Neutrophil gelatinase-associated lipocalin (NGAL) in heart transplant recipients after conversion to everolimus therapy

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Original

article

Neutrophil

gelatinase-associated

lipocalin

(NGAL)

in

heart

transplant

recipients

after

conversion

to

everolimus

therapy

Jo¨rg

Stypmann

(MD)

a

,

Manfred

Fobker

(PhD)

b

,

Katharina

Rosing

(PhD)

b

,

Markus

Engelen

(MD)

a

,

Stefan

Gunia

(MD)

c

,

Angelo

Maria

Dell’Aquila

(MD)

d

,

Jerzy-Roch

Nofer

(MD,

MBA)

b,

*

a

DepartmentofCardiovascularMedicine,UniversityHospitalMu¨nster,Mu¨nster,Germany

b

CenterforLaboratoryMedicine,UniversityHospitalMu¨nster,Mu¨nster,Germany

c

DepartmentofNephrology,HypertensionandRheumatology,UniversityHospitalMu¨nster,Mu¨nster,Germany

d

DepartmentofCardiothoracicSurgery,UniversityHospitalMu¨nster,Mu¨nster,Germany

Introduction

Cardiactransplantation is now an established treatment for patients with end-stage heart failure. The introduction of

calcineurin inhibitors (CNI) into immunosuppressive therapy regimens has significantly improved the survival rate among hearttransplantrecipients.However,thelong-termoutcomeafter heart transplantation is adversely affected by CNI-induced nephrotoxicity, which leads to progressive renal deterioration and ultimately to end-stage renal disease. Proliferation signal inhibitors(PSI),suchaseverolimusorsirolimus,exert immuno-suppressiveeffectsbyblockingthegrowthfactor-driven prolifer-ationofTandBcells[1].ThemajoradvantageofPSIoverCNIis

ARTICLE INFO

Articlehistory:

Received17June2014

Receivedinrevisedform7December2014

Accepted11December2014

Availableonline9January2015

Keywords:

Neutrophilgelatinase-associatedlipocalin

Everolimus

Immunosuppressiontherapy

Renalfunction

Hearttransplantation

ABSTRACT

Background: Due to the lack of nephrotoxic activity, proliferation signal inhibitors (PSI) such as

everolimusarerecommendedforimmunosuppressionafterhearttransplantation,buttheassessment

ofrenalfunctioninpatientsreceivingPSIhasledtoconflictingresults.Weexaminedrenalintegrityand

functionusingneutrophilgelatinase-associatedlipocalin(NGAL) andconventionalmarkers [plasma

creatinine, cystatin C, urine albumin, a1-microglobulin (a1M)] in heart transplant patients, who

underwentconversiontoeverolimusduetoallograftvasculopathy,graftrejectionepisodes,orrenal

functiondeterioration,andinpatientsmaintainedoncalcineurininhibitors(CNI).

Methods:Thiscross-sectionalstudyincluded121consecutivehearttransplantrecipients:44patients

receivedCNI-freeimmunosuppressivetherapywitheverolimusand77patientsreceivedCNI.Renal

parametersweredeterminedinplasmaandurinesamplesusingstandardenzymaticor

immunochemi-calmethods.

Results:Heart transplant recipients receiving everolimus therapy had significantly lower NGAL

concentrations in plasma [median (95% CI): 128 (97–176)ng/mL vs. 252 (224–283)ng/mL,

p<0.001] and urine [median (95% CI): 6.4 (4.5–7.6)ng/g vs. 15.7 (10.2–25.9)ng/g creatinine,

p<0.001].Incontrast,nosignificantdifferenceswereobservedbetweeneverolimus-andCNI-treated

groupswithregardtocreatinineandcystatinC,aswellasurinealbuminanda1Mlevels.Significant

correlationswerenotedbetweenplasmaNGALandcreatinine(r=0.42,p<0.001),cystatinC(r=0.44,

p<0.001), N-terminal brain natriuretic propeptide (r=0.31, p<0.01) and indicators of chronic

inflammation[lipoprotein-associatedphospholipaseA2(Lp-PLA2),r=0.31,p<0.01]andsolubleCD40

ligand(sCD40L,r=0.22,p<0.05),andbetweenurinaryNGALanda1M(r=0.21,p<0.05).Multiple

regressionanalysisindicatedthatcystatinCandLp-PLA2werethebestpredictorsofplasmaNGAL.

Conclusion:Thepresentstudydocuments reducedplasmaandurinaryNGAL levelsin theabsence

ofdifferencesinconventionalrenalparametersinpatientsonCNI-freeimmunosuppressivetherapy

witheverolimus. Theseresults supportfavorable effectsofeverolimus onrenal integrityin heart

transplantrecipients.

ß2015JapaneseCollegeofCardiology.PublishedbyElsevierLtd.Allrightsreserved.

* Correspondingauthorat:Centrumfu¨rLaboratoriumsmedizin,

Universita¨tsklinikumMu¨nster,AlbertSchweizerCampus1,Geba¨udeA1,

48149Mu¨nster,Germany.Tel.:+492518347228;fax:+492518347225.

E-mailaddress:nofer@uni-muenster.de(J.-R.Nofer).

ContentslistsavailableatScienceDirect

Journal

of

Cardiology

j our na l ho me pa g e : w ww . e l se v i e r . com / l oca t e / j j cc

http://dx.doi.org/10.1016/j.jjcc.2014.12.010

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theirlackof intrinsicnephrotoxic activity.Accordingly, mainte-nanceprotocolsthatpartiallyorfullyreplaceCNIwithPSIhave beenreportedtoalleviaterenalimpairment([2–4]andreferences therein). However, the conversion from CNI to PSI in heart transplant recipients is not uniformly effective, with a lack of improvementorevendeterioration ofkidneyfunctionobserved inafewstudies[2–4].

The overwhelming majority of previously published reports have used plasma creatinine concentrations and/or calculated creatinineclearancefortheassessmentofrenalstatusafterheart transplantation. Unfortunately, neither variable reliably reflects renalfunction,themarkeddeteriorationofwhichcanoccurprior toanincreaseinserumcreatininelevels.Neutrophil gelatinase-associatedlipocalin(NGAL),alsoknownassiderocalin,lipocalin-2, orLCN2, is a 25-kDaglycoprotein with bactericidal properties, which was initially isolated from activated neutrophils and subsequentlyidentified in theepithelialcells of thealimentary and respiratory tracts, cardiomyocytes, and renal tubular cells [5,6].UpregulatedNGALproductionhasbeenobservedinseveral inflammatoryconditions,includinginflammatoryboweldisease, chronicobstructivepulmonarydisease,andmyocarditis[5–7].In thesettingofacuterenalfailure,serumandurineNGALlevelsare elevatedfrom7-to16-foldandfrom25-to100-fold,respectively [8]. Consequently, NGAL has been suggested as a sensitive biomarker of acute kidney injury [5,6,8]. However, increased NGALconcentrationshavealsobeenreported inchronickidney disease, which presumably reflects the protracted damage to tubularcells irrespective oftheglomerular filtrationrate(GFR) [5,9–11].Therefore,thedeterminationofNGALlevelsmayaidthe earlydetection of renal function changes that occur alongside chronic pathological conditions and can be overlooked using conventionalindicatorssuchascreatinine,cystatinC,orestimated GFR(eGFR).

Few previous observational studies have reported increased plasmaNGAL concentrationsinadultor infanthearttransplant recipients[12–14].However,theeffectofCNItoPSIconversionin the course of the immunosuppressive therapy after heart transplantation on plasma or urine NGAL levels has not been examinedtodate.Thiscurrent studyreportsthenovel findings of reduced plasma and urine NGAL concentrations in heart transplantrecipientswhoreceivedaCNI-freeimmunosuppressive maintenanceregimenthatincludedeverolimus.

Materialsandmethods

Patients,studydesign,andsamplecollection

Thestudywasofacross-sectionaldesign.Ethicsboardapproval wasgainedforthestudyprotocol,andwritteninformedconsent wasobtained fromall patients. The examined cohort included 121 patients after cardiac transplantation recruited at the DepartmentofCardiothoracicSurgery,UniversityHospital Mu¨n-ster,betweenJanuary1990andDecember2010.Excludedpatients includedthosetemporarilytreatedinothercardiacsurgerycenters as wellas heart–lung transplants, repeat transplants, pediatric transplants (<18 years), patients within their first year after transplantation,patientswithahistoryofdiabetesthatrequired insulin,and patients witha diagnosed malignancy.Allpatients underwentyearly monitoring forcardiac allograftvasculopathy (CAV)withdobutaminestressechocardiographyandsinglephoton emissioncomputedtomography.Incaseofasuspiciousresultin either examination, coronary angiography was subsequently performed. For thepresent study,patients were examined and bloodandurinesamplesweretakenduring2011inconsecutive visits to the outpatients department at the Department of Cardiology. At the time of examination, all patients were free

from acute rejection, acute heart failure, and clinical signs of infection.Initially,allpatientsreceivedthemaintenance immu-nosuppressive therapy, which consisted of CNI [tacrolimus or cyclosporineA(CsA)],mycophenolatemofetil,and/oralow-dose steroid(2–5mgprednisolonedaily).Thesteroidwaswithdrawn onlyifpatientsdevelopedosteoporosisordiabetes.Beginningin April 2004, CNI was successively replaced by everolimus in 44patients.Theindicationsforconversiontoeverolimusincluded CAV (6%), repetitive episodes of graft rejection (6%), and a deterioration in renal function (88%). Serum creatinine levels beforeconversiontoeverolimusaveraged1.75(1.50–1.99)mg/dL anddecreasedslightlybutnotsignificantlythereafter.Atthetime ofthestudy,patients hadbeenreceivingeverolimusforatleast 6 months. The target trough levels for CsA, tacrolimus, and everolimus were 120–150ng/mL, 6–8ng/mL, and 4–6ng/mL, respectively.Statins(withorwithoutezetimibe)wereroutinely giventoallpatients.

Bloodandurinesamplecollection

Bloodsampleswerecollectedfrompatientsbyvenipuncture duringcontrolvisits.Sampleswerecentrifugedimmediatelyafter bloodcollectionandplasmaorserawerealiquotedandstoredat 708C until analysis. Overnight urine samples were collected intovialscontaining5.0mmol/LEDTAtopreventoxidationand werekeptat 708Cuntilanalysis.

Analyticalprocedures

Standard clinical chemistry parameters, including serum creatinine, cystatin C, and C-reactiveprotein (CRP) andurinary creatinineweredeterminedattheCenterforLaboratoryMedicine usingenzymatic,photometric,orimmunoturbidimetricassayson a routinelaboratoryanalyzer(ADVIA1800,SiemensHealthcare Diagnostics, Eschborn, Germany). All interassay coefficients of variation(iCVs)were<6.8%. eGFRwascalculatedbyconverting creatinine (eGFRCrea) or cystatin C (eGFRCyst) values in plasma usingthemodificationofdiet inrenal disease(MDRD)formula [15], or the formula described by Hoek et al. [16]. Plasma concentrations of troponin I (TnI), N-terminal brain natriuretic propeptide(NT-proBNP),andinterleukin-6(IL-6)weremeasured usingchemiluminescenceimmunoassaysonlaboratoryanalyzers (ADVIACentaurXPorImmulite2500,bothSiemens).AlliCVswere <12%. Urine concentrations of albumin and

a

1-microglobulin (

a

1M) were determined on a BN-II System nephelometer (Siemens)withiCVs<7.7%.MeasurementsofNGALconcentrations in theplasma and urine wereperformed using a turbidimetric immunoassay (BioPorto, Gentofte, Denmark) adapted on the ADVIA1800laboratoryanalyzer.Thedetectionlimitoftheassay was12ng/mLandtheiCVswere3.6%and2.5%atlow(198ng/mL) and high(497ng/mL)concentrations, respectively. Lipoprotein-associated phospholipaseA2 (Lp-PLA2) activities in the plasma samples were determined using a colorimetric assay (Cayman Chemical, Ann Arbor, MI, USA) using 2-thio-platelet activating factor as a substrate. Lp-PLA2 activity wascalculated from the slope of the kinetic absorption curve and expressed in

m

molmin 1mL 1.TheiCVswere5.1%foralowcontroland 3.8%foracontrolclosetothesamplemean.SolubleCD40ligand (sCD40L) concentrations in plasma were determined using a commerciallyavailableELISAkit(IBL,Hamburg,Germany). Statisticalanalysis

Exploratory statistics were performed using the MedCalc StatisticalSoftwareversion12.7.7(MedCalcSoftwarebvba,Ostend, Belgium). The level of dependence between two categorical

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variableswasanalyzedbytheChi-squaretest.Thedistributionof continuousvariableswasassessedfornormalityusingtheShapiro– Wilktest.TheStudent’st-test(2-sided)orMann–WhitneyU-test (2-sided)wasusedtocomparegroupswithnormallyornon-normally distributed data, respectively. Spearman’s rank correlation was calculatedtoexaminecorrelations betweennumerical variables. Stepwise multiple regression analysis was used to determine independentfactorsthataffectedNGALconcentrationsinplasma andurine.Non-normallydistributeddatawereLn-transformed.The localsignificancelevel(p)wassetto0.05.Fordescriptivepurposes, therelativerates(inpercent)wereprovidedforcategoricalvariables andmeans(SD)ormedians(95%CI)werecalculatedfornormally ornon-normallydistributedcontinuousvariables,respectively. Results

Thegeneralcharacteristicsofgroupstreatedwitheverolimusor CNIareshowninTable1.Bothgroupsdidnotdiffersignificantly withregardtoageandsex,aswellasbodymassindex,systolicand diastolic blood pressure, and left ventricular ejection fraction (LVEF).Comparablepercentagesofpatientsineachgroupsuffered fromtype2diabetesmellitus.Themeantimeelapsedfromcardiac transplantationfortheentirestudypopulationwas8.55.6years and there was no significant difference between both groups. The everolimus-treated group had received this treatment for 3.81.8 years. The indications for transplantation were mainly coronary heart disease and dilated cardiomyopathy in the ever-olimus-andCNI-treatedgroups,respectively.Similarratesofpatients in each group received mycophenolate mofetil and/or low-dose steroidtherapyinadditiontoeverolimusorCNI(CsAortacrolimus). Theobserved troughconcentrationsofCsA, tacrolimus, and ever-olimuswerewithinthetargetranges.Noneofthepatientsexamined wasreceiving sirolimustherapyoranti-oxidative drugtherapyat the time of the study. No significant differences withrespect to lipid-lowering, anti-hypertensive, or anti-coagulant therapy were presentbetweenbothgroups.

Thedistributionsofvariablesthatreflectedrenalfunctionand integrityinhearttransplantpatientsreceiving immunosuppres-sivetherapy witheverolimus orCNIare shown in Table2.No significantdifferences between the groups weredetected with regards to plasma creatinine and cystatin C levels, as well as eGFRCreaandeGFRCyst.Likewise,urinary

a

1Mconcentrationsand theurinary albumin/creatinine ratio, which reflect tubular and glomerularkidneyinjury,respectively,didnotsignificantlydiffer between the everolimus- and CNI-treated groups. However, patients who received everolimus presented with significantly

lowerplasmaNGALconcentrationsandurinaryNGAL/creatinine ratios compared to the group that received CNI. Conventional biomarkersofsystemicinflammation,suchasplasmaCRPandIL-6 levels,werecomparablebetween bothgroupsexamined inthis study. In contrast, heart transplant recipients that received everolimusshowedsignificantly lowerlevelsofplasmaLp-PLA2 activity and sCD40L concentrations. These parameters reflect chronic inflammatory processes that are dependent on macro-phage activation. Biomarkers that reflect cardiac integrity and function(TnI,NT-proBNP)werenotsignificantlydifferentbetween theeverolimus-andtheCNI-treatedgroups.

Table1

Baselinecharacteristicsofthestudypopulation.

Everolimus

(n=44)

CNI(n=77) p

Age(years) 5714 5615 0.75

Femalegender 8(18) 19(25) 0.54

Bodymassindex(kg/m2

) 25.44.6 24.94.2 0.51 Diabetes 10(22) 12(16) 0.46 Pre-transplantcondition -Ischemiccardiopathy 21(48) 48(62) 0.17 -Dilatedcardiomyopathy 18(40) 19(25) 0.15 -Other 5(12) 10(13) 0.95

Timesincetransplant(years) 84 96 0.99

Timeoneverolimus(years) 3.81.8 – –

Systolicpressure(mmHg) 12110 1249 0.31

Diastolicpressure(mmHg) 777 798 0.19

Leftventricularejection

fraction(LEVF,%) 61.512.1 62.16.9 0.69 Medication -Everolimus 44(100) – – -Tacrolimus – 56(73) – -Cyclosporine – 21(27) – -Mycophenolatemofetil 32(72) 60(80) 0.67 -Azathioprine 1(2) 5(6) 0.61 Steroids 43(98) 67(87) 0.11 Lipidlowering 43(98) 74(96) 0.96 Anti-hypertensive 43(98) 74(96) 0.96

Observedtroughconcentrations

-Everolimus(ng/mL) 5.22.1 – –

-Tacrolimus(ng/mL) – 6.94.1 –

-CyclosporinA(ng/mL) – 133.771.5 –

Accordingtotreatmentgroups:dataaregivenasmeanSDorrates[relative

rates (%) are shown in parentheses]. CNI (calcineurin inhibitors) include

cyclosporineAortacrolimus.Medicationwithsteroidsincludesprednisoneor

prednisolone. Medication with lipid-lowering drugs includes simvastatin or

fluvastatin or pravastatin or atorvastatin combined or not with ezetimibe.

Medication with anti-hypertensive drugs includes b-blockers,

angiotensin-convertingenzymeinhibitors,sartans,orCa2+

-channelblockers.

Table2

Renal,cardiac,andinflammationmarkersinthestudypopulation.

Everolimus(n=44) CNI(n=77) p

NGALinplasma(ng/mL) 128(97–176) 252(224–283) <0.001

NGALinurine(ng/mgcreatinine) 6.4(4.5–7.6) 15.7(10.2–25.9) <0.001

Creatinine(mg/dL) 1.38(1.27–1.53) 1.48(1.28–1.71) 0.36

eGFRCrea(mL/min/1.73m2) 48.4(43.8–57.5) 55.5(44.5–59.6) 0.52

CystatinC(mg/L) 1.72(1.40–2.19) 1.72(1.48–2.02) 0.90

eGFRCyst(mL/min/1.73m2) 43.3(32.3–53.1) 43.4(35.4–50.0) 0.63

Albumin(mg/gcreatinine) 69.5(37.4–112.3) 56.9(29.3–64.9) 0.65 a1-Microglobulin(mg/L) 15.6(11.4–24.3) 17.7(12.9–24.9) 0.51 CRP(mg/dL) 0.68(0.56–0.74) 0.64(0.57–0.82) 0.34 IL-6(ng/mL) 5.01(3.83–6.55) 4.06(3.35–4.90) 0.13 sCD40L(ng/mL) 0.81(0.44–0.98) 1.21(0.82–1.67) <0.05 Lp-PLA2(mmol/mL/min) 0.008(0.007–0.009) 0.010(0.009–0.011) <0.001 NT-proBNP(mg/mL) 601(396–1083) 737(507–972) 0.79 TroponinI(ng/mL) 0.005(0.002–0.008) 0.006(0.004–0.010) 0.35

Dataaregivenasmedian(95%CI).CNI(calcineurininhibitors)includecyclosporineAortacrolimus.

NGAL,neutrophilgelatinase-associatedlipocalin;eGFR,estimatedglomerularfiltrationrate;Crea,creatinine;Cyst,cystatinC;CRP,C-reactiveprotein;IL,interleukin;

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To further assess the reciprocal relationships between renal functionandintegritymarkers,correlationsbetweenparameters that reflected renal function, systemic and local inflammation, aswellascardiacbiomarkerswereexamined.AsshowninFig.1, plasmaNGALconcentrationssignificantlycorrelatedwithplasma levelsof creatinine, cystatin C, eGFRCrea, eGFRCyst,sCD40L, and NT-proBNP,aswellas Lp-PLA2 activity.In contrast,theurinary NGAL/creatinine ratio correlated solely with the urinary

a

1M concentration. Parameters that tended to correlate with NGAL (p<0.10)weresubsequentlyincludedin thestepwisemultiple regression analysis. For circulating levels of NGAL, the best predictor was plasma cystatin C (

b

=0.32, p<0.01), which accountedfor11% oftheplasmaNGAL concentration.This was followedbyLp-PLA2 (

b

=0.28,p<0.01),whichexplained 8%of theplasma NGAL concentration. Similar results were obtained when eGFRCyst was substituted for plasma cystatin C. No predictors of urinary NGAL could be identified in themultiple regressionanalysis.

Discussion

Asaresultofthelackofintrinsicnephrotoxicactivity,theuseof PSI such as everolimus has been frequently recommended for immunosuppressivetherapyafterhearttransplantation.However, theresultsofclinicalstudiesthatcomparedPSIwithCNIinheart transplant recipients have been supportive, neutral, or even negative withregard toadverse effects on renal function after patientswereswitchedtoPSI-basedregimens[2–4].Promptedby theongoingcontroversyregardingtheconversionofCNItoPSIin hearttransplantmaintenancetherapy,weassessedrenalintegrity andfunctionalstatusinhearttransplantpatientswhounderwent conversiontoeverolimusorweremaintainedonCNItherapy.We assessed levels of NGAL, a sensitive indicator of early tubular injury,aswellasconventionalrenalbiomarkers(urinealbumin,

a

1M,plasma creatinine,cystatin C,eGFRCrea,andeGFRCyst). The novelfindingwasobservedoflowerNGALconcentrationsinboth theurineandplasmaofpatientsreceivingCNI-freetherapywith everolimus. Importantly, reduced NGAL levels in everolimus-treatedhearttransplantrecipientsweredeterminedintheabsence of significant differences between both groups withrespect to plasma creatinine, cystatin C, and eGFR, as well as urinary concentrations of albumin and

a

1M. These results could be interpretedas anindicationofthebeneficial effects exerted by everolimus,whichwerenot adequatelyreflected bychangesin conventionalmarkersofrenalintegrityandfunction.

NGAL expression is markedly upregulated within the thick ascendinglimbofHenleandthecollectingductduringischemicor toxicrenalinjury,andtheresultingreleaseofNGALintotheurine accountsforthemajorfractionofurinaryNGAL[5,6,9,17]. Accord-ingly,increasedNGALexcretionintotheurinehasbeenobserved in a variety of renal diseases (e.g. polycystic kidney disease, immunoglobulinAnephropathy,humanimmunodeficiencyvirus nephropathy),inwhichitcloselyreflectsthemagnitudeofrenal tubularinjury[18–20].Inthesettingofchronicheartfailure(CHF), elevated levels of urinary NGAL paralleled by the increased excretionofkidneyinjurymolecule-1andN-acetyl-

b

-D -glucosa-minidase,sensitiveandspecificmarkersoftubulardamage,have beenobservedinseveralstudies[21–24].Importantly,increased NGALexcretioncouldbeusedtopredictunfavorableoutcomes, eveninpatientswithnormalrenalfunctionasassessedbyplasma creatininelevels,eGFR,andtheconcentrationofurinaryalbumin [23]. These findings were corroborated by the results of the presentstudy,whichfailedtoobserve correlationsbetweenthe conventionalindicesofrenalfunctionandNGALconcentrationsin urineandtoidentifypredictorsofurinaryNGALinthemultiple regression analysis. Given the capability of urinary NGAL to specifically predict kidney injury and the close relationship betweenNGALexcretionandtheextentoftubulardamage,itis reasonabletoassumethatpatientsthatreceivedCNI-freetherapy witheverolimushavealowerriskofdevelopingrenalimpairment. InamajorcontrasttoNGALexcretioninurineplasma,NGAL levels were found to be closely related to indicators of renal function (serum creatinine, cystatin C levels, eGFRCrea, and eGFRCyst),aswellasmarkersofheartfunction(NT-proBNP)and chronicinflammation(Lp-PLA2andsCD40L).Thedistinctoriginsof excreted and circulating NGAL mayaccount for these differing findings.WhereasNGALconcentrationsintheurinearedirectly determinedbytubulardamage,thedirectcontributionofkidney injury to circulating NGAL has been questioned; indeed, other tissues and cells may represent its main source in plasma [5,6,25]. For example, NGAL expression in cardiomyocytes is upregulatedbyseveralfoldinthefailingmyocardiuminresponse tohypoxiaorpro-inflammatoryfactors[26].Accordingly, correla-tionsbetweenplasmaNGALlevelsandCHFseverity,asreflectedby New YorkHeartAssociation class, LVEF,and NT-proBNP levels,

-1.0 1.0 2.0 0.0

Ln[Cr

e

a

tnine

]

(mg

/dL)

2.0 4.0 5.0 3.0

Ln

[eGFR

Crea

]

(mL/min/1.73 m

2

)

-1.0 1.0 2.0 0.0

Ln

[C

ys

ta

n

C

]

(mg

/dL)

2.0 4.0 5.0 3.0

Ln[e

GFR

Cy st

]

(mL/min/1.73 m

2

)

-6.0 -4.0 -3.0 -5.0

Ln[Lp

-PLA

2

]

(μmol/mL/min)

-3.0 1.0 3.0 -1.0

Ln

[sCD40L]

(ng

/

mL)

4.0 8.0 10.0 6.0 3.0 5.0 1.0 2.0 4.0 6.0 8.0 2.0 4.0 6.0 8.0

Ln[N

GAL in plasma

] (ng/mL)

2.0 4.0 6.0 8.0

Ln[N

GAL in plasma

] (ng/mL)

Ln[N

GAL in urine] (ng/mg

crea.)

-1.0 1.0 3.0 5.0 7.0

Ln[NT-pr

oBNP]

(mg

/dL)

Ln[

α

1

-micr

o

globul

in]

(mg

/dL)

r=0.42 p<0.001 r=-0.45 p<0.001 r=0.44 p<0.001 r=-0.44 p<0.001 r=0.31 p<0.01 r=0.22 p<0.05 r=0.31 p<0.01 r=0.21 p<0.05

A

B

C

D

E

F

G

H

Fig.1.Correlations betweenneutrophil gelatinase-associatedlipocalin (NGAL)

concentrationsinserumandurineandrenal,cardiac,andinflammatorymarkersin

everolimus-andcalcineurin-inhibitor(CNI)-treatedhearttransplant recipients.

Correlations between variables were examined using the Spearman rank

correlation test. Non-parametric data are presented after Ln-transformation.

Solidlines:correlation; dottedlines: confidenceintervals. (A)Creatinine; (B)

estimatedglomerularfiltrationrateforcreatinine(eGFRCrea);(C)cystatinC;(D)

estimatedglomerularfiltration ratefor cystatin C(eGFRCyst); (E)

lipoprotein-associatedphospholipaseA2(Lp-PLA2);(F)solubleCD40ligand(sCD40L);(G)

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havebeenreportedpreviouslyandthelatterfindingis recapitu-latedbythefindings ofthepresent study[26–29].In addition, activated inflammatory cells, such as neutrophils and macro-phages,mayrepresentanimportantsourceofcirculatingNGAL. Theclose relationshipbetween NGAL concentrationsin plasma (butnoturine)andindicatorsofsystemicinflammation,suchas tumornecrosisfactor

a

,havebeendescribedindiverse clinical settingsthat include CHF and ischemiccerebrovascular disease [26,29,30]. The presentinvestigation extends thesefindings by showingthenovelassociationbetweenplasmaNGALlevelsand markersofchronicinflammatoryprocesses,suchasLp-PLA2and sCD40L.BothLp-PLA2andsCD40Lareindependentriskpredictors ofcoronaryischemicdisease,inwhichtheyarebelievedtoreflect themagnitudeofmacrophageactivationinatheroscleroticlesions. Inthiscontext,itisworthobservingthatincreasedplasmaNGAL levelshavebeenreportedincoronaryischemicdiseaseandthat NGALcanbedetectedwithinatheroscleroticplaques[31,32].The close relationship between sCD40L, Lp-PLA2, and NGAL levels revealedin thepresent study may indicatecommon upstream mechanisms, which locally regulate these factors during the pathogenesisofatheroscleroticvasculopathyand whichmaybe somewhatdistinctfrommechanismsthatgoverntheexpression ofsystemicinflammationmarkers,suchasIL-6andCRP.

Nodefinitiveconclusioncanbedrawnfromthepresentstudy astowhich mechanisms mightaccount for thelowerlevelsof urinaryandplasmaNGALobservedinhearttransplantrecipients afterconversiontoeverolimus.Althoughitisconceivablethatthe discontinuation of nephrotoxic CNI therapy directly limits the extent of tubular damage, the contribution of other, indirect mechanisms cannot be dismissed. The development of renal impairmentasaconsequenceofdiversepathologicalprocessesin themyocardiumandcoronaryvasculatureculminatingincardiac dysfunction,aconditionknownascardiorenalsyndrome,hasbeen describedinCHF,inwhichitisaccompaniedbyelevatedplasma NGALlevels[33,34].Everolimushasbeenpreviouslydemonstrated topreventendomyocardialremodelingandreducedevelopmentof vascularlesionsinbothhearttransplantrecipientsandinanimal models of CAV and atherosclerosis [35–39]. In addition, this compoundwasfoundtoinhibittheinfiltrationofmacrophages intovascular walland therelease of pro-inflammatory factors, suchaschemokinesandLp-PLA2[39,40].Basedonthesefindings andtheresultsofthepresentstudy,itistemptingtohypothesize that everolimus helps to sustain renal integrity indirectly via limitinginflammatoryprocessesintheheartandthereby counter-acting thedevelopment of cardiac dysfunctionand cardiorenal syndrome.

Severallimitationsofthepresentstudyhavetobe acknowl-edged. Firstly, as the patient recruitment was confined to a singletransplantation center,onlya moderatenumber ofheart transplantrecipientscouldbeincludedinthestudy.Thepowerof thestudyisthereforelimitedandathoroughstatisticalanalysis of confounding factors is precluded. Secondly, the study was performedinacross-sectionaldesignand,withtheexceptionof creatinine, no information was available regarding parameters of renal function prior tothe initiation of everolimus therapy. Accordingly,thepresentfindings donot constituteunequivocal evidence that established NGAL as an early indicator of renal deterioration during CNI-based immunosuppression. Neverthe-less,thepresentfindingsclearlydemonstratereducedplasmaand urinaryNGALlevelsineverolimus-treatedpatientsirrespectiveof the time elapsed from their conversion to CNI-free therapy. Consequently, they mandate the establishment of further pro-spectiveobservationalstudiesthatwillaimtoappraisetheutility ofNGALasamarkerofearlykidneyinjuryinpatientswhoreceive post-transplantimmunosuppressivetherapy.Additionalresearch willalsobenecessarytofullyelucidatethedirectand/orindirect

mechanisms that underlie the beneficial renal effects of ever-olimusinpatientsafterhearttransplantation.

Fundingsources

ThisworkwassupportedbyNovartisPharmaGmbH,Hamburg, Germany(toJ.S.andJ.-R.N.)andintramuralresourcesoftheCenter forLaboratoryMedicine(toJ.-R.N.).

Conflictofinterest

Theauthorsdeclarethattherearenoconflictsofinterest. Acknowledgment

The expert technical assistanceof Elke Boerger is gratefully acknowledged.

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