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WO2015153860A1 - Biomarqueurs de débit de filtration glomérulaire et leurs utilisations - Google Patents

Biomarqueurs de débit de filtration glomérulaire et leurs utilisations Download PDF

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WO2015153860A1
WO2015153860A1 PCT/US2015/024040 US2015024040W WO2015153860A1 WO 2015153860 A1 WO2015153860 A1 WO 2015153860A1 US 2015024040 W US2015024040 W US 2015024040W WO 2015153860 A1 WO2015153860 A1 WO 2015153860A1
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biomarker
gfr
subject
level
capture reagent
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Stephen Alaric Williams
David Sterling
Robert Kirk Delisle
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Somalogic Inc
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Somalogic Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present disclosure relates generally to the detection of biomarkers and the characterization of glomerular filtration rate (GFR) and renal function. For example, to determine the GFR in a subject or whether renal function in a subject has declined and/or decreased below previous levels or a control standard level, and to predict or estimate GFR in a subject.
  • the invention relates to one or more biomarkers, methods, devices, reagents, systems, and kits for characterizing GFR and/or renal function in an individual.
  • Glomerular filtration rate is a measure of the volume of fluid filtered from the kidney per unit time and is an accurate marker of kidney function. Assessment of actual GFR may be accomplished through usage of the contrast agent iohexol. Iohexol is a low molecular weight compound (-820 Daltons), with low blood protein binding that is excreted unchanged by the kidney. Measuring iohexol (or iothalamate) clearance periods provides a physiological method for measuring GFR, however the procedure is complicated, time consuming, and requires intravenous administration of an iodinated compound. For these reasons, this direct method of measuring GFR is not used for general, routine assessment.
  • Blood creatinine concentration has long been used to assess kidney function, but factors such as muscle mass, red meat ingestion, and recent vigorous exercise are known to cause higher than expected creatinine levels, leading to misinterpretations of actual kidney function. Additionally, with measurements such as blood creatinine concentration (and blood urea nitrogen), changes in these markers do not occur until kidney function has declined to at least 60% of normal. This lack of sensitivity at GFR levels closer to the accepted normal level of 100 ml/min/1.73 m 2 , limit their use in early detection of declining renal function.
  • Cystatin-C is an endogenous cysteine protease inhibitor produced ubiquitously by nucleated cells. Being of low molecular weight (-13 kD) and thus freely filtered by the glomerulus, not actively excreted by the kidney, and fully metabolized by the tubular epithelial cells, Cystatin-C represents an ideal candidate for an endogenous marker of kidney function. It has been suggested, however, to be altered in cardiovascular disease in a kidney-independent manner (Stevens LA, 2005) as well as Alzheimer's disease (Mi W, 2007) (Kaeser SA, 2007).
  • the present disclosure meets such needs by providing a method for measuring biomarkers in the blood that may be used to assesses and/or characterize GFR and/or kidney function in an individual, particularly at earlier stages of decline that existing clinical tools.
  • the present disclosure provides for a method for estimating the glomerular filtration rate (GFR) in a subject from a biomarker panel comprising a number (N) of biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • GFR glomerular filtration rate
  • the present disclosure further provides for a method for determining whether the renal function in a subject is declining comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • the biomarker panel comprises the biomarker proteins Trefoil Factor 3 (TFF3) and Endostatin (COL18A1).
  • the biomarker panel further comprises at least one additional biomarker protein selected from the group consisting of UNC5D, CST3, AMH, CHRDLl, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • the biomarker panel further comprises the biomarker protein UNC5D.
  • biomarker panel further comprises the biomarker protein CST3 and AMH or FCN2.
  • biomarker panel further comprises the biomarker protein CST3,
  • the biomarker panel further comprises the biomarker protein CST3, AMH and CTSL2.
  • biomarker panel further comprises the biomarker protein CST3, FCN2 and CTSL2.
  • the biomarker panel further comprises the biomarker protein UNC5D and PI3.
  • the biomarker panel comprises the biomarker proteins Trefoil Factor 3 (TFF3) and Endostatin (COL18A1), and optionally one or more additional biomarker proteins selected from the group consisting of UNC5D, CST3, AMH, CHRDL1, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • TNF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • the present disclosure further provides a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3) and Endostatin (COL18A1) in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6.
  • GFR glomerular filtration rate
  • the correspondence correlation is at least about 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the method further comprises detecting the level of at least one additional biomarker protein selected from the group consisting of UNC5D, CST3, AMH, CHRDL1, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • the method further comprises detecting the level of the biomarker protein UNC5D.
  • the method further comprises detecting the level of the biomarker protein CST3 and AMH or FCN2. In another aspect, the method further comprises detecting the level of the biomarker protein CST3, AMH and CHRDL1 or PI3.
  • the method further comprises detecting the level of the biomarker protein CST3, AMH and CTSL2.
  • the method further comprises the biomarker protein CST3, FCN2 and CTSL2.
  • the method further comprises detecting the level of the biomarker protein UNC5D and PI3.
  • the method comprises detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3) and Endostatin (COL18A1), and optionally one or more additional biomarker proteins selected from the group consisting of UNC5D, CST3, AMH, CHRDL1, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • TNF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • the present disclosure further provides a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 and NBL1 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6.
  • GFR glomerular filtration rate
  • the correspondence correlation is at least about 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the method further comprises detecting the level of at least one additional biomarker protein selected from the group consisting of CST3, B2M and IGFBP6.
  • the method further comprises detecting the level of the biomarker proteins CST3, B2M and IGFBP6.
  • the present disclosure further provides a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins CST3 and Endostatin (COL18A1) in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6.
  • GFR glomerular filtration rate
  • the correspondence correlation is at least about 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the method further comprises detecting the level of the biomarker protein CHRDL1.
  • compositions and kits are provided, wherein the composition or kit comprises N capture reagents, wherein each capture reagent specifically binds to a biomarker protein selected from the biomarker proteins listed in Table 2.
  • a composition or kit comprises a first capture reagent that specifically binds to Trefoil Factor 3 (TFF3) and a second capture reagent that specifically binds to Endostatin (COL18A1).
  • composition or kit further comprises at least one capture reagent that specifically binds to a biomarker protein selected from the group consisting of UNC5D, CST3, AMH, CHRDL1, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • a composition or kit comprises a first capture reagent that specifically binds to Trefoil Factor 3 (TFF3) and a second capture reagent that specifically binds to Endostatin (COL18A1), and optionally at least one additional capture reagent that specifically binds to a biomarker protein selected from the group consisting of UNC5D, CST3, AMH, CHRDL1, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • TNF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • a composition or kit comprises a first capture reagent that specifically binds to TFF3, a second capture reagent that specifically binds to COL18A1, and a third capture reagent that specifically binds to the biomarker protein UNC5D.
  • a composition or kit comprises a first capture reagent that specifically binds to TFF3, a second capture reagent that specifically binds to COL18A1, a third capture reagent that specifically binds the biomarker protein CST3 and a fourth capture reagent that specifically binds the biomarker protein AMH or FCN2.
  • a composition or kit comprises a first capture reagent that specifically binds to TFF3, a second capture reagent that specifically binds to COL18A1, a third capture reagent that specifically binds the biomarker proteins CST3, a fourth capture reagent that specifically binds the biomarker protein AMH, and a fifth capture reagent that specifically binds the biomarker protein CHRDL1 or PI3.
  • a composition or kit comprises a first capture reagent that specifically binds to TFF3, a second capture reagent that specifically binds to COL18A1, a third capture reagent that specifically binds the biomarker protein CST3, a fourth capture reagent that specifically binds the biomarker protein FCN2, and a fifth capture reagent that specifically binds the biomarker protein CTSL2.
  • a composition or kit comprises a first capture reagent that specifically binds to TFF3, a second capture reagent that specifically binds to COL18A1, a third capture reagent that specifically binds the biomarker protein UNC5D, and a fourth capture reagent that specifically binds the biomarker protein PI3.
  • a composition comprises proteins of a biological sample.
  • the biological sample is selected from blood, plasma, serum, and urine.
  • the method further comprises that the biomarker proteins are detected with a capture reagent.
  • the biomarker proteins are detected by contacting the sample from the subject with the capture reagent.
  • the capture reagent is selected from the group consisting of an aptamer, antibody, small molecule, nucleic acid ligand and any combination thereof.
  • the biomarker proteins are detected by contacting the sample from the subject with the aptamer.
  • the aptamer comprises a C-5 modified pyrimidine.
  • the C-5 modified pyrimidine is independently, and for each occurrence, selected from the group consisting of the C-5 modified pyrimidines of Figure 20.
  • the C-5 modified pyrimidine is independently, and for each occurrence, selected from the group consisting of 5-(N-benzylcarboxamide)-2'-deoxycytidine (BndC); 5-(N-2-phenylethylcarboxamide)-2'-deoxycytidine (PEdC); 5-(N-3- phenylpropylcarboxamide)-2'-deoxycytidine (PPdC); 5-(N-l-naphthylmethylcarboxamide)- 2'-deoxycytidine (NapdC); 5-(N-2-naphthylmethylcarboxamide)-2'-deoxycytidine (2NapdC); 5-(N- 1 -naphthyl-2-ethylcarboxamide)-2'-deoxycytidine (NEdC); 5-(N-2-naphthyl-2- ethylcarboxamide)-2'-deoxycytidine (2NEdC); 5-(N
  • the aptamer is from about 25 to about 100 nucleotides in length.
  • the biomarker proteins are detected with an aptamer-based assay.
  • the correspondence correlation is selected from the group consisting of at least about 0.6 or more, 0.62 or more, 0.63 or more, 0.64 or more, 0.65 or more, 0.66 or more, 0.67 or more, 0.68 or more, 0.69 or more, 0.7 or more, 0.71 or more, 0.72 or more, 0.73 or more, 0.74 or more, 0.75 or more, 0.76 or more, 0.77 or more, 0.78 or more, 0.79 or more and 0.8 or more, when the actual GFR is equal to or more than about 60 mL/min./1.73m 2 .
  • the correspondence correlation is selected from the group consisting of at least about 0.7 or more, 0.71 or more, 0.72 or more, 0.73 or more, 0.74 or more, 0.75 or more, 0.76 or more, 0.77 or more, 0.78 or more, 0.79 or more, 0.8 or more, 0.81 and 0.82 or more when the actual GFR is equal to or more than about 40 mL/min./1.73m 2 .
  • the correspondence correlation is selected from the group consisting of at least about 0.9 or more, 0.91 or more, 0.92 or more, 0.93 or more, 0.94 or more, 0.95 or more, 0.96 or more and 0.97 or more when the actual GFR is equal to or more than about 1 mL/min./1.73m 2 .
  • N may be selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In any of the embodiments described herein, N may be selected from 2, 3, 4, 5, and 6. In any of the embodiments described herein, N may be 4 or 5.
  • the subject is male.
  • FIG. 1 shows the correlation of Cystatin-C measurements with actual GFR.
  • Timepoints A and B correspond to pre- and post-iohexol treatment samples, respectively.
  • FIG. 2 shows the plot for CST3 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 3 shows the plot for B2M protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 4 shows the plot for TFF3 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 5 shows the plot for NBL1 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 6 shows the plot for AMH protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 7 shows the plot for IGFP6 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 8 shows the plot for CHRDL1 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 9 shows the plot for Elafin (PI3) protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 10 shows the plot for UNC5D protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 11 shows the plot for COL18A1 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 12 shows the plot for FCN2 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 13 shows the plot for CTSL2 protein measured with an aptamer based assay over the full range of GFR (X axis is GFR and Y axis is the RFU as measured by an aptamer based assay).
  • FIG. 14 shows the predicted GFR vs. actual GFR for the five (CST3, B2M, NBL1,
  • TFF3 and IGFBP6 marker variable model The dotted line is the best fit for the plot.
  • FIG. 15 shows the predicted GFR vs. actual GFR for the single (CST3) marker variable model.
  • the line is the 45 degree line (ideal line), which is provided as a reference for comparison purposes of what a perfect set of predicted GFR value would like relative to the actual GFR values.
  • FIG. 16 shows the predicted GFR vs. actual GFR for a two (TFF3 and NBL1) marker variable model.
  • the line is the 45 degree line (ideal line), which is provided as a reference for comparison purposes of what a perfect set of predicted GFR value would like relative to the actual GFR values.
  • FIG. 17 shows the predicted GFR vs. actual GFR for a five (TFF3, CST3, COL18A1, AMH and CHRDL1) marker variable model.
  • the line is the 45 degree line (ideal line), which is provided as a reference for comparison purposes of what a perfect set of predicted GFR value would like relative to the actual GFR values.
  • FIG. 18 shows the predicted GFR vs. actual GFR for a five (TFF3, CST3, COL18A1, AMH and CHRDL1) marker variable model.
  • the line is the 45 degree line (ideal line), which is provided as a reference for comparison purposes of what a perfect set of predicted GFR value would like relative to the actual GFR values.
  • FIG. 19 shows the predicted GFR vs. actual GFR for a three (CST3, COL18A1 and
  • CHRDL1 marker variable model.
  • the line is the 45 degree line (ideal line), which is provided as a reference for comparison purposes of what a perfect set of predicted GFR value would like relative to the actual GFR values.
  • FIG. 20 shows certain exemplary modified pyrimidines that may be incorporated into aptamers, such as slow off-rate aptamers.
  • FIG. 21 illustrates a non- limiting exemplary computer system for use with various computer-implemented methods described herein.
  • ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (as well as fractions thereof unless the context clearly dictates otherwise).
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the terms “include” and “comprise” are open ended and are used synonymously.
  • the terms “comprises,” “comprising,” “includes,” “including,” “contains,” “containing,” and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements may include other elements not expressly listed.
  • the number and identity of biomarkers in a panel are selected based on the sensitivity and specificity for the particular combination of biomarker values.
  • sensitivity and “specificity” are used herein with respect to the ability to correctly classify an individual, based on one or more biomarker levels detected in a biological sample, as having a decreased GFR, decreased determined GFR or decreased GFR range or reduced kidney function or not having a decreased GFR or reduced kidney function.
  • Sensitivity indicates the performance of the biomarker(s) with respect to correctly classifying individuals with a decreased GFR or reduced kidney function.
  • Specificity indicates the performance of the biomarker(s) with respect to correctly classifying individuals who do not have a decreased GFR or reduced kidney function.
  • 85% specificity and 90% sensitivity for a panel of markers used to test a set of control samples (such as samples from healthy individuals or subjects known not to have a decreased GFR or reduced kidney function) and test samples (such as samples from individuals having a decreased GFR or reduced kidney function) indicates that 85% of the control samples were correctly classified as control samples by the panel, and 90% of the test samples were correctly classified as test samples by the panel.
  • overall performance of a panel of one or more biomarkers is represented by the area-under-the-curve (AUC) value.
  • the AUC value is derived from a receiver operating characteristic (ROC) plot.
  • the ROC curve is the plot of the true positive rate (sensitivity) of a test against the false positive rate (1 -specificity) of the test.
  • area under the curve or "AUC” refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art.
  • AUC measures are useful for comparing the accuracy of a classifier across the complete data range.
  • Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest (e.g., a decreased GFR or reduced kidney function and non-decreased GFR or non-reduced kidney function).
  • ROC curves are useful for plotting the performance of a particular feature (e.g., any of the biomarkers described herein and/or any item of additional biomedical information) in distinguishing between two populations (e.g., cases having a decreased GFR or reduced kidney function and controls, which may be cases without a decreased GFR or reduced kidney function).
  • the feature data across the entire population e.g., all decreased GFR or reduced kidney function cases
  • the true positive and false positive rates for the data are calculated.
  • the true positive rate is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases.
  • the false positive rate is determined by counting the number of controls above the value for that feature and then dividing by the total number of controls.
  • ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single sum value, and this single sum value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve.
  • GFR Standardular filtration rate
  • GFR is the flow rate of filtered fluid through the kidney. More specifically, it is the volume of fluid filtered from
  • GFR renal glomerular capillaries into the Bowman's capsule per unit time.
  • the normal range of GFR, adjusted for body surface area, is 100-130 ml/min/1.73m 2 in men and women.
  • GFR measured by inulin clearance is 110 ml/min/1.73m 2 until 2 years of age in both sexes, and then it progressively decreases. After age 40, GFR decreases progressively with age, by about 0.4 - 1.2 mL/min per year.
  • a decline or decrease of the GFR from a previous test result or predetermined standard e.g., see GFR and associated chronic kidney disease
  • kidney disease chronic kidney disease
  • MDRD-eGFR the most severe three are defined by the MDRD-eGFR value, and first three also depend on whether there is other evidence of kidney disease (e.g., proteinuria):
  • CKD5D is another classification for stage 5 patients requiring dialysis.
  • many patients in CKD5 are not yet on dialysis.
  • Glomerular filtration rate is equal to the Clearance Rate when any solute is freely filtered and is neither reabsorbed nor secreted by the kidneys.
  • the "actual GFR" is a rate that may be measured by the quantity of the substance in the urine that originated from a calculable volume of blood. For example, relating this principle to the below equation - for the substance used, the product of urine concentration and urine flow equals the mass of substance excreted during the time that urine has been collected. This mass equals the mass filtered at the glomerulus as nothing is added or removed in the nephron.
  • GFR glomerular filtration rate
  • Another method for determining the "actual GFR" is by injecting inulin or the inulin- analog sinistrin into the plasma. Since both, inulin and sinsitrin, are neither reabsorbed nor secreted by the kidney after glomerular filtration, their rate of excretion is directly
  • the inulin clearance may slightly overestimate the glomerular function. In early stage renal disease, the inulin clearance may remain normal due to hyperfiltration in the
  • actual GFR may be determined by iohexol injection and monitoring. For example, a individual is administered an iohexol injection (IV injection), and the blood and urine levels are monitored to determine how much of the iohexol is cleared per unit of time.
  • IV injection iohexol injection
  • the actual GFR is based on the clearance rate over time for an injected substance.
  • the injected substance is selected from the group consisting of iohexol, inulin and sinistrin.
  • Bio sample “sample”, and “test sample” are used interchangeably herein to refer to any material, biological fluid, tissue, or cell obtained or otherwise derived from an individual. This includes blood (including whole blood, leukocytes, peripheral blood mononuclear cells, plasma, and serum), sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, lymph fluid, nipple aspirate, bronchial aspirate (e.g., bronchoalveolar lavage), bronchial brushing, synovial fluid, joint aspirate, organ secretions, cells, a cellular extract, and cerebrospinal fluid.
  • blood including whole blood, leukocytes, peripheral blood mononuclear cells, plasma, and serum
  • sputum tears, mucus
  • nasal washes nasal aspirate, breath, urine, semen, saliva
  • a blood sample can be fractionated into serum, plasma, or into fractions containing particular types of blood cells, such as red blood cells or white blood cells (leukocytes).
  • a sample can be a combination of samples from an individual, such as a combination of a tissue and fluid sample.
  • biological sample also includes materials containing homogenized solid material, such as from a stool sample, a tissue sample, or a tissue biopsy, for example.
  • biological sample also includes materials derived from a tissue culture or a cell culture.
  • exemplary methods for obtaining a biological sample can be employed; exemplary methods include, e.g., phlebotomy, swab (e.g., buccal swab), and a fine needle aspirate biopsy procedure.
  • tissue susceptible to fine needle aspiration include lymph node, lung, lung washes, BAL (bronchoalveolar lavage), thyroid, breast, pancreas, and liver.
  • Samples can also be collected, e.g., by micro dissection (e.g., laser capture micro dissection (LCM) or laser micro dissection (LMD)), bladder wash, smear (e.g., a PAP smear), or ductal lavage.
  • a "biological sample” obtained or derived from an individual includes any such sample that has been processed in any suitable manner after being obtained from the individual.
  • a biological sample is selected from blood, plasma, serum, and urine.
  • a biological sample may be derived by taking biological samples from a number of individuals and pooling them, or pooling an aliquot of each individual's biological sample.
  • the pooled sample may be treated as described herein for a sample from a single individual, and, for example, if a poor prognosis is established in the pooled sample, then each individual biological sample can be re -tested to determine which individual(s) have a decreased GFR, a GFR value or range that is considered below a normal value or range (e.g., 90mL/min/1.73m 2 or below) or reduced kidney function.
  • Target refers to any molecule of interest that may be present in a biological sample.
  • a "molecule of interest” includes any minor variation of a particular molecule, such as, in the case of a protein, for example, minor variations in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component, which does not substantially alter the identity of the molecule.
  • target molecule refers to a set of copies of one type or species of molecule or multi-molecular structure.
  • target molecules refer to more than one type or species of molecule or multi-molecular structure.
  • exemplary target molecules include proteins, polypeptides, nucleic acids, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, affybodies, antibody mimics, viruses, pathogens, toxic substances, substrates, metabolites, transition state analogs, cofactors, inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, and any fragment or portion of any of the foregoing.
  • a target molecule is a protein, in which case the target molecule may be referred to as a "target protein.”
  • a “capture agent' or “capture reagent” refers to a molecule that is capable of binding specifically to a biomarker.
  • a “target protein capture reagent” refers to a molecule that is capable of binding specifically to a target protein.
  • Nonlimiting exemplary capture reagents include aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, nucleic acids, lectins, ligand-binding receptors, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, synthetic receptors, and modifications and fragments of any of the aforementioned capture reagents.
  • a capture reagent is selected from an aptamer and an antibody.
  • antibody refers to full-length antibodies of any species and fragments and derivatives of such antibodies, including Fab fragments, F(ab') 2 fragments, single chain antibodies, Fv fragments, and single chain Fv fragments.
  • antibody also refers to synthetically-derived antibodies, such as phage display-derived antibodies and fragments, affybodies, nanobodies, etc.
  • marker and “biomarker” are used interchangeably to refer to a target molecule that indicates or is a sign of a normal or abnormal process in an individual or of a disease or other condition in an individual. More specifically, a “marker” or
  • biomarker is an anatomic, physiologic, biochemical, or molecular parameter associated with the presence of a specific physiological state or process, whether normal or abnormal, and, if abnormal, whether chronic or acute. Biomarkers are detectable and measurable by a variety of methods including laboratory assays and medical imaging. In some embodiments, a biomarker is a target protein.
  • biomarker level and “level” refer to a measurement that is made using any analytical method for detecting the biomarker in a biological sample and that indicates the presence, absence, absolute amount or concentration, relative amount or concentration, titer, a level, an expression level, a ratio of measured levels, or the like, of, for, or corresponding to the biomarker in the biological sample.
  • level depends on the specific design and components of the particular analytical method employed to detect the biomarker.
  • a “control level” of a target molecule refers to the level of the target molecule in the same sample type from an individual that does not have the disease or condition, or from an individual that is not suspected or at risk of having the disease or condition, or from an individual that has a non-progressive form of the disease or condition. Further, a “control level” may refer to a reference based on the average or what is considered within normal or healthy parameters. A “control level” may also refer to a reference level taken at a previous time and thus used to compare to a later measured or detected level of a target. For example, the level of a target may be detected at time point A, and then detected at time point B, where time point B is sometime after time point A.
  • time point A may be considered time zero (0) or day zero (0) and time point B may be minutes (e.g., 10, 20, 30, 40, 50, 60 minutes after time point A), hours (e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after time point A), days (e.g, 1, 2, 3, 4, 5, 6 or 7 days after time point A), weeks (e.g., 1, 2, 3 or 4 weeks after time point A), months (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months after time point A) and even years (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 years after time point A) after time point A.
  • minutes e.g., 10, 20, 30, 40, 50, 60 minutes after time point A
  • hours e.g, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after time point A
  • a "control level" of a target molecule need not be determined each time the present methods are carried out, and may be a previously determined level that is used as a reference or threshold to determine whether the level in a particular sample is higher or lower than a normal level.
  • a control level in a method described herein is the level that has been observed in one or more subjects with a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function.
  • a control level in a method described herein is the average or mean level, optionally plus or minus a statistical variation, that has been observed in a plurality of subjects without a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function.
  • Providing a biomarker panel” or “forming a biomarker panel”, as used herein, refers to identifying or selecting at least one, two, three, four, five, six, seven, eight, nine, ten, eleven or twelve biomarker proteins for purposes of detecting the level of the proteins in a sample from an individual.
  • the individual can be a mammal or a non-mammal. In various embodiments, the individual is a mammal.
  • a mammalian individual can be a human or non- human. In various embodiments, the individual is a human.
  • a healthy or normal individual is an individual in which the disease or condition of interest (such as decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function) is not detectable by conventional diagnostic methods.
  • Diagnose refers to the detection, determination, or recognition of a health status or condition of an individual on the basis of one or more signs, symptoms, data, or other information pertaining to that individual.
  • the health status of an individual can be diagnosed as healthy / normal (i.e., a diagnosis of the absence of a disease or condition) or diagnosed as ill / abnormal (i.e., a diagnosis of the presence, or an assessment of the characteristics, of a disease or condition).
  • diagnosis encompass, with respect to a particular disease or condition, the initial detection of the disease; the characterization or classification of the disease; the detection of the progression, remission, or recurrence of the disease; and the detection of disease response after the administration of a treatment or therapy to the individual.
  • the diagnosis of decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function includes distinguishing individuals who have a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function from individuals who do not.
  • Prognose refers to the prediction of a future course of a disease or condition in an individual who has the disease or condition (e.g., predicting patient survival), and such terms encompass the evaluation of disease response after the administration of a treatment or therapy to the individual.
  • “Evaluate”, “evaluating”, “evaluation”, and variations thereof encompass both “diagnose” and “prognose” and also encompass determinations or predictions about the future course of a disease or condition in an individual who does not have the disease as well as determinations or predictions regarding the likelihood that a disease or condition will recur in an individual who apparently has been cured of the disease.
  • the term “evaluate” also encompasses assessing an individual's response to a therapy, such as, for example, predicting whether an individual is likely to respond favorably to a therapeutic agent or is unlikely to respond to a therapeutic agent (or will experience toxic or other undesirable side effects, for example), selecting a therapeutic agent for administration to an individual, or monitoring or determining an individual's response to a therapy that has been administered to the individual.
  • evaluating decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function can include, for example, any of the following: predicting whether decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function in an individual will progress.
  • detecting or “determining” with respect to a biomarker level includes the use of both the instrument used to observe and record a signal corresponding to a biomarker level and the material/s required to generate that signal.
  • the level is detected using any suitable method, including fluorescence, chemiluminescence, surface plasmon resonance, surface acoustic waves, mass spectrometry, infrared
  • microscopy electrochemical detection methods, nuclear magnetic resonance, quantum dots, and the like.
  • Solid support refers herein to any substrate having a surface to which molecules may be attached, directly or indirectly, through either covalent or non-covalent bonds.
  • a “solid support” can have a variety of physical formats, which can include, for example, a membrane; a chip (e.g., a protein chip); a slide (e.g., a glass slide or coverslip); a column; a hollow, solid, semi-solid, pore- or cavity- containing particle, such as, for example, a bead; a gel; a fiber, including a fiber optic material; a matrix; and a sample receptacle.
  • Exemplary sample receptacles include sample wells, tubes, capillaries, vials, and any other vessel, groove or indentation capable of holding a sample.
  • a sample receptacle can be contained on a multi-sample platform, such as a microtiter plate, slide, micro fluidics device, and the like.
  • a support can be composed of a natural or synthetic material, an organic or inorganic material. The composition of the solid support on which capture reagents are attached generally depends on the method of attachment (e.g., covalent attachment).
  • Other exemplary receptacles include microdroplets and micro fluidic controlled or bulk oil/aqueous emulsions within which assays and related manipulations can occur.
  • Suitable solid supports include, for example, plastics, resins, polysaccharides, silica or silica-based materials, functionalized glass, modified silicon, carbon, metals, inorganic glasses, membranes, nylon, natural fibers (such as, for example, silk, wool and cotton), polymers, and the like.
  • the material composing the solid support can include reactive groups such as, for example, carboxy, amino, or hydroxyl groups, which are used for attachment of the capture reagents.
  • Polymeric solid supports can include, e.g., polystyrene, polyethylene glycol tetraphthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile, polymethyl
  • polytetrafluoroethylene butyl rubber, styrenebutadiene rubber, natural rubber, polyethylene, polypropylene, (poly)tetrafluoroethylene, (poly)vinylidenefluoride,
  • Suitable solid support particles that can be used include, e.g., encoded particles, such as Luminex ® -type encoded particles, magnetic particles, and glass particles.
  • linear regression refers to an approach for modeling the relationship between a scalar dependent variable y and one or more explanatory variables denoted X.
  • the case of one explanatory variable is called simple linear regression.
  • multiple linear regression it is called multiple linear regression.
  • linear regression may be used to fit a predictive model to an observed data set of y and x values. After developing such a model, if additional value of x is then given without its
  • the fitted model can be used to make a prediction of the value of y.
  • x represents the level of a protein biomarker
  • y represents the GFR
  • a prediction, estimation and/or determination of the GFR may be made.
  • Orderary least squares or “OLS” or “linear least squares”, as used herein, refers to a method for estimating the unknown parameters in a linear regression model. This method minimizes the sum of squared vertical distances between the observed responses in the dataset and the responses predicted by the linear approximation.
  • the resulting estimator can be expressed by a simple formula, especially in the case of a single regressor on the right- hand side.
  • Correspondence correlation or “concordance correlation coefficient” measures the agreement between two continuous variables X and F(e.g., predicted, estimated or determined GFR and actual GFR).
  • the “correspondence correlation” evaluates the degree to which pairs fall on the 45° line, and contains measurements of accuracy and precision (or the “Lin's Concordance”). Additional information may be found in Lin, Biometrics, Vol. 45, No. 1 (March, 1989), 255-268, which is hereby incorporated by reference.
  • a decrease, decline or reduced renal function is an indication that the kidney(s) are not functioning properly and that the individual is likely suffering from kidney disease.
  • a reduced or "low” e.g., a GFR of at or below about 90 mL/min./1.73m 2
  • GFR is indicative of a reduced renal function or kidney disease.
  • a biomarker level for the biomarkers described herein can be detected using any of a variety of known analytical methods.
  • a biomarker level is detected using a capture reagent.
  • the capture reagent can be exposed to the biomarker in solution or can be exposed to the biomarker while the capture reagent is immobilized on a solid support.
  • the capture reagent contains a feature that is reactive with a secondary feature on a solid support. In these embodiments, the capture reagent can be exposed to the biomarker in solution, and then the feature on the capture reagent can be used in conjunction with the secondary feature on the solid support to immobilize the biomarker on the solid support.
  • Capture reagent is selected based on the type of analysis to be conducted.
  • Capture reagents include but are not limited to aptamers, antibodies, adnectins, ankyrins, other antibody mimetics and other protein scaffolds, autoantibodies, chimeras, small molecules, F(ab') 2 fragments, single chain antibody fragments, Fv fragments, single chain Fv fragments, nucleic acids, lectins, ligand-binding receptors, affybodies, nanobodies, imprinted polymers, avimers, peptidomimetics, hormone receptors, cytokine receptors, and synthetic receptors, and modifications and fragments of these.
  • a biomarker level is detected using a biomarker/capture reagent complex.
  • the biomarker level is derived from the biomarker/capture reagent complex and is detected indirectly, such as, for example, as a result of a reaction that is subsequent to the biomarker/capture reagent interaction, but is dependent on the formation of the biomarker/capture reagent complex.
  • the biomarker level is detected directly from the biomarker in a biological sample.
  • biomarkers are detected using a multiplexed format that allows for the simultaneous detection of two or more biomarkers in a biological sample.
  • capture reagents are immobilized, directly or indirectly, covalently or non-covalently, in discrete locations on a solid support.
  • a multiplexed format uses discrete solid supports where each solid support has a unique capture reagent associated with that solid support, such as, for example quantum dots.
  • an individual device is used for the detection of each one of multiple biomarkers to be detected in a biological sample. Individual devices can be configured to permit each biomarker in the biological sample to be processed simultaneously. For example, a microtiter plate can be used such that each well in the plate is used to analyze one or more of multiple biomarkers to be detected in a biological sample.
  • a fluorescent tag can be used to label a component of the biomarker/capture reagent complex to enable the detection of the biomarker level.
  • the fluorescent label can be conjugated to a capture reagent specific to any of the biomarkers described herein using known techniques, and the fluorescent label can then be used to detect the corresponding biomarker level.
  • Suitable fluorescent labels include rare earth chelates, fluorescein and its derivatives, rhodamine and its derivatives, dansyl, allophycocyanin, PBXL-3, Qdot 605, Lissamine, phycoerythrin, Texas Red, and other such compounds.
  • the fluorescent label is a fluorescent dye molecule.
  • the fluorescent dye molecule includes at least one substituted indolium ring system in which the substituent on the 3 -carbon of the indolium ring contains a chemically reactive group or a conjugated substance.
  • the dye molecule includes an AlexFluor molecule, such as, for example, AlexaFluor 488, AlexaFluor 532, AlexaFluor 647, AlexaFluor 680, or AlexaFluor 700.
  • the dye molecule includes a first type and a second type of dye molecule, such as, e.g., two different AlexaFluor molecules.
  • the dye molecule includes a first type and a second type of dye molecule, and the two dye molecules have different emission spectra.
  • Fluorescence can be measured with a variety of instrumentation compatible with a wide range of assay formats.
  • spectrofluorimeters have been designed to analyze microtiter plates, microscope slides, printed arrays, cuvettes, etc. See Principles of Fluorescence Spectroscopy, by J.R. Lakowicz, Springer Science + Business Media, Inc., 2004. See Bioluminescence & Chemiluminescence: Progress & Current Applications; Philip E. Stanley and Larry J. Kricka editors, World Scientific Publishing Company, January 2002.
  • a chemiluminescence tag can optionally be used to label a component of the biomarker/capture complex to enable the detection of a biomarker level.
  • Suitable chemiluminescent materials include any of oxalyl chloride, Rodamin 6G,
  • the detection method includes an enzyme/substrate
  • the enzyme catalyzes a chemical alteration of the chromogenic substrate which can be measured using various techniques, including spectrophotometry, fluorescence, and chemiluminescence.
  • Suitable enzymes include, for example, luciferases, luciferin, malate dehydrogenase, urease, horseradish peroxidase (HRPO), alkaline phosphatase, beta- galactosidase, glucoamylase, lysozyme, glucose oxidase, galactose oxidase, and glucoses- phosphate dehydrogenase, uricase, xanthine oxidase, lactoperoxidase, microperoxidase, and the like.
  • the detection method can be a combination of fluorescence, chemiluminescence, radionuclide or enzyme/substrate combinations that generate a measurable signal.
  • multimodal signaling could have unique and advantageous characteristics in biomarker assay formats.
  • the biomarker levels for the biomarkers described herein can be detected using any analytical methods including, singleplex aptamer assays, multiplexed aptamer assays, singleplex or multiplexed immunoassays, mR A expression profiling, miRNA expression profiling, mass spectrometric analysis, histological/cytological methods, etc. as discussed below. Determination of Biomarker Levels using Aptamer-Based Assays
  • Assays directed to the detection and quantification of physiologically significant molecules in biological samples and other samples are important tools in scientific research and in the health care field.
  • One class of such assays involves the use of a microarray that includes one or more aptamers immobilized on a solid support.
  • the aptamers are each capable of binding to a target molecule in a highly specific manner and with very high affinity. See, e.g., U.S. Patent No. 5,475,096 entitled “Nucleic Acid Ligands”; see also, e.g., U.S. Patent No. 6,242,246, U.S. Patent No. 6,458,543, and U.S. Patent No. 6,503,715, each of which is entitled "Nucleic Acid Ligand Diagnostic Biochip".
  • the aptamers bind to their respective target molecules present in the sample and thereby enable a determination of a biomarker level corresponding to a biomarker.
  • an "aptamer” refers to a nucleic acid that has a specific binding affinity for a target molecule. It is recognized that affinity interactions are a matter of degree; however, in this context, the "specific binding affinity" of an aptamer for its target means that the aptamer binds to its target generally with a much higher degree of affinity than it binds to other components in a test sample.
  • An “aptamer” is a set of copies of one type or species of nucleic acid molecule that has a particular nucleotide sequence.
  • An aptamer can include any suitable number of nucleotides, including any number of chemically modified nucleotides. "Aptamers" refers to more than one such set of molecules.
  • aptamers can have either the same or different numbers of nucleotides.
  • Aptamers can be DNA or RNA or chemically modified nucleic acids and can be single stranded, double stranded, or contain double stranded regions, and can include higher ordered structures.
  • An aptamer can also be a photoaptamer, where a photoreactive or chemically reactive functional group is included in the aptamer to allow it to be covalently linked to its corresponding target. Any of the aptamer methods disclosed herein can include the use of two or more aptamers that specifically bind the same target molecule. As further described below, an aptamer may include a tag.
  • the aptamer may include up to about 100 nucleotides, up to about 95 nucleotides, up to about 90 nucleotides, up to about 85
  • nucleotides up to about 80 nucleotides, up to about 75 nucleotides, up to about 70
  • nucleotides up to about 65 nucleotides, up to about 60 nucleotides, up to about 55
  • nucleotides up to about 50 nucleotides, up to about 45 nucleotides, up to about 40
  • nucleotides up to about 35 nucleotides, up to about 30 nucleotides, up to about 25
  • the aptamer may be from about 25 to about 100 nucleotides in length (or from about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 nucleotides in length) or from about 25 to 50 nucleotides in length (or from about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,
  • An aptamer can be identified using any known method, including the SELEX process. Once identified, an aptamer can be prepared or synthesized in accordance with any known method, including chemical synthetic methods and enzymatic synthetic methods.
  • SELEX and “SELEX process” are used interchangeably herein to refer generally to a combination of (1) the selection of aptamers that interact with a target molecule in a desirable manner, for example binding with high affinity to a protein, with (2) the amplification of those selected nucleic acids.
  • the SELEX process can be used to identify aptamers with high affinity to a specific target or biomarker.
  • SELEX generally includes preparing a candidate mixture of nucleic acids, binding of the candidate mixture to the desired target molecule to form an affinity complex, separating the affinity complexes from the unbound candidate nucleic acids, separating and isolating the nucleic acid from the affinity complex, purifying the nucleic acid, and identifying a specific aptamer sequence.
  • the process may include multiple rounds to further refine the affinity of the selected aptamer.
  • the process can include amplification steps at one or more points in the process. See, e.g., U.S. Patent No. 5,475,096, entitled "Nucleic Acid Ligands".
  • the SELEX process can be used to generate an aptamer that covalently binds its target as well as an aptamer that non-covalently binds its target. See, e.g., U.S. Patent No. 5,705,337 entitled “Systematic Evolution of Nucleic Acid Ligands by Exponential Enrichment: Chemi- SELEX.”
  • the SELEX process can be used to identify high-affinity aptamers containing modified nucleotides that confer improved characteristics on the aptamer, such as, for example, improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX process-identified aptamers containing modified nucleotides are described in U.S. Patent No. 5,660,985, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides", which describes oligonucleotides containing nucleotide derivatives chemically modified at the 5'- and 2'-positions of pyrimidines. U.S. Patent No.
  • SELEX can also be used to identify aptamers that have desirable off-rate
  • an aptamer comprises at least one nucleotide with a modification, such as a base modification.
  • an aptamer comprises at least one nucleotide with a hydrophobic modification, such as a hydrophobic base modification, allowing for hydrophobic contacts with a target protein.
  • hydrophobic contacts in some embodiments, contribute to greater affinity and/or slower off- rate binding by the aptamer.
  • Nonlimiting exemplary nucleotides with hydrophobic modifications are shown in Figure 20.
  • an aptamer comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 nucleotides with hydrophobic modifications, where each hydrophobic modification may be the same or different from the others.
  • at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least 10 hydrophobic modifications in an aptamer may be
  • a slow off-rate aptamer (including an aptamers comprising at least one nucleotide with a hydrophobic modification) has an off-rate (t 1 ⁇ 2 ) of > 30 minutes, > 60 minutes, > 90 minutes, > 120 minutes, > 150 minutes, > 180 minutes, > 210 minutes, or > 240 minutes.
  • an assay employs aptamers that include photoreactive functional groups that enable the aptamers to covalently bind or "photocrosslink" their target molecules.
  • photoreactive aptamers are also referred to as photoaptamers. See, e.g., U.S. Patent No. 5,763,177, U.S. Patent No. 6,001,577, and U.S. Patent No. 6,291,184, each of which is entitled "Systematic Evolution of Nucleic Acid Ligands by Exponential
  • the aptamers are immobilized on the solid support prior to being contacted with the sample. Under certain circumstances, however, immobilization of the aptamers prior to contact with the sample may not provide an optimal assay. For example, pre-immobilization of the aptamers may result in inefficient mixing of the aptamers with the target molecules on the surface of the solid support, perhaps leading to lengthy reaction times and, therefore, extended incubation periods to permit efficient binding of the aptamers to their target molecules. Further, when photoaptamers are employed in the assay and depending upon the material utilized as a solid support, the solid support may tend to scatter or absorb the light used to effect the formation of covalent bonds between the photoaptamers and their target molecules.
  • immobilization of the aptamers on the solid support generally involves an aptamer-preparation step (i.e., the immobilization) prior to exposure of the aptamers to the sample, and this preparation step may affect the activity or functionality of the aptamers.
  • aptamer assays or "aptamer based assay(s)" that permit an aptamer to capture its target in solution and then employ separation steps that are designed to remove specific components of the aptamer-target mixture prior to detection have also been described (see U.S. Publication No. 2009/0042206, entitled “Multiplexed Analyses of Test Samples”).
  • the described aptamer assay methods enable the detection and quantification of a non-nucleic acid target (e.g., a protein target) in a test sample by detecting and quantifying a nucleic acid (i.e., an aptamer).
  • the described methods create a nucleic acid surrogate (i.e, the aptamer) for detecting and quantifying a non-nucleic acid target, thus allowing the wide variety of nucleic acid technologies, including amplification, to be applied to a broader range of desired targets, including protein targets.
  • a nucleic acid surrogate i.e, the aptamer
  • Aptamers can be constructed to facilitate the separation of the assay components from an aptamer biomarker complex (or photoaptamer biomarker covalent complex) and permit isolation of the aptamer for detection and/or quantification.
  • these constructs can include a cleavable or releasable element within the aptamer sequence.
  • additional functionality can be introduced into the aptamer, for example, a labeled or detectable component, a spacer component, or a specific binding tag or immobilization element.
  • the aptamer can include a tag connected to the aptamer via a cleavable moiety, a label, a spacer component separating the label, and the cleavable moiety.
  • a cleavable element is a photocleavable linker.
  • the photocleavable linker can be attached to a biotin moiety and a spacer section, can include an NHS group for derivatization of amines, and can be used to introduce a biotin group to an aptamer, thereby allowing for the release of the aptamer later in an assay method.
  • the molecular capture reagents comprise an aptamer or an antibody or the like and the specific target may be a biomarker shown in Table 2.
  • a method for signal generation takes advantage of anisotropy signal change due to the interaction of a fluorophore-labeled capture reagent with its specific biomarker target.
  • the labeled capture reacts with its target, the increased molecular weight causes the rotational motion of the fiuorophore attached to the complex to become much slower changing the anisotropy value.
  • binding events may be used to quantitatively measure the biomarkers in solutions.
  • Other methods include fluorescence polarization assays, molecular beacon methods, time resolved fluorescence quenching, chemiluminescence, fluorescence resonance energy transfer, and the like.
  • An exemplary solution-based aptamer assay that can be used to detect a biomarker level in a biological sample includes the following: (a) preparing a mixture by contacting the biological sample with an aptamer that includes a first tag and has a specific affinity for the biomarker, wherein an aptamer affinity complex is formed when the biomarker is present in the sample; (b) exposing the mixture to a first solid support including a first capture element, and allowing the first tag to associate with the first capture element; (c) removing any components of the mixture not associated with the first solid support; (d) attaching a second tag to the biomarker component of the aptamer affinity complex; (e) releasing the aptamer affinity complex from the first solid support; (f) exposing the released aptamer affinity complex to a second solid support that includes a second capture element and allowing the second tag to associate with the second capture element; (g) removing any non-complexed aptamer from the mixture by partitioning the non-complex
  • protein concentration or levels in a sample may be expressed as relative fluorescence units (RFU), which may be a product of detecting the aptamer component of the aptamer affinity complex (e.g., aptamer complexed to target protein create the aptamer affinity complex). That is, for an aptamer-based assay, the protein concentration or level correlates with the RFU.
  • RFU relative fluorescence units
  • Example 6 A nonlimiting exemplary method of detecting biomarkers in a biological sample using aptamers is described in Example 6. See also Kraemer et al., PLoS One 6(10): e26332.
  • Immunoassay methods are based on the reaction of an antibody to its corresponding target or analyte and can detect the analyte in a sample depending on the specific assay format.
  • monoclonal antibodies and fragments thereof are often used because of their specific epitope recognition.
  • Polyclonal antibodies have also been successfully used in various immunoassays because of their increased affinity for the target as compared to monoclonal antibodies.
  • Immunoassays have been designed for use with a wide range of biological sample matrices. Immunoassay formats have been designed to provide qualitative, semi-quantitative, and quantitative results.
  • Quantitative results are generated through the use of a standard curve created with known concentrations of the specific analyte to be detected.
  • the response or signal from an unknown sample is plotted onto the standard curve, and a quantity or level corresponding to the target in the unknown sample is established.
  • ELISA or EIA can be quantitative for the detection of an analyte. This method relies on attachment of a label to either the analyte or the antibody and the label component includes, either directly or indirectly, an enzyme. ELISA tests may be formatted for direct, indirect, competitive, or sandwich detection of the analyte. Other methods rely on labels such as, for example, radioisotopes (I 125 ) or fluorescence. Additional techniques include, for example,
  • Exemplary assay formats include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, fluorescent, chemiluminescence, and fluorescence resonance energy transfer (FRET) or time resolved-FRET (TR-FRET) immunoassays.
  • ELISA enzyme-linked immunosorbent assay
  • FRET fluorescence resonance energy transfer
  • TR-FRET time resolved-FRET
  • biomarkers include biomarker immunoprecipitation followed by quantitative methods that allow size and peptide level discrimination, such as gel electrophoresis, capillary electrophoresis, planar electrochromatography, and the like.
  • detectable label can be, without limitation, fluorescent, luminescent, or radioactive or they may absorb visible or ultraviolet light.
  • detectors suitable for detecting such detectable labels include, without limitation, x-ray film, radioactivity counters, scintillation counters, spectrophotometers, colorimeters, fluorometers, luminometers, and densitometers.
  • Any of the methods for detection can be performed in any format that allows for any suitable preparation, processing, and analysis of the reactions. This can be, for example, in multi-well assay plates (e.g., 96 wells or 386 wells) or using any suitable array or microarray. Stock solutions for various agents can be made manually or robotically, and all subsequent pipetting, diluting, mixing, distribution, washing, incubating, sample readout, data collection and analysis can be done robotically using commercially available analysis software, robotics, and detection instrumentation capable of detecting a detectable label. Determination of Biomarker Levels using Gene Expression Profiling
  • Measuring mRNA in a biological sample may, in some embodiments, be used as a surrogate for detection of the level of the corresponding protein in the biological sample.
  • a biomarker or biomarker panel described herein can be detected by detecting the appropriate RNA.
  • mRNA expression levels are measured by reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR).
  • RT-PCR is used to create a cDNA from the mRNA.
  • the cDNA may be used in a qPCR assay to produce fluorescence as the DNA amplification process progresses. By comparison to a standard curve, qPCR can produce an absolute measurement such as number of copies of mRNA per cell.
  • Northern blots, microarrays, Invader assays, and RT-PCR combined with capillary electrophoresis have all been used to measure expression levels of mRNA in a sample. See Gene Expression Profiling: Methods and Protocols, Richard A. Shimkets, editor, Humana Press, 2004. Detection of Biomarkers Using In Vivo Molecular Imaging Technologies
  • a biomarker described herein may be used in molecular imaging tests.
  • an imaging agent can be coupled to a capture reagent, which can be used to detect the biomarker in vivo.
  • In vivo imaging technologies provide non-invasive methods for determining the state of a particular disease in the body of an individual. For example, entire portions of the body, or even the entire body, may be viewed as a three dimensional image, thereby providing valuable information concerning morphology and structures in the body. Such technologies may be combined with the detection of the biomarkers described herein to provide information concerning the biomarker in vivo.
  • in vivo molecular imaging technologies are expanding due to various advances in technology. These advances include the development of new contrast agents or labels, such as radiolabels and/or fluorescent labels, which can provide strong signals within the body; and the development of powerful new imaging technology, which can detect and analyze these signals from outside the body, with sufficient sensitivity and accuracy to provide useful information.
  • the contrast agent can be visualized in an appropriate imaging system, thereby providing an image of the portion or portions of the body in which the contrast agent is located.
  • the contrast agent may be bound to or associated with a capture reagent, such as an aptamer or an antibody, for example, and/or with a peptide or protein, or an oligonucleotide (for example, for the detection of gene expression), or a complex containing any of these with one or more macromolecules and/or other particulate forms.
  • a capture reagent such as an aptamer or an antibody, for example, and/or with a peptide or protein, or an oligonucleotide (for example, for the detection of gene expression), or a complex containing any of these with one or more macromolecules and/or other particulate forms.
  • the contrast agent may also feature a radioactive atom that is useful in imaging.
  • Suitable radioactive atoms include technetium-99m or iodine- 123 for scintigraphic studies.
  • Other readily detectable moieties include, for example, spin labels for magnetic resonance imaging (MRI) such as, for example, iodine- 123 again, iodine-131, indium- 111, fluorine- 19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron.
  • MRI magnetic resonance imaging
  • Standard imaging techniques include but are not limited to magnetic resonance imaging, computed tomography scanning, positron emission tomography (PET), single photon emission computed tomography (SPECT), and the like.
  • PET positron emission tomography
  • SPECT single photon emission computed tomography
  • the type of detection instrument available is a major factor in selecting a given contrast agent, such as a given radionuclide and the particular biomarker that it is used to target (protein, mRNA, and the like).
  • the radionuclide chosen typically has a type of decay that is detectable by a given type of instrument.
  • its half-life should be long enough to enable detection at the time of maximum uptake by the target tissue but short enough that deleterious radiation of the host is minimized.
  • Exemplary imaging techniques include but are not limited to PET and SPECT, which are imaging techniques in which a radionuclide is synthetically or locally administered to an individual. The subsequent uptake of the radiotracer is measured over time and used to obtain information about the targeted tissue and the biomarker. Because of the high-energy (gamma-ray) emissions of the specific isotopes employed and the sensitivity and
  • the two-dimensional distribution of radioactivity may be inferred from outside of the body.
  • positron-emitting nuclides in PET include, for example, carbon- 11, nitrogen-13, oxygen- 15, and fluorine- 18.
  • Isotopes that decay by electron capture and/or gamma-emission are used in SPECT and include, for example iodine- 123 and technetium- 99m.
  • An exemplary method for labeling amino acids with technetium-99m is the reduction of pertechnetate ion in the presence of a chelating precursor to form the labile technetium- 99m-precursor complex, which, in turn, reacts with the metal binding group of a
  • Antibodies are frequently used for such in vivo imaging diagnostic methods.
  • the preparation and use of antibodies for in vivo diagnosis is well known in the art.
  • aptamers may be used for such in vivo imaging diagnostic methods.
  • an aptamer that was used to identify a particular biomarker described herein may be appropriately labeled and injected into an individual to detect the biomarker in vivo.
  • the label used will be selected in accordance with the imaging modality to be used, as previously described.
  • Aptamer-directed imaging agents could have unique and advantageous characteristics relating to tissue penetration, tissue distribution, kinetics, elimination, potency, and selectivity as compared to other imaging agents.
  • Such techniques may also optionally be performed with labeled oligonucleotides, for example, for detection of gene expression through imaging with antisense oligonucleotides. These methods are used for in situ hybridization, for example, with fluorescent molecules or radionuclides as the label. Other methods for detection of gene expression include, for example, detection of the activity of a reporter gene.
  • optical imaging Another general type of imaging technology is optical imaging, in which fluorescent signals within the subject are detected by an optical device that is external to the subject. These signals may be due to actual fluorescence and/or to bioluminescence. Improvements in the sensitivity of optical detection devices have increased the usefulness of optical imaging for in vivo diagnostic assays.
  • the biomarkers described herein may be detected in a variety of tissue samples using histological or cytological methods.
  • endo- and trans- bronchial biopsies, fine needle aspirates, cutting needles, and core biopsies can be used for histology.
  • Bronchial washing and brushing, pleural aspiration, and sputum, can be used for cyotology.
  • Any of the biomarkers identified herein can be used to stain a specimen as an indication of disease.
  • one or more capture reagent/s specific to the corresponding biomarker/s are used in a cytological evaluation of a sample and may include one or more of the following: collecting a cell sample, fixing the cell sample, dehydrating, clearing, immobilizing the cell sample on a microscope slide, permeabilizing the cell sample, treating for analyte retrieval, staining, destaining, washing, blocking, and reacting with one or more capture reagent/s in a buffered solution.
  • the cell sample is produced from a cell block.
  • one or more capture reagent/s specific to the corresponding biomarkers are used in a histological evaluation of a tissue sample and may include one or more of the following: collecting a tissue specimen, fixing the tissue sample, dehydrating, clearing, immobilizing the tissue sample on a microscope slide, permeabilizing the tissue sample, treating for analyte retrieval, staining, destaining, washing, blocking, rehydrating, and reacting with capture reagent/s in a buffered solution.
  • fixing and dehydrating are replaced with freezing.
  • the one or more aptamer/s specific to the corresponding biomarker/s are reacted with the histological or cytological sample and can serve as the nucleic acid target in a nucleic acid amplification method.
  • amplification methods include, for example, PCR, q-beta replicase, rolling circle
  • the one or more capture reagent/s specific to the corresponding biomarkers for use in the histological or cytological evaluation are mixed in a buffered solution that can include any of the following: blocking materials, competitors, detergents, stabilizers, carrier nucleic acid, polyanionic materials, etc.
  • a “cytology protocol” generally includes sample collection, sample fixation, sample immobilization, and staining.
  • Cell preparation can include several processing steps after sample collection, including the use of one or more aptamers for the staining of the prepared cells. Determination of Biomarker Levels using Mass Spectrometry Methods
  • mass spectrometers can be used to detect biomarker levels.
  • a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument- control system, and a data system. Difference in the sample inlet, ion source, and mass analyzer generally define the type of instrument and its capabilities.
  • an inlet can be a capillary-column liquid chromatography source or can be a direct probe or stage such as used in matrix-assisted laser desorption.
  • Common ion sources are, for example, electrospray, including nanospray and microspray or matrix-assisted laser desorption.
  • Mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time- of-flight mass analyzer. Additional mass spectrometry methods are well known in the art (see Burlingame et al. Anal. Chem. 70:647 R-716R (1998); Kinter and Sherman, New York (2000)).
  • Protein biomarkers and biomarker levels can be detected and measured by any of the following: electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS, ESI-
  • MS/(MS)n matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), desorption/ionization on silicon (DIOS), secondary ion mass spectrometry (SIMS), quadrupole time-of-flight (Q-TOF), tandem time-of-flight (TOF/TOF) technology, called ultraflex III TOF/TOF, atmospheric pressure chemical ionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS) N , atmospheric pressure photoionization mass spectrometry (APPI-MS), APPI-MS/MS, and APPI-(MS) N , quadrupole mass spectrometry, Fourier transform mass spectrometry (FTMS), quantitative mass spectrometry, and ion trap mass spectrometry.
  • Labeling methods include but are not limited to isobaric tag for relative and absolute quantitation (iTRAQ) and stable isotope labeling with amino acids in cell culture (SILAC).
  • Capture reagents used to selectively enrich samples for candidate biomarker proteins prior to mass spectroscopic analysis include but are not limited to aptamers, antibodies, nucleic acid probes, chimeras, small molecules, an F(ab') 2 fragment, a single chain antibody fragment, an Fv fragment, a single chain Fv fragment, a nucleic acid, a lectin, a ligand-binding receptor, affybodies, nanobodies, ankyrins, domain antibodies, alternative antibody scaffolds (e.g.
  • a biomarker "signature" for a given diagnostic test contains a set of markers, each marker having characteristic levels in the populations of interest.
  • Characteristic levels may refer to the mean or average of the biomarker levels for the individuals in a particular group.
  • a diagnostic method described herein can be used to assign an unknown sample from an individual into one of two groups, either having a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function or not having a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function; or alternatively, responding to treatment or not responding to treatment.
  • the assignment of a sample into one of two or more groups is known as classification, and the procedure used to accomplish this assignment is known as a classifier or a classification method. Classification methods may also be referred to as scoring methods.
  • classification methods can be used to construct a diagnostic classifier from a set of biomarker levels.
  • classification methods are performed using supervised learning techniques in which a data set is collected using samples obtained from individuals within two (or more, for multiple classification states) distinct groups one wishes to distinguish. Since the class (group or population) to which each sample belongs is known in advance for each sample, the classification method can be trained to give the desired classification response. It is also possible to use unsupervised learning techniques to produce a diagnostic classifier.
  • diagnostic classifiers include decision trees; bagging + boosting + forests; rule inference based learning; Parzen Windows; linear models; logistic; neural network methods; unsupervised clustering; K-means; hierarchical ascending/ descending; semi-supervised learning; prototype methods; nearest neighbor; kernel density estimation; support vector machines; hidden Markov models; Boltzmann Learning; and classifiers may be combined either simply or in ways which minimize particular objective functions.
  • Pattern Classification R.O. Duda, et al, editors, John Wiley & Sons, 2nd edition, 2001
  • training data includes samples from the distinct groups (classes) to which unknown samples will later be assigned.
  • samples collected from individuals in a control population and individuals in a particular disease population can constitute training data to develop a classifier that can classify unknown samples (or, more particularly, the individuals from whom the samples were obtained) as either having the disease or being free from the disease.
  • the development of the classifier from the training data is known as training the classifier. Specific details on classifier training depend on the nature of the supervised learning technique.
  • Training a na ' ive Bayesian classifier is an example of such a supervised learning technique (see, e.g., Pattern Classification, R.O. Duda, et al, editors, John Wiley & Sons, 2nd edition, 2001; see also, The Elements of Statistical Learning - Data Mining, Inference, and Prediction, T.
  • Over-fitting occurs when a statistical model describes random error or noise instead of the underlying relationship. Over-fitting can be avoided in a variety of way, including, for example, by limiting the number of markers used in developing the classifier, by assuming that the marker responses are independent of one another, by limiting the complexity of the underlying statistical model employed, and by ensuring that the underlying statistical model conforms to the data.
  • An illustrative example of the development of a diagnostic test using a set of biomarkers includes the application of a na ' ive Bayes classifier, a simple probabilistic classifier based on Bayes theorem with strict independent treatment of the biomarkers.
  • Each biomarker is described by a class-dependent probability density function (pdf) for the measured RFU values or log RFU (relative fluorescence units) values in each class.
  • the joint pdfs for the set of markers in one class is assumed to be the product of the individual class- dependent pdfs for each biomarker.
  • Training a naive Bayes classifier in this context amounts to assigning parameters ("parameterization") to characterize the class dependent pdfs. Any underlying model for the class-dependent pdfs may be used, but the model should generally conform to the data observed in the training set.
  • the performance of the naive Bayes classifier is dependent upon the number and quality of the biomarkers used to construct and train the classifier.
  • a single biomarker will perform in accordance with its KS-distance (Kolmogorov-Smirnov).
  • the addition of subsequent markers with good KS distances (>0.3, for example) will, in general, improve the classification performance if the subsequently added markers are independent of the first marker.
  • KS-distance Kolmogorov-Smirnov
  • KS distances >0.3, for example
  • many high scoring classifiers can be generated with a variation of a greedy algorithm. (A greedy algorithm is any algorithm that follows the problem solving metaheuristic of making the locally optimal choice at each stage with the hope of finding the global optimum.)
  • ROC receiver operating characteristic
  • TPR true positive rate
  • FPR false positive rate
  • the area under the ROC curve (AUC) is commonly used as a summary measure of diagnostic accuracy. It can take values from 0.0 to 1.0.
  • the AUC has an important statistical property: the AUC of a classifier is equivalent to the probability that the classifier will rank a randomly chosen positive instance higher than a randomly chosen negative instance (Fawcett T, 2006. An introduction to ROC analysis. Pattern Recognition Letters .27: 861-874). This is equivalent to the Wilcoxon test of ranks (Hanley, J.A., McNeil, B.J., 1982. The meaning and use of the area under a receiver operating characteristic (ROC) curve.
  • ROC receiver operating characteristic
  • Radiology 143, 29- Exemplary embodiments use any number of the biomarkers listed in Table 1 in various combinations to produce diagnostic tests for identifying individuals with a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function.
  • the markers listed in Table 2 can be combined in many ways to produce classifiers or used to predict, estimate and/or determine GFR in a subject.
  • exemplary embodiments use any number of the biomarkers listed in Table 2 in various combinations to produce diagnostic tests for identifying individuals with r declining GFR, reduced kidney function and/or kidney disease..
  • panels of biomarkers are comprised of different sets of biomarkers depending on a specific diagnostic performance criterion that is selected. For example, certain combinations of biomarkers may produce tests that are more sensitive (or more specific) than other combinations.
  • the diagnostic test parameters are complete.
  • a biological sample is run in one or more assays to produce the relevant quantitative biomarker levels used for
  • the measured biomarker levels are used as input for the classification method that outputs a classification and an optional score for the sample that reflects the confidence of the class assignment.
  • a biological sample is optionally diluted and run in a multiplexed aptamer assay, and data is assessed as follows.
  • the data from the assay are optionally normalized and calibrated, and the resulting biomarker levels are used as input to a Bayes classification scheme.
  • the log-likelihood ratio is computed for each measured biomarker individually and then summed to produce a final classification score, which is also referred to as a diagnostic score. The resulting assignment as well as the overall
  • classification score can be reported.
  • the individual log-likelihood risk factors computed for each biomarker level can be reported as well.
  • any combination of the biomarkers described herein can be detected using a suitable kit, such as for use in performing the methods disclosed herein.
  • any kit can contain one or more detectable labels as described herein, such as a fluorescent moiety, etc.
  • a kit includes (a) one or more capture reagents (such as, for example, at least one aptamer or antibody) for detecting one or more biomarkers in a biological sample, and optionally (b) one or more software or computer program products for predicting whether the individual from whom the biological sample was obtained has a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function and/or whether an individual with decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function is responding to treatment.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • kits comprises a solid support, a capture reagent, and a signal generating material.
  • the kit can also include instructions for using the devices and reagents, handling the sample, and analyzing the data. Further the kit may be used with a computer system or software to analyze and report the result of the analysis of the biological sample.
  • the kits can also contain one or more reagents (e.g., solubilization buffers, detergents, washes, or buffers) for processing a biological sample. Any of the kits described herein can also include, e.g., buffers, blocking agents, mass spectrometry matrix materials, antibody capture agents, positive control samples, negative control samples, software and information such as protocols, guidance and reference data.
  • kits are provided for the analysis of GFR or reduced kidney function, wherein the kits comprise PCR primers for one or more biomarkers described herein.
  • a kit may further include instructions for use and correlation of the biomarkers with GFR or reduced kidney function prognosis.
  • a kit may include a DNA array containing the complement of one or more of the biomarkers described herein, reagents, and/or enzymes for amplifying or isolating sample DNA.
  • the kits may include reagents for real-time PCR, for example, TaqMan probes and/or primers, and enzymes.
  • a kit can comprise (a) reagents comprising at least one capture reagent for determining the level of one or more biomarkers in a test sample, and optionally (b) one or more algorithms or computer programs for performing the steps of comparing the amount of each biomarker quantified in the test sample to one or more predetermined cutoffs.
  • an algorithm or computer program assigns a score for each biomarker quantified based on said comparison and, in some embodiments, combines the assigned scores for each biomarker quantified to obtain a total score.
  • an algorithm or computer program compares the total score with a predetermined score, and uses the comparison to determine whether the patient is responding to treatment or not responding to treatment, or the likelihood of reoccurrence of a decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function in an individual treated and considered cured of the disease.
  • one or more instructions for manually performing the above steps by a human can be provided.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from a subject comprising at least a first, second and third capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the COL18A1 protein, and the third capture reagent has binding affinity for the UNC5D protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and AMH in a sample from a subject comprising at least a first, second, third and fourth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the CST3 protein, and the third capture reagent has binding affinity for the COL18A1 protein and the fourth capture reagent has binding affinity for the AMH protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDL1 in a sample from a subject comprising at least a first, second, third, fourth and fifth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the CST3 protein, and the third capture reagent has binding affinity for the COL18A1 protein, the fourth capture reagent has binding affinity for the AMH protein and the fifth capture reagent has binding affinity for CHRDL1.
  • TFF3 Trefoil Factor 3
  • CST3 Endostatin
  • CHRDL1 Endostatin
  • At least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and PI3 in a sample from a subject comprising at least a first, second, third, fourth and fifth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the CST3 protein, and the third capture reagent has binding affinity for the COL18A1 protein, the fourth capture reagent has binding affinity for the AMH protein and the fifth capture reagent has binding affinity for PI3.
  • TFF3 Trefoil Factor 3
  • CST3 Endostatin
  • PI3 Endostatin
  • At least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and FCN2 in a sample from a subject comprising at least a first, second, third and fourth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the CST3 protein, and the third capture reagent has binding affinity for the COL18A1 protein and the fourth capture reagent has binding affinity for the FCN2 protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), FCN2 and CTSL2 in a sample from a subject comprising at least a first, second, third, fourth and fifth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the CST3 protein, and the third capture reagent has binding affinity for the COL18A1 protein, the fourth capture reagent has binding affinity for the FCN2 protein and the fifth capture reagent has binding affinity for CTSL2.
  • TFF3 Trefoil Factor 3
  • CST3 Endostatin
  • CTSL2 Endostatin
  • At least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from a subject comprising at least a first, second, third and fourth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the COL18A1 protein, the third capture reagent has binding affinity for the PI3 protein and the fourth capture reagent has binding affinity for the UNC5D protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3), CST3, NBLl, B2M and IGFBP6 in a sample from a subject comprising at least a first, second, third, fourth and fifth capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein, the second capture reagent has binding affinity for the CST3 protein, and the third capture reagent has binding affinity for the NBLl protein, the fourth capture reagent has binding affinity for the B2M protein and the fifth capture reagent has binding affinity for IGFBP6.
  • TFF3 Trefoil Factor 3
  • CST3 CST3 protein
  • the third capture reagent has binding affinity for the NBLl protein
  • the fourth capture reagent has binding affinity for the B2M protein
  • the fifth capture reagent has binding affinity for IGFBP6.
  • At least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins CST3, Endostatin (COL18A1) and CHRDL1 in a sample from a subject comprising at least a first, second and third capture reagent, wherein the first capture reagent has binding affinity for the CST23 protein, the second capture reagent has binding affinity for the COL18A1 protein, and the third capture reagent has binding affinity for the CHRDL1 protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3) and NBLl in a sample from a subject comprising at least a first and second capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein and the second capture reagent has binding affinity for the NBLl protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer-based assay.
  • the disclosure provides for a kit for detecting the level of the proteins Trefoil Factor 3 (TFF3) and Endostatin (COL18Al)in a sample from a subject comprising at least a first and second capture reagent, wherein the first capture reagent has binding affinity for the TFF3 protein and the second capture reagent has binding affinity for the COL18A1 protein.
  • at least one capture reagent is selected from the group consisting of an aptamer, an antibody, a nucleic acid ligand and any combination thereof.
  • the kit further comprises reagents for performing an aptamer- based assay.
  • the kit further comprises at least one additional capture reagent that has binding affinity for a protein selected from the group consisting of UNC5D, CST3, AMH, CHRDL1, PI3, FCN2, CTSL2, NBL1, B2M and IGFBP6.
  • the kit further comprises at least one additional capture reagent that has binding affinity for the UNC5D protein.
  • the kit further comprises a third, fourth and fifth capture reagent, wherein the third capture reagent has binding affinity for the CST3 protein, the fourth capture reagent as binding affinity for the AMH protein and the fifth capture reagent has binding affinity for the FCN2 protein.
  • the kit further comprises at least one additional capture reagent that has binding affinity for a protein selected from the group consisting of CST3, AMH and CHRDL1 or PI3.
  • the kit further comprises a third, fourth and fifth capture reagent, wherein the third capture reagent has binding affinity for the CST3 protein, the fourth capture reagent as binding affinity for the AMH protein and the fifth capture reagent has binding affinity for the CTSL2 protein.
  • the kit further comprises a third and fourth capture reagent, wherein the third capture reagent has binding affinity for the UNC5D protein and the fourth capture reagent as binding affinity for the PI3 protein.
  • the kit is for estimating the GFR of a subject. In another aspect, the kit is for predicting the GFR of a subject. In another aspect, the kit is for determining the GFR of a subject. In another aspect, the kit is for determining whether the renal function of a subject is declining relative to a control or reference measurement. In another aspect, the kit is for determining whether the renal function of a subject is decreasing relative to a control or reference measurement.
  • the kit provides for a method for determining, estimating and/or predicting the GFR in a subject, wherein the determination, estimation and/or prediction of the GFR to the actual GFR has a correspondence correlation of at least about 0.6, 0.7, 0.8 or 0.9.
  • the correspondence correlation is at least 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8.
  • the correspondence correlation for the method is at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8 when the actual GFR is equal to or above 60 mL/min./l .73m 2 .
  • a method for assessing GFR or kidney function in an individual may comprise the following: 1) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; and 3) report the results of the biomarker levels.
  • the results of the biomarker levels are reported qualitatively rather than quantitatively, such as, for example, a proposed diagnosis ("prostate cancer", “tumor regression”, etc.) or simply a positive / negative result where "positive" and
  • a method for assessing GFR or kidney function in an individual may comprise the following: 1) collect or otherwise obtain a biological sample; 2) perform an analytical method to detect and measure the biomarker or biomarkers in the panel in the biological sample; 3) perform any data normalization or standardization; 4) calculate each biomarker level; and 5) report the results of the biomarker levels.
  • the biomarker levels are combined in some way and a single value for the combined biomarker levels is reported.
  • the reported value may be a single number determined from the sum of all the marker calculations that is compared to a pre - set threshold value that is an indication of the presence or absence of disease.
  • the diagnostic score may be a series of bars that each represent a biomarker value and the pattern of the responses may be compared to a pre-set pattern for determination of the presence or absence of disease.
  • FIG. 21 An example of a computer system 100 is shown in Figure 21.
  • system 100 is shown comprised of hardware elements that are electrically coupled via bus 108, including a processor 101, input device 102, output device 103, storage device 104, computer-readable storage media reader 105a, communications system 106 processing acceleration (e.g., DSP or special-purpose processors) 107 and memory 109.
  • Computer-readable storage media reader 105 a is further coupled to computer- readable storage media 105b, the combination comprehensively representing remote, local, fixed and/or removable storage devices plus storage media, memory, etc. for temporarily and/or more permanently containing computer-readable information, which can include storage device 104, memory 109 and/or any other such accessible system 100 resource.
  • System 100 also comprises software elements (shown as being currently located within working memory 191) including an operating system 192 and other code 193, such as programs, data and the like. With respect to Figure 21, system 100 has extensive flexibility and configurability. Thus, for example, a single architecture might be utilized to implement one or more servers that can be further configured in accordance with currently desirable protocols, protocol variations, extensions, etc. However, it will be apparent to those skilled in the art that embodiments may well be utilized in accordance with more specific application
  • system elements might be implemented as sub- elements within a system 100 component (e.g., within communications system 106).
  • Customized hardware might also be utilized and/or particular elements might be implemented in hardware, software or both. Further, while connection to other computing devices such as network input/output devices (not shown) may be employed, it is to be understood that wired, wireless, modem, and/or other connection or connections to other computing devices might also be utilized.
  • network input/output devices not shown
  • wired, wireless, modem, and/or other connection or connections to other computing devices might also be utilized.
  • the system can comprise a database containing features of biomarkers characteristic of decreased GFR, a GFR value or range that is considered below a normal value or range or reduced kidney function.
  • the biomarker data (or biomarker information) can be utilized as an input to the computer for use as part of a computer implemented method.
  • the biomarker data can include the data as described herein.
  • system further comprises one or more devices for providing input data to the one or more processors.
  • the system further comprises a memory for storing a data set of ranked data elements.
  • the device for providing input data comprises a detector for detecting the characteristic of the data element, e.g., such as a mass spectrometer or gene chip reader.
  • the system additionally may comprise a database management system.
  • User requests or queries can be formatted in an appropriate language understood by the database
  • the system may be connectable to a network to which a network server and one or more clients are connected.
  • the network may be a local area network (LAN) or a wide area network (WAN), as is known in the art.
  • the server includes the hardware necessary for running computer program products (e.g., software) to access database data for processing user requests.
  • the system may include an operating system (e.g., UNIX ® or Linux) for executing instructions from a database management system.
  • the operating system can operate on a global communications network, such as the internet, and utilize a global communications network server to connect to such a network.
  • the system may include one or more devices that comprise a graphical display interface comprising interface elements such as buttons, pull down menus, scroll bars, fields for entering text, and the like as are routinely found in graphical user interfaces known in the art.
  • Requests entered on a user interface can be transmitted to an application program in the system for formatting to search for relevant information in one or more of the system databases.
  • Requests or queries entered by a user may be constructed in any suitable database language.
  • the graphical user interface may be generated by a graphical user interface code as part of the operating system and can be used to input data and/or to display inputted data.
  • the result of processed data can be displayed in the interface, printed on a printer in communication with the system, saved in a memory device, and/or transmitted over the network or can be provided in the form of the computer readable medium.
  • the system can be in communication with an input device for providing data regarding data elements to the system (e.g., expression values).
  • the input device can include a gene expression profiling system including, e.g., a mass spectrometer, gene chip or array reader, and the like.
  • the methods and apparatus for analyzing biomarker information may be implemented in any suitable manner, for example, using a computer program operating on a computer system.
  • a conventional computer system comprising a processor and a random access memory, such as a remotely-accessible application server, network server, personal computer or workstation may be used.
  • Additional computer system components may include memory devices or information storage systems, such as a mass storage system and a user interface, for example a conventional monitor, keyboard and tracking device.
  • the computer system may be a stand-alone system or part of a network of computers including a server and one or more databases.
  • the biomarker analysis system can provide functions and operations to complete data analysis, such as data gathering, processing, analysis, reporting and/or diagnosis.
  • the computer system can execute the computer program that may receive, store, search, analyze, and report information relating to the biomarkers.
  • the computer program may comprise multiple modules performing various functions or operations, such as a processing module for processing raw data and generating supplemental data and an analysis module for analyzing raw data and supplemental data to generate a disease status and/or diagnosis. Identifying a GFR value or range that is considered below a normal value or range or reduced kidney function may comprise generating or collecting any other information, including additional biomedical information, regarding the condition of the individual relative to the disease, identifying whether further tests may be desirable, or otherwise evaluating the health status of the individual.
  • a computer program product may include a computer readable medium having computer readable program code embodied in the medium for causing an application program to execute on a computer with a database.
  • a "computer program product” refers to an organized set of instructions in the form of natural or programming language statements that are contained on a physical media of any nature (e.g., written, electronic, magnetic, optical or otherwise) and that may be used with a computer or other automated data processing system. Such programming language statements, when executed by a computer or data processing system, cause the computer or data processing system to act in accordance with the particular content of the statements.
  • Computer program products include without limitation: programs in source and object code and/or test or data libraries embedded in a computer readable medium.
  • the computer program product that enables a computer system or data processing equipment device to act in pre-selected ways may be provided in a number of forms, including, but not limited to, original source code, assembly code, object code, machine language, encrypted or compressed versions of the foregoing and any and all equivalents.
  • a computer program product for indicating whether decreased GFR or reduced kidney function is likely to progress, regress or reoccur.
  • the computer program product includes a computer readable medium embodying program code executable by a processor of a computing device or system, the program code comprising: code that retrieves data attributed to a biological sample from an individual, wherein the data comprises biomarker levels that correspond to one or more of the biomarkers described herein, and code that executes a classification method that indicates the a decreased GFR or reduced kidney function status of the individual as a function of the biomarker levels.
  • the embodiments may be embodied as code stored in a computer- readable memory of virtually any kind including, without limitation, RAM, ROM, magnetic media, optical media, or magneto-optical media. Even more generally, the embodiments could be implemented in software, or in hardware, or any combination thereof including, but not limited to, software running on a general purpose processor, microcode, programmable logic arrays (PLAs), or application-specific integrated circuits (ASICs).
  • a general purpose processor microcode, programmable logic arrays (PLAs), or application-specific integrated circuits (ASICs).
  • PLAs programmable logic arrays
  • ASICs application-specific integrated circuits
  • embodiments could be accomplished as computer signals embodied in a carrier wave, as well as signals (e.g., electrical and optical) propagated through a transmission medium.
  • signals e.g., electrical and optical
  • the various types of information discussed above could be formatted in a structure, such as a data structure, and transmitted as an electrical signal through a transmission medium or stored on a computer readable medium.
  • methods of monitoring GFR or kidney function are provided.
  • the present methods of predicting whether an individual with a decreased GFR or reduced kidney function will respond to a particular treatment is carried out again at a time 1, and optionally, a time 2, and optionally, a time 3, etc., in order to monitor the status of the GFR or kidney function; or to monitor the effectiveness of one or more treatments of decreased GFR or reduced kidney function at slowing the progression of decreased GFR or reduced kidney function.
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8.
  • the actual GFR is about equal to or more than 60 mL/min./1.73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and AMH in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • CST3, Endostatin COL18
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and AMH in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDL1 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDLl in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), AMH and PI3 in a sample from the subject wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and PI3 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1) and FCN2 in a sample from the subject wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and FCN2 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7. In a related aspect, the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), FCN2 and CTSL2 in a sample from the subject wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), FCN2 and CTSL2 in a sample from the subject wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • COL18A1 Endo
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin
  • the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82 or 0.83.
  • the actual GFR is about equal to or more than 40 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TGF3 Trefoil Factor 3
  • CST3, NBL1, B2M and I
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the actual GFR is about equal to or more than 1 mL/min./1.73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDLl in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and
  • the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8.
  • the actual GFR is about equal to or more than 40 mL/min./l .73m 2 .
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 and NBL1 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 and NBL1 in a sample from the subject, wherein the GFR of the subject is estimated based on the level of each of the biomarker proteins, and wherein the estimated GFR and actual GFR are related by a correspondence correlation of at least about 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the actual GFR is about equal to or more than 1 mL/min./1.73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8.
  • the actual GFR is about equal to or more than 60 mL/min./1.73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1) and AMH in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74,
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1) and AMH in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDL1 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), AMH and CHRDLl in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal to or more than 60 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and PI3 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), AMH and PI3 in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal or more than 60 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and FCN2 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • CST3, Endostatin glomer
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1) and FCN2 in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal or more than 60 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), FCN2 and CTSL2 in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), FCN2 and CTSL2 in a sample from the subject wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69 or 0.7.
  • the actual GFR is about equal or more than 60 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TDF3 Trefoil Factor 3
  • COL18A1 Endo
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82 or 0.83.
  • the actual GFR is about equal or more than 40 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • TGF3 Trefoil Factor 3
  • CST3, NBL1, B2M and I
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the actual GFR is about equal or more than 1 mL/min./l .73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDL1 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDL1 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79 or 0.8.
  • the actual GFR is about equal or more than 40 mL/min./1.73m 2 .
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 and NBL1 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 and NBL1 in a sample from the subject, wherein the GFR of the subject is determined based on the level of each of the biomarker proteins, and wherein the determined GFR and actual GFR are related by a correspondence correlation of at least about 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 or 0.96.
  • the actual GFR is about equal to or more than 1 mL/min./l .73m 2 .
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins TFF3, COL18A1 and UNC5D are higher than a control level of the respective biomarkers.
  • TFF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins TFF3, COL18A1 and UNC5D are higher than a control level of the respective biomarkers.
  • TFF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1) and UNC5D in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins TFF3, COL18A1 and UNC5D are higher than a control level of the respective biomarkers.
  • TFF3 Trefoil Factor 3
  • COL18A1 Endostatin
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and AMH in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • TNF3 glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and AMH in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and AMH in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • TNF3 Trefoil Factor 3
  • CST3 Endostatin
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDLl in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and CHRDLl are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDLl in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and CHRDLl are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and CHRDLl in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and CHRDL1 are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and PI3 in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and PI3 are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • TNF3 glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), AMH and PI3 in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and PI3 are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1), AMH and PI3 in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and PI3 are higher than a control level of the respective biomarkers, and the biomarker protein AMH is lower than a control level of the respective biomarker.
  • TNF3 Trefoil Factor 3
  • CST3 Endostatin
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and FCN2 in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and the biomarker protein FCN2 is lower than a control level of the respective biomarker.
  • TNF3 glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1) and FCN2 in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and the biomarker protein FCN2 is lower than a control level of the respective biomarker.
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • Endostatin (COL18A1) and FCN2 in a sample from the subject wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and the biomarker protein FCN2 is lower than a control level of the respective biomarker.
  • TNF3 Trefoil Factor 3
  • CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers
  • the biomarker protein FCN2 is lower than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), FCN2 and CTSL2 in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and each of the biomarker proteins FCN2 and CTSL2 is lower than a control level of the respective biomarker.
  • TNF3 glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, Endostatin (COL18A1), FCN2 and CTSL2 in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers, and each of the biomarker proteins FCN2 and CTSL2 is lower than a control level of the respective biomarker.
  • TNF3 glomerular filtration rate
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3,
  • TNF3 Trefoil Factor 3
  • CST3 and Endostatin (COL18A1) are higher than a control level of the respective biomarkers
  • each of the biomarker proteins FCN2 and CTSL2 is lower than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D are higher than a control level of the respective biomarkers.
  • TNF3 glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D are higher than a control level of the respective biomarkers.
  • TNF3 glomerular filtration rate
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), Endostatin (COL18A1), PI3 and UNC5D are higher than a control level of the respective biomarkers.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 are higher than a control level of the respective biomarkers.
  • TNF3 glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 are higher than a control level of the respective biomarkers.
  • TNF3 glomerular filtration rate
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBL1, B2M and IGFBP6 in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins Trefoil Factor 3 (TFF3), CST3, NBLl, B2M and IGFBP6 are higher than a control level of the respective biomarkers.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDL1 in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDLlare higher than a control level of the respective biomarkers.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDLl in a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDLl are higher than a control level of the respective biomarkers.
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDLl in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins CST3, Endostatin (COL18A1) and CHRDLlare higher than a control level of the respective biomarkers.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3) and NBLl in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of each of the biomarker proteins TFF3 and NBLl are higher than a control level of the respective biomarkers.
  • GFR glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is decreasing comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3) and NBLlin a sample from the subject, wherein the GFR of the subject is determined to be decreasing if the level of each of the biomarker proteins TFF3 and NBLl are higher than a control level of the respective biomarkers.
  • GFR glomerular filtration rate
  • a method for determining whether renal function in a subject is declining comprising detecting the level of each of the biomarker proteins Trefoil Factor 3 (TFF3) and NBLl in a sample from the subject, wherein the renal function of the subject is determined to be declining if the level of each of the biomarker proteins TFF3 and NBLl are higher than a control level of the respective biomarkers.
  • TFF3 Trefoil Factor 3
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for predicting whether the glomerular filtration rate (GFR) in a subject is declining comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for determining the glomerular filtration rate (GFR) in a subject comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subj ect.
  • a method for predicting the glomerular filtration rate (GFR) in a subject comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for estimating the glomerular filtration rate (GFR) in a subject comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for determining whether the renal function in a subject is declining comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for predicting whether the renal function in a subject is declining comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for determining the status of renal function in a subject comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for predicting the status of renal function in a subject comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • a method for estimating the status of renal function in a subject comprising forming a biomarker panel having N biomarker proteins from the biomarker proteins listed in Table 2, and detecting the level of each of the N biomarker proteins of the panel in a sample from the subject.
  • the method for detecting the biomarker protein is performed by contacting the sample with the capture reagent.
  • the capture reagent has binding affinity for the biomarker protein.
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein B2M in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein B2M, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein TFF3 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein TFF3, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein NBL1 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein NBL1, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein AMH in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein AMH, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein IGFBP6 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein IGFBP6, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein CHRDLl in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein CHRDLl, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein PI3 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein PI3, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • GFR in a subject comprising detecting the level of the biomarker protein UNC5D in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein UNC5D, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein COL18A1 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein COL18A1, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • a method for estimating, predicting and/or determining the glomerular filtration rate (GFR) in a subject comprising detecting the level of the biomarker protein FCN2 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein FCN2, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95 and 0.96).
  • GFR in a subject comprising detecting the level of the biomarker protein CTSL2 in a sample from the subject, wherein the GFR of the subject is estimated, predicted and/or determined based on the level of the biomarker protein CSTL2, and wherein the estimated, predicted and/or determined GFR and actual GFR are related by a correspondence correlation of at least about 0.6 (or 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71,
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein Trefoil Factor 3 (TFF3), in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein TFF3 is higher than a control level of the respective biomarker.
  • GFR glomerular filtration rate
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein B2M, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein B2M is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein NBL1, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein NBL1 is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein IGFBP6, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein IGFBP6 is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein CHRDLl, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein CHRDLl is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein PI3, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein PI3 is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein UNC5D, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein UNC5D is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein COL18A1, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein COL18A1 is higher than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein AMH, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein AMH is lower than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein FCN2, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein FCN2 is lower than a control level of the respective biomarker.
  • a method for determining whether the glomerular filtration rate (GFR) in a subject is declining comprising detecting the level of the biomarker protein CTSL2, in a sample from the subject, wherein the GFR of the subject is determined to be declining if the level of the biomarker protein CSTL2 is lower than a control level of the respective biomarker.
  • This example provides an overview of the study designed to identify biomarkers used to assess GFR and/or kidney function in individuals.
  • the sample set consists of 137 samples before iohexol administration (time point A) and 220 samples after administration (time point B).
  • time point A the measured glomular filtration rate
  • time point B the measured glomular filtration rate
  • reference Cystatin-C and creatinine measurements were included as additional meta-data.
  • the final set consists of 300 samples, 128 labeled as “pre -iohexol,” and 172 labeled as “post-iohexol.” Briefly, some samples were dropped from the dataset due to missing GFR values, data duplication, evidence of hemolysis, or aberrant normalization scale factors. Table 1 gives a summary of the samples in this study. Table 1 : Summary of GFR samples
  • an external test set of 60 observations (20% of the dataset) was chosen randomly, held out from the modeling procedure, and only used to assess predictive abilities of the models.
  • the 240 observations remaining in the training set were randomly distributed to 10 sub-groups which were used for internal cross-validation studies as needed. These studies may be used to evaluate and select different methods during the modeling process while still only using the training set and not the external prediction set.
  • the role of the external prediction set is solely to evaluate the final selected models in predictive capacity. In all set assignments, an effort was made to ensure that each sub-group contains uniform coverage of the range of GFR values as much as possible without manual selection of which observations belong to which group.
  • Figure 1 shows a scatter plot of Cystatin-C versus GFR levels, with Cystatin-C measured with an aptamer based assay (left column) and clinical ELISA (right column) at time points A (pre-iohexol - top) and B (post-iohexol - bottom).
  • a single outlier (Sample Id 21, identified with an arrow) can be seen in the Timepoint B graph for Cystatin-C measured by aptamer based assay versus GFR - the bottom- left panel of Figure 1.
  • Other observations with similar GFR values (93) have Cystatin-C RFU (relative fluorescence units; measured via aptamer based assay) values in the range of 2100 - 2700, while Sample Id has a value of 4751.
  • Cystatin-C measurements exhibit a "lowest level of detection” effect as GFR increases and the effect is much more pronounced in the ELISA measurement than in the aptamer based measurements with the slope of the "point cloud” clearly steeper at lower levels (less than 2.0) of ELISA measured Cystatin-C.
  • GFR levels decrease, clinical Cystatic-C measurements show a clear signal saturation effect, illustrating why Cystatin-C may not detect small changes in GFR that could be clinically relevant.
  • This example provides the list of selected proteins whose level of expression changed with changes in GFR.
  • the protein biomarkers Elafin, TFF3, CST3, Endostatin and Chordin-Like 1 were elevated relative to a control level or reference level.
  • the protein biomarkers Cathepsin L2, Ficolin-2 and Anti-Muellerian Hormone were decreased relative to a control level or reference level.
  • FCN2 Ficolin-2 (FCN2) X3313.21_2.FCN2 0.389 3.20E-09 2.65E-12
  • This example provides an overview of the methods used to develop biomarker models for predicting, estimating and/or determining GFR in a subject for the full range of GFR (e.g., from about 0 to about 120+ mL/min./1.73m 2 ).
  • the curve for EGFR was not included in the set of 6 proteins due to its curve being much lower overall and its general location being in the 'noisy' set of curves at the far right of the graph.
  • the most frequently occurring protein is Cystatin-C (CST3) as detected by the aptamer based assay.
  • a five (5) marker panel was developed comprising CST3, B2M, NBL1, TFF3 and
  • the result of this equation is a scaled, log 10-trans formed GFR that can be converted to actual GFR.
  • the values for GFR on both axes range from -2.0 to 1.0 rather than 0 to 120 mL/min./1.72m 2 .
  • This -2.0 to 1.0 range results from the loglO transformation and scaling, and corresponds to the original GFR range.
  • One of ordinary skill in the art can predict, estimate and/or determine GFR based on the given equation.
  • the detected level of the protein biomarkers along with a linear regression approach may be used to predict, estimate and/or determine GFR in a subject.
  • This example provides an overview of the methods used to develop biomarker models for predicting GFR or determining a decrease or decline of GFR in a subject when the actual GFR values are equal to or greater than 40 mL/min./l .73m 2 .
  • these models were developed to be predictive for GFR when actual GFR values are equal to or greater than 40 mL/min./l .73m 2 .
  • One of ordinary skill in the art can predict, estimate and/or determine GFR based on the given equation.
  • Example 5 Model Prediction for GFR Values of Equal to or Greater than 60 mL/min./1.73m 2
  • This example provides an overview of the methods used to develop biomarker models for predicting GFR or determining a decrease or decline of GFR in a subject when actual GFR values are equal to or greater than 60 mL/min./l .73m 2 .
  • these models were developed to be predictive for GFR when actual GFR values are equal to or greater than 60 mL/min./l .73m 2 .
  • ⁇ OFR range units are mL/minute/1.73m
  • biomarker measured values (RFU) and GFR are log 10 transformed and scaled.
  • An exemplary method of detecting one or more biomarkers in a sample is described, e.g., in Kraemer et al, PLoS One 6(10): e26332, and is described below.
  • HEPES NaCl, KC1, EDTA, EGTA, MgCl 2 and Tween-20 may be purchased, e.g., from Fisher Biosciences.
  • Dextran sulfate sodium salt (DxS04), nominally 8000 molecular weight, may be purchased, e.g., from AIC and is dialyzed against deionized water for at least 20 hours with one exchange.
  • KOD EX DNA polymerase may be purchased, e.g., from VWR.
  • Tetramethylammonium chloride and CAPSO may be purchased, e.g., from Sigma- Aldrich and streptavidin-phycoerythrin (SAPE) may be purchased, e.g., from Moss Inc.
  • SAPE streptavidin-phycoerythrin
  • AEBSF 4-(2- Aminoethyl)-benzenesulfonylfluoride hydrochloride
  • AEBSF 4-(2- Aminoethyl)-benzenesulfonylfluoride hydrochloride
  • Streptavidin-coated 96-well plates may be purchased, e.g., from Thermo Scientific (Pierce Streptavidin Coated Plates HBC, clear, 96-well, product number 15500 or 15501).
  • NHS-PE04-biotin may be purchased, e.g., from Thermo Scientific (EZ- Link NHS-PE04-Biotin, product number 21329), dissolved in anhydrous DMSO, and may be stored frozen in single-use aliquots.
  • IL-8, MIP-4, Lipocalin-2, RANTES, MMP-7, and MMP-9 may be purchased, e.g., from R&D Systems.
  • Resistin and MCP-1 may be purchased, e.g., from PeproTech, and tPA may be purchased, e.g., from VWR.
  • Z-Block is a single-stranded oligodeoxynucleotide of sequence 5'- (AC-BnBn)7-AC-3', where Bn indicates a benzyl- substituted deoxyuridine residue.
  • Z-block may be synthesized using conventional
  • phosphoramidite chemistry may also be synthesized by conventional phosphoramidite chemistry, and may be purified, for example, on a 21.5x75 mm PRP-3 column, operating at 80°C on a Waters Autopurification 2767 system (or Waters 600 series semi-automated system), using, for example, a timberline TL-600 or TL-150 heater and a gradient of triethylammonium bicarbonate (TEAB) / ACN to elute product. Detection is performed at 260 nm and fractions are collected across the main peak prior to pooling best fractions. Buffers
  • Buffer SB 18 is composed of 40 mM HEPES, 101 mM NaCl, 5 mM KC1, 5 mM MgC12, and 0.05% (v/v) Tween 20 adjusted to pH 7.5 with NaOH.
  • Buffer SB 17 is SB 18 supplemented with 1 mM trisodium EDTA.
  • Buffer PB1 is composed of 10 mM HEPES, 101 mM NaCl, 5 mM KC1, 5 mM MgC12, 1 mM trisodium EDTA and 0.05% (v/v) Tween-20 adjusted to pH 7.5 with NaOH.
  • CAPSO elution buffer consists of 100 mM CAPSO pH 10.0 and 1 M NaCl.
  • Neutralization buffer contains of 500 mM HEPES, 500 mM HCl, and 0.05% (v/v) Tween-20.
  • Agilent Hybridization Buffer is a proprietary formulation that is supplied as part of a kit (Oligo aCGH/ChlP-on-chip Hybridization Kit).
  • Agilent Wash Buffer 1 is a proprietary formulation (Oligo aCGH/ChlP-on-chip Wash Buffer 1, Agilent).
  • Agilent Wash Buffer 2 is a proprietary formulation (Oligo aCGH/ChlP-on-chip Wash Buffer 2, Agilent).
  • TMAC hybridization solution consists of 4.5 M tetramethylammonium chloride, 6 mM trisodium EDTA, 75 mM Tris-HCl (pH 8.0), and 0.15% (v/v) Sarkosyl.
  • KOD buffer (10-fold concentrated) consists of 1200 mM Tris-HCl, 15 mM MgS04, 100 mM KC1, 60 mM
  • Serum (stored at -80°C in 100 aliquots) is thawed in a 25°C water bath for 10 minutes, then stored on ice prior to sample dilution. Samples are mixed by gentle vortexing for 8 seconds.
  • a 6%> serum sample solution is prepared by dilution into 0.94x SB 17 supplemented with 0.6 mM MgC12, 1 mM trisodium EGTA, 0.8 mM AEBSF, and 2 ⁇ Z- Block. A portion of the 6% serum stock solution is diluted 10-fold in SB 17 to create a 0.6% serum stock.
  • 6%> and 0.6%> stocks are used, in some embodiments, to detect high- and low- abundance analytes, respectively.
  • Aptamers are grouped into 2 mixes according to the relative abundance of their cognate analytes (or biomarkers). Stock concentrations are 4 nM for each aptamer, and the final concentration of each aptamer is 0.5 nM. Aptamer stock mixes are diluted 4-fold in SB 17 buffer, heated to 95°C for 5 min and cooled to 37°C over a 15 minute period prior to use. This denaturation-renaturation cycle is intended to normalize aptamer conformer distributions and thus ensure reproducible aptamer activity in spite of variable histories. Streptavidin plates are washed twice with 150 buffer PB1 prior to use.
  • Heat-cooled 2x Aptamer mixes (55 ⁇ ) are combined with an equal volume of 6% or 0.6%) serum dilutions, producing equilibration mixes containing 3%> and 0.3%> serum.
  • the plates are sealed with a Silicone Sealing Mat (Axymat Silicone sealing mat, VWR) and incubated for 1.5 h at 37°C. Equilibration mixes are then transferred to the wells of a washed 96-well streptavidin plate and further incubated on an Eppendorf Thermomixer set at 37°C, with shaking at 800 rpm, for two hours.
  • liquid is removed by dumping, followed by two taps onto layered paper towels. Wash volumes are 150 ⁇ and all shaking incubations are done on an Eppendorf Thermomixer set at 25°C, 800 rpm. Equilibration mixes are removed by pipetting, and plates are washed twice for 1 minute with buffer PBl supplemented with 1 mM dextran sulfate and 500 ⁇ biotin, then 4 times for 15 seconds with buffer PBl . A freshly made solution of 1 mM NHS-PE04-biotin in buffer PBl (150 ⁇ ) is added, and plates are incubated for 5 minutes with shaking.
  • the NHS-biotin solution is removed, and plates washed 3 times with buffer PBl supplemented with 20 mM glycine, and 3 times with buffer PBl .
  • Eighty- five ⁇ ⁇ of buffer PBl supplemented with 1 mM DxS04 is then added to each well, and plates are irradiated under a BlackRay UV lamp (nominal wavelength 365 nm) at a distance of 5 cm for 20 minutes with shaking.
  • Samples are transferred to a fresh, washed streptavidin-coated plate, or an unused well of the existing washed streptavidin plate, combining high and low sample dilution mixtures into a single well. Samples are incubated at room temperature with shaking for 10 minutes.
  • Streptavidin plates bearing adsorbed equilibration mixes are placed on the deck of a
  • BioTek EL406 plate washer which is programmed to perform the following steps:
  • unadsorbed material is removed by aspiration, and wells are washed 4 times with 300 ⁇ , of buffer PBl supplemented with 1 mM dextran sulfate and 500 ⁇ biotin. Wells are then washed 3 times with 300 ⁇ ⁇ buffer PBl .
  • One hundred fifty ⁇ ⁇ of a freshly prepared (from a 100 mM stock in DMSO) solution of 1 mM NHS-PE04-biotin in buffer PB 1 is added. Plates are incubated for 5 minutes with shaking. Liquid is aspirated, and wells are washed 8 times with 300 ⁇ , buffer PBl supplemented with 10 mM glycine.
  • thermoshaker mounted under a UV light source (BlackRay, nominal wavelength 365 nm) at a distance of 5 cm for 20 minutes.
  • the thermoshaker is set at 800 rpm and 25°C.
  • samples are manually transferred to a fresh, washed streptavidin plate (or to an unused well of the existing washed plate).
  • High-abundance (3% serum+3% aptamer mix) and low-abundance reaction mixes (0.3% serum+0.3% aptamer mix) are combined into a single well at this point.
  • This "Catch- 2" plate is placed on the deck of BioTek EL406 plate washer, which is programmed to perform the following steps: the plate is incubated for 10 minutes with shaking. Liquid is aspirated, and wells are washed 21 times with 300 ⁇ , buffer PB1 supplemented with 30% glycerol. Wells are washed 5 times with 300 ⁇ , buffer PB 1 , and the final wash is aspirated. One hundred ⁇ , CAPSO elution buffer are added, and aptamers are eluted for 5 minutes with shaking. Following these automated steps, the plate is then removed from the deck of the plate washer, and 90 ⁇ ⁇ aliquots of the samples are transferred manually to the wells of a HybAid 96-well plate that contains 10 ⁇ ⁇ neutralization buffer.
  • Agilent Block (Oligo aCGH/ChlP-on-chip Hybridization Kit, Large Volume, Agilent 5188- 5380), containing a set of hybridization controls composed of 10 Cy3 aptamers is added to each well. Thirty ⁇ ⁇ 2x Agilent Hybridization buffer is added to each sample and mixed. Forty ⁇ ⁇ of the resulting hybridization solution is manually pipetted into each "well" of the hybridization gasket slide (Hybridization Gasket Slide, 8-microarray per slide format,
  • Agilent Custom Agilent microarray slides, bearing 10 probes per array complementary to 40 nucleotide random region of each aptamer with a 20 x dT linker, are placed onto the gasket slides according to the manufacturers' protocol.
  • the assembly Hybridization Chamber Kit - SureHyb-enabled, Agilent is clamped and incubated for 19 hours at 60°C while rotating at 20 rpm.
  • Microarray slides are imaged with an Agilent G2565CA Microarray Scanner System, using the Cy3 -channel at 5 ⁇ resolution at 100% PMT setting, and the XRD option enabled at 0.05.
  • the resulting TIFF images are processed using Agilent feature extraction software version 10.5.1.1 with the GEl_105_Dec08 protocol.
  • Primary Agilent data is available as Supplementary Information (Figure S6).
  • Probes immobilized to beads have 40 deoxynucleotides complementary to the 3' end of the 40 nucleotide random region of the target aptamer.
  • the aptamer complementary region is coupled to Luminex Microspheres through a hexaethyleneglycol (HEG) linker bearing a 5' amino terminus.
  • HOG hexaethyleneglycol
  • Biotin moieties are appended to the 3' ends of detection oligos.
  • Probes are coupled to Luminex Microplex Microspheres essentially per the manufacturer's instructions, but with the following modifications: amino -terminal oligonucleotide amounts are 0.08 nMol per 2.5x 106 microspheres, and the second EDC addition is 5 at 10 mg/mL. Coupling reactions are performed in an Eppendorf
  • ThermoShaker set at 25°C and 600 rpm.
  • Microsphere stock solutions (about 40000 microspheres ⁇ L) are vortexed and sonicated in a Health Sonics ultrasonic cleaner (Model: T1.9C) for 60 seconds to suspend the microspheres.
  • Suspended microspheres are diluted to 2000 microspheres per reaction in 1.5x TMAC hybridization solutions and mixed by vortexing and sonication.
  • Thirty-three per reaction of the bead mixture are transferred into a 96-well HybAid plate.
  • Seven of 15 nM biotinylated detection oligonucleotide stock in 1 x TE buffer are added to each reaction and mixed.
  • Ten ⁇ of neutralized assay sample are added and the plate is sealed with a silicon cap mat seal.
  • the plate is first incubated at 96°C for 5 minutes and incubated at 50°C without agitation overnight in a conventional hybridization oven.
  • a filter plate (Dura pore, Millipore part number MSBVN1250, 1.2 ⁇ pore size) is prewetted with 75 ⁇ 1 x TMAC
  • hybridization solution supplemented with 0.5% (w/v) BSA.
  • the entire sample volume from the hybridization reaction is transferred to the filter plate.
  • the hybridization plate is rinsed with 75 ⁇ l x TMAC hybridization solution containing 0.5% BSA and any remaining material is transferred to the filter plate.
  • Samples are filtered under slow vacuum, with 150 ⁇ , buffer evacuated over about 8 seconds.
  • the filter plate is washed once with 75 ⁇ , 1 x TMAC hybridization solution containing 0.5% BSA and the microspheres in the filter plate are resuspended in 75 l x TMAC hybridization solution containing 0.5% BSA.
  • the filter plate is protected from light and incubated on an Eppendorf Thermalmixer R for 5 minutes at 1000 rpm.
  • the filter plate is then washed once with 75 1 x TMAC hybridization solution containing 0.5% BSA.
  • 75 ⁇ . of 10 ⁇ g/mL streptavidin phycoerythrin (SAPE-100, MOSS, Inc.) in 1 x TMAC hybridization solution is added to each reaction and incubated on
  • microspheres in the filter plate are resuspended in 75 ⁇ 1 x TMAC hybridization solution containing 0.5% BSA.
  • the filter plate is then incubated protected from light on an Eppendorf Thermalmixer R for 5 minutes, 1000 rpm.
  • the filter plate is then washed once with 75 ⁇ 1 x TMAC hybridization solution containing 0.5%> BSA.
  • Microspheres are resuspended in 75 ⁇ , l x TMAC hybridization solution supplemented with 0.5% BSA, and analyzed on a Luminex 100 instrument running XPonent 3.0 software. At least 100 microspheres are counted per bead type, under high PMT calibration and a doublet discriminator setting of 7500 to 18000.
  • Standard curves for qPCR are prepared in water ranging from 108 to 102 copies with 10-fold dilutions and a no-template control. Neutralized assay samples are diluted 40-fold into diH20.
  • the qPCR master mix is prepared at 2x final concentration (2x KOD buffer, 400 ⁇ dNTP mix, 400 nM forward and reverse primer mix, 2x SYBR Green I and 0.5 U KOD EX). Ten ⁇ of 2x qPCR master mix is added to 10 ⁇ of diluted assay sample.
  • qPCR is run on a BioRad MylQ iCycler with 2 minutes at 96°C followed by 40 cycles of 96°C for 5 seconds and 72°C for 30 seconds.

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Abstract

L'invention concerne des méthodes et des kits destinés à la détection de biomarqueurs et à la caractérisation d'un taux de filtration glomérulaire (GFR) et/ou d'une fonction rénale. L'invention concerne en outre des méthodes permettant de déterminer, de prédire et/ou d'estimer le GFR chez un sujet, ou si la fonction rénale chez un sujet a chuté et/ou diminué en dessous des niveaux précédents ou d'un niveau de contrôle standard chez un sujet.
PCT/US2015/024040 2014-04-04 2015-04-02 Biomarqueurs de débit de filtration glomérulaire et leurs utilisations Ceased WO2015153860A1 (fr)

Applications Claiming Priority (2)

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