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
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 inm
molmin 1mL 1.TheiCVswere5.1%foralowcontroland 3.8%foracontrolclosetothesamplemean.SolubleCD40ligand (sCD40L) concentrations in plasma were determined using a commerciallyavailableELISAkit(IBL,Hamburg,Germany). StatisticalanalysisExploratory statistics were performed using the MedCalc StatisticalSoftwareversion12.7.7(MedCalcSoftwarebvba,Ostend, Belgium). The level of dependence between two categorical
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 significantlylowerplasmaNGALconcentrationsandurinaryNGAL/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;
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 anda
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 3.0Ln
[eGFR
Crea]
(mL/min/1.73 m
2)
-1.0 1.0 2.0 0.0Ln
[C
ys
ta
n
C
]
(mg
/dL)
2.0 4.0 5.0 3.0Ln[e
GFR
Cy st]
(mL/min/1.73 m
2)
-6.0 -4.0 -3.0 -5.0Ln[Lp
-PLA
2]
(μmol/mL/min)
-3.0 1.0 3.0 -1.0Ln
[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 8.0Ln[N
GAL in plasma
] (ng/mL)
2.0 4.0 6.0 8.0Ln[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<0.05A
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)
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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