Combination) of) Biomarkers) for) Diagnosis) of) Acute) Kidney) Injury) after) Cardiopulmonary)Bypass) Prowle,(John(R1,2,#((Calzavacca,(Paolo1((Licari,(Elisa1((Ligabo,(E(Valentina1( Echeverri,(Jorge(E1((Bagshaw,(Sean(M3(((HaaseBFielitz,(Anja4((Haase,(Michael4(( Ostland,( Vaughn5( ( Noiri,( Eisei6( ( ( Westerman,( Mark5( ( Devarajan,( Prasad7( ( Bellomo,( Rinaldo1!! ( ( 1.( Department(of(Intensive(Care,(Austin(Hospital,(Melbourne,(Australia.( 2.( Present( address:( Adult( Critical( Care( Unit,( The( Royal( London( Hospital,( Barts( Health(NHS(Trust,(London,(UK.( 3.( Division( of( Critical( Care( Medicine,( Faculty( of( Medicine( and( Dentistry,( University(of(Alberta,(Edmonton,(Alberta,(Canada.( 4.( Department(of(Nephrology(and(Hypertension(&(Diabetes(and(Endocrinology,( OttoBvonBGuerickeBUniversity,(Magdeburg,(Germany.( 5.( Intrinsic(LifeSciences(LLC,(La(Jolla,(CA,(USA.( 6.( Department( of( Nephrology( and( Endocrinology,( Department( of( Hemodialysis( and(Apheresis,(University(Hospital,(The(University(of(Tokyo,(Tokyo,(Japan.( 7.( Cincinnati(Children's(Hospital(Medical(Center,(Cincinnati,(OH,(USA( #( Author(responsible(for(statistical(design(and(analysis( ( Address)correspondence)to:( Prof.(Rinaldo(Bellomo,(( Department(of(Intensive(Care,(Austin(Health,(( 145(Studley(Road,(Heidelberg,(( Victoria(3084,(Australia.( Tel:(61B3B9496(5992;(Fax:(61B3B9496(3932(((((( rinaldo.bellomo@austin.org.au( ( Funding)&)Disclosures) Funding:(Austin(Health(ICU(Research(Fund.((Dr(M.W.(is(a(shareholder,(President(and( CEO( of( Intrinsic( LifeSciences,( developer( and( distributor( of( an( ELISA( assay( for( hepcidin.(Dr(V.A.(is(a(shareholder(and(officer(of(Intrinsic(LifeSciences.(Dr(J.R.P.(and( Professor(R.B.(are(named(in(a(US(patent(in(conjunction(with(Dr(M.W.(Prof(PD(is(a(coB inventor(on(NGAL(patents(for(the(diagnosis(of(acute(kidney(injury.(SB,(PD,(JP(and(RB( have( received( consulting/speaking( fees( from( Alere( Inc.( RB( has( received( consulting( fees(from(Abbot(Diagnostics(Inc.( ) Keywords:) Acute( Kidney( Injury,( Biomarkers,( Cardiac( Bypass,( neutrophil( gelatinase( associated( lipocalin,( glutathioneBSBtransferase,( liver( fatty( acid( binding( protein,( hepcidin( ( ! 2! ! ( Abstract) Novel( AKI( biomarkers( offer( promise( of( earlier( diagnosis( and( risk( stratification,( but( have( yet( to( find( widespread( clinical( application.( We( measured( urinary( α( and( π( glutathione( SBtransferases( (αBGST( and( πBGST),( urinary( LBtype( fatty( acidBbinding( protein( (LBFABP),( urinary( neutrophil( gelatinaseBassociated( lipocalin( (NGAL),( urinary( hepcidin( and( serum( cystatin( c( (CysC)( before( surgery,( postBoperatively( and( at( 24h( after(surgery(in(93(high(risk(patient(undergoing(CPB(and(assessed(the(ability(of(these( biomarkers( alone( and( in( combination( to( predict( RIFLEBR( defined( AKI( in( the( first( 5( postBoperative( days.( TwentyBfive( patients( developed( AKI.( πBGST( (ROCAUC=0.75),( lower(urine(Hepcidin:Creatine(ratio(at(24h((0.77),(greater(urine(NGAL:Cr(ratio(postB op( (0.73),( and( greater( serum( CysC( at( 24h( (0.72)( best( predicted( AKI.(Linear( combinations( with( significant( improvement( in( AUC( were:( Hepcidin:Cr( 24h( +( postB operative( πBGST( ( (AUC=0.86,( p=0.01),( Hepcidin:Cr( 24h( +( NGAL:Cr( post( op( (0.84,( p=0.03)( and( CysC( 24h( +( postBoperative( πBGST( (0.83,( p=0.03),( notably( these( significant( biomarkers( combinations( all( involved( a( tubular( injury( ( and( a( glomerular( filtration( biomarker.( Despite( statistical( significance( in( ROC( analysis,( when( assessed( by(ability(to(define(patients(to(two(groups(at(high(and(low(risk(of(AKI,(combinations( failed( to( significantly( improve( classification( of( risk( compared( to( the( best( single( biomarkers.( In( an( alternative( approach( using( Classification( and( Regression( Tree( (CART)( analysis( a( model( involving( NGAL:Cr( measurement( postBop( followed( by( Hepcidin:Cr( at( 24h( was( developed( which( identified( high,( intermediate( and( low( risk( groups( for( AKI.( Regression( tree( analysis( has( the( potential( produce( models( with( greater(clinical(utility(than(single(combined(scores.! ) ! 3! ! ( Introduction) Acute( kidney( injury( (AKI)( is( associated( with( increased( morbidity( and( mortality1( and( is( of( particular( clinical( significance( in( the( intensive( care( unit.( Conventional(diagnosis(remains(dependent(on(measurement(of(plasma(creatinine.2(( However,( plasma( creatinine( has( many( limitations,( in( particular,( diagnosis( is( often( delayed(and(imprecise(in(the(setting(of(critical(illness.3,4(Delay(in(diagnosis(of(AKI(may( preclude( early( and( effective( intervention.( A( number( of( early( markers( of( AKI( have( been(identified(by(proteomic(analysis(of(plasma(and(urine(from(patients(who(go(on( to( develop( AKI.5,6( These( biomarkers( may( accelerate( AKI( diagnosis( and( provide( insights( into( its( pathogenesis.( ( However,( while( performance( can( be( very( good( in( homogeneous( patient( groups( with( predictable( timing( of( renal( injury,( such( as( pediatric( cardiopulmonary( bypass( –( CPB,7( diagnostic( utility( can( be( less( good( in( a( mixed(populations(of(adults(with(chronic(kidney(disease,8(or(when(renal(insults(vary( in(timing,(nature(and(severity.9((Furthermore,(biomarkers(of(AKI(are(heterogeneous( molecules.( Some( appear( produced( directly( within( the( kidney( as( a( result( of( tubular( injury(or(stress((urinary(neutrophil(gelatinaseBassociated(lipocalin(–(NGAL10);(others( may(appear(in(the(urine(due(to(failure(of(proximal(tubular(uptake((urinary(cystatin(c( 11 )( while( others( again( may( simply( be( more( precise( markers( of( alteration( in( glomerular(filtration(rate((serum(cystatin(c(–(CysC(11).(Finally,(some(biomarkers(of(AKI( may(also(be(markers(of(systemic(inflammation,(complicating(their(interpretation;(for( instance(the(major(source(of(plasma(NGAL(may(be(neutrophils(in(normal(conditions( and(during(systemic(infection,(but(can(be(kidney(derived(during(AKI.12( ! 4! ! ( Thus,( markers( of( tubular( injury( may( specifically( detect( some( patients( with( early( AKI( and( relatively( preserved( GFR,( while( markers( of( GFR( changes( may( detect( hemodynamically(mediated(decreases(in(glomerular(filtration(rate(in(the(absence(of( significant( tubular( injury.( Logically,( therefore,( examination( of( multiple( biomarkers( may( inform( us( not( only( of( the( likely( occurrence( of( AKI,( but( its( nature,( extent( and( pathogenesis.(( Accordingly,(we(examined(the(diagnostic(utility(of(a(panel(of(six(biomarkers( measured( at( baseline( and( at( two( postBoperative( timeBpoints( in( a( singleBcentre( randomized( pilot( controlled( trial( conducted( in( higherBrisk( patients( undergoing( surgery( involving( CPB.13( We( hypothesized( that( some( combinations( of( biomarkers( would( provide( better( diagnostic( accuracy( than( either( alone( and( that( these( combinations(might(involve(biomarkers(that(reflect(differing(aspects(and(phases(of( the(pathogenesis(of(AKI(in(this(population.( ( Materials)&)Methods) Patient!Population! Samples( were( obtained( from( 93( patients( enrolled( in( the( Cardiopulmonary(( bypass,(REnal(injury(and(Atorvastatin(Trial((CREAT—Clinicaltrials.Gov((Nct00910221),( a(singleBcentre(randomized(controlled(trial(of(periB(and((postBoperative(atorvastatin( versus(placebo(for(the(prevention(of(AKI(in(higher(risk(patients(undergoing(CPB.(This( study(was(approved(by(Human(Research(Ethics(Committee(of(the(Austin(Hospital.(In( this( study,( statin( therapy( did( not( influence( the( incidence( of( postBoperative( AKI( or( biomarkers( of( renal( injury.13,14( ( Renal( function( was( monitored( for( 5( days( postB ! 5! ! ( operatively.( ( Samples( of( serum( and( urine( were( obtained( preBoperatively,( immediately(on(return(to(ICU(after(surgery((mean(time(of(4.50(h;(3.58–9.13(h(after( CPB)( and( 24( h( after( surgery.( Of( the( 100( patients( randomized,( 5( withdrew( or( had( surgery( rescheduled( and( in( two( patients,( full( sets( of( serum( and( urine( were( not( collected( for( clinical( reasons( so( that( 93( complete( sets( of( urine( samples( were( available(for(analysis.((((Demographic(and(clinical(data(were(collected(preBoperatively( and(over(the(first(24(h(postBsurgery.(Serum(creatinine(was(measured(preBoperatively( and(daily(for(5(days(after(surgery.((AKI(was(defined(using(the(creatinine(criteria(of(the( Risk,( Injury,( Failure,( Loss,( EndBstage( kidney( disease( (RIFLE)( consensus( definition( of( AKI.15( ( As( data( on( hourly( urine( output( were( not( available( for( the( complete( study( period,(and(in(keeping(with(previous(studies(of(CPBBassociated(AKI,16,17(urine(output( definitions(in(the(RIFLE(criteria(were(not(employed.(Primary(definition(of(AKI(was(the( occurrence(of(RIFLE(Class(R(or(greater((a(>50%(rise(in(creatinine(from(baseline)(in(the( five( postBoperative( days.( Secondary( biomarker( analysis( for( RIFLEBInjury( or( greater( and( a( composite( of( need( for( renal( replacement( therapy( or( death( was( also( conducted.( ! Biomarkers!measurements! Samples( for( biomarker( analysis( were( stored( at( B70˚C( until( the( end( of( the( study( period( and( dispatched( on( dry( ice( for( analysis.( CoBinvestigators( performing( biomarker(assays(were(blinded(to(patient(details(and(AKI(classification.(We(assessed( four(biomarkers(of(tubular(injury:(urinary(α(and(π(glutathione(SBtransferases((αBGST( and( πBGST),17B19( urinary( LBtype( fatty( acidBbinding( protein( (LBFAB)20( and( urinary( ! 6! ! ( neutrophil( gelatinaseBassociated( lipocalin( (NGAL),5,21,22( and( two( biomarkers( that( potentially( reflect( changes( in( glomerular( filtration:( urinary( hepcidin23B25( and( serum( CysC.11(We(have(reported(on(the(utility(of(urinary(hepcidin(and(NGAL(for(diagnosis(of( AKI(in(this(study(cohort(previously.13,14(αBGST(and(πBGST(were(measured(by(enzyme( immunoassay((Argutus(Medical,(Ltd.,(Dublin,(Ireland)26(and(expressed(in(ng/ml.(For( urinary( LBFAB( all( samples( were( assayed( using( a( sandwich( ELISA27( and( reported( in( ng/ml.( Urinary( NGAL( was( measured( by( enzymeBlinked( immunosorbent( assay7( and( reported( in( ng/ml.( Urinary( hepcidin( was( measured( by( competitive( enzymeBlinked( immunoassay((CBELISA)(as(previously(described((at(Intrinsic(LifeSciences,(LLC((La(Jolla,( CA)( and( reported( in( ng/ml.( Serum( CysC( was( measured( using( nephelometric( technology(on(a(Beckman(Image(Analyzer((Beckman(Coulter,(Brea,(CA)(and(reported( in( mg/L.( Serum( and( urinary( creatinine( was( measured( using( the( modified( Jaffe( method( standardized( by( isotope( dilution( mass( spectroscopy.( Urinary( biomarkers( were( assessed( both( with( and( without( normalization( against( urinary( creatinine( concentration(to(control(for(urinary(dilution(where(recommended.((( ( Statistical!analysis! ROC(analysis,(logistic(regression(analysis,(net(reclassification(index(calculation( and(decision(tree(analysis(were(performed(using(R:!A!language!and!environment!for! statistical! computing! (R( Development( Core( Team,( R( Foundation( for( Statistical( Computing,( Vienna,( Austria.( http://www.RBproject.org)( utilizing( the( packages( pROC,28!rpart(and(rms!(http://biostat.mc.vanderbilt.edu/rms).( ! 7! ! ( ( Categorical(data(were(reported(as(percentages,(and(compared(using(Fisher’s( exact(test.((Continuous(data(were(reported(as(median(with(interBquartile(range((IQR)( and( compared( using( the( MannBWhitney( U( test.( For( comparisons,( statistical( significance( was( denoted( by( two( sided( p( values( of( <0.05,( adjustment( for( multiple( comparison(was(performed(using(the(Bonferroni(correction.( The( ability( of( biomarkers( to( predict( AKI( was( assessed( by( plotting( receiverB operator( characteristic( (ROC)( curves( and( reported( as( area( under( the( curve( (AUC)( with(95%(confidence(intervals( 29(and(p(value(for(significance(deviation(from(the(null( model(AUC(of(0.5.(ROC(curve(optimal(cutBoff(values(for(diagnosis,(for(curves(with(a( statistically(significant(AUC,(were(defined(as(the(point(which(maximized(the(Youden( index,( defined( as:( sensitivity( +( specificity( –( 1.30( Biomarkers( that( were( associated( significantly( differed( between( No( AKI( and( RIFLE( R( or( greater( AKI( on( univariate( comparison( after( correction( for( multiple( comparisons( were( considered( in( combination.( When( assessing( combinations( NGAL( and( Hepcidin( were( considered( normalized( to( urinary( creatinine.( ( Combinations( of( biomarkers( were( screened( by( multiple(logistic(regression(analysis(and(considered(significant(if(the(pBvalue(for(the( least( significant( biomarker( reached( significance( level( after( correction( for( multiple( comparisons.( ( Predictive( models( for( significant( biomarker( combinations( were( then( derived( from( the( optimal( weighted( linear( combination( of( the( two( biomarker( measurements( (see( supplementary( material),( an( approach( that( makes( no( assumptions(about(the(distributions(of(sample(data.31(Assessment(of(the(statistical( difference( between( paired( ROC( curves( for( optimal( combination( of( biomarkers( and( individual( biomarkers( was( performed( using( DeLong’s( test( for( two( correlated( ROC( curves32( with( a( oneBtail( comparison( of( ROC( curves.( For( significant( biomarker( ! 8! ! ( combinations,( the( enhancement( predictive( ability( after( addition( of( a( second( biomarker( was( assessed( by( calculation( of( the( Net( Reclassification( Index( (NRI)( and( Integrated(Discrimination(Improvement((IDI).33,34(((In(addition,(the(ROC(curve(optimal( cutBoff(for(AKI(was(assessed(against(the(cutBoff(of(the(best(individual(biomarker(by( calculation( of( the( twoBcategory( Net( Reclassification( Index.( Finally( all( individual( biomarkers( significantly( predictive( of( AKI( univariate( analysis( were( considered( in( a( classification(and(regression(tree((CART)(analysis(for(combined(prediction(of(AKI.(The( CART(method(involves(the(segregation(of(different(values(of(classification(variables( through(a(decision(tree(composed(of(progressive(binary(splits(and(has(been(applied( to( risk( prediction( in( critical( illness.35( To( avoid( overBfitting,( the( tree( was( pruned( to( minimize( the( crossBvalidated( error( and( risk( of( AKI( was( calculated( for( each( of( the( terminal(nodes(in(the(CART(tree(to(generate(the(risk(stratification(model.( ( Results( Patient!characteristics! Of(the(patients(in(this(study,(25(of(93(developed(AKI(as(defined(by(RIFLE(≥(R( (27%).(Fourteen(patients(developed(RIFLE(≥(I(AKI((15%)(and(10(RIFLE(F((9.3%).(Five( patients(received(RRT(and(two(died(in(hospital((1(received(RRT(and(died).(Of(the(25( patients(with(RIFLEBR(or(greater(AKI,(16(had(achieved(RIFLEBR(by(creatinine(criteria( (50%( rise( in( serum( creatinine)( by( postBoperative( day( 1.( However,( only( 8( of( 14( patients(going(on(to(develop(RIFLE(I(or(F(and(only(5(of(10(developing(RIFLE(F(satisfied( creatinine( criteria( for( RIFLEBR( on( postBoperative( day( 1.( Patient( characteristics( are( summarised(in(Table(1.(( ! 9! ! ( ( Individual!biomarkers! Individual(biomarkers(performance(is(provided(in(Table(2.((No(biomarker(was( significantly( associated( with( AKI( when( measured( preBoperatively( (Table( 2).( All( urinary(biomarkers(rose(significantly(after(CPB(both(in(patients(who(developed(AKI( and( those( that( did( not.( Immediately( after( surgery,( urinary( NGAL,( urinary( NGAL:Cr( ratio,(urinary(πBGST,(urinary(LBFAB,(and(serum(CysC(were(associated(with(statistically( significant(ROCBAUC’s((Table(2).(The(best(ROCBAUC(was(for(πBGST(at(0.75.((At(the(24h( timeBpoint,(urinary(LBFAB(and(πBGST(were(not(associated(with(AKI;(however(higher( urinary( NGAL,( NGAL:Cr( ratio( and( serum( CysC( remained( associated( with( AKI,( while( lower( urinary( hepcidin( and( hepcidin:creatinine( ratios( were( significantly( associated( with(AKI.(Again(the(best(AUC(was(0.77(for(the(hepcidin:Cr(ratio(at(24h(( Most( biomarkers( showed( lesser( ability( to( predict( RIFLE( I( or( F( (see( Supplemental( Table( 1).( However,( urinary( hepcidin( at( 24h( and( serum( CysC( at( 24h( were(associated(with(increased(ability(to(predict(more(severe(AKI((AUC(0.84(&(0.80( respectively).(Due(to(the(small(number(of(patients(with(RIFLE(I(this(category(was(not( considered(separately.(When(considering(patients(who(required(RRT(and/or(died(in( hospital((6/93),(urinary(hepcidin(at(24h,(urinary(NGAL(at(24h(and(serum(CysC(at(24h( best( predicted( these( outcomes( during( ICU( admission( with( AUCs( of( 0.8B0.91,( however,( the( number( of( outcomes( was( small( and( confidence( intervals( wide( (Supplemental(Table(2).( ( ! ! ! 10! ! ( Biomarker!combinations( PostBoperative( urinary( NGAL:Cr( ratio( and( urinary( πBGST,( and( 24h( urinary( hepcidin:Cr(ratio(and(serum(CysBc(significantly(differed(between(AKI(and(noBAKI(after( correction( for( multiple( comparison( (Table( 2)( and( were( considered( in( combination.( Three( combinations( were( significant:( urinary( NGAL:Cr( postBoperatively( with(( Hepcidin:Cr( atio( at( 24h( (AUC( 0.84,( p=0.03);( urinary( πBGST( postBoperatively( with( urinary( Hepcidin:Cr( ratio( at( 24h( (AUC( 0.86,( p=0.01)( and( urinary( πBGST( postB operatively( with( serum( CysC( at( 24h( (AUC( 0.83,( p=0.03)( (Supplementary( Table( 3).( ROC(curves(for(the(three(best(combinations(of(biomarkers(NGAL:Cr((postBop)(–(1.32( x(Hepcidin:Cr((24hr);(πBGST((postBop)(–(0.03(x(Hepcidin:Cr((24hr)(and(πBGST((postBop)( +(87.35(x(CysC((24hr)(are(shown((Fig(1).( Biomarker( combinations( demonstrated( increased( discrimination( of( AKI( by( uncategorized(NRI(against(the(prediction(provided(by(the(best(individual(biomarker( in( the( combination( (Table( 3);( for( combinations( involving( 24h( urinary( hepcidin,( this( was(predominantly(by(enhancement(of(the(ability(to(exclude(AKI((significant(NRI(for( nonBevents(only).(While(proportion(of(patients(with(probability(of(AKI(reclassified(in( the(correct(direction(appears(good((NRIs(0.45(to(1.02(out(of(a(maximum(2.00),(the( absolute( change( in( probability( was( low( with( IDI( ranging( from( 0.06( to( 0.16( for( biomarker( combinations( (Table( 3).( Furthermore,( when( the( clinical( utility( of( the( combinations(was(assessed(by(examining(ability(to(categorize(higher(or(lower(risk(of( AKI(using(the(optimal(cutBoff(value(of(the(combination(compared(against(the(cutBoff( value( of( the( best( individual( biomarker,( the( twoBcategory( NRI( was( not( significantly( different(from(zero.(( ! 11! ! ( When( we( assessed( the( predictive( ability( of( all( biomarkers( in( CART( analysis,( only( NGAL:Cr( post( operatively( and( Hep:Cr( at( 24h( remained( in( the( final( pruned( decision(tree((Fig(2).(Terminal(nodes(regression(tree(identified(high,(medium(and(low( risk(of(AKI(groups(in(our(cohort(and(in(particular(enabled(classification(of(a(high(risk( group( immediately( postBoperatively( and( a( very( low( risk( group( at( 24h( after( surgery( (Fig.( 2).( However( reducing( the( decision( tree( to( binary( classification( (High( &( Intermediate(vs.(Low(Risk)(resulted(in(Sensitivity(and(Specificity(similar(to(that(at(cutB offs(of(the(best(linear(combinations(of(biomarkers(and(nonBsignificant(NRI(compared( to(Hepcidin:Cr(24h(alone((Fig.(2,(Table(3).( ( Discussion( Summary!of!findings! Individual( biomarkers( alone( had( only( fair( ability( to( predict( RIFLEBR( defined( AKI( after( cardiopulmonary( bypass.( However,( some( markers( (hepcidin( and( CysC)( performed( better( at( predicting( more( severe( AKI( (RIFLE( ≥I).( Certain( pairs( of( biomarkers((urinary(hepcidin(at(24hr(or(serum(CysC(at(24hr(combined(with(urinary( NGAL( at( 6hr( or( urinary( πBGST( at( 6hr)( produced( significantly( improved( combined( ROCBAUCs( and( achieved( values( above( 0.8.( The( best( combinations( involved( one( biomarker(of(tubular(injury((NGAL(or(πBGST)(and(one(biomarker(glomerular(filtration( rate((GFR)((Hepicidin(or(CysC)(and/or(one(biomarker(measured(early,(and(one(later( in( the( postBoperative( course.( This( suggests( combination( of( markers( of( different( aspects( and( stages( in( the( pathophysiology( of( AKI( improves( prediction.( However,( combinations( did( not( significantly( improve( reclassification( of( risk( category.( CART( ! 12! ! ( analysis(identified(a(higher(cutBoff(for(6h(NGAL:Cr(as(defining(a(high(risk(of(AKI(while,( in(those(without(early(elevated(NGAL,(a(higher(level(of(hepcidin(at(24h(was(effective( at(excluding(AKI.,(suggesting(that(CART(analysis(has(the(potential(for(more(efficient( and(clinically(applicable(diagnostic(modelling.( ( Relationship!to!previous!biomarker!research( ( Moderate(diagnostic(performance(of(urinary(NGAL(and(LBFABP(in(this(cohort( is(in(keeping(with(previous(studies(in(similar(groups(of(adults(after(CPB.36,37(Urinary( NGAL(remained(associated(with(AKI(at(the(24h(timeBpoint,(in(keeping(with(reported( performance( an( older( patient( population( and( associated( chronic( kidney( disease.38(( Results( regarding( hepcidin( changes( and( AKI( in( this( cohort( confirmed( in( two( other( studies( of( postBCPB( AKI.39,40( Finally,( CysC( has( been( shown( to( predict( CPBBAKI( very( early( in( the( postBoperative( course41( with( similar( predictive( value( to( that( shown( in( this(study.( Combinations( of( AKI( biomarkers( have( been( described( in( a( several( publications.42B44( Han( et( al43( showed( that( a( combination( of( proximal( tubular( injury( biomarkers( kidney( injury( molecule( 1( (KIMB1)( and( NBacetylBβBDBglucosaminidase( (NAG)( had( no( additive( ability( to( predict( AKI( in( children( undergoing( CPB.( The( same( authors( demonstrated( that( combination( of( 3( biomarkers( (KIMB1,( NAG,( and( NGAL)( was(significantly(improved(prediction(or(early(AKI(at(3(h(after(cardiac(surgery(in(the( however,(there(was(no(added(value(for(diagnosis(of(late(AKI((>24h).44((A(combination( of(LBFABP(and(NAG(at(4h(after(surgery,(was(associated(with(an(improved(ROCBAUC( for(the(prediction(of(AKI(and(addition(to(a(clinical(risk(prediction(model(produced(a( ! 13! ! ( further(significant(increase(in(AUC.42(Most(recently(the(ability(of(32(potential(urinary( AKI(biomarkers(to(predict(worsening(of(AKI(or(death,(was(studied(in(95(patients(with( mild( AKI( after( cardiac( surgery.( In( this( study( a( combination( of( KIMB1( (a( tubular( damage(marker)(and(ILB18((an(inflammatory(mediator)(was(most(predictive(of(death( or(advanced(AKI.45( (( Strengths!and!limitations!! As(a(small(single(centre(study(conclusions(regarding(clinical(risk(classification( for( AKI( are( limited.( However( it( is( similar( in( size( to( other( examining( multiple( combinations( of( AKI( biomarkers( and( makes( important( points( about( the( biological( nature( of( complementary( biomarkers( and( the( potential( disconnect( between( statistical( and( clinically( significant( improvement( in( risk( prediction.( We( employed( a( wide(panel(of(candidate(biomarkers(and(measured(biomarkers(at(two(postBoperative( time( points.( Predictive( ability( of( biomarkers( was( only( fair,( but( was( comparable( to( many( studies( in( similar( patient( populations,( including( many( older( patients( with( patients(with(CKD.(This(study(was(restricted(to(PostBCPB(AKI(and(findings(may(not(be( generalizable.( However,( postBCPB( AKI( is( a( clinically( relevant( setting( for( the( investigation( of( biomarkers( of( AKI.( ( We( only( used( creatinineBnormalized( values( for( urinary(biomarkers(where(recommended(by(the(developers.(However,(normalization( did( not( significantly( improve( ROCBAUC( of( individual( biomarkers( and( the( validity( of( normalization( against( urinary( creatinine( has( been( questioned.46,47( Our( clinical( trial( patient( population( was( preBselected( for( high( risk( of( AKI( this( provided( a( high( event( rate( to( permit( analysis( of( biomarkers( in( combination( in( a( small( overall( sample;( ! 14! ! ( however(as(patients(were(selected(for(AKI(risk(factors(it(was(not(designed(to(permit( analysis(of(biomarkers(in(combination(with(clinical(risk(prediction(models.(( Statistical(analysis(of(combined(diagnostic(predictors(is(complex.(Our(analysis( is(strengthened(by(use(of(a(robust(method(to(combine(biomarkers,(weighted(linear( combination.(Use(of(an(uncategorized(NRI(has(been(criticised(as(overBsensitive(and( selfBfulfilling,48( However,( at( least( it( is( likely( to( be( an( indicator( that( certain( combinations(of(biomarkers(provide(complementary(information(and(thus(potential( insights( into( the( pathogenesis( of( AKI.( Moreover,( this( analysis( was( tempered( by( examination(of(the(2(category(NRI,(a(better(measure(of(clinical(utility.(CART(models( can( capture( nonBlinearity( much( more( easily( than( regression( models,( are( very( well( suited(to(predictive(modeling(and(are(easy(to(interpret,(however(they(are(prone(to( overBfitting( and( results( can( be( variable( depending( on( the( dataset( used( to( develop( the( tree.( To( address( this( concern( our( model( was( subject( to( pruning,( a( form( of( internal(crossBvalidation(to(minimise(overBfitting.(The(model( performed(similarly(to( linear(combination(of(predictors(when(considered(as(a(binary(outcome,(supporting( our(primary(finding(with(an(alternative(methodology.(Any(model(developed(from(a( single(sample(set(does(require(external(validation(and(the(CART(model(is(presented( to(highlight(the(potential(of(this(methodology(to(develop(more(clinically(applicable( models(for(examining(multiple(biomarkers(and/or(clinical(risk(factors(in(combination( and(to(support(our(primary(analysis.( Finally(while(certain(biomarker(combinations(resulted(in(better(assignment(of( risk( of( AKI( practical( ability( to( better( categorize( groups( high( and( low( risk( of( AKI( did( not( eventuate.( However,( serum( creatinine( has( significant( limitations( as( a( gold( ! 15! ! ( standard( for( diagnosis( of( renal( tubular( injury( due( to( alterations( in( creatinine( production,( volume( of( distribution( in( acute( systemic( illness( and( the( indirect( relationships( between( tubular( injury,( GFR( changes( and( alteration( in( serum( creatinine.(An(imprecise(gold(standard(will(intrinsically(limit(the(performance(of(even( the( best( biomarkers49( and( thus( might( prevent( meaningful( improvement( of( prediction( when( they( are( examined( in( combination.( One( way( to( escape( this( limitation( is( to( examine( creatinine( independent( outcomes( such( as( need( for( RRT( and/or( mortality.50,51( In( this( study( some( individual( biomarkers( (urinary( hepcidin( at( 24h,(urinary(NGAL(at(24h(and(serum(CysC(at(24h)(did(predict(need(for(RRT(or(death,( however( as( the( event( rate( was( small( statistical( analysis( of( combinations( was( not( feasible.( ( Conclusions! AKI(biomarkers(may(aid(in(the(early(diagnosis(of(AKI,(however(performance(in( this(groups(of(highBrisk(adult(patients(was,(at(best,(fair.(Combinations(of(biomarkers( are(able(to(increase(diagnostic(discrimination,(but(this(may(not(result(in(significantly( better( reclassification( of( risk( of( AKI( when( applied( as( a( simple( diagnostic( cutBoff.(( Biomarkers(that(indicate(tubular(injury(and(those(that(may(be(related(to(functional( changes(in(GFR(may(be(most(informative(in(combination(and(should(be(the(target(of( future(research.52( ( ! 16! ! ( 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! 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Transplant.! 2003;18(3):543B551.( ! 19! ! ( 27.( Ferguson(MA,(Vaidya(VS,(Waikar(SS,(et(al.(Urinary(liverBtype(fatty(acidBbinding( protein( predicts( adverse( outcomes( in( acute( kidney( injury.( Kidney! Int.! 2010;77(8):708B714.( 28.( Robin(X,(Turck(N,(Hainard(A,(et(al.(pROC:(an(openBsource(package(for(R(and( S+(to(analyze(and(compare(ROC(curves.(BMC!Bioinformatics.!2011;12:77.( 29.( Hanley( JA,( McNeil( BJ.( The( meaning( and( use( of( the( area( under( a( receiver( operating(characteristic((ROC)(curve.(Radiology.!1982;143(1):29B36.( 30.( Youden(WJ.(Index(for(rating(diagnostic(tests.(Cancer.!1950;3(1):32B35.( 31.( Pepe(MS,(Cai(T,(Longton(G.(Combining(predictors(for(classification(using(the( area( under( the( receiver( operating( characteristic( curve.( Biometrics.! 2006;62(1):221B229.( 32.( DeLong(ER,(DeLong(DM,(ClarkeBPearson(DL.(Comparing(the(areas(under(two( or(more(correlated(receiver(operating(characteristic(curves:(a(nonparametric( approach.(Biometrics.!1988;44(3):837B845.( 33.( Pencina( MJ,( D'Agostino( RB,( Sr.,( D'Agostino( 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(2):157B172;( discussion( 207B 112.( 34.( Pencina( MJ,( D'Agostino( RB,( Sr.,( Steyerberg( EW.( Extensions( of( net( reclassification( improvement( calculations( to( measure( usefulness( of( new( biomarkers.(Stat!Med.!2011;30(1):11B21.( 35.( Wong( HR,( Lindsell( CJ,( Pettila( V,( et( al.( A( multibiomarkerBbased( outcome( risk( stratification( model( for( adult( septic( shock*.( Critical! care! medicine.! 2014;42(4):781B789.( 36.( Haase(M,(Bellomo(R,(Devarajan(P,(Schlattmann(P,(HaaseBFielitz(A,(Group(NMB aI.(Accuracy(of(neutrophil(gelatinaseBassociated(lipocalin((NGAL)(in(diagnosis( ! 20! ! ( and(prognosis(in(acute(kidney(injury:(a(systematic(review(and(metaBanalysis.( Am!J!Kidney!Dis.!2009;54(6):1012B1024.( 37.( Matsui( K,( KamijoBIkemori( A,( Sugaya( T,( Yasuda( T,( Kimura( K.( Usefulness( of( urinary( biomarkers( in( early( detection( of( acute( kidney( injury( after( cardiac( surgery(in(adults.(Circ!J.!2012;76(1):213B220.( 38.( Endre( ZH,( Pickering( JW,( Walker( RJ,( et( al.( Improved( performance( of( urinary( biomarkers(of(acute(kidney(injury(in(the(critically(ill(by(stratification(for(injury( duration(and(baseline(renal(function.(Kidney!Int.!2011.( 39.( HaaseBFielitz(A,(Mertens(PR,(Plass(M,(et(al.(Urine(hepcidin(has(additive(value( in( ruling( out( cardiopulmonary( bypassBassociated( acute( kidney( injury( B( an( observational(cohort(study.(Crit!Care.!2011;15(4):R186.( 40.( Ho(J,(Reslerova(M,(Gali(B,(et(al.(Urinary(hepcidinB25(and(risk(of(acute(kidney( injury( following( cardiopulmonary( bypass.( Clin! J! Am! Soc! Nephrol.! 2011;6(10):2340B2346.( 41.( Wald( R,( Liangos( O,( Perianayagam( MC,( et( al.( Plasma( cystatin( C( and( acute( kidney( injury( after( cardiopulmonary( bypass.( Clin! J! Am! Soc! Nephrol.! 2010;5(8):1373B1379.( 42.( Katagiri( D,( Doi( K,( Honda( K,( et( al.( Combination( of( two( urinary( biomarkers( predicts( acute( kidney( injury( after( adult( cardiac( surgery.( Ann! Thorac! Surg.! 2012;93(2):577B583.( 43.( Han( WK,( Waikar( SS,( Johnson( A,( et( al.( Urinary( biomarkers( in( the( early( diagnosis(of(acute(kidney(injury.(Kidney!Int.!2008;73(7):863B869.( 44.( Han(WK,(Wagener(G,(Zhu(Y,(Wang(S,(Lee(HT.(Urinary(biomarkers(in(the(early( detection(of(acute(kidney(injury(after(cardiac(surgery.(Clin!J!Am!Soc!Nephrol.! 2009;4(5):873B882.( ! 21! ! ( 45.( Arthur(JM,(Hill(EG,(Alge(JL,(et(al.(Evaluation(of(32(urine(biomarkers(to(predict( the( progression( of( acute( kidney( injury( after( cardiac( surgery.( Kidney! Int.! 2014;85(2):431B438.( 46.( Ralib( AM,( Pickering( JW,( Shaw( GM,( et( al.( Test( characteristics( of( urinary( biomarkers(depend(on(quantitation(method(in(acute(kidney(injury.(J!Am!Soc! Nephrol.!2012;23(2):322B333.( 47.( Waikar(SS,(Sabbisetti(VS,(Bonventre(JV.(Normalization(of(urinary(biomarkers( to( creatinine( during( changes( in( glomerular( filtration( rate.( Kidney! Int.! 2010;78(5):486B494.( 48.( Pepe( MS.( Problems( with( risk( reclassification( methods( for( evaluating( prediction(models.(Am!J!Epidemiol.!2011;173(11):1327B1335.( 49.( Waikar(SS,(Betensky(RA,(Emerson(SC,(Bonventre(JV.(Imperfect(gold(standards( for(kidney(injury(biomarker(evaluation.(J!Am!Soc!Nephrol.!2012;23(1):13B21.( 50.( Haase( M,( Devarajan( P,( HaaseBFielitz( A,( et( al.( The( outcome( of( neutrophil( gelatinaseBassociated( lipocalinBpositive( subclinical( acute( kidney( injury:( a( multicenter( pooled( analysis( of( prospective( studies.( J! Am! Coll! Cardiol.! 2011;57(17):1752B1761.( 51.( Nickolas( TL,( SchmidtBOtt( KM,( Canetta( P,( et( al.( Diagnostic( and( prognostic( stratification( in( the( emergency( department( using( urinary( biomarkers( of( nephron(damage:(a(multicenter(prospective(cohort(study.(J!Am!Coll!Cardiol.! 2012;59(3):246B255.( 52.( McCullough( PA,( Shaw( AD,( Haase( M,( et( al.( Diagnosis( of( acute( kidney( injury( using( functional( and( injury( biomarkers:( workgroup( statements( from( the( tenth(Acute(Dialysis(Quality(Initiative(Consensus(Conference.(Contributions!to! nephrology.!2013;182:13B29.( ( ) ) ! 22! ! ( ) Table)1:)Patient(Characteristics) Variable) All) AKI)(n=25)) No)AKI)(n=68)) ) Baseline)) ( ( ( Age((yrs)( 70((61B76)( 72((67B78)( 70((61B75)( 0.14( 31%(( 32%(( 30%(( 1.0( 91((76B113)( 86((72B111)( 95((78B119)( 0.29( 29%(( 24%(( 31%(( 0.61( 8%( 16%( 4%( 0.08( 7%( 8%(( 7%( 1.0( 16%( 20%( 15%( 0.53( 6%( 16%(( 3%( 0.04( ( ( ( 59%( 76%( 53%( 0.06( 62%( 68%( 60%( 0.63( 25%( 48%( 16%( 0.003( 11%( 20%( 7%( 0.13( 139((11B202)( 210((146B240)( 125((106B177)( ( ( ( 50((41B57)( 52((47B69)( 47((40B56)( 0.003( 41%( 56%( 35%( 0.096( 48%( 58%( 44%( 0.35( 41%( 44%( 40%( 0.81( +190((B886(to(+1366)( +43((B640(to(+726)( +310((B103(to(+723)( 0.73( Female(Sex( PreBoperative( Creatinine( Chronic(Kidney( Disease(Stage(3( Chronic(Kidney( Disease(Stage(4( InsulinBrequiring( diabetes( Previous(cardiac( surgery( L(ventricular(ejection( fraction(<35%( ) Surgery) Coronary(bypass( grafts( Valve(replacement( or(repair( Coronary(bypass( grafts(+(Valve( Thoracic(Aortic( Surgery( Bypass(time((min)( ) PostQoperative) APACHE(III(score( %(Blood(transfusion( in(theatre(or(first( 24h( Any(Vasopressor(in( first(24h(of(ICU( Any(Inotrope(in(first( 24h(of(ICU( Fluid(balance( (first(24h(postBop)( p) ( ( <0.001( ( Characteristics!of!93!patients!undergoing!cardiopulmonary!bypass!by!AKIRRisk! category.!Continuous!variables!are!expressed!as!medians!(interquartile!range),!for! categorical!variables!95%!confidence!intervals!are!shown ! 23! !