This invention is related to the field of the prevention and treatment of kidney disease. The treatment of kidney disease may be tailored depending upon the need for, or expectation of, renal recovery. For example, prediction of renal recovery can be determined by monitoring urine biomarkers related to the development of chronic kidney disease. For example, differential expression platforms can be used to identify biomarker proteins in order to establish the risk of renal recovery versus renal non-recovery in patient's having suffered an acute kidney injury.
Acute Kidney Injury (AKI) has an estimated incidence rate of approximately 2000 per million population and this rate is increasing. Ali et al. “Incidence and outcomes in acute kidney injury: a comprehensive population-based study” J Am Soc Nephrol 18:1292-1298 (2007). Approximately 5% of all people admitted to intensive care units around the world develop severe AKI requiring dialysis. Uchino et al., “Acute renal failure in critically ill patients: a multinational, multicenter study” JAMA 294:813-818 (2005). A recent, multi-center study found that fewer than only about 60% patients surviving severe AKI recovered renal function by two months. Palevsky et al., “Intensity of renal support in critically ill patients with acute kidney injury” N Engl J Med 359:7-20 (2008). Thus, a large number of patients with AKI go on to have end-stage renal disease (ESRD).
However, since only a fraction of patients with AKI fail to recover renal function, interventions aimed at improving recovery or providing renal support (e.g. early dialysis) cannot be targeted appropriately without some means of determining which patients will recover and which will not. Unfortunately, clinical risk prediction for recovery after AKI is extremely limited. Research efforts to treat AKI and prevent ESRD could be tailored according to long-term-prognosis. In other words, with an accurate prediction of which patients will not recover kidney function, medical efforts could focus the development and application of aggressive treatment interventions on just these patients. Conversely, patients with a favorable prognosis would be spared from more aggressive interventions and their potential adverse effects.
Thus, development of a biomarker or biomarker panel that allows early prediction of recovery of kidney function would be an extremely valuable clinical tool. What is needed in the art are a panel of biomarkers to predict renal recovery after AKI.
This invention is related to the field of the prevention and treatment of kidney disease. The treatment of kidney disease may be tailored depending upon the need for, or expectation of, renal recovery. For example, prediction of renal recovery can be determined by monitoring urine biomarkers related to the development of chronic kidney disease. For example, differential expression platforms can be used to identify biomarker proteins in order to establish the risk of renal recovery versus renal non-recovery in patient's having suffered an acute kidney injury.
In one embodiment, the present invention contemplates a composition comprising an renal injury biomarker, wherein said biomarker comprises at least a fragment of a protein selected from the group consisting of ferritin, beta globin, catalase, alpha globin, epidermal growth factor receptor pathway substrate 8, mucin isoform precursor, ezrin, delta globin, moesin, phosphoprotein isoform, annexin A2, myoglobin, hemopexin, serine proteinase inhibitor, serpine peptidase inhibitor, CD14 antigen precursor, fibronectin isoform preprotein, angiotensinogen preprotein, complement component precursor, carbonic anhydrase, uromodulin precursor, complement factor H, complement component 4 BP, heparan sulfate proteoglycan 2, olfactomedian-4, leucine rich alpha-2 glycoprotein, ring finger protein 167, inter-alpha globulin inhibitor H4, heparan sulfate proteoglycan 2, N-acylshingosine aminohydrolase, serine proteinase inhibitor clade A member 1, mucin 1, clusterin isoform 1, brain abundant membrane attached signal protein 1, dipeptidase 1, fibronectin 1 isoform 5 preprotein, angiotensinogen preproprotien, carbonic anhydrase, and uromodulin precursor. In one embodiment, the composition further comprises a urine sample. In one embodiment, the urine sample is collected between 1 day and 14 days after a kidney injury. In one embodiment, the urine sample is a human urine sample. In one embodiment, the biomarker is at least 2.5 fold higher as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 2.0 fold higher as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 1.5 fold higher as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 1.25 fold higher as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 2.5 fold lower as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 2.0 fold lower as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 1.5 fold lower as compared to an expected level in a renal recovery group. In one embodiment, the biomarker is at least 1.25 fold lower as compared to an expected level in a renal recovery group.
In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a patient exhibiting at least one symptom of an acute renal injury; and ii) a biological fluid sample obtained from said patient, wherein said sample comprises a renal injury biomarker; b) measuring a renal recovery biomarker value; c) comparing said renal biomarker value to an expected value from a renal recovery group; and d) predicting a probability of renal recovery for said patient based upon said comparison. In one embodiment, the probability of renal recovery is greater than 90%. In one embodiment, the probability of renal recovery is greater than 75%. In one embodiment, the probability of renal recovery is greater than 50%. In one embodiment, the probability of renal recovery is less than 50%. In one embodiment, the probability of renal recovery is less than 25%. In one embodiment, the probability of renal recovery is less than 10%. In one embodiment, the biomarker comprises at least a fragment of a protein selected from the group consisting of ferritin, beta globin, catalase, alpha globin, epidermal growth factor receptor pathway substrate 8, mucin isoform precursor, ezrin, delta globin, moesin, phosphoprotein isoform, annexin A2, myoglobin, hemopexin, serine proteinase inhibitor, serpine peptidase inhibitor, CD14 antigen precursor, fibronectin isoform preprotein, angiotensinogen preprotein, complement component precursor, carbonic anhydrase, uromodulin precursor, complement factor H, complement component 4 BP, heparan sulfate proteoglycan 2, olfactomedian-4, leucine rich alpha-2 glycoprotein, ring finger protein 167, inter-alpha globulin inhibitor H4, heparan sulfate proteoglycan 2, N-acylshingosine aminohydrolase, serine proteinase inhibitor clade A member 1, mucin 1, clusterin isoform 1, brain abundant membrane attached signal protein 1, dipeptidase 1, fibronectin 1 isoform 5 preprotein, angiotensinogen preproprotien, carbonic anhydrase, and uromodulin precursor.
In one embodiment, the present invention contemplates a kit, comprising; a) a first container comprising an antibody specifically directed to an renal injury biomarker, wherein said biomarker comprises at least a fragment of a protein selected from the group consisting of ferritin, beta globin, catalase, alpha globin, epidermal growth factor receptor pathway substrate 8, mucin isoform precursor, ezrin, delta globin, moesin, phosphoprotein isoform, annexin A2, myoglobin, hemopexin, serine proteinase inhibitor, serpine peptidase inhibitor, CD14 antigen precursor, fibronectin isoform preprotein, angiotensinogen preprotein, complement component precursor, carbonic anhydrase, uromodulin precursor, complement factor H, complement component 4 BP, heparan sulfate proteoglycan 2, olfactomedian-4, leucine rich alpha-2 glycoprotein, ring finger protein 167, inter-alpha globulin inhibitor H4, heparan sulfate proteoglycan 2, N-acylshingosine aminohydrolase, serine proteinase inhibitor clade A member 1, mucin 1, clusterin isoform 1, brain abundant membrane attached signal protein 1, dipeptidase 1, fibronectin 1 isoform 5 preprotein, angiotensinogen preproprotien, carbonic anhydrase, and uromodulin precursor; b) instructions for determining whether said biomarker is overexpressed as compared to an expected value from a renal recovery group; c) instructions for determining whether said biomarker is underexpressed as compared to an expected value from a renal recovery group; and d) instructions for determining the probability of renal recovery. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the monoclonal antibody is specifically directed to said biomarker protein fragment.
In one embodiment, the present invention contemplates a signature expression profile comprising a urinary protein biomarker panel, wherein said profile predicts renal recovery. In one embodiment, the biomarker panel comprises a plurality of overexpressed urinary proteins. In one embodiment, the biomarker panel comprises a plurality of underexpressed urinary proteins. In one embodiment, the plurality of overexpressed urinary proteins are selected from the group consisting of beta globin, catalase, alpha globin, mucin isoform precursor, ezrin, delta globin, moesin, phosphoprotein isoform, and annexin A2. In one embodiment, the plurality of underexpressed urinary proteins are selected from the group consisting of myoglobin, hemopexin, serine proteinase inhibitor, serpine peptidase inhibitor, CD14 antigen precursor, fibronectin isoform preprotein, angiotensinogen preprotein, complement component precursor, carbonic anhydrase, and uromodulin precursor.
In one embodiment, the present invention contemplates a signature expression profile comprising a urinary protein biomarker panel, wherein said profile predicts renal non-recovery. In one embodiment, the biomarker panel comprises a plurality of overexpressed urinary proteins. In one embodiment, the biomarker panel comprises a plurality of underexpressed urinary proteins. In one embodiment, the plurality of overexpressed urinary proteins are selected from the group consisting of beta globin, catalase, alpha globin, mucin isoform precursor, ezrin, delta globin, moesin, phosphoprotein isoform, and annexin A2. In one embodiment, the plurality of underexpressed urinary proteins are selected from the group consisting of myoglobin, hemopexin, serine proteinase inhibitor, serpine peptidase inhibitor, CD14 antigen precursor, fibronectin isoform preprotein, angiotensinogen preprotein, complement component precursor, carbonic anhydrase, and uromodulin precursor.
In one embodiment, the present invention contemplates a method, comprising: a) providing; i) a patient exhibiting at least one symptom of an acute renal injury; and ii) a biological fluid sample obtained from said patient, wherein said sample comprises a plurality of renal biomarker nucleic acids; b) expressing said plurality of renal biomarker nucleic acids, thereby creating a signature expression profile; and c) predicting a probability of renal recovery for said patient based upon said signature expression profile. In one embodiment, the signature expression profile predicts a probability of renal recovery of greater than 90%. In one embodiment, the signature expression profile predicts a probability of renal recovery of greater than 75%. In one embodiment, the signature expression profile predicts a probability of renal recovery of greater than 50%. In one embodiment, the signature expression profile predicts a probability of renal recovery of less than 50%. In one embodiment, the signature expression profile predicts a probability of renal recovery of less than 25%. In one embodiment, the signature expression profile predicts a probability of renal recovery of less than 10%. In one embodiment, the signature expression profile comprises a plurality of overexpressed urinary proteins. In one embodiment, the signature expression profile comprises a plurality of underexpressed urinary proteins. In one embodiment, the plurality of overexpressed urinary proteins are selected from the group consisting of beta globin, catalase, alpha globin, mucin isoform precursor, ezrin, delta globin, moesin, phosphoprotein isoform, and annexin A2. In one embodiment, the plurality of underexpressed urinary proteins are selected from the group consisting of myoglobin, hemopexin, serine proteinase inhibitor, serpine peptidase inhibitor, CD14 antigen precursor, fibronectin isoform preprotein, angiotensinogen preprotein, complement component precursor, carbonic anhydrase, and uromodulin precursor.
In one embodiment, the present invention contemplates a kit, comprising; a) a first container comprising reagents for creating a signature expression profile using a biological sample, wherein said signature expression profile comprises a plurality of renal biomarker nucleic acids; b) a second container comprising monoclonal antibodies specific for said renal biomarker nucleic acids; c) a set of instructions for creating said signature expression profile; d) a set of instructions for determining overexpressed renal biomarker nucleic acids; e) a set of instructions for determining underexpressed renal biomarker nucleic acids; f) a set of instructions for predicting the probability of renal recovery; and g) a set of instructions for predicting the probability of renal non-recovery.
As used herein, an “injury to renal function” is an abrupt (i.e., for example, within 14 Days, preferably within 7 Days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury to renal function may be identified, for example, by a decrease in glomerular filtration rate (GFR) or estimated GFR (eGFR), a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy (i.e., for example, dialysis), etc.
As used herein, an “improvement in renal function” is an abrupt (i.e., for example, within 14 Days, preferably within 7 Days, more preferably within 72 hours, and still more preferably within 48 hours) measurable 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 (i.e., for example, 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 greater than or equal to 0.1 mg/dL (≧8.8 μmol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-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 (i.e., for example, 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 greater than or equal to 0.3 mg/dl (≧26.4 μmol/l), a percentage increase in serum creatinine of greater than or equal to 50% (1.5-fold from baseline), or a reduction 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.”
As used herein, the term “relating a signal to the presence or amount” of an analyte refers to assay measurements using a standard curve calculated with known concentrations of the analyte of interest. The skilled artisan will understand that the signals obtained from an assay are often a direct result of complexes formed between, for example, one or more antibodies and a target biomolecule (i.e., for example, an analyte) and/or polypeptides containing an epitope(s) to which, for example, antibodies bind. While such assays may detect a full length biomarker and the assay result may be expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such “immunoreactive” polypeptides present in the sample.
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 physiologically relevant concentration of the analyte. For example, an antibody epitope is usually on the order of 8 amino acids, such that an immunoassay can be configured to detect a marker of interest that 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” or “biomarker” as used herein with regard to a physiological substance such as one of the proteins as described herein. A related marker may also refer to one or more fragments, variants, etc., of a particular protein and/or peptide 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 polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.
The term “subject” or “patient” as used herein, refers to a human or non-human organism. Thus, the methods and compositions described herein are equally applicable to both human and veterinary disease. Further, while a subject or patient is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects or patients are humans, which as used herein refer to living humans that are receiving medical care for a disease or condition.
The term “analyte” as used herein, refers to any measured compound or molecule. Preferably, an analyte is measured in a sample (i.e., for example, a body fluid sample). Such a sample may be obtained from a subject or patient, or may be obtained from biological materials intended to be provided to the subject or patient. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, such that an analyte measurement may be used to evaluate the kidney for preexisting damage.
The term “body fluid sample” as used herein, refers to any sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing medical condition or the effect of a treatment regimen on a medical condition. Preferred body fluid samples include but are not limited to, blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, or pleural effusions. In addition, certain body fluid samples may be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
The term “diagnosis” as used herein, refers to methods by which trained medical personnel can estimate and/or determine the probability (i.e., for example, a likelihood) of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, “diagnosis” includes correlating the results of an assay (i.e., for example, an immunoassay) for a renal biomarker of the present invention, optionally together with other clinical indicia, to determine the occurrence or nonoccurrence of an acute renal injury or acute renal failure for a subject or patient 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. Thus, for example, a measured biomarker level below a predetermined diagnostic threshold may indicate a greater likelihood of the occurrence of a disease in the subject relative to a measured biomarker level above the predetermined diagnostic threshold may indicate a lesser likelihood of the occurrence of the same disease.
The term “prognosis” as used herein, refers to a probability (i.e., for example, a likelihood) that a specific clinical outcome will occur. For example, a level or a change in level of a prognostic 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 likelihood” of an adverse outcome in a patient.
The term “RIFLE” criteria, as used herein, refers to any quantitative clinical evaluation of renal status used to establish renal classifications of Risk, Injury, Failure, Loss, & End Stage Renal Disease based upon a uniform definition of acute kidney injury (AKI). Kellum, Crit. Care Med. 36: S141-45 (2008); and Ricci et al., Kidney Int. 73, 538-546 (2008), each hereby incorporated by reference in its entirety.
The term, “modified RIFLE criteria”, as used herein, provide alternative classifications for stratifying AKI patients, and may include, Stage I, Stage II, and/or Stage III. Mehta et al., Crit. Care 11:R31 (2007), hereby incorporated by reference in its entirety.
The term, “Stage I”, as used herein, refers to a risk stratification comprising a RIFLE Risk category, characterized by an increase in serum creatinine of more than or equal to 0.3 mg/dL (≧26.4 μmol/L) and/or an increase to more than or equal to 150% (1.5-fold) from baseline. Alternatively, the category may be defined by a urine output less than 0.5 mL/kg per hour for more than 6 hours.
The term, “Stage II”, as used herein, refers a risk stratification comprising a RIFLE Injury category, characterized by an increase in serum creatinine to more than 200% (>2-fold) from baseline. Alternatively, the category may be defined by a urine output less than 0.5 mL/kg per hour for more than 12 hours.
The term, “Stage III”, as used herein, refers to a risk stratification comprising a RIFLE Failure category, characterized by an increase in serum creatinine to more than 300% (>3-fold) from baseline and/or serum creatinine ≧354 μmol/L accompanied by an acute increase of at least 44 μmol/L. Alternatively, the category may be defined by a urine output less than 0.3 mL/kg per hour for 24 hours or anuria for 12 hours.
The term “Risk category”, as used herein, refers to a RIFLE classification wherein, in terms of serum creatinine, means any increase of at least 1.5 fold from baseline, or urine production of <0.5 ml/kg body weight/hr for approximately 6 hours.
The term “Injury category” as used herein includes, refers to a RIFLE classification wherein, in terms of serum creatinine, means any increase of at least 2.0 fold from baseline or urine production <0.5 ml/kg/hr for 12 h.
The term “Failure category” as used herein includes, refers to a RIFLE classification wherein, in terms of serum creatinine means any increase of at least 3.0 fold from baseline or a urine creatinine >355 μmol/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.
The term “Loss category” as used herein, refers to a clinical outcome risk and/or a RIFLE classification wherein the clinical outcome risk is characterized by a persistent need for renal replacement therapy for more than four weeks.
The term “End Stage Renal Disease category” or “ESRD category” as used herein, refers to a clinical outcome risk and/or a RIFLE classification characterized by a need for dialysis for more than 3 months.
The term “clinical outcome risk” as used herein, refers to a medical prognosis directed towards either renal recovery or renal non-recovery.
The term “renal biomarker” as used herein, refers to any biological compound related to the progressive development of chronic kidney disease. In particular, a renal biomarker may be a kidney injury marker. For example, a renal biomarker may comprise a urinary protein, or any metabolite and/or derivative thereof, wherein the renal biomarker is either overexpressed or underexpressed as a result of an AKI.
The term “positive going biomarker” as that term is used herein, refers to any biomarker that is determined to be elevated in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.
The term “negative going biomarker” as that term is used herein, refer to any biomarker that is determined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.
The term “positive going renal biomarker value” as used herein, refers to any increased likelihood (i.e., for example, increased probability) of suffering a future injury to renal function assigned to a subject when the measured biomarker concentration is above a specified threshold value, relative to a likelihood assigned when the measured biomarker concentration is below the specified threshold value. Alternatively, when the measured biomarker concentration is below a specified threshold value, an increased likelihood of a non-occurrence of an injury to renal function may be assigned to the subject relative to the likelihood assigned when the measured biomarker concentration is above the specified threshold value. Alternatively, when the measured biomarker concentration is below the threshold value, an improvement of renal function may be assigned to the subject. A positive going kidney injury marker may include, but not be limited to, 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, myocardial infarction, progression to chronic kidney disease, etc.
The term “negative going renal biomarker value” as used herein, refers to any increased likelihood (i.e., for example, an increased probability) of suffering a future injury to renal function assigned to the subject when the measured biomarker concentration is below a specified threshold value, relative to a likelihood assigned when the measured biomarker concentration is above the threshold value. Alternatively, when the measured biomarker concentration is above the threshold value, an increased likelihood of a non-occurrence of an injury to renal function may be assigned to the subject relative to the likelihood assigned when the measured biomarker concentration is below the threshold value. Alternatively, when the measured biomarker concentration is above the threshold value, an improvement of renal function may be assigned to the subject. A negative going kidney injury marker may include, but not be limited to, 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, myocardial infarction, progression to chronic kidney disease, etc.
The term “pre-existing” and “pre-existence” as used herein, means any risk factor (i.e., for example, a renal biomarker) existing at the time a body fluid sample is obtained from the subject.
The term “predicting” as used herein, refers to a method of forming a prognosis and/or a stratification risk assignment, wherein a medically trained person analyzes biomarker information, and optionally with relevant clinical indicia and/or demographic information.
The term “acute renal disease/failure/injury” as used herein, refers to any progressive worsening of renal function over hours to Days, resulting in the retention of nitrogenous wastes (such as urea nitrogen) and creatinine in the blood. Retention of these substances may also be referred to as, azotemia. In: Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, herein incorporated by reference in their entirety.
The term “chronic renal disease/failure/injury” as used herein, refers to a medical condition wherein exemplary symptoms may include, but are not limited to, hyperphosphatemia (i.e., for example, >4.6 mg/dl) or low glomerular filtration rates (i.e., for example, <90 ml/minute per 1.73 m2 of body surface). However, many CKD patients may have normal serum phosphate levels in conjunction with a sustained reduction in glomerular filtration rate for 3 or more months, or a normal GFR in conjunction with sustained evidence of a structural abnormality of the kidney. In some cases, patients diagnosed with chronic kidney disease are placed on hemodialysis to maintain normal blood homeostasis (i.e., for example, urea or phosphate levels). Alternatively, “chronic kidney disease” refers to a medical condition wherein a patients has either i) a sustained reduction in GFR<60 mi/min per 1.73 m2 of body surface for 3 or more months; or ii) a structural or functional abnormality of renal function for 3 or more months even in the absence of a reduced GFR. Structural or anatomical abnormalities of the kidney could be defined as, but not limited to, persistent microalbuminuria or proteinuria or hematuria or presence of renal cysts. Chronic renal failure (chronic kidney disease) may also result from an abnormal loss of renal function over months to years. In: Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, herein incorporated by reference in their entirety.
The term “about” as used herein in the context of any of any assay measurements refers to +/−5% of a given measurement.
The term “asymptomatic” as used herein, refers to a patient and/or subject that does not have a renal disease and/or injury, wherein a renal disease and/or injury symptom may include, but is not limited to, having a reduced glomerular filtration rate (i.e., for example, between approximately 70-89 ml/min per 1.73 m2 of body surface) for less than three months.
The term “glomerular filtration rate” as used herein, refers to any measurement capable of determining kidney function. In general, a normal glomerular filtration rate ranges between approximately 120-90 ml/minute per 1.73 m2 of body surface. Compromised kidney function is assumed when glomerular filtration rates are less than 90 ml/minute per 1.73 m2 of body surface. Kidney failure is probable when glomerular filtration rates fall below approximately 30 ml/minute per 1.73 m2 of body surface. Dialysis is frequently initiated when glomerular filtration rates fall below approximately 15 ml/minute per 1.73 m2 of body surface.
The term “renal failure” as used herein, refers to any acute, sudden, and/or chronic loss of the ability of the kidneys to remove waste and concentrate urine without losing electrolytes.
The term “biological sample” as used herein, refers to any substance derived from a living organism. For example, a sample may be derived from blood as a urine sample, serum sample, a plasma sample, and or a whole blood sample. Alternatively, a sample may be derived from a tissue collected, for example, by a biopsy. Such a tissue sample may comprise, for example, kidney tissue, vascular tissue and/or heart tissue. A biological sample may also comprise body fluids including, but not limited to, urine, saliva, or perspiration.
The term “reagent” as used herein, refers to any substance employed to produce a chemical reaction so as to detect, measure, produce, etc., other substances. The term “antibody” as used herein refers to any 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. In: Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson et al., J. Immunol. Methods 175:267-273 (1994); and Yarmush et al., J. Biochem. Biophys. Methods 25:85-97 (1992). The term antibody includes, but is not limited to, antigen-binding portions, i.e., “antigen binding sites” exemplified by fragments, subsequences, and/or complementarity determining regions (CDRs)) that retain capacity to bind antigen, including, but not limited to: (i) a Fab fragment, a monovalent fragment comprising VL, VH, CL or CHI domains; (ii) a F(ab′)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment comprising VH and CHI domains; (iv) a Fv fragment comprising VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546 (1989)), which comprises a VH domain; or (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term “antibody.”
The term “epitope” as used herein, refers to any antigenic determinant capable of specific binding to an antibody. Epitopes usually display chemically active surface 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 epitopes may be distinguished in that the binding to the former but not the latter can be lost in the presence of denaturing solvents.
The term “correlating” as used herein, in reference to the use of biomarkers, refers to comparing the presence and/or amount of any biomarker(s) in a patient to its presence and/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 likelihood of some future outcome.
This invention is related to the field of the prevention and treatment of kidney disease. The treatment of kidney disease may be tailored depending upon the need for, or expectation of, renal recovery. For example, prediction of renal recovery can be determined by monitoring urine biomarkers related to the development of chronic kidney disease. For example, differential expression platforms can be used to identify biomarker proteins in order to establish the risk of renal recovery versus renal non-recovery in patient's having suffered an acute kidney injury.
Despite significant advances in the epidemiology of acute kidney injury (AKI), prognostication remains a major clinical challenge. Unfortunately, there is no reliable method to predict renal recovery. The discovery of biomarkers to aid in clinical risk prediction for recovery after AKI would represent a significant advance over current practice.
I. Kidney Injury and/or Disease
The kidney is responsible for water and solute excretion from the body. Its functions include maintenance of acid-base balance, regulation of electrolyte concentrations, control of blood volume, and regulation of blood pressure. As such, loss of kidney function through injury and/or disease results in substantial morbidity and mortality. A detailed discussion of renal injuries is provided in Harrison's Principles of Internal Medicine, 17th Ed., McGraw Hill, New York, pages 1741-1830, which are hereby incorporated by reference in their entirety. The kidneys are located in the flank (back of the upper abdomen at either side of the spinal column). They are deep within the abdomen and are protected by the spine, lower rib cage, and the strong muscles of the back. This location protects the kidneys from many external forces. They are well-padded for a reason—kidneys are highly vascular organs, which means that they have a large blood supply. If injury occurs, severe bleeding may result.
Kidneys may be injured by damage to the blood vessels that supply or drain them. This may be in the form of aneurysm, arteriovenous fistula, arterial blockage, or renal vein thrombosis. The extent of bleeding depends on the location and the degree of injury. Kidneys may also bleed profusely if they are damaged centrally (on the inside)—this is a life-threatening injury. Fortunately, most kidney injuries caused by blunt trauma occur peripherally, only causing bruising of the kidney (usually a self-limiting process).
People with undiagnosed kidney conditions—such as angiomyolipoma (benign tumor), ureteropelvic junction obstruction (congenital or acquired UPJ Obstruction), and other disorders—are more susceptible to kidney injuries and more likely to have serious complications if they occur. Other causes of kidney injury and bleeding are medical procedures. Kidney biopsies, nephrostomy tube placements, or other surgeries can cause an abnormal connection between an artery and vein (arteriovenous fistula). This is usually a self-limiting problem, but close observation is usually needed. Injury to the kidney can also disrupt the urinary tract, causing leakage of the urine from the kidney.
Each kidney filters about 1700 liters of blood per Day and concentrates fluid and waste products into about 1 liter of urine per Day. Because of this, the kidneys receive more exposure to toxic substances in the body than almost any other organ. Therefore, they are highly susceptible to injury from toxic substances. Analgesic nephropathy is one of the most common types of toxic damage to the kidney. Exposure to lead, cleaning products, solvents, fuels, or other nephrotoxic chemicals (those which can be toxic to the kidney) can damage kidneys. Excessive buildup of body waste products, such as uric acid (that can occur with gout or with treatment of bone marrow, lymph node, or other disorders) can also damage the kidneys.
Inflammation (irritation with swelling and presence of extra immune cells) caused by immune responses to medications, infection, or other disorders may also injure the structures of the kidney, usually causing various types of glomerulonephritis or acute tubular necrosis (tissue death). Autoimmune disorders may also damage the kidneys. Injury to the kidney may result in short-term damage with minimal or no symptoms. Alternately, it can be life-threatening because of bleeding and associated shock, or it may result in acute renal failure or chronic renal failure.
Ureteral injuries (injuries to the tubes which carry urine from the kidneys to the bladder) can also be caused by trauma (blunt or penetrating), complications from medical procedures, and other diseases in the retroperitoneum such as retroperitoneal fibrosis (RPF), retroperitoneal sarcomas, or metastatic lymph node positive cancers. Medical therapies (such as OB/GYN surgeries, prior radiation or chemotherapy, and previous abdominopelvic surgeries) increase the risk for ureteral injuries.
A. Acute Kidney Failure
Acute (sudden) kidney failure is the sudden loss of the ability of the kidneys to remove waste and concentrate urine without losing electrolytes. There are many possible causes of kidney damage including, but are not limited to, decreased blood flow, which may occur with extremely low blood pressure caused by trauma, surgery, serious illnesses, septic shock, hemorrhage, burns, or dehydration, acute tubular necrosis (ATN), infections that directly injury the kidney such as acute pyelonephritis or septicemia, urinary tract obstruction (obstructive uropathy), autoimmune kidney disease such as interstitial nephritis or acute nephritic syndrome, disorders that cause clotting within the thin blood vessels of the kidney, idiopathic thrombocytopenic thrombotic purpura (ITTP), transfusion reaction, malignant hypertension, scleroderma, hemolytic-uremic syndrome, disorders of childbirth, such as bleeding placenta abruptio or placenta previa
Symptoms of acute kidney failure may include, but are not limited to, decrease in amount of urine (oliguria), urination stops (anuria), excessive urination at night, ankle, feet, and leg swelling, generalized swelling, fluid retention, decreased sensation, especially in the hands or feet, decreased appetite, metallic taste in mouth, persistent hiccups, changes in mental status or mood, agitation, drowsiness, lethargy, delirium or confusion, coma, mood changes, trouble paying attention, hallucinations, slow, sluggish, movements, seizures, hand tremor (shaking), nausea or vomiting, may last for Days, bruising easily, prolonged bleeding, nosebleeds, bloody stools, flank pain (between the ribs and hips), fatigue, breath odor, or high blood pressure.
Acute renal failure (ARF) may also be referred to as acute kidney injury (AKI) and may be characterized by an abrupt (i.e., for example, typically detected within about 48 hours to 1 week) reduction in glomerular filtration rate (GFR). This loss of filtration capacity results in retention of nitrogenous (urea and creatinine) 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. Major causes of ARF are described in association with their respective risk factors are summarized below. See, Table 4; In: Merck Manual, 17th ed., Chapter 222, and which is hereby incorporated by reference in their entirety.
In the case of ischemic ARF, the course of the disease may be divided into four phases. During an initiation phase, which lasts hours to Days, reduced perfusion of the kidney is evolving into injury. Glomerular ultrafiltration reduces, the flow of filtrate 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 vascular 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 including 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 thought to be caused by intrarenal vasoconstriction (leading to ischemic injury) and from the generation of reactive oxygen species that are directly toxic to renal tubular epithelial cells. CIN classically presents as an acute (onset within 24-48 h) but reversible (peak 3-5 Days, resolution within 1 week) rise in blood urea nitrogen and serum creatinine.
A commonly reported criteria for defining and detecting AKI is an abrupt (typically within about 2-7 Days or within a period of hospitalization) elevation of serum creatinine. Although the use of serum creatinine elevation to define and detect AKI is well established, the magnitude of the serum creatinine elevation and the time over which it is measured to define AKI varies considerably among publications. 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.
For example, relationships between elevated serum creatinine and AKI has been reported to be associated with health risks. Praught et al., Curr Opin Nephrol Hypertens 14:265-270 (2005); and Chertow et al., J Am Soc Nephrol 16:3365-3370 (2005) (both references are herein incorporated by reference in their entirety). As described in these publications, acute worsening renal function (AKI) and increased risk of death and other detrimental outcomes are now known to be associated with very small increases in serum creatinine. These creatinine increases 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 increase 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 indicate worsening renal function and increased 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 variable 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 continuous increase in risk with increasing magnitude of serum creatinine rise.
Another study correlated serum creatinine levels with post-surgical mortality rates. Following heart surgery, patients with a mild fall in serum creatinine (i.e., for example, between approximately −0.1 to −0.3 mg/dL) had the lowest mortality rate, wherein patients had a larger mortality rate associated with either large falls in serum creatinine (i.e., for example, more than or equal to −0.4 mg/dL), or an increase in serum creatinine. Lassnigg et al., J Am Soc Nephrol 15:1597-1605 (2004), herein incorporated by reference in its entirety. These findings suggested that even very subtle changes in renal function, as detected by small creatinine changes within 48 hours of surgery, can be predictive of a patient's outcome.
A unified classification system using serum creatinine to define AKI in clinical trials and in clinical practice was proposed to stratify AKI patients. Bellomo et al., Crit Care 8(4):R204-212 (2004), which is herein incorporated by reference in its entirety. For example, a serum creatinine rise of 25% may define contrast-induced nephropathy. McCollough et al, Rev Cardiovasc Med. 7(4): 177-197 (2006), herein incorporated by reference in its entirety. 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 (i.e., for example, approximately 25%) are sufficient to detect AKI that characterizes a worsening renal function and that the magnitude of the serum creatinine change may be 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 patients, serum creatinine is generally regarded to have several limitations in the diagnosis, assessment and monitoring of AKI patients. The time period for serum creatinine to rise to approximately 0.3 mg/dL (25%) is considered diagnostic for AKI can be 48 hours or longer depending on the definition 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 injury, and relying on serum creatinine 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 changing rapidly. Until defined by some embodiments of the present invention, there were no methods to determine whether some patients with AKI would recover fully, or whether some would need dialysis (either short term or long term), or whether some would have other detrimental outcomes including, but not limited to, death, major adverse cardiac events or 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 obstruction, atheroembolic, 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.
These limitations underscore the need for better methods to detect and assess AKI, particularly 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.
B. Chronic Kidney Failure
Unlike acute renal failure, chronic renal failure slowly gets worse. It most often results from any disease that causes gradual loss of kidney function. It can range from mild dysfunction to severe kidney failure. Chronic renal failure may lead to end-stage renal disease (ESRD).
Chronic renal failure usually occurs over a number of years as the internal structures of the kidney are slowly damaged. In the early stages, there may be no symptoms. In fact, progression may be so slow that symptoms do not occur until kidney function is less than one-tenth of normal.
Chronic renal failure and ESRD affect more than 2 out of 1,000 people in the United States. Diabetes and high blood pressure are the two most common causes and account for most cases. Other major causes include, but are not limited to, Alport syndrome, analgesic nephropathy, glomerulonephritis of any type (one of the most common causes), kidney stones and infection, obstructive uropathy, polycystic kidney disease, or reflux nephropathy. Chronic renal failure results in an accumulation of fluid and waste products in the body, leading to a build up of nitrogen waste products in the blood (azotemia) and general ill health. Most body systems are affected by chronic renal failure.
Initial symptoms may include, but are not limited to, fatigue, frequent hiccups, general ill feeling, generalized itching (pruritus), headache, nausea, vomiting, or unintentional weight loss. Further, later symptoms may include, but are not limited to, blood in the vomit or in stools,
decreased alertness, including drowsiness, confusion, delirium, orcoma, decreased sensation in the hands, feet, or other areas, easy bruising or bleeding, increased or decreased urine output, muscle twitching or cramps, seizures, or white crystals in and on the skin (uremic frost).
Circulating levels of cytokines and other inflammation markers are markedly elevated in patients with chronic renal failure. This could be caused by increased generation, decreased removal, or both. However, it is not well established to what extent renal function per se contributes to the uremic proinflammatory milieu. Relationships between inflammation and glomerular filtration rate (GFR) were reported in 176 patients (age, 52+/−1 years; GFR, 6.5+/−0.1 mL/min) close to the initiation of renal replacement therapy. Pecoits-Filho et al., “Associations between circulating inflammatory markers and residual renal function in CRF patients” Am J Kidney Dis. 41(6):1212-1218 (2003). For example, circulating levels of high-sensitivity C-reactive protein (hsCRP), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), hyaluronan, and neopterin were measured after an overnight fast. Patients subsequently were subdivided into two groups according to median GFR (6.5 mL/min). Despite the narrow range of GFR (1.8 to 16.5 mL/min), hsCRP, hyaluronan, and neopterin levels were significantly greater in the subgroup with lower GFRs, and significant negative correlations were noted between GFR and IL-6 (rho=−0.18; P<0.05), hyaluronan (rho=−0.25; P<0.001), and neopterin (rho=−0.32; P<0.0005). In a multivariate analysis, age and GFR were associated with inflammation but cardiovascular disease and diabetes mellitus were not. These results show that a low GFR per se is associated with an inflammatory state, suggesting impaired renal elimination of proinflammatory cytokines, increased generation of cytokines in uremia, or an adverse effect of inflammation on renal function.
C. Dialysis
Dialysis (i.e., for example, renal replacement therapy) is a method of removing toxic substances (impurities or wastes) from the blood when the kidneys are unable to do so and can be performed using several different methods. For example, peritoneal dialysis may filter waste by using the peritoneal membrane inside the abdomen. The abdomen is filled with special solutions that help remove toxins. The solutions remain in the abdomen for a time and then are drained out. This form of dialysis can be performed at home, but must be done every Day. Alternatively, hemodialysis may be performed by circulating the blood through special filters outside the body. The blood flows across a filter, along with solutions that help remove toxins.
Dialysis uses special ways of accessing the blood in the blood vessels. The access can be temporary or permanent. Temporary access takes the form of dialysis catheters—hollow tubes placed in large veins that can support acceptable blood flows. Most catheters are used in emergency situations for short periods of time. However, catheters called tunneled catheters can be used for prolonged periods of time, often weeks to months. Permanent access is created by surgically joining an artery to a vein. This allows the vein to receive blood at high pressure, leading to a thickening of the vein's wall. This vein can handle repeated puncture and also provides excellent blood flow rates. The connection between an artery and a vein can be made using blood vessels (an arteriovenous fistula, or AVF) or a synthetic bridge (arteriovenous graft, or AVG). Blood is diverted from the access point in the body to a dialysis machine. Here, the blood flows counter-current to a special solution called the dialysate. The chemical imbalances and impurities of the blood are corrected and the blood is then returned to the body. Typically, most patients undergo hemodialysis for three sessions every week. Each session lasts 3-4 hours.
The purpose of dialysis is to assist kidney functions including, filters for the blood, removing waste products, regulating body water, maintaining electrolyte balance, or maintaining blood pH remains between 7.35 and 7.45. Further, dialysis may replace some of the functions for kidneys that aren't working properly that would otherwise result in the death of a patient.
Dialysis is most often used for patients who have kidney failure, but it can also quickly remove drugs or poisons in acute situations. This technique can be life saving in people with acute or chronic kidney failure.
Currently, no effective treatments exist to improve renal recovery, or to improve short and long-term renal outcome, after AKI. Furthermore, methods to predict recovery are also lacking. The emerging role of biomarkers for early detection of renal disease and/or renal injury may help identify new prognostic tools to predict renal clinical outcomes. Potential candidates for biomarkers of renal recovery include, but are not limited to, molecules expressed in pathways leading to regeneration and proliferation as well as markers of fibrosis and apoptosis. In addition, renal injury biomarkers may also serve to distinguish early resolution, and hence increased odds of recovery.
Acute kidney injury (AKI) has an estimated incidence rate of approximately 2000 per million population and this rate is increasing. Ali et al., “Incidence and outcomes in acute kidney injury: a comprehensive population-based study” J Am Soc Nephrol 18:1292-1298 (2007). Approximately 5% of all people admitted to intensive care units around the world develop severe AKI requiring dialysis. Uchino et al., “Acute renal failure in critically ill patients: a multinational, multicenter study” JAMA 294:813-818 (2005). A recent, United States multi-center study found that fewer than only about 60% patients surviving severe AKI recovered renal function by two months. Palevsky et al., “Intensity of renal support in critically ill patients with acute kidney injury” N Engl J Med 359:7-20 (2008). Thus, a large number of patients with AKI progress into end-stage renal disease (ESRD).
However, since only a fraction of patients with AKI fail to recover renal function, interventions aimed at improving recovery or at providing renal support (e.g. early dialysis) cannot be selectively targeted appropriately without some means of determining which patients will recover and which will not recover (i.e., for example, the availability of non-invasive biomarkers). Currently, clinical risk prediction for recovery after AKI is extremely limited. Thus, development of a non-invasive biomarker that allows early prediction of recovery of kidney function is a long felt need in the art of renal disease management.
The identification of such non-invasive biomarkers (i.e., for example, a urinary biomarker) would greatly improve long-term prognosis thereby tailoring research efforts to treat AKI and prevent ESRD. In other words, having the ability to predict which patients will not recover kidney function allows a clinician to focus limited resources on the development and application of aggressive treatment interventions on these predicted at-risk patients. Conversely, patients with a favorable prognosis would be spared from more aggressive interventions and their potential adverse effects, thereby releasing medical resources to those in need and reducing overall medical costs.
In one embodiment, the present invention contemplates methods and compositions for evaluating renal function in a subject. As described herein, measurement of various kidney injury markers described herein can be used for diagnosis, prognosis, risk stratification, staging, monitoring, categorizing and a determination of further diagnosis and treatment regimens in subjects suffering or at risk of suffering from an injury to renal function, reduced renal function, and/or acute renal failure (also called acute kidney injury).
Renal biomarkers as described herein may be used individually, or in panels, comprising a plurality of renal biomarkers, for risk stratification. In one embodiment, risk stratification identifies subjects at risk for a future: i) injury to renal function; ii) progression to reduced renal function; iii) progression to ARF; or iv) improvement in renal function, etc. In one embodiment, risk stratification diagnoses an existing disease, comprising identifying subjects who have: i) suffered an injury to renal function; ii) progressed to reduced renal function; or iii) progressed to ARF, etc. In one embodiment, risk stratification monitors for deterioration and/or improvement of renal function. In one embodiment, risk stratification predicts a future medical outcome including, but not limited to, an improved or worsening renal function, a decreased or increased mortality risk, a decreased or increased risk that a subject will require initiation or continuation of renal replacement therapy (i.e., hemodialysis, peritoneal dialysis, hemofiltration, and/or renal transplantation, a decreased or increased risk that a subject will recover from an injury to renal function, 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 increased 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.
The results of a large multicenter clinical trial has recently been reported comparing two intensities of renal support for critically ill patients with acute kidney injury (AKI) in which recovery of renal function was less than 25% at 28 days and not different between the two treatment strategies. Palevsky et al., “Intensity of renal support in critically ill patients with acute kidney injury” N Engl J Med 359:7-20 (2008). These results emphasize that incomplete renal recovery is a common problem in the patients who survive severe AKI. Uchino et al., “Acute renal failure in critically ill patients: a multinational, multicenter study” JAMA, 294: 813-818 (2005). Failure to recover renal function can have tremendous negative effects on quality of life and health care costs. Manns et al., “Cost of acute renal failure requiring dialysis in the intensive care unit: clinical and resource implications of renal recovery” Crit Care Med, 31:449-455 (2003). Therefore, treatments to hasten and facilitate renal recovery are eagerly being sought by both the critical care and nephrology communities. Unfortunately, there are no effective treatments to improve renal recovery. One possible barrier to progress in this area has been the inability to forecast recovery in individual patients. The ability to prognosticate in an AKI patient population would be extremely valuable both for clinical decisions as well as to guide future research on therapy to promote recovery of renal function.
One clinical study reported that patients who recovered from AKI did not appear to differ in clinical characteristics (i.e., for example, age, gender, mechanical ventilation status, or clinical severity scores) from the non-recovery group. Bhandari et al., “Survivors of acute renal failure who do not recover renal function” QJM, 89:415-421 (1996). Secondary analysis from three randomized controlled trials (RCTs) comparing efficacy of continuous renal replacement therapy (RRT) versus intermittent RRT found that: i) APACHE III scores >100; ii) cardiovascular instability; and iii) pre-existing renal impairment were all associated with renal non-recovery. Mehta et al., “A randomized clinical trial of continuous versus intermittent dialysis for acute renal failure” Kidney Int, 60:1154-1163 (2001); Augustine et al., “A randomized controlled trial comparing intermittent with continuous dialysis in patients with ARF” Am J Kidney Dis, 44:1000-1007 (2004); and Uehlinger et al., “Comparison of continuous and intermittent renal replacement therapy for acute renal failure” Nephrol Dial Transplant, 20:1630-1637 (2005), respectively. However, these studies did not adhere to a uniform definition of, or standard timing, to assess renal recovery.
Other studies have suggested that baseline creatinine and urine output at the time of discontinuation of RRT were most predictive of recovery. Uchino et al., “Discontinuation of continuous renal replacement therapy: a post hoc analysis of a prospective multicenter observational study” Crit Care Med, 37:2576-2582 (2009). However, urine output was analyzed after RRT had ended based on a clinical decision rather than at a fixed time point (e.g. 14 Days post AKI) to predict renal recovery. Therefore, these data are compromised to suggest that urine output was predictive of renal recovery, and further, baseline creatinine might have been less valuable because patients with stage 4 and 5 CKD were excluded.
Recently, a number of urinary biomarkers have been investigated for the purpose of early diagnosis of AKI. Since these markers correlate with renal tubular cell injury or function, their patterns in the urine, either alone or in combination, could provide new prognostic information regarding renal recovery. For example, several reports have suggested possible candidate renal biomarkers relating to three aspects of the physiology of renal recovery:
i) inflammatory markers including; a) urinary neutrophil gelatinase-associated lipocalin (uNGAL), which has been extensively studied for predicting AKI (Supavekin et al., “Differential gene expression following early renal ischemia/reperfusion” Kidney Int, 63:1714-1724 (2003); Mishra et al., “Kidney NGAL is a novel early marker of acute injury following transplantation” Pediatr Nephrol, 21:856-863 (2006); Hirsch et al., “NGAL is an early predictive biomarker of contrast-induced nephropathy in children” Pediatr Nephrol, 22: 2089-2095 (2007); and Zappitelli et al., “Urine neutrophil gelatinase-associated lipocalin is an early marker of acute kidney injury in critically ill children: a prospective cohort study” Crit Care, 11: R84 (2007); b) matrix metalloproteinase protein-9 (MMP-9), a matrix degradation enzyme which is up-regulated after ischemic injury in animal models and links to NGAL by a disulfide bond forming urinary NGAL/MMP-9 (uNGAL/MMP-9) (Ronco et al., “Matrix metalloproteinases in kidney disease progression and repair: a case of flipping the coin” Semin Nephrol, 27:352-362 (2007); and c) urinary interleukin-18 (uIL-18), an inflammatory cytokine which is found to potentiate ischemic AKI and has been tested in many clinical settings (Parikh et al., “Urine IL-18 is an early diagnostic marker for acute kidney injury and predicts mortality in the intensive care unit” J Am Soc Nephrol, 16:3046-3052 (2005); and Parikh et al., “Urinary IL-18 is an early predictive biomarker of acute kidney injury after cardiac surgery” Kidney Int, 70:199-203 (2006);
ii) growth factors including urinary hepatocyte growth factor (uHGF), a biomarker linked to renal tubular epithelial cell regeneration (Liu et al., “Hepatocyte growth factor: new arsenal in the fights against renal fibrosis? Kidney Int, 70:238-240 (2006); and
iii) filtration and tubular reabsorption markers, such as cystatin C, which is freely filtered and is normally completely reabsorbed by proximal tubular epithelial cells and urine creatinine. Herget-Rosenthal et al., “Measurement of urinary cystatin C by particle-enhanced nephelometric immunoassay: precision, interferences, stability and reference range” Ann Clin Biochem, 41:111-118 (2004).
Despite these reports, only a few suggest biomarkers having an ability to predict AKI severity. But no study has identified a biomarker as a predictor of renal recovery. Coca et al., “Biomarkers for the diagnosis and risk stratification of acute kidney injury: a systematic review” Kidney Int, 73:1008-1016 (2008). The data presented herein provide heretofore unknown renal biomarkers identified by proteomic gene expression analysis. The data was obtained from urine samples collected during a clinical study as described below.
The data presented herein was collected from 109 patients in the BioMaRK clinical study where 76 patients had complete data available including urine samples. Exactly half (38 patients) recovered renal function (alive and without requirement for dialysis) by day 60. See,
aAccording to the method of Charlson et al.23
bDefined as sepsis plus acute organ dysfunction according to 2001 international consensus criteria for sever sepsis.26
cAccording to the method of Knaus et al.29
dNon renal SOFA score, excluding the renal part, assessed on the first day according to the method of Vincent et al.30
eAccording to the method of Thakar et al.31
fIntensive strategy, intermittent hemodialysis and sustained low-efficiency dialysis were provided six times per week (every day except Sunday), and continuous venovenous hemodiafiltration was prescribed to provide a flow rate of the total effluent (the sum of the dialysate and ultrafiltrate) of 35 ml per kilogram of body weight per hour, based on the weight before the onset of acute illness.1
Patients recovering from renal injury were more likely to be younger, had a shorter length of intensive care unit (ICU) stay before randomization, lower Charlson comorbidity index, and lower nonrenal SOFA score as compared to those not recovering renal function. By contrast, there were no statistical differences in gender, ethnicity, baseline serum creatinine, blood urea nitrogen (BUN) at initiation of RRT, length of hospital stay, length of ICU stay, requirement for mechanical ventilation, Cleveland Clinic ICU Acute Renal Failure (ARF) score, or intensity of RRT. The primary etiology of AKI was ischemia in both groups. However, a significantly lower percentage of ischemia (76.3%) was noted as the cause of AKI in the recovery group compared to 97.4% in non-recovery group. Of the 38 participants who recovered renal function, 26 (68.4%) had complete recovery. Among those failing to recover renal function, 25 patients (65.8%), did not survive past day 60.
In one embodiment, the present invention contemplates a method for identifying urinary biomarkers using a proteomics platform. In one embodiment, the proteomics platform detects protein expression profiles. In one embodiment, the method further comprises comparing a first protein expression profile to a second protein expression profile. In one embodiment, the comparing identifies an overexpressed protein in the first protein expression profile relative to the second protein expression profile. In one embodiment, the comparing identifies an underexpressed protein in the first protein expression profile relative to the second protein expression profile.
A Introduction
In one embodiment, the present invention contemplates a method comprising a proteomics platform (i.e., for example, iTRAQ) capable of summarizing an analysis of relative protein biomarker expression. For example, the proteomics platform may use reporter ion peak area measurements (i.e., for example, supplied by ABI software) to estimate treatment-dependent peptide and protein biomarker relative expression. Such estimations may be accomplished using a Bayesian approach. The proteomics platform described herein includes a protein biomarker relative expression summary and a per-protein biomarker detailed analysis.
B Experiment and Model Description
1. Experiment Design
Proteomic platforms contemplated herein may summarize data from one or more experiments addressing a common comparison. For example, a possible experimental design for such an analysis is presented below. See, Table 5.
2. Input Files
Data for proteomic analyses may be extracted from input files including but not limited to a tandem mass spectra (MSMS) summary file, such as:
3. Statistical Modeling Statistical models to estimate the treatment-dependent effects may including but not limited to: LogIntensity˜Channel+Spectrum+Protein+Peptide+Protein:Treatment+Peptide:Treatment.
4. Data Summarization
The proteomics platform may comprise filtering the data supplied in the MSMS summary to remove unidentified proteins, contaminants, and/or peptides containing selected modifications. A representative analysis may provide a data summary as presented below. See, Table 6.
C. Protein Biomarker Summary
In one embodiment, the proteomics platform may identify each protein biomarker in one or more of the MSMS summaries, for example, in decreasing order of expression change magnitude. See,
D. Protein Biomarker Details
A detailed summary of each protein biomarker is given below, wherein each protein biomarker is designated as 5.###. These sections include protein biomarker relative expression estimates in addition to protein-level estimates.
5.1 beta globin [Homo sapiens]
Protein Accession gi|4504349
5.2 catalase [Homo sapiens]
Protein Accession gi|4557014
5.3 myoglobin [Homo sapiens]; myoglobin [Homo sapiens]; myoglobin [Homo sapiens]
Protein Accession gi|4885477 gi|44955888 gi|44955885
5.4 alpha 1 globin [Homo sapiens]; alpha 2 globin [Homo sapiens]
Protein Accession gi|4504347 gi|4504345
5.5 hemopexin [Homo sapiens]
Protein Accession gi|11321561
5.6 serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]; serine proteinase inhibitor, clade A, member 1 [Homo sapiens]
Protein Accession gi|50363221 gi|50363219 gi|50363217 gi|189163542 gi|189163540 gi|189163538 gi|189163536
5.7 mucin 1 isoform 3 precursor [Homo sapiens]; mucin 1 isoform 2 precursor [Homo sapiens]; mucin 1 isoform 1 precursor [Homo sapiens]
Protein Accession gi|67189069 gi|67189007 gi|65301117
5.8 serpin peptidase inhibitor, clade A, member 3 precursor [Homo sapiens]
Protein Accession gi|50659080
5.9 CD14 antigen precursor [Homo sapiens]; CD14 antigen precursor [Homo sapiens]
Protein Accession gi|91105159 gi|4557417
5.10 brain abundant, membrane attached signal protein 1 [Homo sapiens]
Protein Accession gi|30795231
5.11 dipeptidase 1 [Homo sapiens]; dipeptidase 1 [Homo sapiens]
Protein Accession gi|4758190 gi|89458885
5.12 fibronectin 1 isoform 5 preproprotein [Homo sapiens]
Protein Accession gi|47132553
5.13 angiotensinogen preproprotein [Homo sapiens]
Protein Accession gi|4557287
5.14 complement component 3 precursor [Homo sapiens]
Protein Accession gi|115298678
5.15 carbonic anhydrase I [Homo sapiens]; carbonic anhydrase I [Homo sapiens]; carbonic anhydrase I [Homo sapiens]; carbonic anhydrase I [Homo sapiens]
Protein Accession gi|4502517 gi|92447434 gi|92447432 gi|92447430
5.16 uromodulin precursor [Homo sapiens]; uromodulin precursor [Homo sapiens]
Protein Accession gi|59850812 gi|56550049
5.17 heparan sulfate proteoglycan 2 [Homo sapiens]
Protein Accession gi|126012571
5.18 clusterin isoform 1 [Homo sapiens]
Protein Accession gi|42716297
5.19 ezrin [Homo sapiens]; ezrin [Homo sapiens]
Protein Accession gi|21614499 gi|161702986
5.20 delta globin [Homo sapiens]
Protein Accession gi|4504351
5.21 fibrinogen, beta chain preproprotein [Homo sapiens]
Protein Accession gi|70906435
5.22 moesin [Homo sapiens]
Protein Accession gi|4505257
5.23 transferrin [Homo sapiens]
Protein Accession gi|4557871
5.24 secreted phosphoprotein 1 isoform c [Homo sapiens]; secreted phosphoprotein 1 isoform a [Homo sapiens]; secreted phosphoprotein 1 isoform b [Homo sapiens]
Protein Accession gi|91598939 gi|91206462 gi|4759166
5.25 pancreatic amylase alpha 2A precursor [Homo sapiens]
Protein Accession gi|4502085
5.26 annexin A2 isoform 1 [Homo sapiens]
Protein Accession gi|50845388
5.27 complement component 4 binding protein, alpha chain precursor [Homo sapiens]
Protein Accession gi|4502503
5.28 albumin preproprotein [Homo sapiens]
Protein Accession gi|4502027
5.29 epidermal growth factor receptor pathway substrate 8 [Homo sapiens]
Protein Accession gi|56682953
5.30 apolipoprotein B precursor [Homo sapiens]
Protein Accession gi|105990532
5.31 peroxiredoxin 2 isoform a [Homo sapiens]
Protein Accession gi|32189392
5.32 sphingomyelin phosphodiesterase 1, acid lysosomal isoform 1 precursor [Homo sapiens]
Protein Accession gi|56117840
5.33 proteasome beta 2 subunit [Homo sapiens]
Protein Accession gi|4506195
5.34 complement component 4B preproprotein [Homo sapiens]
Protein Accession gi|178557739
5.35 manganese superoxide dismutase isoform A precursor [Homo sapiens];
manganese superoxide dismutase isoform A precursor [Homo sapiens]
Protein Accession gi|67782307 gi|67782305
5.36 transthyretin [Homo sapiens]
Protein Accession gi|4507725
5.37 haptoglobin isoform 1 preproprotein [Homo sapiens]; haptoglobin isoform 2 preproprotein [Homo sapiens]
Protein Accession gi|4826762 gi|186910296
5.38 fibulin 1 isoform D [Homo sapiens]
Protein Accession gi|34734066
5.39 serine (or cysteine) proteinase inhibitor, clade C (antithrombin), member 1
[Homo sapiens]
Protein Accession gi|4502261
5.40 hypothetical protein LOC148362 [Homo sapiens]
Protein Accession gi|124244088
5.41 ferritin, heavy polypeptide 1 [Homo sapiens]
Protein Accession gi|56682959
5.42 serum amyloid A1 preproprotein [Homo sapiens]; serum amyloid A1
preproprotein [Homo sapiens]
Protein Accession gi|40316912 gi|40316910
5.43 programmed cell death 6 [Homo sapiens]
Protein Accession gi|7019485
5.44 complement component 1 inhibitor precursor [Homo sapiens]; complement component 1 inhibitor precursor [Homo sapiens]
Protein Accession gi|73858570 gi|73858568
5.45 chromatin modifying protein 5 [Homo sapiens]
Protein Accession gi|189409150
5.46 vacuolar protein sorting factor 4B [Homo sapiens]
Protein Accession gi|17865802
5.47 prominin 1 [Homo sapiens]
Protein Accession gi|5174387
5.48 alpha 3 type VI collagen isoform 1 precursor [Homo sapiens]
Protein Accession gi|55743098
5.49 actin, gamma 1 propeptide [Homo sapiens]; beta actin [Homo sapiens]
Protein Accession gi|4501887 gi|4501885
5.50 solute carrier family 5 (sodium/glucose cotransporter), member 12 [Homo sapiens]
Protein Accession gi|157671931
5.51 vacuolar protein sorting 25 [Homo sapiens]
Protein Accession gi|14150155
5.52 annexin A11 [Homo sapiens]; annexin A11 [Homo sapiens]; annexin A11 [Homo sapiens]
Protein Accession gi|4557317 gi|22165433 gi|22165431
5.53 chromatin modifying protein 4B [Homo sapiens]
Protein Accession gi|28827795
5.54 fatty acid binding protein 4, adipocyte [Homo sapiens]
Protein Accession gi|4557579
5.55 chloride intracellular channel 6 [Homo sapiens]
Protein Accession gi|27894378
5.56 proteasome alpha 3 subunit isoform 1 [Homo sapiens]; proteasome alpha 3 subunit isoform 2 [Homo sapiens]
Protein Accession gi|4506183 gi|23110939
5.57 gelsolin isoform a precursor [Homo sapiens]; gelsolin isoform b [Homo sapiens]; gelsolin isoform c [Homo sapiens]; gelsolin isoform c [Homo sapiens]; gelsolin isoform b [Homo sapiens]; gelsolin isoform b [Homo sapiens]; gelsolin isoform b [Homo sapiens]; gelsolin isoform b [Homo sapiens]
Protein Accession gi|4504165 gi|38044288 gi|89083782 gi|89083780 gi|89083778 gi|189083776 gi|189083774
5.58 vacuolar protein sorting 37B [Homo sapiens]
Protein Accession gi|13375926
5.59 programmed cell death 6 interacting protein [Homo sapiens]
Protein Accession gi|22027538
5.60 prosaposin isoform a preproprotein [Homo sapiens]
Protein Accession gi|11386147
5.61 ferritin, light polypeptide [Homo sapiens]
Protein Accession gi|20149498
5.62 galectin 3 binding protein [Homo sapiens]
Protein Accession gi|5031863
5.63 peptidoglycan recognition protein 1 [Homo sapiens]
Protein Accession gi|4827036
5.64 ubiquitin and ribosomal protein S27a precursor [Homo sapiens]; ubiquitin and ribosomal protein S27a precursor [Homo sapiens]
Protein Accession gi|4506713 gi|208022622
5.65 complement component 4A preproprotein [Homo sapiens]
Protein Accession gi|67190748
5.66 hypothetical protein LOC169693 [Homo sapiens]
Protein Accession gi|23397518
5.67 nidogen 1 precursor [Homo sapiens]
Protein Accession gi|115298674
5.68 CD9 antigen [Homo sapiens]
Protein Accession gi|4502693
5.69 solute carrier family 5 (sodium/glucose cotransporter), member 10 isoform 2 [Homo sapiens]
Protein Accession gi|109659836
5.70 sorting nexin 18 isoform a [Homo sapiens]; sorting nexin 18 isoform b 30 [Homo sapiens]
5.71 syntenin isoform 1 [Homo sapiens]; syntenin isoform 1 [Homo sapiens]
Protein Accession gi|56243522 gi|55749490
5.72 glutamyl aminopeptidase (aminopeptidase A) [Homo sapiens]
Protein Accession gi|132814467
5.73 histone cluster 2, H4b [Homo sapiens]; histone cluster 2, H4a [Homo sapiens]; histone cluster 1, H4i [Homo sapiens]; histone cluster 1, H41 [Homo sapiens]; histone cluster 1, H4e [Homo sapiens]; histone cluster 1, H4b [Homo sapiens]; histone cluster 1, H4h [Homo sapiens]; histone cluster 1, H4c [Homo sapiens]; histone cluster 1, H4k [Homo sapiens]; histone cluster 1, H4f [Homo sapiens]; histone cluster 1, H4d [Homo sapiens]; histone cluster 1, H4a [Homo sapiens]; histone cluster 4, H4 [Homo sapiens]; histone cluster 1, H4j [Homo sapiens]
Protein Accession gi|77539758 gi|4504323 gi|4504321 gi|4504317 gi|4504315 gi|4504313 gi|4504311 gi|4504309
5.74 guanine nucleotide binding protein (G protein), q polypeptide [Homo sapiens]
Protein Accession gi|40254462
5.75 syndecan 1 precursor [Homo sapiens]; syndecan 1 precursor [Homo sapiens]
Protein Accession gi|55749480 gi|29568086
5.76 copine III [Homo sapiens]
Protein Accession gi|4503015
5.77 annexin VII isoform 2 [Homo sapiens]; annexin VII isoform 1 [Homo sapiens]
Protein Accession gi|4809279 gi|4502111
5.78 cubilin [Homo sapiens]
Protein Accession gi|126091152
5.79 guanine nucleotide binding protein (G protein), alpha 11 (Gq class) [Homo sapiens]
Protein Accession gi|115511049
5.80 proteasome alpha 1 subunit isoform 2 [Homo sapiens]; proteasome alpha 1 subunit isoform 1 [Homo sapiens]
Protein Accession gi|4506179 gi|23110935
5.81 myeloperoxidase [Homo sapiens]
Protein Accession gi|4557759
5.82 solute carrier family 5 (iodide transporter), member 8 [Homo sapiens]
Protein Accession gi|167466278
5.83 ras homolog gene family, member C precursor [Homo sapiens]; ras homolog gene family, member C precursor [Homo sapiens]; ras homolog gene family, member C precursor [Homo sapiens]; ras homolog gene family, member A [Homo sapiens]
Protein Accession gi|28395033 gi|11494251 gi|11494248 gi|0835049
5.84 complement factor H isoform a precursor [Homo sapiens]
Protein Accession gi|62739186
5.85 flotillin 1 [Homo sapiens]
Protein Accession gi|5031699
5.86 hypothetical protein LOC91894 [Homo sapiens]
Protein Accession gi|131889517
5.87 ring finger protein 152 [Homo sapiens]
Protein Accession gi|27734873
5.88 WIRE protein [Homo sapiens]
Protein Accession gi|18959210
5.89 putative MAPK activating protein PM28 [Homo sapiens]
Protein Accession gi|41281489
5.90 flotillin 2 [Homo sapiens]
Protein Accession gi|94538362
5.91 ephrin receptor EphA1 [Homo sapiens]
Protein Accession gi|56119207
5.92 proteasome beta 3 subunit [Homo sapiens]
Protein Accession gi|22538465
5.93 cathepsin D preproprotein [Homo sapiens]
Protein Accession gi|4503143
5.94 hypothetical protein LOC58527 [Homo sapiens]
Protein Accession gi|24308273
5.95 annexin 5 [Homo sapiens]
Protein Accession gi|4502107
5.96 carboxypeptidase M precursor [Homo sapiens]; carboxypeptidase M precursor [Homo sapiens]; carboxypeptidase M precursor [Homo sapiens]
Protein Accession gi|6631081 gi|53832021 gi|38327526
5.97 profilin 1 [Homo sapiens]
Protein Accession gi|4826898
5.98 cortactin isoform b [Homo sapiens]; cortactin isoform a [Homo sapiens]
Protein Accession gi|20357556 gi|20357552
5.99 solute carrier family 3, member 1 [Homo sapiens]
Protein Accession gi|187423904
5.100 cell division cycle 42 isoform 1 [Homo sapiens]; cell division cycle 42 isoform 1 [Homo sapiens]
Protein Accession gi|89903012 gi|4757952
5.101 CD59 antigen preproprotein [Homo sapiens]; CD59 antigen preproprotein 15 [Homo sapiens]; CD59 antigen preproprotein [Homo sapiens]; CD59 antigen preproprotein [Homo sapiens]; CD59 antigen preproprotein [Homo sapiens]; CD59 antigen preproprotein [Homo sapiens]; CD59 antigen preproprotein [Homo sapiens]; CD59 antigen preproprotein [Homo sapiens]
Protein Accession gi|42761474 gi|42716302 gi|42716299 gi|187829185 gi|187829183 gi|187829070 gi|187828910
5.102 amnionless protein precursor [Homo sapiens]
Protein Accession gi|110611172
5.103 von Willebrand factor A domain containing 1 isoform 1 [Homo sapiens]
Protein Accession gi|40068485
5.104 vacuolar protein sorting 28 isoform 1 [Homo sapiens]
Protein Accession gi|7705885
5.105 mannosidase, alpha, class 2B, member 1 precursor [Homo sapiens]
Protein Accession gi|51873064
5.106 chloride intracellular channel 1 [Homo sapiens]
Protein Accession gi|14251209
5.107 complement factor D preproprotein [Homo sapiens]
Protein Accession gi|42544239
5.108 scavenger receptor class B, member 2 [Homo sapiens]
Protein Accession gi|5031631
5.109 plasminogen [Homo sapiens]
Protein Accession gi|4505881
5.110 ficolin 2 isoform a precursor [Homo sapiens]
Protein Accession gi|61744445
5.111 glycophorin C isoform 2 [Homo sapiens]; glycophorin C isoform 1 [Homo sapiens]
Protein Accession gi|8051605 gi|4504229
5.112 prolylcarboxypeptidase isoform 1 preproprotein [Homo sapiens]; prolylcarboxypeptidase isoform 2 preproprotein [Homo sapiens]
Protein Accession gi|4826940 gi|117306169
5.113 collagen, type VI, alpha 1 precursor [Homo sapiens]
Protein Accession gi|87196339
5.114 calmodulin 1 [Homo sapiens]; calmodulin 3 [Homo sapiens]; calmodulin 2 [Homo sapiens]
Protein Accession gi|5901912 gi|58218968 gi|4502549
5.115 annexin I [Homo sapiens]
Protein Accession gi|4502101
5.116 ras homolog gene family, member G [Homo sapiens]
Protein Accession gi|46249393
5.117 immunoglobulin superfamily, member 8 [Homo sapiens]
5.118 Fc fragment of IgG binding protein [Homo sapiens]
Protein Accession gi|154146262
5.119 hypothetical protein LOC284422 [Homo sapiens]
Protein Accession gi|211059425
5.120 vacuolar protein sorting 37D [Homo sapiens]
Protein Accession gi|117938318
5.121 protease, serine, 1 preproprotein [Homo sapiens]
Protein Accession gi|4506145
5.122 prominin 2 [Homo sapiens]
Protein Accession gi|21389623
5.123 cathepsin B preproprotein [Homo sapiens]; cathepsin B preproprotein [Homo sapiens]; cathepsin B preproprotein [Homo sapiens]; cathepsin B preproprotein [Homo sapiens]; cathepsin B preproprotein [Homo sapiens]
Protein Accession gi|4503139 gi|22538437 gi|22538435 gi|22538433 gi|22538431
5.124 radixin [Homo sapiens]
Protein Accession gi|4506467
5.125 annexin VI isoform 2 [Homo sapiens]; annexin VI isoform 1 [Homo sapiens]
Protein Accession gi|71773415 gi|71773329
5.126 cystatin C precursor [Homo sapiens]
Protein Accession gi|4503107
5.127 tweety 3 [Homo sapiens]
Protein Accession gi|51100978
5.128 hypothetical protein LOC84418 [Homo sapiens]
Protein Accession gi|14165278
5.129 CD82 antigen isoform 2 [Homo sapiens]; CD82 antigen isoform 1 [Homo sapiens]
Protein Accession gi|67782354 gi|4504813
5.130 napsin A preproprotein [Homo sapiens]
Protein Accession gi|4758754
5.131 acid phosphatase 2, lysosomal isoform 1 precursor [Homo sapiens]
Protein Accession gi|4557010
5.132 apolipoprotein E precursor [Homo sapiens]
Protein Accession gi|4557325
5.133 transmembrane protein 176B isoform b [Homo sapiens]; transmembrane protein 176B isoform a [Homo sapiens]; transmembrane protein 176B isoform a [Homo sapiens]; transmembrane protein 176B isoform a [Homo sapiens]; transmembrane protein 176B isoform a [Homo sapiens]
Protein Accession gi|156416022 gi|56416020 gi|156416018 gi|156416016 gi|156416014
5.134 GM2 ganglioside activator precursor [Homo sapiens]
Protein Accession gi|39995109
5.135 mercaptopyruvate sulfurtransferase isoform 2 [Homo sapiens]; mercaptopyruvate sulfurtransferase isoform 2 [Homo sapiens]; mercaptopyruvate sulfurtransferase isoform 1 [Homo sapiens]
Protein Accession gi|61835204 gi|194473681 gi|194473668
5.136 mannosidase, alpha, class 1A, member 1 [Homo sapiens]
Protein Accession gi|24497519
5.137 integral membrane protein 2B [Homo sapiens]
Protein Accession gi|11527402
5.138 tetraspan 1 [Homo sapiens]
Protein Accession gi|21264578
5.139 apolipoprotein D precursor [Homo sapiens]
Protein Accession gi|4502163
5.140 stomatin isoform a [Homo sapiens]
Protein Accession gi|38016911
5.141 eukaryotic translation initiation factor 6 isoform c [Homo sapiens]
Protein Accession gi|31563374
5.142 kininogen 1 isoform 2 [Homo sapiens]
Protein Accession gi|4504893
5.143 syntenin isoform 3 [Homo sapiens]; syntenin isoform 3 [Homo sapiens]
Protein Accession gi|55749523 gi|55749515
5.144 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 2 [Homo sapiens]
Protein Accession gi|4504041
5.145 guanine nucleotide binding protein, alpha z polypeptide [Homo sapiens]
Protein Accession gi|4504051
5.146 cytochrome P450, family 4, subfamily A, polypeptide 11 [Homo sapiens]
Protein Accession gi|158937242
5.147 olfactomedin 4 precursor [Homo sapiens]
Protein Accession gi|32313593
5.148 peptidoglycan recognition protein 2 precursor [Homo sapiens]
Protein Accession gi|56616294
5.149 SMAD family member 9 isoform b [Homo sapiens]; SMAD family member 9 isoform a [Homo sapiens]
Protein Accession gi|5174519 gi 187828357
5.150 brain creatine kinase [Homo sapiens]
Protein Accession gi|21536286
5.151 chromatin modifying protein 2A [Homo sapiens]; chromatin modifying protein 2A [Homo sapiens]
Protein Accession gi|7656922 gi|38372933
5.152 solute carrier family 4, sodium bicarbonate cotransporter, member 4 isoform 2 [Homo sapiens]
Protein Accession gi|4507025
5.153 phospholipid transfer protein isoform a precursor [Homo sapiens]
Protein Accession gi|5453914
5.154 ceruloplasmin precursor [Homo sapiens]
Protein Accession gi|4557485
5.155 glycerophosphodiester phosphodiesterase domain containing 3 [Homo sapiens]
Protein Accession gi|146198640
5.156 glucuronidase, beta [Homo sapiens]
Protein Accession gi|4504223
5.157 solute carrier family 4, anion exchanger, member 1 [Homo sapiens]
Protein Accession gi|4507021
5.158 arylsulfatase A isoform a precursor [Homo sapiens]; arylsulfatase A isoform a precursor [Homo sapiens]; arylsulfatase A isoform a precursor [Homo sapiens]; arylsulfatase A isoform b [Homo sapiens]; arylsulfatase A isoform a precursor [Homo sapiens]
Protein Accession gi|6005990 gi|146229331 gi|146229329 gi|146229327 gi|146229324
5.159 guanine nucleotide binding protein (G protein) alpha 12 [Homo sapiens]
Protein Accession gi|42476111
5.160 solute carrier family 1, member 1 [Homo sapiens]
Protein Accession gi|66773030
5.161 keratin 9 [Homo sapiens]
Protein Accession gi|55956899
5.162 pyruvate kinase, muscle isoform M2 [Homo sapiens]
Protein Accession gi|33286418
5.163 solute carrier family 13 member 3 isoform b [Homo sapiens]; solute carrier family 13 member 3 isoform a [Homo sapiens]
Protein Accession gi|58761541 gi|31377715
5.164 GTPase Rab14 [Homo sapiens]
Protein Accession gi|19923483
5.165 coagulation factor II preproprotein [Homo sapiens]
Protein Accession gi|4503635
5.166 proteasome alpha 6 subunit [Homo sapiens]
Protein Accession gi|23110944
5.167 tubulin, beta, 2 [Homo sapiens]
Protein Accession gi|5174735
5.168 solute carrier family 6, member 19 [Homo sapiens]
Protein Accession gi|51468073
5.169 decay accelerating factor for complement isoform 2 precursor [Homo sapiens]
Protein Accession gi|168693643
5.170 membrane alanine aminopeptidase precursor [Homo sapiens]
Protein Accession gi|157266300
5.171 transmembrane protein 9 [Homo sapiens]
Protein Accession gi|7705999
5.172 toll interacting protein [Homo sapiens]
Protein Accession gi|21361619
5.173 arylsulfatase F precursor [Homo sapiens]
Protein Accession gi|31742482
5.174 calcium binding protein 39 [Homo sapiens]; calcium binding protein 39 [Homo sapiens]; calcium binding protein 39 [Homo sapiens]
Protein Accession gi|7706481 gi|195927022 gi|195927020
5.175 hemicentin 1 [Homo sapiens]
Protein Accession gi|118572606
5.176 prostate stem cell antigen preproprotein [Homo sapiens]
Protein Accession gi|5031995
5.177 solute carrier family 47, member 1 [Homo sapiens]
Protein Accession gi|22907060
5.178 polymeric immunoglobulin receptor [Homo sapiens]
Protein Accession gi|31377806
5.179 cathepsin C isoform a preproprotein [Homo sapiens]
Protein Accession gi|189083844
5.180 biliverdin reductase B (flavin reductase (NADPH)) [Homo sapiens]
Protein Accession gi|4502419
5.181 prostatic binding protein [Homo sapiens]
Protein Accession gi|4505621
5.182 proteasome alpha 4 subunit isoform 1 [Homo sapiens]; proteasome alpha 4 subunit isoform 1 [Homo sapiens]
Protein Accession gi|4506185 gi|156713442
5.183 palate, lung and nasal epithelium associated precursor [Homo sapiens]; palate, lung and nasal epithelium associated precursor [Homo sapiens]
Protein Accession gi|7706119 gi|18765705
5.184 vacuolar protein sorting factor 4A [Homo sapiens]
Protein Accession gi|7019569
5.185 transmembrane protein 192 [Homo sapiens]
Protein Accession gi|154240704
5.186 vitronectin precursor [Homo sapiens]
Protein Accession gi|88853069
5.187 von Willebrand factor preproprotein [Homo sapiens]
Protein Accession gi|89191868
5.188 tripartite motif protein TRIM14 isoform alpha [Homo sapiens]; tripartite motif protein TRIM14 isoform alpha [Homo sapiens]; tripartite motif protein TRIM14 isoform alpha [Homo sapiens]
Protein Accession gi|15208667 gi|15208665 gi|15208663
5.189 PREDICTED: similar to hCG1642538 [Homo sapiens]; PREDICTED: similar to hCG1642538 [Homo sapiens]
Protein Accession gi|169216113 gi|169215489
5.190 actin, alpha 1, skeletal muscle [Homo sapiens]
Protein Accession gi|4501881
5.191 RAP2B, member of RAS oncogene family [Homo sapiens]
Protein Accession gi|38201690
5.192 ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) [Homo sapiens]
Protein Accession gi|7662358
5.193 transmembrane BAX inhibitor motif containing 1 [Homo sapiens]
Protein Accession gi|50593008
5.194 mucolipin 1 [Homo sapiens]
Protein Accession gi|10092597
5.195 annexin IV [Homo sapiens]
Protein Accession gi|4502105
5.196 PREDICTED: similar to kappa immunoglobulin (subgroup V kappa I) [Homo sapiens]
Protein Accession gi|169218204
5.197 diazepam binding inhibitor isoform 2 [Homo sapiens]; diazepam binding inhibitor isoform 3 [Homo sapiens]; diazepam binding inhibitor isoform 1 [Homo sapiens]
Protein Accession gi|120433593 gi|120433590 gi|10140853
5.198 CD63 antigen isoform B [Homo sapiens]; CD63 antigen isoform A [Homo sapiens]
Protein Accession gi|91199546 gi|4502679
5.199 tubulin, alpha 4a [Homo sapiens]
Protein Accession gi|17921989
5.200 MARVEL domain containing 3 isoform 1 [Homo sapiens]
Protein Accession gi|65301122
5.201 sialin [Homo sapiens]
Protein Accession gi|6912666
5.202 solute carrier family 1 (neutral amino acid transporter), member 5 [Homo sapiens]
Protein Accession gi|5032093
5.203 claudin 2 [Homo sapiens]
Protein Accession gi|9966781
5.204 myoferlin isoform a [Homo sapiens]; myoferlin isoform b [Homo sapiens]
Protein Accession gi|7305053 gi|9718759
5.205 tubulin, alpha 1a [Homo sapiens]
Protein Accession gi|17986283
5.206 PDZ domain containing 1 [Homo sapiens]
Protein Accession gi|21361142
5.207 deleted in malignant brain tumors 1 isoform c precursor [Homo sapiens]; deleted in malignant brain tumors 1 isoform b precursor [Homo sapiens]
Protein Accession gi|148539844 gi|148539842
5.208 galactosylceramidase isoform a precursor [Homo sapiens]
Protein Accession gi|83281450
5.209 target of myb1 isoform 1 [Homo sapiens]; target of myb1 isoform 2 [Homo sapiens]
Protein Accession gi|4885637 gi|209180457
5.210 hypothetical protein LOC55194 [Homo sapiens]; PREDICTED: hypothetical protein [Homo sapiens]
Protein Accession gi|8922567 gi|169160626
5.211 cystin 1 [Homo sapiens]
Protein Accession gi|81158080
5.212 CD151 antigen [Homo sapiens]; CD151 antigen [Homo sapiens]; CD151 antigen [Homo sapiens]; CD151 antigen [Homo sapiens]
Protein Accession gi|87159824 gi|87159822 gi|21237751 gi|21237748
5.213 RAB2A, member RAS oncogene family [Homo sapiens]
Protein Accession gi|4506365
5.214 CNDP dipeptidase 2 [Homo sapiens]
Protein Accession gi|8922699
5.215 syntaxin binding protein 2 isoform b [Homo sapiens]; syntaxin binding protein 2 isoform a [Homo sapiens]
Protein Accession gi|188528901 gi|188528689
5.216 actinin, alpha 4 [Homo sapiens]
Protein Accession gi|12025678
5.217 matrix metalloproteinase 7 preproprotein [Homo sapiens]
Protein Accession gi|4505219
5.218 keratin 1 [Homo sapiens]
Protein Accession gi|119395750
5.219 lactotransferrin precursor [Homo sapiens]
Protein Accession gi|54607120
5.220 tumor differentially expressed protein 1 [Homo sapiens]; tumor differentially expressed protein 1 [Homo sapiens]
Protein Accession gi|5803193 gi|39812106
5.221 phosphodiesterase 8A isoform 2 [Homo sapiens]; phosphodiesterase 8A isoform 1 [Homo sapiens]
Protein Accession gi|47132537 gi|27734721
5.222 SH3 domain and tetratricopeptide repeats 1 [Homo sapiens]
Protein Accession gi|1145386551
5.223 aminoacylase 1 [Homo sapiens]
Protein Accession gi|4501901
5.224 cathepsin A isoform a precursor [Homo sapiens]
Protein Accession gi|119395729
5.225 pleckstrin homology domain containing, family B (evectins) member 2 isoform 3 [Homo sapiens]; pleckstrin homology domain containing, family B (evectins) member 2 isoform 2 [Homo sapiens]
Protein Accession gi 154800478 gi|54800476
5.226 BH3 interacting domain death agonist isoform 2 [Homo sapiens]; BH3 interacting domain death agonist isoform 3 [Homo sapiens]; BH3 interacting domain death agonist isoform 1 [Homo sapiens]
Protein Accession gi|4557361 gi|37574728 gi|37574726
5.227 enolase 1 [Homo sapiens]
Protein Accession gi|4503571
5.228 hypothetical protein LOC64855 isoform 2 [Homo sapiens]; hypothetical protein LOC64855 isoform 1 [Homo sapiens]
Protein Accession gi|79750824 gi|51093863
5.229 ribophorin II isoform 2 precursor [Homo sapiens]
Protein Accession gi|209413738
5.230 urate anion exchanger 1 isoform a [Homo sapiens]
Protein Accession gi|24497485
5.231 chromatin modifying protein 4C [Homo sapiens]
Protein Accession gi|22748643
5.232 sacsin [Homo sapiens]
Protein Accession gi|163659918
5.233 lumican precursor [Homo sapiens]
Protein Accession gi|4505047
5.234 chromatin modifying protein 1B [Homo sapiens]
Protein Accession gi|31542306
5.235 SHC (Src homology 2 domain containing) transforming protein 1 isoform 2 [Homo sapiens]; SHC (Src homology 2 domain containing) transforming protein 1 isoform 3 [Homo sapiens]
Protein Accession gi|32261324 gi|194239664
5.236 hypothetical protein LOC28970 [Homo sapiens]
Protein Accession gi|21361495
5.237 ATPase, H+ transporting, lysosomal V0 subunit a4 [Homo sapiens]; ATPase, H+ transporting, lysosomal V0 subunit a4 [Homo sapiens]; ATPase, H+ transporting, lysosomal V0 subunit a4 [Homo sapiens]
Protein Accession gi|85386547 gi|85386056 gi|85386053
5.238 chloride channel 7 isoform b [Homo sapiens]; chloride channel 7 isoform a [Homo sapiens]
Protein Accession gi|167466160 gi|14149607
5.239 solute carrier family 34 (sodium phosphate), member 2 [Homo sapiens]
Protein Accession gi|110611906
5.240 UBX domain protein 6 [Homo sapiens]
Protein Accession gi|13376854
5.241 tetratricopeptide repeat domain 38 [Homo sapiens]
5.242 crystallin, alpha B [Homo sapiens]
Protein Accession gi|4503057
5.243 dual oxidase 2 precursor [Homo sapiens]
Protein Accession gi|132566532
5.244 DnaJ (Hsp40) homolog, subfamily C, member 5 [Homo sapiens]
Protein Accession gi|45504382
5.245 vacuolar H+ATPase B2 [Homo sapiens]
Protein Accession gi|19913428
5.246 angiotensin I converting enzyme 2 precursor [Homo sapiens]
Protein Accession gi|11225609
5.247 aquaporin 1 [Homo sapiens]
Protein Accession gi|37694062
5.248 secretoglobin, family 1A, member 1 (uteroglobin) [Homo sapiens]
Protein Accession gi|4507809
5.249 structural maintenance of chromosomes 2 [Homo sapiens]; structural maintenance of chromosomes 2 [Homo sapiens]; structural maintenance of chromosomes 2 [Homo sapiens]
Protein Accession gi 110347425 gi 110347420 gi|110347418
5.250 superoxide dismutase 1, soluble [Homo sapiens]
Protein Accession gi|4507149
5.251 phospholipid scramblase 1 [Homo sapiens]
Protein Accession gi|10863877
5.252 ATPase, H+ transporting, lysosomal V0 subunit al isoform c [Homo sapiens]; ATPase, H+ transporting, lysosomal V0 subunit al isoform b [Homo sapiens]; ATPase, H+ transporting, lysosomal V0 subunit al isoform a [Homo sapiens]
Protein Accession gi|19913418 gi|194097403 gi|194097401
5.253 solute carrier family 44, member 2 [Homo sapiens]
Protein Accession gi|31377727
5.254 guanine nucleotide binding protein (G protein), alpha inhibiting activity polypeptide 3 [Homo sapiens]
Protein Accession gi|5729850
5.255 PREDICTED: hypothetical protein [Homo sapiens]
Protein Accession gi|1169217813
5.256 dipeptidyl peptidase 7 preproprotein [Homo sapiens]
Protein Accession gi|62420888
5.257 tropomyosin 4 [Homo sapiens]
Protein Accession gi|4507651
5.258 keratin 10 [Homo sapiens]
Protein Accession gi|195972866
5.259 myosin VI [Homo sapiens]
Protein Accession gi|92859701
5.260 heat shock protein 90 kDa alpha (cytosolic), class A member 1 isoform 1 [Homo sapiens]
Protein Accession gi|153792590
5.261 RAB1A, member RAS oncogene family isoform 1 [Homo sapiens]; RAB1B, member RAS oncogene family [Homo sapiens]
Protein Accession gi|4758988 gi 13569962
5.262 villin 1 [Homo sapiens]
Protein Accession gi|194394237
5.263 PREDICTED: similar to hCG1992647, partial [Homo sapiens]
Protein Accession gi|169217452
5.264 aldehyde dehydrogenase 1A1 [Homo sapiens]
Protein Accession gi|21361176
5.265 phosphoglycerate kinase 1 [Homo sapiens]
Protein Accession gi|4505763
5.266 eukaryotic translation initiation factor 5A isoform A [Homo sapiens]
Protein Accession gi|219555707
5.267 heat shock 70 kDa protein 1A [Homo sapiens]; heat shock 70 kDa protein 1B [Homo sapiens]
Protein Accession gi 194248072 gi|167466173
5.268 hypothetical protein LOC126321 isoform b [Homo sapiens]; hypothetical protein LOC126321 isoform c [Homo sapiens]; hypothetical protein LOC126321 isoform a [Homo sapiens]
Protein Accession gi|111378395 gi|111378391 gi|111378386
5.269 PDZK1 interacting protein 1 [Homo sapiens]
Protein Accession gi|5031657
5.270 hexosaminidase A preproprotein [Homo sapiens]
Protein Accession gi|189181666
5.271 paralemmin isoform 1 [Homo sapiens]
Protein Accession gi|93141031
5.272 mannosidase, alpha, class 2B, member 2 [Homo sapiens]
Protein Accession gi|50659093
5.273 CD74 antigen isoform a [Homo sapiens]; CD74 antigen isoform c [Homo sapiens]; CD74 antigen isoform b [Homo sapiens]
Protein Accession gi|68448544 gi|68448537 gi|10835071
5.274 dipeptidylpeptidase IV [Homo sapiens]
Protein Accession gi|18765694
5.275 lipocalin 2 [Homo sapiens]
Protein Accession gi|38455402
5.276 zinc finger protein 262 [Homo sapiens]
Protein Accession gi|44890068
5.277 angiotensin I converting enzyme isoform 1 precursor [Homo sapiens]
Protein Accession gi|4503273
5.278 skeletal muscle receptor tyrosine kinase [Homo sapiens]
Protein Accession gi|5031927
5.279 spastin isoform 2 [Homo sapiens]; spastin isoform 1 [Homo sapiens]
Protein Accession gi|40806170 gi|11875211
5.280 allograft inflammatory factor 1 isoform 3 [Homo sapiens]
Protein Accession gi|14574568
5.281 carbonyl reductase 1 [Homo sapiens]
Protein Accession gi|4502599
5.282 sodium/hydrogen exchanger regulatory factor 1 [Homo sapiens]
Protein Accession gi|4759140
5.283 argininosuccinate synthetase 1 [Homo sapiens]; argininosuccinate synthetase 1 [Homo sapiens]
Protein Accession gi|53759107 gi|16950633
5.284 spinster homolog 1 isoform 1 [Homo sapiens]; spinster homolog 1 isoform 1 [Homo sapiens]
Protein Accession gi|215490096 gi|14042968
5.285 quinolinate phosphoribosyltransferase [Homo sapiens]
Protein Accession gi|45269149
5.286 transmembrane protein 106A [Homo sapiens]; PREDICTED: similar to Transmembrane protein 106A [Homo sapiens]; PREDICTED: similar to Transmembrane protein 106A isoform 3 [Homo sapiens]
Protein Accession gi|21450796 gi|169211133 gi 113427131
5.287 uroplakin 1A [Homo sapiens]
Protein Accession gi|5902148
5.288 secretory carrier membrane protein 3 isoform 1 [Homo sapiens]
Protein Accession gi|16445419
5.289 guanine nucleotide binding protein (G protein), gamma 2 [Homo sapiens]
Protein Accession gi|54114974
5.290 sorbitol dehydrogenase [Homo sapiens]
Protein Accession gi|156627571
5.291 major facilitator superfamily domain containing 1 [Homo sapiens]
Protein Accession gi|112232393
5.292 superoxide dismutase 3, extracellular precursor [Homo sapiens]
Protein Accession gi|118582275
5.293 nuclear transport factor 2 [Homo sapiens]
Protein Accession gi|5031985
5.294 hexosaminidase B preproprotein [Homo sapiens]
Protein Accession gi|4504373
5.295 nucleoporin 188 kDa [Homo sapiens]
Protein Accession gi|62955803
5.296 hypothetical protein LOC54978 [Homo sapiens]
Protein Accession gi|31542711
5.297 disrupted in renal carcinoma 2 [Homo sapiens]
Protein Accession gi|14249552
5.298 adenine phosphoribosyltransferase isoform b [Homo sapiens]
Protein Accession gi|71773201
5.299 GNAS complex locus XLas [Homo sapiens]
Protein Accession gi|117938759
5.300 A kinase (PRKA) anchor protein 12 isoform 2 [Homo sapiens]
Protein Accession gi|21493024
5.301 orosomucoid 1 precursor [Homo sapiens]
Protein Accession gi|167857790
5.302 lysozyme precursor [Homo sapiens]
Protein Accession gi|4557894
5.303 vacuolar protein sorting 37C [Homo sapiens]
Protein Accession gi|57863314
5.304 NAD(P)H dehydrogenase, quinone 2 [Homo sapiens]
Protein Accession gi|156564357
5.305 cystatin B [Homo sapiens]
Protein Accession gi|4503117
5.306 triosephosphate isomerase 1 [Homo sapiens]
Protein Accession gi|4507645
5.307 nucleoside phosphorylase [Homo sapiens]
Protein Accession gi|157168362
5.308 guanine nucleotide binding protein (G protein), alpha 13 [Homo sapiens]
Protein Accession gi|24111250
5.309 phospholipid scramblase 3 [Homo sapiens]
Protein Accession gi|31543417
5.310 plasma glutamate carboxypeptidase [Homo sapiens]
Protein Accession gi|7706387
5.311 bone marrow stromal cell antigen 1 precursor [Homo sapiens]
Protein Accession gi|168229159
5.312 solute carrier organic anion transporter family, member 4C1 [Homo sapiens]
Protein Accession gi|38679890
5.313 glucose transporter 14 [Homo sapiens]
Protein Accession gi|23592238
5.314 PREDICTED: hypothetical protein [Homo sapiens]
Protein Accession gi|169218200
5.315 serine hydroxymethyltransferase 1 (soluble) isoform 2 [Homo sapiens]; serine hydroxymethyltransferase 1 (soluble) isoform 1 [Homo sapiens]
Protein Accession gi|22547189 gi|22547186
5.316 calcium binding protein P22 [Homo sapiens]
Protein Accession gi|6005731
5.317 sushi domain containing 2 [Homo sapiens]
Protein Accession gi|10092665
5.318 occludin [Homo sapiens]
Protein Accession gi|4505487
5.319 CD81 antigen [Homo sapiens]
Protein Accession gi|4757944
5.320 ectonucleotide pyrophosphatase/phosphodiesterase 6 [Homo sapiens]
Protein Accession gi|23503267
5.321 solute carrier family 5 (sodium/glucose cotransporter), member 2 [Homo sapiens]
Protein Accession gi|4507033
5.322 MIT, microtubule interacting and transport, domain containing 1 [Homo sapiens]
Protein Accession gi|20270349
5.323 ring finger protein 167 [Homo sapiens]
Protein Accession gi|14149702
5.324 ubiquitin protein ligase E3C [Homo sapiens]
Protein Accession gi|187960100
5.325 syntaxin 8 [Homo sapiens]
Protein Accession gi|4759188
5.326 sorting nexin 3 [Homo sapiens]
Protein Accession gi|4507143
5.327 RAB7, member RAS oncogene family [Homo sapiens]
Protein Accession gi|34147513
5.328 prenylcysteine oxidase 1 [Homo sapiens]
Protein Accession gi|166795301
5.329 glucose phosphate isomerase [Homo sapiens]
Protein Accession gi|18201905
5.330 galactosidase, beta 1 isoform a preproprotein [Homo sapiens]
Protein Accession gi|119372308
5.331 GDP dissociation inhibitor 2 isoform 1 [Homo sapiens]
Protein Accession gi|6598323
5.332 solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform c [Homo sapiens]; solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform f [Homo sapiens]; solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform e [Homo sapiens]; solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform d [Homo sapiens]; solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform b [Homo sapiens]; solute carrier family 3 (activators of dibasic and neutral amino acid transport), member 2 isoform a [Homo sapiens]
Protein Accession gi|65506891 gi|61744483 gi|61744481 gi|61744479 gi|61744477 gi|61744475
5.333 heat shock 70 kDa protein 8 isoform 1 [Homo sapiens]
Protein Accession gi|5729877
5.334 RAB9A, member RAS oncogene family [Homo sapiens]
Protein Accession gi|4759012
5.335 complement component 8, gamma polypeptide [Homo sapiens]
Protein Accession gi|166197660
5.336 MAPK scaffold protein 1 [Homo sapiens]
Protein Accession gi|11496277
5.337 transketolase isoform 1 [Homo sapiens]; transketolase isoform 2 [Homo sapiens]; transketolase isoform 1 [Homo sapiens]
Protein Accession gi|4507521 gi|205277465 gi|205277463
5.338 complement factor B preproprotein [Homo sapiens]
Protein Accession gi|67782358
5.339 cyclin M3 isoform 1 [Homo sapiens]; cyclin M3 isoform 2 [Homo sapiens]
Protein Accession gi|40068049 gi|40068047
5.340 solute carrier family 9 (sodium/hydrogen exchanger), isoform 3 [Homo sapiens]
Protein Accession gi|194239733
5.341 solute carrier family 12 (potassium/chloride transporters), member 9 [Homo sapiens]
Protein Accession gi|31881740
5.342 RAB5C, member RAS oncogene family isoform a [Homo sapiens]; RAB5C, member RAS oncogene family isoform b [Homo sapiens]
Protein Accession gi|41393614 gi|41393545
5.343 pyruvate kinase, liver and RBC isoform 1 [Homo sapiens]
Protein Accession gi|10835121
5.344 glutathione transferase [Homo sapiens]
Protein Accession gi|4504183
5.345 ATPase, H+ transporting, lysosomal 70kD, V1 subunit A, isoform 1 [Homo sapiens]
Protein Accession gi|19913424
5.346 DnaJ (Hsp40) homolog, subfamily A, member 1 [Homo sapiens]
Protein Accession gi|4504511
5.347 aquaporin 2 [Homo sapiens]
Protein Accession gi|4502179
5.348 serine incorporator 1 [Homo sapiens]
Protein Accession gi|24308213
5.349 syntaxin 7 [Homo sapiens]
Protein Accession gi|170932494
5.350 carbonic anhydrase XII isoform 2 precursor [Homo sapiens]; carbonic anhydrase XII isoform 1 precursor [Homo sapiens]
Protein Accession gi|45935383 gi|4502515
5.351 major histocompatibility complex, class II, DR alpha precursor [Homo sapiens]
Protein Accession gi|52426774
5.352 cytosolic phosphoenolpyruvate carboxykinase 1 [Homo sapiens]
Protein Accession gi|187281517
5.353 glutathione peroxidase 3 precursor [Homo sapiens]
Protein Accession gi|6006001
5.354 transmembrane 7 superfamily member 3 [Homo sapiens]
Protein Accession gi|7706575
5.355 cathelicidin antimicrobial peptide [Homo sapiens]
Protein Accession gi|39753970
5.356 transmembrane protein 55B isoform 1 [Homo sapiens]; transmembrane protein 55B isoform 2 [Homo sapiens]
Protein Accession gi|154816186 gi|154816184
5.357 aconitase 1 [Homo sapiens]
Protein Accession gi|8659555
5.358 nicastrin precursor [Homo sapiens]
Protein Accession gi|24638433
5.359 carboxymethylenebutenolidase [Homo sapiens]
Protein Accession gi|20270371
5.360 major histocompatibility complex, class II, DR beta 3 precursor [Homo sapiens]
Protein Accession gi|17986005
5.361 hypothetical protein LOC51571 [Homo sapiens]
Protein Accession gi|42734438
The ability of a particular renal biomarker assay measurement to distinguish between two populations can be established using ROC analysis. For example, ROC curves established from a “first” subpopulation (i.e., for example, a population predisposed to one or more future changes in renal status) and a “second” subpopulation (i.e., for example, a population not predisposed to one or more future changes in renal status). Calculation of these ROC curves and establishing the area under these ROC curves quantitate the predictive power of the specific assay measurement. In some embodiments, predictive power established by assay measurements described herein comprise an AUC ROC 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.
A. Immunoassays
In general, immunoassays involve contacting a sample containing, or suspected of containing, a biomarker of interest with at least one antibody that specifically binds to the biomarker. A detectable signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The detectable signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices have been reported regarding the detection and analysis of biological biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is herein incorporated by reference in its entirety, including all tables, figures and claims.
Numerous immunoassay devices and methods can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein “blotting” methods. Additionally, certain methods and devices, such as biosensors 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. Pat. Nos. 5,631,171; and 5,955,377, each of which is herein incorporated by reference in its entirety, including all tables, figures and claims. Robotic instrumentation for performing these immunoassays are commercially available including, but not limited to, Beckman ACCESS®, Abbott AXSYM®, Roche ELECSYS®, Dade Behring STRATUS® systems. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.
Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in immunoassays. Solid phases that may be used to immobilize specific binding members include, but are not limited to those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include, but are not limited to, membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. For example, 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 quickly 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 directly to an assay device surface, or by indirect binding. In an example 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.
In certain embodiments, a urinary renal biomarker assay method comprises an immunoassay. For example, antibodies for use in such assays may specifically bind an epitope of a renal biomarker of interest, and may also bind one or more polypeptides that are “related” thereto, as that term is defined hereinafter. In one embodiment, the renal biomarker of interest is a fully length marker (i.e., for example, a protein). In one embodiment, the renal biomarker of interest is a protein fragment marker (i.e., for example, a peptide). Numerous immunoassay formats are available compatible with body fluid samples including, but not limited to, urine, blood, serum, saliva, tears, and plasma.
In this regard, detectable signals obtained from an immunoassay may be a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., for example, an analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay may actually be 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 measurements (i.e., for example, dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quantitation). This list is not meant to be limiting.
The foregoing method steps should not be interpreted to mean that the renal biomarker assay measurements 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, risk stratification, diagnostic, classification, monitoring, etc. methods as described herein may be combined with one or more clinical indicia relevant to the patient population including, but not limited to, demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, 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, foscarnet, 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), a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, 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, a renal failure 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 concentration, 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 renal biomarker assay measurements are described hereinafter. In: Harrison's Principles of Internal Medicine, 17th Ed., McGraw Hill, New York, pages 1741-1830; and In: Current Medical Diagnosis & Treatment 2008, 47th Ed, McGraw Hill, New York, pages 785-815, each of which are herein incorporated by reference in their entirety.
When more than one biomarker is measured, the individual biomarkers 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 biomarkers may also be measured on the same or different body fluid samples. For example, one renal biomarker may be measured in a serum or plasma sample and another renal biomarker may be measured in a urine sample. In addition, assignment of a likelihood may combine a renal biomarker assay measurement with temporal changes in one or more additional variables.
B. Detectable Labels
Generation of a detectable signal from the detectable label can be performed using various optical, acoustical, and electrochemical methods. Examples of detection modes include, but are not limited to, fluorescence, radiochemical detection, reflectance, absorbance, amperometry, conductance, impedance, interferometry, ellipsometry, etc. In certain of these methods, the solid phase antibody may be 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 fluorometer that employs an excitation light source and an optical detector). This list is not meant to be limiting. Antibody-based biosensors may also be employed to determine the presence or amount of analytes that optionally eliminate the need for a labeled molecule.
Biological assays require methods for detection, and one of the most common methods for quantitation of assay measurements 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 used in the immunoassays described above may include, but are not limited to, molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, ecl (electrochemical luminescence) labels, metal chelates, colloidal metal particles, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or through 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.).
Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents may involve at least two reactive groups, 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, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups and are believed to 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.
D. Assay Correlations
In some embodiments, the renal biomarker assay measurement is/are correlated to one or more future changes in renal function. In one embodiment, risk stratification comprises determining a subject's likelihood (i.e., for example, probability) for a future improvement in renal function.
In one embodiment, the renal biomarker assay measurement is/are correlated to a likelihood of such a future improvement in renal function. In one embodiment, the method correlates a likelihood of such a future injury to renal function. In one embodiment, the risk stratification comprises determining a subject's risk for progression to acute renal failure (ARF).
In one embodiment, the renal biomarker assay measurement is/are correlated to a likelihood of such progression to acute renal failure (ARF). In one embodiment, the risk stratification method comprises determining a subject's outcome risk.
In one embodiment, the assay measurement is/are correlated to a likelihood of the occurrence of a clinical outcome related to a renal injury suffered by the subject.
Consequently, the measured concentration value(s) may each be compared to a threshold value, wherein either a “positive going kidney injury marker”, or a “negative going kidney injury marker” is identified. In one embodiment, the risk stratification comprises determining a subject's risk for future reduced renal function. In some embodiments, the method assigns a likelihood, risk, or probability that such 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 some embodiments, the assigned likelihood, risk, or probability relates to an event of interest occurring within a time period including, but not limited to, 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. Alternatively, assigning 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.
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 efficacious 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, a cost/benefit analysis is involved in selecting a diagnostic threshold.
1. Thresholds
Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction uses cardiac troponin, wherein the diagnostic threshold is set at the 97.5th percentile of the cardiac troponin concentration measured in a normal population. Another method to determine a diagnostic threshold comprises measuring 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 thresholds. For example, Receiver Operating Characteristic (“ROC”) arose from the field of signal detection theory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold to distinguish a “diseased” subpopulation from a “nondiseased” subpopulation. Predictive power balances the occurrences of false positives (i.e., for example, when the person tests positive, but actually does not have the disease) and false negatives (i.e., for example, when the person tests negative, 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 sensitivity 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 value is selected to provide an acceptable level of specificity and sensitivity usually determined by summing specificity values with sensitivity values. Consequently, the larger the calculated threshold value the greater the predicative power of the specific assay measurement under analysis.
In this context, “diseased” is meant to refer to a population having one characteristic (i.e., for example, the presence of a disease or condition or the occurrence of some outcome) and “nondiseased” population lacking the same characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.
In addition to threshold value comparisons, other methods for correlating assay measurements to a patient classification (i.e., for example, occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include, but are not limited to, decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject or patient belongs to one classification out of a plurality of classifications.
Multiple thresholds may also be used to assess renal status in a subject and/or patient. For example, a multiple thresholding method may combine a “first” subpopulation which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc., with a “second” subpopulation which is not so predisposed into a single group. This combination group is then subdivided into three or more equal parts (i.e., for example, 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 embodiment, 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 1. 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.
2. Specificity and Sensitivity
In some embodiments, a measured concentration of one or more renal biomarkers, or a composite of such biomarkers, may be treated as continuous variables. For example, any particular biomarker concentration can be converted into a corresponding probability of a future reduction in renal function for the subject, the occurrence of an injury, a classification, etc. Alternatively, a threshold value can provide an acceptable level of specificity and sensitivity in separating a population of subjects into “bins” such as a “first” subpopulation (e.g., which is predisposed to one or more future changes in renal status, the occurrence of an injury, a classification, etc.) and a “second” subpopulation which is not so predisposed.
In one embodiment, a threshold value is selected to separate a first and a second population by one or more of the following measures of test accuracy:
Various measures of test accuracy have been reported and used to determine the effectiveness of a given biomarker. Fischer et al., Intensive Care Med. 29:1043-1051 (2003). These accuracy measures include, but are not limited to, sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and AUC ROC values. For example, AUC ROC values are equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. Consequently, an AUC ROC value 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 discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, 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; 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 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, 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 greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an 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 greater 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 0.1.
G. Conventional Renal Diagnostics
As noted above, the terms “acute renal (or kidney) injury” and “acute renal (or kidney) failure” as used herein are generally defined, in part, in terms of changes in serum creatinine from a baseline value. Most conventional definitions of ARF have common elements, including but not limited to 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 assuming the patient initially had a normal GFR.
1. Glomerular Filtration Rate And Creatinine
Glomerular filtration rate (GFR) is generally defined as the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman'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:
By normalizing the GFR to the body surface 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 estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively 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 (PCr) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (UCr×V) divided by its plasma concentration. This is commonly represented mathematically as:
Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:
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 corrected for their actual BSA:
The accuracy of a creatinine clearance measurement (even when collection is complete) 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 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 provided, minor modifications of the RIFLE urine output criteria have been described. For example, some have assumed an average patient weight of 70 kg, wherein patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure). Bagshaw et al., Nephrol. Dial. Transplant. 23:1203-1210 (2008).
2. Treatment Regimen Selection
Once a renal 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, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. Various appropriate treatments for numerous diseases have been previously 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, N J, 1999. In addition, since the methods and compositions described herein provide prognostic information, the renal biomarkers of the present invention may be used to monitor a course of treatment. For example, an improved prognostic state or a worsened prognostic state may indicate that a particular treatment is or is not efficacious.
Antibodies used in the immunoassays described herein preferably specifically 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 intended 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 molecule which does not display the appropriate epitope(s). Preferably 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 some embodiments, antibodies bind with affinities of at least about 107 M−1, and preferably between about 108 M−1 to about 109 M−1, about 109 M−1 to about 1010 M−1, or about 1010 M−1 to about 1012 M−1.
Affinity may be calculated as Kd=koff/kon (koff is the dissociation rate constant, Kon is the association rate constant and Kd is the equilibrium constant). Affinity can be determined 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 receptor 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 Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12:425-443 (1991); and Nelson et al., Comput. Methods Programs Biomed. 27: 65-68 (1988).
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: 6378-6382 (1990); Devlin et al., Science 249:404-406 (1990); Scott et al., Science 249:386-388 (1990); and Ladner et al., U.S. Pat. No. 5,571,698 (all references herein incorporated by reference). A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic 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 genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.
Antibodies generated 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 affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate 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 conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.
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 antibody 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 specificity of an antibody.
In some embodiments, the present invention also contemplates 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 performing such assays are provided in an assay device, 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 detectable label. Additional optional elements that may be provided as part of an assay device are described hereinafter.
In some embodiments, the present invention provides kits for the analysis of the described kidney injury markers. The kit comprises reagents for the analysis of at least one test sample which comprise 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 sandwich 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, wherein 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 correlations 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.
BioMaRK was an observational cohort study conducted as an ancillary study to the Veterans Affairs/National Institutes of Health (VA/NIH) Acute Renal Failure Trial Network study (ATN study). The ATN study was a multicenter, prospective trial of two strategies for renal replacement therapy in critically ill patients with acute kidney injury. Coca et al., “Biomarkers for the diagnosis and risk stratification of acute kidney injury: a systematic review” Kidney Int, 73:1008-1016 (2008). Adult patients (18 years or older) with AKI and requiring renal-replacement therapy (RRT), as well as failure of one or more non-renal organ systems or sepsis were eligible. As a sub-study to the ATN study, 109 patients were enrolled at The University of Pittsburgh Medical Center, The VA Pittsburgh Healthcare System, The Cleveland Clinical Foundation, The University of Texas Health Science Center at Houston, and Washington University Medical Center to undergo serial blood and urine sampling. Incomplete data including unavailability of urine samples precluded inclusion of 33 subjects; consequently the remaining 76 formed the analysis cohort. Approval from the Institutional Review Boards was received from the University of Pittsburgh and all participating sites.
Medical records of study participants were prospectively reviewed to retrieve hospitalization data including baseline demographic characteristics, serial renal function, daily urine volume, and severity of illness scores. The presence of sepsis was defined by international consensus criteria. Levy et al., “2001 SCCM/ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference” Crit Care Med, 31:1250-1256 (2003). Recovery of renal function was defined by survival and dialysis independence at Day 60 post AKI. For purposes of primary analysis, partial recovery (i.e. failure to return to baseline renal function but free of dialysis) was included in the recovery group. Similarly, all deaths were included in the non-recovery group.
Fresh urine samples were obtained on Days 1, 7, and 14 post AKI. Immediately upon obtaining a well-mixed 30 ml sample, a protease inhibitor tablet (Roche Diagnostics Corporation, IN, USA) was added. After processing, the sample was frozen (at −80° C.) until analyzed. Samples were assayed in duplicate, and data were analyzed using Bio-Rad Bio-Plex Manager Software (version 4.1). Urine creatinine concentrations were measured using a non-enzymatic assay (DICT-500, BioAssay Systems, CA, USA).
Urine collected in accordance with Example I from fourteen (14) patients with severe AKI was evaluated with an unbiased proteomics discovery platform.
Data collected from seven (7) patients that did not recover renal function after AKI was compared to data collected from seven (7) patients that did recover renal function after AKI. The two groups were matched for age (e.g., +/−5 yrs) and gender.
The data presented herein show that approximately thirty (30) proteins were differentially expressed between the Recovery Group and the Non-Recovery Group. A preliminary analysis has categorized these proteins into groups including, but not limited to:
Although it is not necessary to understand the mechanism of an invention, it is believed that the differential expression of proteins categorized in Group 3 and Group 4 might be directly involved in renal recovery because of their involvement with cell proliferation and/or rebuilding of the basement membrane.
This invention was made with government support under grant #DK 070910 awarded by the National Institutes of Health. The government has certain rights in the invention.
Number | Date | Country | |
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61436364 | Jan 2011 | US |
Number | Date | Country | |
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Parent | 13981747 | Aug 2013 | US |
Child | 15399994 | US |