ORIGINAL ARTICLEDNA methylation changes between relapse and remissionof minimal change nephrotic syndromeYasuko Kobayashi & Akira Aizawa & Takumi Takizawa &Chikage Yoshizawa & Hiromi Horiguchi & Yuka Ikeuchi &Satoko Kakegawa & Toshio Watanabe &Kenichi Maruyama & Akihiro Morikawa &Izuho Hatada & Hirokazu ArakawaReceived: 5 January 2012 / Revised: 31 May 2012 / Accepted: 11 June 2012#The Author(s) 2012. This article is published with open access at Springerlink.comAbstractBackgroundDNA methylation of gene promoters is associ-atedwithtranscriptional inactivation. Changes inDNAmethylation can lead to differences in gene expression levelsandtherebyinfluencediseasedevelopment. Wehypothe-sized that epigenetics underlies the pathogenesis of minimalchange nephrotic syndrome (MCNS).MethodsGenome-wide DNA methylation changes betweenrelapseandremissioninmonocytes (n06) andnaiveThelper cells (Th0s) (n04) isolated from patients with MCNSwere investigated using the microarray-based integrated anal-ysisofmethylationbyisochizomers(MIAMI)method. Weconfirmed the MIAMI results using bisulfite-pyrosequencinganalysis. Expression analysis was performed using quantita-tive real-time PCR.ResultsThreegeneloci (GATA2, PBX4, andNYX) weresignificantlylessmethylatedinTh0sduringrelapsethaninremission, comparedtonone inmonocytes. Inaddi-tion, thedistancedistributionfromtheregressionlineofall probesinMIAMIwassignificantlydifferent betweenmonocytes and Th0s. The mRNAlevels of the threegenes in Th0s were not significantly different betweenrelapseandremission.ConclusionsOur results demonstrate that thechangeinDNAmethylationpatterns fromremissiontorelapseinMCNSoccurspredominantlyinTh0sratherthaninmono-cytes and suggest that epigenetic regulation in Th0s underliesthe pathogenesis of MCNS.KeywordsDNAmethylation.Nephrotic syndrome.Monocytes .Naive T helper cells .Microarray-basedintegratedanalysis of methylationbyisoschizomers (MIAMI)method .Genome-wide .ChildrenIntroductionEpigeneticsisthestudyofmitoticallyheritablechangesingeneexpressionthat occur without direct DNAsequencealterations. DNA methylation, one of the principal epigenet-ic mechanisms in mammals, involves the covalent additionof a methyl group to a cytosine residue that is followed by aElectronic supplementary material The online version of this article(doi:10.1007/s00467-012-2248-z) contains supplementary material,which is available to authorized users.Y. Kobayashi (*): A. Aizawa:T. Takizawa:S. Kakegawa:T. Watanabe: H. ArakawaDepartment of Pediatrics,Gunma University Graduate School of Medicine,3-39-22 Showa-machi,Maebashi, Gunma 371-8511, Japane-mail: kobayasu@gunma-u.ac.jpC. Yoshizawa: H. Horiguchi: Y. IkeuchiDepartment of Pediatrics, Gunma Chuo General Hospital,Maebashi, Gunma, JapanK. MaruyamaGunma Childrens Medical Center,Hokkitsumura, Seta-gun, Gunma, JapanA. MorikawaKitakanto Allergy Institute,Midorishi, Gunma, JapanI. HatadaLaboratory of Genome Science,Biosignal Genome Resource Center,Gunma University Institute for Molecular and Cellular Regulation,Maebashi, Gunma, JapanPediatr NephrolDOI 10.1007/s00467-012-2248-zguanine (CpG) [1]. DNA methylation regulates gene expres-sion and is essential for differentiation, embryonic develop-ment [2], genomic imprinting[3], andX-chromosomeinactivation[4].DNAmethylationwithinthepromoterre-gion of a gene is commonly associated with transcriptionalinactivation, whereas demethylationcontributes totran-scriptional activation. Changes intheDNAmethylationprofilecanalsoleadtodifferencesingeneexpressionpat-terns andtherebyinfluencethedevelopment of diseases,such as cancer [5].Minimal change nephrotic syndrome (MCNS) is themost commoncause of nephrotic syndrome inchildrenandis characterizedbymassiveproteinuriaandhypoal-buminemia ina relapse/remissioncourse without histo-logical evidence of immune-mediated inflammatorydamage. These manifestations are typically reversiblewith the use of corticosteroid therapy. Although thepathogenesisof MCNSremainstobeelucidated, immu-nological disruptionhas beenimplicatedinthis disease[6] as Tcell-derivedvascular permeabilityfactors havebeenshowntoberesponsiblefor alterations inglomer-ular permeability [79]. The incidence of MCNS inchildhoodis twofoldhigher inboys, witha prevalencethat is inversely proportional to age, and recurrent re-lapse tendstolessenafteradolescence[10,11].Sincethecharacteristic features of MCNSinclude (1) a recurrentrelapse/remissioncourse, (2) gender preference, (3) agepreferenceofonset andrelapse, and(4)steroidresponsein most patients, a genetic defect cannot explain thepathogenesis of this disease; however, epigenetic alter-ationsmayoccur without adirect changeinthegeneticsequence. DNAmethylationchangeswithageandenvi-ronmental factors, even in the same individual, and isinvolvedwithX-chromosomeinactivation.Audard et al. reported that NFRKB (nuclear factor relatedtokappaBbindingprotein) was highlyexpressedinthenuclearcompartmentduringrelapseandthatNFRKBpro-moteshypomethylationofgenomicDNA, suggestingepi-genetic involvement in the pathogenesis of MCNS [12]. Theepigenotype is influenced by the environment and alters theregulation of gene expression, leading Elie et al. to suggest aprobable impact of epigenetic modifications in infected cellssince MCNS relapses are frequently triggered by external orinternal environmental factors, includingviral infection[13]. Zhang et al. reported significant differences in histoneH3 lysine 4 tri-methylation of peripheral blood mononuclearcells(PBMCs) fromadult patientswithMCNScomparedwith those from healthy subjects. Their results indicate thatalterations in epigenotype are associated with the pathogenesisof MCNS [14].The aimof this study is to elucidate whether theDNAmethylation profile changes between relapse andremission in MCNS cases and whether this process isimmune-competent cell-type-specific. Ultimately, wewished to determine whether epigenetics underlies thepathogenesis of MCNS.Patients and methodsPatientsSamplesformicroarray-basedintegratedanalysisofmeth-ylationbyisochizomers(MIAMI) analysiswereobtainedfrom six male patients with MCNS (Table 1), while samplesfor quantitativereal-timePCR(qRT-PCR) wereobtainedfromanadditional sevenpatientswithMCNS(3boys, 4girls) (Table 2) at relapse andalsofollowingcompleteremission. All patients werediagnosedaccordingtothecriteriaof theInternational Studyof KidneyDiseaseinChildren [15] and had developed nephrotic syndrome priorto 16 years of age. Informed consent was obtained from theparents of each child and from older children/adolescents asnecessary. This study was approved by the Ethics Commit-teeof GunmaUniversityGraduateSchool of Medicine,Japan (Receipt Number 89).Cell separationWeusedmonocytes, whichareprecursorsoftheantigen-presentingcellsderivedfromthemyeloidcell series, andnaiveThelper cells (Th0s), whicharederivedfromthelymphoid system, as material for the analyses. PBMCs wereisolatedfrom20-mL samples of anti-coagulated blood thathad been obtained by gradient separation using the Lymph-prep Tube system (Axis-Shield PoC AS, Oslo, Norway).Monocytes and Th0s were separated from PBMCs that hadbeen magnetically labeled with CD14, CD4, and CD45ROmicroBeads (Miltenyi Biotec) using an autoMACS Pro Sep-arator (Miltenyi Biotec, Bergisch Gladbach, Germany) . Toobtain monocytes, the CD14-positive (CD14+) fraction wascollected as monocytes, whereas the CD14-negative, CD4-positive, andCD45RO-negative (CD14-CD4+CD45RO-)fractionswerecollectedasTh0s.FlowcytometricanalysiswithaMACSQuantAnalyzer(MiltenyiBiotec)revealedthat the precision of cell separation was 96.2 % for CD14+cellsand94.46%forCD4-positiveandCD45RA-positivecells as a CD45RO-negative fraction.CD14+monocytes were obtained from six patients, whileCD14-CD4+CD45RO-Th0cells werealsoobtainedfromfour of these patients (Table 1) for the MIAMI analysis bothat relapse and following complete remission. Genomic DNA(gDNA) wasextractedfromthesecellsaspreviouslyde-scribed [16]. Consistent amounts of extracted gDNA(300 ng from monocytes and 250 ng from Th0s) from eachselectedcell typewerepooledandusedfor subsequentPediatr NephrolDNA methylation analysis using MIAMI in order to excludethe influences of epigenetic differences amongthe celltypes, toidentifyspecificchangesduetoclinical coursesof MCNS, andtoreducethenoisecausedbyindividualdifferences. Total RNAwasextractedfromTh0spurifiedfromsevenother patients withMCNS(Table2) usingaToTALLY RNA kit (Ambion, Austin, TX). Complemen-tary DNA (cDNA) was synthesized from total RNA of eachTh0s sample using the High Capacity RNA-to-cDNA Mas-ter Mix (Applied Biosystems, Foster City, CA) for the qRT-PCR.MIAMI analysisThe MIAMI method, which provides high-throughput globalanalysisofDNAmethylation, wasperformedasdescribedpreviouslyusing1.8and1.0gofpooledgDNAisolatedfrom the monocytes of six patients and from the Th0s of fourpatients, respectively [17, 18]. Briefly, this technique utilizesisoschizomers(HpaII andMspI) that recognizethesamerecognitionsite(CCGG). PooledgDNA wasdigestedwithHpaII, a methylation-sensitive restriction enzyme that cleavesonly unmethylated DNA, and then adapter-ligated and ampli-fiedbyPCRwithprimers designedagainst theadaptersequences. The samples were then further digested with MspI,amethylation-insensitiveenzymethat digestsCCGGsitesirrespectiveoftheirmethylationstatus,andamplifiedagainwiththesameset ofprimers(HpaIIMspI treatment). Thesecond treatment with MspI yields amplicons from unmethy-latedDNAfragments only. Hence, onlyHpaII cleavableunmethylatedDNAfragmentsareamplified, andthesecanthenbequantifiedbasedontheir respectivefluorescenceintensity by microarray analysis. The amplified products werethenlabeledwithCy3(remissionsamples)orCy5(relapsesamples) andco-hybridizedtoamicroarrayspottedwith38,172sixty-mer oligonucleotidescoveringthevicinityofthe transcription start sites (TSSs) of 14,978 genes.Following hybridization, the microarray was scanned, andthe obtained fluorescence intensities were quantified and nor-malized. The same pooled gDNA samples were treated firstwith MspI instead of HpaII (MspIMspI treatment) and ana-lyzed on a duplicate array to correct for false-positives causedby single nucleotide polymorphisms or incomplete digestion.Bisulfite-pyrosequencing analysisIn the bisulfite-pyrosequencing method, unmethylated cyto-sine residues are converted into uracil, whereas methylatedcytosines remainunchanged. Analysisof themethylationstatus inthis manner exploits thequantitativenatureofpyrosequencing by reporting the ratio of cytosine to thymineateachanalyzedCpGsite,whichreflectstheproportionofmethylated DNA. This analysis was performed using pooled Table1CharacteristicsofpatientswithminimalchangenephroticsyndromeatsamplingwhoseDNAwasisolatedformethylationanalysisPatientno.SexAgeatonsetTotalfollow-upperioda(months)FrequentrelapserBiopsySamplingageatrelapseRelapsetimesatsamplingIntervalsb(weeks)PSLatrelapse(mg/day)PLSatremission(mg/day)ISatrelapse(mg/day)ISatremission(mg/day)CD14+(n06)RO(n04)1M10y4m78YesMC13y0m3rd5401000TakenTaken2M5y4m35YesMC6y0m3rd160350CyA90TakenTaken3M5y11m186YesMC18y6m9th23101500TakenTaken4M7y3m188YesMC19y10m13th2360500TakenTaken5M9y9m109YesND11y7m4th1200CyA180CyA160TakenNT6M11y11m61YesND12y5m4th71020MZV125MZV75TakenNTMean8y4m109.513y5m6th25PSL,prednisoloneIS,immunosuppressant;M,Male;y,years;m,months;MC,minimalchange;ND,notdone;CyA,cyclosporinA;MZB,mizoribine;NT,nottakenaFollow-upperiodtillJune2011bIntervalsofsamplingbetweenrelapseandremissionPediatr Nephrolsamplesinaccordancewithestablishedprotocols. Briefly,gDNAextracts frompatients weredigestedwithEcoRI(Takara Bio, Otsu, Japan) and subjected to bisulfite treatmentusingtheEZDNAMethylation-GoldKit(ZymoResearch,Orange, CA). TheanalyzedCpGsiteswereintheclosestHpaII recognition sites at the 5- and 3- ends of the probesdesigned in the TSSs vicinity of GATA2 (A_17_P02574948),PBX4(A_17_P10909964), andNYX(A_17_P11717994)usedintheMIAMIanalysis.Amplificationandsequencingprimersfor pyrosequencingweredesignedwithPyroMarkAssayDesign software ver.2.0(Qiagen, Venlo,the Nether-lands) (Electronic Supplementary Material Table 1).ThetargetedDNAsegmentswereamplifiedusingahotstart protocol with a touchdown PCR system (Veriti Ther-malCycler;AppliedBiosystems),andthe strand servingasthe pyrosequencing template was biotinylated. Following de-naturation, thebiotinylatedsingle-strandedPCRampliconswereisolatedandallowedtohybridizewithasequencingprimer. Pyrosequencing (PyroMark Gold Q96 Reagents; Qia-gen)wasthenperformedusingthePyroMarkQ24system(Qiagen)accordingtothemanufacturersprotocol. These-quencing assay was validated using an internal control (a non-CpG cytosine within the target methylation sequence region).Quantitative real-time PCRTheqRT-PCRanalysiswasperformedusingtheTaqManPCRmethodwitha7900HTFastReal-TimePCRSystem(AppliedBiosystems) withcDNAfromTh0s separatedfromtheother patientswithMCNSat relapseandsubse-quent remission. The three genes previously analyzed usingMIAMI, namely, GATA2 (Hs00231119_m1), PBX4(Hs00257935_m1), andNYX(Hs00360869_m1), wereassayed. Each gene was assayed four times for each sample.Aliquots of cDNA equivalent to 300 ng of total RNA wereusedinthe RT-PCR reactions. GAPDH(Hs99999905_m1)was used as an endogenous control for normalizing the RNAconcentrations. DifferencesintheCTvaluesbetweenthetested genes and endogenous control (CT) were calculatedand used for subsequent statistical analyses.Statistical analysisStatistical analyses of thedistancedistributions fromtheMIAMIregressionlineforeachoftheprobesusedintheassays was performed using a non-parametric MannWhit-neyUtest (Fig. 1). Comparisonsbetweentheexpressionlevels for eachgroupwereanalyzedusingtheWilcoxonmatched-pairs signed-ranktest (Fig. 4a, b). All statisticalanalyses were performed using GraphPad PRISM5 software(GraphPadSoftware, LaJolla, CA) withthesignificancelevel set at P