CN1791797A - A method and kit for detecting the early onset of renal tubular cell injury - Google Patents

Abstract

An early-onset method and kit for detecting renal tubular cell damage, using NGAL as an early urinary biomarker. NGAL is a small secreted polypeptide that is protease resistant and thus readily detected in urine following tubular cell injury. NGAL protein expression was mainly detected in the proximal tubule cells, which resembled a secreted protein with a punctate distribution in the cytoplasm. Apparent NGAL in urine correlates with the amount and duration of renal ischemia and nephrotoxicemia and is a diagnostic feature of tubular cell injury and renal failure. NGAL detection is also a useful marker for monitoring nephrotoxic side effects of drugs or other therapeutic agents.

Description

Methods and kits for detecting early-onset renal tubular cell injury

Background technique

Acute renal failure (ARF) secondary to renal tubular cell injury, including ischemic or nephrotoxic injury, remains a major issue in clinical medicine and nephrology ( nephrology) with persistently high mortality and morbidity. Decades of previous studies have illustrated the role of persistent vasoconstriction, tubular obstruction, cellular structural and metabolic changes, and inflammatory responses in the pathogenesis of ARF. While these studies have shown possible therapeutic approaches in animal models, efforts to translate them into humans have yielded disappointing results. Reasons for this may include the multifaceted renal response to ischemic injury and nephrotoxins, and the lack of early biomarkers of ARF, resulting in delayed initiation of treatment.

When the patient's serum creatinine (creatinine) value or: (1) when the baseline serum creatinine level is less than 2.0mg/dL, an increase of at least 0.5mg/dL; (2) when the baseline serum creatinine level is greater than or equal to 2.0mg/dL In dL, an increase of at least 1.5 mg/dL; or (3) an increase of at least 0.5 mg/dL as a result of exposure to a radiographic agent, regardless of baseline serum creatinine level, is considered to have acute renal function Exhausted.

It is believed that early intervention of therapy during the disease process will reduce ARF-related mortality and shorten the time to treatment for various types of tubular cell injury, including but not limited to ischemic and nephrotoxic kidney injury. Identification of reliable early biomarkers of tubular cell injury would be useful to facilitate early therapeutic intervention and help guide drug development by providing indicators of nephrotoxicity.

Traditional laboratory methods for detecting kidney disease include measurement of serum creatinine, blood urea nitrogen, creatinine clearance, urine electrolytes, microscopic examination of urine sediment, and radiologic studies. These indicators have neither sensitivity nor specificity, nor can they be used for early detection of the disease. Indeed, although elevated serum creatinine is generally considered the "gold standard" for detecting ARF, it is clear that as much as 50% of renal function is lost within the time period of serum creatinine change.

A small number of urinary biomarkers of ischemic kidney injury have been described earlier, including kidney injury molecule-1 (KIM-1) and cysteine-rich protein 61 (Cyr61). KIM-1 is a putative adhesion molecule involved in kidney regeneration. In a rat model of ischemia-reperfusion injury, KIM-1 was found to be upregulated 24-48 hours after the initial injury, making it a reliable but somewhat late marker. Recent studies have shown that KIM-1 can be detected in kidney biopsies and in the urine of patients with ischemic acute tubular necrosis. However, such detection has also been demonstrated in patients who have developed established ischemic renal injury, at a later stage of the disease. The utility of measuring urinary KIM-1 for the detection of early ARF or subclinical renal injury is far from proven.

The protein Cyr61 was found to be a secreted cysteine-rich protein that could be detected in the urine of animal models 3-6 hours after ischemic kidney injury. However, this assay requires bioaffinity purification and a concentration step using heparin-agarose beads, followed by a Western blotting step. Even after bioaffinity purification, several nonspecific cross-reactive peptides were evident. Therefore, detection of Cyr61 in urine is problematic in terms of specificity as well as the tedious nature of the procedure.

Therefore, there remains an urgent need to identify improved biomarkers for early ischemic and nephrotoxic kidney injury.

Contents of the invention

The present invention relates to a method for detecting renal tubular cell damage in mammals, comprising the following steps: 1) obtaining a urine sample from a mammalian test body; 2) contacting the urine sample with an antibody for a renal tubular cell damage biomarker, the The tubular cell injury biomarker comprises NGAL to allow complex formation of the antibody and the tubular cell injury biomarker; and 3) detection of said antibody-biomarker complex.

The present invention relates to a method for monitoring the effectiveness of treating renal tubular cell damage, comprising the steps of: 1) providing a treatment to a mammalian subject suffering from renal tubular cell damage; 2) obtaining at least one treatment from said subject and 3) detecting the presence of biomarkers of tubular cell injury in post-treatment urine samples.

The present invention further relates to a kit for detecting the presence of immediate or early onset biomarkers of renal tubular cell injury in the urine of a subject, comprising: 1) a method for obtaining a certain amount of urine sample; 2) a medium having attached thereto a capture antibody capable of complexing with a biomarker of renal tubular cell injury, said biomarker being NGAL; and 3) detecting the tubular injury biomarker and capture antibody Determination of complexes.

The present invention also relates to a competitive enzyme-linked immunosorbent assay (ELISA) kit for determining the status of renal tubular cell damage in mammalian subjects, comprising specific for renal tubular cell damage biomarkers A primary antibody to determine its presence in the subject's urine sample.

The present invention further relates to a method for identifying the extent of renal tubular cell damage caused by an event, comprising the steps of: 1) obtaining at least one urine sample from said mammalian test subject; 2) detecting in said urine sample Presence of biomarkers of tubular cell injury. 3) Determining the extent of tubular cell damage based on the time of occurrence of the presence of the IRI biomarker in the urine sample relative to the time of the event.

The present invention further relates to a method for detecting renal tubular cell damage in a mammal, comprising the steps of: 1) obtaining a urine sample containing up to 1 ml of the first urine from a mammalian subject suspected of having renal tubular cell damage; 2) ) contacting said urine sample with an antibody to a biomarker of renal tubular cell injury to allow formation of a complex of the antibody and biomarker; and 3) detecting said antibody-biomarker complex.

A preferred tubular cell injury biomarker is NGAL.

Description of drawings

Figure 1 shows the induction of mouse kidney NGAL mRNA after ischemia. Upper panels show representative RT-PCR with primers for mouse actin and NGAL using RNA extracted from kidneys of control (C) mice or after various reperfusion times (hours) as indicated . Lane M contains molecular weight standard markers. The lower panel shows the fold increase in NGAL mRNA expression from the control (CON) at different time points. Values from microarray (solid line) vs. RT-PCR (dashed line) are mean +/- standard deviation (SD) from at least 3 experiments.

Figure 2A shows the induction of mouse kidney NGAL protein following unilateral ischemia. The upper panel shows a representative Western blot using intact kidney samples obtained from control (Con) mice or after reperfusion times (hours) as indicated, expressed as NGAL Polyclonal antibodies or monoclonal antibodies to tubulin were probed (to show the same amount of protein loaded). Molecular weight markers are on the far left. The lower panel shows the fold increase in NGAL protein expression from the control (CON) at different time points. Values from densitometry are mean +/- SD from at least 3 experiments.

Figure 2B shows the induction of mouse kidney NGAL protein following bilateral ischemia. The upper panel shows a representative Western blot using intact kidney samples obtained from control (Con) mice or after reperfusion times (hours) as indicated, expressed as NGAL Polyclonal antibodies or monoclonal antibodies to tubulin were used as probes (to show the same protein loading). Molecular weight markers are on the far left. The lower panel shows the fold increase in NGAL protein expression from the control (CON) at different time points. Values obtained from densitometry are estimates +/- SD from at least 3 experiments.

Figure 3 shows the induction of mouse kidney NGAL protein after ischemia. Representative immunohistochemical results were obtained on frozen sections of mouse kidneys obtained from control mice or after various reflow times (hours) as indicated in the figure. The polyclonal antibody to NGAL was used as a probe for detection. "G" indicates a glomerulus. The rightmost image is at 100X magnification, while the others are at 20X magnification.

Figure 4A shows early determination of NGAL protein in urine of mice with unilateral ischemic ARF. Representative representation of untreated urine samples (1-2 μl per channel, normalized to creatinine content) obtained at the indicated reperfusion times (hours) after unilateral renal artery clamping. Western blot. Molecular weight markers are shown on the right. The Western blot was probed with NGAL (upper panel) or β2-microglobulin (Beta2-M) (middle panel). Urine of N-acetyl-[beta]-D-glucosaminidase (NAG) was tested from a total of five animals at different reperfusion times as indicated in the graph. Values are mean +/-SD. ^* P<0.05 compared with control at each time period, analysis of variance (ANOVA).

Figure 4B shows early detection of NGAL protein in the urine of mice with bilateral ischemic ARF. Representative Western blots of untreated urine samples (1-2 [mu]l per channel, normalized to creatinine content) after bilateral renal artery clamping at reperfusion times (hours) as indicated. Molecular weight markers are shown on the right. The Western blot was probed with NGAL (upper panel) or β2-microglobulin (Beta2-M) (middle panel). Urine N-acetyl-β-D-glucosaminidase was assayed from five animals out of a total of eight animals at different reperfusion times as indicated. Values are mean +/- SD. ^* P<0.05 compared with control at each time period, analysis of variance (ANOVA).

Figure 5 shows the determination of NGAL protein in the urine of mice with subclinical renal ischemia. Representative Western blots were performed on untreated urine samples (1-2 μl per channel, normalized to creatinine content) 5, 10 or 20 minutes after bilateral renal artery clamping. Samples were obtained at reperfusion times (hours) as indicated. Molecular weight markers are shown on the left. These animals showed normal serum creatinine at 24 hours of reflux.

Figure 6 shows the determination of NGAL protein in the urine of rats with ischemic ARF. After 30 minutes of bilateral renal artery clamping in rats, representative Western blots were performed on untreated urine samples (1-2 μl per channel, normalized to creatinine content) that were Obtained at reperfusion times (hours) as indicated. Molecular weight markers are shown on the left. These animals showed a significant increase in serum creatinine at 24 hours of reflux.

Figure 7 shows the in vitro induction of NGAL mRNA following ischemia. The upper panel shows a representative RT-PCR with primers for human NGAL applied to cells extracted from renal proximal tubular epithelial cells (RPTEC) after various times (hours) of partial ATP depletion as indicated. RNA. Lane M contains a 100 bp DNA ladder. The middle panel shows the fold increase in NGAL mRNA expression at various time points from the control (0), normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Values shown are mean +/- SD from at least three experiments for each point. The lower panels show representative Western blots (of three independent experiments) with RPTEC samples obtained from cell pellets (Pel) or medium ( Sup) was obtained in the same amount of cells, and the Western blot was probed with the polyclonal antibody to NGAL. Molecular weight markers are shown on the left.

Figure 8A shows early detection of NGAL protein in urine as described in mice with cisplatin-induced injury. Representative Western blots were performed on untreated urine samples (1-2 μl per channel, normalized to creatinine content) after administration of cisplatin at the indicated The Western blot was probed with an antibody to β-2-microglobulin (upper panel) and NGAL (middle panel) as probes obtained on days of . Molecular weight markers are shown on the left.

Figure 8B shows urinary NAG measurements at different days (n=4) after administration of cisplatin (n=4) in Figure 8A. Values represent mean +/- SD. ^* P<0.05 compared with Day 0.

Figure 9 shows that administration of cisplatin resulted in tubular cell necrosis and apoptosis. In a cisplatin-treated kidney (upper center panel), hematoxylin-eosin staining shows tubular dilation, luminal fragments, and flattened epithelium. At high magnification, the asterisk-marked renal tubules show condensed, intensely stained nuclei (arrowheads), indicating apoptosis (upper right panel). TUNEL staining revealed TUNEL-positive nuclei in cisplatin-treated kidneys (bottom middle panel). At high magnification, the renal tubules indicated by asterisks show condensed fragmented nuclei (arrowheads) that are characteristic of apoptosis (lower right panel). Figures marked high magnification are 100× magnifications, while others are 20×. Results for control mice are shown in the upper and lower left panels.

Figure 10 shows the results of rapid induction of renal NGAL by administration of cisplatin. Representative Western blots were performed on kidney lysates from mice treated with cisplatin (20 μg/kg) intraperitoneally and at the various time points (hours) indicated. Blots were probed with a polyclonal antibody to NGAL or a monoclonal antibody to tubulin. Molecular weight markers are shown on the left.

Figure 11 shows that cisplatin administration results in a rapid induction of NGAL in renal tubular epithelial cells. Representative immunohistochemistry was performed on frozen kidney sections from mice treated with cisplatin (20 μg/kg) intraperitoneally and obtained at various time points (hours) as indicated , the immunohistochemistry was detected with NGAL as a probe. G, Glomerulus. Figures labeled HP are at 100X magnification, while others are at 20X magnification.

Figure 12 shows that administration of cisplatin at 20 μg/kg resulted in rapid emergence of NGAL in urine. Representative Western blot (upper panel) of untreated urine samples (3-5 μl/channel, normalized to creatinine content) treated with cisplatin as indicated After injection, before or at different time points. The same urine samples were analyzed for NAG secretion (middle panel) and serum from the same animals were assayed for creatinine (lower panel). ^* P<0.05 compared with control.

Figure 13 shows that administration of cisplatin at 5 μg/kg resulted in rapid emergence of NGAL in urine. Representative Western blot (upper panel) of untreated urine samples (3-5 μl/lane, normalized to creatinine content) treated with cisplatin as indicated After injection, before or at different time points. The same urine samples were analyzed for NAG secretion (middle panel) and serum from the same animals were assayed for creatinine (lower panel). ^* P<0.05 compared with control.

Figure 14 shows the quantification of urinary NGAL following cisplatin injection. Known amounts of purified recombinant NGAL were subjected to Coomassie Blue (CB) staining (upper left panel) and Enhanced Chemiluminescence (ECL) analysis (upper right panel). Quantification of NGAL secretion at different time points following administration of cisplatin at 20 μg/kg or 5 μg/kg by densitometric analysis of Western blots and comparison with established standards of Western blots of purified NGAL performed under the same conditions Determination.

Figure 15 shows in panel A the urinary NGAL ratio in patients with cadaveric kidney transplants (CAD, n=4) versus patients with living related donor transplants (LRD, n=6). Assay (p<0.005). Panel B shows the correlation between cold ischemia time and urinary NGAL in CAD (p<0.001, r=0.98, Spearman analysis). Panel C shows the correlation between peak serum creatinine and urinary NGAL in CAD (p<0.001, r=0.96, Spearman analysis).

Figure 16 shows in panel A the results of serial measurements of urinary NGAL in patients after open heart surgery, plotted against time (hours) after bypass (n=15). Panel B shows the correlation between bypass time and 2-hour urinary NGAL in patients with ARF (n=5) (p<0.01, r=0.76, Spearman analysis). Panel C shows the correlation between changes in serum creatinine and 2-hour urinary NGAL in patients with ARF (p<0.01, r=0.66, Spearman analysis).

Detailed description of the invention

Throughout this application, various publications and unpublished manuscripts are referenced within parentheses. The disclosure of said publication in its entirety is hereby incorporated by reference into this application to more fully describe the state of the art to which this application pertains. Full bibliographic citations for these references can be found at the end of this application immediately preceding the claims.

The present invention provides a method and a kit for detecting the presence of biomarkers of renal tubular cell injury in the urine of subjects with early renal tubular cell injury. Early detection of the onset of this injury can reduce the time to treat the injury and can reduce the risk of developing clinical acute renal failure (ARF). The renal tubular cell injury may include, but not limited to, ischemic renal injury (IRI) or nephrotoxic renal injury (NRI).

A simple point-of-care kit for the rapid detection of urinary NGAL at the bedside using a principle similar to that of the widely used urine pregnancy test kit, which enables clinical Doctors can quickly diagnose ARF, quickly carry out laboratory verification, and use effective methods for treatment and prevention. Use of this kit may represent standard of care given to all patients at risk of developing ARF, including for cardiac surgery, kidney transplantation, stroke, trauma, sepsis, dehydration, and nephrotoxins (antibiotics, anti-inflammatory agents, radiocontrast agents (radiocontrast agents) -contrast agent) and chemotherapeutic agents). In current clinical practice, when ARF occurs in the context of these predisposing conditions, diagnosis is very delayed and the associated mortality and morbidity are unacceptably high. Ironically and even tragically, although effective preventive and therapeutic approaches are universally available, they are almost never administered in a timely fashion due to the lack of early biomarkers of ARF. It is anticipated that multiplex serial measurements of NGAL will be indispensable not only for the diagnosis and quantification of initial renal injury, but also for monitoring early treatment response and predicting long-term outcomes.

A biomarker of tubular cell injury (also referred to as an RTCI biomarker) may be an immediate RTCI biomarker, such as NGAL, which appears in urine within 2 hours of tubular cell injury. Immediate RTCI biomarkers can, as in the case of NGAL, appear immediately in the first urine output of subjects with tubular cell injury. RTCI biomarkers can also be early-onset RTCI biomarkers, which can appear within the first 24 hours of tubular cell injury. Likewise, NGAL is also an example of an early-onset RTCI biomarker.

Effective RTCI biomarkers are usually secreted proteins that can be excreted into the urine by the kidneys. Effective RTCI biomarkers are also usually protease-resistant proteins such as NGAL. However, the RTCI biomarker can also be a protease-sensitive protein, as long as a stable fragment of the protein can be detected in urine, eg, using an antibody to NGAL as described below.

The RTCI biomarker can be a biomarker of ischemic renal injury (IRI biomarker), a biomarker of nephrotoxic renal injury (NRI biomarker), or a mixture thereof. NGAL is an example of both an IRI biomarker and an NRI biomarker.

The method of the present invention can be used to detect the occurrence of renal tubular cell injury and monitor its treatment for various events, including various renal insufficiency, impaired cardiac function, surgical procedures, in intensive care unit A patient in the test, and administering drugs, radioactive contrast dyes or other pharmaceutical substances to said test subjects. The injury to tubular cells may be ischemic renal injury, nephrotoxic renal injury, or other injury affecting tubular cells. Such events may include the administration or ingestion of large or multiple nephrotoxins including, but not limited to, cancer chemotherapy (cisplatin, cyclophosphamide, isosfamide, formazan isosfamide), antibiotics (gentamicin, vancomycin, tobramycin), antifungals (amphotericin), anti-inflammatory agents ( NSAIDs), immunosuppressants (cyclosporine, tacrolimus) and radiocontrast media. The method can be used to evaluate the nephrotoxicity of newly developed compounds as well as known compounds.

The present invention also provides a method and a kit, which are used to evaluate the degree of renal damage according to the proportional relationship between the degree of damage and the amount of NGAL present in the urine excreted by the test subject, said The degree of injury can range from the onset of renal tubular cell injury to clinical ARF. The present invention provides clinicians with a means to estimate the extent of kidney injury at an initial assessment and to monitor changes in injury status (worsening, improvement of symptoms or remaining unchanged) based on the amount of NGAL detected in the urine.

Typically, a clinician will establish a protocol for collecting and analyzing a volume of fresh urine samples from a patient at selected intervals. Typically, the urine samples are obtained intermittently over a defined period of time. The period of time for intermittent sampling can be dictated by the conditions of the subject and can range from sampling every 24 hours to continuous sampling, more typically from every 4 hours to every 30 minutes.

Using the methods and techniques described herein, not only qualitative levels of RTCI biomarkers present in urine can be analyzed and estimated, but also quantitative levels of RTCI biomarkers present in urine can be analyzed and determined. The clinician can choose the qualitative method, the quantitative method or both according to the state of the patient. Typically, the volume of urine collected is less than 1 ml, more usually less than 10 μl. Typical samples may range from about 1 μl to about 1 ml. Usually, larger urine samples (approximately 1 ml) are used for quantitative determinations. Often these small amounts of urine are readily and rapidly obtained from clinical subjects who are prone to or have developed ARF.

Once indicators of tubular cell damage or acute renal failure are detected, intervention and treatment of the disease or condition is initiated and the clinician can monitor the progress of said treatment or intervention using the methods and kits of the invention. Typically, one or more subsequent post-treatment urine samples may be obtained and analyzed for the presence of RTCI biomarkers as treatment for renal injury is initiated and continued. The treatment is continued until the presence of RTCI biomarkers is no longer detectable in subsequent post-treatment urine samples. As treatment and intervention improve the condition, there is a corresponding decrease in the expression of RTCI biomarkers and their presence in urine. The degree of improvement can be expressed as a corresponding decrease in the levels of RTCI biomarkers, such as NGAL, measured in the sample. When the renal injury is nearly completely healed, the method can be used to detect the complete absence of RTCI biomarkers, which marks the completion of the treatment process.

Monoclonal and polyclonal antibodies that bind to RTCI biomarkers are useful in the methods and kits of the invention. Said antibodies can be prepared by methods known in the art. For example, in "Characterization of two ELISAs for NGAL, a newly described lipocalinin human neutrophils", Lars Kjeldsen et al., (1996) Journal of Immunological Methods, Vol. 198, 155-16, describes the preferred RTCI biomarker NGAL monoclonal antibody for , which is hereby incorporated by reference. Examples of monoclonal antibodies to NGAL such as HYB-211-01, HYB-211-02 and NYB-211-05 are available from the Antibody Shop, Copenhagen, Denmark. In general, HYB-211-01 and HYB-211-02 can be used with reduced and non-reduced forms of NGAL. An example of a polyclonal antibody to NGAL is described in "An Iron Delivery Pathway Mediated by a Lipocalin", Jun Yang et al., Molecular Cell, (2002), Vol. 10, 1045-1056, incorporated herein by reference. To prepare this polyclonal antibody, rabbits were immunized with recombinant gel-filtered NGAL protein. Sera were incubated with GST-Sepharose 4B beads to remove contaminants and polyclonal antibodies were obtained in the sera as described by applicants in JunYang et al., Molecular Cell (2002).

Typically, the step of detecting the complex of the capture antibody and the RTCI biomarker comprises contacting the complex with a second antibody to detect the biomarker.

The method for detecting complexes of RTCI biomarkers and primary antibodies comprises the steps of: separating any unbound material in a urine sample from the capture antibody-biomarker complexes; separating the capture antibody-biomarker complexes from the detection RTCI biological contacting the marker with a secondary antibody to allow formation of a complex between the RTCI biomarker and the secondary antibody; separating any unbound secondary antibody from the RTCI biomarker-secondary antibody complex; and detecting the RTCI biomarker Secondary antibody in marker-secondary antibody complex.

Kits for use in the methods generally include a medium having a capture antibody attached thereto, whereby a urine sample is contacted with the medium to expose the capture antibody to NGAL contained in the sample. The kit includes a method of acquisition which may include an implement, such as a spatula or a common stick, having a surface containing the medium. The obtaining method may also include a container for receiving a urine sample, wherein the container has a urine-contacting surface containing the medium. In another specific embodiment, the assay for detecting complexes of RTCI biomarkers and said antibody may comprise an ELISA and may be used for the quantification of NGAL in a urine sample. In an alternative embodiment, the method of obtaining may comprise a kit comprising a cassette containing the medium.

Early detection of RTCI biomarkers can provide an indication of the presence of the protein in a urine sample in the short term. Generally, the methods and kits of the present invention can detect RTCI biomarkers in urine samples within 4 hours, more typically within 2 hours, and most typically within 1 hour after renal tubular cell injury. Preferably, said RTCI biomarker is detectable within 30 minutes after renal tubular cell injury.

The method and kit for detecting RTCI biomarkers of the present invention can be prepared by modifying the methods and kits known in the art, so as to be used for the rapid detection of other proteins and ligands in biological samples. Examples of methods and kits that can be modified into the present invention are in May et al., US Patent 5,656,503, issued August 12, 1997; As described in US Patent 4,870,007 issued September 26, 1993 to Smith-Lewis, US Patent 5,273,743 issued December 28, 1993 to Ahlem et al., and US Patent 4,632,901 issued December 30, 1986 to Valkers et al. All of the above references are hereby incorporated by reference.

A rapid one-step method for detecting RTCI biomarkers can reduce the time to detect tubular cell damage. A general method may comprise the steps of: obtaining a urine sample suspected of containing an RTCI biomarker; mixing a portion of the sample with a detection antibody that specifically binds to the RTCI biomarker to initiate detection of the antibody and the RTCI biomarker in the sample contacting the mixture of sample and detection antibody with an immobilized capture antibody that specifically binds to the RTCI biomarker, wherein the capture antibody does not cross-react with the detection antibody, to bind the detection antibody to the RTCI biomarker, Instead, the RTCI biomarker is bound to the capture antibody to form a detectable complex; unbound detection antibody and any unbound sample are removed from the complex; and the detection antibody assays the complex. The detectable antibody is labeled with a detectable label, such as a radioactive label, an enzyme, a biological dye, magnetic beads, or biotin, as is well known in the art.

To identify potential genes and their proteins that can accompany and mark the earliest onset of tubular cell injury, such as ischemic and nephrotoxic kidney injury, cDNA microarrays can be used to detect which of a large number of potential gene targets are significantly upregulated . Using this screening approach, neutrophil gelatinase-associated lipocalin (NGAL) was identified as a gene whose expression in a mouse Increased by more than 10 times within an hour.

NGAL belongs to the lipocalin superfamily of more than 20 structurally related secreted proteins thought to transport various ligands in the β-barreled calyx. Human NGAL was originally identified as a 25 kDa protein covalently bound to gelatinase from human neutrophils, where it represents one of the neutrophil secondary granule protiens. Molecular cloning studies revealed that human NGAL is similar to the mouse 24p3 gene that was originally identified in primary cultures of mouse kidneys induced to proliferate. NGAL is expressed at very low levels in other human tissues, including kidney, trachea, lung, stomach and colon. NGAL expression was significantly induced in stimulated epithelium. For example, it is upregulated in inflammatory or neoplastic areas of colonic epithelial cells, but absent in untreated areas or within metastatic lesions. NGAL concentrations in serum of patients with acute bacterial infection, in sputum of subjects with asthma or chronic obstructive pulmonary disease, and in bronchial fluid from emphysematous lungs rise. In all these cases, NGAL induction was assumed to be the result of an interaction between inflammatory cells and the epithelial lining, in which upregulation of NGAL expression was evident.

It is believed that the detected induction of NGAL represents a novel intrinsic response of renal proximal tubular cells to tubular cell injury, including ischemic and nephrotoxic injury, and does not originate solely from activated neutrophils. First, the response is rapid, with NGAL appearing in the urine within 2 hours of injury together with the first urine output after renal artery occlusion, whereas renal neutrophils accumulate in this model of ischemic ARF Usually most pronounced 4 hours after injury. Second, the temporal pattern of NGAL induction and neutrophil accumulation is divergent. NGAL mRNA and protein expression was most pronounced at 12 hours of reflux, whereas neutrophil accumulation peaked at 24 hours, by which time NGAL expression had been significantly reduced. Third, no NGAL-expressing neutrophils were detected by immunofluorescence in the kidney samples examined (Fig. 3). Fourth, we demonstrate that NGAL mRNA and protein induction following ischemia in vitro occurs in cultured human proximal tubule cells, and that NGAL is secreted into the medium within 1 h of ATP depletion in a system completely absent of neutrophils. However, there may be a role for infiltrated neutrophils in the observed upregulation of NGAL. Upregulation of NGAL in tubular cells can be induced by the local release of cytokines from neutrophils trapped in the microcirculation early after ischemic injury.

A proper explanation for the induction of NGAL by stimulated epithelial cells is lacking, and it remains unclear whether NGAL is protective or proximate to injury, or even a harmless bystander. Recent evidence suggests that NGAL may represent a pro-apoptotic molecule in at least a subset of cell types. In a mouse pro-B lymphocyte cell line, cytokine withdrawal resulted in marked NGAL induction and apoptosis. Purified NGAL produced the same pro-apoptotic response as cytokine depletion, including activation of Bax, suggesting that NGAL is contextual to programmed cell death. NGAL has also been linked to apoptosis in reproductive tissues. Regenerated mammary and uterine epithelial cells express high levels of NGAL, timed to coincide with the phase of maximal apoptosis. It is likely that NGAL regulates a subset of cell populations by inducing apoptosis. Stimulated epithelial cells can upregulate NGAL to induce apoptosis of infiltrated neutrophils, thereby allowing resident cells to survive the destruction of the inflammatory response. Alternatively, epithelial cells can use this mechanism to regulate their own demise. However, it has been interestingly shown that NGAL induction following renal ischemia-reperfusion injury occurs predominantly in proximal tubular cells, whereas apoptosis under the same conditions is predominantly a distal tubular phenomenon.

Other recent studies have revealed that NGAL enhances the epithelial phenotype. NGAL is expressed by penetrating rat ureteric buds and triggers nephrogenesis by stimulating the transformation of mesenchymal cells into renal epithelial cells. Another lipocalin, glycodelin, has been shown to induce an epithelial phenotype when expressed in human breast cancer cells. These findings are particularly applicable to the mature kidney, where one of the well documented responses to ischemic injury is the prominent appearance of dedifferentiated epithelial cells lining the proximal tubules. An important aspect of renal regeneration and repair following ischemic injury involves reacquisition of the epithelial cell phenotype, a process that recapitulates several aspects of normal development. This suggests that NGAL can be expressed by damaged renal tubules to induce re-epithelialization. Support for this view comes from the recent identification of NGAL as an iron transporter that complements transferrin during nephrogenesis. Delivery of iron to cells is known to be critical for cell growth and development, and this may be critical for kidney regeneration after ischemia, as it is during ontogeny. Since NGAL appears to bind and transport iron, it is also likely that NGAL acts as a sink for iron efflux from damaged proximal tubular epithelial cells. Since NGAL has been observed to be endocytose by proximal tubules, this protein may recycle iron into living cells. This stimulates growth and development, as well as removes the reactive molecule, iron, from the site of tissue damage, thereby limiting iron-mediated cytotoxicity.

NGAL is a novel urinary biomarker of cisplatin-induced nephrotoxic kidney injury that is more sensitive than previously described biomarkers. An example is kidney injury molecule-1 or KIM-1, a putative adhesion molecule involved in kidney regeneration. In a rat model of cisplatin nephrotoxicity, KIM-1 was quantified 24-48 hours after the initial injury, making it a somewhat late marker of tubular cell injury. In contrast, NGAL was readily and quantitatively detected within 3 hours of administration of cisplatin at doses known to cause renal failure. Furthermore, urine NGAL detection precedes the appearance of other markers such as NAG in urine. The appearance of NGAL in urine also precedes an increase in serum creatinine, which has been widely used to diagnose nephrotoxic renal failure.

Urinary NGAL was evident even after mild "subclinical" cisplatin doses, although serum creatinine remained normal. Therefore, the present invention has important implications for the clinical management of patients undergoing cisplatin therapy. The effectiveness of cisplatin is dose-dependent, but the occurrence of nephrotoxicity often prevents the use of higher doses to maximize its antitumor potential. Nephrotoxicity following cisplatin therapy is common and acute renal failure can occur after a single dose. Although several therapeutic approaches have proven effective in the treatment of cisplatin-induced nephrotoxicity in animals, successful human trials have remained largely anecdotal. One reason for this may be the lack of early markers of nephrotoxic acute renal failure, thereby delaying the initiation of treatment. In current clinical practice, acute kidney injury is usually diagnosed by measuring serum creatinine. However, creatinine is known to be an unreliable and delayed indicator of acute changes in renal function. First, serum creatinine concentration does not change until approximately 50% of kidney function has been lost. Second, serum creatinine does not accurately describe renal function until it may take several days to reach a steady state. Therefore, the use of serum creatinine measurements impairs the ability to detect and quantify renal injury during the early stages of renal injury. However, animal studies have shown that while nephrotoxic acute renal failure can be prevented and/or treated, there is only a narrow "window of opportunity" to achieve it, and treatment must begin very early after the initial injury. The lack of early biomarkers of kidney injury impairs the ability of clinicians to initiate potentially effective treatments in a timely fashion. In experimental systems, the application of NGAL is also valuable for the detection of existing or developed therapeutic or prophylactic interventions, as well as for the early evaluation of potential nephrotoxicity of other agents. NGAL assay is a novel, non-invasive, early urinary biomarker for cisplatin-induced renal injury. Early detection enables clinicians to initiate timely therapeutic intervention and establish methods to prevent the development of overt nephrotoxic renal failure.

It has been found that NGAL is readily and rapidly detected as a relatively clean immunoreactive peptide in Western blots in mice and rats with as little as 1 [mu]l of initially untreated urine following renal ischemia. Furthermore, urinary NGAL is evident even after very mild "subclinical" renal ischemia, despite normal levels of serum creatinine. Urinary NGAL detection also far precedes the appearance of traditional markers in urine, including β2-microglobulin and NAG.

The upregulation and urinary excretion of NGAL may illustrate the rapid response of renal tubular cells to various injuries, and the detection of NGAL in urine may provide a widely used non-invasive clinical tool for early diagnosis of tubular cell injury.

NGAL is a sensitive, non-invasive, urinary biomarker of renal tubular cell injury, including renal ischemia and nephrotoxicemia. Detecting the expression of NGAL in the urine of patients suffering from acute, mild and early forms of renal tubular cell injury, using the rapid and simple detection method and kit of the present invention, can alert and enable clinicians to experience acute renal failure. Initiate intervention efforts in a timely manner in patients and alert clinicians to implement approaches aimed at preventing patients with mild subclinical tubular cell injury such as nephrotoxicity, renal transplantation, vascular surgical and cardiovascular events Developed into overt ARF.

In the United States alone, approximately 16,000 kidney transplants are performed each year. This number is steadily increasing year by year. Approximately 10,000 of these are cadaveric kidney transplants and are at risk of ARF. Each of these patients would greatly benefit from serial NGAL testing, which can represent routine care.

Ischemic kidney injury has also been associated with open heart surgery due to the transient interruption of blood flow inherent in the procedure. The number of open heart surgeries performed each year can be estimated. In any moderately busy adult hospital, approximately 500 such procedures are performed each year. Assuming that there are at least 400 such moderately busy hospitals in the United States alone, it can be conservatively estimated that 200,000 open heart surgeries are performed each year. Serial NGAL determinations in these patients are again invaluable and may represent the standard of care.

Experimental procedure

1. Mouse model of renal ischemia-reperfusion injury:

We used an established murine model of renal ischemia-reperfusion injury in which the short-term structural and functional consequences of renal ischemia had previously been demonstrated (3-7). Briefly, male Swiss-Webster mice (Taconic Farm, Germantown, NY) weighing 25-35 g were housed with a 12:12 hour light:dark cycle and allowed free access to food and water. The animals were anesthetized with sodium pentobarbital (50 mg/kg ip) and placed on a warming table to maintain a rectal temperature of 37°C. Three separate procedures were used: (a) unilateral ischemia, (b) bilateral ischemia with ARF, and (c) bilateral mild subclinical ischemia. For the first set (unilateral ischemia) experiments, the left renal pedicle was occluded with a non-traumatic vascular clamp for 45 minutes, during which time the kidney was kept warm and moist. The clamps were then removed, the kidney's blood return was observed, and the incision was sutured. The mice were allowed to recover in a warm cage. After 0, 3, 12 or 24 hours of reperfusion, the animals were anesthetized again, their abdominal cavity was opened, and blood obtained from inferior vena cava puncture was analyzed by quantitative colorimetric assay kit (Sigma, St. Louis, MO). Determination of creatinine. The mice were sacrificed with intraperitoneal pentobarbital. The left ventricle was then perfused with 10 ml of 1X PBS followed by 10 ml of 4% paraformaldehyde in PBS to achieve in situ fixation of the kidney. Two kidneys were harvested (the right kidney of each animal served as a control). At each reflux period, at least three separate animals were examined. One half of each kidney was snap frozen in liquid nitrogen and stored at -70°C until further processing; samples were fixed in formalin, embedded in paraffin and sectioned (4 μm). Paraffin sections were stained with hematoxylin-eosin and examined histologically. Clamped kidneys showed characteristic morphological changes resulting from ischemia-reperfusion injury, as previously published by others (3-6) and by us (2). The other half of each kidney was embedded in OCT compound (Tissue-Tek) and frozen sections (4 μm) were obtained for immunohistochemistry.

For the second set of experiments (bilateral ischemia), both kidneys were clamped for 30 min and examined at different reflow periods as detailed previously. This group of eight animals was designed to represent ARF and showed a significant rise in serum creatinine 24 hours after injury.

For the third set of experiments (bilateral mild subclinical ischemia), both kidneys of different animals were clamped for only 5, 10 or 20 minutes and examined at different refluxing periods as previously detailed. This very mild level of injury was designed to mimic subclinical renal ischemia, and the mice in this group did not show any rise in serum creatinine measured 24 hours after injury.

2. Rat model of renal ischemia-reperfusion injury:

We used an established rodent model of renal ischemia-reperfusion injury (2). Briefly, male Sprague-Dawley rats (Taconic Farm, Germantown, NY) weighing 200-250 g were anesthetized with ketamine (150 μg/g) and xylazine (3 μg/g) and performed with microvascular clamps. Bilateral renal animals were blocked for 30 min as detailed in the mouse procedure. Timed urine collections were obtained at 3, 6, 9, 12 and 24 hours of reperfusion, and blood was collected at lethal time (24 hours) for creatinine determination.

3. Isolate RNA:

Whole mouse kidney tissue (or cultured human proximal tubular cells, see below) was disrupted with Tissue Tearor (Biospec Products, Racine, WI). Total RNA from control and ischemic kidneys was isolated using the RNeasy Mini Kit (Qiagen, Valencia, CA) and quantified spectrophotometrically.

4. Microarray steps:

A detailed description of the microarray hardware and procedure has been published previously (3). Briefly, for each experiment, 100 μg of purified total mouse kidney RNA was analyzed with Superscript II in the presence of Cy3-dUTP (Amersham, Piscataway, NJ) for controls and Cy5-dUTP for ischemic samples. Reverse transcriptase (Life Technologies, Rockville, MD) was used for reverse transcription. The cDNA samples were purified using Microcon YM-50 filters (Millipore, Madison, WI) and hybridized to a microarray plate containing 8,979 unique sequence-verified mouse probes (3). During each reflux period, at least three independent animals were examined, and at least two independent microarray experiments were performed for each animal. The array slides were scanned with a microarray scanner (GenePix 4000B, Axon Instruments, Foster City, CA) to obtain separate TIFF images for Cy3 and Cy5 fluorescence, respectively. The signal intensity of Cy3 and Cy5 of a single gene was detected with GenePix Pro 3.0 data extraction software (AxonInstruments). Quality control and data analysis were done as previously described (3).

5. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR):

Equal amounts (1 μg) of total RNA from control and experimental mouse kidneys were reverse transcribed with Superscript II reverse transcriptase (Life Technologies) in the presence of random hexamers according to the instruction manual. PCR was performed using a kit (Roche, Indianapolis, IN) and the following primers:

Mouse NGAL sense 5'-CACCACGGACTACAAACCAGTTCGC-3';

Mouse NGAL antisense 5'-TCAGTTGTCAATGCATTGGTCGGTG-3';

Human NGAL sense 5'-TCAGCCGTCGATACACTGGTC-3'; and

Human NGAL antisense 5'-CCTCGTCCGAGTGGTGAGCAC-3';

Primer pairs for mouse and human β-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Clontech (La Jolla, CA). A mock reaction without cDNA served as a negative control. PCR products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining and quantified by densitometry. Fold change in NGAL mRNA expression in ischemic versus control kidneys is shown after normalization for β-actin or GAPDH amplification.

6. Immunohistochemistry:

Frozen sections were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, blocked with goat serum for 1 hour, and incubated with primary antibody to NGAL (1:500 dilution) for 1 hour. Slides were then exposed to a secondary Cy5-conjugated antibody (Amersham, Arlington Heights, IL) for 30 minutes in the dark and observed with a fluorescence microscope (Zeiss Axiophot) equipped with a rhodamine filter.

For co-localization of NGAL and Rab11, serial sections were first incubated with NGAL antibody or Rab11 monoclonal antibody (1:500 dilution; Transduction Laboratories), followed by Cy5-conjugated (for NGAL) or Cy3-conjugated Secondary antibodies (for Rab11) were incubated together and visualized with rhodamine or fluorescein filters, respectively. To co-localize NGAL with proliferating cell nuclear antigen (PCNA), sections were incubated with monoclonal antibodies to NGAL and PCNA (1:500 dilution; Upstate) and stained by immunoperoxidase staining ( ImmunoCruz Staining System, Santa Cruz Biotechnology) completed the detection. For the TUNEL assay we applied ApoAlert DNA Fragmentation (Assay Kit Clontech). Paraffin sections were deparaffinized with xylene and descending grade ethanol, fixed with 4% formaldehyde/PBS at 4°C for 30 minutes, permeabilized with proteinase K for 15 minutes at room temperature, and treated with 0.2% Triton X-100 /PBS for 15 minutes at 4°C and incubated with a mixture of nucleotides and TdT enzyme for 60 minutes at 37°C. The reaction was terminated with 2X SSC, and the sections were washed with PBS and mounted with Crystal/mount (Biomeda, Foster City, CA). TUNEL-positive apoptotic nuclei were detected by fluorescence microscopy.

7. Urine collection:

Mice or rats were placed in metabolic cages (Nalgene, Rochester, NY) and urine was collected before and every hour after bilateral renal artery occlusion. Urine samples were centrifuged at 5000 xg to remove debris and the resulting supernatants were analyzed by Western blot. Urinary creatinine was determined by a quantitative colorimetric assay kit (Sigma) to normalize samples for urinary NGAL detection. A colorimetric assay kit for the detection of N-acetyl-β-D-glucosaminidase (NAG) in urine was obtained from Roche.

8. Cell culture:

Human renal proximal tubular epithelial cells (RPTEC) were obtained from Clonetics (San Diego, CA). Cells were grown in Renal Epithelial CellBasal Medium supplemented with REGM complex (0.5μl/ml hydrocortisone, 10pg/ml hEGF, 0.5μg/ml epinephrine, 6.5pg/ml triiodomethyl Adenine, 10 μg/ml transferrin, 5 μg/ml insulin, 1 μg/ml gentamicin sulfate and 2% FBS) as recommended by the manufacturer.

9. Gentle ATP depletion of cultured cells:

We modified a previously described protocol for in vitro ischemia induced by ATP depletion with inhibitors of oxidative phosphorylation (8, 9). On the second day post-confluence, RPTEC cells were incubated with 1 μl antimycin A (Sigma) for various times up to 6 hours. We have previously shown that in other types of cultured renal epithelial cells, such as MDCK(8) and 786-O(9) cells, this results in a mild and partially reversible ATP depletion without loss of cell viability. ATP levels were monitored using a luciferase-based assay kit (Sigma) and expressed as a percentage of the control value. Cells were harvested at different time points of ATP depletion and analyzed for NGAL mRNA expression by RT-PCR, whereas NGAL protein expression was analyzed by Western analysis. The secretion of NGAL into the medium was also monitored.

10. Mouse model of cisplatin nephrotoxicity:

We used an established murine model in which the structural and functional consequences of cisplatin-induced nephrotoxicity had been previously demonstrated (12-14, 18). Briefly, male Swiss-Webster mice (Tconic Farm, Germantown, NY) weighing 25-30 g were housed with a 12:12 hour light:dark cycle and allowed free access to food and water. The mice were given a single intraperitoneal injection of cisplatin at a dose of 5 μg/kg or 20 μg/kg body weight. It has been previously shown that larger doses lead to necrosis and apoptosis of tubular cells and impair renal function within 3-4 days of cisplatin injection (12-14, 18). Animals were placed in metabolic cages (Nalgene, Rochester, NY) and urine was collected before and at various time points after cisplatin injection (3, 12, 24, 48, 72 and 96 hours). At similar time points, the animals were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally), their abdominal cavity was opened, and the blood obtained by puncture of the inferior vena cava was quantified by a quantitative colorimetric assay kit (Sigma, St. .Louis, MO) for measurement of serum creatinine. The mouse was sacrificed, its kidney was perfusion fixed in situ with 4% paraformaldehyde in PBS, and both kidneys were harvested. One half of each kidney was snap frozen in liquid nitrogen and stored at -70°C until further processing: samples were fixed in formalin, embedded in paraffin and sectioned (4mm). Paraffin sections were stained with hematoxylin-eosin and subjected to TUNEL assay. The remainder was used for Western blotting. Whole kidneys were homogenized with a Polytron homogenizer in ice-cold lysis buffer (20 mM Tris, pH 7.4, 250 mM sucrose, 150 mM NaCl, 1% NP-40 and 1X Complete(R ⁾ protease inhibitors). Homogenates were incubated on ice for 30 minutes, centrifuged at 1,000 xg for 5 minutes at 4°C to remove nuclei and cell debris, and analyzed for protein content by the Bradford method (Bio-Rad, Hercules, CA). The other half of each kidney was embedded in OCT compound (Tissue-Tek) and the resulting frozen sections (4 μm) were used for immunohistochemistry.

11. Expression, purification and Western blotting of recombinant murine NGAL:

The full-length mouse NGAL cDNA was cloned into the pGEX expression vector (Pharmacia, Nutley, NJ), expressed in bacteria as a fusion protein with glutathione-S-transferase (GST), and treated with glutathione- Agarose column (Amersham) purification followed by thrombin cleavage was performed as previously described (16, 19, 20). Proteins were analyzed by SDS-PAGE followed by Coomassie blue staining or Western blotting with a polyclonal antibody to NGAL. Protein concentration was determined by the Bradford method (Bio-Rad, Hercules, CA).

12. Quantification of urinary NGAL by Western blotting:

The amount of NGAL in urine was determined by comparison with a defined standard of recombinant purified NGAL. Densitometric analysis of Western blots was performed under the same conditions of transfer and exposure using known recombinant NGAL concentrations and known urine volumes.

All chemicals were purchased from Sigma unless otherwise noted. For Western blotting, protein concentration was determined by the Bradford method (Bio-Rad, Hercules, CA), and an equal amount of total protein was loaded on each lane. Unless otherwise indicated, a 1:10,000 dilution of monoclonal antibody to α-tubulin was used to confirm equal protein loading, while polyclonal antibody to NGAL was used at 1:500 (15). Unless otherwise indicated, immunodetection of transferred proteins was accomplished using enhanced chemiluminescence (Amersham).

Example 1

NGAL is a small protease-resistant, secreted polypeptide that can be detected in urine. Significant upregulation of NGAL mRNA and protein levels has been shown early after renal ischemia in mice. NGAL protein expression was mainly detected in the proximal tubule cells, which resembled a secreted protein with a punctate distribution in the cytoplasm. Indeed, in mouse and rat models of ARF, NGAL is readily and rapidly detectable in urine (in the first urination) after ischemic injury, when no renal leukocytes are observed penetration. The origin of NGAL from tubular cells was further confirmed in cultured human proximal tubular cells subjected to in vitro ischemic injury, where NGAL mRNA was significantly and immediately Inside, NGAL protein was readily detectable in the culture medium. Our results illustrate that NGAL may represent a new early urinary biomarker of ischemic kidney injury.

Identification of novel genes upregulated early after renal ischemia-reperfusion injury:

A genome-wide search for transcripts later induced by renal ischemia-reperfusion injury in a mouse model identified seven early biomarkers. Three independent mice were examined at each reperfusion time (3, 12 and 24 hours) and at least two independent microarray experiments were performed for each examined animal. Comparison of the transcriptome profiles of control and ischemic kidneys yielded a small subset of 7 genes that were consistently induced more than 10-fold. It has recently been identified that one of these transcripts, cysteine-rich protein 61 (Cyr61 ), is induced by renal ischemia (1). Surprisingly, the behavior of the other 6 differentially expressed genes was new to the ARF literature. We chose to further characterize one of these previously unidentified genes, neutrophil gelase-associated lipocalin (NGAL).

Characteristics of animal models of early renal failure:

Short-term structural and functional consequences of renal ischemia have been demonstrated using a murine model of ischemia-reperfusion injury (3-7). Characteristic histopathological features of ischemic injury were evident in 24 h reperfusion samples following unilateral (45 min) and bilateral ischemia (30 min). These include loss of the brush border membrane, tubular dilation, flattened tubular epithelium, luminal debris, and interstitial infiltrate (Figure 1). The presence of apoptotic cells was demonstrated by TUNEL assay. Apoptosis is mainly localized in the cells of the distal tubule and the ascending limb of the Henle’s loop, both in detached and attached cells in the lumen. Proximal tubule cells were also occasionally apoptotic, but glomeruli were largely apoptotic. No TUNEL-positive cells were detected in control kidneys or in ischemic samples neglecting TdT (not shown). The above histological and apoptotic changes were absent in kidneys subjected to less severe degrees of ischemia (5, 10 or 20 minutes of bilateral ischemia) (not shown). Serum creatinine levels reflected observed histopathological changes. Thus, mice with unilateral renal ischemia or mild subclinical bilateral ischemia showed serum creatinine levels indistinguishable from control animals, whereas mice with 30 minutes of bilateral ischemia showed serum creatinine levels. Anhydride significantly increased (Figure 1).

NGAL mRNA is significantly induced early after renal ischemia:

By microarray analysis, NGAL was found to be consistently induced 3.2 ± 0.5-fold, 11.1 ± 1.2-fold and 4.3 ± 0.6-fold in ischemic mouse kidneys reperfused at 3, 12 and 24 hours compared to control kidneys of the same animals (Mean +/- SD from three animals at each time point). This finding was confirmed by semi-quantitative RT-PCR using normalization with β-actin or GAPDH. No significant changes in β-actin or GAPDH mRNA expression were recorded at any of the reperfusion times examined, as previously described (3). However, applying mouse-specific primers, we detected a significant upregulation of NGAL mRNA expression (4.1±0.5-fold, 9±0.6-fold and 4.2±0.4-fold at 3, 12 and 24 hours of reperfusion, respectively, where the values Represents the mean +/- SD of three individual animals). These results are illustrated in Figure 1 and are generally consistent with the changes detected by transcriptome analysis.

NGAL protein is significantly overexpressed in the early proximal tubules of the ischemic mouse kidney:

NGAL protein expression paralleled mRNA expression after ischemia in the kidney. By Western analysis, NGAL was just detectable as a 25 kDa immunoreactive peptide in control mouse kidneys. The identity of this band as NGAL was established in an independent set of experiments in which pre-incubation of the primary antibody with recombinant mouse lipocalin completely blocked this immunoreactivity (not shown). In unilateral ischemia experiments, NGAL expression was induced 3-4 fold by densitometry in ischemic kidneys from three independent animals within 3 hours of injury, as shown in Figure 2, panel A. This response was significantly enhanced in bilateral ischemia experiments from eight independent animals. NGAL in these mice was induced 3-fold at 3 h after reperfusion, peaked at 12-fold more in the 24-h sample, and decreased to normal levels during the 72-h recovery period (Fig. 2, panel B) .

Using immunohistochemical techniques, NGAL protein was hardly detected in the kidney of control mice, but it was significantly upregulated in the proximal tubule within 3 hours of ischemia, as shown in Figure 3. Proximal tubules in these sections were identified based on the presence of a brush border membrane, the ratio of nuclear to cell size, and cell morphology. Induced NGAL appears in the proximal tubule cells in a punctate cytoplasmic distribution, which resembles a secreted protein. This expression pattern was identical in unilateral and bilateral models of ischemia-reperfusion injury and was consistently evident in every animal studied. Glomeruli lacked NGAL expression, and NGAL-expressing neutrophils were not evident. Since NGAL has been shown to colocalize at least partially with endosomes in cultured Wilms tumor kidney cells (11), NGAL and Rab11, a late recirculating endosome, were examined in serial kidney sections. body markers) distribution. Merged images show significant co-localization of NGAL with Rab11 (not shown). To determine the functional significance of enhanced NGAL expression after ischemia, serial kidney sections were examined for NGAL expression, TUNEL-positive nuclei, or PCNA-positive nuclei. Whereas tubular cells overexpressing NGAL were not TUNEL-positive (not shown), significant co-localization of NGAL with PCNA was evident in proliferating and regenerating cells during the 48 hr refluxing period (not shown).

NGAL protein was readily detected in urine immediately after induction of ARF in mice:

This experiment illustrates the utility of detecting urinary NGAL as an early non-invasive biomarker of ischemic kidney injury. Urine creatinine concentrate was used for consistent loading and NGAL was absent in urine prior to ischemia. In marked contrast, NGAL appeared as a 25 kDa band within 2 hours of injury (in the first urine after ischemia) in all animals examined, as shown in Figures 4A and 4B. The identity of this band as NGAL was established in an independent set of experiments in which pre-incubation of the primary antibody with recombinant mouse lipocalin completely blocked this immunoreactivity (not shown). NGAL was readily detectable by Western analysis in as little as 1 [mu]l of untreated urine and persisted throughout the assay period (24 hours of reperfusion). We then compared urinary NGAL secretion to that of previously established markers of damage, such as β2-microglobulin and NAG. Although urinary NGAL was evident within 2 hours of ischemia, in the same urine samples 12 hours after only unilateral ischemia (Fig. 4, panel A) and 8 hours after bilateral ischemia (Fig. 4, Panel B), β2-microglobulin is detectable. Similarly, urinary NAG secretion was only after 12 hours of unilateral ischemia (lower panel of Figure 4, panel A) and after 8 hours of bilateral ischemia (lower panel of Figure 4, panel B), compared with non-ischemic significantly increased compared to control animals.

NGAL protein was readily detected in urine after even mild renal ischemia in mice:

To determine the sensitivity of urinary NGAL detection in the absence of overt ARF, we applied an experimental procedure in which different groups of mice were subjected to bilateral renal artery occlusion for only 5, 10 or 20 minutes. These studies were designed to evaluate urinary NGAL secretion after mild subclinical renal ischemia. Serum creatinine measured 24 hours after reflux was within normal limits in all these mice. Surprisingly, NGAL was readily detected in as little as 1 [mu]l of untreated urine in these animals (Fig. 5), although its appearance was somewhat delayed compared to animals with ARF. Thus, while 30 minutes of bilateral ischemia resulted in urinary NGAL secretion within 2 hours (Figure 4), mice with 20 or 10 minutes of bilateral ischemia showed urinary NGAL after 4 hours, whereas mice with 5 minutes of ischemia only NGAL was secreted after 6 hours (Fig. 5). Thus, the presence of NGAL in urine appears to correlate with the dose and duration of renal ischemia.

Example 2

NGAL protein was readily detected in urine immediately after induction of ARF in rats:

Because of the debate regarding species differences in the response to renal artery occlusion, we below examined the behavior of NGAL in a different animal model, an established rat model of renal ischemia-reperfusion injury. Urinary creatinine concentrations were used to make loading consistent, and NGAL was absent in urine prior to renal ischemia in rats. In marked contrast, NGAL showed 25 kDa immunoreactivity within 3 hours of injury (in the first post-ischemic urine effusion), as shown in FIG. 6 . In comparison, serum creatinine in this model of ischemic injury was elevated only after 24 hours of reperfusion (not shown). Again, NGAL was detectable in as little as 1 [mu]l of untreated urine and persisted throughout the period examined (24 hours of reperfusion).

Example 3

NGAL mRNA is induced in cultured human proximal tubule cells after early minimal ischemia:

To confirm the source of NGAL from ischemic proximal tubular cells, we modified a previously described procedure for in vitro ischemia induced by ATP depletion in cultured human proximal tubular cells (RPTEC). Incubation in 1 μM antimycin produced a mild partial ATP depletion, which became about 83±3% of the control within 1 hour and decreased more slowly to about 75±3% of the control by 6 hours (from four Mean +/- SD of experiments). The morphological consequences of this mild ATP depletion were not discernible. NGAL mRNA is barely detectable in resting cells. However, after partial ATP depletion, a rapid duration-dependent induction of NGAL mRNA was clearly visible by RT-PCR, as shown in Figure 7.

NGAL protein was readily detected in culture medium following early ischemia in vitro:

We next examined NGAL protein expression in RPTEC cells and media after mild ATP depletion. NGAL protein was detectable in control RPTEC cells, and its expression increased in a duration-dependent manner after ATP depletion, as shown in FIG. 7 . No NGAL immunoreactive protein was found in the medium from control cells, but NGAL was readily detectable within 1 hour of slight ATP depletion. Note that further increases in NGAL protein abundance correlate with the duration of ATP depletion. These results demonstrate that the induced NGAL protein is rapidly secreted into the medium, similar to the rapid appearance of NGAL in urine following renal ischemia in vivo.

Example 4

NGAL protein was readily detected in the urine of mice at the early stage of mild nephrotoxicemia:

Cisplatin-induced nephrotoxemia was induced in mice in order to determine whether nephrotoxemia resulted in the expression of NGAL protein in urine, thereby elucidating its use as an early non-invasive biomarker of nephrotoxic kidney injury. In an established mouse model of cisplatin nephrotoxicity, NGAL was readily detectable in urine within one day of cisplatin administration (Fig. 8A, descending trace). In contrast, urinary β2-microglobulin was barely detectable after 2 days of cisplatin administration, and urinary β2-microglobulin was not reliably detectable until 4–5 days after cisplatin administration ( Figure 8, panel A, uplink trace). Similarly, increased urinary NAG secretion was not evident until 4 and 5 days after cisplatin administration (Figure 8, panel B).

Cisplatin nephrotoxicity is characterized by apoptosis and necrosis of tubular cells:

Mice were given a single intraperitoneal injection of cisplatin at a dose of 5 mg/kg or 20 mg/kg body weight. Results in control mice and those mice that received higher doses of cisplatin are shown in FIG. 9 . Larger doses resulted in tubular cell necrosis, evidenced by the presence of tubular dilation, luminal fragments, and flattened epithelium in hematoxylin-eosin-stained sections (upper center panel). Canalicular cells were also demonstrated to be undergoing programmed cell death by showing condensed intensely-stained nuclei (upper right panel). This was confirmed by TUNEL experiments showing condensed, fragmented nuclei typical of apoptosis (lower middle and lower right panels). No necrosis or apoptosis was detected in control kidneys (upper and lower left panels). Kidneys of mice treated with smaller doses of cisplatin also appeared normal, similar to untreated controls (not shown). Figure 9 is a representative of 5 separate experiments.

NGAL protein is rapidly induced by cisplatin in renal tubules:

Since NGAL is induced following ischemic injury of the kidney (17), the response to cisplatin-induced nephrotoxic injury was examined. By Western analysis, NGAL was barely detectable in kidney lysates from control mice, but was rapidly induced within 3 hours of cisplatin administration (20 mg/kg), as shown in Figure 10 . Renal NGAL expression was increased, which was duration dependent, with a peak at 48 hours and continued upregulation until 96 hours. These results were confirmed by immunofluorescent staining, shown in Figure 11. Kidneys harvested 3 hours (3h) (upper right panel) and 12 hours (12h) (lower left panel) after cisplatin injection showed induction of NGAL protein. A high magnification (HP) of a section harvested at 12 hours (lower right panel) is also shown in FIG. 11 . Arrows in the lower left panel indicate the region shown in the HP plot. NGAL was mainly induced in proximal tubule cells within 3 hours of cisplatin injection, but was absent in cells from control mice (Con) (upper left panel). Proximal tubules in these sections were identified based on the presence of a brush border membrane, the ratio of nuclear to cell size, and cell morphology. Induced NGAL appears in the proximal tubule cells in a punctate cytoplasmic distribution, which resembles a secreted protein. Induced NGAL appears in the proximal tubule cells in a punctate cytoplasmic distribution, which resembles a secreted protein. This expression pattern is similar to that observed in a mouse model of ischemia-reperfusion injury (17). Glomeruli lacked NGAL expression, and NGAL-expressing neutrophils were not evident. Figure 11 represents 5 animals at each time point.

NGAL protein is readily detectable in urine following high-dose cisplatin:

NGAL protein was detected in urine following high-dose cisplatin (20 mg/kg), thereby demonstrating its use as an early non-invasive biomarker of nephrotoxic kidney injury. Urine creatinine concentrations were used to make loading consistent and NGAL was essentially absent in urine prior to ischemia. In marked contrast, urinary NGAL was readily detectable within 3 hours of cisplatin injury (20 mg/kg) in all animals examined, as shown in Figure 12 (upper panel). The identity of this band as NGAL was established in an independent set of experiments in which primary antibodies were pre-incubated with recombinant mouse lipocalin to completely block this immunoreactivity (not shown). NGAL was readily detectable by Western analysis in as little as 5 μl of untreated urine. Urinary NGAL secretion was increased, which was duration dependent, with a peak at 48 hours and continued upregulation until 96 hours. We then compared urinary NGAL secretion to previously established markers of injury, such as NAG. Whereas urinary NGAL was evident within 3 hours of cisplatin, whereas urinary NAG secretion increased significantly only 96 hours after injury (middle panel). Furthermore, renal function, assessed by serum creatinine measurement, showed significant changes only after 96 hours of cisplatin (lower panel). This graph is representative of 5 independent experiments at each time point.

NGAL protein can be detected in urine even after small doses of cisplatin:

Cisplatin injections of only 5 μg/kg were performed on different groups of mice to determine the sensitivity of urinary NGAL detection after subclinical nephrotoxic injury, shown in FIG. 13 . NGAL was detectable in as little as 5 μl of untreated urine from these animals (upper panel), although its presence appeared quantitatively smaller compared to animals with 20 μg/kg cisplatin (Figure 12, upper panel). Thus, the presence of NGAL in urine is dose-related to the nephrotoxin. Whereas urinary NGAL secretion was evident within 3 hours of cisplatin, urinary NAG secretion in this group of animals did not increase significantly even after 96 hours of injury (middle panel). Furthermore, evaluation of renal function by serum creatinine measurement showed that serum creatinine was not significantly changed even after 96 hours of low dose cisplatin (bottom panel). This example demonstrates that NGAL is a more sensitive biomarker of nephrotoxicity than currently employed biomarkers.

Urinary NGAL secretion following cisplatin is dose and duration dependent:

Quantification of urinary NGAL secretion to determine its use as an indicator of the severity of renal injury following cisplatin administration is shown in FIG. 14 . This requires expression and purification of known amounts of NGAL to be used as standards. Analysis of recombinant NGAL protein by SDS-PAGE after Coomassie blue staining revealed a single protein band of appropriate size (upper left panel). Western blots of aliquots of known concentrations showed a linear increase in signal intensity over the range 3-100 ng/ml (upper right panel). The amount of NGAL in urine was then determined by comparison with these defined standards of recombinant purified NGAL. Urinary NGAL secretion increased after 20 μg/kg cisplatin, which was duration-dependent (lower panel). A similar, albeit somewhat blunted, response was evident in animals treated with cisplatin doses that resulted in clinically insignificant nephrotoxic lesions.

Example 5

Urine samples were obtained from patients 2 hours after kidney transplantation, a predictive human model of ischemic kidney injury, is shown in FIG. 15 . Compared with patients (n=6) who received kidneys from living relative donors (Panel A), patients (n=4) who received kidneys from cadavers had increased urinary NGAL, which was readily detected by Western blot and ELISA , the cadaveric kidneys were stored on ice for a period of time prior to transplantation. There was a significant correlation between urinary NGAL and cold ischemia time, indicating that NGAL secretion was directly proportional to the degree of renal injury (Panel B) (r=0.98, Spearman analysis). There was also a significant association between urinary NGAL and peak serum creatinine (Panel C) (r = 0.96, Spearman analysis). It is important to emphasize that urinary NGAL measured within 2 hours of transplantation was predictive of ARF as reflected by peak serum creatinine, which occurred several days later. Urine from normal controls or from patients with chronic renal failure contained almost undetectable amounts of NGAL, suggesting that urinary NGAL upregulation is specific for acute kidney injury (not shown). Likewise, urine from patients with urinary tract infections and renal transplant rejection (two neutrophil-associated conditions) contained only minimal amounts of NGAL (not shown), readily as was evident in cadaveric renal transplants Larger amounts (>100ng/ml) were distinguished. These data show that NGAL is a novel early urinary biomarker of acute kidney injury after kidney transplantation.

Example 6

Serial urine samples were prospectively obtained from 15 post-open heart surgery patients with the results shown in FIG. 16 . Urinary NGAL was quantified by Western blot and ELISA and was found to be elevated in 5 of 15 patients (Panel A). Each line represents a patient. The % change from baseline in serum creatinine is shown on the right in Panel A. The same 5 patients developed postoperative acute renal failure, defined as a 50% or more increase in serum creatinine from baseline, resulting in an incidence of approximately 33%. In 10 patients who did not develop acute renal failure, there was a small early increase in urinary NGAL secretion (2-hour creatinine value 6.0 ± 2.0 ng/mg), which was within 12 hours after surgery Rapidly normalized to barely detectable levels (Panel A). In marked contrast, patients who subsequently developed acute renal failure showed a greater than 10-fold increase in the 2-hour value of urinary NGAL (75 ± 10 ng/mg creatinine) and a greater than 20-fold increase in the 4-hour value of urinary NGAL ( 120±12ng/mg creatinine). There is a correlation between the amount of urinary NGAL and the time of cardiopulmonary bypass (cardiopulmonary bypass), which shows that NGAL secretion is directly proportional to the degree of renal injury (Figure B) (r = 0.76, Spearman analysis). There was also a significant association between urinary NGAL and peak serum creatinine (Panel C) (r=0.66, Spearman analysis). It is important to emphasize again that urinary NGAL measured within 2 hours of cardiac surgery can predict ARF as reflected by peak serum creatinine that occurs hours or even days later. These data show that NGAL is a novel early urinary biomarker of acute kidney injury after open-heart surgery and that its quantification can predict acute kidney failure in this susceptible population.

Although the invention has been described in conjunction with preferred embodiments, it will be apparent to those skilled in the art, after reading the foregoing description, that various changes, substitutions of equivalents and modifications can be made to the subject matter set forth herein. Accordingly, the present invention may be practiced by methods other than those specifically described herein. Accordingly, protection here is limited only by the appended claims and their equivalents.

references

1. Muramatsu Y, Tsuji M, KohdaY, Pham B, Perantoni AO, Zhao H, JoS-K, Yuen PST, Craig L, Hu X, Star RA: Early detection of cysteine rich protein61(CYR61, CCN1) in urine following renal ischemia reperfusion injury. Kidney Int 62:1601-1610, 2002

2. Yoshida T, Kurelia M, Beato F, Min H, Ingelfinger JR, Stears RL, Swinford RD, Gullans SR, Tang S-S: Monitoring changes in gene expression renal ischemia-reperfusion in the rat. Kidney Int 61:1646-1654, 2002

3. Supavekin S, Zhanh W, Kucherlapati R, Kaskel FJ, Moore LC, DevarajanP: Differential gene expression folloWing early renal ischemia-reperfusion. Kidney Int 63: 1714-1724, 2003

4. Nogae S, Miyazaki M, Kobayashi N, Saito T, Abe K, Saito H, NakanePK, Nakanishi Y, Koji T: Induction of apoptosis in ischemia-reperfusion model of mouse kidney: Possible involvement of Fas. J Am Soc Nephrol 9: 620-631, 1998

5. Daemen MARC, Van de Ven MWCM, Heineman E, Buurman WA: Involvement of endogenous interleukin-10 and tumor necrosis factor-αin renalischemia-reperfusion injury. Transplantation 67: 792-800, 1999

6. Kelly KJ, Plotkin Z, Dagher PC: Guanosine supplementation reduces apoptosis and protects renal function in the setting of ischemic injury. J ClinInvest 108: 1291-1298, 2001

7. Burne NJ, Rabb H: Pathophysiological contributions offucosyltransferases in renal ischemia reperfusion injury. J Immunol169: 2648-2652, 2002

8. Feldenberg LR, Thevananther S, del Rio M, De Leon M, Devarajan P: Partial ATP depletion induces Fas-and caspase-mediated apoptosis in MDCK cells. Am J Physiol 276(Renal physiol 45): F837-F846, 1999

9. Devarajan P, De Leon M, Talasazan F, Schoenfeld AR, Davidowitz EJ, Burk RD: The von Hippel-Lindau gene product inhibits renal cell apoptosis via Bcl-2-dependent pathways. J Biol Chem 276: 40599-401605, 20

10. Molitoris BA, Weinberg JM, Venkatachalam MA, ZagerRA, Nath KA, Goligorsky MS: Acute renal failure II. Experimental models of acute renal failure: imperfect but indispensable. Am J Physiol Renal Physiol 278: F1-F12, 2000

11. Kjeldsen L, Cowland JB, Borregaard N: Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse. Biochim Biophys Acta 1482: 272-283, 2000

12. Megyesi J, Safirstein RL, Price PM: Induction of p21WAF1/CIP/SD1 inkidney tubule cells affects the course of cisplatin-induced acute renal failure. JClin Invest 101: 777-782, 1998

13. Shiraishi F, Curtis LM, Truong L, Poss K, Visner GA, Madsen K, NickHS, Agarwal A: Heme oxygenase-1 gene ablation or expression modulates cisplatin-induced renal tubular apoptosis. Am J Physiol 278: F726-F036, 200

14. Ramesh G, Reeves WB: TNF-a mediates chemokine and cytokine expression and renal injury cisplatin nephrotoxicity. J Clin Invest 110: 835-842, 2002

15. Muramatsu Y, Tsujie M, Kohda Y, Pham B, Perantoni AO, Zhao H, JoS-K, Yuen PST, Craig L, Hu X, Star RA: Early detection of cysteine rich protein61(CYR61, CCN1) in urine following Renalischemic reperfusion injury. Kidney Int 62:1601-1610, 2002

16. Yang J, Goetz D, Li J-Y, Wand W, Mori K, Setlik D, Du T, Erdjument-Bromage H, Tempst P, Strong R, Barasch J: An iron delivery pathway mediated by a lipocalin.Mol Cell 10:1045 -1056, 2002

17. Mishra J, Ma Q, Prada A, Mitsnefes M, Zahedi K, Yang J, Barasch J, Devarajan P: Identification of NGAL as a novel urinary biomarker for ischemicinjury. J Am Soc Nephrol 14: 2534-2543, 2003

18. Tsuruya K, Ninomiya T, Tokumoto M, Hirakawa M, Masutani K, Taniguchi M, Fukuda K, Kanai H, KishiharaK, HirakataH, Iida M: Direct involvement of the receptor-mediated apoptotic pathways in cisplatin-induced renal tubular. Int ney death cell 63:72-82, 2003

19. Bundgaard J, Sengelov H. Borregaard N, Kjeldsen L: Molecular cloning and expression of a cDNA encoding Ngal: a lipocalin expressed in humanneutrophils. Biochem Biophys Res Commun 202: 1468-1475, 1994

20. Del Rio M, Imam A, De Leon M, Gomez G, Mishra J, Ma Q, Parikh S, Devarajan P: The death domain of kidney ankyrin interacts with Fas and promotes Fas-mediated cell death in renal epithelia. J Am Soc Nephrol 15:41-51, 2004

Claims (29)

1. A method for detecting renal tubular cell damage in mammals, said damage comprising ischemic kidney damage and nephrotoxic damage, comprising the following steps: 1) Obtain a urine sample from a mammalian test subject; 2) contacting the urine sample with antibodies to tubular cell damage biomarkers comprising NGAL to allow complex formation of the antibodies and biomarkers; and 3) Detecting said antibody-biomarker complex. 2. The method of claim 1, wherein a majority of the urine samples from said test subject are obtained intermittently. 3. The method of claim 2, wherein said urine samples are obtained continuously. 4. The method of claim 1, wherein the step of detecting the antibody-biomarker complex comprises contacting said complex with a second antibody for detection of the biomarker. 5. The method of claim 1, wherein said mammalian subject is a human patient. 6. A method for monitoring the effectiveness of a treatment for renal tubular cell injury comprising the steps of: 1) Provide treatment to mammalian subjects suffering from renal tubular cell damage; 2) obtaining at least one post-treatment urine sample from said subject; and 3) Detection of the presence of biomarkers of renal tubular cell injury in post-treatment urine samples. 7. The method of claim 6, wherein said biomarker comprises NGAL. 8. The method of claim 6, further comprising the step of obtaining one or more subsequent treated urine samples, wherein the step of providing treatment is continued until all of the treated urine samples are no longer detected in the subsequent treated urine samples. the presence of the biomarkers described above. 9. The method of claim 6, wherein the step of detecting comprises the steps of: i) contacting the urine sample with a capture antibody for the biomarker to allow complex formation of the antibody and the biomarker; and ii) detecting said antibody-biomarker complex. 10. The method of claim 9, wherein the step of detecting the antibody-biomarker complex comprises the steps of: (1) separating any unbound material from the urine sample from the capture antibody-biomarker complex; (2) contacting the capture antibody-biomarker complex with a second antibody for detection of the biomarker to allow formation of a complex between the biomarker and the second antibody; (3) separating any unbound secondary antibody from the biomarker-secondary antibody complex; and (4) Detecting the second antibody in the biomarker-second antibody complex. 11. The method of claim 10, wherein step (i) comprises the step of contacting the urine sample with a medium having the primary antibody attached thereto. 12. A kit for detecting the presence of immediate or early onset biomarkers of renal tubular cell injury, including ischemic renal injury and nephrotoxic injury, in the urine of a subject, comprising: 1) means for obtaining a certain amount of urine sample; 2) a medium having attached thereto a capture antibody capable of complexing a biomarker of renal tubular cell injury, the biomarker comprising NGAL; and 3) Assays for detection of complexes of biomarkers and capture antibodies. 13. The kit of claim 12, wherein the volume of the urine sample is less than 1 ml, more typically less than 10 microliters. 14. The kit of claim 12, wherein said means for obtaining comprises a device comprising a surface, said surface comprising said medium. 15. The kit of claim 12, wherein said means for obtaining comprises a container for receiving a urine sample, wherein the urine-contacting surface of the container contains said medium. 16. The kit of claim 12, wherein said assay comprises an ELISA. 17. The kit of claim 12, wherein said means of obtaining comprises a kit comprising a cartridge containing said medium. 18. The kit according to claim 12, which is a side-by-side test kit. 19. The around-the-patient test kit of claim 18, wherein the volume of the urine sample is less than 1 ml, more typically less than 10 microliters. 20. The on-patient test kit of claim 19, wherein said means of obtaining comprises a device comprising a dip stick having said medium on its surface. 21. The peri-patient test kit of claim 19, wherein said assay comprises a dip stick colorimetric assay. 22. A competitive enzyme-linked immunosorbent assay (ELISA) kit, which is used to determine the status of renal tubular cell damage in a mammalian test body, said damage comprising ischemic kidney damage and nephrotoxic damage, said The kit comprises a primary antibody specific for NGAL to determine its presence in a urine sample of said subject. 23. The ELISA kit of claim 22, wherein said urine sample comprises a volume of about 1 milliliter or less. 24. A method of identifying the extent of renal tubular cell injury caused by an event, including ischemic renal injury and nephrotoxic injury, comprising the steps of: 1) obtaining at least one urine sample from said mammalian subject; 2) detecting the presence of biomarkers of tubular cell injury in said urine sample; and 3) Based on the time of occurrence of the presence of the biomarker in the urine sample, the extent of tubular cell damage is determined relative to the time of the event. 25. The method of claim 24, wherein said biomarker comprises NGAL. 26. The method of claim 24, wherein said event is a surgical procedure. 27. The method of claim 24, wherein said event is a response to renal insufficiency, impaired cardiac function, surgical procedure, patient in an intensive care unit, and administration of a drug, radiocontrast dye, or other pharmaceutical substance to said Experimental body. 28. A method for detecting renal tubular cell injury in mammals, including ischemic renal injury and nephrotoxic injury, comprising the steps of: 1) obtaining a urine sample containing up to 1 ml of first urine from a mammalian test subject; 2) contacting said urine sample with antibodies to biomarkers of renal tubular cell damage to allow complexes of the antibodies and biomarkers to form; and 3) Detecting said antibody-biomarker complex. 29. The method of claim 28, wherein said biomarker comprises NGAL.

Images