WO2012068545A1 - Ngal in acute kidney injury - Google Patents

Ngal in acute kidney injury Download PDF

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Publication number
WO2012068545A1
WO2012068545A1 PCT/US2011/061531 US2011061531W WO2012068545A1 WO 2012068545 A1 WO2012068545 A1 WO 2012068545A1 US 2011061531 W US2011061531 W US 2011061531W WO 2012068545 A1 WO2012068545 A1 WO 2012068545A1
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Prior art keywords
aki
amount
protein
subject
ngal
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PCT/US2011/061531
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French (fr)
Inventor
Jonathan Barasch
Thomas L. Nickolas
Kai Schmidt-Ott
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Jonathan Barasch
Nickolas Thomas L
Kai Schmidt-Ott
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Publication of WO2012068545A1 publication Critical patent/WO2012068545A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/347—Renal failures; Glomerular diseases; Tubulointerstitial diseases, e.g. nephritic syndrome, glomerulonephritis; Renovascular diseases, e.g. renal artery occlusion, nephropathy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease

Definitions

  • Acute kidney injury is a common clinical event with severe consequences.
  • AKI is associated with a 25-80% risk of in-hospital death 4"6 and it has been implicated in the pathogenesis of chronic kidney disease (CKD) 7 .
  • CKD chronic kidney disease
  • the "risk, injury, failure, loss of function, end stage renal disease” (RIFLE) and the Acute Kidney Injury Network (AKIN) definitions of AKI are based on changes in both the levels of serum creatinine (sCr) and urinary output 8 ' 9 . These definitions can be problematic, however, when applied to patients in the emergency department (ED) because the baseline sCr may be unknown, and placement of a urinary catheter may not be indicated.
  • sCr level may not adequately reflect the severity of kidney injury since sCr kinetics are altered by age, gender, muscle mass, nutritional status, and medications.
  • uNGAL urinary neutrophil gelatinase- associated lipocalin
  • uKIM-1 kidney injury molecule 1
  • uIL-18 interleukin 18
  • uL-FABP liver fatty acid binding protein
  • cystatin C uCysC
  • Urinary biomarkers may provide a strategy to improve the diagnosis of iAKI, and predict its severity and its clinical outcomes beyond currently available tests.
  • uNGAL may identify a substantial population with "subclinical AKI” who had escaped detection by sCr measurements but who were at increased risk of a poor clinical
  • the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM-1 protein that exceeds about 2 ng/ml indicate that the subject may have AKI.
  • AKI acute kidney injury
  • the invention described herein relates to a method for predicting in- hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and the amount of KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM-1 protein that exceeds about 2 ng/ml indicate a high risk of subject mortality.
  • the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and an amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM- 1 protein that exceeds about 2 ng/ml indicate that the subject is at high risk of needing in-hospital dialysis.
  • the invention described herein relates to a method for determining whether a subject suspected of having AKI has transient or sustained AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has transient AKI, and an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has sustained AKI.
  • the invention described herein relates to a method for determining whether a subject suspected of having AKI has pre-renal AKI (pAKI) or intrinsic AKI (iAKI), the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has pAKI, and an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has iAKI.
  • pAKI pre-renal AKI
  • iAKI intrinsic AKI
  • the invention described herein relates to a method for determining whether a subject suspected of having AKI has pre-renal azotemia or AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has prerenal azotemia, and an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has AKI.
  • the invention described herein relates to a method for determining the severity of the AKI in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 100 ng/ml indicates that the subject has mild AKI, an amount of NGAL protein in the range of about 100 ng/ml to about 250 ng/ml indicates that the subject has intermediate AKI, and an amount of NGAL protein higher than about 250 ng/ml indicates that the subject has severe AKI, and wherein the risk level is based on the RIFLE scoring system.
  • the invention described herein relates to a method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and the amount of sCr protein present in the sample; wherein an amount of NGAL protein that is below about 100 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate a low risk of an adverse event, an amount of NGAL protein that exceeds or is equal to about 100 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate an intermediate risk of an adverse event, an amount of NGAL protein that is below about 100 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate an intermediate risk of an adverse event, and an amount of NGAL protein that exceeds or is equal to about 100 ng/ml and an amount of sCr protein
  • the invention described herein relates to a method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of KIM- 1 protein and sCr protein present in the sample; wherein an amount of KIM- 1 protein that is below about 2 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate a low risk of an adverse event, an amount of KIM- 1 protein that exceeds or is equal to about 2 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate an intermediate risk of an adverse event, an amount of KIM- 1 protein that is below about 2 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate an intermediate risk of an adverse event, and an amount of KIM- 1 protein that exceeds or is equal to about 2 ng/ml and an amount of sCr protein present in the sample
  • the adverse event is in-hospital mortality, or in-hospital dialysis, or both.
  • other threshold values can be used. Suitable threshold values for each marker are provided in the detailed description and in the Examples.
  • the subject is a human.
  • Some embodiments of the methods described above, in the detailed description, and the claims, further comprise subsequently treating the AKI in the subject.
  • Some embodiments of the methods described above, in the detailed description, and the claims, further comprise determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds about 1 mg/dl, indicates that the subject may have AKI.
  • the determining step comprises contacting the sample with an antibody that binds to the NGAL protein and an antibody that binds to the KIM-1 protein.
  • the method comprises performing an immunoassay.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the antibodies are immobilized on a solid support.
  • the solid support comprises a dipstick or a test strip.
  • the method comprises performing an immunoblotting method.
  • kits for carrying out any of the methods described herein.
  • the kits comprise one or more of (a) a device for detecting NGAL protein present in a bodily fluid sample; (b) a device for detecting KIM-1 protein present in a bodily fluid sample; and (c) a device for detecting sCr in a bodily fluid sample.
  • kits contain instructions indicating threshold levels of one or more of NGAL, KIM-1, and sCr above which a determination can be made regarding whether a subject has a particular AKI status, including, but not limited to, whether or not the subject has AKI or is at risk of developing AKI, or whether or not the subject has, or is likely to develop, transient versus sustained AKI, or whether or not the subject has, or is likely to develop, mild, intermediate, or severe AKI as classified according to the well known RIFLE severity classification system.
  • the kits comprise one or more antibodies capable of binding to one or more of NGAL protein, KIM-1 protein, and serum creatinine (sCr).
  • kits further comprise positive controls for one or more of NGAL protein, KIM-1 protein, and sCr.
  • the positive controls are in the device.
  • the device in the kits is an ELISA plate, a dipstick, or a test strip.
  • Figure 1 shows a study flow chart. Patients from three emergency departments were recruited at the time of hospital admission and urine samples were collected. Urinary biomarkers were measured and correlated with the renal diagnosis and the subsequent hospital course.
  • Figure 2(A-F) shows urinary biomarker levels in the diagnostic classification of ED patients.
  • uNGAL, uKIM-1, uL-FABP, uIL-18, and uCysC were compared by ANOVA in adjudicated patients with normal kidney function (Norm), stable CKD (CKD), pAKI, and iAKI (p values in upper left hand corners of each graph).
  • Biomarkers differed significantly in patients with iAKI, compared to all other adjudicated groups (*p ⁇ 0.05, **p ⁇ 0.01,
  • Figure 3 shows ROC curves for urinary biomarkers of nephron damage for the detection of intrinsic AKI (vs. prerenal AKI, stable CKD, or normal kidney function).
  • a reference line with an area under the curve (AUC-ROC) of 0.5 is shown for comparison.
  • FIG. 4 shows correlation between uNGAL levels quantified by chemiluminescent microparticle immunoassay (CMIA, Abbott ARCHITECT) and by immunoblot analysis of the monomeric form of AKI-associated NGAL.
  • CMIA chemiluminescent microparticle immunoassay
  • Figure 5 shows urinary biomarker levels by duration and maximal severity of AKI.
  • Left panels show by duration of AKI.
  • Biomarker levels were compared between patients with no AKI (None), transient AKI (Trans), and sustained AKI (Sust) using ANOVA (p values in upper left hand corners).
  • uNGAL and uKIM-1 levels were significantly different in patients with sustained AKI when compared to patients with transient AKI (*** p ⁇ 0.001 by post hoc Tukey test; ns, non- significant).
  • Right panels show by severity of AKI.
  • Figure 7 shows box plots of biomarker levels in prediction of outcomes. Boxes indicate median, lower and upper quartiles. Whiskers represent data within 1.5 IQR of the lower quartile, and the within 1.5 IQR of the upper quartile. Circles represent outliers.
  • Biomarker levels are presented on a log 10 scale.
  • Figure 8 shows urinary NGAL level and outcomes. Patients were stratified by NGAL level. Percentages of the subgroups that experience endpoints are shown, p for trend is statistically significant for composite and individual AKI outcomes.
  • Figure 9 shows biomarker expression in different patient diagnostic groups. Boxplots indicate median, inter-quartile range, and whiskers indicate the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum within 1.5 IQR of the upper quartile. Circles indicate outliers. Circles at the top of the Y-axis indicate outliers beyond the range of the graph. Both the chemiluminescent microparticle immunoassay [NGAL CMIA (NGAL- ARCHITECT)] and NGAL-immunoblot (NGAL I-BLOT) were assayed in every sample in order to authentic the monomeric form of uNGAL. Data are expressed in ng/ml units.
  • Figure 10 shows association of the biomarker and the maximal RIFLE class achieved during hospitalization.
  • the data are represented as fold-increase compared with normalized RIFLE negative levels. Note the response of the biomarkers to increasing RIFLE class (* p ⁇ 0.05, n.s.- not significant).
  • Figure 11A shows a luminescent mouse model based on the insertion of luciferase in the NGAL locus. Luciferase expression is activated upon 12-hours of ischemia to the left kidney. Expression of NGAL protein comes from the kidney medulla (as shown by cutting open the left kidney versus the right kidney).
  • Figure 11B shows mRNA situ hybridization, which demonstrates NGAL expression in both the TAL and the collecting ducts.
  • FIG 12 shows all AKI biomarkers were significantly elevated in patients with AKI.
  • uNGAL had a more than 13 -fold elevation in AKI, the most robust response of all biomarkers.
  • All biomarkers had a less than 1-fold elevation in patients with Prerenal
  • FIG. 13 shows biomarker levels are elevated with RIFLE positive azotemia.
  • the pattern of response indicates that biomarkers respond to the severity and duration of RIFLE positive azotemia differently.
  • biomarkers respond to the severity and duration of RIFLE positive azotemia differently.
  • For sCr in addition to rising in AKI - sCr also rose in transient azotemia. This relationship was mirrored by that of KIM- 1 and L-FABP.
  • Cystatin-C levels increased only in persistent RIFLE +/intrinsic AKI. Aspects of these two qualities were also evident for other biomarkers, notably L-FABP and Cy statin C.
  • FIG. 14A shows associations between urinary biomarkers and death and dialysis adjusted for sCr, age, liver disease and study site.
  • Fig. 14B shows associations between urinary biomarkers and death and dialysis adjusted for sCr, age, liver disease and SIRS. All AKI biomarkers independently predicted death and acute inpatient dialysis, howoever only KIM-1 and uNGAL predicted morality. Although sCr was associated with death and dialysis, it was not associated with mortality.
  • sustained AKI is defined as RIFLE-AKI that persisted for 72 hours or more.
  • iAKI intrinsic AKI
  • pAKI pre-renal AKI
  • low risk generally indicates a risk or event rate of about 3% or less.
  • high risk generally indicates a risk or event rate of about 15% or more. Risk levels between low risk and high risk are referred to as
  • the invention described herein relates to methods for using urine biomarkers to independently diagnose AKI and to predicted death or dialysis, independently of sCr.
  • a biomarker useful for distinguishing AKI from other diagnoses is uNGAL.
  • Acute kidney injury is common and has severe consequences and AKI is associated with a 25-80%) increased risk of death.
  • Acute kidney injury also known as "intrinsic AKI”
  • CKD chronic kidney diseases
  • AKI serum creatinine
  • sCr serum creatinine
  • AKI is challenging to diagnose because our clinical standard diagnostic is by serial serum creatinine (sCr) measurement, meaning the diagnosis is retrospective.
  • sCr can be elevated in other forms of kidney dysfunction, such as prerenal azotemia and chronic kidney disease, which in the absence of historical information can mimic AKI.
  • Urinary biomarkers which might prospectively diagnose AKI have not been analyzed in large clinical studies.
  • the invention relates to the finding that urine AKI biomarkers can predict AKI, with urine NGAL anticipating both severity and duration of azotemia, which is also shown in part by other biomarkers.
  • the invention relates to the finding that AKI can be distinguished from other causes of elevated sCr by biomarkers, in particular NGAL.
  • the invention relates to the finding that KIM-1 and NGAL biomarkers can be useful predictors of death.
  • the invention relates to the finding that NGAL protein is highly elevated in critical illness and that NGAL is a highly up- regulated gene in the setting of ischemic AKI.
  • certain embodiments of the invention relate to the finding that a single measurement of urine biomarkers predicted both the development of AKI and a combined outcome of death and dialysis in a heterogeneous cohort of patients being admitted from the ED.
  • both uNGAL and uKIM-1 can be used to predict both the development of AKI and a combined outcome of death and dialysis.
  • the invention described herein is based on testing of a panel of urine biomarkers against classical algorithms of kidney function to separate conditions that acutely elevate sCr (i.e. AKI and prerenal azotemia).
  • the biomarkers described herein can be applied to diverse pathophysiologies, treatments and clinical outcomes that can otherwise confound the diagnosis of true kidney injury and the use of the RIFLE score. 12 ' 15 ' 16 ' 45'
  • the invention relates to the diagnosis of AKI in triage, and in certain embodiments, to findings that: (a) a single measurement of a urinary biomarker can be used to diagnose AKI as defined by serial measurements of sCr, (b) the NPV of urine biomarkers for AKI is high and corresponding negative likelihood ratios are low, in particular for uNGAL; therefore, levels below defined cutoffs described herein make ongoing AKI decidedly unlikely; and (c) uNGAL has significantly better diagnostic test characteristics than other biomarkers, particularly in separating AKI from prerenal azotemia and CKD.
  • biomarker cutoff values described herein can also be used to evaluate combined outcome of death or acute dialysis and can be conducted on all patients regardless of adjudication status.
  • the results described herein show that the biomarker cutoff values described herein can be used to predicte death or acute dialysis independently of sCr.
  • the uNGAL and uKIM-1 cutoff values described herein are independently associated with death after adjustment for other covariates, whereas other biomarkers and sCr are not.
  • the biomarker cutoff values described herein can be used for the prediction of in-hospital mortality in the ED population.
  • the biomarker cutoff values described herein can be used for the prediction of in-hospital mortality, or poor outcome, for patients who cannot be diagnosed using classical algorithms ("Unclear Diagnoses", Table 1).
  • the invention relates to the finding from analyses conducted to evaluate relationships between sCr, urine biomarkers and AKI diagnostics and to identify biomarkers that are useful even without reference to adjudicated diagnoses.
  • Most of the biomarkers described herein (uNGAL, uKIM-1, uL-FABP and uCysC but not uIL-18) rose significantly from baseline in proportion to severity of RIFLE score, but uNGAL
  • the invention relates to findings from stratified ROC analyses showing that the biomarkers described herein consistently detected AKI regardless of site, gender, race, ethnicity, CKD history or the presence of either UTI or prerenal azotemia at presentation (Table 4).
  • the biomarkers and cutoff values described herein are generalizable to heterogeneous populations in gender, race-ethnicity,
  • certain aspects of the invention relate to the use of PC A to determine the relationship between factors that measure or contribute to kidney function and the diagnosis of AKI.
  • the invention relates to the finding that biomarkers of tubular damage make the greatest contribution to the diagnosis of AKI.
  • the invention relates to the finding that markers of glomerular function and physiologic states make a contribution to the diagnosis of AKI.
  • the urinary biomarkers describe herein are diagnostic tools that can be useful for improving the diagnosis of AKI in acute triage settings beyond the classical tests of renal function.
  • the methods described herein can be used to diagnose patients who could not be diagnosed using classical algorithms ("Unclear Diagnoses", Table 1) and identify patients that will to benefit from the use of urinary biomarkers.
  • the present invention provides that neutrophil gelatinase-associated lipocalin (NGAL) levels are higher in AKI patients than in patients that do not have AKI.
  • amounts of NGAL protein in a bodily fluid, such as urine or blood that exceed a certain threshold amount, may be used to diagnose AKI in a subject, predict in-hospital mortality of a subject suspected of having AKI, predict the need for in- hospital dialysis in a subject suspected of having AKI, determine whether a subject suspected of having AKI has transient or sustained AKI, determine the severity of the AKI in subject suspected of having AKI, and/or determine the risk of an adverse event in a subject suspected of having AKI.
  • the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein present in the sample, wherein an amount of NGAL protein that exceeds a threshold amount indicates that the subject may have AKI.
  • AKI acute kidney injury
  • the invention described herein relates to a method for predicting in- hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein present in the sample, wherein an amount of NGAL protein that exceeds a threshold amount indicates a high risk of subject mortality.
  • the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein present in the sample, wherein an amount of NGAL protein that exceeds a threshold amount indicates that the subject is at high risk of needing in-hospital dialysis.
  • the invention described herein relates to a diagnostic kit for diagnosing AKI in a subject, the kit comprising a) a device for detecting the amount of NGAL protein present in a sample of a bodily fluid; and b) instructions indicating threshold amounts of the NGAL protein, above which a determination can be made that the patient has AKI or is at risk of developing AKI.
  • the kit comprises an antibody capable of binding to the NGAL protein.
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the kit further comprises a positive control for the NGAL protein.
  • the positive control is in the device.
  • the device is an ELISA plate, a dipstick, or a test strip.
  • the threshold amount of NGAL protein is about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/
  • the method further comprises subsequently treating the AKI in the subject.
  • Treatment can comprise the use of any treatment well known to one of skill in the art.
  • the method further comprises determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds a threshold amount indicates that the subject may have AKI.
  • the amount of sCr protein is about 0.1 mg/dl, or about 0.2 mg/dl, or about 0.3 mg/dl, or about 0.4 mg/dl, or about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1.0 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2.0 mg/dl, or about 2.1 mg/dl, or about 2.2 mg/dl, or about 2.3 mg/dl, or about 2.4 mg/dl, or about 2.5 mg/dl, or about 2.6 mg/dl, or about 2.7 mg/dl, or about
  • any amount that is greater than the threshold amount is to be considered to exceed the threshold amount.
  • an amount of one or more marker(s) may exceed the threshold by about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%>, or about 12%>, or about 13%>, or about 14%>, or about 15%o, or about 16%>, or about 17%>, or about 18%>, or about 19%>, or about 20%>, or about 21%>, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%), or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%), or about 35%, or about 36%, or about 37%, or about 38%, or about 39%, or about 40%, or about 41%, or about 42%, or about 43%, or about 44%, or about 45%, or about 46%, or about 4
  • the determining step comprises contacting the sample with an antibody that binds to the NGAL protein.
  • the antibody is immobilized on a solid support.
  • the solid support comprises a dipstick or a test strip.
  • the method comprises performing an immunoassay.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
  • the method comprises performing an immunoblotting method.
  • the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody.
  • the invention described herein relates to a method for predicting the severity of AKI in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject at a first point in time; (b) determining the amount of NGAL protein at the first point in time in the sample; (c) obtaining a sample of a bodily fluid from the subject at a second point in time; (d) determining the amount of NGAL protein at the second point in time in the sample; wherein the subject does not show any signs of kidney disease at the first point in time, and wherein an increase in the amount of NGAL protein present in the sample at the second point in time compared to the amount of NGAL protein present in the sample at the first point in time indicates that the subject has or is at risk of developing AKI.
  • an increase of about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold indicates that the subject is at risk of developing AKI.
  • an increase of about 1-2 fold, 2-3 fold, 3-4 fold, 4-5 fold, or about 1-3 fold, 2-4 fold, 3-5 fold, 1-4 fold, or 2- 5 fold indicates that the subject is at risk of developing AKI.
  • an increase of about 5-fold, 6-fold, 7-fold or 8-fold indicates that the subject has AKI.
  • an increase of about 5-6 fold, 6-7 fold, 7-8 fold, or about 5-7 fold, 6-8 fold, or about 5-8 fold indicates that the subject has AKI.
  • an increase of about 9-fold, or 10-fold, or 11 -fold, or 12-fold, or more indicates that the subject is at risk of kidney failure due to AKI.
  • an increase of about 9-10 fold, 10-11 fold, 11-12 fold, or more, or about 9-11 fold or 9-12 fold indicates that the subject is at risk of kidney failure due to AKI.
  • the invention described herein relates to a method for determining whether a subject suspected of having AKI has transient or sustained AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein in the sample.
  • the invention described herein relates to a method for determining the severity of the AKI in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein in the sample.
  • the severity level is based on the RIFLE scoring system. In another embodiment, the severity level is not based on the RIFLE scoring system.
  • the invention described herein relates to a method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and the amount of sCr protein present in the sample.
  • an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng
  • an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng
  • the adverse event comprises in-hospital mortality. In another embodiment, the adverse event comprises in-hospital dialysis. In another embodiment, the adverse event comprises both in-hospital mortality and in-hospital dialysis.
  • the subject is a mammal. In another embodiment, the subject is a human. In one embodiment, the subject has no signs of kidney disease. In another embodiment, the subject has signs of kidney disease. In one embodiment, the subject has undergone or will undergo hemodialysis treatment.
  • the bodily fluid used in accordance with the invention is blood.
  • the bodily fluid is urine.
  • the bodily fluid is plasma.
  • the bodily fluid is blood serum.
  • the diagnostic methods described herein can be combined in various ways. For example, in some embodiments only one marker is assessed. In other embodiments, more than one of the markers may be assessed, in any combination, and optionally together with one or more additional markers.
  • the present invention provides that kidney injury molecule 1 (KIM-1) levels are higher in AKI patients than in patients that do not have AKI.
  • KIM-1 protein in a bodily fluid such as urine or blood, that exceed a certain threshold amount, may be used to diagnose AKI in a subject, predict in-hospital mortality of a subject suspected of having AKI, predict the need for in-hospital dialysis in a subject suspected of having AKI, and/or determine the risk of an adverse event in a subject suspected of having AKI.
  • the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM-1 protein present in the sample, wherein an amount of KIM-1 protein that exceeds a threshold amount indicates that the subject may have AKI.
  • AKI acute kidney injury
  • the invention described herein relates to a method for predicting in- hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM- 1 protein present in the sample, wherein an amount of KIM- 1 protein that exceeds a threshold amount indicates a high risk of subject mortality.
  • the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM-1 protein present in the sample, wherein an amount of KIM- 1 protein that exceeds a threshold amount indicates that the subject is at high risk of needing in-hospital dialysis.
  • the invention described herein relates to a diagnostic kit for diagnosing AKI in a subject, the kit comprising a) a device for detecting the amount of KIM - 1 protein present in a sample of a bodily fluid; and b) instructions indicating threshold amounts of the KIM-1 protein, above which a determination can be made that the patient has AKI or is at risk of developing AKI.
  • the kit comprises an antibody capable of binding to the KIM-1 protein.
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the kit further comprises a positive control for the KIM-1 protein.
  • the positive control is in the device.
  • the device is an ELISA plate, a dipstick, or a test strip.
  • the threshold amount of KIM-1 protein is about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 0.5 ng/ml,
  • the method further comprises determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds a threshold amount indicates that the subject may have AKI.
  • the amount of sCr protein is about 0.1 mg/dl, or about 0.2 mg/dl, or about 0.3 mg/dl, or about 0.4 mg/dl, or about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1.0 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2.0 mg/dl, or about 2.1 mg/dl, or about 2.2
  • any amount that is greater than the threshold amount is to be considered to exceed the threshold amount.
  • an amount of one or more marker(s) may exceed the threshold by about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%>, or about 12%>, or about 13%>, or about 14%>, or about 15%o, or about 16%>, or about 17%>, or about 18%>, or about 19%>, or about 20%>, or about 21%>, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%), or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%), or about 35%, or about 36%, or about 37%, or about 38%, or about 39%, or about 40%, or about 41%, or about 42%, or about or about 43%, or about 44%, or about 45%, or about 46%), or or about
  • the determining step comprises contacting the sample with an antibody that binds to the KIM-1 protein.
  • the antibody is immobilized on a solid support.
  • the solid support comprises a dipstick or a test strip.
  • the method comprises performing an immunoassay.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
  • the method comprises performing an immunoblotting method.
  • the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody.
  • the invention described herein relates to a method for
  • determining the risk of an adverse event in a subject suspected of having AKI comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM- 1 protein and the amount of sCr protein present in the sample.
  • an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or
  • an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or
  • 1.4 ng/ml or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or
  • the adverse event comprises in-hospital mortality. In another embodiment, the adverse event comprises in-hospital dialysis. In another embodiment, the adverse event comprises both in-hospital mortality and in-hospital dialysis.
  • the subject is a mammal. In another embodiment, the subject is a human. In one embodiment, the subject has no signs of kidney disease. In another embodiment, the subject has signs of kidney disease. In one embodiment, the subject has undergone or will undergo hemodialysis treatment.
  • the bodily fluid used in accordance with the invention is blood.
  • the bodily fluid is urine.
  • the bodily fluid is plasma.
  • the bodily fluid is blood serum.
  • the diagnostic methods described herein can be combined in various ways. For example, in some embodiments only one marker is assessed. In other embodiments, more than one of the markers may be assessed, in any combination, and optionally together with one or more additional markers.
  • the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about
  • the method further comprises subsequently treating the AKI in the subject.
  • Treatment can comprise the use of any treatment well known to one of skill in the art.
  • the method further comprises determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds about 0.1 mg/dl, or about 0.2 mg/dl, or about 0.3 mg/dl, or about 0.4 mg/dl, or about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1.0 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2.0 mg/dl, or about 2.1 mg/dl, or about 2.2 mg/dl, or about 2.3 mg/dl, or about 2.4 mg/dl, or about 2.5
  • the determining step comprises contacting the sample with an antibody that binds to the NGAL protein and an antibody that binds to the KIM-1 protein.
  • the method comprises performing an immunoassay.
  • the immunoassay is an enzyme-linked immunosorbent assay (ELISA).
  • the antibodies are immobilized on a solid support.
  • the solid support comprises a dipstick or a test strip.
  • the method comprises performing an immunoblotting method.
  • the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody.
  • the invention described herein relates to a method for predicting in-hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and the amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/
  • the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and the amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, about 100 ng/ml, or
  • the invention described herein relates to a method for
  • determining the risk of an adverse event in a subject suspected of having AKI comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein, and the amount of KIM- 1 protein, and the amount of sCr protein present in the sample.
  • an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng
  • an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng
  • 3.3 ng/ml or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/
  • an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about
  • 2.4 ng/ml or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml,
  • the adverse event comprises in-hospital mortality.
  • the adverse event comprises in-hospital dialysis.
  • the adverse event comprises both in-hospital mortality and in-hospital dialysis.
  • the invention described herein relates to a diagnostic kit for diagnosing AKI in a subject, the kit comprising (a) a device for detecting the amount of NGAL protein present in a sample of a bodily fluid; (b) a device for detecting the amount of KIM-1 protein present in a sample of a bodily fluid; and (c) instructions indicating threshold levels of the NGAL protein and the KIM-1 protein, above which a determination can be made that the patient has AKI or is at risk of developing AKI, wherein the threshold levels are about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/m
  • threshold levels of NGAL, KIM-1, and sCr can be selected based on the data provided in the Examples section of this application which provides, for example, mean values for the markers that associated with various AKI classifications, and ranges (e.g. amounts within 95% confidential interval boundaries from the mean values), and statistical analyses of AUCs for the various markers. Based on this data other suitable threshold values can be selected.
  • NGAL, KIM-1, and sCr described herein are generally referred to in terms of the amount by mass/volume (e.g.
  • ng/ml other units of measurement can be used.
  • NGAL levels in terms of ⁇ g/g creatinine.
  • Any suitable units or measurements can be used and it should be understood that amounts of NGAL measured and/or represented in other units can be equivalent to the amounts and ranges described herein.
  • the present invention is not limited to methods that comprise measuring NGAL, KIM-1, or sCr using the specific units provided herein. One of skill in the art can readily make the necessary conversions between units.
  • the kit comprises an antibody capable of binding to the NGAL protein and an antibody capable of binding to the KIM-1 protein.
  • the kit further comprises a positive control for each of NGAL protein and KIM-1 protein.
  • the positive control is in the device.
  • the device is an ELISA plate, a dipstick, or a test strip.
  • the antibody is a monoclonal antibody.
  • the antibody is a polyclonal antibody.
  • the subject is a mammal. In another embodiment, the subject is a human. In one embodiment, the subject has no signs of kidney disease. In another embodiment, the subject has signs of kidney disease. In one embodiment, the subject has undergone or will undergo hemodialysis treatment.
  • the bodily fluid used in accordance with the invention is blood.
  • the bodily fluid is urine.
  • the bodily fluid is plasma.
  • the bodily fluid is blood serum.
  • NGAL protein any suitable methods for detecting NGAL protein, KIM-1 protein and serum creatinine known in the art can be used.
  • KIM-1 protein any suitable methods for detecting NGAL protein, KIM-1 protein and serum creatinine known in the art can be used.
  • several antibodies to NGAL and KIM- 1 are known in the art and can be used in accordance with the present invention.
  • Any antibody such as a monoclonal or polyclonal antibody, that binds to NGAL, KIM-1 or sCr can be used.
  • monoclonal antibodies that bind to NGAL are described in "Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils", Lars Kjeldsen et al, (1996) Journal of Immunological Methods, Vol. 198, 155-16, the contents of which are herein incorporated by reference.
  • An example of a polyclonal antibody for NGAL is described in "An Iron Delivery Pathway Mediated by a Lipocalin", Jun Yang et al, Molecular Cell, (2002), Vol.
  • Any method can be used to detect and or measure the levels of NGAL, KIM-1 or sCr, including, but not limited to, immunohistochemistry-based methods, immuno-blotting based methods, immunoprecipitation-based methods, affinity-column based methods
  • methods that enable detection of monomeric NGAL may be used, including, but not limited to, immunoblotting methods.
  • a primary antibody to NGAL, KIM-1 , or sCr may be used in conjunction with a secondary or tertiary antibody labeled with a detectable moiety, such as a fluorescent moiety, a radioactive moiety, or a moiety that is an enzyme substrate and can be used to generate a detectable moiety, such as horse radish peroxidase.
  • a detectable moiety such as a fluorescent moiety, a radioactive moiety, or a moiety that is an enzyme substrate and can be used to generate a detectable moiety, such as horse radish peroxidase.
  • NGAL, KIM-1, or sCr In circumstances where the amount of NGAL, KIM-1, or sCr is to be measured, positive controls containing known amounts of NGAL, KIM-1, or sCr protein can be used, for example for calibration purposes.
  • NGAL, KIM-1, or sCr for use as a positive control can be obtained from any source or produced by any method known in the art.
  • NGAL or KIM-1 protein can be recombinantly produced. Methods for the recombinant production of proteins are well known in the art.
  • a nucleotide sequence encoding NGAL or KIM-1 can be included in an expression vector containing expression control sequences and expressed in, and purified from, any suitable cell type, such as bacterial cells or mammalian cells.
  • recombinant NGAL can be produced as described in Yang, et al. (2002) Mol Cell 10, 1045-1056; Goetz et al. (2002) Mol. Cell 10, 1033-1043; Goetz et al. (2000) Biochemistry 39, 1935-1941; and Mori, et al. (2005) J. Clin Invest. 115, 610-621, the contents of which are hereby incorporated by reference.
  • Methods of determining the serum creatinine level of a subject are well known in the art and are routinely performed by medical professionals. Any method for determining a subject's serum creatinine level can be used in conjunction with the methods of the present invention. A review of some methods for determining serum creatinine levels is provided in: Clin. Biochem. Rev. (2006); 27(4): p 173-184; "Measurement of Serum Creatinine - Current Status and Future Goals" by Peake et al., the contents of which are hereby incorporated by reference.
  • samples of a bodily fluid can be obtained and/or tested using any means.
  • methods for collecting, handling and processing urine, blood, serum and plasma, and other body fluids are well known in the art and can be used in the practice of the present invention.
  • two or more consecutive or subsequent samples of a body fluid can be taken.
  • the subject's body fluid can be sampled daily, or weekly, or within a few weeks, or monthly or within a few months, semiannually, annually, or within several years, and at any interval in between. Repeat sampling can be done at a period of time after treatment to detect any change in AKI status.
  • Sampling need not be continuous, but can be intermittent (e.g., sporadic). In some embodiments, it is not be necessary to obtain and keep a sample of the bodily fluid from the subject.
  • the subject can urinate onto a test strip, for example a test strip of the type used in pregnancy testing kits.
  • a sample of bodily fluid such as blood from a pin prick, can be applied onto a test strip - for example a test strip similar to those used for blood typing.
  • NGAL testing kit e.g. a urine dipstick based kit, or an ELISA based kit
  • home -testing kits are also within the scope of the present invention.
  • the present invention comprises a kit for performing the methods of the invention, containing, for example, a device for detecting one or more of NGAL protein, KMI-1 protein, and serum creatinine.
  • a device for detecting one or more of NGAL protein, KMI-1 protein, and serum creatinine can comprise, for example, an ELISA plate, a dipstick to be dipped in a urine or blood sample, or a stick on which the subject should urinate.
  • such devices are configured such that they give a positive result only if the level of one or more of NGAL, KIM-1, or sCr exceeds a threshold level, such as one of the threshold levels described herein. Methods for making and using such devices are well known in the art.
  • Example 1 Diagnostic and Prognostic Stratification in the Emergency Department Using Urinary Biomarkers of Nephron Damage - A Multicenter Prospective Cohort Study
  • a multi-center trial in three ED was conducted: (1) an inner-city community hospital in Manhattan, NY (Allen Hospital of New York- Presbyterian Hospital, AH-NYPH); (2) a suburban community hospital in Staten Island, NY (Staten Island University Hospital; SIUH); and (3) a tertiary care center in Berlin, Germany (Helios Clinics, Berlin-Buch).
  • Described herein are the diagnostic and the predictive characteristics of a single measurement of several novel urinary biomarkers.
  • AKI The primary etiologies of AKI were hypotension (34%), urinary obstruction (29%>), sepsis (22%), glomerulonephritis or vasculitis (6%>), hepatorenal syndrome (2.1%), rhabdomyolysis (2.1%), and acute interstitial nephritis (biopsy-proven), scleroderma crisis, contrast nephropathy and multiple myeloma (1% each).
  • Patients with AKI differed from other groups with respect to age and race and underlying CKD (Table 1). Diabetes, congestive heart failure, and liver disease were more frequent in patients with AKI. Presenting sCr and its change from baseline were significantly higher in AKI compared to other groups (Table 1, Figure 9). In addition, patients with AKI had longer hospital stays, were more likely to require nephrology consultation or ICU transfer, and experienced higher rates of death, dialysis, and the composite endpoint of death or dialysis (Table 1).
  • Table 1 Patient characteristics by diagnosis and clinical outcome. Cohort Characteristics Total Patients Patients Patients Patients Patients
  • iAKI intrinsic AKI
  • ROC curve analyses were used to determine whether a single measurement of a urinary biomarker could distinguish patients with AKI from patients belonging to the other diagnostic groups (Table 2). To evaluate diagnostic cut-offs, the 60th and 75th percentiles for each biomarker in the adjudicated population were chosen a priori.
  • Table 2 Test characteristics of urinary biomarkers in the diagnosis of iAKI
  • Urinary biomarker levels differed between patients with iAKI and any other diagnosis (by t-test: p ⁇ 0.001 for all biomarkers).
  • subset comparisons showed that urinary biomarker levels were significantly elevated in patients with iAKI compared with each of the other diagnostic categories (pAKI, stable CKD, or normal kidney function; by one-way ANOVA and posthoc Tukey test) (Figure 2).
  • ROC curve analyses indicated good discriminatory ability for uNGAL (AUC-ROC 0.81), fair discriminatory ability for uKIM-1 and uL-FABP (AUC-ROC 0.71 and 0.70, respectively) and poor discriminatory ability for uCysC and uIL-18 (AUC-ROC 0.65 and 0.64, respectively) (Figure 3, Table 2) to distinguish iAKI from other diagnoses.
  • the AUC-ROC of uNGAL was significantly higher than that of other urinary markers (p ⁇ 0.001 each, Table 2).
  • the monomeric form of uNGAL was quantified by immunoblot, it markedly correlated with the standardized clinical platform and performed similarly in ROC analyses ( Figures 3 and 4)
  • AUC-ROCs remained similar when biomarker levels were standardized for urinary creatinine concentrations. Sensitivity, specificity, predictive values, and likelihood ratios for diagnostic cut-offs at the 60 th and 75th percentiles for each biomarker are shown in Table 2.
  • the cut-off value of uNGAL was 104 ng/ml.
  • Sensitivity analyses was performed by selecting subsets of patients based on study site, ethnicity, gender, infection of the urinary tract (UTI) and the co-prevalence of CKD or RIFLEAKI.
  • the AUC-ROCs for the diagnosis of iAKI remained consistent within these subsets (Table 4).
  • Table 4 Sensitivity analysis. Test characteristics of urinary biomarkers in the diagnosis of intrinsic AKI in different subsets of patients. AUC-ROC values are reported (data in parentheses are 95% confidence intervals). The subsets included 3 separate hospital cohorts, different ethnicities, genders, and medical histories including CKD and urinary tract infections. AUC values and their rank ordering for different biomarkers were consistent in all patient subsets. The bottom panel shows discrimination of intrinsic AKI (i-AKI) from other causes of elevated sCr (PR- AKI: prerenal AKI; CKD: stable CKD) by biomarkers.
  • i-AKI intrinsic AKI
  • PR- AKI prerenal AKI
  • CKD stable CKD
  • Table 5 Mean (SD) Biomarker Level for Increasing sCr vs. Unchanging or
  • uKIM-1, uL-FABP, and uIL-18 were not significantly different in sustained and transient AKI.
  • PC3 variables defining hemodynamics
  • PC4 included temperature
  • PC5 included presenting serum sodium
  • Table 7 Association of urinary biomarker levels and peak RIFLE class during 7 days of hospitalization. Note the differential response of urinary biomarkers with increasin
  • uNGAL and uKIM-1 were found to independently add to a combined prediction model (Table 8, Model 7). However, R square and AUC-ROC of the combined model increased only slightly when compared to the single biomarker models.
  • Table 8 Multivariate logistic regression analysis of urinary biomarkers in the prediction of the composite outcome (in-hospital dialysis initiation or mortality) a, b, c
  • c RSQ Nagelkerke R square
  • AUC-ROC Area under the Receiver Operating Characteristic Curve
  • IDI Integrated Discrimination Improvement
  • the uNGAL-assisted model also achieved a significant IDI when compared with models using uL-FABP, uIL-18, or CysC (Models 4, 5 and 6) (p ⁇ 0.05 each).
  • a triple model of sCr, uNGAL and uKIM-1 did not achieve a significant IDI compared with the double model with sCr and uNGAL (Table 8).
  • Table 9 Net reclassification improvement as facilitated by biomarker-aided prediction models.
  • Individual probabilities of experiencing the composite outcome were calculated according to the baseline model (Model 1, see Table 8) and to the biomarker-assisted models (Models 2 and 3, see Table 8) to stratify patients into three risk classes ( ⁇ 2%, 2-15%, >15%).
  • Appropriate biomarker-assisted reclassification was defined as a step-up in risk class in patients with events and a step-down in risk-class in patients without events, while inappropriate reclassification was vice versa.
  • Event rates within sCr+ or sCrpatients were substantially different depending on whether they were biomarker+ or biomarker- (Figure 6). In particular, about 15% of the population had low sCr, but high biomarker levels, placing them at low risk by conventional stratification, but at an increased risk upon application of biomarker-aided stratification.
  • Diagnostic categorization was performed by adjudicators, who were blinded to urinary biomarker levels.
  • a priori defined algorithms assigned patients to one of four renal diagnoses (normal kidney function, stable chronic kidney disease, pAKI, iAKI). Patients were labeled "unclassified” when ambiguity occurred, or when disagreement among two or more investigators could not be resolved by re-evaluation of clinical data.
  • Urine samples were centrifuged (12,000rpm; 10 min) and stored at -80°C within 12 hrs after patient enrollment.
  • uNGAL, uIL-18, uKIM-1, and uCysC were measured by the ARCHITECT platform (Abbott Laboratories) (21).
  • CMIA chemiluminescent microparticle immunoassay
  • the assays include a microparticle reagent prepared by covalently attaching an antianalyte antibody to paramagnetic particles and a conjugate reagent prepared by labeling a second anti-analyte antibody with acridinium.
  • the calibrators for the uNGAL, uIL-18 and uKIM-1 assays were recombinant proteins and the calibrators for the uCysC assay was prepared from human urine.
  • the highest calibrator for each assay was 1500 ng/mL, 1 ng/mL, 10 ng/mL, and 2500 ng/mL for uNGAL, uIL-18, uKIM-1 and CysC, respectively.
  • Specimens were diluted to read within the calibration curve.
  • Coefficients of variation were 3.0% for uNGAL at a 385ng/ml (21); 2.5% for uKIM-1 at 5.8 ng/ml (Abbott Laboratories); 2.2% for uIL-18 at 0.048 pg/ml (Abbott Laboratories); 1.8% for uCysC at 350 ng/ml (Abbott Laboratories) and similar at other cutpoints.
  • uL-FABP was measured using a sandwich-type ELISA kit (CMIC Co., Ltd). The CV was 6.8% for uL-FABP at 13 ng/ml (22).
  • Monomeric uNGAL (23-26 KDa) was measured by immunoblots, which were prepared with non- reducing 4%) to 15%) gradient polyacrylamide gels (Bio-Rad, Hercules, CA) using standards (0.3 to 3 ng) of human recombinant NGAL and NGAL antibody (AntibodyShop,
  • Serum creatinine was assayed at each hospital by the Jaffe reaction, calibrated traceable to isotope dilution mass spectrometry.
  • PC A principal component analysis
  • Urine biomarkers, sCr, BUN, age, presenting temperature, blood pressure and laboratory measurements from the ED were used. Only principal components (PCs) having eigenvalues >1.0 were selected. Associations between PCs and AKI were evaluated by univariate and multiple logistic regression analysis and expressed as ORs (with 95% CIs).
  • IDF Integrated discrimination improvement
  • NRI net reclassification improvement
  • this sample size ensured that differences of 0.1 in AUC- ROC between biomarkers would be detectable at 80%> power using a two-sided z-test at a significance level of 0.05 under the assumption of strong correlation (0.8) between biomarker levels. Given an estimated rate of 20% of unclassifiable cases we aimed to enroll more than 500 patients per study site.
  • Example 2 Test characteristics of urinary neutrophil gelatinase-associated lipocalin for differential diagnosis and risk stratification in patients with established acute kidney injury (AKI)
  • AKI based on serum creatinine levels and urinary output (37).
  • serum creatinine levels do not always reflect the severity of renal damage (38), and they do not differentiate between intrinsic and prerenal AKI.
  • Intrinsic AKI is associated with a higher mortality than prerenal AKI (39).
  • the differential diagnosis is important, because treatment of intrinsic and prerenal AKI differs (38).
  • Prerenal AKI is an increase in serum creatinine caused by factors that compromise renal perfusion, which rapidly improved to baseline with volume repletion or improvement in cardiac output within 3 days of directed therapy or within 7 days, when a more careful therapeutic regimen was warranted (e. g. in congestiveheart failure).
  • Intrinsic AKI is sudden increase in serum creatinine in the presence of a potential acute tubular necrosis-inducing event or specific kidney disease without a response to fluid resuscitation and/or hemodynamic optimization. Current tests, including fractional excretion of sodium, fractional excretion of urea, and urea creatinine ratio are of limited utility in diagnosing intrinsic AKI (38).
  • NGAL a 25kD protein
  • NGAL a 25kD protein
  • NGAL a 25kD protein
  • It is produced in response to epithelial damage and is upregulated in acute kidney injury (41).
  • NGAL discriminated intrinsic AKI from prerenal AKI, chronic kidney disease, and normal renal function (42).
  • NGAL could be useful in the early diagnosis of AKI and prediction of poor clinical outcomes in AKI (43).
  • Urinary neutrophil gelatinase-associated lipocalin was tested to determine whether it predicts adverse outcomes in acute kidney injury (primary endpoint: progression to a higher RIFLE class, dialysis or death) and distinguishes between intrinsic and prerenal acute kidney injury in the cohort of 161 patients.
  • Urinary NGAL levels discriminated intrinsic AKI from prerenal AKI (ROC 0.87, CI 0.81-0.94). Patients with the primary endpoint had higher median urinary NGAL levels (248.2 vs. 68.3 ⁇ g/L, p ⁇ 0.001).
  • NGAL independently predicted worsening AKI, when corrected for demographics, comorbidities, creatinine, and RIFLE class.
  • Urinary NGAL was collected on inclusion and two days later. Diagnosis of intrinsic or prerenal AKI was adjudicated based on medical records by two independent clinicians who were blinded to uNGAL levels.
  • Exclusion criteria unavailable baseline creatinine, incomplete follow-up and postrenal obstruction. 16 patients were excluded. Of the remaining 145 patients, 32 had prerenal AKI, 75 had intrinsic AKI, 38 were unclassifiable. Urinary NGAL was measured using ARCHITECT® (Abbott Laboratories) technology.
  • Table 9 Patient characteristics, by diagnosis and kidney parameters Stable or
  • n (%) (eGFR was calculated using the
  • AUC-ROC operating characteristic curves
  • Table 11 Test characteristics of biomarkers in the prediction of intrinsic AKI vs. prerenal AKI at different cutoff levels
  • Biomarker Level Sensitivity Specificity value predictive value uNGLA >47.35 0.89 (0.8- 0.53 (0.35- 0.82 (0.71- 0.68 (0.46-0.84)
  • Optimal cutoff levels for NGLA were derived from an independent patient cohort in the emergency room. Cutoff levels of the remaining biomarkers represent adjusted percentiles in the current study population to ensure comparability of the results
  • NGAL levels displayed less overlap between the prospective outcome groups when compared to serum creatinine (Figure 7). Median NGAL levels on inclusion and 2 days after inclusion are significantly higher in patients, who later developed worsening AKI in comparison to all others ( Figure 7). NGAL level and outcomes were determined ( Figure 8). Patients were stratified by NGAL level. Percentages of the subgroups that experience endpoints are shown, p for trend is statistically significant for composite and individual AKI outcomes. [00243] Multiple Logistic Regression Models for the predicton of worsening AKI (step-up in RIFLE class, dialysis initiation, or mortality) was performed. Wald score, p value and odds ratio describe the contribution of the individual covariate to the model. R square, AUC-ROC and diagnostic accuracy are measures of the overall performance of the model (Table 12).
  • uNGAL distinguished intrinsic AKI from prerenal AKI and predicted worsening of AKI. uNGAL performed better than conventional laboratory tests in differentiating intrinsic AKI from prerenal AKI. NGAL is an independent predictor of worsening AKI in logistic regression analysis.
  • Example 3 Test Characteristics of Urinary Neutrophil Gelatinase-Assocaited Lipocalin (uNGAL) for Differential Diagnosis and Risk Stratification in Patients with Established Acute Kidney Injury
  • Serum creatinine dynamics are used for the diagnosis of acute kidney injury (AKI) in current classification schemes (e.g. RIFLE), but are temporally and diagnostically poor.
  • Urinary NGLA a biomarker of nephron damage, may help to differentiate intrinsic kidney damage from prerenal AKI.
  • Methods The relationship between uGAL levels at the time of diagnosis of AKI and outcomes was evaluated prospectively. uNGLA levels were determined on the Abbott ACHITECT standardized clinical platform. The primary outcome was worsening of AKI (progression to higher RILE category, dialysis initiation, or death) during hospitalization.
  • uNGLA in was tested distinguishing intrinsic from prerenal AKI. Results: 162 hospitalized AKI patients were studied. 17 pateients with postrenal obstruction or insufficient clinical information were excluded. From the remaining 145 patients, 75 patients had a clinical diagnosis of intrinsic AKI, 32 patients had prerenal AKI, and 38 patients had an uncertain or ambiguous diagnosis. uNGLA levels were found to effectively discriminated intrinsic AKI from prerenal AKI (ROC 0.87, CI 0.81-0.94). NGLA levels at a cutoff
  • uNGLA is useful in the differential diagnosis and prognostic stratification of patients with established AKI.
  • Mehta RL Kellum JA, Shah SV, et al. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit Care 2007; 11 :R31. 10. Mishra J, Ma Q, Prada A, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol
  • Waikar SS Bonventre JV. Creatinine kinetics and the definition of acute kidney injury. J Am Soc Nephrol 2009;20:672-9.
  • gelatinaseassociated lipocalin an independent predictor of adverse outcomes in acute kidney injury. Am J Nephrol 2010;31 :501-9.

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Abstract

The present invention is directed to methods for the diagnosis of AKI, and to methods for predicting in-hospital mortality of a subject suspected of having AKI and the need for in- hospital dialysis in a subject suspected of having AKI, based on the presence in a bodily fluid, such as urine, of a level of NGAL and KIM-1 protein that exceeds a threshold level. The present invention is also directed to methods for determining whether a subject suspected of having AKI has transient or sustained AKI, for determining the severity of the AKI in a subject suspected of having AKI and for determining the risk of an adverse event in a subject suspected of having AKI. The present invention is also directed diagnostic kits that can be used in association with the various diagnostic methods of the invention.

Description

NGAL IN ACUTE KIDNEY INJURY
[0001] This application claims the benefit of and priority to U.S. provisional patent application No. 61/415,233 filed November 18, 2010, the disclosure of all of which is hereby incorporated by reference in its entirety for all purposes.
[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
[0003] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.
BACKGROUND OF THE INVENTION
[0004] Acute kidney injury (AKI) is a common clinical event with severe consequences. In the United States, over one million hospitalized patients are diagnosed yearly with AKI, and its incidence is rising1"4. AKI is associated with a 25-80% risk of in-hospital death4"6 and it has been implicated in the pathogenesis of chronic kidney disease (CKD)7.
[0005] The "risk, injury, failure, loss of function, end stage renal disease" (RIFLE) and the Acute Kidney Injury Network (AKIN) definitions of AKI are based on changes in both the levels of serum creatinine (sCr) and urinary output8'9. These definitions can be problematic, however, when applied to patients in the emergency department (ED) because the baseline sCr may be unknown, and placement of a urinary catheter may not be indicated.
Furthermore, "subclinical AKI", can fail to display diagnostic changes in sCr despite evidence of nephron damage10. Additionally, the sCr level may not adequately reflect the severity of kidney injury since sCr kinetics are altered by age, gender, muscle mass, nutritional status, and medications.
[0006] Also, the RIFLE and AKIN definitions do not consider the presence of structural nephron damage ("intrinsic AKI"), despite its documented association with poor clinical outcomes. In fact, sCr levels may meet these critera in the absence of nephron damage as a result of altered hemodynamics ("prerenal AKI")5'11"16. Some of these shortcomings may be addressed by novel kidney injury biomarkers, which are released into urine following cellular injury. Currently, the most promising candidates include urinary neutrophil gelatinase- associated lipocalin (uNGAL); kidney injury molecule 1 (uKIM-1); interleukin 18 (uIL-18); liver fatty acid binding protein (uL-FABP); and cystatin C (uCysC)17'18. Their diagnostic and prognostic performance has been evaluated but only in single-center studies, usually involving subtypes of AKI in selected clinical settings. As a result, it is unclear: (1) how the biomarkers behave in large, heterogeneous populations; (2) which biomarker successfully discriminates intrinsic AKI (iAKI) from either prerenal AKI (pAKI) or CKD, at the time of patient presentation; and (3) whether combinations of biomarkers have greater diagnostic accuracy than single markers. Urinary biomarkers may provide a strategy to improve the diagnosis of iAKI, and predict its severity and its clinical outcomes beyond currently available tests. uNGAL may identify a substantial population with "subclinical AKI" who had escaped detection by sCr measurements but who were at increased risk of a poor clinical
19
outcome .
[0007] There remains a paucity of prospective data comparing emerging urinary AKI Biomarkers in heterogeneous populations and there is a need for improved urinary biomarkers to diagnose iAKI and to predict the hospital course. This invention addesses this need.
SUMMARY OF THE INVENTION
[0008] In one aspect, the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM-1 protein that exceeds about 2 ng/ml indicate that the subject may have AKI.
[0009] In another aspect, the invention described herein relates to a method for predicting in- hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and the amount of KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM-1 protein that exceeds about 2 ng/ml indicate a high risk of subject mortality. [0010] In yet another aspect, the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and an amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM- 1 protein that exceeds about 2 ng/ml indicate that the subject is at high risk of needing in-hospital dialysis.
[0011] In another aspect, the invention described herein relates to a method for determining whether a subject suspected of having AKI has transient or sustained AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has transient AKI, and an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has sustained AKI.
[0012] In another aspect, the invention described herein relates to a method for determining whether a subject suspected of having AKI has pre-renal AKI (pAKI) or intrinsic AKI (iAKI), the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has pAKI, and an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has iAKI.
[0013] In another aspect, the invention described herein relates to a method for determining whether a subject suspected of having AKI has pre-renal azotemia or AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has prerenal azotemia, and an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has AKI.
[0014] In one aspect, the invention described herein relates to a method for determining the severity of the AKI in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein in the sample; wherein an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI, an amount of NGAL protein in the range of about 50 ng/ml to about 100 ng/ml indicates that the subject has mild AKI, an amount of NGAL protein in the range of about 100 ng/ml to about 250 ng/ml indicates that the subject has intermediate AKI, and an amount of NGAL protein higher than about 250 ng/ml indicates that the subject has severe AKI, and wherein the risk level is based on the RIFLE scoring system.
[0015] In another aspect, the invention described herein relates to a method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of NGAL protein and the amount of sCr protein present in the sample; wherein an amount of NGAL protein that is below about 100 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate a low risk of an adverse event, an amount of NGAL protein that exceeds or is equal to about 100 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate an intermediate risk of an adverse event, an amount of NGAL protein that is below about 100 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate an intermediate risk of an adverse event, and an amount of NGAL protein that exceeds or is equal to about 100 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate a high risk of an adverse event.
[0016] In another aspect, the invention described herein relates to a method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a urine sample from the subject; and (b) determining the amount of KIM- 1 protein and sCr protein present in the sample; wherein an amount of KIM- 1 protein that is below about 2 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate a low risk of an adverse event, an amount of KIM- 1 protein that exceeds or is equal to about 2 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate an intermediate risk of an adverse event, an amount of KIM- 1 protein that is below about 2 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate an intermediate risk of an adverse event, and an amount of KIM- 1 protein that exceeds or is equal to about 2 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate a high risk of an adverse event. In one embodiment, the adverse event is in-hospital mortality, or in-hospital dialysis, or both. [0017] In all of the above embodiments, as well as those described in the detailed description and the claims, other threshold values can be used. Suitable threshold values for each marker are provided in the detailed description and in the Examples.
[0018] In some embodiments of the methods described above, in the detailed description, and the claims, the subject is a human.
[0019] In some embodiments of the methods described above, in the detailed description, and the claims, the subject has no signs of kidney disease.
[0020] Some embodiments of the methods described above, in the detailed description, and the claims, further comprise subsequently treating the AKI in the subject.
[0021] Some embodiments of the methods described above, in the detailed description, and the claims, further comprise determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds about 1 mg/dl, indicates that the subject may have AKI.
[0022] In some embodiments of the methods described above, in the detailed description, and the claims, the determining step comprises contacting the sample with an antibody that binds to the NGAL protein and an antibody that binds to the KIM-1 protein.
[0023] In some embodiments of the methods described above, in the detailed description, and the claims, the method comprises performing an immunoassay. In some embodiments, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the antibodies are immobilized on a solid support. In some embodiments, the solid support comprises a dipstick or a test strip. In some embodiments, the method comprises performing an immunoblotting method.
[0024] In another aspect, the present invention is directed to diagnostic kits for carrying out any of the methods described herein. In some embodiments the kits comprise one or more of (a) a device for detecting NGAL protein present in a bodily fluid sample; (b) a device for detecting KIM-1 protein present in a bodily fluid sample; and (c) a device for detecting sCr in a bodily fluid sample. In some embodiments the kits contain instructions indicating threshold levels of one or more of NGAL, KIM-1, and sCr above which a determination can be made regarding whether a subject has a particular AKI status, including, but not limited to, whether or not the subject has AKI or is at risk of developing AKI, or whether or not the subject has, or is likely to develop, transient versus sustained AKI, or whether or not the subject has, or is likely to develop, mild, intermediate, or severe AKI as classified according to the well known RIFLE severity classification system. In some embodiments, the kits comprise one or more antibodies capable of binding to one or more of NGAL protein, KIM-1 protein, and serum creatinine (sCr). In some embodiments, the kits further comprise positive controls for one or more of NGAL protein, KIM-1 protein, and sCr. In some embodiments, the positive controls are in the device. In some embodiments, the device in the kits is an ELISA plate, a dipstick, or a test strip.
BRIEF DESCRIPTION OF THE FIGURES
[0025] Figure 1 shows a study flow chart. Patients from three emergency departments were recruited at the time of hospital admission and urine samples were collected. Urinary biomarkers were measured and correlated with the renal diagnosis and the subsequent hospital course.
[0026] Figure 2(A-F) shows urinary biomarker levels in the diagnostic classification of ED patients. uNGAL, uKIM-1, uL-FABP, uIL-18, and uCysC were compared by ANOVA in adjudicated patients with normal kidney function (Norm), stable CKD (CKD), pAKI, and iAKI (p values in upper left hand corners of each graph). Biomarkers differed significantly in patients with iAKI, compared to all other adjudicated groups (*p<0.05, **p<0.01,
***p<0.001 by posthoc Tukey test). All diagrams represent geometric means with 95% confidence intervals.
[0027] Figure 3 shows ROC curves for urinary biomarkers of nephron damage for the detection of intrinsic AKI (vs. prerenal AKI, stable CKD, or normal kidney function). A reference line with an area under the curve (AUC-ROC) of 0.5 is shown for comparison.
[0028] Figure 4 shows correlation between uNGAL levels quantified by chemiluminescent microparticle immunoassay (CMIA, Abbott ARCHITECT) and by immunoblot analysis of the monomeric form of AKI-associated NGAL.
[0029] Figure 5 (A-E) shows urinary biomarker levels by duration and maximal severity of AKI. Left panels show by duration of AKI. Biomarker levels were compared between patients with no AKI (None), transient AKI (Trans), and sustained AKI (Sust) using ANOVA (p values in upper left hand corners). uNGAL and uKIM-1 levels were significantly different in patients with sustained AKI when compared to patients with transient AKI (*** p<0.001 by post hoc Tukey test; ns, non- significant). Right panels show by severity of AKI.
Biomarker levels were analyzed by maximal RIFLE severity class within 7 days of hospitalization (by creatinine criteria; 0 = no AKI, R = RIFLE-R, I = RIFLE-I, F = RIFLE-F). ANOVA was significant for all biomarkers except uIL-18 (p values in upper left hand corners). Differences in biomarker levels between adjacent RIFLE classes were analyzed by post hoc Tukey test (* p<0.05; ** p<0.01; *** p<0.001). All diagrams represent geometric means with 95% confidence intervals.
[0030] Figure 6 shows rates of clinical events (initiation of dialysis or mortality in-hospital) in patients stratified by admission serum creatinine and uNGAL (A) or uKIM-1 (B). Cutoffs were applied at the 75th percentile for each biomarker (sCr: 1.4 mg/dl, uNGAL: 104 ng/ml, uKIM-1 : 2.82 ng/ml). Significance level was determined by Pearson's Chi Square test (*** pO.001, ** p<0.01, * p<0.05, n.s.=non-significant).
[0031] Figure 7 (A-B) shows box plots of biomarker levels in prediction of outcomes. Boxes indicate median, lower and upper quartiles. Whiskers represent data within 1.5 IQR of the lower quartile, and the within 1.5 IQR of the upper quartile. Circles represent outliers.
Biomarker levels are presented on a log 10 scale.
[0032] Figure 8 shows urinary NGAL level and outcomes. Patients were stratified by NGAL level. Percentages of the subgroups that experience endpoints are shown, p for trend is statistically significant for composite and individual AKI outcomes.
[0033] Figure 9 (A-H) shows biomarker expression in different patient diagnostic groups. Boxplots indicate median, inter-quartile range, and whiskers indicate the lowest datum still within 1.5 IQR of the lower quartile, and the highest datum within 1.5 IQR of the upper quartile. Circles indicate outliers. Circles at the top of the Y-axis indicate outliers beyond the range of the graph. Both the chemiluminescent microparticle immunoassay [NGAL CMIA (NGAL- ARCHITECT)] and NGAL-immunoblot (NGAL I-BLOT) were assayed in every sample in order to authentic the monomeric form of uNGAL. Data are expressed in ng/ml units.
[0034] Figure 10 shows association of the biomarker and the maximal RIFLE class achieved during hospitalization. The data are represented as fold-increase compared with normalized RIFLE negative levels. Note the response of the biomarkers to increasing RIFLE class (* p<0.05, n.s.- not significant).
[0035] Figure 11A shows a luminescent mouse model based on the insertion of luciferase in the NGAL locus. Luciferase expression is activated upon 12-hours of ischemia to the left kidney. Expression of NGAL protein comes from the kidney medulla (as shown by cutting open the left kidney versus the right kidney). Figure 11B shows mRNA situ hybridization, which demonstrates NGAL expression in both the TAL and the collecting ducts.
[0036] Figure 12 shows all AKI biomarkers were significantly elevated in patients with AKI. uNGAL had a more than 13 -fold elevation in AKI, the most robust response of all biomarkers. All biomarkers had a less than 1-fold elevation in patients with Prerenal
Azotemia and Stable CKD.
[0037] Figure 13 (A-F) shows biomarker levels are elevated with RIFLE positive azotemia. The pattern of response indicates that biomarkers respond to the severity and duration of RIFLE positive azotemia differently. For sCr, in addition to rising in AKI - sCr also rose in transient azotemia. This relationship was mirrored by that of KIM- 1 and L-FABP. However, for uNGAL and urine Cystatin-C, levels increased only in persistent RIFLE +/intrinsic AKI. Aspects of these two qualities were also evident for other biomarkers, notably L-FABP and Cy statin C.
[0038] Figure 14. Fig 14A shows associations between urinary biomarkers and death and dialysis adjusted for sCr, age, liver disease and study site. Fig. 14B shows associations between urinary biomarkers and death and dialysis adjusted for sCr, age, liver disease and SIRS. All AKI biomarkers independently predicted death and acute inpatient dialysis, howoever only KIM-1 and uNGAL predicted morality. Although sCr was associated with death and dialysis, it was not associated with mortality.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The issued patents, applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference.
Abbreviations and Definitions
[0040] The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
[0041] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%. [0042] The term "transient AKI" is defined as RIFLE-AKI that resolved within 72 hours.
[0043] The term "sustained AKI" is defined as RIFLE-AKI that persisted for 72 hours or more.
[0044] The abbreviation "iAKI" refers to intrinsic AKI.
[0045] The abbreviation "pAKI" refers to pre-renal AKI
[0046] The term "low risk," as used herein, generally indicates a risk or event rate of about 3% or less. The term "high risk," as used herein, generally indicates a risk or event rate of about 15% or more. Risk levels between low risk and high risk are referred to as
"intermediate risk."
Detailed Description
[0047] The invention described herein relates to methods for using urine biomarkers to independently diagnose AKI and to predicted death or dialysis, independently of sCr. In certain embodiments, a biomarker useful for distinguishing AKI from other diagnoses is uNGAL.
[0048] Over 1 million hospitalized Americans are diagnosed yearly with AKI. Acute kidney injury (AKI) is common and has severe consequences and AKI is associated with a 25-80%) increased risk of death. Acute kidney injury (AKI, also known as "intrinsic AKI") is a common clinical event with severe consequences. In the United States, over one-million hospitalized patients are diagnosed yearly with AKI, and its incidence is rising.1"4 AKI has been implicated in the pathogenesis of chronic kidney diseases (CKD),5 and it is associated with a 25-80% risk of in hiospital death 4' 6-7
[0049] Despite its prevalence, the diagnosis of AKI in the acute setting is challenging due in large part to the inadequacies of serum creatinine (sCr), the current diagnostic standard. Urinary AKI biomarker levels rise with AKI and biomarker responses to kidney dysfunction vary with severity and duration of RIFLE + Azotemia. AKI is challenging to diagnose because our clinical standard diagnostic is by serial serum creatinine (sCr) measurement, meaning the diagnosis is retrospective. Moreover, sCr can be elevated in other forms of kidney dysfunction, such as prerenal azotemia and chronic kidney disease, which in the absence of historical information can mimic AKI. Urinary biomarkers which might prospectively diagnose AKI have not been analyzed in large clinical studies. [0050] Current definitions of AKI depend on identifying a change in sCr from baseline. This may create several problems: (1) baseline sCr may be unknown when a patient presents for medical care; (2) hours to days may elapse after an injury, before sCr reaches a diagnostic threshold8; and (3) the level of sCr may not adequately reflect kidney injury, since the kinetics of sCr are altered by age, gender, muscle mass, nutritional status, hemodynamics, fluid status, medications and underlying CKD.8 10 These limitations have generated intense interest in the identification of "biomarkers" which provide early diagnosis of AKI and distinguish it from other mechanisms which also elevate SCr.11"12 Several urine biomarkers are elevated by stresses and injuries to the kidney, including urinary neutrophil gelatinase- associated lipocalin (uNGAL), kidney injury molecule 1 (uKIM-I), interleukin 18 (uIL-I8), liver fatty acid binding protein (uL-FABP), cystatinC (uCysC) and the fractional excretion of sodium (FENa).13-16 However, the value of these biomarkers to diagnose AKI has been investigated only in small, single-center studies, usually involving subtypes of AKI in selected clinical settings. Larger studies in heterogeneous populations are lacking and it is unclear which urinary biomarker performs best in prospectively diagnosing AKI at patient presentation.
[0051] In certain aspects, the invention relates to the finding that urine AKI biomarkers can predict AKI, with urine NGAL anticipating both severity and duration of azotemia, which is also shown in part by other biomarkers. In certain aspect, the invention relates to the finding that AKI can be distinguished from other causes of elevated sCr by biomarkers, in particular NGAL. In certain aspects, the invention relates to the finding that KIM-1 and NGAL biomarkers can be useful predictors of death. In certain aspects, the invention relates to the finding that NGAL protein is highly elevated in critical illness and that NGAL is a highly up- regulated gene in the setting of ischemic AKI.
[0052] In certain aspects, certain embodiments of the invention relate to the finding that a single measurement of urine biomarkers predicted both the development of AKI and a combined outcome of death and dialysis in a heterogeneous cohort of patients being admitted from the ED. In certain embodiments, unlike sCr, both uNGAL and uKIM-1 can be used to predict both the development of AKI and a combined outcome of death and dialysis.
[0053] jn cerj-am aspects, the invention described herein is based on testing of a panel of urine biomarkers against classical algorithms of kidney function to separate conditions that acutely elevate sCr (i.e. AKI and prerenal azotemia). In certain embodiments, the biomarkers described herein can be applied to diverse pathophysiologies, treatments and clinical outcomes that can otherwise confound the diagnosis of true kidney injury and the use of the RIFLE score.12' 15' 16' 45'
[0054] In certain aspects, the invention relates to the diagnosis of AKI in triage, and in certain embodiments, to findings that: (a) a single measurement of a urinary biomarker can be used to diagnose AKI as defined by serial measurements of sCr, (b) the NPV of urine biomarkers for AKI is high and corresponding negative likelihood ratios are low, in particular for uNGAL; therefore, levels below defined cutoffs described herein make ongoing AKI decidedly unlikely; and (c) uNGAL has significantly better diagnostic test characteristics than other biomarkers, particularly in separating AKI from prerenal azotemia and CKD.
[0055] The biomarker cutoff values described herein can also be used to evaluate combined outcome of death or acute dialysis and can be conducted on all patients regardless of adjudication status. The results described herein show that the biomarker cutoff values described herein can be used to predicte death or acute dialysis independently of sCr.
[0056] In certain embodiments, the uNGAL and uKIM-1 cutoff values described herein are independently associated with death after adjustment for other covariates, whereas other biomarkers and sCr are not. In certain aspects, the biomarker cutoff values described herein can be used for the prediction of in-hospital mortality in the ED population. In other embodiments, , the biomarker cutoff values described herein can be used for the prediction of in-hospital mortality, or poor outcome, for patients who cannot be diagnosed using classical algorithms ("Unclear Diagnoses", Table 1).
[0057] In certain aspects, the invention relates to the finding from analyses conducted to evaluate relationships between sCr, urine biomarkers and AKI diagnostics and to identify biomarkers that are useful even without reference to adjudicated diagnoses. Most of the biomarkers described herein (uNGAL, uKIM-1, uL-FABP and uCysC but not uIL-18) rose significantly from baseline in proportion to severity of RIFLE score, but uNGAL
demonstrated the most robust dose-response (>10-fold from baseline to RIFLE-F).
[0058] In certain aspects, the invention relates to findings from stratified ROC analyses showing that the biomarkers described herein consistently detected AKI regardless of site, gender, race, ethnicity, CKD history or the presence of either UTI or prerenal azotemia at presentation (Table 4). In certain embodiments, the biomarkers and cutoff values described herein are generalizable to heterogeneous populations in gender, race-ethnicity,
comorbidities, and differing etiologies of AKI. [0059] Because the analysis of multiple glomerular and tubular biomarkers may be confounded by inter-correlations, certain aspects of the invention relate to the use of PC A to determine the relationship between factors that measure or contribute to kidney function and the diagnosis of AKI. In certain embodiments, the invention relates to the finding that biomarkers of tubular damage make the greatest contribution to the diagnosis of AKI. In certain embodiments, the invention relates to the finding that markers of glomerular function and physiologic states make a contribution to the diagnosis of AKI.
[0060] The urinary biomarkers describe herein are diagnostic tools that can be useful for improving the diagnosis of AKI in acute triage settings beyond the classical tests of renal function. In certain embodiments, the methods described herein can be used to diagnose patients who could not be diagnosed using classical algorithms ("Unclear Diagnoses", Table 1) and identify patients that will to benefit from the use of urinary biomarkers.
NGAL
[0061] In one aspect, the present invention provides that neutrophil gelatinase-associated lipocalin (NGAL) levels are higher in AKI patients than in patients that do not have AKI. In one aspect of the invention, amounts of NGAL protein in a bodily fluid, such as urine or blood, that exceed a certain threshold amount, may be used to diagnose AKI in a subject, predict in-hospital mortality of a subject suspected of having AKI, predict the need for in- hospital dialysis in a subject suspected of having AKI, determine whether a subject suspected of having AKI has transient or sustained AKI, determine the severity of the AKI in subject suspected of having AKI, and/or determine the risk of an adverse event in a subject suspected of having AKI.
[0062] In one aspect, the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein present in the sample, wherein an amount of NGAL protein that exceeds a threshold amount indicates that the subject may have AKI.
[0063] In another aspect, the invention described herein relates to a method for predicting in- hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein present in the sample, wherein an amount of NGAL protein that exceeds a threshold amount indicates a high risk of subject mortality. [0064] In another aspect, the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein present in the sample, wherein an amount of NGAL protein that exceeds a threshold amount indicates that the subject is at high risk of needing in-hospital dialysis.
[0065] In another aspect, the invention described herein relates to a diagnostic kit for diagnosing AKI in a subject, the kit comprising a) a device for detecting the amount of NGAL protein present in a sample of a bodily fluid; and b) instructions indicating threshold amounts of the NGAL protein, above which a determination can be made that the patient has AKI or is at risk of developing AKI. In one embodiment, the kit comprises an antibody capable of binding to the NGAL protein. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. In another embodiment, the kit further comprises a positive control for the NGAL protein. In one embodiment, the positive control is in the device. In one embodiment, the device is an ELISA plate, a dipstick, or a test strip.
[0066] In one embodiment, the threshold amount of NGAL protein is about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, or about 155 ng/ml, or about 160 ng/ml, or about 165 ng/ml, or about 170 ng/ml, or about 175 ng/ml, or about 180 ng/ml, or about 185 ng/ml, or about 190 ng/ml, or about 195 ng/ml, or about 200 ng/ml, or about 225 ng/ml, or about 250 ng/ml, or about 275 ng/ml, or about 300 ng/ml, or about 325 ng/ml, or about 350 ng/ml, or about 375 ng/ml, or about 400 ng/ml, or more.
[0067] In one embodiment, the method further comprises subsequently treating the AKI in the subject. Treatment can comprise the use of any treatment well known to one of skill in the art. [0068] In another embodiment, the method further comprises determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds a threshold amount indicates that the subject may have AKI. In one embodiment, the amount of sCr protein is about 0.1 mg/dl, or about 0.2 mg/dl, or about 0.3 mg/dl, or about 0.4 mg/dl, or about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1.0 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2.0 mg/dl, or about 2.1 mg/dl, or about 2.2 mg/dl, or about 2.3 mg/dl, or about 2.4 mg/dl, or about 2.5 mg/dl, or about 2.6 mg/dl, or about 2.7 mg/dl, or about 2.8 mg/dl, or about 2.9 mg/dl, or about 3.0 mg/dl, or more.
[0069] In all of the embodiments herein that deal with making a determination based on whether or not an amount of one or more marker(s) in a bodily fluid exceeds a threshold amount, any amount that is greater than the threshold amount is to be considered to exceed the threshold amount. In general, the further above the threshold the amount of the marker(s) is/are, the greater the likelihood that the subject is at risk of developing AKI or has AKI. For example, an amount of one or more marker(s) may exceed the threshold by about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%>, or about 12%>, or about 13%>, or about 14%>, or about 15%o, or about 16%>, or about 17%>, or about 18%>, or about 19%>, or about 20%>, or about 21%>, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%), or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%), or about 35%, or about 36%, or about 37%, or about 38%, or about 39%, or about 40%, or about 41%, or about 42%, or about 43%, or about 44%, or about 45%, or about 46%, or about 47%, or about 48%, or about 49%, or about 50%, or about 75%, or about 100%, or more.
[0070] In one embodiment, the determining step comprises contacting the sample with an antibody that binds to the NGAL protein. In one embodiment, the antibody is immobilized on a solid support. In one embodiment, the solid support comprises a dipstick or a test strip. In another embodiment, the method comprises performing an immunoassay. In one embodiment, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In another embodiment, the method comprises performing an immunoblotting method. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. [0071] In yet another aspect, the invention described herein relates to a method for predicting the severity of AKI in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject at a first point in time; (b) determining the amount of NGAL protein at the first point in time in the sample; (c) obtaining a sample of a bodily fluid from the subject at a second point in time; (d) determining the amount of NGAL protein at the second point in time in the sample; wherein the subject does not show any signs of kidney disease at the first point in time, and wherein an increase in the amount of NGAL protein present in the sample at the second point in time compared to the amount of NGAL protein present in the sample at the first point in time indicates that the subject has or is at risk of developing AKI.
[0072] In one embodiment, an increase of about 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold indicates that the subject is at risk of developing AKI. In another embodiment, an increase of about 1-2 fold, 2-3 fold, 3-4 fold, 4-5 fold, or about 1-3 fold, 2-4 fold, 3-5 fold, 1-4 fold, or 2- 5 fold, indicates that the subject is at risk of developing AKI.
[0073] In another embodiment, an increase of about 5-fold, 6-fold, 7-fold or 8-fold indicates that the subject has AKI. In another embodiment, an increase of about 5-6 fold, 6-7 fold, 7-8 fold, or about 5-7 fold, 6-8 fold, or about 5-8 fold, indicates that the subject has AKI.
[0074] In another embodiment, an increase of about 9-fold, or 10-fold, or 11 -fold, or 12-fold, or more, indicates that the subject is at risk of kidney failure due to AKI. In another embodiment, an increase of about 9-10 fold, 10-11 fold, 11-12 fold, or more, or about 9-11 fold or 9-12 fold, indicates that the subject is at risk of kidney failure due to AKI.
[0075] In another aspect, the invention described herein relates to a method for determining whether a subject suspected of having AKI has transient or sustained AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein in the sample.
[0076] In one embodiment, an amount of NGAL protein in the range of about 15 ng/ml to about 20 ng/ml, or about 20 ng/ml to about 25 ng/ml, or about 25 ng/ml to about 30 ng/ml, or about 30 ng/ml to about 35 ng/ml, or about 35 ng/ml to about 40 ng/ml, or about 40 ng/ml to about 45 ng/ml, or about 45 ng/ml to about 50 ng/ml, or about 50 ng/ml to about 55 ng/ml, or about 55 ng/ml to about 60 ng/ml, or about 60 ng/ml to about 65 ng/ml, or about 15 ng/ml to about 25 ng/ml, or about 20 ng/ml to about 30 ng/ml, or about 25 ng/ml to about 35 ng/ml, or about 30 ng/ml to about 40 ng/ml, or about 35 ng/ml to about 45 ng/ml, or about 40 ng/ml to about 50 ng/ml, or about 45 ng/ml to about 55 ng/ml, or about 50 ng/ml to about 60 ng/ml, or about 15 ng/ml to about 30 ng/ml, or about 20 ng/ml to about 40 ng/ml, or about 30 ng/ml to about 50 ng/ml, or about 40 ng/ml to about 60 ng/ml, or any range in between, indicates that the subject does not have AKI.
[0077] In one embodiment, an amount of NGAL protein in the range of about 35 ng/ml to about 40 ng/ml, or about 40 ng/ml to about 45 ng/ml, or about 45 ng/ml to about 50 ng/ml, or about 50 ng/ml to about 55 ng/ml, or about 55 ng/ml to about 60 ng/ml, or about 60 ng/ml to about 65 ng/ml, or about 65 ng/ml to about 70 ng/ml, or about 70 ng/ml to about 75 ng/ml, or about 75 ng/ml to about 80 ng/ml, or about 80 ng/ml to about 85 ng/ml, or about 85 ng/ml to about 90 ng/ml, or about 90 ng/ml to about 95 ng/ml, or about 95 ng/ml to about 100 ng/ml, or about 100 ng/ml to about 125 ng/ml, or about 125 ng/ml to about 150 ng/ml, or about 35 ng/ml to about 50 ng/ml, or about 40 ng/ml to about 60 ng/ml, or about 50 ng/ml to about 70 ng/ml, or about 60 ng/ml to about 80 ng/ml, or about 70 ng/ml to about 90 ng/ml, or about 80 ng/ml to about 100 ng/ml, or about 90 ng/ml to about 125 ng/ml, or about 100 ng/ml to about 150 ng/ml, or about 40 ng/ml to about 70 ng/ml, or about 50 ng/ml to about 80 ng/ml, or about 60 ng/ml to about 90 ng/ml, or about 70 ng/ml to about 100 ng/ml, or about 80 ng/ml to about 125 ng/ml, or about 100 ng/ml to about 150 ng/ml, or any range in between, indicates that the subject has transient AKI.
[0078] In one embodiment, an amount of NGAL protein in the range of about 60 ng/ml to about 70 ng/ml, or about 70 ng/ml to about 80 ng/ml, or about 80 ng/ml to about 90 ng/ml, or about 90 ng/ml to about 100 ng/ml, or about 100 ng/ml to about 110 ng/ml, or about 110 ng/ml to about 120 ng/ml, or about 120 ng/ml to about 130 ng/ml, or about 130 ng/ml to about 140 ng/ml, or about 140 ng/ml to about 150 ng/ml, or about 150 ng/ml to about 160 ng/ml, or about 160 ng/ml to about 170 ng/ml, or about 170 ng/ml to about 180 ng/ml, or about 180 ng/ml to about 190 ng/ml, or about 190 ng/ml to about 200 ng/ml, or about 200 ng to about 210 ng/ml, or about 210 ng/ml to about 220 ng/ml, or about 220 ng/ml to about 230 ng/ml, or about 230 ng/ml to about 240 ng/ml, or about 240 ng/ml to about 250 ng/ml, or about 250 ng/ml to about 260 ng/ml, or about 260 ng/ml to about 270 ng/ml, or about 270 ng/ml to about 280 ng/ml, or about 280 ng/ml to about 290 ng/ml, or about 290 ng/ml to about 300 ng/ml, or about 60 ng/ml to about 100 ng/ml, or about 80 ng/ml to about 120 ng/ml, or about 100 ng/ml to about 140 ng/ml, or about 120 ng/ml to about 160 ng/ml, or about 140 ng/ml to about 180 ng/ml, or about 160 ng/ml to about 200 ng/ml, or about 180 ng/ml to about 220 ng/ml, or about 200 ng/ml to about 240 ng/ml, or about 100 ng/ml to about 200 ng/ml, or about 150 ng/ml to about 250 ng/ml, or about 200 ng/ml to about 300 ng/ml, or any range in between, indicates that the subject has sustained AKI.
[0079] In another embodiment, an amount of NGAL protein that exceeds about 100 ng/ml, or about 110 ng/ml, or about 120 ng/ml, or about 130 ng/ml, or about 140 ng/ml, or about 150 ng/ml, or about 160 ng/ml, or about 160 ng/ml, or about 170 ng/ml, or about 180 ng/ml, or about 190 ng/ml, or about 200 ng/ml, or about 210 ng/ml, or about 220 ng/ml, or about 230 ng/ml, or about 240 ng/ml, or about 250 ng/ml, or about 260 ng/ml, or about 270 ng/ml, or about 280 ng/ml, or about 290 ng/ml, or about 300 ng/ml, indicates that the subject has sustained AKI.
[0080] In one aspect, the invention described herein relates to a method for determining the severity of the AKI in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein in the sample.
[0081] In one embodiment, an amount of NGAL protein in the range of about 15 ng/ml to about 20 ng/ml, or about 20 ng/ml to about 25 ng/ml, or about 25 ng/ml to about 30 ng/ml, or about 30 ng/ml to about 35 ng/ml, or about 35 ng/ml to about 40 ng/ml, or about 40 ng/ml to about 45 ng/ml, or about 45 ng/ml to about 50 ng/ml, or about 50 ng/ml to about 55 ng/ml, or about 55 ng/ml to about 60 ng/ml, or about 60 ng/ml to about 65 ng/ml, or about 15 ng/ml to about 25 ng/ml, or about 20 ng/ml to about 30 ng/ml, or about 25 ng/ml to about 35 ng/ml, or about 30 ng/ml to about 40 ng/ml, or about 35 ng/ml to about 45 ng/ml, or about 40 ng/ml to about 50 ng/ml, or about 45 ng/ml to about 55 ng/ml, or about 50 ng/ml to about 60 ng/ml, or about 15 ng/ml to about 30 ng/ml, or about 20 ng/ml to about 40 ng/ml, or about 30 ng/ml to about 50 ng/ml, or about 40 ng/ml to about 60 ng/ml, or any range in between, indicates that the subject does not have AKI.
[0082] In one embodiment, an amount of NGAL protein in the range of about 35 ng/ml to about 40 ng/ml, or about 40 ng/ml to about 45 ng/ml, or about 45 ng/ml to about 50 ng/ml, or about 50 ng/ml to about 55 ng/ml, or about 55 ng/ml to about 60 ng/ml, or about 60 ng/ml to about 65 ng/ml, or about 65 ng/ml to about 70 ng/ml, or about 70 ng/ml to about 75 ng/ml, or about 75 ng/ml to about 80 ng/ml, or about 80 ng/ml to about 85 ng/ml, or about 85 ng/ml to about 90 ng/ml, or about 90 ng/ml to about 95 ng/ml, or about 95 ng/ml to about 100 ng/ml, or about 100 ng/ml to about 125 ng/ml, or about 125 ng/ml to about 150 ng/ml, or about 35 ng/ml to about 50 ng/ml, or about 40 ng/ml to about 60 ng/ml, or about 50 ng/ml to about 70 ng/ml, or about 60 ng/ml to about 80 ng/ml, or about 70 ng/ml to about 90 ng/ml, or about 80 ng/ml to about 100 ng/ml, or about 90 ng/ml to about 125 ng/ml, or about 100 ng/ml to about 150 ng/ml, or about 40 ng/ml to about 70 ng/ml, or about 50 ng/ml to about 80 ng/ml, or about 60 ng/ml to about 90 ng/ml, or about 70 ng/ml to about 100 ng/ml, or about 80 ng/ml to about 110 ng/ml, or about 35 ng/ml to about 85 ng/ml, or about 50 ng/ml to about 100 ng/ml, or about 75 ng/ml to about 125 ng/ml, or any range in between, indicates that the subject has mild AKI.
[0083] In one embodiment, an amount of NGAL protein in the range of about 60 ng/ml to about 70 ng/ml, or about 70 ng/ml to about 80 ng/ml, or about 80 ng/ml to about 90 ng/ml, or about 90 ng/ml to about 100 ng/ml, or about 100 ng/ml to about 110 ng/ml, or about 110 ng/ml to about 120 ng/ml, or about 120 ng/ml to about 130 ng/ml, or about 130 ng/ml to about 140 ng/ml, or about 140 ng/ml to about 150 ng/ml, or about 150 ng/ml to about 160 ng/ml, or about 160 ng/ml to about 170 ng/ml, or about 170 ng/ml to about 180 ng/ml, or about 180 ng/ml to about 190 ng/ml, or about 190 ng/ml to about 200 ng/ml, or about 200 ng to about 210 ng/ml, or about 210 ng/ml to about 220 ng/ml, or about 220 ng/ml to about 230 ng/ml, or about 230 ng/ml to about 240 ng/ml, or about 240 ng/ml to about 250 ng/ml, or about 250 ng/ml to about 260 ng/ml, or about 260 ng/ml to about 270 ng/ml, or about 270 ng/ml to about 280 ng/ml, or about 280 ng/ml to about 290 ng/ml, or about 290 ng/ml to about 300 ng/ml, or about 60 ng/ml to about 100 ng/ml, or about 80 ng/ml to about 120 ng/ml, or about 100 ng/ml to about 140 ng/ml, or about 120 ng/ml to about 160 ng/ml, or about 140 ng/ml to about 180 ng/ml, or about 160 ng/ml to about 200 ng/ml, or about 180 ng/ml to about 220 ng/ml, or about 200 ng/ml to about 240 ng/ml, or about 220 ng/ml to about 260 ng/ml, or about 100 ng/ml to about 200 ng/ml, or about 150 ng/ml to about 250 ng/ml, or about 200 ng/ml to about 300 ng/ml, or about 100 ng/ml to about 250 ng/ml, or about 150 ng/ml to about 300 ng/ml, or about 200 ng/ml to about 350 ng/ml, or any range in between, indicates that the subject has intermediate AKI.
[0084] In one embodiment, an amount of NGAL protein in the range of about 100 ng/ml to about 150 ng/ml, or about 150 ng/ml to about 250 ng/ml, or about 200 ng/ml to about 300 ng/ml, or about 250 ng/ml to about 350 ng/ml, or about 300 ng/ml to about 400 ng/ml, or about 350 ng/ml to about 450 ng/ml, or about 400 ng to about 500 ng/ml, or about 450 ng/ml to about 550 ng/ml, or about 500 ng/ml to about 600 ng/ml, or about 550 ng/ml to about 650 ng/ml, or about 600 ng/ml to about 700 ng/ml, or about 200 ng/ml to about 400 ng/ml, or about 300 ng/ml to about 500 ng/ml, or about 400 ng/ml to about 600 ng/ml, or about 500 ng/ml to about 700 ng/ml, or any range in between, indicates that the subject has severe AKI.
[0085] In another embodiment, an amount of NGAL protein higher than about 150 ng/ml, or about 160 ng/ml, or about 170 ng/ml, or about 180 ng/ml, or about 190 ng/ml, or about 200 ng/ml, or about 210 ng/ml, or about 220 ng/ml or about 230 ng/ml, or about 240 ng/ml, or about 250 ng/ml, or about 260 ng/ml, or about 270 ng/ml, or about 280 ng/ml, or about 290 ng/ml, or about 300 ng/ml, indicates that the subject has severe AKI.
[0086] In one embodiment, the severity level is based on the RIFLE scoring system. In another embodiment, the severity level is not based on the RIFLE scoring system.
[0087] In another aspect, the invention described herein relates to a method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and the amount of sCr protein present in the sample.
[0088] In one embodiment, an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl, indicate a low risk of an adverse event.
[0089] In another embodiment, an amount of NGAL protein that exceeds or is equal to about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl indicate indicate an intermediate risk of an adverse event.
[0090] In another embodiment, an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl indicate an intermediate risk of an adverse event.
[0091] In another embodiment, an amount of NGAL protein that exceeds or is equal to about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl, indicate a high risk of an adverse event.
[0092] In one embodiment, the adverse event comprises in-hospital mortality. In another embodiment, the adverse event comprises in-hospital dialysis. In another embodiment, the adverse event comprises both in-hospital mortality and in-hospital dialysis.
[0093] In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. In one embodiment, the subject has no signs of kidney disease. In another embodiment, the subject has signs of kidney disease. In one embodiment, the subject has undergone or will undergo hemodialysis treatment.
[0094] In one embodiment, the bodily fluid used in accordance with the invention is blood. In another embodiment, the bodily fluid is urine. In another embodiment, the bodily fluid is plasma. In still another embodiment, the bodily fluid is blood serum.
[0095] The diagnostic methods described herein can be combined in various ways. For example, in some embodiments only one marker is assessed. In other embodiments, more than one of the markers may be assessed, in any combination, and optionally together with one or more additional markers.
KIM- 1
[0096] In one aspect, the present invention provides that kidney injury molecule 1 (KIM-1) levels are higher in AKI patients than in patients that do not have AKI. In one aspect of the invention, amounts of KIM-1 protein in a bodily fluid, such as urine or blood, that exceed a certain threshold amount, may be used to diagnose AKI in a subject, predict in-hospital mortality of a subject suspected of having AKI, predict the need for in-hospital dialysis in a subject suspected of having AKI, and/or determine the risk of an adverse event in a subject suspected of having AKI.
[0097] In one aspect, the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM-1 protein present in the sample, wherein an amount of KIM-1 protein that exceeds a threshold amount indicates that the subject may have AKI. [0098] In another aspect, the invention described herein relates to a method for predicting in- hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM- 1 protein present in the sample, wherein an amount of KIM- 1 protein that exceeds a threshold amount indicates a high risk of subject mortality.
[0099] In another aspect, the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM-1 protein present in the sample, wherein an amount of KIM- 1 protein that exceeds a threshold amount indicates that the subject is at high risk of needing in-hospital dialysis.
[00100] In another aspect, the invention described herein relates to a diagnostic kit for diagnosing AKI in a subject, the kit comprising a) a device for detecting the amount of KIM - 1 protein present in a sample of a bodily fluid; and b) instructions indicating threshold amounts of the KIM-1 protein, above which a determination can be made that the patient has AKI or is at risk of developing AKI. In one embodiment, the kit comprises an antibody capable of binding to the KIM-1 protein. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. In another embodiment, the kit further comprises a positive control for the KIM-1 protein. In one embodiment, the positive control is in the device. In one embodiment, the device is an ELISA plate, a dipstick, or a test strip.
[00101] In one embodiment, the threshold amount of KIM-1 protein is about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, or more. [00102] In one embodiment, the method further comprises subsequently treating the AKI in the subject. Treatment can comprise the use of any treatment well known to one of skill in the art.
[00103] In another embodiment, the method further comprises determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds a threshold amount indicates that the subject may have AKI. In one embodiment, the amount of sCr protein is about 0.1 mg/dl, or about 0.2 mg/dl, or about 0.3 mg/dl, or about 0.4 mg/dl, or about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1.0 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2.0 mg/dl, or about 2.1 mg/dl, or about 2.2 mg/dl, or about 2.3 mg/dl, or about 2.4 mg/dl, or about 2.5 mg/dl, or about 2.6 mg/dl, or about 2.7 mg/dl, or about 2.8 mg/dl, or about 2.9 mg/dl, or about 3.0 mg/dl.
[00104] In all of the embodiments herein that deal with making a determination based on whether or not an amount of one or more marker(s) in a bodily fluid exceeds a threshold amount, any amount that is greater than the threshold amount is to be considered to exceed the threshold amount. In general, the further above the threshold the amount of the marker(s) is/are, the greater the likelihood that the subject is at risk of developing AKI or has AKI. For example, an amount of one or more marker(s) may exceed the threshold by about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%>, or about 12%>, or about 13%>, or about 14%>, or about 15%o, or about 16%>, or about 17%>, or about 18%>, or about 19%>, or about 20%>, or about 21%>, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%), or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%), or about 35%, or about 36%, or about 37%, or about 38%, or about 39%, or about 40%, or about 41%, or about 42%, or about or about 43%, or about 44%, or about 45%, or about 46%), or about 47%, or about 48%, or about 49%, or about 50%, or about 75%, or about 100%), or more.
[00105] In one embodiment, the determining step comprises contacting the sample with an antibody that binds to the KIM-1 protein. In one embodiment, the antibody is immobilized on a solid support. In one embodiment, the solid support comprises a dipstick or a test strip. In another embodiment, the method comprises performing an immunoassay. In one embodiment, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In another embodiment, the method comprises performing an immunoblotting method. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody.
[00106] In another aspect, the invention described herein relates to a method for
determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of KIM- 1 protein and the amount of sCr protein present in the sample.
[00107] In one embodiment, an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl, indicate a low risk of an adverse event.
[00108] In another embodiment, an amount of KIM- 1 protein that exceeds or is equal to about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl indicate an intermediate risk of an adverse event.
[00109] In another embodiment, an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about
1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 ng/ml indicate an intermediate risk of an adverse event.
[00110] In another embodiment, an amount of KIM- 1 protein that exceeds or is equal to about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about
1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 ng/ml indicate a high risk of an adverse event.
[00111] In one embodiment, the adverse event comprises in-hospital mortality. In another embodiment, the adverse event comprises in-hospital dialysis. In another embodiment, the adverse event comprises both in-hospital mortality and in-hospital dialysis.
[00112] In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. In one embodiment, the subject has no signs of kidney disease. In another embodiment, the subject has signs of kidney disease. In one embodiment, the subject has undergone or will undergo hemodialysis treatment.
[00113] In one embodiment, the bodily fluid used in accordance with the invention is blood. In another embodiment, the bodily fluid is urine. In another embodiment, the bodily fluid is plasma. In still another embodiment, the bodily fluid is blood serum.
[00114] The diagnostic methods described herein can be combined in various ways. For example, in some embodiments only one marker is assessed. In other embodiments, more than one of the markers may be assessed, in any combination, and optionally together with one or more additional markers.
NGAL andKIM-1
[00115] In one aspect, the invention described herein relates to a method for diagnosing acute kidney injury (AKI) in a subject, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, or about 155 ng/ml, or about 160 ng/ml, and an amount of KIM- 1 protein that exceeds about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, indicate that the subject may have AKI.
[00116] In one embodiment, the method further comprises subsequently treating the AKI in the subject. Treatment can comprise the use of any treatment well known to one of skill in the art.
[00117] In another embodiment, the method further comprises determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds about 0.1 mg/dl, or about 0.2 mg/dl, or about 0.3 mg/dl, or about 0.4 mg/dl, or about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1.0 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2.0 mg/dl, or about 2.1 mg/dl, or about 2.2 mg/dl, or about 2.3 mg/dl, or about 2.4 mg/dl, or about 2.5 mg/dl, or about 2.6 mg/dl, or about 2.7 mg/dl, or about 2.8 mg/dl, or about 2.9 mg/dl, or about 3.0 mg/dl, indicates that the subject may have AKI.
[00118] In one embodiment, the determining step comprises contacting the sample with an antibody that binds to the NGAL protein and an antibody that binds to the KIM-1 protein. In one embodiment, the method comprises performing an immunoassay. In one embodiment, the immunoassay is an enzyme-linked immunosorbent assay (ELISA). In one embodiment, the antibodies are immobilized on a solid support. In one embodiment, the solid support comprises a dipstick or a test strip. In one embodiment, the method comprises performing an immunoblotting method. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody. [00119] In another aspect, the invention described herein relates to a method for predicting in-hospital mortality of a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and the amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, or about 155 ng/ml, or about 160 ng/ml, and an amount of KIM- 1 protein that exceeds about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, indicate a high risk of subject mortality.
[00120] In yet another aspect, the invention described herein relates to a method for predicting the need for in-hospital dialysis in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein and the amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, or about 155 ng/ml, or about 160 ng/ml, and an amount of KIM- 1 protein that exceeds about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml indicate that the subject is at high risk of needing in-hospital dialysis.
[00121] In another aspect, the invention described herein relates to a method for
determining the risk of an adverse event in a subject suspected of having AKI, the method comprising (a) obtaining a sample of a bodily fluid from the subject; and (b) determining the amount of NGAL protein, and the amount of KIM- 1 protein, and the amount of sCr protein present in the sample.
[00122] In one embodiment, an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, or about 155 ng/ml, or about 160 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl, and an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml indicate a low risk of an adverse event.
[00123] In another embodiment, an amount of NGAL protein that exceeds or is equal to about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl indicate indicate an intermediate risk of an adverse event.
[00124] In another embodiment, an amount of NGAL protein that is below about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl indicate an intermediate risk of an adverse event.
[00125] In another embodiment, an amount of KIM- 1 protein that exceeds or is equal to about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about
3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that is below about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl indicate an intermediate risk of an adverse event.
[00126] In another embodiment, an amount of KIM- 1 protein that is below about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about
2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 ng/ml indicate an intermediate risk of an adverse event.
[00127] In another embodiment, an amount of NGAL protein that exceeds or is equal to about 20 ng/ml, or about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, and an amount of sCr protein that exceeds or is equal to about 0.5 mg/dl, or about 0.6 mg/dl, or about 0.7 mg/dl, or about 0.8 mg/dl, or about 0.9 mg/dl, or about 1 mg/dl, or about 1.1 mg/dl, or about 1.2 mg/dl, or about 1.3 mg/dl, or about 1.4 mg/dl, or about 1.5 mg/dl, or about 1.6 mg/dl, or about 1.7 mg/dl, or about 1.8 mg/dl, or about 1.9 mg/dl, or about 2 mg/dl, or about 2.5 mg/dl, or about 3 mg/dl, and an amount of KIM- 1 protein that exceeds or is equal to about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml, indicate a high risk of an adverse event.
[00128] In other embodiments, various combinations of the amounts of NGAL protein, KIM-1 protein and sCr protein can be used to determine the risk of an adverse event in a subject suspected of having AKI. [00129] In one embodiment, the adverse event comprises in-hospital mortality. In another embodiment, the adverse event comprises in-hospital dialysis. In another embodiment, the adverse event comprises both in-hospital mortality and in-hospital dialysis.
[00130] In another aspect, the invention described herein relates to a diagnostic kit for diagnosing AKI in a subject, the kit comprising (a) a device for detecting the amount of NGAL protein present in a sample of a bodily fluid; (b) a device for detecting the amount of KIM-1 protein present in a sample of a bodily fluid; and (c) instructions indicating threshold levels of the NGAL protein and the KIM-1 protein, above which a determination can be made that the patient has AKI or is at risk of developing AKI, wherein the threshold levels are about 25 ng/ml, or about 30 ng/ml, or about 35 ng/ml, or about 40 ng/ml, or about 45 ng/ml, or about 50 ng/ml, or about 55 ng/ml, or about 60 ng/ml, or about 65 ng/ml, or about 70 ng/ml, or about 75 ng/ml, or about 80 ng/ml, or about 85 ng/ml, or about 90 ng/ml, or about 95 ng/ml, or about 100 ng/ml, or about 101 ng/ml, or about 102 ng/ml, or about 103 ng/ml, or about 104 ng/ml, or about 105 ng/ml, or about 106 ng/ml, or about 107 ng/ml, or about 108 ng/ml, or about 109 ng/ml, or about 110 ng/ml, or about 115 ng/ml, or about 120 ng/ml, or about 125 ng/ml, or about 130 ng/ml, or about 135 ng/ml, or about 140 ng/ml, or about 145 ng/ml, or about 150 ng/ml, or about 155 ng/ml, or about 160 ng/ml for the NGAL protein, and about 0.5 ng/ml, or about 0.6 ng/ml, or about 0.7 ng/ml, or about 0.8 ng/ml, or about 0.9 ng/ml, or about 1 ng/ml, or about 1.1 ng/ml, or about 1.2 ng/ml, or about 1.3 ng/ml, or about 1.4 ng/ml, or about 1.5 ng/ml, or about 1.6 ng/ml, or about 1.7 ng/ml, or about 1.8 ng/ml, or about 1.9 ng/ml, or about 2 ng/ml, or about 2.1 ng/ml, or about 2.2 ng/ml, or about 2.3 ng/ml, or about 2.4 ng/ml, or about 2.5 ng/ml, or about 2.6 ng/ml, or about 2.7 ng/ml, or about 2.8 ng/ml, or about 2.9 ng/ml, or about 3 ng/ml, or about 3.1 ng/ml, or about 3.2 ng/ml, or about 3.3 ng/ml, or about 3.4 ng/ml, or about 3.5 ng/ml, or about 3.6 ng/ml, or about 3.7 ng/ml, or about 3.8 ng/ml, or about 3.9 ng/ml, or about 4 ng/ml, or about 4.1 ng/ml, or about 4.2 ng/ml, or about 4.3 ng/ml, or about 4.4 ng/ml, or about 4.5 ng/ml, or about 4.6 ng/ml, or about 4.7 ng/ml, or about 4.8 ng/ml, or about 4.9 ng/ml, or about 5 ng/ml for the KIM-1 protein.
[00131] It should also be noted that, in each of the embodiments described herein, other threshold levels of NGAL, KIM-1, and sCr can be selected based on the data provided in the Examples section of this application which provides, for example, mean values for the markers that associated with various AKI classifications, and ranges (e.g. amounts within 95% confidential interval boundaries from the mean values), and statistical analyses of AUCs for the various markers. Based on this data other suitable threshold values can be selected. [00132] It should also be noted that, although the amounts of NGAL, KIM-1, and sCr described herein are generally referred to in terms of the amount by mass/volume (e.g.
ng/ml), other units of measurement can be used. For example it is common to refer to NGAL levels in terms of μg/g creatinine. Any suitable units or measurements can be used and it should be understood that amounts of NGAL measured and/or represented in other units can be equivalent to the amounts and ranges described herein. The present invention is not limited to methods that comprise measuring NGAL, KIM-1, or sCr using the specific units provided herein. One of skill in the art can readily make the necessary conversions between units.
[00133] In one embodiment, the kit comprises an antibody capable of binding to the NGAL protein and an antibody capable of binding to the KIM-1 protein. In another embodiment, the kit further comprises a positive control for each of NGAL protein and KIM-1 protein. In one embodiment, the positive control is in the device. In another embodiment, the device is an ELISA plate, a dipstick, or a test strip. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the antibody is a polyclonal antibody.
[00134] In one embodiment, the subject is a mammal. In another embodiment, the subject is a human. In one embodiment, the subject has no signs of kidney disease. In another embodiment, the subject has signs of kidney disease. In one embodiment, the subject has undergone or will undergo hemodialysis treatment.
[00135] In one embodiment, the bodily fluid used in accordance with the invention is blood. In another embodiment, the bodily fluid is urine. In another embodiment, the bodily fluid is plasma. In still another embodiment, the bodily fluid is blood serum.
[00136] Any suitable methods for detecting NGAL protein, KIM-1 protein and serum creatinine known in the art can be used. For example, several antibodies to NGAL and KIM- 1 are known in the art and can be used in accordance with the present invention.
[00137] Any antibody, such as a monoclonal or polyclonal antibody, that binds to NGAL, KIM-1 or sCr can be used. For example, monoclonal antibodies that bind to NGAL are described in "Characterization of two ELISAs for NGAL, a newly described lipocalin in human neutrophils", Lars Kjeldsen et al, (1996) Journal of Immunological Methods, Vol. 198, 155-16, the contents of which are herein incorporated by reference. An example of a polyclonal antibody for NGAL is described in "An Iron Delivery Pathway Mediated by a Lipocalin", Jun Yang et al, Molecular Cell, (2002), Vol. 10, 1045-1056, herein incorporated by reference in its entirety. To prepare this polyclonal antibody, rabbits were immunized with recombinant gel-filtered NGAL protein. Sera were incubated with GST-Sepharose 4B beads to remove contaminants, yielding the polyclonal antibodies in serum, as described by the applicants in Jun Yang et al., Molecular Cell (2002). Further non-limiting examples of antibodies that can be used to detect NGAL protein in the methods of the invention are also provided in the Examples. Antibodies that bind to NGAL are also available commercially, for example from the Antibody Shop, Copenhagen, Denmark, as HYB-211-01, HYB-211-02, and NYB-211-05. In addition, one of skill in the art can readily produce antibodies that bind to NGAL or uL-FABP, or can have them produced by an antibody production company.
[00138] Any method can be used to detect and or measure the levels of NGAL, KIM-1 or sCr, including, but not limited to, immunohistochemistry-based methods, immuno-blotting based methods, immunoprecipitation-based methods, affinity-column based methods
(including immunoaffmity column based methods), ELISA-based methods, other methods in which an antibody is immobilized on a solid substrate (such as beads), and the like.
[00139] In some embodiments, methods that enable detection of monomeric NGAL may be used, including, but not limited to, immunoblotting methods.
[00140] In some methods a primary antibody to NGAL, KIM-1 , or sCr may be used in conjunction with a secondary or tertiary antibody labeled with a detectable moiety, such as a fluorescent moiety, a radioactive moiety, or a moiety that is an enzyme substrate and can be used to generate a detectable moiety, such as horse radish peroxidase. Such methods are well known in the art and can be used to detect the presence and/or measure the amount of NGAL, KIM-1, or sCr in a bodily fluid sample, such as urine, without undue experimentation.
[00141] In circumstances where the amount of NGAL, KIM-1, or sCr is to be measured, positive controls containing known amounts of NGAL, KIM-1, or sCr protein can be used, for example for calibration purposes. NGAL, KIM-1, or sCr for use as a positive control can be obtained from any source or produced by any method known in the art. For example, NGAL or KIM-1 protein can be recombinantly produced. Methods for the recombinant production of proteins are well known in the art. For example, a nucleotide sequence encoding NGAL or KIM-1 can be included in an expression vector containing expression control sequences and expressed in, and purified from, any suitable cell type, such as bacterial cells or mammalian cells. For example, for use as a positive control in the methods of the invention, recombinant NGAL can be produced as described in Yang, et al. (2002) Mol Cell 10, 1045-1056; Goetz et al. (2002) Mol. Cell 10, 1033-1043; Goetz et al. (2000) Biochemistry 39, 1935-1941; and Mori, et al. (2005) J. Clin Invest. 115, 610-621, the contents of which are hereby incorporated by reference.
[00142] Methods of determining the serum creatinine level of a subject are well known in the art and are routinely performed by medical professionals. Any method for determining a subject's serum creatinine level can be used in conjunction with the methods of the present invention. A review of some methods for determining serum creatinine levels is provided in: Clin. Biochem. Rev. (2006); 27(4): p 173-184; "Measurement of Serum Creatinine - Current Status and Future Goals" by Peake et al., the contents of which are hereby incorporated by reference.
[00143] According to the methods of the invention, samples of a bodily fluid can be obtained and/or tested using any means. For example, methods for collecting, handling and processing urine, blood, serum and plasma, and other body fluids, are well known in the art and can be used in the practice of the present invention. In some embodiments, two or more consecutive or subsequent samples of a body fluid can be taken. Depending upon the circumstances, including the clinical condition of the patient, the subject's body fluid can be sampled daily, or weekly, or within a few weeks, or monthly or within a few months, semiannually, annually, or within several years, and at any interval in between. Repeat sampling can be done at a period of time after treatment to detect any change in AKI status. Sampling need not be continuous, but can be intermittent (e.g., sporadic). In some embodiments, it is not be necessary to obtain and keep a sample of the bodily fluid from the subject. For example, in some embodiments, the subject can urinate onto a test strip, for example a test strip of the type used in pregnancy testing kits. In other embodiments, a sample of bodily fluid, such as blood from a pin prick, can be applied onto a test strip - for example a test strip similar to those used for blood typing. Although generally the sample of a bodily fluid, such as blood or urine, is obtained from a subject and tested by a laboratory or by a medical professional (for example using an NGAL testing kit, e.g. a urine dipstick based kit, or an ELISA based kit), home -testing kits are also within the scope of the present invention.
[00144] In one aspect, the present invention comprises a kit for performing the methods of the invention, containing, for example, a device for detecting one or more of NGAL protein, KMI-1 protein, and serum creatinine. Such devices can comprise, for example, an ELISA plate, a dipstick to be dipped in a urine or blood sample, or a stick on which the subject should urinate. In some embodiments, such devices are configured such that they give a positive result only if the level of one or more of NGAL, KIM-1, or sCr exceeds a threshold level, such as one of the threshold levels described herein. Methods for making and using such devices are well known in the art.
[00145] It will be readily apparent to those skilled in the relevant arts that other suitable modifications and adaptations to the methods and applications described herein, such as the threshold amounts of the various markers in various bodily fluids, can be made without departing from the scope of the invention or any embodiment thereof.
[00146] The following non-limiting examples illustrate the invention described herein, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.
[00147] The following methods can be used in connection with the embodiments of the invention.
EXAMPLES
Example 1: Diagnostic and Prognostic Stratification in the Emergency Department Using Urinary Biomarkers of Nephron Damage - A Multicenter Prospective Cohort Study
[00148] A multi-center trial in three ED was conducted: (1) an inner-city community hospital in Manhattan, NY (Allen Hospital of New York- Presbyterian Hospital, AH-NYPH); (2) a suburban community hospital in Staten Island, NY (Staten Island University Hospital; SIUH); and (3) a tertiary care center in Berlin, Germany (Helios Clinics, Berlin-Buch).
Described herein are the diagnostic and the predictive characteristics of a single measurement of several novel urinary biomarkers.
[00149] Site-specific race and ethnic composition included: Berlin (100% White); SIUH (7% Hispanic, 9% Black, 79% White, 5% Other); and AH-NYPH (52% Hispanic, 21% Black, 27%o White). 1234 (75.5%) patients could be assigned to one of four diagnostic categories (normal kidney function, stable CKD, prerenal AKI, intrinsic AKI); 401 patients remained unclassified (Figure 1). Baseline characteristics by diagnostic and outcome groups are presented in Table 1. Ninety-six patients (7.8%) had AKI, 254 (20.1%) had prerenal azotemia, 154 (12.5%) had stable CKD, and 730 (59.2%) had normal kidney function. The primary etiologies of AKI were hypotension (34%), urinary obstruction (29%>), sepsis (22%), glomerulonephritis or vasculitis (6%>), hepatorenal syndrome (2.1%), rhabdomyolysis (2.1%), and acute interstitial nephritis (biopsy-proven), scleroderma crisis, contrast nephropathy and multiple myeloma (1% each). Patients with AKI differed from other groups with respect to age and race and underlying CKD (Table 1). Diabetes, congestive heart failure, and liver disease were more frequent in patients with AKI. Presenting sCr and its change from baseline were significantly higher in AKI compared to other groups (Table 1, Figure 9). In addition, patients with AKI had longer hospital stays, were more likely to require nephrology consultation or ICU transfer, and experienced higher rates of death, dialysis, and the composite endpoint of death or dialysis (Table 1).
[00150] Table 1 : Patient characteristics by diagnosis and clinical outcome. Cohort Characteristics Total Patients Patients Patients Patients Patients
Cohort with without with with with clinical clinical intrinsic prerenal unclear events events AKIJ AKI, diagnosis†
(death or stable
dialysis)§ CKD or
normal
function
Sample size 1635 72 1563 96 1138 401
Demographics
Mean Age, years (SD) 64.4 70.9 64.1 67.7 63.2 67.0
(18.7) (16.9)** (18.7) (17.6)* (18.8) (18.3)**
White (%) 67.6 84.7** 68.7 80.2* 68.8 61.9**
Black (%) 9.5 5.6 9.9 7.3 10 8.6
Hispanic Ethnicity (%) 18.1 19.1 11.5 18.8 17.8
Gender (% female) 47.7 58.3 52 47.9 54.4 52.3
Admission Diagnosis
Primary Renal 0.8 0.4 9 4*** 0.2 0.5
Disease (%)
Kidney Stones of 4.8 1.4 2.4 0.6 2.5
Other Postrenal
Processes (%)
Acute Coronary 3.6 0 3.8 2.1 3.7 3.7
Syndrome/Acute
Myocardial
Infarction (%)
Decompensated 8.2 15.3* 7.8 8.3 7.8 9.2
Congestive Heart
Failure (%)
Cardiac Arrythmia 3.6 2.8 3.7 3.1 3.9 3
(%)
Sepsis (%) 3.4 15.3*** 2.8 15.6*** 0.9
Infection without 22.6 13.9 23 17.7 20.5 29 7***
Sepsis (%)
Liver Disease (%) 2.1 11.1 1.7 9 4*** 1.6 1.7
TIA or Stroke (%) 4 8.3 3.8 1 4.8 2.7
Malignancy (%) 4.8 15.3*** 4.3 j2 5*** 4.4 4
Admission Parameters
Mean Systolic Blood 137 129 (28)* 137 (27) 129 (33)* 137 (26) 137 (29)
Pressure mmHg (27)
(SD)
Mean Heart Rate, 88 (21) 91 (25) 88 (21) 92 (21)* 87 (21) 90 (22)* min 1 (SD)
Shock index > 0.8 23.8 29.9 23.5 37.6** 21.7 26.6
(%) CoMorbid Conditions
History of CKD (Stage 25.2 36.1 ** 22.6 37.5*** 19.6
3 or up) (%)a
Diabetes (%) 29.4 37.5 29 36.5* 25.6 38.3***
Hypertension (%) 60.9 69.4 60.5 59.4 59.3 70***
Coronary Artery 23.2 23.9 23.2 20.8 23.6 22.7
Disease (%)
Congestive Heart 27.6 40.8* 27 32.6 24.2 36***
Failure (%)
Peripheral Vascular 5.6 4.2 5.7 5.2 5.3 6.6
Disease (%)
(%) 6.5 8.5 6.4 5.2 6.2 7.6
Kidney Function
Mean Baseline sCr, 0.9 1.0 (0.6)* 0.9 (0.4) 1.1 0.9 (0.3) 1.0 mg/dl (SD)C (0.4) (0.6)*** (0.5)***
(SD) C 1.1 1.9(2.3)** 1.1 (0.7) 2.9 1.0 (0.5) 1.2
(0.8) (3.4)*** (0 7)***
Mean Ratio of
Change in sCr from
Baseline to Adission 1.2 1.9 1.2 (0.4) 2.5 1.1 (0.3) 1.2 (0.4)
(SD) C (0.5) (1.8)*** (2.6)***
Mean eGFR at 70.5 43.3 71.7 28.8 76.4 63.6
Admission, (33.2) (30.6)*** (31.7) (22.1)*** (31.8) (30.8)*** niL/minute/1.73 m2
(SD)
Clinical Events
Death, % 3.4 - - 2i 9*** 1.7 4
In-Hospital Dialysis 1.5 - - Y1 7*** 0.3 1.3
Inititiation, %
Death without prior 2.9 15.6*** 1.6 3.5 dialysis, %
Death or Inpatient 4.4 - - 33.3** 1.8 4.7
Dialysis, %
ICU Admission, % 14.4 - - 29 2*** 11.8 18.2***
[00151] § In comparison to patients without clinical events; * p<0.05; ** p<0.01; *** p<
0.001 (t-test or Chi square test, as appropriate)
[00152] % In comparision to patients with prerenal AKI, stable CDK, or normal function; * p<0.05; ** p<0.01; *** p< 0.001 (t-test or Chi square test, as appropriate)
1001531 † In comparision to all Adjudicated patients; * p<0.05; ** p<0.01; *** p< 0.001 (t- test or Chi square test, as appropriate)
[00154] a defined by baseline eGFR <60 mL/minute
[00155] b sCr = serum creatinine [00156] c geometric mean and SD
[00157] The primary etiologies of intrinsic AKI (iAKI) were hypotension (34%), urinary obstruction (29%), sepsis (22%), glomerulonephritis or vasculitis (6%), hepatorenal
syndrome (2.1%), rhabdomyolysis (2.1%); causes of iAKI present at lower prevalence (1%) included contrast nephropathy, acute interstitial nephritis (biopsy-proven), scleroderma crisis, and multiple myeloma. Seventy-two patients (4.4%) experienced the composite outcome of dialysis initiation or death. Causes of death (n=56) included sepsis (n=22; 39%), metastatic cancer (n=9; 16%), heart failure (n=7; 13%), liver failure (n=5; 9%), stroke or intracranial bleed (n=5; 9%), respiratory failure (n=3; 5%), and GI bleed (n=l; 2%). iAKI was strongly associated with adverse in-hospital outcomes, including death, dialysis initiation, and ICU admission (Table 1).
[00158] ROC curve analyses were used to determine whether a single measurement of a urinary biomarker could distinguish patients with AKI from patients belonging to the other diagnostic groups (Table 2). To evaluate diagnostic cut-offs, the 60th and 75th percentiles for each biomarker in the adjudicated population were chosen a priori.
[00159] Most patients with iAKI had already reached their peak RIFLE class on admission (n=62; 64.6%). Accordingly, both the presenting sCr and its change from baseline were significantly higher in iAKI compared with patients without iAKI (Table 1, Figure 2). sCr at presentation and the ratio of presenting/baseline sCr highly discriminated iAKI patients
[AUC-ROC 0.91 (0.87 - 0.94) and 0.89 (0.84 - 0.94), respectively] (Table 2).
[00160] Table 2: Test characteristics of urinary biomarkers in the diagnosis of iAKI
including ROC-AUC analysis, Predictive Values, and Likelihood Ratios. Two cut-off values were analyzed for each urinary biomarker, corresponding to the 60th and 75th percentile of the biomarker level across the entire cohort.
Urinary AUC* Cutoff Sensitivity Specificity Positive Negative Positive Negative
Bio(95% CI) Level Predictive Predictive Likelihood Likelihood marker (ng/ml) Value Value Ratio Ratio
0.81 (0.76 - uNGAL 0.86) 104 68% 81% 23% 97% 3.64 0.39
47 82% 67% 17% 98% 2.49 0.27
0.71 (0.65 - uKIM-1 0.76) ** 2.817 52% 79% 17% 95% 2.49 0.61
1.665 63% 65% 12% 95% 1.76 0.58
0.64 (0.57 - uIL-18 0.70)*** 0.065 43% 78% 14% 94% 1.95 0.73
0.036 58% 65% 12% 95% 1.67 0.64 uL- 0.70 (0.65 - 12.9 50% 80% 18% 95% 2.56 0.62 FABP 0.76) ***
5.2 67% 66% 14% 96% 1.90 0.50
0.65 (0.58 - uCysC 0.72)*** 171 46% 80% 16% 95% 2.26 0.68
101 59% 64% 12% 95% 1.70 0.60
0.90 (0.86 - sCr 0.93) 1.4 81% 82% 28% 98% 4.49 0.23
1.1 92% 63% 17% 99% 2.48 0.13
[00161] * AUC = Area under the receiver operator characteristic curve
[00162] ** p 0.001; *** p 0.0001 vs. NGLA CIMA
[00163] Upper Cutoff- 75Λ%; Lower Cutoff = 60Λ%
[00164] Positive Likelihood Ratio = Sensitivity/(1-Specificity)
[00165] Negative Likelihood Ratio = (1 - Sensitivity)/Specificity
[00166] Presenting sCr was not predictive of iAKI when its level was in the middle tertile of its range (0.9-1.2 mg/dL), nor could it distinguish those patients whose sCr continued to rise after admission (> 1.5-fold) from the rest of the cohort. Furthermore, only 63.5% of the patients had documented baseline sCr values at the time of presentation to the ED,
highlighting the difficulties of interpreting single measurements of sCr in triage.
[00167] Urinary biomarker levels differed between patients with iAKI and any other diagnosis (by t-test: p<0.001 for all biomarkers). In addition, subset comparisons showed that urinary biomarker levels were significantly elevated in patients with iAKI compared with each of the other diagnostic categories (pAKI, stable CKD, or normal kidney function; by one-way ANOVA and posthoc Tukey test) (Figure 2). ROC curve analyses indicated good discriminatory ability for uNGAL (AUC-ROC 0.81), fair discriminatory ability for uKIM-1 and uL-FABP (AUC-ROC 0.71 and 0.70, respectively) and poor discriminatory ability for uCysC and uIL-18 (AUC-ROC 0.65 and 0.64, respectively) (Figure 3, Table 2) to distinguish iAKI from other diagnoses.
[00168] The AUC-ROC of uNGAL was significantly higher than that of other urinary markers (p<0.001 each, Table 2). When the monomeric form of uNGAL was quantified by immunoblot, it markedly correlated with the standardized clinical platform and performed similarly in ROC analyses (Figures 3 and 4) Also, AUC-ROCs remained similar when biomarker levels were standardized for urinary creatinine concentrations. Sensitivity, specificity, predictive values, and likelihood ratios for diagnostic cut-offs at the 60th and 75th percentiles for each biomarker are shown in Table 2. The cut-off value of uNGAL was 104 ng/ml. [00169] To determine whether the biomarkers could predict the severity of AKI, a post-hoc analysis was conduction to compare urine biomarker levels at presentation with the maximal RIFLE class achieved during 7 days of hospitalization (Table 3, Figure 10). uNGAL had the highest fold increase for each successive RIFLE severity class and in contrast to other biomarkers, the diagnostic accuracy of uNGAL improved robustly as RIFLE severity increased from R to F (AUC uNGAL: 0.68 to 0.88; respectively).
[00170] Table 3 : Prediction of Peak RIFLE after Hospital Admission†
Prediction of RIFLE R
AUC p value vs NGAL
NGAL CMIA 0.68 (0.63 - 0.73) -
KIM-1 0.69 (0.65 - 0.74) NS
IL-18 0.61 (0.56 - 0.66) 0.008
LFABP 0.67 (0.61 - 0.70) NS
Cystatin-C 0.60 (0.54 - 0.65) 0.005
Prediction of RIFLE I
AUC p value vs NGAL
NGAL CMIA 0.79 (0.73 - 0.85) -
KIM-1 0.73 (0.67 - 0.79) NS
IL-18 0.63 (0.56 - 0.70) <0.0001
LFABP 0.71 (0.64 - 0.77) 0.024
Cystatin-C 0.67 (0.59 - 0.74) 0.003
Prediction of RIFLE F
AUC p value vs NGAL
NGAL CMIA 0.88 (0.84 - 0.93) -
KIM-1 0.74 (0.67 - 0.80) 0.0006
IL-18 0.49 (0.40 - 0.58) <0.0001
LFABP 0.80 (0.74 - 0.86) 0.0074
Cystatin-C 0.65 (0.55 - 0.75) <0.0001
[00171] †Post-Hoc analysis of fold-change in biomarker levels compared with a RIFLE negative reference population
[00172] NS - not significant
[00173] Sensitivity analyses was performed by selecting subsets of patients based on study site, ethnicity, gender, infection of the urinary tract (UTI) and the co-prevalence of CKD or RIFLEAKI. The AUC-ROCs for the diagnosis of iAKI remained consistent within these subsets (Table 4).
[00174] Table 4: Sensitivity analysis. Test characteristics of urinary biomarkers in the diagnosis of intrinsic AKI in different subsets of patients. AUC-ROC values are reported (data in parentheses are 95% confidence intervals). The subsets included 3 separate hospital cohorts, different ethnicities, genders, and medical histories including CKD and urinary tract infections. AUC values and their rank ordering for different biomarkers were consistent in all patient subsets. The bottom panel shows discrimination of intrinsic AKI (i-AKI) from other causes of elevated sCr (PR- AKI: prerenal AKI; CKD: stable CKD) by biomarkers.
Figure imgf000045_0001
[00176] *p <0.05 vs. uNGAL
[00177] a Excluding Asian (10) and Other (18) [00178] In the cohort, both the sCr at presentation and the ratio of presenting/baseline sCr highly discriminated adjudicated AKI patients [AUC-ROC 0.91 (0.87 - 0.94) and 0.89 (0.84 - 0.94), respectively]. However, presenting sCr only moderately predicted the maximum RIFLE score (ROC 0.74, CI 0.72-0.77), was not predictive of AKI in the middle tertile of its range (0.9-1.2 mg/dL), and it could not distinguish patients whose sCr continued to rise (at least an additional 1.5 fold) after admission from those whose sCr remained unchanged or fell after admission (Table 5). In contrast, both uNGAL and uKIM-1 were significantly elevated in patients whose sCr continued to rise after hospital admission and were useful across the range of presenting sCr.
[00179] Table 5: Mean (SD) Biomarker Level for Increasing sCr vs. Unchanging or
Decreasing sCr After Hospital Admission
Non-Increasing (n =
Increasing (n = 38) 1226) p-value
Initial sCr 1 .3 (0.6) 1 .4 (1 .7) NS
NGAL CMIA
(ng/ml) 449 (1220) 186 (578) 0.04
KIM 1 (ng.ml) 2.721 (2.291 ) 2.355 (3.752) 0.006
IL 18 (ng/ml) 0.0073 (0.089) 0.078 (0.208) NS
L FABP (ng/ml) 37 (90) 24 (60) NS
Cystatin C
(ng/ml) 1228 (491 1 ) 831 (4218) NS
[00180] Given that iAKI is associated with longer durations and greater severity of azotemia compared to pAKI (16,25), secondary analyses were performed to assess the relationship between biomarker levels and the duration and severity of AKI. To evaluate duration of AKI, the entire cohort was categorized into three subgroups: no AKI, transient AKI (defined as RIFLE -AKI that resolved by 72 hours), and sustained AKI (RIFLE- AKI that persisted for 72 hours or more). Only uNGAL and uCysC levels were significantly higher in patients with sustained AKI when compared to patients with transient AKI episodes (Figure 5).
Conversely, uKIM-1, uL-FABP, and uIL-18 were not significantly different in sustained and transient AKI.
[00181] To further substantiate the concept that biomarkers contribute to the diagnosis of AKI independently of established tests, demographic and clinical parameters, as well as laboratory tests and biomarker results were entered into a PCA model. The analysis revealed 5 independent principal components (PCs) with an eigenvalue threshold of >1.0, which jointly explained 62.5% of the variance across the population (Table 6a, b). PCI incorporated the 5 urine biomarkers that corresponded to tubular damage and explained 21.2% of total variance, while PC2 included both serum BUN and creatinine and corresponded to glomerular function and explained 15.1% of total variance. PC3 (variables defining hemodynamics), PC4 (included temperature) and PC5 (included presenting serum sodium) each described smaller percentages of the variance (11%, 8.5% and 6.7%, respectively). Univariate and multivariate logistic regression demonstrated that PCI (biomarkers of tubular damage) significantly and independently contributed to the diagnosis of AKI (Table 6c). These data corroborate the concept that urinary biomarkers represent an independent class of diagnostics that add to the diagnostic workup of AKI beyond currently available tests.
[00182] The entire cohort was next stratified by peak RIFLE severity class (within 7 days from inclusion) (Figure 5). uNGAL levels progressively and significantly increased in parallel with RIFLE severity class. In contrast, while the other biomarkers were higher in RIFLE -AKI than in non-AKI, these markers did not strictly parallel RIFLE class.
Accordingly, the AUC-ROC for uNGAL progressively increased for the prediction of RIFLE-R, RIFLE -I, and RIFLE-F, while progressive increases were absent or less pronounced for other biomarkers (Table 7). Together, these data indicate that uNGAL is the most powerful indicator of severity and duration of AKI.
A: Extraction of Principal Components C: Association with AKI
Univariate Multiple
Eigen % of Cumulative % Regression Regression
Principal Component value Variance Variance OR (95% CI) OR (95% CI)
2.03 (1.54 - 2.24 (1.62 -
1 : Tubular Damage 3.181 21.2 21 .2 2.67) 3.09)
3.67 (2.80 - 3.53 (2.67 -
2: Glomular Function 2.265 15.1 36.3 4.82) 4.66)
0.79 (0.60 -
3: Hemodynamics 1.643 1 1 47.3 1.03)
1.22 (0.98 -
4: Infection 1.28 8.5 55.8 1.51 )
0.54 (0.43 - 0.58 (0.44 -
5: Water/Volume 1.004 6.7 62.5 0.69) 0.76)
B: Principal Component Loading Matrix after Varimax Rotation
Λ■
Tubular 2: Glomular 3: 5:
Variable Component Damage Function Hemodynamics 4: Infection Water/Volume
Age 5 48 3 -2 55
Systolic Blood
Pressure -5 1 89 -6 15
Diastolic Blood
Pressure -4 -15 89 -3 -1 1
Heart Rate 21 -1 1 15 46 -49
Temperature 1 1 -21 -1 79 4
Serum Na 1 -22 7 -7 72
Serum BUN 10 89 -1 1 -2 -2 Serum WBC 4 18 -13 52 -17
Urine Specific
Gravity 55 -18 2 -34 -28
Presenting Scr 12 88 -5 -2 -6
NGAL CMIA 65 32 -12 5 1
KIM 1 76 2 -16 -1 6
IL18 69 -27 -5 26 -1 1
L FABP 65 29 8 4 -5
Cystatin C 74 12 9 16 5
[00183] Table 7: Association of urinary biomarker levels and peak RIFLE class during 7 days of hospitalization. Note the differential response of urinary biomarkers with increasin
RIFLE class.
Prediction of RIFLE R, I or F
AUC-ROC
0.72 (0.69 - uNGAL 0.75)
0.69 (0.65 - uKIM-1 0.72)
0.58 (0.55 - ulL-18 0.62)
0.68 (0.65 - uL-FABP 0.72)
0.61 (0.58 - uCysC 0.65)
Prediction of RIFLE I or F
AUC-ROC
uNGAL 0.8 (0.76 - 0.84) uKIM-1 0.7 (0.65 - 0.74)
0.56 (0.51 - ulL-18 0.62)
0.72 (0.67 - uL-FABP 0.77)
uCysC 0.64 (0.58 - 0.7)
Prediction of RIFLE F
AUC-ROC
0.83 (0.77 - uNGAL 0.89)
0.69 (0.62 - uKIM-1 0.75)
ulL-18 0.51 (0.43 - 0.6)
0.73 (0.67 - uL-FABP 0.79)
uCysC 0.6 (0.51 - 0.69)
[00184] a RIFLE class ?ased on creatinine criteria
[00185] b RIFLE-R: N= ==167, RIFLE-I=126, RIFLE-F: N=55
[00186] Patients with iAKI experienced higher rates of a composite clinical outcome of death or dialysis during hospitalization (Table 1). Multiple logistic regression was used to construct a conventional baseline prediction model that was adjusted for serum creatinine at inclusion (Table 8, Model 1). Each individual biomarker was then added to the baseline model (Table 8, Models 2-6). Each biomarker added significantly to the predictive ability of the baseline model. In addition, two measures of the overall performance of the regression model, R square and AUC-ROC, improved when each biomarker was added to the baseline model. Stepwise backward selection techniques were then used to identify combinations of biomarkers that independently contributed to the prediction of the composite outcome.
uNGAL and uKIM-1 were found to independently add to a combined prediction model (Table 8, Model 7). However, R square and AUC-ROC of the combined model increased only slightly when compared to the single biomarker models.
[00187] Table 8: Multivariate logistic regression analysis of urinary biomarkers in the prediction of the composite outcome (in-hospital dialysis initiation or mortality) a, b, c
Figure imgf000049_0001
[00188] All cutoffs at the 75% percentile; values represent Exp (B) (the change in the odds ratio associated with the predictor variable), with 95% confidence intervals
[00189] b All multiple Logistic Models are adjusted for age, admission parameters, comorbidities, and location
[00190] c RSQ = Nagelkerke R square, AUC-ROC = Area under the Receiver Operating Characteristic Curve, IDI = Integrated Discrimination Improvement
[00191] d Stepwise Selection Model including sCr, uNGAL, uKIM-1 , uIL-18, uL-FABP, uCysC [00192] e IDI vs. Model 1 (ns, non-signficant; ** p<0.01; *** p<0.001)
[00193] f IDI vs. Model 2
[00194] To examine the incremental utility of urinary biomarkers and their combinations in more detail, the integrated discrimination improvement (IDI) was calculated, comparing the biomarkeraided models (Models 2-5) with the sCr-based baseline model (Model 1) (23). This method compares the risk estimates derived from each model in patients with outcomes (events) and in patients without outcomes (non-events). A biomarker-aided risk estimate will achieve a positive IDI compared to a conventional risk estimate, if patients with events are assigned higher risk estimates and if patients without events are assigned lower risk estimates. The data showed that uNGAL and uKIM-1 -assisted models achieved significant IDIs when compared to the baseline model (Table 8). The uNGAL-assisted model also achieved a significant IDI when compared with models using uL-FABP, uIL-18, or CysC (Models 4, 5 and 6) (p<0.05 each). A triple model of sCr, uNGAL and uKIM-1 (Model 7) did not achieve a significant IDI compared with the double model with sCr and uNGAL (Table 8). These data indicated that uNGAL and uKIM-1 individually improved risk stratification when combined with sCr, while a combination of uNGAL and uKIM-1 did not further improve the predictive ability.
[00195] To estimate the net reclassification improvement (NRI) facilitated by uNGAL and uKIM-1, three risk categories (<2%, 2-15%, and >15%>) of experiencing the composite outcome within our ED population based on the expected rates of in-hospital mortality and in-hospital dialysis initiation were defined (16). Next, each patient was assigned to one of these risk classes based on either the biomarker-assisted models (Models 2 or 3) or based on the conventional model (Model 1). The net number of patients with a classification improvement after uNGAL-stratified assessment was 120 (7.8%) among patients without events and 13 (18.3%) among patients with events (Table 9). The net number of patients with a classification improvement after uKIM-1 -stratified assessment was 204 (13.4%) among patients without events and 7 (10.4%) among patients with events (Table 9). Hence, the introduction of uNGAL and uKIM-1 facilitated net reclassification improvements of 26.1% and 23.8%), respectively.
[00196] Table 9: Net reclassification improvement as facilitated by biomarker-aided prediction models. A. uNGAL-assisted model. B. uKIM-1 -assisted model. Individual probabilities of experiencing the composite outcome were calculated according to the baseline model (Model 1, see Table 8) and to the biomarker-assisted models (Models 2 and 3, see Table 8) to stratify patients into three risk classes (<2%, 2-15%, >15%). Appropriate biomarker-assisted reclassification was defined as a step-up in risk class in patients with events and a step-down in risk-class in patients without events, while inappropriate reclassification was vice versa.
Figure imgf000051_0001
Figure imgf000051_0002
[00197] ** p<0.01; *** p< 0.001
[00198] To translate these findings into a diagnostic strategy, patients were risk stratified by sCr level and used uNGAL or uKIM-1 to further subdivide these categories. Based on 75th percentile cutoffs we separated patients into sCr+ (sCr > 1.4 mg/dl) and sCr- (sCr < 1.4 mg/dl) at the time of inclusion. sCr+ and sCr- patients were tehn subdivided into biomarker+ (biomarker > cutoff at 75th percentile) or biomarker- (biomarker < cutoff at 75th percentile). Event rates within sCr+ or sCrpatients were substantially different depending on whether they were biomarker+ or biomarker- (Figure 6). In particular, about 15% of the population had low sCr, but high biomarker levels, placing them at low risk by conventional stratification, but at an increased risk upon application of biomarker-aided stratification.
[00199] This is the first multicenter study to comprehensively compare the diagnostic and predictive abilities of urinary biomarkers. The goals of the analysis described herein were to evaluate their ability to: (1) distinguish iAKI from pAKI, stable CKD, and normal kidney function; and (2) to facilitate a prospective risk assessment regarding a requirement for dialysis or the death of the patient during subsequent hospitalization.
[00200] Every patient that entered the ED and was subsequently hospitalized for more than 24 hours was included in the study. While the exclusion of patients hospitalized for less than 24 hours may have introduced some bias in favor of sicker patients when compared to the inclusion of all-comers, derivation of the test characteristics of biomarkers compared to standard clinical information necessitated a reasonable period to accumulate follow-up data.
[00201] This study was limited by the absence of a diagnostic gold standard of iAKI. This limitation was addressed by establishing a standardized adjudication procedure to define iAKI, which was based on sCr dynamics, the etiology of AKI and the response to therapy considering kidney physiology in addition to AKI pathogenesis (16). Using this strict approach, analyze three-quarters of the cohort could be analyzed without ambiguity.
However, because a definitive diagnosis could not be assigned to approximately one-quarter, adjudication-independent secondary analyses across the entire cohort and these approaches were found to be complementary: the utility to diagnose iAKI in a strictly defined cohort paralleled the biomarkers' ability to predict the intensity and duration of AKI in all comers. For instance, uNGAL performed significantly better than the other biomarkers in diagnosing iAKI and consistently, uNGAL displayed a closer association with severity and duration of AKI compared with the other biomarkers. Importantly, uNGAL was progressively more effective in predicting increasing RIFLE classes, a finding consistent with previous studies (26,27). It is noteworthy that both the presenting sCr and its change from baseline highly discriminated iAKI patients from other diagnostic groups. This can be because that sCr was a major determinant of the diagnostic adjudication procedure itself and that most patients had already achieved their peak RIFLE severity class at presentation to the ED. This also shows that urinary biomarkers can be useful when sCr dynamics are unknown or when sCr is in the middle of its range.
[00202] The results described herein confirmed the known association of the type of AKI with the clinical outcome. While less than 4% of patients with normal kidney function, stable CKD or pAKI experienced the composite outcome of in-hospital mortality or the requirement to initiate in-hospital hemodialysis, 33.5% of the patients with iAKI experienced this outcome. Several smaller studies have linked high urinary biomarker levels with these unfavorable clinical events (16,28-31) and here we confirmed this association. However, the large sample size enabled characterization of the contribution of each biomarker: uNGAL along with uKIM-1 were the most accurate predictors of subsequent clinical events, and each markedly improved risk assessment when combined with the conventional sCr-assisted prediction models, as determined by IDI and NRI. [00203] In contrast, the combination of uNGAL and uKIM-1 did not further improve risk classification, nor was there evidence of superiority of one of the two markers in head-to- head comparisons.
[00204] In sum, the results described herein characterize the performance of different urinary biomarkers obtained on patient entry to the hospital to diagnose iAKI (as defined by strict, retrospective criteria) and to determine its severity and clinical sequela. This analysis prospectively validated the concept that the addition of urinary biomarkers and their interpretation together with sCr levels identified patients at risk, who otherwise would have been missed during triage.
[00205] Identification of the Monomeric uNGAL in Patients with Intrinsic AKI
[00206] Several studies have reported multiple molecular forms of NGAL, including the predominant monomeric 23-26-kD form, a disulfide-linked 45 -kD homodimeric form, and a 125-kD heterodimeric form representing NGAL covalently conjugated with gelatinase (33- 35). Recently, it was reported that the monomeric form of NGAL was secreted by kidney epithelial cells and detected in the urine of patients with AKI, but that the homodimer was derived from neutrophils and was found in the urine of patients with urinary tract infections (36). In the current study, a standardized clinical platform using a chemiluminescent microparticle assay (Abbott ARCHITECT) was used to detect uNGAL. Derived test characteristics were observed that (while individual uNGAL measurement were increased by UTI) were largely unaffected in patients with urinary tract infections (Table 7). To clarify the species of uNGAL that pedicted the diagnosis of AKI, immunoblots were performed on every urinary sample and quantified the intensity of the 23-26-kD band corresponding to the monomeric form of uNGAL compared to recombinant NGAL. A marked correlation between uNGAL levels measured by this technique and by the standardized clinical platform was observed (Pearson correlation 0.93, p<0.0001, Figure 5). In addition, the AUC-ROC for the detection of intrinsic AKI of uNGAL determined by the standardized clinical platform (0.81, CI 0.76-0.86) was virtually identical to that of monomeric uNGAL determined by
immunoblot (0.81, CI 0.75-0.86; Figure 4). These data confirm that monomeric uNGAL is the epidemiologic significant form, which can be measured equivalently by the
chemiluinescent microparticle immunoassay or by immunoblots.
[00207] Enrollment [00208] The study was carried out in three centers: 1. Allen Hospital of New York- Presbyterian Hospital (AH-NYPH), an inner-city community hospital including a designated chest pain and stroke center; 2. Staten Island University Hospital (SIUH), a community hospital including a designated trauma center, chest pain center and stroke center; 3. Helios Clinics, Berlin-Buch, a community hospital including a designated trauma center, chest pain center and stroke center.
[00209] Recruiting study personnel, during their work hours, enrolled all available patients over 18 years old irrespective of their condition, who were in the process of admission to the hospital from the ED (September 2008 - March 2009). Patients, who had less than 24 hours of follow up, or who were on chronic renal replacement therapy, were excluded. One urine sample was collected in the ED and medical records were accessed. Treating physicians were neither aware of urinary biomarker levels, nor did our study hospitals routinely determine serum cystatin C levels. The study was approved by each site's Institutional Review Board and performed in compliance with HIPAA at all study sites. Informed consent was obtained for all participants. This trial was registered at ClinicalTrials.gov (identifier NCT00786708).
[00210] This observational study was intended to derive diagnostic test characteristics of urinary biomarkers to predict the development of inpatient iAKI. Secondary analyses included severity of AKI, duration of AKI, and a composite clinical outcome of dialysis initiation or mortality. Baseline sCr was determined by review of the prior 12 months of records, or if unavailable, baseline sCr was assumed from the lowest recorded sCr during the hospital course. Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease (MDRD) formula (20).
[00211] Diagnostic categorization was performed by adjudicators, who were blinded to urinary biomarker levels. A priori defined algorithms assigned patients to one of four renal diagnoses (normal kidney function, stable chronic kidney disease, pAKI, iAKI). Patients were labeled "unclassified" when ambiguity occurred, or when disagreement among two or more investigators could not be resolved by re-evaluation of clinical data.
[00212] Definitions of diagnostic categories were applied as follows:
[00213] Normal kidney function (n=730): Patients met the following criteria: 1. Baseline eGFR > 60 ml/min/1.73m2; 2. Failure to meet minimal RIFLE criteria for AKI (8); 3. absence of fluctuations in sCr during the first three days of hospitalization (>0.3 mg/dl when baseline sCr was >1.0 mg/dl, or >0.2 mg/dl when the baseline sCr was <1.0 mg/dl); 4. absence of recent exposures to stimuli which typically cause iAKI (e. g. shock requiring vasopressors, positive blood cultures, SIRS or sepsis, nephrolithiasis, recent chemotherapy, nephrotoxins, rhabdomyolysis, glomerulonephritis, interstitial nephritis, vasculitis, preeclampsia, multiple myeloma, or thrombotic microangiopathy). Patients who met criteria 1 and 2, but did not meet criterion 3 or 4 were labeled "unclassified".
[00214] Stable CKD (n=154): Patients met criteria 1. baseline eGFR <60 ml/min/1.73m2; as well as criteria 2-4 as defined in "normal kidney function". Patients who met criteria 1 and 2, but did not meet criterion 3 or 4 were placed into the "unclassified" category.
[00215] Prerenal AKI (n=254): Patients met the criteria: 1. minimal RIFLE sCr criteria for AKI (1.5 -fold increase in sCr or 25% decrease in eGFR from baseline; urine output criteria were not considered due to difficulty in obtaining accurate measurements in the ED); 2. normalized their values below the RIFLE 'R' threshold within three days; 3. historical and/or clinical data suggesting decreased kidney perfusion, but no exposure to stimuli which induce iAKI (see above); 4. response to measures to restore renal perfusion, such as fluid application or diuretic withdrawal. Patients who met criteria 1 and 2, but did not meet criterion 3 or 4 were labeled "unclassified".
[00216] Intrinsic AKI (n=96): Patients met the criteria: 1. minimal RIFLE sCr criteria for AKI; 2. sCr failed to normalize below the RIFLE 'R' threshold by three days following admission. 3. Patients had evidence of exposure to stimuli, which induce iAKI (see above). Patients who met criteria 1 and 2, but did not meet criterion 3 or who were exposed to factors that may have changed the creatinine course subsequent to inclusion (e. g. contrast administration in hospital) were labeled "unclassified".
[00217] Laboratory Measurements
[00218] Urine samples were centrifuged (12,000rpm; 10 min) and stored at -80°C within 12 hrs after patient enrollment. uNGAL, uIL-18, uKIM-1, and uCysC were measured by the ARCHITECT platform (Abbott Laboratories) (21). These assays utilized a chemiluminescent microparticle immunoassay (CMIA) using a non-competitive, two anti-analyte antibody sandwich. The assays include a microparticle reagent prepared by covalently attaching an antianalyte antibody to paramagnetic particles and a conjugate reagent prepared by labeling a second anti-analyte antibody with acridinium. The calibrators for the uNGAL, uIL-18 and uKIM-1 assays were recombinant proteins and the calibrators for the uCysC assay was prepared from human urine. The highest calibrator for each assay was 1500 ng/mL, 1 ng/mL, 10 ng/mL, and 2500 ng/mL for uNGAL, uIL-18, uKIM-1 and CysC, respectively. Specimens were diluted to read within the calibration curve. Coefficients of variation (CV) were 3.0% for uNGAL at a 385ng/ml (21); 2.5% for uKIM-1 at 5.8 ng/ml (Abbott Laboratories); 2.2% for uIL-18 at 0.048 pg/ml (Abbott Laboratories); 1.8% for uCysC at 350 ng/ml (Abbott Laboratories) and similar at other cutpoints. uL-FABP was measured using a sandwich-type ELISA kit (CMIC Co., Ltd). The CV was 6.8% for uL-FABP at 13 ng/ml (22). Monomeric uNGAL (23-26 KDa) was measured by immunoblots, which were prepared with non- reducing 4%) to 15%) gradient polyacrylamide gels (Bio-Rad, Hercules, CA) using standards (0.3 to 3 ng) of human recombinant NGAL and NGAL antibody (AntibodyShop,
Copenhagen, Denmark). The coefficient of variation was <5% at different cutpoints (16). Serum creatinine was assayed at each hospital by the Jaffe reaction, calibrated traceable to isotope dilution mass spectrometry.
[00219] Statistics
[00220] Statistical analyses were performed with SAS v9.2 (SAS Institute, Cary, North Carolina) and SPSS vl6.0-vl9.0 (SPSS, Chicago, Illinois). Sample sizes were estimated based on previous data (16) and were calculated to detect within each study center differences in biomarker performance between patients with iAKI and others.
[00221] Continuous variables were log-transformed and presented as geometric means and SD, where appropriate. Comparisons between two conditions used the Students t-Test, comparisons between three or more conditions used ANOVA and post-hoc Tukey test.
[00222] Categorical variables were compared by chi-square test. The null hypothesis was rejected at p<0.05. Biomarker diagnostic test characteristics for iAKI were determined by receiver operator characteristic (ROC) curve analysis. For clinical outcomes, univariate logistic regression was used to identify independent predictors, including demographic parameters (age, gender, race), comorbidities (congestive heart failure, vascular disease, hypertension, diabetes, HIV, CKD), admission parameters (heart rate, blood pressure, shock index), kidney function parameters, and study site.
[00223] Predictors significantly associated with clinical outcomes from univariate models were entered into a multiple logistic regression model and backward selection techniques were used to determine a baseline risk model. Biomarkers were then sequentially entered into the baseline model, including an adjustment for sCr, to determine independent relationships between biomarkers and the composite clinical outcome. A principal component analysis (PC A) was used to evaluate the contribution of biomarkers to predict AKI without consequences of multicollinearity (44). Urine biomarkers, sCr, BUN, age, presenting temperature, blood pressure and laboratory measurements from the ED were used. Only principal components (PCs) having eigenvalues >1.0 were selected. Associations between PCs and AKI were evaluated by univariate and multiple logistic regression analysis and expressed as ORs (with 95% CIs).
[00224] Integrated discrimination improvement (IDI) and net reclassification improvement (NRI) were calculated according to Pencina et al. (23).
[00225] Sample size estimates
[00226] Sample sizes were calculated to detect within each study center differences in biomarker performance between patients with iAKI and others. Given that approximately 5% of patients in the ED experience iAKI, we estimated that the inclusion of 20 patients with iAKI and 400 without iAKI per center would yield near 100% power to detect a significant difference by twosided t-test at a significance level of 0.05 under the assumption of an effect size of 2.2 based on log transformed biomarker means and standard deviations for uNGAL published earlier (32). In addition, this sample size ensured that differences of 0.1 in AUC- ROC between biomarkers would be detectable at 80%> power using a two-sided z-test at a significance level of 0.05 under the assumption of strong correlation (0.8) between biomarker levels. Given an estimated rate of 20% of unclassifiable cases we aimed to enroll more than 500 patients per study site.
[00227] Example 2: Test characteristics of urinary neutrophil gelatinase-associated lipocalin for differential diagnosis and risk stratification in patients with established acute kidney injury (AKI)
[00228] The RIFLE-classification from the Acute Dialysis Quality Initiative (ADQI) and the Acute Kidney Injury network (AKIN) define AKI based on serum creatinine levels and urinary output (37). However, serum creatinine levels do not always reflect the severity of renal damage (38), and they do not differentiate between intrinsic and prerenal AKI. Intrinsic AKI is associated with a higher mortality than prerenal AKI (39). In addition, the differential diagnosis is important, because treatment of intrinsic and prerenal AKI differs (38). Prerenal AKI is an increase in serum creatinine caused by factors that compromise renal perfusion, which rapidly improved to baseline with volume repletion or improvement in cardiac output within 3 days of directed therapy or within 7 days, when a more careful therapeutic regimen was warranted (e. g. in congestiveheart failure). Intrinsic AKI is sudden increase in serum creatinine in the presence of a potential acute tubular necrosis-inducing event or specific kidney disease without a response to fluid resuscitation and/or hemodynamic optimization. Current tests, including fractional excretion of sodium, fractional excretion of urea, and urea creatinine ratio are of limited utility in diagnosing intrinsic AKI (38).
[00229] NGAL, a 25kD protein, is expressed by epithelia and functions as a component of the innate immune system (40). It is produced in response to epithelial damage and is upregulated in acute kidney injury (41). In a previous study, NGAL discriminated intrinsic AKI from prerenal AKI, chronic kidney disease, and normal renal function (42). Several studies suggested that NGAL could be useful in the early diagnosis of AKI and prediction of poor clinical outcomes in AKI (43).
[00230] Urinary neutrophil gelatinase-associated lipocalin (NGAL) was tested to determine whether it predicts adverse outcomes in acute kidney injury (primary endpoint: progression to a higher RIFLE class, dialysis or death) and distinguishes between intrinsic and prerenal acute kidney injury in the cohort of 161 patients. Urinary NGAL levels discriminated intrinsic AKI from prerenal AKI (ROC 0.87, CI 0.81-0.94). Patients with the primary endpoint had higher median urinary NGAL levels (248.2 vs. 68.3 μg/L, p<0.001). In logistic regression analysis, NGAL independently predicted worsening AKI, when corrected for demographics, comorbidities, creatinine, and RIFLE class.
[00231] 161 adult hospitalized patients with confirmed AKI according to RIFLE criteria were included. Blood samples for creatinine and AKI indices were taken on inclusion.
Urinary NGAL was collected on inclusion and two days later. Diagnosis of intrinsic or prerenal AKI was adjudicated based on medical records by two independent clinicians who were blinded to uNGAL levels.
[00232] Exclusion criteria: unavailable baseline creatinine, incomplete follow-up and postrenal obstruction. 16 patients were excluded. Of the remaining 145 patients, 32 had prerenal AKI, 75 had intrinsic AKI, 38 were unclassifiable. Urinary NGAL was measured using ARCHITECT® (Abbott Laboratories) technology.
[00233] Patient characteristics were assessed by diagnosis and kidney parameters (Table 9)
[00234] Table 9: Patient characteristics, by diagnosis and kidney parameters Stable or
All improved Intrinsic Prerenal patients Worsening AKI (n- AKI AKI
Characteristic (n=145) AKI (n=49) 95) (n=75) (n=32)
Demographics
67.7 68.8 71.1
Mean age (SD), y (14.4) 65.6 (14.4) (14.4) 64 (15.5) (14.2)
59 40 14
Women, n (%) (40.7%) 19 (38.8%) (41.7%) 27 (36%) (43.8%)
Black race, n(%) 1 (0.7%) 0 (0%) 1 (1 %) 0 (0%) 1 (3.1 %)
Comorbidities
Congestive Heart 101 67 26
Failure (%) (69.7%) 34 (69.4%) (69.8%) 42 (56%) (81.3%)
Diabetes mellitus, 30 19 1 1
n (%) 42 (29%) 12 (24.5%) (31.3%) (25.3%) (34.4%)
Hypertension, n 1 18 78 28
(%) (81.4%) 40 (81.6%) (81.3%) 54 (72%) (87.5%)
Coronary artery 44 27 1 1
disease, n (%) (30.3%) 17 (34.7%) (28.1 %) 18 (24%) (34.4%)
Peripheral artery 21 12 10
disease, n (%) (14.5%) 9 (18.4%) (12.5%) (13.3%) 6 (18.8%) cerebrovascular
disease, n (%) 13 (9%) 4 (8.2%) 9 (9.4%) 7 (9.3%) 2 (6.3%)
CKD stage at baseline (based on GFR), n (%) (eGFR was calculated using the
Modification of Diet in Renal Disease Formular (MDRD))
43 30 28
1 :>90: n(%) (29.75) 13 (26.5%) (31.3%) (37.3%) 7 (21 .9%)
64 45 28 1 1
2: 60-89 (44.1 %) 19 (38.8%) (46.9%) (37.3%) (34.4%)
33 18 14 14
3: 30-59 (22.8%) 15 (30.6%) (18.8%) (18.7%) (43.8%)
4: 14-29 5 (3.4%) 2 (4.1 %) 3 (3.1 %) 5 (6.7%) 0 (0%)
[00235] ROC analyis was performed of the different biomarkers in the differenetial diagnosis of AKI was performed (Table 10)
[00236] Table 10: ROC analysis of different biomarkers in the differential diagnosis of AKI
Test (Data represent areas under the receiver- Diagnosis of
operating characteristic curves (AUC-ROC) intrinsic AKI vs.
with 95% confidence interval) prerenal AKI
Urinary NGAL 0.87 (0.81 - 0.94)
Urinary NGLA/urinary creatinine 0.89 (0.82 0.95)**
0.74 (0.63 0.84)**,
Serum creatine #
0.72 (0.62 0.82)**,
RIFLE class #
Fractional excretion of urea 0.59 (0.48 0.71 )# Fractional excretion of sodium 0.54 (0.42 0.65)# Serum urea / serum creatinine ratio 0.71 (0.59 0.82)*, #
[00237] * p<0.01 vs null hypothesis: true area = 0.5
[00238] ** p< 0.001 vs null hypothesis: true area [00239] # p< 0.05 vs urinary NGAL
[00240] The test characteristics of biomarkers in the prediction of intrinsic AKI versus prerenal AKI at different cutoff levels was determined (Table 11).
[00241] Table 11 : Test characteristics of biomarkers in the prediction of intrinsic AKI vs. prerenal AKI at different cutoff levels
Positive
Cutoff predictive Negative
Biomarker Level Sensitivity Specificity value predictive value uNGLA >47.35 0.89 (0.8- 0.53 (0.35- 0.82 (0.71- 0.68 (0.46-0.84)
(absolute 0.95) 0.7) 0.89)
level)
(ug/i) >104.43 0.75 (0.63- 0.88 (0.7- 0.93 (0.83- 0.6 (0.44-0.73)
0.84) 0.96) 0.98)
uNGAL (per >45.28 0.88 (0.78- 0.55 (0.36- 0.82 (0.72- 0.65 (0.44-0.82) urinary 0.94) 0.72) 0.9)
creatine)
(ug/g) >127.6 0.72 (0.6- 0.87 (0.69- 0.93 (0.82- 0.56 (0.41-0.7)
0.81 ) 0.96) 0.98)
Serum >166.6 0.85 (0.75- 0.44 (0.27- 0.78 (0.67- 0.56 (0.35-0.75) creatinine 0.92) 0.62) 0.86)
(umol/l) >204.84 0.63 (0.51- 0.66 (0.47- 0.81 (0.68- 0.43 (0.29-0.58)
0.73) 0.81 ) 0.9)
RIFLE 2 0.88 (0.78- 0.34 (0.19- 0.76 (0.65- 0.55 (0.32-0.76) severity class 0.94) 0.53) 0.84)
3 0.49 (0.38- 0.88 (0.7- 0.9 (0.76- 0.42 (0.31-0.55)
(R=1 , l=2, F3)
0.61 ) 0.96) 0.97)
Serum <63.26 0.45 (0.34- 0.89 (0.7- 0.92 (0.76- 0.38 (0.26-0.51 ) urea/serum 0.57) 0.97) 0.98)
creatinine
ratio (both in <83.88 0.63 0.63 0.82 0.39 mmol/l) (0.51-0.74) (0.42-0.8) (0.69-0.91 ) (0.25-0.54)
Optimal cutoff levels for NGLA were derived from an independent patient cohort in the emergency room. Cutoff levels of the remaining biomarkers represent adjusted percentiles in the current study population to ensure comparability of the results
[00242] NGAL levels displayed less overlap between the prospective outcome groups when compared to serum creatinine (Figure 7). Median NGAL levels on inclusion and 2 days after inclusion are significantly higher in patients, who later developed worsening AKI in comparison to all others (Figure 7). NGAL level and outcomes were determined (Figure 8). Patients were stratified by NGAL level. Percentages of the subgroups that experience endpoints are shown, p for trend is statistically significant for composite and individual AKI outcomes. [00243] Multiple Logistic Regression Models for the predicton of worsening AKI (step-up in RIFLE class, dialysis initiation, or mortality) was performed. Wald score, p value and odds ratio describe the contribution of the individual covariate to the model. R square, AUC-ROC and diagnostic accuracy are measures of the overall performance of the model (Table 12).
[00244] Table 12: Multiple Logistic Regression Models for the prediction of worsening AKI
Figure imgf000061_0001
[00245] In AKI patients uNGAL distinguished intrinsic AKI from prerenal AKI and predicted worsening of AKI. uNGAL performed better than conventional laboratory tests in differentiating intrinsic AKI from prerenal AKI. NGAL is an independent predictor of worsening AKI in logistic regression analysis.
[00246] Example 3: Test Characteristics of Urinary Neutrophil Gelatinase-Assocaited Lipocalin (uNGAL) for Differential Diagnosis and Risk Stratification in Patients with Established Acute Kidney Injury
[00247] Serum creatinine dynamics are used for the diagnosis of acute kidney injury (AKI) in current classification schemes (e.g. RIFLE), but are temporally and diagnostically poor. Urinary NGLA, a biomarker of nephron damage, may help to differentiate intrinsic kidney damage from prerenal AKI. Methods: The relationship between uGAL levels at the time of diagnosis of AKI and outcomes was evaluated prospectively. uNGLA levels were determined on the Abbott ACHITECT standardized clinical platform. The primary outcome was worsening of AKI (progression to higher RILE category, dialysis initiation, or death) during hospitalization. In those patients with a likely or definitive diagnostic attribution based on clinical criteria, uNGLA in was tested distinguishing intrinsic from prerenal AKI. Results: 162 hospitalized AKI patients were studied. 17 pateients with postrenal obstruction or insufficient clinical information were excluded. From the remaining 145 patients, 75 patients had a clinical diagnosis of intrinsic AKI, 32 patients had prerenal AKI, and 38 patients had an uncertain or ambiguous diagnosis. uNGLA levels were found to effectively discriminated intrinsic AKI from prerenal AKI (ROC 0.87, CI 0.81-0.94). NGLA levels at a cutoff
>100ng/ml had a sensitivity of 0.75, specificity of 0.88, a positive predictive value of 0.93, and a diagnostic accuracy of 0.78 in separating intrinsic AKI versus prerenal AKI. Patients with the primary endpoint had significantly higher uNGAL (p<0.001). In logistic regression analysis, uNGAL levels independently predicted worsening AKI, when corrected for demographic, comorbidities, serum creatinine, and RIFLE class. Conculsion: uNGLA is useful in the differential diagnosis and prognostic stratification of patients with established AKI.
[00248] REFERENCES
1. Chertow GM, Burdick E, Honour M, Bonventre JV, Bates DW. Acute kidney injury, mortality, length of stay, and costs in hospitalized patients. J Am Soc Nephrol
2005;16:3365-70.
2. Hsu CY, McCulloch CE, Fan D, Ordonez JD, Chertow GM, Go AS. Community-based incidence of acute renal failure. Kidney Int 2007;72:208-12.
3. Waikar SS, Curhan GC, Wald R, McCarthy EP, Chertow GM. Declining mortality in patients with acute renal failure, 1988 to 2002. J Am Soc Nephrol 2006;17: 1143-50.
4. Xue JL, Daniels F, Star RA, et al. Incidence and mortality of acute renal failure in
Medicare beneficiaries, 1992 to 2001. J Am Soc Nephrol 2006;17: 1135-42.
5. Liano F, Pascual J. Epidemiology of acute renal failure: a prospective, multicenter, community-based study. Madrid Acute Renal Failure Study Group. Kidney Int 1996;50:811- 8.
6. Nash K, Hafeez A, Hou S. Hospital-acquired renal insufficiency. Am J Kidney Dis 2002;39:930-6.
7. Venkatachalam MA, Griffin KA, Lan R, Geng H, Saikumar P, Bidani AK. Acute kidney injury: a springboard for progression in chronic kidney disease. Am J Physiol Renal Physiol 2010.
8. Bellomo R, Ronco C, Kellum JA, Mehta RL, Palevsky P. Acute renal failure - definition, outcome measures, animal models, fluid therapy and information technology needs: the Second International Consensus Conference of the Acute Dialysis Quality Initiative (ADQI) Group. Crit Care 2004;8:R204-12.
9. Mehta RL, Kellum JA, Shah SV, et al. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit Care 2007; 11 :R31. 10. Mishra J, Ma Q, Prada A, et al. Identification of neutrophil gelatinase-associated lipocalin as a novel early urinary biomarker for ischemic renal injury. J Am Soc Nephrol
2003;14:2534-43.
11. Perrone RD, Madias NE, Levey AS. Serum creatinine as an index of renal function: new insights into old concepts. Clin Chem 1992;38: 1933-53.
12. Star RA. Treatment of acute renal failure. Kidney Int 1998;54: 1817-31.
13. Waikar SS, Bonventre JV. Creatinine kinetics and the definition of acute kidney injury. J Am Soc Nephrol 2009;20:672-9.
14. Macedo E, Mehta RL. Prerenal failure: from old concepts to new paradigms. Curr Opin Crit Care 2009;15:467-73.
15. Lameire N, Van Biesen W, Vanholder R. Acute renal failure. Lancet 2005;365:417-30.
16. Nickolas TL, O'Rourke MJ, Yang J, et al. Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelatinase-associated lipocalin for diagnosing acute kidney injury. Ann Intern Med 2008;148:810-9.
17. Szczech LA. The development of urinary biomarkers for kidney disease is the search for our renal troponin. J Am Soc Nephrol 2009;20: 1656-7.
18. Honore PM, Joannes-Boyau O, Boer W. The early biomarker of acute kidney injury: in search of the Holy Grail. Intensive Care Med 2007;33: 1866-8.
19. Haase M, Devarajan P, Haase-Fielitz A, et al. The outcome of neutrophil
gelatinaseassociated lipocalin-positive subclinical acute kidney injury a multicenter pooled analysis of prospective studies. J Am Coll Cardiol 2011;57: 1752-61.
20. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation.
Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999;130:461-70.
21. Grenier FC, Ali S, Syed H, et al. Evaluation of the ARCHITECT urine NGAL assay: assay performance, specimen handling requirements and biological variability. Clin Biochem 2010;43:615-20.
22. Kamijo A, Kimura K, Sugaya T, et al. Urinary fatty acid-binding protein as a new clinical marker of the progression of chronic renal disease. J Lab Clin Med 2004;143:23-30.
23. Pencina MJ, DAgostino RB, Sr., DAgostino RB, Jr., Vasan RS. Evaluating the added predictive ability of a new marker: from area under the ROC curve to reclassification and beyond. Stat Med 2008;27:157-72; discussion 207-12.
24. Singer E, Elger A, Elitok S, et al. Urinary neutrophil gelatinase-associated lipocalin distinguishes pre -renal from intrinsic renal failure and predicts outcomes. Kidney Int 2011;80:405-14. 25. Parikh CR, Coca SG. Acute kidney injury: defining prerenal azotemia in clinical practice and research. Nat Rev Nephrol 2010;6:641-2.
26. de Geus HR, Bakker J, Lesaffre EM, le Noble JL. Neutrophil Gelatinase-associated Lipocalin at ICU Admission Predicts for Acute Kidney Injury in Adult Patients. Am J Respir Crit Care Med 2010;Epub 10/12/2010.
27. Koyner JL, Vaidya VS, Bennett MR, et al. Urinary biomarkers in the clinical prognosis and early detection of acute kidney injury. Clin J Am Soc Nephrol 2010;5:2154-65.
28. Haase M, Bellomo R, Devarajan P, Schlattmann P, Haase-Fielitz A. Accuracy of neutrophil gelatinase-associated lipocalin (NGAL) in diagnosis and prognosis in acute kidney injury: a systematic review and meta-analysis. Am J Kidney Dis 2009;54: 1012-24.
29. Yang HN, Boo CS, Kim MG, Jo SK, Cho WY, Kim HK. Urine neutrophil
gelatinaseassociated lipocalin: an independent predictor of adverse outcomes in acute kidney injury. Am J Nephrol 2010;31 :501-9.
30. Kumpers P, Hafer C, Lukasz A, et al. Serum neutrophil gelatinase-associated lipocalin at inception of renal replacement therapy predicts survival in critically ill patients with acute kidney injury. Crit Care 2010;14:R9.
31. Ferguson MA, Vaidya VS, Waikar SS, et al. Urinary liver-type fatty acid-binding protein predicts adverse outcomes in acute kidney injury. Kidney Int;77:708-14.
32. Nickolas TL, O'Rourke MJ, Yang J, et al. Sensitivity and specificity of a single emergency department measurement of urinary neutrophil gelatinase-associated lipocalin for diagnosing acute kidney injury. Ann Intern Med 2008;148:810-9.
33. Mori K, Lee HT, Rapoport D, et al. Endocytic delivery of lipocalin-siderophore-iron complex rescues the kidney from ischemia-reperfusion injury. J Clin Invest
2005;115:610-21.
34. Cai L, Rubin J, Han W, Venge P, Xu S. The origin of multiple molecular forms in urine of HNL/NGAL. Clin J Am Soc Nephrol;5:2229-35.
35. Kjeldsen L, Johnsen AH, Sengelov H, Borregaard N. Isolation and primary structure of NGAL, a novel protein associated with human neutrophil gelatinase. J Biol Chem
1993;268: 10425-32.
36. Cai L, Rubin J, Han W, Venge P, Xu S. The Origin of Multiple Molecular Forms in Urine of HNL/NGAL. Clin J Am Soc Nephrol 2010.
37. Bellomo, R et al, Critical Care, 2004. 8: R 204-212.
38. Lameire, N et al, Lancet 2005; 365: 417-430.
39. Liano, F et al, Kidney Int 1996; 50:811-818. 40. Flo TH, et al. Nature. 2004 Dec 16; 432(7019): 917-21.
41. Mishra J, et al.Lancet. 2005 Apr 2-8;365(9466): 1231-8.
42. Nickolas, TL et al, Annals of internal medcine 2008; 148: 810-819.
43. Bennett, M at al, Clin J Am Soc Nephrol 2008; 3: 665-673.
44. Jolliffe IT. Principal component analysis. 2nd ed. New York: Springer; 2002.
45. Van Biesen W, Vanholder R, Lameire N. Defining acute renal failure: RIFLE and beyond. Clin J Am Soc Nephrol. 2006 Nov;l(6):1314-9.

Claims

WHAT IS CLAIMED IS: In the Claims:
1. A method for diagnosing acute kidney injury (AKI) in a subject, the method
comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of NGAL protein and KIM-1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM-1 protein that exceeds about 2 ng/ml indicate that the subject may have AKI.
2. The method of claim 1 , wherein the subject is a human.
3. The method of claim 1, wherein the subject has no signs of kidney disease.
4. The method of claim 1, further comprising subsequently treating the AKI in the
subject.
5. The method of claim 1, further comprising determining the amount of sCR protein present in the sample, wherein an amount of sCr protein that exceeds about 1 mg/dl indicates that the subject may have AKI.
6. The method of claim 1, wherein the determining step comprises contacting the sample with an antibody that binds to the NGAL protein and an antibody that binds to the KIM-1 protein.
7. The method of claim 6, comprising performing an immunoassay.
8. The method of claim 7, wherein the immunoassay is an enzyme-linked
immunosorbent assay (ELISA).
9. The method of claim 6, wherein the antibodies are immobilized on a solid support.
10. The method of claim 9, wherein the solid support comprises a dipstick or a test strip.
11. The method of claim 6, comprising performing an immunoblotting method.
12. A method for predicting mortality of a subject suspected of having AKI, the method comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of NGAL protein and the amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM- 1 protein that exceeds about 2 ng/ml indicate a high risk of mortality.
13. A method for predicting the need for dialysis in a subject suspected of having AKI, the method comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of NGAL protein and the amount of KIM- 1 protein present in the sample, wherein an amount of NGAL protein that exceeds about 100 ng/ml and an amount of KIM- 1 protein that exceeds about 2 ng/ml indicate that the subject is at high risk of needing dialysis.
14. A method for determining whether a subject suspected of having AKI has transient or sustained AKI, the method comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of NGAL protein in the sample; wherein
(i) an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI,
(ii) an amount of NGAL protein in the range of about 50 ng/ml to about 80 ng/ml indicates that the subject has transient AKI, and (iii) an amount of NGAL protein in the range of about 100 ng/ml to about 200 ng/ml indicates that the subject has sustained AKI.
15. A method for determining the severity of AKI, the method comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of NGAL protein in the sample; wherein
(i) an amount of NGAL protein in the range of about 25 ng/ml to about 35 ng/ml indicates that the subject does not have AKI,
(ii) an amount of NGAL protein in the range of about 50 ng/ml to about 100 ng/ml indicates that the subject has mild AKI,
(iii) an amount of NGAL protein in the range of about 100 ng/ml to about 250 ng/ml indicates that the subject has intermediate AKI, and
(iv) an amount of NGAL protein higher than about 250 ng/ml indicates that the subject has severe AKI, and wherein the the classification of the AKI as either mild, intermediate, or severe is based on the RIFLE severity classification system.
16. A method for determining the risk of an adverse event in a subject suspected of
having AKI, the method comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of NGAL protein and the amount of sCr protein present in the sample; wherein (i) an amount of NGAL protein that is below about 100 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate a low risk of an adverse event,
(ii) an amount of NGAL protein that exceeds or is equal to about 100 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate an intermediate risk of an adverse event,
(iii) an amount of NGAL protein that is below about 100 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate an intermediate risk of an adverse event, and
(iv) an amount of NGAL protein that exceeds or is equal to about 100 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate a high risk of an adverse event.
A method for determining the risk of an adverse event in a subject suspected of having AKI, the method comprising: a) obtaining a urine sample from the subject; and
(b) determining the amount of KIM- 1 protein and the amount of sCr protein present in the sample; wherein
(i) an amount of KIM- 1 protein that is below about 2 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate a low risk of an adverse event,
(ii) an amount of KIM- 1 protein that exceeds or is equal to about 2 ng/ml and an amount of sCr protein that is below about 1 mg/dl indicate an intermediate risk of an adverse event,
(iii) an amount of KIM- 1 protein that is below about 2 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate an intermediate risk of an adverse event, and (iv) an amount of KIM- 1 protein that exceeds or is equal to about 2 ng/ml and an amount of sCr protein that exceeds or is equal to about 1 mg/dl indicate a high risk of an adverse event.
18. The method of claim 16 or 17, wherein the adverse event comprises in-hospital
mortality, or in-hospital dialysis, or both.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8592170B2 (en) 2008-03-12 2013-11-26 The Trustees Of Columbia University In The City Of New York High molecular weight Ngal as a biomarker for chronic kidney disease
CN104730250A (en) * 2013-12-20 2015-06-24 北京义翘神州生物技术有限公司 Enzyme linked immunosorbent assay kit for detecting human kidney injury molecule-1
US9534027B2 (en) 2010-05-24 2017-01-03 The Trustees Of Columbia University In The City Of New York Mutant NGAL proteins and uses thereof
WO2017027858A1 (en) * 2015-08-12 2017-02-16 The Trustees Of Columbia University In The City Of New York Methods of treating volume depletion and kidney injury
US9624281B2 (en) 2012-11-21 2017-04-18 The Trustees Of Columbia University In The City Of New York Mutant NGAL proteins and uses thereof
RU2702023C1 (en) * 2018-08-17 2019-10-04 Федеральное государственное бюджетное учреждение "Ростовский научно-исследовательский онкологический институт" Министерства здравоохранения Российской Федерации Diagnostic technique for acute renal injury following organ-preserving surgical treatment of localized renal cell carcinoma
US11181523B2 (en) 2015-10-15 2021-11-23 Universidad De Chile Method for the early detection of acute kidney injury in critical patients, using fibroblast growth factor 23, klotho and erythropoietin as biomarkers
US11350887B2 (en) 2019-08-07 2022-06-07 Fresenius Medical Care Holdings, Inc. Systems and methods for detection of potential health issues
WO2022192751A3 (en) * 2021-03-12 2022-12-01 The Trustees Of Columbia University In The City Of New York Urine test predicts kidney injury and death in covid-19

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100233728A1 (en) * 2003-03-27 2010-09-16 Prasad Devarajan Method and kit for detecting the early onset of renal tubular cell injury

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100233728A1 (en) * 2003-03-27 2010-09-16 Prasad Devarajan Method and kit for detecting the early onset of renal tubular cell injury

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HALL ET AL.: "IL-18 and urinary NGAL predict dialysis and graft recovery after kidney transplantation.", J AM SOC NEPHROL., vol. 21, no. 1, 27 September 2009 (2009-09-27), pages 190 - 195 *
HAN ET AL.: "Urinary biomarkers in the early diagnosis of acute kidney injury.", KIDNEY INT., vol. 73, no. 7, April 2008 (2008-04-01), pages 863 - 869 *
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, vol. 11, no. 4, 2007, pages R84 *

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US9534027B2 (en) 2010-05-24 2017-01-03 The Trustees Of Columbia University In The City Of New York Mutant NGAL proteins and uses thereof
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US11181523B2 (en) 2015-10-15 2021-11-23 Universidad De Chile Method for the early detection of acute kidney injury in critical patients, using fibroblast growth factor 23, klotho and erythropoietin as biomarkers
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US11350887B2 (en) 2019-08-07 2022-06-07 Fresenius Medical Care Holdings, Inc. Systems and methods for detection of potential health issues
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