METHODS AND COMPOSITIONS FOR DIAGNOSIS AND PROGNOSIS OF RENAL INJURY AND RENAL FAILURE

Abstract

The present invention relates to methods and compositions for monitoring, diagnosis, prognosis, and determination of treatment regimens in subjects suffering from or suspected of having a renal injury. In particular, the invention relates to using assays that detect one or more markers selected from the group consisting of Clusterin, Heart-type fatty acid binding protein, Hepatocyte growth factor, Interferon gamma, Interleukin-12 subunit beta, Interleukin-16, Interleukin-2, 72 kDa type IV collagenase, Matrix metalloproteinase-9, Midkine, and Serum amyloid P-component as diagnostic and prognostic biomarkers in renal injuries.

Claims

1. A method for evaluating renal status in a subject, comprising: performing one or more assays configured to detect a kidney injury marker selected from the group consisting of Clusterin, Heart-type fatty acid binding protein, Hepatocyte growth factor, Interferon gamma, Interleukin-12 subunit beta, Interleukin-16, Interleukin-2, 72 kDa type IV collagenase, Matrix metalloproteinase-9, Midkine, and Serum amyloid P-component by introducing the urine sample obtained from the subject into an assay instrument which (i) contacts the urine sample with one or more antibodies which specifically bind for detection the biomarker(s) which are assayed, and (ii) generates one or more assay results indicative of binding of each biomarker which is assayed to a respective antibody to provide one or more assay results; using the one or more assay results, or one or more values derived therefrom, to assign a probability that a future acute renal injury will occur within 48 hours of the time the urine sample is obtained to the subject, wherein the assignment is made by comparing the assay result or value derived therefrom to a predetermined threshold value selected in a population study to separate the population of individuals in the population study into a first subpopulation which is at an increased probability for acute renal failure within 48 hours relative to a second subpopulation; and if the one or more assay results, or one or more values derived therefrom, assign the subject into the first subpopulation, treating the subject by one or more of initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, delaying or avoiding procedures that are known to be damaging to the kidney, and modifying diuretic administration.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0065] FIG. 1 provides data tables determined in accordance with Example 6 for the comparison of marker levels in urine samples collected for Cohort 1 (patients that did not progress beyond RIFLE stage 0) and in urine samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0066] FIG. 2 provides data tables determined in accordance with Example 7 for the comparison of marker levels in urine samples collected for Cohort 1 (patients that did not progress beyond RIFLE stage 0 or R) and in urine samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage I or F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0067] FIG. 3 provides data tables determined in accordance with Example 8 for the comparison of marker levels in urine samples collected for Cohort 1 (patients that reached, but did not progress beyond, RIFLE stage R) and in urine samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage I or F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0068] FIG. 4 provides data tables determined in accordance with Example 9 for the comparison of marker levels in urine samples collected for Cohort 1 (patients that did not progress beyond RIFLE stage 0) and in urine samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0069] FIG. 5 provides data tables determined in accordance with Example 6 for the comparison of marker levels in plasma samples collected for Cohort 1 (patients that did not progress beyond RIFLE stage 0) and in plasma samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0070] FIG. 6 provides data tables determined in accordance with Example 7 for the comparison of marker levels in plasma samples collected for Cohort 1 (patients that did not progress beyond RIFLE stage 0 or R) and in plasma samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage I or F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0071] FIG. 7 provides data tables determined in accordance with Example 8 for the comparison of marker levels in plasma samples collected for Cohort 1 (patients that reached, but did not progress beyond, RIFLE stage R) and in plasma samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage I or F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

[0072] FIG. 8 provides data tables determined in accordance with Example 9 for the comparison of marker levels in plasma samples collected for Cohort 1 (patients that did not progress beyond RIFLE stage 0) and in plasma samples collected from subjects at 0, 24 hours, and 48 hours prior to reaching stage F in Cohort 2. Tables provide descriptive statistics, AUC analysis, and sensitivity, specificity and odds ratio calculations at various threshold (cutoff) levels for the various markers.

DETAILED DESCRIPTION OF THE INVENTION

[0073] The present invention relates to methods and compositions for diagnosis, differential diagnosis, risk stratification, monitoring, classifying and determination of treatment regimens in subjects suffering or at risk of suffering from injury to renal function, reduced renal function and/or acute renal failure through measurement of one or more kidney injury markers. In various embodiments, a measured concentration of one or more markers selected from the group consisting of Clusterin, Heart-type fatty acid binding protein, Hepatocyte growth factor, Interferon gamma, Interleukin-12 subunit beta, Interleukin-16, Interleukin-2, 72 kDa type IV collagenase, Matrix metalloproteinase-9, Midkine, and Serum amyloid P-component, or one or more markers related thereto, are correlated to the renal status of the subject.

[0074] For purposes of this document, the following definitions apply: As used herein, an injury to renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable reduction in a measure of renal function. Such an injury may be identified, for example, by a decrease in glomerular filtration rate or estimated GFR, a reduction in urine output, an increase in serum creatinine, an increase in serum cystatin C, a requirement for renal replacement therapy, etc. Improvement in Renal Function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) measurable increase in a measure of renal function. Preferred methods for measuring and/or estimating GFR are described hereinafter.

[0075] As used herein, reduced renal function is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.1 mg/dL (8.8 mol/L), a percentage increase in serum creatinine of greater than or equal to 20% (1.2-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour).

[0076] As used herein, acute renal failure or ARF is an abrupt (within 14 days, preferably within 7 days, more preferably within 72 hours, and still more preferably within 48 hours) reduction in kidney function identified by an absolute increase in serum creatinine of greater than or equal to 0.3 mg/dl (26.4 mol/l), a percentage increase in serum creatinine of greater than or equal to 50% (1.5-fold from baseline), or a reduction in urine output (documented oliguria of less than 0.5 ml/kg per hour for at least 6 hours). This term is synonymous with acute kidney injury or AKI.

[0077] In this regard, the skilled artisan will understand that the signals obtained from an immunoassay are a direct result of complexes formed between one or more antibodies and the target biomolecule (i.e., the analyte) and polypeptides containing the necessary epitope(s) to which the antibodies bind. While such assays may detect the full length biomarker and the assay result be expressed as a concentration of a biomarker of interest, the signal from the assay is actually a result of all such immunoreactive polypeptides present in the sample. Expression of biomarkers may also be determined by means other than immunoassays, including protein measurements (such as dot blots, western blots, chromatographic methods, mass spectrometry, etc.) and nucleic acid measurements (mRNA quatitation). This list is not meant to be limiting.

[0078] As used herein, the term Clusterin refers to one or more polypeptides present in a biological sample that are derived from the Clusterin precursor (Swiss-Prot P10909 (SEQ ID NO: 1)).

TABLE-US-00002 10203040 MMKTLLLFVGLLLTWESGQVLGDQTVSDNELQEMSNQGSK 50607080 YVNKEIQNAVNGVKQIKTLIEKTNEERKTLLSNLEEAKKK 90100110120 KEDALNETRESETKLKELPGVCNETMMALWEECKPCLKQT 130140150160 CMKFYARVCRSGSGLVGRQLEEFLNQSSPFYFWMNGDRID 170180190200 SLLENDRQQTHMLDVMQDHFSRASSIIDELFQDRFFTREP 210220230240 QDTYHYLPFSLPHRRPHFFFPKSRIVRSLMPFSPYEPLNF 250260270280 HAMFQPFLEMIHEAQQAMDIHFHSPAFQHPPTEFIREGDD 290300310320 DRTVCREIRHNSTGCLRMKDQCDKCREILSVDCSTNNPSQ 330340350360 AKLRRELDESLQVAERLTRKYNELLKSYQWKMLNTSSLLE 370380390400 QLNEQFNWVSRLANLTQGEDQYYLRVTTVASHTSDSDVPS 410420430440 GVTEVVVKLFDSDPITVTVPVEVSRKNPKFMETVAEKALQ EYRKKHREE

[0079] The following domains have been identified in Clusterin:

TABLE-US-00003 Residues Length Domain ID 1-22 22 Signal peptide 23-449 427 Clusterin 23-227 205 Clusterin beta chain 228-449 222 Clusterin alpha chain

[0080] As used herein, the term Heart-type fatty acid-binding protein refers to one or more polypeptides present in a biological sample that are derived from the Heart-type fatty acid-binding protein precursor (Swiss-Prot P05413 (SEQ ID NO: 2)).

TABLE-US-00004 10203040 MVDAFLGTWKLVDSKNFDDYMKSLGVGFATRQVASMTKPT 50607080 TIIEKNGDILTLKTHSTFKNTEISFKLGVEFDETTADDRK 90100110120 VKSIVTLDGGKLVHLQKWDGQETTLVRELIDGKLILTLTH 130 GTAVCTRTYEKEA

[0081] The following domains have been identified in Heart-type fatty acid-binding protein:

TABLE-US-00005 Residues Length Domain ID 1 1 Initiator methionine 2-133 132 Heart-type fatty acid-binding protein

[0082] As used herein, the term Hepatocyte growth factor refers to one or more polypeptides present in a biological sample that are derived from the Hepatocyte growth factor precursor (Swiss-Prot P14210 (SEQ ID NO: 3)).

TABLE-US-00006 10203040 MWVTKLLPALLLQHVLLHLLLLPIAIPYAEGQRKRRNTIH 50607080 EFKKSAKTTLIKIDPALKIKTKKVNTADQCANRCTRNKGL 90100110120 PFTCKAFVFDKARKQCLWFPFNSMSSGVKKEFGHEFDLYE 130140150160 NKDYIRNCIIGKGRSYKGTVSITKSGIKCQPWSSMIPHEH 170180190200 SFLPSSYRGKDLQENYCRNPRGEEGGPWCFTSNPEVRYEV 210220230240 CDIPQCSEVECMTCNGESYRGLMDHTESGKICQRWDHQTP 250260270280 HRHKFLPERYPDKGFDDNYCRNPDGQPRPWCYTLDPHTRW 290300310320 EYCAIKTCADNTMNDTDVPLETTECIQGQGEGYRGTVNTI 330340350360 WNGIPCQRWDSQYPHEHDMTPENFKCKDLRENYCRNPDGS 370380390400 ESPWCFTTDPNIRVGYCSQIPNCDMSHGQDCYRGNGKNYM 410420430440 GNLSQTRSGLTCSMWDKNMEDLHRHIFWEPDASKLNENYC 450460470480 RNPDDDAHGPWCYTGNPLIPWDYCPISRCEGDTTPTIVNL 490500510520 DHPVISCAKTKQLRVVNGIPTRTNIGWMVSLRYRNKHICG 530540550560 GSLIKESWVLTARQCFPSRDLKDYEAWLGIHDVHGRGDEK 570580590600 CKQVLNVSQLVYGPEGSDLVLMKLARPAVLDDFVSTIDLP 610620630640 NYGCTIPEKTSCSVYGWGYTGLINYDGLLRVAHLYIMGNE 650660670680 KCSQHHRGKVTLNESEICAGAEKIGSGPCEGDYGGPLVCE 690700710720 QHKMRMVLGVIVPGRGCAIPNRPGIFVRVAYYAKWIHKII LTYKVPQS

[0083] The following domains have been identified in Hepatocyte growth factor:

TABLE-US-00007 Residues Length Domain ID 1-31 31 signal sequence 32-494 463 Hepatocyte growth factor alpha chain 495-728 234 Hepatocyte growth factor beta chain

[0084] As used herein, the term Interferon gamma refers to one or more polypeptides present in a biological sample that are derived from the Interferon gamma precursor (Swiss-Prot P01579 (SEQ ID NO: 4)).

TABLE-US-00008 10203040 MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNA 50607080 GHSDVADNGTLFLGILKNWKEESDRKIMQSQIVSFYFKLF 90100110120 KNFKDDQSIQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN 130140150160 YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFR GRRASQ

[0085] The following domains have been identified in Interferon gamma:

TABLE-US-00009 Residues Length Domain ID 1-23 23 Signal peptide 24-161 138 Interferon gamma 162-166 5 Propeptide

[0086] As used herein, the term Interleukin-12 subunit beta (also known as Interleukin-12 p40) refers to one or more polypeptides present in a biological sample that are derived from the Interleukin-12 subunit beta precursor (Swiss-Prot P29460 (SEQ ID NO: 5)).

TABLE-US-00010 10203040 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPD 50607080 APGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVK 90100110120 EFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQ 130140150160 KEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSR 170180190200 GSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACP 210220230240 AAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKN 250260270280 LQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGK 290300310320 SKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWS EWASVPCS

[0087] The following domains have been identified in Interleukin-12 subunit beta:

TABLE-US-00011 Residues Length Domain ID 1-22 22 Signal peptide 23-328 306 Interleukin-12 subunit beta

[0088] As used herein, the term Interleukin-16 refers to one or more polypeptides present in a biological sample that are derived from the Interleukin-16 precursor (Swiss-Prot Q14005 (SEQ ID NO: 6)).

TABLE-US-00012 10203040 MDYSFDTTAEDPWVRISDCIKNLFSPIMSENHGHMPLQPN 50607080 ASLNEEEGTQGHPDGTPPKLDTANGTPKVYKSADSSTVKK 90100110120 GPPVAPKPAWFRQSLKGLRNRASDPRGLPDPALSTQPAPA 130140150160 SREHLGSHIRASSSSSSIRQRISSFETFGSSQLPDKGAQR 170180190200 LSLQPSSGEAAKPLGKHEEGRFSGLLGRGAAPTLVPQQPE 210220230240 QVLSSGSPAASEARDPGVSESPPPGRQPNQKTLPPGPDPL 250260270280 LRLLSTQAEESQGPVLKMPSQRARSFPLTRSQSCETKLLD 290300310320 EKTSKLYSISSQVSSAVMKSLLCLPSSISCAQTPCIPKEG 330340350360 ASPTSSSNEDSAANGSAETSALDTGFSLNLSELREYTEGL 370380390400 TEAKEDDDGDHSSLQSGQSVISLLSSEELKKLIEEVKVLD 410420430440 EATLKQLDGIHVTILHKEEGAGLGFSLAGGADLENKVITV 450460470480 HRVFPNGLASQEGTIQKGNEVLSINGKSLKGTTHHDALAI 490500510520 LRQAREPRQAVIVTRKLTPEAMPDLNSSTDSAASASAASD 530540550560 VSVESTAEATVCTVTLEKMSAGLGFSLEGGKGSLHGDKPL 570580590600 TINRIFKGAASEQSETVQPGDEILQLGGTAMQGLTRFEAW 610620630 NIIKALPDGPVTIVIRRKSLQSKETTAAGDS 1-1332 1332 Pro-interleukin-16 1212-1332 121 Interleukin-16

[0089] As used herein, the term Interleukin-2 refers to one or more polypeptides present in a biological sample that are derived from the Interleukin-2 precursor (Swiss-Prot P60568 (SEQ ID NO: 7)).

TABLE-US-00013 10203040 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLD 50607080 LQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLE 90100110120 EELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE 130140150 TTFMCEYADETATIVEFLNRWITFCQSIISTLT

[0090] The following domains have been identified in Interleukin-2:

TABLE-US-00014 Residues Length Domain ID 1-20 20 Signal peptide 21-153 133 Interleukin-2

[0091] As used herein, the term 72 kDa type IV collagenase (also known as Matrix metalloproteinase-2) refers to one or more polypeptides present in a biological sample that are derived from the 72 kDa type IV collagenase precursor (Swiss-Prot P08253 (SEQ ID NO: 8)).

TABLE-US-00015 10203040 MEALMARGALTGPLRALCLLGCLLSHAAAAPSPIIKFPGD 50607080 VAPKTDKELAVQYLNTFYGCPKESCNLFVLKDTLKKMQKF 90100110120 FGLPQTGDLDQNTIETMRKPRCGNPDVANYNFFPRKPKWD 130140150160 KNQITYRIIGYTPDLDPETVDDAFARAFQVWSDVTPLRFS 170180190200 RIHDGEADIMINFGRWEHGDGYPFDGKDGLLAHAFAPGTG 210220230240 VGGDSHFDDDELWTLGEGQVVRVKYGNADGEYCKFPFLFN 250260270280 GKEYNSCTDTGRSDGFLWCSTTYNFEKDGKYGFCPHEALF 290300310320 TMGGNAEGQPCKFPFRFQGTSYDSCTTEGRTDGYRWCGTT 330340350360 EDYDRDKKYGFCPETAMSTVGGNSEGAPCVFPFTFLGNKY 370380390400 ESCTSAGRSDGKMWCATTANYDDDRKWGFCPDQGYSLFLV 410420430440 AAHEFGHAMGLEHSQDPGALMAPIYTYTKNFRLSQDDIKG 450460470480 IQELYGASPDIDLGTGPTPTLGPVTPEICKQDIVFDGIAQ 490500510520 IRGEIFFFKDRFIWRIVTPRDKPMGPLLVATFWPELPEKI 530540550560 DAVYEAPQEEKAVFFAGNEYWIYSASTLERGYPKPLTSLG 570580590600 LPPDVQRVDAAFNWSKNKKTYIFAGDKFWRYNEVKKKMDP 610620630640 GFPKLIADAWNAIPDNLDAVVDLQGGGHSYFFKGAYYLKL 650660 ENQSLKSVKFGSIKSDWLGC

[0092] The following domains have been identified in 72 kDa type IV collagenase:

TABLE-US-00016 Residues Length Domain ID 1-29 29 Signal peptide 30-109 90 Activation peptide 110-660 551 72 kDa type IV collagenase

[0093] As used herein, the term Matrix metalloproteinase-9 refers to one or more polypeptides present in a biological sample that are derived from the Matrix metalloproteinase-9 precursor (Swiss-Prot P14780 (SEQ ID NO: 9)).

TABLE-US-00017 10203040 MSLWQPLVLVLLVLGCCFAAPRQRQSTLVLFPGDLRTNLT 50607080 DRQLAEEYLYRYGYTRVAEMRGESKSLGPALLLLQKQLSL 90100110120 PETGELDSATLKAMRTPRCGVPDLGRFQTFEGDLKWHHHN 130140150160 ITYWIQNYSEDLPRAVIDDAFARAFALWSAVTPLTFTRVY 170180190200 SRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQG 210220230240 DAHFDDDELWSLGKGVVVPTRFGNADGAACHFPFIFEGRS 250260270280 YSACTTDGRSDGLPWCSTTANYDTDDRFGFCPSERLYTQD 290300310320 GNADGKPCQFPFIFQGQSYSACTTDGRSDGYRWCATTANY 330340350360 DRDKLFGFCPTRADSTVMGGNSAGELCVFPFTFLGKEYST 370380390400 CTSEGRGDGRLWCATTSNFDSDKKWGFCPDQGYSLFLVAA 410420430440 HEFGHALGLDHSSVPEALMYPMYRFTEGPPLHKDDVNGIR 450460470480 HLYGPRPEPEPRPPTTTTPQPTAPPTVCPTGPPTVHPSER 490500510520 PTAGPTGPPSAGPTGPPTAGPSTATTVPLSPVDDACNVNI 530540550560 FDAIAEIGNQLYLFKDGKYWRFSEGRGSRPQGPFLIADKW 570580590600 PALPRKLDSVFEEPLSKKLFFFSGRQVWVYTGASVLGPRR 610620630640 LDKLGLGADVAQVTGALRSGRGKMLLFSGRRLWRFDVKAQ 650660670680 MVDPRSASEVDRMFPGVPLDTHDVFQYREKAYFCQDRFYW 690700 RVSSRSELNQVDQVGYVTYDILQCPED

[0094] The following domains have been identified in Matrix metalloproteinase-9:

TABLE-US-00018 Residues Length Domain ID 1-19 19 Signal peptide 20-93 74 Activation peptide 107-707 601 Matrix metalloproteinase-9 82 kDa form

[0095] In addition, a 67 kDa form of Matrix metalloproteinase-9 has been identified.

[0096] As used herein, the term Midkine refers to one or more polypeptides present in a biological sample that are derived from the Midkine precursor (Swiss-Prot P21741 (SEQ ID NO: 10)).

TABLE-US-00019 10203040 MQHRGFLLLTLLALLALTSAVAKKKDKVKKGGPGSECAEW 50607080 AWGPCTPSSKDCGVGFREGTCGAQTQRIRCRVPCNWKKEF 90100110120 GADCKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQETI 130140 RVTKPCTPKTKAKAKAKKGKGKD

[0097] The following domains have been identified in Midkine:

TABLE-US-00020 Residues Length Domain ID 1-20 20 Signal peptide 21-143 123 Midkine

[0098] As used herein, the term Serum amyloid P-component refers to one or more polypeptides present in a biological sample that are derived from the Serum amyloid P-component precursor (Swiss-Prot P02743 (SEQ ID NO: 11)).

TABLE-US-00021 10203040 MNKPLLWISVLTSLLEAFAHTDLSGKVFVFPRESVTDHVN 50607080 LITPLEKPLQNFTLCFRAYSDLSRAYSLFSYNTQGRDNEL 90100110120 LVYKERVGEYSLYIGRHKVTSKVIEKFPAPVHICVSWESS 130140150160 SGIAEFWINGTPLVKKGLRQGYFVEAQPKIVLGQEQDSYG 170180190200 GKFDRSQSFVGEIGDLYMWDSVLPPENILSAYQGTPLPAN 210220 ILDWQALNYEIRGYVIIKPLVWV

[0099] The following domains have been identified in Serum amyloid P-component:

TABLE-US-00022 Residues Length Domain ID 1-19 19 Signal peptide 20-223 204 Serum amyloid P-component 20-222 203 Serum amyloid P-component (1-203)

[0100] As used herein, the term relating a signal to the presence or amount of an analyte reflects this understanding. Assay signals are typically related to the presence or amount of an analyte through the use of a standard curve calculated using known concentrations of the analyte of interest. As the term is used herein, an assay is configured to detect an analyte if an assay can generate a detectable signal indicative of the presence or amount of a physiologically relevant concentration of the analyte. Because an antibody epitope is on the order of 8 amino acids, an immunoassay configured to detect a marker of interest will also detect polypeptides related to the marker sequence, so long as those polypeptides contain the epitope(s) necessary to bind to the antibody or antibodies used in the assay. The term related marker as used herein with regard to a biomarker such as one of the kidney injury markers described herein refers to one or more fragments, variants, etc., of a particular marker or its biosynthetic parent that may be detected as a surrogate for the marker itself or as independent biomarkers. The term also refers to one or more polypeptides present in a biological sample that are derived from the biomarker precursor complexed to additional species, such as binding proteins, receptors, heparin, lipids, sugars, etc.

[0101] The term positive going marker as that term is used herein refer to a marker that is determined to be elevated in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition. The term negative going marker as that term is used herein refer to a marker that is determined to be reduced in subjects suffering from a disease or condition, relative to subjects not suffering from that disease or condition.

[0102] The term subject as used herein refers to a human or non-human organism. Thus, the methods and compositions described herein are applicable to both human and veterinary disease. Further, while a subject is preferably a living organism, the invention described herein may be used in post-mortem analysis as well. Preferred subjects are humans, and most preferably patients, which as used herein refers to living humans that are receiving medical care for a disease or condition. This includes persons with no defined illness who are being investigated for signs of pathology.

[0103] Preferably, an analyte is measured in a sample. Such a sample may be obtained from a subject, or may be obtained from biological materials intended to be provided to the subject. For example, a sample may be obtained from a kidney being evaluated for possible transplantation into a subject, and an analyte measurement used to evaluate the kidney for preexisting damage. Preferred samples are body fluid samples.

[0104] The term body fluid sample as used herein refers to a sample of bodily fluid obtained for the purpose of diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient or transplant donor. In certain embodiments, such a sample may be obtained for the purpose of determining the outcome of an ongoing condition or the effect of a treatment regimen on a condition. Preferred body fluid samples include blood, serum, plasma, cerebrospinal fluid, urine, saliva, sputum, and pleural effusions. In addition, one of skill in the art would realize that certain body fluid samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

[0105] The term diagnosis as used herein refers to methods by which the skilled artisan can estimate and/or determine the probability (a likelihood) of whether or not a patient is suffering from a given disease or condition. In the case of the present invention, diagnosis includes using the results of an assay, most preferably an immunoassay, for a kidney injury marker of the present invention, optionally together with other clinical characteristics, to arrive at a diagnosis (that is, the occurrence or nonoccurrence) of an acute renal injury or ARF for the subject from which a sample was obtained and assayed. That such a diagnosis is determined is not meant to imply that the diagnosis is 100% accurate. Many biomarkers are indicative of multiple conditions. The skilled clinician does not use biomarker results in an informational vacuum, but rather test results are used together with other clinical indicia to arrive at a diagnosis. Thus, a measured biomarker level on one side of a predetermined diagnostic threshold indicates a greater likelihood of the occurrence of disease in the subject relative to a measured level on the other side of the predetermined diagnostic threshold.

[0106] Similarly, a prognostic risk signals a probability (a likelihood) that a given course or outcome will occur. A level or a change in level of a prognostic indicator, which in turn is associated with an increased probability of morbidity (e.g., worsening renal function, future ARF, or death) is referred to as being indicative of an increased likelihood of an adverse outcome in a patient.

[0107] Marker Assays

[0108] In general, immunoassays involve contacting a sample containing or suspected of containing a biomarker of interest with at least one antibody that specifically binds to the biomarker. A signal is then generated indicative of the presence or amount of complexes formed by the binding of polypeptides in the sample to the antibody. The signal is then related to the presence or amount of the biomarker in the sample. Numerous methods and devices are well known to the skilled artisan for the detection and analysis of biomarkers. See, e.g., U.S. Pat. Nos. 6,143,576; 6,113,855; 6,019,944; 5,985,579; 5,947,124; 5,939,272; 5,922,615; 5,885,527; 5,851,776; 5,824,799; 5,679,526; 5,525,524; and 5,480,792, and The Immunoassay Handbook, David Wild, ed. Stockton Press, New York, 1994, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims.

[0109] The assay devices and methods known in the art can utilize labeled molecules in various sandwich, competitive, or non-competitive assay formats, to generate a signal that is related to the presence or amount of the biomarker of interest. Suitable assay formats also include chromatographic, mass spectrographic, and protein blotting methods. Additionally, certain methods and devices, such as bio sensors and optical immunoassays, may be employed to determine the presence or amount of analytes without the need for a labeled molecule. See, e.g., U.S. Pat. Nos. 5,631,171; and 5,955,377, each of which is hereby incorporated by reference in its entirety, including all tables, figures and claims. One skilled in the art also recognizes that robotic instrumentation including but not limited to Beckman ACCESS, Abbott AXSYM, Roche ELECSYS, Dade Behring STRATUS systems are among the immunoassay analyzers that are capable of performing immunoassays. But any suitable immunoassay may be utilized, for example, enzyme-linked immunoassays (ELISA), radioimmunoassays (RIAs), competitive binding assays, and the like.

[0110] Antibodies or other polypeptides may be immobilized onto a variety of solid supports for use in assays. Solid phases that may be used to immobilize specific binding members include include those developed and/or used as solid phases in solid phase binding assays. Examples of suitable solid phases include membrane filters, cellulose-based papers, beads (including polymeric, latex and paramagnetic particles), glass, silicon wafers, microparticles, nanoparticles, TentaGels, AgroGels, PEGA gels, SPOCC gels, and multiple-well plates. An assay strip could be prepared by coating the antibody or a plurality of antibodies in an array on solid support. This strip could then be dipped into the test sample and then processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot. Antibodies or other polypeptides may be bound to specific zones of assay devices either by conjugating directly to an assay device surface, or by indirect binding. In an example of the later case, antibodies or other polypeptides may be immobilized on particles or other solid supports, and that solid support immobilized to the device surface.

[0111] Biological assays require methods for detection, and one of the most common methods for quantitation of results is to conjugate a detectable label to a protein or nucleic acid that has affinity for one of the components in the biological system being studied. Detectable labels may include molecules that are themselves detectable (e.g., fluorescent moieties, electrochemical labels, metal chelates, etc.) as well as molecules that may be indirectly detected by production of a detectable reaction product (e.g., enzymes such as horseradish peroxidase, alkaline phosphatase, etc.) or by a specific binding molecule which itself may be detectable (e.g., biotin, digoxigenin, maltose, oligohistidine, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, etc.).

[0112] Preparation of solid phases and detectable label conjugates often comprise the use of chemical cross-linkers. Cross-linking reagents contain at least two reactive groups, and are divided generally into homofunctional cross-linkers (containing identical reactive groups) and heterofunctional cross-linkers (containing non-identical reactive groups). Homobifunctional cross-linkers that couple through amines, sulfhydryls or react non-specifically are available from many commercial sources. Maleimides, alkyl and aryl halides, alpha-haloacyls and pyridyl disulfides are thiol reactive groups. Maleimides, alkyl and aryl halides, and alpha-haloacyls react with sulfhydryls to form thiol ether bonds, while pyridyl disulfides react with sulfhydryls to produce mixed disulfides. The pyridyl disulfide product is cleavable. Imidoesters are also very useful for protein-protein cross-links. A variety of heterobifunctional cross-linkers, each combining different attributes for successful conjugation, are commercially available.

[0113] In certain aspects, the present invention provides kits for the analysis of the described kidney injury markers. The kit comprises reagents for the analysis of at least one test sample which comprise at least one antibody that a kidney injury marker. The kit can also include devices and instructions for performing one or more of the diagnostic and/or prognostic correlations described herein. Preferred kits will comprise an antibody pair for performing a sandwich assay, or a labeled species for performing a competitive assay, for the analyte. Preferably, an antibody pair comprises a first antibody conjugated to a solid phase and a second antibody conjugated to a detectable label, wherein each of the first and second antibodies that bind a kidney injury marker. Most preferably each of the antibodies are monoclonal antibodies. The instructions for use of the kit and performing the correlations can be in the form of labeling, which refers to any written or recorded material that is attached to, or otherwise accompanies a kit at any time during its manufacture, transport, sale or use. For example, the term labeling encompasses advertising leaflets and brochures, packaging materials, instructions, audio or video cassettes, computer discs, as well as writing imprinted directly on kits.

[0114] Antibodies

[0115] The term antibody as used herein refers to a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope. See, e.g. Fundamental Immunology, 3rd Edition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes antigen-binding portions, i.e., antigen binding sites, (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term antibody.

[0116] Antibodies used in the immunoassays described herein preferably specifically bind to a kidney injury marker of the present invention. The term specifically binds is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody specifically binds if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). Preferably the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In preferred embodiments, Preferred antibodies bind with affinities of at least about 10.sup.7 M.sup.1, and preferably between about 10.sup.8 M.sup.1 to about 10.sup.9 M.sup.1, about 10.sup.9 M.sup.1 to about 10.sup.10 M.sup.1, or about 10.sup.10 M.sup.1 to about 10.sup.12M.sup.1 .

[0117] Affinity is calculated as K.sub.d=k.sub.off/k.sub.on (k.sub.off is the dissociation rate constant, K.sub.on is the association rate constant and K.sub.d is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labeled ligand at various concentrations (c). The data are graphed using the Scatchard equation: r/c=K(nr): where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n=number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art. See, e.g., van Erp et al., J. Immunoassay 12: 425-43, 1991; Nelson and Griswold, Comput. Methods Programs Biomed. 27: 65-8, 1988.

[0118] The term epitope refers to an antigenic determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.

[0119] Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-82, 1990; Devlin et al., Science 249, 404-6, 1990, Scott and Smith, Science 249, 386-88, 1990; and Ladner et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. See, e.g., U.S. Pat. No. 6,057,098, which is hereby incorporated in its entirety, including all tables, figures, and claims.

[0120] The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labeled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a color reaction will appear where antibody to the immobilized polypeptide(s) are present.

[0121] The antibodies so identified may then be further analyzed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.

Assay Correlations

[0122] The term correlating as used herein in reference to the use of biomarkers refers to comparing the presence or amount of the biomarker(s) in a patient to its presence or amount in persons known to suffer from, or known to be at risk of, a given condition; or in persons known to be free of a given condition. Often, this takes the form of comparing an assay result in the form of a biomarker concentration to a predetermined threshold selected to be indicative of the occurrence or nonoccurrence of a disease or the likelihood of some future outcome.

[0123] Selecting a diagnostic threshold involves, among other things, consideration of the probability of disease, distribution of true and false diagnoses at different test thresholds, and estimates of the consequences of treatment (or a failure to treat) based on the diagnosis. For example, when considering administering a specific therapy which is highly efficacious and has a low level of risk, few tests are needed because clinicians can accept substantial diagnostic uncertainty. On the other hand, in situations where treatment options are less effective and more risky, clinicians often need a higher degree of diagnostic certainty. Thus, cost/benefit analysis is involved in selecting a diagnostic threshold.

[0124] Suitable thresholds may be determined in a variety of ways. For example, one recommended diagnostic threshold for the diagnosis of acute myocardial infarction using cardiac troponin is the 975.sup.th percentile of the concentration seen in a normal population. Another method may be to look at serial samples from the same patient, where a prior baseline result is used to monitor for temporal changes in a biomarker level.

[0125] Population studies may also be used to select a decision threshold. Reciever Operating Characteristic (ROC) arose from the field of signal dectection therory developed during World War II for the analysis of radar images, and ROC analysis is often used to select a threshold able to best distinguish a diseased subpopulation from a nondiseased subpopulation. A false positive in this case occurs when the person tests positive, but actually does not have the disease. A false negative, on the other hand, occurs when the person tests negative, suggesting they are healthy, when they actually do have the disease. To draw a ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision threshold is varied continuously. Since TPR is equivalent with sensitivity and FPR is equal to 1-specificity, the ROC graph is sometimes called the sensitivity vs (1-specificity) plot. A perfect test will have an area under the ROC curve of 1.0; a random test will have an area of 0.5. A threshold is selected to provide an acceptable level of specificity and sensitivity.

[0126] In this context, diseased is meant to refer to a population having one characteristic (the presence of a disease or condition or the occurrence of some outcome) and nondiseased is meant to refer to a population lacking the characteristic. While a single decision threshold is the simplest application of such a method, multiple decision thresholds may be used. For example, below a first threshold, the absence of disease may be assigned with relatively high confidence, and above a second threshold the presence of disease may also be assigned with relatively high confidence. Between the two thresholds may be considered indeterminate. This is meant to be exemplary in nature only.

[0127] In addition to threshold comparisons, other methods for correlating assay results to a patient classification (occurrence or nonoccurrence of disease, likelihood of an outcome, etc.) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can produce probability values representing the degree to which a subject belongs to one classification out of a plurality of classifications.

[0128] Measures of test accuracy may be obtained as described in Fischer et al., Intensive Care Med. 29: 1043-51, 2003, and used to determine the effectiveness of a given biomarker. These measures include sensitivity and specificity, predictive values, likelihood ratios, diagnostic odds ratios, and ROC curve areas. The area under the curve (AUC) of a ROC plot is equal to the probability that a classifier will rank a randomly chosen positive instance higher than a randomly chosen negative one. The area under the ROC curve may be thought of as equivalent to the Mann-Whitney U test, which tests for the median difference between scores obtained in the two groups considered if the groups are of continuous data, or to the Wilcoxon test of ranks.

[0129] As discussed above, suitable tests may exhibit one or more of the following results on these various measures: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding sensitivity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, still more preferably at least 0.8, even more preferably at least 0.9 and most preferably at least 0.95, with a corresponding specificity greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, still more preferably at least 0.5, even more preferably 0.6, yet more preferably greater than 0.7, still more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; at least 75% sensitivity, combined with at least 75% specificity; a ROC curve area of greater than 0.5, preferably at least 0.6, more preferably 0.7, still more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or more or about 0.5 or less, more preferably at least about 3 or more or about 0.33 or less, still more preferably at least about 4 or more or about 0.25 or less, even more preferably at least about 5 or more or about 0.2 or less, and most preferably at least about 10 or more or about 0.1 or less; a positive likelihood ratio (calculated as sensitivity/(1-specificity)) of greater than 1, at least 2, more preferably at least 3, still more preferably at least 5, and most preferably at least 10; and or a negative likelihood ratio (calculated as (1-sensitivity)/specificity) of less than 1, less than or equal to 0.5, more preferably less than or equal to 0.3, and most preferably less than or equal to 0.1

[0130] Additional clinical indicia may be combined with the kidney injury marker assay result(s) of the present invention. These include other biomarkers related to renal status. Examples include the following, which recite the common biomarker name, followed by the Swiss-Prot entry number for that biomarker or its parent: Actin (P68133); Adenosine deaminase binding protein (DPP4, P27487); Alpha-1-acid glycoprotein 1 (P02763); Alpha-1-microglobulin (P02760); Albumin (P02768); Angiotensinogenase (Renin, P00797); Annexin A2 (P07355); Beta-glucuronidase (P08236); B-2-microglobulin (P61679); Beta-galactosidase (P16278); BMP-7 (P18075); Brain natriuretic peptide (proBNP, BNP-32, NTproBNP; P16860); Calcium-binding protein Beta (S100-beta, P04271); Carbonic anhydrase (Q16790); Casein Kinase 2 (P68400); Cathepsin B (P07858); Ceruloplasmin (P00450); Clusterin (P10909); Complement C3 (P01024); Cysteine-rich protein (CYR61, O00622); Cytochrome C (P99999); Epidermal growth factor (EGF, P01133); Endothelin-1 (P05305); Exosomal Fetuin-A (P02765); Fatty acid-binding protein, heart (FABP3, P05413); Fatty acid-binding protein, liver (P07148); Ferritin (light chain, P02793; heavy chain P02794); Fructose-1,6-biphosphatase (P09467); GRO-alpha (CXCL1, (P09341); Growth Hormone (P01241); Hepatocyte growth factor (P14210); Insulin-like growth factor I (P01343); Immunoglobulin G; Immunoglobulin Light Chains (Kappa and Lambda); Interferon gamma (P01308); Lysozyme (P61626); Interleukin-lalpha (P01583); Interleukin-2 (P60568); Interleukin-4 (P60568); Interleukin-9 (P15248); Interleukin-12p40 (P29460); Interleukin-13 (P35225); Interleukin-16 (Q14005); Ll cell adhesion molecule (P32004); Lactate dehydrogenase (P00338); Leucine Aminopeptidase (P28838); Meprin A-alpha subunit (Q16819); Meprin A-beta subunit (Q16820); Midkine (P21741); MIP2-alpha (CXCL2, P19875); MMP-2 (P08253); MMP-9 (P14780); Netrin-1 (O95631); Neutral endopeptidase (P08473); Osteopontin (P10451); Renal papillary antigen 1 (RPA1); Renal papillary antigen 2 (RPA2); Retinol binding protein (P09455); Ribonuclease; S100 calcium-binding protein A6 (P06703); Serum Amyloid P Component (P02743); Sodium/Hydrogen exchanger isoform (NHE3, P48764); Spermidine/spermine N1-acetyltransferase (P21673); TGF-Beta1 (P01137); Transferrin (P02787); Trefoil factor 3 (TFF3, Q07654); Toll-Like protein 4 (O00206); Total protein; Tubulointerstitial nephritis antigen (Q9UJW2); Uromodulin (Tamm-Horsfall protein, P07911).

[0131] For purposes of risk stratification, Adiponectin (Q15848); Alkaline phosphatase (P05186); Aminopeptidase N (P15144); CalbindinD28k (P05937); Cystatin C (P01034); 8 subunit of FIFO ATPase (P03928); Gamma-glutamyltransferase (P19440); GSTa (alpha-glutathione-S-transferase, P08263); GSTpi (Glutathione-S-transferase P; GST class-pi; P09211); IGFBP-1 (P08833); IGFBP-2 (P18065); IGFBP-6 (P24592); Integral membrane protein 1 (Itm1, P46977); Interleukin-6 (P05231); Interleukin-8 (P10145); Interleukin-18 (Q14116); IP-10 (10 kDa interferon-gamma-induced protein, P02778); IRPR (IFRD1, O00458); Isovaleryl-CoA dehydrogenase (IVD, P26440); I-TAC/CXCL11 (O14625); Keratin 19 (P08727); Kim-1 (Hepatitis A virus cellular receptor 1, O43656); L-arginine:glycine amidinotransferase (P50440); Leptin (P41159); Lipocalin2 (NGAL, P80188); MCP-1 (P13500); MIG (Gamma-interferon-induced monokine Q07325); MIP-la (P10147); MIP-3a (P78556); MIP-1beta (P13236); MIP-1d (Q16663); NAG (N-acetyl-beta-D-glucosaminidase, P54802); Organic ion transporter (OCT2, O15244); Osteoprotegerin (O14788); P8 protein (O60356); Plasminogen activator inhibitor 1 (PAI-1, P05121); ProANP(1-98) (P01160); Protein phosphatase 1-beta (PPI-beta, P62140); Rab GDI-beta (P50395); Renal kallikrein (Q86U61); RT1.B-1 (alpha) chain of the integral membrane protein (Q5Y7A8); Soluble tumor necrosis factor receptor superfamily member 1A (sTNFR-I, P19438); Soluble tumor necrosis factor receptor superfamily member 1B (sTNFR-II, P20333); Tissue inhibitor of metalloproteinases 3 (TIMP-3, P35625); uPAR (Q03405) may be combined with the kidney injury marker assay result(s) of the present invention.

[0132] Other clinical indicia which may be combined with the kidney injury marker assay result(s) of the present invention includes demographic information (e.g., weight, sex, age, race), medical history (e.g., family history, type of surgery, pre-existing disease such as aneurism, congestive heart failure, preeclampsia, eclampsia, diabetes mellitus, hypertension, coronary artery disease, proteinuria, renal insufficiency, or sepsis, type of toxin exposure such as NSAIDs, cyclosporines, tacrolimus, aminoglycosides, foscarnet, ethylene glycol, hemoglobin, myoglobin, ifosfamide, heavy metals, methotrexate, radiopaque contrast agents, or streptozotocin), clinical variables (e.g., blood pressure, temperature, respiration rate), risk scores (APACHE score, PREDICT score, TIMI Risk Score for UA/NSTEMI, Framingham Risk Score), a urine total protein measurement, a glomerular filtration rate, an estimated glomerular filtration rate, a urine production rate, a serum or plasma creatinine concentration, a renal papillary antigen 1 (RPA1) measurement; a renal papillary antigen 2 (RPA2) measurement; a urine creatinine concentration, a fractional excretion of sodium, a urine sodium concentration, a urine creatinine to serum or plasma creatinine ratio, a urine specific gravity, a urine osmolality, a urine urea nitrogen to plasma urea nitrogen ratio, a plasma BUN to creatnine ratio, and/or a renal failure index calculated as urine sodium/(urine creatinine/plasma creatinine). Other measures of renal function which may be combined with the kidney injury marker assay result(s) are described hereinafter and in Harrison's Principles of Internal Medicine, 17.sup.th Ed., McGraw Hill, New York, pages 1741-1830, and Current Medical Diagnosis & Treatment 2008, 47.sup.th Ed, McGraw Hill, New York, pages 785-815, each of which are hereby incorporated by reference in their entirety.

[0133] Combining assay results/clinical indicia in this manner can comprise the use of multivariate logistical regression, loglinear modeling, neural network analysis, n-of-m analysis, decision tree analysis, etc. This list is not meant to be limiting.

[0134] Diagnosis of Acute Renal Failure

[0135] As noted above, the terms acute renal (or kidney) injury and acute renal (or kidney) failure as used herein are defined in part in terms of changes in serum creatinine from a baseline value. Most definitions of ARF have common elements, including the use of serum creatinine and, often, urine output. Patients may present with renal dysfunction without an available baseline measure of renal function for use in this comparison. In such an event, one may estimate a baseline serum creatinine value by assuming the patient initially had a normal GFR. Glomerular filtration rate (GFR) is the volume of fluid filtered from the renal (kidney) glomerular capillaries into the Bowman's capsule per unit time. Glomerular filtration rate (GFR) can be calculated by measuring any chemical that has a steady level in the blood, and is freely filtered but neither reabsorbed nor secreted by the kidneys. GFR is typically expressed in units of ml/min:

[00001]$\mathrm{GFR}=\frac{\mathrm{Urine}.Math..Math.\mathrm{Concentration}\mathrm{Urine}.Math..Math.\mathrm{Flow}}{\mathrm{Plasma}.Math..Math.\mathrm{Concentration}}$

[0136] By normalizing the GFR to the body surface area, a GFR of approximately 75-100 ml/min per 1.73 m.sup.2 can be assumed. The rate therefore measured is the quantity of the substance in the urine that originated from a calculable volume of blood.

[0137] There are several different techniques used to calculate or estimate the glomerular filtration rate (GFR or eGFR). In clinical practice, however, creatinine clearance is used to measure GFR. Creatinine is produced naturally by the body (creatinine is a metabolite of creatine, which is found in muscle). It is freely filtered by the glomerulus, but also actively secreted by the renal tubules in very small amounts such that creatinine clearance overestimates actual GFR by 10-20%. This margin of error is acceptable considering the ease with which creatinine clearance is measured.

[0138] Creatinine clearance (CCr) can be calculated if values for creatinine's urine concentration (U.sub.Cr), urine flow rate (V), and creatinine's plasma concentration (P.sub.Cr) are known. Since the product of urine concentration and urine flow rate yields creatinine's excretion rate, creatinine clearance is also said to be its excretion rate (U.sub.CrV) divided by its plasma concentration. This is commonly represented mathematically as:

[00002]$C_{\mathrm{Cr}}=\frac{U_{\mathrm{Cr}}V}{P_{\mathrm{Cr}}}$

[0139] Commonly a 24 hour urine collection is undertaken, from empty-bladder one morning to the contents of the bladder the following morning, with a comparative blood test then taken:

[00003]$.Math.C_{\mathrm{Cr}}=\frac{U_{\mathrm{Cr}}24.Math.\text{-}.Math.\mathrm{hour}.Math..Math.\mathrm{volume}}{P_{\mathrm{Cr}}2460.Math..Math.\mathrm{mins}}$

[0140] To allow comparison of results between people of different sizes, the CCr is often corrected for the body surface area (BSA) and expressed compared to the average sized man as ml/min/1.73 m2. While most adults have a BSA that approaches 1.7 (1.6-1.9), extremely obese or slim patients should have their CCr corrected for their actual BSA:

[00004]$C_{\mathrm{Cr}.Math.\text{-}.Math.\mathrm{corrected}}=\frac{C_{\mathrm{Cr}}1.73}{\mathrm{BSA}}$

[0141] The accuracy of a creatinine clearance measurement (even when collection is complete) is limited because as glomerular filtration rate (GFR) falls creatinine secretion is increased, and thus the rise in serum creatinine is less. Thus, creatinine excretion is much greater than the filtered load, resulting in a potentially large overestimation of the GFR (as much as a twofold difference). However, for clinical purposes it is important to determine whether renal function is stable or getting worse or better. This is often determined by monitoring serum creatinine alone. Like creatinine clearance, the serum creatinine will not be an accurate reflection of GFR in the non-steady-state condition of ARF. Nonetheless, the degree to which serum creatinine changes from baseline will reflect the change in GFR. Serum creatinine is readily and easily measured and it is specific for renal function.

[0142] For purposes of determining urine output on a Urine output on a mL/kg/hr basis, hourly urine collection and measurement is adequate. In the case where, for example, only a cumulative 24-h output was available and no patient weights are provided, minor modifications of the RIFLE urine output criteria have been described. For example, Bagshaw et al., Nephrol. Dial. Transplant. 23: 1203-1210, 2008, assumes an average patient weight of 70 kg, and patients are assigned a RIFLE classification based on the following: <35 mL/h (Risk), <21 mL/h (Injury) or <4 mL/h (Failure).

[0143] Selecting a Treatment Regimen

[0144] Once a diagnosis is obtained, the clinician can readily select a treatment regimen that is compatible with the diagnosis, such as initiating renal replacement therapy, withdrawing delivery of compounds that are known to be damaging to the kidney, kidney transplantation, delaying or avoiding procedures that are known to be damaging to the kidney, modifying diuretic administration, initiating goal directed therapy, etc. The skilled artisan is aware of appropriate treatments for numerous diseases discussed in relation to the methods of diagnosis described herein. See, e.g., Merck Manual of Diagnosis and Therapy, 17th Ed. Merck Research Laboratories, Whitehouse Station, N.J., 1999. In addition, since the methods and compositions described herein provide prognostic information, the markers of the present invention may be used to monitor a course of treatment. For example, improved or worsened prognostic state may indicate that a particular treatment is or is not efficacious.

[0145] One skilled in the art readily appreciates that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

EXAMPLE 1

Contrast-Induced Nephropathy Sample Collection

[0146] The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after receiving intravascular contrast media. Approximately 250 adults undergoing radiographic/angiographic procedures involving intravascular administration of iodinated contrast media are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

[0147] males and females 18 years of age or older; [0148] undergoing a radiographic/angiographic procedure (such as a CT scan or coronary intervention) involving the intravascular administration of contrast media; [0149] expected to be hospitalized for at least 48 hours after contrast administration. [0150] able and willing to provide written informed consent for study participation and to comply with all study procedures.

Exclusion Criteria

[0151] renal transplant recipients; [0152] acutely worsening renal function prior to the contrast procedure; [0153] already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; [0154] expected to undergo a major surgical procedure (such as involving cardiopulmonary bypass) or an additional imaging procedure with contrast media with significant risk for further renal insult within the 48 hrs following contrast administration; [0155] participation in an interventional clinical study with an experimental therapy within the previous 30 days; [0156] known infection with human immunodeficiency virus (HIV) or a hepatitis virus.

[0157] Immediately prior to the first contrast administration (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL) and a urine sample (10 mL) are collected from each patient. Blood and urine samples are then collected at 4 (0.5), 8 (1), 24 (2) 48 (2), and 72 (2) hrs following the last administration of contrast media during the index contrast procedure. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

[0158] Serum creatinine is assessed at the site immediately prior to the first contrast administration (after any pre-procedure hydration) and at 4 (0.5), 8 (1), 24 (2) and 48 (2)), and 72 (2) hours following the last administration of contrast (ideally at the same time as the study samples are obtained). In addition, each patient's status is evaluated through day 30 with regard to additional serum and urine creatinine measurements, a need for dialysis, hospitalization status, and adverse clinical outcomes (including mortality).

[0159] Prior to contrast administration, each patient is assigned a risk based on the following assessment: systolic blood pressure <80 mm Hg=5 points; intra-arterial balloon pump=5 points; congestive heart failure (Class III-IV or history of pulmonary edema)=5 points; age >75 yrs=4 points; hematocrit level <39% for men, <35% for women=3 points; diabetes=3 points; contrast media volume=1 point for each 100 mL; serum creatinine level >1.5 g/dL=4 points OR estimated GFR 40-60 mL/min/1.73 m.sup.2=2 points, 20-40 mL/min/1.73 m.sup.2=4 points, <20 mL/min/1.73 m.sup.2=6 points. The risks assigned are as follows: risk for CIN and dialysis: 5 or less total points=risk of CIN7.5%, risk of dialysis0.04%; 6-10 total points=risk of CIN14%, risk of dialysis0.12%; 11-16 total points=risk of CIN26.1%, risk of dialysis1.09%; >16 total points=risk of CIN=57.3%, risk of dialysis12.8%.

EXAMPLE 2

Cardiac Surgery Sample Collection

[0160] The objective of this sample collection study is to collect samples of plasma and urine and clinical data from patients before and after undergoing cardiovascular surgery, a procedure known to be potentially damaging to kidney function. Approximately 900 adults undergoing such surgery are enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

[0161] males and females 18 years of age or older; [0162] undergoing cardiovascular surgery; [0163] Toronto/Ottawa Predictive Risk Index for Renal Replacement risk score of at least 2 (Wijeysundera et al., JAMA 297: 1801-9, 2007); and [0164] able and willing to provide written informed consent for study participation and to comply with all study procedures.

Exclusion Criteria

[0165] known pregnancy; [0166] previous renal transplantation; [0167] acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); [0168] already receiving dialysis (either acute or chronic) or in imminent need of dialysis at enrollment; [0169] currently enrolled in another clinical study or expected to be enrolled in another clinical study within 7 days of cardiac surgery that involves drug infusion or a therapeutic intervention for AKI; [0170] known infection with human immunodeficiency virus (HIV) or a hepatitis virus.

[0171] Within 3 hours prior to the first incision (and after any pre-procedure hydration), an EDTA anti-coagulated blood sample (10 mL), whole blood (3 mL), and a urine sample (35 mL) are collected from each patient. Blood and urine samples are then collected at 3 (0.5), 6 (0.5), 12 (1), 24 (2) and 48 (2) hrs following the procedure and then daily on days 3 through 7 if the subject remains in the hospital. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

EXAMPLE 3

Acutely Ill Subject Sample Collection

[0172] The objective of this study is to collect samples from acutely ill patients. Approximately 900 adults expected to be in the ICU for at least 48 hours will be enrolled. To be enrolled in the study, each patient must meet all of the following inclusion criteria and none of the following exclusion criteria:

Inclusion Criteria

[0173] males and females 18 years of age or older; [0174] Study population 1: approximately 300 patients that have at least one of: [0175] shock (SBP <90 mmHg and/or need for vasopressor support to maintain MAP >60 mmHg and/or documented drop in SBP of at least 40 mmHg); and [0176] sepsis; [0177] Study population 2: approximately 300 patients that have at least one of: [0178] IV antibiotics ordered in computerized physician order entry (CPOE) within 24 hours of enrollment; [0179] contrast media exposure within 24 hours of enrollment; [0180] increased Intra-Abdominal Pressure with acute decompensated heart failure; and [0181] severe trauma as the primary reason for ICU admission and likely to be hospitalized in the ICU for 48 hours after enrollment; [0182] Study population 3: approximately 300 patients [0183] expected to be hospitalized through acute care setting (ICU or ED) with a known risk factor for acute renal injury (e.g. sepsis, hypotension/shock (Shock=systolic BP <90 mmHg and/or the need for vasopressor support to maintain a MAP >60 mmHg and/or a documented drop in SBP>40 mmHg), major trauma, hemorrhage, or major surgery); and/or expected to be hospitalized to the ICU for at least 24 hours after enrollment.

Exclusion Criteria

[0184] known pregnancy; [0185] institutionalized individuals; [0186] previous renal transplantation; [0187] known acutely worsening renal function prior to enrollment (e.g., any category of RIFLE criteria); [0188] received dialysis (either acute or chronic) within 5 days prior to enrollment or in imminent need of dialysis at the time of enrollment; [0189] known infection with human immunodeficiency virus (HIV) or a hepatitis virus; [0190] meets only the SBP <90 mmHg inclusion criterion set forth above, and does not have shock in the attending physician's or principal investigator's opinion.

[0191] After providing informed consent, an EDTA anti-coagulated blood sample (10 mL) and a urine sample (25-30 mL) are collected from each patient. Blood and urine samples are then collected at 4 (0.5) and 8 (1) hours after contrast administration (if applicable); at 12 (1), 24 (2), and 48 (2) hours after enrollment, and thereafter daily up to day 7 to day 14 while the subject is hospitalized. Blood is collected via direct venipuncture or via other available venous access, such as an existing femoral sheath, central venous line, peripheral intravenous line or hep-lock. These study blood samples are processed to plasma at the clinical site, frozen and shipped to Astute Medical, Inc., San Diego, Calif. The study urine samples are frozen and shipped to Astute Medical, Inc.

EXAMPLE 4

Immunoassay Format

[0192] Analytes are is measured using standard sandwich enzyme immunoassay techniques. A first antibody which binds the analyte is immobilized in wells of a 96 well polystyrene microplate. Analyte standards and test samples are pipetted into the appropriate wells and any analyte present is bound by the immobilized antibody. After washing away any unbound substances, a horseradish peroxidase-conjugated second antibody which binds the analyte is added to the wells, thereby forming sandwich complexes with the analyte (if present) and the first antibody. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution comprising tetramethylbenzidine and hydrogen peroxide is added to the wells. Color develops in proportion to the amount of analyte present in the sample. The color development is stopped and the intensity of the color is measured at 540 nm or 570 nm. An analyte concentration is assigned to the test sample by comparison to a standard curve determined from the analyte standards.

[0193] Concentrations are expressed in the following examples as follows: Clusterin ng/mL, Heart-type fatty acid binding protein ng/mL, Hepatocyte growth factor pg/mL, Interferon gamma pg/mL, Interleukin-12 subunit beta pg/mL, Interleukin-16 pg/mL, Interleukin-2 pg/mL, 72 kDa type IV collagenase ng/mL, Matrix metalloproteinase-9 pg/mL (urine) and ng/mL (plasma), Midkine ng/mL, and Serum amyloid P-component ng/mL.

EXAMPLE 5

Apparently Healthy Donor and Chronic Disease Patient Samples

[0194] Human urine samples from donors with no known chronic or acute disease (Apparently Healthy Donors) were purchased from two vendors (Golden West Biologicals, Inc., 27625 Commerce Center Dr., Temecula, Calif. 92590 and Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, Va. 23454). The urine samples were shipped and stored frozen at less than 20 C. The vendors supplied demographic information for the individual donors including gender, race (Black/White), smoking status and age.

[0195] Human urine samples from donors with various chronic diseases (Chronic Disease Patients) including congestive heart failure, coronary artery disease, chronic kidney disease, chronic obstructive pulmonary disease, diabetes mellitus and hypertension were purchased from Virginia Medical Research, Inc., 915 First Colonial Rd., Virginia Beach, Va. 23454. The urine samples were shipped and stored frozen at less than 20 degrees centigrade. The vendor provided a case report form for each individual donor with age, gender, race (Black/White), smoking status and alcohol use, height, weight, chronic disease(s) diagnosis, current medications and previous surgeries.

EXAMPLE 6

Kidney Injury Markers for Evaluating Renal Status in Patients at RIFLE Stage 0

[0196] Patients from the intensive care unit (ICU) were classified by kidney status as non-injury (0), risk of injury (R), injury (I), and failure (F) according to the maximum stage reached within 7 days of enrollment as determined by the RIFLE criteria.

[0197] Two cohorts were defined as (Cohort 1) patients that did not progress beyond stage 0, and (Cohort 2) patients that reached stage R, I, or F within 10 days. To address normal marker fluctuations that occur within patients at the ICU and thereby assess utility for monitoring AKI status, marker levels were measured in urine samples collected for Cohort 1. Marker concentrations were measured in urine samples collected from a subject at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. In the following tables, the time prior max stage represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/12 hours. For example, 24 hr prior for this example (0 vs R, I, F) would mean 24 hr (+/12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).

[0198] Each marker was measured by standard immunoassay methods using commercially available assay reagents. A receiver operating characteristic (ROC) curve was generated for each marker and the area under each ROC curve (AUC) was determined. Patients in Cohort 2 were also separated according to the reason for adjudication to stage R, I, or F as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output. That is, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage was used.

[0199] The ability to distinguish cohort 1 (subjects remaining in RIFLE 0) from Cohort 2 (subjects progressing to RIFLE R, I or F) was determined using ROC analysis. SE is the standard error of the AUC, n is the number of sample or individual patients (pts, as indicated). Standard errors were calculated as described in Hanley, J. A., and McNeil, B. J., The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology (1982) 143: 29-36; p values were calculated with a two-tailed Z-test. An AUC <0.5 is indicative of a negative going marker for the comparison, and an AUC >0.5 is indicative of a positive going marker for the comparison.

[0200] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0201] The results of these three analyses for various markers of the present invention are presented in FIG. 1.

EXAMPLE 7

Kidney Injury Markers for Evaluating Renal Status in Patients at RIFLE Stages 0 and R

[0202] Patients were classified and analyzed as described in Example 6. However, patients that reached stage R but did not progress to stage I or F were grouped with patients from non-injury stage 0 in Cohort 1. Cohort 2 in this example included only patients that progressed to stage I or F. Marker concentrations in urine samples were included for Cohort 1. Marker concentrations in urine samples collected within 0, 24, and 48 hours of reaching stage I or F were included for Cohort 2.

[0203] The ability to distinguish cohort 1 (subjects remaining in RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.

[0204] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0205] The results of these three analyses for various markers of the present invention are presented in FIG. 2.

EXAMPLE 8

Kidney Injury Markers for Evaluating Renal Status in Oatients Progressing from Stage R to Stages I and F

[0206] Patients were classified and analyzed as described in Example 6, but only those patients that reached Stage R were included in this example. Cohort 1 contained patients that reached stage R but did not progress to stage I or F within 10 days, and Cohort 2 included only patients that progressed to stage I or F. Marker concentrations in urine samples collected within 12 hours of reaching stage R were included in the analysis for both Cohort 1 and 2.

[0207] The ability to distinguish cohort 1 (subjects remaining in RIFLE R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.

[0208] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0209] The results of these three analyses for various markers of the present invention are presented in FIG. 3.

EXAMPLE 9

Kidney Injury Markers for Evaluating Renal Status in Patients at RIFLE Stage 0

[0210] Patients were classified and analyzed as described in Example 6. However, patients that reached stage R or I but did not progress to stage F were eliminated from the analysis. Patients from non-injury stage 0 are included in Cohort 1. Cohort 2 in this example included only patients that progressed to stage F. The maximum marker concentrations in urine samples were included for each patient in Cohort 1. The maximum marker concentrations in urine samples collected within 0, 24, and 48 hours of reaching stage F were included for each patient in Cohort 2.

[0211] The ability to distinguish cohort 1 (subjects remaining in RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.

[0212] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0213] The results of these three analyses for various markers of the present invention are presented in FIG. 4.

Example 10

Kidney Injury Markers for Evaluating Renal Status in Patients at RIFLE Stage 0

[0214] Patients from the intensive care unit (ICU) were classified by kidney status as non-injury (0), risk of injury (R), injury (I), and failure (F) according to the maximum stage reached within 7 days of enrollment as determined by the RIFLE criteria.

[0215] Two cohorts were defined as (Cohort 1) patients that did not progress beyond stage 0, and (Cohort 2) patients that reached stage R, I, or F within 10 days. To address normal marker fluctuations that occur within patients at the ICU and thereby assess utility for monitoring AKI status, marker levels were measured in the plasma component of blood samples collected for Cohort 1. Marker concentrations were measured in the plasma component of blood samples collected from a subject at 0, 24 hours, and 48 hours prior to reaching stage R, I or F in Cohort 2. In the following tables, the time prior max stage represents the time at which a sample is collected, relative to the time a particular patient reaches the lowest disease stage as defined for that cohort, binned into three groups which are +/12 hours. For example, 24 hr prior for this example (0 vs R, I, F) would mean 24 hr (+/12 hours) prior to reaching stage R (or I if no sample at R, or F if no sample at R or I).

[0216] Each marker was measured by standard immunoassay methods using commercially available assay reagents. A receiver operating characteristic (ROC) curve was generated for each marker and the area under each ROC curve (AUC) was determined. Patients in Cohort 2 were also separated according to the reason for adjudication to stage R, I, or F as being based on serum creatinine measurements (sCr), being based on urine output (UO), or being based on either serum creatinine measurements or urine output. That is, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of urine output; for those patients adjudicated to stage R, I, or F on the basis of urine output alone, the stage 0 cohort may have included patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements; and for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the stage 0 cohort contains only patients in stage 0 for both serum creatinine measurements and urine output. Also, for those patients adjudicated to stage R, I, or F on the basis of serum creatinine measurements or urine output, the adjudication method which yielded the most severe RIFLE stage was used.

[0217] The ability to distinguish cohort 1 (subjects remaining in RIFLE 0) from Cohort 2 (subjects progressing to RIFLE R, I or F) was determined using ROC analysis. SE is the standard error of the AUC, n is the number of sample or individual patients (pts, as indicated). Standard errors were calculated as described in Hanley, J. A., and McNeil, B. J., The meaning and use of the area under a receiver operating characteristic (ROC) curve. Radiology (1982) 143: 29-36; p values were calculated with a two-tailed Z-test. An AUC <0.5 is indicative of a negative going marker for the comparison, and an AUC >0.5 is indicative of a positive going marker for the comparison.

[0218] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0219] The results of these three analyses for various markers of the present invention are presented in FIG. 5.

EXAMPLE 11

Kidney Injury Markers for Evaluating Renal Status in Patients at RIFLE Stages 0 and R

[0220] Patients were classified and analyzed as described in Example 10. However, patients that reached stage R but did not progress to stage I or F were grouped with patients from non-injury stage 0 in Cohort 1. Cohort 2 in this example included only patients that progressed to stage I or F. Marker concentrations in the plasma component of blood samples were included for Cohort 1. Marker concentrations in the plasma component of blood samples collected within 0, 24, and 48 hours of reaching stage I or F were included for Cohort 2.

[0221] The ability to distinguish cohort 1 (subjects remaining in RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.

[0222] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0223] The results of these three analyses for various markers of the present invention are presented in FIG. 6.

EXAMPLE 12

Kidney Injury Markers for Evaluating Renal Status in Patients Progressing from Stage R to Stages I and F

[0224] Patients were classified and analyzed as described in Example 10, but only those patients that reached Stage R were included in this example. Cohort 1 contained patients that reached stage R but did not progress to stage I or F within 10 days, and Cohort 2 included only patients that progressed to stage I or F. Marker concentrations in the plasma component of blood samples collected within 12 hours of reaching stage R were included in the analysis for both Cohort 1 and 2.

[0225] The ability to distinguish cohort 1 (subjects remaining in RIFLE R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.

[0226] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0227] The results of these three analyses for various markers of the present invention are presented in FIG. 7.

EXAMPLE 13

Kidney Injury Markers for Evaluating Renal Status in Patients at RIFLE Stage 0

[0228] Patients were classified and analyzed as described in Example 10. However, patients that reached stage R or I but did not progress to stage F were eliminated from the analysis. Patients from non-injury stage 0 are included in Cohort 1. Cohort 2 in this example included only patients that progressed to stage F. The maximum marker concentrations in the plasma component of blood samples were included from each patient in Cohort 1. The maximum marker concentrations in the plasma component of blood samples collected within 0, 24, and 48 hours of reaching stage F were included from each patient in Cohort 2.

[0229] The ability to distinguish cohort 1 (subjects remaining in RIFLE 0 or R) from Cohort 2 (subjects progressing to RIFLE I or F) was determined using ROC analysis.

[0230] Various threshold (or cutoff) concentrations were selected, and the associated sensitivity and specificity for distinguishing cohort 1 from cohort 2 were determined. OR is the odds ratio calculated for the particular cutoff concentration, and 95% CI is the confidence interval for the odds ratio.

[0231] The results of these three analyses for various markers of the present invention are presented in FIG. 8.

[0232] While the invention has been described and exemplified in sufficient detail for those skilled in this art to make and use it, various alternatives, modifications, and improvements should be apparent without departing from the spirit and scope of the invention. The examples provided herein are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Modifications therein and other uses will occur to those skilled in the art. These modifications are encompassed within the spirit of the invention and are defined by the scope of the claims.

[0233] It will be readily apparent to a person skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.

[0234] All patents and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0235] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms comprising, consisting essentially of and consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0236] Other embodiments are set forth within the following claims.