Associations between microRNA (miR-21, 126, 155 and 221), albuminuria and heavy metals

AssociationsbetweenmicroRNA(miR-21,126,155and221),albuminuriaandheavy

metalsinHongKongChineseadolescents

AliceP.S.Konga,1,KangXiaoa,1,KaiChowChoib,GangWanga,MichaelH.M.Chanc,ChungShunHoc,

IrisChanc,ChunKwokWongc,JulianaC.N.Chana,d,CheukChunSzetoa,⁎

aDepartmentofMedicineandTherapeutics,TheChineseUniversityofHongKong,HongKongSAR,ChinabNethersoleSchoolofNursing,TheChineseUniversityofHongKong,HongKongSAR,ChinacDepartmentofChemicalPathology,TheChineseUniversityofHongKong,HongKongSAR,ChinadLiKaShingInstituteofHealthSciences,TheChineseUniversityofHongKong,HongKongSAR,China

abstractarticleinfo

Articlehistory:Received9January2012Receivedinrevisedform27January2012Accepted16February2012Availableonline1March2012

Keywords:ArsenicLeadCadmiumMercuryAlbuminuriaAdolescentsBackgroundandaim:Pathogeneticmechanismsunderlyingalbuminuriaarenotcompletelyunderstood.Heavymetalsmightleadtoatherosclerosisandkidneydamage.miR-21,126,155and221regulatedendothelialfunctionandmightcontributetothedevelopmentofalbuminuria.Todate,noclinicaltrialhasexploredtherelationshipbetweenmiRNAs,microalbuminuriaandheavymetalsinhuman.Inthisstudy,weaimedtoexaminetheassociationbetweenmicroalbuminuria,miRNAsandheavymetalsinadolescents.Materialsandmethods:Fromacross-sectional,population-recruitedstudy,weidentified60schoolchildrenaged12–19yearswithmicroalbuminuria(definedasspoturinealbumin–creatinineratio>3.5mg/mmol).Wecomparedtheurineheavymetals(arsenic,mercury,cadmiumandlead)andmiRNAslevels(miR-21,126,155and221)withanother60age-andsex-matchednormoalbuminuricadolescentsascontrol.Results:Meanageofthestudycohortwas15.5±2.1years.43%wereboys.AmongthefourmiRNAstested,onlymiR-21wasassociatedwithmicroalbuminuria(p=0.02).UrinaryarsenicandleadlevelshadanegativeassociationwithbothmiR-21andmiR-221.Nosignificantassociationwasfoundbetweenheavymetalsexaminedandmicroalbuminuria.Conclusion:Theresultsofourstudysuggestanassociationbetweenmicroalbuminuria,miR-21andheavymetals(arsenicandlead).ThismightimplythatmiR-21isinvolvedinthepathogeneticmechanismslinkingheavymetalsexposureandalbuminuria.©2012ElsevierB.V.Allrightsreserved.

1.Introduction

Urbanizationandtechnologyhaveledtorapidglobaltransition

andmaycontributetothepandemicofnon-communicablediseases

includingcardiovasculardiseases(CVD)andchronickidneydisease

(CKD).Earlyidentificationofat-riskindividualsisimportantto

preventprogressionofCVDandCKD.Albuminuria,amarkerofvascular

andrenaldamage,isasimple,commonlyusedbiochemicaltoolto

identifyindividualsatriskofdevelopmentofCVDandCKD.

Apartfromchangesinhabitsandlifestyle,exposuretoheavy

metalsisincreasinglyrecognizedasaconsequenceofurbanization.

Mostheavymetalscannotbemetabolizedbyourbody,andexcessive

accumulationinthebodywilldisturbthenormalfunctionsofcells.Kidneyisthekeyorgantoeliminateheavymetalsfromthebody.

Heavymetalsmightleadtoalbuminuriathroughinducingoxidative

stresstorenaltubularcells[1,2].Certainheavymetalshaveadditive

effectininducingnephrotoxicity.Forexample,synergisticeffectof

arsenic(As)andcadmium(Cd)incausingrenaldamagehasbeen

demonstratedinChinesegeneralpopulation[3].Inaddition,chronic

exposuretotoxicheavymetalsmaypromoteatherosclerosisand

contributetothedevelopmentofCKDandCVD[2,4].

MicroRNAs(miRNAs)areendogenous,smallnon-codingRNAs

whichareinvolvedinregulationofgeneexpressionandmanycrucial

biologicalprocesses,includingdevelopment,differentiation,apoptosis,

andproliferation[5].FourmiRNAs,miR-21,miR-126,miR-155and

miR-221,hadbeenreportedtohaveregulatoryfunctionsingene

expressionofvascularproliferationfactorssuchasprogrammedcell

deathprotein4(PDCD4),phosphataseandtensin(PTEN),vascular

endothelialgrowthfactor(VEGF),angiotensinIItype1receptor

(AT1R)andvascularsmoothmusclecell(VSMC)[6–9].Whetherthese

fourmiRNAsareinvolvedinthepathogeneticprocessesofalbuminuria

throughtheirrolesinregulatinggeneexpressionsinvasculaturehave

notbeenexplored.Itisplausiblethatheavymetalsinduced

theClinicaChimicaActa413(2012)1053–1057

⁎Correspondingauthorat:DepartmentofMedicineandTherapeutics,TheChineseUniversityofHongKong,PrinceofWalesHospital,Shatin,N.T.,HongKongSAR,China.Tel.:+85226323146;fax:+85226373852.E-mailaddress:ccszeto@cuhk.edu.hk(C.C.Szeto).1APSKandKX:co-firstauthorofthe

manuscript.

0009-8981/$–seefrontmatter©2012ElsevierB.V.Allrightsreserved.doi:

10.1016/j.cca.2012.02.014ContentslistsavailableatSciVerseScienceDirect

ClinicaChimicaActa

journalhomepage:www.elsevier.com/locate/clinchimdevelopmentofalbuminuriathroughdamagetothevascularfunctions

viathesemiRNAs.

Againstthisbackground,weconductedthisstudyaimingto

examinetheassociationsbetweenmicroalbuminuria,urinarymiRNA,

namelymiR-21,miR-126,miR-155andmiR-221whichhadknown

regulatoryroleinvascularfunctions,andheavymetalslevelsina

population-recruitedcohortofHongKongChineseadolescents.

2.Subjectsandmethod

2.1.Studypopulation

SubjectswererecruitedfromtheHongKongSchoolChildren

Projectconductedin2003andthemethodologyhadbeenpreviously

described[10,11].Inbrief,itwasacross-sectional,population-

recruitedstudyincluding2115HongKongChineseadolescentsaged

12–19yearswhowererandomlyselectedfromallsecondaryschools

inHongKong.Onlyhealthyschoolchildrenwithoutanyknownmedical

illnessesoronlong-termmedicationswereinvitedtoparticipateinthis

study.Informedwrittenconsentsfromboththeparticipantsandtheir

parentsorguardiansweresoughtbeforetheyenteredthestudy.The

studywasapprovedbytheClinicalResearchEthicsCommitteeofthe

ChineseUniversityofHongKong.

Ateamoftrainedresearchnursesandresearchassistants

measuredtheanthropometricparametersandsampledbloodand

urinespecimensfromtheparticipantsintheschool.Anthropometric

indices,includingbodyweight(kg),height(m),percentageofbody

fatbybioimpedance,waistandhipcircumstances,weremeasured.

Bloodpressure(BP)wasmeasuredbyOmronbloodpressuredevice

(OmronHealthcareInc.,Tokyo,Japan).Eachstudenthadrestedfor

morethan5minandtheaverageoftworeadingswasusedforthe

analysis.

Afterfastingforatleast8h,bloodsampleswerecollectedandspot

morningurinesampleswerealsocollected.Plasmaglucose(PG)and

lipidprofileincludingtotalcholesterol(TC),triglyceride(TG),high

densitylipoproteincholesterol(HDL-C)andlowdensitylipoprotein

cholesterol(LDL-C)wereassayed.UrinarymiRNAsandheavymetals

levelsweremeasuredfromthestoredurinealiquots.

Wedefinedmicroalbuminuriaasspoturinealbumincreatinine

ration(ACR)>3.5mg/mmol[10,12].Amongaboutonetenthof

adolescentsinthetotalcohortwithmicroalbuminuria[10],60urine

aliquotswithmicroalbuminuriawereavailableforthisstudy.We

identifiedage-andsex-matchedcontrols(i.e.withnormalurine

ACRlevels)fromthesamecohortforcomparison.

2.2.Laboratoryassays

Serumandurinarycreatinine(Jaffekineticmethod),aswellas

urinaryalbumin(immunoturbidimetrymethod)weremeasuredby

DPModularAnalytics(RocheDiagnosticsCorp,Indianapolis,IN,

USA).Theurinesampleswerecentrifugedat3000gfor30minat

4°C.Weused400μLsupernatantfortotalRNAisolationbythe

mirVanaTMPARISTMKit(Ambion,Inc.Austin,TX,USA)according

tothemanufacturer'sinstruction.Then60μLRNAwascollectedand

storedat−80°Cuntilreversetranscription.

TaqMan®miRNAreversetranscriptionKit(AppliedBiosystems,

FosterCity,CA,USA)wasusedforreversetranscription.Inbrief,

1.67μLtotalRNAwasmixedwith1μLspecificprimers,0.05μL

100mMdNTPs(withdTTP),0.5μL10×reversetranscriptionbuffer,

0.33μL(50U)MultiScribe™ReverseTranscriptase,0.05μLRNase

inhibitor(20U/μL)andmadeupto5μLwithH2O.Reversetranscription

(RT)wasperformedat16°Cfor30min,42°Cfor30minand85°Cfor

5min.TheresultingcDNAwasstoredin−80°Cuntiluse.

ForRT-QPCR(reversetranscription-quantitativepolymerase

chainreaction),weadded0.25μLprimer,0.33μLRTproduction,

2.5μLTaqManUniversalPCRmastermixand1.92μLnuclease-freewatertogethertoget5μLfinalreactionvolume.Theexpressionof

miR-21,miR-126,miR-155,andmiR-221werequantifiedbyRT-

QPCR.ABIPrism7900SequenceDetectionSystem(AppliedBiosystems,

FosterCity,CA)wasusedfortheRT-QPCRreaction.Allprimersand

probesarecommerciallyavailablefromthemanufacturer.Eachsample

wasrunintriplicate.RT-QPCRwasperformedat50°Cfor2min,95°C

for10min,followedby40cyclesat95°Cfor15sand60°Cfor1min.

ThesmallnucleolarRNARNU48(AppliedBiosystems)wasusedas

housekeepinggenetonormalizethemiRNAexpression.Datawere

analyzedwithSDSrelativequantificationsoftwareversion2.2.2

(AppliedBiosystems).Thesamebaselineandthresholdofthreshold

cycleweresetforeachtarget.

Arsenic(As),mercury(Hg),cadmium(Cd)andlead(Pb)levelsin

urinewereanalyzedinstoredurinesamplesusingthestate-of-the-

artinductivelycoupledplasmamassspectrometry(ICPMS)technique

(ICPMS7500c,AgilentTechnologies,Tokyo,Japan)[13].

2.3.Statisticalanalysis

Continuousvariableswithskewedandnormallikedistributionwere

presentedasmedians(inter-quartileranges)andmeans(standard

deviations)respectively.Categoricaldatawerepresentedascounts

andpercentages.Thenormalityofthecontinuousvariableswas

assessedusingskewnessstatisticandnormalQ–Qplotgraphically.

BMIandTGwerepositivelyskewedandthereforelog-transformedto

correcttheirskewnessbeforesubjectedtostatisticalanalysis.

Log-transformationofmiRNAdatadidnotrendertheirnormalitysatis-

factorily;theywereanalyzedwithnon-parametricmethods.Since

undetectableurineheavymetallevelwassettozero,theurinelevels

ofthefourheavymetalswerethusagainanalyzedwithnon-

parametricmethods.Thecomparisonsbetweenthenormoalbuminuric

andalbuminuricgroupsondemographicandclinicalcharacteristics,

cardiovascularriskfactors,miRNAdataandurineheavymetalswere

doneusingt-test,Mann–Whitneytest,PearsonChi-squaretestand

Fisher'sexacttest,asappropriate.Inter-correlationsbetweencardiovas-

cularriskfactors,ACR,miRNAdataandurineheavymetalswere

assessedbyPearsonorSpearmancorrelationcoefficients,depending

onwhethermiRNAdataandurineheavymetalswereinvolvedor

not.AllstatisticalanalyseswereperformedusingSPSS17.0(SPSSInc.,

Chicago,IL).Allstatisticaltestsweretwo-sidedandapvalueb0.05

wasconsideredstatisticallysignificant.

3.Results

Baselineclinicaldemographicdataofthealbuminuricandcontrol

groupsaresummarizedandcomparedinTable1.Inshort,therewas

nosignificantdifferencebetweenanybaselinebiochemicalnor

anthropometriccharacteristicsbetweenthealbuminuricandcontrol

groups.

3.1.RelationbetweenmicroalbuminuriaandurinarymiRNA

UrinarymiRNAlevelswerecomparedbetweenalbuminuricand

controlgroupsandsummarizedinFig.1.Wefoundthaturinary

miR-21levelwassignificantlyhigherinthealbuminuricthanthe

controlgroup(3234.3[772.8–5248.7]versus1028.7[265.6–2999.6]

copyper100,000copiesofthehousekeepinggene,p=0.020).Similarly,

urinarymiR-221levelwasmarginallyhigherinthealbuminuricthan

thecontrolgroup(102.1[29.2–324.9]versus66.6[27.8–129.5]copy

per100,000copiesofthehousekeepinggene,p=0.077),buttheresult

didnotreachstatisticalsignificance.Incontrast,urinarymiR-126and

miR-155levelsweresimilarbetweenthealbuminuricandcontrol

groups.Theactuallevelofurinaryalbuminexcretionalsocorrelated

withtheurinarylevelofmiR-21(Spearman'sr=0.223,p=0.015),

butnotmiR-221(r=0.155,p=0.09),miR-126(r=0.040,p=0.7)or

miR-155(r=0.081,p=0.4).1054A.P.S.Kongetal./ClinicaChimicaActa413(2012)1053–1057