HK1213320B - Methods and compositions for diagnosis and prognosis of renal injury and renal failure - Google Patents

Abstract

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect one or more markers selected from the group consisting of soluble p-selectin, protein NOV homolog, soluble epidermal growth factor receptor, netrin-4, haptoglobin, heat shock protein beta-1, alpha- 1 -antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, active caspase-3, and soluble platelet endothelial cell adhesion molecule as diagnostic and prognostic biomarkers in renal injuries.

Description

Methods and compositions for diagnosis and prognosis of renal injury and renal failure

The present application is a divisional application of a chinese national phase application having an application number of 200980140805.3 after entering the chinese national phase of PCT application having an international application number of PCT/US2009/055449 filed as "aspettem pharmaceutical company", entitled "method and composition for diagnosing and prognosing renal injury and renal failure.

The present invention claims priority of U.S. provisional patent application No. 61/092,733 filed on 28/8/2008, priority of U.S. provisional patent application No. 61/092,905 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/092,912 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/092,926 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/093,154 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/093,247 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/093,249 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/093,262 filed on 29/8/2008, priority of U.S. provisional patent application No. 61/093,263 filed on 29/8/29/2008, priority of, Priority of U.S. provisional patent application No. 61/093,264 filed on day 8/29 of 2008, priority of U.S. provisional patent application No. 61/093,266 filed on day 8/29 of 2008, priority of U.S. provisional patent application No. 61/093,244 filed on day 8/29 of 2008, priority of U.S. provisional patent application No. 61/093,272 filed on day 8/29 of 2008, the entire contents of each of the aforementioned U.S. patent applications, including all tables, figures, and claims, are hereby incorporated by reference.

Background

The background of the invention discussed below is for the purpose of assisting the reader in understanding the invention only and is not admitted to be a description of or constitute prior art to the present invention.

The kidneys are responsible for the drainage of water and solutes from the body. Renal functions include maintaining acid-base balance, regulating electrolyte concentration, controlling blood volume, and regulating blood pressure. Thus, loss of renal function due to injury and/or disease results in a significant amount of morbidity and mortality. A detailed discussion of renal injury is provided in Harrison's Principles of Internalmedicine,17th Ed., McGraw Hill, New York, pp 1741-1830, the entire contents of which are incorporated herein by reference. The renal disease and/or renal injury may be acute or chronic. Acute and chronic kidney diseases are described below (Current Medical Diagnosis & Treatment 2008,47th Ed, McGraw Hill, New York, pp 785-: "acute renal failure is a deterioration in renal function that lasts from several hours to several days, resulting in the retention of nitrogenous waste products (e.g., urea nitrogen) and creatinine in the blood. These retained substances are called azotemia. Chronic renal failure (chronic kidney disease) is caused by abnormal loss of renal function that lasts for months to years.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) is a sudden (typically detected within about 48 hours to 1 week) decrease in glomerular filtration. This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) waste products and non-nitrogenous waste products normally excreted by the kidneys, a decrease in urine output, or both. ARF is reported to be associated with admission rates of about 5%, cardiopulmonary bypass surgery of 4-15% and intensive care admission rates as high as 30%. ARF can be classified as prerenal, intrinsic renal, or postrenal in causal relationship. Intrarenal diseases can be further classified into glomerular abnormalities, tubular abnormalities, interstitial abnormalities, and vascular abnormalities. The major causes of ARF are described in the following table, which is available from Merck Manual,17th ed., chapter 222 and incorporated herein by reference in its entirety.

In the case of ischemic ARF, the etiology of the disease can be divided into four stages. During the initial phase, which lasts from several hours to several days, the decrease in renal perfusion evolves into injury. Glomerular ultrafiltration is reduced, fluid filtration is reduced due to debris in the tubules, and filtrate rewet through the damaged epithelium occurs. Renal injury can be mediated at this stage by reperfusion of the kidney. The initiation phase is followed by an extension phase characterized by continued ischemic injury and inflammation, and may involve endothelial injury and vascular congestion. During the maintenance phase, which lasts for 1 to 2 weeks, renal cell injury occurs and glomerular filtration and urine output reach a minimum. This is followed by a recovery phase in which the renal epithelial cells are repaired and GFR gradually recovers. Despite this, the survival rate of subjects with ARF is only about 60%.

Acute kidney injury caused by radiocontrast agents (also known as contrast agents) and other nephrotoxins such as cyclosporine, antibiotics including aminoglycosides, and anticancer drugs such as cisplatin, appears to last from several days to about a week. Contrast-induced nephropathy (CIN, AKI induced by radiocontrast agents) is believed to be caused by intrarenal vasoconstriction and by the production of reactive oxygen species that are directly toxic to renal tubular epithelial cells. CIN typically appears as an acute (onset within 24-48 hours) but increases reversibly (peak 3 to 5 days, breakdown within a week) in blood urea nitrogen and serum creatinine.

A commonly reported criterion for determining and detecting AKI is a sudden (typically within about 2-7 days or during hospitalization) increase in serum creatinine. Although the use of increases in serum creatinine to determine and detect AKI is an established approach, the extent of serum creatinine increase and the time of measurement to determine AKI vary significantly in publications. Traditionally, a relatively large increase in serum creatinine (e.g., 100%, 200%, at least 100%) relative to a 2mg/dL value was used to determine AKI. However, recent trends have tended to use smaller serum creatinine increases to determine AKI. The relationship between serum creatinine, AKI and associated health risks is reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14: 265. RTH. -. 270,2005 and Chertow et al, J Am Soc Nephrol 16: 3365. RTH. -. 3370,2005, the entire contents of which are incorporated herein by reference. As described in these publications, acute worsening renal function (AKI) is known to be associated with a very small increase in serum creatinine and an increased risk of death and other adverse outcomes. These increases may be determined as relative (percent) values or nominal values. It has been reported that a relative increase in serum creatinine of as little as 20% relative to the pre-injury value is indicative of acute renal function deterioration (AKI) and increased health risk, but more commonly the value determining AKI and increased health risk is a relative increase of at least 25%. Nominal increases of only 0.3mg/dL, as little as 0.2mg/dL or even 0.1mg/dL have been reported to indicate an increased risk of renal function deterioration and death. Different periods of serum creatinine rise to these thresholds have been used to determine AKI, e.g., 2 days, 3 days, 7 days or variable periods defined as the time of patient admission or at intensive care. These studies indicate that there is no specific threshold (or period of time) for serum creatinine rise for worsening renal function or AKI, but rather a continuous increase in risk with an increase in the degree of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605,2004, incorporated herein by reference) investigated the increase and decrease in serum creatinine. Patients with mild decreases in serum creatinine-0.1 to-0.3 mg/dL after cardiac surgery have the lowest mortality rates. Patients with a greater decline in serum creatinine (greater than or equal to-0.4 mg/dL) or any increase in serum creatinine had greater mortality. These findings led the authors to conclude that even very minor changes in renal function (detected by small creatinine changes within 48 hours of surgery) severely affected the patient's outcome. Efforts were made to achieve a unified classification system in using serum creatinine to determine AKI in clinical trials and clinical practice, Bellomo et al, Crit care.8(4): R204-12,2004, incorporated herein by reference, proposing the following classifications to classify AKI patients:

"hazardous": serum creatinine increased 1.5 fold relative to baseline, or urine volume less than 0.5ml/kg body weight/hour for six hours;

"Damage": serum creatinine increased 2-fold relative to baseline, or urine volume less than 0.5 ml/kg/hr for 12 hours;

"exhaustion": a 3-fold increase in serum creatinine over baseline, or creatinine >355 μmol/l (and an increase greater than 44) or urine output of less than 0.3ml/kg/hr for 24 hours or anuria for at least 12 hours;

and includes two clinical outcomes

"loss": there is a continuing need for renal replacement therapy for more than four weeks.

"ESRD": late stage of kidney disease-dialysis is required for more than 3 months.

These criteria became RIFLE criteria, which provided a useful clinical tool to classify renal conditions. The RIFLE standard provides a uniform definition of AKI, which has been validated in various studies, as discussed in Kellum, Crit.Care Med.36: S141-45,2008 and Ricci et al, Kidney Int.73,538-546,2008 (each incorporated herein by reference).

Recently, Mehta et al, crit.Care 11: R31(doi:10.1186.cc5713),2007 (incorporated herein by reference) proposed the following similar classifications for AKI patients, which have improved RIFLE:

"stage I": an increase in serum creatinine of greater than or equal to 0.3mg/dL (≧ 26.4 μmol/L) or an increase of greater than or equal to 150% (1.5-fold) relative to baseline or a urine output of less than 0.5 mL/kg/hr for greater than 6 hours;

"stage II": serum creatinine increased greater than 200% (2-fold) or urine output less than 0.5 mL/kg/hr for greater than 12 hours relative to baseline;

"stage III": serum creatinine increased greater than 300% (3 fold) relative to baseline or serum creatinine greater than or equal to 354 μmol/L with an acute increase of at least 44 μmol/L or urine output of less than 0.3mL/kg/hr for 24 hours or anuria for 12 hours.

The CIN co-working group (McCollough et al, Rev Cardiovasc Med.2006; 7(4):177- & 197, incorporated herein by reference) determined contrast-induced nephropathy (one of the AKIs) using a 25% increase in serum creatinine. Although different groups proposed slightly different criteria for serum creatinine to detect AKI, it is common that small changes in serum creatinine (e.g., 0.3mg/dL or 25%) are sufficient to detect AKI (worsening renal function) and that the degree of change in serum creatinine is an indicator of AKI severity and risk of death,

although continuous measurements of serum creatinine over several days are an acceptable method for detecting and diagnosing AKI and are considered one of the most important tools for assessing AKI patients, serum creatinine is generally considered to have several limitations in diagnosing, evaluating, and monitoring AKI patients. The period of time for serum creatinine to rise to a value considered diagnostic for AKI (0.3 mg/dL or 25% rise) may be 48 hours or more, depending on the definition used. Because cell damage can occur in AKI for a period of several hours, measuring the rise in serum creatinine at 48 hours or more can be a later indicator of damage and, therefore, dependence on serum creatinine can delay diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact renal status, and treatment requires an indicator of the most severe stage of AKI when renal function is rapidly changing. Some patients with AKI will recover completely, some will require dialysis (short or long term) and some will have other adverse consequences including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, the cause of AKI (prerenal, intrarenal, postrenal obstruction, atheroembolic, etc.) or the type or location of intrarenal disease (e.g., renal tubules, glomeruli, or renal space) cannot be distinguished. Similarly, urine output is defined, which is known to be critical for the management and treatment of patients with AKI.

These limitations underscore the need for better methods of detecting and assessing AKI, particularly at the early and subclinical stages, as well as at the late stages where renal recovery and repair can occur. Moreover, there is a need for better identification of patients at risk for AKI.

Summary of The Invention

As described herein, measurement of one or more markers selected from the group consisting of soluble p-selectin, a protein homologous to NOV, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial cell adhesion molecule (collectively referred to herein as "kidney injury markers" and individually as "kidney injury markers") can be used to diagnose, prognose, risk stratification, staging, monitor, classify, and determine a further diagnostic and therapeutic regimen for a subject suffering from an injury to renal function, reduced renal function, and/or acute renal failure (also referred to as acute renal injury).

These kidney injury markers can be used alone or in combination, including a plurality of kidney injury markers, for risk stratification (i.e., identifying an injury to renal function, a gradual decrease in renal function, a future progression to ARF, a future improvement in renal function, etc.) of a subject at risk, diagnosing an existing disease (i.e., identifying a subject with an injury to renal function, identifying a subject with a gradual decrease in renal function, a subject with a progression to ARF, etc.), for monitoring deterioration or improvement in renal function, for predicting a future medical outcome, such as an improvement or deterioration in renal function, a decreased or increased risk of death, a decreased or increased risk of a subject requiring renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or kidney transplantation), a decreased or increased risk of a subject recovering from an injury to renal function, a decreased or increased risk of a subject recovering from ARF, etc.), A reduction or increase in the risk of the subject developing end-stage renal disease, a reduction or increase in the risk of the subject developing chronic renal failure, a reduction or increase in the risk of the subject having a rejection of transplanted kidneys, and the like.

In a first aspect, the present invention relates to methods for assessing the status of a kidney in a subject comprising performing an assay configured to detect one or more kidney injury markers of the present invention in a sample of bodily fluid obtained from the subject, then correlating the results of the assay, e.g., measured concentrations of one or more markers selected from the group consisting of soluble p-selectin, a protein homologous to NOV, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3) and soluble platelet endothelial adhesion molecule, to the status of the kidney.

In some embodiments, the methods of assessing renal status described herein are risk stratification methods of a subject, i.e., assigning one or more future changes in renal status, if any, to the subject. In some embodiments, the analysis results are correlated with these future changes. The following are preferred embodiments of risk stratification.

In preferred risk stratification embodiments, the methods comprise determining the risk of a future injury to renal function in the subject, and correlating the results of the analysis with a likely injury to renal function. For example, each measured concentration may be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of a future injury to renal function corresponds to a subject having a measured concentration greater than a threshold relative to a likelihood of a corresponding measured concentration being less than the threshold. For a "negative going" kidney injury marker, an increased likelihood that an injury to renal function will result corresponds to a patient having a measured concentration less than a threshold relative to a likelihood that the corresponding measured concentration is greater than the threshold.

In other preferred risk stratification embodiments, the methods comprise determining the risk of future reduced renal function in the subject and correlating the analytical structure with the likelihood of such reduced renal function. For example, each measured concentration may be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of future reduced renal function corresponds to a subject having a measured concentration above a threshold relative to a likelihood of a corresponding measured concentration below the threshold. For a "negative going" kidney injury marker, an increased likelihood of future reduced renal function corresponds to a subject having a measured concentration below a threshold relative to a likelihood that the corresponding measured concentration is below the threshold.

In other preferred risk stratification embodiments, the methods comprise determining the likelihood of a future improvement in renal function in the subject, and correlating the results of the analysis with the likelihood of such improvement in renal function. For example, each measured concentration may be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of future improvement in renal function corresponds to a subject having a measured concentration below a threshold relative to a likelihood that the corresponding measured concentration is above the threshold. For a "negative going" kidney injury marker, an increased likelihood of future improvement in renal function corresponds to a subject having a measured concentration above a threshold relative to a likelihood that the corresponding measured concentration is below the threshold.

In other preferred risk stratification embodiments, the methods comprise determining the risk of the subject developing ARF and correlating the outcome with the likelihood of developing ARF. For example, each test concentration may be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of progression to ARF corresponds to a subject with a measured concentration above a threshold relative to a likelihood that the corresponding measured concentration is below the threshold. For a "negative going" kidney injury marker, an increased likelihood of developing ARF corresponds to a subject having a measured concentration below a threshold relative to a likelihood that the measured concentration is above the threshold.

In other preferred risk stratification embodiments, the methods comprise determining the risk of outcome of the subject and correlating the outcome of the analysis with the likelihood of occurrence of a clinical outcome associated with the renal injury from which the subject is suffering. For example, each measured concentration may be compared to a threshold value. For a "positive going" kidney injury marker, an increased likelihood of one or more of the following outcomes, relative to the likelihood of the corresponding measured concentration being below the threshold, corresponds to a subject whose measured concentration is above the threshold: acute kidney injury, progression to a worsening stage of AKI, death, need for renal replacement therapy, need for withdrawal of nephrotoxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, and the like. For a "negative going" kidney injury marker, an increased likelihood of one or more of the following results is assigned to the subject for a measured concentration below a threshold relative to the likelihood assigned for a measured concentration above the threshold: acute kidney injury, progression to a worsening stage of AKI, death, need for renal replacement therapy, need for withdrawal of nephrotoxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, and the like.

In these risk stratification embodiments, the corresponding likelihood or risk is preferably a more or less relevant event occurring within 180 days of obtaining a body fluid sample from a subject. In particularly preferred embodiments, the likelihood or risk addressed is associated with a relevant event occurring within a relatively short period of time, e.g., 18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 ethylamine, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours or less. The risk of obtaining a body fluid sample from a subject at 0 hour corresponds to the current diagnosis.

In a preferred risk stratification embodiment, the subject is selected for risk stratification based on one or more known prerenal, intrinsic renal, or postrenal risk factors that are pre-existing in the subject. For example, subjects undergoing or having undergone major vascular surgery, coronary artery bypass surgery, or other cardiac surgery, subjects with congenital congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, or sepsis, or subjects exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, heme, myosin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin, are all preferred subjects for monitoring risk according to the methods described herein. This list is not meant to limit the invention. Herein, "pre-existing" means that a risk factor is present when a body fluid sample is obtained from a subject. In a particularly preferred embodiment, the subject is selected for risk stratification based on an existing diagnosis of impaired renal function, reduced renal function, or ARF.

In other embodiments, the methods described herein for assessing renal status are methods for diagnosing renal injury in a subject, i.e., assessing whether a subject has impaired renal function, reduced renal function, or ARF. in these embodiments, the results of the assessment, e.g., the measured concentration of one or more markers selected from the group consisting of soluble p-selectin, a protein NOV homolog, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet endothelial adhesion molecule, are correlated with whether a change in renal status has occurred.

In preferred diagnostic embodiments, the methods comprise diagnosing the occurrence or nonoccurrence of an injury to renal function and correlating the results of the analysis with the occurrence or nonoccurrence of such an injury. For example, each measured concentration is compared to a threshold value. For a positive marker, an increased likelihood of developing an injury to renal function corresponds to a subject having a measured concentration greater than a threshold value (relative to a likelihood that the corresponding measured concentration is below the threshold value); alternatively, an increased likelihood of the nonoccurrence of an injury to renal function may correspond to the subject when the measured concentration is below the threshold (relative to a likelihood corresponding when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of developing an injury to renal function corresponds to the subject when the measured concentration is below the threshold (relative to the likelihood corresponding to when the measured concentration is above the threshold); alternatively, an increased likelihood of not developing an injury to renal function corresponds to the subject when the measured concentration is above the threshold (relative to a likelihood corresponding when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, the methods comprise diagnosing the occurrence or nonoccurrence of reduced renal function and correlating the results of the analysis with the occurrence or nonoccurrence of an injury causing the reduction in renal function. For example, each measured concentration may be compared to a threshold value. For a positive marker, an increased likelihood of an injury causing reduced renal function occurring corresponds to the subject when the measured concentration is greater than the threshold (relative to the likelihood corresponding to the measured concentration being below the threshold); alternatively, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may correspond to the subject when the measured concentration is below the threshold (relative to the likelihood corresponding to when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of an injury causing reduced renal function occurring corresponds to the subject when the measured concentration is below the threshold (relative to a likelihood corresponding to when the measured concentration is above the threshold); alternatively, an increased likelihood of not developing an injury that causes reduced renal function may correspond to the subject when the measured concentration is above the threshold (relative to a likelihood corresponding when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, the methods comprise diagnosing the occurrence of ARF and correlating the results of the analysis with the occurrence of damage that causes ARF. For example, each measured concentration may be compared to a threshold value. For positive markers, an increased likelihood of developing damage that causes ARF corresponds to the subject when the measured concentration is greater than the threshold (relative to the likelihood of corresponding measured concentrations being below the threshold); alternatively, an increased likelihood of no occurrence of injury causing ARF may correspond to the subject when the measured concentration is below the threshold (relative to the likelihood of corresponding measured concentrations being above the threshold). For a negative going marker, an increased likelihood of developing damage that causes ARF corresponds to the subject when the measured concentration is below the threshold (relative to a likelihood corresponding to when the measured concentration is above the threshold); alternatively, an increased likelihood of not developing an injury that causes ARF may correspond to the subject when the measured concentration is above the threshold (relative to corresponding to a likelihood when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, the methods comprise diagnosing the subject as in need of renal replacement therapy and correlating the results of the analysis with the need for renal replacement therapy. For example, each measured concentration is compared to a threshold value. For a positive marker, an increased likelihood of an injury occurring that produces a need for renal replacement therapy corresponds to the subject when the measured concentration is greater than the threshold (relative to the likelihood when the corresponding measured concentration is below the threshold); alternatively, an increased likelihood of the nonoccurrence of an injury that produces a need for renal replacement therapy may correspond to the subject when the measured concentration is below the threshold (relative to the likelihood of the corresponding measured concentration being above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury that produces a need for renal replacement therapy corresponds to the subject when the measured concentration is below the threshold (relative to a likelihood corresponding when the measured concentration is above the threshold); alternatively, an increased likelihood of not developing an injury that results in need of renal replacement therapy may correspond to the subject when the measured concentration is above the threshold (relative to a likelihood corresponding when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, the methods comprise diagnosing the subject as in need of kidney transplantation and correlating the results of the analysis with the need for kidney transplantation. For example, each measured concentration is compared to a threshold value. For positive markers, an increased likelihood of the occurrence of an injury that requires kidney transplantation corresponds to the subject when the measured concentration is greater than the threshold (relative to the likelihood when the corresponding measured concentration is below the threshold); alternatively, an increased likelihood of nonoccurrence of an injury that would result in need of kidney transplantation may correspond to the subject when the measured concentration is below the threshold (relative to the likelihood corresponding to when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury that produces a need for kidney transplantation corresponds to the subject when the measured concentration is below the threshold (relative to a likelihood corresponding when the measured concentration is above the threshold); alternatively, an increased likelihood of not developing an injury that requires kidney transplantation may correspond to the subject when the measured concentration is above the threshold (relative to a likelihood corresponding when the measured concentration is below the threshold).

In other embodiments, the methods described herein for assessing renal status are methods for monitoring renal injury in a subject, i.e., assessing whether renal function is improving or worsening in a subject who has suffered an injury, decrease or arf, in which embodiments the assay results, e.g., the measured concentration of one or more markers selected from the group consisting of soluble p-selectin, a protein homologous to NOV, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3) and soluble platelet endothelial adhesion molecule, are correlated with whether a change in renal status occurs.

In preferred monitoring embodiments, the methods comprise monitoring the renal status of a subject having an impairment of renal function, and correlating the results of the analysis to whether the renal status of the subject has changed. For example, the measured concentration is compared to a threshold value. For a positive marker, when the measured concentration is above a threshold, a worsening renal function may correspond to a subject; alternatively, an improvement in renal function may correspond to the subject when the measured concentration is below the threshold. For a negative going marker, a worsening renal function may correspond to a subject when the measured concentration is below the threshold; alternatively, the improvement in renal function may correspond to the subject when the measured concentration is above the threshold.

In other preferred monitoring embodiments, the methods comprise monitoring the renal status of a subject having reduced renal function and correlating the results of the analysis to whether the renal status of the subject has changed. For example, the measured concentration is compared to a threshold value. For a positive marker, when the measured concentration is above a threshold, a worsening renal function may correspond to a subject; alternatively, an improvement in renal function may correspond to the subject when the measured concentration is below the threshold. For a negative going marker, a worsening renal function may correspond to a subject when the measured concentration is below the threshold; alternatively, the improvement in renal function may correspond to the subject when the measured concentration is above the threshold.

In other preferred monitoring embodiments, the methods comprise monitoring the renal status of a subject having acute renal failure, and correlating the results of the analysis to whether the renal status of the subject has changed. For example, the measured concentration is compared to a threshold value. For a positive marker, when the measured concentration is above a threshold, a worsening renal function may correspond to a subject; alternatively, an improvement in renal function may correspond to the subject when the measured concentration is below the threshold. For a negative going marker, a worsening renal function may correspond to a subject when the measured concentration is below the threshold; alternatively, the improvement in renal function may correspond to the subject when the measured concentration is above the threshold.

In other preferred monitoring embodiments, the methods comprise monitoring the renal status of the subject at risk for an injury to renal function caused by the pre-renal, intra-renal, or post-renal presence of one or more known risk factors for ARF, and correlating the results of the analysis with whether the renal status of the subject has changed. For example, the measured concentration is compared to a threshold value. For a positive marker, when the measured concentration is above a threshold, a worsening renal function may correspond to a subject; alternatively, an improvement in renal function may correspond to the subject when the measured concentration is below the threshold. For a negative going marker, a worsening renal function may correspond to a subject when the measured concentration is below the threshold; alternatively, the improvement in renal function may correspond to the subject when the measured concentration is above the threshold.

In other embodiments, the methods of assessing renal status described herein are methods for classifying a subject's renal injury, i.e., determining whether the subject's renal injury is pre-, intra-, or post-renal, and/or further subdividing these classes into subclasses, such as acute tubular injury, acute glomerulonephritis, acute tubulointerstitial nephritis, acute vascular nephropathy, or osmotic disease, and/or corresponding likelihood of developing a particular stage of RIFLE, in these embodiments, the results of the analysis, e.g., the results of the analysis of one or more markers selected from the group consisting of soluble p-selectin, a protein NOV homolog, a soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, β -1, α -1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble tumor necrosis factor 2-adhesion-caspase, heat shock protein adhesion-3-like enzyme (HSP), and the following preferred embodiments are platelet adhesion-3 adhesion-like molecules.

In preferred classified embodiments, these methods comprise determining whether the renal injury in the subject is pre-, intra-or post-renal; and/or further classifying these into subclasses, such as acute tubular injury, acute glomerulonephritis, acute tubulointerstitial nephritis, acute vascular renal disease, or osmotic disease; and/or corresponding the likelihood to the subject that will progress to a particular RIFLE stage, and correlating the results of the analysis to the subject's classification of injury. For example, the measured concentration is compared to a threshold value, and when the measured concentration is higher than the threshold value, corresponds to a specific category; alternatively, a different category may be assigned to the subject when the measured concentration is below the threshold.

Various methods may be used by those skilled in the art to achieve the desired threshold for these methods. For example, the threshold may be determined by selecting 75th, 85 th that represents the percentage of renal injury markers measured in normal subjects^th，90^th，95^thOr 99th concentration, was determined from a population of normal subjects. Alternatively, the threshold may be determined from a "diseased" population of subjects, e.g., those suffering from an injury or having a predisposition to suffer an injury (e.g., progression to ARF or some other clinical outcome, such as death, dialysis, kidney transplantation, etc.), by selecting 75th, 85 th representative of the percentage of kidney injury markers measured in the diseased subject^th，90^th，95^thOr 99th concentration. In another alternative embodiment, the threshold value may be determined from a previously measured renal injury marker for the same subject; that is, a temporary change in the level of a kidney injury marker in a subject can be used to correlate risk to the subject.

The foregoing discussion is not intended to suggest, however, that the kidney injury markers of the present invention must be compared to corresponding individual thresholds. Methods for combining the analysis results may include using multivariate log regression, log linear simulation, neural network analysis, n-of-m analysis, hierarchical decision analysis, calculating the proportion of markers, and the like. This list is not intended to be limiting. In these methods, the composition result determined by combining the individual markers can be processed as long as it is itself a marker; that is, a threshold value for the composition result as described herein can be determined for the individual marker, and the composition result for the individual patient is compared to the threshold value.

The ability to distinguish between specific tests of two populations can be established using ROC analysis. For example, a ROC curve established from a "first" subpopulation that is preset for one or more future changes in renal status and a "second" subpopulation that is not so preset and the area under the curve provides a measurement test quality can be used to evaluate the ROC curve. Preferably, the test described herein provides a ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95.

In some aspects, the measured concentration of one or more kidney injury markers or the composition of these markers can be treated as a continuous variable. For example, any particular concentration may translate into a corresponding likelihood of a subject having reduced renal function in the future, having a renal injury, being classified, and in an alternative embodiment, a threshold may provide an acceptable level of specificity and sensitivity, dividing the subject population into "bins," such as a "first" subpopulation (e.g., pre-placed in one or more future changes in renal function, having an injury, being classified, etc.) and a "second" subpopulation that is not pre-placed. The threshold is selected to separate the first and second populations by one or more of the following measures of accuracy:

an odd ratio of greater than 1, preferably at least about 2 or greater or about 5 or less, more preferably at least about 3 or greater or about 0.33 or less, still more preferably at least about 4 or greater or about 0.25 or less, even more preferably at least about 5 or greater or about 0.2 or less, and most preferably at least about 10 or greater or about 0.1 or less;

a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9, and most preferably at least about 0.95, with a corresponding sensitivity of greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, still more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably greater than about 0.95;

a sensitivity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, still more preferably at least about 0.8, even more preferably at least about 0.9 and most preferably about 0.95, with a corresponding specificity of greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, still more preferably greater than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9 and most preferably greater than about 0.95;

at least about 75% sensitivity, combining at least about 75% specificity;

a forward likelihood ratio (estimated as sensitivity/(1-specificity)) of greater than 1, at least about 2, more preferably at least about 3, still more preferably at least about 5, and most preferably at least about 10; or

The negative likelihood ratio (estimated as (1-sensitivity)/specificity) is less than 1, less than or equal to about 0.5, more preferably less than or equal to about 0.3, and most preferably less than or equal to about 0.1.

The term "about" for any of the measurements described herein above refers to +/-5% of the given measurement.

Multiple thresholds can be used to assess the renal status of a subject. For example, a "first" subpopulation that is predisposed to one or more future changes in renal status, the occurrence of injury, classification, etc., and a "second" subpopulation that is not predisposed may be combined into a single group. The group is then divided into three or more equal divisions (called tertiles, quartiles, quintiles, etc., based on the number of subdivisions). Odd ratios correspond to subjects based on falling into a reclassification. If one is considered to be divided into triplicates, the lowest or highest tertile may be used as a reference for comparison to other reclassifications. This reference reclassification corresponds to an odd ratio of 1. the second tertile corresponds to the odd ratio associated with the first tertile. That is, some of the second tertile may be 3 times more likely to suffer from one or one future change in renal status than some of the first tertile. The third tertile also corresponds to the odd scale associated with the first tertile.

In some embodiments, the assay method is an immunoassay. The antibodies used in these assays will specifically bind to the full-length kidney injury marker of interest, and may also bind to one or more polypeptides "associated" therewith, as that term is defined below. A variety of immunoassay formats are known to those of skill in the art. Preferred body fluid samples are selected from: urine, blood, serum, saliva, tears, and plasma.

The above method steps should not be disturbed to mean that the renal injury marker assay results are used for isolation in the methods described herein. However, other variables or other clinical markers may be included in the methods described herein. For example, methods of risk stratification, diagnosis, classification, monitoring, etc., may combine the results of the analysis with one or more measured subject variables selected from the group consisting of: demographic information (e.g., weight, gender, age, race), medical history (e.g., family history, type of surgery, pre-existing disease, such as aneurysm, congestive heart failure, preeclampsia, eclampsia, diabetes, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, species of toxin exposure, such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, heme, myosin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, body temperature, number of breaths), risk scores (APACHE score, predxt score, TIMI risk score for UA/NSTEMI, Framingham risk score), glomerular filtration rate, estimated glomerular filtration rate, urine production, serum or plasma creatinine concentration, blood glucose concentration, glucose concentration, glucose, Urine creatinine concentration, sodium excretion fraction, urine sodium concentration, ratio of urine creatinine to serum or plasma creatinine, urine specific gravity, urine isotonicity, ratio of urine urea nitrogen to plasma urea nitrogen, ratio of plasma BUN to creatinine, renal failure index estimated as urine sodium/(urine creatinine/plasma creatinine), serum or plasma Neutrophil Gelatinase (NGAL) concentration, urine NGAL concentration, serum or plasma cystatin C concentration, serum or plasma cardiac troponin concentration, serum or plasma BNP concentration, serum or plasma NTproBNP concentration, serum or plasma proBNP concentration. Other measures of renal function that can be combined with the results of one or more renal injury marker assays are described below and described in Harrison's Principles of Internal Medicine,17th Ed, McGraw Hill, New York, pages 1741-.

When more than one marker is measured, the separate markers may be measured in samples taken at the same time or may be determined by obtaining samples at different times (e.g., earlier or later). Separate markers may also be measured on the same or different body fluid samples. For example, one kidney injury marker can be measured in a serum or plasma sample and another kidney injury marker can be measured in a urine sample. Furthermore, the corresponding likelihood may combine the results of a single kidney injury marker assay with temporal changes in one or more additional variables.

In various related aspects, the invention also relates to devices and kits for carrying out the methods described herein. Suitable kits include reagents sufficient to perform an assay for at least one of the kidney injury markers, together with instructions for performing the threshold comparison.

In some embodiments, the reagents for performing these assays are provided in assay devices, and these assay devices may include kits. Preferred reagents may include one or more solid phase antibodies including antibodies that detect the binding of a desired biomarker target to a solid support. Under sandwich immunoassay conditions, these reagents may also include one or more detectably labeled antibodies, including antibodies that detect the binding of the desired biomarker target to the detectable label. Other optional elements provided as part of the analytical device are described below.

Detectable labels may include molecules that are themselves detectable (e.g., fluorophores, electrochemical labels, ecl (electrochemiluminescent) labels, metal chelates, colloidal metal particles, etc.) and molecules that are indirectly detected by the production of detectable reaction products (e.g., enzymes such as horseradish, peroxidase, alkaline phosphatase) or by using specific binding molecules that are themselves detectable (e.g., labeled antibodies bound to secondary antibodies, biotin, digoxigenin, maltose, oligonucleotide histidine, 2, 4-dinitrobenzene, phenyl arsenate, ssDNA, dsDNA).

The signal generated by the signal generating element may be represented using various optical, acoustic, and electrochemical methods known in the art. Examples of detection models include fluorescence, radiochemical detection, reflection, absorption, amperometric, conductance, impedance, interference measurements, ellipsometric measurements, and the like. In some of these methods, the solid phase antibody is coupled to a transducer (e.g., a diffraction grating, an electrochemical sensor, etc.) for generating a signal, while in other embodiments, the signal is generated by a transducer that is spatially separated from the solid phase antibody (e.g., a fluorometer using an excitation light source and an optical detector). This list is not meant to be limiting. Antibody-based biosensors may also be used to determine the presence and amount of an analyte, which optionally obviates the need for a label molecule.

Detailed Description

The present invention relates to methods and compositions for diagnosing, differentially diagnosing, risk staging, monitoring, classifying, and determining a treatment regimen for a patient having or at risk of developing an injury to renal function, reduced renal function, and/or acute renal failure by measuring one or more markers of renal injury, reduced renal function, and/or acute renal failure in various embodiments, a measured concentration of one or more markers, or one or more markers associated therewith, associated with a renal status of a subject is selected from the group consisting of soluble p-selectin, a protein homologous to protein NOV, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, leukocyte elastase, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 (and most preferably active caspase-3), and soluble platelet adhesion molecule.

For the purposes herein, the following definitions apply:

as used herein, an "injury to renal function" is a sudden (within 14 days, preferably within 7 days, more preferably within 72 hours, still more preferably within 48 hours) measurable decrease in measured renal function. Such an injury can be identified, for example, by a decrease in tubular filtration rate or an assessment of GRR, a decrease in urine output, an increase in serum creatinine, an increase in serum cystatin C, a need for renal replacement therapy, and the like. An "improvement in renal function" is a sudden (within 14 days, preferably within 7 days, more preferably within 72 hours, still more preferably within 48 hours) measurable increase in the time renal function is measured. Preferred methods for measuring and/or assessing GFR are described below.

As used herein, "reduced renal function" is a sudden (within 14 days, preferably within 7 days, more preferably within 72 hours, still more preferably within 48 hours) decrease in renal function, as identified by an absolute increase in serum creatinine of greater than or equal to 0.1mg/dl (. gtoreq.8.8. mu. mol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold over baseline), or a decrease in urine output (recorded oliguria of less than 0.5 ml/kg/h).

As used herein, "acute renal failure" or "ARF" is a sudden (within 14 days, preferably within 7 days, more preferably within 72 hours, still more preferably within 48 hours) decrease in renal function that is identified by an absolute increase in serum creatinine of greater than or equal to 0.3mg/dl (. gtoreq.26.4. mu. mol/L), a percentage increase in serum creatinine of greater than or equal to 50% (1.5 times baseline) or a decrease in urine output (recorded oliguria of less than 0.5ml/kg/h for 6 hours). The term is synonymous with "acute kidney injury" or "AKI".

In this regard, it will be understood by those skilled in the art that the signal obtained from the immunoassay is a direct result of the complex formed between one or more antibodies and the target biomolecule (i.e., analyte) and the polypeptide containing the requisite epitope attached to the antibody. However, such assays can detect full-length biomarkers and the assay results are expressed as the concentration of the relevant biomarker, the signal from the assay being the result of virtually all such "immunologically active" polypeptides present in the sample. Expression of biomarkers can also be determined by non-immunoassay methods, including protein measurements (e.g., dot blot, western blot, chromatography, mass spectrometry, etc.) and nucleic acid measurements (mRNA quantitation). This list is not meant to be limiting.

The term "P-selectin" as used herein refers to one or more polypeptides derived from a P-selectin precursor (Swiss-Prot P16109(SEQ ID NO:1)) present in a biological sample.

Most preferably, the p-selectin assay detects one or more soluble forms of p-selectin. The P-selectin is a single-pass type I membrane protein, with a large extracellular domain, most or all of which is present in soluble form as a result of an alternative splicing event that deletes all or part of the transmembrane domain or as a result of proteolytic membrane-bound form. In the case of immunoassays, one or more antibodies that bind to an epitope in the extracellular domain can be used to detect these soluble forms. The following domains have been identified in p-selectin:

the term "protein NOV homologue" as used herein refers to one or more polypeptides derived from a precursor of the protein NOV homologue (Swiss-Prot P48745(SEQ ID NO:2)) present in a biological sample.

The following domains have been identified in proteins homologous to the protein NOV:

the term "epithelial cell growth factor receptor" as used herein refers to one or more polypeptides derived from precursors of epithelial cell growth factor receptors present in a biological sample (Swiss-Prot P00533(SEQ ID NO: 3)).

Most preferably, the epithelial growth factor receptor assay detects soluble forms of one or more epithelial growth factor receptors. The epithelial cell growth factor receptor is a single pass type I membrane protein, with a large extracellular domain, most or all of which is present in a soluble form of the epithelial cell growth factor receptor produced by an alternative splicing event that deletes all or part of the transmembrane domain or by proteolysis of a membrane-bound form. In the case of an immunoassay, one or more antibodies that bind to an epitope in the extracellular domain can be used to detect these soluble forms. The following domains have been identified in the epithelial cell growth factor receptor:

the term "nerve growth factor-4" as used herein refers to one or more polypeptides derived from a nerve growth factor-4 precursor (Swiss-Prot Q9HB63(SEQ ID NO:4)) present in a biological sample.

The following domains are identified in nerve growth factor-4:

the term "haptoglobin" as used herein refers to one or more polypeptides derived from haptoglobin precursor (Swiss-Prot P00738(SEQ ID NO:5)) present in a biological sample.

The following domains have been identified in haptoglobin:

the term "α -1-antitrypsin" as used herein refers to one or more polypeptides derived from α -1-antitrypsin precursor (Swiss-Prot P01009(SEQ ID NO:6)) present in a biological sample.

The following domains have been identified in α -1-antitrypsin:

the term "leukocyte elastin" as used herein refers to one or more polypeptides derived from a leukocyte elastin precursor (Swiss-Prot P08246(SEQ ID NO:7)) present in a biological sample.

The following domains have been identified in leukocyte elastin:

the term "tumor necrosis factor receptor superfamily member 6" as used herein refers to one or more polypeptides derived from a tumor necrosis factor receptor superfamily member 6 precursor (Swiss-Prot P25445(SEQ ID NO:8)) present in a biological sample.

Most preferably, the tumor necrosis factor receptor superfamily member 6 assay detects one or more soluble forms of tumor necrosis factor receptor superfamily member 6. Tumor necrosis factor receptor superfamily member 6 is a single pass type I membrane protein with a large extracellular domain, most or all of which is present in a soluble form of tumor necrosis factor receptor superfamily member 6, produced by an alternative splicing event that deletes all or part of the transmembrane domain or by proteolysis of a membrane-bound form. In the case of immunoassays, one or more antibodies in the extracellular domain that bind to an epitope can be used to detect these soluble forms. The following domains have been identified in member 6 of the tumor necrosis factor receptor superfamily:

the term "member 6 of the tumor necrosis factor ligand superfamily" as used herein refers to one or more polypeptides derived from a precursor of member 6 of the tumor necrosis factor ligand superfamily present in a biological sample (Swiss-Prot P48023(SEQ ID NO: 9)).

Most preferably, the tumor necrosis factor ligand superfamily member 6 assay detects one or more soluble forms of tumor necrosis factor ligand superfamily member 6. Tumor necrosis factor ligand superfamily member 6 is a single pass type II membrane protein with a large extracellular domain, most or all of which is present in a soluble form of tumor necrosis factor ligand superfamily member 6, produced by an alternative splicing event that deletes all or part of the transmembrane domain or by proteolysis of the membrane-bound form. In the case of immunoassays, one or more antibodies in the extracellular domain that bind to an epitope can be used to detect these soluble forms. The following domains have been identified in member 6 of the tumor necrosis factor ligand superfamily:

the term "intercellular adhesion molecule 2" as used herein refers to one or more polypeptides derived from precursors of intercellular adhesion molecule 2 (Swiss-Prot P13598(SEQ ID NO:10)) present in a biological sample.

Most preferably, the intercellular adhesion molecule 2 assay detects soluble forms of the intercellular adhesion molecule 2. The intercellular adhesion molecule 2 is a single-pass type I membrane protein, with a large extracellular domain, most or all of which is present in the soluble form of the intercellular adhesion molecule 2, produced by alternative splicing events that delete all or part of the transmembrane domain or produced by proteolysis of the membrane-bound form. In the case of immunoassays, one or more antibodies in the extracellular domain that bind to an epitope can be used to detect these soluble forms. The following domains have been identified in intercellular adhesion molecule 2:

the term "caspase-3" as used herein refers to one or more polypeptides derived from a precursor of caspase-3 (Swiss-Prot P42574(SEQ ID NO:11)) present in a biological sample.

The following domains have been identified in caspase-3:

suitable assays may be used to recognize only the p17 subunit of caspase-3, may recognize only the p12 subunit of caspase-3 (24kDa) instead of full-length caspase-3, may recognize only full-length caspase-3, or may recognize one subunit of full-length caspase-3. In this regard, it will be understood by those skilled in the art that the signal obtained from the immunoassay is the direct structure of a complex formed by one or more antibodies and the target biomolecule (i.e., analyte) and a polypeptide containing the epitope to which the antibody binds as necessary. However, this assay detects the full length caspase-3 molecule, the assay structure is expressed as the concentration of caspase-3, and the signal from the assay is actually the result of all such "immunoreactive" polypeptides present in the sample.

The term "platelet endothelial cell adhesion molecule" as used herein refers to one or more polypeptides derived from a precursor of platelet endothelial cell adhesion molecule (Swiss-Prot P16284(SEQ ID NO:12)) present in a biological sample.

Most preferably, the platelet endothelial cell adhesion molecule assay detects one or more soluble forms of platelet endothelial cell adhesion molecules. Platelet endothelial cell adhesion molecules are single-pass type I membrane proteins with large extracellular domains, most or all of which are present in soluble forms of platelet endothelial cell adhesion molecules, produced by alternative splicing events that delete all or part of the transmembrane domain or by proteolysis of membrane-bound forms. In the case of immunoassays, one or more antibodies in the extracellular domain that bind to an epitope can be used to detect these soluble forms. The following domains have been identified in platelet endothelial cell adhesion molecules:

the term "heat shock protein β -1" as used herein refers to one or more polypeptides derived from a precursor of heat shock protein β -1 (Swiss-Prot P04792(SEQ ID NO:13)) present in a biological sample.

The term "epidermal growth factor receptor" as used herein refers to one or more polypeptides derived from a precursor of epidermal growth factor receptor (Swiss-Prot P00533(SEQ ID NO:14)) present in a biological sample.

Most preferably, the epidermal growth factor receptor assay detects one or more soluble forms of epidermal growth factor receptor. The epidermal growth factor receptor is a single-pass type I membrane protein, with a large extracellular domain, most or all of which is present in a soluble form of the epidermal growth factor receptor, produced by an alternative splicing event that deletes all or part of the transmembrane domain or by proteolysis of a membrane-bound form. In the case of immunoassays, one or more antibodies in the extracellular domain that bind to an epitope can be used to detect these soluble forms. The following domains have been identified in epidermal growth factor receptors:

the term "signal related to the presence or amount" of an analyte as used herein reflects this understanding. The analytical signal is typically related to the presence and amount of analyte by using a standard curve estimated using known concentrations of the analytes of interest. The term analyte, as used herein, is "configured to detect" an analyte if the analyte can produce a detectable signal indicative of the amount or presence of a physiologically relevant concentration of the analyte. Because the epitope of the antibody is at the 8 amino acid position, an immunoassay configured to detect the relevant label will also detect polypeptides associated with the label sequence, such that those polypeptides contain an epitope that must bind to the antibody or to the antibody for analysis. The term "relevant marker" as used herein in relation to a biomarker described herein, such as one of the kidney injury markers, refers to one or more fragments, variants, etc. of a particular marker or its biosynthetic parent that can be detected as a surrogate for the marker itself or as an independent biomarker. The term also refers to one or more polypeptides in a biological sample derived from biomarker precursors complexed with additional substances such as binding proteins, receptors, heparin, lipids, carbohydrates, and the like.

The term "positive going" marker, as used herein, refers to a marker that is determined to be elevated in a subject having a disease or disorder (relative to a subject not having a disease or disorder). The term "negative going" marker, as used herein, refers to a marker that is determined to be decreased in a subject having a disease or disorder (relative to a subject not having a disease or disorder).

The term "subject" as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are useful for human and veterinary disease. Further, the present invention can also be used for post mortem analysis when the subject is preferably a living organism. Preferably the subject is a human, and most preferably is a "patient", as used herein, refers to a living human receiving medical care for a disease or condition. This includes persons without established disease who are being examined for pathological phenomena.

Preferably, the analyte is measured in the sample. Such a sample may be obtained from the subject or may be obtained from a biological substance that is desired to be provided to the subject. For example, a sample may be obtained from a kidney being evaluated for potential transplantation to a subject and an analyte measurement used to evaluate pre-existing damage to the kidney. Preferably the sample is a body fluid sample.

The term "body fluid sample" as used herein refers to a body fluid sample obtained for the purpose of diagnosis, prognosis, classification or evaluation of the relevant subject, e.g., a patient or transplant donor. In some embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen for a condition. Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural effusion. Furthermore, those skilled in the art will appreciate that some bodily fluid samples may be more readily analyzed after fractionation and purification steps, e.g., separation of whole blood into serum or plasma components.

The term "diagnosis" as used herein refers to a method by which one of skill in the art can estimate and/or determine the likelihood of whether a patient has a given disease or condition. In the context of the present invention, "diagnosis" includes the use of the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical features, to obtain a sample and assay from a patient to achieve a diagnosis (i.e., the occurrence or non-occurrence) of acute kidney injury or ARF. Such a diagnosis is "determined", which is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of a variety of conditions, and clinicians in the field do not use biomarkers to cause a vacuum of information, rather than using test results with other clinical indicators to effect a diagnosis. Thus, a measured level of a biomarker on one side of the predetermined diagnostic threshold indicates a greater likelihood of the subject developing a disease relative to a measured level on the other side of the predetermined diagnostic threshold.

Similarly, prognostic risk represents the likelihood that a given process or outcome will occur. Changes in the level or level of a prognostic indicator, which in turn is associated with an increased likelihood of disease (e.g., worsening renal function, future ARF, or death), refers to "indicating an increased likelihood" of an adverse outcome in a patient.

Marker analysis

In general, an immunoassay comprises contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicating the presence or amount of complexes formed by the binding of the polypeptides in the sample to the antibodies. The signal is then correlated with the presence or amount of the biomarker in the sample. Many methods and devices for detecting and analyzing biomarkers are well known to those skilled in the art. See, for example, U.S. patent 6,143,576; 6,113,855; 6,019,944, respectively; 5,985,579, respectively; 5,947,124, respectively; 5,939,272, respectively; 5,922,615, respectively; 5,885,527, respectively; 5,851,776, respectively; 5,824,799, respectively; 5,679,526, respectively; 5,525,524, respectively; and 5,480,792 and the immunological Handbook, David Wild, ed.stockton Press, New York,1994, each of which is incorporated herein by reference, including tables, drawings and claims.

Assay devices and assay methods known in the art can use labeled molecules in various sandwich, competitive or non-competitive assay formats to generate signals related to the presence and amount of relevant biomarkers. Suitable analytical formats also include chromatography, mass spectrometry and protein "imprinting" methods. In addition, some methods and devices, such as biosensors and optical immunoassays, can be used to determine the presence and amount of an analyte without the need for a labeling molecule. See U.S. Pat. nos. 5,631,171; and 5,955,377, each of which is incorporated herein by reference, including all tables, figures, and claims. Those skilled in the art will also appreciate that robotic devices include, but are not limited to, BeckmanAbbottRocheDade BehringThe system is also in an immunoassay analyzer capable of performing an immunoassay. However, any suitable immunoassay may be used, e.g., enzyme linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), competitive binding assays, etc.

Antibodies or other polypeptides may be immobilized on a variety of solid supports for analysis. Solid phases useful for immobilizing specific binding members include those developed in solid phase binding assays and/or used as solid phases. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, Tenta gel, Agro gel, PEGA gel, SPOCC gel and multiwell plates. The assay strip may be prepared by coating the antibody or antibodies under assay on a solid support. The strip may then be immersed in a test sample and then subjected to rapid washing and detection steps to produce a measurable signal, such as a coloured spot. The antibody or other polypeptide may be bound to a specific region of the assay device by direct conjugation to the surface of the assay device or by indirect binding. In the latter case, the antibody or other polypeptide may be immobilized on a particle or other solid support, and the solid support is immobilized on the surface of the device.

Bioassays require methods for detection and one of the most common methods for quantifying the results is to conjugate a detectable label to a protein or nucleic acid that has an affinity for one of the components in the biological system under study. Detectable labels can include molecules that are themselves detectable (e.g., fluorophores, electrochemical labels, metal chelates, etc.) and molecules that are indirectly detected by the production of detectable reaction products (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or molecules that are indirectly detected by their own detectable specific binding molecules (e.g., biotin, digoxigenin, maltose, oligohistidine, 2, 4-dinitrobenzene, phenyl arsenate, ssDNA, dsDNA, etc.).

The preparation of solid phase and detectably labeled conjugates typically involves the use of chemical cross-linkers, which comprise at least two reactive groups and can be divided into homofunctional cross-linkers (comprising identical reactive groups) and heterofunctional cross-linkers (comprising non-identical reactive groups), homofunctional cross-linkers, which are non-specifically coupled by amine, thiol or reaction, are available from a number of commercial sources, maleimides, alkyl and aryl halides, and α -haloacyl groups react with thiol groups to form thioether linkages, while pyridyl disulfides react with thiol groups to form mixed disulfides.

In some aspects, the invention provides kits for analyzing the kidney injury markers. The kit comprises reagents for analyzing at least one test sample comprising at least one kidney injury marker antibody. The kit may also include equipment and instructions for performing one or more of the diagnostic and/or prognostic relationships described herein. Preferred kits include antibody pairs for performing a sandwich assay or labeled species for performing a competition assay for analysis. Preferably, the antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies binds to a kidney injury label. Most preferably, each of the antibodies is a monoclonal antibody. The instructions for using the kit and performing the association may be in the form of a label, which refers to any writing or recording material that can be adhered to or placed with the kit at any time during the manufacturing process, shipping, sale, or use. For example, the term label includes advertising leaflets and brochures, packaging materials, instructions, audio or video tapes, compact discs and printed matter directly printed on the kit.

Antibodies

The term "antibody" as used herein refers to a peptide or polypeptide derived from substantially an immunoglobulin based on coding or post-immunoglobulin gene simulation or immunoglobulin genes or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g., Fundamental Immunology,3rd Edition, w.e.paul, ed., Raven Press, n.y. (1993); wilson (1994; J.Immunol.methods 175: 267-273; Yarmush (1992) J.biochem.Biophys.Methodss 25: 85-97. the term antibody includes antigen binding proteins, i.e., "antigen binding sites" (e.g., fragments, subsequences, Complementarity Determining Regions (CDRs)) that retain the ability to bind to an antigen, including i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains, ii) F (ab') 2 fragments, bivalent fragments including two Fab fragments linked by a disulfide bridge at the hinge region; iii) Fd fragment consisting of the VH and CH1 domains; iv) Fv fragments, consisting of the single-arm VL and VH domains of an antibody; v) dAb fragments (Ward et al, (1989) Nature 341:544-546) which are composed of VH domains; and vi) an isolated Complementarity Determining Region (CDR). Single chain antibodies may be included in the term "antibody" by reference.

The antibodies used in the immunoassays described herein preferably specifically bind to the kidney injury markers of the present invention. The term "specifically binds" is not meant to indicate that the antibody binds exclusively to its desired target, as described above, the antibody binds to any polypeptide exhibiting binding of the antibody to an epitope. However, an antibody "specifically binds" 5-fold greater than its affinity for a non-target molecule that does not display a suitable epitope if it has an affinity for the desired target. Preferably, the affinity of the antibody for the target molecule is at least about 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more greater than the affinity for the non-target molecule. In preferred embodiments, preferred antibody binding affinities are at least about 10⁷M^-1And preferably about 10⁸M^-1To about 10⁹M^-1About 10⁹M^-1To about 10¹⁰M^-1Or about 10¹⁰M^-1To about 10¹²M^-1。

Affinity was estimated as K_d＝k_off/k_on(k_offIs the dissociation rate constant, k_onIs the binding rate constant and Kd is the equilibrium constant). Affinity can be determined by measuring the fractional range (r) of labeled ligand at different concentrations (c) at equilibrium. Data were generated using Scatchard equation: r/c is plotted as K (n-r), where r is the number of moles of acceptor/moles of ligand range at equilibrium; c is the free ligand concentration at equilibrium; k is the equilibrium binding constant; n is the number of ligand binding sites per receptor molecule. By mapping analysis, r/c is plotted on the Y-axis and r is plotted on the X-axis, thus generating a Scatchard plot. Measurement of antibody affinity by Scatchard analysis is well known in the art. See, e.g., van Erp et al, J.Immunoassasay 12: 425-; methods program, Comput, Nelson and Griswoldms Biomed.27:65-8,1988

The term "epitope" refers to an antigenic determinant capable of specifically binding to an antibody. Epitopes are typically composed of chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics. The difference between the structural and non-structural epitopes is that binding to the former, but not to the latter, is lost in the presence of denaturants.

Various publications discuss the use of phage display technology to generate and screen polypeptide libraries for binding selected analytes. See, for example, Cwirla et al, Proc. Natl. Acad. Sci. USA 87,6378-82, 1990; devlin et al, Science 249, 404-; and Ladner et al, U.S. Pat. No.5,571,698. The basic concept of the phage display method is to establish a physical bond between the polypeptide-encoding DNA to be screened and the polypeptide. This physical binding is provided by the phage particle, which displays the polypeptide as part of a capsid that encapsulates the phage genome encoding the polypeptide. The physical binding established between the polypeptide and its genetic material allows for simultaneous mass screening of a very large number of bacteriophages carrying different polypeptides. Phage displaying polypeptides with affinity for the target bind to the target, and these phage are enriched by affinity screening of the target. The recognition of the polypeptides displayed by these phages can be determined by their respective genomes. Using these methods, polypeptides identified as having binding affinity to a desired target can be synthesized in large quantities by conventional methods. See, for example, U.S. patent No. 6,057,098, which is incorporated herein by reference, including all tables, figures, and claims.

Antibodies generated by these methods can be screened by a first screen for affinity and specificity and related purified polypeptides, and if desired, the results compared to the affinity and specificity of antibodies having polypeptides that are desired to be excluded from binding. The screening step may comprise immobilizing the purified polypeptide in separate wells of a microtiter plate. The solution containing the potential antibody or group of antibodies is then placed in the respective microtiter wells and incubated for about 30 minutes to 2 hours. The microtiter wells are then washed and a labeled secondary antibody (e.g., an anti-rat antibody conjugated to alkaline phosphatase if the ascending antibody is a rat antibody) is added to the wells for incubation for about 30 minutes, followed by washing. By adding the matrix to the wells, a color reaction will occur in which the antibody is present to the immobilized polypeptide.

Such recognized antibodies can then be further analyzed for affinity and specificity in selected assay designs. In the development of immunoassays for target proteins, the purified target protein functions as a standard by which the sensitivity and specificity of immunoassays using selected antibodies are judged. Because the binding affinities of the various antibodies differ, some antibody pairs (e.g., in a sandwich assay) can spatially interfere with each other, etc., the performance of an assay for an antibody can be a more important measure than the absolute affinity and specificity of the antibody.

Analyzing correlations

As used herein, the term "correlate," when using biomarkers, refers to comparing the presence or amount of a biomarker in a patient's body to the presence or amount of a biomarker known to have or be in a human having a given condition or known not to have a given condition. Typically, the results in the form of biomarker concentrations are compared to a predetermined threshold selected to reveal the likelihood of whether the disease has occurred or some future outcome.

The selection of a diagnostic threshold includes consideration of the likelihood of disease among other factors, factors for correct and incorrect diagnosis at different test thresholds, assessment of the outcome of treatment (failure to treat) based on diagnosis. For example, when considering administering a specific treatment with high efficiency and low risk level, little testing is required because the clinician can accept a large amount of diagnostic uncertainty. On the other hand, clinicians often require more certainty of diagnosis where the selected treatment is inefficient and at greater risk. Thus, the cost/benefit analysis includes selecting a diagnostic threshold.

Suitable thresholds may be determined in various ways. For example, one recommended diagnostic threshold for diagnosing acute myocardial infarction using cardiac troponin is 97.5 of the concentration seen in normal populations^thPercentage (D). Another method may be to observe a continuous sample of the same patient, where a previous "baseline" is used to monitor temporal changes in biomarker levels.

Population studies can also be used to select decision thresholds. Receiver operating characteristics ("ROC") arise from the field of signal detection theory developed during world war ii for analyzing radar images, and ROC analysis is often used to select a threshold that best distinguishes "diseased" subpopulations from "non-diseased" subpopulations. False positives in this case occur when individuals detect positivity but actually have no disease. False positives, on the other hand, occur when an individual tests negative, indicating that it is healthy, but in fact does have a disease. To plot the ROC curve, the True Positive Rate (TPR) and the False Positive Rate (FPR) are determined as continuously varying decision thresholds. Since TPR corresponds to sensitivity and FPR equals 1-specificity, the ROC plot is sometimes referred to as the sensitivity vs (1-specificity) plot. The full test will have an area under the ROC curve of 1.0; the random test would have an area of 0.5. The threshold is selected to provide an acceptable level of specificity and sensitivity.

As used herein, "diseased" refers to a population having one characteristic (either present in a disease or condition or with some consequence), and "non-diseased" refers to a population lacking the characteristic. While a single decision threshold is the simplest application of this method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may correspond to a relatively high degree of confidence, and above a second threshold, the presence of disease may correspond to a relatively high degree of confidence. An uncertainty may be considered between the two thresholds. This is meant to be exemplary in nature.

In addition to threshold comparisons, other methods of correlating analysis results for patient classification (whether disease is occurring, likelihood of outcome, etc.) include hierarchical decision graphs, rule sets, Bayesian methods, and neural network methods. These methods can generate a likelihood value representing the degree to which the degree of the receptor belongs in one of a plurality of classifications.

Measurement test accuracy, as described in Fischer et al, Intensive Care Med.29: 1043-. These measurements include sensitivity and specificity, predictive value, probability ratio, diagnostic odd ratio and ROC curve area. The area under the curve (AUC) of this ROC plot corresponds to the likelihood of a ranker ranking randomly selected positive instances higher than randomly selected negative instances. The area under the ROC curve can be considered to be equivalent to the Mann-Whitney U test, which tests for media differences between the scores obtained in the two groups considered if they were consecutive data sets, or the area under the ROC curve can be considered to be equivalent to the Wilcoxon test rating.

As noted above, suitable tests may exhibit one or more of the following results with respect to various measurements: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95, with a corresponding sensitivity of greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, still more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95, with a corresponding specificity of greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, still more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity combined with at least 75% specificity, ROC curve area greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95; an odd ratio is other than 1, preferably at least about 2 or greater or about 0.5 or less, more preferably at least about 3 or greater or about 0.33 or less, still more preferably at least about 4 or greater or about 0.25 or less, even more preferably at least about 5 or greater or about 0.2 or less, and most preferably at least about 10 or greater or about 0.1 or less; the positive likelihood ratio (estimated as sensitivity/(1-specificity)) is greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10, and the negative likelihood ratio (estimated as (1-sensitivity)/specificity) is less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1.

Additional clinical indices may be combined with the results of the kidney injury marker assays of the invention these include the biomarkers associated with renal status examples include the following, which describe the common biomarker name, followed by the biomarker or its parent protein Swiss-Prot entry number actin (P68133), adenosine deaminase binding protein (DPP4, P27487), α -1-acid glycoprotein 1(P02763), α -1-microglobulin (P02760), albumin (P02768), angiotensinogen (rennin P00797), annexin A2(P07355), α -glucuronidase (P08236), B-2-microglobulin (P61679), β -galactosidase (P78), BMP-7(P18075), natriuretic peptide (proBNP, BNP 32, proNTBNP, P60), calbindin protein (S100-betalin, Ph, EP 06563757, EP.

For risk stratification purposes, adiponectin (Q15848), alkaline phosphatase (P05186), aminopeptidase N (P15144), calbindin D28k (P05937), cystatin C (P01034), 8 subunit of F1FO ATPase (P03), gamma-glutamyltransferase (P19440), GSTa (α -glutathione-S-transferase, P08263), GSTpi (glutathione-S-transferase P; GSTclass-pi; P09211), IGFBP-1(P08833), IGFBP-2(P18065), IGFBP-2452 (P245692), integral membrane protein 1(Itm1, P46977), interleukin-6 (P05231), interleukin-8 (P10145), interleukin-18 (Q16), IP-10 (IP-14110), soluble protein of TNF-. gamma.1 protein, EP 4526, alpha. -Pro-P, EP-Asp-P-2000 receptor, EP-Asp-9, protein kinase P-Asp-III, (EP-P) and protein kinase P-7, protein kinase P-IV-8 (EP-P-Asp-7), protein kinase P-Asp 7, protein kinase P-Asp-7, protein kinase protein III, GSTcp-S-P-7, protein III, protein.

Other clinical indicators that can be combined with the renal injury marker assay results of the invention include demographic information (e.g., weight, gender, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurysm, congestive heart failure, preeclampsia, eclampsia, diabetes, hypertension, coronary artery disease, proteinuria, renal insufficiency or sepsis, exposure to toxins such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, heme, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents or streptozotocin), clinical variables (e.g., blood pressure, body temperature, respiration rate), risk scores (APACHE score, pret score, TIMI risk score for UA/USTEMI, Framingham risk score), total urine protein measurements, glomerular filtration rate, estimated glomerular filtration rate, urine volume, serum or plasma creatinine concentration, renal papillary antigen 1(RPA1) measurement, renal papillary antigen 2(RPA2) measurement, urine creatinine concentration, sodium excretion fraction, urea sodium concentration, ratio of urine creatinine to serum or plasma creatinine, urine specific gravity, urine osmolality, ratio of urine urea nitrogen to plasma urea nitrogen, ratio of plasma BUN to creatinine, and/or renal failure index are estimated as urea sodium/(urine creatinine/plasma creatinine). Other measurements that can be combined with the results of the renal injury marker analysis are described below and are described in Harrison's Principles of Internal Medicine,17^thEd., McGrawHill, New York, p. 1741-1830 and in Current Medical diagnostics&Treatment2008,47^thEd, McGraw Hill, New York, th785 pages 815, the entire contents of each of which are incorporated herein by reference.

Combining the analysis results/clinical indices in this manner may include using multivariate log regression, log linear modeling, neural network analysis, n-of-m analysis, hierarchical decision graphs, and the like. Such list is not meant to be limiting.

Diagnosis of acute renal failure

As noted above, the terms "acute renal injury" and "acute renal failure" as used herein are defined, in part, in terms of changes in serum creatinine relative to a baseline value. Most definitions of ARF have common elements including the use of serum creatinine and urine output. The patient may have renal insufficiency without an available baseline measurement of renal function for comparison. In this event, one method can estimate baseline serum creatinine by assuming that the patient initially has a normal GFR. Glomerular Filtration Rate (GFR) is the volume of fluid filtered by the glomerular capillaries into the Bowman's capsule per unit time. Glomerular Filtration Rate (GFR) can be estimated by measuring any chemical that has a stable level in the blood and is freely filtered from reabsorption and excretion by the kidneys. GFR is expressed by units of ml/min:

by normalizing the GFR to body surface area, it can be assumed that the GFR is approximately 75-100 ml/min per 1.73m 2. Thus, the measurement speed is the amount of substance in the urine resulting from the volume of blood that can be estimated.

There are many different techniques for estimating or evaluating glomerular filtration rate (GFR or eGFR). However, in clinical practice, creatinine clearance is used to measure GFR. Creatinine is essentially produced by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered through the glomerulus, but is excreted in small amounts by the tubules, so creatinine clearance is overestimated by GFR 10% to 20%. This margin of error is acceptable and is considered to measure the filtrate with creatinine clearance.

Creatinine clearance (CCr) can be estimated if the urinary concentration value of creatinine (Ucr), the urine flow rate (V), and the plasma concentration of creatinine (Pcr) are known. Since the resulting urine concentration and urine flow rate produce the rate of excretion of creatinine, creatinine clearance may also be referred to as its excretion rate (UcrXV) divided by plasma concentration. This is generally expressed by the following mathematical formula:

a 24 hour urine collection is typically performed from bladder contents of an empty bladder one morning to the next morning, and then compared to a blood test:

to allow comparison of results for different body sizes, the CCr was calculated by calibration to Body Surface Area (BSA) and expressed as ml/min/1.73m 2 compared to average body size men. However, most adults have BAS close to 1.7(1.6-1.9), and essentially obese or young patients should have a CCr calibrated by their actual BSA:

the accuracy of creatinine clearance measurements (even when collection is complete) is limited because glomerular filtration rates decrease, creatinine excretion increases, and thus, there is less rise in serum creatinine. As a result, creatinine excretion is much greater than the filtration load, resulting in a potentially large overestimation of GFR (a two-fold large difference). However, for clinical purposes, it is important to determine whether renal function is stable or worsening or improving. This is usually determined by measuring serum creatinine alone. As with serum creatinine clearance, serum creatinine does not accurately reflect the GFR of the non-steady state symptoms of ARF. Nevertheless, the degree of change in serum creatinine relative to baseline reflects changes in GFR. Serum creatinine is easily and readily measured, and is specific for renal function.

For the purpose of determining urine output on a mL/kg/hour basis, it is sufficient to collect and measure urine hourly. In this case, for example, only 24-hour cumulative output is available, no patient body weight is provided, and a small change in the RIFLE urine output index has been described. For example, Bagshaw et al, Nephrol. Dial. Transplant.23: 1203-: <35mL/h (Risk), <21mL/h (Injury) or <4mL/h (failure).

Selecting a treatment regimen

Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, delivering a compound that eliminates known damage to the kidney, kidney transplantation, delaying or avoiding a procedure known damage to the kidney, modifying diuretic administration, initiating targeted therapy, and the like. One skilled in the art will recognize that the appropriate treatments for a variety of diseases discussed are relevant to the diagnostic methods described herein. See, e.g., Merck Manual of diagnosis and Therapy,17th Ed. In addition, because the methods and compositions described herein provide prognostic information, the markers of the invention can be used to detect a course of treatment. For example, an improved or worsened prognostic status may indicate whether a particular treatment is effective.

It will be readily understood by those skilled in the art that the present invention is fully adapted to be carried out with the objects and obtaining the ends and advantages mentioned, as well as those inherent therein. Examples are provided herein to represent preferred embodiments, and are provided herein for illustrative purposes and are not intended to limit the scope of the present invention.

Example 1: contrast-induced nephropathy sample collection

The purpose of this sample collection study is to collect plasma and urine samples as well as clinical data from patients before and after receiving intravascular contrast media. About 250 adults undergoing a radiographic/angiographic procedure involving intravascular administration of iodinated contrast media are involved. To participate in this study, each patient must meet and not meet the following exclusion criteria:

selection criteria

Males and females 18 years or older;

undergoing a radiological imaging/angiography procedure (e.g., CT scan or coronary intervention) including intravascular administration of a contrast medium;

it is desirable to admit the subject at least 48 hours after administration of the contrast agent.

Written informed consent can and will be provided for participation in the study and adherence to all study steps.

Exclusion criteria

A renal transplant recipient;

acute worsening of renal function prior to the contrast agent step;

already receiving dialysis (acute or chronic) or about to require dialysis when involved;

would be expected to undergo a major surgical procedure (e.g. cardiopulmonary extracorporeal circulation) or an additional imaging step with contrast media to have a significant risk of further renal injury within 48 hours after administration of the contrast media;

participating in interventional clinical studies with experimental treatment within the first 30 days;

infection with Human Immunodeficiency Virus (HIV) or hepatitis virus is known.

EDTA anti-conjugated blood samples (10mL) and urine samples (10mL) were collected from each patient immediately prior to the first administration of the contrast agent (and after any prior step hydration). Blood and urine samples are then collected at 4 (+ -0.5), 8 (+ -1), 24 (+ -2), 48 (+ -2) and 72 (+ -2) hours after the last administration of contrast medium during the step of labeling the contrast agent. Blood is collected by direct venipuncture or by other available venous access, such as the presence of a femoral sheath, central venous line, peripheral intravascular line, or hep-lock. The blood samples thus studied were processed into plasma at the clinical site, frozen and transported to the asset Medical, inc. Study urine samples were frozen and transported to asset Medical, Inc.

Serum creatinine was administered on site immediately prior to the first administration of the contrast agent (after any pre-step hydration) and immediately 4 (+ -0.5) hours, 8 (+ -1) hours, 24 (+ -2) hours, 48 (+ -2) hours and 72 (+ -2) hours after the last administration of the contrast agent (ideally at the same time as the study sample was obtained). In addition, the status of each patient was assessed by day 30 with associated additional serum creatinine measurements and urine creatinine measurements, requiring dialysis, admission status and adverse clinical outcomes (including death).

Prior to administration of the contrast agent, each patient corresponded to a risk based on the following assessments: systolic pressure<80mm Hg is 5 min; the aortic inner capsule reverse balloon pump is divided into 5 points; congestive heart failure (grade III-IV or pulmonary edema history) score 5; age (age)>4 points after 75 years old; hematometer level male<39% of women<3 points for 35 percent; diabetes mellitus is divided into 3 points; the volume of the 100mL contrast medium is 1 min; serum creatinine levels>1.5g/dL 4 min or estimated GFR 40-60 mL/min/1.73m²2 min, 20-40 mL/min/1.73m²The number of the segments is 4,<20mL/min/1.73m²this risk is corresponded to the risk of CIN and dialysis as follows: 5 or less total score-7.5% CIN risk, dialysis risk-0.04%; 6-10 total points are CIN risk-14%, dialysis risk-0.12%; 11-16 total points-26.1% of CIN risk and-1.09% of dialysis risk;total score>16-57.3% risk of CIN and-12.8% risk of dialysis.

Example 2: cardiac surgery sample collection

The purpose of this sample collection study is to collect plasma and urine samples as well as clinical data of patients before and after cardiovascular surgery (a step known to potentially impair renal function). Approximately 900 adults underwent this procedure. In order to participate in the study, each patient must meet all of the following selection criteria and not the exclusion criteria:

selection criteria

Males and females 18 years or older;

undergoing cardiovascular surgery;

the Toronto/Ottawa predicted risk index has a renal replacement risk score of at least 2 (Wijeysundersea et al, JAMA 297:1801-9, 2007); and

written informed consent can and will be provided for participation in the study and adherence to all study steps.

Exclusion criteria

The pregnancy is already carried out;

prior kidney transplantation;

involvement in prior acute renal function deterioration (e.g., RIFLE of any kind);

already receiving dialysis (acute or chronic) or requiring dialysis immediately upon participation;

currently participating in another clinical study or another clinical study expected to participate in cardiac surgery within seven days, including drug infusion or AKI treatment intervention;

infection with Human Immunodeficiency Virus (HIV) or hepatitis virus is known.

EDTA anti-conjugated blood samples (10mL), whole blood (3mL) and urine samples (35mL) were collected from each patient within 3 hours prior to the first incision (and after any pre-step hydration). Any blood and urine samples were collected 4 (+ -0.5), 8 (+ -1), 24 (+ -2), 48 (+ -2) hours after surgery, and seven days after each day if the subject was in the hospital and then on the third day. Blood is collected by direct venipuncture or by other available venous access (e.g., the presence of a femoral sheath, central venous line, peripheral intravascular line, or hep-lock). The blood samples thus studied were frozen and transported to an asset Medical, inc. Study urine samples were frozen and transported to astute medical, Inc.

Example 3: acute disease subject sample collection

The aim of this study was to collect samples from patients with acute disease. About 900 adults are expected to participate in ICU for at least 48 hours. To participate in the study, each patient must meet all of the following selection criteria and not meet the exclusion criteria:

selection criteria

Males and females 18 years or older;

study population 1: 300 patients with at least one of the following symptoms:

shock (SBP <90mmHg and/or booster support is required to maintain MAP >60mmHg and/or a recorded drop in SBP of at least 40 mmHg); and

sepsis;

study population 2: 300 patients with at least one of the following symptoms:

participants recorded the specified 24-hour IV antibiotics in a Computer Program Order Entry (CPOE);

participants were exposed to the contrast medium within 24 hours;

increased intra-abdominal pressure is associated with acute decompensated heart failure; and

enter the ICU due to a previously severe trauma and may require 48 hours of admission at the ICU;

study population 3: about 300 patients

Known risk factors and acute kidney injury are expected to accompany hospital admissions through intensive care (ICU or ED) (e.g., sepsis, hypotension/shock (shock ═ contracting BP <90mmHg and/or requiring booster support to maintain MAP >60mmHg and/or recorded SBP reduction >40mmHg), severe trauma, hemorrhage or major surgery); and/or is expected to be admitted to the ICU for at least 24 hours.

Exclusion criteria

The pregnancy is already carried out;

individuals under public care;

involvement in prior acute renal function deterioration (e.g., RIFLE indicators of any kind);

dialysis (acute or chronic) was received within the first 5 days of participation or required immediately upon participation;

known to be infected with Human Immunodeficiency Virus (HIV) or hepatitis virus;

only the above criteria encompassed by SBP <90mmHg were met, with no shock in the opinion of the attending physician or researcher.

After providing informed consent, EDTA-anti-conjugated blood samples (10mL) and urine samples (25-30mL) were collected from each patient. Blood and urine samples were then collected at 4 (+ -0.5) and 8 (+ -1) hours after administration of the contrast agent (if used), at 12 (+ -1), 24 (+ -2), and 48 (+ -2) hours after participation, and thus daily to day 7 to day 14, when the subject was hospitalized. Blood is collected by direct venipuncture or by other available venous access (e.g., the presence of a femoral sheath, central venous line, peripheral intravascular line, or hep-lock). The blood samples thus studied were clinically processed into plasma, frozen and transported to an asset Medical, inc. Study urine samples were frozen and transported to asset Medical, Inc.

Example 4 immunoassay format

The analyte is measured using standard sandwich enzyme immunoassay techniques. The primary antibody bound to the analyte is immobilized in the wells of a 96-well polystyrene microplate. Analyte standards and test samples are dropped into appropriate wells and any analyte is surrounded by immobilized antibody. After washing to remove unencapsulated material, horseradish peroxidase-conjugated secondary antibody is added to the wells and the remaining analyte binds, thereby forming a sandwich complex (if present) and primary antibody with the analyte. After washing to remove any unencapsulated antibody-enzyme reagent, a matrix solution containing tetramethylbenzidine and hydrogen peroxide was added to the wells. The spread of the color bands is proportional to the amount of analyte present in the sample. The band evolution stops and the bands are measured at 540nm or 570 nm. The analyte concentration corresponds to the test sample, compared to a standard curve measured by an analyte standard.

The concentrations in the samples were expressed as soluble p-selectin-ng/mL, protein NOV homologous protein-pg.mL, neurogenic factor 4-ng/mL, haptoglobin-mg/mL, α -1-antitrypsin-mg/mL, leukocyte elastase-ng/mL, soluble tumor necrosis factor receptor superfamily member 6-pg/mL, soluble tumor necrosis factor ligand superfamily member 6-pg/mL, soluble intercellular adhesion molecule 2-units/mL, caspase 3 (active) -ng/mL, soluble platelet endothelial adhesion molecule-ng/mL, heat shock protein β -1-ng/mL, soluble epidermal growth factor receptor-pg/mL.

Example 5 overt healthy donors and Chronic disease patient samples

Human urine samples from donors without known chronic or acute disease were purchased from two suppliers (golden west Biologicals, inc.,27625 Commerce Center Dr., Temecula, CA 92590 and Virginia medical Research, inc.,915 First clinical Rd., Virginia Beach, VA 23454). Urine samples were transported and stored frozen below-20 ℃. Demographic information provided by the supplier for each donor included gender, race (black/white), smoking status and age.

Human urine samples from donors with various chronic diseases including congestive heart failure, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, diabetes and hypertension were purchased from Virginia Medical Research, inc.,915 First renal Rd., Virginia Beach, VA 23454. The urine samples were transported and stored frozen at temperatures below 20 ℃. The vendor provides a case report with age, sex, race (black/white), smoking status, alcohol use, height, weight, diagnosis of chronic disease, current medications and previous surgical conditions for individual donors.

Example 6 renal injury markers for assessing renal status in RIFLE stage 0 patients

Based on the maximal phase reached within 7 days, which is involved by RIFLE criteria, patients in Intensive Care Unit (ICU) are classified as non-injured (0), injured risk (R), injured (I) and exhausted (F) according to renal status.

Two groups of patients were defined, the first group of patients did not progress beyond stage 0, and the second group of patients reached stage R, I, F within ten days. To express normal marker fluctuations that occur in patients in the ICU and thereby assess the utility of markers for monitoring AKI status, marker levels in urine samples from a first population of patients were collected. The concentration of the marker in urine samples from patients who reached 0 hours, 24 hours, 48 hours before the R, I, or F phase in patients in the second population was measured. In the following table, the time "before maximum stage" indicates that samples were taken at this time, divided into three groups of +/-12 hours relative to the time before the particular patient reached the lowest disease stage. For example, 24 hours before this example (0 and R, I, F) refers to 24 hours (+/-12 hours) before stage R is reached (or I if no sample is in stage R, or F if no sample is in stage R or I).

Each marker was measured by standard immunoassay methods using commercially available assay reagents. Receiver Operator Characteristic (ROC) curves are generated and the area under each ROC curve (AUC) is determined. Patients in the second population are separated according to a reason for R, I or F stage based on serum creatinine measurements (sCr), based on Urine Output (UO) or based on determinations of serum creatinine measurements or urine output. That is, for those patients judged to be in the R, I, or F stage based on serum creatinine measurements alone, the stage 0 population may have included patients judged to be in the R, I, or F stage based on urine output, for those patients judged to be in the R, I, or F stage based on urine output alone, the stage 0 population may have included patients judged to be in the R, I, or F stage based on serum creatinine measurements, and for those patients judged to be in the R, I, or F stage based on serum creatinine measurements or urine output, the stage 0 population may include only patients in the stage 0 based on serum creatinine and urine output. Furthermore, for those patients judged to be in the R, I or F stage based on serum creatinine measurements or urine output, the judgment method that resulted in the most severe RIFLE stage was used.

The following descriptive statistics were obtained:

soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

Binding to globin:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Caspase 3 (active):

sCr or UO

sCr only

UO only

Soluble platelet endothelial cell adhesion molecule:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

Soluble epidermal growth factor receptor:

sCr or UO

sCr only

UO only

In the table below, the ability to distinguish between the first population (subjects who remained RIFLE 0) and the second population (subjects who developed RIFLE R, I or F) was determined using ROC analysis. SE is the standard deviation of AUC, and n is the number of samples ("pts", as shown) from a single patient. Standard deviations are calculated as described in Hanley, j.a., and McNeil, b.j., The means and use of The area a Receiver Operating Characteristics (ROC) curve radio (1982)143: 29-36; p-values were calculated using a two-tailed Z-test. AUC <0.5 indicates a negative marker for comparison, and AUC >0.5 indicates a positive marker for comparison.

Soluble p-selectin:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.42	0.072	51	22	1.727
24 hours	0.59	0.072	51	24	0.214
						48 hours	0.40	0.269	51	1	1.285

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.41	0.114	94	6	1.561
24 hours	0.58	0.117	94	7	0.479
						48 hours	0.58	0.214	94	2	0.710

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.49	0.083	42	17	1.120
24 hours	0.66	0.074	42	22	0.031
						48 hours	0.65	0.156	42	4	0.320

Protein NOV homologs:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.62	0.060	101	31	0.050
24 hours	0.69	0.062	101	26	0.003
						48 hours	0.79	0.192	101	2	0.129

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.46	0.092	173	10	1.299

24 hours	0.54	0.096	173	10	0.652
						48 hours	0.40	0.153	173	3	1.500

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.70	0.064	78	25	0.002
24 hours	0.76	0.062	78	23	0.000
						48 hours	0.71	0.134	78	5	0.116

Nerve growth factor 4:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.57	0.095	52	12	0.462
24 hours	0.40	0.192	52	2	1.402
						48 hours	0.88	0.221	52	1	0.082

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.56	0.152	86	4	0.689
24 hours	0.39	0.186	86	2	1.458
						48 hours	0.24	0.191	86	1	1.828

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.54	0.101	38	11	0.678
24 hours	0.51	0.176	38	3	0.941
						48 hours	0.92	0.189	38	1	0.026

Binding to globin:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.52	0.051	216	38	0.743
24 hours	0.55	0.046	216	51	0.291
						48 hours	0.65	0.065	216	23	0.017

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.40	0.067	375	16	1.865
24 hours	0.52	0.065	375	21	0.777
						48 hours	0.56	0.091	375	11	0.519

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.57	0.055	181	34	0.202
24 hours	0.56	0.049	181	45	0.224
						48 hours	0.60	0.066	181	23	0.116

α -1-antitrypsin:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.44	0.049	216	38	1.816
24 hours	0.45	0.044	216	51	1.741
						48 hours	0.49	0.063	216	23	1.134

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.40	0.067	375	16	1.864
24 hours	0.38	0.058	375	21	1.966
						48 hours	0.43	0.084	375	11	1.581

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.48	0.054	181	34	1.261
24 hours	0.52	0.049	181	45	0.692
						48 hours	0.50	0.064	181	23	1.019

Leukocyte elastase:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.60	0.056	103	36	0.071
24 hours	0.58	0.052	103	46	0.121
						48 hours	0.52	0.067	103	23	0.771

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.50	0.086	226	12	0.988
24 hours	0.45	0.069	226	18	1.494
						48 hours	0.59	0.092	226	11	0.344

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.56	0.061	88	31	0.303

24 hours	0.59	0.055	88	41	0.113
						48 hours	0.49	0.069	88	22	1.155

Soluble tumor necrosis factor receptor superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.63	0.062	117	28	0.040
24 hours	0.65	0.061	117	28	0.015
						48 hours	0.76	0.163	117	3	0.106

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.49	0.098	194	9	1.065
24 hours	0.61	0.093	194	11	0.221
						48 hours	0.58	0.151	194	4	0.580

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.69	0.068	92	22	0.005
24 hours	0.71	0.063	92	25	0.001
						48 hours	0.80	0.112	92	6	0.008

Soluble tumor necrosis factor ligand superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.70	0.108	78	8	0.065

24 hours	0.78	0.082	78	12	0.001
						48 hours	0.54	0.212	78	2	0.868

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.39	0.133	118	4	1.578
24 hours	0.57	0.125	118	6	0.553
						48 hours	0.68	0.173	118	3	0.293

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.72	0.114	60	7	0.050
24 hours	0.69	0.099	60	10	0.061
						48 hours	0.50	0.209	60	2	1.016

Soluble intercellular adhesion molecule 2:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.36	0.056	92	29	1.987
24 hours	0.44	0.062	92	26	1.671
						48 hours	0.38	0.258	92	1	1.356

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.41	0.088	154	10	1.692
24 hours	0.50	0.095	154	10	0.992
						48 hours	0.48	0.166	154	3	1.098

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.29	0.057	70	24	2.000
24 hours	0.41	0.065	70	24	1.811
						48 hours	0.49	0.149	70	4	1.029

Caspase 3 (active):

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.57	0.075	51	22	0.351
24 hours	0.64	0.071	51	24	0.043
						48 hours	0.94	0.166	51	1	0.008

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.53	0.124	94	6	0.813
24 hours	0.55	0.116	94	7	0.680
						48 hours	0.75	0.203	94	2	0.223

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.59	0.084	42	17	0.302
24 hours	0.69	0.073	42	22	0.010

48 hours

0.77

0.143

42

4

0.058

Soluble platelet endothelial cell adhesion molecule:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.37	0.093	43	10	1.832
24 hours	0.37	0.154	43	3	1.593
						48 hours	nd	nd	43	0	0.211

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.36	0.118	65	5	1.766
24 hours	0.41	0.158	65	3	1.452
						48 hours	0.57	0.215	65	2	0.747

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.32	0.092	30	10	1.952
24 hours	0.36	0.156	30	3	1.627
						48 hours	nd	nd	30	0	0.211

Heat shock protein β -1:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.54	0.088	54	14	0.674
24 hours	0.38	0.187	54	2	1.481

48 hours

0.56

0.302

54

1

0.854

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.70	0.123	88	6	0.095
24 hours	0.43	0.195	88	2	1.295
						48 hours	0.28	0.152	88	2	1.854

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.55	0.094	40	13	0.581
24 hours	0.43	0.166	40	3	1.312
						48 hours	0.63	0.306	40	1	0.683

Soluble epidermal growth factor receptor:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.52	0.074	51	22	0.756
24 hours	0.60	0.073	51	23	0.163
						48 hours	0.53	0.299	51	1	0.922

First group v second group, determination of serum creatinine

Time before max phase

AUC

SE

nFirst group

nSecond group

p

0 hour	0.37	0.108	93	6	1.768
						24 hours	0.49	0.113	93	7	1.038
48 hours	0.87	0.163	93	2	0.023

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.61	0.084	42	17	0.169
24 hours	0.69	0.074	42	21	0.011
						48 hours	0.66	0.155	42	4	0.301

Various threshold (or "cutoff") concentrations were selected and the relative sensitivity and specificity used to distinguish the first population from the second population is shown in the table below. OR is the odd ratio calculated for a particular cut-off concentration, and 95% CI is the confidence interval for the odd ratio.

Soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

Binding to globin:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Caspase 3 (active):

sCr or UO

sCr only

UO only

Soluble platelet endothelial cell adhesion molecule:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

Soluble epidermal growth factor receptor:

sCr or UO

sCr only

UO only

Example 7 evaluation of renal injury markers for renal status in patients with RIFLE stages 0 and R

Patients were classified and analyzed as described in example 6. However, in the first cohort, patients who reached stage R but did not progress to stage I or F were grouped with patients from non-injury stage 0. In this example, the second population includes only patients who progressed to stage I or F. For the first population, the concentration of the marker in the urine sample is included. For the second population, marker concentrations in urine samples collected within 0, 24 and 48 hours of reaching expiration I or F are included.

The following descriptive statistics were obtained:

soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

In the table below, the ability to distinguish between the first population (subjects who remained RIFLE 0 or R) and the second population (subjects who developed RIFLE I or F) was determined using ROC analysis.

Soluble p-selectin:

First group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.49	0.290	99	1	1.028
24 hours	0.65	0.079	99	16	0.059
						48 hours	0.98	0.099	99	1	0.000

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	nd	nd	114	0	0.211

24 hours	0.78	0.161	114	3	0.085
						48 hours	nd	nd	114	0	0.211

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.55	0.300	82	1	0.855
24 hours	0.66	0.082	82	15	0.046
						48 hours	0.95	0.107	82	2	0.000

NOV homologs of proteins:

First group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.45	0.096	167	9	1.376
24 hours	0.58	0.074	167	18	0.301
						48 hours	0.43	0.159	167	3	1.345

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.64	0.301	198	1	0.638
24 hours	0.76	0.141	198	4	0.067
						48 hours	0.50	0.206	198	2	1.000

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.46	0.097	133	9	1.350
24 hours	0.57	0.078	133	16	0.393
						48 hours	0.46	0.164	133	3	1.187

Nerve growth factor 4:

First group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.41	0.101	75	8	1.614
24 hours	0.40	0.189	75	2	1.415
						48 hours	0.44	0.198	75	2	1.238

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.23	0.188	92	1	1.842
24 hours	nd	nd	92	0	0.211
						48 hours	0.14	0.093	92	2	2.000

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.44	0.106	58	8	1.411
24 hours	0.42	0.195	58	2	1.325
						48 hours	0.87	0.231	58	1	0.109

α -1-antitrypsin:

First group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.49	0.064	351	21	1.156
24 hours	0.43	0.057	351	25	1.782

48 hours

0.41

0.075

351

13

1.763

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.122	428	5	1.430
24 hours	0.37	0.103	428	6	1.804
						48 hours	0.35	0.110	428	5	1.829

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.54	0.068	294	20	0.557
24 hours	0.46	0.062	294	22	1.519
						48 hours	0.44	0.078	294	13	1.524

Leukocyte elastase:

First group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.62	0.075	202	17	0.107
24 hours	0.72	0.063	202	23	0.000
						48 hours	0.57	0.085	202	13	0.424

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.25	0.139	260	2	1.925
24 hours	0.48	0.129	260	5	1.135
						48 hours	0.54	0.133	260	5	0.782

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.60	0.076	174	17	0.165
24 hours	0.71	0.066	174	21	0.002
						48 hours	0.58	0.086	174	13	0.357

Soluble tumor necrosis factor receptor superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.31	0.104	189	5	1.925
24 hours	0.61	0.073	189	18	0.122
						48 hours	0.72	0.131	189	5	0.092

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	nd	nd	225	0	0.211
24 hours	0.97	0.060	225	4	0.000
						48 hours	0.68	0.211	225	2	0.406

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.29	0.100	148	5	1.961
24 hours	0.55	0.078	148	16	0.553
						48 hours	0.72	0.132	148	5	0.101

Soluble tumor necrosis factor ligand superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.60	0.153	109	4	0.523

24 hours	0.66	0.115	109	7	0.154
						48 hours	0.76	0.143	109	4	0.073

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	nd	nd	133	0	0.211
24 hours	0.58	0.213	133	2	0.718
						48 hours	0.73	0.206	133	2	0.274

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.57	0.153	82	4	0.654
24 hours	0.65	0.137	82	5	0.266
						48 hours	0.74	0.167	82	3	0.152

Soluble intercellular adhesion molecule 2:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.22	0.063	148	9	2.000
24 hours	0.44	0.070	148	18	1.582
						48 hours	0.45	0.163	148	3	1.218

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.08	0.080	179	1	2.000
24 hours	0.40	0.134	179	4	1.536
						48 hours	0.24	0.137	179	2	1.938

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.22	0.063	117	9	2.000
24 hours	0.44	0.074	117	16	1.591
						48 hours	0.49	0.169	117	3	1.040

Heat shock protein β -1:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.102	78	8	1.509
24 hours	0.54	0.174	78	3	0.835
						48 hours	0.57	0.214	78	2	0.742

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.81	0.263	96	1	0.235
24 hours	0.90	0.211	96	1	0.061
						48 hours	0.33	0.169	96	2	1.691

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.46	0.107	61	8	1.285
24 hours	0.39	0.188	61	2	1.443
						48 hours	0.67	0.301	61	1	0.568

Various threshold (or "cutoff") concentrations were selected and the relative sensitivity and specificity used to distinguish the first population from the second population is shown in the table below. OR is the odd ratio calculated for a particular cut-off concentration, and 95% CI is the confidence interval for the odd ratio.

Soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

Example 8 evaluation of renal injury markers for renal status in patients progressing from stage R to stages I and F

Patients were classified and analyzed as described in example 6, but only those patients in the arrival period R were included in this example. The first population included patients who reached stage R but did not develop to stage I or F within 10 days, and the second population included only patients who developed to stage I or F. The concentration of marker in urine samples collected 12 hours within the arrival period R was included in the analysis for both the first and second populations.

The following descriptive statistics were obtained:

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

In the table below, the ability to distinguish between the first population (subjects who remained RIFLE R) and the second population (subjects who developed RIFLE I or F) was determined using ROC analysis.

α -1-antitrypsin:

First group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.48	0.088	33	16	1.153
24 hours	0.48	0.088	33	16	1.153
						48 hours	0.48	0.088	33	16	1.153

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.81	0.127	14	5	0.013
24 hours	0.81	0.127	14	5	0.013
						48 hours	0.81	0.127	14	5	0.013

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.39	0.095	25	13	1.750
24 hours	0.39	0.095	25	13	1.750
						48 hours	0.39	0.095	25	13	1.750

Leukocyte elastase:

First group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.70	0.082	33	17	0.015
24 hours	0.70	0.082	33	17	0.015
						48 hours	0.70	0.082	33	17	0.015

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.58	0.159	12	5	0.600
24 hours	0.58	0.159	12	5	0.600
						48 hours	0.58	0.159	12	5	0.600

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.67	0.094	24	14	0.077
24 hours	0.67	0.094	24	14	0.077
						48 hours	0.67	0.094	24	14	0.077

Soluble tumor necrosis factor receptor superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.58	0.101	29	12	0.442
24 hours	0.58	0.101	29	12	0.442
						48 hours	0.58	0.101	29	12	0.442

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.91	0.106	11	4	0.000
24 hours	0.91	0.106	11	4	0.000
						48 hours	0.91	0.106	11	4	0.000

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.40	0.116	21	8	1.589

24 hours	0.40	0.116	21	8	1.589
						48 hours	0.40	0.116	21	8	1.589

Various threshold (or "cutoff") concentrations were selected and the relative sensitivity and specificity used to distinguish the first population from the second population is shown in the table below. OR is the odd ratio calculated for a particular cut-off concentration, and 95% CI is the confidence interval for the odd ratio.

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Example 9 evaluation of renal injury markers for renal status in patients in RIFLE stage 0

Patients in Intensive Care Unit (ICU) were classified as non-injured (0), injured (R), injured (I) and exhausted (F) according to renal status based on participation in the maximal phase reached within 7 days as determined by RIFLE criteria.

Two groups of patients were defined, the first group of patients did not develop beyond stage 0, and the second group of patients reached stage R, I or F within ten days. To express the normal marker fluctuations that occur in patients in the ICU and thereby assess the utility of the marker for monitoring AKI status, marker levels in blood samples of a first population of patients were collected. The concentration of the marker in the plasma fraction of blood samples from patients who reached 0 hour, 24 hours, 48 hours before the R, I or F phase in the second group of patients was measured. In the following table, the "time before maximum phase" indicates that the sample was taken at this time, divided into three groups of +/-12 hours, relative to the time before the particular patient reached the minimum disease phase. For example, 24 hours prior to the example (0 for R, I, F) means 24 hours (+/-12 hours) prior to reaching the R phase (or I if no sample is in the R phase, or F if no sample is in the R or I phase).

Each marker was measured by standard immunoassay methods using commercially available assay reagents. Receiver Operator Characteristic (ROC) curves are generated and the area under each ROC curve (AUC) is determined. Patients in the second population were separated according to the reason for R, I or F stage based on serum creatinine measurements (sCr), based on Urine Output (UO) or based on determinations of serum creatinine measurements or urine output. That is, for those patients judged to be in the R, I, or F phase based on serum creatinine measurements alone, the phase 0 population may have included patients judged to be in the R, I, or F phase based on urine output, for those patients judged to be in the R, I, or F phase based on urine output alone, the phase 0 population may have included patients judged to be in the R, I, or F phase based on serum creatinine measurements, and for those patients judged to be in the R, I, or F phase based on serum creatinine measurements or urine output, the phase 0 population may include only patients in the phase 0 based on serum creatinine and urine output. Furthermore, for those patients judged to be in the R, I or F phase based on serum creatinine measurements or urine output, the judgment method that resulted in the most severe RIFLE phase was used.

The following descriptive statistics were obtained:

soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

Binding to globin:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Soluble platelet endothelial cell adhesion molecule:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

In the table below, the ability to distinguish between the first population (subjects who remained RIFLE 0) and the second population (subjects who developed RIFLE R, I or F) was determined using ROC analysis. SE is the standard deviation of AUC, and n is the number of samples ("pts", as shown) from a single patient. Standard deviations are calculated as described in Hanley, j.a., and McNeil, b.j., The means and use of The area a Receiver Operating Characteristics (ROC) curve radio (1982)143: 29-36; p-values were calculated using a two-tailed Z-test. AUC <0.5 indicates a negative marker for comparison, and AUC >0.5 indicates a positive marker for comparison.

Soluble p-selectin:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.63	0.119	26	8	0.293
24 hours	0.47	0.091	26	17	1.236
						48 hours	nd	nd	26	0	0.211

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.198	47	2	1.293
24 hours	0.41	0.100	47	9	1.626
						48 hours	0.81	0.267	47	1	0.247

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.67	0.123	27	7	0.161
24 hours	0.51	0.105	27	11	0.962
						48 hours	0.56	0.307	27	1	0.856

Protein NOV homologs:

first group v second group, determination of serum creatinine measurement or urine output

Max periodTime of day	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.56	0.072	82	21	0.379
24 hours	0.56	0.073	82	20	0.447
						48 hours	0.74	0.287	82	1	0.396

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.70	0.106	135	8	0.057
24 hours	0.60	0.090	135	12	0.262
						48 hours	0.85	0.142	135	3	0.014

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.60	0.073	68	21	0.187
24 hours	0.58	0.087	68	14	0.370
						48 hours	0.74	0.204	68	2	0.235

Nerve growth factor 4:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.56	0.091	56	13	0.482
24 hours	0.63	0.178	56	3	0.482
						48 hours	0.79	0.275	56	1	0.299

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.73	0.120	88	6	0.050
24 hours	0.81	0.153	88	3	0.042
						48 hours	0.35	0.175	88	2	1.619

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.63	0.090	41	14	0.147
24 hours	0.60	0.180	41	3	0.572
						48 hours	0.83	0.257	41	1	0.200

Binding to globin:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.40	0.049	221	35	1.955
24 hours	0.41	0.042	221	51	1.971
						48 hours	0.45	0.062	221	22	1.601

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.40	0.068	386	16	1.843
24 hours	0.45	0.062	386	21	1.620
						48 hours	0.37	0.081	386	10	1.888

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.44	0.052	183	34	1.716
24 hours	0.48	0.048	183	45	1.396
						48 hours	0.54	0.068	183	21	0.589

α -1-antitrypsin:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.29	0.042	221	35	2.000
24 hours	0.25	0.033	221	51	2.000
						48 hours	0.28	0.049	221	22	2.000

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.39	0.066	386	16	1.905
24 hours	0.34	0.054	386	21	1.997
						48 hours	0.38	0.083	386	10	1.842

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.35	0.048	183	34	1.998
24 hours	0.28	0.038	183	45	2.000
						48 hours	0.26	0.049	183	21	2.000

Leukocyte elastase:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.63	0.061	84	31	0.027
24 hours	0.70	0.050	84	46	0.000
						48 hours	0.73	0.066	84	22	0.001

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.64	0.089	200	12	0.117
24 hours	0.54	0.073	200	18	0.564
						48 hours	0.61	0.097	200	10	0.254

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.57	0.066	76	27	0.282
24 hours	0.65	0.057	76	37	0.006
						48 hours	0.67	0.072	76	20	0.020

Soluble tumor necrosis factor receptor superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.67	0.076	97	17	0.027
24 hours	0.62	0.068	97	23	0.077
						48 hours	0.52	0.210	97	2	0.922

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.74	0.119	155	6	0.046

24 hours	0.57	0.083	155	14	0.403
						48 hours	0.66	0.174	155	3	0.371

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.70	0.080	78	15	0.013
24 hours	0.65	0.080	78	16	0.071
						48 hours	0.58	0.176	78	3	0.662

Soluble tumor necrosis factor ligand superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.45	0.095	77	10	1.368
24 hours	0.62	0.092	77	12	0.193
						48 hours	0.60	0.215	77	2	0.651

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.30	0.103	117	5	1.945
24 hours	0.54	0.115	117	7	0.703
						48 hours	0.38	0.151	117	3	1.554

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.52	0.111	58	8	0.876
24 hours	0.57	0.102	58	10	0.492
						48 hours	0.57	0.216	58	2	0.749

Soluble intercellular adhesion molecule 2:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.50	0.098	47	11	0.976
24 hours	0.17	0.093	47	3	2.000
						48 hours	nd	nd	47	0	0.211

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.36	0.117	72	5	1.764
24 hours	0.27	0.124	72	3	1.938
						48 hours	0.61	0.216	72	2	0.607

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.50	0.098	33	12	1.020
24 hours	0.31	0.143	33	3	1.808
						48 hours	nd	nd	33	0	0.211

Soluble platelet endotheliumCell adhesion molecule:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.49	0.097	47	11	1.087
24 hours	0.39	0.157	47	3	1.515
						48 hours	nd	nd	47	0	0.211

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.38	0.120	72	5	1.686
24 hours	0.55	0.175	72	3	0.762
						48 hours	0.64	0.215	72	2	0.508

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.52	0.099	33	12	0.848
24 hours	0.35	0.153	33	3	1.661
						48 hours	nd	nd	33	0	0.211

Heat shock protein β -1:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.34	0.076	57	14	1.965
24 hours	0.26	0.122	57	3	1.953
						48 hours	0.08	0.081	57	1	2.000

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.52	0.123	91	6	0.894
24 hours	0.45	0.163	91	3	1.255
						48 hours	0.84	0.176	91	2	0.053

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.25	0.067	42	15	2.000
24 hours	0.42	0.164	42	3	1.372
						48 hours	0.04	0.044	42	1	2.000

Various threshold (or "cutoff") concentrations were selected and the relative sensitivity and specificity used to distinguish the first population from the second population is shown in the table below. OR is the odd ratio calculated for a particular cut-off concentration, and 95% CI is the confidence interval for the odd ratio.

Soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

Binding to globin:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Soluble platelet endothelial cell adhesion molecule:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

Example 10 evaluation of renal injury markers for renal status in patients with RIFLE stages 0 and R

Patients were classified and analyzed as described in example 9. However, in the first cohort, patients who reached stage R but did not progress to stage I or F were grouped with patients from non-injury stage 0. In this example, the second population includes only patients who progressed to stage I or F. For the first population, the concentration of the marker in the plasma component of the blood sample is included. For the second population, the concentration of marker in plasma components of blood samples collected within 0, 24 and 48 hours up to expiration I or F is included.

The following descriptive statistics were obtained:

soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

Soluble epidermal growth factor receptor:

sCr or UO

sCr only

UO only

In the table below, the ability to distinguish between the first population (subjects who remained RIFLE 0 or R) and the second population (subjects who developed RIFLE I or F) was determined using ROC analysis.

Soluble p-selectin:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	nd	nd	46	0	0.211
24 hours	0.47	0.090	46	13	1.233
						48 hours	0.54	0.301	46	1	0.885

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	nd	nd	59	0	0.211
24 hours	0.53	0.175	59	3	0.859
						48 hours	nd	nd	59	0	0.211

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	nd	nd	40	0	0.211
24 hours	0.44	0.090	40	13	1.491
						48 hours	0.55	0.303	40	1	0.869

Protein NOV homologs:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.55	0.102	126	9	0.602
24 hours	0.49	0.075	126	17	1.062
						48 hours	0.62	0.175	126	3	0.483

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.93	0.128	157	2	0.001
24 hours	0.77	0.125	157	5	0.028
						48 hours	0.82	0.185	157	2	0.088

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.53	0.102	102	9	0.786
24 hours	0.41	0.075	102	15	1.792
						48 hours	0.55	0.212	102	2	0.809

Nerve growth factor 4:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.67	0.103	80	9	0.099
24 hours	0.67	0.152	80	4	0.258
						48 hours	0.72	0.208	80	2	0.285

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.80	0.190	98	2	0.113
24 hours	0.89	0.154	98	2	0.012
						48 hours	0.69	0.211	98	2	0.372

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.71	0.102	62	9	0.041

24 hours	0.51	0.210	62	2	0.969
						48 hours	0.73	0.291	62	1	0.421

α -1-antitrypsin:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.36	0.054	356	23	1.991
24 hours	0.28	0.045	356	25	2.000
						48 hours	0.24	0.054	356	13	2.000

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.111	441	6	1.494
24 hours	0.43	0.104	441	7	1.480
						48 hours	0.46	0.126	441	5	1.265

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.39	0.058	293	22	1.954
24 hours	0.27	0.045	293	24	2.000
						48 hours	0.17	0.042	293	13	2.000

Leukocyte elastase:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.63	0.075	176	17	0.081

24 hours	0.57	0.072	176	19	0.296
						48 hours	0.62	0.086	176	13	0.152

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.94	0.094	230	3	0.000
24 hours	0.61	0.136	230	5	0.417
						48 hours	0.69	0.133	230	5	0.155

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.57	0.076	154	17	0.333
24 hours	0.56	0.072	154	19	0.410
						48 hours	0.60	0.089	154	12	0.269

Soluble tumor necrosis factor receptor superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.83	0.113	143	5	0.003
24 hours	0.61	0.076	143	17	0.136
						48 hours	0.60	0.136	143	5	0.476

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.90	0.149	178	2	0.008
24 hours	0.72	0.131	178	5	0.093
						48 hours	0.58	0.213	178	2	0.721

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.84	0.125	111	4	0.007
24 hours	0.55	0.081	111	15	0.530
						48 hours	0.59	0.153	111	4	0.535

Soluble tumor necrosis factor ligand superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.26	0.094	109	5	1.990
24 hours	0.53	0.115	109	7	0.763
						48 hours	0.56	0.152	109	4	0.672

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.35	0.174	132	2	1.623
24 hours	0.46	0.164	132	3	1.213
						48 hours	0.29	0.154	132	2	1.831

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.19	0.084	81	4	2.000
24 hours	0.45	0.129	81	5	1.320
						48 hours	0.56	0.175	81	3	0.734

Soluble intercellular adhesion molecule 2:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.60	0.106	60	9	0.365
24 hours	0.38	0.134	60	4	1.632
						48 hours	0.41	0.193	60	2	1.365

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.40	0.189	79	2	1.407
24 hours	0.14	0.091	79	2	2.000
						48 hours	0.53	0.212	79	2	0.881

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.59	0.108	45	9	0.419
24 hours	0.59	0.218	45	2	0.683
						48 hours	0.62	0.306	45	1	0.689

Heat shock protein β -1:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.30	0.080	82	9	1.989
24 hours	0.42	0.140	82	4	1.415
						48 hours	0.26	0.147	82	2	1.895

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.51	0.208	101	2	0.962
24 hours	0.53	0.211	101	2	0.870
						48 hours	0.70	0.210	101	2	0.333

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.28	0.079	64	9	1.995
24 hours	0.32	0.168	64	2	1.714
						48 hours	0.14	0.129	64	1	1.995

Soluble epidermal growth factor receptor:

first group v second group, determination of serum creatinine measurement or urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.60	0.119	26	8	0.398
24 hours	0.58	0.091	26	17	0.369
						48 hours	nd	nd	26	0	0.211

First group v second group, determination of serum creatinine

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.31	0.167	47	2	1.749
24 hours	0.48	0.105	47	9	1.188
						48 hours	0.00	0.000	47	1	n/a

The first group v and the second group, determining the urine output

Time before Max period	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.67	0.123	27	7	0.176

24 hours	0.73	0.097	27	11	0.019
						48 hours	0.63	0.309	27	1	0.675

Various threshold (or "cutoff") concentrations were selected and the relative sensitivity and specificity used to distinguish the first population from the second population is shown in the table below. OR is the odd ratio calculated for a particular cut-off concentration, and 95% CI is the confidence interval for the odd ratio.

Soluble p-selectin:

sCr or UO

sCr only

UO only

Protein NOV homologs:

sCr or UO

sCr only

UO only

Nerve growth factor 4:

sCr or UO

sCr only

UO only

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor ligand superfamily member 6:

sCr or UO

sCr only

UO only

Soluble intercellular adhesion molecule 2:

sCr or UO

sCr only

UO only

Heat shock protein β -1:

sCr or UO

sCr only

UO only

Soluble epidermal growth factor receptor:

sCr or UO

sCr only

UO only

Example 11 evaluation of renal injury markers for renal status in patients progressing from stage R to stages I and F

Patients were classified and analyzed as described in example 9, but only those patients in the arrival period R were included in this example. The first population included patients who reached stage R but did not develop to stage I or F within 10 days, and the second population included only patients who developed to stage I or F. The concentration of marker in the plasma component of the blood sample collected at 12 hours within the arrival period R was included in the analysis for both the first population and the second population.

The following descriptive statistics were obtained:

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

In the table below, the ability to distinguish between the first population (subjects who remained RIFLE R) and the second population (subjects who developed RIFLE I or F) was determined using ROC analysis.

α -1-antitrypsin:

First group v second group, determination of serum creatinine measurement or urine output

Time before max phase

AUC

SE

nFirst group

nSecond group

p

0 hour	0.38	0.088	28	15	1.842
						24 hours	0.38	0.088	28	15	1.842
48 hours	0.38	0.088	28	15	1.842

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.154	12	5	1.336
24 hours	0.43	0.154	12	5	1.336
						48 hours	0.43	0.154	12	5	1.336

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.100	22	13	1.538
24 hours	0.43	0.100	22	13	1.538
						48 hours	0.43	0.100	22	13	1.538

Leukocyte elastase:

First group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.64	0.098	25	13	0.163
24 hours	0.64	0.098	25	13	0.163
						48 hours	0.64	0.098	25	13	0.163

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.43	0.171	10	4	1.339
24 hours	0.43	0.171	10	4	1.339

48 hours

0.43

0.171

10

4

1.339

The first group v and the second group, determining the urine output

Before max phaseTime of day	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.79	0.090	18	12	0.001
24 hours	0.79	0.090	18	12	0.001
						48 hours	0.79	0.090	18	12	0.001

Soluble tumor necrosis factor receptor superfamily member 6:

first group v second group, determination of serum creatinine measurement or urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.39	0.124	10	12	1.619
24 hours	0.39	0.124	10	12	1.619
						48 hours	0.39	0.124	10	12	1.619

First group v second group, determination of serum creatinine

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.88	0.185	4	2	0.043
24 hours	0.88	0.185	4	2	0.043
						48 hours	0.88	0.185	4	2	0.043

The first group v and the second group, determining the urine output

Time before max phase	AUC	SE	nFirst group	nSecond group	p
						0 hour	0.38	0.132	9	10	1.646
24 hours	0.38	0.132	9	10	1.646
						48 hours	0.38	0.132	9	10	1.646

Various threshold (or "cutoff") concentrations were selected and the relative sensitivity and specificity used to distinguish the first population from the second population is shown in the table below. OR is the odd ratio calculated for a particular cut-off concentration, and 95% CI is the confidence interval for the odd ratio.

α -1-antitrypsin:

sCr or UO

sCr only

UO only

Leukocyte elastase:

sCr or UO

sCr only

UO only

Soluble tumor necrosis factor receptor superfamily member 6:

sCr or UO

sCr only

UO only

While the invention has been described or illustrated in sufficient detail to enable those skilled in the art to make and use it, various alterations, changes, and modifications should be apparent without departing from the spirit and scope of the invention. The examples provided herein represent preferred embodiments, which are illustrative and not intended to limit the scope of the invention. Variations thereof and other uses will occur to those skilled in the art. Such variations are included within the spirit of the invention and are defined by the claims.

Various substitutions and alterations will become apparent to those skilled in the art without departing from the spirit and scope of the invention.

All patents and publications mentioned in this specification are indicative of the levels of those of ordinary skill in the art. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was individually and individually indicated to be incorporated by reference.

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

Other embodiments are within the scope of the following claims.

Claims (27)

1. Use of an agent for performing one or more assays in the manufacture of a diagnostic agent for assessing renal status of a subject, wherein:

the assay is configured to detect the renal injury marker leukocyte elastase in a bodily fluid sample obtained from the subject or to detect the renal injury marker leukocyte elastase and one or more additional renal injury markers in a bodily fluid sample obtained from the subject to provide one or more assay results, and the assay results are correlated with the renal status of the subject,

the one or more additional markers of renal injury are selected from the group consisting of soluble p-selectin, a protein homologous to protein NOV, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 and soluble platelet endothelial adhesion molecule,

wherein the correlating step comprises assigning the likelihood of one or more future changes in renal status to the subject based on the analysis; and is

Wherein the one or more future changes in renal status comprise future Acute Renal Failure (ARF) within 48 hours of the time at which the body fluid sample is obtained from the subject.

2. The use of claim 1, wherein said correlating step comprises correlating the assay results with one or more of risk stratification, diagnosis, status, prognosis, classification and monitoring the renal status of the subject.

3. The use of claim 1, wherein the analysis results comprise the following (vii) or one or more of the following (vii) and (i) - (vi) and (viii) - (xiii):

(i) the concentration of soluble p-selectin measured;

(ii) the concentration of the protein NOV homologous protein measured;

(iii) measured concentration of soluble epidermal growth factor receptor;

(iv) measured nerve growth factor-4 concentration;

(v) the measured concentration of haptoglobin;

(vi) measured concentrations of α -1-antitrypsin;

(vii) the measured concentration of leukocyte elastin;

(viii) (ii) the measured concentration of soluble tumor necrosis factor receptor superfamily member 6;

(ix) (ii) the measured concentration of soluble tumor necrosis factor ligand superfamily member 6;

(x) The measured concentration of heat shock protein β -1;

(xi) The concentration of soluble intercellular adhesion molecule 2 measured;

(xii) The measured concentration of active caspase-3;

(xiii) Measured concentration of soluble platelet endothelial cell adhesion molecule;

and the correlating step for each analysis result comprises comparing the measured concentration with a threshold concentration, an

For a positive going marker, when the measured concentration is above the threshold, the subject will have an injury to renal function in the future, reduced renal function in the future, increased likelihood of future ARF or improvement in renal function relative to the likelihood that the measured concentration is below the threshold, or when the measured concentration is below the threshold, the corresponding subject will have an injury to renal function in the future, reduced likelihood of future ARF or improvement in renal function in the future, relative to the likelihood that the measured concentration is above the threshold, or

For a negative going marker, a subject will have an injury to renal function in the future, a decrease in renal function in the future, an increase in the likelihood of ARF or improvement in renal function in the future when the measured concentration is below the threshold, relative to the likelihood that the concentration should be measured above the threshold or when the measured concentration is above the threshold, the corresponding subject will have an injury to renal function in the future, a decrease in renal function in the future, an decrease in ARF or improvement in renal function in the future, relative to the corresponding likelihood when the measured concentration is below the threshold.

4. The use of claim 1, wherein the one or more future changes in renal status comprise a clinical outcome associated with a subject having a renal injury.

5. The use of claim 1, wherein the analysis results comprise the following (vii) or the following (vii) and one or more of (i) - (vi) and (viii) - (xiii):

(i) the concentration of soluble p-selectin measured;

(ii) the concentration of the protein NOV homologous protein measured;

(iii) measured concentration of soluble epidermal growth factor receptor;

(iv) measured nerve growth factor-4 concentration;

(v) the measured concentration of haptoglobin;

(vi) measured concentrations of α -1-antitrypsin;

(vii) the measured concentration of leukocyte elastin;

(viii) (ii) the measured concentration of soluble tumor necrosis factor receptor superfamily member 6;

(ix) (ii) the measured concentration of soluble tumor necrosis factor ligand superfamily member 6;

(x) The measured concentration of heat shock protein β -1;

(xi) The concentration of soluble intercellular adhesion molecule 2 measured;

(xii) The measured concentration of active caspase-3;

(xiii) Measured concentration of soluble platelet endothelial cell adhesion molecule;

and the correlating step for each analysis result comprises comparing the measured concentration with a threshold concentration, an

For a positive marker, when the measured concentration is above the threshold, the subject has a subsequent acute kidney injury, a worsening stage of AKI status, a death, a need for renal replacement therapy, a need for withdrawal of a nephrotoxin, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic renal disease that is increased, a decreased likelihood of a subsequent acute kidney injury, worsening stage of AKI status, a death, a need for withdrawal of a nephrotoxin, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic renal disease, relative to the likelihood that the measured concentration is below the threshold, or a corresponding subject has a subsequent acute kidney injury, worsening stage of AKI status, a death, a need for renal replacement therapy, a need for withdrawal of a nephrotoxin, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic renal disease, relative to the likelihood that

For a negative going marker, when the measured concentration is below the threshold, the subject has a decreased likelihood of subsequent acute kidney injury, worsening AKI status, death, need for renal replacement therapy, need for withdrawal of nephrotoxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease relative to the likelihood that the corresponding subject should have a measured concentration above the threshold or when the measured concentration is above the threshold, the corresponding subject has a decreased likelihood of subsequent acute kidney injury, worsening AKI status, death, need for renal replacement therapy, need for withdrawal of nephrotoxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease relative to the likelihood that the corresponding measured concentration is below the threshold.

6. Use according to claim 1, wherein the likelihood of one or more future changes in renal status is a related event that may occur more or less within a time period selected from the group consisting of: 48 hours, 36 hours, 24 hours and 12 hours.

7. A use according to claim 1, wherein the subject is selected for evaluation of renal status based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF.

8. The use of claim 1, wherein the subject is selected for evaluation based on a diagnosis of the presence of one or more of the following diseases: congestive heart failure, proteinuria, renal insufficiency, glomerular filtration below the normal range, liver cirrhosis, serum creatinine above the normal range, injury to renal function, reduced renal function or ARF, or selecting subjects for evaluation based on the following undergoing or having undergone surgery: major vascular surgery, coronary artery bypass, or cardiovascular surgery thereof, or selecting a subject for evaluation based on exposure to NSAIDs, cyclophosphamide, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, heme, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin.

9. A use according to claim 1, wherein said correlating step comprises assigning a diagnosis of the occurrence or nonoccurrence of one or more of an injury to renal function, reduced renal function, or ARF to the subject based on the assay result.

10. The use of claim 9, wherein the analysis results comprise the following (vii) or one or more of the following (vii) and (i) - (vi) and (viii) - (xiii):

(i) the concentration of soluble p-selectin measured;

(ii) the concentration of the protein NOV homologous protein measured;

(iii) measured concentration of soluble epidermal growth factor receptor;

(iv) measured nerve growth factor-4 concentration;

(v) the measured concentration of haptoglobin;

(vi) measured concentrations of α -1-antitrypsin;

(vii) the measured concentration of leukocyte elastin;

(viii) (ii) the measured concentration of soluble tumor necrosis factor receptor superfamily member 6;

(ix) (ii) the measured concentration of soluble tumor necrosis factor ligand superfamily member 6;

(x) The measured concentration of heat shock protein β -1;

(xi) The concentration of soluble intercellular adhesion molecule 2 measured;

(xii) The measured concentration of active caspase-3;

(xiii) Measured concentration of soluble platelet endothelial cell adhesion molecule;

and the correlating step for each analysis result comprises comparing the measured concentration with a threshold concentration, an

For a positive going marker, when the measured concentration is above the threshold, the subject will have an injury to renal function in the future, reduced renal function in the future, increased likelihood of future ARF or improvement in renal function relative to the likelihood that the measured concentration is below the threshold, or when the measured concentration is below the threshold, the corresponding subject will have an injury to renal function in the future, reduced likelihood of future ARF or improvement in renal function in the future, relative to the likelihood that the measured concentration is above the threshold, or

For a negative going marker, a subject will have an injury to renal function in the future, a decrease in renal function in the future, an increase in the likelihood of ARF or improvement in renal function in the future when the measured concentration is below the threshold, relative to the likelihood that the concentration should be measured above the threshold or when the measured concentration is above the threshold, the corresponding subject will have an injury to renal function in the future, a decrease in renal function in the future, an decrease in ARF or improvement in renal function in the future, relative to the corresponding likelihood when the measured concentration is below the threshold.

11. A use according to claim 1, wherein said correlating step comprises assessing whether renal function is worsening or improving in a subject having an injury to renal function, reduced renal function, or ARF based on the assay result.

12. The use of claim 11, wherein the analysis results comprise the following (vii) or the following (vii) and one or more of (i) - (vi) and (viii) - (xiii):

(i) the concentration of soluble p-selectin measured;

(ii) the concentration of the protein NOV homologous protein measured;

(iii) measured concentration of soluble epidermal growth factor receptor;

(iv) measured nerve growth factor-4 concentration;

(v) the measured concentration of haptoglobin;

(vi) measured concentrations of α -1-antitrypsin;

(vii) the measured concentration of leukocyte elastin;

(viii) (ii) the measured concentration of soluble tumor necrosis factor receptor superfamily member 6;

(ix) (ii) the measured concentration of soluble tumor necrosis factor ligand superfamily member 6;

(x) The measured concentration of heat shock protein β -1;

(xi) The concentration of soluble intercellular adhesion molecule 2 measured;

(xii) The measured concentration of active caspase-3;

(xiii) Measured concentration of soluble platelet endothelial cell adhesion molecule;

and the correlating step for each analysis result comprises comparing the measured concentration with a threshold concentration, an

For a positive marker, when the measured concentration is above the threshold, the renal function of the subject is worsening, or when the measured concentration is below the threshold, the renal function of the subject is improving, or

For a negative going marker, a measured concentration below a threshold corresponds to a worsening renal function in the subject, or a measured concentration above a threshold corresponds to an improvement in renal function in the subject.

13. A use according to claim 1, wherein said assessment is a diagnosis of whether said subject has suffered an injury to renal function.

14. A use according to claim 1, wherein said assessment is a diagnosis of whether said subject has reduced renal function.

15. The use of claim 1, wherein said assessment is a diagnosis of whether said subject has developed acute renal failure.

16. The use of claim 1, wherein said assessment is a diagnosis of whether said subject is in need of renal replacement therapy.

17. The use of claim 1, wherein said assessment is a diagnosis of whether said subject requires kidney transplantation.

18. A use according to claim 1, wherein said assessment is determining whether said subject is at risk of developing an injury to renal function in the future.

19. A use according to claim 1, wherein said assessment is a determination of whether said subject is at risk of developing a future decrease in renal function.

20. A use according to claim 1, wherein said assessment is the determination of whether the subject is at risk of developing acute renal failure in the future.

21. The use of claim 1, wherein said assessing is carried out to determine whether said subject is at risk for developing a future renal replacement therapy.

22. A use according to claim 1, wherein said assessment is the determination of whether the subject is at risk of developing a future renal transplant need.

23. The use of claim 3, wherein the one or more future changes in renal status comprise future Acute Renal Failure (ARF) within 24 hours of obtaining the body fluid sample.

24. Use of an agent for the detection of the kidney injury marker leukocyte elastase or the detection of leukocyte elastase and one or more additional kidney injury markers selected from the group consisting of soluble p-selectin, a homologous protein to protein NOV, soluble epidermal growth factor receptor, nerve growth factor-4, haptoglobin, heat shock protein β -1, α -1-antitrypsin, soluble tumor necrosis factor receptor superfamily member 6, soluble tumor necrosis factor ligand superfamily member 6, soluble intercellular adhesion molecule 2, caspase-3 and soluble platelet endothelial adhesion molecule for the preparation of a diagnostic agent for the assessment of future acute kidney injury.

25. A use according to claim 5, wherein the increased or decreased likelihood of subsequent acute kidney injury, worsening AKI status, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease assigned to the subject is a likelihood that an event of interest is more or less likely to occur within 30 days of the time at which the body fluid sample is obtained from the subject.

26. A use according to claim 5, wherein the increased or decreased likelihood of subsequent acute kidney injury, worsening AKI status, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease assigned to the subject is a likelihood that an event of interest is more or less likely to occur within 72 hours of the time at which the body fluid sample is obtained from the subject.

27. A use according to claim 5, wherein the increased or decreased likelihood of subsequent acute kidney injury, worsening AKI status, mortality, need for renal replacement therapy, need for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, or chronic kidney disease assigned to the subject is a likelihood that an event of interest is more or less likely to occur within 24 hours of the time at which the body fluid sample is obtained from the subject.