Assay with an igfbp-1 peptide or protein to predict severe acute kidney injury

Abstract

An assay for predicting severe acute kidney injury is disclosed. The assay detects cleavage of a peptide or protein with an internal sequence that mimics all or some of the central linker region of insulin-like growth factor binding protein 1. The amount of cleavage of the peptide or protein predicts the likelihood that a patient will develop severe acute kidney injury. The peptide or protein used in the assay may contain a terminal His tag and a terminal Flag tag to immobilize the amino-terminal and carboxy-terminal ends of the peptide on a test strip of the assay after cleavage.

Description

Attorney Docket No.22206-82964 ASSAY WITH AN IGFBP-1 PEPTIDE OR PROTEIN TO PREDICT SEVERE ACUTE KIDNEY INJURY CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 641,118, entitled “Assay with an IGFBP-1 Peptide or Protein to Predict Severe Acute Kidney Injury” and filed on May 1, 2024. The complete disclosure of said provisional application is hereby incorporated by reference. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under grant numbers 1R01DK101034, R01DK080234, and UM1TR004909 awarded by the National Institute of Health. The Government has certain rights in the invention. BACKGROUND OF THE INVENTION Acute kidney injury can be divided into three classes: mild (AKI-1), moderate (AKI-2) and severe (AKI-3). When AKI-3 patients require dialysis, it is AKI-D. The search for urinary AKI biomarkers has resulted in the discovery of NGAL, KIM-1, IL- 18, and many other candidates. NephroCheck, the combination of IGFBP-7 and TIMP-2, received US FDA approval to predict the development of moderate to severe AKI (AKI 2 or 3) within 12 hours in critically ill ICU patients with a sensitivity of 92%. However, the test has a false positive rate of 50% so half the patients that will not develop stage 2 / 3 AKI will have a positive test. This limits NephroCheck’s clinical usefulness. The National Institute for Health and Care Excellence (NICE) in the UK recently reviewed the evidence and recommended against the use of NephroCheck in the National Health Service due to its failure in any studies to demonstrate a change in clinical outcomes, the variability in response, the high false positive rate, and the lack of data for cost effectiveness. The American Association for Clinical Chemistry also states that NephroCheck is not yet recommended for routine risk assessment in the US due to the lack of evidence of benefit, suboptimal specificity, and limited performance outside the ICU. The reference range in patientswithout AKI for the NephroCheck assay is 0.04–2.22 (ng / mL)2 / 1000 which overlaps with the cutoff value used for the assay which is 0.3. In addition to its high false positive rate, another factor that may contribute to the lack of benefit of the NephroCheck assay is what it is designed to predict. NephroCheck is designed and approved to predict the development of AKI-2 and AKI-3. The pivotal manuscript did not report the number of patients among the 71 with moderate to severe AKI that had AKI-3 but given the natural course of AKI, there were probably more stage 2 patients than stage 3 patients in the positive outcome group. In a study comparing stage 2 to stage 3 AKI, patients with more severe AKI had more than twice the in hospital mortality rate. Patients with AKI-3 often require dialysis while patients with AKI-2 generally do not. Thus, distinguishing patients that will develop AKI-D from other patients is a very important and NephroCheck cannot do this. It would therefore be desirable to develop a rapid, easy to implement assay that can differentiate No AKI / AKI-1 / AKI-2 / AKI-3 without dialysis from AKI-D and predict severe AKI. The inventors have developed an assay to predict severe AKI. This assay can help guide treatment (including the initiation of dialysis) and define sepsis in patients that have a demonstrated infection but have not yet manifested end-organ damage. Additionally, the assay can be useful to guide enrollment in clinical trials and monitor responses to therapy, which is important because there are multiple drug development programs underway in preclinical programs. Finally, when new drug treatments become available, biomarkers will be used to guide drug therapies. Thus, the development of an accurate point of care test to predict who will develop severe AKI and / or dialysis requiring AKI satisfies a major need. BRIEF SUMMARY OF THE INVENTION The inventors have discovered complete or nearly complete cleavage of a protein (insulin-like growth factor binding protein 1 (IGFBP-1)) that is often present in the urine of patients that develop severe AKI. Cleavage of IGFBP-1 does not occur in urine from healthy controls and rarely in patients with mild or moderate AKI. The protein cleavage occurs both in the native protein which is present naturally in the urine of many patients with severe AKI but also when exogenous IGFBP-1 is addedto the urine. The inventors have shown that this cleavage is specific for patients with severe AKI and that the enzyme activity is robust, occurring even after 10 years of storage at -80 C. Data shows that not all patients who will develop severe AKI produce the protein (about 70%), but all the patients who will develop severe AKI requiring dialysis (12 / 12) and half the patients that will develop stage 3 AKI but not require dialysis (4 / 8) have the enzymatic activity. In contrast, no patients that will not develop AKI (0 / 20), 11% of patients that will develop stage 1 AKI (3 / 27) and 15% of patients that will develop stage 2 AKI (2 / 13) have the enzyme activity. These data suggest that an assay that measures the amount of cleavage of IGFBP-1 is a sensitive and specific test for severe AKI. In one embodiment, the present invention is directed to a lateral flow assay (LFA) that measures the ability of enzymes in the urine to cleave a peptide or protein containing an IGFBP-1 cleavage site. The protein or peptide can be synthetic or recombinant. In another embodiment, the present invention is directed to assay that can predict the development of AKI-3 or AKI-D in patients. In yet another embodiment, the present invention is directed to a synthetic IGFBP-1 peptide or recombinant protein that can be used in a lateral flow assay to predict the development of AKI-3 or AKI-D in patients. In another embodiment, the present invention is directed to a peptide or protein containing the IGFBP-1 cleavage sequence that can be used in a fluorescence assay. The peptide or protein has one or more fluorescent tag(s) that change fluorescence after cleavage of the peptide or protein. In yet another embodiment, the present invention is directed to an assay that measures the cleavage of a protein or peptide with an IGFBP-1 cleavage site to predict severe AKI in patients. The lateral flow assays and fluorescence assay of the present invention utilize a peptide or protein having at least a portion of the IGFBP-1 linker sequence that may be natural, recombinant, or synthetic. These and other features, objects and advantages of the present invention will become better understood from a consideration of the following detaileddescription of the preferred embodiments and appended claims in conjunction with the drawings as described following: BRIEF DESCRIPTION OF THE DRAWINGS FIG.1 is a schematic of an example of patients selected by two tests: Test 1 and Test 2. Test 1 selects for stage 2 or stage 3 AKI and has 50% false positive rate (FPR). Test 2 selects for AKI-D and has 0% false positive rate in patients that will not develop AKI, 11% in patients that will develop a maximum of stage 1 AKI, 15% for that will develop a maximum of stage 2 AKI and 50% for patients that will develop stage 3 not requiring dialysis. Test 2 has 100% accuracy to predict AKI-D. FIG.2 is a Western blot using an antibody against the c-terminal end of IGFBP-1 of urine from 4 healthy control subjects (HC), 20 subjects with AKI stage 1 that did not progress, and 10 subjects with AKI stage 1 that progressed to AKI stage 3. The dotted line marks the 25 kDa range to make it easier to see the intact, 28 kDa IGFBP-1 in the blots. The arrow at the lower right indicates the C-terminal fragment that is seen only in urine from subjects that progressed to AKI stage 3. FIG.3 is a Western blot showing cleavage of recombinant human IGFBP-1 by urines from patients with AKI. Patients had stage 1 AKI at the time of collection. The X axis label shows the maximum stage of AKI achieved. The band at 15kDa is the result of cleavage by urine enzymes. Five of six patients with maximum stage 1 are negative as are the patients with maximum stage 2 and 3. Both patients requiring renal replacement therapy (RRT) are positive. FIG.4 illustrates the overall schema for one embodiment of the synthetic peptide or intact protein. The protein or peptide contains a His tag binding site, a 20 amino acid peptide sequence, a biotinylation site for gold nanoparticle binding and a Flag tag site. Since the label is at the C-terminus, only peptides or peptide fragments that include the label can be visualized in the assay. Uncleaved protein or peptide will bind to the first line and be visualized. The amino terminus of cleaved protein or peptide will bind to the first line but will not be visualized because it does not contain the label. The carboxy-terminus of cleaved protein or peptide will bind to the second line and be visualized. The amount of label at the second line or the ratio of label at the second line to label at the first line can be used. In this embodiment, cleavage ofthe peptide occurs before the labeled peptide is added to the lateral flow assay. Cleavage of the peptide or protein could occur at any point within the sequence of the central linker region of the IGFBP-1 protein. The red arrow shows a potential cleavage site. The antibody binding moieties could be replaced by any means of binding the peptide. The gold tag could be replaced by any means of detecting the peptide in the assay. FIG.5 is a schematic showing the design of one embodiment of the lateral flow assay of the present invention. In this embodiment, the labeled peptide or protein is added prior to deposition on the lateral flow assay for cleavage to occur and cleavage by the urine enzyme occurs as the urine migrates through the conjugate pad. FIG.6 is a schematic showing the design of another embodiment of the lateral flow assay of the present invention. In this embodiment, the labeled peptide is included in the conjugate pad and cleavage by the urine enzymes occurs as the urine migrates through the pad. FIG.7 is a schematic showing the design of yet another embodiment of the lateral flow assay of the present invention. FIG.8 is a table showing the number of positive and negative results from the testing of urine of 80 patients for the presence of cleaved IGFBP-1. The results are separated according to the AKI stage of the patient. FIG.9 is a table showing the characteristics of patients from which urine samples were used for proteomic analysis. Statistical significance was determined by the chi-squared test for categorical variables and the Mann-Whitney U test for continuous variables. EF means ejection fraction; PVD means peripheral vascular disease; DM means diabetes mellitus; COPD means chronic obstructive pulmonary disease; CABG means coronary artery bypass grafting; IABP means intra-aortic balloon pump; and RRT means Renal replacement therapy. FIG.10 is a table showing the six proteins that were significantly increased in subjects who needed KRT. FIG.11A is a scatter plot showing the relative abundance of spectra for IGFBP-1 in urine from twenty control subjects that did not progress beyond AKIstage 1 (-) and ten subjects that required renal replacement therapy (+). Quantitative value is a normalized representation of spectral counts. Each dot represents the normalized abundance of IGFBP-1 in urine for one subject. FIG.11B is a graph that shows the number of peptides observed for each peptide sequence in control subjects and subjects that required renal replacement therapy. The numbers under the X-axis show the position of the amino acid sequence within the IGFBP-1 protein for the corresponding peptides. FIG.12 is a schematic showing the design of a fluorescence assay of the present invention for prediction of severe AKI. A protein or peptide that contains the cleavage site from IGFBP-1 is created and tagged with a fluorescent compound on one end and a quencher on the other. When the protein or peptide is cleaved, the quencher is separated from the fluorescent tag and fluorescence is observed. The urine and the protein or peptide are combined. The urine and protein or peptide are incubated to allow cleavage if the enzyme is present. In the absence of the enzyme that is predictive of the development of severe AKI, there is no fluorescence measured. In the presence of the enzyme, fluorescence is observed. Larger amounts of enzyme cause more fluorescence and are predictive of more severe AKI. FIG.13 is a table showing the characteristics of 206 subjects that developed AKI following cardiothoracic surgery. Twenty-five of the subjects met the combined primary outcome of death or stage 3 AKI. Validation of the characteristics of IGFBP- 1 as a predictor of severe AKI was done in this cohort. FIG.14A is a box plot showing the urinary IGFBP1 concentration for negative (-) and positive (+) patients for the primary outcome. The biomarker concentration is presented as median and interquartile ranges in the box plot and as individual dots in the scatter plot. The white bar labeled (-) represent the biomarker concentrations in subjects who did not meet the primary outcome. The grey bar labeled (+) represents the biomarker concentration in the subjects who met the outcome. The dotted line on the scatter plot represents the optimal cutoff value for urine IGFBP 1 concentration. FIG.14B shows the median concentrations and interquartileranges for patients with stage 1 AKI 24 hours after cardiac surgery that reached a maximum of stage 1, stage 2 and stage 3 AKI within 10 days after surgery. FIG.15 is a table that shows the median values and interquartile ranges of IGFBP-1 and IGFBP-1 / creatinine for patients with negative and positive outcomes. It also shows the P value for the comparison of the negative and positive groups and the AUC value for IGFBP-1 and IGFBP-1 / creatinine. FIG.16 is a Western blot showing the cleavage of recombinant IGFBP-1 by urine from a patient that required renal replacement therapy (RRT). Recombinant IGFBP-1 was incubated with urine for 30 minutes at 37º C and then the protein was separated by polyacrylamide gel electrophoresis and western blotting was done with an antibody that recognized the C-terminus of IGFBP-1. The first lane shows the recombinant protein without the addition of urine. No cleavage of the protein occurs and the protein is visualized at its native molecular weight slightly above 25 kDa. Similarly, urine from a healthy control did not cleave the protein. In lane 3, the protein was incubated with urine from the patient that required RRT and the protein is cleaved to a fragment that is slightly above 15 kDa. Use of the serine protease inhibitor AEBSF and denaturing the urine at 97º C abolished the cleavage by the urine enzymes. The use of the calcium binder EDTA, the calpain inhibitor leupeptin, the aspartic acid protease inhibitor pepstatin A, and the broad spectrum inhibitor HALT did not. FIG.17 is a schematic showing the protein regions of IGFBP-1. The IGFBP-1 molecule includes a signal peptide which is cleaved and is not part of the circulating protein. The remaining 234 amino acid residues includes an 82 amino acid N- terminal region which begins with APWQ and ends with CVQE, a 68 amino acid central linker region which begins with SDAS and ends with WKEP and an 84 amino acid C-terminal region which begins with CRIE and goes to the end of the molecule. Cleavage of IGFBP-1 in the urine occurs within the central linker region (SDASAPHAAEAGSPESPESTEITEEELLDNFHLMAPSEEDHSILWDAISTYDGSKA LHVTNIKKWKEP). The native IGFBP-1 molecule includes cysteine cross links within both the N-terminal region and the C-terminal region. These crosslinks cause the protein to fold and bring the amino terminal region closer to the C-terminalregion. This folding may be required for cleavage by the urine enzyme. In this case, a synthetic peptide or recombinant protein will have to fold in a similar way as the native protein in order to be cleaved. FIG.18 is a table showing the performance of prognostic biomarkers of AKI. Area under the curve (AUC) from the receiver operating characteristics, optimal cut- off values, sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio, and p-value are shown for urinary concentrations of IGFBP-1, NGAL, and [TIMP-2]*[IGFBP7]. FIG.19 is a flowchart of patient enrollment in the validation study. Samples from 225 patient with stage 1 AKI were collected within 72 hours of cardiothoracic surgery. Twelve samples were insufficient for analysis, leaving a total of 213 patients for analysis of which 27 patients met criteria for inclusion in the composite primary outcome group. FIGS.20A-20D are plots of urinary concentrations of urinary biomarkers. Shown are the measured concentrations of urinary IGFBP-1 (panel A), [TIMP- 2]*[IGFBP7] (panel C), and NGAL (panel D) for patients the met the primary outcome compared to controls. Each individual data point represents a measured value (IGFBP-1 n = 213, [TIMP2]*[IGFBP7] n = 154, NGAL n = 150). Urinary concentrations of IGFBP-1 separated by stage of AKI and each individual inclusion criteria of the composite outcome are shown in (panel B). The horizontal bars represent the median value and the surrounding boxes represent the interquartile range. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant by unpaired T-test (Panels A, C, D) or one-way ANOVA (Panel B). FIGS.21A-21B are receiver operating characteristics (ROC) curves for biomarkers of AKI. Shown in (Panel A) is the ROC comparison of urinary IGFBP-1 against the traditional clinical biomarker, creatinine. Values for the AUC, optimal cutoff, sensitivity, specificity, and likelihood ratio for IGFBP-1 are shown on the graph. The ROC for IGFBP-1 (n = 213), [TIMP-2]*[IGFBP7] (n = 154), and NGAL (n = 150) are shown in (Panel B). AUC values for each biomarker are displayed on the graph.FIGS.22A-22B are plots showing the effect of IGFBP-1 knockout on CIAKI. Serum creatinine values (Panel A) and blood urea nitrogen (BUN) (Panel B) measured from mouse serum following cisplatin or saline (control) injection are displayed. Each dot (control) or triangle (cisplatin injection) represent a measurement for an individual mouse. Control = saline injected wild type, Control- KO = saline injected IGFBP-1 knockout, Cisplatin-WT = cisplatin injected wild type, Cisplatin-KO = cisplatin injected IGFBP-1 knockout. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant by one-way ANOVA. FIGS.23A-23D show the effect of IGFBP-1 KO on renal tubular cell damage during CIAKI. Representative images of PAS staining of mice kidney tissue are shown at 200x magnification in FIGS.23A-C. Wild type sham mice are represented in panel (A), wild type mice that received cisplatin injection are found in panel (B), and IGFBP-1 KO mice that received cisplatin injections are represented in panel (C). Slides from each animal were scored blinding by a renal pathologist (panel D) for degree of tubular injury. Histology scoring was evaluated by Kruskal-Wallis with Dunn’s multiple comparison test. Each dot (control) or triangle (cisplatin injection) represent scoring for an individual mouse. Control = saline injected wild type, Control-KO = saline injected IGFBP-1 knockout, Cisplatin-WT = cisplatin injected wild type, Cisplatin-KO = cisplatin injected IGFBP-1 knockout. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant. FIGS.24A-24F show the effect of IGFBP-1 KO on cell death and apoptosis during CIAKI. Representative images of TUNEL staining from wild type saline injected mice (panel A), wild type cisplatin injected mice (panel B), and IGFBP-1 KO cisplatin injected mice (panel C) are shown in FIGS.24A-C. The percentage of TUNEL positive cells (panel D), caspase 3 / 7 activity in renal tissue homogenates (panel E) and BCL-xl protein concentrations of renal tissue homogenates (panel F) are shown below the representative images in FIGS.24D-F. Each dot (control) or triangle (cisplatin injection) represent a measurement for an individual mouse, (n = 6-12). Control = saline injected wild type, Control-KO = saline injected IGFBP-1 knockout, Cisplatin-WT = cisplatin injected wild type, Cisplatin-KO = cisplatininjected IGFBP-1 knockout. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant by one-way ANOVA. FIG.25 is a chart showing proteomic analysis of mouse renal tissue homogenates following cisplatin injection. Changes in the renal proteome between saline injected control mice (blue columns) and cisplatin injected CIAKI mice (red columns) are shown. IGFBP-1 was not detected in the analysis. Known biomarkers of AKI including NGAL, AGT, and IGFBP7 were significantly increased following cisplatin injection. Exact p-values are displayed on the graph above the respective columns. AGT = angiotensinogen. FIG.26A-26B are plots showing measurement of phosphorylate AKT and FAK in renal tissue homogenates. The percentage of total AKT that is phosphorylated is displayed in (Panel A). The total concentration of p-FAK is displayed in (Panel B). Values are normalized for tissue homogenate protein concentrations. Each dot (control) or triangle (cisplatin injection) represent a measurement for an individual mouse. Control = saline injected wild type, Control- KO = saline injected IGFBP-1 knockout, Cisplatin-WT = cisplatin injected wild type, Cisplatin-KO = cisplatin injected IGFBP-1 knockout. * p < 0.05 and ns = not significant by one-way ANOVA. FIG.27 is a schematic representation of proposed mechanism of IGFBP-1 signaling in AKI. IGFBP-1 modulates cellular signaling in renal proximal tubular epithelial cell by one of two mechanisms: sequestration of IGF-1 or direct binding of the C-terminus RGD domain to the α5β1 integrin receptor. Binding of IGF-1 to its receptor activates a phosphorylation cascade through AKT via Phosphoinositide 3- kinase (PI3K) ultimately leading to increased synthesis of the anti-apoptotic BCL-xl protein. Binding of IGFBP-1 to the α5β1 integrin receptor activates a separate phosphorylation cascade through FAK via SRC kinases ultimately leading to increased cell migration and increased luminal pressure worsening AKI. The decreased apoptotic markers, increased BCL-xl, and increased ratio of p-AKT in our studies suggest that IGFBP-1 KO mice are protected against CIAKI due to inability to sequester IGF-1 and subsequent increased activation of the IGF-1r.FIG.28 shows alternative embodiments of the synthetic peptides of the present invention that contain overlapping portions of the IGFBP-1 central linker region where cleavage of the IGFBP-1 protein occurs. These peptides could be substitutes for the IGFBP-1 internal sequence shown in FIG.5. DETAILED DESCRIPTION OF THE INVENTION With reference to FIGS.1-28, the preferred embodiments of the present invention may be described. As an initial matter, FIG.1 shows an example of why it is necessary for a biomarker to predict severe AKI that will require dialysis instead of the combination of stage 2 and 3 AKI and why low false positive rate is important in the test. The left side of the figure shows 100 septic ICU patients of whom 40 (40%) will develop AKI and 60% will not. Twenty of the patients will develop AKI-1 (mild), 12 patients will develop AKI-2 (moderate), and 8 patients will develop AKI-3 (severe). From the 8 AKI-3 patients, half (four) will require dialysis. Test 1 predicts stage 2 / 3 AKI with 92% sensitivity and a 50% false positive rate. Because of the high false positive rate 40 of the 80 patients that will not develop stage 2 or 3 AKI will test positive. For Test 1, 58 patients have a positive test of which only seven will have AKI-3 and three-and-a- half will require dialysis (6%). In contrast, Test 2 has characteristics of the test of the present invention. It has 50% sensitivity to predict stage 3 AKI without dialysis and 100% for AKI-D. It has 0% false positive rate for patients without AKI, 11% for patients that will develop stage 1, and 15% for patients that will develop stage 2. Ten patients have a positive test and six of them will have stage 3 AKI or AKI-D. Four of those patients require dialysis (40%). These numbers were obtained from preliminary data shown in FIG.8. The improved prediction allows the correct treatment to be targeted to the appropriate patient to achieve more cost-effective care and improved outcomes. As another example, without biomarker enrichment for a clinical trial of an AKI drug to prevent progression to AKI stage 3, 8% of patients will progress to stage 3 in the control group. In the group that receives a drug with a 20% effect size, 6% would progress. For a power of 80% and alpha of 0.05, 2554 patients per group for a total of 5108 need to be enrolled. When test 1 is used for enrollment, it is positive in 58patients of which 7 progress to stage 3 (12%).20% improvement with the drug means that the progression rate is 80% of the control or 9.6%.1833 patients per group for a total of 3666 need to be enrolled. Test 2 is positive in 10 patients of which 6 progress (60%). In the drug group 48% progress.270 patients per group for a total of 540 will need to be enrolled. Moore calculated the average per patient cost of a clinical trial in the US between 2015-2017 was $41,413. Thus, the cost of performing the trial without biomarkers would be $211,500,000, using test 1 for enrichment would be $90,600,000 and using test 2 would be $22,300,000. The large false positive rate and prediction of stage 2 / 3 AKI explains why NephroCheck has not improved mortality or the need for RRT (dialysis and kidney transplantation) in the studies in which it is used to select patients to be enrolled in AKI treatment bundles. In contrast, the inventors’ preliminary data shows that a test using cleavage of a sequence within IGFBP-1 has a much lower false positive rate and predicts severe AKI (stage 3). Identification of IGFBP-1 cleavage and resulting peptides by mass spectrometry: The inventors identified the protein insulin like growth factor binding protein 1 (IGFBP-1) as an AKI biomarker and confirmed these findings using an ELISA. Urine samples were obtained from a cohort of patients enrolled in the SAKInet consortium between 2008 and 2013. Approval by the local Institutional Review Boards of the collecting centers was obtained at each center, and informed consent was obtained from all subjects. Urine samples were collected from 322 subjects who underwent cardiac surgery, of which 225 subjects met the inclusion criteria for this study of baseline creatinine less than (<) 3 mg / dl, development of KDIGO stage 1 AKI within 72 hours of the surgery, and urine sample collection within 72 hours of surgery. All subjects were followed until hospital discharge or death. Nineteen patients were excluded as they did not develop AKI, were already at least AKI stage 2 at the time of collection, or if there was not sufficient sample to be processed. Discovery Analysis: In the discovery analysis, the inventors performed a proteomic analysis of urine samples obtained from 30 patients; 10 who needed kidney replacement therapy within 10 days of surgery (KRT, +) and 20 patientswhose serum creatinine increased by less than or equal to (^) 0.3 mg / dl (post urine collection) and did not die within 30 days of surgery. Each subject requiring KRT was matched to two control subjects with the same surgery type and baseline serum creatinine within 0.2 mg / dl (FIG.9). Samples were then randomized into two blocks of 5 patients that required KRT and 10 matched controls. Candidate biomarkers were identified using LC-MS / MS. Following sample collection, urine samples were frozen at -80oC. Prior to LC- MS / MS, samples were thawed on ice. Protein concentration was measured with “QuanTtest Red” (Quantimetrix: Redondo Beach, CA) by assaying urine samples in duplicates in a 96 well plate. Urine proteins were digested using the Filter-Assisted Sample Preparation (FASP) protocol13on YM-10 Microcon filter units. Proteins were denatured and alkylated, followed by digestion with typsin. The peptides were separated online by reverse phase at 350 nl / min using a 5x0.2 mm guard column and a 100×0.1 mm analytical C18 BEH 1.7µm column (Waters) with a nanoACQUITY UPLC system (Waters). Peptides were eluted onto the Thermo Scientific Fusion Tribrid mass spectrometer using a 120 min gradient from 98:2 to 40:60 buffer A:B ratio (Buffer A=0.1% formic acid, 0.05% acetonitrile; buffer B=0.1% formic acid, 75% acetonitrile). Eluted peptides were ionized by electrospray (2.0 kV) followed by MS / MS analysis using collision induced dissociation. Survey scans of peptide precursors from 300 to 1500 m / z was performed at 500K resolution (at 400 m / z) in the orbitrap with a 5×105ion count target. Tandem MS was performed by isolation at 1.6 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2ion count target was set to 104and the maximum injection time at 35 milliseconds. For each fragmentation cycle a full MS scan was acquired once every three seconds, the nested MS2scans were performed at a maximum rate between the MS scans. LC-MS / MS data were analyzed using MS-Amanda Proteome Discoverer (Research Institute of Molecular Pathology, Vienna, Austria; version 2.1.0.81) and Mascot (Matrix Science, London, UK; version 2.1.0.81). Search engines used the December 2015 version of uniprot human FASTA database with the addition of common contaminants using a semi-tryptic search with a fragment ion masstolerance of 0.50 Da and a parent ion tolerance of 2.0 PPM. Carbamidomethyl of cysteine was specified as a fixed modification. Pyro-Glu of glutamine, deamidatation of asparagine, oxidation of histidine and acetyl of the n-terminus were specified as variable modifications. Scaffold (version 4.8.1, Proteome Software Inc., Portland, OR) was used to validate MS / MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 99.0% probability to achieve an FDR less than 0.1% by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 3 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm. Proteins that contained similar peptides and could not be differentiated based on MS / MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Normalized intensity values for each protein were exported from Scaffold to excel. Mean values, fold change values and p values using an unpaired t-test assuming unequal variance were calculated. From the 30 patient samples selected for the discovery cohort, the inventors identified 2065 high confidence proteins of which 126 had p-values of less than 0.01 for comparison between the groups. Six of these were increased in subjects who needed KRT and 120 were decreased. Of the six proteins that increased, IGFBP-1, heme-binding protein 2, myoglobin, adipocyte-type fatty acid binding protein, thymosin beta-4, and heart-type fatty acid binding protein all had a p value <0.01 (FIG.10). The protein that exhibited the largest increase in cases of severe AKI (+) was IGFBP-1 (14-fold, p < 0.01, FIG.11A). The mean spectral counting assessment of normalized abundance (quantitative value) in patients with mild AKI vs those requiring KRT were 1±0.4 and 15±4.1, respectively. Median amino acid coverage was 0% and 26%, respectively. Total amino acid coverage across all samples was 47%. Twenty-seven spectra for 12 unique peptides were identified from IGFBP-1. The inventors compared the number of peptides observed between control and KRT patients (FIG.11B). A larger number of peptides were seen in those patients who progressed to the composite outcome.Because the discovery analysis identified IGFBP1 as the prime candidate to predict the progression to severe AKI, the inventors validated urinary IGFBP-1 concentrations in 206 subjects that developed AKI following cardiothoracic surgery. Of the 206 patients included in the validation cohort, 25 met the combined primary outcome of death or stage 3 AKI (FIG.13). There was no difference between the control group and subjects that progressed to the primary endpoint in regards to age (66.1± 0.9 VS 69.6± 2.9, p = 0.197), gender (66.5 VS 64 % males, p = 0.665), or race (68.7 VS 76 % Caucasian, p > 0.461). There was no difference between controls and progressors regarding pre-existing medical comorbidities including diabetes (39 VS 44 %, p > 0.603), systolic heart failure with ejection fraction less than (<) 35% (16.2 % VS 32 %, p = 0.055), or baseline serum creatinine (1.17 ± 0.03 VS 1.32 ± 0.08 mg / dl, p = 0.08). Surgical parameters including history of previous cardiac surgery (40 VS 44%, p = 0.633), the need for pre-operative IABP (10.1% VS 16%, p = 0.379), the mean cardiac bypass time (134.5 ± 6.2 VS 139.9 ± 20.5 minutes, p = 0.7655), or type of surgical procedure was not statistically different between controls and patients that progressed to severe AKI There was no difference in the average time of sample collection between the two groups (26.3 ± 1.0 VS 27.5 ± 2.8 hours, p = 0.694) (FIG.13). The primary outcome used to validate the performance of the biomarker in the validation study was the composite of death within 30 days, or progression to KDIGO stage 3 AKI within 10 days. Urine IGFBP-1 concentrations were measured using a sandwich ELISA in each sample. The urine IGFBP-1 concentration was assessed using a commercially available ELISA kit (Raybiotech, Norcross, GA). The urine creatinine concentrations were determined by kinetic Jaffe assay using the Creatinine Reagent Set (Pointe Scientific, Canton, MI). IGFBP-1 concentration was normalized to urine creatinine to minimize effects of biomarker concentration from urine volume. The IGFBP-1 and urine creatinine concentrations were assessed in duplicates. Urinary IGFBP-1 concentrations were significantly higher in the positive primary outcome group. The median urinary IGFBP-1 concentration in the positive primary outcome group was 48.2 ng / ml (IQR: 14.4-296.6) and in the negative group 2.8 ng / ml (IQR: 1.1-11.2, p<0.0001) (FIG.13). The median concentration of IGFBP-1 normalized to urinecreatinine (IGFBP-1 / uCr) was significantly higher in the positive primary outcome group when compared to the negative primary outcome group at 95.9 ng / mg (IQR: 38.5-1863) vs 3.8 ng / mg (1.5-23.9, p<0.0001), respectively (FIG.14). The prognostic ability of urinary IGFBP-1 to predict the progression to severe AKI was determine by the AUC of ROC (Figure 21) and was 0.85 (95% CI: 0.80- 0.93), and that of IGFBP- 1 normalized to uCr was 0.85 (95% CI: 0.77-0.93). The prognostic ability of urine IGFBP-1 was significantly better than baseline serum creatinine (AUC: 0.63, p < 0.05) and collection time serum creatinine (AUC: 0.70, p<0.05). Since proteolysis has been shown to affect the function of insulin-like growth factor binding proteins, gel slices from urine proteins were analyzed by semitryptic proteomic analysis. The inventors found fully tryptic and semitryptic IGFBP-1 cleavage products from patients with AKI-3 in gel bands at molecular weights between 11 and 22 kDa. In contrast, very little IGFBP-1 was observed in the mild AKI patients and the protein that was observed was present at 28 kDa. These studies suggest that IGFBP-1 is completely cleaved during severe AKI and that activity of the protease that cleaves IGFBP-1 could be a good marker of severe AKI. Sequence analysis suggested that IGFBP-1 cleavage was not caused by trypsin, could occur at H114, and could be catalyzed by MMP-9. As discussed in more detail below, not all AKI-3 patients have IGFBP-1 fragments in their urine, but all AKI-D patients tested have enzymatic activity in their urine that cleaves IGFBP-1. Demonstration of cleavage products of endogenous IGFBP-1 by western blot: To confirm the inventors’ observations from proteomic studies described above, the inventors performed western blot analysis of urine from healthy controls and two groups of patients with AKI after cardiac surgery. All subjects had AKI-1 at the time urine was collected (24 hours after surgery). Twenty patients did not progress further and ten patients progressed through AKI-3 to AKI-D. The western blots are shown in FIG.2. No IGFBP-1 was seen in urine from any healthy controls (marked as HC in FIG.2). Bands at the molecular weight of intact IGFBP-1 (28 kDa) (directly above the dotted lines in FIG.2) were seen in most AKI-3 subjects and in some that did not progress beyond stage 1 (AKI 1 in FIG.2). A monoclonal antibody to the C-terminus of IGFBP-1 was used to identify IGFBP-1 and itsfragments. The C-terminal fragment was seen in most of the AKI-3 subjects. This C-terminal fragment was not observed in any of the non-progressors that did not develop greater than stage 1 AKI nor in the healthy controls. However, this C- terminal fragment was seen in seven of the ten patients that progressed on to stage 3 AKI and in all of the stage 3 AKI patients where any IGFBP-1 could be visualized. This suggests that the protease that cleaves IGFBP-1 is active in the urine of AKI-1 patients that will develop severe AKI (AKI-3 or AKI-D) but not in those that will not progress beyond mild AKI. For patients with severe AKI that did not have IGFBP1 present, the study design did not allow the inventors to determine if there is enzyme activity. This was a good start but only 70% of AKI-3 patients had the cleavage products of interest. However, other studies the inventors have conducted that are described below suggested that the catalytic enzyme activity is present in urine of patients who will go on to AKI-D, even if the cleavage products are not seen. Recombinant IGFBP-1 cleavage. To determine if the enzyme in urine could cleave exogenous IGFBP-1, we added 1 µl of urine from subjects that progressed to mild or severe AKI, 10 ng of full-length, recombinant human IGFBP-1, and 13 µl of PBS to an Eppendorf tube and incubated at 37˚ C. The digested protein was analyzed by western blot with an antibody against IGFBP-1. As shown in FIG.3, when urine from patients with AKI-1 at the time of collection that progressed to RRT is combined with the recombinant IGFBP-1, a cleavage fragment of the IGFBP-1 is seen. Only one of the patients that did not progress beyond stage 1 had a positive test in this example. The activity in urine is destroyed by high heat because when AKI urine is denatured at 95˚ C for five minutes, the cleaved band disappears and only the intact IGFBP-1 is seen, as shown in FIG.16. The enzyme activity is also blocked by the nonspecific protease inhibitor AEBSF. Importantly, the inventors never observed cleavage of the recombinant IGFBP-1 using urine from healthy controls and only rarely urine from patients that would remain at AKI-1 or progress to AKI-2. This demonstrates that the presence of the enzyme activity is specific to severe AKI (i.e., low false positive rate). FIG.16 shows the effect of heat denaturation and protease inhibitors on cleavage of exogenous IGFBP-1 by urine from a patient that developedsevere AKI. The band at 28 kDa shows the uncleaved protein and the band just above 15 kDa shows the cleaved protein. HALT is a combination of six broad- spectrum protease inhibitors: AEBSF, aprotinin, bestatin, E-64, leupeptin and pepstatin A. Pepstatin is an inhibitor of aspartic proteinases. Leupeptin is an inhibitor of calpain proteases. EDTA is a binder of divalent cations. AEBSF is an irreversible inhibitor of serine proteases. Predictive characteristics of test. The inventors evaluated urine samples from 80 patients to determine the ability of urine enzyme cleavage of recombinant human IGFBP-1 to predict severe AKI. Each lane on the blot was scored as positive (a distinct band is observed at 15 kDa) or negative (no band or faint band). The results are shown in FIG.8. The AKI stage of each patient was linked to the presence or absence of the band. FIG.8 shows the outcome for each stage (based on maximum creatinine). RRT was defined as patients that required RRT plus a maximum increase in creatinine ≥150%. No patients without AKI had a positive test (0% False positive). False positive rates were 11% among patients with stage 1 AKI and 15% in stage 2. Half of patients with stage 3 AKI that did not require dialysis had a positive test and all 12 patients that required RRT had a positive test. The overall false positive rate in this cohort is 8.3%. This demonstrates that the false positive rate is low and the sensitivity for predicting the most severe cases of AKI is high. Specificity of test. Among the 20 samples from patients without AKI, 16 were collected from patients in the medical ICU. The patients had a number of comorbidities including hypertension (5 patients), diabetes (4), trauma (4), sepsis (2), cancer (2), COPD (2), heart failure, lupus, pneumonia, infective endocarditis, liver transplant, CABG, encephalopathy and seizures (1 each). None of the urine samples from patients without AKI showed the ability to cleave recombinant IGFBP- 1. This study shows that cleavage of IGFBP-1 by urine enzymes does not appear to be caused by these comorbidities. Activation of matrix metalloprotease 9 / MMP-9 as a biomarker: Matrix metalloproteinases are a family of endopeptidases which are important in health and disease. MMP-9 is secreted as an inactive prohormone that requires activation in the extracellular space. The inactive state of the prohormone is maintained by a cysteineresidue in the N-terminal pro-domain bound to a zinc atom that blocks the active site. Activation can be achieved through cleavage of the pro-domain by MMP-2, MMP-3, MMP-7, MMP-10, MMP-13, Cathepsin K and urokinase / plasmin. MMP-9 is also degraded by autodigestion which is inhibited by binding to NGAL. NGAL is an AKI biomarker that can predict the presence of AKI but is not commonly used in the clinic. MMP-9 binding to NGAL protects MMP-9 from autodegradation. The inventors propose that each of these steps is necessary for the enzyme biomarker prediction of severe AKI. For digestion of IGFBP1 to occur, there must be sufficient injury to the tubule for MMP-9 to be produced. There must be production of enzymes that can activate secreted MMP-9. Finally, for activity to be maintained, MMP-9 must be protected from autolysis by the presence of NGAL. Previous reports have suggested that MMP-9 increases in the kidney of patients with AKI but urinary MMP- 9 alone has not proven to be a good biomarker. The MMP-9-like activity in urine, however, is a good predictor based on the additional mechanisms of activation and preventing degradation of MMP-9. The enzyme that cleaves IGFBP-1 may be MMP- 9. Assay to quantify the cleavage of an IGFBP1 peptide by urine enzymes: In one embodiment, the present invention is directed to a lateral flow assay that can detect cleavage of a synthetic peptide or recombinant protein with an internal sequence that mimics all or a portion of the central linker region of IGFBP-1 (SDASAPHAAEAGSPESPESTEITEEELLDNFHLMAPSEEDHSILWDAISTYDGSKA LHVTNIKKWKEP). The purpose of the assay is to detect the activity of an enzyme in urine that cleaves the protein. The amount of cleavage of the peptide or protein as determined by the lateral flow assay is used to predict the likelihood that a patient will develop severe acute kidney injury. Cleavage of the peptide or protein may require an appropriate 3-D structure of the peptide or protein. As shown in FIG.4, one synthetic peptide of the present invention contains a His tag recognition sequence on the amino-terminal end and a flag tag recognition sequence and a biotinylation site on the carboxy-terminal end. The His tag and the flag tag are used to immobilize the amino- and carboxy-terminal ends of the peptide after cleavage. The enzymatic biotinylation site is used to label the peptide at the C-terminal endwith streptavidin-bound, gold nanoparticles. The overall schema for the synthetic peptide is shown in FIG.4. The red arrow above the peptide sequence shows the site where cleavage could be expected to occur. Western blotting is used to determine that the peptide is cleaved by the urine protease, the appropriate amount of urine peptide to add for visualization (0.1-10 ng), the appropriate length of time to incubate the peptide with urine for cleavage to occur (15 seconds – 2 minutes), and the temperature dependence of the assay (4º- 37º C). In one embodiment, a concentration of 10 ng of peptide in a volume of 10 microliters of PBS added to 10 microliters of urine and incubated for one hour at 37° C is used. These parameters were selected based on the studies shown in FIG.3 and discussed above. Urine from a control subject without AKI is compared to urine from patients that had stage 1 AKI at the time of collection but went on to require kidney replacement therapy (dialysis). After incubation, the peptide is separated on a 16% gel and transferred to PVDF and visualized by western blotting using the anti- Flag tag antibody. Intact (uncleaved) peptide is visualized at 5.8 kDa on the blot whereas the C-terminal portion of the cleaved peptide is visualized at 4.3 kDa. The appropriate ratio of urine to peptide or protein concentration to include in the assay must be determined. In addition, the appropriate time of incubation over the range from 15 seconds to 2 hours must be determined. Based on the western blot studies, the inventors expect the appropriate ratio of urine to peptide concentration to be 1-50 microliters of urine to 0.1-10 ng of peptide or protein. The percent cleavage is estimated by dividing the image intensity from the smaller (4.3 kDa) band by the sum of the intensities of the smaller and larger bands. The percent cleavage vs the time is plotted and this plot is used to select the time point from near the top of the steepest part of the curve. The temperature dependence of the reaction will also be determined by performing the assay at 4°, 10°, 20°, 30° and 37° C. After the initial characterizations of the assay are completed, the percent cleavage as determined by the western blot assay and the proteomic analysis are compared. The proteomic assay is performed on the synthetic peptide (without gold- labeling at the biotinylation site) that has been purified using a nickel affinity resinwhich binds the His tag region on the amino terminal end. The His tagged peptide is captured using Ni-NTA Affinity Resin (Abcam). Binding, wash and elution are performed as described in the product materials using 50 mM NaH2PO4 buffer with 250 mM imidazole for elution of the peptide. Nickel-resin pull down and proteomic analysis are performed after digestion with trypsin to shorten the peptides. As can be seen in FIG.4, the first lysine (K) peptide in the peptide occurs in biotinylation site which is after the His-tag and the entire internal IGFBP-1 sequence. There are no arginine (R) residues. The peptides identified after nickel affinity resin pull down are either 38 amino acids (intact or not cleaved by the urine enzyme) or 15 amino acids long (cleaved by the urine enzyme) resulting in peptides that are 3993.1 or 1706.7 Daltons. Proteomic analysis is performed using liquid chromatography tandem mass spectrometry on a Thermo Scientific Eclipse Tribrid mass spectrometer. The percent cleavage as calculated from the Western blot analysis is compared to the percent cleavage as determined by the proteomic analysis. The studies described above are performed using the peptide that was not biotinylated or labeled with gold nanoparticles. It is possible that the gold labeling may interfere with cleavage. To ensure that cleavage of the gold-labeled peptide occurs, the key studies are repeated at the selected peptide concentration and time point after biotinylation and gold nanoparticle labeling. The peptide is biotinylated at the 15 amino acid, “AviTag” sequence using enzymatic biotinylation with the E coli biotin ligase (BirA). Biotinylation is done using the Enzymatic Protein Biotinylation Kit (Sigma-Aldrich) according to the manufacturer’s instructions. This biotinylation reaction is highly specific and ensures that only the expected site near the C- terminus of the peptide is biotinylated. After biotinylation, streptavidin bound 10 nm gold nanoparticles (Cytodiagnostics) are incubated at a 1:10 dilution with the biotinylated peptide resulting in gold particles bound specifically near the C-terminus of the peptide. The use of a specific enzymatic biotinylation site and streptavidin bound gold particles will help ensure that the gold is bound only at the C-terminus since binding at other sites will make the lateral flow assay inaccurate. Western blotting and proteomic analysis after trypsin digestion is performed to confirm that cleavage is similar to that seen prior to biotinylation and gold labeling.Assembly of lateral flow assay: To assemble the strip of the lateral flow assay (LFA), a high-flow nitrocellulose membrane is mounted on a backing card. Antibodies are printed on the strip using a Biostriper liquid dispensing system (Radetec Diagnostics) at 200 µl / minute at a concentration of 200 µg / min. Two lines are printed on the nitrocellulose strip: the first line contains the anti-His tag antibody (R&D Biosystems) and the second line contains the anti-Flag tag antibody (R&D Biosystems). Next, the absorbent pad is mounted on the backing card. The absorbent pad is located at the opposite end from sample port, and it is configured to absorb the unbound portions of the sample at the conclusion of the assay. Finally, the conjugate pad and sample pad are added to the backing card. The sample pad is positioned beneath the sample port so that the sample is dropped on the sample pad. The sample flows from the sample pad to the conjugate pad before reaching the strip. The basic design of the assay is shown in FIG.5. The steps of using the assay of FIG.5 may be described as follows. In step 1, urine and peptide are incubated in the tube. In step 2, incubation of the peptide results in the presence of three peptides. Uncleaved peptide contains both the His and Flag tags and a gold label. Cleaved N-terminus contains the His tag but no label and will not be visualized in the assay. Cleaved C-terminus contains the Flag tag and the gold label. In step 3, peptides migrate through assay device. Peptides with amino terminal His tag bind to the first line and the intact peptide is visualized by its gold tag. In step 4, unbound C- terminal peptide migrates to second line, binds to the anti-Flag antibody and is visualized by gold tag. In step 5, the amount of intact peptide is quantified by intensity at the first line and amount of cleaved peptide is quantified by intensity at the second line. While FIG.5 illustrates the anti-His tag antibodies positioned on the test strip before the anti-Flag tag antibodies, it should be understood that relative positioning of the anti-His tag antibodies and anti-Flag tag antibodies could be changed. In this description, “first position” and “second position” simply refer to different positions on the test strip. In alternative embodiments, nickel and other divalent metals are used to bind to the His tag instead of anti-His tag antibodies. In yet other alternative embodiments, other tags are used are substitutes for the Histag and Flag tag, such as hemagglutinin, Myc, V5, GST, maltose binding protein, and Strep-tag. Demonstrate that lateral flow assay can detect cleavage of peptide: The LFA will be tested using six banked samples of urine. Three samples are from healthy control subjects and three are from patients that developed severe AKI after cardiac surgery. Intensity at each line is read using a lateral flow reader. The percent cleavage is calculated by dividing the intensity of the second line (cleaved peptide) by the summed intensity of the first and second lines (total peptide). Potential pitfalls and alternative approaches – Gold labeling occurs at sites other than the C-terminus: To ensure that gold labeling occurs only at the C- terminal BirA biotinylation site, studies will be performed in which a peptide is used that includes an enterokinase site immediately after the IGFBP-1 sequence. Enterokinase is a serine protease that recognizes the amino acid sequence -Asp- Asp-Asp-Asp-Lys-|-X. The peptide will be completely cleaved with enterokinase. Complete cleavage can be confirmed by proteomic analysis of an aliquot after Ni- NTA Affinity Resin pulldown. The cleaved peptide will be run on the lateral flow assay of the present invention. If all of the labeling is at the C-terminus biotinylation site, only the second line will have signal. A visible line on the first line means that there is non-specific labeling. The gold labeling may interfere with cleavage. To ensure that cleavage of the gold-labeled peptide occurs, the inventors will repeat the western blot studies at the selected peptide concentration and time point after biotinylation and gold nanoparticle labeling. The His-tag line (i.e. the first line) may be saturated resulting in uncleaved peptide being deposited on the second line. This would result in a falsely high measured amount of peptide cleavage. To test for this, a second His tag antibody line is printed on the device. If the first line is saturated, labeling on the second line is observed. If this is the case, a double thickness His tag antibody line will be printed. Ability of the assay to predict severe AKI in banked specimens: Three embodiments of the lateral flow assay and one of a fluorescence assay have been designed. The first preferred embodiment is shown in FIG.5 and was described above. The second preferred embodiment of the assay is shown in FIG.6 in whichthe conjugate pad contains the labeled peptide. The steps of using the assay of FIG.6 may be described as follows. In step 1, urine is added to the sample well and migrates into the conjugate pad which contains the labeled peptide. In step 2, enzymes in the urine digest the peptide in the pad. In step 3, intact and digested peptide migrate through device. In step 4, digested and intact peptide with amino terminal His tags bind to antibodies. Only intact peptide has the gold particle and is visualized. In step 5, unbound peptide migrates to second line where the Flag tag binds and is visualized. The third preferred embodiment of the assay is shown in FIG.7. In this embodiment, the peptide is printed directly on the first line of nitrocellulose membrane. The steps of using the assay of FIG.7 may be described as follows. In step 1, urine containing endogenous enzyme is added to the assay device and migrates to the first line. The amount of peptide that is cleaved depends on the activity of the enzyme in the urine. Uncleaved peptide that remains on the line is visualized using the gold particle. In step 2, cleaved carboxyl ends of the peptide migrate to the second line, bind to the Flag antibodies and are visualized. In the first three embodiments, imaging of the peptide or protein can be done using gold or another visual substrate or it could be done using a fluorescent substrate. In a different embodiment of FIG.7, anti-His tag antibodies or another binding molecule is substituted for the anti-Flag tag antibodies. In a fourth embodiment (FIG.12), a protein or peptide that has a fluorescent moiety on one end and a quencher moiety on the other end is incubated with the urine that is being tested. If no cleavage of the protein or peptide occurs, no fluorescence is visualized and the patient is predicted not to develop severe AKI. If cleavage occurs, fluorescence is visualized and severe AKI is predicted. The amount of fluorescence that occurs is proportional to the amount of active enzyme present and correlates with the severity of the AKI that will develop. The goal of further studies is to test and select one of the three versions for the lateral flow assay, determine if a fluorescence readout is needed, and determine if the assay can predict the development of severe AKI in banked urine specimens from patients with AKI after cardiac surgery. Three methods are used for cleaving the peptide in the lateral flow assay: (1) cleavage of the peptide in a separate tube(see FIG.5), (2) cleavage of the peptide as it progresses through the assay (see FIG.6), and (3) cleavage of the peptide from a preprinted line in the assay (see FIG. 7). If cleavage is performed in the lateral flow assay (methods 2 or 3), it will be easier for ICU personnel to use the point of care test. An additional method (FIG.12) uses cleavage of a peptide or protein that contains a fluorescence moiety and a quencher moiety. In method 1, which is illustrated in FIG.5, the peptide and urine are incubated together in an Eppendorf tube at the peptide-to-urine ratio, time and temperature determined as optimum in the studies above. Then, 50 µl of this solution is added to the sample pad through the sample port of the lateral flow assay followed by 50 µl of buffer. The sample moves through the strip and ultimately to the absorbent pad. The results are read after 5, 10, 15, 20, 30, 40, 50 and 60 minutes. In method 2, which is illustrated in FIG.6, the peptide is added to the conjugate pad during construction of the lateral flow assay. Fifty µl of urine is added to the sample pad through the sample port followed by 50 µl of sample buffer. Affordable, disposable, exact volume transfer pipettes are preferably used. In this method, cleavage of the peptide occurs as the urine migrates through the conjugate pad and continues through the device. In method 3, which is illustrated in FIG.7, the intact peptide is printed on the first line of the nitrocellulose in the lateral flow device. Urine is added to the sample pad through the sample well followed by buffer. As the enzyme migrates past the line of printed peptide, it is digested. The free C-terminal end of the peptide, which contains the Flag tag and the gold label, migrates through the device and binds to the printed Flag tag antibody line. The amount of peptide to be printed, the temperature dependence of cleavage, the effect of the amount of urine added, and the time required for the second line to appear will be evaluated. This third method is the simplest method to use at the point of care. In method 4, which is illustrated in FIG.12, urine is incubated with a protein or peptide that contains a fluorescent substrate on one end and a quencher molecule on the other end. The protein or peptide is incubated with urine from the patient. If the enzyme is present, the protein or peptide is cleaved. When the quencher isseparated from the fluorescent moiety, fluorescence can be induced. The absence of fluorescence indicates that no cleavage has occurred and the presence of fluorescence indicates that cleavage has occurred. The intensity of fluorescent is proportional to the amount of enzyme (and cleavage) that is present and predicts progression of AKI. The results in each of these embodiments of the assays will be determined using banked urine samples from three healthy controls and six patients that had AKI stage 1 at the time the sample was collected. Three of the AKI patients did not progress beyond stage 1 over the next 7 days and three progressed to stage 3 AKI and needed dialysis within 7 days. It is expected that the healthy control patients will show only labeled peptide on the first line and none on the second line. It is also expected that patients that did not progress beyond stage 1 will have more intensity on the first line than the second line, and patients that progressed to stage 3 will have more labeled peptide intensity on the second line than the first line. If needed, parameters of the assay, such as time, volumes, temperatures, and printed amounts of antibodies or peptide, can be adjusted. After the inventors have refined the test with these initial nine samples, the inventors will test in a larger cohort that includes patients with AKI after cardiac surgery and ICU patients with other causes of AKI. Readout: The amount of peptide present at each line is digitally read using lateral flow reader or fluorescence reader. The peptide may be a gold-labeled peptide or a fluorescently-labeled peptide. Total intensity of each line will be determined by subtracting background intensity from the intensity reading of the line. The percent cleavage is estimated by dividing the intensity of the second line (Flag tag) by the sum of the intensity of the first and second line. The assay that shows the largest increase in intensity second line will be selected for further evaluation. Alternatively, the peptide may include a fluorescent tag instead of a gold tag. A streptavidin bound Alexa Fluor 488 conjugate (Thermo Fisher) may be used. The amount of fluorescence at each line is read using the reader. For the fluorescence assay in method 4, fluorescence is read in a fluorescence reader or plate reader. Initial validation: The presence of peptide or protein cleavage using the three embodiments of the lateral flow assay and one embodiment of thefluorescence assay will be determined in 50 banked urine samples. The presence of peptide cleavage in the assay will be determined in 10 controls, 40 urine samples from patients that had cardiac surgery, and 40 samples of patients in the medical ICU when they developed AKI. The samples include 10 healthy control subjects and 40 cardiac surgery patients that had AKI stage 1 at the times the samples were collected. These samples are banked in a freezer from a previous study. The 40 cardiac surgery AKI patients include 10 patients that did not progress beyond stage 1, 10 patients that progressed to stage 2, 10 patients that progressed to stage 3 but did not require dialysis, and 10 patients that progressed to stage 3 and required dialysis (AKI-D). The samples from the medical ICU includes 10 subjects that progressed to AKI-D and 10 with stage 3 AKI without RRT. The patients that had a new insult that could cause AKI (for instance, a new cardiac arrest) will be excluded. These patients have acute and chronic comorbidities that will allow us to assess the impact of comorbidities on the results. The percent cleavage is determined in each assay. An ROC curve for the ability to predict severe AKI will be constructed by plotting the false positive rate versus the true positive rate for each value of the percent cleavage. An ideal cutoff will be selected using the Youden index. Sensitivity and specificity will also be evaluated. Measurement of urinary biomarkers and creatinine concentrations in human samples: Human urine IGFBP-1, NGAL, and [TIMP-2]*[IGFBP7] concentrations were measured using commercially available ELISA kits (Raybiotech, Norcross, GA). The urine creatinine concentrations were determined by kinetic Jaffe assay, using the Creatinine Reagent Set (Pointe Scientific, Canton, MI). All samples were assessed in duplicates. Breeding and genotyping of wild type and IGFBP-1 KO mice: B6.129X1- Igfbp1tm1Taub / J (IBP-1 knockout C57Bl / 6J background) mice were obtained from The Jackson Laboratory (Bar Harbor, MN) and bred to maintain a homozygote colony of IGFBP1–KO mice. C57Bl / 6J (wild type) mice originally obtained from The Jackson Laboratory were used as controls. Offspring were weaned at 21 days of age and marked with numbered ear tags. For genotyping, a small piece of tail (<5mm) was cut with sterile surgical scissors while the mouse was immobilized byhand. Southern blot was performed to identify the presence of wild type (550 bp) and KO (320 bp) alleles using the following primers: Common AAC AAC TGT GGG CAT TGT CA; Knockout TGG ATG TGG AAT GTG TGC GAG; and wild type AGC AGG CTG TGG ATG AGA CT. Cisplatin-induced AKI (CIAKI) in mice: 8-10 weeks old male mice underwent a single Cisplatin (20 mg / kg) or saline (control) injection administered Intraperitoneal (IP) with a 1cc syringe and 25-gauge needle. Analgesic (buprenorphine, 0.1 mg / kg via SQ injection) was administered after cisplatin injection if the animal showed signs of pain or distress. Seventy-two hours after the cisplatin or saline injections, mice were euthanized via overdose of carbon dioxide followed by cervical dislocation. Blood and kidneys were collected at the time of sacrifice. Determination of mouse blood urea nitrogen (BUN) and creatinine (CRE) levels: BUN and Creatinine were determined from serum collected at the time of sacrifice using diagnostic kits from Pointe Scientific (Canton, MI). Periodic acid-Schiff histology (PAS) of mouse kidney tissue: PAS- stained sections of kidney tissue were scored blindly by a renal pathologist in a semi-quantitative manner. For each animal, at least 10 high power fields of view (400X) were examined. The percentage of tubules that displayed cellular necrosis, loss of brush border, cast formation, vacuolization, or tubular dilation were scored as follows: 0 = no evidence, 1 = < 25% involvement, 2 = 26-50% involvement, 3 = >50% involvement. TUNEL assay: Mouse kidney samples were embedded in paraffin and 5- micron sections were prepared. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed and quantified as previously described. Briefly, the samples were stained using the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s protocol. Cells were counterstained with 4',6-diamidino-2-phenylindol (DAPI) to visualize cell nuclei, mounted under cover slips with Prolong® Antifade kit (Invitrogen, Carlsbad, CA) and images were acquired using an Olympus IX-81 inverted microscope (Olympus America, Center Valley, PA) equipped with a Hamamatsu ORCA-ER monochrome camera (Hamamatsu Photonics K.K., Hamamatsu City, Japan).Measurement of caspase 3 / 7 activity: Caspase 3 / 7 activity was measured in kidney tissue homogenates with Promega Caspase-Glo® 3-7 Assay (Madison, WI) according to the manufacturers protocol. All samples were run in triplicate and analyzed with a SpectramaxM5e Multimode plate reader (Molecular Devices, LLC, San Jose, CA) Measurement of BCL-xl protein concentrations in kidney tissue homogenates: BCL-xl protein concentrations were measured using a commercially available ELISA kit (Abcam, Boston, MA). Samples were run in duplicate on a 96- well plate. BCL-XL protein concentrations were normalized for total protein concentration of the tissue homogenate. Total tissue homogenate protein concentrations were measured using the BCA assay. Measurement of phosphorylated protein kinase B (p-AKT) and phosphorylated focal adhesion kinase (p-FAK) in mouse kidney tissue homogenates: Tissue concentrations of pan-AKT and p-AKT were measured simultaneously in the same sample using a commercially available ELISA kit (Raybiotech, Norcross, GA). The measured OD450 for p-AKT was compared against pan-AKT to determine the percentage of p-AKT (p-AKT / pan-AKT * 100). P- FAK levels were measured using a commercially available ELISA kit (Raybiotech, Norcross, GA). For pan-AKT, p-AKT, and p-FAK were ran in duplicates on a 96-well plate. Values were normalized for tissue homogenate protein concentration as determined by a BCA assay. Statistical Analysis: Normalized intensity values (Quantitative Value) for each protein were exported from Scaffold to Microsoft Excel. Mean values, fold change values and p-values were assessed using an unpaired t-test assuming unequal variance. We compared the mean spectral count values for each protein between the control and case groups. We set a p-value threshold <0.01 to consider the proteins to be valuable prognostic biomarkers. Statistical analyses of the validation cohort were performed using IBM SPSS version 24 software and GraphPad Prism 9.2.0. For patient demographic and validation study data, an unpaired T-test was used to compare parametric variables and an unpaired T-test with Mann-Whitney correction was used to compare non-parametric data betweentwo groups. GraphPad Prism 9.2.0 was used to analyze data from animal studies. A one-way ANOVA with Tukey post-hoc analysis was used to compare parametric data when multiple groups were present. Data for histological scoring of mouse PAS staining was analyzed using a non-parametric Kruskal-Wallis with Dunn’s multiple comparison test. Statistical significance was set at p < 0.05. Receiver operator characteristic (ROC) curves were used to quantify the ability of a biomarker to predict the primary outcome. A Youden’s test was used to set the optimal cutoff values for urinary IGFBP-1 and NGAL. The optimal cutoff value for [TIMP- 2]*[IGFBP7] was set at 0.3, as is used commercially. Youden’s test of our patient cohort also confirmed the optimal cutoff as 0.3 for [TIMP-2]*[IGFBP7]. We compared Area Under the Curve (AUC) values of receiver operator characteristics (ROC) between biomarkers using GraphPad Prism 9.2.0. Schematic figures were generated using BioRender.com. Measurement of urinary concentrations of IGFBP-1, NGAL, and [TIMP- 2]*[IGFBP7]: Urinary IGFBP-1 concentrations were significantly higher in the patients that met the primary composite outcome compared to patients that did not progress beyond stage 1 or 2 AKI (134.2 ± 37.7 VS 18.8 ± 8 ng / ml, p < 0.0001, n = 213) (FIG.20A). Urinary concentrations of IGFBP-1 also increased progressively with each maximum stage of AKI (stage 1: median 2.8 ng / ml, interquartile range (IQR) 1.34-10.9 ng / ml, 95% confidence interval (CI) 2.01-3.72 ng / ml, n = 163 VS stage 2: median 2.39 ng / ml, IQR: 0.86-23.26 ng / ml, 95% CI: 0.95-22.74, n = 22 VS stage 3: median 44.17 ng / ml, IQR: 9.74-208.8 ng / ml, 95% CI: 10.2-162 ng / ml, n = 22, stage 1 VS stage 2 p = 0.968, Stage 1 VS Stage 3, p < 0.0001, Stage 2 VS Stage 3, p < 0.001 ) (FIG.20B). There was no difference between the individual groups that comprised the composite primary outcome (Stage 3 AKI with RRT median: 44.17 ng / ml, IQR: 9.75 – 208.8 ng / ml, 95% CI: 10.2 – 162.0 ng / ml, n = 22 VS Stage 3 AKI with RRT median: 81.1 ng / ml, IQR: 13.38 – 353.2 ng / ml, 95% CI: 10.2 – 365.3 ng / ml, n = 14 VS 30-day mortality median: 40.41 ng / ml, IQR: 9.72 – 333.1 ng / ml, 95% CI: 4.99 – 422.2 ng / ml, n = 9). Please note that some subjects qualified for inclusion in the primary outcome cohort by meeting more than one inclusion criteria. Urinary concentrations of [TIMP-2]*[IGFBP7] (0.87 ± 0.1 VS 1.92 ±0.5, p < 0.01, n = 154) (FIG.20C) and NGAL (82.8 ± 16.6 VS 220.5 ± 101, p < 0.05, n = 150) (FIG.20D) were also increased, but not to the degree of IGFBP-1. Comparison of urinary IGFBP-1 concentrations against creatinine: The prognostic ability of urinary IGFBP-1 concentration to predict the progression to severe AKI was determined by the AUC of the ROC. The AUC for urinary IGFBP-1 concentrations was 0.85 (95% CI: 0.77 - 0.91, p < 0.0001). Using an optimal set point for urinary IGFBP-1 concentration of 10.2 ng / ml, we were able to predict the progression to the primary outcome with a sensitivity of 76.9%, specificity of 74.1%, and a likelihood ratio of 2.97 (FIG.18). Similarly, the AUC of IGFBP-1 / uCr was 0.83 (95% CI: 0.75-0.91, p < 0.0001). The ability of urinary IGFBP-1 to predict progression to the primary outcome was better than serum creatinine at the time of collection (AUC: 0.69, 95% CI: 0.57-0.80, p < 0.01) and the change in serum creatine from time of collection (AUC: 0.70, 95% CI: 0.59-0.82, p<0.001) (FIG.21A). Comparison of urinary IGFBP-1 against other prognostic biomarkers of AKI: Additionally, we performed a direct comparison of urinary IGFBP-1 concentrations against two established prognostic biomarkers of moderate to severe AKI: urinary NGAL and [TIMP-2]*[IGFBP7]. Urinary IGFBP-1 concentrations (AUC: 0.85, 95% CI: 0.77 - 0.91, p < 0.0001, n = 213) were able to more accurately predict the progression to the composite outcome when compared to urinary concentrations of NGAL (AUC: 0.72, 95% CI: 0.60-0.83, p < 0.05, n = 150) and [TIMP-2]*[IGFBP7] (AUC: 0.71, 95% CI: 0.60-0.83, p < 0.01, n = 154) (FIG.21B). Additionally, IGFBP-1 displayed a higher sensitivity (76.9 VS 70.6%) and specificity than NGAL (74.1 VS 66.2%). Although, [TIMP-2]*[IGFBP7] showed a slight increase in sensitivity (80% VS 76.9%) compared to IGFBP-1, [TIMP-2]*[IGFBP7] had a dramatically lower specificity (44% VS 74.1%) and positive predictive value (17.2% VS 29.4%) compared to IGFBP-1 (FIG.18). IGFBP-1 also showed a higher positive predictive value, negative predictive value, and likelihood ratio when compared to both NGAL and [TIMP-2]*[IGFBP7] (FIG.18). The effects of IGFBP-1 KO on renal function following cisplatin injection in mice: To determine the effects of IGFBP-1 on CIAKI in mice, we measured serum creatinine and BUN levels. Wild type (WT) mice injected with cisplatinshowed a significant increase in serum creatinine levels when compared to saline injected controls (0.3 ± 0.06 mg / dl VS 1.5 ± 0.29 mg / dl, p < 0.0001, n = 10-12), while IGFBP-1 KO mice that received cisplatin showed an attenuated rise in serum creatinine levels when compared to their WT counterparts (0.5 ± 0.08 mg / dl VS 1.5 ± 0.29 mg / dl, p < 0.001, n = 10-11) (FIG.22A). Similarly, WT mice injected with cisplatin showed a significant increase in BUN levels compared to controls (24.2 ± 0.6 mg / dl VS 150.1 ± 13.3 mg / dl, p < 0.0001, n = 10-12). IGFBP-1 KO mice that received cisplatin showed a blunted increase in BUN levels when compared to WT mice that received cisplatin (91.7 ± 15.8 mg / dl VS 150.1 ± 13.3 mg / dl, p < 0.001, n = 10-11) (FIG.22B). The Effect of IGFBP-1 KO on renal tubular damage: To examine the effects of IGFBP-1 KO on renal tubular damage, Period Acid Schiff (PAS) stained sections of kidney tissue were examined and scored blindly by a renal pathologist. Saline injected control WT mice (FIG.23A) showed no renal tubular damage, whereas WT mice that received cisplatin (FIG.23B) showed significantly increased renal tubular damage (mean pathology score: 0±0 VS.2.75±0.08, p < 0.0001, n = 12) (FIG.23D). IGFBP-1 KO mice injected with cisplatin (FIG.23C) showed attenuated renal tubular damage compared to WT mice that received cisplatin (1.36 ± 0.36 VS 2.75 ± 0.08, n = 12, p < 0.05) (FIG.23D). Effects of IGFBP-1 KO on cell death and markers of apoptosis: We next applied the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay to qualitatively and quantitatively assess the degree of cell death induced by cisplatin in mouse kidneys. TUNEL assay measures the presence of DNA 3’OH ends produced by DNA cleavage by endogenous DNases. The kidney is known to have high activity of DNase I that makes tubular epithelium sensitive to injury, particularly by cisplatin and permits the use of TUNEL as a mechanistic and quantitative marker of irreversible cell death. In our experiments, TUNEL assay showed no irreversible cell death in control mice, markedly induced cell death in wild-type mice treated with cisplatin, and statistically significant (~3-fold) mitigation of that injury in KO mice (FIGS.24A-D). Because TUNEL does not distinguish between apoptotic and necrotic injury, we used caspase 3 / 7 activity as a pro-apoptotic markerand Bcl-xL as an anti-apoptotic marker to determine whether cisplatin injury and its amelioration was apoptotic by nature. Our experiments clearly showed cisplatin induced apoptosis in WT mice and that it was ameliorated in IGFBP-1 KO mice (FIG. 24E-F). Taken together, these data indicate that inactivation of IGFBP-1 significantly mitigates kidney cell death and apoptosis induced by cisplatin. Comparison of biomarkers in kidney tissue between saline and cisplatin injected WT mice: To determine if the development of CIAKI altered the expression of IGFBP-1 in the kidney proteomic analysis was performed on mouse renal tissue homogenates. We compared protein expression between saline injected control mice and CIAKI mice. We were unable to detect IGFBP-1 in renal tissue homogenates by proteomic analysis. However, changes in the renal proteome following CIAKI showed significant changes in expression of other known biomarkers of AKI including NGAL (p < 0.0001), angiotensinogen (p < 0.00001), and IGFBP7 (p < 0.05). There was an insignificant change in expression of the only other IGFBP (IGFBP-4) that was detected in our analysis. Measurement of phosphorylated protein kinase B (p-AKT) and phosphorylated focal adhesion kinase (p-FAK) in renal tissue homogenates: To further investigate the signaling mechanism of renal protection in IGFBP-1 KO mice, we measured concentrations of p-AKT and p-FAK in mouse renal tissue homogenates. IGFBP-1 modulates cellular signaling by one of two mechanisms: sequestration of IGF-1 and binding of its C-terminus RGD domain to the α5β1 integrin receptor. The immediate downstream effect of IGF-1 binding the IGF-1r is phosphorylation of AKT by phosphoinositide 3 kinase (PIP3K). Our data show a significant increase in the ratio of p-AKT to pan-AKT in IGFBP-1 KO mice compared to WT controls (4.04 ± 0.22% VS 14.39 ± 3.83%, p < 0.05, n = 7-10) and cisplatin injected WT mice (5.57 ± 0.25% VS 14.39 ± 3.83%, p < 0.05, n = 9-10). Although there was an increase in the ratio of p-AKT to pan-AKT in IGFBP-1 KO controls compared to WT controls, this difference was not statistically different (4.04 ± 0.22% VS 11.47 ± 3.37%, p = 0.11, n = 7-11). We found that total levels of p-FAK were reduced in IGFBP-1 KO controls (102.3 ± 21.96 OD450 / mg VS 48.65 ± 24.53 OD450 / mg, p < 0.05, n = 6-7), WT cisplatin injected mice (102.3 ± 21.96 OD450 / mgVS 55.75 ± 13.91 OD450 / mg, p < 0.05, n = 7-8), and IGFBP-1 KO cisplatin injected mice compared to WT controls (102.3 ± 21.96 OD450 / mg VS 58.24 ± 20.16 OD450 / mg, p < 0.05, n = 7-8). Collectively, these data suggest that attenuating of CIAKI in IGFBP-1 KO mice stems from increased activation of the IGF-1r. Discussion: In this study, we have identified IGFBP-1 as a prognostic biomarker to predict progression to severe AKI. Proteomic analysis of urine samples from a selected group of subjects enrolled in the SAKInet cohort identified IGFBP-1 as having the largest fold-increase among patients that required RRT within 10 days of surgery. In our previously published proteomic study we observed that IGFBP-1 spectral counts were higher in subjects who developed severe AKI compared to controls. But in that study, there was limited coverage of the IGFBP-1 protein by LC MS / MS (27%), so it did not appear to be a strong predictor of severe AKI. Improved instrumentation allowed us to identify a more robust protein coverage (47%) of IGFBP-1 in the current study and increased confidence of its ability to predict progression to the composite outcome. The improved study design and data quality in this study identified IGFBP-1 as a biomarker that is likely to predict severe AKI. There have been several promising prognostic biomarkers of AKI proposed, however, to date no single biomarker has demonstrated the ability for widespread clinical implementation. Urinary concentrations of NGAL collected at the time of cardiothoracic surgery in pediatric patients are highly accurate (AUC: 0.998) at predicting AKI progression, however its effectiveness in adult patients is less impressive. [TIMP-2]*[IGFBP7] collected within the first 24 hours of ICU admission can predict the progression to moderate or severe AKI (KDIGO stage 2 to 3) in critically ill patients within 72 hours. However, [TIMP-2]*[IGFBP7] does not distinguish which patients will progress to the most severe stage of AKI or require RRT. Due to its high false positive rate and several other factors, it is not recommended for use by the The American Association for Clinical Chemistry or the National Health Service in the United Kingdom. The furosemide stress test (FST), a test of renal tubular function, has been proposed to address this issue as it can predict progression to severe AKI and the future need for RRT with high sensitivity and specificity, but its usefulness is limited as it cannot be followed serially due tothe duration of the diuretic response. Despite significant progress in the field of prognostic biomarkers, it remains challenging to reliably predict the progression to severe AKI and the need for RRT. Our validation studies demonstrated that the ability of urinary IGFBP-1 concentrations to predict the composite outcome is very good (AUC: 0.85). Urinary IGFBP-1 was dramatically superior to the traditional clinical biomarker, serum creatinine. Additionally, IGFBP-1 was superior when compared against two other established prognostic biomarkers of AKI: urinary NGAL and [TIMP-2]*[IGFBP7]. Our findings show that urinary IGFBP-1 was a stronger predictor of the progression to the composite outcome regarding AUC, specificity, PPV, NPV, likelihood ratio, and p-value. Although, [TIMP-2]*[IGFBP7] showed a mild increase in sensitivity compared to IGFBP-1, IGFBP-1 was superior in regards to specificity, PPV, NPV, likelihood ratio, and p-value. The slightly higher sensitivity of [TIMP- 2]*[IGFBP7] is to be expected as both urinary NGAL and [TIMP-2]*[IGFBP7] are designed to be collected prior to renal injury to predict the progression to moderate or severe AKI. Whereas our study was designed to detect progression to severe AKI in patients that had already developed stage 1 AKI. These differences in study design somewhat limit the direct comparisons between biomarkers and could explain the higher sensitivity of urinary [TIMP-2}*[IGFBP7] compared to IGFBP-1. In our study, urine concentration of IGFBP-1 in subjects that progressed to stage 2 AKI were not different from those that remained at stage 1. In contrast, concentrations were significantly increased in patients that progressed to the Stage 3 AKI by KDIGO creatinine criteria, the need for RRT, and 30-day mortality. This demonstrates that IGFBP-1 can discriminate patients that will reach moderate AKI from patients that will achieve more severe clinical outcomes. The ability to distinguish between subjects that will have moderate vs severe AKI will be important in guiding treatment, as well as enrollment and enrichment of clinical trials. A lack of clinical therapies for AKI stems from a poor understanding of the molecular mechanism of AKI. Reverse translation of novel clinical biomarkers to preclinical animal studies could be a tool to shed light on the pathophysiology of AKI, ultimately leading to the development of new clinical therapies. Several previouslydiscovered biomarkers of AKI have also been implicated in the pathophysiology of AKI. NGAL is a 25-kDa protein of the lipocalin family. In the kidney, NGAL is basally expressed at low levels but is quickly upregulated within 3 hours of the initial insult and readily detectable in the urine in both clinical studies and animal models AKI. Mechanistically, it is believed that NGAL makes use of its ability to bind iron- siderophore complexes to regulate innate immunity

[3839] and modulate function of iron-responsive genes. Kidney Injury Molecule 1 (KIM-1) is a 38 kDa type I transmembrane glycoprotein with an extracellular immunoglobulin-like domain that is expressed at low basal levels in renal proximal tubule cells, however it is dramatically upregulated following AKI. Rat models of AKI have shown increased KIM-1 following ischemia and administration of nephrotoxins. KIM-1 has been implicated to play an important role in proximal tubular cell recovery and regeneration by mediating phagocytosis of apoptotic bodies and cell debris. IGFBP- 7 and TIMP-2 have been shown to play a role in cell cycle arrest in human microvascular cells and cancer cell lines, but their mechanistic role during AKI has not been established. It should be noted that IGFBP7 shares only 20-25% homology with IGFBPs 1-6 and binds IGF-1 and IGF-2 with much lower affinity, thus its function as a true IGFBP is debatable. In parallel with our clinical data showing patients with higher levels of urinary IGFBP-1 suffer more severe AKI, our animal studies mirror these findings by showing that IGFBP-1 KO mice have attenuation of CIAKI. Our data implicates IGFBP-1 as a mediator of AKI severity in CIAKI. IGFBP-1 mice show a blunted decline in renal function, decreased renal tubular damage, and decreased cell death by apoptosis. IGFBP-1 regulates cell signaling through two signaling mechanisms: sequestration of IGF-1 from binding the IGF-1r and binding of its C-terminus RGD domain to the α5β1integrin receptor. The immediate downstream effect of IGF-1 binding the IGF-1r is phosphorylation of AKT by PIP3K. Likewise, the immediate downstream effect of the RGD domain of IGFBP-1 binding the α5β1integrin receptor is phosphorylation of FAK by SRC kinases, thus p-AKT and p-FAK can be used as surrogate markers of IGF-1r and α5β1 integrin receptor activation, respectively (FIG.27). In our studies,IGFBP-1 KO mice showed an increased ratio of p-AKT to pan-AKT, suggesting that the lack of IGFBP-1 to sequester IGF-1 binding to the IGF-1r enriches downstream anti-apoptotic mediators to attenuate renal injury. Conversely, p-FAK was decreased in IGFBP-1 KO controls, cisplatin injected WT mice, and cisplatin injected IGFBP-1 KO mice. The lack of p-FAK in IGFBP-1 KO mice is expected since the absence of IGFBP-1 removes the ligand for binding the α5β1integrin receptor. The decrease in p-FAK in WT mice with CIAKI is interesting, as activation of the α5β1integrin receptor is generally considered to worsen renal injury in other models of AKI. However, to our knowledge the role of p-FAK signaling in CIAKI has not been explored. One possible explanation for the decrease in p-FAK among WT mice with CIAKI is that our study was designed to look at only a single time point (72 hours post-injection). It is possible that p-FAK formation occurs early in the onset of CIAKI and is depleted by 72 hours. Another explanation could be that cisplatin injection has a direct effect on the α5β1 integrin receptor as cisplatin injection in cancer models has been shown to decrease total FAK expression and block FAK phosphorylation at specific tyrosine residues. Future studies will be needed to gain a firm grasp on the role of α5β1integrin receptor and p-FAK signaling in CIAKI. Interestingly, our proteomic analysis of mouse renal tissue homogenates did not show an increase in IGFBP-1 expression following cisplatin injection. This finding suggests that IGFBP-1 is upregulated in other organs. The liver is known to be the major site of IGFBP-1 synthesis in the body and given the size of IGFBP-1 (28 kDa) it is likely freely filtered from the serum into the tubular lumen. It is tempting to speculate that renal injury stimulates a renal-hepatic crosstalk that upregulates IGFBP-1 in the liver, which is then secreted into the blood and freely filtered across the glomerulus to reach the tubular lumen. As our studies showed attenuated CIAKI in IGFBP-1 KO mice, extrapolation of our animal studies to our clinical findings could suggest that patients with higher levels of urinary IGFBP-1 experienced more severe AKI due to increased sequestration of IGF-1 and subsequent activation of anti-apoptotic pathways. Cleavage of the IGFBP-1 protein may play an important role in the biological response to acute kidney injury. Thismay be why cleavage of IGFBP-1 is a better biomarker of severe AKI than IGFBP-1 concentrations in the urine and better than other available biomarkers. In conclusion, cleavage of urinary IGFBP-1 is highly predictive of severe AKI in subjects after cardiac surgery. It may identify those who will progress to require RRT thus facilitating the planning of clinical interventions (e.g. placing dialysis catheters, initiating RRT in a non-emergent manner, etc.) or avoiding the use of aggressive and invasive interventions in those who are not likely to progress to severe AKI. Moreover, it may help in identifying high risk patients allowing for objective enrichment of study populations in AKI clinical trials. Perhaps most exciting is the fact that we have implicated a mechanistic role of IGFBP-1 during AKI which could lead to a more robust understanding of the pathophysiological mechanism and the eventual development of novel therapeutic treatments targets. Utility: Another potential use for this biomarker test is to use it to enroll patients in clinical trials for drugs to treat AKI. A drug that could improve the ability of patients with AKI to avoid kidney failure requiring dialysis could dramatically improve clinical outcomes. Several drugs have been tested for this purpose, but none have been successful. A major impediment to completion of a successful clinical trial in AKI is that many of the patients that receive the drug in the trial will not progress to severe AKI regardless of the treatment. These patients have no chance to benefit from the drug. This means that many more patients need to be enrolled in the study, adding additional cost, effort, duration of the study and risk of drug related side effects. An example demonstrating the importance of using a biomarker with a lower false positive rate is shown in figure 4. If the expected outcome of a drug is to reduce the number of patients that will develop AKI-D, we can use the hypothetical ICU population shown in figure 3. In this population 4 of 100 patients enrolled will require dialysis. If the drug can reduce the number of patients that reach AKI-D by 25%, 3 in the drug group will develop AKI-D. To adequately power this study, 10,602 subjects will need to be enrolled. A recent study found that the median cost per patient for a clinical trial is $41,413 (19). Thus, the cost for a study of this size is $439,060,626. By using biomarkers with characteristics of NephroCheck™, the number can be reduced to 6938 and the cost to $287,323,394. By using a biomarkerwith the characteristics of the cleavage assay described here, the number can be reduced to 712 and the cost to $29,486,056. This reflects an almost 10-fold improvement in the cost and number of patients needed to enroll. In this description, “KRT” for Kidney Replacement Therapy and “RRT” for Renal Replacement Therapy are used interchangeably. The present invention has been described with reference to certain preferred and alternative embodiments that are intended to be exemplary only and not limiting to the full scope of the present invention as set forth in the appended claims.

Claims

WE CLAIM:

1. An assay for predicting severe acute kidney injury, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample comprising urine from a patient at risk for severe acute kidney injury and a plurality of labeled proteins, peptides or peptide fragments, wherein at least some of said proteins or said peptides comprise a terminal His tag and a terminal Flag tag, wherein at least some of said peptide fragments comprise a terminal His tag or a terminal Flag tag; a test strip positioned in said cassette for receiving at least a portion of said sample, wherein said test strip comprises a plurality of anti-His tag antibodies at a first position and a plurality of anti-Flag tag antibodies at a second position, wherein said plurality of anti-His tag antibodies are operable for binding said His tag of said at least some of said proteins and said peptides or said His tag of said at least some of said peptide fragments, wherein said plurality of anti-Flag tag antibodies are operable for binding said Flag tag of said at least some of said peptide fragments; an indicator at said first position of said test strip when at least one of said anti-His tag antibodies is bound to said His tag of said at least some of said proteins or said peptides; and an indicator at said second position of said test strip when at least one of said anti-Flag tag antibodies is bound to said Flag tag of said at least some of said peptide fragments.

2. The assay of claim 1, wherein said test strip is a nitrocellulose membrane, cellulose membrane, nylon membrane, polyethylene membrane, or silica membrane.

3. The assay of claim 1, wherein said labeled proteins and labeled peptides comprise a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1.

4. An assay for predicting severe acute kidney injury, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample comprising urine from a patient at risk for severe acute kidney injury and a plurality of labeled proteins, peptides or peptide fragments, wherein at least some of said proteins or said peptides comprise a first terminal tag and a second terminaltag, wherein at least some of said peptide fragments comprise a first terminal tag or a second terminal tag; a test strip positioned in said cassette for receiving at least a portion of said sample, wherein said test strip comprises a plurality of anti-first terminal tag molecules at a first position and a plurality of anti-second terminal tag molecules at a second position, wherein said plurality of anti-first terminal tag molecules are operable for binding said first terminal tag of said at least some of said proteins or said peptides or said first terminal tag of said at least some of said peptide fragments, wherein said plurality of anti-second terminal tag molecules are operable for binding said second terminal tag of said at least some of said peptide fragments; an indicator at said first position of said test strip when at least one of said anti-first terminal tag molecules is bound to said first terminal tag of said at least some of said proteins or said peptides; and an indicator at said second position of said test strip when at least one of said anti-second terminal tag molecules is bound to said second terminal tag of said at least some of said peptide fragments.

5. An assay for predicting severe acute kidney injury, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample of urine from a patient at risk for severe acute kidney injury; a conjugate pad positioned in said cassette, wherein said conjugate pad comprises a plurality of labeled peptides, wherein each of said plurality of labeled peptides comprises a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1, wherein said labeled peptide further comprises a terminal His tag and a terminal Flag tag; a test strip positioned in said cassette, wherein said test strip comprises a plurality of anti-His tag antibodies at a first position and a plurality of anti-Flag tag antibodies at a second position, wherein said plurality of anti-His tag antibodies are operable for binding said His tag of at least some of said plurality of labeled peptides or a His tag of a plurality of peptide fragments of said plurality of labeled peptides, wherein said plurality of anti-Flag tag antibodies are operable for binding a Flag tag of said plurality of peptide fragments of said plurality of labeled peptides.

6. The assay of claim 5, wherein said labeled peptide comprises a gold label or a fluorescent label.

7. The assay of claim 5, wherein said test strip is a nitrocellulose membrane, cellulose membrane, nylon membrane, polyethylene membrane, or silica membrane.

8. An assay for predicting severe acute kidney injury, comprising: a cassette, wherein said cassette comprises a sample well for receiving a sample of urine from a patient at risk for severe acute kidney injury; and a test strip positioned in said cassette, wherein said test strip comprises a plurality of labeled peptides at a first position and a plurality of anti-Flag tag antibodies at a second position, wherein each of said plurality of labeled peptides comprises a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1, wherein said labeled peptide further comprises a terminal His tag and a terminal Flag tag, wherein said plurality of anti-Flag tag antibodies are operable for binding a Flag tag of peptide fragments of said plurality of labeled peptides.

9. The assay of claim 8, wherein said labeled peptide comprises a gold label or a fluorescent label.

10. The assay of claim 8, wherein said test strip is a nitrocellulose membrane, cellulose membrane, nylon membrane, polyethylene membrane, or silica membrane.

11. A method of predicting severe acute kidney injury in a patient using an assay, comprising the steps of: supplying an assay device comprising a test strip positioned in a cassette, wherein said cassette comprises a sample well, wherein said test strip comprises a plurality of anti-His tag antibodies at a first position and a plurality of anti-Flag tag antibodies at a second position; incubating a urine sample from a patient at risk for severe acute kidney injury with a plurality of labeled peptides to form a urine-peptide mixture, wherein said labeled peptide comprises a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1, wherein said labeled peptide further comprises a terminal His tag and a terminal Flag tag;introducing at least some of said urine-peptide mixture to said sample well of said cassette; binding said His tag of at least one of said labeled peptides to at least one of said plurality of anti-His tag antibodies to indicate that at least one of said labeled peptides has not been cleaved, and / or binding said Flag tag of at least one of said labeled peptides to at least one of said plurality of anti-Flag tag antibodies to indicate that at least one of said labeled peptides has been cleaved; and quantifying an amount of said labeled peptide that has not been cleaved by an intensity of an indicator at said first position of said test strip or quantifying an amount of said labeled peptide that has been cleaved by an intensity of an indicator at said second position.

12. The method of claim 11, wherein said labeled peptide comprises a gold label or a fluorescent label.

13. A method of predicting severe acute kidney injury in a patient using an assay, comprising the steps of: supplying an assay device comprising a conjugate pad and a test strip positioned in a cassette, wherein said cassette comprises a sample well, wherein said conjugate pad comprises a plurality of labeled peptides, wherein said labeled peptide comprises a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1, wherein said labeled peptide further comprises a terminal His tag and a terminal Flag tag, wherein said test strip comprises a plurality of anti-His tag antibodies at a first position and a plurality of anti-Flag tag antibodies at a second position; introducing a urine sample from a patient at risk for severe acute kidney injury to said sample well of said cassette; binding said His tag of at least one of said labeled peptides to at least one of said plurality of anti-His tag antibodies to indicate that at least one of said labeled peptides has not been cleaved, and / or binding said Flag tag of at least one of said labeled peptides to at least one of said plurality of anti-Flag tag antibodies to indicate that at least one of said labeled peptides has been cleaved; andquantifying an amount of said labeled peptide that has not been cleaved by an intensity of an indicator at said first position of said test strip or quantifying an amount of said labeled peptide that has been cleaved by an intensity of an indicator at said second position.

14. The method of claim 13, wherein said labeled peptide comprises a gold label or a fluorescent label.

15. A method of predicting severe acute kidney injury in a patient using an assay, comprising the steps of: supplying an assay device comprising a test strip positioned in a cassette, wherein said cassette comprises a sample well, wherein said test strip comprises a plurality of labeled peptides at a first position and a plurality of anti-Flag tag antibodies at a second position, wherein said labeled peptide comprises a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1, wherein said labeled peptide comprises a terminal Flag tag; introducing a urine sample from a patient at risk for severe acute kidney injury to said sample well of said cassette; binding said Flag tag of at least one of said labeled peptides to at least one said plurality of anti-Flag tag antibodies to indicate that at least one of said labeled peptides has been cleaved; and quantifying an amount of said labeled peptide that has been cleaved by an intensity of an indicator at said second position of said test strip.

16. The method of claim 15, wherein said labeled peptide comprises a gold label or a fluorescent label.

17. A method of predicting severe acute kidney injury in a patient: incubating a urine sample from a patient at risk for severe acute kidney injury with a plurality of labeled peptides to form a urine-peptide mixture, wherein said labeled peptide comprises a cleavage site corresponding to a cleavage site of insulin-like growth factor binding protein 1, wherein said labeled peptide further comprises a terminal fluorescent tag and a terminal quencher; detecting a fluorescence of said urine-peptide mixture; andcorrelating an intensity of said fluorescence to an amount of said labeled peptide that has been cleaved.

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