal sensitivity and specificity of uPTM-FetA / UCr cutoff values ​​(19). According to the established cutoffs, patients were classified into low-risk (uPTM-FetA / UCr < optimal cutoff) or high-risk (uPTM-FetA / UCr ≥ optimal cutoff) groups. The statistical significance of differences between groups was assessed using Student's t-test, Mann-Whitney U test, or Fisher's exact test, depending on the data distribution.

[0753] To visualize the occurrence of renal endpoints during follow-up between groups, Kaplan-Meier curves were presented, and the log-rank test was used to assess the statistical significance of differences between groups. The association between uPTM-FetA / UCr and the risk of eGFR decline was analyzed using Cox proportional hazards regression analysis. Covariates with non-normal distributions were natural logarithm-transformed before analysis. Hazard ratios and 95% confidence intervals were presented for each relative increase of 1 standard deviation (for continuous risk factors) or for each change compared with the reference group (for categorical risk factors). In Model 1, we performed a multivariate-adjusted analysis using traditional risk factors (age, sex, eGFR, UACR, and HbA1c). We then calculated additional models with additional adjustments to Model 1, including lipid-related parameters (i.e., total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering medications (Model 2)), blood pressure-related parameters (systolic blood pressure, diastolic blood pressure, and use of antihypertensive medications (Model 3)), other clinical parameters (body mass index, smoking status (Model 4)), liver function abnormalities (Model 5), and a combination of all the above factors (Model 6).

[0754] To evaluate the prognostic performance of our biomarker, uPTM-FetA / UCr, we incorporated it into a clinical-plus-biomarker model. Various evaluation scales were used to assess the additive value of this biomarker in predicting the risk of worsening renal function. Model fit was assessed using the Akaike information criterion (AIC) and likelihood ratio test (LRT), with lower AIC values ​​or larger χ2 values ​​indicating better overall fit. Model calibration was assessed using the Hosmer-Lemeshow goodness-of-fit test, with a p-value >0.05 indicating good agreement between observed and predicted results. Model discrimination was determined by calculating the area under the receiver operating characteristic curve (AUC). The optimal cutoffs for maximizing sensitivity and specificity in each model were determined using the Youden index. The improvement in AUC after incorporating the biomarker was calculated.

[0755] To assess model reclassification, we used a continuous / category-free NRI (NRI > 0) because there was no established risk cutoff that justified the use of categorical net reclassification improvement. The overall NRI measured the proportion of predicted probabilities that shifted upward or downward when the biomarker was added to the clinical model. In addition, the absolute integrated discrimination improvement (IDI) was employed to quantify the mean increase in predicted probability for subjects with worsening renal function and the mean decrease in predicted probability for subjects without worsening renal function after adding the biomarker.

[0756] All data were analyzed using R version 4.1.2 (R Foundation for Statistical Computing, Vienna, Austria). For all statistical analyses, a p-value <0.05 was considered significant.

[0757] DIALECT (validation) cohort analysis

[0758] Similar to the primary analysis, patients in the DIALECT validation cohort (DIALECT) were stratified into high- and low-risk groups based on the uPTM-FetA / UCr cutoff obtained from the NTUH cohort study. Survival analyses, including Cox proportional hazards regression models, described for the primary analysis were repeated in this DIALECT validation cohort with the same renal endpoints as outcome variables.

[0759] Data and results management

[0760] Baseline eGFR 3 ≥30% decrease from baseline, eGFR <15 mL / min / 1.73 m 2The data will be collected from a series of clinical records provided by the NTUH and DIALECT cohorts, corresponding to all patients diagnosed with renal failure or worsening renal function accompanied by the need for renal replacement therapy. All Excel files containing these data will be manually downloaded by the principal investigator and stored in a specific folder on a computer with an encrypted disk and other privacy and security measures in line with the EU General Data Protection Regulation (GDPR). This folder will be accessible only to the principal investigator and statisticians. Patient urine samples will be transferred from NTUH and UMCG to BPM and then stored at -80°C according to BPM's sample management procedures and will not be removed until use. After testing, test results will be recorded in an Excel file and stored in a specific folder by BPM's study laboratory staff, as previously described.

[0761] Analysis of data and results

[0762] For the primary (NTUH) cohort, we will conduct screening of recruited patients over a 3.5-year period. During the screening period, urine samples were collected every 6 months, and for each patient, baseline was defined as the time when the E103 value first exceeded the assay's limit of quantitation (LoQ = 5.428 ng / mL). For patients with E103 measurements below the LoQ but above the limit of detection (LoD = 1.901 ng / mL), the E103 value was set as the LoD. Patients with E103 values ​​below the LoD were defined as "undetectable." Changes in eGFR from baseline to 5 years of follow-up were calculated as 100% * (eGFR - baseline eGFR) / baseline eGFR. Study endpoints were a ≥ 30% decline in eGFR from baseline and an eGFR < 15 mL / min / 1.73 m 2 or the need for renal replacement therapy (i.e., kidney transplantation, peritoneal dialysis, or initiation of hemodialysis). Data were censored if the outcome had not occurred at the end of follow-up, and the recording time was set to the end of follow-up. Patients with a follow-up shorter than 2 years or fewer than five eGFR measurements were excluded from the analysis.

[0763] Then, for analysis, the E103 test results were divided by urinary creatinine (UCr) to obtain the uPTM-FetA / UCr value. IVD103=log10(uPTM-FetA / UCr) is the logarithmic version to base 10. We adopted the receiver operating characteristic (ROC) curve value that maximizes the sum of sensitivity and specificity by using the Youden Index method and set it as the clinical cutoff. According to the clinical cutoff, subjects were divided into two groups: low-risk group (uPTM-FetA / UCr below the clinical cutoff) and high-risk group (uPTM-FetA / UCr above the clinical cutoff). Hazard ratios were evaluated using the Cox proportional hazards model, and survival curves of the two groups were compared using Kaplan-Meier graphs and log-rank tests.

[0764] To assess whether clinical status influences the ability of uPTM-FetA / UCr to predict the risk of renal deterioration, we added other variables to the univariate Cox proportional hazards model and fitted a multivariate Cox proportional hazards model to observe the change in the hazard ratio of uPTM-FetA / UCr and consider clinical factors. In Model 1, we performed a multivariate-adjusted analysis using traditional risk factors (age, sex, eGFR, urinary albumin / creatinine ratio (UACR), and HbA1c). We then calculated further models with additional adjustments for Model 1, including lipid-related parameters (i.e., total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering medications (Model 2)), blood pressure-related parameters (systolic blood pressure, diabetic blood pressure, and use of antihypertensive medications (Model 3)), other clinical parameters (body mass index, smoking status (Model 4)), presence or absence of liver function abnormalities (aspartate transaminase (AST) or alanine transaminase (ALT) < 5 U / L or AST or ALT > 40 U / L (Model 5)), and a combination of all the above factors (Model 6).

[0765] UACR is generally used as a clinical index to evaluate renal function. UACR > 30 mg / g is considered an important index for assessing the progression of renal function decline and is generally considered to be a sign of chronic kidney disease. 10 It is used to detect and monitor renal function deterioration, which may require further evaluation and treatment. To demonstrate the ability of uPTM-FetA / UCr to provide additional interpretive information in clinical use, we performed a comparative analysis of uPTM-FetA / UCr (above and below the clinical cutoff) compared with UACR (above and below 30 mg / g) by using a Cox regression model after adjusting for baseline risk factors age and sex to evaluate the event incidence of renal function deterioration.

[0766] In a validation analysis, the DIALECT cohort (validation) was used to validate the uPTM-FetA / UCr clinical cutoff derived from the primary analysis. Patients were divided into high-risk (uPTM-FetA / UCr above the cutoff) and low-risk (uPTM-FetA / UCr below the cutoff) groups, and hazard ratios were estimated using a univariate Cox model. Survival curves for the two groups were then compared using Kaplan-Meier plots and the log-rank test.

[0767] Example 6 Results of the primary analysis

[0768] Of all participants enrolled in the NTUH cohort, 294 were eligible for inclusion in this study. The mean age was 61 ± 10 years, 163 (55%) were men, and the eGFR was 88 ± 16 mL / min / 1.73 m. 2 The median UACR was 9.9 [5.2-25.3] mg / g.

[0769] Determination of cutoffs and association with renal endpoints - NTUH cohort

[0770] During a median follow-up of 4.6 years (range, 4.2-4.8), 42 participants (14%) in the NTUH cohort achieved a renal endpoint. The optimal uPTM-FetA / UCr cutoff for risk stratification of renal endpoints was 7.53 ng / mg (equivalent to an IVD103 cutoff of 0.877) based on the Youden index. Population characteristics in the uPTM-FetA / UCr stratified groups are presented in Table 4. Figure 5A illustrates Kaplan-Meier curves for the NTUH cohort, indicating that adding uPTM-FetA / UCr to the prediction model according to non-limiting embodiments improved the ability to predict worsening renal function. Referring to Figure 5A, the curves show a correlation between a 30% decline in eGFR or <15 mL / min / 1.73 m in high-risk and low-risk groups stratified by NTUH uPTM-FetA / Ucr levels. 2 In the high-risk uPTM-FetA / Ucr group, 21% experienced a renal endpoint, which was significantly higher than the 3% observed in the low-risk group (p<0.001).

[0771] According to this clinical cutoff, subjects were divided into a low-risk group (uPTM-FetA / UCr<7.53ng / mg) and a high-risk group (uPTM-FetA / UCr≥7.53ng / mg) (Table 4). Table 4 shows that there was a significant difference in the frequency of worsening renal function between the low-risk and high-risk groups (2.7% and 21%, p<0.001), and the survival time of the high-risk group was significantly shorter than that of the low-risk group (4.56 [4.14, 4.77] vs. 4.63 [4.24, 4.78], p=0.026).

[0772] A univariate Cox proportional hazards model showed that the high-risk group had a significantly higher risk of worsening renal function than the low-risk group, with a hazard ratio of 8.94 (95% confidence interval 2.76-28.9, p-value <0.001) (Table 5). Figure 3 illustrates Kaplan-Meier curves for 294 subjects from the NTUH cohort of Examples 5-7 in some embodiments. Referring to Figure 3, the Kaplan-Meier curves also showed a significant event risk in the high-risk group compared with the low-risk group, with a log-rank test p-value <0.0001. The ... 2 The endpoint is 0.01 mg / g. Figure 4A illustrates the Kaplan-Meier curves for subjects with UCr < 30 mg / g in the NTUH cohort in Examples 5-7, according to non-limiting embodiments. Referring to Figures 4A and 3, in the high-risk uPTM-FetA / UCr group, 21% experienced a renal endpoint, which was significantly higher than the 3% observed in the low-risk group (P < 0.001). Additionally, the cumulative event probability by baseline uPTM-FetA / UCr risk category at 1-4 years post-baseline in the NTUH cohort is displayed in Table 6. [Table 7] JPEG2026505167000009.jpg144159 [Table 8] JPEG2026505167000011.jpg86159

[0773] Additional prognostic value of current markers including UACR - NTUH cohort

[0774] The association between the high-risk uPTM-FetA / UCr group and renal endpoints was also observed in univariate Cox regression analysis (HR: 8.94, 95% CI: 2.76–28.90, P<0.001) and became even more pronounced in the fully adjusted model 6 (HR: 9.94, 95% CI: 2.96–33.40, P<0.001; Table 5).

[0775] According to Table 5, after performing multivariate Cox proportional hazards models with the addition of various clinical factors, the ability of uPTM-FetA / UCr to predict worsening renal function remained significant in models 1–6 (p-values ​​< 0.05), with hazard ratios ranging from 7.67 to 11.1. [Table 9] [Table 10]

[0776] The additional value of UACR was also illustrated by further analyses of subgroups with uPTM-FetA / UCr levels below or above 7.53 ng / mg and below or above 30 mg / g. In the subgroup of patients with UACR < 30 mg / g, who are generally considered to be at low risk for renal function deterioration, patients with uPTM-FetA / UCr > 7.53 ng / mg had a higher risk of renal function deterioration compared with patients with uPTM-FetA / UCr < 7.53 ng / mg (event rate: 17% vs. 3%, respectively; HR: 5.14, 95% CI: 1.54-17.20, P = 0.008). Similarly, in the subgroup of patients with a UACR >30 mg / g, who are generally considered to be at high risk of worsening renal function, patients with a uPTM-FetA / UCr >7.53 ng / mg were at higher risk of worsening renal function, whereas none of the patients with a uPTM-FetA / UCr <7.53 ng / mg had any events (event incidence: 35% vs. 0%, respectively; HR: 13.00; 95% CI: 3.76-44.80; P < 0.001; Table 7 and Figures 4A and 4B).

[0777] Figure 4A illustrates Kaplan-Meier curves for subjects with a UACR < 30 mg / g in the NTUH cohort of Examples 5-7, according to some embodiments. Figure 4B illustrates Kaplan-Meier curves for subjects with a UACR ≥ 30 mg / g in the NTUH cohort of Examples 5-7, according to some embodiments, by uPTM-FetA / UCr risk category. Referring to Figures 4A and 4B, the Kaplan-Meier curves show that the event risk in patients with a uPTM-FetA / UCr ≥ 7.53 ng / mg was significant compared to the low uPTM-FetA / UCr group for both UACR < 30 mg / g and UACR ≥ 30 mg / g, with log-rank p-values ​​of 0.0011 and 0.007, respectively. This suggests that uPTM-FetA / UCr may provide additional information to UACR when assessing the risk of progression of renal function decline. [Table 11]

[0778] In line with these findings, we further evaluated the additive prognostic value of uPTM-FetA / UCr added to the current clinical model including age, sex, eGFR, UACR, and HbA1c, as detailed in Supplementary Table 4. [Table 12] Values ​​are presented as OR (95% CI), P value, AIC (Akaike Information Criterion), AUC (Area Under the Curve), HL (Hosmer-Lemeshow), IDI (Integrated Discrimination Improvement Index), LRT (Likelihood Ratio Test), NI (Not Included), NRI (Net Reclassification Improvement).

[0779] Figure 6 illustrates ROC curves, indicating that adding uPTM-FetA / UCr to a prediction model according to a non-limiting embodiment improves the performance of predicting worsening renal function. Referring to Figure 6, the area under the receiver operating characteristic curve (AUROC) of the clinical model including age, sex, eGFR, UACR, and HbA1c is 0.70 (0.62-0.78), and the AUROC of adding uPTM-FetA / Ucr to the clinical model is 0.80 (0.73-0.87). The AUROCs of the clinical model and clinical plus uPTM-FetA / UCr are 0.70 (0.62-0.78) and 0.80 (0.73-0.87), respectively. The addition of biomarkers to the clinical model improved the model fit (ΔLRT χ 2 = 21.85, P < 0.001), calibration (Hosmer-Lemeshow test P = 0.574), and discrimination ability (AUC increased from 0.70 to 0.80, P < 0.001, Figure 6). Adding biomarkers improved risk classification (continuous NRI 0.72 [95% CI 0.52-0.92]) and the overall predictive ability of the model (IDI 7.8% [5.9-9.8%]).

[0780] Example 7 DIALECT (validation) cohort

[0781] Overall, 376 participants from the DIALECT cohort were eligible for inclusion in this study. The mean age was 64 ± 11 years, 249 (66%) were men, and the eGFR was 76 ± 24 mL / min / 1.73 m. 2 The median UACR was 9.0 [3.5–47.0] mg / g (Table 8).

[0782] Although baseline eGFR was statistically lower than that of the NTUH cohort, there was no significant difference in UACR (p<0.001 and p=0.657, respectively).

[0783] Positive validation was obtained in the DIALECT cohort analysis, confirming the utility of the uPTM-FetA / Ucr clinical cutoff with good predictive power for patients at risk of worsening renal function.

[0784] Validation Study Results - DIALECT Cohort

[0785] In the DIALECT cohort, 125 participants (33%) experienced a renal endpoint during a median follow-up of 3.7 years (range, 2.5-5.3). In the high-risk uPTM-FetA / Ucr group, 44% experienced a renal endpoint compared with 26% observed in the low-risk group (P<0.001).

[0786] Of the 376 subjects, 111 (30%) experienced a deterioration in renal function during follow-up, and their median survival time was 3.65 years (IQR, 2.51-5.33 years). According to the clinical cutoff of uPTM-FetA / Ucr 7.53 ng / mg obtained in the NTUH cohort, subjects were divided into low-risk (uPTM-FetA / Ucr < 7.53 ng / mg) and high-risk (uPTM-FetA / Ucr ≥ 7.53 ng / mg) groups (Table 8). There was a significant difference in the frequency of deterioration in renal function (26% and 44%, respectively, p < 0.001).

[0787] The results of the Cox proportional hazards model showed that the risk of worsening renal function was significantly higher in the high-risk group than in the low-risk group, with a hazard ratio of 1.87 (95% confidence interval 1.31-2.66, p-value <0.001). Figure 5B illustrates Kaplan-Meier curves for the DIALECT cohort, indicating that adding uPTM-FetA / Ucr to the prediction model according to non-limiting embodiments improves the ability to predict worsening renal function. Figure 5B illustrates Kaplan-Meier curves for 376 subjects in the DIALECT (validation) cohort of Examples 5-7 in some embodiments. Referring to Figure 5B, the curves show the association between a 30% decline in eGFR or <15 mL / min / 1.73 m in the high-risk and low-risk groups stratified by uPTM-FetA / Ucr levels at NTUH. 2 The endpoint was . The Kaplan-Meier curves also showed a significant event risk in the high-risk group compared with the low-risk group, with a log-rank p-value of 0.00041. Table 9 also shows the cumulative event probability by baseline uPTM-FetA / Ucr risk category at 1-4 years post-baseline in the DIALECT (validation) cohort. [Table 13] JPEG2026505167000017.jpg76159 [Table 14] JPEG2026505167000019.jpg88159 [Table 15]

[0788] Analysis of Examples 4-7 In this study, uPTM-FetA was associated with the risk of eGFR decline in patients with type 2 diabetes, independent of age, sex, eGFR, UACR, baseline HbA1c, and several other potential confounders. These findings, which have been validated in a second, larger cohort of patients with type 2 diabetes, highlight the potential clinical use of uPTM-Fet-A as a predictive marker for DKD.

[0789] The human fetuin-A precursor protein consists of three parts: the A chain, the connecting peptide, and the B chain, which are 321, 40, and 27 amino acids long, respectively. The connecting peptide of the precursor is removed by limited proteolysis, a post-translational modification, after which only the A and B chains form the active fetuin-A protein. The monoclonal antibody in the ELISA kit used in this study (Human uPTM3-DKD ELISA kit, BPM Corp.) detects only the connecting peptide-containing fetuin-A.

[0790] We found that elevated uPTM-FetA levels correlate with progressive renal function decline in patients with type 2 diabetes. These findings are generally consistent with previous studies demonstrating an association between urinary fetuin A and albuminuria and poorer renal function in patients with type 2 diabetes and DKD. In some embodiments, uPTM-FetA levels are unrelated to serum c-FetA levels among patients (internal data from the NTUH cohort, data not shown). Other studies have also observed discrepancies between circulating and urinary fetuin A levels. This suggests that fetuin A may be produced locally in the kidneys of patients with kidney disease, including DKD. uPTM-FetA specifically accumulates in the urine of patients with progressive renal function decline. This may be due to impaired tubular reabsorption of the molecule rather than increased filtration or secretion. In some embodiments, uPTM-FetA may be a predictive marker of progressive kidney disease. In some embodiments, uPTM-FetA may have a direct pathogenic effect that needs to be further investigated in future preclinical and interventional studies. These results show that uPTM-FetA was analyzed against UACR in multivariate-adjusted Cox regression in type 2 diabetes patients without CKD. This suggests that uPTM-FetA may be more suitable for predicting renal function decline than UACR. Furthermore, the use of antihypertensive drugs, such as angiotensin receptor blockers and angiotensin-converting enzyme inhibitors, which are frequently prescribed in type 2 diabetes patients, reduces albuminuria, limiting the utility of UACR as a predictive marker for DKD. uPTM-FetA may not have this drawback.

[0791] Although there have been studies using clinical parameters with or without the assistance of machine learning to establish DKD prediction models for patients with type 2 diabetes, the follow-up period was quite short. Because of the high intra-individual variability of eGFR among patients with early DKD, assessment of eGFR within a period of only 5 years is considered insufficient to assess the deterioration of renal function in patients with type 2 diabetes. Given the fact that global renal hyperfiltration with increasing eGFR is observed in a significant proportion of this population and that hyperfiltration may mask the development of early-stage kidney disease, long-term follow-up of renal function in patients with type 2 diabetes is essential.

[0792] This study has several strengths. It included the use of two large, well-characterized cohorts, allowing extensive adjustment for potential confounders. The long follow-up period in the NTUH cohort allowed for a proper assessment of the association between renal outcomes and uPTM-FetA, which is not underestimated by the issue of renal hyperfiltration in early DKD. Finally, the use of two independent, bi-ethnic cohorts, including patients from different geographic areas, is encouraging regarding the external validity of these findings to other type 2 diabetes populations worldwide.

[0793] In conclusion, this study demonstrates that uPTM-FetA is strongly associated with worsening renal function, independent of albuminuria, eGFR, age, sex, and multiple other risk factors, in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes. The additive prognostic value of uPTM-FetA over albuminuria and other markers is particularly clear in patients without CKD at baseline. These findings, validated in a second large, independent cohort, offer promise regarding the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0794] The results of this study demonstrate that uPTM-FetA / UCr can effectively predict the risk of worsening renal function in patients with type 2 diabetes, independently of albuminuria, eGFR, age, sex, HbA1c, lipid-related parameters, blood pressure-related parameters, BMI, smoking status, and liver function status. The additive prognostic value of uPTM-FetA / UCr in addition to uACR is particularly clear. The clinical validity of the uPTM-FetA / UCr clinical cutoff (=7.53 ng / mg) was validated in an independent type 2 diabetes cohort.

[0795] These results indicate that uPTM-FetA / UCr can effectively predict actual eGFR decline, and high-risk patients can receive early intervention or relevant adjustments to achieve preventive effects, which will bring many clinical benefits and benefit type 2 diabetes patients.

[0796] In this study, uPTM-FetA was associated with the risk of eGFR decline in patients with type 2 diabetes and provided significant additional prognostic value independent of age, sex, eGFR, UACR, baseline HbA1c, and several other potential confounders. These findings, which have been validated in a second, larger cohort of patients with type 2 diabetes, highlight the potential clinical use of uPTM-FetA as a predictive marker for DKD.

[0797] The human fetuin-A precursor protein consists of three parts: the A chain, the connecting peptide, and the B chain, which are 321, 40, and 27 amino acids long, respectively. The connecting peptide of the precursor is removed by limited proteolysis, a post-translational modification, after which only the A and B chains form the active fetuin-A protein. The monoclonal antibody in the ELISA kit used in this study (Human uPTM3-DKD ELISA kit, BPM Corp.) detects only the connecting peptide-containing fetuin-A.

[0798] We found that elevated uPTM-FetA levels correlate with the progression of renal function decline in patients with type 2 diabetes. These findings are generally consistent with previous studies that have shown an association between urinary fetuin A and albuminuria and poorer renal function in patients with type 2 diabetes and DKD. Interestingly, among patients, uPTM-FetA levels are not related to serum PTM-FetA levels (internal data from the NTUH cohort, data not shown). Other studies have also observed discrepancies between circulating and urinary fetuin A levels. This indicates that fetuin A is produced locally in the kidneys of patients with kidney disease, including DKD.

[0799] Fetuin A is not synthesized by the kidney in humans or animal models under healthy conditions. However, upon injury, proximal tubular epithelial cells (PTECs) can synthesize fetuin A and release it into the apical side of the tubular cells. In animal studies using rat models of cisplatin-induced and ischemia / reperfusion-induced acute kidney injury, fetuin A was detected in the urinary exosomal fraction but not in the soluble non-exosomal fraction, indicating that urinary fetuin A is produced by PTECs. In addition, urinary fetuin A levels increase before a rapid increase in serum creatinine and before morphological damage to the tubules occurs. It is hypothesized that local production of fetuin A by tubular cells protects the tubules from ongoing inflammation and fibrosis. Whether uPTM-FetA can simply be a predictive marker or also has a direct pathogenic effect requires further investigation in future preclinical and interventional studies.

[0800] Nevertheless, our findings indicate that in patients with type 2 diabetes, uPTM-FetA, in contrast to UACR, was strongly associated with declining renal function in multivariate-adjusted Cox regression analysis. Furthermore, this marker has clear additional prognostic value in addition to current clinical models that include UACR. This suggests that uPTM-FetA may be better suited to predicting declining renal function than UACR, or may at least provide additional clinical value. Furthermore, the use of antihypertensive drugs, such as angiotensin receptor blockers and angiotensin-converting enzyme inhibitors, which are frequently prescribed in patients with type 2 diabetes, reduces albuminuria, limiting the utility of UACR as a predictive marker for DKD. Potentially, uPTM-FetA may not have this drawback, but future studies are needed to evaluate changes in uPTM-FetA after the initiation of these antihypertensive drugs.

[0801] This study has several strengths. The inclusion of two large, well-characterized cohorts allowed for validation and extensive adjustment for potential confounders. The two-ethnic cohort, including patients from different geographic areas, is encouraging regarding the external validity of these findings to other type 2 diabetes populations worldwide. Furthermore, despite differences in baseline eGFR and albuminuria between these two cohorts, the prognostic predictive ability of uPTM-FetA remains significant. Therefore, this biomarker may be potentially useful in type 2 diabetes patients with various disease states. Our study also has several limitations. The follow-up period in the validation cohort was significantly shorter than that in the primary cohort. Furthermore, due to the observational design of this study, we were only able to evaluate the prognostic predictive ability of uPTM-FetA and were unable to confirm the causative effect of uPTM-FetA on kidney disease progression. Furthermore, comparison of other markers, such as kidney injury molecule-1 and neutrophil gelatinase-binding lipocalin, with uPTM-FetA may provide useful information regarding its meaning and interpretation. Finally, none of the patients were using SGLT2 inhibitors at baseline, and only a small proportion of the included patients started using SGLT2 inhibitors during the follow-up period. The use of these drugs (inhibitors) may not be expected to change the additional value of uPTM-FetA / Ucr in predicting eGFR decline in patients with type 2 diabetes.

[0802] This study demonstrates that uPTM-FetA is strongly associated with worsening renal function, independent of albuminuria, eGFR, age, sex, and multiple other risk factors, in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes. These findings have been validated in a second large, independent cohort, providing promise regarding the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0803] Example 8

[0804] Based on the cohort data analyzed in Examples 4-7, multiple risk categories for the prognosis of renal function decline based on biomarkers were developed. The biomarker in this example was human-specific PTM fetuin-A (E103). As shown in Table 8-1 below, two "cutoff" values ​​were developed as threshold points for the multiple risk categories. [Table 16]

[0805] In this example, worsening renal function was defined as the need for renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal dialysis, or hemodialysis) or a persistent increase in serum creatinine >50% for at least 3 months from the baseline visit.

[0806] Based on two threshold values, Cutoff 1 and Cutoff 2, three risk categories were developed as shown in Table 8-2. [Table 17]

[0807] Cohort data were analyzed as indicated in Tables 8-3, 8-4, 8-5, 8-6 below and Figures 7 and 8. Figure 7 illustrates a Kaplan-Meier curve indicating survival probability over time in years for values ​​of uPTM-FetA / UCr cutoff 1, according to a non-limiting embodiment. Figure 8 illustrates a Kaplan-Meier curve indicating event-free probability over time in years for values ​​of uPTM-FetA / UCr cutoff 2, according to a non-limiting embodiment. [Table 18] JPEG2026505167000024.jpg56159 [Table 19] JPEG2026505167000026.jpg116159 [Table 20]

[0808] Based on the above analysis, we developed a biomarker-based scoring system for predicting renal function decline. The scoring system is based on a urine test measuring a single biomarker (DNlite-IVD103). The urine test was combined with five clinical factors that can be considered relatively easy to manage: age, sex, eGFR, UACR, and HbA1c.

[0809] Scoring is based on the following formula, based on Table 8-7: Occurrence score = exp(y) / (1+exp(y)), During the ceremony, y=a0+a1*Age+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53ng / mg) Here, if E103 / UCr>7.53ng / mg, then I(E103 / UCr>7.53ng / mg)=1, or if E103 / UCr is 7.53ng / mg or less, then I(E103 / UCr>7.53ng / mg)=0. [Table 21]

[0810] The thresholds and risk categories based on these scores are shown in Tables 8-8 and 8-9: [Table 22] [Table 23]

[0811] The predictive performance was as indicated in Tables 8-10 and 8-11 and Figures 9 and 10. Figure 9 illustrates a Kaplan-Meier curve indicating the progression-free probability over time in years for two cutoff values ​​of the incidence score, according to a non-limiting embodiment. Figure 10 illustrates a cumulative incidence plot against the incidence score, according to a non-limiting embodiment. [Table 24] [Table 25]

[0812] Additional data analyses are provided below in Tables 8-12, 8-13, 8-14, 8-15, 8-16, and 8-17, as well as in Figures 11 and 12. Figure 11 illustrates a Kaplan-Meier curve for the DIALECT cohort, showing the progression-free probability over time in years for two cutoff values ​​of the incidence score, according to a non-limiting embodiment. Figure 12 illustrates a cumulative incidence plot against incidence score for the DIALECT cohort, according to a non-limiting embodiment. [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31]

[0813] Purpose of use

[0814] The human uPTM3-DKD ELISA is a colorimetric immunoassay designed to quantitatively measure the intrinsic specific post-translational modification (PTM) fetuin A in human urine. It must be performed in a qualified clinical laboratory by a licensed medical professional, such as a clinical laboratory technician. This product is intended for in vitro diagnostic use as an aid in assessing the risk of renal complications in diabetic patients. Furthermore, the uPTM3-DKD test may also be applied to accurately identify progressive renal decline in type 2 diabetes with microalbuminuria. [Table 32] [Table 33]

[0815] Reagent preparation

[0816] 1. Human-specific PTM Fetuin A (E103) Calibrator

[0817] Reconstitute the E103 calibrator with 0.2 mL of distilled or deionized water and allow to stand at room temperature for 10 minutes until completely dissolved, mixing gently. At that point, the reconstituted E103 calibrator will have a concentration of 5 μg / mL. Dilute the 5 μg / mL E103 calibrator 5-fold with diluent and then gently mix to obtain the 1 μg / mL E103 calibrator. The serial dilution procedure to generate all the calibrators for establishing the E103 calibration curve is shown in the table below. All calibrators should be prepared and mixed thoroughly immediately before use. [Table 34]

[0818] 2. 1X Wash Buffer

[0819] Before use, bring the 10X Wash Buffer to room temperature until all salt crystals have dissolved. 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.

[0820] 3. 1X mAb Anti-Specific PTM Fetuin A (E103)

[0821] Calculate the amount of 1X mAb anti-specific PTM fetuin-A (E103) needed for each assay and mix the required amount of 1000X mAb anti-specific PTM fetuin-A (E103) with diluent. For each microplate, mix 8 μL of 1000X mAb anti-specific PTM fetuin-A (E103) with 8 mL of diluent. Mix evenly but gently.

[0822] 4. 1X HRP Conjugate

[0823] Calculate the amount of 1X HRP conjugate needed for each assay and mix the 4000X HRP conjugate with diluent accordingly. For each microplate, mix 3 μL of 4000X HRP conjugate with 12% diluent. Mix evenly but gently.

[0824] Materials needed but not provided

[0825] Precision single micropipettes (1-10μL, 20-100μL, 100-200μL, 200-1000μL) and multichannel pipettes (100μL)

[0826] Microcentrifuge tubes and disposable tips

[0827] ·500mL measuring cylinder

[0828] Vortex mixer and microcentrifuge

[0829] Orbital shaker

[0830] Plastic containers for preparing reagents

[0831] ·Microplate reader capable of endpoint measurement at 450±10nm

[0832] Distilled or deionized water

[0833] Adhesive plate seal

[0834] Reagent reservoir

[0835] Warnings and Cautions

[0836] This test is for in vitro diagnostic use only by specialists.

[0837] Do not use reagents after the expiration date.

[0838] Exposure to improper temperatures during all storage and assay procedures may adversely affect results.

[0839] Do not reuse microplate wells.

[0840] · Wear protective gloves during all assay procedures.

[0841] Being photosensitive and a skin irritant, TMB substrate should be protected from direct light exposure and skin contact during all storage and assay procedures.

[0842] Avoid skin contact with the stop solution containing 0.5N sulfuric acid, which may cause skin irritation and burns.

[0843] · Consider all clinical specimens to be potentially infectious.

[0844] Disposal of any waste materials should comply with local requirements and existing good laboratory practice regulations.

[0845] Specimen collection and handling

[0846] Morning urine samples should be collected in a clean, dry container. Avoid cross-contamination.

[0847] · No additives or preservatives are required for urine sample integrity.

[0848] Store urine samples at -20°C until use. Avoid repeated freezing and thawing of urine samples.

[0849] Allow the urine sample to come to room temperature before performing the assay. Centrifuge the urine sample at 1,000 ± 20 x g for 5 minutes. Remove the supernatant and assay immediately.

[0850] If necessary, use a diluent to dilute the sample.

[0851] Assay procedure

[0852] Prepare as many microplate modules as needed for all calibrators and urine samples and secure them in the holder. Allow all reagents to come to room temperature before use.

[0853] 1. Mix Calibrators 1-8 and each centrifuged urine sample with 1X mAb anti-E103 in a 1:1 ratio in a microcentrifuge tube (for triplicate testing, it is recommended to mix 180 μL of each calibrator / sample with 180 μL of 1X mAb anti-E103). Incubate on an orbital shaker at 25°C and 200 rpm for 2 hours.

[0854] 2. Transfer 100 μL of each incubated mixture to the assigned well of the E103-coated microplate and incubate for 1.5 hours at 25°C and 200 rpm on an orbital shaker. Keep the microplate covered and level throughout the incubation.

[0855] 3. After incubation, discard the contents of the wells.

[0856] 4. Wash each well with 300-400 μL of 1X Wash Buffer. Discard the contents and tap the well vigorously on absorbent paper to remove any residual liquid. Wash a total of four times.

[0857] 5. Add 100 μL of 1× HRP conjugate into each well. Incubate the microplate on an orbital shaker at 25° C. and 200 rpm for 60 minutes.

[0858] 6. After incubation, discard the contents of the wells and wash the wells as described in step 4.

[0859] 7. Add 100 μL of TMB substrate to each well. Incubate in the dark at room temperature for 30 minutes.

[0860] 8. Add 100 μL of Stop Solution into each well. Mix by shaking briefly until the mixture is homogenous.

[0861] 9. Measure the absorbance at 450±10 nm within 30 minutes and calculate the results.

[0862] Calculating the results

[0863] 1. Establish a calibration curve using a 5-parameter or 4-parameter logistic curve fit. The concentration of the specific PTM fetuin-A in the patient's urine sample can be calculated from the calibration curve by interpolation. The range of this assay is 7.813-500 ng / mL.

[0864] 2. Calculate the amount of unique PTM fetuin-A corrected for urinary creatinine (ng / mg Cr).

[0865] Performance characteristics

[0866] accuracy

[0867] 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 used. Samples were tested in triplicate twice daily. Intra-run precision: The coefficient of variation (CV) for each of the eight samples was calculated from the results of three measurements within each run. Inter-run precision: The coefficient of variation (CV) for each of the eight samples was calculated from the results of three measurements within each of 10 separate runs.

[0868] The accuracy results are summarized in the table below. [Table 35]

[0869] Measurement range

[0870] 7.813-500ng / mL

[0871] linearity

[0872] The test was evaluated and found to be linear between 1.769 and 500 ng / mL (R 2 =0.99).

[0873] Detection limit

[0874] Upper limit of blank (LoB) = 0.873 ng / mL

[0875] Limit of detection (LoD) = 4.510 ng / mL

[0876] Limit of quantification (LoQ)=18.9830ng / mL [Table 36]

[0877] For example, Figure 2 illustrates a standard curve for the human uPTM3-DKD ELISA (calibration range 7.813-500 ng / mL), according to a non-limiting embodiment.

[0878] While preferred embodiments of the present invention 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 invention. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. An assay kit for determining the likelihood of progressive renal function decline, comprising: a first solution comprising a first reagent that interacts with urinary post-translationally modified fetuin A fragment (uPTM-FetA) for indicating the level of said fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution comprising a second reagent that interacts with the urinary creatinine to indicate the level of urinary creatinine in the urine sample; a device for measuring the level of fetuin-A fragment and the level of urinary creatinine in the urine sample to determine a ratio of the measured level of fetuin-A fragment to the measured level of urinary creatinine; wherein the ratio greater than about 7.5 ng / mg indicates a higher likelihood of progressive decline in renal function leading to a renal endpoint in the subject over a period of about 10 years; The assay kit, wherein the first solution and the second solution are the same or different from each other.

2. 10. The assay kit of claim 1, wherein said ratio greater than about 7.5 ng / mg triggers medical intervention to forestall said progressive decline in renal function.

3. 2. The assay kit of claim 1, wherein the higher likelihood is greater than the lower likelihood of progressive renal function decline over the approximately 10-year period in a second subject having a lower ratio of the measured level of the fetuin-A fragment to the measured level of the urinary creatinine of less than about 7.5 ng / mg.

4. 2. The assay kit of claim 1, wherein the higher likelihood is lower than the likelihood of progressive renal decline over the approximately 10-year period in a third subject having a higher ratio of the measured level of fetuin-A fragment to the measured level of urinary creatinine of less than about 29.0 ng / mg.

5. 2. The assay kit of claim 1, wherein a ratio value greater than 7.5 ng / mg indicates that the progressive renal failure is at a higher probability.

6. The assay kit of claim 1, wherein the progressive decline in renal function is a decline in estimated glomerular filtration rate (eGFR).

7. The subject's eGFR is about 60 milliliters per minute per 1.73 square meters (mL / min / 1.73 m ２ 7. The assay kit of claim 6, wherein the assay kit is indicated to have a nucleotide sequence of 0.1 or more.

8. 10. The assay kit of claim 1, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for the 12 months prior to collection of the urine sample or both before and after the time of collection of the urine sample.

9. 10. The assay kit of claim 1, wherein the subject's urinary albumin / creatinine ratio (UACR) is indicated to be less than about 30 milligrams per gram (mg / g) for the 12 months prior to collection of the urine sample or both before and after the time of collection of the urine sample.

10. 10. The assay kit of claim 1, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus about 7 to about 20 years prior to collection of the urine sample.

11. 2. The assay kit of claim 1, wherein the ratio greater than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg.

12. 2. The assay kit of claim 1, wherein the ratio greater than about 7.5 ng / mg is greater than about 29 ng / mg.

13. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 10% or greater.

14. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 15% or greater.

15. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 20% or greater.

16. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 25% or greater.

17. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 30% or greater.

18. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 35% or greater.

19. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 40% or greater.

20. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 45% or greater.

21. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 50% or greater.

22. 10. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is between about 10% and about 50%.

23. 2. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 30% to about 40%.

24. 10. The assay kit of claim 1, wherein the higher likelihood of progressive renal decline leading to the renal endpoint is about 40% to about 50%.

25. 24. The assay kit of claims 1-23, wherein the progressive decline in renal function is expected to lead to the renal endpoint after four years.

26. 2. The assay kit of claim 1, wherein the progressive decline in renal function is expected to lead to the renal endpoint after five years.

27. 2. The assay kit of claim 1, wherein said interaction with said fetuin A fragment comprises binding to said fetuin A fragment.

28. 2. The assay kit of claim 1, wherein said interaction with said urinary creatinine comprises binding to said urinary creatinine.

29. The assay kit of claim 1 , wherein at least one of the first reagent and the second reagent comprises an antibody.

Images