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

ClinicaChimicaActa413(2012)1053–1057

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ClinicaChimicaActa

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

AliceP.S.Konga,1,KangXiaoa,1,KaiChowChoib,GangWanga,MichaelH.M.Chanc,ChungShunHoc,IrisChanc,ChunKwokWongc,JulianaC.N.Chana,d,CheukChunSzetoa,

a

DepartmentofMedicineandTherapeutics,TheChineseUniversityofHongKong,HongKongSAR,ChinaNethersoleSchoolofNursing,TheChineseUniversityofHongKong,HongKongSAR,Chinac

DepartmentofChemicalPathology,TheChineseUniversityofHongKong,HongKongSAR,Chinad

LiKaShingInstituteofHealthSciences,TheChineseUniversityofHongKong,HongKongSAR,China

b

articleinfoabstract

Backgroundandaim:Pathogeneticmechanismsunderlyingalbuminuriaarenotcompletelyunderstood.Heavymetalsmightleadtoatherosclerosisandkidneydamage.miR-21,126,155and221regulatedendothelialfunctionandmightcontributetothedevelopmentofalbuminuria.Todate,noclinicaltrialhasexploredtherelationshipbetweenmiRNAs,microalbuminuriaandheavymetalsinhuman.Inthisstudy,weaimedtoexaminetheassociationbetweenmicroalbuminuria,miRNAsandheavymetalsinadolescents.Materialsandmethods:Fromacross-sectional,population-recruitedstudy,weidenti ed60schoolchildrenaged12–19yearswithmicroalbuminuria(de nedasspoturinealbumin–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.Nosigni cantassociationwasfoundbetweenheavymetalsexaminedandmicroalbuminuria.

Conclusion:Theresultsofourstudysuggestanassociationbetweenmicroalbuminuria,miR-21andheavymetals(arsenicandlead).ThismightimplythatmiR-21isinvolvedinthepathogeneticmechanismslinkingheavymetalsexposureandalbuminuria.

©2012ElsevierB.V.Allrightsreserved.

Articlehistory:

Received9January2012

Receivedinrevisedform27January2012Accepted16February2012Availableonline1March2012Keywords:ArsenicLead

CadmiumMercuryAlbuminuriaAdolescents

1.Introduction

Urbanizationandtechnologyhaveledtorapidglobaltransitionandmaycontributetothepandemicofnon-communicablediseasesincludingcardiovasculardiseases(CVD)andchronickidneydisease(CKD).Earlyidenti cationofat-riskindividualsisimportanttopreventprogressionofCVDandCKD.Albuminuria,amarkerofvascularandrenaldamage,isasimple,commonlyusedbiochemicaltooltoidentifyindividualsatriskofdevelopmentofCVDandCKD.

Apartfromchangesinhabitsandlifestyle,exposuretoheavymetalsisincreasinglyrecognizedasaconsequenceofurbanization.Mostheavymetalscannotbemetabolizedbyourbody,andexcessiveaccumulationinthebodywilldisturbthenormalfunctionsofcells.

Correspondingauthorat:DepartmentofMedicineandTherapeutics,TheChineseUniversityofHongKong,PrinceofWalesHospital,Shatin,N.T.,HongKongSAR,China.Tel.:+85226323146;fax:+85226373852.

E-mailaddress:ccszeto@cuhk.edu.hk(C.C.Szeto).1

APSKandKX:co- rstauthorofthe

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

10.1016/a.2012.02.014

Kidneyisthekeyorgantoeliminateheavymetalsfromthebody.Heavymetalsmightleadtoalbuminuriathroughinducingoxidativestresstorenaltubularcells[1,2].Certainheavymetalshaveadditiveeffectininducingnephrotoxicity.Forexample,synergisticeffectofarsenic(As)andcadmium(Cd)incausingrenaldamagehasbeendemonstratedinChinesegeneralpopulation[3].Inaddition,chronicexposuretotoxicheavymetalsmaypromoteatherosclerosisandcontributetothedevelopmentofCKDandCVD[2,4].

MicroRNAs(miRNAs)areendogenous,smallnon-codingRNAswhichareinvolvedinregulationofgeneexpressionandmanycrucialbiologicalprocesses,includingdevelopment,differentiation,apoptosis,andproliferation[5].FourmiRNAs,miR-21,miR-126,miR-155andmiR-221,hadbeenreportedtohaveregulatoryfunctionsingeneexpressionofvascularproliferationfactorssuchasprogrammedcelldeathprotein4(PDCD4),phosphataseandtensin(PTEN),vascularendothelialgrowthfactor(VEGF),angiotensinIItype1receptor(AT1R)andvascularsmoothmusclecell(VSMC)[6–9].WhetherthesefourmiRNAsareinvolvedinthepathogeneticprocessesofalbuminuriathroughtheirrolesinregulatinggeneexpressionsinvasculaturehavenotbeenexplored.Itisplausiblethatheavymetalsinducedthe

1054A.P.S.Kongetal./ClinicaChimicaActa413(2012)1053–1057

developmentofalbuminuriathroughdamagetothevascularfunctionsviathesemiRNAs.

Againstthisbackground,weconductedthisstudyaimingtoexaminetheassociationsbetweenmicroalbuminuria,urinarymiRNA,namelymiR-21,miR-126,miR-155andmiR-221whichhadknownregulatoryroleinvascularfunctions,andheavymetalslevelsinapopulation-recruitedcohortofHongKongChineseadolescents.2.Subjectsandmethod2.1.Studypopulation

SubjectswererecruitedfromtheHongKongSchoolChildrenProjectconductedin2003andthemethodologyhadbeenpreviouslydescribed[10,11].Inbrief,itwasacross-sectional,population-recruitedstudyincluding2115HongKongChineseadolescentsaged12–rmedwrittenconsentsfromboththeparticipantsandtheirparentsorguardiansweresoughtbeforetheyenteredthestudy.ThestudywasapprovedbytheClinicalResearchEthicsCommitteeoftheChineseUniversityofHongKong.

Ateamoftrainedresearchnursesandresearchassistantsmeasuredtheanthropometricparametersandsampledbloodandurinespecimensfromtheparticipantsintheschool.Anthropometricindices,includingbodyweight(kg),height(m),percentageofbodyfatbybioimpedance,waistandhipcircumstances,weremeasured.Bloodpressure(BP)wasmeasuredbyOmronbloodpressuredevice(OmronHealthcareInc.,Tokyo,Japan).Eachstudenthadrestedformorethan5minandtheaverageoftworeadingswasusedfortheanalysis.

Afterfastingforatleast8h,bloodsampleswerecollectedandspotmorningurinesampleswerealsocollected.Plasmaglucose(PG)andlipidpro leincludingtotalcholesterol(TC),triglyceride(TG),highdensitylipoproteincholesterol(HDL-C)andlowdensitylipoproteincholesterol(LDL-C)wereassayed.UrinarymiRNAsandheavymetalslevelsweremeasuredfromthestoredurinealiquots.

Wede nedmicroalbuminuriaasspoturinealbumincreatinineration(ACR)>3.5mg/mmol[10,12].Amongaboutonetenthofadolescentsinthetotalcohortwithmicroalbuminuria[10],60urinealiquotswithmicroalbuminuriawereavailableforthisstudy.Weidenti edage-andsex-matchedcontrols(i.e.withnormalurineACRlevels)boratoryassays

Serumandurinarycreatinine(Jaffekineticmethod),aswellasurinaryalbumin(immunoturbidimetrymethod)weremeasuredbyDPModularAnalytics(RocheDiagnosticsCorp,Indianapolis,IN,USA).Theurinesampleswerecentrifugedat3000gfor30minat4°C.Weused400μLsupernatantfortotalRNAisolationbythemirVanaTMPARISTMKit(Ambion,Inc.Austin,TX,USA)accordingtothemanufacturer'sinstruction.Then60μLRNAwascollectedandstoredat 80°Cuntilreversetranscription.

TaqMan®miRNAreversetranscriptionKit(AppliedBiosystems,FosterCity,CA,USA)wasusedforreversetranscription.Inbrief,1.67μLtotalRNAwasmixedwith1μLspeci cprimers,0.05μL100mMdNTPs(withdTTP),0.5μL10×reversetranscriptionbuffer,0.33μL(50U)MultiScribe ReverseTranscriptase,0.05μLRNaseinhibitor(20U/μL)andmadeupto5μLwithH2O.Reversetranscription(RT)wasperformedat16°Cfor30min,42°Cfor30minand85°Cfor5min.TheresultingcDNAwasstoredin 80°Cuntiluse.

ForRT-QPCR(reversetranscription-quantitativepolymerasechainreaction),weadded0.25μLprimer,0.33μLRTproduction,2.5μLTaqManUniversalPCRmastermixand1.92μLnuclease-free

watertogethertoget5μL nalreactionvolume.TheexpressionofmiR-21,miR-126,miR-155,andmiR-221werequanti edbyRT-QPCR.ABIPrism7900SequenceDetectionSystem(AppliedBiosystems,FosterCity,CA)wasusedfortheRT-QPCRreaction.Allprimersandprobesarecommerciallyavailablefromthemanufacturer.Eachsamplewasrunintriplicate.RT-QPCRwasperformedat50°Cfor2min,95°Cfor10min,followedby40cyclesat95°Cfor15sand60°Cfor1min.ThesmallnucleolarRNARNU48(AppliedBiosystems)wasusedashousekeepinggenetonormalizethemiRNAexpression.DatawereanalyzedwithSDSrelativequanti cationsoftwareversion2.2.2(AppliedBiosystems).Thesamebaselineandthresholdofthresholdcycleweresetforeachtarget.

Arsenic(As),mercury(Hg),cadmium(Cd)andlead(Pb)levelsinurinewereanalyzedinstoredurinesamplesusingthestate-of-the-artinductivelycoupledplasmamassspectrometry(ICPMS)technique(ICPMS7500c,AgilentTechnologies,Tokyo,Japan)[13].2.3.Statisticalanalysis

Continuousvariableswithskewedandnormallikedistributionwerepresentedasmedians(inter-quartileranges)andmeans(standarddeviations)respectively.Categoricaldatawerepresentedascountsandpercentages.ThenormalityofthecontinuousvariableswasassessedusingskewnessstatisticandnormalQ–Qplotgraphically.BMIandTGwerepositivelyskewedandthereforelog-transformedtocorrecttheirskewnessbeforesubjectedtostatisticalanalysis.Log-transformationofmiRNAdatadidnotrendertheirnormalitysatis-factorily;theywereanalyzedwithnon-parametricmethods.Sinceundetectableurineheavymetallevelwassettozero,theurinelevelsofthefourheavymetalswerethusagainanalyzedwithnon-parametricmethods.Thecomparisonsbetweenthenormoalbuminuricandalbuminuricgroupsondemographicandclinicalcharacteristics,cardiovascularriskfactors,miRNAdataandurineheavymetalsweredoneusingt-test,Mann–Whitneytest,PearsonChi-squaretestandFisher'sexacttest,asappropriate.Inter-correlationsbetweencardiovas-cularriskfactors,ACR,miRNAdataandurineheavymetalswereassessedbyPearsonorSpearmancorrelationcoef cients,dependingonwhethermiRNAdataandurineheavymetalswereinvolvedornot.AllstatisticalanalyseswereperformedusingSPSS17.0(SPSSInc.,Chicago,IL).Allstatisticaltestsweretwo-sidedandapvalueb0.05wasconsideredstatisticallysigni cant.3.Results

BaselineclinicaldemographicdataofthealbuminuricandcontrolgroupsaresummarizedandcomparedinTable1.Inshort,therewasnosigni cantdifferencebetweenanybaselinebiochemicalnoranthropometriccharacteristicsbetweenthealbuminuricandcontrolgroups.

3.1.RelationbetweenmicroalbuminuriaandurinarymiRNA

UrinarymiRNAlevelswerecomparedbetweenalbuminuricandcontrolgroupsandsummarizedinFig.1.WefoundthaturinarymiR-21levelwassigni cantlyhigherinthealbuminuricthanthecontrolgroup(3234.3[772.8–5248.7]versus1028.7[265.6–2999.6]copyper100,000copiesofthehousekeepinggene,p=0.020).Similarly,urinarymiR-221levelwasmarginallyhigherinthealbuminuricthanthecontrolgroup(102.1[29.2–324.9]versus66.6[27.8–129.5]copyper100,000copiesofthehousekeepinggene,p=0.077),buttheresultdidnotreachstatisticalsigni cance.Incontrast,urinarymiR-126andmiR-155levelsweresimilarbetweenthealbuminuricandcontrolgroups.TheactuallevelofurinaryalbuminexcretionalsocorrelatedwiththeurinarylevelofmiR-21(Spearman'sr=0.223,p=0.015),butnotmiR-221(r=0.155,p=0.09),miR-126(r=0.040,p=0.7)ormiR-155(r=0.081,p=0.4).

A.P.S.Kongetal./ClinicaChimicaActa413(2012)1053–1057

Table1

Clinicalandbiochemicalcharacteristicsofthestudycohort.

Albuminuricgroup

NumberofcaseSex(M:F)Age(years)

UrineACR(mg/mmol) Weight(kg)Height(cm)

BodyMassIndex(kg/m2) Waistcircumference(cm)Hipcircumference(cm)Waisttohipratio

Bloodpressure(mmHg)SystolicDiastolic

Serumcreatinine(μmol/L)HDL-cholesterol(mmol/L)LDL-cholesterol(mmol/L)Triglyceride(mmol/L) FPG(mmol/L)

6026:3415.5(2.1)

7.49(5.58–16.75)48.7(9.7)160.7(8.6)

18.4(17.3–19.8)65.1(5.8)86.6(7.0)0.75(0.04)114.0(13.2)71.2(9.8)71.4(14.9)1.7(0.3)2.3(0.6)0.7(0.6–1.0)4.7(0.3)

Controlgroup6026:3415.5(2.1)

0.43(0.30–0.82)50.9(10.3)160.2(8.5)

19.3(18.0–21.2)67.1(7.6)88.4(7.1)0.76(0.05)115.2(13.8)73.8(10.3)73.7(13.6)1.7(0.4)2.2(0.5)0.7(0.6–1.0)4.7(0.3)

pvalue–––

p=0.23p=0.79p=0.07p=0.10p=0.17p=0.39p=0.63p=0.15p=0.38p=0.45p=0.56p=0.76p=0.32

No.ofpatientArsenic(nmol)Mercury(nmol)Cadmium(nmol)Lead(nmol)

Table2

Comparisonofurinaryheavymetallevelsbetweengroups.

Albuminuricgroup6054.20.260.180.36

(32.8–102.8)(0.10–0.60)(0.13–0.29)(0.25–0.54)

Controlgroup6059.40.290.200.41

(36.1–117.0)(0.19–0.52)(0.16–0.27)(0.28–0.52)

1055

pvaluep=0.60p=0.71p=0.83p=0.70

Dataarepresentedinconcentrationpermmolofcreatinine,asmedian(inter-quartilerange),andcomparedusingMann–WhitneyUtest.

Datamarkedwith werepresentedasmedian(inter-quartilerange);allotherswerepresentedasmean(standarddeviation)andcomparedbyStudent'sttest.ACR,albumin–creatinineratio;FPG,fastingplasma

glucose.

Weobservedsigni cantcorrelationsbetweentheurinarylevelofsomeheavymetalsandmiRNAtargetsinthetotalcohort(Table3).Speci cally,urinaryarseniclevelsigni cantlycorrelatedwithurinarylevelofmiR-21(r= 0.288,p=0.001)andmiR-221(r= 0.253,p=0.005);urinaryleadlevelalsocorrelatedwithurinarylevelofmiR-21(r= 0.184,p=0.048)andmiR-221(r= 0.303,p=0.001)(Table3).4.Discussion

Fromtheresultsofthisstudy,wehadfoundthaturinarymiR-21wasassociatedwithmicroalbuminuria,andmiR-21wasassociatedwithurinaryarsenicandleadlevels.Toourknowledge,ourstudyisthe rstclinicalstudytoexaminetherelationshipofmiRNAs,micro-albuminuriaandheavymetalsinhuman.

Fromourresults,therewerehigherlevelsofmiR-21inthemicro-albuminuricgroupcomparedtothenormoalbuminuricgroup,signi-fyingtheincreasedexpressionofmiR-21inschoolchildrenwithmicroalbuminuria.miR-21waspostulatedtobeprotectiveagainstthedevelopmentofalbuminuriabecauseitmightinhibitapoptosis

3.2.Urinaryheavymetal

UrinaryheavymetallevelswerecomparedbetweenalbuminuricandcontrolgroupsandsummarizedinTable2.Inshort,therewasnosigni cantdifferenceinurinaryarsenic,mercury,cadmium,orleadlevelbetweenalbuminuricandcontrolgroups.Similarly,theactuallevelofurinaryalbuminexcretiondidnotcorrelatewiththeurinarylevelofanyoftheheavymetal(detailsnotshown).

parisonofurinarymiRNAlevelsbetweenalbuminuricgroupandcontrolgroup:(A)miR-21;(B)miR-126;(C)miR-155;and(D)miR-221.DataarecomparedbyMann–WhitneyUtest.

1056A.P.S.Kongetal./ClinicaChimicaActa413(2012)1053–1057

Table3

RelationbetweenurinaryheavymetalandmiRNAlevelsinthetotalcohort.a

Arsenic

MercuryCadmiumLeadmiR-21r= 0.288,r= 0.177,r= 0.098,r= 0.184,p=0.001p=0.08p=0.29p=0.048miR-126r= 0.128,r= 0.146,r= 0.168,r= 0.037,p=0.16p=0.15p=0.07p=0.70miR-155r=0.049,r= 0.006,r= 0.094,r= 0.118,p=0.59p=0.95p=0.31p=0.21miR-221

r= 0.253,r= 0.181,r= 0.114,r= 0.303,p=0.005

p=0.075

p=0.22

p=0.001

a

Spearman'scorrelationcoef cientsarepresented.

ofpodocytes[14].miR-21alsoinhibitsmesangialcellsproliferationinanimalmodelandinvitroexperiments[15].Contrarytotheresultsfromanimalstudy[15],inwhichreducedexpressionofmiR-21indb/dbdiabeticnephropathy(DN)micewerefound,ourresultsinhumansubjectsfoundthatadolescentswithmicroalbuminuriahadincreasedexpressionofmiR-21levelcomparedtonormoalbuminuriccontrol.OurresultssuggestthatmiR-21mightbeinvolvedinthepathogeneticmechanismsofalbuminuria.Sincemorethan1000miRNAshavebeenidenti edinhumanbeings,itisplausiblethatothermiRNAs,ratherthanthe4testedinourexperiments,areinvolvedintheprocessofdevelopmentofalbuminuria.Forexamples,miR-216aandmiR-217alsotargetPTENandmaybetheplayersinlinkingalbuminuria,CKDandCVD[16].OtherexampleincludesmiR-377whichdecreasestheexpressionofSuperoxidedismutase(SOD)1,SOD2andp21-Activatedproteinkinase(PAK)1,andinturnleadstoenhancedsusceptibilitytooxidantstressandaccumulationoftheextracellularmatrixproteinand bronectin[17].

Arsenicmightdecreasekidneyfunctionbyinducingoxidativestressinkidney.Inanimalexperiment,micewithsubchronicexposuretoarsenichadoxidativedamagetoDNAinkidneytissue,especiallyintheproximaltubularcellsandBowman'scapsule,andtheseledtomore8-OHdGexpression[18].Ithadalsobeendemonstratedin290adults(86menand204women)thaturinaryarseniclevelwaspositivelycorrelatedwithbothkidneybiomarkeroftoxicity,suchasβ2-microglobulinandN-acetyl-β-D-glucosaminidase(NAG),andoxidativestressindices,suchasurinarymalondialdehyde(MDA)and8-hydroxy-2′-deoxyguanosine(8-OHdG)[1].Inacase-control,hospital-basedstudyinTaiwan,including125CKDpatientsand229controls,urinaryarseniclevelwasfoundtopositivelyassociatedwithCKD[19].Contrarytothesepreviousreportedstudies,ourresultsdidnotshowanysigni paringmicroalbuminurictonormoalbuminuricgroup,therewasnodifferencebetweenurinaryarseniclevels.Wealsodidnot ndanysigni cantassociationbetweenmicroalbuminuriaandotherheavymetalsincludingmercury,leadandcadmium.Indeed,theassoci-ationbetweenmercuryandalbuminuriawasunclearandtheresultsfrompreviouspublishedliteratureswerecon icting.Ournegativeresultsareinkeepingwiththenegative ndingsfromotherpublishedstudies[20].Occupationalexposuretomercuryhadinsigni cantimpactonurinealbuminexcretion[20].Among89chloralkaliworkerswhohadbeenexposedtomercuryvaporand75controls,whosemedianurinarymercurylevelsbeing14.3nmol/mmolcreatinine(25.4μg/gcreatinine)and1.1nmol/mmolcreatinine(1.9μg/gcreatinine)respec-tively,theirurinaryalbuminlevelshadnodifference[20].Incontrast,someresearchfoundanassociationbetweenurinarymercuryandurinaryalbuminlevels[21].Dentalamalgamisasourceofmercuryexposure.Inaresearchinvolving534childrenaged6–10years,childreninthedentalamalgamgrouphadasigni cantlyincreasedprevalenceofmicroalbuminuriacomparedtochildrenwithoutpriordentalamalgamrestorations[22].Apositiveassociationbetweenleadandalbuminuriahadpreviouslybeendescribed[23].InNHANES(1999–2006),among14,778participantsanalyzed,comparedsubjects

fromthetopquartiles(>2.4μg/dL,>1.6–2.4μg/dL,>1.1–1.6μg/dL)tothelowestquartiles(b1.1μg/dL)ofbloodleadlevels,theadjustedoddratiosforthesubjectstohavealbuminuria(≥30mg/gcreatinine)were1.19(95%CI=0.96–1.47),0.92(95%CI=0.76–1.12),0.83(95%CI=0.66–1.04)respectively[23].Ournegative ndingoftheassociationbetweenurinarycadmiumandmicroalbuminuriaechoeswiththe ndingofnon-diabeticsubjectsinanAustralianstudy[24].However,somestudiesreportedapositiveassociationbetweencadmiumandalbuminuria.InNHANES(1999–2006)involving14,778participants,comparedsubjectsfromthetopquartiles(>0.6μg/L,>0.4–0.6μg/L,>0.2–0.4μg/L)tothelowestquartileofbloodcadmiumlevels(≤0.2μg/L),theadjustedoddratiosforthesubjectstohavealbuminuriawere1.92(95%CI=1.53–2.43),1.32(95%CI=1.07–1.64),1.10(95%CI=0.89–1.36)respectively[23].AccordingtoanAustralianstudy(n=182,43withdiabetes,139withoutdiabetes,37.19±0.92yearsofage),amongpeoplewithdiabetes,urinarycadmiumlevelwasassociatedwithalbuminuria,whileinpeoplewithoutdiabetes,therewasnosuchassociation[24].InastudyinChina,peoplewhoworkedandlivedincadmium-pollutedareahadhigherurinecadmiumlevel(11.86μg/gcreatinine,range:1.69–55.72)andhigheralbumin(6.4mg/gcreatinine,range:1.1–85.3)comparingtopeopleinnon–cadmium-pollutedarea(urinecadmium1.81μg/gcreatinine,range:0.09–5.72;albumin2.9mg/gcreatinine,range:0.1–37.8)[25].Inanotherstudyof361peopleagedfrom6to74years,forchildrenagedfrom6to17years,urinaryalbuminlevelwasassociatedwithurinecadmium.However,afteradjustedforcreatinineandotherpoten-tialconfounders,theseassociationbecameinsigni cant,exceptforadultsaged18yearsorolder[26].

Severallimitationsofthisstudyhavetobeaddressed.First,ourstudysubjectswerecommunityrecruitedanddidnothavetoxicrangeofurinaryheavymetallevels.Therefore,itmightbecauseoftheselowlevelsofexposuretoheavymetals,noclinicallysigni cantalterationinkidneyfunctionisfoundortherenaleffectistoosubtletobedetected.Second,stly,thecross-sectionaldesignofthisstudyprecludestheexam-inationofcausalrelationshipsbetweenalbuminuria,miRNAstargetsandurinaryheavymetallevels.

Inconclusion,fromthiscase-controlcohortrecruitedfromapopulationwidesurvey,wefoundthatmiR-21wasassociatedwithmicroalbuminuria.IncreasedexpressionofmiR-21levelinthemicro-albuminuricgroupmightbethereactionsecondarytothedevelop-mentofmicroalbuminuria.Furthermore,miR-21wasalsoassociatedwithurinaryarsenicandleadlevels.TheresultsmightimplythatmiR-21isinvolvedinthepathogeneticmechanismslinkingheavymetalsexposureandmicroalbuminuria.Furtherstudiesandexperi-mentsarerequiredtoexaminethecausalrelationshipbetweenmiR-21,microalbuminuriaandheavymetals.Con ictofinterest

Allauthorsdeclarenocon ictofinterest.Acknowledgements

Wethankallschoolpersonnel,parentsandparticipantsformakingthisstudypossible.ThisstudywassupportedbyfundingfromtheResearchGrantCommittee(CUHK4055/01MandCUHK4465/06M),LiKaShingInstituteofHealthScienceandHongKongInstituteofDiabetesandObesity,undertheauspicesofTheChinese

A.P.S.Kongetal./ClinicaChimicaActa413(2012)1053–10571057

UniversityofHongKong.Theresultspresentedinthispaperhavenotbeenpublishedpreviouslyinwholeorpart,exceptinabstractformat.References

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