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!
!
(
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! 18!
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! 19!
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(
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! 21!
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(
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(
)
)
! 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!
!