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TheTuberin/mTORPathwayPromotesApoptosisofTubularEpithelialCellsinDiabetes

ChakradharVelagapudi,*BasantS.Bhandari,*SherryAbboud-Werner,†SimonaSimone,*‡HannaE.Abboud,*andSamyL.Habib*§Departmentsof*Medicineand†Pathology,UniversityofTexasHealthScienceCenter,SanAntonio,Texas;‡DepartmentofEmergencyandTransplantation,UniversityofBari,Policlinico,Bari,Italy;and§GeriatricResearch,Education,andClinicalCenter,SouthTexasVeteransHealthcareSystem,SanAntonio,Texas

ABSTRACT

Apoptosiscontributestothedevelopmentofdiabeticnephropathy,butthemechanismbywhichhighglucose(HG)inducesapoptosisisnotfullyunderstood.Becausethetuberin/mTORpathwaycanmodulateapoptosis,westudiedtheroleofthispathwayinapoptosisintypeIdiabetesandinculturedproximaltubularepithelial(PTE)cellsexposedtoHG.Comparedwithcontrolrats,diabeticratshadmoreapoptoticcellsinthekidneycortex.InductionofdiabetesalsoincreasedphosphorylationoftuberininassociationwithmTORactivation(measuredbyp70S6Kphosphorylation),inactivationofBcl-2,increasedcytosoliccytochromecexpression,activationofcaspase3,andcleavageofPARP;insulintreatmentpreventedthesechanges.Invitro,exposureofPTEcellstoHGincreasedphosphorylationoftuberinandp70S6K,phosphorylationofBcl-2,expressionofcytosoliccytochromec,andcaspase3activity.HighglucoseinducedtranslocationofthecaspasesubstrateYY1fromthecytoplasmtothenucleusandenhancedcleavageofPARP.PretreatmentthecellswiththemTORinhibitorrapamycinreducedthenumberofapoptoticcellsinducedbyHGandthedownstreameffectsofmTORactivationnotedabove.Furthermore,genesilencingoftuberinwithsiRNAdecreasedcleavageofPARP.Thesedatashowthatthetuberin/mTORpathwaypromotesapoptosisoftubularepithelialcellsindiabetes,mediatedinpartbycleavageofPARPbyYY1.

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Apoptosisoftubularepithelialcellsisamajorfea-tureofdiabetickidneydisease,andhyperglycemiatriggersthegenerationoffreeradicalsandoxidantstressintubularcells.1–3Hyperglycemiaandhighglucoseinvitroalsoleadtoapoptosis,aformofprogrammedcelldeathcharacterizedbycellshrinkage,chromatincondensation,andDNAfragmentationinvarietyofcelltypesincludingproximaltubularepithelialcells.2,3Prolongedex-posureofproximaltubularepithelialcellstohighglucoseinhibitscellproliferationandinducesgrowtharrestorcellularapoptosis.2–5Highglucoseactivatessignalingpathwaysthatincludethephosphotidylinostiol3kinase(PI3ki-nase)andproteinkinaseB(Akt).6Moreover,highglucosephosphorylatestuberinatThr1462inmouserenalproximaltubularepithelialcellsandin

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corticalkidneytissueoftypeIdiabeticrats.6Tu-berin,aproductofthetumorsuppressorgeneTSC-2,existsinanactivestatetoinhibitthemamma-liantargetofrapamycin(mTOR)signalingpath-way.7–10Tuberinexpressionincreasesthesuscepti-bilityofrenalcellstoapoptosisinducedbythetumorpromoterokadaicacid.11Inaddition,tu-berintriggersapoptosisaccompaniedbyphosphor-ReceivedApril6,2010.AcceptedSeptember8,2010.

Publishedonlineaheadofprint.Publicationdateavailableatwww.jasn.org.

Correspondence:Dr.SamyL.Habib,TheUniversityofTexasHealthScienceCenter,DepartmentofMedicine-MSC7882,7703FloydCurlDrive,SanAntonio,TX78229.Phone:210-567-4699;Fax:210-567-4712;E-mail:habib@uthscsa.edu

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Table1.Physicalandmetabolicparametersincontrolanddiabeticrats

ControlDiabetesDiabetes؉InsulinwhereasinsulintreatmentofthediabeticratsdecreasedthenumberoftheapoptoticcellsbutBloodglucose(mg/dl)108Ϯ5.9426.8Ϯ49.3aBodyweight(g)391.7Ϯ22.7284.0Ϯ9.6aKidneyweight(g)2.51Ϯ0.173.33Ϯ0.25bKidney/bodyweight(mg/g)6.5Ϯ0.3911.6Ϯ0.68aProteinurea(mgprotein/24hurine)7.5Ϯ2.5549.2Ϯ14.4bValuesaremeansϮSEM:nϭ4.aPϽ0.01andbPϽ0.05versuscontrolbyANOVA.

ylationofBadonSer136andincreasestheassociationofBAD/Bcl2andBAD/Bcl-XL.12mTORphosphorylatesandactivatesp70S6K(p70ribo-somalproteinS6kinase)onThr389.13mTORisalsoinvolvedinthephosphorylation/inactivationofBcl-2inmicrotubulestreatedwithapoptoticagents.14Bcl-2–relatedproteinscom-priseafamilyofpositiveandnegativeregulatorsofapoptosis.PhosphorylationofBcl-2inactivatesitsanti-apoptoticeffectandtriggersthereleaseofcytochromecfromthemitochon-dria,leadingtotheactivationofdownstreamcaspases.15,16Caspase3istheproteasethatdoesmostoftheDNArepairenzyme,poly(ADP-ribose)polymerase(PARP),cleavingdur-ingapoptosis.17–19Thesequenceatwhichcaspases,granzymeA,orcalpaincleavePARPisverywellconservedinthePARPproteininmanydistinctspecies,indicatingthepotentialim-portanceofPARPcleavageinapoptosis.Diabetesorhighglu-cosecleavesPARPinthethoracicaortaofadultmaleBALB/cmice,20bovineaorticendothelialcells,21andmesangialcellsintherenalglomerulus.22YingYang1(YY1)isknowntobeasubstrateofcaspases,theintracellularexecutionersofapopto-sis.23Themechanismbywhichhighglucoseinducesapoptosisisnotfullyunderstood.Inthisstudy,weinvestigatedthepoten-tialroleofthetuberin/mTORpathwayinactivatinganapo-ptoticsignalcascadeinaratmodeloftypeIdiabetesandinproximaltubularepithelialcells.

RESULTS

At4weeksafterstreptozotocin(STZ)injection,bloodglucoselevels,kidneyweight,kidney/bodyweight,andproteinureaweresignifi-cantlyincreasedinthediabeticgroup,whereasinsulintreatmentofdiabeticanimalsnormalizedtheseparameters(Table1).Bodyweightwassignificantlydecreasedinthediabeticgroupandnormalizedtocontrollevelsinanimalstreatedwithinsulin.

DiabetesIsAssociatedwithIncreasedNumberofApoptoticCells

Diabetesinducedanincreaseinterminaldeoxynucleotidyltrans-ferase(TdT)–mediateddUTPnick-endlabeling(TUNEL)-positivetubularcellsinthekidneysectionofdiabeticrats,andinsulin(fourtofiveunitsdaily;Novolin)reversedthesechanges(Figure1A).ThepercentageofTUNEL-positivetubularcellsstainedinthekidneysec-tionswashigherindiabeticratscomparedwiththecontrolrats,

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83Ϯ30.4nottocontrollevels(Figure1B).

391.3Ϯ112.97Ϯ0.13HyperglycemiaEnhancesTuberin7.6Ϯ0.48Phosphorylation

8.3Ϯ0.5andActivationofthemTORPathwaytoIncreaseApoptosis

Tostudytheeffectofdiabetesontheup-streamsignalofmTOR,theAkt-depen-dentphosphorylationsiteontuberin,Thr1462,wasexam-inedinkidneycortexbyWesternblotanalysis.Tuberinphosphorylationandp-tuberin/totaltuberinlevelswerein-creasedindiabeticratscomparedwithcontrolrats,whereasinsulintreatmentreversedthesechangestocontrollevels(Figure2,AandB).Totaltuberinwasincreasedindiabeticratscomparedwithcontrolrats,andinsulintreatmentre-versedthesechangestocontrollevels(Figure2C).

TostudytheroleofdiabetesindownstreamtargetofmTOR,p70S6KonThr389wasexaminedbyWesternblotandimmunostaininganalysis.PhosphorylationoftuberinleadstoactivationofmTORthroughincreaseexpressionofphospho-p70S6KatThr389indiabeticratscomparedwithcontrolrats,whereasinsulintreatmentreversedthesechangestocontrollevels(Figure2,DandE).

DiabetesActivatesApoptosisCascadeSignals

Toelucidatetheinvolvementoftuberin/S6Kcascadesignalsintheactivationoftheapoptoticpathwayindiabetes,wefirst

Figure1.Diabetesisassociatedwithincreasedapoptosisinkidneycortexofrats,andinsulintreatmentsignificantlydecreasesthesechanges.(A)Kidneysectionsofcontrol,diabetes,anddiabetesϩinsulinofratswerestainedbyTUNEL.ThereisanincreaseinTUNEL-positivecellstofive-tosixfoldindiabeticratscomparedwithcontrolrats.Insulintreatmentofdiabeticrats(seeResults)significantlydecreasestheapoptosis.(B)PercentageoftotalnumberofTUNEL-positivecellscountedinkidneysectionsofcontrol,dia-betes,anddiabetesϩinsulinofrats.Significantdifferencefromcon-trolanimalsisindicatedby**PϽ0.01and*PϽ0.05.

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tionandincreasedcytosoliccytochromecexpressionindiabeticrats(Figure3,CandD)comparedwithcontrolrats.Treatmentofthediabeticratswithinsulinreversesthesechangestocontrollevels(Figure3,A–D).Caspase3hasbeenshowntobepri-marilyresponsibleforthecleavageofPoly(ADP-Ribose)polymerase(PARP)duringcelldeath.Diabetesactivatesandcleavescaspase3at17and11kD,whichwhencleaved/activatedplaysacentralroleintheexecutionofthecelldeathprogram.Weassessedtheactivityofcaspase3inthekidneycortexofcontrol,diabetes,anddiabetesϩinsulinratsbyWesternblotanalysis.Diabetesenhancesactiva-tion/cleavageofcaspase3.

PARPcleavesbycaspases,granzymeA,orcalpainandthatleadstoapoptosis.ThecleavingofPARPat116and85kDwasincreasedindi-abeticrats.Treatmentofdiabeticratswithinsu-linreversestheapoptosiscascadesignalstocon-trollevels(Figure3,A–H),suggestingthatdiabetesactivatestheapoptosiscascadesignalsthroughtuberin/p70S6K/Bcl2phosphoryla-tion,whichreleasescytochromectoactivate/cleavecaspasesandPARP.Collectively,thesedatasuggestthattuberin/p70S6K/Bcl2phos-phorylationplaysanimportantroleintheacti-vationofcelldeathcascadesignalsindiabetes.

HighGlucoseIncreasesCellDeathinProximalTubularCells

Tofurtherdeterminetheabovehypothe-sis,apoptosiswasinducedbyhighglu-Figure2.DiabetesincreasesthephosphorylationoftuberinandactivatesthemTORcoseinHK2cells.Cellswereexaminedbypathway,andinsulintreatmentreversesittocontrollevels.(A)Representativeimmu-doublestainingwithannexinVandpro-noblotshowsthatdiabetes(d)enhancesthephosphorylationoftuberinatThr1462inpidiumiodide(PI)usingflowcytometry.kidneycortexofrats.Treatmentofthediabeticratswithinsulin(dϩi)reversedtheInthisassay,wedeterminedtheabilityofchangestocontrol(c)levels.Actinwasusedasaloadingcontrol.(B)HistogramsintheFITCconjugateofannexinVtostainbottompanelshowincreasedphosphotuberin/tuberinexpressionindiabeticanimalshighglucose–exposedcellsindepen-and(C)increasedtotaltuberin/actinexpressionindiabeticanimals.(D)Increaseindently(duringearlyapoptosis)andinphosphorylationtuberin(P-Tuberin)isassociatedwithincreasedactivationofthecombinationwithPI(duringsecondarymTORpathwaybyphosphorylationS70S6K(P-S70S6K)atThr389inkidneycortexin

necrosis).Incellsexposedtohighglu-diabeticratscomparedwithcontrolrats.Insulintreatment(dϩi)reversedthese

cose,thepercentageofcellsinearlyapo-changestocontrollevels.Actinwasusedasaloadingcontrol.(E)Histogramshows

levelsofphospho-p70S6K/totalp-70S6K.HistogramsrepresentmeansϮSEoffourptosiswassignificantlyincreasedcom-animals.Significantdifferencefromcontrolanimalsisindicatedby**PϽ0.01.Westernparedwithnormalglucoseexposure.

DatafromFigure4Ashowthatcellsex-blotwasrepeatedtwotimesforeachanimal.

posedtohighglucoseattheindicated

examinedtheeffectofdiabetesonphosphorylationofBcl-2.timepointsshowedagradualincreaseinthenumberofPhosphorylationofBcl-2atSer87andexpressionofcyto-apoptoticcellsreachingapeakat36hoursandadecreaseinchromecinthekidneycortexofcontrol,diabetes,andthepercentageofapoptoticcellsat48hours,consistentwithdiabetesϩinsulinratswasmeasuredbyWesternblot.DatainthedatashownbytheapoptoticcascadesignalsintheWest-Figure3,AandB,showthatdiabetesenhancesthephosphor-ernblotanalysis.Increaseinthenumberofapoptoticcellsylation/inactivationofBcl-2atSer87thatresultsinitsinactiva-aftertreatmentwithhighglucosewasalsoconfirmedby

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Figure3.Diabetesisassociatedwithincreasedapoptosiscas-cadesignals,andinsulintreatmentrestoresthesechangestothecontrollevels.(AandB)Diabetesenhancedphosphorylationof

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Hoechststaining(Figure4B).AnnexinVandHoechststainingdatashowthatexposureofthecellstohighglucosehasamaximumapoptoticeffectat36hours.

HighGlucoseEnhancesTuberinPhosphorylationandmTORActivationinProximalTubularCells

Toshowinvolvementofthetuberin/mTORpathwayintheapo-ptosissignalcascade,HK2cellswereincubatedfortheindicatedtimeperiodsinserum-freemediumcontainingeithernormalglucoseorhighglucose.Highglucosecausedanincreaseintu-berinphosphorylationatThr1462withamaximumexpressionat24hoursandthendecreasedat48hours(Figure5,AandB).However,highglucosecausedanincreaseintotaltuberinatalltimepoints(Figure5,AandC),suggestingthattuberinpromotesthecellstogoapoptosispathway.TheeffectofhighglucoseonthedownstreamregulatorofmTORwasdeterminedbymeasuringthephosphorylationlevelsofp70S6KatThr389.Phosphorylationofp70S6Kwasincreasedincellsincubatedwithhighglucosefor24to48hours,andamaximumeffectwasobservedat36hours(Figure5,DandE).

ActivationofthemTORPathwaybyHighGlucoseActivatestheApoptoticCascadeSignalsinProximalTubularEpithelialCells

ActivationofthemTORpathwaybyhighglucoseisassociatedwithanincreaseinthephosphorylationofBcl-2.Phosphory-lationofBcl-2atSer87wasincreasedincellsincubatedwithhighglucosefor24to48hoursandreachedamaximumat36hours(Figure5,FandG).ConsistentwithphosphorylationofBcl-2,weobservedanincreaseincytosoliccytochromecex-pressionat24to48hoursthatreachedamaximumat36hours,suggestingthatphosphorylation/inactivationofBcl-2triggersthereleaseofcytochromecfromthemitochondriaintothecytosol(Figure5,HandI).

Highglucosealsoactivatesandcleavescaspase3at17and11kD(Figure5,JandK).ThemaximumeffectofhighglucoseoncleavingPARPat116and85kDwasobservedat36hours(Figure5,LandM).Thesedatashowthatthisprocessstartsasearlyas24hours,reachesamaximumat36hours,andgraduallystartstosubsideby48hoursafterhighglucosetreatment(Figure5,LandM).Theabovedatacol-lectivelyshowthathighglucoseincreasesphosphorylationoftuberin,resultingintheactivationofthemTORpathwaythatphosphorylates/activatesp70S6K,phosphorylates/in-activatesBCL-2,andtriggersthereleaseofcytochromecfrommitochondriaintothecytosoltoactivatethedown-Bcl-2atSer87(CandD),increasedincytosoliccytochromecexpression(EandF),increasedcleavageofcaspase-3at11kD,and(GandH)increasedcleavageofPARPat85kD,andinsulintreatmentreversedtheseschangestocontrollevelsinrats.Actinwasusedasaloadingcontrol.HistogramshowsrepresentativemeansϮSEoffouranimals.Significantdifferencefromcontrolanimalsisindicatedby**PϽ0.01.Westernblotwasrepeatedtwotimesforeachanimal.

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Figure4.Highglucoseinducesapoptosisofproximaltubularepithelialcells.(A)DatarepresentannexinVbindingandPIstainingofcellsexposedtohighglucoseconcentration(25mM).Serum-starvedcellswereexposedtoglucoseforthetimeperiodsindicated.CellswereharvestedandstainedwithFITC-conjugatedannexinVandPIfor15minutes.ThecellswereanalyzedbyflowcytometerasdescribedinConciseMethods.Maximumnumberofapoptoticcellswasshownat36hoursafterhighglucosetreatment.(B)Cellswereplatedonatwo-chamberslide.Serum-starvedcellsweretreatedwithnormalorhighglucoseforvarioustimepointsasindicated.ApoptoticnucleiweredetectedusingHoechst33258(Sigma)stainingandanalyzedbyfluorescencemicroscopyat350-nmexcitationand460-nmemission.AnnexinandHoechststain-ingwererepeatedfourtimesforeachtimepoint.*PϽ0.05,**PϽ0.01.266

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streamcaspasesandcleavePARP,leadingtoanincreaseincellapoptosis.

DownregulationofTuberinbysiRNAPreventsApoptosis

Toconfirmtheroleoftuberininapoptosis,cellstransfectedwithcontrolsiRNAandtreatedwithhighglucoseshowasignificantincreaseintuberinproteinexpressionandin-creaseincleavageofPARP(Figure5,NandO).Ontheotherhand,cellstransfectedwithsiRNAagainsttuberin(TSC2)andtreatedwithhighglucoseshowasignificantdecreaseintuberinproteinexpressionandadecreaseincleavageofPARP(Figure5,NandO).Thesedataindicatethattuberinactivatesapoptosis.

InhibitionofmTORActivityDecreasedApoptosisinProximalTubularEpithelialCells

TostudywhetherinhibitionofthemTORpathwaycouldpreventtheactivationoftheapoptosiscascadepathway,serum-starvedHK2cellswerepretreatedwith20nMrapa-mycinfor24hoursbeforeexposuretohighglucose.Apo-ptosiswasexaminedat36and48hoursusingflowcytometry,afterstainingthecellswithannexinVandPI.ApoptoticcellswerealsomeasuredbyHoechststaining.Thenumberinthebottomrightofthequadrantrepresentscellsinearlyapoptosis.InhibitionofmTORactivationbyrapamycinsignificantlyde-creasesapoptosisat36and48hoursincellstreatedwithhighglucose(Figure6,AandB).Adecreaseinapoptoticcellspre-treatedwithrapamycinwasconfirmedbyHoechststaining(Figure6C).

TostudywhetheradecreaseinapoptosisisrelatedtothemTORpathway,wemeasuredtheexpressionofphospho-p70S6Kanddownstreamsignalscascadeofapoptosis,Bcl2,caspase3,cytochromec,andPARP.Pretreatmentofcellswithrapamycinbeforeexposuretohighglucoseabolishedphos-phorylationofp70S6KatThr389(Figure6DandE)andphos-phorylationofBcl2atSer87(Figure6FandG),decreasedcyto-soliccytochromecexpression(Figure6HandI),abolishedtheactivityofcaspase3(Figure6JandK),anddecreasedcleavageofPARP(Figure6LandM).Collectively,thesedatasuggestthatblockingmTORactivityisanimportantpathwayinvolvedinactivationoftheapoptosiscascadesignals.

HighGlucoseRedistributesYY1fromCytoplasmintoNucleus

TodeterminethemechanismbywhichhighlucoseenhancesapoptosiscascadesignalsinHK2cells,weshowthecytoplas-micandnucleardistributionofthetranscriptionalregulatorYY1byimmunohistochemicalstainingandWesternblotanal-ysis.ThestainingofthecellsshowsthelocationofYY1inthecytoplasmofcellsgrowninnormalglucose(Figure7A).Trans-locationofYY1fromthecytoplasmintothenucleusreachesamaximumat36hoursinhighglucose–treatedcells(Figure7A).Moreover,pretreatmentofthecellswithrapamycinbe-forehighglucoseblockeditstranslocation(Figure7A).We

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confirmedtheredistributionofYY1byWesternblotanalysisincytoplasmicandnuclearfractions(Figure7,BandC).ThesedatasuggestthathighglucoseincreasesthenuclearredistributionofYY1thatmayresultincleavageofPARPandanincreaseinpro-grammedcelldeath.However,inhibitionofmTORactivitybyrapamycinreversedYY1localization,indicatingthatactivationofthemTORpathwayplaysanimportantroleintheactivationofapoptoticsignalingincellstreatedwithhighglucose.

DISCUSSION

Figure5.HighglucoseincreasestuberinphsophorylationandmTORactivationtoacti-vateapoptosissignalpathwaysinproximaltubularepithelialHK2cells.(A)Representativeimmunoblotshowsanincreaseinphospho-tuberin(p-tuberin)andtotaltuberin.(BandC)Histogramsinthebottompanelshowincreaseinphospho-tuberin/tuberinexpressionandincreaseintotaltuberin/actinexpressionincellstreatedwithhighglucoseforthetimeperiodsindicated.(D)Representativeimmunoblotshowsanincreaseinphopho-p70S6K(P-p70S6K)inHK2cellstreatedwithhighglucose(25mMglucoseD-glucose)forthetimeperiodsindicated.(E)HistogramsshowincreasedP-p70S6K/p70S6K.(FandG)HighglucoseenhancesphosphorylationofBcl-2,

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Inthisstudy,weidentifiedanovelmech-anismoftubularepithelialcellapoptosisindiabetes.Ourdatashowedforthefirsttimethatdiabetesisassociatedwithen-hancedphosphorylationoftuberin,in-creasetotaltuberin,phosphorylation/activationofthemTORsubstratep70S6K,phosphorylation/inactivationofBcl-2,anincreaseincytosoliccyto-chromecexpression,activationofcaspase3,andcleavageofPARP,leadingtoapoptosisoftubularepithelialcellsinthekidneycortexofrats.TUNELstainingshowedthatdiabetescausesapoptosis,mainlyintubularepithelialcellsofthekidneycortexofrats.Wealsoshowedthatthispathwayisactivatedinculturedhumanproximaltubularepithelialcellsexposedtohighglucose.Inculturedprox-imaltubularepithelialcells,highglucoseen-hancesphosphorylationoftuberin/p70S6K/Bcl-2,releaseofcytochromecfrommitochondria,andactivationofcaspase3.Inaddition,downregulationoftuberinwithsiRNAabolishedtuberinexpressionandsig-nificantlydecreasedcleavageofPARP.Rapa-mycintreatmentofcellsexposedtohighglu-coseresultedin1)inhibitionofmTOR,2)decreaseinp70S6K/Bcl-2phosphoryla-tion,3)decreaseincytochromeCexpres-sion,4)increaseincleavageofcaspase3,5)cytoplasmiclocalizationofYY1,and6)adecreaseincleavageofPARP.Thesedataindicatethatthetuberin/mTORpathwayplaysanimportantroleintheactivationofthisapoptoticsignalingcascade.Tuberinhasbeenshowntoincreasethesusceptibil-ityofthetumorcellstoapoptosisandtotriggerapoptosisthroughactivationofthe

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HIJKLMNTSC2-SiRNA ----+ +Control-siRNA --+ + --NGNG HGHGNGHGHG NG HGNGHGTuberinPARP116KDaCleaved PARP85KDaGAPDHO1.5 stinu1.0****rartibrA0.5********0.0NGHGNGHG NG HGNGHGNGHG NG HGControl siRNA --+ + ----+ + --TSC2 siRNA ----+ + ----+ +Tuberin/GAPDHC-PARP/GAPDHFigure5(cont.).(HandI)increasescytosoliccytochromecexpression,(JandK)increasescleavageofcaspaseat11kD,and(LandM)increasescleavageofPARP268

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proapoptoticproteinBAD.11,12Ourdata

showedthatanincreaseintuberinexpres-sionincellstreatedwithhighglucosere-sultsinapoptosis,whereasdownregulationoftuberinwithsiRNAinhibitstheproapo-ptoticsignals,indicatingtheimportantroleoftuberinintheproapoptoticsignalcas-cade.

Thereisanevidenceoftheinvolve-mentofthemTORpathwayincelldeath/survival24,25andinautophagy.26,27mTORinhibitionplaysapreponderantroleinthecontrolofnetproteinsynthe-sisandcellsize.Inaddition,mTORmayalsohaveapleiotropicfunctionintheregulationofcelldeath.Thisfunctionseemstobedictatedbythecellularcon-textandbymultipledownstreamtargetsincludingapoptosis-regulatoryproteinssuchp53,Bad,andBcl-2.Anotherexpla-nationisthatrapamycinpreventstrans-locationofmTORfromthecytoplasmtothenucleusandpreventstranscriptionalactivationandphosphorylationofp53.Thatmayleadtoinhibitionofthepro-apoptoticproteinssuchasBaxandBcl2andactivationofthemitochondrialcelldeathpathway.InactivatedBcl-2isknowntotriggerthereleaseofcytochromecfrommitochondriaandtoactivatethedown-streamcaspases.15,16Ourdatashowedanin-creaseinthephosphorylationofBcl-2,tu-berin,and70S6KinthekidneycortexofratswithtypeIdiabetes.Thisisassociatedwithanincreaseincytosoliccytochromecex-pressionandenhancedcleavageofcaspase3.CleavedPARPisalsosignificantlyhigherinthekidneycortexofdiabeticrats.Takentogether,thesefindingsindicatethat,indi-abetes,apoptoticsignalsareactivatedthroughphosphorylationoftuberinandactivationofthemTORpathway.Activa-tionofmTORanddownstreamtargetp70S6Kisassociatedwithincreasedphos-at85kDinHK2cellstreatedforthetimeperiodsindicated.Glyceraldehyde3-phos-phatedehydrogenase(GAPDH)wasusedasalodingcontrol.Westernblotwasrepeatedthreetimesforeachtimepoint.(NandO)CellstransfectedwithsiRNAagainsttuberin(TSC2)andtreatedwithloworhighglucoseshowsignificantdecreaseintuberinproteinexpressionanddecreaseincleavageofPARP.*PϽ0.05,**PϽ0.01.

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Figure6.RapamycininhibitsmTORactivationanddecreasesapoptosisofproximaltubularepithelialcellstreatedwithhighglucose.(A)Serum-starvedcellsweretreatedwithrapamycin(20nM)for24hoursbeforeexposuretohighglucoseforthetimeperiodsindicated.DatarepresentannexinVbindingandPIstainingofcellsexposedtohighglucoseconcentration(25mM).CellswereharvestedandstainedwithFITC-conjugatedannexinVandPIfor15minutes.ThecellswereanalyzedbyflowcytometerasdescribedinConciseMethods.HistogramrepresentsmeanϮSEofthreeindependentexperiments.Significantdifferencefromuntreatedcellsisindicatedby*PϽ0.05and**PϽ0.01.(B)Flowcytometrydatarepresentoneofthreeexperiments.(C)Cellswereplatedontwo-chamberslides.Serum-starvedcellsweretreatedwithnormalorhighglucoseinthepresenceorabsenceofrapamycinforvarioustimepointsasindicated.ApoptoticnucleiweredetectedusingHoechst33258stainingandanalyzedbyfluorescencemicroscopyat350-nmexcitationand460-nmemission.

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alsoshowedthatchronicexposureofHK2cellstohighglucoseincreasesthenumberofapopto-ticcellsat24hours,withamaximumeffectat36hours.Highglucosealsoenhancesphosphory-lationoftuberinandp70S6Kat24hours,withamaximumeffectat36hours.Theseeffectsweremarkedlyreducedbyrapamycin.

Thecaspases,afamilyofcysteine-de-pendent,aspartate-directedproteases,areprominentamongapoptosis-associatedmolecules.28Onceactivated,caspasescleaveavarietyofintracellularpolypep-tides,includingmajorstructuralelementsofthecytoplasmandnucleus,whicharecompo-nentsofDNArepairmachinery.Caspase-3isexpressedincellsasaninactiveprecursorfromwhichthep11subunitofthematurecaspase-3isproteolyticallycleavedduringapoptosis.Ourdatashowedthecleavageofcaspase3andgenerationofthe11-kDac-tivefragmentinthekidneycortexofdia-beticrats.Caspase3wasalsocleavedat11kDinproximaltubularepithelialcellstreatedwithhighglucose,consistentwithincreasedcytochromecexpression,sug-gestingthatcytochromecactivatescaspase3.YY1isasubstrateofcaspasesandoneofthefewproteinsthatmodifiesPARP-1inresponsetoDNAdamagingagents.23,29OurdatashowedthatYY1istranslocatedfromthecytoplasmtothenu-cleusandisassociatedwithcleavageofPARP,resultinginapoptosisofproximaltubularcellstreatedwithhighglucose.Treatmentofthecellswithrapamycinblocksthetransloca-tionofYY1fromthecytoplasmtonucleus,reducescleavageofPARP,anddecreasesthenumberoftheapoptoticcells.

PARP,aDNArepairenzyme,whencleaved,playsacriticalroleinapoptosisinseveralcells,includingrenalcells.20–22PARPiscleavedbycaspase3earlyduringapoptosisinmanydifferentcelltypes.ThecleavageofPARPisthoughttopre-Figure6.(DandE)ImmunoblotandhistogramshowthatpretreatmentofHK2cellsventdepletionofenergy,NAD,andATPwithrapamycin(20nM)for24hoursbeforeexposuretohighglucosefor36hoursrequiredatlaterstagesofapoptosis.29abolishesphosphorylationofp70S6K,(FandG)decreasesBcl2phosphoryaltionatSer

PARP,whencleavedintoa85-kDfrag-87,(HandI)decreasescytosoliccytochromecexpression,(JandK)decreases

ment,isinactivatedandisunabletore-cleavageofcaspase-3at11kD,and(LandM)decreasesthecleavageofPARPat85

kD.GAPDHwasusedasloadingcontrol.Annexin,Hoechststaining,andWesternblotpairDNAbreaksorfragmentation.We

showthatphosphorylationoftuberinandac-wererepeatedthreetimesforeachtimepoint.

tivationofmTORisassociatedwiththecleav-phorylation/inactivationofBcl-2andreleaseofcyotchromecageofPARPindiabeticratsandinproximaltubularepithelialfromthemitochondriatothecytoplasm,suggestingthatthecellstreatedwithhighglucose,suggestingthatthemTORpathwaytuberin/mTORpathwayplaysacriticalroleintheapoptosisofplaysanimportantroleinactivationoftheapoptosissignalingtubularepithelialcellsinthekidneycortexofratswithdiabetes.Wecascade.

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Figure7.HighglucoseresultsintheredistributionofYY1fromthecytoplasmtonucleus,andrapamycinreversestheseeffects.ImmunostainingandWesternblotanalysiswereusedtodetectthelocalizationofYY1incellsexposedtonormalglucoseorhighglucosefor36hoursorcellstreatedwithrapamycin(20nM)for24hoursbeforeexposuretohighglucosefor36hours.(A)AlexaFluro594redsignalsforYY1weredetectedusingafilterwithexcitationrangeof535nmandDAPIbluesignalsfornuclearDNAusingafilterwithexcitationat488nm.Toshowstainingspecificity,controlcellswerestainedwithoutprimaryantibody.(B)HighglucosedecreasesYY1inthecytoplasmicfraction.(C)HighglucoseincreasesYY1inthenuclearfraction.Pretreatmentofthecellswithrapamycinreversedthesechanges.LaminBwasusedasanuclearmarker.ImmunostainingandWesternblotwererepeatedthreetimes.

Insummary,ourdataprovidethefirstevidencethathyperglyce-miaindiabetesandhighglucoseconcentrationleadstoincreasetotaltuberinexpressionandphosphorylation,therebyactivatingthemTORpathway.ActivationofmTORinturnsphosphorylates/inactivatesBCL-2withincreasedreleaseofcytochromecfromthemitochondriatothecytoplasm.Cytochromeccleavescaspase3,andtheactivatedcaspase3likelyresultsintranslocationofYY1fromthecytoplasmtothenucleustocleavePARPoranotherdeathsubstrate,resultinginanincreaseinpro-grammedcelldeath(Figure8).Thissignalingcascademayplayanimportantroleinapoptosisinducedbyhyperglyce-miaduringdiabeticnephropathy.Furthermore,ourresultshighlightthenotionthatoptimalmanagementofdiabeticcomplications,particularlydiabetickidneydisease,willre-

quireafullunderstandingofthechroniceffectsofhyper-glycemia.

CONCISEMETHODSAnimals

Two-month-oldmaleLongEvansratsweighingbetween200and225gwerepurchasedfromabreedingcolonymaintainedattheUni-versityofTexasM.D.AndersonCancerCenter,Smithville,TX.Theanimalswereallowedfoodandwateradlibitumbeforeandduringtheexperiments.Theratsweredividedintothreegroupsoffourratspergroup.Group1(controls)wasinjectedwithanequivalentamountofsodiumcitratebufferalone.Group2wasinjectedintravenouslyvia

thetailveinwith55mg/kgbodyweightSTZ

High glucose(Sigma,St.Louis,MO)insodiumcitratebuffer

(0.01M,pH4.5)underisofluoraneinhalation

Mitochondriaanesthesia(Abbott,AbbottPark,IL)toinduce

Ptype1diabetes.Group3wasinjectedwithSTZ

Cytochrome CTuberinCaspasesasingroup2andwastreatedwithfourtofiveP

Bcl2unitsofinsulindaily.Averageserumglucose

mTORlevelsandbodyweightofbothgroupswere

YY1

measuredat4weeksofSTZinjection.Animalswerekilledat4weeks,andthekidneyswerere-P

p70S6K

movedrapidly.Thekidneysweredissectedlongitudinally,onehalfwaspreservedin10%formalininPBS,pH7.4,forTUNELassay,

PARPandtheremainingtissuewasusedforbio-chemicalanalysis.TheInstitutionalAnimal

CareandUseCommitteeoftheUniversityofApoptosis

Nucleus

TexasHealthScienceCenteratSanAntonio

Figure8.Proposedmodelofactivationoftheapoptosiscascadesignalsindiabetes.approvedtheseanimalstudies.

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ProximalTubularEpithelialCells

HK-2cells,aproximaltubulecellline(AmericanTypeCellCollection,Rock-ville,MD),weregrowninDulbecco’sMEMandNutrientMixtureF-12con-taining10%FBSand5mmol/Lglucose.Confluentcellsweregrowth-ar-restedovernightinserum-freeDulbecco’sMEMbeforetheexperiments.

InVivoExperiments

TUNELAssay.

TUNELstainingusingtheTUNELApoptosisDetectionKit(Upstate)wasperformedaccordingtothemanufacturer’sinstructions.Thesec-tionswereexaminedusinglightmicroscopy.SectionsincubatedwithPBS,insteadofTDTenzymesolution,servedasthenegativecontrols.ThenumberofTUNEL-positivecellswascountedinfiverandomlyselectedfieldsunder400ϫmagnificationforeachanimal.Fourani-malswerestudiedpergroup.

WesternBlotAnalysis.

Homogenatesofkidneycortexwerepreparedasdescribedpreviou-sly.30ProteinconcentrationsweredeterminedwiththeBradfordassay31usingBSAasastandard.Westernblotanalysiswasperformedasde-scribedpreviously.32Phospho-tuberin,tuberin,phospho-p70S6k,p70S6K,phospho-Bcl2,cytochromec,caspase3,andPARPantibodieswerefromCellSignaling(Beverly,MA);actinandGAPDHantibodieswereobtainedfromSantaCruzBiotechnology.AfterextensivewashingofmembranewithTris-bufferedsalineTween-20buffer,anti-rabbitIgconjugatedwithhorseradishperoxidasewasaddedata1:5000dilutionandincubatedfor1houratroomtemperature.Anenhancedchemilu-minescencekit(Amersham)wasusedtoidentifyproteinexpression.Ex-pressionofeachproteinwasquantifiedbydensitometryusingNationalInstitutesofHealthimage1.62softwareandnormalizedtoaloadingcontrol.

DownregulationofTuberinbysiRNA.

HEK293cellsweregrownin6-wellplatesinnormalglucosemedium.Se-lectedsiRNAduplexeswith“UU”overhangsand5Јphosphateontheanti-sensestrandagainstTSC2orcontrolsiRNAwereobtainedasakitfromSantaCruz.CellswereinfectedwithsiRNAspecificforTSC2orcontrolnonspecificsiRNAduplexesasdescribedpreviously.33Cellsweretreatedwithhighglu-cosefor36hoursbeforeharvestingforWesternblotanalysis.

InVitroExperiments

HK2cellswereseededatadensityof0.5ϫ106cellson60-mmpetridishesin5mMglucose.Whencellsreached90%confluency,serumwaswithdrawnfor24hours,andcellsweretreatedwithhighglucose(25mM)aloneorpreincubatedwithrapamycin(20nM)for24hoursunderserum-freeconditionsbeforeexposuretohighglucoseforvarioustimepointsasindicated.Thecellswerelysedinradioimmuneprecipitationassaybufferasdescribedabove.ProteinconcentrationsweredeterminedwiththeBradfordassay.31Protein(40␮g)wassubjectedto8%SDS-PAGEandWesternblot.

CellLysatesandTissueHomogenatesFractionation

Cytosolicproteinfractionswereextractedfromthecelllysatesandkidneycortexhomogenatesusingmitochondriaandcytosolfractionationkit(Pierce).Cytoplasmicandnuclearfractionswerealsoextractedfromthecelllysatesusinganuclearandcytoplasmicfractionationkit(Pierce).Theprotein

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concentrationofthenuclearextractswasdeterminedusingtheBradfordmethod.31ApoptosisAssay

AnnexinAssay.

TheapoptoticcellsweremeasuredusingannexinV-FITCconjugatedtoPIbyflowcytometry.Serum-starvedcellsweretreatedwithnormalorhighglucoseinthepresenceorabsenceofrapamycinforvarioustimepointsasindicated.HarvestedcellswereassayedforapoptosisusinganannexinV-FITCapoptosisdetectionkit(Calbiochem)accordingtothemanufacturer’sinstructions.Topreparethecellsamplesforflowcytom-etry,cellsweregentlywashedtwotimeswithannexin-bindingbuffer.Toeachplate,0.5mlofTrypsin(0.05%)wasadded,andtheplateswereincubateduntilthecellsappeardetachedbymicroscopicevaluation.Cellswerereleasedfromtheplatewithgentletappingandaddedtothecollectedcellsfromthemedium.CellsweresuspendedincoldbindingbufferandstainedwithannexinVFITCandPI.Analysiswasconductedfor20,000cellsusingaflowcytometerwithCellQuestsoftware(Becton-Dickinson,Rutherford,NJ)usingFL1andFL2rangesforannexinVFITCandPI,respectively.Ineachofthegraphs,thebottomleftquadrantrepresentslivecells,thebottomrightquad-rantrepresentscellsinearlyapoptosis,thetoprightquadrantrepre-sentscellsinsecondarynecrosis,andthetopleftquadrantrepresentscellsstainedwithPIalone.

HoechstStaining.

Cellswereplatedontwo-chamberslides.Serum-starvedcellsweretreatedwithnormalorhighglucoseinthepresenceorabsenceofrapamycinforvarioustimepointsasindicated.ApoptoticnucleiweredetectedusingHoechst33258(Sigma)staining(10␮g/ml,30minat37°C)incellsfixedwith4%paraformaldehydeandanalyzedviaflu-orescencemicroscopyat350-nmexcitationand460-nmemission.

ImmunostainingofYY1

Adoublefluorescencelabelingmethodwasusedasdescribedpreviously30withminormodifications.HK2cellsinchamberslideswereserum-starvedfor24hoursandincubatedwith25mMglucoseortreatedwithrapamycinfor24hoursbeforeexposedtohighglucosefortheindicatedtimeperiods.ThecellswerewashedwithPBS,fixed,andstainedwithmouseantibodyagainstYY1(SantaCruz),followedbytreatmentwithanti-mouseIgG(1:200)conjugatedwithFITC.TheslideswerereactedwithVectashieldMount-ingMediumwith4’,6-diamidino-2-phenylindole(DAPI)(VectorLaborato-ries).Inthisassay,DNAwaslabeledwithDAPI,andYY1wasidentifiedbytheprimarymonoclonalantibodyandAlexaFluro594conjugatedsecondaryantibody.AlexaFluro594redsignalsforYY1weredetectedusingafilterwithanexcitationrangeof535nmandDAPIbluesignalsfornuclearDNAusingafilterwithexcitationat488nm.AlexaFluro594andDAPIweredetectedusingOlympusFV-500laserscanningconfocalmicroscopy.Toshowstain-ingspecificity,controlcellswerestainedwithoutprimaryantibody.

StatisticalAnalysis

DataarepresentedasmeanϮSE.StatisticaldifferencesweredeterminedusingANOVAfollowedbyDunnett’s(Exp.versusControl)testusingonetrialanalysis.SignificantassociationwasdefinedwhenPϽ0.05and0.01ascomparedwithcontrol.

JAmSocNephrol22:262–273,2011

ACKNOWLEDGMENTS

ConfocalfluorescencemicroscopyandFACSanalysiswereper-formedinthecoreopticalimagingfacilitysupportedbyUTHSCSAandSanAntonioCancerInstituteandtheimmunostainingattheMorphologycoreoftheGeorgeO’BrienKidneyResearchCenter.WethankDanRiley,M.D.,forreadingthemanuscript.ThisworkwassupportedinpartbygrantsfromaVAmeritreviewawardandNIHRO1(DK078971)(toH.E.A.),aResearchScholarAwardfromTheItalianSocietyofNephrology(toS.S.),andtheAmericanHeartAs-sociation,NewInvestigatorAwardandMeritReviewAwardfromSouthTexasVeteransHealthcareSystem(toS.L.H.).

DISCLOSURES

None.

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