Assay and related methods, kits and devices

Abstract

The present disclosure is related to correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a progressive renal decline to a kidney endpoint over a period of time in the subject.

Description

Attorney Docket No.132417-0001WO01 ASSAY AND RELATED METHODS, KITS AND DEVICES CROSS REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U.S. provisional patent application App. No. 63 / 441,745, filed on January 27, 2023, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," and U.S. provisional patent application App. No.63 / 532,636, filed on August 14, 2023, entitled "ASSAY AND RELATED METHODS, KITS AND DEVICES," each of which is incorporated herein by this reference in its entirety. BACKGROUND

[0002] Diabetic nephropathy (DN), also known as Diabetic Kidney Disease (DKD), is a progressive kidney disease associated with longstanding diabetes mellitus. Over time, poorly controlled diabetes can cause damage to blood vessel clusters in your kidneys that filter waste from your blood. This can lead to kidney damage. It causes abnormal fluid filtration and increased urinary albumin excretion, eventually leading to kidney failure. SUMMARY

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

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

[0005] In some embodiments, the present disclosure relates to a method of preparing an assay, including: providing a first solution including a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, 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 indicates a higher likelihood of the progressive renal decline to the kidney endpoint, and wherein the first solution and the second solution are the same solution or different solution from each other. Page: 1 of 115Attorney Docket No.132417-0001WO01

[0006] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg, more preferably higher than about 7.53ng / 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 a medical intervention to obviate the progressive renal decline.

[0007] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg.

[0008] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg.

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

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

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

[0012] In some embodiments, the present disclosure relates to a method, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients.

[0013] In some embodiments, the present disclosure relates to a method, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer.

[0014] In some embodiments, the present disclosure relates to a method, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about Page: 2 of 115Attorney Docket No.132417-0001WO01 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7.

[0015] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint.

[0016] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint.

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

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

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

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

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

[0022] In some embodiments, the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

[0023] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0024] In some embodiments, the present disclosure relates to a method, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0025] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0026] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample. Page: 3 of 115Attorney Docket No.132417-0001WO01

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

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

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

[0030] In some embodiments, the present disclosure relates to a method, wherein the second ratio being 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 being 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 being 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 being 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 being 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 being lower than about 7.5 ng / mg is lower than about 1 ng / mg. In some embodiments, the present disclosure relates to Page: 4 of 115Attorney Docket No.132417-0001WO01 a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

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

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

[0033] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%. In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%. In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%. In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%. In some embodiments, the present disclosure relates to a Page: 5 of 115Attorney Docket No.132417-0001WO01 method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%.

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

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

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

[0037] In some embodiments, the present disclosure relates to a method, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. In some embodiments, the present disclosure relates to a method, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. In some embodiments, the present disclosure relates to a method, wherein at least one reagent among the first reagent and the second reagent includes an antibody. In some embodiments, the present disclosure relates to an assay kit to determine a likelihood of a progressive renal decline, including: a first solution including a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA) to indicate the level of the Fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, 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, indicates a higher likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, and wherein the first solution and the second solution are the same solution or different solution from each other. Page: 6 of 115Attorney Docket No.132417-0001WO01

[0038] In some embodiments, the present disclosure relates to an assay kit, 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 a medical intervention to obviate the progressive renal decline.

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

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

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

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

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

[0044] In some embodiments, the present disclosure relates to an assay kit, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients.

[0045] In some embodiments, the present disclosure relates to an assay kit, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer.

[0046] In some embodiments, the present disclosure relates to an assay kit, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about Page: 7 of 115Attorney Docket No.132417-0001WO01 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7.

[0047] In some embodiments, the present disclosure relates to an assay kit, wherein, the method further includes, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint.

[0048] In some embodiments, the present disclosure relates to an assay kit, wherein, the method further includes, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint.

[0049] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

[0050] In some embodiments, the present disclosure relates to an assay kit, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0051] In some embodiments, the present disclosure relates to an assay kit, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0052] In some embodiments, the present disclosure relates to an assay kit, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0053] In some embodiments, the present disclosure relates to an assay kit, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

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

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

[0056] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than Page: 8 of 115Attorney Docket No.132417-0001WO01 about 14 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg.

[0057] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 29 ng / mg.

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

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

[0060] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being 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 being 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 being 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 being 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 being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg. In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg.

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

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

[0064] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher. Page: 9 of 115Attorney Docket No.132417-0001WO01

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0083] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%. Page: 10 of 115Attorney Docket No.132417-0001WO01

[0084] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 4 years.

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

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

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

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

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

[0090] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint within 7 years.

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

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

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

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

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

[0096] In some embodiments, the present disclosure relates to a method of correlating a likelihood of a progressive renal decline, including: determining the level 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 fragment of a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), wherein the second biomarker is a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, 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, indicates a higher likelihood of the progressive renal decline to the kidney endpoint.

[0097] In some embodiments, the present disclosure relates to a method, 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 / , triggers a medical intervention to obviate the progressive renal decline. Page: 11 of 115Attorney Docket No.132417-0001WO01

[0098] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg.

[0099] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg.

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

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

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

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

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

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

[0106] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint. Page: 12 of 115Attorney Docket No.132417-0001WO01

[0107] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint.

[0108] In some embodiments, the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

[0109] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0110] In some embodiments, the present disclosure relates to a method, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0111] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0112] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

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

[0114] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg. In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg. In some embodiments, Page: 13 of 115Attorney Docket No.132417-0001WO01 the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 29 ng / mg.

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

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

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

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

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

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

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

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

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

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

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

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

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

[0128] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher. Page: 14 of 115Attorney Docket No.132417-0001WO01

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0145] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 4 years.

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

[0147] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 6 years. Page: 15 of 115Attorney Docket No.132417-0001WO01

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

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

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

[0151] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 7 years.

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

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

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

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

[0156] In some embodiments, the present disclosure relates to a method, wherein at least one reagent among the first reagent and the second reagent includes an antibody. BRIEF DESCRIPTION OF THE FIGURES

[0157] Figures 1A-1B illustrate Kaplan-Meier curves with end point of 30% decline or <15 in eGFR, where Figure 1A is directed to Non-CKD NTUH patients with classified as high-risk and low-risk by IVD103, and where Figure 1B is directed to Patients in DIALECT study classified as high-risk and low-risk by IVD103 according to a non-limiting embodiment.

[0158] Figure 2 illustrates a calibration curve of the Human uPTM3-DKD ELISA (calibration range is 7.813 - 500 ng / mL) according to a non-limiting embodiment.

[0159] Figure 3 illustrates a Kaplan-Meier curve for 294 subjects in the NTUH cohort in Examples 5-7 according to a non-limiting embodiment.

[0160] Figure 4A illustrates Kaplan-Meier curves for subjects in the NTUH cohort with UCr<30 mg / g by uPTM-FetA / UCr risk category in Examples 5-7 according to a non-limiting embodiment.

[0161] Figure 4B illustrates Kaplan-Meier curves for subjects in the NTUH cohort with UCr≥30 mg / g by uPTM-FetA / UCr risk category in Examples 5-7 according to a non-limiting embodiment.

[0162] Figure 5A illustrates a Kaplan-Meier curve for NTUH cohort indicating increase in performance of predicting kidney function deterioration by adding uPTM-FetA / UCr in prediction model according to a non-limiting embodiment. Page: 16 of 115Attorney Docket No.132417-0001WO01

[0163] Figure 5B illustrates a Kaplan-Meier curve for the DIALECT cohort indicating increase in performance of predicting kidney function deterioration by adding uPTM-FetA / UCr in prediction model according to a non-limiting embodiment.

[0164] Figure 6 illustrates a ROC curve indicating increase in performance of predicting kidney function deterioration by adding uPTM-FetA / UCr in prediction model according to a non- limiting embodiment.

[0165] Figure 7 illustrates a Kaplan-Meier curve indicating survival probability over time in years, regarding the cut-off 1 value of uPTM-FetA / UCr, according to a non-limiting embodiment.

[0166] Figure 8 illustrates a Kaplan-Meier curve indicating event-free probability over time in years, regarding the cut-off 2 value of uPTM-FetA / UCr, according to a non-limiting embodiment.

[0167] Figure 9 illustrates a Kaplan-Meier curve indicating progressive-free probability over time in years, regarding the two cut off values of occurrence scores, according to a non-limiting embodiment.

[0168] Figure 10 illustrates a cumulative incidence plot over occurrence score, according to a non-limiting embodiment.

[0169] Figure 11 illustrates a Kaplan-Meier curve for the DIALECT cohort indicating progressive-free probability over time in years, regarding the two cut off values of occurrence scores, according to a non-limiting embodiment.

[0170] Figure 12 illustrates a cumulative incidence plot over occurrence score for the DIALECT cohort, according to a non-limiting embodiment. DETAILED DESCRIPTION OF THE DISCLOSURE

[0171] The present invention is based on unexpected discoveries that urine and serum protein and their fragments, either alone or in combination, are differentially presented in diabetic kidney disease (DKD) patients as compared to DKD-free subjects. These protein molecules are therefore useful markers for diagnosing early stage DKD. It is of great importance to identify reliable biomarkers useful in diagnosing early stage DKD. DKD or DN is a kidney disorder associated with diabetes.

[0172] 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 develops in up to 40% of patients with T2DM, and its progression may be slowed or prevented through intervention. Therefore, timely identification of patients at high risk of kidney function decline is of utmost importance.

[0173] In some embodiments, DKD can have five progression phases:

[0174] Stage 1: characterized by diabetic mellitus with normal GFR and normal albuminuria (e.g., ACR<30 mg / g);

[0175] Stage 2: characterized by glomerular hyperfiltration (greater than 120 mL / minute / 1.73 m2) and renal enlargement accompanying with normal GFR and normal albuminuria (e.g., ACR<30 mg / g); Page: 17 of 115Attorney Docket No.132417-0001WO01

[0176] Stage 3: characterized by microalbuminuria;

[0177] Stage 4: characterized by overt albuminuria and a progressive decline in GFR; and

[0178] Stage 5: characterized by a GFR of less than 15 mL / minute / 1.73 m2. Commonly, stages 1-3 are deemed as early stage and stages 4 and 5 are deemed as late stage.

[0179] In other words, DKD may display no symptoms in its early course. As such, it may be difficult to detect the incipiency of this disease. In fact, present diagnosis of DKD depends on development of microalbuminuria, which occurs when kidney damage is already in place. The lack of an early diagnostic test prevents effective treatment of early stage DKD.

[0180] The marker that is generally used in current clinical practice to assess risk of progressing to CKD, is the creatinine-based estimated glomerular filtration rate (eGFR). Unfortunately, eGFR is burdened by confounding by muscle mass, its intra- individual trajectories are highly variable, and eGFR may not reliably predict progression of kidney disease, particularly in the early stages of DKD. Albuminuria is another commonly used biomarker to assess risk of DKD. However, up to 40% of patients with T2DM who suffer from renal function worsening remain normoalbuminuric. Thus, due to the low sensitivity of albuminuria, its use in predicting DKD progression is limited.

[0181] Consequently, eGFR and albuminuria are considered insufficient to predict an individual's kidney function decline. Moreover, these measures may not adequately be used to assess response to renoprotective treatments in DKD. An explanation for the limited ability of these and other existing markers of kidney function decline, is that they are "late" markers that show kidney injury / dysfunction when the damage has already occurred. Therefore, there is a need for biomarkers closely representing underlying molecular mechanisms involved in disease progression, that can ideally be targeted by therapies. However, at this point, there is a lack of such biomarkers with proven additional predictive value of DKD.

[0182] As used herein, the term "kidney end point" is a point of deterioration in the kidney function that needs a renal replacement therapy, such as kidney transplantation, initiation of peritoneal dialysis or hemodialysis. The deterioration in the kidney function can be indicated by, for example, the estimated glomerular filtration rate (eGFR), such as eGFR calculated with the Chronic Kidney Disease Epidemiology Collaboration equation. For example, reaching an eGFR<15 mL / min / 1.73 m2is considered as an indication of the kidney end point.

[0183] As used herein, the term "medical intervention" for kidney function decline means a treatment of the recipient in attempt to stabilize, slow down, delay, or suppress the deterioration, which results in a decrease in the rate of the deterioration in the recipient. Various types of medical interventions can be implemented. Examples of medical intervention includes guided diets, restrictive diets, limiting sodium and other ion intake, reducing or avoiding alcohol consumption, reducing or avoiding smoking cigarette or consuming related products, intaking of various medications, managing weight, kidney transplant or retransplant, dialysis, other medically recognizable treatments, and any combination thereof. Page: 18 of 115Attorney Docket No.132417-0001WO01

[0184] Accordingly, the present disclosure is related to a diagnostic method using a biomarker, which can be a protein or a protein that can be considered as a fragment of the protein. A urine sample, a serum sample, or both the urine and serum samples, can be collected from a subject and the urine level, serum level or both levels of the biomarker can be determined via various methods, e.g., mass spectrometry and immune analysis.

[0185] When a biomarker contains a single protein molecule, its level in a subject can be compared with a reference point to determine a corresponding indication or likelihood of a target condition. The reference point, such as a single reference point or a plurality of reference points, which represents the level of the same biomarker such as a level of a biomarker in a DKD-free subject, can be determined based on the representative levels of the biomarker in groups of DKD patients and DKD-free subjects. For example, it can be the middle point between the mean levels of the two groups of DKD patients and DKD-free subjects. For example, it can be a plurality of points, mean points or arbirary points among a plurality of groups of DKD patients (e.g., groups of patients respectively in different progress stages of DKD) and a group of DKD-free subjects. In some embodiments, a biomarker level higher than the reference point is indicative of the target condition, such as DKD. In some embodiments, biomarker levels among the plurality of points are indicative of different stages or progresses of the target condition, such as DKD.

[0186] There is a significant clinical need for novel markers to predict a kidney function decline in patients with type 2 diabetes mellitus (T2DM).

[0187] A post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), which can include a urinary connecting peptide-containing Fetuin A fragments (uC-FetA), can be a biomarker to indicate indicates a likelihood of the progressive renal decline to the kidney endpoint and can predict a kidney function decline in patients with T2DM.

[0188] Fetuin-A, encoded by AHSG gene, is a hepatokine associated with insulin resistance and is an independent risk factor of T2DM. As a hepatic secretory protein, Fetuin-A has been known to bind to the insulin receptor in muscle and fat and inhibits insulin action In an insulin-resistant state, there is a shift of insulin signaling from the PI3K axis to the MAPK axis in endothelial cells of renal arterioles, causing renal vasoconstriction. Fetuin-A also stimulates proinflammatory cytokine secretion by perivascular fat tissue and may thus cause impairment of renal function by affecting renal sinus fat which is located around renal arteries. Besides the renal vasculature, insulin resistance has a direct impact on podocyte viability and tubular function.

[0189] Fetuin-A such as post-translationally modified Fetuin-A fragments in urine (uPTM- FetA) (also known as alpha-2- Heremans–Schmid glycoprotein) or a Fetuin-A-based fragment has or can have characteristics that suggest that it could serve as such an alternative, independent non- invasive marker. Fetuin-A is elevated in the urine of patients with acute kidney injury (AKI), and recent evidence suggests that it is also linked to (degree of) interstitial fibrosis / tubular atrophy (IFTA). Urinary Fetuin-A also elevated in patients with chronic kidney diseases such as autosomal dominant polycystic kidney disease and focal segmental glomerular sclerosis. Next to that, urinary Fetuin-A has Page: 19 of 115Attorney Docket No.132417-0001WO01 been shown previously to be associated with kidney function decline in patients with chronic kidney disease including diabetic nephropathy, and this protein has been proposed to be used as a biomarker for early detection in these diseases.

[0190] As such, Fetuin-A or its fragment may be a marker that is causally involved and may have an added predictive value in the development and progression of DKD. This was supported by the results of large-scale urine proteomic profiling of among participants from the Taiwan Renal Biomarker Study, in which urinary concentrations of a post-translationally modified connecting peptide-containing Fetuin- A fragments (uPTM-FetA) was identified as a candidate marker of DKD in T2DM patients. This study supports the identified marker uPTM-FetA as an early marker of kidney function deterioration among two large, independent, bi- ethnic, prospective cohort studies in patients with T2DM. The present disclosure supports that uPTM-FetA is independently associated with kidney function decline in patients with type 2 diabetes, which highlights the potential clinical use of uPTM-FetA as a prognostic marker for kidney disease in patients with T2DM.

[0191] In some embodiments, the present disclosure relates to a method of preparing an assay, including: providing a first solution including a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, 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 indicates a higher likelihood of the progressive renal decline to the kidney endpoint, and wherein the first solution and the second solution are the same solution or different solution from each other.

[0192] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg, more preferably higher than about 7.53ng / 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 a medical intervention to obviate the progressive renal decline.

[0193] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg.

[0194] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years Page: 20 of 115Attorney Docket No.132417-0001WO01 in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg.

[0195] In some embodiments, the present disclosure relates to a method, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure.

[0196] In some embodiments, the present disclosure relates to a method, further including assessing a probability of an occurrence of the progressive renal decline to the kidney endpoint, based on associating the ratio with at least one among age, gender, eGFR, UACR, and HbA1c level.

[0197] In some embodiments, the present disclosure relates to a method, wherein at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level is adjusted based on at least one respective coefficient respectively corresponding to at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level.

[0198] In some embodiments, the present disclosure relates to a method, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients.

[0199] In some embodiments, the present disclosure relates to a method, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer.

[0200] In some embodiments, the present disclosure relates to a method, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7.

[0201] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint.

[0202] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint. Page: 21 of 115Attorney Docket No.132417-0001WO01

[0203] In some embodiments, the present disclosure relates to a method, wherein the first threshold value is about 0.1.

[0204] In some embodiments, the present disclosure relates to a method, wherein the first threshold value is about 0.11.

[0205] In some embodiments, the present disclosure relates to a method, wherein the second threshold value is about 0.25.

[0206] In some embodiments, the present disclosure relates to a method, wherein the second threshold value is about 0.3.

[0207] In some embodiments, the present disclosure relates to a method, wherein the second threshold value is about 0.27.

[0208] In some embodiments, the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

[0209] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0210] In some embodiments, the present disclosure relates to a method, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0211] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0212] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0213] In some embodiments, the present disclosure relates to a method, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample.

[0214] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg.

[0215] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg.

[0216] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg.

[0217] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg. Page: 22 of 115Attorney Docket No.132417-0001WO01

[0218] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg.

[0219] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg.

[0220] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg.

[0221] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg.

[0222] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 25 ng / mg.

[0223] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 30 ng / mg.

[0224] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 40 ng / mg.

[0225] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 45 ng / mg.

[0226] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 47 ng / mg.

[0227] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 48 ng / mg.

[0228] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 49 ng / mg.

[0229] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 50 ng / mg.

[0230] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 6 ng / mg.

[0231] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4.5 ng / mg.

[0232] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4 ng / mg.

[0233] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.5 ng / mg.

[0234] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg.

[0235] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg.

[0236] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg. Page: 23 of 115Attorney Docket No.132417-0001WO01

[0237] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg.

[0238] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

[0239] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher.

[0240] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher.

[0241] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher.

[0242] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher.

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

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

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

[0246] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher.

[0247] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher.

[0248] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%.

[0249] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 45%.

[0250] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 40%.

[0251] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 35%.

[0252] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%.

[0253] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%.

[0254] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%.

[0255] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%. Page: 24 of 115Attorney Docket No.132417-0001WO01

[0256] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%.

[0257] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % to about 45%.

[0258] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%.

[0259] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 4 years.

[0260] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 5 years.

[0261] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 6 years.

[0262] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 7 years.

[0263] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 8 years.

[0264] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 9 years.

[0265] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 7 years.

[0266] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 8 years.

[0267] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 9 years.

[0268] In some embodiments, the present disclosure relates to a method, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment.

[0269] In some embodiments, the present disclosure relates to a method, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine.

[0270] In some embodiments, the present disclosure relates to a method, wherein at least one reagent among the first reagent and the second reagent includes an antibody.

[0271] In some embodiments, the present disclosure relates to an assay kit to determine a likelihood of a progressive renal decline, including: a first solution including a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA) to indicate the level of the Fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being Page: 25 of 115Attorney Docket No.132417-0001WO01 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, indicates a higher likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, and wherein the first solution and the second solution are the same solution or different solution from each other.

[0272] In some embodiments, the present disclosure relates to an assay kit, 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 a medical intervention to obviate the progressive renal decline.

[0273] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg.

[0274] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg.

[0275] In some embodiments, the present disclosure relates to an assay kit, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure.

[0276] In some embodiments, the present disclosure relates to an assay kit, further including assessing a probability of an occurrence of the progressive renal decline to the kidney endpoint, based on associating the ratio with at least one among age, gender, eGFR, UACR, and HbA1c level.

[0277] In some embodiments, the present disclosure relates to an assay kit, wherein at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level is adjusted based on at least one respective coefficient respectively corresponding to at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level.

[0278] In some embodiments, the present disclosure relates to an assay kit, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients.

[0279] In some embodiments, the present disclosure relates to an assay kit, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint Page: 26 of 115Attorney Docket No.132417-0001WO01 is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer.

[0280] In some embodiments, the present disclosure relates to an assay kit, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7.

[0281] In some embodiments, the present disclosure relates to an assay kit, wherein, the method further includes, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint.

[0282] In some embodiments, the present disclosure relates to an assay kit, wherein, the method further includes, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint.

[0283] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

[0284] In some embodiments, the present disclosure relates to an assay kit, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0285] In some embodiments, the present disclosure relates to an assay kit, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0286] In some embodiments, the present disclosure relates to an assay kit, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0287] In some embodiments, the present disclosure relates to an assay kit, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0288] In some embodiments, the present disclosure relates to an assay kit, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample.

[0289] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg.

[0290] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg. Page: 27 of 115Attorney Docket No.132417-0001WO01

[0291] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg.

[0292] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg.

[0293] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg.

[0294] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg.

[0295] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg.

[0296] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg.

[0297] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 25 ng / mg.

[0298] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 30 ng / mg.

[0299] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 40 ng / mg.

[0300] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 45 ng / mg.

[0301] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 47 ng / mg.

[0302] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 48 ng / mg.

[0303] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 49 ng / mg.

[0304] In some embodiments, the present disclosure relates to an assay kit, wherein the ratio being higher than about 7.5 ng / mg is higher than about 50 ng / mg.

[0305] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 6 ng / mg.

[0306] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 5 ng / mg.

[0307] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4 ng / mg.

[0308] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.5 ng / mg.

[0309] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg. Page: 28 of 115Attorney Docket No.132417-0001WO01

[0310] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg.

[0311] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg.

[0312] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg.

[0313] In some embodiments, the present disclosure relates to an assay kit, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

[0314] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher.

[0315] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher.

[0316] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher.

[0317] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher.

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

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

[0320] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % or higher.

[0321] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher.

[0322] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher.

[0323] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%.

[0324] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 45%.

[0325] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 40%.

[0326] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 35%.

[0327] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%.

[0328] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%. Page: 29 of 115Attorney Docket No.132417-0001WO01

[0329] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%.

[0330] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%.

[0331] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%.

[0332] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % to about 45%.

[0333] In some embodiments, the present disclosure relates to an assay kit, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%.

[0334] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 4 years.

[0335] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 5 years.

[0336] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 6 years.

[0337] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 7 years.

[0338] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 8 years.

[0339] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 9 years.

[0340] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint within 7 years.

[0341] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint within 8 years.

[0342] In some embodiments, the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint within 9 years.

[0343] In some embodiments, the present disclosure relates to an assay kit, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment.

[0344] In some embodiments, the present disclosure relates to an assay kit, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine.

[0345] In some embodiments, the present disclosure relates to an assay kit, wherein at least one reagent among the first reagent and the second reagent includes an antibody.

[0346] In some embodiments, the present disclosure relates to a method of correlating a likelihood of a progressive renal decline, including: determining the level 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 fragment of a post-translationally modified Fetuin-A fragments in urine Page: 30 of 115Attorney Docket No.132417-0001WO01 (uPTM-FetA), wherein the second biomarker is a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, 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, indicates a higher likelihood of the progressive renal decline to the kidney endpoint.

[0347] In some embodiments, the present disclosure relates to a method, 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 / , triggers a medical intervention to obviate the progressive renal decline.

[0348] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg.

[0349] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg.

[0350] In some embodiments, the present disclosure relates to a method, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure.

[0351] In some embodiments, the present disclosure relates to a method, further including assessing a probability of an occurrence of the progressive renal decline to the kidney endpoint, based on associating the ratio with at least one among age, gender, eGFR, UACR, and HbA1c level.

[0352] In some embodiments, the present disclosure relates to a method, wherein at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level is adjusted based on at least one respective coefficient respectively corresponding to at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level.

[0353] In some embodiments, the present disclosure relates to a method, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients. Page: 31 of 115Attorney Docket No.132417-0001WO01

[0354] In some embodiments, the present disclosure relates to a method, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer.

[0355] In some embodiments, the present disclosure relates to a method, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7.

[0356] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint.

[0357] In some embodiments, the present disclosure relates to a method, wherein, the method further includes, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint.

[0358] In some embodiments, the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

[0359] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0360] In some embodiments, the present disclosure relates to a method, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0361] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0362] In some embodiments, the present disclosure relates to a method, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0363] In some embodiments, the present disclosure relates to a method, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample.

[0364] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg. Page: 32 of 115Attorney Docket No.132417-0001WO01

[0365] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg.

[0366] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg.

[0367] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg.

[0368] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg.

[0369] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg.

[0370] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg.

[0371] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg.

[0372] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 25 ng / mg.

[0373] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 30 ng / mg.

[0374] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 40 ng / mg.

[0375] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 45 ng / mg.

[0376] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 47 ng / mg.

[0377] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 48 ng / mg.

[0378] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 49 ng / mg.

[0379] In some embodiments, the present disclosure relates to a method, wherein the ratio being higher than about 7.5 ng / mg is higher than about 50 ng / mg.

[0380] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 6 ng / mg.

[0381] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4.5 ng / mg.

[0382] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4 ng / mg.

[0383] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.5 ng / mg. Page: 33 of 115Attorney Docket No.132417-0001WO01

[0384] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg.

[0385] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg.

[0386] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg.

[0387] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg.

[0388] In some embodiments, the present disclosure relates to a method, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg.

[0389] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher.

[0390] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher.

[0391] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher.

[0392] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher.

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

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

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

[0396] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher.

[0397] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher.

[0398] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%.

[0399] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 45%.

[0400] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 40%.

[0401] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 35%.

[0402] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%. Page: 34 of 115Attorney Docket No.132417-0001WO01

[0403] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%.

[0404] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%.

[0405] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%.

[0406] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%.

[0407] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % to about 45%.

[0408] In some embodiments, the present disclosure relates to a method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%.

[0409] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 4 years.

[0410] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 5 years.

[0411] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 6 years.

[0412] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 7 years.

[0413] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 8 years.

[0414] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 9 years.

[0415] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 7 years.

[0416] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 8 years.

[0417] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 9 years.

[0418] In some embodiments, the present disclosure relates to a method, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment.

[0419] In some embodiments, the present disclosure relates to a method, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine.

[0420] In some embodiments, the present disclosure relates to a method, wherein at least one reagent among the first reagent and the second reagent includes an antibody.

[0421] In some embodiments, a method of preparing an assay may comprise providing a first solution including a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first Page: 35 of 115Attorney Docket No.132417-0001WO01 reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, wherein the ratio being higher than about 3.3 ng / mg, more preferably higher than about 3.5 ng / mg, more preferably higher than about 4 ng / mg, more preferably higher than about 5 ng / mg, more preferably higher than about 6 ng / mg, more preferably higher than about 7 ng / mg, more preferably higher than about 7.5 ng / mg, indicates a higher likelihood of the progressive renal decline to the kidney endpoint, and wherein the first solution and the second solution are the same solution or different solution from each other.

[0422] In some embodiments, an assay kit to determine a likelihood of a progressive renal decline may comprise a first solution including a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA) to indicate the level of the Fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the determined level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being higher than about 3.3 ng / mg, more preferably higher than about 3.5 ng / mg, more preferably higher than about 4 ng / mg, more preferably higher than about 5 ng / mg, more preferably higher than about 6 ng / mg, more preferably higher than about 6.5 ng / mg, more preferably higher than about 7 ng / mg, more preferably higher than about 7.2 ng / mg, more preferably higher than about 7.3 ng / mg, more preferably higher than about 7.4 ng / mg, more preferably higher than about 7.45 ng / mg.

[0423] In some embodiments, the ratio can be higher than about 7.5 ng / mg, more preferably higher than about 7.51 ng / mg, more preferably higher than about 7.52 ng / mg, more preferably higher 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 a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, and wherein the first solution and the second solution are the same solution or different solution from each other.

[0424] In some embodiments, a method of correlating a likelihood of a progressive renal decline may comprise determining the level 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 may be a Page: 36 of 115Attorney Docket No.132417-0001WO01 fragment of a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), wherein the second biomarker may be a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, wherein the ratio being higher than about 7.5 ng / mg, more preferably higher than about 7.51 ng / mg, more preferably higher than about 7.52 ng / mg, more preferably higher 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 the progressive renal decline to the kidney endpoint.

[0425] In some embodiments, the ratio being higher than about 7.5 may trigger a medical intervention to obviate the progressive renal decline.

[0426] In some embodiments, the higher likelihood may be higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than lower than about 7.53, lower than about 7.5 ng / mg, lower than about 7 ng / mg, 6 ng / mg, lower than about 5.95 ng / mg, lower than about 5.5 ng / mg, lower than about 5 ng / mg, lower than about 4.5 ng / mg, lower than about 4.4 ng / mg, lower than about 4.38 ng / mg, lower than about 4.3 ng / mg, lower than about 4.2 ng / mg, lower than about 4.1 ng / mg, lower than about 4.0 ng / mg, lower than about 3.95 ng / mg, lower than about 3.9 ng / mg, lower than about 3.5 ng / mg, lower than about 3.4 ng / mg, lower than about 3.3 ng / mg, lower than about 3 ng / mg, lower than about 2.5 ng / mg, lower than about 2 ng / mg, lower than about 1 ng / mg, lower than about 0.5 ng / mg, or lower than about 0.1 ng / mg.

[0427] In some embodiments, the progressive renal decline may be a decline in estimated Glomerular Filtration Rate (eGFR).

[0428] In some embodiments, the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

[0429] In some embodiments, the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2).

[0430] In some embodiments, the subject has been indicated to have a Urinary Albumin-to- creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0431] In some embodiments, the subject has been indicated to have a Urinary Albumin-to- creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

[0432] In some embodiments, the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample. Page: 37 of 115Attorney Docket No.132417-0001WO01

[0433] In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7 ng / mg. In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7.1 ng / mg. In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7.2 ng / mg. In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7.3 ng / mg. In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7.4 ng / mg. In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7.45 ng / mg. In some embodiments, the ratio being higher than about 6 ng / mg may be higher than about 7.5 ng / mg.

[0434] In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 7.51 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 7.52 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 7.53 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 7.55 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 7.6 ng / mg.

[0435] In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 9 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 10 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 12 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 14 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 15 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 20 ng / mg. In some embodiments, the ratio being higher than about 7.5 ng / mg may be higher than about 24 ng / mg.

[0436] In some embodiments, the higher likelihood may be higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg, such as 7.53 ng.mg.

[0437] In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 7 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be 6 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 5.95 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 5.5 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 5 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 4.5 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 4.4 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 4.38 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 4.3 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about Page: 38 of 115Attorney Docket No.132417-0001WO01 4.2 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 4.1 ng / mg.

[0438] In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 4.0 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 3.95 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 3.9 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 3.5 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 3.4 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 3.3 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 3 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 2.5 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 2 ng / mg. In some embodiments, the second ratio being lower than about 7.5 ng / mg may be lower than about 1 ng / mg, lower than about 0.5 ng / mg, or lower than about 0.1 ng / mg.

[0439] In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 5 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 10 % or higher.

[0440] In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 11 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 12 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 13 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 15 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 16 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 18 % or higher.

[0441] In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 20 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 22 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 24 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 25 % or higher.

[0442] In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 30 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 33 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 35 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 38 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 39 % or higher. In some embodiments, the higher Page: 39 of 115Attorney Docket No.132417-0001WO01 likelihood of the progressive renal decline to the kidney endpoint may be about 40 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 45 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 46 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 48 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 50 % or higher. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 10 % to about 50%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 10 % to about 40%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 11 % to about 39%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 5 % to about 20%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 6 % to about 16%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 10 % to about 25%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 12 % to about 24%.In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 15 % to about 45%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 20 % to about 40%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 25 % to about 35%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 25 % to about 40%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 25 % to about 30%.

[0443] In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 10 % to about 20%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 15 % to about 25%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 20 % to about 30%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 30 % to about 50%.nIn some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 30 % to about 46%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 35 % to about 45%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 30 % to about 40%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 30 % to about 45%. In some embodiments, the higher likelihood of the progressive renal decline to the kidney endpoint may be about 40 % to about 50%. Page: 40 of 115Attorney Docket No.132417-0001WO01

[0444] In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 1 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 2 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 3 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 4 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 5 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 6 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 7 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 8 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint after about 9 years.

[0445] In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 1 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 2 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 3 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 4 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 5 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 6 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 7 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 8 years. In some embodiments, the progressive renal may be to decline to the kidney endpoint within about 9 years.

[0446] In some embodiments, the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. In some embodiments, the interacting with the urinary creatinine includes binding to the urinary creatinine. In some embodiments, at least one reagent among the first reagent and the second reagent includes an antibody.

[0447] As supported in the present disclosure, uPTM-FetA can be associated with risk of eGFR decline in patients with type 2 diabetes, which can be independent or substantially less dependent of age, sex, eGFR, UACR, baseline HbA1c, multiple other potential confounders, or any combination thereof. These findings were validated in a second, larger cohort of patients with type 2 diabetes, highlighting the potential clinical use of uPTM-Fet-A as a predictive marker for DKD. The human precursor protein of fetuin A comprises three parts, namely the A chain, connecting peptide, and B chain, which are 321, 40, and 27 amino acids in length, respectively. The connecting peptide of the precursor is removed by a posttranslational modification, limited proteolysis, after which only the A and B chain form the active fetuin A protein. The monoclonal antibody in the ELISA kit that can be used (e.g., Human uPTM3-DKD ELISA kit, BPM Corp.) to detect the connecting peptide-containing fetuin A. The present disclosure supports that elevated uPTM-FetA is associated with progression of renal function decline in type 2 diabetes patients. In some embodiments, the concentration of uPTM- FetA may not be substantially related to serum C-FetA among patients (in-house data from NTUH cohort, data not shown). In some embodiments, the incongruence between circulating and urine fetuin Page: 41 of 115Attorney Docket No.132417-0001WO01 A concentrations can be observed. Seemingly, uPTM-FetA specifically accumulates in urine of patients with progressive renal decline. This is likely attributable to impaired tubular reabsorption of the molecule, rather than increased filtration or secretion. uPTM-FetA can be a predictive marker of progressive kidney disease. uPTM-FetA can have direct pathogenic effects.

[0448] In non-CKD type 2 diabetes patients, uPTM-FetA was to UACR multivariable- adjusted Cox regression analyses. This suggests that uPTM-FetA may be better suitable for predicting renal function decline than UACR. Moreover, the use of antihypertensives such as angiotensin receptor blockers and angiotensin converting enzyme inhibitors, which are frequently prescribed in type 2 diabetes patients, reduces albuminuria, thus limited usefulness of UACR as a predictive marker of DKD. Potentially, uPTM-FetA may not have this disadvantage.

[0449] There were studies establishing prediction model DKD for type 2 diabetes patients using clinical parameters with or without aid of machine learning, but the follow-up duration was rather short. Evaluation of eGFR within only a period of 5 years is considered insufficient for assessing renal function deterioration in type 2 diabetes patients, as intra‑individual variability of eGFR is high among early DKD patients. Long-term follow up of renal function in type 2 diabetes patients is essential, given the fact that whole- kidney hyperfiltration with increased eGFR is seen in substantial portion of this population and may mask the development of kidney disease in early phase. There are several strengths of this study. The current study include use of two large, well- characterized cohorts allowed for extensive adjustments for potential confounders. Follow-up duration in NTUH cohort is long, which allows adequate assessment of associations of uPTM-FetA with renal outcome not underestimated by the issue of renal hyperfiltration in early DKD. Finally, the use of two independent, bi- ethnic cohorts including patients from different geographical areas, is reassuring with regard to the external validity of these findings to other type 2 diabetes populations worldwide.

[0450] Accordingly, it has been demonstrated in the present disclosure that in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes, uPTM-FetA is strongly associated with renal function deterioration, independent of albuminuria, eGFR, age, sex, and multiple other risk factors. The added prognostic value of uPTM-FetA on top of albuminuria and other markers is particularly clear in patients without CKD at baseline. These findings are validated in a large, independent, second cohort, which is promising with regard to the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0451] ASSAY

[0452] In some embodiments, various methods, reagents, devices, and kits can be used to measure the level of Fetuin-A or its fragment in various mediums, such as in a urine, serum, and other bodily fluids.

[0453] In some embodiments, the enzyme-linked immunosorbent assay (ELISA) can be used to measure the level of Fetuin-A. For example, the ELISA can be used for quantitative measurement of Fetuin-A with specific post translational modification (PTM) in human urine and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Page: 42 of 115Attorney Docket No.132417-0001WO01 Technologists. For example, calibrators or unknown urine samples are mixed with an antibody having a binding affinity to a part of Fetuin-A, such as anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate pre-bounded with Fetuin-A or its segment, such as unique PTM Fetuin-A. The monoclonal antibody recognizes Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, in some embodiments, signal-generating molecule such as signal-molecule-tagged antibody can be used. In some embodiments, a Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3',5,5'- tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve. NON-LIMITING EMBODIMENTS

[0454] The present disclosure is also described by way of the following non-limiting embodiments. However, the use of these and other embodiments anywhere in the specification is illustrative only and in no way limits the scope and meaning of the disclosure. Likewise, the disclosure is not limited to any particular preferred embodiment or aspect described herein. Indeed, modifications and variations may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the disclosure in spirit or in scope. 1. A method of preparing an assay, comprising: providing a first solution including a urine sample from a subject diagnosed with type 2 diabetes mellitus and a first reagent to interact with a post-translationally modified Fetuin-A fragments in urine (uPTM-FetA), to determine the level of the Fetuin A fragment in the urine sample; providing a second solution including the urine sample and a second reagent to interact with a urinary creatinine, to determine the level of the urinary creatinine in the urine sample; in response to the determined level of the Fetuin A fragment and the determined level of the urinary creatinine, correlating a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, 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 indicates a higher likelihood of the progressive renal decline to the kidney endpoint, and wherein the first solution and the second solution are the same solution or different solution from each other. 2. The method of Embodiment 1, wherein the ratio being higher than about 7.5 ng / mg, more preferably higher than about 7.53ng / 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 a medical intervention to obviate the progressive renal decline. Page: 43 of 115Attorney Docket No.132417-0001WO01 3. The method of any of Embodiments 1-2, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg. 4. The method of any of Embodiments 1-3, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg. 5. The method of Embodiment 1-4, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure. 6. The method of Embodiments 1-5, further comprising assessing a probability of an occurrence of the progressive renal decline to the kidney endpoint, based on associating the ratio with at least one among age, gender, eGFR, UACR, and HbA1c level. 7. The method of Embodiment 6, wherein at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level is adjusted based on at least one respective coefficient respectively corresponding to at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level. 8. The method of Embodiment 6, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients. 9. The method of embodiment 8, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer. 10. The method of Embodiment 20-24, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about 1.2; a3 value is from about -0.1 to about 0.0; Page: 44 of 115Attorney Docket No.132417-0001WO01 a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7. 11. The method of Embodiments 9-10, wherein, the method further comprises, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint. 12. The method of Embodiment 11, wherein, the method further comprises, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint. 13. The method of Embodiment 11-12, wherein the first threshold value is about 0.1. 14. The method of Embodiment 11-12, wherein the first threshold value is about 0.11. 15. The method of Embodiment 12, wherein the second threshold value is about 0.25. 16. The method of Embodiment 12, wherein the second threshold value is about 0.3. 17. The method of Embodiment 12, wherein the second threshold value is about 0.27. 18. The method of any of Embodiments 1-3, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR). 19. The method of Embodiment 18, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher. 20. The method of Embodiment 19, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2). 21. The method of any of Embodiments 1-20, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample. 22. The method of any of Embodiments 1-21, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample. 23. The method of any of Embodiments 1-22, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample. 24. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg. 25. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg. Page: 45 of 115Attorney Docket No.132417-0001WO01 26. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg. 27. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg. 28. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg. 29. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg. 30. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg. 31. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg. 32. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg. 33. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 29 ng / mg. 34. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 40 ng / mg. 35. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 45 ng / mg. 36. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 47 ng / mg. 37. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 48 ng / mg. 38. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 49 ng / mg. 39. The method of any of Embodiments 1-23, wherein the ratio being higher than about 7.5 ng / mg is higher than about 50 ng / mg. 40. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 6 ng / mg. 41. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4.5 ng / mg. 42. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4 ng / mg. 43. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.5 ng / mg. 44. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg. Page: 46 of 115Attorney Docket No.132417-0001WO01 45. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg. 46. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg. 47. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg. 48. The method of any of Embodiments 1-39, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg. 49. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher. 50. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher. 51. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher. 52. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher. 53. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % or higher. 54. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % or higher. 55. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % or higher. 56. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher. 57. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher. 58. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%. 59. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 45%. 60. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 40%. 61. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 35%. 62. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%. 63. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%. Page: 47 of 115Attorney Docket No.132417-0001WO01 64. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%. 65. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%. 66. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%. 67. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % to about 45%. 68. The method of any of Embodiments 1-48, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%. 69. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint after 4 years. 70. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint after 5 years. 71. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint after 6 years. 72. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint after 7 years. 73. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint after 8 years. 74. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint after 9 years. 75. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint within 7 years. 76. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint within 8 years. 77. The method of any of Embodiments 1-68, wherein the progressive renal is to decline to the kidney endpoint within 9 years. 78. The method of any of Embodiments 1-77, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. 79. The method of any of Embodiments 1-78, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. 80. The method of any of Embodiments 1-79, wherein at least one reagent among the first reagent and the second reagent includes an antibody. 81. An assay kit to determine a likelihood of a progressive renal decline, comprising: a first solution including a first reagent to interact with a post-translationally modified Fetuin- A fragments in urine (uPTM-FetA) to indicate the level of the Fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; Page: 48 of 115Attorney Docket No.132417-0001WO01 a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, 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, indicates a higher likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, and wherein the first solution and the second solution are the same solution or different solution from each other. 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 a medical intervention to obviate the progressive renal decline. 83. The assay kit of any of Embodiments 81-82, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg. 84. The assay kit of any of Embodiments 81-83, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg. 85. The assay kit of Embodiment 81-84, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure. 86. The assay kit of Embodiments 81-85, further comprising assessing a probability of an occurrence of the progressive renal decline to the kidney endpoint, based on associating the ratio with at least one among age, gender, eGFR, UACR, and HbA1c level. 87. The assay kit of Embodiment 86, wherein at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level is adjusted based on at least one respective coefficient respectively corresponding to at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level. 88. The assay kit of Embodiment 86, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein Page: 49 of 115Attorney Docket No.132417-0001WO01 I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients. 89. The assay kit of embodiment 88, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer. 90. The assay kit of Embodiment 20-24, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7. 91. The assay kit of Embodiments 89-90, wherein, the method further comprises, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint. 92. The assay kit of Embodiment 91, wherein, the method further comprises, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint. 93. The assay kit of any of Embodiments 81-83, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR). 94. The assay kit of Embodiment 93, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher. 95. The assay kit of Embodiment 94, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2). 96. The assay kit of any of Embodiments 81-95, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample. 97. The assay kit of any of Embodiments 81-96, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) Page: 50 of 115Attorney Docket No.132417-0001WO01 in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample. 98. The assay kit of any of Embodiments 81-97, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample. 99. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg. 100. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg. 101. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg. 102. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg. 103. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg. 104. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg. 105. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg. 106. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg. 107. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg. 108. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 29 ng / mg. 109. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 40 ng / mg. 110. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 45 ng / mg. 111. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 47 ng / mg. 112. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 48 ng / mg. 113. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 49 ng / mg. 114. The assay kit of any of Embodiments 81-98, wherein the ratio being higher than about 7.5 ng / mg is higher than about 50 ng / mg. Page: 51 of 115Attorney Docket No.132417-0001WO01 115. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 6 ng / mg. 116. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 5 ng / mg. 117. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4 ng / mg. 118. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.5 ng / mg. 119. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg. 120. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg. 121. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg. 122. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg. 123. The assay kit of any of Embodiments 81-114, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg. 124. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher. 125. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher. 126. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher. 127. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher. 128. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % or higher. 129. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % or higher. 130. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % or higher. 131. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher. 132. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher. 133. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%. Page: 52 of 115Attorney Docket No.132417-0001WO01 134. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 45%. 135. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 40%. 136. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 35%. 137. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%. 138. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%. 139. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%. 140. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%. 141. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%. 142. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % to about 45%. 143. The assay kit of any of Embodiments 81-123, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%. 144. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint after 4 years. 145. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint after 5 years. 146. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint after 6 years. 147. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint after 7 years. 148. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint after 8 years. 149. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint after 9 years. 150. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint within 7 years. 151. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint within 8 years. 152. The assay kit of any of Embodiments 81-143, wherein the progressive renal is to decline to the kidney endpoint within 9 years. Page: 53 of 115Attorney Docket No.132417-0001WO01 153. The assay kit of any of Embodiments 81-152, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. 154. The assay kit of any of Embodiments 81-153, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. 155. The assay kit of any of Embodiments 81-154, wherein at least one reagent among the first reagent and the second reagent includes an antibody. 156. A method of correlating a likelihood of a progressive renal decline, comprising: determining the level 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 fragment of a post- translationally modified Fetuin-A fragments in urine (uPTM-FetA), wherein the second biomarker is a urinary creatinine; determining the ratio of the level of the first biomarker to the level of the second biomarker; correlating the ratio with a likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, 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, indicates a higher likelihood of the progressive renal decline to the kidney endpoint. 157. The method of Embodiment 156, 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 / , triggers a medical intervention to obviate the progressive renal decline. 158. The method of any of Embodiments 156-157, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg. 159. The method of any of Embodiments 156-158, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg. 160. The method of Embodiment 156-159, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure. 161. The method of Embodiments 156-160, further comprising assessing a probability of an occurrence of the progressive renal decline to the kidney endpoint, based on associating the ratio with at least one among age, gender, eGFR, UACR, and HbA1c level. 162. The method of Embodiment 161, wherein at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level is adjusted based on at least one respective coefficient Page: 54 of 115Attorney Docket No.132417-0001WO01 respectively corresponding to at least one among the ratio, the age, the gender, the eGFR, the UACR, and HbA1c level. 163. The method of Embodiment 161, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (1): y = a0 + a1*AGE+a2*Gender(M)+a3*eGFR+a4*UACR+a5*HbA1c+a6*I(E103 / UCr>7.53 ng / mg) ………. Equation (1) wherein I(E103 / UCr>7.53 ng / mg)=1, if E103 / UCr>7.53 ng / mg or I(E103 / UCr>7.53 ng / mg)= 0 if E103 / UCr is equal or less than 7.53 ng / mg, y is a first probabilistic value to be based on to assess the probability of the occurrence of the progressive renal decline to the kidney endpoint, a0, a1, a2, a3, a4, a5 and a6 are the assigned coefficients. 164. The method of embodiment 163, wherein the step of assessing the probability of the occurrence of the progressive renal decline to the kidney endpoint is based on Equation (2): p = exp(y) / (1+ exp(y)) ………. Equation (2) wherein p is a second probabilistic value to be based on to assess the probability of the occurrence of the cancer. 165. The method of Embodiment 20-24, wherein a0 value is from about -10.0 to about 0.5; a1 value is from about -0.0 to about 0.1; a2 value is from about -0.3 to about 1.2; a3 value is from about -0.1 to about 0.0; a4 value is from about 0.1 to about 0.7; a5 value is from about -0.3 to about 0.3; and a6 value is from about 1.2 to about 3.7. 166. The method of Embodiments 164-165, wherein, the method further comprises, in response to the second probabilistic value exceeding a first threshold value, determining that a patient from whom the urine was derived from has an elevated risk of the progressive renal decline to the kidney endpoint. 167. The method of Embodiment 166, wherein, the method further comprises, in response to the second probabilistic value exceeding a second threshold value higher than the first threshold value, determining that a patient from whom the urine was derived from has a higher elevated risk higher than the elevated risk of the progressive renal decline to the kidney endpoint. 168. The method of any of Embodiments 156-158, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR). 169. The method of Embodiment 168, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher. Page: 55 of 115Attorney Docket No.132417-0001WO01 170. The method of Embodiment 169, wherein the second subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2). 171. The method of any of Embodiments 156-170, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample. 172. The method of any of Embodiments 156-171, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample. 173. The method of any of Embodiments 156-172, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample. 174. The method of any of Embodiments 156-173, wherein the ratio being higher than about 7.5 ng / mg is higher than about 9.5 ng / mg. 175. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 10 ng / mg. 176. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 11 ng / mg. 177. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 12 ng / mg. 178. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 14 ng / mg. 179. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 15 ng / mg. 180. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 20 ng / mg. 181. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 24 ng / mg. 182. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg. 183. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 29 ng / mg. 184. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 40 ng / mg. 185. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 45 ng / mg. Page: 56 of 115Attorney Docket No.132417-0001WO01 186. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 47 ng / mg. 187. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 48 ng / mg. 188. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 49 ng / mg. 189. The method of any of Embodiments 156-174, wherein the ratio being higher than about 7.5 ng / mg is higher than about 50 ng / mg. 190. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 6 ng / mg. 191. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4.5 ng / mg. 192. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 4 ng / mg. 193. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.5 ng / mg. 194. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 3.3 ng / mg. 195. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 1 ng / mg. 196. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.5 ng / mg. 197. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.2 ng / mg. 198. The method of any of Embodiments 156-189, wherein the second ratio being lower than about 7.5 ng / mg is lower than about 0.1 ng / mg. 199. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher. 200. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher. 201. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher. 202. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher. 203. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % or higher. 204. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % or higher. Page: 57 of 115Attorney Docket No.132417-0001WO01 205. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % or higher. 206. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher. 207. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher. 208. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%. 209. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 45%. 210. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 40%. 211. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 35%. 212. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % to about 30%. 213. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 20%. 214. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % to about 25%. 215. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % to about 30%. 216. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%. 217. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % to about 45%. 218. The method of any of Embodiments 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%. 219. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint after 4 years. 220. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint after 5 years. 221. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint after 6 years. 222. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint after 7 years. 223. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint after 8 years. Page: 58 of 115Attorney Docket No.132417-0001WO01 224. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint after 9 years. 225. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint within 7 years. 226. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint within 8 years. 227. The method of any of Embodiments 156-218, wherein the progressive renal is to decline to the kidney endpoint within 9 years. 228. The method of any of Embodiments 156-227, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. 229. The method of any of Embodiments 156-228, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. 230. The method of any of Embodiments 156-229, wherein at least one reagent among the first reagent and the second reagent includes an antibody. NON-LIMITING EXAMPLES

[0455] 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 illustrative and representative thereof. Thus, the examples provided below, while illustrated with a particular medical device or active agent, are applicable to the range of medical devices and active agents described herein. Example 1

[0456] Study design

[0457] For the current study, we included patients from two independent, non-overlapping prospective cohort studies including patients with type 2 diabetes from different geographical areas. All analyses were first performed in the NTUH cohort initiated in the National Taiwan University Hospital (NTUH) in Taiwan. These findings were then independently replicated in a cohort from the DIAbetes and LifEstyle Cohort Twente (DIALECT) in the Netherlands.

[0458] Regarding Examples 1-3, Supplemental Table 1 indicates Cohort characteristics and inclusion and exclusion criteria. Supplemental Table 1 | Cohort characteristics and inclusion and exclusion criteriaPage: 59 of 115Attorney Docket No.132417-0001WO01

[0459] uPTM-FetA measurements

[0460] Spot urine (NTUH) or 24h collection urine (DIALECT) samples were collected at baseline. These samples were stored at -80 degrees Celsius until measurement. uPTM- FetA was measured using the 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.

[0461] Definition of the Kidney Endpoint

[0462] Kidney function was assessed using the estimated glomerular filtration rate (eGFR) calculated with the Chronic Kidney Disease Epidemiology Collaboration equation. For both cohorts, the primary outcome of kidney function deterioration was defined as an eGFR decline ≥30% from baseline, reaching an eGFR<15 mL / min / 1.73 m2, or a need of renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal dialysis or hemodialysis). Follow-up was censored at the end of follow-up, or after the date that the kidney endpoint was met.

[0463] Covariables

[0464] At baseline, clinical, anthropometric and demographic data were retrieved though electronic patient records and / or questionnaires. The severity of albuminuria was determined on the basis of the urinary albumin to creatinine ratio (UACR). All other laboratory measurements were performed using routine laboratory methods.

[0465] Statistical Analyses

[0466] Baseline characteristics were presented as mean ± standard deviation (SD), median [interquartile range (IQR)], or frequency (percentage), depending on data distribution. We performed receiver operating characteristic (ROC) analysis and determined a cut-off value for IVD103 with optimal sensitivity and specificity using the Youden index. According to the established cut-off, patients were categorized into low-risk (IVD103<optimal cut-off) or high-risk (IVD103≥optimal cut- off) groups. Statistical significance of differences between the groups were assessed using Student's t- tests, Mann-Whitney U tests, or Fisher's exact test, depending on data distribution. Page: 60 of 115Attorney Docket No.132417-0001WO01

[0467] Kaplan-Meier curves are presented to visualize the occurrence of the kidney endpoint during follow-up between groups, where log-rank tests were used to assess statistical significance of differences between the groups. The associations of IVD103 with risk of eGFR decline were analyzed using Cox proportional-hazards regression analyses. Non-normally distributed covariables were natural log-transformed prior to analysis. Hazard ratios and 95% confidence intervals were presented per one standard deviation relative increment (risk factors of continuous nature) or per change compared with a reference group (risk factors of categorical nature). In model 1, we performed multivariable adjusted analyses with traditional risk factors (age, sex, eGFR, UACR, HbA1c). Thereafter, we computed further models with additive adjustments to Model 1, including lipid-related parameters (i.e. total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid- lowering drugs; Model 2); blood pressure-related parameters (systolic blood pressure, diabetic blood pressure, and use of anti-hypertensive drugs; Model 3); other clinical parameters (body mass index, smoking status; Model 4); and a combination of all factors above (Model 5).

[0468] All data were analyzed using R version 4.1.2 (R Foundation for Statistical Computing, Vienna Austria). For all statistical analyses, p-value<0.05 was considered significant. Example 2

[0469] NTUH cohort (NTUH)

[0470] This prospective cohort study (project title: Validation of Early Diagnostics Biomarkers for Diabetic Nephropathy and its extension; registered project No.: 201107004RC and 201705062RIPD) was conducted in patients with type 2 diabetes at the National Taiwan University Hospital (NTUH) in Taiwan between November 2011 and February 2020. This study included adult patients with type 2 diabetes according to the criteria of the American Diabetes Association. Considering the predefined hypothesis of uPTM- FetA as an early marker of DKD, we aimed to verify whether the prospective associations of uPTM-FetA with the kidney endpoint are observed in patients without CKD at baseline. Therefore, the NTUH cohort of NTUH study only included individuals with baseline eGFR>60 ml / min / 1.73m2and UACR < 30 mg / g. A detailed overview of inclusion and exclusion criteria is provided in Supplemental Table 1. The study was approved by the NTUH institutional review board (IRB No.:1063703310), and the study was carried out in accordance with the guidelines of good clinical practice and the WMA Declaration of Helsinki. All subjects provided written informed consent prior to study participation. RESULTS

[0471] Baseline characteristics

[0472] NTUH cohort

[0473] Among all participants enrolled in the NTUH cohort, eventually 223 were eligible for inclusion in the current study. Mean age was 60.38 ± 9.66 years, 125 (56%) were male, eGFR was 90.00 ± 13.87 ml / min / 1.73m2, and median UACR was 5.87 [3.49 to 11.59] mg / g (Table 1).

[0474] In Table 1, Normally distributed data are presented as mean ± standard deviation, skewed data as median [interquartile range], and categorical data as number (percentage). Non- Page: 61 of 115Attorney Docket No.132417-0001WO01 detectable u-PTM-FetAs were set to the lowest value available. Abbreviations: eGFR, estimated glomerular filtration rate as calculated using the creatinine-based CKD-EPI formula; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index. Table 1. Baseline characteristics of the non-CKD population according to IVD103 levels

[0475] Cut-off determination and associations with kidney endpoint – NTUH cohort

[0476] During a median follow-up of 9.25 [7.52 to 9.66] years, 59 participants (26%) from the NTUH cohort experienced the kidney endpoint. Based on the Youden index, the optimal IVD103 cut-off for kidney endpoint risk stratification was 0.644. Characteristics of the population in strata of IVD103 groups are presented in Table 1. Figure 1A illustrates a Kaplan-Meier curve with end point of 30% decline or <15 in eGFR, where Figure 1A is directed to Non-CKD NTUH patients with classified as high-risk and low-risk by IVD103. In the high-risk IVD103 group, 36% experienced the kidney endpoint, which was significantly higher compared to the 19% observed in the low-risk group (P = 0.0054; Figure 1A).

[0477] The associations of the high-risk IVD103 group with the kidney endpoint were also observed in univariable Cox regression analyses (HR: 2.06; 95% CI: 1.23 to 3.47; P = 0.006) and became even more pronounced in the fully adjusted model 5 (HR: 2.96; 95% CI: 1.64 to 5.34; P < 0.001), while UACR did not have significant association with the kidney endpoint (Table 2). Page: 62 of 115Attorney Docket No.132417-0001WO01

[0478] In Table 2, Cox proportional-hazard model were performed to assess the association of IVD103 with risk of kidney function deterioration (eGFR of ≥30% from baseline, eGFR<15 mL / min / 1.73 m2 or a need of renal replacement therapy). Model 1 was adjusted age, sex, eGFR, UACR, and HbA1c. Model 2 was additionally adjusted for total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering drugs. 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) drugs. Model 4 was additionally adjusted for body mass index, smoking status. Model 5 was adjusted for factors above. HR = Hazard Ratio, CI = Confidence Interval. Table 2. Multivariable-adjusted association between IVD103 and risk of eGFR decline in 223 non-CKD type 2 diabetes patients from the NTUH cohortExample 3

[0479] DIALECT (validation) cohort (DIALECT)

[0480] To validate the optimal cut-off in assessing risk of renal function loss, data from DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort study was used, which has been described in detail in this disclosure. In brief, DIALECT is a prospective cohort study in patients with type 2 diabetes, performed in the Ziekenhuisgroep Twente Hospital, which is located in Almelo and Hengelo, The Netherlands. Adult patients with type 2 diabetes treated in the outpatient clinic of this secondary care hospital were eligible. Patients were recruited between March 2009 and May 2019. The study was performed according to the guidelines of good clinical practice and the WMA Declaration of Helsinki, and all subjects provided written informed consent before study participation. The study was approved by the local institutional review boards (METC registration numbers NL57219.044.16 and 1009.68020) and is registered in The Netherlands Trial Register (NTR trial code 5855). RESULTS Page: 63 of 115Attorney Docket No.132417-0001WO01

[0481] Baseline characteristics

[0482] DIALECT cohort

[0483] In total, 621 participants enrolled in the DIALECT cohort were eligible for inclusion in the current study. Mean age was 64.11 ± 9.97 years, 381 (61%) were male,

[0484] eGFR was 75.90 ± 24.49 ml / min / 1.73m2, and median UACR was 8.85 [3.54 to 48.67] mg / g (Table 3).

[0485] In Table 3, normally distributed data are presented as mean ± standard deviation, skewed data as median [interquartile range], and categorical data as number (percentage). Non- detectable u-PTM-FetAs were set to the lowest value available. Abbreviations: eGFR, estimated glomerular filtration rate as calculated using the creatinine-based CKD-EPI formula; HbA1c, glycated hemoglobin; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index. Page: 64 of 115Attorney Docket No.132417-0001WO01 Table 3. Baseline characteristics of the DIALECT cohort population according to IVD103 levels

[0486] Results of validation study – DIALECT cohort

[0487] In the DIALECT cohort, 277 participants (45%) experienced the kidney endpoint during the median follow-up of 4.16 [2.53, 6.41] years. Figure 1B illustrates a Kaplan-Meier curve with end point of 30% decline or <15 in eGFR, where Figure 1B is directed to Patients in DIALECT study classified as high-risk and low-risk by IVD103 according to a non-limiting embodiment.

[0488] In the high-risk IVD103 group, 46% experienced the kidney endpoint, which was higher compared to the 42% observed in the low-risk group (P = 0.0039; Figure 1B).

[0489] DIALECT (validation) cohort analyses

[0490] Similar to the primary analyses, patients in the DIALECT (validation) cohort (DIALECT) were stratified into high-risk and low-risk groups, based on the IVD103 cut-off obtained from NTUH cohort study. Survival analyses including Cox proportional hazard regression models as Page: 65 of 115Attorney Docket No.132417-0001WO01 described for the primary analyses were repeated in this DIALECT (validation) cohort, with the same kidney endpoint as outcome variable. ANALYSIS OF EXAMPLES 1-3

[0491] In this study, uPTM-FetA was associated with risk of eGFR decline in patients with type 2 diabetes, independent of age, sex, eGFR, UACR, baseline HbA1c, and multiple other potential confounders. These findings were validated in a second, larger cohort of patients with type 2 diabetes, highlighting the potential clinical use of uPTM-Fet-A as a predictive marker for DKD.

[0492] The human precursor protein of fetuin A consists of three parts, namely the A chain, connecting peptide, and B chain, which are 321, 40, and 27 amino acids in length, respectively. The connecting peptide of the precursor is removed by a posttranslational modification, limited proteolysis, after which only the A and B chain form the active fetuin A protein. The monoclonal antibody in the ELISA kit that was used in the current study (Human uPTM3-DKD ELISA kit, BPM Corp.) only detects the connecting peptide-containing fetuin A.

[0493] We found that elevated uPTM-FetA is associated with progression of renal function decline in type 2 diabetes patients. These findings are generally consistent with previous studies that showed associations of urinary fetuin A with albuminuria and lower kidney function type 2 diabetes patients with DKD. In some embodiments, the concentration of uPTM-FetA is not related to serum C-FetA among patients (in-house data from NTUH cohort, data not shown). The incongruence between circulating and urine fetuin A concentrations was also observed in other studies. uPTM-FetA specifically accumulates in urine of patients with progressive renal decline. This is likely attributable 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 needs to be further investigated in future preclinical and interventional studies. These results show that in non-CKD type 2 diabetes patients, uPTM-FetA was to UACR multivariable-adjusted Cox regression analyses. This suggests that uPTM-FetA may be better suitable for predicting renal function decline than UACR. Moreover, the use of antihypertensives such as angiotensin receptor blockers and angiotensin converting enzyme inhibitors, which are frequently prescribed in type 2 diabetes patients, reduces albuminuria, thus limited usefulness of UACR as a predictive marker of DKD. uPTM-FetA may not have this disadvantage.

[0494] There were studies establishing prediction model DKD for type 2 diabetes patients using clinical parameters with or without aid of machine learning, but the follow-up duration was rather short. Evaluation of eGFR within only a period of 5 years is considered insufficient for assessing renal function deterioration in type 2 diabetes patients, as intra‑individual variability of eGFR is high among early DKD patients. Long-term follow up of renal function in type 2 diabetes patients is essential, given the fact that whole- kidney hyperfiltration with increased eGFR is seen in substantial portion of this population and may mask the development of kidney disease in early phase. Page: 66 of 115Attorney Docket No.132417-0001WO01

[0495] There are several strengths of this study. The current study include use of two large, well-characterized cohorts allowed for extensive adjustments for potential confounders. Follow-up duration in NTUH cohort is long, which allows adequate assessment of associations of uPTM-FetA with renal outcome not underestimated by the issue of renal hyperfiltration in early DKD. Finally, the use of two independent, bi- ethnic cohorts including patients from different geographical areas, is reassuring with regard to the external validity of these findings to other type 2 diabetes populations worldwide.

[0496] In conclusion, this study demonstrates that in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes, uPTM-FetA is strongly associated with renal function deterioration, independent of albuminuria, eGFR, age, sex, and multiple other risk factors. The added prognostic value of uPTM-FetA on top of albuminuria and other markers is particularly clear in patients without CKD at baseline. These findings are validated in a large, independent, second cohort, which is promising with regard to the potential scientific and clinical use of uPTM-FetA in type 2 diabetes. Example 4

[0497] The Human uPTM3-DKD ELISA is a colorimetric immunoassay intended for quantitative measurement of unique Fetuin-A with specific post translational modification (PTM) in human urine and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Technologists.

[0498] PRINCIPLE OF THE TEST

[0499] The Human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrators or unknown urine samples are mixed with anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate pre-bounded with unique PTM Fetuin-A. The monoclonal antibody recognizes unique PTM Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, a Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve. Example 5

[0500] Study design

[0501] For the current study of Examples 5 through 7, we included patients from two independent, non-overlapping prospective cohort studies including patients with type 2 diabetes from different geographical areas. All analyses were first performed in the NTUH cohort initiated in the National Taiwan University Hospital (NTUH) in Taiwan. These findings were then independently replicated in a cohort from the DIAbetes and LifEstyle Cohort Twente (DIALECT) in the Netherlands.

[0502] For this study, the following set up indicated in Supplemental Table 2 was used: Supplemental Table 2 Page: 67 of 115Attorney Docket No.132417-0001WO01

[0503] The Human uPTM3-DKD ELISA Kit is a colorimetric immunoassay to quantify unique Fetuin-A with specific post translational modification (hereafter called as E103) in urine. This product is non-automated IVD for prognosis use and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Technologists. The concentration of E103 should be corrected by urine creatinine before applying to the clinic. The Human uPTM3-DKD ELISA Kit is to be used in conjunction with clinical evaluation as an aid in assessing the prognosis of kidney function in type 2 diabetes.

[0504] The Human uPTM3-DKD ELISA is a competitive immunoassay. In this assay, calibrators or unknown urine samples are mixed with anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate precoated with unique PTM Fetuin-A. The monoclonal antibody recognizes unique PTM Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, the Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nm wavelength and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve.

[0505] Parameters of clinical performance determined

[0506] Hazard ratios and 95% confidence intervals calculated based on Cox proportional hazards regression analysis will be used to evaluate the association between E103 and kidney function deterioration with a decline in estimated glomerular filtration rate (eGFR) of ≥30% from baseline, eGFR<15 mL / min / 1.73 m2or a need of renal replacement therapy.

[0507] Statistics & Sample Size Calculation

[0508] the required sample size was estimated based on the following assumptions: • The hazard ratio (θ) of the high-risk group relative to the low-risk group is expected to be a value greater than 1, and this study sets a hazard ratio of 2.0; • According to literature statistics, about 25% to 40% of patients with type 2 diabetes will develop diabetic nephropathy. Therefore, this study estimated that the incidence of renal function deterioration was 25% (pE= 0.25); • The proportion of subjects in the low-risk group and the high-risk group is the same (pL=pH=0.5); • Type I error(α) is 0.05; • power (1-β) is 80%; • The number of samples is calculated as ((z1-α / 2+z1-β) / (lnθ-ln(1)))2 / (pLpHpE). Under the assumptions above, 261 patients are required. Page: 68 of 115Attorney Docket No.132417-0001WO01

[0509] Study Population

[0510] We used data from National Taiwan University Hospital (NTUH) cohort in Taiwan and DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort in Netherlands. The NTUH cohort (project title: Validation of Early Diagnostics Biomarkers for Diabetic Nephropathy and its extension; registered project No.: 201107004RC and 201705062RIPD) was conducted in patients with type 2 diabetes at the NTUH between November 2011 and February 2020. This study included adult patients with type 2 diabetes according to the criteria of the American Diabetes Association. The exclusion criteria are diagnosis of type 1 diabetes mellitus, current pregnancy, current known malignancy, current acute or chronic infections, other primary or secondary renal diseases and Illicit drug use. DIALECT is a prospective cohort study in patients with type 2 diabetes, performed in the Ziekenhuisgroep Twente Hospital, which is located in Almelo and Hengelo, The Netherlands. Adult patients with type 2 diabetes treated in the outpatient clinic of this secondary care hospital were eligible. Patients were recruited between March 2009 and May 2019. The study was performed according to the guidelines of good clinical practice and the WMA Declaration of Helsinki, and all subjects provided written informed consent before study participation.

[0511] NTUH cohort (NTUH)

[0512] This prospective cohort study (project title: Validation of Early Diagnostics Biomarkers for Diabetic Nephropathy and its extension; registered project with institutional review board approval No.: 201107004RC and 201705062RIPD) was conducted in patients with type 2 diabetes at the National Taiwan University Hospital (NTUH) in Taiwan between November 2011 and February 2018. A detailed overview of inclusion and exclusion criteria is provided in Supplemental Table 3. Supplemental Table 3 | Cohort characteristics and detailed inclusion and exclusion criteriaPage: 69 of 115Attorney Docket No.132417-0001WO01

[0513] The study was carried out in accordance with the guidelines of good clinical practice and the WMA Declaration of Helsinki. All subjects provided written informed consent prior to study participation.

[0514] DIALECT (validation) cohort (DIALECT)

[0515] To validate the optimal cut-off in assessing risk of renal function loss, data from DIAbetes and LifEstyle Cohort Twente (DIALECT) cohort study was used, which has been described in detail elsewhere (16). In brief, DIALECT is a prospective cohort study in patients with type 2 diabetes, performed in the Ziekenhuisgroep Twente Hospital, which is located in Almelo and Hengelo, The Netherlands. Adult patients with type 2 diabetes treated in the outpatient clinic of this secondary care hospital were eligible and recruited between March 2009 and May 2019 (Supplemental Table 3). The study was performed according to the guidelines of good clinical practice and the WMA Declaration of Helsinki, and all subjects provided written informed consent before study participation. The study was approved by the local institutional review boards (METC registration numbers NL57219.044.16 and 1009.68020) and is registered in The Netherlands Trial Register (NTR trial code 5855).

[0516] Test procedure

[0517] uPTM-FetA measurements

[0518] Spot urine (NTUH) or 24h collection urine (DIALECT) samples were collected at baseline. These samples were stored at -80 degrees Celsius until measurement. uPTM-FetA was measured using the 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 (UCr) into uPTM-FetA / UCr for normalization.

[0519] 1. Prepare enough microplate modules for all calibrators and urine samples and secure them in a holder. Recover all reagents to room temperature prior to use.

[0520] 2. Add 50 μL Calibrator 1-8 and urine sample into each well of E103 Coated Microplate respectively.

[0521] 3. Add 50 μL 1X mAb anti-E103 into each well of E103 Coated Microplate.

[0522] 4. Incubate for 2 hours at 25°C and 200 rpm on an orbital shaker.

[0523] 5. After the incubation, discard the contents in the wells.

[0524] 6. Wash each well with 300-400 μL 1X Wash Buffer. Discard the contents and sharply strike the wells on absorbent paper to remove residual liquid. Wash for a total of 4 times.

[0525] 7. Add 100 μL 1X HRP Conjugate into each well. Incubate the microplate for 30 minutes at 25°C and 200 rpm on an orbital shaker.

[0526] 8. After the incubation, discard the contents in the wells and wash the wells as described in Step 4 and 5.

[0527] 9. Add 100 μL TMB Substrate into each well. Incubate in the dark for 30 minutes at room temperature. Page: 70 of 115Attorney Docket No.132417-0001WO01

[0528] 10. Add 100 μL Stop Solution into each well. Mix by a brief shaking until the mixture become homogeneous.

[0529] 11. Determine the absorbance at 450±10 nm within 30 minutes and calculate the results.

[0530] Definition of the Kidney Endpoint

[0531] Kidney function was assessed using the estimated glomerular filtration rate (eGFR) calculated with the Chronic Kidney Disease Epidemiology Collaboration equation. For both cohorts, the primary outcome of kidney function deterioration was defined as an eGFR decline ≥30% from baseline, reaching an eGFR<15 mL / min / 1.73 m2, or a need of renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal dialysis or hemodialysis). Follow-up was censored at the end of follow-up, or after the date that the kidney endpoint was met.

[0532] Covariables

[0533] At baseline, clinical, anthropometric and demographic data were retrieved though electronic patient records and / or questionnaires. The severity of albuminuria was determined on the basis of the urinary albumin to creatinine ratio (UACR). All other laboratory measurements were performed using routine laboratory methods.

[0534] Statistical Analyses

[0535] Baseline characteristics were presented as mean ± standard deviation (SD), median [interquartile range (IQR)], or frequency (percentage), depending on data distribution. We performed receiver operating characteristic (ROC) analysis and determined a cut-off value for uPTM-FetA / UCr with optimal sensitivity and specificity using the Youden index (19). According to the established cut- off, patients were categorized into low-risk (uPTM-FetA / UCr < optimal cut-off) or high-risk (uPTM- FetA / UCr ≥ optimal cut-off) groups. Statistical significance of differences between the groups were assessed using Student's t-tests, Mann-Whitney U tests, or Fisher's exact test, depending on data distribution.

[0536] Kaplan-Meier curves are presented to visualize the occurrence of the kidney endpoint during follow-up between groups, where log-rank tests were used to assess statistical significance of differences between the groups. The associations of uPTM-FetA / UCr with risk of eGFR decline were analysed using Cox proportional-hazards regression analyses. Non-normally distributed covariables were natural log-transformed prior to analysis. Hazard ratios and 95% confidence intervals were presented per one standard deviation relative increment (risk factors of continuous nature) or per change compared with a reference group (risk factors of categorical nature). In model 1, we performed multivariable adjusted analyses with traditional risk factors (age, sex, eGFR, UACR, HbA1c). Thereafter, we computed further models with additive adjustments to Model 1, including lipid-related parameters (i.e. total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering drugs; Model 2); blood pressure-related parameters (systolic blood pressure, diastolic blood pressure, and use of anti-hypertensive drugs; Model 3); other clinical parameters (body Page: 71 of 115Attorney Docket No.132417-0001WO01 mass index, smoking status; Model 4); liver function abnormality (Model 5); and a combination of all factors above (Model 6).

[0537] To evaluate the prognostic performance of our biomarker, uPTM-FetA / UCr, we incorporated it into the clinical plus biomarker model. Various measures were utilized to assess the added value of the biomarker in predicting the risk of kidney function deterioration. Model fit was assessed using the Akaike information criterion (AIC) and the likelihood ratio test (LRT), where lower AIC values or higher χ2 values indicated superior global fit. Model calibration was evaluated using the Hosmer-Lemeshow goodness-of-fit test, with p-values > 0.05 indicating good agreement between observed and predicted outcomes. Model discrimination was determined by calculating the area under the curve (AUC) of the receiver operating characteristic. The optimal cut-off for maximum sensitivity and specificity in each model was determined using the Youden index. The improvement in AUC after incorporating the biomarker was calculated.

[0538] To assess model reclassification, we used continuous / category-free net reclassification improvement (NRI > 0) since there were no established risk cut-offs that justified the use of categorical NRI. The overall NRI measured the proportion of predicted probabilities that shifted upward or downward when the biomarker was added to the clinical model. Additionally, the absolute integrated discrimination improvement (IDI) was employed to quantify the average increase in predicted probabilities for subjects with kidney function deterioration and the reduction for those without, following the addition of the biomarker.

[0539] All data were analysed using R version 4.1.2 (R Foundation for Statistical Computing, Vienna Austria). For all statistical analyses, p-value<0.05 was considered significant.

[0540] DIALECT (validation) cohort analyses

[0541] Similar to the primary analyses, patients in the DIALECT (validation) cohort (DIALECT) were stratified into high-risk and low-risk groups, based on the uPTM-FetA / UCr cut-off obtained from NTUH cohort study. Survival analyses including Cox proportional hazard regression models as described for the primary analyses were repeated in this DIALECT (validation) cohort, with the same kidney endpoint as outcome variable.

[0542] Data and results management

[0543] A series of clinical records provided by the NTUH cohort and DIALECT cohort corresponding to all the patients with the diagnosis of kidney function deterioration with a decline in eGFR of ≥30% from baseline3, eGFR<15 mL / min / 1.73 m2or a need of renal replacement therapy will be used. All the excel files containing these data will be manually downloaded and saved by the Principal Investigator, in a specific folder on a computer with an encrypted disk and other privacy and safety measures, in alignment with General Data Protection Regulation (GDPR). The folder will only be accessible to the PI and the Statistician. The patient's urine sample will be transferred to BPM from NTUH and UMCG, and then be stored at -80°C in accordance with the sample management procedure of BPM, and will not be taken out until it is used. After testing, test results will be recorded Page: 72 of 115Attorney Docket No.132417-0001WO01 in excel files and saved by the BPM testing laboratory personnel, in a specific folder as previously described.

[0544] Data and results analysis

[0545] For the Primary (NTUH) Cohort, we conduct a screening period of 3.5 years for recruited patients. Urine samples were taken every 6 months during the screening period, and for each patient, the baseline was defined as the time with E103 value first greater than assay Limit of Quantitation (LoQ = 5.428 ng / mL). For patients with E103 measurements smaller than LoQ but greater than Limit of Detection (LoD = 1.901 ng / mL), the E103 values were set to as LoD. Patients with E103 values smaller than LoD were set to as "non-detectable. During the 5-year follow-up period from baseline, the change in eGFR was calculated as 100%*(eGFR-baseline eGFR) / baseline eGFR. The study endpoint was kidney function deterioration defined as an eGFR decline ≥30% from baseline, reaching an eGFR<15 mL / min / 1.73 m2, or a need of renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal dialysis or haemodialysis). If no outcome occurred at the end of the follow-up time, it was censored data, and the record-setting time was the end of the follow-up. Patients with follow-up shorter than 2 years or the number of eGFR measurements were less than 5 were excluded from the analysis.

[0546] When analyzing, E103 test results were then divided by urine creatinine (UCr) to obtain the uPTM-FetA / UCr values and IVD103=log10(uPTM-FetA / UCr) is the logarithm version of base 10. By using Youden Index method, we take the value of the receiver operating characteristic (ROC) curve that maximizes the sum of the sensitivity and specificity as the clinical cut-off. According to the clinical cut-off, subjects were divided into two groups: low-risk group (uPTM- FetA / UCr less than the clinical cut-off) and high-risk group (uPTM-FetA / UCr greater than or equal to the clinical cut-off). The Cox proportional hazard model was used to assess the hazard ratio, and the Kaplan-Meier graph and log-rank test to compare the survival curves of the two groups.

[0547] To assess whether the clinical status has an impact on the ability of uPTM-FetA / UCr in predicting the risk of worsening renal function, other variables were added to the univariate Cox proportional hazards model to fit the multivariate Cox proportional hazards model, and changes in the hazard ratio of uPTM-FetA / UCr were observed, and clinical factors were discussed. In Model 1, we performed multivariable adjusted analyses with traditional risk factors (age, sex, eGFR, urine albumin-to-creatinine ratio (UACR), HbA1c). Thereafter, we computed further models with additive adjustments to Model 1, including lipid-related parameters (i.e. total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering drugs; Model 2); blood pressure-related parameters (systolic blood pressure, diabetic blood pressure, and use of anti-hypertensive drugs; Model 3); other clinical parameters (body mass index, smoking status; Model 4); liver function abnormal or not (Aspartate transaminase (AST) or Alanine Transaminase (ALT) <5 U / L or AST or ALT >40 U / L, Model 5), and a combination of all factors above (Model 6).

[0548] UACR is commonly used as a clinical indicator to evaluate renal function. UACR > 30 mg / g is considered an important indicator for evaluating the progression of renal function decline Page: 73 of 115Attorney Docket No.132417-0001WO01 and commonly used to detect and monitor chronic kidney disease10, and may require further evaluation and treatment. To demonstrate the ability of uPTM-FetA / UCr in providing additional interpretive information in clinical use, we conduct a comparative analysis to evaluate the event rate of kidney function deterioration in comparison to uPTM-FetA / UCr (above and below the clinical cut- off) and UACR (above and below 30 mg / g) by using Cox regression model upon adjustment for baseline risk factors of age and gender.

[0549] In the validating analysis, DIALECT (validation) cohort (DIALECT) were used to validate the uPTM-FetA / UCr clinical cut-off derived from the primary analysis. The patients were divided into high-risk group (uPTM-FetA / UCr greater than or equal to the cut-off) and low-risk group (uPTM-FetA / UCr less than the cut-off) and univariate Cox model was used to estimate the hazard ratio. Survival curves of the two groups were then compared by using Kaplan-Meier plot and log-rank test. Example 6

[0550] Results of primary analysis

[0551] Among all participants enrolled in the NTUH cohort, 294 were eligible for inclusion in the current study. Mean age was 61 ± 10 years, 163 (55%) were male, eGFR was 88 ± 16 mL / min / 1.73m2, and median UACR was 9.9 [5.2 to 25.3] mg / g.

[0552] Cut-off determination and associations with kidney endpoint – NTUH cohort

[0553] During a median follow-up of 4.6 [4.2 to 4.8] years, 42 participants (14%) from the NTUH cohort reached the kidney endpoint. Based on the Youden index, the optimal uPTM-FetA / UCr cut-off for kidney endpoint risk stratification was 7.53 ng / mg (equivalent to IVD103 cut-off 0.877). Characteristics of the population in strata of uPTM-FetA / UCr groups are presented in Table 4. Figure 5A illustrates a Kaplan-Meier curve for NTUH cohort indicating increase in performance of predicting kidney function deterioration by adding uPTM-FetA / UCr in prediction model according to a non-limiting embodiment. Referring to Figure 5A, the curve is with endpoint of 30% decline or <15mL / min / 1.73 m2in eGFR in the high- and low-risk groups stratified by uPTM-FetA / Ucr levels for the NTUH. In the high-risk uPTM-FetA / Ucr group, 21% experienced the kidney endpoint, which was significantly higher compared to the 3% observed in the low-risk group (p < 0.001).

[0554] . According to this clinical cut-off, subjects were divided into low-risk group (uPTM- FetA / UCr<7.53 ng / mg) and high-risk group (uPTM-FetA / UCr≥7.53 ng / mg) (Table 4). From Table 4, there was a significant difference in the frequency of kidney function deterioration between the low- risk group and the high-risk group (2.7% and 21%, p-value<0.001) and the survival time is significantly shorter for high-risk group than that of low-risk (4.56[4.14, 4.77] vs.4.63[4.24, 4.78], p- value=0.026).

[0555] Univariate Cox proportional hazards model showed that the high-risk group had a significant 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 curve for 294 subjects in the NTUH cohort in Examples 5-7 in some embodiments. Referring to Figure 3, the Page: 74 of 115Attorney Docket No.132417-0001WO01 Kaplan-Meier curve also showed that the high-risk group had a significant risk of events relative to the low-risk group, with a log-rank test p-value<0.0001. The Kaplan-Meier curve with end point of 30% decline or <15 ml / min / 1.73m2in eGFR in the high- and low-risk groups stratified by uPTM- FetA / UCr levels for the NTUH. Figure 4A illustrates Kaplan-Meier curves for subjects in the NTUH cohort with UCr<30 mg / g in Examples 5-7 according to a non-limiting embodiment. Referring to Figure 4A and Figure 3, in the high-risk uPTM-FetA / UCr group, 21% experienced the kidney endpoint, which was significantly higher compared to the 3% observed in the low-risk group (P < 0.001). The cumulative probabilities for events in the NTUH cohort by baseline uPTM-FetA / UCr risk category at time points of 1-4 years after baseline were also displayed in Table 6. Table 4. (1) Baseline characteristics of the NTUH cohort according to uPTM-FetA / UCr grouping Risk group1VariableOverall Low-riskHigh-riskp-value2(n=294) (n=112) (n=182) Age, years 61.18 (9.86) 59.92 (10.37) 61.96 (9.49) 0.093 Gender (Male), n (%) 163 (55%) 65 (58%) 98 (54%) 0.546 Diabetes duration, years 11.00 [7.00, 16.00] 11.00 [7.75, 17.00] 11.00 [7.00, 16.00] 0.879 Baseline eGFR, ml / min / 1.73m² 88.00 (16.03) 88.97 (16.20) 87.41 (15.93) 0.418 UACR, mg / g 9.90 [5.16, 25.30] 9.02 [4.69, 21.04] 10.95 [5.39, 32.33] 0.105 UACR Category 0.060 Normal 229 (78%) 94 (84%) 135 (74%) Microalbuminuria 65 (22%) 18 (16%) 47 (26%)uPTM-FetA / UCr, ng / mg 9.52 [5.65, 20.34] 4.38 [3.34, 5.95]16.42 [10.09,30.19] <0.001HbA1c, % 7.32 (1.12) 7.24 (1.08) 7.36 (1.15) 0.388 HDL, mg / dl 46.42 (11.13) 46.49 (11.00) 46.37 (11.24) 0.930 LDL, mg / dl 101.90 (26.77) 107.23 (25.64) 98.62 (26.99) 0.007 SBP, mmHg 137.51 (18.02) 136.80 (17.59) 137.95 (18.32) 0.594 DBP, mmHg 81.37 (11.00) 80.91 (10.04) 81.65 (11.57) 0.566 Cholesterol, mg / dl 171.14 (32.94) 176.39 (30.65) 167.91 (33.95) 0.028 Triglyceride, mg / dl 139.99 (93.17) 136.23 (71.37) 142.30 (104.47) 0.555 BMI, kg / m² 24.79 (3.71) 25.24 (4.14) 24.50 (3.40) 0.114 Glucose AC, mg / dl 141.50 (37.18) 142.77 (39.39) 140.73 (35.85) 0.656 Hypertension medication, n(%)Diuretic 15 (5.1%) 4 (3.6%) 11 (6.0%) 0.423 ACEI 5 (1.7%) 2 (1.8%) 3 (1.6%) >0.999 ARB 142 (48%) 50 (45%) 92 (51%) 0.339 Beta blocker 44 (15%) 19 (17%) 25 (14%) 0.502 CCB 72 (24%) 23 (21%) 49 (27%) 0.264 Lipid medication, n (%) 109 (37%) 39 (35%) 70 (38%) 0.619 Liver function abnormality3, n 33 (11%) 9 (8.0%) 24 (13%) 0.189 (%)1uPTM-FetA / UCr > 7.53 ng / mg denotes high-risk group while uPTM-FetA / UCr < 7.53 ng / mg denote low-risk group. Page: 75 of 115Attorney Docket No.132417-0001WO01 Risk group1VariableOverall Low-riskHigh-riskp-value2(n=294) (n=112) (n=182)2Welch Two Sample t-test; Fisher's exact test; Kruskal-Wallis rank sum test.3AST / ALT<5 U / L or >40 U / L Data were presented as mean ± standard deviation (SD), median [interquartile range (IQR)], or frequency (percentage), depending on data distribution. Non-detectable u-PTM-FetAs were set to the lowest value available. Abbreviations: eGFR, estimated glomerular filtration rate as calculated using the creatinine-based CKD-EPI formula; HbA1c, glycated haemoglobin; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP,diastolic blood pressure; BMI, body mass index.Table 4. (2) Summary of 294 subjects in the NTUH cohort by kidney function decline risk RiskIVD103 1.04 (0.44) 0.61 (0.22) 1.29 (0.34) <0.001 HDL, mg / dl 46.42 (11.13) 46.49 (11.00) 46.37 (11.24) 0.930 LDL, mg / dl 101.90 (26.77) 107.23 (25.64) 98.62 (26.99) 0.007 SBP, mmHg 137.51 (18.02) 136.80 (17.59) 137.95 (18.32) 0.594 DBP, mmHg 81.37 (11.00) 80.91 (10.04) 81.65 (11.57) 0.566 Cholesterol, mg / dl 171.14 (32.94) 176.39 (30.65) 167.91 (33.95) 0.028 Triglyceride, mg / dl 139.99 (93.17) 136.23 (71.37) 142.30 (104.47) 0.555 BMI, kg / m² 24.79 (3.71) 25.24 (4.14) 24.50 (3.40) 0.114 Glucose AC, mg / dl 141.50 (37.18) 142.77 (39.39) 140.73 (35.85) 0.656 Glucose PC, mg / dl 179.50 (57.17) 174.30 (53.34) 182.70 (59.33) 0.223 Liver function abnormal, n (%) 33 (11%) 9 (8.0%) 24 (13%) 0.189 kidney function deterioration, n (%) 42 (14%) 3 (2.7%) 39 (21%) <0.001 Survival time, years 4.58 [4.16, 4.77] 4.63 [4.24, 4.78] 4.56 [4.14, 4.77] 0.0261Mean (SD); n (%); Median [IQR] 2Welch Two Sample t-test; Fisher's exact test; Kruskal-Wallis rank sum test*IVD103=log10[E103 / Urine Creatinine]

[0556] Added prognostic value on top of current markers, including UACR – NTUH cohort

[0557] The associations of the high-risk uPTM-FetA / UCr group with the kidney endpoint were also observed in univariable Cox regression analyses (HR: 8.94; 95% CI: 2.76 to 28.90; P < 0.001) and became even more pronounced in the fully adjusted model 6 (HR: 9.94; 95% CI: 2.96 to 33.40; P < 0.001, Table 5).

[0558] According to Table 5, after adding various clinical factors to carry out the multivariate Cox proportional hazards model, the ability of uPTM-FetA / UCr to predict the deterioration of renal function remained significant (p-value < 0.05) in Models 1 to 6, with a hazard ratio of 7.67-11.1. Page: 76 of 115Attorney Docket No.132417-0001WO01 Table 5. Hazard ratios of kidney function deterioration for 294 subjects in the NTUH cohort

[0559] Added value on top of UACR was also illustrated by additional analyses in subgroups depending on uPTM-FetA / UCr below or above 7.53 ng / mg and UACR below or above 30 mg / g. In the subgroup of patients with UACR < 30 mg / g, who would generally be considered at low risk of kidney function deterioration, patients with uPTM-FetA / UCr > 7.53 ng / mg had a higher risk of kidney function deterioration compared to patients with uPTM-FetA / UCr < 7.53 ng / mg (event rates: 17% vs.3%, respectively; HR: 5.14; 95%CI: 1.54 to 17.20; P = 0.008). Similarly, in the subgroup of Page: 77 of 115patients with UACR > 30 mg / g, who would generally be considered at high risk of kidney function deterioration, patients with uPTM-FetA / UCr > 7.53 ng / mg had a higher risk of kidney function deterioration, whereas there were no events at all among patients with uPTM-FetA / UCr < 7.53 ng / mg (event rates: 35% vs.0%, respectively; HR: 13.00; 95%CI: 3.76 to 44.80; P < 0.001; Table 7 and Figure 4A and 4B).

[0560] Figure 4A illustrates Kaplan-Meier curves for subjects in the NTUH cohort with UACR<30 mg / g in Examples 5-7 in some embodiments. Figure 4B. illustrates Kaplan-Meier curves for subjects in the NTUH cohort with UACR≥30 mg / g by uPTM-FetA / UCr risk category in Examples 5-7 in some embodiments. Referring to Figures 4A and 4B, the Kaplan-Meier curves showed that the patients with uPTM-FetA / UCr≥7.53 ng / mg had a significant risk of events relative to the low uPTM- FetA / UCr group for both UACR<30 mg / g and UACR≥30 mg / g with log-rank test p-values 0.0011 and 0.007, respectively. This suggested that uPTM-FetA / UCr can provide additional information to UACR in assessing risk of kidney function progression. Table 7. Hazard ratios of kidney function deterioration by uPTM-FetA / UCr and UACR category for 294 subjects in the NTUH cohort Category*N Number of 1 p-events HR (95% CI) value1. uPTM-FetA / UCr<7.53 ng / mg & UACR 90 3 Reference --- < 30 mg / g 2. uPTM-FetA / UCr≥7.53 ng / mg & UACR 135 23 5.14 (1.54, 0.008 < 30 mg / g 17.2) 3. uPTM-FetA / UCr<7.53 ng / mg & UACR 16 0 NA†NA ≥ 30 mg / g 4. uPTM-FetA / UCr≥7.53 ng / mg & UACR 47 16 13.0 (3.76, <0.001 ≥ 30 mg / g 44.8) 1HR = Hazard Ratio, CI = Confidence Interval†The HR can’t be estimated due to without event occurrence. *Adjusted for age and sex

[0561] In line with these findings, added prognostic value of uPTM-FetA / UCr on top of a current clinical model including age, sex, eGFR, UACR and HbA1c was further assessed, as shown in detail in Supplemental Table 4. Supplemental Table 4. Performance of the clinical and clinical plus biomarker prediction models for kidney function deterioration Clinical model Clinical plus biomarker model Variable Age 1.38 (0.89-2.20), 0.161 1.35 (0.85-2.18), 0.209 Gender 1.36 (0.68-2.79), 0.391 1.52 (0.73-3.23), 0.267 eGFR 0.73 (0.49-1.09), 0.115 0.70 (0.46-1.06), 0.086 UACR 1.62 (1.16-2.28), 0.005 1.56 (1.11-2.22), 0.012 HbA1c 1.04 (0.74-1.44), 0.806 1.02 (0.71-1.44), 0.909 uPTM-FetA / UCr NI 9.53 (3.27-40.7), <0.001Performance measure AIC 233.56 213.71 LRT χ2test, P 19.59, <0.001 41.44, <0.001 ΔLRT χ2test, P Reference 21.85, <0.001 H-L test χ2, P 8.16, 0.518 7.61, 0.574 AUC (95%CI) 0.70 (0.62-0.78) 0.80 (0.73-0.87) ΔAUC, P Reference 0.09, <0.001 NRI Reference 0.72 (0.52-0.92) IDI (%) Reference 7.8 (5.9-9.8) Values are given as OR (95%CI), P-value. AIC, Akaike information criterion; AUC, area under the curve; H-L, Hosmer-Lemeshow; IDI, integrated discrimination index; LRT, likelihood ratio test; NI, not included; NRI, net reclassification improvement.

[0562] Figure 6 illustrates a ROC curve indicating increase in performance of predicting kidney function deterioration by adding uPTM-FetA / UCr in prediction model according to a non- limiting embodiment. Referring to Figure 6, the clinical model including age, sex, eGFR, UACR, and HbA1c has an area under the receiver operating characteristic (AUROC) curve of 0.70 (0.62-0.78), while the AUROC curve of the clinical model plus uPTM-FetA / Ucr is 0.80 (0.73-0.87). The AUROC of clinical model and clinical plus uPTM-FetA / UCr is 0.70 (0.62-0.78) and 0.80 (0.73-0.87), respectively. The addition of the biomarker to the clinical model improved model fit (ΔLRT χ2=21.85, P<0.001), calibration (Hosmer-Lemeshow test P = 0.574), discrimination (AUC increase from 0.70 to 0.80, P < 0.001; Figure 6). The addition of biomarker improves risk classification (continuous NRI 0.72 [95%CI 0.52-0.92]) and also resulting in an overall gain in predictive ability of the model (IDI 7.8% [5.9-9.8%]). Example 7

[0563] DIALECT (validation) cohort

[0564] In total, 376 participants enrolled in the DIALECT cohort were eligible for inclusion in the current study. Mean age was 64 ± 11 years, 249 (66%) were male, eGFR was 76 ± 24 mL / min / 1.73m2, and median UACR was 9.0 [3.5 to 47.0] mg / g (Table 8).

[0565] Baseline eGFR was statistically lower than in NTUH cohort, while UACR was not significantly different (p < 0.001 and p = 0.657, respectively).

[0566] Analyses by DIALECT cohort provided positive validation, confirming that the utility of the uPTM-FetA / Ucr clinical cut-off has good predictive power for patients at risk of kidney function deterioration.

[0567] Results of validation study – DIALECT cohort

[0568] In the DIALECT cohort, 125 participants (33%) experienced the kidney endpoint during the median follow-up of 3.7 [2.5 to 5.3] years. In the high-risk uPTM-FetA / Ucr group, 44% experienced the kidney endpoint, which was higher compared to the 26% observed in the low-risk group (P < 0.001).

[0569] Of the 376 subjects, 111(30%) experienced kidney function deterioration during follow-up, and their median survival time was 3.65 years (IQR, 2.51-5.33 years). According to the clinical cut-off of uPTM-FetA / Ucr obtained by NTUH cohort of 0.7.53 ng / mg, subjects were divided into low-risk (uPTM-FetA / Ucr<7.53 ng / mg) group and high-risk (uPTM-FetA / Ucr≥7.53 ng / mg) group (Table 8). There was a significant difference in the frequency of kidney function deterioration (26 % and 44%, p-value<0.001).

[0570] The result of 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 1.87 (95% confidence interval 1.31-2.66, p-value<0.001). Figure 5B illustrates a Kaplan-Meier curve for the DIALECT cohort indicating increase in performance of predicting kidney function deterioration by adding uPTM-FetA / Ucr in prediction model according to a non-limiting embodiment. Figure 5B illustrates Kaplan-Meier curve for 376 subjects in the DIALECT (validation) cohort in Examples 5-7 in some embodiments. Referring to Figure 5B, the curve is with endpoint of 30% decline or <15mL / min / 1.73 m2in eGFR in the high- and low-risk groups stratified by uPTM- FetA / Ucr levels for the NTUH. The Kaplan-Meier curve also showed that the high-risk group had a significant risk of events relative to the low-risk group, with a log-rank test p-value=0.00041. The cumulative probabilities for events in the DIALECT (validation) cohort by baseline uPTM-FetA / Ucr risk category at time points of 1-4 years after baseline were also displayed in Table 9. Table 8. (1) Baseline characteristics of the DIALECT cohort according to uPTM-FetA / Ucr grouping Risk group1VariableOverall1Low-riskHigh-riskp-value2(n=376) (n=223) (n=153) Age, years 64.44 (10.75) 62.44 (9.94) 67.36 (11.25) <0.001 Gender (Male), n (%) 249 (66%) 131 (59%) 118 (77%) <0.001Diabetes duration, years13.00 [7.00,13.00 [7.00, 19.00] 19.00]13.00 [7.00, 22.00] 0.163Baseline eGFR, ml / min / 1.73m² 76.30 (23.98) 81.80 (20.16) 68.28 (26.77) <0.001UACR, mg / g 8.99 [3.52, 46.95] 6.80 [3.07, 18.21]25.31 [5.55,205.69] <0.001uPTM-FetA / Ucr, ng / mg 5.76 [0.76, 17.16] 1.30 [0.06, 3.94]25.75 [14.24,48.77] <0.001HbA1c, % 7.57 (1.11) 7.57 (1.11) 7.58 (1.11) 0.926 HDL, mg / dl 43.17 (12.33) 44.20 (11.92) 41.67 (12.79) 0.054 LDL, mg / dl 80.96 (32.03) 81.00 (32.81) 80.90 (30.96) 0.976 SBP, mmHg 131.46 (15.87) 131.45 (14.55) 131.48 (17.66) 0.986 DBP, mmHg 74.25 (9.82) 74.45 (8.70) 73.95 (11.28) 0.646 Cholesterol, mg / dL 165.35 (39.95) 167.98 (40.12) 161.52 (39.53) 0.123 Triglyceride, mg / dL 218.25 (156.65) 226.76 (173.30) 205.85 (128.14) 0.180 BMI, kg / m² 32.05 (5.78) 32.21 (5.56) 31.81 (6.08) 0.519 Glucose AC, mg / dL 174.62 (103.55) 171.12 (89.47) 179.71 (121.31) 0.4551uPTM-FetA / Ucr > 7.53 ng / mg denotes high-risk group while uPTM-FetA / Ucr < 7.53 ng / mg denote low-risk group.Risk group1VariableOverall1Low-riskHigh-riskp-value2(n=376) (n=223) (n=153)2Welch Two Sample t-test; Fisher’s exact test; Kruskal-Wallis rank sum test. Data were presented as mean ± standard deviation (SD), median [interquartile range (IQR)], or frequency (percentage), depending on data distribution. Non-detectable u-PTM-FetAs were set to the lowest value available. Abbreviations: eGFR, estimated glomerular filtration rate as calculated using the creatinine-based CKD-EPI formula; HbA1c, glycated haemoglobin; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index. Table 8. (2) Summary of DIALECT 376 subjects grouped by kidney function decline risk Risk groupVariableOverall1 Low-riskHigh-riskp-2 (n=376)(n=223)(n=153)valueAge, years 64.44 (10.75) 62.44 (9.94) 67.36 (11.25) <0.001 Gender (Male), n (%) 249 (66%) 131 (59%) 118 (77%) <0.001Diabetes duration, years13.00 [7.00,13.00 [7.00, 19.00] 19.00]13.00 [7.00, 22.00] 0.163eGFR, ml / min / 1.73m² 76.30 (23.98) 81.80 (20.16) 68.28 (26.77) <0.001UACR, mg / g 9.03 [3.52, 47.17] 6.86 [3.06, 18.38]25.31 [5.55,205.69] <0.001HbA1c, % 7.57 (1.11) 7.57 (1.11) 7.58 (1.11) 0.926E103, ng / mL11.65 [1.90,24.93] 1.90 [1.90, 5.82]20.25 [12.46, 35.16]<0.001E103 / Ucr, ng / mg 5.83 [0.77, 17.45] 1.30 [0.06, 3.94]25.75 [14.24,48.77] <0.001IVD103* 1.09 (0.60) 0.47 (0.27) 1.47 (0.41) <0.001 HDL, mg / dl 43.17 (12.33) 44.20 (11.92) 41.67 (12.79) 0.054 LDL, mg / dl 80.96 (32.03) 81.00 (32.81) 80.90 (30.96) 0.976 SBP, mmHg 131.46 (15.87) 131.45 (14.55) 131.48 (17.66) 0.986 DBP, mmHg 74.25 (9.82) 74.45 (8.70) 73.95 (11.28) 0.646 Cholesterol, mg / dL 165.35 (39.95) 167.98 (40.12) 161.52 (39.53) 0.123 Triglyceride, mg / dL 218.25 (156.65) 226.76 (173.30) 205.85 (128.14) 0.180 BMI, kg / m² 32.05 (5.78) 32.21 (5.56) 31.81 (6.08) 0.519 Glucose, mg / dL 174.62 (103.55) 171.12 (89.47) 179.71 (121.31) 0.455 kidney function deterioration, n(%)125 (33%) 57 (26%) 68 (44%) <0.001Survival time, years 3.65 [2.51, 5.33] 3.83 [2.79, 5.69] 3.44 [2.12, 5.25] 0.1471Mean (SD); n (%); Median [IQR]2Welch Two Sample t-test; Fisher’s exact test; Kruskal-Wallis rank sum test*IVD103=log10[E103 / Urine Creatinine] Table 9. Cumulative probability (with 95% confidence intervals) of event for kidney function deterioration at various time points for subjects in the DIALECT (validation) cohort. Category Time points1Year 2 Year 3 Year 4 YearCategory Time points1Year 2 Year 3 Year 4 Year(6%, 16%) (12%, 24%) (25%, 40%) (30%, 46%) ANALYSIS OF EXAMPLE 4-7

[0571] In this study, uPTM-FetA was associated with risk of eGFR decline in patients with type 2 diabetes, independent of age, sex, eGFR, UACR, baseline HbA1c, and multiple other potential confounders. These findings were validated in a second, larger cohort of patients with type 2 diabetes, highlighting the potential clinical use of uPTM-Fet-A as a predictive marker for DKD.

[0572] The human precursor protein of fetuin A consists of three parts, namely the A chain, connecting peptide, and B chain, which are 321, 40, and 27 amino acids in length, respectively. The connecting peptide of the precursor is removed by a posttranslational modification, limited proteolysis, after which only the A and B chain form the active fetuin A protein. The monoclonal antibody in the ELISA kit that was used in the current study (Human uPTM3-DKD ELISA kit, BPM Corp.) only detects the connecting peptide-containing fetuin A.

[0573] We found that elevated uPTM-FetA is associated with progression of renal function decline in type 2 diabetes patients. These findings are generally consistent with previous studies that showed associations of urinary fetuin A with albuminuria and lower kidney function type 2 diabetes patients with DKD. In some embodiments, the concentration of uPTM-FetA is not related to serum C-FetA among patients (in-house data from NTUH cohort, data not shown). The incongruence between circulating and urine fetuin A concentrations was also observed in other studies. This suggests that there may be local production of fetuin-A in the kidney in patients with kidney in patients with kidney diseases including DKD. uPTM-FetA specifically accumulates in urine of patients with progressive renal decline. This is likely attributable 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 needs to be further investigated in future preclinical and interventional studies. These results show that in non-CKD type 2 diabetes patients, uPTM-FetA was to UACR multivariable-adjusted Cox regression analyses. This suggests that uPTM-FetA may be better suitable for predicting renal function decline than UACR. Moreover, the use of antihypertensives such as angiotensin receptor blockers and angiotensin converting enzyme inhibitors, which are frequently prescribed in type 2 diabetes patients, reduces albuminuria, thus limited usefulness of UACR as a predictive marker of DKD. uPTM-FetA may not have this disadvantage.

[0574] There were studies establishing prediction model DKD for type 2 diabetes patients using clinical parameters with or without aid of machine learning, but the follow-up duration was rather short. Evaluation of eGFR within only a period of 5 years is considered insufficient for assessing renal function deterioration in type 2 diabetes patients, as intra‑individual variability ofeGFR is high among early DKD patients. Long-term follow up of renal function in type 2 diabetes patients is essential, given the fact that whole- kidney hyperfiltration with increased eGFR is seen in substantial portion of this population and may mask the development of kidney disease in early phase.

[0575] There are several strengths of this study. The current study include use of two large, well-characterized cohorts allowed for extensive adjustments for potential confounders. Follow-up duration in NTUH cohort is long, which allows adequate assessment of associations of uPTM-FetA with renal outcome not underestimated by the issue of renal hyperfiltration in early DKD. Finally, the use of two independent, bi- ethnic cohorts including patients from different geographical areas, is reassuring with regard to the external validity of these findings to other type 2 diabetes populations worldwide.

[0576] In conclusion, this study demonstrates that in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes, uPTM-FetA is strongly associated with renal function deterioration, independent of albuminuria, eGFR, age, sex, and multiple other risk factors. The added prognostic value of uPTM-FetA on top of albuminuria and other markers is particularly clear in patients without CKD at baseline. These findings are validated in a large, independent, second cohort, which is promising with regard to the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0577] The results of this study demonstrate that, for type 2 diabetes patients, uPTM- FetA / UCr can effectively predict the risk of renal function deterioration, independent of albuminuria, eGFR, age, sex, HbA1c, lipid-related parameters, blood pressure-related parameters, BMI, smoking status, and liver function status. The added prognostic value of uPTM-FetA / UCr on top of UACR is particularly clear. The clinical effectiveness of the uPTM-FetA / UCr clinical cut-off (=7.53 ng / mg) was validated in an independent type 2 diabetes cohort.

[0578] These result shows that uPTM-FetA / UCr can effectively predict the actual decrease of eGFR. Patients with high risk can take early treatment or related adjustments to achieve the effect of prevention, which has considerable clinical benefits and will be a boon for type 2 diabetes patients.

[0579] In this study, uPTM-FetA was associated with risk of eGFR decline in patients with type 2 diabetes, and provided significant added prognostic value independent of age, sex, eGFR, UACR, baseline HbA1c, and multiple other potential confounders. These findings were validated in a second, larger cohort of patients with type 2 diabetes, highlighting the potential clinical use of uPTM- FetA as a predictive marker for DKD.

[0580] The human precursor protein of fetuin-A consists of three parts, namely the A chain, connecting peptide, and B chain, which are 321, 40, and 27 amino acids in length, respectively. The connecting peptide of the precursor is removed by a posttranslational modification, limited proteolysis, after which only the A and B chain form the active fetuin-A protein. The monoclonalantibody in the ELISA kit that was used in the current study (Human uPTM3-DKD ELISA kit, BPM Corp.) only detects the connecting peptide-containing fetuin-A.

[0581] We found that elevated uPTM-FetA is associated with progression of renal function decline in patients with type 2 diabetes. These findings are generally consistent with previous studies that showed associations of urinary fetuin-A with albuminuria and lower kidney function in patients with type 2 diabetes and DKD. Intriguingly, the concentration of uPTM-FetA is not related to serum PTM-FetA among patients (in-house data from NTUH cohort, data not shown). The incongruence between circulating and urine fetuin-A concentrations was also observed in other studies. This indicates that there is a local production of fetuin-A in the kidney in patients with kidney diseases including DKD.

[0582] Fetuin-A is not synthesized by the kidney of human or animal model under healthy conditions. However, upon injury, proximal tubule epithelial cells (PTEC) are able to synthesize fetuin-A and release it to the apical side of the tubule. In an animal study using cisplatin-induced and ischemia / reperfusion-induced acute kidney injury rat models, fetuin-A is detected in the urinary exosome fraction instead of in the soluble non-exosomal fraction, indicating that fetuin-A in the urine is produced by the PTEC. In addition to that, urinary fetuin-A level increases before serum creatinine surge and also before tubular injury present morphologically. It is hypothesized that local production of fetuin-A by the tubular cells protects the tubules from ongoing inflammation and fibrosis. Whether uPTM-FetA may be merely a predictive marker or also has direct pathogenic effects needs to be further investigated in future preclinical and interventional studies.

[0583] Nevertheless, our findings show that in patients with type 2 diabetes, uPTM-FetA was strongly associated with renal function decline, in contrast to UACR in the multivariable-adjusted Cox regression analyses. Moreover, marker has clear added prognostic value on top of a current clinical model including UACR. This suggests that uPTM-FetA may be better suitable for predicting kidney function decline than UACR, or at least provide added clinical value. Moreover, the use of antihypertensives such as angiotensin receptor blockers and angiotensin converting enzyme inhibitors, which are frequently prescribed in patients with type 2 diabetes, reduces albuminuria, thus limited usefulness of UACR as a predictive marker of DKD. Potentially, uPTM-FetA may not have this disadvantage, although future research is needed to assess the changes in uPTM-FetA after initiation of these antihypertensives.

[0584] There are several strengths of this study. The current study included two large, well- characterized cohorts, which allowed for validation and extensive adjustments for potential confounders. Bi-ethnic cohorts including patients from different geographical areas, is reassuring with regard to the external validity of these findings to other type 2 diabetes populations worldwide. Also, despite the difference between eGFR and albuminuria baseline status in these two cohorts, the prognostic ability of uPTM-FetA remains significant. Thus, this biomarker may be potentially usefulin patients with type 2 diabetes in various condition. Our study also has some limitations. The follow- up time in the validation cohort is considerably shorter than that in the primary cohort. Also, due the observational design of the current study, we are only able to assess the prognostic abilities of uPTM- FetA and cannot confirm its causal effects on kidney disease progression. Moreover, how other markers such as kidney injury molecule-1 and neutrophil gelatinase- associated lipocalin compare to uPTM-FetA can provide helpful information on its meaning and interpretation. Finally, none of the patients used SGLT2-inhibitors at baseline, and only a small fraction of the included patients started SGLT2- inhibitor use during the follow-up period. The use of these drugs may not be expected to change the added value of uPTM-FetA / Ucr in predicting eGFR decline in patients with type 2 diabetes, inhibitors.

[0585] This study demonstrates that in two large, independent, bi-ethnic prospective cohorts of patients with type 2 diabetes, uPTM-FetA is strongly associated with renal function deterioration, independent of albuminuria, eGFR, age, sex, and multiple other risk factors. These findings were validated in a large, independent, second cohort, which is promising with regard to the potential scientific and clinical use of uPTM-FetA in type 2 diabetes.

[0586] EXAMPLE 8

[0587] A plurality of risk categories was developed for prognosis renal function decline based on a biomarker based on the cohort data analyzed in EXAMPLES 4-7. The biomarker in this example was Human unique PTM Fetuin-A (E103). For the plurality of risk categories, two “cut-off” values as a plurality of points as threshold values were developed as shown in Table 8-1 below. Table 8-1. Develop risk categories for prognosis renal function decline based on a novel biomarker

[0588] In this example, renal function deterioration was defined as need for renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal- or haemodialysis) or a persistent increase of >50% in serum creatinine from baseline visit for at least 3 months.

[0589] Based on the two Cut-off 1 and Cut-off 2 values as the two threshold values, the three risk categories as indicated in Table 8-2 were developed. Table 8-2

[0590] Cohort data were analyzed as indicated in Table 8-3, 8-4, 8-5, 8-6 and Figures 7 and 8 below. Figure 7 illustrates a Kaplan-Meier curve indicating survival probability over time in years, regarding the cut-off 1 value of uPTM-FetA / UCr, according to a non-limiting embodiment. Figure 8 illustrates a Kaplan-Meier curve indicating event-free probability over time in years, regarding the cut-off 2 value of uPTM-FetA / UCr, according to a non-limiting embodiment. Table 8-3. Performance for Cut-off 1 – DemographicTable 8-4. Performance for Cut-off 2 – DemographicTable 8-5. Cut-off 1Table 8-6. Cut-off 2

[0591] Based on the analysis above, biomarker-based scoring for predicting renal function decline was developed. The scoring is based on a urine test that measures a single biomarker (DNlite- IVD103). The urine test was combined with five clinical factors that can be considered relatively simple. The five clinical factors were age, gender, eGFR, UACR, and HbA1c.

[0592] The scoring was based on the following equations based on Table 8-7: Occurrence score=exp(y) / (1+exp(y)), y = a0 whereinE103 / UCr is equal or less than 7.53 ng / mg Table 8-7

[0593] Thresholds and risk categories based on these scoring were as in Tables 8-8 and 8-9: Table 8-8. Thresholds for risk groupsFigures 9 and 10 Figure 9 illustrates a Kaplan-Meier curve indicating progressive-free probability over time in years, regarding the two cut off values of occurrence scores, according to a non-limiting embodiment. Figure 10 illustrates a cumulative incidence plot over occurrence score, according to a non-limiting embodiment. Table 8-10. CoxPH modelTable 80-11, Risk Ratio

[0595] Additional data analysis are provided as below in Tables 8-12, 8-13, 8-14, 8-15, 8-16, 8-17, and Figures 11 and 12. Figure 11 illustrates a Kaplan-Meier curve for the DIALECT cohort indicating progressive-free probability over time in years, regarding the two cut off values of occurrence scores, according to a non-limiting embodiment. Figure 12 illustrates a cumulativeincidence plot over occurrence score for the DIALECT cohort, according to a non-limiting embodiment. Table 8-12. Predictive Model based on NTUH cohort (N=294) Clinical Model Clinical plus E1R103 / UCr ModelO95% CI1 p-value OR1 95% CI1 p-valueAGE 1.03 0.99, 1.08 0.161 1.03 0.98, 1.08 0.209 SEX (M) 1.36 0.68, 2.79 0.391 1.52 0.73, 3.23 0.267 eGFR 0.98 0.96, 1.01 0.115 0.98 0.95, 1.00 0.086 UACR 1.50 1.13, 2.00 0.005 1.45 1.09, 1.96 0.012 HbA1c 1.04 0.76, 1.39 0.806 1.02 0.73, 1.39 0.909 E103 / UCr>7.53 ng / mg 9.53 3.27, 40.7 <0.001AUC 0.704 0.797 1 OR = Odds Ratio, CI = Confidence Interval Table 8-13, Summary of thresholdsTable 8-14. NTUH cohort Risk group VariableOverall,Low riskN = 294 N = 147 N = 103AGE 61.18 (9.86) 58.26 (10.38) 62.32 (7.93) 68.26 (8.06) <0.001 SEX(M) 163 (55%) 80 (54%) 54 (52%) 29 (66%) 0.294 eGFR 88.00 (16.03) 92.13 (15.96) 88.89 (11.39) 72.13 (16.11) <0.001 UACR 2.29 [1.64, 3.23]1.95 [1.35,2.77] 2.36 [1.73, 3.08] 3.92 [2.91, 4.58] <0.001HbA1c 7.32 (1.12) 7.25 (1.09) 7.37 (1.16) 7.40 (1.16) 0.637 IVD103 1.04 (0.44) 0.78 (0.38) 1.26 (0.33) 1.33 (0.37) <0.001 E103 / UCr9.52 [5.64,5.59 [3.50, 16.43 [10.04, 17.23 [10.13, 20.34]7.53] 27.24]40.18]<0.001Score 0.14 (0.13) 0.04 (0.03) 0.18 (0.04) 0.39 (0.10) <0.001Risk group VariableOverall,N = 294Event 42 (14%) 6 (4.1%) 18 (17%) 18 (41%) <0.001 Time 4.58 [4.16, 4.77]4.63 [4.23,4.54 [4.15, 4.78] 4.56 [2.85, 4.78] 0.0271Mean (SD); n (%); Median [IQR] 2One-way ANOVA; Fisher’s exact test; Kruskal-Wallis rank sum test Table 8-15. DIALECT Risk group VariableOverall,Low riskN = 376 N = 239 N = 47AGE 64.44 (10.75) 61.64 (9.92) 65.57 (8.88) 71.29 (10.67) <0.001 SEX(M) 249 (66%) 139 (58%) 38 (81%) 72 (80%) <0.001 eGFR 76.30 (23.98) 84.73 (18.90) 77.86 (20.83) 53.10 (22.54) <0.001 UACR 2.20 [1.26, 3.85] 1.72 [1.05, 2.69] 1.92 [1.37, 3.61] 4.74 [3.38, 5.91] <0.001 HbA1c 7.57 (1.11) 7.58 (1.08) 7.43 (1.13) 7.64 (1.17) 0.566 IVD103 1.35 (0.47) 1.00 (0.37) 1.27 (0.30) 1.60 (0.43) <0.001 E103 / UCr18.80 [10.19,7.27 [5.83, 15.35 [11.70, 35.42 [19.86, 43.63]14.72] 31.45]68.01]<0.001Score 0.19 (0.25) 0.03 (0.03) 0.17 (0.04) 0.59 (0.18) <0.001 Event 125 (33%) 57 (24%) 17 (36%) 51 (57%) <0.001 Time 3.65 [2.51, 5.33] 4.05 [2.84, 5.89] 3.83 [2.13, 5.60] 2.96 [1.22, 4.36] <0.001 1Mean (SD); n (%); Median [IQR] 2One-way ANOVA; Fisher’s exact test; Kruskal-Wallis rank sum test Table 8-16, Cox modelTable 8-17 Baseline risk Risk Cumulative incidence Risk ratio

[0596] INTENDED USE

[0597] The Human uPTM3-DKD ELISA is a colorimetric immunoassay intended for quantitative measurement of unique Fetuin-A with specific post translational modification (PTM) in human urine and should be performed at qualified clinical laboratories by certified medical professionals, such as Medical Technologists. This product is for in vitro diagnostic use as an aid in risk assessment of renal complications in diabetic patients. Furthermore, the uPTM3-DKD test could also be applied to accurately differentiate progressive decliners of renal function in type 2 diabetes with microalbuminuria.

[0598] REAGENTS PREPARATION

[0599] 1.Human unique PTM Fetuin-A (E103) Calibrators

[0600] Reconstitute the E103 Calibrator with 0.2 mL distilled or deionized water, sit for 10 minutes at room temperature until completely dissolved and mix gently. The reconstituted E103 Calibrator is now at a concentration of 5 μg / mL. Dilute the 5 μg / mL E103 Calibrator with Diluent by 5-fold, then mix gently to geta 1 μg / mL E103 Calibrator. Procedures for the serial dilution to generate all calibrators for establishing E103calibration curve are shown in the following table and all calibrators must be prepared and mixed well immediately prior to use.

[0601] 2.1X Wash Buffer

[0602] Recover 10X Wash Buffer to room temperature prior to use until all the salt crystals are dissolved. Calculate the required amount of 1X Wash Buffer for each assay. For each microplate, mix 50 mL 10X Wash Buffer with 450 mL distilled or deionized water. Mix uniformly but gently.

[0603] 3.1X mAb anti-unique PTM Fetuin-A (E103)

[0604] Calculate the required amount of 1X mAb anti-unique PTM Fetuin-A (E103) for each assay, and mix 1000XmAb anti-unique PTM Fetuin-A (E103) with Diluent according to the amount required. For each microplate, mix 8 μL 1000X mAb anti-unique PTM Fetuin-A (E103) with 8 mL Diluent. Mix uniformly but gently.

[0605] 4.1X HRP Conjugate

[0606] Calculate the required amount of 1X HRP Conjugate for each assay, and mix 4000X HRP Conjugate with Diluent according to the amount required. For each microplate, mix 3 μL 4000X HRP Conjugate with 12 diluents. Mix uniformly but gently.

[0607] MATERIALS REQUIRED BUT NOT PROVIDED

[0608] ․ Precision single micropipettes (1-10 μL, 20-100 μL, 100-200 μL, 200-1000 μL) and multi-channel pipettes (100 μL)

[0609] ․ Microcentrifuge tubes and disposable tips

[0610] ․ Graduated cylinder 500 mL

[0611] ․ Vortex mixer and microcentrifuge

[0612] ․ Orbital shaker

[0613] ․ Plastic container for the preparation of reagents

[0614] ․ Microplate reader capable of endpoint measurement at 450±10 nm

[0615] ․ Distilled or deionized water

[0616] ․ Adhesive plate seals

[0617] ․ Reagent reservoirs

[0618] WARNINGS AND PRECAUTIONS

[0619] ․ This test is for professional in vitro diagnostic use only.

[0620] ․ Do not use reagents after expiration date.

[0621] ․ Improper temperature exposure during all storage and assay procedures can adversely affect results.

[0622] ․ Do not reuse the microplate wells.

[0623] ․ Wear protective gloves during all assay procedures.

[0624] ․ Being light sensitive and skin irritative, TMB Substrate should avoid direct light exposure and skin contact during all storage and assay procedures.

[0625] ․ Avoid skin contact with Stop Solution containing 0.5 N sulfuric acid. It may cause skin irritation and burns.

[0626] ․ Consider all clinical specimens potentially infectious.

[0627] ․ Disposal of any discarded materials should be in accordance with local requirements and existing regulations for good laboratory practice.

[0628] SPECIMEN COLLECTION AND HANDLING

[0629] ․ Morning urine samples must be collected in clean and dry containers. Avoid cross contamination.

[0630] ․ No additives or preservatives are necessary for integrity of urine samples.

[0631] ․ Urine samples are stored at -20°C until to be used. Avoid repeated freezing and thawing of urine samples.

[0632] ․ Before performing the assay, recover urine samples to room temperature. Centrifuge urine samples for 5 minutes at 1,000±20 x g. Take supernatant and assay immediately.

[0633] ․ Use Diluent for sample dilution if necessary.

[0634] ASSAY PROCEDURE

[0635] Prepare enough microplate modules for all calibrators and urine samples and secure them in a holder. Recover all reagents to room temperature prior to use.

[0636] 1. Mix Calibrator 1-8 and each centrifuged urine sample with 1X mAb anti-E103 in a 1:1 ratio in microcentrifuge tubes (for triplicate tests, mixing 180 μL of each Calibrator / sample with 180 μL 1X mAb anti-E103 is recommended). Incubate for 2 hours at 25°C and 200 rpm on an orbital shaker.

[0637] 2. Transfer 100 μL of each incubated mixture into assigned wells of 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 during all incubations.

[0638] 3. After the incubation, discard the contents in the wells.

[0639] 4. Wash each well with 300-400 μL 1X Wash Buffer. Discard the contents and sharply strike the wells on absorbent paper to remove residual liquid. Wash for a total of 4 times.

[0640] 5. Add 100 μL 1X HRP Conjugate into each well. Incubate the microplate for 60 minutes at 25°C and 200 rpm on an orbital shaker.

[0641] 6. After the incubation, discard the contents in the wells and wash the wells as described in Step 4.

[0642] 7. Add 100 μL TMB Substrate into each well. Incubate in the dark for 30 minutes at room temperature.

[0643] 8. Add 100 μL Stop Solution into each well. Mix by a brief shaking until the mixture become homogeneous.

[0644] 9. Determine the absorbance at 450±10 nm within 30 minutes and calculate the results.

[0645] CALCULATION OF RESULTS

[0646] 1. Use a 5- or 4- parameter logistic curve fit to establish a calibration curve. The concentration of unique PTM Fetuin-A in patient urine samples can be calculated from the calibration curve by interpolation. The range of this assay is 7.813 - 500 ng / mL.

[0647] 2. Calculate the unique PTM Fetuin-A quantity with a urinary creatinine correction (ng / mg Cr).

[0648] PERFORMANCE CHARACTERISTICS

[0649] Precision

[0650] Precision of the Human uPTM3-DKD ELISA was evaluated in a 5 day study. Using 3 human urine samples from known diabetic patients and 5 spiked human urine samples, the samples were tested twice a day in triplicates. Within-Run precision: Coefficient of variation (CV) was calculated for each of eight samples from the results of 3 determinations in each run. Between-Run precision: Coefficient of variation (CV) was calculated for each of eight samples from the results of 3 determinations each in 10 different runs.

[0651] The precision results are summarized in the following table:

[0652] Measuring range

[0653] 7.813 - 500 ng / mL

[0654] Linearity

[0655] The test was assessed and found to be linear from 1.769 to 500 ng / mL (R2=0.99).

[0656] Detection limit

[0657] Limit of Blank (LoB) = 0.873 ng / mL

[0658] Limit of Detection (LoD) = 4.510 ng / mL

[0659] Limit of Quantitation (LoQ) = 18.9830 ng / mL

[0660] For example, Figure 2 illustrates a calibration curve of the Human uPTM3-DKD ELISA (calibration range is 7.813 - 500 ng / mL) according to a non-limiting embodiment.

[0661] While preferred embodiments of the present invention have been shown and described herein, it will be obvious 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 should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed:

1. An assay kit to determine a likelihood of a progressive renal decline, comprising: a first solution including a first reagent to interact with a post-translationally modified Fetuin- A fragments in urine (uPTM-FetA) to indicate the level of the Fetuin A fragment in a urine sample from a subject diagnosed with type 2 diabetes mellitus; a second solution including a second reagent to interact with a urinary creatinine to indicate the level of the urinary creatinine in the urine sample; a device to determine the level of the Fetuin A fragment and the level of the urinary creatinine in the urine sample, to determine a ratio of the determined level of the Fetuin A fragment to the determined level of the urinary creatinine, wherein the ratio being higher than about 7.5 ng / mg indicates a higher likelihood of a progressive renal decline to a kidney endpoint over a period of about ten years in the subject, and wherein the first solution and the second solution are the same solution or different solution from each other.

2. The assay kit of claim 1, wherein the ratio being higher than about 7.5 ng / mg triggers a medical intervention to obviate the progressive renal decline.

3. The assay kit of claim 1, wherein the higher likelihood is higher than a lower likelihood of the progressive renal decline over the period of about ten years in a second subject having a lower ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 7.5 ng / mg.

4. The assay kit of claim 1, wherein the higher likelihood is lower than a likelihood of the progressive renal decline over the period of about ten years in a third subject having a higher ratio of the determined level of the Fetuin A fragment to the level of the urinary creatinine lower than about 29.0 ng / mg.

5. The assay kit of claim 1, wherein a value of the ratio higher than 7.5 ng / mg indicates a degree of the higher likelihood of the progressive renal failure.

6. The assay kit of claim 1, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).

7. The assay kit of claim 6, wherein the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL / min / 1.73 m2) or higher.

8. The assay kit of claim 1, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

9. The assay kit of claim 1, wherein the subject has been indicated to have a Urinary Albumin-to-creatinine ratio (UACR) lower than about 30 milligram per gram (mg / g) in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.

10. The assay kit of claim 1, wherein the subject or the second subject was diagnosed with type 2 diabetes mellitus from about 7 years to about 20 years prior to collecting the urine sample.

11. The assay kit of claim 1, wherein the ratio being higher than about 7.5 ng / mg is from about 7.5 ng / mg to about 29 ng / mg.

12. The assay kit of claim 1, wherein the ratio being higher than about 7.5 ng / mg is higher than about 29 ng / mg.

13. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher.

14. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 15 % or higher.

15. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 20 % or higher.

16. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 25 % or higher.

17. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % or higher.

18. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 35 % or higher.

19. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % or higher.

20. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 45 % or higher.

21. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 50 % or higher.

22. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % to about 50%.

23. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%.

24. The assay kit of claim 1, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 40 % to about 50%.

25. The assay kit of claim 1-23, wherein the progressive renal is to decline to the kidney endpoint after 4 years.

26. The assay kit of claim 1, wherein the progressive renal is to decline to the kidney endpoint after 5 years.

27. The assay kit of claim 1, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment.

28. The assay kit of claim 1, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine.

29. The assay kit of claim 1, wherein at least one reagent among the first reagent and the second reagent includes an antibody.

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