USE OF INSULIN-LIKE GROWTH FACTOR-BINDING PROTEIN 7 AND TISSUE INHIBITOR OF METALLOPROTEINASE 2 IN THE MANAGEMENT OF RENAL REPLACEMENT THERAPY

Abstract

The present invention provides methods and compositions for managing renal replacement therapy. A risk score, which is determined from a urinary concentration of IGFBP7 (insulin-like growth factor-binding protein 7) and/or a urinary concentration of TIMP-2 (tissue inhibitor of metalloproteinase 2), is determined obtained from the patient, and is used to manage patient treatment.

Claims

1. A method for treating renal stress in a subject in need of renal replacement therapy, comprising: calculating a risk score which is (i) a urinary concentration of IGFBP7 (insulin-like growth factor-binding protein 7), (ii) a urinary concentration of TIMP-2 (tissue inhibitor of metalloproteinase 2), or (iii) a composite of a urinary concentration of IGFBP7 and a urinary concentration of TIMP-2, by measuring an IGFBP7 concentration and/or a TIMP-2 concentration in a urine sample obtained from the subject to provide the risk score; comparing the risk score to a risk score threshold value, wherein when the risk score is above the risk score threshold value the subject is determined to be in renal stress; and if the comparing step indicates that the subject is in renal stress, treating the subject with a method of renal replacement therapy that produces less renal stress relative to treatment with intermittent hemodialysis.

2. A method according to claim 1, wherein the method of renal replacement therapy that produces less renal stress relative to treatment with intermittent hemodialysis is continuous renal replacement therapy or prolonged intermittent renal replacement therapy (PIRRT).

3. A method according to claim 1 or 2, wherein the risk score is calculated by multiplication of the concentrations of IGFBP7 and TIMP-2.

4. The method according to claim 3, wherein the risk score is [TIMP-2][IGFBP7]/1000, where the concentrations of IGFBP7 and TIMP-2 are each measured in ng/mL.

5. The method according to claim 4, wherein the threshold is about 2.0.

6. A method according to one of claims 1-5, wherein the urinary concentration of IGFBP7 and/or the urinary concentration of TIMP-2 are measured by introducing the urine sample obtained from the subject into an immunoassay instrument; wherein the immunoassay instrument comprises a solid phase, and one or both of an IGFBP7 antibody immobilized at a first location on the solid phase and a TIMP-2 antibody immobilized at a second location on the solid phase; wherein the instrument causes the urine sample to contact one or both of the first location and the second location; wherein the instrument measures the amount of IGFBP7 which binds to the IGFBP7 antibody immobilized at the first location and determines therefrom the concentration of IGFBP7 in the urine sample; and/or wherein the instrument measures the amount of TIMP-2 which binds to the TIMP-2 antibody immobilized at the second location and determines therefrom the concentration of TIMP-2 in the urine sample; wherein the instrument optionally mathematically combines the concentration of IGFBP7 and the concentration of TIMP-2 in the urine sample into the risk score; and wherein the instrument reports the risk score in a human readable form.

7. A method according to claim 6, wherein the urine sample obtained from the subject is further contacted with a second IGFBP7 antibody conjugated to detectable label and a second TIMP-2 antibody conjugated to detectable label; wherein first sandwich complexes are formed between the IGFBP7 antibody, IGFBP7 present in the urine sample, and the second IGFBP7 antibody; wherein second sandwich complexes are formed between the TIMP-2 antibody, TIMP-2 present in the urine sample, and the second TIMP-2 antibody; wherein the amount of IGFBP7 which binds to the IGFBP7 antibody is determined by the instrument detecting the detectable label bound at the first location; and wherein the amount of TIMP-2 which binds to the TIMP-2 antibody is determined by the instrument detecting the detectable label bound at the second location.

8. A method according to one of claims 1-7, wherein the subject is an intensive care unit patient.

9. A method according to one of claims 1-8, wherein the patient is in acute renal failure.

10. A method according to one of claims 1-9, wherein the subject has sepsis.

11. A method according to one of claims 1-9, wherein the subject is recovering from surgery.

12. A method according to one of claims 1-11, wherein the subject is undergoing renal replacement therapy at the time the urine sample is obtained from the subject to provide the risk score.

13. A method according to claim 12, wherein the risk score is used to monitor ongoing renal replacement therapy, wherein if the risk score is above the threshold, the rate or amount of fluid volume being removed from the subject by the ongoing renal replacement therapy is reduced, and/or the clearance rate of solutes by the ongoing renal replacement therapy is reduced.

14. A method according to claim 12, wherein the risk score is used to monitor ongoing renal replacement therapy, wherein if the risk score is above the threshold, the ongoing renal replacement therapy protocol is adjusted to reduce hypotensive effects associated with the ongoing renal replacement therapy or dose.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0070] For purposes of this document, the following definitions apply:

[0071] As used herein, an injury to renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc. Improvement in Renal Function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable increase in a measure of renal function. Preferred methods for measuring and/or estimating GFR are described hereinafter.

[0072] As used herein, reduced renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.1 mg/dL (8.8 .sub.Ilmol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0. 5 ml/kg per hour).

[0073] As used herein, acute renal failure or ARF is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.3 mg/dl (26.4 mol/l), a percentage increase in serum creatinine of greater than or equal to 50% (1. 5-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours). This term is synonymous with acute kidney injury or AKI.

[0074] As used herein, chronic kidney disease or CKD is CKD is defined as abnormalities of kidney structure or function, present for >3 months, with implications for health. Approximately 11% of U.S. adults reportedly have CKD, many of whom are elderly. The condition is usually asymptomatic until its advanced stages.

[0075] The term subject as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably patients, which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.

[0076] Preferably, an analyte is measured in a sample. Such a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement used to evaluate the kidney for preexisting damage. Preferred samples are body fluid samples.

[0077] The term body fluid sample as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components. A body fluid sample is obtained immediately prior to a procedure if it is obtained within 72 hours of initiating the procedure, and preferably within 48 hours, 24 hours, 18 hours, 12 hours, or 6 hours thereof.

[0078] The term diagnosis as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability (a likelihood) of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, diagnosis includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed. That such a diagnosis is determined is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.

[0079] Similarly, a prognostic risk signals a probability (a likelihood) that a given course or outcome will occur. A level or a change in level of a prognostic indicator, which in turn is associated with an increased probability of morbidity (e.g., worsening renal function, future ARF, or death) is referred to as being indicative of an increased likelihood of an adverse outcome in a patient.

[0080] IGFBP7 and TIMP-2 Assays

[0081] In general, immunoassays are specific binding assay that involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.

[0082] The assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein blotting methods. Additionally, certain methods and devices, such as biosensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman ACCESS, Abbott AXSYM, Roche ELECSYS, Dade Behring STRATUS systems are among the immunoassay analyzers that are capable of performing immunoassays. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), lateral flow assays, competitive binding assays, and the like.

[0083] Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.

[0084] Such assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

[0085] Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

[0086] In certain aspects, the present invention provides kits for the analysis of IGFBP7 and/or TIMP-2. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that bind each biomarker being assayed. The kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein. Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte. Preferably, an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies that bind a kidney injury marker. Most preferably each of the antibodies are monoclonal antibodies. The instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.

[0087] Antibodies

[0088] The term antibody as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3 rd Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., antigen binding sites, (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term antibody.

[0089] Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker of the present invention. The term specifically binds is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody specifically binds if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, Preferred antibodies bind with affinities of at least about 10.sup.7 M.sup.1, and preferably between about 10.sup.8 M.sup.1 to about 10.sup.9 M.sup.1, about 10.sup.9 M.sup.1 to about 10.sup.10 M.sup.1, or about 10.sup.10 M.sup.1 to about 10.sup.12 M.sup.1 .

[0090] Affinity is calculated as K.sub.d=k.sub.off/k.sub.on (koff is the dissociation rate constant, K.sub.on is the association rate constant and Kd is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c=K(nr): where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K =equilibrium association constant; and n=number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

[0091] The term epitope refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0092] Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

[0093] The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.

[0094] The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.

[0095] While the present application describes antibody-based binding assays in detail, alternatives to antibodies as binding species in assays are well known in the art. These include receptors for a particular target, aptamers, etc. Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist. High-affinity aptamers containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions, and may include amino acid side chain functionalities.

[0096] Assay Correlations

[0097] The term correlating as used herein in reference to the use of biomarkers refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.

[0098] Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.

[0099] Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5th percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior baseline result is used to monitor for temporal changes in a biomarker level.

[0100] Population studies may also be used to select a decision threshold. Reciever Operating Characteristic (ROC) arose from the field of signal dectection therory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold able to best distinguish a diseased subpopulation from a nondiseased subpopulation. A false positive in this case occurs when the person tests positive, but actually does not have the disease. A false negative, on the other hand, occurs when the person tests negative, suggesting they are healthy, when they actually do have the disease. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1specificity, the ROC graph is sometimes called the sensitivity vs (1specificity) plot. A perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5. A threshold is selected to provide an acceptable level of specificity and sensitivity.

[0101] In this context, diseased is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and nondiseased is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.

[0102] In addition to threshold comparisons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.

[0103] Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (AUC) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

[0104] As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1-specificity)) of greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1-sensitivity)/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1

[0105] Clinical indicia which may be combined with the kidney injury marker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total protein measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal papillary antigen 1 (RPA1) measurement; a renal papillary antigen 2 (RPA2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine). Other measures of renal function which may be combined in the methods of the present invention are described hereinafter and in Harrison's Principles of Internal Medicine, 17.sup.th Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47.sup.th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.

[0106] Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.

[0107] Diagnosis of Acute Renal Failure

[0108] As noted above, the terms acute renal (or kidney) injury and acute renal (or kidney) failure as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:

[00001]$\mathrm{GFR}=\frac{\mathrm{Urine}.Math..Math.\mathrm{Concentration}\mathrm{Urine}.Math..Math.\mathrm{Flow}}{\mathrm{Plasma}.Math..Math.\mathrm{Concentration}}$

[0109] By normalizing the GFR to the body surface area, a GFR of approximately 75-100 ml/min per 1.73 m.sup.2 can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.

[0110] There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.

[0111] Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (U.sub.Cr), urine flow rate (V), and creatinine's plasma concentration (P.sub.Cr) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (U.sub.CrV) divided by its plasma concentration. This is commonly represented mathematically as:

[00002]$C_{\mathrm{Cr}}=\frac{U_{\mathrm{Cr}}V}{P_{\mathrm{Cr}}}$

[0112] Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:

[00003]$C_{\mathrm{Cr}}=\frac{U_{\mathrm{Cr}}24.Math.\text{-}.Math.\mathrm{hour}.Math..Math.\mathrm{volume}}{P_{\mathrm{Cr}}2460.Math..Math.\mathrm{mins}}$

[0113] To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:

[00004]$C_{\mathrm{Cr}-\mathrm{corrected}}=\frac{C_{\mathrm{Cr}}1.73}{\mathrm{BSA}}$

[0114] The accuracy of a creatinine clearance measurement (even when collection is complete) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.

[0115] For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are provided, minor modifications of the RIFLE urine output criteria have been described. For example, Bagshaw et al., Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure).

[0116] Selecting a Treatment Regimen

[0117] Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, N.J., 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.

[0118] The distinction between prerenal AKI and instrinsic AKI is an important clinical assessment that directs the therapeutic intervention(s). Patients who are prerenal need therapies directed at hemodynamics to improve renal blood flow. These therapies are often involve inotropes, intravenous fluids and/or vasopressors. Each of these interventions have potential side effects (e.g. arrhythmias, volume overload, vasoconstriction) and would not be advisable to implement these therapies if they are not destined to improve renal function. Thus, the distinction between prerenal AKI and intrinsic AKI helps determine the therapy which should be prescribed. If prerenal AKI is not present, therapy is directed at mitigating AKI and providing supportive care.

[0119] Prerenal acute renal failure occurs when a sudden reduction in blood flow to the kidney camera (renal hypoperfusion) causes a loss of kidney function. Causes can include low blood volume, low blood pressure, shunting of blood from the kidney, heart failure, and local changes to the blood vessels supplying the kidney. In prerenal acute renal failure, there is nothing wrong with the kidney itself. Treatment focuses on correcting the cause of the prerenal acute renal failure.

[0120] In prerenal AKI without fluid overload, administration of intravenous fluids is typically the first step to improve renal function. This is particularly used in patients in whom prerenal AKI develops as the result of intravascular volume depletion in order to restore normal circulating blood volume. Volume status may be monitored to avoid over- or under-replacement of fluid as described herein. Fluids with colloidal particles such as albumin may be preferred over simple saline infusion. In a prerenal condition wherein the forward flow is compromised, drugs directed at augmenting cardiac output are typically employed.

[0121] In patients with congestive heart failure in whom AKI has developed as a result of excessive diuresis, withholding of diuretics and cautious volume replacement may be sufficient to restore kidney function. Inotropes such as norepinephrine and dobutamine may be given to improve cardiac output and hence renal perfusion.

[0122] Hospitalized fluid overload patients are typically treated with fluid restriction, IV diuretics, inotropes (e.g., milrinone or dobutamine) and combination therapies. The loop diuretic furosemide is the most frequently prescribed diuretic for treatment of volume overload in HF. Initial oral doses of 20 to 40 mg once a day should be administered to patients with dyspnea on exertion and signs of volume overload who do not have indications for acute hospitalization. Severe overload and pulmonary edema are indications for hospitalization and intravenous furosemide. Some patients with mild HF can be treated effectively with thiazide diuretics. Those who have persistent volume overload on a thiazide diuretic should be switched to an oral loop diuretic. In patients with severe kidney injury, diuretics may not result in significant diuresis. Ultrafiltration, also called aquapheresis, may be used to treat fluid overload in such cases.

[0123] In contrast to prerenal AKI, the main goal of treatment of acute tubular necrosis (ATN) is to prevent further injury to the kidney. Ischemic ATN can be caused when the kidneys are not sufficiently perfused for a long period of time (e.g. due to renal artery stenosis) or by shock. Sepsis causes 30% to 70% of deaths in patients with ATN; therefore, avoidance of intravenous lines, bladder catheters, and respirators is recommended. Because septic patients are vasodilated, large volumes of administered fluid accumulate in the lung interstitium of these patients. Extracellular fluid volume should be assessed promptly, and repletion of any deficit should be initiated promptly. Hemodynamic status should be modified by appropriate fluid therapy, giving vasopressors and/or inotropes and treating any underlying sepsis. All possible nephrotoxic drugs should be stopped. In addition, doses of all medications that are eliminated by the kidney should be adjusted.

[0124] Renal replacement therapy refers to therapy that replaces the normal blood-filtering function of the kidneys. Various types of RRT are used by clinicians, including the following: [0125] continuous renal replacement therapy (CRRT) [0126] continuous hemodialysis (CHD) [0127] continuous arteriovenous hemodialysis (CAVHD) [0128] continuous venovenous hemodialysis (CVVHD) [0129] continuous hemofiltration (CHF) [0130] continuous arteriovenous hemofiltration (CAVH or CAVHF) [0131] continuous venovenous hemofiltration (CVVH or CVVHF) [0132] continuous hemodiafiltration (CHDF) [0133] continuous arteriovenous hemodiafiltration (CAVHDF) [0134] continuous venovenous hemodiafiltration (CVVHDF) [0135] intermittent renal replacement therapy (IRRT) [0136] intermittent hemodialysis (IHD) [0137] intermittent venovenous hemodialysis (IVVHD) [0138] intermittent hemofiltration (IHF) [0139] intermittent venovenous hemofiltration (IVVH or IVVHF) [0140] intermittent hemodiafiltration (IHDF)

[0141] Acute dialysis-dependent renal failure is a common problem in the intensive care unit (ICU) and, despite significant improvements in the care of critically ill patients, the mortality from this complication remains over 50%. The development of renal failure is an independent predictor of mortality in this patient population.

[0142] The precise timing of RRT initiation is usually a matter of clinical judgment. The classic indications for dialysis include:

[0143] diuretic resistant pulmonary edema

[0144] hyperkalemia (refractory to medical therapy)

[0145] metabolic acidosis (refractory to medical therapy)

[0146] uremic complications (pericarditis, encephalopathy, bleeding)

[0147] dialyzable intoxications (eg, lithium, toxic alcohols, and salicylates).

[0148] While many of these indications are typically used in the setting of chronic renal failure, the consequences of these complications are likely to be more severe in critically ill patients; therefore, there has been a growing trend to start dialysis prior to the development of these indications. Delays in the initiation of treatment have often been based on a concern that dialysis itself may delay recovery of renal function.

[0149] IGFBP7 and TIMP-2 have been described for risk assessment of AKI. Kellum and Chawla, Neohrol. Dial.. Transplant 31(1):16-22, 2016. The present invention demonstrates that these biomarkers can also be used for assessing whether kidney function is under stress for purposes of managing the administration of renal replacement therapy to minimize further stress that will lead to additional renal damage.

[0150] CRRT is any renal replacement therapy that is intended to be applied for 24 h per day in an ICU. The term CRRT describes a variety of blood purification techniques, which may differ significantly according to the mechanism of solute transport, the type of membrane, the presence or absence of dialysate solution, and the type of vascular access. CRRT provides slower solute clearance per unit time as compared with intermittent therapies but over 24 h may even exceed clearances with IHD. The choice of CRRT is thought to provide better hemodynamic tolerability, more efficient solute clearance, better control of intravascular volume, and better clearance of middle and large molecular weight substances relative to intermittent dialysis. Pannu and Gibney, Ther. Clin. Risk. Manag. 1: 141-50, 2005, which is hereby incorporated by reference in its entirety.

[0151] Hypotension is one of the most common complications associated with intermittent hemodialysis, occurring in approximately 20%-30% of all treatments. Some of the causes are dialysis specific, such as excessive or rapid volume removal, changes in plasma osmolality, and autonomic dysfunction. In critically ill patients who may be hemodynamically unstable, it would be desirable to minimize this complication, as it may lead to further organ ischemia and injury. The risk scores of the present invention may be used to determine if a shift is necessary between, for example, intermittent hemodialysis, and a method of renal replacement therapy that produces less renal stress. In this regard, when the risk score is elevated above the applicable threshold, one may reduce the rate or amount of fluid being removed. Additionally, the clearance rate of small solutes (e.g., urea) is slower per unit time with CRRT (17 mL/min vs more than 160 mL/min with intermittent hemodialysis).

[0152] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

EXAMPLE 1

Longer Duration of RRT for Patients with Elevated Biomarkers

[0153] ICU patients with acute kidney injury (AKI) and receiving renal replacement therapy (RRT) were included in the analysis. A urine sample was collected from each patient during RRT and within 48 hours after initiation of RRT. TIMP2, IGFBP7, and TIMP2IGBFP7 (multiplication of the concentrations of the two biomarkers) were measured in the urine samples by immunoassay with the NephroCheck Test kit on the Astute140 Meter. Patients were divided into two groups by their biomarker concentrations, being either less than or equal to or greater than the specified threshold. The range and median of the number of days on RRT, length of hospital stay, and length of ICU stay were determined for each patient group. Patients with biomarker concentrations greater than the threshold received RRT for more days and had a longer length of stay in the hospital and in the ICU than patients with biomarker concentrations less than or equal to the threshold.

TABLE-US-00003 [TIMP2] Endpoint [IGFBP7], Kruskal_Wallis Endpoint (ng/mL).sup.2/1000 n Range Median(IQR) P-value Days on RRT 2 19 1.00 to 9.00 3.00(2.00-4.00) 0.007 >2 24 1.00 to 9.00 5.00(4.00-6.00) Hospital Length 2 19 3.00 to 95.00 21.00(9.00-47.00) 0.561 of Stay >2 23 2.00 to 69.00 32.00(17.00-45.00) ICU Length 2 18 3.00 to 95.00 9.50(6.00-19.00) 0.185 of Stay >2 21 3.00 to 50.00 19.00(7.00-26.00)

TABLE-US-00004 Endpoint [IGFBP7], Kruskal_Wallis Endpoint ng/mL n Range Median(IQR) P-value Days on RRT 150 18 1.00 to 9.00 3.50(2.00-5.00) 0.121 >150 25 1.00 to 9.00 5.00(3.00-6.00) Hospital Length 150 18 3.00 to 95.00 20.00(9.00-45.00) 0.438 of Stay >150 24 2.00 to 69.00 31.50(18.00-49.50) ICU Length 150 18 3.00 to 95.00 9.50(6.00-19.00) 0.171 of Stay >150 21 3.00 to 50.00 20.00(7.00-26.00)

TABLE-US-00005 Endpoint [TIMP2], Kruskal_Wallis Endpoint ng/mL n Range Median(IQR) P-value Days on RRT 12 17 1.00 to 9.00 3.00(2.00-4.00) 0.002 >12 26 1.00 to 9.00 5.00(4.00-6.00) Hospital Length 12 17 3.00 to 95.00 23.00(14.00-47.00) 0.980 of Stay >12 25 2.00 to 69.00 31.00(14.00-45.00) ICU Length 12 16 3.00 to 95.00 11.50(5.50-23.00) 0.501 of Stay >12 23 3.00 to 50.00 18.00(6.00-26.00)

EXAMPLE 2

Use of Biomarkers for Choosing RRT Modality

[0154] A 65 year-old male is admitted to the intensive care unit (ICU) after presenting to the emergency department with a diagnosis of a severe, community acquired pneumonia. Due to worsening respiratory insufficiency and an inability to maintain adequate oxygenation, he is intubated and placed on mechanical ventilation. He also is noted to have a low blood pressure and received several liters of intravenous (IV) crystalloid intravenous fluid for volume resuscitation. He does not respond and as a result, vasopressor therapy is started to maintain systemic blood pressure. He is also pancultured and placed on broad-spectrum antimicrobial therapy.

[0155] His urine output remains persistently below than 0.3 mL/kg/hr since his admission despite the aggressive volume resuscitation that he receives and his serum creatinine rises from an admission level of 1.3 mg/dL to 5.1 mg/dL, suggesting AKI stage III. He requires significant positive pressure ventilatory support, including elevated FiO2 and PEEP. Of note, his pulmonary compliance is decreased, his central venous pressure is persistently elevated, and he is becoming increasingly edematous, all of which suggest significant total body fluid overload. He is evaluated with a transthoracic echo (TTE) to assess his cardiac function and performance and also, shortly after admission to the ICU, he has a central venous line (CVL) placed for intravenous assess and for assessment of central venous pressure (CVP), which remains consistently elevated.

[0156] Based on his clinical status, the patient is a candidate for RRT. A urine sample is collected for measurement of [TIMP2][IGFBP7]. The [TIMP2][IGFBP7] is >2.0, indicating high levels of kidney stress. The elevated [TIMP2][IGFBP7] level (high kidney stress) indicates the patient's kidneys have a low tolerance for the hemodynamic instability and/or other systemic physiological derangements associated with the patient's condition. In addition, the elevated [TIMP2][IGFBP7] level indicates risk of a prolonged course of RRT. Therefore, the clinical team selects continuous renal replacement therapy (rather than intermittent renal replacement therapy), which is recommended in situations in which shifts in fluid balance and metabolic fluctuations are poorly tolerated.

[0157] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[0158] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0159] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.

[0160] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of and consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0161] Other embodiments are set forth within the following claims.