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EP4655585A1 - Dosage et procédés, kits et dispositifs associés - Google Patents

Dosage et procédés, kits et dispositifs associés

Info

Publication number
EP4655585A1
EP4655585A1 EP24747962.9A EP24747962A EP4655585A1 EP 4655585 A1 EP4655585 A1 EP 4655585A1 EP 24747962 A EP24747962 A EP 24747962A EP 4655585 A1 EP4655585 A1 EP 4655585A1
Authority
EP
European Patent Office
Prior art keywords
kidney
endpoint
decline
progressive renal
present disclosure
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24747962.9A
Other languages
German (de)
English (en)
Inventor
Tzu-Ling Tseng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bio Preventive Medicine Corp
Original Assignee
Bio Preventive Medicine Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bio Preventive Medicine Corp filed Critical Bio Preventive Medicine Corp
Publication of EP4655585A1 publication Critical patent/EP4655585A1/fr
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/493—Physical analysis of biological material of liquid biological material urine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4713—Plasma globulins, lactoglobulin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • Diabetic nephropathy also known as Diabetic Kidney Disease (DKD)
  • DKD Diabetic Kidney Disease
  • DN Diabetic nephropathy
  • DKD Diabetic Kidney Disease
  • DN Diabetic nephropathy
  • DKD Diabetic Kidney Disease
  • SUMMARY 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney 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.
  • 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.
  • 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.
  • the present disclosure relates to a method, wherein the first threshold value is about 0.1.
  • 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).
  • eGFR estimated Glomerular Filtration Rate
  • 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.
  • 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).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to Page: 4 of 115 Attorney 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.
  • 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.
  • 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%.
  • 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%.
  • 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 115 Attorney Docket No.132417-0001WO01 method, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 30 % to about 40%.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney 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.
  • 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.
  • 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.
  • the present disclosure relates to an assay kit, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).
  • 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.
  • 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).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • 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 115 Attorney 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).
  • 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.
  • 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).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney 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.
  • 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.
  • 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. [00116] 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. [00117] 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.
  • 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. [00119] 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. [00120] 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. [00121] 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.
  • 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. [00123] 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. [00124] 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. [00125] 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.
  • 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. [00127] 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. [00128] 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 115 Attorney Docket No.132417-0001WO01 [00129] 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.
  • 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. [00131] 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. [00132] 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. [00133] 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.
  • 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%. [00135] 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%. [00136] 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%. [00137] 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%.
  • 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%. [00139] 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%. [00140] 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%. [00141] 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%.
  • 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%. [00143] 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%. [00144] 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%. [00145] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 4 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 5 years. [00147] 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 115 Attorney Docket No.132417-0001WO01 [00148] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 7 years. [00149] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 8 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 9 years. [00151] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 7 years. [00152] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 8 years. [00153] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 9 years. [00154] 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.
  • the present disclosure relates to a method, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. [00156] 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.
  • 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.
  • 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.
  • Figure 3 illustrates a Kaplan-Meier curve for 294 subjects in the NTUH cohort in Examples 5-7 according to a non-limiting embodiment.
  • 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.
  • 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.
  • 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 115 Attorney Docket No.132417-0001WO01
  • 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 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.
  • 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.
  • 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.
  • 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 cumulative incidence plot over occurrence score for the DIALECT cohort, according to a non-limiting embodiment.
  • DKD diabetic kidney disease
  • CKD chronic kidney disease
  • EKD end-stage kidney disease
  • 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.
  • DKD can have five progression phases: [00174] Stage 1: characterized by diabetic mellitus with normal GFR and normal albuminuria (e.g., ACR ⁇ 30 mg/g); [00175] Stage 2: characterized by glomerular hyperfiltration (greater than 120 mL/minute/1.73 m 2 ) and renal enlargement accompanying with normal GFR and normal albuminuria (e.g., ACR ⁇ 30 mg/g); Page: 17 of 115 Attorney Docket No.132417-0001WO01 [00176] Stage 3: characterized by microalbuminuria; [00177] Stage 4: characterized by overt albuminuria and a progressive decline in GFR; and [00178] Stage 5: characterized by a GFR of less than 15 mL
  • stages 1-3 are deemed as early stage and stages 4 and 5 are deemed as late stage.
  • 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.
  • 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).
  • 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.
  • up to 40% of patients with T2DM who suffer from renal function worsening remain normoalbuminuric.
  • due to the low sensitivity of albuminuria its use in predicting DKD progression is limited. [00181] 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.
  • 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 m 2 is considered as an indication of the kidney end point.
  • 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 115 Attorney Docket No.132417-0001WO01 [00184] 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.
  • 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.
  • it can be the middle point between the mean levels of the two groups of DKD patients and DKD-free subjects.
  • 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.
  • a biomarker level higher than the reference point is indicative of the target condition, such as DKD.
  • biomarker levels among the plurality of points are indicative of different stages or progresses of the target condition, such as DKD.
  • 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.
  • Fetuin-A encoded by AHSG gene, is a hepatokine associated with insulin resistance and is an independent risk factor of T2DM.
  • Fetuin-A 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.
  • 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.
  • urinary Fetuin-A has Page: 19 of 115 Attorney 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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 115 Attorney 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to a method, wherein the first threshold value is about 0.1. [00204] In some embodiments, the present disclosure relates to a method, wherein the first threshold value is about 0.11. [00205] In some embodiments, the present disclosure relates to a method, wherein the second threshold value is about 0.25. [00206] In some embodiments, the present disclosure relates to a method, wherein the second threshold value is about 0.3. [00207] In some embodiments, the present disclosure relates to a method, wherein the second threshold value is about 0.27.
  • the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).
  • eGFR estimated Glomerular Filtration Rate
  • 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.
  • 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).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney Docket No.132417-0001WO01 [00218] 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. [00219] 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.
  • 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. [00221] 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. [00222] 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. [00223] 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.
  • 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. [00225] 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. [00226] 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. [00227] 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.
  • 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. [00229] 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. [00230] 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. [00231] 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.
  • 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. [00233] 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. [00234] 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. [00235] 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.
  • 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 115 Attorney Docket No.132417-0001WO01 [00237] 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. [00238] 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.
  • 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. [00240] 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. [00241] 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. [00242] 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.
  • 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. [00244] 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. [00245] 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. [00246] 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.
  • 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.
  • 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%.
  • 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%.
  • 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%.
  • 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%. [00252] 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%. [00253] 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%. [00254] 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%.
  • 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 115 Attorney Docket No.132417-0001WO01 [00256] 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%. [00257] 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%.
  • 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%. [00259] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 4 years. [00260] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 5 years. [00261] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 6 years. [00262] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 7 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 8 years. [00264] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 9 years. [00265] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 7 years. [00266] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 8 years. [00267] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 9 years.
  • the present disclosure relates to a method, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. [00269] In some embodiments, the present disclosure relates to a method, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. [00270] 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.
  • 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 115 Attorney Docket No.132417-0001WO01 higher than about 7.5 ng/mg, more preferably higher than about 7.53 ng/mg, more
  • 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.
  • 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.
  • 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.
  • 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. [00276] 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present disclosure relates to an assay kit, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).
  • eGFR estimated Glomerular Filtration Rate
  • 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.
  • 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).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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. [00289] 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. [00290] 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.
  • 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. [00292] 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. [00293] 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. [00299] 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. [00300] 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. [00301] 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.
  • 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. [00303] 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. [00304] 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. [00305] 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.
  • 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. [00307] 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. [00308] 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.
  • 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 115 Attorney Docket No.132417-0001WO01 [00310] 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. [00311] 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.
  • 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. [00313] 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. [00314] 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. [00315] 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.
  • 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. [00317] 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. [00318] 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. [00319] 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.
  • 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. [00321] 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. [00322] 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. [00323] 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%.
  • 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%. [00325] 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%. [00326] 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%. [00327] 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%.
  • 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 115 Attorney Docket No.132417-0001WO01 [00329]
  • 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%.
  • 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%.
  • 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%. [00332] 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%. [00333] 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%. [00334] 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.
  • the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint after 5 years. [00336] 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. [00337] 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. [00338] 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. [00339] 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.
  • the present disclosure relates to an assay kit, wherein the progressive renal is to decline to the kidney endpoint within 7 years. [00341] 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. [00342] 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. [00343] 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.
  • the present disclosure relates to an assay kit, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. [00345] 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.
  • 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 115 Attorney 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
  • 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.
  • 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.
  • 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.
  • 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. [00351] 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.
  • 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.
  • 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.
  • 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. [00357] 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.
  • the present disclosure relates to a method, wherein the progressive renal decline is a decline in estimated Glomerular Filtration Rate (eGFR).
  • eGFR estimated Glomerular Filtration Rate
  • 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.
  • 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).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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.
  • 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 115 Attorney Docket No.132417-0001WO01 [00365]
  • 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.
  • 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. [00367] 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. [00368] 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. [00369] 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.
  • 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. [00371] 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. [00372] 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. [00373] 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.
  • 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. [00375] 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. [00376] 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. [00377] 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.
  • 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. [00379] 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. [00380] 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. [00381] 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.
  • 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.
  • 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 115 Attorney Docket No.132417-0001WO01 [00384]
  • 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.
  • 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. [00386] 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. [00387] 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. [00388] 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.
  • 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. [00390] 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. [00391] 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. [00392] 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.
  • 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. [00394] 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. [00395] 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. [00396] 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.
  • 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. [00398] 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%. [00399] 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%. [00400] 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%.
  • 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%. [00402] 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 115 Attorney Docket No.132417-0001WO01 [00403] 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%.
  • 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%. [00405] 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%. [00406] 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%. [00407] 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%.
  • 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%. [00409] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 4 years. [00410] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 5 years. [00411] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 6 years. [00412] In some embodiments, the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 7 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 8 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint after 9 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 7 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 8 years.
  • the present disclosure relates to a method, wherein the progressive renal is to decline to the kidney endpoint within 9 years.
  • the present disclosure relates to a method, wherein the interacting with the Fetuin A fragment includes binding to the Fetuin A fragment. [00419] In some embodiments, the present disclosure relates to a method, wherein the interacting with the urinary creatinine includes binding to the urinary creatinine. [00420] 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.
  • 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 115 Attorney 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
  • 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
  • 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
  • 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 115 Attorney 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
  • the ratio being higher than about 7.5 may trigger a medical intervention to obviate the progressive renal decline.
  • 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/m
  • the progressive renal decline may be a decline in estimated Glomerular Filtration Rate (eGFR).
  • eGFR estimated Glomerular Filtration Rate
  • the subject has been indicated to have the eGFR of about 60 milliliter per minute per 1.73 square meter (mL/min/1.73 m 2 ) or higher.
  • 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 m 2 ).
  • 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.
  • UCR Urinary Albumin-to- creatinine ratio
  • 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.
  • 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.
  • 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.
  • 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. [00434] 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.
  • the ratio being higher than about 7.5 ng/mg may be higher than about 7.6 ng/mg.
  • the ratio being higher than about 7.5 ng/mg may be higher than about 9 ng/mg.
  • the ratio being higher than about 7.5 ng/mg may be higher than about 10 ng/mg.
  • the ratio being higher than about 7.5 ng/mg may be higher than about 12 ng/mg.
  • the ratio being higher than about 7.5 ng/mg may be higher than about 14 ng/mg.
  • the ratio being higher than about 7.5 ng/mg may be higher than about 15 ng/mg.
  • 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.
  • 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.
  • the second ratio being lower than about 7.5 ng/mg may be lower than about 7 ng/mg.
  • 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.
  • 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 115 Attorney 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. [00438] In some embodiments, the second ratio being lower than about 7.5 ng/mg may be lower than about 4.0 ng/mg.
  • 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.
  • 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. [00439] 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.
  • 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.
  • 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. [00442] 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.
  • 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 115 Attorney 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.
  • 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%.
  • 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%.
  • 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%. [00443] 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%.
  • 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%.
  • 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.
  • 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. [00445] 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.
  • 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. [00446] 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.
  • 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.
  • the present disclosure supports that elevated uPTM-FetA is associated with progression of renal function decline in type 2 diabetes patients.
  • 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).
  • the incongruence between circulating and urine fetuin Page: 41 of 115 Attorney 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. [00448] In non-CKD type 2 diabetes patients, uPTM-FetA was to UACR multivariable- adjusted Cox regression analyses.
  • uPTM-FetA may be better suitable for predicting renal function decline than UACR.
  • 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.
  • 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.
  • ASSAY 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.
  • the enzyme-linked immunosorbent assay ELISA
  • ELISA enzyme-linked immunosorbent assay
  • 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 115 Attorney Docket No.132417-0001WO01 Technologists.
  • PTM post translational modification
  • 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.
  • mAb anti-unique PTM Fetuin-A monoclonal antibody
  • the monoclonal antibody recognizes Fetuin-A in calibrators or unknown samples under competition in microplate wells.
  • signal-generating molecule such as signal-molecule-tagged antibody can be used.
  • a Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3',5,5'- tetramethylbenzidine (TMB) substrate.
  • HRP horse Radish Peroxide
  • TMB 3,3',5,5'- tetramethylbenzidine
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney 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 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 m 2 ) or higher. 20.
  • eGFR estimated Glomerular Filtration Rate
  • 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 m 2 ).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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.
  • 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 115 Attorney 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • eGFR estimated Glomerular Filtration Rate
  • 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 m 2 ) 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 m 2 ).
  • 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.
  • UCR Urinary Albumin-to-creatinine ratio
  • 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 115 Attorney Docket No.132417-0001WO01 in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.
  • UCR Urinary Albumin-to-creatinine ratio
  • mg/g milligram per gram Page: 50 of 115 Attorney Docket No.132417-0001WO01 in the previous 12 months before collecting the urine sample or around the time of collecting the urine sample.
  • 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 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 47 ng/mg. 112.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • at least one reagent among the first reagent and the second reagent includes an antibody.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • eGFR estimated Glomerular Filtration Rate
  • UCR Urinary Albumin-to-creatinine ratio
  • 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 156-189, 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 156-198, wherein the higher likelihood of the progressive renal decline to the kidney endpoint is about 10 % or higher.
  • 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.
  • 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.
  • 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.
  • 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 115 Attorney 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.
  • Example 1 Study design [00457] 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. [00458] Regarding Examples 1-3, Supplemental Table 1 indicates Cohort characteristics and inclusion and exclusion criteria.
  • uPTM-FetA measurements [00460] 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.
  • 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 m 2 , or a need of renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal dialysis or hemodialysis).
  • eGFR estimated glomerular filtration rate
  • IVD103 ⁇ optimal cut-off 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.
  • 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).
  • 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.
  • Model 2 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).
  • Model 5 body mass index, smoking status; Model 5
  • 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.
  • NTUH cohort [00470]
  • 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.
  • uPTM- FetA as an early marker of DKD
  • NTUH cohort of NTUH study only included individuals with baseline eGFR>60 ml/min/1.73m 2 and 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 [00471] Baseline characteristics [00472] NTUH cohort [00473] Among all participants enrolled in the NTUH cohort, eventually 223 were eligible for inclusion in the current study.
  • 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 [00475] Cut-off determination and associations with kidney endpoint – NTUH cohort [00476] 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.
  • FIG. 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.
  • 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.
  • DIALECT validation
  • DIALECT validation cohort
  • 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.
  • DIALECT (validation) cohort analyses 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 115 Attorney Docket No.132417-0001WO01 described for the primary analyses were repeated in this DIALECT (validation) cohort, with the same kidney endpoint as outcome variable.
  • 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.
  • 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.
  • We found that elevated uPTM-FetA is associated with progression of renal function decline in type 2 diabetes patients.
  • 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.
  • 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.
  • 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.
  • 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 [00497] 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.
  • the Human uPTM3-DKD ELISA is a competitive immunoassay.
  • 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.
  • a Horse Radish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate.
  • HRP Horse Radish Peroxide
  • Example 5 Study design [00501] 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.
  • NTUH National Taiwan University Hospital
  • DIALECT LifEstyle Cohort Twente
  • 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.
  • E103 specific post translational modification
  • 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.
  • 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.
  • mAb monoclonal antibody
  • HRP Horse Radish Peroxide
  • TMB 3,3’,5,5’-tetramethylbenzidine
  • 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.
  • NTUH cohort (NTUH) [00512] 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
  • DIALECT (validation) cohort (DIALECT) [00515] 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.
  • uPTM-FetA concentrations were divided by urinary creatinine (UCr) into uPTM-FetA/UCr for normalization.
  • Ur urinary creatinine
  • [00519] 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.
  • [00520] 2. Add 50 ⁇ L Calibrator 1-8 and urine sample into each well of E103 Coated Microplate respectively.
  • [00521] 3. Add 50 ⁇ L 1X mAb anti-E103 into each well of E103 Coated Microplate.
  • [00522] 4. Incubate for 2 hours at 25°C and 200 rpm on an orbital shaker. [00523] 5. After the incubation, discard the contents in the wells.
  • 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 m 2 , or a need of renal replacement therapy (i.e., kidney transplantation, initiation of peritoneal dialysis or hemodialysis).
  • eGFR estimated glomerular filtration rate
  • ROC receiver operating characteristic
  • Model 2 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 115 Attorney Docket No.132417-0001WO01 mass index, smoking status; Model 4); liver function abnormality (Model 5); and a combination of all factors above (Model 6). [00537] To evaluate the prognostic performance of our biomarker, uPTM-FetA/UCr, we incorporated it into the clinical plus biomarker model.
  • Model fit was assessed using the Akaike information criterion (AIC) and the likelihood ratio test (LRT), where lower AIC values or higher ⁇ 2 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.
  • DIALECT (validation) cohort analyses 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.
  • LoQ 5.428 ng/mL
  • LoD Limit of Detection
  • the E103 values were set to as LoD.
  • Patients with E103 values smaller than LoD were set to as "non-detectable.
  • 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 m 2 , 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.
  • 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. [00547] 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).
  • Model 1 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).
  • lipid-related parameters i.e. total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, and use of lipid-lowering drugs
  • 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
  • UACR > 30 mg/g is considered an important indicator for evaluating the progression of renal function decline Page: 73 of 115 Attorney Docket No.132417-0001WO01 and commonly used to detect and monitor chronic kidney disease 10 , and may require further evaluation and treatment.
  • 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.
  • DIALECT (validation) cohort 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.
  • Results of primary analysis [00551] Among all participants enrolled in the NTUH cohort, 294 were eligible for inclusion in the current study.
  • 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.
  • the curve is with endpoint of 30% decline or ⁇ 15mL/min/1.73 m 2 in eGFR in the high- and low-risk groups stratified by uPTM-FetA/Ucr levels for the NTUH.
  • the Page: 74 of 115 Attorney 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/1.73m 2 in 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.
  • 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
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • Results of validation study – DIALECT cohort [00568] 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). [00569] 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).
  • FIG. 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.
  • the curve is with endpoint of 30% decline or ⁇ 15mL/min/1.73 m 2 in eGFR in the high- and low-risk groups stratified by uPTM- FetA/Ucr levels for the NTUH.
  • 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.
  • 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
  • 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.
  • 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.
  • the concentration of uPTM-FetA is not related to serum C-FetA among patients (in-house data from NTUH cohort, data not shown).
  • 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 may be a predictive marker of progressive kidney disease.
  • uPTM-FetA may have direct pathogenic effects needs to be further investigated in future preclinical and interventional studies.
  • 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.
  • 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.
  • uPTM-FetA/UCr The added prognostic value of uPTM-FetA/UCr on top of UACR is particularly clear.
  • 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.
  • 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.
  • 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.
  • 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.
  • PTEC proximal tubule epithelial cells
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • uPTM-FetA may not have this disadvantage, although future research is needed to assess the changes in uPTM-FetA after initiation of these antihypertensives.
  • 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 useful in 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.
  • kidney injury molecule-1 and neutrophil gelatinase- associated lipocalin compare to uPTM-FetA can provide helpful information on its meaning and interpretation.
  • kidney injury molecule-1 and neutrophil gelatinase- associated lipocalin can provide helpful information on its meaning and interpretation.
  • the biomarker in this example was Human unique PTM Fetuin-A (E103).
  • E103 Human unique PTM Fetuin-A
  • 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 [00588] 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.
  • the three risk categories as indicated in Table 8-2 were developed.
  • Table 8-2 [00590] 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 – Demographic Table 8-4. Performance for Cut-off 2 – Demographic Table 8-5. Cut-off 1 Table 8-6.
  • Cut-off 2 [00591] 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.
  • CoxPH model Table 80-11, Risk Ratio [00595] 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 cumulative incidence plot over occurrence score for the DIALECT cohort, according to a non-limiting embodiment. Table 8-12.
  • E103 Human unique PTM Fetuin-A (E103) Calibrators
  • E103 Calibrator 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 ⁇ g/mL. Dilute the 5 ⁇ g/mL E103 Calibrator with Diluent by 5-fold, then mix gently to geta 1 ⁇ g/mL E103 Calibrator.
  • SPECIMEN COLLECTION AND HANDLING [00629] . Morning urine samples must be collected in clean and dry containers. Avoid cross contamination. [00630] . No additives or preservatives are necessary for integrity of urine samples. [00631] . Urine samples are stored at -20°C until to be used. Avoid repeated freezing and thawing of urine samples. [00632] . 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. [00633] . Use Diluent for sample dilution if necessary.
  • ASSAY PROCEDURE 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. [00636] 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 ⁇ L of each Calibrator/sample with 180 ⁇ L 1X mAb anti-E103 is recommended). Incubate for 2 hours at 25°C and 200 rpm on an orbital shaker. [00637] 2.
  • 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.
  • calibration range is 7.813 - 500 ng/mL

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Abstract

La présente invention concerne la corrélation d'un rapport du niveau déterminé du fragment de Fétuine A au niveau déterminé de la créatinine urinaire avec une probabilité d'un déclin rénal progressif à un point final rénal sur une période de temps chez le sujet.
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