WO2003082258A1 - Use of cyr61 for the diagnosis of acute renal failure - Google Patents

Abstract

The present invention relates to the early diagnosis of acute renal failure. Specifically, the invention provides a method of diagnosing the onset of acute renal failure in a subject after the subject suffers an injury to the kidneys, comprising detecting an elevated level of Cyr61 in a body fluid sample from the subject. Also provided is a kit for detecting Cyr61 in a body fluid sample from the subject.

Description

USE OF Cyr61 FOR THE DIAGNOSIS OF ACUTE RENAL FAILURE

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 60/367,411, filed March 25, 2002, the disclosure of which application is hereby incorporated in its entirety by this reference.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates to the early diagnosis of kidney disease. Specifically, the present invention relates to the diagnosis of the onset of acute renal failure by detecting an elevated level of Cyrβl in a sample from a subject with acute renal failure.

BACKGROUND ART

Acute renal failure (ARF) is a devastating disease that has a mortality rate of 25- 70% [25]. In about 50% of patients diagnosed with ARF, the disease is caused or aggravated by hypotension or renal ischemia [19;25]. Although much is known about the pathogenesis of ischemia/reperfusion in animals, translation of an effective therapy for acute renal failure from laboratory bench to bedside has been difficult [11;22;23;25]. Numerous agents that are effective in animals (for example, atrial natriuretic peptide (ANP), insulin-like growth factor- 1 (IGF-1), thyroid hormone, endothelin antagonists) have failed in clinical trials [10;11;14;23]. The failure to translate results from bench to bedside could be due to a heterogeneous patient population, multiple and diverse types of renal insults, failure to enroll patients early in the course of their disease, or by choosing incorrect disease mechanisms to target for treatment [11 ;23]. three original members of this family of genes, [Cyrόl, Connective tissue growth factor and Nephroblastoma overexpressed (NON)]. Cyrόl can promote the proliferation, migration, and adhesion of endothelial cells and fibroblasts [15], stimulate growth and differentiation of chondrocytes [27], and induce neovascularization of the cornea in vivo [2]. Cyrόl may play a role in wound repair and has been detected at sites of vascular injury [28]. Fibroblasts adhere to a surface treated with Cyrόl [7], which then induces actin cytoskeleton reorganization, formation of filopodia and focal adhesive complexes, and adhesive signaling events [6]. Cyrόl stimulates directed migration and adhesion of endothelial cells, platelets, and fibroblasts via an integrin α_vβ [2], αn β₃ [13], and α₆βι integrin pathways [7], respectively. Cyrόl promoter-driven LacZ expression is induced in granulation tissue induced by a cutaneous wound, and Cyrόl promotes healing of a wounded fibroblast monolayer [16].

Currently, acute renal failure is diagnosed by finding an abnormally elevated plasma creatinine level. Plasma creatinine rises about 1 -2 mg % per day following an injury to the kidneys. In most studies of acute renal failure [3], the mean plasma creatinine is 3-4 mg % [1] at the time the diagnosis of acute renal failure is first made. Thus, acute renal failure is usually not diagnosed until after 2-3 days following an injury to the kidneys. Because animal studies have shown that therapeutic agents are more effective when given early in the course of the disease [23], it is important to find a means of diagnosing acute renal failure as early as possible. A similar pattern of renal function and kidney injuries has been noted in human and laboratory animals.

The present invention provides a method for early diagnosis of the onset of acute renal failure in a subject following an ischemic injury to the kidneys, comprising detecting an elevated level of Cyrόl in a body fluid sample from the subject. SUMMARY OF THE INVENTION

The present invention provides a method and kit for diagnosing acute renal failure in a subject, comprising detecting an elevated level of Cyrόl in a biological sample from the subject, whereby the detection of the elevated level of Cyrόl diagnoses the presence of acute renal failure in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a Northern blot analysis of Cyrόl expression following ischemia/reperfusion. Rat kidney tissue was obtained from normal or sham-operated animals, or 2, 4, 24, and 72 hours after 40 min of bilateral renal ischemia. Northern blots containing 7 μg of total RNA from whole kidney (A) or outer medulla (B) were hybridized with a ³²P-labeled portion of rat Cyrόl, washed, and the signal detected by autoradiography.

Figure 2 shows a Northern blot analysis of Cyrόl expression in adult rat tissues following bilateral renal ischemia. Organs were obtained from rats subjected to sham surgery or 40 min ischemia and 4 hrs reperfusion. H, heart; Lu, lung; K, kidney; Sp, spleen; M, skeletal muscle (pretibial muscle); Li, liver. Northern blotting and hybridization as in Figure 1.

Figure 3 shows heparin bead capture of Cyrόl. Kidney tissues were obtained from normal mice or 4 hrs after 40 min of bilateral renal ischemia (I/R 4 hr). Kidney tissue homogenates were run as is (10 ug per lane; Lanes 1), or were purified by incubating 200 ug protein with heparin beads, washing with 150 mM (lanes 2) or 400 mM (lanes 3) NaCl-RIPA buffer, then eluting with 800 mM NaCl-RIPA buffer followed by Laemmli sample buffer. Samples were analyzed by SDS-PAGE and Western blotting, using an affinity purified antibody to mouse/rat Cyrόl. Figure 4 shows the time course of Cyrόl protein upregulation following renal injury in mice. Balb/c mice were subjected to 40 min ischemia then 0.5, 1, 2, 4, 6, 8, 12, 18, 24 hrs reperfusion. Kidney tissue homogenates (200 μg total protein per lane) were incubated with heparin beads, washed with 400 mM NaCl RIPA buffer, then eluted with 800 mM NaCl RIPA buffer, and Laemmli sample buffer. Samples were analyzed by SDS-PAGE and Western blotting using an affinity purified antibody to mouse/rat Cyrόl . N, normal kidney. S, sham operated kidney obtained 4 hrs after the surgery.

Figure 5 shows the organ specificity of Cyrόl protein upregulation after renal ischemia. Organs were obtained from SD male rats subjected to 40 min ischemia / 6 hrs reperfusion (I/R όhr) or 6 hrs after sham surgeiy (Sham). Tissue homogenates (300 μg total protein per lane) were purified as described in Figure 4, and analyzed by SDS- PAGE and Western blotting. H, heart; Lu, lung; K, kidney; Sp, spleen; M, skeletal muscle (pretibial muscle); Li, liver. For comparison, Vi, 1/5, 1/10, or 1/20 of the I/R 6hr kidney homogenates were loaded to the right three lanes.

Figure 6 shows localization of Cyrόl mRNA by in situ hybridization. Organs were obtained from male Sprague-Dawley rats subjected to sham surgery or 40 min ischemia and 2 hrs reperfusion. Each block was probed for anti-sense and sense probe on serial sections. Granule counts using the sense probe were at background levels for each treatment. A: control normal kidney; B: 2 hrs after sham surgery; C: 2 hrs after renal ischemia; D: higher power of outer stripe of outer medulla.

Figure 7 shows urine Cyrόl before and after renal ischemia. Twenty-four hoururine collections were obtained from three rats (1-3) before and after 30 min bilateral renal ischemia. Portions of the urine supernatant (volume factored for urine creatinine; see below) were purified by binding to heparin beads and analyzed by SDS- PAGE and Western blotting, as in Figure 4. Two duplicate membranes were probed with affinity purified anti-Cyrόl antibody (A) or primary antibody that had been pre- incubated with excess peptide (B).

Figure 8 shows the temporal appearance of Cyrόl in urine after volume depletion or renal ischemia. Rats were subjected to renal ischemia or volume depletion, and kidney homogenates processed as above. Urine collections were obtained either after volume depletion, or within 3 or 6 hr after renal ischemia. Portions of the urine supernatant (volume factored for urine creatinine, see Examples) were purified by binding to heparin beads and analyzed by SDS-PAGE and Western blotting, as in Figure 4. Two duplicate membranes were probed with affinity purified anti-Cyrόl antibody (A) or primary antibody that had been pre-incubated with excess peptide (B).

DETAILED DESCRIPTION OF THE INVENTION

It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes multiple copies of the agent and can also include more than one particular species of agent.

The present invention provides a method of diagnosing acute renal failure (ARF) in a subject, comprising detecting an elevated level of Cyrόl in a biological sample from the subject, whereby the detection of the elevated level of Cyrόl diagnoses the presence of ARF in the subject.

As used herein, "acute renal failure" means a sudden and severe loss of function of the kidneys usually resulting from ischemia of the kidneys that is associated with a rise in serum or plasma creatinine levels. This rise is in creatinine is not normalized by a trial of fluid volume expansion or relief of urinary obstruction. As used herein, a "sample" means a part representative of the subject. For example, a sample includes, but is not limited to, a quantity of the subject's skin, blood, saliva, urine, cerebrospinal fluid, hair, semen or any tissue obtainable for study and examination.

As used herein, a "subject" means an individual. Thus, the "subject" can include domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds. Preferably, the subject is a mammal such as a primate, and, more preferably, a human. A human subject can be a patient in whom acute renal failure is suspected. Moreover, the human subject can be a potential donor of a kidney for transplantation. Thus, the present invention can be used to detect subtle acute renal failure in an asymptomatic kidney donor to determine whether the donor's kidneys are suitable for transplantation.

Acute renal failure is usually caused by ischemia or nephrotoxins and is often seen in subjects with sepsis, septic shock or multiple organ failure (23, 25). As used herein, "ischemia" means an interrupted or diminished supply of blood to an organ or tissue. Ischemia can be caused by a mechanical obstruction (i.e., a thrombus or embolus) in an artery, external compression of an artery, iatrogenic blocking of blood flow in an artery to an organ that is to be surgically removed from one subject and subsequently transplanted into another subject, sepsis or hypotension (low blood pressure). Hypotension can result from hemorrhage, sepsis, septic shock, multiple organ failure, a cardiac arrhythmia and/or a neurogenic reflex causing vasodilation and subsequent pooling of blood in the lower extremities (e.g., a vasovagal reflex). "Ischemic injury" refers to damage to an organ that results from an interrupted or diminished blood supply to the organ. An "ischemic/reperfusion" injury is the damage that occurs in an organ following the restoration of blood flow to the organ after it has had an ischemic injury. As used herein, "nephrotoxic injury" means damage to a kidney caused by drugs, for example, antibiotics, chemo herapeutic agents, and non- steroidal anti-inflammatory drugs (NSAID) or other chemical agents including, but not limited to, radiocontrast agents, mercury and other heavy metals. It is often difficult for a medical practitioner to diagnose ARF early in its course because the signs of the disease are subtle and frequently overshadowed by signs of the underlying disease that is the cause of the ARF. For example, a subject can present to an emergency medical facility complaining of chest pain and shortness of breath associated with nausea and vomiting, obvious signs and symptoms of a heart attack. Associated with the heart attack, there can be a cardiac arrhythmia that can cause hypotension and subsequent ARF. During prolonged periods of recurring arrhythmias and hypotension, the kidneys can suffer ischemic injury. An ischemic injury of the kidneys can cause ARF.

Currently, there is no way of detenriining the onset of ARF. Thus, an opportunity to treat ARF in its earliest stages, when intervention is most effective, is lost. Typically, by the time serum or plasma creatinine levels reach the range of 3-4 mg %, the subject has been in ARF for at least 2 or 3 days.

The present invention provides a method of detecting early ARF in a subject by detecting an elevated level of Cyrόl in a body fluid sample from the subject. For example, a person of skill can take a sample of blood from a subject by venipuncture, or from an indwelling catheter that has been previously placed in a vein of the subject, using techniques well known in the art. Alternatively, a person of skill can collect a urine sample from the subject, using standard techniques. Other bodily fluids that can be tested for an elevated level of Cyrόl include, but are not limited to, saliva, semen and cerebrospinal fluid.

After the body fluid sample is obtained from the subject, the level of Cyrόl in the sample can be deteπnined by, for example, Western blot or heparin-binding ELISA or a traditional ELISA, as taught in the Examples below. In Western blot analysis, body fluid samples can be incubated with heparin beads, washed with 400 mM NaCl RIPA buffer, then eluted with 800 mM NaCl RIPA buffer and Laemmli sample buffer. Samples can be analyzed by SDS-PAGE and Western blotting using an affinity purified antibody to mouse/rat or human Cyrόl.

In a heparin binding ELISA assay for detecting Cyrόl, heparin is used as the capture surface. A heparin binding ELISA differs from a traditional "sandwich" ELISA because heparin (rather than a capture antibody) is used as the capture surface [34]. This can be accomplished, for example, by using heparin chemically coupled to bovine serum albumen (Heparin-BSA). Heparin-BSA is chemically coupled to the surface of a well or bead or magnetic bead. The capture surface is incubated successively with blocking agent (for example, milk, BSA, fish gelatin, liver homogenate, etc.), sample, washing solution, primary antibody, and excess primary antibody is washed away. The amount of primary antibody is detected by standard means (for example, using a directly labeled fluorescent primary antibody, or with a secondary antibody coupled to chemical or enzymatic amplification system).

In a traditional "sandwich" ELISA, a capture antibody is bound to the surface of a well or bead or magnetic bead. The capture surface is incubated successively with blocking agent, sample, wash, detection antibody, and excess detection antibody is washed away. The amount of detection antibody is detected by standard means (for example, using a directly labeled detection antibody, or with secondary antibody coupled to chemical or enzymatic amplification system).

Measurements of the levels of Cyrόl, for example in blood and/or urine, can be made when the subject is first seen, for example, upon admission to an emergency facility or upon transfer to an intensive care unit. Sequential samples of blood and/or urine can be obtained and tested at intervals ranging from about every 30 minutes to about every 12-24 hours. A rise in the level of Cyrόl can be detected in a body fluid, for example blood and/or urine, from about 30 minutes following an ischemic injury to the kidneys to about 48 hours after the injury. Thus, detection of Cyrόl can also be made from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13....24, 32, 40... hours after ischemic injury to the kidneys to about 48 hours after the injury and all times in between. A rise in the level of Cyrόl in the sample indicates the presence of ARF. Thus, if Cyrόl is found to be elevated in a first test of a body fluid sample, or if the level of Cyrlόl in a later sample is higher than the level of Cyrόl in an earlier sample, the diagnosis of can be made.

The concentration of Cyrόl in plasma or serum can be determined by western blot, heparin-binding ELISA, or traditional sandwich ELISA, using recombinant Cyrόl as a standard. An ELISA assay for Cyrόl is described in the Examples below. The concentration of Cyrόl in urine can be similarly determined and the results factored for dilution or concentration of the urine. This typically is accomplished, for example, by dividing the urine Cyrόl concentration by the creatinine concentration of the urine sample. Thus, the units will be reported as mg Cyrόl per mg creatinine. The measurement of Cyrόl can be made in normal healthy subjects as well as in subjects who may be at risk for ARF.

The present invention provides a kit for detecting Cyrόl in a biological sample, comprising: a) a capture surface for specifically capturing Cyrόl, b) a reagent for detecting Cyrόl on the capture surface, and c) a Cyrόl standard. As used herein, a "capture surface" is a surface that can hold or bind a substance, for example, a protein, for detection. The capture surface of the present invention can be a microtiter plate or any surface known in the art that holds or binds a substance for detection. The capture surface of the present invention can comprise heparin-BSA, Cyrόl -specific monoclonal antibodies or polyclonal antibodies that hold or bind Cyrόl. Thus, the microtiter plate of the kit can be precoated with heparin-BSA or Cyrόl -specific monoclonal antibodies, lyophilized and sealed with foil, with optional removable 16- well racks.

An example of a reagent that can detect Cyrόl is a labeled detector, such as a biotinylated primary or secondary antibody. For example, the detector can be rabbit IgG which binds to Cyrόl captured on the capture surface and is detected using a labeled secondary antibody. When the detector is rabbit IgG, it can be in a vial containing about 60 μl pre-diluted antibody. The detector can be labeled antibody that specifically binds Cyrόl. It is contemplated that other marker molecules known in the art can be used to detect Cyrόl bound to or held by the capture surface of the kit.

The Cyrόl standard of the kit can include one or more vials (e.g., two, three, four, five, six or more vials), each containing about 6 ng of lyophilized human or mouse Cyrόl.

The kit of the present invention can further comprise additional components, including 1) a wash buffer, including 50 mM Tris-Cl, 0.2% and TWEEN 20®, pH 8.0, 2) an assay buffer and 3) a sample diluent. For example, a diluent can be TBS-T, which is 20mM Tris pH 7.4 or 8.0, 137 or 150 mM NaCl (when Cyrόl -specific antibody is the capture surface) or 400 mM NaCl (when heparin-BSA is the capture surface), 0,1% TWEEN 20®. The kit of the present invention can also include pre- diluted poly-HRP conjugated streptavidin, (e.g., 120 μl), a color reagent, for example, tetramethyl-benzidine (TMB) (e.g., 12 ml), and a stop solution, for example, 12 ml of 1 M sulphuric acid.

In addition to the kit components described above, other materials known in the art may be used with the kit or for practicing the method of this invention. Examples of other materials include, but are not limited to, multichannel or repeating pipettes, pipettes capable of accurately measuring 1 to 1,000 μl, orbital shakers, clean 10-15 ml serological tubes and Eppendorf tubes for preparation of working dilutions, 96-well microtiter plate reader with 450 nm and 650 nm filters, a plate sealer (re-sealable bag containing two adhesive strips and manual), distilled water, and computerized data plotting or graph paper for manual plotting of data.

Because it is known that Cyrόl is a growth factor that can play a role in cutaneous wound repair and has been detected at sites of vascular injury, it is within the scope of the present invention that Cyrόl can be used in the treatment and/or prevention of ARF. A practitioner can administer to a subject diagnosed with ARF, or likely to develop ARF, an effective amount of Cyrόl in a pharmaceutical carrier. In general, an "effective amount" of an agent is that amount needed to achieve the desired result or results. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the Cyrόl without causing substantial deleterious biological effects or interacting in a deleterious manner with any of the other components of the composition in which it is contained.

The practitioner can administer the Cyrόl to the subject orally, rectally or parenterally. Parenteral administration of the Cyrόl, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. As used herein, "parenteral administration" includes intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous and intratracheal routes. For example, Cyrόl can be administered as a continuous infusion. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.

For example, a human subject (patient) diagnosed with ARF can be treated by parenteral administration of Cyrόl in a dosage range from about 0.1 mg to about 100 mg/Kg of body weight, 1 to 4 times a day, depending on the age, gender and weight of the patient. The patient is monitored for general physical signs to evaluate nonspecific effects of treatment. Efficacy of the treatment is evaluated using standard indices for monitoring urinary output and by analyzing blood chemistry to identify the return of plasma or serum creatinine levels to normal. If Cyrόl is administered to a subject to prevent ARF in a clinical setting likely to cause ARF, efficacy of treatment can be determined by detecting no elevation in the level of plasma or serum creatinine in the subject.

The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLES

Surgery: Sprague-Dawley rats and BALB/c mice were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN) or the National Institutes of Health (Frederick, MD). All animals had free access to water and food (4%) mouse-rat diet; Harlan Sprague Dawley Inc). Animal care followed the criteria of the University of Texas Southwestern Medical Center and the NIH for the care and use of laboratory animals in research. Animals were anesthetized with an I.M. injection of 100 mg/kg ketamine, 10 mg/kg xylazine, and 1 mg/kg acepromazine, and placed on a heating table kept at 39°C to maintain constant body temperature. Both renal pedicles were cross-clamped for 40 min. After the clamps were removed, 1-6 mL of prewarmed (37°C) normal saline was instilled into the abdominal cavity. The abdomen was closed, and the animal was placed in a 29°C incubator. The animal was sacrificed at 0.5-72 hours after surgery. Sham-treated animals went through the same surgical procedure as the other animals, including dissection of the renal pedicles; however, renal clamps were not applied.

UNA blot analysis. Samples of total RNA (7-10 Dg) were fractionated via electrophoresis on 0.9% agarose-formaldehyde gel and transferred onto a nylon membrane. The equality of RNA samples after transfer to the membrane was substantiated by UV illumination of ethidium bromide. Membranes were fixed by baking at 80 °C, then prehybridized at 42 °C in 50% formamide, 0.5 % SDS and 5x Denhardts, 5 x SSC, and 0.5 mg/ml salmon sperm DNA. The membranes were hybridized with [³²P]dCTP-labeled cDNA clones. 0.5-2 kB portions of mouse Cyrόl were generated by PCR, and the products confirmed by automated sequencing. The hybridized membranes were washed twice in O.lx SSC and 0.1% SDS at 50 °C. Loading of RNA was normalized by rehybridizing with GAPDH.

Protein blot analysis. Kidney tissue was homogenized in tissue extraction buffer (T-PER, #78510; Pierce, Rockford, IL) containing protease inhibitors (complete mini protease inhibitor cocktail tablets, #1 836 153; Roche, Mannheim, Germany). The homogenate was centrifuged at 13000 rpm for 10 min, and the supernatant was equilibrated with RIPA buffer (150 mM NaCl, 50 mM Tris pH 8.0, 0.1% SDS, 1% Nonident P-40, 0.5%> deoxycholoate, 1 mM PMSF). The cell lysate was incubated with heparin beads (Heparin Sepharose 6 Fast Flow, #17-0998-01 Amersham Pharmacia Biotech, Piscataway, NJ) at a volume ratio of 1:20 for 1 hr at 4°C; then the beads were washed twice with excess RIPA buffer containing 150-400 mM NaCl. In some cases, protein was eluted by adding RIPA buffer containing 800 mM NaCl. Then Laemmli sample buffer (Bio-Rad, # 161-0737; Hercules, CA) with 20 mM dithiothreitol was added, incubated at 60°C for 15 min, and the samples stored in 4°C until the electrophoresis. The protein content of the homogenate (before bead purification) was determined according to the Bradford method. Purified protein (corresponding to 200- 1,000 micrograms total protein of the unpurified homogente per lane) was fractionated on a 12%o polyacrylamide gel and transferred to a PVDF membrane (Immobilon-P, Millipore, Bedford, MA). The membranes were blocked by incubation in 5%> (w/v) nonfat dry milk in Tris-buffered saline containing 0.1 %> Tween 20 (TBS-T) for overnight. The membranes were incubated with a primary antibody (usually diluted without dry milk, only with TBS-T) for 2 hr at a dilution of 0.75 Dg/ mL, washed, and then incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibody for 1 hr at a dilution of 1 :2000. The reaction products were visualized by using chemiluminescence (ECL-plus, Amersham, # RPN2132). Blots were exposed to a BioMax MR film (Kodak, Rochester, NY) for 1-15 min until bands were clearly visible. The primary rabbit anti-mouse/rat Cyrόl antibody was made to a 23 amino acid peptide (CPHPNEASFRLYSLFNDIHKFRD) and affinity purified by Quality Controlled Biochemicals, Inc. (Hopkinton, MA).

Temporal expression of Cyrόl mRNA. The results of the representational difference analysis were confirmed by Northern blotting with RNA obtained from whole kidney or outer medulla (Figure 1). Following ischemia, there was a dramatic upregulation of Cyrόl in the outer medulla and whole kidney. Ischemia increased Cyrόl mRNA abundance in whole kidney at 2 hour, with a fall off by 4 hrs, and return towards normal at 24-72 hrs.

Organ specific expression of Cyrόl mRNA. Cyrόl is expressed in muscle, placenta, epidermis, tongue, submandibular gland, bone, and cortical tubules in the newborn mouse kidney (refSchutze/Jakob; Kireeva/Lau). The expression of Cyrόl in adult rat tissues was analyzed (Figure 2). In animals subjected to sham surgery, Cyrόl was expressed in lung, and at very low levels in heart, kidney, and skeletal muscle. Following renal ischemia, the abundance of Cyrόl mRNA was increased markedly in the kidney, whereas there was no increase in heart, lung, and muscle. Cyrόl mRNA was not detected in spleen and liver.

Regulation of Cyrόl protein after ischemia. To determine if the amount of Cyrόl protein is increased after injury, Western analysis using an affinity purified anti- peptide antibody to Cyrόl was performed. The antibody detected several non-specific bands in normal and 4 hour reperfused kidney (Figure 3; first and last lanes). However, partial purification of Cyrόl using heparin beads followed by a 150 mM NaCl wash yielded a doublet in normal kidney, and an increased expression of the lower molecular weight species following ischemia. By washing the beads under more stringent conditions (400 mM NaCl), a single band in either normal or 4 hour reperfused kidney could be obtained. Elution with 800 mM NaCl prior to boiling in Lamelli buffer slightly increased the recovery of Cyrόl. Using these optimized conditions, the appearance of Cyrόl following 40 min of bilateral renal ischemia was studied. Cyrόl was barely detectable in normal and sham-operated animals, increased to detectable levels at 30 min, peaked at 8 hours after ischemia, and was still expressed at high levels 24 hours after ischemia.

Induction of Cyrόl in distant organs after renal ischemia. To determine whether Cyrόl is induced in distant organs after renal ischemia, Cyrόl was measured in rat heart, lung, kidney, spleen, and muscle 6 hrs after sham or bilateral renal ischemia (Figure 5). Cyrόl was induced mainly in kidney, with smaller increases seen in heart and lung tissue samples.

Detection of Cyrόl in urine and serum after ischemia. Since Cyrόl is a secreted protein and apparently produced in the proximal tubule following renal ischemia, urine and serum were examined following renal injury to determine whether Cyrόl could be detected. Heparin bead affinity purified Cyrόl was not detected in normal urine (Figure 7), or in urine from volume depleted rats (Figure 8), but was detected in the first 24 hour urine sample after renal ischemia (Figure 7). Urine also contained a number of non-specific higher molecular weight bands; however, only the 40 kDa band was blocked with Cyrόl peptide (Figures 7 and 8, bottom). The temporal appearance of Cyrόl in urine was then examined. Heparin bead affinity purified Cyrόl was detected as early as 3-6 hrs after renal ischemia (Figure 8). Urinary Cyrόl appeared to be maximal at 6-9 hrs after renal ischemia but was detectable in all the urine collections during the first 24 hrs.

Kit and method for enzyme linked immunosorbent assay (ELISA) for the detection of naturally occurring Cyrόl in cell culture supernatants or complex biological fluids. Materials: The present kit includes enough reagents for at least one 96-well ELISA plate. The standard and the samples should be run in duplicate. In a specific, non-limiting example, the kit includes the following materials: Microtiter plate: One precoated and saturated 96-well microtiter ELISA plate, lyophilized and sealed with foil, removable 16- well racks Plate sealer/Manual: Resealable bag containing two adhesive strips and manual Wash buffer: 30 ml of 20x concentrate (lx Wash buffer: 50mM Tris-Cl, 0.2%

TWEEN 20®, pH 8.0) Assay buffer: 10 ml of 1 Ox concentrate

Sample diluent: 12 ml of lx diluent

Cyrόl standard: Two vials containing όng lyophilized human or mouse Cyrόl

Biotinylated detector: One vial containing 60μl pre-diluted rabbit IgG Streptavidin enzyme: One vial containing 120μl pre-diluted poly-HRP conjugated streptavidin Color reagent: 12 ml one-component TMB (tetramethyl-benzidine) solution

Stop solution: 12 ml 1M Sulphuric acid (H₂SO4)

Other materials used for detecting Cyrόl can include the following: Multichannel or repeating pipettes, Pipettes capable of accurately measuring 1-1000 μl, Orbital shaker, Clean 10- 15ml serological tubes and Eppendorf tubes for preparation of working dilutions, 96-well microtiter plate reader with 450nm and 650nm filters, Distilled water, and

Computerized data plotting or graph paper for manual plotting of data.

Method:

Pre-coated mouse monoclonal antibodies are used to capture Cyrόl from the samples. A biotinylated, polyclonal rabbit antiserum, which is incubated together with the samples, is used to detect bound cyrόl . After incubation with peroxidase conjugated streptavidin, the color developing reagent is added. Horseradish peroxidase catalyzes the oxidation of tetramethyl-benzidine (TMB) into a blue oxidation product of TMB. By addition of 1M H₂SO4, the reaction is stopped and the color changes to yellow. Manual plate washing

Washing and complete removal of all liquid at the end of each incubation step is very important to obtain low background values. The following washing procedure is recommended:

1. Remove existing fluid from each well by flicking the plate over a sink.

2. Blot the plate on clean paper towels.

3. Forcefully pipette 250μl diluted wash buffer into each well.

4. Repeat steps 1-3 twice.

5. Always remove wash buffer immediately. Do not incubate plate in wash buffer.

Preparation of reagents and dilutions

Reagents supplied as small volumes should be spun down before opening the tubes to avoid loss of reagents. Warm up buffers to room temperature before use.

1. Wash and assay buffer: Dilute lOx assay buffer concentrate and 20x wash buffer concentrate with distilled water. 2. Microtiter plate: Unpack ELISA plate and remove foil seal. Reconstitute the plate by pipetting lOOμl lx wash buffer into each well, wait 5 min, flick and blot the plate. Use plate sealer to avoid drying of plate.

3. Standard: Reconstitute Cyrόl by addition of 300μl assay buffer to make up 20ng/ml. Mix well. Serial dilutions are prepared within the wells. The reconstituted standard should not be stored for longer than 24h at 4°C.

4. Biotinylated detector: Dilute 1/100 in assay buffer (final volume 6ml).

5. Streptavidin enzyme: Dilute 1/100 in assay buffer (final volume 12ml).

Half of the volume of the solutions is needed for working with a half plate. A new lyophilized standard protein should be used every time.

Assay procedure

1. Start with the reconstituted plate. Remove the plate sealer and add lOOμl sample diluent in duplicate to the standard wells (A1/A2 to H1/H2).

2.. Add sample diluent to the sample wells. Samples should be measured diluted V or 1/4. For Vi dilution, add 50μl diluent to all sample wells; for 1/4 dilution add 75μl diluent to all sample wells (Some samples may be diluted higher, e.g. 1/8).

3. Add 1 OOμl of the reconstituted standard to wells H1/H2. Prepare Vi dilutions within the wells by mixing and pipetting lOOμl to wells G1/G2, F1/F2, ..., B1/B2. Discard lOOμl from wells B1/B2. Wells A1/A2 are background controls. Do not add standard protein to wells A1/A2. Avoid touching the bottom of the wells with the pipette-tips.

4. Add samples to the sample wells. Use 50μl of the samples for V dilution and 25 μl of the samples for 1/4 dilution.

5. Add 50μl biotin-conjugate (diluted 1/100 in assay buffer) to each well including the background controls (A1/A2). Seal the plate and incubate for 2h at room temperature.

6. Wash plate four times. 7. Add lOOμl streptavi din-enzyme (diluted 1/100) in assay buffer) to each well. Seal plate and incubate for lh at room temperature.

8. Wash plate four times.

9. Add lOOμl TMB-substrate solution to each well. Allow the blue color to develop for 10-30 minutes. DO NOT shake plate during this incubation step. Stop the reaction by adding 50μl stop-solution to each well. The blue color turns to yellow as a result of the pH shift.

10. Measure plate in a 96-well microplate reader using 450nm as measuring and 650nm as reference wavelength.

Calculation of results

Plot the standard curve on semi-logarithmic paper. Known concentrations of Cyrόl are plotted on the log-scale (X-axis), the corresponding OD is plotted on the linear scale. The concentrations of Cyrόl in unknown samples may be determined by plotting the sample OD on the Y-axis and drawing a horizontal line that intersects with the standard curve. A vertical line dropped from the point of intersection with the standard curve to the X-axis intersects the X-axis at the point of the concentration of the unknown sample. Multiply this value with the dilution factor of the sample to get the original concentration of the undiluted sample.

Although the present compositions and processes have been described with reference to specific details of certain embodiments thereof, it is not intended that such details should be regarded as limitations upon the scope of the invention except as and to the extent that they are included in the accompanying claims.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. REFERENCES

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Claims

What is claimed is:

1. A method of diagnosing acute renal failure in a subject, comprising detecting an elevated level of Cyrόl in a biological sample from the subject, whereby the detection of the elevated level of Cyrόl diagnoses the presence of acute renal failure in the subject.

2. The method of claim 1, wherein the subject is a mammal.

3. The method of claim 2, wherein the mammal is a human.

4. The method of claim 1, wherein the renal failure is caused by ischemia.

5. The method of claim 4, wherein the ischemia is caused by hypotension.

6. The method of claim 1 , wherein the sample is selected from the group consisting of blood and urine.

7. The method of claim 1, wherein the detection occurs from about 30 minutes to about 48 hours after acute renal failure begins.

8. The method of claim 7, wherein the detection occurs from about 2 hours to about 40 hours after acute renal failure begins.

9. The ethod of claim 8, wherein the detection occurs from about 3 hours to about 32 hours after acute renal failure begins.

10. The method of claim 9, wherein the detection occurs from about 6 hours to about 24 hours after acute renal failure begins.

11. A method of treating acute renal failure in a subject diagnosed with acute renal failure, comprising administering to the subject an effective amount of Cyrόl in a pharmaceutically acceptable carrier.

12. The method of claim 11, wherein the Cyrόl is administered parenterally.

13. A kit for detecting Cyrόl in a biological sample, comprising: a) a capture surface for specifically capturing Cyrόl; b) a reagent for detecting Cyrόl on the capture surface; and c) a Cyrόl standard.

14. The kit of claim 13, wherein the capture surface is a microtiter plate.

15. The kit of claim 13, wherein the capture surface is heparin-BSA.

16. The kit of claim 13, wherein the capture surface comprises Cyrόl -specific monoclonal antibodies.

17. The kit of claim 13, wherein the capture surface comprises Cyrόl -specific polyclonal antibodies.

18. The kit of claim 13, wherein the reagent for detecting Cyrόl is biotinylated.

19. The kit of claim 18, wherein the biotinylated reagent is an antibody.

20. The kit of claim 13, further comprising a wash buffer.

21. The kit of claim 13, further comprising an assay buffer.

22. The kit of claim 13, further comprising a sample diluent.

23. The kit of claim 13, further comprising streptavidin.

24. The kit of claim 13, further comprising a color reagent.

25. The kit of claim 24, wherein the color reagent is tetramethyl-benzidine solution.

26. The kit of claim 13, further comprising a stop solution.

27. The kit of claim 26, wherein the stop solution is sulphuric acid.