NZ625725B2 - Methods and compositions for diagnosis and prognosis of renal injury and renal failure - Google Patents

Abstract

method for evaluating renal status in a subject, comprises performing one or more assays configured to detect Growth/differentiation factor 15 (GDF-15) in a body fluid sample obtained from the subject to provide an assay result; and correlating the assay result(s) to the renal status of the subject. The correlation step comprises correlating the assay result(s) to one or more of diagnosis, risk stratification, prognosis, classifying and monitoring of the renal status of the subject. t. The correlation step comprises correlating the assay result(s) to one or more of diagnosis, risk stratification, prognosis, classifying and monitoring of the renal status of the subject.

Description

METHODS AND COMPOSITIONS FOR DIAGNOSIS AND PROGNOSIS OF RENAL INJURY AND RENAL FAILURE The present application claims ty to US. Provisional Application Nos. 61/562,778 filed November 22, 2011, 61/562,802 filed November 22, 2011, 61/562,813 filed November 22, 2011, 61/562,817 filed November 22, 2011, 61/562,824 filed November 22, 2011, 61/562,829 filed er 22, 2011, 61/562,872 filed November 22, 2011, 61/562,879 filed November 22, 2011, ,883 filed November 22, 2011, 61/562,885 filed November 22, 2011, 61/562,916 filed November 22, 2011, 61/562,943 filed November 22, 2011, 61/562,947 filed November 22, 2011, and 61/562,951 filed November 22, 2011, each of which is hereby orated in its entirety including all , figures, and claims.

OUND OF THE INVENTION The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not ed to describe or constitute prior art to the present invention.

The kidney is responsible for water and solute excretion from the body. Its functions include maintenance of ase balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function h injury and/or e results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison’s Principles of Internal Medicine, 17Lh Ed., McGraw Hill, New York, pages 1741-1830, which are hereby incorporated by reference in their entirety. Renal disease and/or injury may be acute or chronic. Acute and chronic kidney disease are described as follows (from Current Medical Diagnosis & Treatment 2008, 47Lh Ed, McGraw Hill, New York, pages 785-815, which are hereby incorporated by reference in their entirety): “Acute renal failure is worsening of renal function over hours to days, resulting in the retention of nitrogenous wastes (such as urea en) and creatinine in the blood. Retention of these substances is called ia. Chronic renal failure (chronic kidney disease) results from an abnormal loss of renal function over months to years”.

Acute renal failure (ARF, also known as acute kidney injury, or AKI) is an abrupt ally detected within about 48 hours to 1 week)reduction in glomerular 2012/066152 tion. This loss of filtration capacity s in retention of nitrogenous (urea and nine) and non-nitrogenous waste products that are normally excreted by the kidney, a reduction in urine output, or both. It is reported that ARF complicates about 5% of hospital admissions, 4-15% of cardiopulmonary bypass surgeries, and up to 30% of intensive care admissions. ARF may be categorized as prerenal, intrinsic renal, or postrenal in causation. Intrinsic renal disease can be further divided into glomerular, tubular, interstitial, and vascular abnormalities. Maj or causes of ARF are described in the following table, which is adapted from the Merck Manual, 17Lh ed., Chapter 222, and which is hereby incorporated by reference in their entirety: Type Risk Factors Prerenal ECF volume depletion Excessive diuresis, hemorrhage, GI losses, loss of intravascular fluid into the extravascular space (due to s, peritonitis, pancreatitis, or burns), loss of skin and mucus membranes, renal salt- and water-wasting states Low cardiac output Cardiomyopathy, MI, cardiac tamponade, pulmonary embolism, pulmonary hypertension, positive-pressure mechanical ventilation Low systemic vascular Septic shock, liver failure, antihypertensive drugs resistance Increased renal vascular NSAIDs, cyclosporines, tacrolimus, hypercalcemia, resistance anaphylaxis, etics, renal artery obstruction, renal vein thrombosis, sepsis, hepatorenal syndrome Decreased efferent ACE inhibitors or angiotensin II or blockers arteriolar tone (leading to decreased GFR from d glomerular transcapillary pressure, especially in patients with bilateral renal artery stenosis) Acute tubular injury Ischemia nged or severe prerenal state): surgery, hage, arterial or venous ction; Toxins: NSAIDs, cyclosporines, imus, lycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, streptozotocin Acute glomerulonephritis ANCA-associated: Crescentic glomerulonephritis, polyarteritis nodosa, Wegener's granulomatosis; Anti- GBM glomerulonephritis: Goodpasture's syndrome; Immune-complex: Lupus glomerulonephritis, postinfectious glomerulonephritis, cryoglobulinemic glomerulonephritis Type Risk Factors Acute tubulointerstitial Drug reaction (eg, ams, NSAIDs, sulfonamides, nephritis ciprofloxacin, thiazide diuretics, furosemide, phenytoin, allopurinol, pyelonephritis, papillary necrosis Acute vascular Vasculitis, malignant hypertension, thrombotic nephropathy microangiopathies, scleroderma, atheroembolism Infiltrative diseases Lymphoma, sarcoidosis, leukemia Postrenal r precipitation Uric acid (tumor lysis), sulfonamides, triamterene, acyclovir, vir, methotrexate, ethylene glycol ingestion, myeloma n, myoglobin Ureteral obstruction Intrinsic: Calculi, clots, sloughed renal tissue, fungus ball, edema, malignancy, congenital defects; Extrinsic: Malignancy, retroperitoneal fibrosis, al trauma during surgery or high impact injury Bladder obstruction Mechanical: Benign prostatic hyperplasia, prostate cancer, bladder cancer, urethral strictures, phimosis, imosis, al valves, obstructed indwelling urinary catheter; Neurogenic: Anticholinergic drugs, upper or lower motor neuron lesion In the case of ischemic ARF, the course of the disease may be d into four phases. During an initiation phase, which lasts hours to days, reduced perfusion of the kidney is evolving into injury. Glomerular iltration reduces, the flow of te is reduced due to debris within the tubules, and back leakage of filtrate through injured epithelium occurs. Renal injury can be mediated during this phase by reperfusion of the kidney. Initiation is followed by an extension phase which is characterized by continued ischemic injury and inflammation and may involve endothelial damage and ar congestion. During the maintenance phase, lasting from 1 to 2 weeks, renal cell injury occurs, and glomerular filtration and urine output reaches a minimum. A recovery phase can follow in which the renal epithelium is repaired and GFR gradually recovers. Despite this, the survival rate of subjects with ARF may be as low as about 60%.

Acute kidney injury caused by radiocontrast agents (also called contrast media) and other nephrotoxins such as cyclosporine, antibiotics ing aminoglycosides and anticancer drugs such as cisplatin manifests over a period of days to about a week. Contrast induced nephropathy (CIN, which is AKI caused by radiocontrast agents) is t to be caused by intrarenal vasoconstriction (leading to ic injury) and from the generation of reactive oxygen species that are ly toxic to renal tubular epithelial cells. CIN classically presents as an acute (onset within 24-48h) but reversible (peak 3-5 days, resolution within 1 week) rise in blood urea nitrogen and serum nine.

A commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 days or within a period of alization) elevation of serum creatinine. gh the use of serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum nine elevation and the time over which it is measured to define AKI varies considerably among ations. Traditionally, relatively large increases in serum creatinine such as 100%, 200%, an increase of at least 100% to a value over 2 mg/dL and other definitions were used to define AKI. However, the recent trend has been towards using smaller serum creatinine rises to define AKI. The relationship between serum creatinine rise, AKI and the associated health risks are reviewed in Praught and Shlipak, Curr Opin Nephrol Hypertens 14:265-270, 2005 and Chertow et al, JAm Soc Nephrol 16: 3365-3370, 2005, which, with the references listed therein, are hereby incorporated by reference in their entirety. As described in these publications, acute worsening renal function (AKI) and increased risk of death and other ental outcomes are now known to be associated with very small increases in serum creatinine. These ses may be determined as a relative (percent) value or a nominal value. Relative increases in serum creatinine as small as 20% from the pre-injury value have been reported to indicate acutely worsening renal function (AKI) and increased health risk, but the more commonly reported value to define AKI and increased health risk is a relative se of at least 25%. Nominal increases as small as 0.3 mg/dL, 0.2 mg/dL or even 0.1 mg/dL have been reported to te worsening renal function and sed risk of death. Various time periods for the serum creatinine to rise to these threshold values have been used to define AKI, for example, ranging from 2 days, 3 days, 7 days, or a le period defined as the time the patient is in the hospital or intensive care unit. These studies indicate there is not a particular threshold serum creatinine rise (or time period for the rise) for worsening renal function or AKI, but rather a uous increase in risk with increasing magnitude of serum creatinine rise.

One study (Lassnigg et all, J Am Soc Nephrol 15:1597-1605, 2004, hereby incorporated by nce in its entirety) investigated both increases and decreases in serum creatinine. Patients with a mild fall in serum creatinine of -0.1 to -0.3 mg/dL following heart surgery had the lowest mortality rate. Patients with a larger fall in serum creatinine (more than or equal to -0.4 mg/dL) or any increase in serum creatinine had a larger mortality rate. These findings caused the authors to conclude that even very subtle changes in renal function (as detected by small nine changes within 48 hours of surgery) seriously effect patient’s outcomes. In an effort to reach sus on a unified classification system for using serum creatinine to define AKI in clinical trials and in clinical practice, o et al., Crit Care. 8(4):R204-l2, 2004, which is hereby incorporated by reference in its entirety, proposes the following classifications for stratifying AKI patients: “Risk”: serum creatinine increased 1.5 fold from baseline OR urine production of <05 ml/kg body /hr for 6 hours; “Injury”: serum creatinine increased 2.0 fold from baseline OR urine production <0.5 ml/kg/hr for 12 h; “Failure”: serum creatinine sed 3.0 fold from baseline OR nine >355 umol/l (with a rise of >44) or urine output below 0.3 ml/kg/hr for 24 h or anuria for at least 12 hours; And included two clinical outcomes: “Loss”: persistent need for renal replacement therapy for more than four weeks.

“ESRD”: end stage renal disease—the need for dialysis for more than 3 .

These criteria are called the RIFLE criteria, which provide a useful clinical tool to classify renal status. As discussed in Kellum, Crit. Care Med. 36: 8141-45, 2008 and Ricci et al., Kidney Int. 73, 538-546, 2008, each hereby incorporated by reference in its entirety, the RIFLE criteria provide a uniform definition of AKI which has been validated in numerous studies.

More ly, Mehta et al., Crit. Care ll:R3l (doi:10.1186.cc57l3), 2007, hereby incorporated by reference in its entirety, proposes the following r classifications for stratifying AKI patients, which have been modified from RIFLE: “Stage I”: increase in serum creatinine of more than or equal to 0.3 mg/dL (2 26.4 umol/L) or increase to more than or equal to 150% (1.5-fold) from ne OR urine output less than 0.5 mL/kg per hour for more than 6 hours; “Stage II”: increase in serum creatinine to more than 200% (> 2-fold) from baseline OR urine output less than 0.5 mL/kg per hour for more than 12 hours; “Stage 111”: increase in serum creatinine to more than 300% (> 3-fold) from baseline OR serum creatinine 2 354 umol/L accompanied by an acute se of at least 44 umol/L OR urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.

The CIN Consensus Working Panel (McCollough et al, Rev Cardiovasc Med. 2006; 7(4)3] 77-197, hereby incorporated by reference in its entirety) uses a serum creatinine rise of 25% to define Contrast induced nephropathy (which is a type of AKI).Although various groups propose slightly different criteria for using serum creatinine to detect AKI, the consensus is that small changes in serum creatinine, such as 0.3 mg/dL or 25%, are sufficient to detect AKI (worsening renal function) and that the magnitude of the serum creatinine change is an indicator of the severity of the AKI and mortality risk.

Although serial measurement of serum creatinine over a period of days is an accepted method of detecting and diagnosing AKI and is considered one of the most important tools to evaluate AKI patients, serum creatinine is generally ed to have several limitations in the sis, ment and monitoring of AKI patients. The time period for serum creatinine to rise to values (e. g., a 0.3 mg/dL or 25% rise) considered diagnostic for AKI can be 48 hours or longer depending on the tion used. Since cellular injury in AKI can occur over a period of hours, serum creatinine elevations detected at 48 hours or longer can be a late indicator of , and relying on serum nine can thus delay diagnosis of AKI. Furthermore, serum creatinine is not a good indicator of the exact kidney status and treatment needs during the most acute phases of AKI when kidney function is ng rapidly. Some patients with AKI will recover fully, some will need dialysis (either short term or long term) and some will have other detrimental outcomes including death, major adverse cardiac events and chronic kidney disease. Because serum creatinine is a marker of filtration rate, it does not differentiate between the causes of AKI (pre-renal, intrinsic renal, post-renal ction, embolic, etc) or the category or location of injury in intrinsic renal disease (for example, tubular, glomerular or interstitial in origin). Urine output is similarly limited, Knowing these things can be of vital importance in managing and treating patients with AKI.

These limitations underscore the need for better methods to detect and assess AKI, ularly in the early and subclinical stages, but also in later stages when recovery and repair of the kidney can occur. Furthermore, there is a need to better identify patients who are at risk of having an AKI.

BRIEF SUMMARY OF THE INVENTION Provided herein are methods and compositions for evaluating renal function in a subject. As described herein, measurement of one or more biomarkers selected from the group consisting of Stanniocalcin-1, Antithrombin-III, Toll-like receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, Estradiol, Growth/differentiation factor 15, and tein convertase subtilisin/kexin type 9 (each referred to herein as a y injury marker”) can be used for diagnosis, prognosis, risk stratification, staging, monitoring, categorizing and determination of further diagnosis and treatment regimens in ts ing or at risk of suffering from an injury to renal function, reduced renal function, and/or acute renal e (also called acute kidney injury).

The kidney injury s of the present invention may be used, individually or in panels comprising a plurality of kidney injury markers, for risk stratification (that is, to identify subjects at risk for a future injury to renal function, for future ssion to reduced renal on, for future progression to ARF, for future ement in renal function, etc.); for diagnosis of existing disease (that is, to identify subjects who have suffered an injury to renal function, who have progressed to reduced renal function, who have progressed to ARF, etc.); for monitoring for deterioration or improvement of renal function; and for predicting a future medical outcome, such as improved or worsening renal function, a decreased or increased mortality risk, a decreased or increased risk that a subject will require renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or renal transplantation, a sed or increased risk that a subject will recover from an injury to renal on, a decreased or increased risk that a subject will recover from ARF, a decreased or increased risk that a subject will progress to end stage renal disease, a decreased or sed risk that a subject will progress to chronic renal failure, a decreased or increased risk that a subject will suffer rejection of a transplanted kidney, etc.

In a first aspect, the present invention relates to methods for evaluating renal status in a subject. These methods comprise performing an assay method that is configured to detect one or more biomarkers selected from the group ting of Stanniocalcin-1, rombin-III, Toll-like receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, Estradiol, Growth/differentiation factor 15, and Proprotein convertase subtilisin/kexin type 9 is/are then correlated to the renal status of the subject. This correlation to renal status may include ating the assay result(s) to one or more of risk stratification, diagnosis, prognosis, staging, classifying and monitoring of the subject as described herein. Thus, the present invention utilizes one or more kidney injury markers of the present invention for the evaluation of renal injury. [0015A] In a related aspect provided herein is a method for evaluating renal status in a subject, sing: performing one or more assays ured to detect Growth/differentiation factor 15 in a body fluid sample obtained from the subject to provide an assay result; and correlating the assay result(s) to the renal status of the subject, wherein said correlation step comprises correlating the assay (s) to one or more of diagnosis, risk stratification, prognosis, fying and monitoring of the renal status of the subject. [0015B] In a further aspect provided herein is a method for evaluating biomarker levels in a body fluid sample, comprising: ing a urine sample from a subject selected for evaluation based on a determination that the t is at risk of a future or current acute renal injury; and performing a plurality of analyte g assays configured to detect a ity of kers comprising Growth/differentiation factor by introducing the urine sample obtained from the subject into an assay ment which (i) contacts a plurality of ts which specifically bind for detection the plurality of biomarkers with the urine sample, and (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to a respective specific binding reagent in the plurality of reagents, wherein the subject is selected for evaluation based on a determination that the subject is in need of diagnosis, risk stratification, staging, prognosis, classifying or monitoring of the renal status of the subject. [0015C] In a further aspect provided herein is a kit, comprising: reagents for performing one or more assays configured to detect one or more kidney injury markers comprising at least Growth/differentiation factor 15 when used according to a method of the invention. [0015D] In another aspect provided herein is a a plurality of reagents which specifically bind for detection a plurality of biomarkers comprising Growth/differentiation factor 15; and an assay instrument configured to receive a urine sample and contact the ity of reagents with the urine sample and to generate one or more assay results indicative of binding of each biomarker which is assayed to a respective specific binding reagent in the plurality of reagents when used according to a method of the invention.

In certain embodiments, the methods for evaluating renal status described herein are methods for risk stratification of the t; that is, assigning a likelihood of one or more future s in renal status to the subject. In these embodiments, the assay result(s) is/are correlated to one or more such future changes. The following are preferred risk stratification embodiments.

In preferred risk stratification embodiments, these methods se determining a subject’s risk for a future injury to renal function, and the assay result(s) is/are correlated to a likelihood of such a future injury to renal function. For example, the measured concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of suffering a future injury to renal on is assigned to the subject when the measured concentration is above the threshold, ve to a likelihood assigned when the ed concentration is below the threshold. For a ive going” kidney injury marker, an increased likelihood of suffering a future injury to renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the ed concentration is above the threshold.

In other preferred risk fication embodiments, these methods comprise determining a t’s risk for future reduced renal function, and the assay result(s) is/are correlated to a likelihood of such reduced renal function. For example, the measured concentrations may each be compared to a threshold value. For a ive going” kidney injury marker, an increased likelihood of suffering a future reduced renal function is assigned to the subject when the measured concentration is above the threshold, relative to a likelihood assigned when the measured concentration is below threshold. For a “negative going” kidney injury marker, an increased likelihood of future reduced renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In still other preferred risk stratification embodiments, these s comprise determining a subject’s likelihood for a future ement in renal function, and the assay result(s) is/are correlated to a likelihood of such a future improvement in renal function. For e, the ed concentration(s) may each be compared to a threshold value. For a “positive going” kidney injury , an sed likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold. For a “negative going” kidney injury marker, an increased likelihood of a future improvement in renal function is assigned to the subject when the measured concentration is above the old, relative to a likelihood assigned when the measured concentration is below the threshold.

In yet other preferred risk stratification embodiments, these methods comprise determining a subject’s risk for progression to ARF, and the result(s) is/are correlated to a likelihood of such ssion to ARF. For e, the measured tration(s) may each be compared to a threshold value. For a “positive going” kidney injury , an increased likelihood of progression to ARF is assigned to the subject when the ed concentration is above the threshold, ve to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of progression to ARF is assigned to the subject when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

And in other preferred risk stratification embodiments, these methods comprise determining a subject’s outcome risk, and the assay result(s) is/are correlated to a likelihood of the occurrence of a clinical outcome related to a renal injury suffered by the subject. For example, the measured concentration(s) may each be ed to a threshold value. For a “positive going” kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement y, a ement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, myocardial infarction, progression to chronic kidney disease, etc., is assigned to the subject when the measured concentration is above the old, ve to a likelihood assigned when the measured concentration is below the threshold. For a “negative going” kidney injury marker, an increased likelihood of one or more of: acute kidney injury, progression to a worsening stage of AKI, mortality, a requirement for renal replacement therapy, a requirement for withdrawal of renal toxins, end stage renal disease, heart failure, stroke, dial infarction, progression to chronic kidney disease, etc., is assigned to the t when the measured concentration is below the threshold, relative to a likelihood assigned when the measured concentration is above the threshold.

In such risk stratification embodiments, preferably the likelihood or risk assigned is that an event of interest is more or less likely to occur within 180 days of the time at which the body fluid sample is obtained from the subject. In particularly preferred embodiments, the likelihood or risk assigned relates to an event of interest occurring within a shorter time period such as 18 months, 120 days, 90 days, 60 days, 45 days, 30 days, 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, 12 hours, or less. A risk at 0 hours of the time at which the body fluid sample is obtained from the subject is equivalent to diagnosis of a current condition.

In preferred risk stratification embodiments, the subject is selected for risk stratification based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF. For e, a subject undergoing or having undergone major ar surgery, coronary artery bypass, or other cardiac y; a subject having pre-existing congestive heart e, preeclampsia, sia, diabetes mellitus, ension, ry artery disease, proteinuria, renal insufficiency, glomerular filtration below the normal range, cirrhosis, serum creatinine above the normal range, or sepsis; or a subject exposed to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, aque contrast , or streptozotocin are all preferred subjects for monitoring risks according to the methods described herein. This list is not meant to be limiting. By “pre-existence” in this t is meant that the risk factor exists at the time the body fluid sample is ed from the subject. In particularly preferred embodiments, a subject is chosen for risk stratification based on an existing diagnosis of injury to renal function, reduced renal function, or ARF.

In other embodiments, the methods for evaluating renal status described herein are methods for diagnosing a renal injury in the subject; that is, ing whether or not a t has suffered from an injury to renal function, reduced renal function, or ARF. In these embodiments, the assay result(s), for example measured concentration(s) of one or more biomarkers selected from the group consisting of ocalcin-l, Antithrombin-III, Toll-like receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, Estradiol, Growth/differentiation factor 15, and tein convertase subtilisin/kexin type 9 is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are red diagnostic embodiments.

In red diagnostic embodiments, these methods comprise diagnosing the occurrence or urrence of an injury to renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of such an injury. For example, each of the measured concentration(s) may be compared to a threshold value. For a ve going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured tration is below the threshold); alternatively, when the measured concentration is below the old, an increased likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury to renal function is assigned to the subject when the measured concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the ed concentration is above the threshold, an sed likelihood of the nonoccurrence of an injury to renal function may be assigned to the subject (relative to the likelihood ed when the measured concentration is below the threshold).

In other preferred diagnostic embodiments, these s comprise diagnosing the occurrence or nonoccurrence of reduced renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of an injury causing reduced renal function. For example, each of the measured tration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury causing reduced renal on is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased hood of the occurrence of an injury causing d renal function is assigned to the subject when the measured concentration is below the threshold (relative to the hood assigned when the measured tration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of an injury causing reduced renal function may be assigned to the subject (relative to the likelihood assigned when the measured tration is below the threshold).

In yet other preferred diagnostic embodiments, these methods comprise diagnosing the occurrence or urrence of ARF, and the assay result(s) is/are ated to the occurrence or nonoccurrence of an injury causing ARF. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased hood of the occurrence of ARF is assigned to the subject when the measured concentration is above the threshold (relative to the hood assigned when the measured tration is below the threshold); atively, when the measured concentration is below the threshold, an sed likelihood of the nonoccurrence of ARF may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of ARF is ed to the subject when the ed concentration is below the threshold (relative to the likelihood assigned when the measured concentration is above the threshold); alternatively, when the measured concentration is above the threshold, an increased likelihood of the nonoccurrence of ARF may be assigned to the subject ive to the likelihood assigned when the measured concentration is below the threshold).

In still other preferred diagnostic embodiments, these methods comprise diagnosing a subject as being in need of renal replacement therapy, and the assay result(s) is/are correlated to a need for renal replacement therapy. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an increased likelihood of the occurrence of an injury creating a need for renal replacement therapy is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal ement therapy is assigned to the subject when the measured concentration is below the threshold (relative to the hood assigned when the measured concentration is above the old); alternatively, when the measured concentration is above the old, an increased likelihood of the nonoccurrence of an injury creating a need for renal replacement therapy may be assigned to the t ive to the likelihood assigned when the measured concentration is below the threshold).

In still other preferred stic embodiments, these methods comprise diagnosing a subject as being in need of renal transplantation, and the assay result(s0 is/are correlated to a need for renal transplantation. For example, each of the measured concentration(s) may be compared to a threshold value. For a positive going marker, an sed likelihood of the ence of an injury creating a need for renal transplantation is assigned to the subject when the measured concentration is above the threshold (relative to the likelihood assigned when the measured concentration is below the threshold); alternatively, when the measured concentration is below the threshold, an increased hood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is above the threshold). For a negative going marker, an increased likelihood of the occurrence of an injury creating a need for renal transplantation is assigned to the t when the measured concentration is below the threshold (relative to the likelihood assigned when the measured tration is above the old); alternatively, when the measured concentration is above the threshold, an sed likelihood of the nonoccurrence of an injury creating a need for renal transplantation may be assigned to the subject (relative to the likelihood assigned when the measured concentration is below the threshold).

In still other ments, the methods for evaluating renal status described herein are methods for monitoring a renal injury in the subject; that is, assessing whether or not renal function is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF. In these ments, the assay result(s), for e measured concentration(s) of one or more biomarkers selected from the group consisting of Stanniocalcin-l, rombin-III, ike receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, Estradiol, Growth/differentiation factor 15, and Proprotein convertase subtilisin/kexin type 9 is/are correlated to the occurrence or nonoccurrence of a change in renal status. The following are preferred monitoring ments. [003 1] In red monitoring embodiments, these s comprise monitoring renal status in a subject suffering from an injury to renal function, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; atively, when the measured concentration is below the threshold, an improvement of renal function may be ed to the subject. For a negative going marker, when the measured tration is below the threshold, a worsening of renal on may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from reduced renal function, and the assay result(s) is/are correlated to the occurrence or urrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the old, an improvement of renal function may be ed to the subject. For a negative going marker, when the measured concentration is below the threshold, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is above the threshold, an ement of renal function may be assigned to the subject.

In yet other preferred monitoring embodiments, these methods comprise monitoring renal status in a subject suffering from acute renal failure, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going , when the measured concentration is above the threshold, a ing of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be assigned to the t. For a negative going marker, when the measured tration is below the threshold, a worsening of renal function may be ed to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be ed to the subject.

In other additional preferred monitoring embodiments, these methods comprise monitoring renal status in a subject at risk of an injury to renal function due to the istence of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF, and the assay result(s) is/are correlated to the occurrence or nonoccurrence of a change in renal status in the subject. For example, the measured concentration(s) may be compared to a threshold value. For a positive going marker, when the measured concentration is above the old, a worsening of renal function may be assigned to the subject; alternatively, when the measured concentration is below the threshold, an improvement of renal function may be ed to the subject. For a negative going marker, when the measured tration is below the threshold, a worsening of renal function may be ed to the subject; alternatively, when the measured concentration is above the threshold, an improvement of renal function may be assigned to the subject.

In still other embodiments, the methods for evaluating renal status described herein are methods for classifying a renal injury in the subject; that is, determining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or r subdividing these classes into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage. In these embodiments, the assay (s), for example ed concentration(s) of one or more biomarkers selected from the group consisting of Stanniocalcin-l, Antithrombin-III, Toll-like receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, iol, Growth/differentiation factor 15, and Proprotein tase subtilisin/kexin type 9 is/are correlated to a particular class and/or subclass. The following are preferred classification embodiments.

In preferred classification embodiments, these methods comprise ining whether a renal injury in a subject is prerenal, intrinsic renal, or postrenal; and/or further subdividing these s into subclasses such as acute tubular injury, acute glomerulonephritis acute tubulointerstitial nephritis, acute vascular nephropathy, or infiltrative disease; and/or assigning a likelihood that a subject will progress to a particular RIFLE stage, and the assay result(s) is/are correlated to the injury classification for the subject. For example, the measured concentration may be compared to a threshold value, and when the measured tration is above the threshold, a particular classification is ed; alternatively, when the measured concentration is below the old, a different classification may be assigned to the subject.

A variety of methods may be used by the skilled artisan to arrive at a d threshold value for use in these methods. For example, the threshold value may be determined from a population of normal ts by selecting a concentration representing the 75th, 85th, 90th, 95th, or 99th percentile of a kidney injury marker measured in such normal subjects. Alternatively, the threshold value may be determined from a “diseased” population of subjects, e. g., those suffering from an injury or having a predisposition for an injury (e.g., progression to ARF or some other clinical outcome such as death, dialysis, renal transplantation, etc.), by selecting a concentration representing the 75th, 85th, 90th, 95th, or 99th tile of a kidney injury marker ed in such subjects. In another alternative, the threshold value may be ined from a prior measurement of a kidney injury marker in the same subject; that is, a temporal change in the level of a kidney injury marker in the t may be used to assign risk to the subject.

The foregoing discussion is not meant to imply, however, that the kidney injury markers of the present invention must be compared to corresponding individual thresholds. Methods for combining assay results can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree is, calculating ratios of s, etc. This list is not meant to be limiting. In these methods, a composite result which is determined by combining individual markers may be treated as if it is itself a marker; that is, a threshold may be determined for the composite result as described herein for individual markers, and the composite result for an individual patient compared to this old. [003 9] The ability of a particular test to distinguish two populations can be ished using ROC analysis. For example, ROC curves established from a “first” subpopulation which is posed to one or more future changes in renal status, and a “second” subpopulation which is not so predisposed can be used to calculate a ROC curve, and the area under the curve provides a measure of the quality of the test.

Preferably, the tests described herein provide 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 certain aspects, the measured concentration of one or more kidney injury markers, or a composite of such markers, may be treated as continuous variables. For example, any particular concentration can be converted into a corresponding probability of a future ion in renal function for the subject, the occurrence of an injury, a classification, etc. In yet another ative, a threshold that can provide an acceptable level of specificity and sensitivity in ting a population of ts into “bins” such as a “first” ulation (e. g., which is predisposed to one or more future changes in renal status, the occurrence of an injury, a fication, etc.) and a “second” subpopulation which is not so predisposed. A threshold value is ed to separate this first and second population by one or more of the following measures of test accuracy: an odds ratio greater than 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a 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 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 ably about 0.6, yet 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 at least about 0.95, with a corresponding specificity greater than 0.2, preferably greater than about 0.3, more ably r than about 0.4, still more preferably at least about 0.5, even more preferably about 0.6, yet more preferably r than about 0.7, still more preferably greater than about 0.8, more preferably greater than about 0.9, and most preferably r than about 0.95; at least about 75% sensitivity, combined with at least about 75% specificity; a positive likelihood ratio (calculated as sensitivity/(l-specificity)) of greater than 1, at least about 2, more preferably at least about 3, still more preferably at least about 5, and most ably at least about 10; or a negative likelihood ratio (calculated as (l-sensitivity)/specificity) of 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” in the context of any of the above measurements refers to +/- 5% of a given measurement. le thresholds may also be used to assess renal status in a subject. For example, a “first” ulation which is predisposed to one or more future s in renal status, the ence of an injury, a fication, etc., and a “second” subpopulation which is not so predisposed can be combined into a single group. This group is then subdivided into three or more equal parts (known as tertiles, quartiles, quintiles, etc., depending on the number of subdivisions). An odds ratio is assigned to subjects based on which subdivision they fall into. If one considers a tertile, the lowest or highest tertile can be used as a reference for comparison of the other subdivisions. This reference subdivision is assigned an odds ratio of l. The second tertile is assigned an odds ratio that is relative to that first tertile. That is, someone in the second tertile might be 3 times more likely to suffer one or more future changes in renal status in comparison to someone in the first tertile. The third tertile is also assigned an odds ratio that is relative to that first tertile.

In certain embodiments, the assay method is an immunoassay. Antibodies for use in such assays will specifically bind a full length kidney injury marker of interest, and may also bind one or more polypeptides that are “related” thereto, as that term is defined hereinafter. Numerous immunoassay s are known to those of skill in the art.

Preferred body fluid samples are ed from the group consisting of urine, blood, serum, saliva, tears, and plasma. In the case of those kidney injury markers which are membrane proteins as described hereinafter, preferred assays detect soluble forms thereof.

The foregoing method steps should not be interpreted to mean that the kidney injury marker assay result(s) is/are used in isolation in the methods described herein.

Rather, additional variables or other clinical indicia may be included in the methods described herein. For example, a risk fication, diagnostic, classification, monitoring, etc. method may combine the assay result(s) with one or more variables ed for the subject selected from the group consisting of demographic information (e. g., weight, sex, age, race), medical y (e. g., family history, type of surgery, pre-existing disease such as sm, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, nuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscamet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e. g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score, risk scores of Thakar et al. (J. Am. Soc.

Nephrol. 16: 162-68, 2005), Mehran et al. (J. Am. Coll. Cardiol. 44: 1393-99, 2004), Wijeysundera et al. (JAMA 297: 1801-9, 2007), Goldstein and Chawla (Clin. J. Am. Soc.

Nephrol. 5: 943-49, 2010), or Chawla et al. (Kidney Intl. 68: 2274-80, 2005)), a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine tration, a urine creatinine concentration, a fractional excretion of , a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea en to plasma urea nitrogen ratio, a plasma BUN to ine ratio, a renal e index calculated as urine sodium / (urine creatinine / plasma creatinine), a serum or plasma neutrophil gelatinase (NGAL) concentration, a urine NGAL concentration, a serum or plasma cystatin C tration, a serum or plasma cardiac troponin concentration, a serum or plasma BNP concentration, a serum or plasma NTproBNP concentration, and a serum or plasma proBNP concentration. Other measures of renal function which may be combined with one or more kidney injury marker assay result(s) are described hereinafter and in Harrison’s Principles of Internal Medicine, l7Lh Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47Lh Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by nce in their entirety.

When more than one marker is measured, the individual s may be measured in samples obtained at the same time, or may be determined from samples obtained at different (e. g., an earlier or later) times. The individual markers may also be ed on the same or ent body fluid samples. For example, one kidney injury marker may be measured in a serum or plasma sample and another kidney injury marker may be ed in a urine sample. In addition, assignment of a likelihood may combine an individual kidney injury marker assay result with temporal changes in one or more additional variables.

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

In certain embodiments, reagents for ming such assays are provided in an assay , and such assay devices may be included in such a kit. Preferred reagents can comprise one or more solid phase antibodies, the solid phase antibody comprising antibody that detects the intended biomarker target(s) bound to a solid support. In the case of sandwich immunoassays, such reagents can also include one or more detectably labeled antibodies, the detectably labeled antibody comprising antibody that detects the intended biomarker target(s) bound to a able label. Additional optional ts that may be provided as part of an assay device are described hereinafter.

Detectable labels may include molecules that are themselves detectable (e. g., fluorescent moieties, electrochemical labels, ecl (electrochemical luminescence) labels, metal chelates, dal metal particles, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e. g., enzymes such as adish dase, alkaline phosphatase, etc.) or h the use of a specific binding molecule which itself may be detectable (e. g., a labeled antibody that binds to the second antibody, biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Generation of a signal from the signal development element can be performed using various optical, acoustical, and electrochemical methods well known in the art. es of detection modes include fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ometry, etc. In certain of these methods, the solid phase antibody is coupled to a transducer (e. g., a diffraction grating, electrochemical sensor, etc) for generation of a signal, while in others, a signal is generated by a transducer that is spatially separate from the solid phase antibody (e. g., a eter that employs an excitation light source and an optical or). This list is not meant to be limiting. Antibody-based biosensors may also be employed to ine the presence or amount of analytes that optionally eliminate the need for a labeled molecule.

DETAILED DESCRIPTION OF THE INVENTION The present ion relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in subjects suffering or at risk of suffering from injury to renal function, reduced renal on and/or acute renal failure through ement of one or more kidney injury markers. In various embodiments, a measured concentration of one or more biomarkers selected from the group consisting of Stanniocalcin-l, Antithrombin-III, ike receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, ation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, Estradiol, Growth/differentiation factor 15, and Proprotein convertase subtilisin/kexin type 9 or one or more markers related thereto, are correlated to the renal status of the subject.

For purposes of this nt, the following tions apply: [005 1] As used herein, an “injury to renal function” is an abrupt (within 14 days, ably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) able reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc. “Improvement in Renal Function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) able increase in a measure of renal function.

Preferred methods for measuring and/or estimating GFR are described hereinafter.

As used herein, “reduced renal function” is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of r than or equal to 0.1 mg/dL (2 8.8 umol/L), a percentage increase in serum creatinine of greater than or equal to 20% (l .2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0. 5 ml/kg per hour).

As used herein, “acute renal failure” or “ARF” is an abrupt n 14 days, preferably within 7 days, more ably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of r than or equal to 0.3 mg/dl (2 26.4 umol/l), a percentage increase in serum creatinine of greater than or equal to 50% (l. 5-fold from baseline), or a ion in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours).

This term is synonymous with “acute kidney injury” or “AKI.” The following biomarkers (listed with the Swiss-Prot entry number of the human precursor) find use in the present invention as kidney injury markers: Swiss-Prot Entry # Name P52823 Stanniocalcin-l P01008 Antithrombin-III 060603 Toll-like rece tor 2 na Triiodothyronine (T3) na Thyroxine (T4) Q16610 Extracellular matrix rotein l P00488 Coagulation factor XIII A chain P05160 Coagulation factor XIII B chain Q96PD4 Interleukin-17F Q9GZX6 Interleukin-22 P04004 Vitronectin na Progesterone na iol Q99988 Growth/differentiation factor 15 Q8NBP7 Proprotein convertase subtilisin/kexin type 9 na — not applicable As used herein, the term “Triiodothyroxine” refers to the thyroid hormone also known as T3, while “Thyroxine” refers to the thyroid hormone known as T4. T3 is a product of deiodination of T4 by enzymes in the thyroid. T3 and T4 are carried in the circulation by thyroxine-binding globulins, thyroxine binding prealbumins, and albumins.

As a consequence of structural differences, T3 and T4 may be distinguished from one another, for example using antibodies that are ic for differences between the hormones.

Estradiol ((178)-estra-l,3,5(10)-triene-3,l7-diol) is a steroid e that is the predominant estrogen in females during uctive years both in terms of absolute serum levels as well as in terms of estrogenic activity. During menopause, estrone is the predominant circulating estrogen and during pregnancy estriol is the inant circulating estrogen in terms of serum levels. Estradiol is also present in males, being produced as an active metabolic product of testosterone. The serum levels of estradiol in males (14 -55 pg/mL) are roughly comparable to those of postmenopausal women (< 35 Progesterone (pregnene-3,20-dione) is a C-21 steroid e. In women, progesterone levels are relatively low during the preovulatory phase of the menstrual cycle, rise after ovulation, and are elevated during the luteal phase. Progesterone levels are relatively low in children and postmenopausal women, while adult males have levels similar to those in women during the follicular phase of the menstrual cycle.

As used herein, the term “Stanniocalcin-l” refers to one or more polypeptides present in a biological sample that are derived from the Stanniocalcin-l precursor (human precursor: Prot P52823 (SEQ ID NO: 1)) 20 3O 4O 50 6O WLQNSAVLLV LVISASAiHfl AflQNDSVSPR KSRVAAQNSA EVVRCLNSAL FACL 7O 8O 90 100 110 120 ENSTCDTDGM YDICKSFLYS AAKED.QGKA bVKLSLKCIA VGVTSKVFJA IQQCSTFQQM 130 140 150 160 170 180 IAflVQflfiCYS KLNVCSIAKR NPfiAI_flVVQ LPNiFSNQYY VQLVRS.LfiC DfiDiVSiIQD 190 200 210 220 230 240 SLWEKIGPNM LQTD PRAD FNRQRTN?PQ QNIR GfifiDSPSHIK RTSiESA [005 9] The following domains have been identified in Stanniocalcin-l: Residues Length Domain ID 1-17 17 Signal peptide 18-33 16 Propeptide 34-247 214 Stanniocalcin-l As used herein, the term “Proprotein convertase subtilisin/kexin type 9” refers to one or more polypeptides present in a biological sample that are derived from the Proprotein convertase subtilisin/kexin type 9 precursor (human precursor: Swiss-Prot Q8NBP7 (SEQ ID NO: 2)) 20 3O 4O 50 6O MGTVSSRRSW WP.PLTJJJJ.T.1GPAGARA DYfifl LVLAIRSfifiD fiHGi 7O 8O 90 100 110 120 TATFHRCAKD LYVV VuKflfiLHLSQ R.QA QAARRGYITK IIHVFHGL.P 130 140 150 160 170 180 GFLVKMSGDL L?.A.K.PHV DYIflfiDSSVb N.?R ITPPQYRADE YQPPDGGSJV 190 200 210 220 230 240 TSIQ SDiRfiIfiGRV MVlDbfiNVPfi fiDGiRhHQQA SKCDSHGTHA AGVVSGQDAG 250 260 270 280 290 300 VAKGASMRSL QVJNCQGKGT VSGTLIGLEF IQKSQLVQPV LPLA GGYSRVJNAA 310 320 330 340 350 360 CQRLARAGVV JVTAAGNFRD DACLYSPASA PEVITVGATN AQDQPVTIGT .GTNFGRCVD 370 380 390 ~00 ~10 ~20 LFAPGEDIIG ASSDCSTCFV SQSGTSQAAA AWML SAfiPflL_IAfl .RQRLliFSA ~30 440 450 ~60 ~70 ~80 KDVINflAWbP flDQRV._PVL VAALPPSTHG AGWQLFCQTV WSAHSGPTQW ATAVARCAPD ~90 500 510 520 530 540 *flILSCSSFS RSGKRRGfiQM IVCR AHNAFGGEGV YAIARCCLJP QAVCSVHTAP 550 560 570 580 590 600 PAEASMGTRV {CHQQGHVJT GCSSHWflVflD LG_HKPPVLR PRGQPNQCVG HREASIHASC 610 620 630 640 650 660 CHAPGLflCKV KfiHGIPAPQL QViVACfifiGW 1L_GCSALPG TSHVLGAYAV DNTCVVRSRD 670 680 690 VSTTGSTSEG AVTAVAICCR SRHLAQASQE LQ The ing domains have been identified in Proprotein convertase subtilisin/kexin type 9: Residues Length Domain ID 1-30 30 Signal peptide 3 1- 152 122 Propeptide 153-692 540 Proprotein convertase subtilisin/kexin type 9 1-174 —> MSPWK (SEQ ID NO: 3) in isoform 2 333-365 —> GRTSLVPPATAAPALCHRVGHHRLLPTWLALQP (SEQ ID NO: 4) in isoform 2 366-692 Missing in isoform 2 As used herein, the term “Interleukin-22” refers to one or more polypeptides present in a biological sample that are derived from the eukin-22 precursor (human precursor: Swiss-Prot Q9GZX6 (SEQ ID NO: 5)) 20 3O 4O 50 6O MAALQKSVSS FLMGTLATSC LLLLALDVQG GAAAPISSHC QDDKSNFQQP YITNQTFMLA 7O 8O 90 100 110 120 KEASIADNNT DVRLIGEKIF HGVSMSEQCY LMKQVTVEII flflVTbPQSDR FQPYWQEVVP 130 140 150 160 170 FIARISNRLS TCHI?GDD.H IQRNVQKDKD IVKKLGfiSGfi IKAIG?LDL. ACI The following domains have been identified in Interleukin-22: Residues Length Domain ID 1-33 33 Signal peptide 34- 179 146 Interleukin-22 As used herein, the term “Coagulation factor XIII A chain” refers to one or more polypeptides present in a biological sample that are derived from the Coagulation factor XIII A chain precursor (human precursor: Swiss-Prot P00488 (SEQ ID NO: 6)) 20 3O 4O 50 6O MSLISR_AEG GRRAVPPNNS NAAflDDLPIV flLQGVVPRGV NLQLELNVIS RWDI 7O 8O 90 100 110 120 NKVDHH_DKY LNNKLIVRRG QSFYVQIDFS RDLF RVEYVIGRYP QENKGTYIPV 130 140 150 160 170 180 PIVSELQSGK WGAKIVMRED QSVQLSIQSS PKCIVGKFRM YVAVWTPYGV LQTSRNPETD 190 200 210 220 230 240 TYIIFNPWC? DDAVYLDNfiK flRflflYVLNDI GVIFYGEVND IKTRSWSYGQ F?DGILDTC. 250 260 270 280 290 300 YVMDRAQMDD SGRGNPIKVS QVGSAMVNAK DDEGVLVGSW GVPP SAWTGSVDID 310 320 330 340 350 360 LfiYQSSflNPV RYGQCWVFAG VFNTFDRCLG IPARIVTRYF SAHDNDARDQ flDGV 370 380 390 ~00 ~10 ~20 VVSKLTKDSV WNYHCWNEAW MTRPDDPVGF GGWQAVDS_P QLNSDGMYQC GPASVQAIKi ~30 440 450 ~60 ~70 ~80 GiVCFQFDAP FVFAEVNSDL IYIIAKKDGI HVVLNVDA.H IGKDIVTKQI GGDGMMDITD ~90 500 510 520 530 540 iYKbQfiGQfifi fiRLAIfiIALM YGAKKPLVTE GVMKSRSVVD MDbflVflNAVL SITF 550 560 570 580 590 600 QNNSHVRYTI NIIE YIGVPKAflbK KflibDVi.flP LSbKKflAVLI QAG?YMGQ.L 610 620 630 640 650 660 LQASLitiI ARINLIRDVL AKQKSIV._I PfiIIIKVRGI MIVI VQEINPLKLI 670 680 690 700 710 720 DQNVWVHDDG PGVTQPMKKM bRLIRPNS_V QWfifiVCRPWV SGHRKDIASM HVYG ?.DVQIQ?RP SM The following domains have been identified in ation factor XIII A chain: Residues Length Domain ID 1 l Initiator methionine 2-38 37 Activation peptide 39-732 694 Coagulation factor XIII A chain As used herein, the term “Toll-like receptor 2” refers to one or more polypeptides present in a biological sample that are d from the Toll-like receptor 2 precursor (human sequence: Swiss-Prot 060603 (SEQ ID NO: 7)): 20 30 40 50 60 MPHTLWMVWV LGVIISLSKfl flSSNQASLSC DRNGICKGSS GSLNSIPSGL TEAVKSLDLS 70 80 90 100 110 120 NNRITYISNS D.QRCVNLQA .VLISNGINI InnDStSSLG SL?HLD.SYN YLSNLSSSWF 130 140 150 160 170 180 KPISSITFLV L.GVPYKTLG ?TSLFSHLTK LQI.RVGNMD TFTKIQRKDb AGLibLflflLfl 190 200 210 220 230 240 IDASD.QSY? PKS.KSIQNV SHLILHMKQH I. .nItVDV _SSVnC.n.R DID.DIb{bS 250 260 270 280 290 300 n.81GniNs. RNVK ITDESLFQVM K. VQISGT nLntDDCi.N GVGVFRASDN 310 320 330 340 350 360 DRVIDPGKV? TLTIRR.HIP RFY.FYDLST RVKR ITV?VSKVF. VPC..SQP.K 370 380 390 ~00 ~10 ~20 LSfiV LMVnnY.KNs AC?DAWPSIQ TLI.RQNI.A s.nKiG«i.. iLKV.iNIDI ~30 440 450 ~60 ~70 ~80 SKVStHSMPL .CQWPnKMKY .N.SSTRI{S V.GCIPKi.n I.DVSNVR.V LFS.VLPQ.K ~90 500 510 520 530 540 ?.YISRVKLW TLPDASLLPM JLVLKISRVA I.itSKnQ.D ShiILKILLA GGNVFICSCE 550 560 570 580 590 600 nQQA .AKV.IDWPA VYLCDSPSiV RGQQVQDVR. SVS?CHRTA. VSGWCCA.F. 610 620 630 640 650 660 LI..TGVLCH RFHGJWYWKM WWAWLQAKRK PRKAPSRNIC YDAFVSYSER DAYWV?N.MV 670 680 690 700 710 720 Qn.nNtVPPt KLCniKRDFI PGKWIIDNII IKIV bV.SfiNbVKS nWCKYnLDtS 730 740 750 760 770 780 HFR.FD?NND AAI.IL.«PI nKKAIPQRFC VIKI YLnWPMDnAQ RnGtWVN.RA AIKS In n embodiments, the Toll-like receptor 2 assay detects one or more soluble forms of Toll-like receptor 2. Toll-like receptor 2 is a single-pass membrane protein having an extracellular domain which may be found in soluble forms of Toll-like receptor 2 generated by proteolysis of the membrane-bound form or by alternative splicing. In the case of an immunoassay, one or more antibodies that bind to epitopes within an extracellular domain may be used to detect these soluble form(s). The ing domains have been fied in ike receptor 2: Residues Length Domain ID 1-18 18 Signal e 19-784 766 Toll-like receptor 2 9 21 transmembrane domain 610-784 175 cytoplasmic domain 19-588 570 ellular domain As used , the term “Antithrombin-III” refers to one or more polypeptides present in a biological sample that are derived from the Antithrombin-III precursor (human precursor: Swiss-Prot P01008 (SEQ ID NO: 8)) 20 30 40 50 60 MYSVVIGTVT SGKQKVYIIS LIIIGFWDCV TCHGSPVDIC TAKPRDIPMN PMCIYRSPEK 70 80 90 100 110 120 KAIfiDflGSfiQ NRQV W?.SKANSQF ATTFYQHAAD SKNDNDVIFL SPLSISTAFA 130 140 150 160 170 180 WTKAGACVDT LQQIWfiVEKh DIISLKISDQ IiFFFAKnNC RLYQKAVKSS KLVSANQLFG 190 200 210 220 230 240 DKSITFN?TY QDIS?IVYGA KLQPIDbKfiV AfiQSRAAINK WVSVKILGRI LAIN 250 260 270 280 290 300 ?.TVLVLVNT IYFKGAWKSK bSPfiVIRKfi. bYKADGLSCS ASMWYQEGKF RYRQVAEGTQ 310 320 330 340 350 360 V.?LPFKGDD ITMV.ILPKP ?KSIAKVfiKfi QfiWL DflLflflWWLVV HMPQFRIEDG 370 380 390 ~00 410 420 FSIK?Q.QDM GLVDIFSP?K SKLPGIVA?G QDDIYVSDAt HKAtIflVNfifi GSflAAASIAV 430 440 450 ~60 VIAGRSANPN RVIbKANRPb LVtIRflVPIN IIIbMGRVAN PCVK The following domains have been fied in Antithrombin-III: Residues Length Domain ID 1-32 32 Signal peptide 33-464 432 Antithrombin-III As used herein, the term “Vitronectin” refers to one or more polypeptides present in a biological sample that are derived from the Vitronectin precursor (human precursor: Prot P04004 (SEQ ID NO: 9)) 20 3O 4O 50 6O MAPIRPLLIL A.LAWVALAD Q?SCKGRCTE GFNVDKKCQC DELCSYYQSC CTDYTAECKP 7O 8O 90 100 110 120 QVIQGDVEIM PfiDfiYIVYDD GunKNNAIVi LQVGGPSLTS DnQAQSKGNP flQlPVLKPfifl 130 140 150 160 170 180 fiAPAPflVGAS KPEGIDSRPE PQPP AflflflLCSGKP FDAFTDLKNG SDFAFRGQYC 190 200 210 220 230 240 YfiLDflKAVRP RDVW DAAF TQIVCQGKTY DFKGSQYWRF EDGVLDPDYP 250 260 270 280 290 300 RVISDGFDGI PDNVDAAIA. GRE? VYFFKGKQYW EYQFQHQPSQ SLSA 310 320 330 340 350 360 VFEHFAMMQ? DSWflDItflL. tWGRiSAGi? QPQEISRDWH GVPGQVDAAM AGRIYISGMA 370 380 390 ~00 ~10 420 PRPSDAKKQ? FRHQNRKGY? SQRGHSRGQV QNSRRPSRAi WLSLESSflfiS NLGANNYDDY 430 440 450 ~60 ~70 RMDWDVPATC EPIQSVFFFS GDKYYRVNDQ TRRVDTVDPP YPRSIAQYWL GCPAPGHL The following domains have been identified in Vitronectin: Residues Length Domain ID 1-19 19 Signal peptide -478 459 ectin -398 379 Vitronectin V65 subunit -63 44 Somatomedin-B 399-478 80 Vitronectin V10 subunit As used herein, the term “Coagulation factor XIII B chain” refers to one or more polypeptides present in a biological sample that are derived from the Coagulation factor XIII B chain sor (human precursor: Swiss-Prot P05160 (SEQ ID NO: 10)) 20 30 40 50 60 MRLKNDTFII IIIISGflLYA fiflKPCGbPHV LNGRIAQYYY TFKSFYFPMS IDKKDSFFCL 70 80 90 100 110 120 AGY..flSGRQ flflQilCliflG WSPflPRCtKK SVGY LYKI QENMQYGCAS 130 140 150 160 170 180 GYK..GGKDfi fiVVQCISDGW SSQP_CRKflH fi_CLAPfl.YV GNYSTTQKTF KVKDKVQYEC 190 200 210 220 230 240 ATGYYTAGGK KiflfiVficbiY GWSLTPKCTK IKCSSLQIIfi NGYbHPVKQ. YfifiGDVVbe 250 260 270 280 290 300 CHENYYLSGS DLIQCYNEGW YPflSPVCflGR QNRCPPPPLP INSKIQiHS. 1YQHGLIVHI 310 320 330 340 350 360 fiCfiLNbflIHG SAflIQCflDGK WIfiPPKCIfiG QflKVACfiflPP tIfiNGAAkLH SKIYYNGDKV 370 380 390 ~00 ~10 ~20 TYACKSGYL. HGSN?ITCN? GKWILPPfiCV flVNflNCK-PP VVflNGAVADG TGSS ~30 440 450 ~60 ~70 ~80 VflYRCNfiYY. LQGSKISQCE QGKWSSPPVC 3PCTVWVDY EMKW LFGD ~90 500 510 520 530 540 LIDFVCKQGY DISPLTPIS? LSVQCNRG?V KYPLCTRK?S KGWCTSPPLI KHGVIISSTV 550 560 570 580 590 600 DlYfiNGSSVfi HbLfl GSRflAYCLDG MWIIPPICIfi PClLSbifiMfi KWDF 610 620 630 640 650 660 DNRPHILHGfi YIflbICRGDI YPALLYIIGS DRGQ LKYPRCIPRQ STLSYQEPLR The following domains have been identified in Coagulation factor XIII B chain: Residues Length Domain ID 1-20 20 Signal peptide 21-661 641 Coagulation factor XIII B chain As used herein, the term “Interleukin-17F” refers to one or more polypeptides present in a biological sample that are derived from the Interleukin-17F precursor (human sor: Swiss-Prot Q96PD4 (SEQ ID NO: 11)) 20 30 40 50 60 MTVKTLHGPA MVKY.L.SIL GLAFLS?AAA RKIPKVGHTF FQKPESCPPV PGGSMKLDIG 70 80 90 100 110 120 IINENQRVSM SQNIESQSTS PWNYTVTWDP NRYPSEVVQA QCRNDGCINA QGKEDISMNS 130 140 150 160 VPIQQETLVV RQKHQGCSVS FQLEKVLVTV GCTCVTPVIH HVQ The following domains have been identified in Interleukin-17F: Residues Length Domain ID 1-30 30 Signal peptide 31-163 133 Interleukin-17F As used herein, the term “Extracellular matrix protein 1” refers to one or more polypeptides present in a biological sample that are derived from the Extracellular matrix protein 1 precursor (human precursor: Swiss-Prot Q16610 (SEQ ID NO: 12)) 20 30 40 50 60 MGTTARAALV LTYLAVASAA ATGQ QQLRPfiHhQfi VGYAAPPSPP LSQSLPWDHP 70 80 90 100 110 120 DSSQiGPPFE GQSQVQPPPS QQflK .LPAQIPAfiK fiVGPPLPQ?A VPIQK?.PSL 130 140 150 160 170 180 KflGl PAPEGDQSHP fiPfiSWNAAQH CQQDRSQGGW GiRnDGFPPG QPSPDVDVQI 190 200 210 220 230 240 CLPNQQHVVY GPWNLPQSSY SHIIRQGfiIL NbufiIGYSRC CiCRSHTNR. ECAKIVWfifiA 250 260 270 280 290 300 MSRbCfiAflbS WCCI RQGLARESCE QfifiAPQPdYQ nRACPSiQPD ISSGIELPFP 310 320 330 340 350 360 PGVPTEDVIK NICHLRRFRS ATDP LQ??LLA.IQ RCC? QGNNiTCTWK 370 380 390 ~00 ~10 ~20 DKYC DQEYAVKTHH PPSP TQDECFAQRA PYPVYDQDIA TIDIGQVTPN ~30 440 450 ~60 ~70 ~80 IMGHICGRQR VATKHKHIPG LIHNWTARCC DIPFP?QACC ibIV DLCGPQRRIW ~90 500 510 520 530 540 RDPAICCYLS PGDEQVNCFN INYLQNVALV SGDTEVAKGQ GLQGSIGGIV ISSiSfiPKflfl The following domains have been identified in Extracellular matrix protein 1: Residues Length Domain ID 1-19 19 Signal peptide -540 521 Extracellular matrix protein 1 237-361 Missing in isoform 2 1-71 Missing in isoform 3 72-81 —> MALPLRDRVK (SEQ ID NO: 13) in isoform 3 237-241 —> VRLGS (SEQ ID NO: 14) in isoform 3 242-250 Missing in isoform 3 74 —> GKEGRGPRPHSQPWLGERVGCSHIPPSI (SEQ ID NO: 15) in isoform 4 As used herein, the term “Growth/differentiation factor 15” refers to one or more polypeptides present in a biological sample that are derived from the Growth/differentiation factor 15 precursor (human precursor: Swiss-Prot Q99988 (SEQ ID NO: 16)) 20 3O 4O 50 6O WPGQ?IQTVN GSQWLLVL.V LSWLPHGGAL SIAflASRASb PGPSflLHSflD SQEQflIRKQY 7O 8O 90 100 110 120 ?DL.T?.RAN QSWLDSN_DL VPAPAVRIIT P?VR.GSGGH LHLRISRAA. PflGIPfiASQ. 130 ..40 150 160 170 180 {QAIFQISPT ASQSWDVTQP IRRQISIAQP QAPAIHIRLS DQL. RPQ4 190 200 210 220 230 240 ?.H.RPQAAR GRQRARARVG DHCPAGPGQC CRLHTVQASL DWV. SPR?VQVTMC 250 260 270 280 290 300 IGACPSQFRA ANWHAQIKTS DHRLKPDTVP APCCVPASYN PMVLIQKTDT GVSDQTYDDL LAKDCHCI The following domains have been identified in Growth/differentiation factor Residues Length Domain ID 1-29 29 Signal peptide - 194 165 Propeptide 195 -308 1 14 /differentiation factor 15 As used herein, the term ing a signal to the presence or amount” of an analyte reflects the ing tanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is “configured to detect” an analyte if an assay can generate a detectable signal indicative of the presence or amount of a logically relevant concentration of the analyte.

Because an antibody epitope is on the order of 8 amino acids, an immunoassay configured to detect a marker of st will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay. The term “related marker” as used herein with regard to a biomarker such as one of the kidney injury markers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers. The term also refers to one or more ptides present in a biological sample that are derived from the biomarker precursor complexed to additional s, such as binding ns, receptors, heparin, lipids, sugars, etc.

In this regard, the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and ptides containing the necessary epitope(s) to which the dies bind. While such assays may detect the full length ker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay is ly a result of all such “immunoreactive” polypeptides present in the sample. Expression of biomarkers may also be determined by means other than immunoassays, including protein ements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting.

As used herein, the term “hydrocortisone” (also known as cortisol) refers to (l l B)-l 1,17,21 -trihydroxypregnene-3,20-dione. Hydrocortisone is a steroid hormone, or glucocorticoid, produced by the adrenal gland. It is released in response to stress and a low level of blood glucocorticoids. Its primary functions are to increase blood sugar through gluconeogenesis; suppress the immune system; and aid in fat, protein and carbohydrate metabolism.

The term “positive going” marker as that term is used herein refer to a marker that is determined to be elevated in ts suffering from a disease or condition, relative to subjects not suffering from that disease or condition. The term ive going” marker as that term is used herein refer to a marker that is ined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.

The term “subject” as used herein refers to a human or non-human organism.

Thus, the methods and compositions described herein are applicable to both human and nary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably “patients,” which as used herein refers to living humans that are receiving medical care for a disease or ion. This includes persons with no defined illness who are being igated for signs of ogy.

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

The term “body fluid sample” as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or tion of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the e of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid s include blood, serum, plasma, cerebrospinal fluid, urine, , sputum, and l effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma ents.

The term “diagnosis” as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability (“a likelihood”) of whether or not a patient is suffering from a given disease or ion. In the case of the present invention, “diagnosis” includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical teristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed.

That such a diagnosis is “determined” is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The d clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the ence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.

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

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

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

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

Biological assays require methods for detection, and one of the most common s for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied.

Detectable labels may include molecules that are lves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable on product (e. g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e. g., biotin, digoxigenin, maltose, oligohistidine, 2,4- dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

Preparation of solid phases and able label conjugates often comprise the use of al cross-linkers. Cross-linking reagents contain at least two reactive , and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups).

Homobifunctional cross-linkers that couple through amines, dryls or react non- specifically are available from many commercial sources. Maleimides, alkyl and aryl s, alpha-haloacyls and pyridyl disulfides are thiol ve groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

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

Antibodies The term “antibody” as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e. g. Fundamental Immunology, 3rd Edition, W.E. Paul, ed., Raven Press, NY. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J.

Biochem. Biophys. s 25:85-97. The term dy includes n-binding portions, i.e., "antigen binding sites," (e. g., fragments, subsequences, complementarity determining s (CDRs)) that retain capacity to bind antigen, ing (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a 2 fragment, a bivalent fragment comprising two Fab nts linked by a disulfide bridge at the hinge ; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." 2012/066152 Antibodies used in the immunoassays described herein preferably ically bind to a kidney injury marker of the present invention. The term “specifically binds” is not intended to indicate that an antibody binds exclusively to its ed target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target le which does not display the appropriate epitope(s). ably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, Preferred antibodies bind with affinities of at least about 107 M'l, and preferably between about 108 M"1 to about 109 M'l, about 109 M'1 to about 1010 M'l, or about 1010 M'1 to about 1012 M'1 .

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

The term “epitope” refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active e groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

Conformational and nonconformational es are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e. g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 698. A basic concept of phage display methods is the ishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide.

This al association is provided by the phage particle, which displays a polypeptide as part of a capsid ing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their c material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective s. Using these methods a polypeptide identified as having a binding affinity for a d target can then be synthesized in bulk by conventional means. See, e. g., U.S.

Patent No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

The antibodies that are ted by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the ty and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the ed polypeptides in te wells of microtiter plates.

The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody ated to alkaline phosphatase if the raised dies are mouse antibodies) is added to the wells and ted for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where dy to the immobilized polypeptide(s) are present.

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

While the t application bes antibody-based binding assays in detail, alternatives to antibodies as binding species in assays are well known in the art.

These include receptors for a particular , rs, etc. rs are oligonucleic acid or peptide molecules that bind to a specific target molecule. rs are usually created by selecting them from a large random sequence pool, but natural aptamers also exist. High-affinity aptamers containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. es of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions, and may include amino acid side chain functionalities.

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

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

Suitable thresholds may be determined in a variety of ways. For example, one ended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 97.5th percentile of the concentration seen in a normal population.

Another method may be to look at serial samples from the same patient, where a prior “baseline” result is used to monitor for temporal changes in a biomarker level.

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

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

] In addition to threshold isons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) e decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications. es of test accuracy may be obtained as described in Fischer et 61]., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures e ivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (“AUC”) of a ROC plot is equal to the ility that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

As sed above, suitable tests may exhibit one or more of the following results on these s measures: a specificity of r than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 02, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably r than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 02, preferably r than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of r than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more ably at least 0.9, and most ably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1-specificity)) of r than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1-sensitivity)/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention. These include other biomarkers related to renal status. Examples include the following, which recite the common biomarker name, followed by the Swiss-Prot entry number for that biomarker or its parent: Actin (P68133); Adenosine deaminase binding protein (DPP4, P27487); l-acid glycoprotein 1 3); Alpha-l-microglobulin (P02760); Albumin 8); Angiotensinogenase (Renin, P00797); AnneXin A2 (P07355); Beta-glucuronidase (P08236); B microglobulin 9); Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta (S100-beta, P04271); Carbonic anhydrase (Ql6790); Casein Kinase 2 (P68400); Ceruloplasmin (P00450); Clusterin (P10909); Complement C3 (P01024); Cysteine-rich protein (CYR61, 000622); Cytochrome C (P99999); Epidermal growth factor (EGF, P01133); Endothelin-l 5); Exosomal -A (P02765); Fatty acid-binding protein, heart (FABP3, ); Fatty acid-binding protein, liver (P07148); Ferritin (light chain, P02793; heavy chain P02794); Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCLl, (P09341); Growth e (P01241); Hepatocyte growth factor (P14210); Insulin-like growth factor I 3); Immunoglobulin G; Immunoglobulin Light Chains (Kappa and Lambda); eron gamma (P01308); Lysozyme (P61626); eukin- 1alpha (P01583); Interleukin-2 (P60568); Interleukin-4 (P60568); Interleukin-9 (P15248); Interleukin-12p40 (P29460); Interleukin-13 (P35225); Interleukin-16 (Ql4005); L1 cell adhesion molecule (P32004); Lactate ogenase (P00338); Leucine Aminopeptidase (P28838); Meprin A-alpha t (Ql6819); Meprin A-beta subunit (Ql6820); Midkine (P21741); MIP2-alpha (CXCL2, Pl9875); MMP-2 (P08253); MMP-9 (P14780); Netrin-l (095631); Neutral endopeptidase (P08473); 0steopontin (P10451); Renal papillary antigen 1 ; Renal papillary antigen 2 (RPA2); Retinol binding protein (P09455); Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum Amyloid P Component (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764); Spermidine/spermine Nl-acetyltransferase (P21673); TGF-Betal (P01137); Transferrin (P02787); Trefoil factor 3 (TFF3, 007654); Toll-Like protein 4 (000206); Total n; Tubulointerstitial nephritis n 2); Uromodulin (Tamm-Horsfall protein, P07911).

For purposes of risk stratification, Adiponectin (Ql5848); Alkaline atase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937); Cystatin C (P01034); 8 t of F1F0 ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa (alpha-glutathione-S-transferase, P08263); GSTpi (Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-l (P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itml, P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18 (Ql4116); IP-10 (10 kDa interferon-gamma-induced n, P02778); IRPR , 000458); Isovaleryl-CoA dehydrogenase (IVD, P26440); I- TAC/CXCLll (014625); Keratin 19 7); Kim-1 (Hepatitis A Virus cellular receptor 1, 043656); L-arginine:glycine otransferase (P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-l 0); MIG (Gamma-interferon-induced monokine 007325); MIP-la (P10147); MIP-3a (P78556); MIP-lbeta (P13236); MIP-ld (Ql6663); NAG (N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter (0CT2, ); 0steoprotegerin (014788); P8 protein (060356); Plasminogen activator inhibitor 1 (PAI-l, ); ProANP(1-98) (P01160); Protein phosphatase 1- beta (PPI-beta, P62140); Rab GDI-beta (P50395); Renal rein (QS6U61 ); RT1.B-1 (alpha) chain of the integral membrane protein (Q5Y7A8); Soluble tumor necrosis factor receptor superfamily member 1A (sTNFR-I, P19438); Soluble tumor necrosis factor or superfamily member 1B (sTNFR-II, ); Tissue inhibitor of metalloproteinases 3 3, P35625); uPAR (003405) may be combined with the kidney injury marker assay result(s) of the present invention.

Other clinical indicia which may be combined with the kidney injury marker assay (s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family y, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscamet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, aque contrast agents, or streptozotocin), clinical variables (e. g., blood pressure, temperature, ation rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total n measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal ary antigen 1 (RPAl) measurement; a renal papillary antigen 2 (RPA2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium / (urine creatinine / plasma creatinine). Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison’s Principles of Internal ne, 17Lh Ed., McGraw Hill, New York, pages 1741-1830, and Current 2012/066152 Medical Diagnosis & Treatment 2008, 47Lh Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by nce in their entirety.

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

Diagnosis of Acute Renal Failure As noted above, the terms “acute renal (or ) injury” and “acute renal (or kidney) failure” as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by ng the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the 's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min: Urine Uonctznfirution >< e Flow rFR :—«a Plasnm Concentration By normalizing the GFR to the body e area, a GFR of approximately 75—100 ml/min per 1.73 m2 can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.

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

Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (UCr), urine flow rate (V), and creatinine's plasma concentration (Pct) are known. Since the product of urine concentration and urine flow rate yields nine's ion rate, nine clearance is also said to be its excretion rate (UCrxV) divided by its plasma concentration. This is commonly represented mathematically as: Commonly a 24 hour urine collection is aken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken: {55, X 24— hour volume {'3‘hath: l}. 5 _.

N; 34 x filj‘?fl.fifl§_. _ \ H g ‘ To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr ted for their actual BSA: (ff-,ZT'E‘M‘{TI31‘3"$",‘3?¥3E‘$31L$ [01 l l] The accuracy of a creatinine nce measurement (even when collection is te) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.

For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are ed, minor modifications of the RIFLE urine output ia have been described.

For e, Bagshaw et al., Nephrol. Dial. Transplant. 23: 1203–1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the ing: <35 mL/h , <21 mL/h (Injury) or <4 mL/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, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, ng or avoiding procedures that are known to be damaging to the , ing ic administration, initiating goal directed therapy, etc. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, NJ, 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.

The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Example 1: Contrast-induced nephropathy sample collection The objective of this sample tion study is to collect samples of plasma and urine and clinical data from patients before and after receiving ascular contrast media. Approximately 250 adults undergoing radiographic/angiographic procedures involving intravascular administration of ted contrast media are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria: ion Criteria males and females 18 years of age or older; undergoing a radiographic / angiographic ure (such as a CT scan or coronary intervention) involving the intravascular administration of st media; expected to be alized for at least 48 hours after contrast administration. able and willing to provide written informed consent for study participation and to comply with all study procedures.

Exclusion Criteria renal transplant recipients; acutely worsening renal function prior to the contrast procedure; already receiving dialysis (either acute or chronic) or in nt need of dialysis at enrollment; expected to o a major surgical procedure (such as involving cardiopulmonary bypass) or an additional imaging procedure with contrast media with significant risk for further renal insult within the 48 hrs ing contrast administration; participation in an interventional clinical study with an experimental therapy within the previous 30 days; known ion with human immunodeficiency virus (HIV) or a hepatitis virus.

Immediately prior to the first contrast administration (and after any pre- procedure ion), an EDTA anti-coagulated blood sample (10 mL) and a urine sample (10 mL) are collected from each patient. Blood and urine samples are then collected at 4 (i0.5), 8 (i1), 24 (i2) 48 (i2), and 72 (i2) hrs following the last administration of contrast media during the index contrast procedure. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral , central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and d to Astute Medical, Inc.

Serum creatinine is assessed at the site immediately prior to the first contrast administration (after any pre-procedure hydration) and at 4 (i0.5), 8 (i1), 24 (i2) and 48 (i2) ), and 72 (i2) hours following the last administration of contrast (ideally at the same time as the study s are obtained). In addition, each patient’s status is evaluated through day 30 with regard to additional serum and urine creatinine measurements, a need for dialysis, hospitalization status, and adverse clinical outcomes (including mortality).

Prior to contrast administration, each patient is assigned a risk based on the following assessment: ic blood pressure <80 mm Hg = 5 points; intra-arterial balloon pump = 5 points; congestive heart failure (Class III-IV or history of pulmonary edema) = 5 points; age >75 yrs = 4 points; hematocrit level <39% for men, <35% for women = 3 points; diabetes = 3 points; contrast media volume = 1 point for each 100 mL; serum creatinine level >1.5 g/dL = 4 points OR estimated GFR 40—60 mL/min/ 1.73 m2 = 2 points, 20—40 /1.73 m2 = 4 points, < 20 mL/min/1.73 m2 = 6 points. The risks ed are as follows: risk for CIN and is: 5 or less total points = risk of CIN - 7.5%, risk of dialysis - 0.04%; 6—10 total points = risk of CIN - 14%, risk of dialysis - 0.12%; 11—16 total points = risk of CIN - 26.1%, risk of dialysis - 1.09%; >16 total points = risk of CIN - 57.3%, risk of is - 12.8%.

Example 2: Cardiac surgery sample tion The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after undergoing cardiovascular surgery, a procedure known to be potentially damaging to kidney function.

Approximately 900 adults undergoing such surgery are enrolled. To be enrolled in the study, each patient must meet all of the ing inclusion criteria and none of the following ion criteria: Inclusion ia males and females 18 years of age or older; undergoing cardiovascular surgery; o/Ottawa Predictive Risk Index for Renal Replacement risk score of at least 2 (Wijeysundera et al., JAMA 297: 1801-9, 2007); and able and willing to provide written informed consent for study participation and to comply with all study procedures.

Exclusion Criteria known pregnancy; previous renal transplantation; acutely worsening renal on prior to enrollment (e. g., any category of RIFLE criteria); already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; currently enrolled in another clinical study or expected to be enrolled in another clinical study within 7 days of cardiac surgery that involves drug infusion or a therapeutic intervention for AKI; known infection with human immunodeficiency virus (HIV) or a hepatitis virus.

Within 3 hours prior to the first incision (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3 mL), and a urine sample (35 mL) are collected from each patient. Blood and urine samples are then collected at 3 (i0.5), 6 (i0.5), 12 (i1), 24 (i2) and 48 (i2) hrs following the procedure and then daily on days 3 through 7 if the subject remains in the hospital. Blood is collected via direct ncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and d to Astute Medical, Inc.

Example 3: Acutely ill subject sample collection The objective of this study is to collect samples from y ill ts.

Approximately 1900 adults ed to be in the ICU for at least 48 hours will be enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the ing ion criteria: Inclusion Criteria males and females 18 years of age or older; Study population 1: approximately 300 patients that have at least one of: shock (SBP < 90 mmHg and/or need for vasopressor support to maintain MAP > 60 mmHg and/or documented drop in SBP of at least 40 mmHg); and sepsis; Study tion 2: approximately 300 patients that have at least one of: IV otics ordered in computerized physician order entry (CPOE) within 24 hours of ment; contrast media exposure within 24 hours of enrollment; increased Intra-Abdominal Pressure with acute decompensated heart failure; and severe trauma as the primary reason for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment; Study tion 3: approximately 300 patients expected to be hospitalized through acute care setting (ICU or ED) with a known risk factor for acute renal injury (e. g. sepsis, hypotension/shock (Shock = systolic BP < 90 mmHg and/or the need for vasopressor support to maintain a MAP > 60 mmHg and/or a documented drop in SBP > 40 mmHg), major trauma, hemorrhage, or major surgery); and/or ed to be hospitalized to the ICU for at least 24 hours after enrollment; Study tion 4: approximately 1000 patients that are 21 years of age or older, within 24 hours of being admitted into the ICU, expected to have an indwelling urinary catheter for at least 48 hours after enrollment, and have at least one of the following acute conditions within 24 hours prior to enrollment: (i) respiratory SOFA score of 2 2 (PaO2/FiO2 <300), (ii) cardiovascular SOFA score of 2 1 (MAP < 70 mm Hg and/or any vasopressor required). ion Criteria known pregnancy; institutionalized individuals; previous renal lantation; known acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); received dialysis (either acute or chronic) within 5 days prior to ment or in imminent need of dialysis at the time of enrollment; known infection with human immunodeficiency virus (HIV) or a hepatitis virus; meets any of the following: (i) active bleeding with an anticipated need for > 4 units PRBC in a day; (ii) obin < 7 g/dL; (iii) any other condition that in the ian’s opinion would contraindicate drawing serial blood s for clinical study purposes; meets only the SBP < 90 mmHg inclusion criterion set forth above, and does not have shock in the attending physician’s or principal investigator’s opinion; After obtaining informed consent, an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-50 mL) are collected from each patient. Blood and urine samples are then collected at 4 (i 0.5) and 8 (i 1) hours after contrast administration (if applicable); at 12 (i 1), 24 (i 2), 36 (i 2), 48 (i 2), 60 (i 2), 72 (i 2), and 84 (i 2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Blood is collected via direct ncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the al site, frozen and shipped to Astute Medical, Inc., San Diego, CA. The study urine samples are frozen and shipped to Astute Medical, Inc. e 4. Immunoassay format Analytes are measured using standard sandwich enzyme immunoassay techniques. A first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate. Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish dase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody. ing a wash to remove any unbound antibody-enzyme reagent, a substrate solution sing tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte t in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards.

Units for the concentrations reported in the following data tables are as follows: nocalcin-l - ng/mL, Antithrombin-III - ng/mL, Toll-like receptor 2 - ng/mL, Triiodothyronine (T3) - ng/mL, Thyroxine (T4) - ng/mL, Extracellular matrix 2012/066152 protein 1 - ng/mL, ation factor XIII A chain/Coagulation factor XIII B chain - ng/mL (assay detects both chains), Interleukin-17F - ng/mL, Interleukin-22 - ng/mL, Vitronectin - ng/mL, Progesterone - ng/mL, Estradiol - ng/mL, Growth/differentiation factor 15 - pg/mL, Proprotein convertase subtilisin/kexin type 9.

In the case of those kidney injury markers which are membrane proteins as described herein, the assays used in these examples detect soluble forms thereof.

Example 5. Apparently Healthy Donor and Chronic Disease Patient Sal—mm Human urine samples from donors with no known chronic or acute disease (“Apparently Healthy Donors”) were purchased from two vendors n West Biologicals, Inc., 27625 Commerce Center Dr., Temecula, CA 92590 and Virginia Medical Research, Inc., 915 First al Rd., Virginia Beach, VA 23454). The urine samples were d and stored frozen at less than -200 C. The vendors ed demographic information for the individual donors including gender, race (Black /White), smoking status and age.

Human urine samples from donors with various chronic diseases (“Chronic Disease Patients”) ing tive heart failure, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, diabetes mellitus and hypertension were purchased from Virginia l Research, Inc., 915 First Colonial Rd., Virginia Beach, VA 23454. The urine samples were d and stored frozen at less than -20 degrees centigrade. The vendor provided a case report form for each individual donor with age, gender, race (Black/White), smoking status and alcohol use, height, weight, chronic disease(s) diagnosis, current medications and previous surgeries.

Example 6. Use of Kidney Injury Markers for evaluating renal status in patients Patients from the intensive care unit (ICU) were enrolled in the following study. Each patient was classified by kidney status as non-injury (0), risk of injury (R), injury (I), and failure (F) according to the maximum stage d within 7 days of ment as determined by the RIFLE criteria. EDTA anti-coagulated blood samples (10 mL) and a urine samples (25-30 mL) were collected from each patient at ment, 4 (i 0.5) and 8 (i 1) hours after contrast administration (if applicable); at 12 (i 1), 24 (i 2), and 48 (i 2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Markers were each measured by standard immunoassay methods using cially ble assay reagents in the urine samples and the plasma component of the blood samples collected.

Two s were defined to represent a “diseased” and a “normal” population. While these terms are used for convenience, “diseased” and “normal” simply represent two cohorts for comparison (say RIFLE 0 vs RIFLE R, I and F; RIFLE 0 vs RIFLE R; RIFLE 0 and R vs RIFLE I and F; etc.). The time “prior max stage” represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/- 12 hours. For example, “24 hr prior” which uses 0 vs R, I, F as the two s would mean 24 hr (+/— 12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).

A receiver operating characteristic (ROC) curve was generated for each biomarker measured and the area under each ROC curve (AUC) is determined. ts in Cohort 2 were also ted according to the reason for adjudication to cohort 2 as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output. Using the same example sed above (0 vs R, I, F), for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements alone, the stage 0 cohort may include patients adjudicated to stage R, I, or F on the basis of urine ; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may include ts adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, in the data for patients adjudicated on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage is used.

The ability to distinguish cohort 1 from Cohort 2 was determined using ROC analysis. SE is the standard error of the AUC, n is the number of sample or individual patients (“pts,” as indicated). rd errors are calculated as described in Hanley, J. A., and McNeil, B.J., The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology (1982) 143: 29-36; p values are calculated with a two-tailed Z-test. An AUC < 0.5 is indicative of a negative going marker for the comparison, and an AUC > 0.5 is indicative of a positive going marker for the comparison.

Various threshold (or “cutoff’) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 are determined. OR is the odds ratio ated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

Table 1: Comparison of marker levels in urine samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0) and in urine samples ted from subjects at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2.

Stanniocalcin- 1 24hr prior to AKl stage 48hr prior to AKl stage Cohort 2 Cohort 1 Cohort 2 0.131 0.0962 0.218 0.267 0.385 0.297 0.334 0.978 0.241 0.60 0.84 0.0176 0.00189 0.0874 1.25 6.00 0.682 47 5 22 5 t 1sCr only 0hr prior to AKl stage 24hr prior to AKl stage 48hr prior to AKl stage Cohort 2 Cohort 1 Cohort 2 0.106 0.120 0.160 0.169 0.388 0.169 0.140 0.914 0.133 0.50 0.60 0.0295 0.00189 0.0371 0.410 6.00 0.367 8 98 5 8 47 5 24hr prior to AKl stage 48hr prior to AKl stage Cohort 2 Cohort 1 Cohort 2 0.131 0.0894 0.156 0.249 0.364 0.196 0.333 0.976 0.213 0.61 0.63 0.0176 0.00189 0.0181 1.25 6.00 0.682 47 8 22 8 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.57 0.11 0.0592 0.0760 80% 75% 29% 9% 0.0191 0.0152 100% 0.146 0.307 0.675 OR Quart 2 1.0 0.96 1.5 2.4 >1.1 0.96 0.42 p Value 1.0 0.98 0.68 0.29 <O.96 0.98 0.51 95% CI of 0.17 0.057 0.22 0.48 0.034 OR Quart2 5.8 16 10 12 5.3 OR Quart 3 6.7 2.0 7.9 1.8 1.5 p Value 0.018 0.58 0.022 0.48 0.69 95% CI of 1.4 0.17 1.4 0.35 0.21 OR Quart3 32 24 46 9.2 11 OR Quart 4 2.3 0.96 3.8 2.4 0.92 p Value 0.30 0.98 0.14 0.29 <O.51 0.98 0.94 95% CI of 0.48 0.057 0.64 0.48 >O.19 0.057 0.11 OR Quart4 11 16 23 12 na 16 7.7 Extracellular matrix protein 1 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.736 0.347 0.337 1.10 0.701 0.686 1.12 0.991 0.829 0.0041 0.96 0.000528 0.000528 0.0222 4.80 7.81 2.46 74 212 14 74 106 14 WO 78253 48hr prior to AKI stage 0.000528 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.0075 0.000528 0.000528 0.0222 4.11 7.81 6.57 76 259 16 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.61 0.55 0.61 0.62 0.58 0.62 0.49 0.58 0.51 0.038 0.080 0.081 0.075 0.0012 0.91 0.30 0.86 0.137 0.0928 % 18% 0.0290 0.860 Cutoff5 1.01 1.46 1.15 1.01 1.46 . . . 1.15 Sens 5 39% 27% 37% 39% 28% 38% 29% 36% 25% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff6 2.04 2.35 2.11 2.04 2.35 2.11 2.04 2.35 2.11 Sens 6 18% 10% 18% 20% 28% 18% 14% 21% 19% Spec 6 90% 90% 90% 90% 90% 90% 90% 90% 90% OR Quart 2 1.7 0.82 2.2 0.80 0.48 0.99 0.75 1.5 0.73 p Value 0.13 0.75 0.037 0.63 0.31 0.97 0.72 0.66 0.68 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value . . . 95% C1 0f 1.9 0.60 1.8 1.4 0.53 1.5 0.24 0.48 0.32 OR Quart4 7.3 4.8 7.6 6.5 4.4 6.7 4.3 13 A .9 Coagulation factor XIII A and chains 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 4.61 2.98 3.06 8.20 6.77 6.87 8.99 10.7 8.40 0.30 0.97 0.000120 0.000303 0.000120 0.000324 84.2 46.4 84.2 29.4 74 212 14 74 106 14 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 7.73 4.00 7.48 .5 7.90 8.65 11.0 12.0 10.00 0.29 0.82 58 0.000120 0.000180 .4 143 30.6 509 14 217 14 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 4.34 3.28 2.58 7.55 6.66 6.87 8.30 10.0 8.26 0.48 0.93 0.000303 0.000120 0.000324 46.4 84.2 22.9 76 259 16 76 117 16 l 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage WO 78253 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.57 0.46 0.56 0.58 0.57 0.57 0.54 0.53 0.51 0.075 0.93 1.79 1.58 71% 71% 75% 34% 30% 32% 0.826 0.258 1.39 0.000324 0.435 7.10 Cutoff 5 . 9.23 Sens 5 31% 17% 31% 36% 32% 31% Spec 5 80% 80% 80% 80% 80% 80% Cutoff 6 18.0 19.9 17.9 18.0 19.9 17.9 Sens 6 18% 7% 17% 12% 16% 19% Spec 6 90% 90% 90% 90% 90% 90% OR Quart 20.77 0.85 0.99 1.2 0.99 . . 2.4 p Value 0.46 0.78 0.97 0.71 0.99 0.35 0.72 0.41 0.21 95% CI Of 0.39 0.28 0.50 0.52 0.28 0.67 0.28 0.088 0.61 OR Quart2 1.5 2.6 1.9 2.6 3.5 9.9 OR Quart 3 1.2 1.3 1.1 1.3 0.59 0.32 p Value 0.66 0.61 0.73 0.54 0.48 0.33 95% CI of 0.60 0.47 0.57 0.58 0.14 0.032 OR Quart3 2.2 3.6 2.2 2.9 2.5 3.1 OR Quart 4 1.5 1.2 1.6 2.4 2.5 1.7 p Value 0.21 0.78 0.16 0.020 0.091 . 0.48 95% CI Of 0.79 0.41 0.84 1.1 0.86 0.94 0.28 0.24 0.39 OR Quart4 2.9 3.3 3.1 5.2 7.4 A .2 6.2 4.1 7.4 Vitronectin sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 33.1 25.1 25.2 80.0 62.1 39.1 152 121 37.5 0.31 0.48 2.95 0.112 4.01 750 750 140 74 212 14 74 106 14 WO 78253 48hr prior to AKI stage 1 stage 48hr prior to AK1 stage Cohort 2 Cohort 1 Cohort 2 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.60 0.43 0.65 0.54 0.47 0.54 0.50 0.55 0.44 0.038 0.080 0.080 0.077 0.96 0.50 0.40 .1 8.64 8% 14% A .35 A 0.3 Cutoff 5 64.6 76.6 59.7 64.6 76.6 . . . 59.7 Sens 5 31% 10% 35% 24% 20% 25% 21% 29% 6% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6 104 169 99.3 104 169 99.3 104 169 99.3 Sens 6 23% 0% 27% 15% 8% 17% 7% 14% 0% Spec 6 90% 90% 90% 90% 90% 90% 90% 90% 90% OR Quart 2 1.2 1.2 1.5 0.77 1.2 0.98 0.15 0.66 0.49 p Value 0.64 0.78 0.28 0.52 0.75 0.97 0.089 0.65 0.41 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value . . . 95% C1 0f 1.1 0.69 1.6 0.76 0.61 0.63 0.17 0.50 0.33 OR Quart4 4.1 5.4 6.6 3.2 5.7 2.6 2.5 8.3 5.0 Estradiol 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 9.50 6.48 8.04 .5 9.98 8.04 7.34 16.6 2.54 0.89 0.87 1.18 0.143 6.25 28.3 128 9.84 18 62 2 18 50 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 7.90 7.13 14.0 11.2 11.2 14.0 .5 14.8 5.89 0.99 0.79 1.18 0.143 9.84 28.3 128 18.2 101 2 81 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 9.28 5.88 6.93 11.9 7.34 7.41 8.70 6.17 3.06 0.017 0.98 1.23 0.143 4.27 34.2 31.6 11.5 19 53 4 19 42 4 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage 2012/066152 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.57 0.69 0.62 0.53 0.70 0.58 0.75 0.58 0.16 0.58 9.74 5.88 100% 100% 75% 45% 64% 51% .88 9.74 3.62 100% 3.62 100% 7.96 Cutoff 5 . . . . . 9.96 Sens 5 37% 20% 47% 28% 20% 25% SpecS 81% 80% 81% 81% 80% 81% Cutoff 6 17.0 24.4 14.2 17.0 24.4 14.2 Sens 6 26% 0% 27% 17% 20% 0% Spec 6 90% 90% 91% 90% 90% 91% OR Quart 20.63 0 1.0 2.2 0.96 . . >1.1 p Value 0.63 na 1.0 0.38 0.98 0.63 <0.96 <na <0.96 95% C1 0f 0.093 na 0.12 0.36 0.057 0.23 >0.061 >na >0.061 OR Quart2 4.2 na 8.1 14 16 na OR Quart 3 2.4 3.3 2.3 3.0 1.0 >2.3 p Value 0.26 0.32 0.38 0.23 1.0 <0.51 95% C1 0f 0.51 0.32 0.36 0.51 0.059 >0.19 OR Quart3 12 34 15 18 17 na OR Quart43.5 0.96 5.2 4.8 2.0 >1.0 p Value 0.11 0.98 0.065 0.073 0.58 . . <10 95% C1 0f 0.77 0.057 0.90 0.86 0.17 1.1 >na >0.059 >0.057 OR Quart4 16 16 31 27 23 36 na na na Progesterone sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 21.1 34.6 21.1 81.5 28.4 42.7 28.4 81.5 .5 28.2 25.5 88.8 0.044 0.0098 2.91 4.04 2.91 18.7 152 113 152 144 —§§_13_5062 18 62 2 18 so 2 WO 78253 48hr prior to AKI stage 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 Median 18.8 27.5 Average 30.4 38.1 ——44 25291 34098 —:—_ 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.51 0.19 0.60 0.68 0.41 0.73 0.71 0.70 0.45 SE 0.077 0.12 0.086 0.076 0.14 0.072 0.21 0.21 0.15 p 0.85 0.0085 0.24 0.018 0.51 0.0015 0.32 0.35 0.76 Cutoff 5 35.0 57.6 35.0 35.0 57.6 . . . .

Sens 5 37% 0% 47% 50% 20% 53% 50% 50% 25% Spec5 81% 80% 81% 81% 80% 81% 81% 80% 81% Cutoff 6 61.1 75.2 68.1 61.1 75.2 68.1 61.1 75.2 68.1 Sens 6 11% 0% 13% 17% 0% 16% 50% 50% 0% Spec 6 90% 90% 91% 90% 90% 91% 90% 90% 91% OR Quart 2 1.0 >0 1.0 0.63 0 0.47 >1.1 >1.0 1.1 p Value 1.0 <na 1.0 0.63 na 0.55 <0.96 <1.0 0.96 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . p Value . . 95% C1 0f 0.39 >0.34 0.67 0.83 0.062 1.1 >0.061 >0.059 0.061 OR Quart4 5.8 na 16 17 18 36 na na 19 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 .70 3.12 2.72 6.00 4.28 2.72 3.89 4.22 1.07 0.74 0.13 0.61 0.533 0.569 0.000958 1.97 .2 14.4 20.4 3.48 18 62 2 18 50 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2.59 3.57 1.98 2.89 4.74 1.98 1.78 3.92 0.0187 0.30 0.32 0.569 0.000958 1.97 4.81 20.4 2.00 101 2 81 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 6.52 2.96 3.44 6.14 3.93 3.72 3.82 3.53 1.74 0.025 0.91 0.757 0.00599 1.92 14.4 19.3 6.10 19 53 4 19 42 4 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage WO 78253 sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.50 0.30 0.56 0.67 0.36 0.71 0.45 0.25 0.58 0.16 0.58 1.95 3.35 100% 100% 75% 32% 25% 60% 1.95 1.95 1.78 100% 1.78 100% A .54 Cutoff 5 . . . . 5.79 Sens 5 26% 0% 33% 50% 0% 25% SpecS 81% 80% 81% 81% 80% 81% Cutoff 6 7.12 8.80 7.09 7.12 8.80 7.09 Sens 6 16% 0% 20% 33% 0% 0% Spec 6 90% 90% 91% 90% 90% 91% OR Quart 20.75 >0 0.43 1.6 >2.2 . . >1.1 p Value 0.71 <na 0.38 0.63 <0.52 0.63 <0.96 <na <0.96 95% C1 0f 0.17 >na 0.068 0.24 >0.19 0.23 >0.061 >na >0.061 OR Quart2 3.3 na 2.8 11 na na OR Quart 3 1.0 >3.4 1.0 2.2 >10 >23 p Value 1.0 <0.31 1.0 0.38 <0.98 <0.51 95% C1 0f 0.24 >0.33 0.20 0.36 >0.062 >0.19 OR Quart3 4.2 na 4.9 14 na na OR Quart 4 0.94 >2.2 1.4 7.4 >22 >10 p Value 0.93 <0.52 0.70 0.022 <0.52 . <10 95% C1 0f 0.23 >0.19 0.29 1.3 >0.19 1.8 >na >na >0.057 OR Quart4 3.9 na 6.3 41 na 57 na na na Growth/differentiation factor 15 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 58000 18600 54200 95300 73200 54200 95600 151000 48900 0.56 0.86 1700 641 19600 341000 810000 88800 —§§_13_50 18 62 2 18 so 2 2012/066152 48hr prior to AKI stage Cohort 1 Cohort 1 40200 83500 83500 130000 810000 101 2 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 Median 17400 33300 88 1 00 95700 Average 1 4400 80000 112000 44400 110000 97799 79499 0.0020 0.097 641 5180 641 13700 326000 341000 326000 235000 19 53 4 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.57 0.39 0.64 0.68 0.44 0.81 0.67 0.44 0.79 SE 0.077 0.14 0.085 0.076 0.14 0.065 0.21 0.21 0.14 p 0.40 0.43 0.090 0.021 0.67 1.8E—6 0.43 0.78 0.034 70800 12900 100% 1 3% 12900 100% 1 3% 49600 88800 49600 88800 37400 Sens 4 42% 20% 47% 61% 0% 0% 75% Spec 4 71% 70% 72% 71% 70% 70% 72% Cutoff 5 76400 116000 49500 76400 116000 116000 49500 Sens 5 42% 0% 47% 39% 0% 74% 50% 0% 75% Spec5 81% 80% 81% 81% 80% 81% 81% 80% 81% Cutoff 6 205000 218000 93200 205000 218000 93200 205000 218000 93200 Sens 6 11% 0% 47% 17% 0% A 7% 0% 0% 50% Spec 6 90% 90% 91% 90% 90% 91% 90% 90% 91% OR Quart 2 1.0 >2.2 2.3 1.6 >3.5 1.0 >0 >10 >1.1 p Value 1.0 <0.52 0.38 0.63 <0.29 1.0 <na <0.98 <0.96 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . p Value . 95% C1 0f 0.60 >0.064 0.90 0.86 >0.064 2.9 >0.061 >na >0.32 OR Quart4 10 na 31 27 na 250 na na na Proprotein convertase subtilisin/kexin type 9 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 882 359 580 3140 3220 580 3890 13100 0 0.98 0.78 70.6 81.8 580 11400 96400 580 18 62 2 18 50 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 3260 419 586 4450 2890 586 4180 10600 8.37 0.74 0.76 121 48.1 580 11400 96400 592 101 2 81 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 897 366 1740 2550 2730 2600 3270 13200 2560 0.95 0.98 70.6 95.1 580 11400 96400 6340 19 53 4 19 42 4 l 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.61 0.71 0.58 0.62 0.73 0.65 0.67 0.63 0.83 0.13 0.010 575 1360 100% 100% 75% 66% 61% 85% 575 575 575 100% 100% Cutoff 5 1300 Sens 5 37% 40% 33% 44% 80% 75% SpecS 81% 80% 81% 81% 80% 81% Cutoff 6 2690 4940 2690 2690 4940 2690 Sens 6 26% 20% 27% 39% 20% 25% Spec 6 90% 90% 91% 90% 90% 91% OR Quart 2 1.0 >1.0 0.70 0.16 0 . >0 p Value 1.0 <1.0 0.67 0.11 na 0.38 <na <na <na 95% C1 Of 0.21 >0.059 0.13 0.017 na 0.069 >na >na >na OR Quart2 4.7 na 3.7 1.5 na na OR Quart 3 0.71 >2.2 0.43 0.75 0 >1.1 p Value 0.68 <0.54 0.38 0.71 na <0.96 95% C1 Of 0.14 >0.18 0.068 0.17 na >0.061 OR Quart3 3.7 na 2.8 3.3 na na OR Quart42.5 >2.1 1.8 2.0 4.3 >3.5 p Value 0.21 <0.56 0.45 0.31 0.20 . <0.30 95% C1 Of 0.60 >0.18 0.40 0.52 0.45 0.52 >na >na >0.32 OR Quart4 10 na 7.9 7.7 42 9.6 na na na Table 2: Comparison of marker levels in urine samples collected from Cohort 1 (patients that did not ss beyond RIFLE stage 0 or R) and in urine samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage I or F in Cohort 2.

Toll-like receptor 2 Cohort 2 0.592 0.690 0.516 0.10 0.0621 WO 78253 0hr prior to AKI stage sCr only 0.31 0.21 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 Antithrombin-III sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 121 1 1 1 54. 3 590 340 198 1350 89 6 435 0.056 0.43 4.48 0.0182 0.347 6000 6000 2060 61 465 25 61 212 25 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 0.0182 6000 48hr prior to AKI stage 2012/066152 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 6000 6000 2060 59 486 23 59 209 23 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 _ _ _ _ OR Quart41.2 1.0 1.2 1.8 . 1.9 3.2 . 2.8 p Value 0.68 1.0 0.68 0.13 0.27 0.12 0.088 0.34 0.13 95% C1 0f 0.51 0.062 0.51 0.84 0.48 0.85 0.84 0.31 0.73 OR Quart4 2.8 16 2.8 4.0 A .3 12 11 Extracellular matrix protein 1 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 1.18 0.409 0.316 1.85 0.871 1.11 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2.39 1.19 1.82 2.9E—7 0.34 0.00789 28 0.0117 13.8 10.9 6.57 61 466 25 61 212 25 I stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1.31 0.495 1.01 1.52 1.76 2.27 1.30 10.2 2.77 0.93 0.89 0.128 0.000528 0.272 M(Sxamp) 4.67 150 7.31 n 638 7 638 14 638 8 11 (Patient) 267 7 267 14 267 8 U0 only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.795 0.463 0.311 1.82 0.893 0.986 2.45 1.20 1.59 1.7E—6 0.72 0.00419 0.000528 0.0117 13.8 10.9 6.57 59 486 23 59 209 23 0hr prior to AKI stage 48hr prior to AKI stage sCr only 0.49 0.11 0.94 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . . . . . . . . . p Value 0.0081 1.00 0.012 5.0E—4 0.11 0.0019 0.76 <0.21 0.77 95% C1 Of 1.4 0.14 1.3 1.9 0.74 1.6 0.39 >0.45 0.39 OR Quart4 11 7.2 10 9.9 18 8.6 3.6 na 3.6 Coagulation factor XIII A and B chains 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 . 6.25 3.84 2.26 774 11.6 7.04 6.20 .1 9.23 8.57 0.0027 0.66 —_0.000180 0.000189 0.000120 80 143 84.2 30.6 61 466 25 61 212 25 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.000120 0.000180 UO only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 3.74 .80 3.74 6.27 3.74 2.54 Average 6.96 8.29 6.96 11.7 6.96 5.53 WO 78253 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 .5 9.13 7.09 0.0017 0.46 0.000189 0.000120 0.000327 143 84.2 22.9 59 486 23 59 209 23 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.51 0.30 0.53 0.57 0.53 0.57 0.43 0.52 0.43 SE 0.044 0.11 0.046 0.040 0.079 0.041 0.061 0.10 0.063 p 0.75 0.076 0.45 0.068 0.68 0.086 0.23 0.85 0.29 nCohort 1 466 638 486 466 638 A 86 466 638 A 86 nCohort 2 48 7 45 61 14 59 25 8 23 0.844 0.470 80% 88% 83% 9% 13% 29% 13% 0.000189 0.000324 0.000145 0.427 96% 100% 91% 7.46 13.2 10.8 % 25% 17% 80% 80% 80% Cutoff 6 18.9 20.0 18.8 18.9 20.0 18.8 18.9 20.0 18.8 Sens 6 10% 0% 13% 16% 0% Spec 6 90% 90% 90% 90% 90% OR Quart21.2 0 1.4 0.83 4.1 p Value 0.68 na 0.51 0.65 0.21 . . 95% C1 0f 0.51 na 0.55 0.36 0.45 0.26 0.21 0.31 0.33 OR Quart2 2.8 na 3.3 1.9 37 OR Quart 3 1.0 2.0 1.2 1.3 6.2 p Value 1.0 0.57 0.65 0.45 0.093 95% CI of 0.42 0.18 0.49 0.62 0.74 OR Quart3 2.4 23 3.1 2.9 52 OR Quart41.2 4.1 1.5 1.6 3.0 p Value 0.68 0.21 0.39 0.20 0.34 . . 95% C1 0f 0.51 0.45 0.61 0.77 0.31 0.67 0.61 0.31 0.61 OR Quart4 2.8 37 3.6 3.4 30 2.9 5.8 29 7.1 WO 78253 Vitronectin sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 36.0 29.7 16.5 77.7 71.8 66.6 125 137 152 0.75 0.86 3.33 0.112 0.237 750 750 750 61 466 25 61 212 25 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 55.7 31 . 1 22. 5 63.6 73.4 211 60.3 131 334 0.78 0.0042 .44 0.0795 4.15 228 750 750 14 638 8 14 267 8 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 36.0 29.1 16.5 78.5 70.4 39.3 127 137 55.5 0.67 0.28 2.03E—5 0.112 0.237 750 750 216 59 486 23 59 209 23 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 0.43 . . 0.58 Cutoff 3 6.27 0.112 6.27 14.1 8.72 14.1 4.09 4.09 1.94 Sens 3 92% 100% 91% 90% 93% 92% 92% 100% 91% 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only A 8.3 . 74.8 67.1 16% 38% 13% 80% 80% 80% Cutoff 6 147 168 139 147 168 139 147 168 139 Sens 6 21% 0% 24% 10% 7% Spec 6 90% 90% 90% 90% 90% OR Quart 20.99 2.0 0.99 2.6 2.0 p Value 0.99 0.57 0.99 0.031 0.42 95% CI of 0.38 0.18 0.36 1.1 0.37 OR Quart2 2.6 23 2.7 6.1 11 OR Quart 3 0.88 1.0 0.86 1.8 1.0 . . . p Value 0.80 1.00 0.78 0.19 1.0 0.032 0.76 na 0.73 95% CI of 0.33 0.062 0.30 0.74 0.14 OR Quart3 2.4 16 2.4 4.5 7.2 OR Quart42.7 3.1 3.1 2.7 3.1 p Value 0.017 0.34 0.0095 0.021 0.17 . . . . 95% C1 0f 1.2 0.31 1.3 1.2 0.61 1.3 0.70 0.25 0.81 OR Quart4 6.2 30 7.2 6.5 15 8.5 6.4 9.1 8.7 eukin-17F O—hrprior to AKI stage 24hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 0.00406 0.00382 0.00406 0.00530 000000 000000 0.00500 0.00780 0.00841 0.034 9.15E—5 0.0300 Cohort 2 0.00238 0.00811 0.0102 0.37 0.00208 Cohort 2 . 0.00604 000001 000800 StdeV 0.00427 0.00495 0.00854 —_0100-0 0000-0 0.0300 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 0.60 0.075 0.16 0.00282 0.00238 2 0.00232 0.00198 0.00232 72% 100% 71% 80% 100% 79% 37% 29% 37% 30% 24% 30% 0.00238 0.00238 0.00238 0.00232 0.00198 0.00198 89% 100% 88% 80% 100% 84% % 29% 31% 30% 24% 27% 0.00232 0.00238 0.00232 0.000770 0.00198 9.15E—5 % 29% 30% 13% 24% 8% 0.00555 0.00579 0.00555 0.00555 0.00579 0.00555 71% 70% 70% 71% 70% 70% 0.00770 0.00770 0.00770 0.00770 0 0 17% 0% 18% 30% 33% 32% 80% 81% 81% 80% 81% 81% 0.0100 OR Quart212 >10 11 1.8 0 1.3 p Value 0.022 <1.0 0.029 0.45 na 0.73 95% CI of 1.4 >0.060 1.3 0.39 na 0.27 OR Quart2 110 na 99 8.4 na 6.7 OR Quart 34.5 >1.0 4.6 1.8 2.1 1.7 p Value 0.19 <0.98 0.19 0.45 0.56 0.48 95% CI of 0.47 >0.062 0.47 0.39 0.18 0.37 OR Quart3 43 na 44 8.4 24 8.2 OR Quart 44.3 >0 4.4 2.8 0 2.7 p Value 0.20 <na 0.20 0.17 na 0.19 95% C1 Of 0.45 >na 0.45 0.64 na 0.61 OR Quart4 42 na 42 12 na 12 Interleukin-22 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage Cohort 2 1.78E—5 52 000799 StdeV 0.00339 0.0192 p(t—ntest) 9.1E4 1 stage Cohort 2 1 stage Cohort 2 1.78E—5 0.0145 0.0535 Sens 3 100% 100% 100% 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 1.78E—5 Cutoff 6 1.78E—5 0.000334 1.78E—5 1.78E—5 0.000334 1.78E—5 Sens 6 22% 0% 24% 30% 33% 32% Spec 6 93% 90% 95% 93% 90% 95% OR Quart2>15 >1.1 >12 >16 >21 >11 p Value <0.013 <0.96 <0.026 <0.012 <0.56 <0.029 95% C10f >1.8 >0O64 >13 >18 >018 >1.3 OR Quart2 na na na na na na OR Quart 3 >47 >10 >62 >47 >0 >60 p Value <0.18 <0.98 <0.11 <0.18 <na <0.12 95% C1 of >049 >0O62 >067 >049 >na >064 OR Quart3 na na na na na na OR Quart 4>4.5 >0 >45 >7.6 >10 >75 p Value <0.19 <na <0.19 <0.069 <1.0 <0.072 95% C1 of >047 >na >047 >086 >0O6O >083 OR Quart4 na na na na na na 0—hrprior to AKI stage Cohort l Cohort 2 Median ———.

WO 78253 Cohort 2 2.84 3.81 2.82 0.53 0.792 9.76 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 0.45 0.088 0.56 nd nd nd 14 13 1.97 1.60 0.862 .32 6.71 7.57 OR Quart 2 nd nd nd 1.4 >1.1 1.0 p Value nd nd nd 0.69 <0.96 1.0 95% CI of nd nd nd 0.28 >0.064 0.18 OR Quart2 nd nd nd 6.8 na 5.5 OR Quart 3 nd nd nd 1.4 >1.1 1.4 p Value nd nd nd 0.69 <O.96 0.69 95% CI of nd nd nd 0.28 >0.064 0.28 OR Quart3 nd nd nd 6.8 na 7.0 OR Quart 4 nd nd nd 1.0 >1.1 1.0 p Value nd nd nd 1.0 <O.96 0.96 95% CI of nd nd nd 0.19 >0.064 0.19 OR Quart4 nd nd nd 5.4 na 5.7 WO 78253 Cohort 2 4.67 .73 4.58 0.83 0.428 .5 I stage Cohort 2 I stage Cohort 2 4.01 .63 4.55 0.92 0.428 .5 0hr prior to AKI stage sCr only sCr only nd 0.56 nd nd nd 1.19 1.19 1.19 nd nd nd 93% 100% 92% 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 6.89 8.80 Cutoff 6 nd nd nd 12.5 12.5 12.5 Sens 6 nd nd nd 7% 0% 8% Spec 6 nd nd nd 91% 91% 90% OR Quart 2nd nd nd 7.2 >1.0 7.2 p Value nd nd nd 0.076 <0.98 0.078 95% CI of nd nd nd 0.82 >0.062 0.80 OR Quart2 nd nd nd 64 na 65 OR Quart 3 nd nd nd 2.1 >1.0 2.0 p Value nd nd nd 0.56 <0.98 0.58 95% CI of nd nd nd 0.18 >0.062 0.17 OR Quart3 nd nd nd 24 na 24 OR nd nd nd 5.8 >10 1.4 p Value nd nd nd 0.12 <1.0 0.20 95% CI of nd nd nd 0.63 >0.060 0.45 OR Quart4 nd nd nd 53 na 1 2 Proprotein convertase subtilisin/kexin type 9 Cohort 2 1190 2012/066152 Cohort 2 1930 3190 0.8 1 11400 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 0.58 0.088 0.36 nCohort 2 nd nd nd 14 3 13 OR Quart 2 nd nd nd >6.0 >0 1.4 p Value nd nd nd <0.11 <na 0.20 95% C1 Of nd nd nd >0.66 >na 0.45 OR Quart2 nd nd nd na na ‘ 2 OR Quart 3 nd nd nd >75 >21 5.7 p Value nd nd nd <0.071 <0.54 0.12 95% C1 0f nd nd nd >0.84 >0.18 0.62 OR Quart3 nd nd nd na na 53 OR Quart4nd nd nd >33 >10 3.1 p Value nd nd nd <0.31 <1.0 0.34 95% CI of nd nd nd >0.33 >0.060 0.30 OR Quart4 nd nd nd na na 32 Table 3: Comparison of marker levels in urine samples collected within 12 hours of reaching stage R from Cohort 1 (patients that reached, but did not progress beyond, RIFLE stage R) and from Cohort 2 (patients that reached RIFLE stage I or F).

Estradiol UO only Cohort 2 Cohort 1 Cohort 2 nd 11.6 6.20 Average 14.5 9. 36 nd 16.6 6.58 _—_ nd 014 Min 1.46 A .27 nd 1.46 4.77 55. 8 34.2 nd 55.8 9.47 nd 17 5 nd 17 5 At Enrollment sCr or UO sCr only UO only AUC 0.36 nd 0.21 SE 0.11 nd 0.13 p 0.17 nd 0.028 t 1 23 nd 17 nCohort 2 11 nd 5 Cutoff1 5.56 nd 4.77 Sens 1 73% nd 80% Spec 1 22% nd 12% Cutoff 2 4.77 nd 4.77 Sens 2 82% nd 80% Spec 2 17% nd 12% Cutoff 3 4.27 nd 3.57 Sens 3 91% nd 100% Spec 3 17% nd 12% Cutoff 4 19.9 nd 20.2 Sens 4 9% nd 0% Spec 4 74% nd 71% Cutoff 5 24.6 nd 25.9 Sens 5 9% nd 0% Spec 5 83% nd 82% Cutoff 6 26.7 nd 38.9 Sens 6 9% nd 0% Spec 6 91% nd 94% OR Quart 2 2.7 nd >1.5 p Value 0.46 nd <0.79 95% CI of 0.19 nd >0.071 OR Quart2 37 nd na OR Quart 3 10 nd >3.0 p Value 0.067 nd <0.43 95% CI of 0.85 nd >0.20 OR Quart3 120 nd na At Enrollment sCr or UO sCr only UO only OR Quart 4 4.8 nd >4.0 p Value 0.22 nd <0.33 95% C1 0f 0.38 nd >0.25 OR Quart4 60 nd na /differentiation factor 15 U0 only Cohort 2 Cohort 1 Cohort 2 nd 108000 11400 nd 105000 28900 nd 83100 31300 nd 0.061 nd 5720 5560 nd 284000 79100 nd 17 5 nd 17 5 At Enrollment sCr or UO sCr only UO only AUC 0.44 nd 0.19 SE 0.11 nd 0.13 p 0.60 nd 0.013 nCohort 1 23 nd 17 nCohort 2 11 nd 5 Cutoff 1 11 100 nd 5720 Sens 1 73% nd 80% Spec 1 22% nd 6% Cutoff 2 5720 nd 5720 Sens 2 82% nd 80% Spec 2 13% nd 6% Cutoff 3 5170 nd 0 Sens 3 91% nd 100% Spec 3 9% nd 0% Cutoff 4 115000 nd 116000 Sens 4 9% nd 0% Spec 4 74% nd 71% Cutoff 5 151000 nd 172000 Sens 5 9% nd 0% Spec 5 83% nd 82% Cutoff 6 208000 nd 221000 Sens 6 9% nd 0% Spec 6 91% nd 94% OR Quart 2 13 nd >0 p Value 0.044 nd <na 95% C1 0f 1.1 nd >na OR Quart2 170 nd na OR Quart 3 2.3 nd >6.0 At Enrollment sCr or UO sCr only UO only p Value 0.53 nd <0.19 95% CI of 0.17 nd >0.42 OR Quart3 31 nd na OR Quart 4 4.8 nd >4.0 p Value 0.22 nd <0.33 95% C1 0f 0.38 nd >0.25 OR Quart4 60 nd na Proprotein convertase isin/kexin type 9 ——Cron1y wordy Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Average 2310 1320 nd nd 2820 364 nd nd 0.29 nd nd 120 176 nd nd 19200 521 nd nd 17 5 nd nd 17 5 At Enrollment sCr or UO sCr only UO only AUC 0.49 nd 0.38 SE 0.11 nd 0.15 p 0.93 nd 0.41 nCohort 1 23 nd 17 nCohort 2 11 nd 5 Cutoff 1 353 nd 293 Sens 1 73% nd 80% Spec 1 39% nd 35% Cutoff 2 293 nd 293 Sens 2 82% nd 80% Spec 2 35% nd 35% Cutoff 3 170 nd 170 Sens 3 91% nd 100% Spec 3 22% nd 24% Cutoff 4 2060 nd 2250 Sens 4 18% nd 0% Spec 4 74% nd 71% Cutoff 5 2510 nd 3030 Sens 5 18% nd 0% Spec 5 83% nd 82% Cutoff 6 4940 nd 10700 Sens 6 9% nd 0% Spec 6 91% nd 94% OR Quart 2 2.1 nd >1.5 p Value 0.49 nd <0.79 At ment sCr or UO sCr only UO only 95% CI of 0.25 nd >0.071 OR Quart2 18 nd na OR Quart 3 2.8 nd >6.0 p Value 0.32 nd <0.19 95% CI of 0.36 nd >0.42 OR Quart3 22 nd na OR Quart 4 1.2 nd >1.5 p Value 0.89 nd <0.79 95% CI of 0.12 nd >0.071 OR Quart4 11 nd na Table 4: Comparison of the maximum marker levels in urine samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0) and the maximum values in urine samples collected from subjects between enrollment and 0, 24 hours, and 48 hours prior to reaching stage F in Cohort 2.

Toll-like receptor 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 0.399 0.926 0.399 0.378 48hr prior to AKI stage 8 22 3 8 22 3 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only nd nd 0.69 nd nd . p 0.067 nd 0.087 0.087 nd 0.11 0.87 nd 0.87 nCohort 1 22 nd 22 22 nd 22 22 nd 22 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.346 100% 0.346 100% 0.346 100% 0.678 0.838 1.48 >3.0 <0.43 95% CI of >0.20 OR Quart2 Ila OR Quart 3 >0 p Value <0.45 nd <0.45 <0.45 nd <na nd <na 95% CI of >0.20 nd >0.20 >0.20 nd >na OR Quart3 na nd na na nd na OR Quart 4 >7.0 nd >7.0 >7.0 nd >1.0 p Value <0.13 nd <0.13 <0.13 nd <1.0 nd <1.0 95% CI of >0.57 nd >O.57 >O.57 nd >0.050 nd >0.050 OR Quart4 na nd Ila Ila nd Ila Ila nd Ila Antithrombin-III 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 22 106 13 22 106 13 48hr prior to AKI stage WO 78253 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 697 644 761 1520 1360 1740 0.89 0.79 11.5 3.56 10.2 5660 6000 5660 13 217 10 13 217 10 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 268 148 219 1040 610 992 1940 1360 2060 0.30 0.49 54.7 3.56 36.0 5660 6000 5660 13 117 7 13 117 7 0hr prior to AKI stage sCr only 0.57 0.085 .7 4.1 0.47 5.7 1.5 0.47 1.5 . 0 p Value 0.12 0.21 0.54 0.12 0.66 0.54 0.67 0.65 ha 95% CI of 0.63 0.45 0.040 0.63 0.24 0.040 0.23 0.25 ha 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of . . . . . .

OR Quart4 85 30 15 75 12 9.7 7.6 ellular matrix protein 1 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2.06 0.536 1.49 .48 0.998 3.29 12.1 1.23 4.20 2.5E—4 2.5E—5 0.327 0.0273 0.327 57.7 7.81 14.4 22 106 13 22 106 13 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1.49 0.851 1.38 3.31 2.25 3.75 4.16 10.9 4.71 0.73 0.66 0.327 0.0143 0.327 14.4 150 14.4 13 217 10 13 217 10 -Cohort 1U0 only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 ' 2.68 0.678 2.18 6.95 1.11 2.57 .4 1.26 3.11 7.3E—5 0.0087 0.327 0.0273 0.327 57.7 7.81 9.15 13 117 7 13 117 7 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.66 0.12 0.16 23 13 14 22 13 10 7 0.721 0.451 70% 71% 46% 39% 0.348 0.324 100% 1.21 2.04 2.56 OR Quart 2 >3.3 2.0 2.0 >3.3 >32 >42 >33 p Value <0.31 0.58 0.58 <0.31 <0.32 <0.20 <0.31 95% CI of >O.33 0.18 0.17 >O.33 >O.33 OR Quart2 Ila 23 23 Ila Ila OR Quart 3 >9.0 5.4 0.97 >11 >0 p Value <0.047 0.13 0.98 <0.031 <na 95% CI of >1.0 0.61 0.058 >1.2 >na OR Quart3 Ila 48 16 Ila Ila OR Quart 4 >21 5.3 13 >17 >4.6 p Value <0.0048 0.13 0.016 <0.0091 0.21 <0.049 <0.20 <0.18 95% CI of >2.5 0.60 1.6 >2.0 0.45 >1.0 >O.46 >0.48 OR Quart4 Ila 47 110 Ila 38 Ila Ila Ila Coagulation factor XIII A and B chains 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 8.53 5.92 7.60 13.1 9.60 8.49 13.1 13.5 8.58 0.27 0.77 0.579 0.000189 0.0238 46.4 84.2 30.6 22 106 13 22 106 13 WO 78253 48hr prior to AKI stage 0.000189 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.745 0.000189 . 46.4 84.2 14.5 13 117 7 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.65 0.51 0.65 0.62 0.50 0.63 0.51 0.46 0.47 SE 0.067 0.083 0.083 0.069 0.083 0.087 0.086 0.095 0.11 p 0.026 0.88 0.073 0.074 0.97 0.15 0.86 0.69 0.82 2.94 2.04 18% 22% 0.809 100% .0 Cutoff5 16.0 18.5 16.3 16.0 18.5 . . . 16.3 Sens 5 30% 8% 36% 27% 8% 31% 15% 10% 0% Spec 5 80% 80% 80% 80% 80% 80% 80% 80% 80% Cutoff 6 20.7 24.7 20.7 20.7 24.7 20.7 20.7 24.7 20.7 Sens 6 26% 8% 36% 23% 8% 31% 8% 10% 0% Spec6 91% 90% 91% 91% 90% 91% 91% 90% 91% OR Quart 2 1.4 2.0 0.97 0.72 2.7 0.97 0.22 1.5 3.2 p Value 0.69 0.42 0.97 0.69 0.25 0.97 0.18 0.65 0.32 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value . 95% C1 0f 0.88 0.13 0.62 0.62 0.25 0.48 0.14 0.37 0.18 OR Quart4 15 7.2 18 8.7 9.7 15 3.4 12 24 Vitronectin 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 87.4 35.2 56.6 131 90.6 136 173 148 220 0.26 0.33 3.33 2.17 0.119 750 750 750 22 106 13 22 106 13 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 .6 86.8 45.6 71.7 148 122 165 214 181 244 0.61 0.47 6.79 1.96 6.79 750 750 750 13 217 10 13 217 10 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 114 36.5 101 139 87.8 139 132 145 171 0.23 0.37 3.33 2.17 0.119 462 750 462 13 117 7 13 117 7 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.59 0.66 0.63 0.59 0.64 0.55 0.56 0.55 SE 0.067 0.085 0.083 0.069 0.085 0.087 0.087 0.096 0.12 p 0.036 0.28 0.056 0.069 0.29 0.12 0.56 0.55 0.65 nCohort1 106 217 117 106 217 117 106 217 117 nCohort 2 23 13 14 22 13 13 13 10 7 23.8 . 2.83 80% 86% % 22% 2% 2.83 23.0 0 92% 90% 100% 64.8 148 96.7 31% 20% 57% 80% 80% 80% OR Quart 20.17 0.48 0.30 0.17 0.48 0.48 p Value 0.12 0.56 0.31 0.12 0.56 0.56 95% CI Of 0.019 0.043 0.030 0.019 0.043 0.042 OR Quart2 1.6 5.5 3.1 1.6 5.5 5.6 OR Quart 3 1.2 3.8 0.30 1.2 3.8 0 p Value 0.74 0.10 0.31 0.74 0.10 na 95% CI of 0.34 0.76 0.030 0.34 0.76 na OR Quart3 4.6 19 3.1 4.6 19 na OR Quart42.7 1.5 3.6 2.5 1.5 2.1 p Value 0.10 0.66 0.074 0.15 0.66 . . . 0.40 95% CI Of 0.82 0.24 0.88 0.73 0.24 0.74 0.49 0.10 0.36 OR Quart4 8.9 9.3 15 8.2 9.3 13 9.6 4.0 13 Estradiol 48hr prior to AKI stage Cohort 1 Cohort 2 6.69 18.2 11.2 19.0 18.3 14.8 0.47 0.143 4.59 128 34.2 50 3 WO 78253 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 9.48 nd nd 13.0 nd nd .5 nd nd 0.93 nd nd 4.59 nd nd 28.3 nd nd 4 nd nd 4 nd nd 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 14.0 6.61 18.2 16.7 8.23 19.0 13.0 6.60 14.8 0.030 0.016 4.59 0.143 4.59 34.2 31.6 34.2 4 42 3 4 42 3 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.58 0.15 0.60 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only Sens 6 29% 25% 50% 29% 25% 67% Spec 6 90% 90% 90% 90% 90% 90% OR Quart2>1.1 0 >10 >1.1 0 >1.1 p Value <0.96 na <1.0 <0.96 na . <0.95 95% C1 0f >0.061 na >0.055 >0.061 na >0.055 >0.061 nd >0.060 OR Quart2 na na na na na na na nd na OR Quart 3 >38 2.1 >1.1 >3.8 2.1 >1.1 >0 nd >0 p Value <0.27 0.56 <0.95 <0.27 0.56 <0.95 <na nd <na 95% C1 0f >0.35 0.18 >0.060 >0.35 0.18 >na OR Quart3 na 25 na na 25 na OR Quart 4 >35 0.95 >22 >35 0.95 >2.2 p Value <0.30 0.97 <0.54 <0.30 0.97 . <0.54 95% C1 0f >0.32 0.056 >0.17 >0.32 0.056 >0.17 >0.17 nd >0.17 OR Quart4 na 16 na na 16 na na nd na Progesterone 48hr prior to AKI stage Cohort 1 Cohort 2 22.9 53.1 31.4 43.9 27.4 20.1 0.44 2.91 20.9 152 57.8 50 3 50 3 sCr only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 31.1 nd nd 31.9 nd nd 21.7 nd nd 0.69 nd nd 7.44 nd nd 57.8 nd nd 4 nd nd 4 nd nd 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 55.5 21.1 53.1 51.7 32.2 43.9 22.7 32.9 20.1 0.25 0.55 .9 2.91 20.9 75.2 152 57.8 4 42 3 WO 78253 48hr prior to AKI stage 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.62 0.48 0.77 0.62 0.48 0.77 0.69 nd 0.72 SE 0.12 0.15 0.14 0.12 0.15 0.14 0.18 nd 0.17 0.20 19.3 100% 19.3 100% 19.3 100% .0 70% nd 71% ‘ 0.1 68.1 >1.1 <O.95 95% CI of >0.060 OR Quart2 na OR Quart 3 . >0 p Value <na 95% CI of >na OR Quart3 na OR Quart 4 3.2 1.0 >3.7 3.2 1.0 >37 >22 nd >2.2 p Value 0.33 0.97 <0.29 0.33 0.97 <0.29 <0.55 nd <0.54 95% C1 Of 0.30 0.061 >0.32 0.30 0.061 >0.32 >0.17 nd >0.17 OR Quart4 36 18 na 36 18 na na nd na 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 3.41 3.38 3.41 3.91 4.72 3.29 2.20 4.52 2.45 0.65 0.59 48hr prior to AKI stage Cohort 1 Cohort 2 58 0.792 .4 5.68 50 3 50 3 48hr prior to AKI stage Cohort 1 Cohort 2 nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd nd I stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 4.54 3.33 3.41 4.17 4.28 3.29 3.80 2.45 0.66 0.00599 0.792 19.3 5.68 42 3 42 3 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only sCr or UO sCr on1y 0.32 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 7.09 OR Quart22.2 >1.1 0 2.2 >1.1 0 >25 nd 1.1 p Value 0.55 <0.95 na 0.55 <0.95 na <0.47 nd 0.95 95% C1 0f 0.17 >0.064 na 0.17 >0.064 na >0.20 nd 0.060 OR Quart2 27 na na 27 na 20 OR Quart33.5 >1.1 1.0 3.5 >1.1 0 p Value 0.30 <0.95 1.0 0.30 <0.95 na 95% CI of 0.32 >0.064 0.055 0.32 >0.064 na OR Quart3 39 na 18 39 na na OR Quart40.93 >2.3 2.0 0.93 >2.3 1.1 p Value 0.96 <0.51 0.59 0.96 <0.51 . . 0.95 95% C1 0f 0.053 >0.19 0.16 0.053 >0.19 0.16 >0.066 nd 0.060 OR Quart4 16 na 26 16 na 26 na nd 20 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 8.98 4.61 10.1 7.92 5.53 9.58 .20 5.47 6.40 0.28 0.22 1.20 0.00501 2.96 .7 28.5 15.7 7 50 3 7 50 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 3.83 nd nd 4.46 nd nd 3.33 nd nd 0.52 nd nd 1.20 nd nd 8.98 nd nd 4 nd nd 4 nd nd 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 .9 4.43 10.1 .1 5.17 9.58 .34 4.67 6.40 0.050 0.13 2012/066152 48hr prior to AKI stage Cohort 1 Cohort 2 0.00501 2.96 16.8 15.7 42 3 42 3 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 0.43 0.43 0.15 0.15 0.66 0.66 81 81 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 Growth/differentiation factor 15 sCr or UO 0hr prior to AKI stage Cohort 1 Cohort 1 Cohort 2 —26900 38600 26900 2012/066152 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 51100 71300 50900 35000 131000 52900 0.69 0.79 MH—E:109000 5180 641 5180 109000 810000 109000 11 (Sarnp) 7 50 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 48500 nd nd 45500 nd nd 32700 nd nd 0.52 nd nd 5180 nd nd 79700 nd nd 4 nd nd 4 nd nd 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 33400 20300 38600 45200 53100 50900 44700 80600 52900 0.85 0.96 64 5180 5180 641 5180 326000 109000 109000 326000 109000 A 2 4 42 3 —:—_ 4 42 s 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only sCr or UO sCr only 0.59 0.45 0.59 0.51 0.12 0.15 0.12 0.17 nCohort 1 nCohort 2 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only U0 only 0% 0% 70% 70% 124000 124000 0% 0% 205000 218000 205000 205000 218000 205000 205000 0% 0% 0% 0% 0% 0% 0% 90% 90% 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 Proprotein convertase isin/kexin type 9 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1 9 920 406 920 Average 3910 1530 1530 3910 1530 ”9— 0.67 0.78 95.1 592 95.1 592 96400 3260 3260 96400 3080 7 50 3 7 50 3 sCr only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1050 nd nd 1550 nd nd 1 150 nd nd 0.75 nd nd 81 3 nd nd 3260 nd nd 4 nd nd 4 nd nd UO only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 86 882 86 882 486 920 WO 78253 48hr prior to AKI stage Cohort 1 Cohort 2 3360 1530 14800 1350 0.83 _—000 95.1 592 00400 3000 n (Sarnp) ‘ 2 42 3 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.75 0.73 0.70 0.75 0.73 0.70 0.74 nd 0.71 0.11 0.15 0.15 0.11 0.15 0.15 0.17 nd 0.17 0.022 0.13 0.20 0.022 0.13 0.20 0.16 nd 0.22 50 81 42 50 81 50 nd 1 2 100% 100% 100% 1060 1360 2850 95% CI of >na OR Quart2 Ila OR Quart 3 >2.4 p Value <O.15 <0.30 <O.25 <O.15 <O.49 95% CI of >O.54 >O.33 >036 >O.54 >O.19 OR Quart3 na na Ila Ila Ila OR Quart 4 >3.5 >1.0 >1.0 >3.5 >1.0 p Value <0.30 <1.0 <1.0 <0.30 <1.0 <1.0 <1.0 nd <1.0 95% CI of >0.32 >0.059 >0.055 >0.32 >0.059 >0.055 >0.056 nd >0.055 OR Quart4 Ila Ila Ila Ila Ila Ila Ila nd Ila Table 5: Comparison of marker levels in EDTA samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0) and in EDTA samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. ocalcin- 1 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.0730 0.0607 0.0406 0.00673 0.283 6.00 . 18 50 5 18 25 5 0.990 0.000985 0.0265 6.00 0.119 99 5 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 0.0701 0.109 0.0701 0.0983 0.547 0.198 0.547 0.122 p(t—ntest) 0.27 Mi 0 0.000985 0.00830 0.0150 0.00830 _m10-68n (Samp) 11 (Patient) 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only sCr or UO sCr only 71% 75% 70% 72% 80% 28% 69% 25% 32% 18% 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value . . . . . 95% CI of 0.29 na 0.27 0.20 >0.32 0.27 >0.20 na 0.13 OR Quart4 5.0 na 5.7 4.9 na 5.7 na na 8.9 Extracellular matrix protein 1 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1350 1530 1400 1490 1610 1490 478 462 437 0.29 0.45 846 779 1110 Max 3060 2350 3060 2630 3060 2570 n (Samp) 54 13 54 23 54 9 11 (Patient) 53 13 53 23 53 9 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1930 1490 2100 1930 1550 2030 982 449 568 0.24 0.070 1240 535 1440 WO 78253 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2630 3060 2570 2 110 3 2 92 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1450 1490 1400 1520 1570 1490 478 466 436 0.67 0.64 846 779 1110 2630 3060 2570 48 9 44 9 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 0.52 0.60 0.21 0.21 0.94 0.63 95% CI of OR Quart2 OR Quart 3 0.70 0 1.0 0.83 0 0.49 3.5 >0 1.1 p Value 0.67 ha 1.0 0.80 ha 0.33 0.31 <na 0.96 95% CI of 0.13 ha 0.17 0.21 ha 0.11 0.32 >na 0.061 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only sCr only OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 Coagulation factor XIII A and B chains 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 ior to AKI stage Cohort 1 Cohort 2 Median 10900 12200 92 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 10800 10300 9150 48 44 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.39 0.59 0.44 0.41 0.84 0.50 0.37 0.62 0.45 SE 0.091 0.21 0.095 0.073 0.18 0.072 0.11 0.18 0.11 p 0.23 0.68 0.52 0.20 0.050 1.0 0.23 0.51 0.65 nCohort 1 54 110 48 54 1 10 8 54 110 8 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 8080 9800 7150 100% 89% 44% 15% 9800 6730 100% 12900 15400 80% 81% 22900 0.50 0.59 95% CI of 0.040 OR Quart2 6.2 OR Quart 3 3.1 >1.0 1.6 1.4 >0 3.5 >1.0 2.6 p Value 0.22 <0.98 0.63 0.64 <na 0.31 <0.98 0.32 95% CI of 051 >0.062 0.23 0.32 >113. 0.39 OR Quart3 19 na 11 6.4 na 17 OR Quart 4 1.7 >0 1.6 2.9 >22 . 1.1 p Value 0.58 <na 0.63 0.14 <0.54 0.28 <1.0 0.94 95% CI of 025 >113. 0.23 0.70 >0.18 0.34 >0.060 0.13 OR Quart4 12 na 11 12 na 41 na 8.9 ectin 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 120000 104000 120000 1 1 3000 123000 108000 123000 121000 32800 42000 32800 35300 0.098 0.86 65900 35100 65900 84900 184000 205000 197000 23 54 23 53 48hr prior to AKI stage Cohort 2 —119000 116000 119000 99400 119000 108000 2012/066152 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 120000 120000 105000 36900 6820 36900 16700 0.44 0.50 35100 94600 35100 87500 _205000 116000 104000 205000 121000 n (Samp) 1 10 11 (Patient) 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 119000 133000 119000 104000 119000 113000 105000 120000 120000 43900 30100 35300 21800 65900 84900 184000 205000 197000 48 9 44 9 0hr prior to AKI stage 48hr prior to AKI stage sCr only 0.48 0.21 13 2 12 115000 115000 115000 77% 100% 75% 46% 48% 115000 115000 85% 100% 46% 48% 115000 92% 100% 92% 91% 100% % 48% 27% 0% 9% 132000 137000 128000 132000 132000 54% 0% 58% 39% 22% 70% 70% 71% 70% 70% 158000 153000 140000 158000 158000 23% 0% 33% 9% 11% 173000 173000 167000 173000 173000 23% 0% 25% 4% 11% 4.6 >0 1.0 0.44 >0 0.17 1.0 >1.1 0.50 p Value 0.20 <na 1.0 0.30 <na 0.046 1.0 <0.96 0.59 95% CI of 0.46 >na 0.12 0.092 >na 0.030 0.12 >0.064 0.040 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of _ _ OR Quart4 47 na 20 9.6 6.3 8.8 8.9 Estradiol sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1.37 1.24 1.48 2.75 1.80 1.61 3.95 1.84 0.932 0.16 0.77 0.288 0.513 0.541 .4 10.8 3.68 22 53 9 22 52 9 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1.00 1.40 1.48 1.00 2.32 1.42 0.514 2.86 0.529 0.52 0.59 0.638 0.288 0.871 1.37 17.4 1.93 2 108 3 2 91 3 -Cohort 1U0 only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 ' 1.41 1.16 1.40 2.86 1.97 1.55 3.80 2.00 0.926 0.20 0.55 0.288 0.513 0.541 .4 10.8 3.68 24 47 9 24 43 9 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.5 1 0.1 1 0.864 1.16 100% 78% 19% 51% 0.552 OR Quart 2 >49 >1.1 >2.2 0.93 >0 0.69 0.43 >2.2 0.46 p Value <0.18 <0.96 <0.55 0.92 <na 0.63 0.51 <0.54 0.55 95% C1 Of >0.49 >0.064 >0.17 0.22 >na 0.037 OR Quart2 na na na 4.0 na 5-8 OR Quart 3 >37 >10 >35 0.93 >1.0 2.4 p Value <0.28 <0.98 <0.30 0.92 <0.98 0.36 95% C1 Of >0.34 >0.062 >0.32 0.22 >0.062 0.36 OR Quart3 na na na 4.0 na 16 OR Quart4>8.7 >0 >12 1.5 >1.1 . . . 1.0 p Value <0.059 <na <0.030 0.56 <0.96 0.36 0.68 <0.96 1.0 95% C1 Of >0.92 >na >1.3 0.38 >0.064 0.47 0.21 >0.064 0.12 OR Quart4 na na na 6.1 na 7.8 11 na 8.3 Progesterone sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 3.87 4.43 3.90 21.9 6.21 5.65 52.7 7.20 4.28 0.036 0.82 1.65 1.35 2.45 194 46.5 15.8 22 53 9 22 52 9 WO 78253 48hr prior to AKI stage 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.64 0.73 0.62 0.48 0.15 0.50 0.49 0.51 0.47 SE 0.090 0.21 0.095 0.074 0.17 0.073 0.11 0.17 0.11 p 0.13 0.27 0.20 0.76 0.040 0.96 0.93 0.96 0.80 3.41 3.09 2.06 100% 6.28 Cutoff5 7.12 8.62 7.77 7.12 8.62 . . . 7.77 Sens 5 38% 0% 25% 27% 0% 29% 22% 33% 22% Spec5 81% 81% 81% 81% 81% 81% 81% 81% 81% Cutoff6 9.61 11.6 9.73 9.61 11.6 9.73 9.61 11.6 9.73 Sens 6 15% 0% 17% 23% 0% 17% 11% 33% 0% Spec6 91% 91% 91% 91% 91% 91% 91% 91% 91% OR Quart 2 >67 >0 >70 0.58 >0 0.45 0.50 0.96 0.46 p Value <0.10 <na <0.097 0.46 <na 0.28 0.59 0.98 0.55 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 p Value . . 95% C1 0f >0.69 >0.059 >0.50 0.36 >0.064 0.28 0.041 0.057 0.037 OR Quart4 na na na 5.3 na 1.3 6.2 16 5.8 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 1.15 1.00 1.15 1.34 0.902 1.25 1.14 0.493 0.601 0.550 0.019 0.59 0.000162 0.302 0.0946 1.59 3.76 1.61 22 53 9 22 52 9 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.641 1.07 1.34 0.641 1.13 1.42 0.906 0.602 0.169 0.26 0.41 27 0.000162 1.30 1.28 3.76 1.61 2 108 3 2 91 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.946 1.13 1.01 0.868 1.21 1.07 0.506 0.520 0.521 0.011 0.46 0.000162 0.344 0.0946 1.59 2.78 1.61 24 47 9 24 43 9 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.54 0.79 0.54 0.36 0.34 0.36 0.48 0.73 0.45 SE 0.091 0.19 0.095 0.073 0.21 0.071 0.11 0.17 0.11 p 0.68 0.14 0.68 0.046 0.46 0.043 0.88 0.16 0.65 nCohort 1 53 108 53 108 7 53 108 7 nCohort 2 13 2 22 2 24 9 3 9 100% 89% 4% 2% 4% 68% 2% 0.000227 62 0 1.27 0 92% 100% 100% 100% 1.39 1.57 1.66 0% 33% 0% 81% 81% 81% 1.88 OR Quart20 >0 0.18 6.2 >1.1 1.0 p Value na <na 0.15 0.038 <0.96 1.0 95% C1 Of na >na 0.017 1.1 >0.064 0.12 OR Quart2 na na . 35 na 8.3 OR Quart 3 0.73 >0 0.38 1.6 >0 1.6 p Value 0.69 <na 0.32 0.63 <na 0.62 95% C1 Of 0.16 >na 0.058 0.23 >na 0.23 OR Quart3 3.5 na 11 na 12 OR Quart40.68 >2.1 . 8.5 >1.1 1.0 p Value 0.62 <0.56 0.78 0.015 <0.96 . . . 1.0 95% C1 Of 0.15 >0.18 0.26 1.5 >0.064 1.5 0.095 >0.059 0.12 OR Quart4 3.2 na 48 na 34 4.7 na 8.3 Growth/differentiation factor 15 48hr prior to AKI stage Cohort 1 Cohort 2 1820 1820 2320 2480 1700 1550 0.79 271 733 7790 5690 53 9 2012/066152 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 2080 1150 2080 4700 2080 1010 Average 2650 1150 2650 4700 2650 1180 3700 1830 551 0.12 0.17 2090 271 733 7320 8110 1800 2 108 3 2 91 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2370 2030 1900 3430 2360 2700 2260 1640 1400 0.025 0.57 1010 452 1400 8110 7790 5690 24 47 9 24 43 9 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only 0.74 0.66 0.20 0.071 0.25 0.028 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only Sens 6 8% 0% 8% 18% 50% 11% Spec6 91% 91% 91% 91% 91% 91% OR Quart 20.75 >0 0.26 1.3 >0 >7.8 p Value 0.74 <na 0.27 0.73 <na . <0.081 95% C1 0f 0.14 >na 0.024 0.25 >na 0.19 0.46 >na >0.78 OR Quart2 4.0 na 2.9 7.0 na 1.5 48 na na OR Quart 3 0.43 >1.0 0.92 2.9 >1.0 2.1 1.0 >1.0 >1.1 p Value 0.38 <0.98 0.92 0.18 <0.98 0.33 1.0 <0.98 <0.96 95% C1 0f 0.068 >0.062 0.15 0.62 >0.062 >0.061 OR Quart3 2.8 na 5.5 14 na na OR Quart41.1 >1.1 1.8 3.6 >10 >38 p Value 0.92 <0.96 0.48 0.100 <1.0 . <0.27 95% C1 0f 0.22 >0.064 0.35 0.78 >0.059 0.76 0.30 >0.19 >0.35 OR Quart4 5.3 na 9.7 17 na 14 35 na na Proprotein convertase subtilisin/kexin type 9 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 443000 462000 543000 456000 513000 569000 208000 226000 189000 0.30 0.48 102000 76300 326000 858000 0 884000 23 54 9 23 53 9 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 321000 491000 529000 321000 504000 466000 213000 124000 213000 122000 0.23 0.76 76300 233000 76300 326000 Max 1100000 698000 1100000 409000 1100000 543000 n(Samp) 110 2 110 2 110 3 11 (Patient) 92 2 92 2 92 3 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 443000 466000 494000 448000 504000 564000 216000 211000 191000 0.29 0.43 80700 76300 326000 858000 867000 884000 11 (Samp) A 8 12 A 8 25 48 9 48hr prior to AKI stage 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.61 0.72 0.60 0.43 0.21 0.43 0.59 0.48 0.58 SE 0.091 0.21 0.095 0.073 0.19 0.072 0.11 0.17 0.11 0.23 0.28 0.29 0.14 0.93 0.44 54 110 54 1 10 54 110 A 8 13 2 23 2 9 3 9 497000 538000 497000 262000 228000 491000 318000 A 91000 77% 100% 75% 74% 100% 78% 100% 78% 56% 64% 54% 11% 9% 15% 56% 20% 54% 491000 538000 491000 246000 228000 248000 326000 318000 326000 80% 89% 100% 89% 56% 64% 54% 9% 9% 20% 20% 19% 442000 538000 442000 228000 228000 318000 318000 318000 92% 100% 92% 91% 100% 100% 100% 100% 48% 64% 48% 7% 9% 20% 20% 19% 642000 597000 667000 642000 597000 667000 642000 597000 667000 16% 33% 0% 33% 71% 70% 70% 71% 786000 692000 698000 786000 692000 698000 786000 692000 698000 8% 50% 17% 9% 0% 11% 0% 22% 81% 80% 81% 81% 80% 81% 80% 81% 835000 826000 834000 835000 826000 835000 826000 834000 8% 0% 8% 4% 0% 11% 0% 11% 90% 92% 0 >22 0 na <0.52 na 95% c1 of —a OR Quart2 na OR Quart 3 2.4 p Value 0.36 95% CI of 0.36 OR Quart3 _ _ 16 OR Quart43.2 >1.0 2.2 2.3 >1.0 3.0 1.5 >1.1 1.5 p Value 0.34 <0.98 0.55 0.25 <0.98 0.14 0.68 <0.96 0.69 95% C1 Of 0.30 >0.062 0.17 0.55 >0.062 0.71 0.21 >0.064 0.21 OR Quart4 35 na 9.8 na 13 11 na 11 Table 6: Comparison of marker levels in EDTA samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0 or R) and in EDTA samples collected from ts at 0, 24 hours, and 48 hours prior to reaching stage I or F in Cohort 2.

Toll-like receptor 2 Cohort 2 0.0983 0.473 1.45 0.25 —1.14E5 114E5 . 0.00107 M(Sxamp) 19.7 8.94 . 6.59 n 91 19 20 sCr only 0hr prior to AKI stage 24hr prior to AKI stage Cohort 2 1 stage Cohort 2 0.0925 0.485 1.49 0.23 6.59 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.49 0.46 0.46 0.54 0.59 0.50 0.00402 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only Cutoff 4 . 0.194 . . 0.194 Sens 4 33% 33% Spec 4 71% 71% Cutoff 5 . 0.398 . . 0.398 Sens 5 0% 0% Spec 5 80% 80% Cutoff 6 . 4.75 . . 4.75 Sens 6 0% 0% Spec 6 90% 90% OR Quart2 . >1.1 . . >1.0 p Value . <0.96 . . <10 95% CI of . >0.064 . . >0.060 OR Quart2 . na . . na OR Quart 3 . >2.1 . . >2.1 p Value . <0.54 95% CI of _ >0.18 OR Quart3 _ na OR Quart 4 . >0 p Value 95% CI of OR Quart4 Antithrombin-III sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage ort2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 -nd115000Median 110000 86200 110000 94500 109000 115000 97000 _nd—_36600 53700 36600 35200 0.66 0.21 36200 49300 36200 32000 252000 206000 252000 140000 —§3—33—91112 9 112 7 9 91 7 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 105000 86200 105000 102000 110000 109000 110000 98400 30700 53700 30700 38400 0.92 0.38 36200 49300 36200 32000 186000 206000 186000 140000 98 9 98 6 76 9 76 6 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only lUO only sCr or UO sCr only lUO only 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only 0.44 0.13 0.62 nCohort 2 nd nd nd 9 nd 9 7 nd 6 83500 83500 100% 100% 100% 1% 0% 0% 123000 123000 121000 71% 71% 70% 142000 142000 141000 33% 0% 0% 80% 80% 82% 163000 163000 151000 22% 0% 0% 90% 90% 91% OR Quart 2nd nd nd 0 nd 0.31 1.0 nd 2.1 p Value nd nd nd na nd 0.32 1.0 nd 0.56 95% C1 Of nd nd nd na nd 0.18 OR Quart2 nd nd nd na nd 25 OR Quart 3 nd nd nd 0.32 nd 1.0 p Value nd nd nd 0.34 nd 1.0 95% CI of nd nd nd 0.032 nd 0.059 OR Quart3 nd nd nd 3.3 nd 17 OR Quart 4 nd nd nd 1.9 nd . . 2.1 p Value nd nd nd 0.42 nd 0.65 0.54 nd 0.56 95% C1 Of nd nd nd 0.40 nd 0.29 0.18 nd 0.18 OR Quart4 nd nd nd 8.6 nd 7.2 25 nd 25 Extracellular matrix protein 1 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1350 1510 1490 1480 1580 1560 434 458 410 0.51 0.87 846 535 945 2280 3060 2100 9 112 7 9 91 7 WO 78253 48hr prior to AKI stage Cohort 1 Cohort 2 1470 1450 1560 1460 468 364 0.61 535 945 3060 1990 98 6 76 6 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only sCr or UO sCr only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 p Value nd nd nd 0.62 nd 0.96 0.97 nd 0.56 95% CI of nd nd nd 0.25 nd 0.19 0.14 nd 0.18 OR Quart4 nd nd nd 10 nd 5.7 7.9 nd 25 WO 78253 Coagulation factor XIII A and B chains 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 10300 11300 10900 12800 12100 12700 6690 5970 3900 3550 881 8160 24900 33300 19200 9 112 7 9 91 7 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 10300 10300 12100 12800 11400 13100 6690 5710 4070 3550 881 8160 24900 33300 19200 9 98 6 9 76 6 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only sCr or UO sCr only nd nd 0.53 nd . 0.56 nd nd 0.10 nd . 0.12 nd nd 0.77 nd . 0.60 OR Quart 2 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only p Value 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . . . p Value nd nd nd 0.97 nd 0.67 0.58 nd <0.54 95% C1 Of nd nd nd 0.13 nd 0.23 0.17 nd >0.18 OR Quart4 nd nd nd 7.3 nd 9.8 23 nd na Vitronectin sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median nd nd 118000 111000 118000 87500 104000 121000 85900 45900 33500 49400 0.15 0.010 35100 57400 21800 167000 205000 182000 9 112 7 9 91 7 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 118000 111000 118000 77500 120000 104000 120000 85700 33200 45900 33200 54100 0.18 0.020 57400 35100 57400 21800 205000 167000 205000 182000 98 9 98 6 76 9 76 6 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only sCr or UO sCr only nd nd 0.40 0.23 nd nd 0.10 0.11 nd nd 0.36 0.011 nd nd nd 35100 57400 nd nd nd 89% 86% 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 134000 133000 134000 22% 14% 70% 71% 150000 149000 150000 22% 14% 80% 80% 172000 172000 0% 14% 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 p Value 95% CI of OR Quart4 Interleukin-17F 1 stage Cohort 2 0.259 0.233 0.0957 0.75 0.126 0.312 UO only 0hr prior to AKI stage 24hr prior to AKI stage Cohort 2 0.233 0.250 0.101 0.36 0.101 0.468 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.37 0.38 0.36 0.51 0.50 0.48 0.074 0.79 Cutoff 5 0.339 0.337 0.356 0.339 0.337 0.356 Sens 5 11% 0% 12% 15% 0% 16% Spec 5 80% 80% 81% 80% 80% 81% Cutoff 6 0.473 0.410 0.529 0.473 0.410 0.529 Sens 6 0% 0% 0% 0% 0% 0% Spec 6 90% 90% 90% 90% 90% 90% OR Quart21.9 >1.1 1.0 1.2 0 0.76 p Value 0.42 <0.96 0.96 0.76 na 0.71 95% CI of 0.41 >0.064 0.19 0.30 na 0.18 OR Quart2 8.9 na 5.7 5.3 na 3.2 OR 1.8 >1.0 1.5 1.6 1.0 1.0 p Value 0.45 <0.98 0.64 0.53 1.0 1.0 95% C1 0f 0.39 >0.062 0.29 0.39 0.060 0.25 OR Quart3 8.4 na 7.3 6.3 17 A .0 OR Quart42.4 >1.1 3.0 1.2 0.97 1.0 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only p Value 0.26 <0.96 0.15 0.76 0.98 0.94 95% CI of 0.53 >0.064 0.67 0.30 0.058 0.26 OR Quart4 11 na 13 5.3 16 A .2 Interleukin-22 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage Cohort 2 0.806 0.815 0.460 0.39 0.195 1.85 sCr only Average 1.11 0.570 .

StdeV 1.91 0.228 . p(t—test) n (Samp) 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 0.34 0.48 0.17 0.17 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 95% CI of OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 24hr prior to AKI stage 48hr prior to AKI stage ort2 Cohort 2 Cohort 1 Cohort 2 Median . 0.984 1.10 0.551 0.550 0.593 0.543 0.36 0.016 0.000162 0.0946 0.000162 1.59 3.76 1.34 9 110 7 9 90 7 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 1.07 0.984 1.07 0.528 1.14 0.988 1.14 0.502 0.554 0.550 0.554 0.495 WO 78253 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 0.44 0.0071 0.0946 0.000162 0.0946 0.000162 3.18 1.59 3.18 1.34 —E§_Z§_7596 9 96 6 9 7s 6 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC nd nd nd 0.44 nd 0.45 0.23 nd 0.17 SE nd nd nd 0.10 nd 0.10 0.11 nd 0.10 p nd nd nd 0.54 nd 0.63 0.012 nd 0.0014 nCohort 1 nd nd nd 110 nd 96 110 nd 96 nCohort 2 nd nd nd 9 nd 9 7 nd 6 0.500 nd 0.000162 71% 83% 8% 0% 0.000162 0.000162 86% 83% 100% 1.34 1.60 1.86 >1.1 <0.96 95% CI of >0.064 OR Quart2 na p Value <na 95% CI of >na OR Quart3 na OR Quart 4 >65 p Value <0.099 95% CI of >070 OR Quart4 na sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage ——————— 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1.79 2.57 1.37 2.20 3.16 1.46 1.92 2.79 1.15 0.31 0.11 0.000327 0.000136 0.000136 4.97 22.2 3.13 9 110 7 9 90 7 U0 only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 1.79 2.53 1.54 2.20 3.01 1.51 1.92 2.71 1.25 0.38 0.18 0.000327 36 0.000136 4.97 22.2 3.13 9 96 6 9 75 6 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC nd nd nd 0.39 nd 0.41 0.26 nd 0.29 SE nd nd nd 0.10 nd 0.10 0.11 nd 0.12 p nd nd nd 0.30 nd 0.36 0.033 nd 0.092 . 0.196 78% 83% % 5% 0.000136 0.196 100% 83% 0.000136 0 100% 100% 1% 0% 3.57 Cutoff 5 nd nd nd 4.47 nd 1 .44 Sens 5 nd nd nd 11% nd 11% 0% nd 0% Spec 5 nd nd nd 80% nd 80% 80% nd 80% Cutoff 6 nd nd nd 6.18 nd 5.67 6.18 nd 5.67 Sens 6 nd nd nd 0% nd 0% 0% nd 0% Spec 6 nd nd nd 90% nd 91% 90% nd 91% OR Quart 2 nd nd nd 1.0 nd 1.0 >2.2 nd >2.3 p Value nd nd nd 1.0 nd 0.97 <0.52 nd <0.52 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 p Value . . 95% C1 Of nd nd nd 0.25 nd 0.25 >0.34 nd >0.19 OR Quart4 nd nd nd 10 nd 11 na nd na Proprotein convertase subtilisin/kexin type 9 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 505000 291000 505000 345000 523000 342000 523000 330000 203000 173000 203000 167000 0.010 0.015 76300 102000 76300 80700 1100000 596000 1100000 529000 112 9 112 7 91 9 91 7 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 A 96000 291000 496000 303000 518000 342000 518000 297000 199000 173000 199000 156000 0.012 0.0090 76300 102000 76300 80700 1000000 596000 1000000 511000 98 9 98 6 76 9 76 6 0hr prior to AKI stage 48hr prior to AKI stage sCr only 261000 246000 176000 78% 71% 83% % 9% 2% 102000 176000 176000 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 76300 76300 100% 100% 1% 1% 620000 620000 71% 71% 698000 698000 80% 80% 826000 826000 0% 0% 90% 90% 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 p Value 95% CI of OR Quart4 Table 7: Comparison of marker levels in EDTA samples ted within 12 hours of reaching stage R from Cohort 1 (patients that reached, but did not progress beyond, RIFLE stage R) and from Cohort 2 (patients that reached RIFLE stage I or F).

Estradiol UO only Cohort 2 Cohort 1 Cohort 2 nd 1.82 1.84 nd 2.74 5.46 nd 2.04 8.00 nd 0.19 nd 0.642 0.774 nd 7.74 17.4 nd 18 4 nd 18 4 At Enrollment sCr or UO sCr only UO only 0.64 nd 0.46 0.13 nd 0.17 0.27 nd 0.80 At Enrollment sCr or UO sCr only UO only nCohort 1 23 nd 18 t 2 7 nd 4 Cutoff1 1.99 nd 0.979 Sens 1 71% nd 75% Spec 1 65% nd 17% Cutoff 2 1.08 nd 0.732 Sens 2 86% nd 100% Spec 2 22% nd 11% Cutoff 3 0.732 nd 0.732 Sens 3 100% nd 100% Spec 3 13% nd 11% Cutoff 4 3.22 nd 3.24 Sens 4 57% nd 25% Spec 4 74% nd 72% Cutoff 5 3.48 nd 4.66 Sens 5 57% nd 25% Spec 5 83% nd 83% Cutoff 6 4.89 nd 6.27 Sens 6 14% nd 25% Spec 6 91% nd 94% OR Quart 2 0 nd 1.2 p Value na nd 0.89 95% CI of na nd 0.058 OR Quart2 na nd 27 OR Quart 3 0.42 nd 0 p Value 0.52 nd na 95% CI of 0.029 nd na OR Quart3 6.1 nd na OR Quart 4 2.5 nd 3.3 p Value 0.40 nd 0.40 95% C1 Of 0.29 nd 0.20 OR Quart4 21 nd 55 Growth/differentiation factor 15 U0 only Cohort 2 Cohort 1 Cohort 2 nd 2600 1500 nd 2610 2340 nd 1580 2240 nd 0.77 nd 437 750 nd 6010 5600 nd 18 4 nd 18 4 At Enrollment ———uoonly At Enrollment sCr or UO sCr only UO only AUC 0.60 nd 0.40 SE 0.13 nd 0.17 p 0.45 nd 0.56 nCohort 1 23 nd 18 nCohort 2 7 nd 4 Cutoff 1 1740 nd 829 Sens 1 71% nd 75% Spec 1 43% nd 17% Cutoff 2 829 nd 660 Sens 2 86% nd 100% Spec 2 17% nd 11% Cutoff 3 660 nd 660 Sens 3 100% nd 100% Spec 3 13% nd 11% Cutoff 4 3120 nd 3120 Sens 4 43% nd 25% Spec 4 74% nd 72% Cutoff 5 3530 nd 3920 Sens 5 43% nd 25% Spec 5 83% nd 83% Cutoff 6 4700 nd 5190 Sens 6 43% nd 25% Spec 6 91% nd 94% OR Quart 2 0.36 nd 0 p Value 0.45 nd na 95% CI of 0.025 nd na OR Quart2 5.1 nd na OR Quart 3 0.42 nd 1.0 p Value 0.52 nd 1.0 95% CI of 0.029 nd 0.048 OR Quart3 6.1 nd 21 OR Quart 4 1.5 nd 3.3 p Value 0.72 nd 0.40 95% CI of 0.17 nd 0.20 0R Q1181rt4 13 nd 55 tein convertase subtilisin/kexin type 9 U0 only Cohort 2 Cohort 1 Cohort 2 nd 523000 351000 nd 526000 334000 nd 210000 169000 nd 0.10 nd 176000 123000 nd 1000000 511000 nd 18 4 nd 18 4 At ment sCr or UO sCr only UO only AUC 0.24 nd 0.19 SE 0.11 nd 0.14 p 0.021 nd 0.030 nCohort 1 23 nd 18 nCohort 2 7 nd 4 Cutoff 1 210000 nd 210000 Sens 1 71% nd 75% Spec 1 13% nd 17% Cutoff 2 80700 nd 0 Sens 2 86% nd 100% Spec 2 0% nd 0% Cutoff 3 0 nd 0 Sens 3 100% nd 100% Spec 3 0% nd 0% Cutoff 4 620000 nd 604000 Sens 4 0% nd 0% Spec 4 74% nd 72% Cutoff 5 654000 nd 654000 Sens 5 0% nd 0% Spec 5 83% nd 83% Cutoff 6 764000 nd 786000 Sens 6 0% nd 0% Spec 6 91% nd 94% OR Quart 2 1.2 nd >1.5 p Value 0.92 nd <0.79 95% CI of 0.059 nd >0.071 OR Quart2 23 nd na OR Quart 3 1.0 nd >1.2 p Value 1.0 nd <0.91 95% CI of 0.052 nd >0.059 OR Quart3 19 nd na OR Quart 4 9.3 nd >4.0 p Value 0.089 nd <0.33 95% CI of 0.71 nd >0.25 OR Quart4 120 nd na Table 8: Comparison of the maximum marker levels in EDTA samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0) and the maximum values in EDTA samples collected from ts between enrollment and 0, 24 hours, and 48 hours prior to reaching stage F in Cohort 2.

Toll-like receptor 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.0648 0.0902 0.0925 0.121 1.96 0.0768 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0. 1 62 5 .50 0.0649 0.36 0.56 0.00551 0.00107 0.00551 0.496 19.7 0.132 1 stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.0648 0.112 0.0925 0.121 2.20 0.0768 0.00107 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only sCr or UO sCr only nCohort 1 nCohort 2 0.00402 100% 0.0366 0.0366 0.0182 2 88% 88% 88% 100% 28% 27% 16% 8% 0.00402 0.00107 0.00402 0.00402 p Value 0.10 nd 0.46 0.22 nd <0.45 nd 95% CI of 0.66 nd 0.19 0.38 nd 0.19 >0.20 nd >0.069 OR Quart2 97 nd 37 60 nd 37 na nd na 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 Antithrombin-III 24hr prior to AKI stage Cohort 1 Cohort 2 110000 49300 119000 49300 A 1300 17300 0.0054 —_23000 61300 32000 252000 66600 252000 66600 53 3 53 3 24hr prior to AKI stage Cohort 1 Cohort 2 103000 49300 114000 49300 33700 17300 0.0022 61300 32000 186000 66600 A 4 3 A 4 3 0hr prior to AKI stage sCr only Sens 3 100% nd 100% 100% 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 0% 0% 0% 0% 130000 130000 130000 130000 0% 0% 0% 0% 72% 70% 72% 70% 147000 145000 147000 145000 Cutoff 6 177000 nd 166000 177000 nd 166000 Sens 6 0% nd 0% 0% nd 0% Spec6 91% nd 91% 91% nd 91% OR Quart 2 >0 nd >0 >0 nd >0 p Value <na nd <na <na nd <na 95% C1 Of >na nd >na >na nd >na OR Quart2 na nd na na nd na OR Quart 3 >0 nd >0 >0 nd >0 p Value <na nd <na <na nd <na 95% C1 Of >na nd >na >na nd >na OR Quart3 na nd na na nd na OR Quart 4 >38 nd >45 >38 nd >4.5 p Value <0.27 nd <0.23 <0.27 nd <0.23 95% C10f >O.35 nd >O.39 >O.35 nd >039 OR Quart4 na nd na na nd na Extracellular matrix protein 1 O—hrprior to AKI stage Cohort 1 Cohort 2 Median 1530 1450 —mo 1340 Cohort 2 1450 0hr prior to AKI stage 24hr prior to AKI stage WO 78253 sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.36 nd 0.38 0.36 nd 0.38 0.18 0.51 100% 100% 100% 1710 Cutoff 5 1950 nd 1910 1950 nd 1910 Sens 5 0% nd 0% 0% nd 0% Spec 5 83% nd 82% 83% nd 82% Cutoff 6 2270 nd 2230 2270 nd 2230 Sens 6 0% nd 0% 0% nd 0% Spec 6 92% nd 91% 92% nd 91% OR Quart2>1.1 nd >1.1 >1.1 nd >1.1 p Value <0.96 nd <0.95 <0.96 nd <0.95 95% Cl Of >0.061 nd >0.061 >0.061 nd >0.061 OR Quart2 na nd na na nd na OR Quart3>1.1 nd >1.1 >1.1 nd >1.1 p Value <0.96 nd <0.95 <0.96 nd <0.95 95% Cl Of >0.061 nd >0.061 >0.061 nd >0.061 OR Quart3 na nd na na nd na OR Quart4>1.1 nd >1.2 >1.1 nd >1.2 p Value <0.96 nd <0.90 <0.96 nd <0.90 95% Cl Of >0.061 nd >0.066 >0.061 nd >0.066 OR Quart4 na nd na na nd na Coagulation factor XIII A and B chains 24hr prior to AKI stage Cohort 1 Cohort 2 12200 13200 13300 10500 6710 6040 0.48 881 3550 33300 14700 53 3 53 3 ———ss 3 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 >1.1 nd 1.1 >1.1 nd 1.1 p Value <0.96 nd 0.95 <0.96 nd 0.95 95% CI of >0.061 nd 0.060 >0.061 nd 0.060 OR Quart4 na nd 20 na nd 20 WO 78253 Vitronectin sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 120000 35100 120000 35100 122000 22100 Stdev 33100 9630 p—(ttest) 1.9E—5 _62200 21800 24hr prior to AKI stage Cohort 1 Cohort 2 120000 121000 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only 0 nd 0 nd <0.001 nd <0.001 nd <1.0E—5 nd <1.0E—5 nd 100% 100% 100% 100% 0% 0% 0% 0% 133000 131000 133000 131000 72% 70% 72% 70% 158000 157000 158000 157000 0% 0% 0% 0% 81% 82% 81% 82% 173000 167000 173000 167000 0% 0% 0% 0% 91% 91% 91% 91% OR Quart 2 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only p Value 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . . . . p Value <0.27 nd <0.23 <0.27 nd <0.23 95% C10f >035 nd >039 >035 nd >039 OR Quart4 na nd na na nd na Estradiol sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage 24hr prior to AKI stage Cohort 2 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only 0.97 nd 0.97 0.065 nd 0.065 3 nd 2.5E—13 3.57 nd 4.47 3.57 100% nd 100% 100% WO 78253 0hr prior to AKI stage 24hr prior to AKI stage sCr only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 p Value 95% CI of OR Quart4 Progesterone 1 stage Cohort 2 31.8 77.0 3.2E—6 .07 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.86 nd 0.86 0.86 nd 0.86 SE 0.14 nd 0.14 0.14 nd 0.14 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 p Value . 95% C1 0f >0.17 nd >0.17 >0.17 nd >0.17 OR Quart4 na nd na na nd na 24hr prior to AKI stage Cohort 2 0.000162 0.425 0.736 0.024 0hr prior to AKI stage 24hr prior to AKI stage —_Cohortz Cohort 1 Cohort 2 0.302 62 0.000162 3.276 1.27 3.76 1.27 52 3 52 3 0hr prior to AKI stage 24hr prior to AK1 stage Cohort 2 0.000162 0.425 0.736 0.017 0.000162 1.27 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.21 nd 0.22 0.21 nd 0.22 SE 0.16 nd 0.16 0.16 nd 0.16 OR Quart2>1.1 nd >1.2 >1.1 nd >1.2 p Value <0.96 nd <0.90 <0.96 nd <0.90 95% CI of >0.061 nd >0.066 >0.061 nd >0.066 OR Quart2 na nd na na nd na OR Quart 3 >0 nd >0 >0 nd >0 p Value <na nd <na <na nd <na 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 95% CI of OR Quart3 OR Quart 4 p Value 95% CI of OR Quart4 na nd na na nd na Cohort 2 0.291 0.572 0.752 0.082 0.000136 1.42 UO only 0hr prior to AKI stage 1 stage Cohort 1 Cohort 2 0.291 0.572 0.752 0.064 0.000136 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 0% nd 3.92 nd 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 p Value 95% CI of OR Quart4 /differentiation factor 15 sCr or UO 1 8 Average 2270 7840 StdeV 1680 1 50 0hr prior to AKI stage 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.99 nd 0.99 0.99 nd 0.99 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 0.036 <1.0E—5 6800 100% 6800 100% 6800 100% 2740 100% Cutoff 5 3190 nd 3190 3190 nd 3190 Sens 5 100% nd 100% 100% nd 100% SpecS 81% nd 81% 81% nd 81% Cutoff 6 4990 nd 4990 4990 nd 1 990 Sens 6 100% nd 100% 100% nd 100% Spec 6 90% nd 91% 90% nd 91% OR Quart 2 >0 nd >0 >0 nd >0 p Value <na nd <na <na nd <na 95% C1 Of >na nd >na >na nd >na OR Quart2 na nd na na nd na OR Quart 3 >0 nd >0 >0 nd >0 p Value <na nd <na <na nd <na 95% C1 Of >na nd >na >na nd >na OR Quart3 na nd na na nd na OR Quart 4 >35 nd >37 >35 nd >3.7 p Value <0.30 nd <0.29 <0.30 nd <0.29 95% C1 0f >0.32 nd >0.32 >0.32 nd >0.32 OR Quart4 na nd na na nd na Proprotein convertase subtilisin/kexin type 9 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage Cohort l Cohort 2 Cohort l Cohort 2 102000 A 68000 102000 158000 515000 158000 228000 115000 0.0099 76300 80700 1100000 291000 —§§_§_5353 3 WO 78253 Cohort 2 102000 158000 115000 0.0070 Min 80700 Max 867000 291000 291000 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 76300 76300 76300 100% 100% 100% 2% 2% 2% 667000 668000 667000 72% 70% 72% 786000 786000 786000 81% 82% 81% 835000 834000 835000 0% 0% 0% 91% 91% 91% 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 >3.8 nd >45 >38 nd >4.5 p Value <0.27 nd <0.23 <0.27 nd <0.23 95% CI of >O.35 nd >O.39 >O.35 nd >039 OR Quart4 na nd na na nd na Table 9: Comparison of marker levels in urine samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0, R, or I) and in urine samples collected from Cohort 2 (subjects who progress to RIFLE stage F) at 0, 24 hours, and 48 hours prior to the subject ng RIFLE stage I.

Toll-like receptor 2 0hr prior to AKI stage 24hr prior to AKI stage Cohort 2 0hr prior to AKI stage sCr only WO 78253 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only 1.43 OR Quart 2 0 nd 0 0 nd 0 p Value na nd na na nd na 95% C1 Of na nd na na nd na OR Quart2 na nd na na nd na OR Quart 3 2.1 nd 2.1 1.0 nd 2.1 p Value 0.56 nd 0.56 1.0 nd 0.56 95% CI of 0.18 nd 0.18 0.060 nd 0.18 OR Quart3 24 nd 24 17 nd 24 OR Quart43.1 nd 3.2 7.2 nd 5.6 p Value 0.34 nd 0.32 0.076 nd 0.13 95% CI of 0.30 nd 0.32 0.81 nd 0.61 OR Quart4 32 nd 33 64 nd 52 Antithrombin-III 48hr prior to AK1 stage 283 0.0182 6000 24hr prior to AKI stage 48hr prior to AKI stage Cohort 1 Cohort 2 Cohort 1 Cohort 2 103 219 nd nd 355 1 140 nd nd 939 2120 nd nd 0.0078 nd nd WO 78253 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 14.7 nd nd 5660 nd nd 1 1 nd nd 1 1 nd nd 0hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only sCr only 0.61 0.53 0.43 0.21 0.10 0.15 0.60 0.78 0.63 696 696 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of Extracellular matrix protein 1 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2.05 1.62 1.18 2.23 9.60 2.37 0.86 0.91 0.124 3.61E—6 0.0117 9.15 150 6.00 689 6 283 6 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 0.924 0.507 0.641 0.953 1.73 1.89 0.692 9.79 2.75 0.82 0.97 0.128 3.61E—6 0.272 1.95 150 6.00 8 697 4 8 288 4 24hr prior to AKI stage 48hr prior to AKI stage Cohort 2 Cohort 1 Cohort 2 2.18 nd nd 2.32 nd nd 2.55 nd nd 0.81 nd nd 9 nd nd 9.15 nd nd 11 nd nd 11 nd nd 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only sCr or UO sCr only 0.75 0.94 0.70 0.35 0.61 0.090 0.12 0.077 0.12 0.15 nCohort 1 nCohort 2 0.00904 100% 1.09 WO 78253 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr only sCr only UO only 95% CI of OR Quart2 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 Vitronectin sCr or UO 48hr prior to AKI stage Cohort 2 Cohort l Cohort 2 UO only 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage Cohort l Cohort 2 Cohort l Cohort 2 Cohort l Cohort 2 Median 31.0 136 31.0 52.9 nd nd 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage —_140Chort2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 73.4 73.4 83.2 nd nd 133 78.0 nd nd 0. 18 0.81 nd nd 0.0795 5.54 0.0795 2.03E—5 nd nd _699—11 150 216 11 11 n ) 699 11 nd nd 0hr prior to AKI stage 24hr prior to AKI stage 48hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.48 0.27 0.55 0.55 0.51 0.59 0.29 0.45 nd SE 0.093 0.20 0.12 0.077 0.10 0.091 0.12 0.15 nd p 0.85 0.27 0.71 0.54 0.93 0.33 0.079 0.73 nd nCohort 1 689 697 699 689 697 699 689 697 nd 95% CI of nd OR Quart2 nd OR Quart 3 . . . . nd p Value 0.42 <1.00 na 0.42 1.0 nd 95% CI of 0.089 >0.062 na 0.089 0.14 nd OR Quart3 2.7 na na 2.7 7.2 nd OR Quart 4 1.0 >1.0 0.99 1.5 0.99 . . . nd p Value 0.99 <0.99 0.99 0.52 1.00 0.48 0.34 1.00 nd 95% C1 Of 0.25 >0.063 0.20 0.42 0.14 0.39 0.31 0.062 nd OR Quart4 4.1 na 5.0 5.5 7.1 7.1 30 16 nd 2012/066152 Interleukin-22 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage Cohort 2 1.78E—5 000124 00007 StdeV 0.00488 0.0295 p(t—ntest) ‘ .8E— 6 1 stage Cohort 2 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only sCr or UO sCr only 0.71 11d 0.67 nd 0.12 11d 0.11 nd 0.083 0.12 nd OR Quart 2 W0 2013/078253 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only p Value 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . . . . p Value <O.56 nd <O.54 <O.31 nd <O.32 95% C1 0f >O.18 nd >O.18 >033 nd >032 OR Quart4 na nd na na nd na Estradiol sCr or UO 24hr prior to AKI stage 2012/066152 24hr prior to AKI stage sCr or UO sCr only UO only 0.68 0.74 0.70 0.12 0.17 0.15 0.15 0.15 0.19 122 126 104 6 3 4 9.25 9.25 9.47 83% 100% 75% 64% 63% 67% 9.25 9.25 4.53 83% 100% 100% 64% 63% 27% 4.53 9.25 4.53 100% 100% 100% 28% 63% 27% 11.0 11.0 10.7 33% 33% 50% 70% 71% 70% 13.3 13.9 12.9 33% 33% 50% 80% 80% 81% 22.4 23.2 22.4 17% 33% 25% 90% 90% 90% >10 >0 >10 <0.98 <na <0.98 95% C10f >0.062 >na >0.062 OR Quart2 na na 113, OR Quart 3 >33 >21 >10 p Value <0.31 <0.54 <0.98 95% CI of >033 >0.18 >0.062 OR Quart3 na na na OR Quart 4 >21 >10 >22 p Value <0.54 <1.0 <0.54 95% CI of >018 >0.060 >0.18 OR Quart4 na na na Progesterone sCr or UO 24hr prior to AKI stage WO 78253 Median 23.5 55.5 p(t—test) 11 (Samp) 104 24hr prior to AKI stage sCr or UO sCr only UO only 0.62 0.42 0.73 0.13 0.17 0.15 0.34 0.66 0.12 122 126 104 6 3 4 .3 7.23 52.2 83% 100% 75% 43% 10% 78% .3 7.23 20.3 83% 100% 100% 43% 10% 44% 7.23 7.23 20.3 100% 100% 100% 11% 10% 44% 37.3 40.0 39.6 67% 0% 75% 70% 71% 70% 57.6 57.6 56.1 33% 0% 50% 80% 80% 81% 79.5 79.5 81.6 0% 0% 0% 90% 90% 90% 1.0 >1.1 >1.0 1.0 <0.97 <0.98 2012/066152 24hr prior to AKI stage sCr or UO sCr only UO only 95% CI of 0.060 >0.064 >0.062 OR Quart2 17 na na OR Quart 3 1.0 >1.1 >0 p Value 1.0 <0.97 <na 95% C1 Of 0.060 >0.064 >na OR Quart3 17 na na OR Quart43.2 >1.1 >3.4 p Value 0.32 <0.97 <0.31 95% CI ofm OR Quart4 33 na na 24hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only 0.63 0.55 0.66 0.13 0.17 0.15 0.30 0.76 0.30 122 126 104 6 3 4 2.67 1.19 3.96 83% 100% 75% 39% 27% 48% 2.67 1.19 2.67 83% 100% 100% 39% 27% 39% 1.19 1.19 2.67 100% 100% 100% 28% 27% 39% 6.44 6.52 6.52 50% 33% 50% 70% 71% 70% 8.06 8.39 8.39 50% 33% 50% 80% 80% 81% 11.7 12.5 12.5 17% 0% 0% 90% 90% 90% >33 >10 >22 <0.31 <0.98 <0.54 95% CI ofm OR Quart2 na na na OR Quart 3 >0 >l.O >0 p Value <na <0.98 <na 95% C1 Of >na >0.062 >na OR Quart3 na na na OR >3.3 >l.O >22 p Value <0.31 <1.0 <0.54 95% CI ofm OR Quart4 na na na Table 10: Comparison of marker levels in EDTA samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0, R, or I) and in EDTA samples collected from Cohort 2 (subjects who progress to RIFLE stage F) at 0, 24 hours, and 48 hours prior to the subject reaching RIFLE stage I.

Toll-like receptor 2 sCr or UO 0hr prior to AKI stage 24hr prior to AKI stage Cohort 1 Cohort 2 Cohort 2 Median 0.0770 0.0681 0.0648 WO 78253 Cohort 2 0.121 0.162 0.35 0.00551 Cohort 2 0.0648 0.121 0.162 0.33 0.00551 0.496 0hr prior to AKI stage sCr only 0.00402 p Value 0.54 nd 0.56 0.32 95% CI of 0.18 nd 0.18 0.32 2012/066152 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only OR Quart2 OR Quart 3 . p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of OR Quart4 17 nd 17 17 nd Antithrombin-III Cohort 1 105000 111000 24hr prior to AKI stage sCr or UO sCr only UO only 0.027 nd 0.031 0.081 nd 0.087 6.2E—9 nd 6.5E—8 128 nd 112 2 nd 2 36200 nd 36200 100% nd 100% 1% nd 1% 36200 nd 36200 100% nd 100% 1% nd 1% 36200 nd 36200 WO 78253 24hr prior to AKI stage sCr or UO sCr only UO only Sens 3 100% nd 100% Spec 3 1% nd 1% Cutoff 4 123000 nd 121000 Sens 4 0% nd 0% Spec 4 70% nd 71% Cutoff 5 142000 nd 141000 Sens 5 0% nd 0% Spec 5 80% nd 81% Cutoff 6 164000 nd 151000 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 >0 nd >0 p Value <na nd <na 95% Cl Of >na nd >na OR Quart2 113 nd na OR Quart 3 >0 nd >0 p Value <na nd <na 95% Cl Of >na nd >na OR Quart3 na nd na OR Quart 4 >22 nd >2.2 p Value <0.53 nd <0.52 95% CI of >019 nd >0.19 OR Quart4 na nd na Extracellular matrix protein 1 24hr prior to AKI stage Cohort 1 Cohort 2 2012/066152 24hr prior to AKI stage sCr or UO sCr only UO only 0.23 nd 0.25 0.20 nd 0.20 0.18 nd 0.23 128 nd 112 2 nd 2 779 nd 779 100% nd 100% 2% nd 3% 779 nd 779 100% nd 100% 2% nd 3% 779 nd 779 100% nd 100% 2% nd 3% 1720 nd 1720 0% nd 0% 70% nd 71% 1950 nd 1910 0% nd 0% 81% nd 80% 2240 nd 2230 0% nd 0% 91% nd 90% >0 nd >0 <na nd <na 95% CI Of >na nd >na OR Quart2 113 nd na OR Quart 3 >10 nd >1.0 p Value <0.98 nd <0.98 95% CI of >0.062 nd >0.062 OR Quart3 na nd na OR Quart4>1.1 nd >1.1 p Value <0.97 nd <0.96 95% CI of >0.064 nd >0.064 OR Quart4 na nd na Vitronectin sCr or UO 24hr prior to AKI stage WO 78253 Min 57400 Max 205000 A 0400 n (Samp) 112 2 24hr prior to AKI stage sCr or UO sCr only UO only AUC 0 nd 0 SE <0.001 nd <0.001 p <1.0E—5 nd <1.0E—5 nCohort 1 128 nd 112 nCohort 2 2 nd 2 Cutoff 1 0 nd 0 Sens 1 100% nd 100% Spec 1 0% nd 0% Cutoff 2 0 nd 0 Sens 2 100% nd 100% Spec 2 0% nd 0% Cutoff 3 0 nd 0 Sens 3 100% nd 100% Spec 3 0% nd 0% Cutoff 4 133000 nd 132000 Sens 4 0% nd 0% Spec 4 70% nd 71% Cutoff 5 150000 nd 149000 Sens 5 0% nd 0% Spec 5 80% nd 80% Cutoff 6 172000 nd 167000 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 >0 nd >0 p Value <na nd <na 95% Cl Of >na nd >na OR Quart2 na nd na OR Quart 3 >0 nd >0 p Value <na nd <na 95% Cl Of >na nd >na OR Quart3 na nd na OR Quart 4 >22 nd >2.2 p Value <0.53 nd <0.52 95% CI of >019 nd >0.19 OR Quart4 na nd na Interleukin-22 2012/066152 1 stage Cohort 2 0hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only sCr or UO sCr only UO only AUC 0.35 nd 0.33 0.54 nd 0.53 SE 0.12 nd 0.12 0.11 nd 0.11 p 0.24 nd 0.18 0.71 nd 0.81 0.413 0.274 0.195 100% Cutoff4 0.881 nd 0.893 0.881 nd 0.893 Sens 4 17% nd 17% 62% nd 62% Spec4 71% nd 71% 71% nd 71% Cutoff 5 1.07 nd 1.07 1.07 nd 1.07 Sens 5 17% nd 17% 25% nd 25% Spec 5 80% nd 80% 80% nd 80% Cutoff 6 1.52 nd 1.60 1.52 nd 1.60 Sens 6 0% nd 0% 12% nd 12% Spec6 91% nd 91% 91% nd 91% OR Quart 2 1.0 nd 1.0 0 nd 0 p Value 0.98 nd 1.0 na nd na 0hr prior to AKI stage 24hr prior to AKI stage sCr only sCr only 95% CI of OR Quart2 OR Quart 3 p Value 95% CI of OR Quart3 OR Quart 4 . p Value 95% CI of 0.18 nd 0.18 0.19 nd OR Quart4 25 nd 24 5.4 nd 24hr prior to AKI stage sCr or UO sCr only UO only 0.99 nd 0.99 0.044 nd 0.048 <1.0E—5 nd <1.0E—5 126 nd 110 2 nd 2 .8 nd 10.8 100% nd 100% 99% nd 99% .8 nd 10.8 100% nd 100% 99% nd 99% 2012/066152 24hr prior to AKI stage sCr or UO sCr only UO only Cutoff 3 10.8 nd 10.8 Sens 3 100% nd 100% Spec 3 99% nd 99% Cutoff 4 1.96 nd 2.01 Sens 4 100% nd 100% Spec 4 71% nd 70% Cutoff 5 2.49 nd 2.94 Sens 5 100% nd 100% Spec 5 80% nd 80% Cutoff 6 4.07 nd 4.47 Sens 6 100% nd 100% Spec 6 90% nd 90% OR Quart 2 >0 nd >0 p Value <na nd <na 95% CI of >na nd >na OR Quart2 11a nd na OR Quart 3 >0 nd >0 p Value <na nd <na 95% CI of >na nd >na OR Quart3 na nd na OR Quart 4 >21 nd >2.2 p Value <0.54 nd <0.54 95% CI of >018 nd >0.18 OR Quart4 na nd na Progesterone 2012/066152 24hr prior to AKI stage sCr or UO sCr only UO only 0.99 nd 0.99 0.054 nd 0.048 <1.0E—5 nd <1.0E—5 126 nd 110 2 nd 2 31.4 nd 31.4 100% nd 100% 98% nd 98% 31.4 nd 31.4 100% nd 100% 98% nd 98% 31.4 nd 31.4 100% nd 100% 98% nd 98% 6.56 nd 6.51 100% nd 100% 71% nd 70% 8.03 nd 8.03 100% nd 100% 80% nd 80% .4 nd 10.4 100% nd 100% 90% nd 90% >0 nd >0 <na nd <na 95% CI Of >na nd >na OR Quart2 11a nd na OR Quart 3 >0 nd >0 p Value <na nd <na 95% CI Of >na nd >na OR Quart3 na nd na OR Quart 4 >21 nd >2.2 p Value <0.54 nd <0.54 95% CI of >018 nd >0.18 OR Quart4 na nd na —24hrprior to AKI stage Cohort 1 Cohort 2 Median 2.57 0.858 Average 3.10 0.858 StdeV 2.71 0. 801 p _ 0.25 0.000136 0.291 Max 22.2 1.42 11 (Samp) __ 2012/066152 24hr prior to AKI stage sCr or UO sCr only UO only 0.16 nd 0.17 0.18 nd 0.18 0.052 nd 0.064 126 nd 110 2 nd 2 0.256 nd 0.256 100% nd 100% 7% nd 8% 0.256 nd 0.256 100% nd 100% 7% nd 8% 0.256 nd 0.256 100% nd 100% 7% nd 8% 3.68 nd 3.39 0% nd 0% 71% nd 70% 4.47 nd 4.31 0% nd 0% 80% nd 80% 6.18 nd 5.44 0% nd 0% 90% nd 90% >0 nd >0 <na nd <na 95% CI Of >na nd >na OR Quart2 11a nd na OR Quart 3 >10 nd >1.0 p Value <0.98 nd <0.98 95% CI of >0.062 nd >0.062 OR Quart3 na nd na OR Quart4>1.0 nd >1.0 24hr prior to AKI stage sCr or UO sCr only UO only p Value <0.98 nd <0.98 95% CI of >0.062 nd >0.062 OR Quart4 na nd na Growth/differentiation factor 15 24hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only 1.0 nd 1.0 0 nd 0 nd <1.0E—5 126 nd 110 2 nd 2 7790 nd 7790 100% nd 100% 100% nd 100% 7790 nd 7790 100% nd 100% 100% nd 100% 7790 nd 7790 100% nd 100% 100% nd 100% 2840 nd 3100 100% nd 100% 71% nd 70% 3750 nd 3880 WO 78253 24hr prior to AKI stage sCr or UO sCr only UO only 100% nd 100% 80% nd 80% 5190 nd 5190 100% nd 100% 90% nd 90% >0 nd >0 <na nd <na 95% Cl Of >na nd >na OR Quart2 11a nd na OR Quart 3 >0 nd >0 p Value <na nd <na 95% CI of >na nd >na OR Quart3 na nd na OR Quart 4 >2.1 nd >2.2 p Value <0.54 nd <0.54 95% CI of >018 nd >0.18 OR Quart4 na nd na Proprotein convertase subtilisin/kexin type 9 24hr prior to AKI stage 24hr prior to AKI stage sCr or UO sCr only UO only 0.074 nd 0.080 0.13 nd 0.13 9.8E—4 nd 0.0017 128 nd 112 24hr prior to AKI stage sCr or UO sCr only UO only nCohort 2 2 nd 2 Cutoff 1 76300 nd 76300 Sens l 100% nd 100% Spec 1 1% nd 1% Cutoff 2 76300 nd 76300 Sens 2 100% nd 100% Spec 2 1% nd 1% Cutoff 3 76300 nd 76300 Sens 3 100% nd 100% Spec 3 1% nd 1% Cutoff 4 598000 nd 598000 Sens 4 0% nd 0% Spec 4 70% nd 71% Cutoff 5 676000 nd 676000 Sens 5 0% nd 0% Spec 5 80% nd 80% Cutoff 6 820000 nd 814000 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 >0 nd >0 p Value <na nd <na 95% CI Of >na nd >na OR Quart2 na nd na OR Quart 3 >0 nd >0 p Value <na nd <na 95% CI Of >na nd >na OR Quart3 na nd na OR Quart 4 >22 nd >2.2 p Value <0.53 nd <0.52 95% CI of >019 nd >0.19 OR Quart4 na nd na Table 11: ison of marker levels in enroll urine samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0 or R within 48hrs) and in enroll urine samples collected from Cohort 2 (subjects reaching RIFLE stage I or F within 48hrs). Enroll samples from patients already at RIFLE stage I or F were included in Cohort 2.

Antithrombin-III UO only Cohort 2 Cohort 1 Cohort 2 172 89.5 164 707 322 97s StdeV 1630 1680 935 1730 —_0347 . 14.7 0.0182 0.347 UO only Cohort 2 Cohort 1 Cohort 2 6000 6000 6000 12 188 47 12 188 47 At Enrollment sCr or UO sCr only UO only AUC 0.65 0.63 0.63 SE 0.045 0.088 0.048 p 8.7E—4 0.13 0.0064 nCohort 1 190 227 188 t 2 54 12 47 Cutoff 1 89.6 114 84.9 Sens 1 70% 75% 70% Spec 1 52% 56% 48% Cutoff 2 56.5 56.5 54.2 Sens 2 81% 83% 81% Spec 2 35% 32% 31% Cutoff 3 42.4 54.2 21.8 Sens 3 91% 92% 91% Spec 3 24% 30% 13% Cutoff 4 152 172 158 Sens 4 57% 50% 51% Spec 4 70% 70% 70% Cutoff 5 223 280 231 Sens 5 37% 33% 36% Spec 5 80% 80% 80% Cutoff 6 435 600 472 Sens 6 24% 17% 28% Spec 6 90% 90% 90% OR Quart 2 2.5 2.0 2.2 p Value 0.087 0.58 0.14 95% CI of 0.88 0.18 0.77 OR Quart2 7.0 23 6.4 OR Quart 3 2.7 4.1 2.0 p Value 0.057 0.21 0.21 95% CI of 0.97 0.45 0.68 OR Quart3 7.7 38 5.8 OR Quart4 4.8 5.3 3.8 p Value 0.0020 0.13 0.0096 95% CI of 1.8 0.60 1.4 OR Quart4 13 47 10 Extracellular matrix protein 1 sCr or UO sCr only UO only Cohort 1 Cohort 2 Cohort 1 Cohort 2 Cohort 1 Cohort 2 Median 0.346 1.26 0.392 1.26 0.371 1.12 ——3.58 3.19 0.923 3.64 UO only Cohort 2 Cohort 1 Cohort 2 .16 1.59 8.75 0.14 7.9E—5 0.00419 0.000528 0.00419 14.4 14.4 57.7 12 188 47 12 188 47 At ment sCr or UO sCr only UO only AUC 0.69 0.67 0.66 SE 0.043 0.088 0.047 p 1.2E—5 0.055 4.6E—4 nCohort 1 190 227 188 nCohort 2 54 12 47 Cutoff 1 0.411 0.689 0.396 Sens 1 70% 75% 70% Spec 1 56% 61% 54% Cutoff 2 0.238 0.362 0.182 Sens 2 81% 83% 81% Spec 2 41% 47% 36% Cutoff 3 0.0936 0.348 0.0837 Sens 3 91% 92% 91% Spec 3 19% 47% 18% Cutoff4 0.857 1.08 0.995 Sens 4 56% 58% 51% Spec 4 70% 70% 70% Cutoff 5 1.28 1.82 1.48 Sens 5 50% 25% 45% Spec 5 80% 80% 80% Cutoff 6 2.15 2.82 2.21 Sens 6 35% 17% 38% Spec 6 90% 90% 90% OR Quart 2 1.3 2.0 1.3 p Value 0.59 0.58 0.62 95% CI of 0.46 0.18 0.45 OR QuartZ 3.8 23 3.8 OR Quart 3 1.9 3.1 1.5 p Value 0.22 0.34 0.46 95% CI of 0.69 0.31 0.52 OR Quart3 5.2 30 4.2 OR Quart 4 5.7 6.4 4.0 p Value 2.6E—4 0.089 0.0042 95% CI of 2.2 0.75 1.6 OR Quart4 15 55 10 Coagulation factor XIII A and B chains —— uomy Cohort 1 Cohort 2 Cohort 2 Cohort 1 Cohort 2 ——15.5 . 16.7 6.13 17.1 44.7 8.20 31.3 0.062 2.9E—5 0.000455 0.000120 0.000145 158 58.0 158 12 188 47 12 188 47 At Enrollment sCr or UO sCr only UO only AUC 0.61 0.49 0.62 SE 0.045 0.086 0.048 p 0.014 0.88 0.014 nCohort 1 190 227 188 nCohort 2 54 12 47 Cutoff1 2.52 1.89 2.73 Sens 1 70% 75% 70% Spec 1 45% 34% 47% Cutoff 2 0.976 0.956 0.956 Sens 2 81% 83% 81% Spec 2 23% 21% 22% Cutoff 3 0.000443 0.701 27 Sens 3 91% 92% 91% Spec 3 5% 19% 4% Cutoff4 6.03 7.34 5.99 Sens 4 46% 25% 51% Spec 4 70% 70% 70% Cutoff5 8.96 11.3 8.96 Sens 5 37% 17% 40% Spec 5 80% 80% 80% Cutoff 6 18.1 20.9 18.1 Sens 6 24% 8% 28% Spec 6 90% 90% 90% OR Quart 2 0.69 2.1 0.40 p Value 0.46 0.41 0.11 95% CI of 0.26 0.36 0.13 OR Quart2 1.8 12 1.2 OR Quart 3 1.5 1.5 1.1 p Value 0.38 0.65 0.85 95% CI of 0.62 0.25 0.44 OR Quart3 3.6 9.5 2.7 OR Quart 4 2.2 1.6 2.0 p Value 0.064 0.64 0.10 95% CI of 0.95 0.25 0.86 2012/066152 At Enrollment sCroon OR Quart4 5.2 9.7 4.8 Vitronectin UO only Cohort 2 Cohort 1 Cohort 2 29.5 23.0 49.4 89.7 48.4 118 177 93.4 162 0.38 1.3E—4 2.03E—5 0.550 2.03E—5 641 750 641 12 188 47 12 188 47 At Enrollment sCr or UO sCr only UO only AUC 0.66 0.52 0.68 SE 0.044 0.087 0.046 p 1.9E—4 0.78 7.8E—5 nCohort 1 190 227 188 nCohort 2 54 12 47 Cutoff 1 24.8 14.1 28.4 Sens 1 70% 75% 70% Spec 1 54% 30% 60% Cutoff 2 16.0 8.80 18.5 Sens 2 81% 83% 81% Spec 2 37% 19% 44% Cutoff 3 7.43 6.91 7.43 Sens 3 91% 92% 91% Spec 3 19% 15% 19% Cutoff4 37.7 45.7 37.7 Sens 4 54% 33% 57% Spec 4 70% 70% 70% Cutoff 5 50.6 64.3 53.9 Sens 5 46% 33% 47% Spec 5 80% 80% 80% Cutoff 6 98.1 138 98.1 Sens 6 28% 8% 32% Spec 6 90% 90% 90% OR Quart 2 1.3 0.98 1.4 p Value 0.61 0.98 0.59 95% CI of 0.47 0.19 0.44 OR QuartZ 3.6 5.1 4.2 OR Quart 3 1.6 0.64 2.0 p Value 0.33 0.64 0.21 95% CI of 0.61 0.10 0.68 OR Quart3 4.3 4.0 5.8 At Enrollment sCr or UO sCr only UO only OR Quart 4 4.3 1.3 5.2 p Value 0.0016 0.71 0.0013 95% CI of 1.7 0.29 1.9 OR Quart4 11 6.2 14 UO only Cohort 2 Cohort 1 Cohort 2 2.60 3.37 3.41 3.40 4.67 3.96 1.99 3.90 2.60 0.61 0.64 1.94 0.569 0.792 .67 19.3 8.37 3 42 7 3 42 7 At Enrollment sCr or UO sCr only UO only AUC 0.53 0.44 0.49 SE 0.11 0.18 0.12 p 0.77 0.72 0.91 t 1 50 55 42 nCohort 2 8 3 7 Cutoff1 2.59 1.87 2.59 Sens 1 75% 100% 71% Spec 1 38% 25% 33% Cutoff 2 1.87 1.87 1.78 Sens 2 88% 100% 86% Spec 2 26% 25% 21% Cutoff 3 0.757 1.87 0.757 Sens 3 100% 100% 100% Spec 3 8% 25% 7% Cutoff4 5.32 5.68 5.68 Sens 4 38% 0% 29% Spec 4 70% 71% 71% Cutoff 5 6.52 6.52 6.56 Sens 5 12% 0% 14% Spec 5 80% 80% 81% Cutoff 6 7.57 8.37 7.57 Sens 6 12% 0% 14% Spec 6 90% 91% 90% OR Quart 2 2.0 >1.2 1.1 p Value 0.59 <0.92 0.93 95% CI of 0.16 >0.065 0.13 OR Quart2 25 na 9.3 OR Quart 3 3.5 >2.3 0.50 At ment sCr or UO sCr only UO only p Value 0.30 <0.51 0.59 95% CI of 0.32 >0.19 0.039 OR Quart3 39 na 6.4 OR Quart 4 2.0 >0 1.1 p Value 0.59 <na 0.93 95% CI of 0.16 >na 0.13 OR Quart4 25 na 9.3 UO only Cohort 2 Cohort 1 Cohort 2 1.20 4.21 3.02 2.11 5.50 4.17 2.27 5.20 3.26 0.27 0.52 0.443 0.00501 0.443 4.69 18.8 10.1 3 42 7 3 42 7 At Enrollment sCr or UO sCr only UO only AUC 0.44 0.33 0.47 SE 0.11 0.18 0.12 p 0.60 0.34 0.78 nCohort 1 50 55 42 nCohort 2 8 3 7 Cutoff 1 1.20 0.428 2.39 Sens 1 75% 100% 71% Spec 1 26% 18% 33% Cutoff 2 1.01 0.428 1.20 Sens 2 88% 100% 86% Spec 2 26% 18% 29% Cutoff 3 0.428 0.428 0.428 Sens 3 100% 100% 100% Spec 3 20% 18% 21% Cutoff4 5.62 6.00 6.52 Sens 4 25% 0% 14% Spec 4 70% 71% 71% Cutoff 5 8.06 8.06 8.89 Sens 5 12% 0% 14% Spec 5 80% 80% 81% Cutoff 6 12.8 12.8 13.5 Sens 6 0% 0% 0% Spec 6 90% 91% 90% OR Quart 2 0.50 >1.2 2.4 p Value 0.59 <0.92 0.50 At Enrollment sCr or UO sCr only UO only 95% CI of 0.040 >0.065 0.19 OR Quart2 6.2 na 31 OR Quart 3 2.4 >1.1 4.0 p Value 0.37 <0.96 0.26 95% CI of 0.36 >0.061 0.35 OR Quart3 15 na 45 OR Quart 4 0.50 >1.2 1.1 p Value 0.59 <0.92 0.95 95% CI of 0.040 >0.065 0.061 OR Quart4 6.2 na 20 Proprotein convertase isin/kexin type 9 U0 only Cohort 2 Cohort 1 Cohort 2 3180 379 1430 2420 1100 1900 1390 1980 1220 0.37 0.31 813 70.6 521 3260 11000 3260 3 42 7 3 42 7 At Enrollment sCr or UO sCr only UO only AUC 0.80 0.84 0.81 SE 0.097 0.15 0.10 p 0.0019 0.022 0.0023 nCohort 1 50 55 42 nCohort 2 8 3 7 Cutoff 1 897 804 919 Sens 1 75% 100% 71% Spec 1 76% 69% 76% Cutoff 2 804 804 897 Sens 2 88% 100% 86% Spec 2 74% 69% 76% Cutoff 3 479 804 479 Sens 3 100% 100% 100% Spec 3 60% 69% 60% Cutoff 4 686 897 745 Sens 4 88% 67% 86% Spec 4 70% 71% 71% Cutoff 5 1260 1300 1260 Sens 5 50% 67% 57% Spec 5 80% 80% 81% Cutoff 6 3040 3080 3040 Sens 6 38% 67% 43% WO 78253 At Enrollment sCr or UO sCr only UO only Spec 6 90% 91% 90% OR Quart 2 >0 >0 >0 p Value <na <na <na 95% C1 Of >na >na >na OR Quart2 na na na OR Quart 3 >5.6 >1.1 >4.0 p Value <0.15 <0.96 <0.26 95% CI of >054 >0.061 >0.35 OR Quart3 na na na OR Quart 4 >5.1 >22 >53 p Value <0.17 <0.55 <0.16 95% CI of >050 >0.17 >0.51 OR Quart4 na na na Table 12: Comparison of marker levels in enroll EDTA samples collected from Cohort 1 (patients that did not progress beyond RIFLE stage 0 or R within 48hrs) and in enroll EDTA samples collected from Cohort 2 (subjects reaching RIFLE stage I or F within 48hrs). Enroll samples from patients already at stage I or F were included in Cohort 2.

Antithrombin-III UO only Cohort 2 Cohort 1 Cohort 2 nd 105000 86000 nd 110000 87900 nd 35300 34600 nd 0.085 nd 36200 32000 nd 186000 146000 nd 41 10 nd 41 10 At Enrollment sCr or UO sCr only UO only 0.32 nd 0.33 0.10 nd 0.10 p 0.072 nd 0.094 t 1 47 nd 41 nCohort 2 10 nd 10 Cutoff 1 81300 nd 81300 Sens 1 70% nd 70% Spec 1 23% nd 24% Cutoff 2 49300 nd 49300 Sens 2 80% nd 80% Spec 2 2% nd 2% Cutoff 3 36200 nd 36200 Sens 3 90% nd 90% At Enrollment sCr or UO sCr only UO only Spec 3 2% nd 2% Cutoff 4 120000 nd 117000 Sens 4 20% nd 20% Spec 4 70% nd 71% Cutoff 5 147000 nd 145000 Sens 5 0% nd 10% Spec 5 81% nd 80% Cutoff 6 164000 nd 163000 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 0.50 nd 0.46 p Value 0.59 nd 0.55 95% CI of 0.040 nd 0.036 OR Quart2 6.2 nd 5.8 OR Quart 3 2.6 nd 2.4 p Value 0.32 nd 0.36 95% CI of 0.39 nd 0.36 OR Quart3 17 nd 17 OR Quart4 1.8 nd 1.8 p Value 0.57 nd 0.55 95% CI of 0.25 nd 0.25 OR Quart4 13 nd 13 Extracellular matrix protein 1 —— uomy Cohort 1 Cohort 2 Cohort 2 Cohort 1 Cohort 2 nd woo woo 341 nd 470 341 nd 1.00 nd 535 945 nd 2570 1990 nd 41 10 nd 41 10 At Enrollment sCr or UO sCr only UO only AUC 0.51 nd 0.52 SE 0.10 nd 0.10 p 0.95 nd 0.85 t 1 47 nd 41 nCohort 2 10 nd 10 Cutoff1 1310 nd 1310 Sens 1 70% nd 70% Spec 1 43% nd 46% Cutoff2 1170 nd 1170 Sens 2 80% nd 80% At Enrollment sCr or UO sCr only UO only Spec 2 23% nd 24% Cutoff3 1110 nd 1110 Sens 3 90% nd 90% Spec 3 21% nd 22% Cutoff4 1710 nd 1710 Sens 4 40% nd 40% Spec 4 70% nd 71% Cutoff 5 1950 nd 1840 Sens 5 10% nd 20% Spec 5 81% nd 80% Cutoff 6 2240 nd 2150 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 0.28 nd 0.91 p Value 0.30 nd 0.93 95% CI of 0.026 nd 0.11 OR QuartZ 3.1 nd 7.7 OR Quart 3 1.0 nd 1.5 p Value 1.0 nd 0.69 95% CI of 0.16 nd 0.20 OR Quart3 6.1 nd 11 OR Quart 4 0.92 nd 1.5 p Value 0.92 nd 0.69 95% CI of 0.15 nd 0.20 OR Quart4 5.5 nd 11 ation factor XIII A and B chains UO only Cohort 2 Cohort 1 Cohort 2 nd 11200 10000 nd 11800 11400 nd 6580 5310 nd 0.84 nd 881 3550 nd 33300 21300 nd 41 10 nd 41 10 At Enrollment sCr or UO sCr only UO only 0.46 nd 0.49 0.10 nd 0.10 0.69 nd 0.89 47 nd 41 nd 10 9230 nd 9230 70% nd 70% At ment sCr or UO sCr only UO only Spec 1 38% nd 41% Cutoff 2 7990 nd 7990 Sens 2 80% nd 80% Spec 2 28% nd 29% Cutoff 3 7150 nd 7150 Sens 3 90% nd 90% Spec 3 23% nd 24% Cutoff 4 14000 nd 13500 Sens 4 20% nd 20% Spec 4 70% nd 71% Cutoff 5 16500 nd 16200 Sens 5 20% nd 20% Spec 5 81% nd 80% Cutoff 6 22500 nd 19800 Sens 6 0% nd 10% Spec 6 91% nd 90% OR Quart 2 0.50 nd 1.0 p Value 0.59 nd 1.0 95% CI of 0.040 nd 0.12 OR Quart2 6.2 nd 8.4 OR Quart 3 3.6 nd 2.4 p Value 0.17 nd 0.36 95% CI of 0.57 nd 0.36 OR Quart3 23 nd 17 ORQuart4 1.1 nd 1.1 p Value 0.94 nd 0.93 95% CI of 0.13 nd 0.13 OR Quart4 8.9 nd 9.3 Vitronectin UO only Cohort 2 Cohort 1 Cohort 2 00 002000 07000 Average 114000 83300 nd 112000 83300 p(t—test) . nd 0.018 Min nd 57400 21800 Max nd 177000 153000 11 (Samp) nd 41 10 11 (Patient) nd 41 10 At Enrollment sCr or UO sCr only UO only 0.27 nd 0.29 0.097 nd 0.099 0.020 nd 0.030 47 nd 41 At Enrollment sCr or UO sCr only UO only t 2 10 nd 10 Cutoff 1 60700 nd 60700 Sens 1 70% nd 70% Spec 1 4% nd 5% Cutoff 2 60500 nd 60500 Sens 2 80% nd 80% Spec 2 4% nd 5% Cutoff 3 21800 nd 21800 Sens 3 90% nd 90% Spec 3 0% nd 0% Cutoff 4 125000 nd 123000 Sens 4 10% nd 10% Spec 4 70% nd 71% Cutoff 5 145000 nd 143000 Sens 5 10% nd 10% Spec 5 81% nd 80% Cutoff 6 158000 nd 154000 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 2.3 nd 2.2 p Value 0.51 nd 0.55 95% CI of 0.19 nd 0.17 OR Q11th 29 nd 28 OR Quart 3 2.3 nd 3.6 p Value 0.51 nd 0.30 95% CI of 0.19 nd 0.32 OR Quart3 29 nd 40 OR Quart 4 7.8 nd 6.0 p Value 0.081 nd 0.14 95% CI of 0.78 nd 0.56 OR Quart4 78 nd 64 U0 only Cohort 2 Cohort 1 Cohort 2 nd 0.946 0.578 nd 1.01 0.674 nd 0.650 0.531 nd 0.14 nd 0.000227 0.000162 nd 4.25 1.59 nd 41 10 nd 41 10 At Enrollment sCr or UO sCr only UO only At Enrollment sCr or UO sCr only UO only SE 0.099 nd 0.10 p 0.039 nd 0.046 nCohort 1 47 nd 41 t 2 10 nd 10 Cutoff 1 0.500 nd 0.500 Sens 1 70% nd 70% Spec 1 15% nd 15% Cutoff 2 0.424 nd 0.424 Sens 2 80% nd 80% Spec 2 11% nd 10% Cutoff 3 0 nd 0 Sens 3 100% nd 100% Spec 3 0% nd 0% Cutoff4 1.18 nd 1.17 Sens 4 20% nd 20% Spec 4 70% nd 71% Cutoff 5 1.32 nd 1.27 Sens 5 20% nd 20% Spec 5 81% nd 80% Cutoff 6 1.60 nd 1.54 Sens 6 0% nd 10% Spec 6 91% nd 90% OR Quart 2 0.50 nd 0.46 p Value 0.59 nd 0.55 95% CI of 0.040 nd 0.036 OR Quart2 6.2 nd 5.8 OR Quart 3 0 nd 0.46 p Value na nd 0.55 95% CI of na nd 0.036 OR Quart3 na nd 5.8 OR Quart 4 6.5 nd 5.5 p Value 0.044 nd 0.076 95% CI of 1.1 nd 0.84 OR Quart4 40 nd 36 UO only Cohort 2 Cohort 1 Cohort 2 nd 2.35 0.996 nd 2.68 1.46 nd 1.92 1.62 nd 0.070 nd 0.0578 0.000136 nd 7.82 4.15 A 7 10 nd 41 10 nd 41 10 At Enrollment sCr or UO sCr only UO only AUC 0.29 nd 0.30 SE 0.099 nd 0.10 p 0.034 nd 0.052 nCohort 1 47 nd 41 nCohort 2 10 nd 10 1 0.196 nd 0.196 Sens 1 70% nd 70% Spec 1 6% nd 7% Cutoff 2 0.000136 nd 0.000136 Sens 2 90% nd 90% Spec 2 0% nd 0% Cutoff 3 0.000136 nd 0.000136 Sens 3 90% nd 90% Spec 3 0% nd 0% Cutoff 4 3.39 nd 3.32 Sens 4 20% nd 20% Spec 4 70% nd 71% Cutoff 5 4.35 nd 3.97 Sens 5 0% nd 10% Spec 5 81% nd 80% Cutoff 6 6.06 nd 5.44 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 0.50 nd 0.46 p Value 0.59 nd 0.55 95% CI of 0.040 nd 0.036 OR QuartZ 6.2 nd 5.8 OR Quart 3 1.1 nd 1.0 p Value 0.94 nd 1.0 95% CI of 0.13 nd 0.12 OR Quart3 8.9 nd 8.4 OR Quart 4 3.6 nd 3.9 p Value 0.17 nd 0.16 95% CI of 0.57 nd 0.59 OR Q1181rt4 23 nd 26 Proprotein convertase subtilisin/kexin type 9 U0 only Cohort 2 Cohort 1 Cohort 2 nd 443000 261000 nd 481000 265000 nd 171000 159000 nd 6.7E—4 nd 182000 80700 nd 858000 596000 nd 41 10 nd 41 10 2012/066152 At ment sCr or UO sCr only UO only AUC 0.17 nd 0.17 SE 0.083 nd 0.084 p 5.3E—5 nd 7.2E—5 nCohort 1 47 nd 41 nCohort 2 10 nd 10 Cutoff 1 123000 nd 123000 Sens 1 70% nd 70% Spec 1 0% nd 0% Cutoff 2 102000 nd 102000 Sens 2 80% nd 80% Spec 2 0% nd 0% Cutoff 3 80700 nd 80700 Sens 3 90% nd 90% Spec 3 0% nd 0% Cutoff 4 538000 nd 580000 Sens 4 10% nd 10% Spec 4 70% nd 71% Cutoff 5 654000 nd 667000 Sens 5 0% nd 0% Spec 5 81% nd 80% Cutoff 6 699000 nd 699000 Sens 6 0% nd 0% Spec 6 91% nd 90% OR Quart 2 0 nd 0 p Value na nd na 95% CI of na nd na OR Quart2 na nd na OR Quart 3 2.3 nd 2.2 p Value 0.51 nd 0.55 95% CI of 0.19 nd 0.17 OR Quart3 29 nd 28 OR Quart 4 14 nd 17 p Value 0.023 nd 0.018 95% CI of 1.4 nd 1.6 OR Quart4 140 nd 170 While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

Modifications therein and other uses will occur to those d in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

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

All patents and publications ned in the specification are tive of the levels of those of ordinary skill in the art to which the ion pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically 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 which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, sting essentially of” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the es shown and described or portions thereof, but it is recognized that s 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 ion 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 set forth within the following claims.

Claims (119)

We claim:

1. A method for evaluating renal status in a subject, sing: performing one or more assays configured to detect Growth/differentiation factor 15 in a body fluid sample obtained from the subject to provide an assay ; correlating the assay result(s) to the renal status of the subject, wherein said ation step comprises correlating the assay (s) to one or more of diagnosis, risk stratification, prognosis, classifying and monitoring of the renal status of the subject.

2. A method according to claim 1, wherein said correlation step ses correlating the assay result(s) to prognosis of the renal status of the subject.

3. A method according to claim 1, wherein said correlating step comprises assigning a likelihood of one or more future changes in renal status to the subject based on the assay result(s).

4. A method according to claim 3, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal on, and future acute renal failure (ARF).

5. A method according to one of claims 1-4, wherein said assay results further comprise at least 2, 3, 4, or 5 of: a measured concentration of Stanniocalcin-1, a measured concentration of Antithrombin-III, a measured concentration of Toll-like or 2, a measured concentration of Triiodothyronine (T3), a measured concentration of Thyroxine (T4), a measured concentration of Extracellular matrix protein 1, a measured concentration of Coagulation factor XIII A chain, a measured concentration of Coagulation factor XIII B chain, a measured concentration of Interleukin-17F, a ed concentration of Interleukin-22, a ed concentration of Vitronectin, a measured concentration of Progesterone, Estradiol, and a measured concentration of Proprotein convertase subtilisin/kexin type 9.

6. A method according to one of claims 1-5, wherein a plurality of assay s are combined using a function that converts the plurality of assay results into a single ite result.

7. A method according to claim 3, wherein said one or more future changes in renal status comprise a clinical outcome related to a renal injury suffered by the subject.

8. A method according to claim 3, wherein the likelihood of one or more future changes in renal status is that an event of interest is more or less likely to occur within 30 days of the time at which the body fluid sample was obtained from the subject.

9. A method according to claim 8, wherein the likelihood of one or more future changes in renal status is that an event of interest is more or less likely to occur within a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours.

10. A method according to one of claims 1-5, wherein the subject is selected for tion of renal status based on the pre-existence in the t of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF.

11. A method according to one of claims 1-5, n the subject is ed for evaluation of renal status based on an existing diagnosis of one or more of 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, sepsis, injury to renal function, d renal function, or ARF, or based on undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery, or based on re to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin.

12. A method according to one of claims 1-5, wherein said correlating step comprises assessing r or not renal on is improving or worsening in a subject who has suffered from an injury to renal function, reduced renal function, or ARF based on the assay result(s).

13. A method according to one of claims 1-5, wherein said method is a method of ing a risk of the future occurrence or nonoccurrence of an injury to renal function in said subject.

14. A method according to one of claims 1-5, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of d renal function in said subject.

15. A method according to one of claims 1-5, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of a need for dialysis in said subject.

16. A method according to one of claims 1-5, wherein said method is a method of ing a risk of the future occurrence or nonoccurrence of acute renal failure in said subject.

17. A method according to one of claims 1-5, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of a need for renal ement therapy in said subject.

18. A method according to one of claims 1-5, wherein said method is a method of assigning a risk of the future occurrence or nonoccurrence of a need for renal transplantation in said t.

19. A method according to one of claims 1-5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 72 hours of the time at which the body fluid sample was ed.

20. A method according to one of claims 1-5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal on, and future acute renal failure (ARF) within 48 hours of the time at which the body fluid sample was obtained.

21. A method ing to one of claims 1-5, wherein said one or more future changes in renal status comprise one or more of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 24 hours of the time at which the body fluid sample was obtained.

22. A method according to one of claims 1-5, wherein the subject is in RIFLE stage 0 or R.

23. A method according to claim 22, wherein the subject is in RIFLE stage 0, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage R, I or F within 72 hours.

24. A method according to claim 23, wherein the subject is in RIFLE stage 0, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 72 hours.

25. A method according to claim 23, wherein the subject is in RIFLE stage 0, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 72 hours.

26. A method according to claim 22, wherein the t is in RIFLE stage 0 or R, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 72 hours.

27. A method ing to claim 26, n the subject is in RIFLE stage 0 or R, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 72 hours.

28. A method according to claim 22, wherein the subject is in RIFLE stage R, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 72 hours.

29. A method according to claim 28, wherein the subject is in RIFLE stage R, and said correlating step comprises assigning a hood that the subject will reach RIFLE stage F within 72 hours.

30. A method according to one of claims 1-5, wherein the subject is in RIFLE stage 0, R, or I, and said correlating step comprises ing a likelihood that the subject will reach RIFLE stage F within 72 hours.

31. A method according to claim 30, wherein the subject is in RIFLE stage I, and said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 72 hours.

32. A method according to claim 23, wherein said ating step comprises assigning a likelihood that the subject will reach RIFLE stage R, I or F within 48 hours.

33. A method according to claim 24, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 48 hours.

34. A method ing to claim 25, n said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 48 hours.

35. A method according to claim 26, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 48 hours.

36. A method according to claim 27, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 48 hours.

37. A method according to claim 28, n said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 48 hours.

38. A method according to claim 29, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 48 hours.

39. A method according to claim 30, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 48 hours.

40. A method according to claim 31, wherein said correlating step comprises assigning a likelihood that the t will reach RIFLE stage F within 48 hours.

41. A method according to claim 23, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage R, I or F within 24 hours.

42. A method according to claim 24, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 24 hours.

43. A method according to claim 25, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 24 hours.

44. A method according to claim 26, wherein said ating step comprises ing a likelihood that the t will reach RIFLE stage I or F within 24 hours.

45. A method according to claim 27, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 24 hours.

46. A method according to claim 28, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage I or F within 24 hours.

47. A method according to claim 29, wherein said ating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 24 hours.

48. A method according to claim 30, wherein said ating step comprises ing a likelihood that the subject will reach RIFLE stage F within 24 hours.

49. A method according to claim 31, wherein said correlating step comprises assigning a likelihood that the subject will reach RIFLE stage F within 24 hours.

50. A method according to one of claims 1-5, wherein the subject is not in acute renal failure.

51. A method according to one of claims 1-5, n the subject has not experienced a ld or greater increase in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

52. A method according to one of claims 1-5, wherein the subject has a urine output of at least 0.5 ml/kg/hr over the 6 hours ing the time at which the body fluid sample was obtained.

53. A method according to one of claims 1-5, wherein the subject has not enced an se of 0.3 mg/dL or greater in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

54. A method ing to one of claims 1-5, wherein the subject (i) has not experienced a 1.5-fold or greater increase in serum nine over a ne value determined prior to the time at which the body fluid sample was obtained, (ii) has a urine output of at least 0.5 ml/kg/hr over the 6 hours preceding the time at which the body fluid sample was obtained, and (iii) has not experienced an increase of 0.3 mg/dL or greater in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

55. A method according to one of claims 1-5, wherein the subject has not experienced a 1.5-fold or greater increase in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

56. A method according to one of claims 1-5, wherein the subject has a urine output of at least 0.5 ml/kg/hr over the 6 hours preceding the time at which the body fluid sample was obtained.

57. A method according to one of claims 1-5, wherein the subject (i) has not experienced a 1.5-fold or greater increase in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained, (ii) has a urine output of at least 0.5 ml/kg/hr over the 12 hours ing the time at which the body fluid sample was obtained, and (iii) has not experienced an increase of 0.3 mg/dL or greater in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

58. A method according to one of claims 1-5, wherein said correlating step comprises assigning one or more of: a likelihood that within 72 hours the subject will (i) experience a 1.5-fold or greater increase in serum creatinine (ii) have a urine output of less than 0.5 hr over a 6 hour period, or (iii) experience an increase of 0.3 mg/dL or r in serum creatinine.

59. A method according to claim 58, wherein said correlating step comprises ing one or more of: a likelihood that within 48 hours the subject will (i) experience a 1.5-fold or greater increase in serum creatinine (ii) have a urine output of less than 0.5 ml/kg/hr over a 6 hour period, or (iii) experience an increase of 0.3 mg/dL or greater in serum creatinine.

60. A method according to claim 58, wherein said correlating step comprises assigning one or more of: a hood that within 24 hours the t will (i) experience a 1.5-fold or greater se in serum creatinine (ii) have a urine output of less than 0.5 ml/kg/hr over a 6 hour period, or (iii) experience an increase of 0.3 mg/dL or greater in serum creatinine.

61. A method according to claim 58, wherein said correlating step comprises ing a likelihood that within 72 hours the t will experience a 1.5-fold or greater increase in serum creatinine.

62. A method according to claim 58, wherein said ating step comprises assigning a likelihood that within 72 hours the subject will have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

63. A method according to claim 58, wherein said correlating step comprises assigning a hood that within 72 hours the subject will experience an increase of 0.3 mg/dL or greater in serum creatinine.

64. A method according to claim 58, wherein said correlating step comprises assigning a likelihood that within 48 hours the subject will experience a 1.5-fold or greater increase in serum creatinine.

65. A method according to claim 58, wherein said correlating step comprises assigning a likelihood that within 48 hours the subject will have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

66. A method according to claim 58, wherein said correlating step comprises assigning a likelihood that within 48 hours the subject will experience an increase of 0.3 mg/dL or greater in serum creatinine.

67. A method according to claim 58, wherein said correlating step comprises assigning a likelihood that within 24 hours the subject will experience a 1.5-fold or greater increase in serum creatinine.

68. A method according to claim 58, wherein said correlating step comprises assigning a hood that within 24 hours the subject will have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

69. A method according to claim 58, wherein said correlating step comprises assigning a likelihood that within 24 hours the subject will experience an increase of 0.3 mg/dL or r in serum creatinine.

70. A method according to one of claims 1-5, wherein the subject has not experienced a 2-fold or r increase in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

71. A method according to one of claims 1-5, wherein the subject has a urine output of at least 0.5 ml/kg/hr over the 12 hours preceding the time at which the body fluid sample was ed.

72. A method according to one of claims 1-5, wherein the subject (i) has not experienced a 2-fold or greater increase in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained, (ii) has a urine output of at least 0.5 hr over the 2 hours preceding the time at which the body fluid sample was obtained, and (iii) has not experienced an increase of 0.3 mg/dL or r in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

73. A method according to one of claims 1-5, wherein the subject has not enced a 3-fold or greater increase in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

74. A method according to one of claims 1-5, wherein the subject has a urine output of at least 0.3 ml/kg/hr over the 24 hours ing the time at which the body fluid sample was obtained, or anuria over the 12 hours ing the time at which the body fluid sample was obtained.

75. A method according to one of claims 1-5, wherein the subject (i) has not experienced a 3-fold or greater increase in serum creatinine over a ne value determined prior to the time at which the body fluid sample was obtained, (ii) has a urine output of at least 0.3 ml/kg/hr over the 24 hours ing the time at which the body fluid sample was obtained, or anuria over the 12 hours preceding the time at which the body fluid sample was obtained, and (iii) has not experienced an se of 0.3 mg/dL or greater in serum creatinine over a baseline value determined prior to the time at which the body fluid sample was obtained.

76. A method according to one of claims 1-5, wherein said correlating step comprises assigning one or more of: a likelihood that within 72 hours the subject will (i) experience a 2-fold or r increase in serum creatinine (ii) have a urine output of less than 0.5 ml/kg/hr over a 12 hour period, or (iii) experience an increase of 0.3 mg/dL or greater in serum creatinine.

77. A method according to claim 76, wherein said correlating step comprises assigning one or more of: a likelihood that within 48 hours the subject will (i) experience a 2-fold or greater increase in serum creatinine (ii) have a urine output of less than 0.5 ml/kg/hr over a 6 hour period, or (iii) experience an increase of 0.3 mg/dL or greater in serum creatinine.

78. A method according to claim 76, wherein said correlating step comprises assigning one or more of: a likelihood that within 24 hours the subject will (i) experience a 2-fold or greater increase in serum creatinine, or (ii) have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

79. A method according to claim 76, n said correlating step comprises assigning a likelihood that within 72 hours the subject will experience a 2-fold or greater increase in serum creatinine.

80. A method according to claim 76, wherein said ating step comprises assigning a likelihood that within 72 hours the subject will have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

81. A method according to claim 76, wherein said correlating step comprises assigning a likelihood that within 48 hours the subject will experience a 2-fold or greater increase in serum creatinine.

82. A method according to claim 76, wherein said correlating step comprises assigning a likelihood that within 48 hours the t will have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

83. A method according to claim 76, wherein said correlating step comprises assigning a likelihood that within 24 hours the subject will experience a 2-fold or greater increase in serum creatinine.

84. A method according to claim 76, n said ating step comprises assigning a hood that within 24 hours the subject will have a urine output of less than 0.5 ml/kg/hr over a 6 hour period.

85. A method according to one of claims 1-5, n said correlating step comprises assigning one or more of: a likelihood that within 72 hours the t will (i) experience a 3-fold or greater increase in serum creatinine, or (ii) have a urine output of less than 0.3 ml/kg/hr over a 24 hour period or anuria over a 12 hour period.

86. A method according to claim 85, wherein said correlating step comprises assigning one or more of: a likelihood that within 48 hours the subject will (i) experience a 3-fold or greater increase in serum creatinine, or (ii) have a urine output of less than 0.3 hr over a 24 hour period or anuria over a 12 hour period.

87. A method according to claim 85, wherein said ating step comprises ing one or more of: a likelihood that within 24 hours the subject will (i) experience a 3-fold or greater increase in serum creatinine, or (ii) have a urine output of less than 0.3 ml/kg/hr over a 24 hour period or anuria over a 12 hour period.

88. A method according to claim 85, wherein said correlating step comprises assigning a likelihood that within 72 hours the subject will experience a 3-fold or greater increase in serum creatinine.

89. A method according to claim 85, wherein said correlating step comprises assigning a likelihood that within 72 hours the subject will have a urine output of less than 0.3 ml/kg/hr over a 24 hour period or anuria over a 12 hour period.

90. A method according to claim 85, wherein said correlating step ses assigning a likelihood that within 48 hours the subject will experience a 3-fold or greater se in serum creatinine.

91. A method according to claim 85, wherein said correlating step comprises assigning a likelihood that within 48 hours the subject will have a urine output of less than 0.3 ml/kg/hr over a 24 hour period or anuria over a 12 hour period.

92. A method according to claim 85, wherein said correlating step comprises assigning a likelihood that within 24 hours the subject will experience a 3-fold or greater increase in serum creatinine.

93. A method according to claim 85, wherein said ating step comprises assigning a likelihood that within 24 hours the subject will have a urine output of less than 0.3 ml/kg/hr over a 24 hour period or anuria over a 12 hour .

94. A method according to one of claims 1-93, wherein the body fluid sample is a urine .

95. A method according to one of claims 1-94, wherein said method further comprises performing assays that detect one, two or three, or more of Stanniocalcin-1, Antithrombin-III, Toll-like or 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, Vitronectin, Progesterone, Estradiol, and Proprotein convertase subtilisin/kexin type 9.

96. A kit, comprising: reagents for performing one or more assays configured to detect one or more kidney injury markers comprising at least Growth/differentiation factor 15 when used ing to the method of any one of claims 1 to 95.

97. A kit according to claim 96, wherein said reagents comprise one or more binding reagents, each of which specifically binds one of said kidney injury markers.

98. A kit according to claim 97, wherein a plurality of binding reagents are contained in a single assay device.

99. A kit according to claim 97, wherein at least one of said assays is configured as a sandwich binding assay.

100. A kit according to claim 97, wherein at least one of said assays is configured as a competitive binding assay.

101. A kit according to any one of claims 96-100, wherein said one or more assays further comprise assays that detect one, two or three, or more of ocalcin-1, Antithrombin-III, Toll-like receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, eukin-22, Vitronectin, Progesterone, Estradiol, and Proprotein convertase subtilisin/kexin type 9.

102. A method for evaluating biomarker levels in a body fluid sample, comprising: obtaining a urine sample from a subject selected for evaluation based on a determination that the subject is at risk of a future or t acute renal injury; and performing a plurality of analyte binding assays configured to detect a plurality of kers comprising Growth/differentiation factor 15 by introducing the urine sample obtained from the subject into an assay ment which (i) contacts a plurality of reagents which specifically bind for detection the plurality of biomarkers with the urine sample, and (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to a respective specific binding t in the plurality of reagents, wherein the subject is selected for evaluation based on a determination that the subject is in need of diagnosis, risk stratification, staging, prognosis, classifying or monitoring of the renal status of the subject.

103. A method ing to claim 102, wherein the subject is selected for evaluation based on a determination that the t is at risk of a future acute renal injury.

104. A method according to claim 103, wherein the subject is selected for evaluation based on a determination that the subject is at risk of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF).

105. A method according to claim 103, n the subject is ed for evaluation based on a ination that the subject is at risk of a future acute renal injury within 30 days of the time at which the urine sample was obtained from the t.

106. A method according to claim 102, wherein the subject is selected for evaluation based on a determination that the subject is at risk of a future acute renal injury within a period selected from the group consisting of 21 days, 14 days, 7 days, 5 days, 96 hours, 72 hours, 48 hours, 36 hours, 24 hours, and 12 hours.

107. A method according to claim 102, wherein the subject is selected for based on the pre-existence in the subject of one or more known risk factors for prerenal, intrinsic renal, or postrenal ARF.

108. A method according to claim 102, wherein the subject is selected for evaluation based on an existing diagnosis of one or more of congestive heart failure, preeclampsia, eclampsia, es mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, glomerular tion below the normal range, cirrhosis, serum creatinine above the normal range, sepsis, injury to renal function, reduced renal function, or ARF, or based on undergoing or having undergone major vascular surgery, coronary artery bypass, or other cardiac surgery, or based on exposure to NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque st agents, or streptozotocin.

109. A method according to claim 102, wherein the plurality of assays are immunoassays performed by (i) introducing the urine sample into an assay device comprising a plurality of antibodies, at least one of which binds to each biomarker which is assayed, and (ii) generating an assay result indicative of binding of each biomarker to its respective dy.

110. A method according to claim 102, wherein the subject is selected for tion based on a determination that the subject is at risk of one or more future changes in renal status ed from the group consisting of a future injury to renal function, future reduced renal function, future improvement in renal on, and future acute renal failure (ARF) within 72 hours of the time at which the urine sample was obtained.

111. A method according to claim 102, wherein the subject is selected for evaluation based on a determination that the subject is at risk of one or more future changes in renal status selected from the group consisting of a future injury to renal function, future reduced renal function, future improvement in renal function, and future acute renal failure (ARF) within 48 hours of the time at which the urine sample was obtained.

112. A method according to claim 102, wherein the subject is selected for evaluation based on a determination that the subject is at risk of one or more future s in renal status selected from the group consisting of a future injury to renal on, future reduced renal function, future ement in renal function, and future acute renal e (ARF) within 24 hours of the time at which the urine sample was obtained.

113. A method according to claim 102, n the subject is in RIFLE stage 0 or R.

114. A method according to claim 102, wherein the subject is in RIFLE stage 0, R, or I.

115. A method according to any one of claims 102 to 114, wherein the at least one assay result further comprises a measured biomarker concentration selected from the group consisting of a measured tration of Stanniocalcin-1, a measured concentration of rombin-III, a measured concentration of Toll-like receptor 2, a measured concentration of Triiodothyronine (T3), a measured concentration of Thyroxine (T4), a measured concentration of Extracellular matrix protein 1, a measured concentration of Coagulation factor XIII A chain, a measured concentration of Coagulation factor XIII B chain, a measured concentration of Interleukin-17F, a measured tration of Interleukin-22, a ed tration of Vitronectin, a measured concentration of Progesterone, Estradiol, and a measured concentration of Proprotein convertase subtilisin/kexin type 9.

116. A system for ting biomarker levels, comprising: a plurality of reagents which specifically bind for detection a plurality of biomarkers comprising Growth/differentiation factor 15; and an assay instrument configured to receive a urine sample and contact the plurality of reagents with the urine sample and to generate one or more assay results indicative of binding of each biomarker which is assayed to a respective specific binding reagent in the plurality of reagents when used according to the method of any one of claims 1 to 95 or 102 to 115.

117. A system according to claim 116, wherein the reagents se a plurality of antibodies, at least one of which binds to each of the kers which are assayed.

118. A system ing to claim 117, n assay instrument comprises an assay device and an assay device , wherein the plurality of antibodies are immobilized at a plurality of predetermined locations within the assay device, wherein the assay device is configured to receive the urine sample such that the urine sample contacts the plurality of predetermined locations, and wherein the assay device reader interrogates the plurality of ermined locations to generate the assay results.

119. A system according to any one of claims 116 to 118, wherein the plurality of biomarkers further comprises one or more biomarkers selected from the group consisting of Stanniocalcin-1, Antithrombin-III, Toll-like receptor 2, Triiodothyronine (T3), Thyroxine (T4), Extracellular matrix protein 1, Coagulation factor XIII A chain, Coagulation factor XIII B chain, Interleukin-17F, Interleukin-22, ectin, Progesterone, Estradiol, and Proprotein convertase subtilisin/kexin type 9. ast8129pct.txt