WO2012112589A2 - Methods and compositions for detecting urinary cholesterol as a biomarker for acute kidney injury - Google Patents

Abstract

The present invention relates to methods and diagnostic tests that are useful to detect the presence of acute kidney injury in a subject. The presence or amount of cholesterol is determined in a urine sample obtained from the subject. The determined presence or amount of cholesterol is compared to a reference standard, wherein the presence or elevated amount of cholesterol in the urine sample from the subject indicates the presence of kidney injury in the subject.

Description

METHODS AND COMPOSITIONS FOR DETECTING URINARY CHOLESTEROL AS A BIOMARKER FOR ACUTE KIDNEY INJURY

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.

61/442,668, filed February 14, 2011, which is incorporated herein in its entirety.

STATEMENT OF GOVERNMENT FUNDING

This invention was made with government support under grant numbers DK384342, R21-DK083315, HL081332, HL-103836, DK082192, awarded by the National Institutes of Health. The government has certain rights in the invention.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into the specification. The name of the text file containing the sequence listing is: 38763_Sequence_Final_2012-02- 08.txt. The file is 7KB; was created on February 8, 2012; and is being submitted via EFS-Web with the filing of the specification. FIELD OF THE INVENTION

The present invention relates to novel methods and reagents for detecting acute kidney injury based on detectable levels of cholesterol in the urine.

BACKGROUND

The onset of acute kidney injury (AKI) is typically a silent clinical event, almost always being documented in its aftermath by the onset of progressive azotemia. This is in striking contrast to acute heart or brain injury, whose onsets are clinically apparent (e.g., angina and myocardial infarction; acute neurological deficits). Given AKI's silent onset, reliable "biomarkers" for it have been sought. The underlying rationale has been that early detection can lead to early therapy, thereby preventing the onset of acute renal failure (ARF). Therefore, a need exists for non-invasive assays to detect and monitor the presence of AKI in patients.

SUMMARY

In one aspect, a method is provided for determining the presence of kidney injury in a mammalian subject. The method comprises (a) performing an assay to determine the presence or amount of cholesterol in a urine sample obtained from the subject; and (b) comparing the results of step (a) with a cholesterol reference standard. The presence or elevated level of cholesterol in step (a) as compared to the cholesterol reference standard indicates the presence of kidney injury in the subject.

In one embodiment, the assay of step (a) determines the amount of cholesterol associated with a cellular component of the urine sample. In one embodiment, the cellular component comprises cell plasma membrane or cell organelle membranes. In one embodiment, the cellular component comprises components of proximal tubular cells, including intact cells.

In one embodiment, the amount of cholesterol in the urine samples of step (a) is normalized using an internal control substance present in the cellular component of the urine samples. In one embodiment, the internal control substance comprises a phospholipid.

In one embodiment, the method comprises obtaining a urine sample from the subject. In one embodiment, the method comprises processing the urine sample obtained from the subject. In one embodiment, the method comprises separating the cellular component from the urine. In one embodiment, the cellular component of the urine sample is separated from the urine by subjecting the urine sample to a sufficient centrifugal force to obtain a pellet containing cholesterol. In another embodiment, the cellular component of the urine sample is separated by filtering the urine sample to permit capture of cell components containing cholesterol.

In one embodiment, the mammal is a rodent, rabbit, cow, horse, dog, cat, or human.

In one embodiment, the cholesterol reference standard is derived from at least one subject with no known kidney injury. DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 is a graphical representation of cholesterol levels in urinary pellets observed in control mice, mice with pre-renal azotemia, and mice with glycerol-mediated and maleate-mediated acute kidney injury (AKI) at 3 and 24 hours; the cholesterol levels were normalized with phospholipid phosphate (PLP) as an internal control; * signifies a significant difference (p<0.01) as compared to normal controls, as described in Example 1;

FIGURE 2 is a graphical representation of urea-derived nitrogen levels per volume of blood, as determined by a blood urea nitrogen (BUN) analysis, for control mice, mice with pre-renal azotemia, and mice with glycerol-mediated and maleate- mediated acute kidney injury (AKI); * signifies a significant difference (p<0.01) as compared to normal controls, as described in Example 1 ;

FIGURE 3 is a graphical representation of cholesterol levels detected in human urine pellet samples obtained from normal control individuals, intensive care unit (ICU) patients without AKI ("ICU / AKI-"), ICU patients with AKI ("ICU / AKI+"), and patients with chronic kidney disease ("CKD"); cholesterol levels are normalized using phospholipid phosphate (PLP) as a control; "NS" signifies a no significant difference as compared to normal controls and p values indicating significant differences are provided, as described in Example 2;

FIGURE 4 is a graphical representation of relative levels of RNA Polymerase II (Pol II) binding to the human 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) gene as determined in normal control individuals, ICU patients without AKI ("ICU / AKI-"), and ICU patients with AKI ("ICU / AKI+"); Pol II binding levels are normalized using Pol II binding to the β actin gene as an internal control; p values indicating significant differences in Pol II binding as compared to normal individuals are indicated, as described in Example 3; and

FIGURE 5 is a graphical representation of relative levels of trimethylated histone H3 at the lysine 4 position ("H3K4m3") at exon 1 of the human 3-hydroxy-3-methyl- glutaryl-CoA reductase (HMGCR) gene, as determined by ChIP analysis in normal control individuals, ICU patients without AKI ("ICU / AKI-"), and ICU patients with AKI ("ICU / AKI+"); H3K4m3 levels are normalized using the H3K4m3 levels to the β actin gene as an internal control; a p value indicating significant differences in H3K4m3 levels as compared to normal individuals is indicated, as described in Example 3.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. For instance, practitioners are particularly directed to Sambrook, J., and Russell, D.W., eds., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2001), which is incorporated herein by reference, for definitions and terms of the art.

The following definitions are presented to provide clarity with respect to the terms as they are used in the specification and claims to describe the present invention.

As used herein, the term "azotemia" refers to abnormally high levels of nitrogen-containing compounds in the blood. These elevated levels are often attributed to insufficient filtering of the compounds from the blood by the kidneys. Azotemia can generally be divided into three classifications: pre-renal, renal (or structural), and post-renal. Pre-renal azotemia is caused by restricted blood flow to the kidneys. Causes can include decreased cardiac output, restriction of the renal artery, and shock. There is no inherent damage to the kidneys that causes the elevated levels of nitrogen in the blood. Renal, or structural, azotemia is typically caused by damage or disease of the kidney that directly impairs the ability of the kidney to properly filter the blood. Such damage is referred to herein as acute kidney injury ("AKI"). Post-renal azotemia is caused by blockage of urine flow after filtration by the kidneys. Such blockage can be caused by such conditions as kidney or bladder stones, and compression of the ureters. There is no inherent disease of the kidney that causes the abnormal nitrogen levels in the blood.

As used herein, the term "gene activation" refers to the enhanced or increased transcription of the gene leading to increased levels of mRNA and, ultimately, increased levels of the corresponding protein.

A used herein, the term "cellular component" refers to the portion of the urine that derives from cellular debris or whole cells that are sloughed off from the kidney tissue during the filtration process. The cellular component can include proximal tubular cells or tubular "brush border" fragments that separate from the kidney tissue and enter tubular lumina.

A seemingly constant consequence of ischemic or toxic acute kidney injury (AKI) is an up-regulation of renal cortical 3-hydroxy-3-methyl-glutaryl-CoA reductase ("HMG CoA reductase" or "HMGCR") gene activity (Zager, R.A., et al, "Increased proximal tubular cholesterol content: Implications for cell injury and the emergence of 'acquired cytoresistance'," Kidney Int. 56: 1788-1797, 1999; Zager, R.A., "Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack," Kidney Int. 55: 193-205, 2000; Zager, R.A. and T. Kalhorn, "Changes in free and esterified cholesterol: Hallmarks of acute tubular injury and acquired cytoresistance," Am. J. Pathol. 757: 1007-1016, 2000; Zager, R.A., et al, "Cholesterol ester accumulation: An immediate consequence of acute ischemic renal injury," Kidney Int. 59: 1750-1761, 2001; Zager, R.A., "P glycoprotein-mediated cholesterol cycling: a potentially important determinant of proximal tubular cell viability," Kidney Int. 60:944-956, 2001; Zager, R.A., et al, "Renal cholesterol accumulation: A durable response following acute and subacute renal insults," Am. J. Pathology 759:743-752, 2001 ; Zager, R.A. and A.C. Johnson, "Renal cortical cholesterol accumulation: An integral component of the systemic stress response," Kidney Int. 60:2229-2310, 2001; Zager, R.A., et al, "The mevalonate pathway during acute tubular injury: selected determinants and consequences," Am. J. Pathol. 767:681-692, 2002; Zager, R.A., et al, "Proximal tubular cholesterol loading following mitochondrial, but not glycolytic blockade," Am. J. Physiol. 2#5:F1092-F1099, 2003; Naito, M. et al, "Renal ischemia-induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 174:54-62, 2009). Within 12-24 hours post AKI induction, this culminates in an approximate 20-40% increase in renal cortical cholesterol content. Furthermore, when cultured human proximal tubular (HK-2) cells are subjected to either toxin- or ATP depletion-mediated injury, increased HMGCR activity and cholesterol accumulation result (Gaudio, K.M., et al, "Role of heat stress response in the tolerance of immature renal tubules to anoxia," Am. J. Physiol. 274:F1029-1036, 1998). The significance of these in vitro findings is that they imply that injury-induced in vivo cholesterol accumulation reflects, at least in part, direct proximal tubule cell events. The "downstream" consequences of proximal tubule cholesterol loading remain incompletely defined. However, the inventor's prior work indicates that it helps mediate the phenomenon of "ischemic preconditioning" (so called "acquired cytoresistance"), whereby previously injured tubular cells become resistant to further ischemic or toxic attack (Zager, R.A., et al, "Increased proximal tubular cholesterol content: Implications for cell injury and the emergence of 'acquired cytoresistance'," Kidney Int. 56: 1788-1797, 1999; Zager, R.A., "Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack," Kidney Int. 55: 193-205, 2000; Zager, R.A., "P glycoprotein-mediated cholesterol cycling: a potentially important determinant of proximal tubular cell viability," Kidney Int. 60:944-956, 2001). Indeed, this sequence of events is analogous to the so-called "heat shock" response: i.e., whereby a renal stress (i.e., heat shock) up-regulates cytoprotective molecules (i.e., heat shock proteins) which confer a cytoresistant state (Gaudio, K.M., et al, "Role of heat stress response in the tolerance of immature renal tubules to anoxia," Am. J. Physiol. 274:F1029-1036, 1998; Van Why, S.K. and N.J. Siegel, "Heat shock proteins in renal injury and recovery," Curr. Opin. Nephrol. Hypertens. 7:407-412, 1998). That transient hyperthermia raises both heat shock protein and renal tubular cholesterol levels directly supports this 'heat shock' analogy (Zager, R.A. and A.C. Johnson, "Renal cortical cholesterol accumulation: An integral component of the systemic stress response," Kidney Int. 60:2229-2310, 2001).

A morphologic correlate of AKI is sloughing of proximal tubular 'brush border' fragments and intact tubular cells into urine (Venkatachalam, M.A., et al, "Ischemic damage and repair in the rat proximal tubule: differences among the SI, S2, and S3 segments," Kidney Int. 4:31-49, 1978; Donohoe, J.F., et al, "Tubular leakage and obstruction after renal ischemia: structural-functional correlations," Kidney Int. 73:208- 22, 1978; Racusen, L.C., et al, "Dissociation of tubular cell detachment and tubular cell death in clinical and experimental 'acute tubular necrosis'," Lab Invest. 64:546-556, 1991; Racusen, L.C., "Epithelial cell shedding in acute renal injury," Clin. Exp. Pharmacol. Physiol. 25:273-275, 1998).

As described herein, the inventor has discovered that evidence of increased HMGCR gene activity detectable in urine, including elevated cholesterol levels, elevated binding of RNA Polymerase II to the HMGCR gene promoter, and elevated chromatin modification corresponding to the HMGCR gene, correlate with AKI. Specifically, the inventor demonstrates herein that (1) acute tubular injury activates the HMGCR gene; (2) this results in increased cholesterol synthesis and proximal tubular cell cholesterol loading; and (3) upon release of tubular cells and tubular cell debris into the urinary space, increases in urinary pellet cholesterol content result. The inventor previously demonstrated that experimental chronic nephropathy does not activate these pathways (Johnson, A.C., et al, "Experimental glomerulopathy alters renal cortical cholesterol," Am. J. Pathol. 162:283-291, 2003). Thus, it is demonstrated that an increase in urinary pellet cholesterol has utility as an AKI biomarker. This discovery satisfies a need for simple, non-invasive assays to detect and monitor the occurrence of AKI in patients.

In accordance with the foregoing, the present invention relates to the discovery that elevated levels of cholesterol detectable in the urine correlate with acute kidney injury (AKI). Elevated levels of cholesterol were detected in urinary pellets in two murine models of structural AKI, but not in a murine model of pre-renal azotemia. Furthermore, elevated levels of cholesterol were detected in urinary pellets from human intensive care unit (ICU) patients with AKI, but not in ICU patients without AKI or patients with chronic kidney disease. Finally, additional evidence of the 3-hydroxy-3- methyl-glutaryl-CoA reductase (HMGCR) gene activation, namely, an increased binding of RNA Polymerase II to the promoter region of the gene and increased histone modification in the chromatin structure corresponding to the gene, was detected in urinary pellets from human ICU patients with AKI, but not in ICU patients without AKI.

As described in the examples, mice were subjected to two models of AKI (rhabomyolysis; maleate toxicity) or to a model of pre-renal azotemia (indomethicin + surgical stress). Urine samples were collected 3 or 24 hours later. Experimental AKI, but not pre-renal azotemia, induced ~2- to 3-fold increases in urinary pellet cholesterol levels, and this occurred within 3 hours of AKI induction.

In addition, urine samples were collected from 29 critically ill patients with or without AKI (n= 14, 15, respectively), from 15 human patients with CKD, and from 15 normal volunteers. The urines were centrifuged, the pellets underwent lipid extraction, and the extracts were assayed for cholesterol and total membrane phospholipid content. The results were expressed as cholesterol/phospholipid ratios. Clinical AKI demonstrated a doubling of pellet cholesterol levels. Conversely, neither critical illness without AKI, nor clinical CKD, had this effect. Noteworthy in regard to this finding is that many urinary protein AKI biomarkers, e.g., NGAL, KIM-1, and MCP-1, are typically elevated with both acute as well as chronic kidney disease. That pellet cholesterol levels were elevated in the AKI+ cohort, but not in the CKD cohort, demonstrates differential diagnostic utility in this regard. HMGCR gene activation in AKI+ patients was supported by findings of increased R A polymerase II binding to, and increased levels of a gene activating histone marker (H3K4m3) at, urinary fragments of the HMGCR gene (exon 1 ; chromatin immunoprecipitation). Because the degree of Pol Il-gene binding correlates with rates of gene transcription, this finding provides clinical support for the experimental observation that AKI increases HMGCR gene activity. That a gene activating histone marker, H3K4m3, was also elevated at the HMGCR gene in the AKI+, but not in the AKI-, provides additional support that AKI induces HMGCR gene activation, and indicates that corresponding increases in urinary pellet cholesterol levels have utility for use as a biomarker for the presence of AKI.

These data provide clinical support for a novel concept: that AKI activates the HMGCR/cholesterol axis and that, with release of tubular cells and tubular cell debris into urine, increases in urinary pellet cholesterol levels result. Particularly noteworthy in this regard are the following: (1) prior experimental data demonstrate that pellet cholesterol levels rise within 3 hours post- AKI induction, indicating potential utility in detecting early AKI; (2) pellet cholesterol levels remain normal with experimental prerenal azotemia; and (3) pellet cholesterol levels are able to differentiate patients with acute versus chronic renal disease.

At least four major advantages accompany the invention described herein. First, cholesterol is highly stable in urine samples, in marked contrast to traditional protein biomarkers that are subject to enzymatic and non-enzymatic degradation. Second, cholesterol is a biomarker for cells that are recoverable in the urine. Traditional urinary protein biomarkers of renal injury reflect a balance between what is generated in the kidney and what is generated outside the kidney with secondary urinary excretion. Thus, urinary protein biomarker levels may not reflect direct kidney events. This drawback is not an issue with cholesterol levels associated with the cell components of urinary samples because circulating cells do not gain access to urine by glomerular filtration (with the possible exception of red blood cells, which are readily detected). Third, cholesterol is the only known lipid biomarker of acute renal injury. The current emphasis in the renal literature is to discover a panel of biomarkers to be used in concert, rather than relying on a single one. The addition of a lipid biomarker to a protein biomarker panel has the potential to expand clinical utility. Fourth, essentially all described protein biomarkers are elevated by both acute as well as chronic kidney disease (CKD). There is a need for a biomarker that can not only detect acute kidney injury, but can also differentiate it from pre-existing chronic kidney disease. The present disclosure satisfies this need.

In accordance with the foregoing, one aspect the present disclosure provides a method for determining the presence of kidney injury in a mammalian subject. The method comprises (A) performing an assay to determine the presence or amount of cholesterol in a urine sample obtained from the subject, and (B) comparing the results of step A with a cholesterol reference standard, wherein the presence or elevated level of cholesterol in step A compared to the reference standard indicates the presence of kidney injury in the subject. In preferred embodiments, the reference standard is a value derived from one or more subjects with no kidney injury. In some embodiments, the assay of step (A) measures cholesterol associated with a cellular component of the urine sample. The cellular component can comprise cell plasma membranes or cell organelle membranes. In further embodiments, the cellular component comprises components of proximal tubular cells, including intact cells.

In one embodiment, the method comprises obtaining a urine sample from the subject. It will be understood that the sample can be obtained from the subject directly by the practitioner of the method from the subject. The sample can also be obtained indirectly from the subject through an intermediary, such as a primary care provider or clinic technician.

In one embodiment, the method comprises processing the urine sample. In some embodiments, the urine sample is processed to remove substantially all of the liquid from the sample. In preferred embodiments, the cellular component of the urine sample is separated from the urine in the sample. In some embodiments, the cellular component of the urine sample is separated from the urine by subjecting the urine sample to a sufficient centrifugal force to obtain a pellet containing cholesterol. In additional embodiments, the cellular component of the urine sample is separated by filtering the urine sample to permit capture of cell components containing cholesterol.

In one embodiment, the assay comprises determining the level of cholesterol in the urine, wherein an elevated amount of cholesterol in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject. The assay to determine the level of cholesterol in the urine can comprise any assay for detection or quantification of cholesterol that are commonly known in the art. For example, as described in Example 1, the Amplex Red assay can be employed. In one embodiment, the cholesterol in the urine is associated with a cellular component derived at least in part from kidney proximal tubule cells. The cellular component can be isolated from the urine by methods such as, for example, centrifugation to form a pellet, or filtration. The assay can further comprise normalizing the level of cholesterol in the urine to the level of an internal control substance in the cellular component of the urine samples. Any internal control substances can be used, for example, that is known in the art to provide a comparison useful to infer differences or fluctuations in the amounts of cholesterol per volume of urine or relative to the amount of cellular component appearing in the urine. For example, internal control substance can comprise a phospholipid, as herein in the Examples.

In one embodiment, the reference cholesterol standard is the level of cholesterol determined in one or more urine samples obtained from one or more mammalian subjects of the same species that do not have any known kidney injury or disease. In one embodiment, the reference standard is the level of cholesterol determined in one or more urine samples obtained from one or more mammalian subjects of the same species that do not have any detectable kidney injury or disease. In one embodiment, the reference standard is the level of cholesterol determined in one or more urine samples obtained from one or more mammalian subjects of the same species that do not have detectable AKI. In one embodiment, the level of cholesterol determined for the reference standard is determined with the same or similar assay used to determine the presence or amount of cholesterol in the subject. The reference standard can be derived from one or more individuals of the same species as the subject, such as 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or more individuals, or any number between. In one embodiment, the reference standard is a numeric value of the cholesterol that represents a median or average level of cholesterol in a plurality of individuals of the same species of the subject that are known to not have AKI. In one embodiment, the reference standard is a value predetermined relative to the performance of the assay step on the urine sample obtained from the subject.

According to this aspect, the results of step (A) are compared with a reference standard to determine the presence of AKI in the subject. In some embodiments, a determination is made when the amount of cholesterol in a urine sample obtained from the subject is elevated compared to the reference standard. In some embodiments, the presence of AKI in the subject is indicated when the amount of cholesterol in the urine sample obtained from the subject exceeds the value of reference standard by approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, wherein percent refers to the reference standard. To illustrate this characterization, a value of 75 exceeds a reference standard value of 50 by 50% of the reference standard value (i.e., 25). In some embodiments, the presence of AKI in the subject is determined when the comparison indicates a statistically significant elevation of cholesterol in a urine sample from the subject compared to the reference standard. Statistically significant differences can be established according to standard statistical analytic methods familiar in the art. For example, as described in Example 2, t tests can be performed to compare values and ascertain statistical differences. Further, Bonferroni corrections can be used for multiple comparisons. Statistical significance can be established at any threshold accepted in the art. For example, as described below in Example 2, statistical significance was judged at a P value of <0.05 when using unpaired t test comparisons.

In another aspect, the disclosure provides an additional method for determining the presence of kidney injury in a mammalian subject. The method comprises: (A) performing an assay on the contents of a urine sample obtained from the subject to determine a relative activity level of the 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR) gene in kidney proximal tubule cells; and (B) comparing the relative activity level of the HMGCR gene in step A to a reference standard, wherein an elevated level in step A compared to the standard indicates the presence of kidney injury in the subject. In preferred embodiments, the reference standard is a value derived from one or more subjects with no known kidney injury. In some embodiments, the method comprises the step of separating a cellular component from the urine in the sample. In further embodiments, the cellular component is separated from the urine in the sample by centrifugation or filtration.

In one embodiment, the assay comprises determining the amount of 3-hydroxy-3- methyl-glutaryl-CoA reductase present in the urine sample, wherein an elevated amount of 3-hydroxy-3-methyl-glutaryl-CoA reductase in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject. In some embodiments, the 3-hydroxy-3-methyl-glutaryl-CoA reductase in the urine is associated with a cellular component of the urine sample. In some embodiments, the amount of 3- hydroxy-3-methyl-glutaryl-CoA reductase is determined by a method selected from the group consisting of mass spectrometry and Western blot employing an antibody or fragment thereof that specifically binds to 3-hydroxy-3-methyl-glutaryl-CoA reductase.

In another embodiment, the assay comprises determining the amount of mRNA corresponding to the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene, wherein an elevated amount of mRNA in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject. In some embodiments, the assay comprises using reverse transcription polymerase chain reaction or Northern blot, using primers or probes specific for the 3-hydroxy-3-methyl-glutaryl-CoA reductase mRNA.

In another embodiment, the assay comprises: (A) measuring the level of RNA Polymerase II (Pol II) bound to the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene; and (B) normalizing the level of Pol II binding in step A to the level of Pol II binding to an internal control gene, wherein an elevated normalized amount of Pol II bound to the 3-hydroxy-3-methyl-glutaryl-CoA reductase gene measured in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject. In some embodiments, the assay comprises a chromatin immunoprecipitation (ChIP) step.

In another embodiment, the assay comprises: (A) measuring the level of histone methylation in the chromatin structure corresponding to the 3-hydroxy-3-methyl-glutaryl- CoA reductase gene; and (B) normalizing the level of histone methylation in step A to the level of histone methylation in the chromatin structure corresponding to an internal control gene, wherein an elevated normalized amount of histone methylation measured in the urine of the subject compared to the reference standard indicates the presence of kidney injury in the subject. In some embodiments, the assay comprises a chromatin immunoprecipitation (ChIP) step.

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. All literature citations are expressly incorporated by reference.

EXAMPLE 1

This Example describes the discovery that urinary pellet cholesterol levels are elevated following two murine experimental acute kidney injury models, but not in response to pre-renal azotemia. Rationale:

As described above, a consequence of ischemic or toxic acute kidney injury (AKI) is an up-regulation of renal cortical 3-hydroxy-3-methyl-glutaryl-CoA reductase ("HMG CoA reductase" or "HMGCR") gene activity (Zager, R.A., et al, "Increased proximal tubular cholesterol content: Implications for cell injury and the emergence of 'acquired cytoresistance'," Kidney Int. 56: 1788-1797, 1999; Zager, R.A., "Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack," Kidney Int. 55: 193-205, 2000; Zager, R.A. and T. Kalhorn, "Changes in free and esterified cholesterol: Hallmarks of acute tubular injury and acquired cytoresistance," Am. J. Pathol. 757: 1007-1016, 2000; Zager, R.A., et al, "Cholesterol ester accumulation: An immediate consequence of acute ischemic renal injury," Kidney Int. 59: 1750-1761, 2001; Zager, R.A., "P glycoprotein-mediated cholesterol cycling: a potentially important determinant of proximal tubular cell viability," Kidney Int. 60:944-956, 2001; Zager, R.A., et al, "Renal cholesterol accumulation: A durable response following acute and subacute renal insults," Am. J. Pathology 759:743-752, 2001 ; Zager, R.A. and A.C. Johnson, "Renal cortical cholesterol accumulation: An integral component of the systemic stress response," Kidney Int. 60:2229-2310, 2001; Zager, R.A., et al, "The mevalonate pathway during acute tubular injury: selected determinants and consequences," Am. J. Pathol. 767:681-692, 2002; Zager, R.A., et al, "Proximal tubular cholesterol loading following mitochondrial, but not glycolytic blockade," Am. J. Physiol. 2#5:F1092-F1099, 2003; Naito, M. et al, "Renal ischemia-induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 174:54-62, 2009). Within 12-24 hours post- AKI induction, this culminates in an approximate 20-40% increase in renal cortical cholesterol content. Furthermore, when cultured human proximal tubular (HK-2) cells are subjected to either toxin- or ATP depletion-mediated injury, increased HMGCR activity and cholesterol accumulation result (Gaudio, K.M., et al, "Role of heat stress response in the tolerance of immature renal tubules to anoxia," Am. J. Physiol. 274:F1029-1036, 1998). Also, the sloughing of proximal tubular 'brush border' fragments and intact tubular cells into urine often occurs as the result of AKI.

The utility of detectable cholesterol in the urine as a biomarker for AKI is examined. Methods:

Urine samples were collected from mice that were subjected to two experimental models of AKI: glycerol induced rhabdomyolysis (Nath, K.A., et al, "Induction of heme oxygenase is a rapid, protective response in rhabdomyolysis in the rat," J. Clin. Invest. 90:267-270, 1992; Nath, K.A., et al, "Renal response to repetitive exposure to heme proteins: chronic injury induced by an acute insult," Kidney Int. 57:2423-2433, 2000), or maleate (ATP depletion induced ARF (Kellerman, P.S., "Exogenous adenosine triphosphate (ATP) preserves proximal tubule microfilament structure and function in vivo in a maleic acid model of ATP depletion," J. Clin. Invest. 92: 1940-1949, 1993; Zager, R.A., et al, "Maleate nephrotoxicity: Mechanisms of injury and correlates with ischemic / hypoxic tubular cell death," Am. J. Physiol. 294 V\ %1-\91 , 2008). Specifically, 14 male CD-I mice were subjected to the glycerol model of AKI, and 13 male CD-I mice were subjected to the maleate model of AKI. Either 3 hours or 24 hours later (approximately half at each time point), the mice were anesthetized with a 50 mg/Kg intraperitoneal administration of pentobarbital, and urine was obtained by gentle pressure on the exposed urinary bladder. Urine extraction was followed by blood sample collection from the inferior vena cava for blood urea nitrogen (BUN) analysis.

As a model of pre-renal azotemia (PRA), 4 mice were subjected to surgical stress by performing a midline abdominal laparotomy with abdominal exploration, simulating sham bilateral renal pedicle occlusion (Naito, M. et al, "Renal ischemia- induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 174:54-62, 2009), followed 30 minutes later by an intraperitoneal injection of 10 mg/Kg indomethicin. At the 24 hour time point, urine and blood samples were obtained as described above.

Urine samples were centrifuged at 12,000 rpm a to produce a pellet of the solid debris and cellular components of the urine. It will be understood that any centrifugation speed can be employed that produces a pellet. The liquid urine was siphoned off. Alternative methods to obtain and isolate the solid debris and/or cellular components of the urine sample include filtering the urine.

Lipid content within the centrifuged urine pellets was extracted in chloroform:methanol. Lipid fractions were assayed for cholesterol content using the Amplex Red assay (Stirewalt, D.L., et al, "Mevastatin can increase toxicity in primary AMLs exposed to standard therapeutic agents, but statin efficacy is not simply associated with ras hotspot mutations or overexpression," Leuk. Res. 27: 133-45, 2003; Li, H.Y., et al, "Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses," Blood 707:3628-34, 2003). As described in Li, H.Y., et al., 2003, the Amplex Red assay (Molecular Probes, Eugene, Oregon) is a fluorometric technique that relies on the oxidation of cholesterol into a ketone and hydrogen peroxide. The hydrogen peroxide reacts stoichiometrically with the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) in the presence of horseradish peroxidase to form the fluorescent compound resorufin. To perform this assay, the obtained pellets or cell associated fraction of the urine were resuspended in the Amplex Red reaction buffer (0.1 M potassium phosphate, 0.05 M NaCl, 5 mM cholic acid, 0.1% Triton X-100, pH 7.4) at a concentration of 2000/μΕ and vortexed. Fifty μϊ_^ aliquots of each of the resulting lysates were pipetted into a 96-well tissue culture plate (e.g., Falcon/Becton Dickinson, Franklin Lakes, New Jersey), a 50 aliquot of an Amplex Red working solution (300 μΜ Amplex Red reagent, 2 U/mL horseradish peroxidase, 2 U/mL cholesterol oxidase, and 0.2 U/mL cholesterol esterase) was added to each well. The plate was then incubated for 90 minutes at 37°C, protected from light. Fluorescence was subsequently measured on a CytoFluor II fluorescent plate reader (e.g., PerSeptive Biosystems, Framingham, Massachusetts) using an excitation wavelength of 530 nm and an emission wavelength of 590 nm. A cholesterol standard curve can be determined for each plate using a cholesterol calibrator (Sigma) diluted at various concentrations in Amplex Red reaction buffer in lieu of cell lysates.

Alternative approaches for assaying cholesterol levels include the use of gas chromatography, as described in Li, H.Y., et al, "Cholesterol-modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses," Blood 707:3628-34, 2003. Briefly, lipids obtained from a urine sample can be extracted in chloroform- methanol (1 :2) and dried under nitrogen. After reconstitution in hexane, samples are transferred to glass tubes containing an internal standard solution (stigmasterol, 100 μg/mL in ethyl acetate [EtOAc]; Sigma), dried under nitrogen, and reconstituted in 100 μϊ_^ bis-(trimethylsilyl)trifluoroacetamide (BSTFA; Sigma) (25% vol/vol EtOAc). These samples are then sealed in an injection vial and heated for 1 hour at 60°C. After completion of BSTFA derivatization, samples are applied to a chromatograph, e.g., Hewlett Packard 5890 Series II gas chromatograph, fitted with a flame ionization detector and a 30 m x 0.32 mm DB-5 (0.25 μιη) column (e.g., J&W Scientific, Folsom, California). The initial temperature (100°C) is maintained for 3 minutes, after which it is increased by 40°C per minute to 290°C and thereafter by 5°C per minute to 300°C for 5 minutes. Cholesterol ethers are quantitated after elution from the gas chromatograph at 12.5 minutes.

To obtain a more precise indication of cholesterol loading in the proximal tubule cells, cholesterol levels from the urinary pellets were normalized by total pellet phospholipid phosphate (PLP) content and expressed as cholesterol/phosphate ratios (Zager, R.A., et al, "Increased proximal tubular cholesterol content: Implications for cell injury and the emergence of 'acquired cytoresistance'," Kidney Int. 56: 1788-1797, 1999; Zager, R.A., "Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack," Kidney Int. 55: 193-205, 2000; Zager, R.A. and T. Kalhorn, "Changes in free and esterified cholesterol: Hallmarks of acute tubular injury and acquired cytoresistance," Am. J. Pathol. 757: 1007-1016, 2000). Phospholipid phosphate (PLP) content can be assayed according to acceptable and known methods in the art; for example, as described in Van Veldhoven, P.P., and G.P Mannaerts, "Inorganic and Organic Phosphate Measurements in the Nanomolar Range," Anal Biochem 161:45- 48, 1987. The PLP content served as an internal standard in each sample assayed to normalize for the overall volume of cellular debris, specifically, cellular membranes found in the urine. Alternatives to PLP for use as internal standards include other phospholipids, such as phosphatylcholine.

Blood urea nitrogen was assayed for the urine donors to confirm the occurrence of azotemia in the experimental AKI and pre-renal kidney azotemia models using standard, well-known techniques based on an autoanalyzer instrument. For the glycerol and maleate AKI models, the BUN analysis results were assayed from blood sample obtained at the 24 hour samples.

All animal experiments were approved by the institution's Institutional Animal Care and Use Committee (IACUC).

Results:

As shown in FIGURE 1, significant increases in cholesterol / phospholipid phosphate ratios were observed as early as 3 hours post maleate or glycerol injection compared to values obtained from 14 normal mice (* = p<0.01). These significant elevations in urinary pellet cholesterol content were also observed at the 24 hour time point. In contrast to the glycerol and maleate models of AKI, the pre-renal azotemia (PRA) model failed to induce any increase in cholesterol levels (FIGURE 1).

To establish the presence of azotemia in the structural AKI and pre-renal azotemia experimental models, a blood urea nitrogen (BUN) analysis was conducted in parallel to the urinalysis. As shown in FIGURE 2, the pre-renal azotemia (PRA) and both structural AKI models demonstrated significantly elevated urea levels in the blood (* = p<0.01). It was noted that while the PRA model induced substantial azotemia (BUN = 64±4 mg/dl), no increase in urinary pellet cholesterol levels resulted (described above; FIGURE 1).

Conclusion:

These results demonstrate that elevated cholesterol levels in urinary pellets correlate with the presence of acute kidney injury in two different murine models. Additionally, the elevated cholesterol levels in urinary pellets were capable of differentiating between structural acute kidney injury (i.e., tubular necrosis) and functional acute kidney failure (i.e., pre-renal azotemia), notwithstanding a demonstrated azotemia in all experimental models.

EXAMPLE 2

This Example describes the discovery that urinary pellet cholesterol levels are elevated in human patients with acute kidney injury (AKI), but not in critically ill intensive care unit (ICU) patients without AKI or patients with chronic kidney disease (CKD).

Rationale:

As described in Example 1, using multiple murine models, the presence of elevated cholesterol levels in urine pellets was correlated with structural AKI but not with pre-renal azotemia. Therefore, urine samples obtained from various human patients suffering from AKI or other diseases were similarly assessed to further ascertain the role of urinary pellet cholesterol levels as a biomarker for AKI.

Methods:

Banked, frozen urine samples were obtained from three groups of patients: (1) critically ill patients with AKI ("AKI+"; n=14); (2) critically ill patients without AKI ("AKI-"; n=15); and (3) patients with chronic kidney disease ("CKD"; n=15). Briefly, the AKI+ and AKI- individuals were subsets of patients who were enrolled in a large, Institutional Review Board (IRB) approved, prospective observational study of critically ill adults treated in multiple intensive care (ICU) units at Vanderbilt University (Ware, L.B., et al, "Renal cortical albumin gene induction and urinary albumin excretion in responses to acute kidney injury," Am. J. Physiol, (in press) 2010 Dec 8. PMID: 21 147844; Siew, E.D., et al, "Urine neutrophil gelatinase-associated lipocalin moderately predicts acute kidney injury in critically ill adults," J. Am. Soc. Nephrol. 20: 1823-32, 2009; Munshi, R., et al, "MCP-1 gene activation marks acute kidney injury. J. Am. Soc. Nephrol. 22: 165-175, 2011). AKI+ was defined as a > 50% (or >26.5 μιηοΐ/ΐ) increase in serum creatinine concentrations from baseline. Creatinine levels can be assayed according to standard methods known in the art. For instance, a standard creatinine assay kit, available from Abeam, Cambridge, MA, incorporates creatininase, which converts creatinine to creatine. Subsequently, the creatine is converted to sarcosine, which is specifically oxidized to produce a product which reacts with a probe to generate red color (detected by spectrophotometry at OD = 570 nm) and fluorescence (Ex/Em = 538/587 nm). All AKI+ patients had a presumptive diagnosis of "acute tubular necrosis," as judged by consulting nephrologists.

The AKI- group was comprised of 15 critically ill, ICU-hospitalized patients who had comparable overall illness severity as the AKI+ group, as determined by APACHE II scores (Knaus, W.A., et al, "APACHE II: A Severity of Disease Classification System," Crit Care Med:.13 %-29, 1985) but who did not have AKI. The AKI+ and AKI- populations were matched for age, race, gender, and sepsis status. Demographic information for these patients and the specifics of urine sample collection have been previously described (Ware, L.B., et al, "Renal cortical albumin gene induction and urinary albumin excretion in responses to acute kidney injury," Am. J. Physiol. 300:F628- F638, 201 1).

The CKD patient population consisted of six individuals with diabetic nephropathy, and nine individuals with non-diabetic CKD. These patients were enrolled in a study of CKD as part of a Seattle kidney study; Kidney Research Institute, Seattle, Washington. Subjects were eligible for the present study if they had a Modification of Diet in Renal Disease estimated GFR <60 ml/min/ 1.73 m², were not receiving dialysis, and were at least 18 years of age. They were selected for study based on serum creatinine levels, i.e., they were chosen to match the approximate degree of serum creatinine elevations that were observed in the AKI+ group at the time of study (AKI+, 2.65±0.31 mg/dl; AKI-, 0.84±0.1 mg/dl; CKD, 2.2±0.08 mg/dl; SEM). In addition to these 44 patient urine samples (14 AKI+, 15 AKI-; 15 CKD), additional urine samples were obtained from 15 healthy volunteers.

Table 1 - Patient characteristics of the ICU patient cohorts reported in this

Clinical characteristics of the ICU patients with and without AKI at the time of enrollment. The serum creatinine concentrations at enrollment (and AKIN scores) were statistically different between the two groups. The other factors did not manifest statistical significance (overlapping 95% confidence intervals). ICU, intensive care unit; APACHE II, Acute Physiology and Chronic Health Evaluation II score; AKIN, Modified AKIN score based on serum creatinine measurements (Ricci, Z., et al, "Classification and staging of acute kidney injury: beyond the RIFLE and AKIN criteria," Nature Reviews Nephrology 7:201-208, 2011). Continuous variables are given as means ± 1 SD.

After thawing the samples at room temperature, the urine samples were centrifuged (5000 rpm x 5 min). The resulting pellets were subject to lipid extraction in chloroform: methanol (Zager, R.A., et al, "Increased proximal tubular cholesterol content: Implications for cell injury and the emergence of 'acquired cytoresistance'," Kidney Int. 56: 1788-1797, 1999; Zager, R.A., "Plasma membrane cholesterol: a critical determinant of cellular energetics and tubular resistance to attack," Kidney Int. 55: 193- 205, 2000; Zager, R.A. and T. Kalhorn, "Changes in free and esterified cholesterol: Hallmarks of acute tubular injury and acquired cytoresistance," Am. J. Pathol. 757: 1007- 1016, 2000). The lipid fractions were assayed for cholesterol by the Amplex Red method (Stirewalt, D.L., et al., "Mevastatin can increase toxicity in primary AMLs exposed to standard therapeutic agents, but statin efficacy is not simply associated with ras hotspot mutations or overexpression," Leuk. Res. 27: 133-45, 2003; Li, H.Y., et al, "Cholesterol- modulating agents kill acute myeloid leukemia cells and sensitize them to therapeutics by blocking adaptive cholesterol responses," Blood 707:3628-34, 2003), with the results being factored by PLP as an internal standard, as described above in Example 1. Statistical assessments were conducted using unpaired Student's t-test with Bonferroni corrections for multiple comparisons. Values were expressed as means ± 1 SEM. Statistical significance was judged at a P value of <0.05.

Results:

Clinical characteristics of the patients who formed the ICU/AKI- and ICU/AKI+ cohorts are presented in Table 1. Other than significantly higher serum creatinine levels and AKIN scores at enrollment for the AKI+ versus AKI- group, no other statistically significant differences existed between these two groups (overlapping 95% confidence intervals). Of particular note, the overall illness severity (as assessed by APACHE II scores) and the frequency of sepsis syndrome for the two groups were highly comparable.

As shown in FIGURE 3, urinary pellet cholesterol levels for the chronic kidney disease (CKD) and ICU/AKI- patients were virtually identical to those of the normal volunteers, as denoted with "NS". In contrast, the ICU/AKI+ patients manifested a near-doubling of urine pellet cholesterol levels compared to the normal controls (p<0.001) or the ICU/AKI- patients (p<0.005). It was noted that the cholesterol levels were factored by the amount of PLP as an internal control for the cellular lipid present in the sample. Thus, the levels of cholesterol represented in FIGURE 3 reflect the cholesterol levels present in the cells recovered in the urine, relative to other lipid constituents of the cells. These results support the mouse urinary cholesterol assessments, as described above in Example 1, indicating that urinary pellet cholesterol levels are useful as an AKI biomarker.

The urine creatinine concentrations for the AKI- and AKI+ groups did not statistically differ (100 ± 16 mg/dl and 137 ± 26 mg/dl, respectively, P = 0.23). There was no significant relationship between the urine creatinine concentrations and the pellet cholesterol/phospholipids ratios in the AKI+ group (r = 0.14). This is consistent with the fact that creatinine excretion reflects GFR, whereas pellet cholesterol levels reflect tubular cell cholesterol loading and tubular cell membrane sloughing. Of note, however, the urine pellet cholesterol levels did significantly correlate with the serum creatinine concentrations (r = 0.5; P < 0.01), suggesting that it could potentially be a reflection of tubular injury severity.

As shown in FIGURE 3, the CKD patients did not manifest any increase in urinary pellet cholesterol content compared with control urine pellet values. Of note, statin therapy, which is common in patients with CKD, could theoretically be a confounding variable in interpreting urinary pellet cholesterol levels. However, the inventor has previously demonstrated that statins do not alter renal cortical cholesterol content (Zager, R.A., et al, "The mevalonate pathway during acute tubular injury: selected determinants and consequences," Am. J. Pathol. 767:681-692, 2002). Here, eight of the 15 CKD patients were receiving statin treatment, and yet their urinary pellet cholesterol levels were slightly, but not statistically, higher than values observed in non- statin treated CKD patients (206 ± 40 versus 128 ± 34, respectively; P = 0.17), not lower. Thus, it appears most unlikely that statin treatment would negate the potential utility of pellet cholesterol levels as a useful AKI biomarker.

Conclusion:

These results indicate that elevated cholesterol levels in human urinary pellets correlate with AKI. Specifically, the elevated cholesterol levels in urinary pellets permit differentiation between human AKI patients and either patients without AKI or patients diagnosed with chronic kidney disease. These results are consistent with results contained in murine models, as described in Example 1, and demonstrate that elevated cholesterol levels in human urinary pellets are useful biomarkers for AKI.

EXAMPLE 3

This Example describes the discovery that patients with acute kidney injury (AKI) exhibit increased levels of RNA polymerase II (Pol II) and of histone H3 lysine 4 trimethylation (H3K4m3) at exon 1 of HMG CoA reductase (HMGCR) gene in urine chromatin samples.

Rationale:

Experimental acute kidney injury (AKI) is known to up-regulate expression of the HMG CoA reductase (HMGCR) gene, resulting in proximal tubule cholesterol loading. AKI also causes sloughing of proximal tubular cell debris into tubular lumina. The inventors recently demonstrated that in vivo gene activation can be non-invasively monitored in patients by measuring RNA polymerase II (Pol II) binding to target gene fragments in urinary pellets (Ware, L.B., et al., "Renal cortical albumin gene induction and urinary albumin excretion in responses to acute kidney injury," Am. J. Physiol. 300:F628-F638, 2011 ; Munshi, R., et al, "MCP-1 gene activation marks acute kidney injury. J. Am. Soc. Nephrol. 22: 165-175, 2011). Three principles underlie the approach: first, Pol II is the enzyme that drives transcription (Kornberg, R.D., "The molecular basis of eukaryotic transcription," Proc. Natl Acad. Sci. USA 704: 12955-12961, 2007); second, degrees of Pol II binding to target genes is an indirect gauge of gene transcription (Kornberg, R.D., "The molecular basis of eukaryotic transcription," Proc. Natl Acad. Sci. USA 704: 12955-12961, 2007; Nikolov, D.B. and S.K. Burley, "RNA polymerase II transcription initiation: A structural view," Proc. Natl Acad. Sci. USA 94: 15-22, 1997); and third, chromatin immunoprecipitation assay (ChIP) can be successfully deployed for measuring Pol II - gene binding, using sheared, formalin fixed, urinary chromatin samples (Ware, L.B., et al, "Renal cortical albumin gene induction and urinary albumin excretion in responses to acute kidney injury," Am. J. Physiol. 300:F628-F638, 201 1; Munshi, R., et al, "MCP-1 gene activation marks acute kidney injury. J. Am. Soc. Nephrol. 22: 165-175, 201 1). Further, the inventors previously demonstrated that one potential reason for increased HMGCR gene activity in response to AKI is the induction of "gene activating" histone modifications at the HMGCR gene (Naito, M. et al, "Renal ischemia-induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 774:54-62, 2009). This allows for enhanced Pol II - gene binding, and hence, increased transcription. One notable example of this altered histone profile is an increase in the amount of trimethylated histone H3 at the lysine 4 position, yielding H3K4m3 (Naito, M. et al, "Renal ischemia-induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 774:54-62, 2009).

Based on the foregoing, and the demonstrated correlation of increased cholesterol levels in urinary pellets with AKI, the up-regulation of the HMGCR gene was investigated for a correlation with the occurrence of AKI. Specifically, levels of Pol II binding to exon 1 of the HMGCR gene were assayed in the context of ICU/AKI+, ICU/AKI-, and normal control patients. Similarly, the trimethylation levels of histone H3 at lysine 4 position (yielding H3K4m3) of the chromatin corresponding to exon 1 of the HMGCR gene were assayed in the context of ICU / AKI +, ICU / AKI-, and normal control patients.

Methods:

Using the approach previously described (Naito, M. et al, "Renal ischemia- induced cholesterol loading: Transcription factor recruitment and chromatin remodeling along the HMG CoA reductase gene," Am. J. Pathol. 174:54-62, 2009; Munshi, R., et al, "MCP-1 gene activation marks acute kidney injury. J. Am. Soc. Nephrol. 22: 165-175, 201 1, both expressly incorporated herein by reference), the degree of Pol II binding to exon 1 of the HMGCR gene was assessed in urinary pellets obtained from the AKI- and AKI+ human patients and from normal volunteers. Specifically, urine pellets obtained from the above centrifugations were fixed in formalin. The pellets were resuspended in 1 ml of IP buffer (containing the following inhibitors: 0.5 mmol/L dithiothreitol, 10 μg/ml leupeptin, 0.5 mmol/L phenylmethyl sulfonyl fluoride, 30 mmol/L p-nitrophenyl phosphate, 10 mmol/L NaF, 0.1 mmol/L a₃V04, 0.1 mmol/L a₂Mo04, and

10 mmol/L β-glycerophosphate) and chromatin was sheared using six rounds of sonication (power 5, 15 seconds, on ice). The suspension was cleared by centrifugation at 12,000 x g (10 minutes at 4°C), and the supernatant, representing sheared chromatin, was aliquoted and stored at -80°C.

Sheared chromatin were aliquoted in wells of 96-well polystyrene high-binding capacity microplates (No. 9018; Corning, Corning, NY). The wells were washed once with 200 μΐ of PBS per well, and were incubated overnight with 0.2 μg of protein A (No. P7837; Sigma, St. Louis, Missouri) in 100 μΐ of PBS per well. After washing (200 μΐ of PBS per well), well walls were blocked with 200 μΐ of blocking buffer (15 to 60 minutes, 22°C). The wells were cleared and 0.25 μg of monoclonal antibody specific for RNA Polymerase II ("Pol II CTD 4h8"; No. GTX25408, Gene Tex, Irvine, California) were added with 100 μΐ of blocking buffer per well (60 minutes, 22°C). Chromatin samples (5.0-μ1 chromatin preparations/ 100 μΐ of blocking buffer) were added (100 μΐ/well) and plates were floated in an ultrasonic water bath (60 minutes, 4°C) to accelerate protein-antibody binding. The wells were washed three times with 200 μΐ of IP buffer and one time with 200 μΐ of TE buffer. Wells were incubated with 100 μΐ of elution buffer (15 minutes at 55°C, followed by 15 minutes at 95°C). Total DNA (input) was isolated using the same plate and concurrently with immunoprecipitated DNA by suspending 5.0 μΐ of chromatin in 100 μΐ of elution buffer (15 minutes at 55°C, followed by 15 minutes at 95°C). DNA samples were stored (-20°C).

For the above protocol, the following buffers were used: phosphate-buffered saline (PBS): 137 mmol/L NaCl, 10 mmol/L Na phosphate, 2.7 mmol/L KC1, pH 7.4; TE buffer: 10 mmol/L Tris, 1 mmol/L ethylene diamine tetraacetic acid, pH 7.0; immunoprecipitation (IP) buffer: 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, 5 mmol/L ethylene diamine tetraacetic acid, NP-40 (0.5% v/v), Triton X-100 (1.0% v/v); blocking buffer: 5% bovine serum albumin, 100 μg/ml sheared salmon sperm DNA in IP buffer; elution buffer: 25 mmol/L Tris base, 1 mmol/L ethylene diamine tetraacetic acid, pH 9.8, 200 μg/ml proteinase K (20 mg/ml stock, stored at -20°C).

DNA samples obtained from chromatin immunoprecipitation were assayed for the HMGCR exon 1 and β-actin by quantitative PCR. The reaction mixture contained 2.5 μΐ of optimized PCR buffer with dye, DNA polymerase, dNTPs with dUTP, and buffer (2* SYBR® Green PCR master mix (SensiMix, Quantace)), 2.3 μΐ of DNA template, and 0.2 μΐ of primers (10 μιηοΙ/L) in 5-μ1 final volume in a 384-well optical reaction plate (Applied Biosystems, Foster City, California). Amplification (three step, 40 cycles), data acquisition, and analyses were done using the 7900HT real-time PCR system and SDS Enterprise Database (Applied Biosystems). Primers for the reactions are listed below in Table 2. All PCR reactions were run in triplicate. PCR calibration curves were generated for each primer pair from a dilution series of total mouse sheared genomic DNA. The PCR primer efficiency curve was fit to cycle threshold (Ct) versus log_e (genomic DNA dilutions) using an r-squared best fit. DNA concentration values for each ChIP and input DNA samples were calculated from their respective average Ct values. Final results were expressed as percent input DNA. All results were factored by Pol II binding at exon 1 of the β-actin 'housekeeping' gene as an internal control.

Table 2 - Primers used in qPCR analysis to assess Pol II binding.

Further, ChIP analysis was performed on urinary pellet samples from AKI- and AKI+ patients and from normal volunteers to assess trimethylation levels of histone H3 at lysine 4 position (yielding H3K4m3) corresponding to exon 1 of the HMGCR gene. Briefly, ChIP analysis was performed as described above in regard to the assessment of Pol II binding. The chromatin was immunoprecipitated using 0.5 μg rabbit polyclonal antibody, specific for H3K4m3 (No. Ab8580, Abeam, Cambridge, MA).

It is noted that the present invention is not limited to the above-described approaches to assess HMGCR activity from the urine. Additional embodiments are contemplated in which relative levels of HMGCR gene product are assayed. For instance, the presence of HMG CoA reductase protein in the urine pellets can be assayed directly using mass spectrometry or Western blot staining employing antibodies, or fragments thereof, that are specific for the protein. In a further embodiment, mRNA corresponding to the HMGCR gene can be quantified from urine samples using well-known techniques such as quantitative reverse transcription PCR ("RT PCR"). Exemplary primers for the RT PCR assays are listed in Table 2 above, which are specific for Exon 1 of the MHGCR gene. Additional primers employed in an RT PCR assay can be synthesized to specifically prime the reaction for the HMGCR mRNA template, which is set forth below in Table 3 and is listed in the GenBank database as accession number NM_000859.2 (SEQ ID NO: l).

Results:

As shown in FIGURE 4, a marked increase in Pol II binding was observed in the AKI+ patients, compared to controls (p<0.001). The AKI- patients also had a significant increase in Pol II - HMGCR gene binding compared to values seen in control samples (p<0.025). However, the degree of elevation of Pol II binding in AKI- patients was only ~l/3 as great as that which was observed in the AKI+ group (p<0.025). Thus, this data is consistent with the concept that AKI induces HMGCR gene activation, culminating in increased renal tubular, and ultimately, urinary pellet cholesterol levels. The reason for the modest increase in Pol II-HMGCR binding in the AKI- patients remains unknown. Without being bound to any particular theory, it is possible that the intermediate level of Pol II-HMGCR binding in the AKI- patients stems from the fact that many critically ill patients sustain sub-clinical renal injury (23, 25) that is insufficient in degree to induce either clinically overt AKI (i.e., as denoted by azotemia), or renal tubular cholesterol loading. As shown in FIGURE 5, a doubling of the H3K4m3 levels at exon 1 of the HMGCR gene was observed in the AKI+ patients, compared to either the AKI- cohort or the normal volunteers (p<0.03). There was no significant difference between the AKI- and normal control patients. This data is consistent with the concept that AKI induces HMGCR gene activation, as evidenced by histone modification, culminating in increased renal tubular, and ultimately, urinary pellet cholesterol levels.

Conclusion:

These Pol II and H3K4m3 results support the concept, developed in rodents, that AKI activates the HMGCR gene. This data also demonstrates that the urinary ChIP assay has utility for detecting AKI in patients via the detection of the transcriptional activation of the HMGCR gene. This supports the utility of these non- intrusive assays for detecting AKI in patients, and monitoring in vivo chromatin remodeling and gene activation in AKI patients.

Table 3 - SEQ ID NO: l; Genbank Accession gi| 196049378|reflNM_000859.2| Homo sapiens 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), transcript variant I, mR A CTCTTATTGGTCGAAGGCTCGTCCAGCTCCGAGCGTGCGTAAGGTGAG GGCTCCTTCCGCTCCGCGACTGCGTTAACTGGAGCCAGGCTGAGCGTCGGCG CCGGGGTTCGGTGGCCTCTAGTGAGATCTGGAGGATCCAAGGATTCTGTAGC TACAATGTTGTCAAGACTTTTTCGAATGCATGGCCTCTTTGTGGCCTCCCATCC CTGGGAAGTCATAGTGGGGACAGTGACACTGACCATCTGCATGATGTCCATG AACATGTTTACTGGTAACAATAAGATCTGTGGTTGGAATTATGAATGTCCAAA GTTTGAAGAGGATGTTTTGAGCAGTGACATTATAATTCTGACAATAACACGAT GCATAGCCATCCTGTATATTTACTTCCAGTTCCAGAATTTACGTCAACTTGGA TCAAAATATATTTTGGGTATTGCTGGCCTTTTCACAATTTTCTCAAGTTTTGTA TTCAGTACAGTTGTCATTCACTTCTTAGACAAAGAATTGACAGGCTTGAATGA AGCTTTGCCCTTTTTCCTACTTTTGATTGACCTTTCCAGAGCAAGCACATTAGC AAAGTTTGCCCTCAGTTCCAACTCACAGGATGAAGTAAGGGAAAATATTGCT CGTGGAATGGCAATTTTAGGTCCTACGTTTACCCTCGATGCTCTTGTTGAATG TCTTGTGATTGGAGTTGGTACCATGTCAGGGGTACGTCAGCTTGAAATTATGT GCTGCTTTGGCTGCATGTCAGTTCTTGCCAACTACTTCGTGTTCATGACTTTCT TCCCAGCTTGTGTGTCCTTGGTATTAGAGCTTTCTCGGGAAAGCCGCGAGGGT CGTCCAATTTGGCAGCTCAGCCATTTTGCCCGAGTTTTAGAAGAAGAAGAAA ATAAGCCGAATCCTGTAACTCAGAGGGTCAAGATGATTATGTCTCTAGGCTTG GTTCTTGTTCATGCTCACAGTCGCTGGATAGCTGATCCTTCTCCTCAAAACAG TACAGCAGATACTTCTAAGGTTTCATTAGGACTGGATGAAAATGTGTCCAAG AGAATTGAACCAAGTGTTTCCCTCTGGCAGTTTTATCTCTCTAAAATGATCAG CATGGATATTGAACAAGTTATTACCCTAAGTTTAGCTCTCCTTCTGGCTGTCA AGTACATCTTCTTTGAACAAACAGAGACAGAATCTACACTCTCATTAAAAAA CCCTATCACATCTCCTGTAGTGACACAAAAGAAAGTCCCAGACAATTGTTGTA GACGTGAACCTATGCTGGTCAGAAATAACCAGAAATGTGATTCAGTAGAGGA AGAGACAGGGATAAACCGAGAAAGAAAAGTTGAGGTTATAAAACCCTTAGT GGCTGAAACAGATACCCCAAACAGAGCTACATTTGTGGTTGGTAACTCCTCCT TACTCGATACTTCATCAGTACTGGTGACACAGGAACCTGAAATTGAACTTCCC AGGGAACCTCGGCCTAATGAAGAATGTCTACAGATACTTGGGAATGCAGAGA AAGGTGCAAAATTCCTTAGTGATGCTGAGATCATCCAGTTAGTCAATGCTAA GCATATCCCAGCCTACAAGTTGGAAACTCTGATGGAAACTCATGAGCGTGGT GTATCTATTCGCCGACAGTTACTTTCCAAGAAGCTTTCAGAACCTTCTTCTCTC CAGTACCTACCTTACAGGGATTATAATTACTCCTTGGTGATGGGAGCTTGTTG TGAGAATGTTATTGGATATATGCCCATCCCTGTTGGAGTGGCAGGACCCCTTT GCTTAGATGAAAAAGAATTTCAGGTTCCAATGGCAACAACAGAAGGTTGTCT TGTGGCCAGCACCAATAGAGGCTGCAGAGCAATAGGTCTTGGTGGAGGTGCC AGCAGCCGAGTCCTTGCAGATGGGATGACTCGTGGCCCAGTTGTGCGTCTTCC ACGTGCTTGTGACTCTGCAGAAGTGAAAGCCTGGCTCGAAACATCTGAAGGG TTCGCAGTGATAAAGGAGGCATTTGACAGCACTAGCAGATTTGCACGTCTAC AGAAACTTCATACAAGTATAGCTGGACGCAACCTTTATATCCGTTTCCAGTCC AGGTCAGGGGATGCCATGGGGATGAACATGATTTCAAAGGGTACAGAGAAA GCACTTTCAAAACTTCACGAGTATTTCCCTGAAATGCAGATTCTAGCCGTTAG TGGTAACTATTGTACTGACAAGAAACCTGCTGCTATAAATTGGATAGAGGGA AGAGGAAAATCTGTTGTTTGTGAAGCTGTCATTCCAGCCAAGGTTGTCAGAG AAGTATTAAAGACTACCACAGAGGCTATGATTGAGGTCAACATTAACAAGAA TTTAGTGGGCTCTGCCATGGCTGGGAGCATAGGAGGCTACAACGCCCATGCA GCAAACATTGTCACCGCCATCTACATTGCCTGTGGACAGGATGCAGCACAGA ATGTTGGTAGTTCAAACTGTATTACTTTAATGGAAGCAAGTGGTCCCACAAAT GAAGATTTATATATCAGCTGCACCATGCCATCTATAGAGATAGGAACGGTGG GTGGTGGGACCAACCTACTACCTCAGCAAGCCTGTTTGCAGATGCTAGGTGTT CAAGGAGCATGCAAAGATAATCCTGGGGAAAATGCCCGGCAGCTTGCCCGAA TTGTGTGTGGGACCGTAATGGCTGGGGAATTGTCACTTATGGCAGCATTGGCA GCAGGACATCTTGTCAAAAGTCACATGATTCACAACAGGTCGAAGATCAATT TACAAGACCTCCAAGGAGCTTGCACCAAGAAGACAGCCTGAATAGCCCGACA GTTCTGAACTGGAACATGGGCATTGGGTTCTAAAGGACTAACATAAAATCTG TGAATTAAAAAAGCTCAATGCATTGTCTTGTGGAGGATGAATAGATGTGATC ACTGAGACAGCCACTTGGTTTTTGGCTCTTTCAGAGAGGTCTCAGGTTCTTTC CATGCAGACTCCTCAGATCTGAACACAGTTTAGTGCTTTACATGCTGTGCTCT TTGAAGAGATTTCAACAAGAATATTGTATGTTAAAGCATCAGAGATGGTAAT CTACAGCTCACCTCTGAAGGCAAATATAAGCTGGGAAAAAAGTTTTGATGAA ATTCTTGAAGTTCATGGTGATCAGTGCAATTGACCTTCTCCCTCACTCCTGCC AGTTGAAAATGGATTTTTAAATTATACTGTAGCTGATGAAACTCCTGATTTTG TAGTTAATTTATTAAGTCTGGGATGTAGAACTTCAAGAAGTAAGAGCTAAGTT CTAAGTTCATGTTTGTAAATTAATACTTCATTTGGTGCTGGTCTATTTTGATTT TGGGGGGTAATCAGCATTATTCTTCAGAAGGGGACCTGTTTTCTTCAAGGGAA GAAACACTCTTATTCCCAAACTACAGAATAATGTGTTAAACATGCTAAATAGT TCTATCAGGAAAACAAATCACTGTATTTATCTCCGCAGGCTATTTGTTCAGAG AGGCCTTTTGTTTAAATATAAATGTTTAAATATAAATGTTTGTCTGGATTGGC TATAACATGTCTTTCAGCATTAGGCTTTTAAGAAACACAGGGTTTTGTATTCT TTACTAAAGATATCAGAGCTCTTAATGTTGCTTAGATGAGGGTGACTGTCAAG TACAAGCAAGACTGGGACCTTAGAAATCATTGTAGAAACACAGTTTTGAAAG AAAAATACCATGTCTCTAAGCCAACTTTAATTGCTTAAAAGACATTTTTATTT AGTTGAAAAATCTAGTTTTTTTTGTAAACTGTATCAAATCTGTATATGTTGTA ATAAAACTTATGCTAGTTTATTGGAAGTGTTCAAGAAATAAAAATCAACTTGT GTACTGATAAAATACTCTAGCCTGGGCCAGAGAAGATAATGTTCTTTAATGTT GTCCAGGAAACCCTGGCTTGCTTGCCGAGCCTAATGAAAGGGAAAGTCAGCT TTCAGAGCCAGTGAAGGAGCCACGTGAATGGCCCTAGAACTGTGCCTAGTTC CTGTGGCCAGGAGGTTGGTGACTGAAACATTCACACAGGGCTCTTTGATGGA CCCACGAACGCTCTTAGCTTTCTCAGGGGGTCAGCAGAGTTATTGAATCTTAA TTTTTTTTAATGTACAAGTTTTGTATAAATAATAAAGAACTCCTTATTTTGTAT TACATCTAATGCTTCAAGTGTTGCTCTTGGAAAGCTGATGATGTCTCTTGTAG AAGATGGACTCTGAAAAACATTCCAGGAAACCATGGCAGCATGGAGAGCCTC TTAGTGATTGTGTCTGCATTGTTATTGTGGAAGATTTACCTTTTCTGTTGTACG TAAAGCTTAAATTGCTTTTGTTGTGACTTTTTAGCCAGTGACTTTTTCTGAGCT TTTCATGGAAGTGGCAGTGAAAAATATGTTGAGTGTTCATTTTAGTGACTGTA ATTAATATCTTGCTGGATTAATGTTTTGTACAATTACTAAATTGTATACATTTT GTTATAGAATACTTTTTTCTAGTTTCAGTAAATAATGAAAAGGAAGTTAATAC CAAAAAAAAA While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

Claims

CLAIMS The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for determining the presence of kidney injury in a mammalian subject, the method comprising:

(a) performing an assay to determine the presence or amount of cholesterol in a urine sample obtained from the subject; and

(b) comparing the results of step (a) with a cholesterol reference standard, wherein the presence or elevated level of cholesterol in step (a) as compared to the reference standard indicates the presence of kidney injury in the subject.

2. The method of Claim 1, wherein the assay of step (a) determines the amount of cholesterol associated with a cellular component of the urine sample.

3. The method of Claim 2, wherein the cellular component comprises cell plasma membrane or cell organelle membranes.

4. The method of Claim 2, wherein the cellular component comprises components of proximal tubular cells, including intact cells.

5. The method of Claim 2, wherein the amount of cholesterol in the urine samples of step (a) is normalized using an internal control substance present in the cellular component of the urine samples.

6. The method of Claim 5, wherein the internal control substance comprises a phospholipid.

7. The method of Claim 2, wherein the assay comprises separating the cellular component from the urine.

8. The method of Claim 7, wherein the cellular component of the urine sample is separated from the urine by subjecting the urine sample to a sufficient centrifugal force to obtain a pellet containing cholesterol.

9. The method of Claim 7, wherein the cellular component of the urine sample is separated by filtering the urine sample to permit capture of cell components containing cholesterol.

10. The method of Claim 1, wherein the mammal is a rodent, rabbit, cow, horse, dog, cat, or human.

1 1. The method of Claim 1, wherein the cholesterol reference standard is derived from at least one subject with no known kidney injury.