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MOLECULAR STUDY OF IDIOPATHIC NEPHROTIC SYNDROME Gemma Bullich Vilanova ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en DOCTORALTHESIS MOLECULARSTUDYOFIDIOPATHIC NEPHROTICSYNDROME GemmaBullichVilanova April2016 Moleculaarstudyofidiiopathiicneph hroticsyndrom me Thesiispresenttedby GemmaaBullichV Vilanova TofulfilthePhDd degreeat UnivversitatA AutònomadeBarcelona ThessisrealizedunderthedireectionofDr.ElisabetArssCriachandDr.RoserTo orraBalcellsaatthe BiologyofFun ndacióPuigvvert,Barcelona laaboratoryofMolecularB ThessisascribedttotheDeparrtmentofCellularBiologyy,PhysiologyyandImmun nology,Facultyof Medicine,UniverrsitatAutòno omadeBarcelona PhDinCellularBiiology RosserTorraBalccells DirecttorofthetheesisDr. Dr..ElisabetArssCriach Direectoroftheth hesisDr. CarmeN NoguésSanm miquel Tuto orofthethessis Gemm maBullichVilanova P PhDCandidat te Barccelona,April2016 AlsmeusparesialTayo, AGRAÏMENTS Aquestatesiéselresultatdelacontribuciódemoltagentsenselaqualnohaguésestatpossiblearribar fins aquí. Tots els que citaré, i espero no deixarͲme ningú, heu posat el vostre granet de sorra (o un sorralsencer)enquèaquestatesistirésendavant,moltesgràcies! Enprimerllocvoliaagrairdetotcoralesmevesdirectoresdetesi,l’ElisabetArsilaRoserTorra,totel quehanfetpermidurantaquestsanys.Al’Elisabet,moltíssimesgràciesperlatevaenormeimplicació entotselsaspectes,perestarsempredisposadaadedicarͲmeunaestona,perensenyarͲmeapensaria profunditzar,pelteuesperitcrític,pertotselsconsellsiperconfiarenmiienelmeucriteri.Detuhe aprèsmoltíssimanivellcientíficperòtambéanivellpersonal,ésunluxetreballarambtu.AlaRoser, moltíssimesgràciesperintentartrobarsempreunforadetenlatevaatapeïdaagenda,perresoldretots els dubtes clínics, per aportar un punt d’optimisme als projectes, per implicarͲme en altres projectes, pervalorarͲmetant,perhaverͲmeconsolatenelsmomentsmésestressants,peraconseguirquetingui unsoudigneiperhaverfetpossiblequed’aquípoquetme’nvagiaHolanda. AlaCarmeNogués,latutorad’aquestatesi,moltesgràciespertotal’ajudaburocràtica,perfacilitarͲme almàximtotselstràmitsiperresoldreelsmeusdubtes. Moltíssimes gràcies a lescompanyesdel laboratoride Biologia Molecular per pintardecolors els dies grisos.APatricia,porestarsiempredispuestaaayudarme,portustoquesdehumorquemeencantany porenseñarmeaserunapersonaordenadayorganizadadentrodellab,estoysegurísimaquedóndesea quevayaatrabajarecharédemenosuna“labmanager”comotú.AlaLaura,“laalegríadelahuerta”, per les teves ganes d’aprendre el que calgui i per encomanarͲnos les teves rialles. A la Luz, l’altra “alegríadelahuerta”,perensenyarͲmequecalmoltpocperserfeliç,pertoteslesxerradesd’ànimsi perestarsempredisposadaaajudarͲmeenl’àmbitcientíficinocientífic.AChiara,porescucharmeen losmomentosbajosyfilosofarsobrelavidaysobresisirveparaalgoestodeldoctorado,seguroque sí!!!Ah!yporresolvermelasmilesdedudassobreelinglés!Al’Elena,pertotal’ajudaatotselsnivells, perpreocuparͲtesemprepercomestic,pelsbonsconsells,ipersertanoptimistaipositiva,etsunsol.A laBea,perhaverͲmeajudattantesvegadesenmiltemes:científics,devestuari,detesi,d’allotjament...i perensenyarͲnosquesinohihariscfísicnoenshemd’estressar,tandebòpoguésferͲho!AlaIrene, perquè si no fos per ella no faríem ni la meitat de sopars, ets la “lab manager” de la vida fora del laboratoriielcotxeoficialquetants“apuros”enshasolucionat,gràciesperestarsempredisposadaa ajudar! A la Judit, pels ànims en el moments tristos i per preocuparͲte per mi. A l’Iván, el profe de bioinformàtica,perferͲmeperdrelaporalapantallanegraipertotal’ajudaenlaNGSilanoNGS,en laborables i festius. A Dani,por su humorandaluz. Ial Toni, la recent incorporaciódel laboratori,per cuidarͲnosdesdeelprimerdiaambcroissantsibraçosdegitano,quinbonfitxatge! Hi ha una sèrie de persones que ja no estan al laboratori però que sens dubte els he d’agrair la seva ajuda. A la Sheila, la meva “profe”, ha estat un autèntic plaer seguir els teus passos i tenirͲte com a referent en laSN, moltesgràcies per tot el que em vas ensenyar abans de marxar i per estar sempre disposadaaresoldre’mtotselsdubtes.ADeborah,migrancompañeradeatardeceresrománticosenla Puigvert,¡cómoteheechadodemenosestosúltimosaños!Muchasgraciasportodostusconsejosypor compartir tantos momentos dulces y amargos. A l’Olga Sancho, la psicòloga, gràcies per ajudarͲme a encarareldoctorat(ilavida)d’unamaneraconstructiva.APaola,¡suertedetuoptimismoduranteel proyectodelospodocitos!Muchasgraciasportodosloshalagosyporvalorarmetanto.AAniaporser unabuenazaytenerungrancorazón.AlaLaia,laXèniailaLluïsa,sempredisposadesaajudariala MònicaVall,peranimarͲmeenelsmomentsdurs. ApartdellaboratorideBiologiaMolecular,laFundaciótégranspersonesalesqualshed’agrairlaseva contribucióenaquestatesi.AJoséBallarín,muchasgraciasportodotuapoyo,porproporcionarmeuna situaciónlaboraldigna,porvalorartantonuestrotrabajoyporestarsiempredispuestoaresolvermis dudas. A la Sílvia Mateu, moltes gràcies per totes les gestions, pel “papeleo” i per tots els ànims. A MontseDíazyArturOliver,porvuestraayudaenelestudiodemembranosa.AlaSílviaylaCharo,pels ànimsiperferͲmesentirquetoteldaltabaixemocionalquecomportaescriurelatesiésnormal.Ales doctorandesnefròlogues,Nàdia,IaraiMónicapertoteslesparaulesdesuportielsànims,enespeciala laNàdia,haestatunplaerajudarͲteenlesnefropatiesintersticials,nomésetquedaescalarunamica mésdemuntanyafinsarribaralcim,moltsànims!Al’OlgaLópezpelselogisilatevamotivació.AlRicard Pallejà,pertotselsarticlesquem’hasaconseguitambunagranrapidesa.AlaNúriaVinyolasperlaseva disposicióaajudarͲmeenelquècalgués.Atotselsnois d’arxius,pertenirsempreapuntunaparaula simpàticaiunsomriure.Al’IgnasiGich,pelsuportestadísticialaCarolynNeweyperlescorreccionsde l’anglès. AgrairtambéalXavierEstivillperacollirͲmealseulaboratoridelCRGdurantlanostracolͼlaboració.Al DaniialJustoperensenyarͲmetotselstrucsisecretsdelaNGS. Hihaalgunespersonesquetotinoestarpresentseneldiaadiahantingutunpapermoltdecisiuen l’inici d’aquesta tesi. Volia agrair al Pep per fer que una simple conversa es convertís en una gran oportunitat.AlJosepFarguesperescoltarͲme,aconsellarͲmeiajudarͲmejaabansd’emprendreaquest camí.Duranttotsaquestsanysmen’herecordatenmoltsmomentsdelatevafrase:eldoctoratésuna cursa de resistència. Quina gran veritat! A la Naiara, moltes gràcies per tots els bons consells i per encomanarͲmelailͼlusióperlaciència. Alesbioquímiques,gràciespernodefallirenorganitzarsopars!AlaGeorgina,moltíssimesgràciesper les trucades, per cuidarͲme i preocuparͲte per mi, pel punt d’optimisme i les ganes de tirar endavant que sempre em dónes, ets una gran amiga, hem compartit tot des de que vam entrar al món de la ciènciainopucestarmésorgullosadel’amigaquehedescobert,esperopoderͲteajudarenlatevatesi tantcomtuhasfetamblameva.Al’Àstrid,l’escriptoradetesiexprés(quinaenveja!)iaventurera,per transmetreaquestamotivacióiganesdetirarͲtealapiscina,esperoqueaviatorganitzemunviatgea París! A la Laura, la primera doctora del grup, gràcies per tots els consells i per compartir molts momentsidubtessobreelfuturdurantelmàster.AlaMireiaperquèlasevamotivacióiganesdefer cosesalegreneldiaaqualsevolialaHannah,quinasortcoincidiralCRGitornarͲnosaretrobar!Itambé a la resta de colla de la uni, Guillem, David, Raquel i Gemma per compartir penes dins i fora del doctoratiperquènoempucimaginarelsanysd’universitatsensevosaltres. Als“runners”moltesgràciespertransmetresuperaciókmdarrerakm.AlaMayteperaguantartotesles vivències del doctorat i de la vida en general. Al Roger, René, Raúl, Lluís, Uri i a les 2 Patris, moltes gràciesperaguantarlesangustiesdeldoctoratitreure’mdecasadetantentant.AlDavidil’Òscarper ajudarͲmeaenderrocartotselsmursiperpreocuparͲvospermi,maioblidaréels100km!!! Elsamicste’lsvastrobantpelcamídelavidaperòsovinteldestíelsseparainoelspotsveuretantcom voldries. Gemma, moltes gràcies per estar sempre disposada a ajudarͲme, per animarͲme en tot momentiperferaquestaportadatantxula,etsgenial!!!Martí,moltesgràciesperestaralmeucostat en els moments difícils i per acceptar les meves decisions. Marta Cuenca, gràcies per no defallir en veure’ns encara que les respectives vides ho posin difícil i per estar sempre disposada a escoltarͲme, espero poder veure’t més sovint a partir d’ara. A les nenes de Ganàpies Eva, Anna Palou, Anneta i Santako, per tots els viatges que hem compartit, pels ànims que m’heu donat tots aquest anys i per entendreels“nopuchedefertesi”.Alsamicsgranollerins,MartaiEdmon,ElisendaiJoan,peraportar riallesibonsmomentsalscapsdesetmanadetesi,moltesgràcies! AlafamíliaCastelluda,moltesgràciesperserlamillorsegonafamíliaquepodriatenir.APepeiImma, peracollirͲmealavostrafamília,percuidarͲmeipreocuparͲvospermiipervalorartantelquefaig.A Santi,Montse,José,Carol,ImmaiToni,gràciespertotelsuportquem’heudonattotsaquestsanys,per interessarͲvos per com m’anava el doctorat i per aguantar més d’una conversa sobre articles i coses rares.AlesxicotiuesJúliaiMireia,perarrancarͲmesempreunsomriureiferͲmepassartotselsmals. AlaIaia,lluitadorairiallera,moltesgràciesperensenyarͲmeunsvalorsiunamanerad’enfocarlavida quehasigutclauperfereldoctorat.Al’AvimoltesgràciesperensenyarͲmelaimportànciad’estudiar, detreurebonesnotesipersentirͲtetantorgullósdemi.Sempreusportaréambmi. Alsmeuspares,RosaiFrancescm’heudonatlesalesperarribarfinsaquí.Gràciespercreuresempreen mi, per valorarͲme tant, per estar al meu costat en els bons i mals moments, per acceptar les meves decisions,perferͲmeaixecarenelsmomentsdifícils.GràciesperensenyarͲmeelvalordelescoses,aser humil,alluitarpelquèunvol.Souelmeureferentalavidaisihearribatfinsaquíésgràciesavosaltres. AlTayo,perlasevaajudainfinitaduranttotsaquestsanys.Etsunapersonaincreïbleiemsentomolt afortunadadecompartirlavidaambtu.Gràciesperestaralmeucostattotsaquestsanys,percreure tantenmiienelquèfaig,perferͲmeenfocarelsproblemesdelamaneramésconstructivaiperajudarͲ meaaixecarentotselsmomentstristos.Etselmeupríncepblau,laparellaqueemcuida,l’amicque escolta, el pallasso que fa riure, l’aventurer amb qui viure experiències inoblidables, un mestre dins i foradel’escolaiunadelespersonesquemésadmirodelmón. Per últim voldria donar les gràcies a tots aquells pacients queens han donat la seva mostra i hanfet possibleaquestatesi,aixícomatotselsclínicsquehancolͼlaboratenl’estudid’aquestspacientsiens hanajudataseguiraprenent.Aquestestuditampochauriaestarpossiblesenseelfinançamentperpart del Ministerio de Sanidad (FIS/FEDER 09/01506, FIS/FEDER 12/01523, FIS/FEDER PI13/01731 i FIS/FEDERPI15/01824)ilaREDinREN. TABLEOFCONTENTS ABBREVIATIONS..................................................................................................................1 PRESENTATION...................................................................................................................5 INTRODUCTION............................................................................................................................. 7 1. KIDNEYANATOMYANDPHYSIOLOGY......................................................................................9 2. THEGLOMERULARFITLRATIONBARRIER..............................................................................11 2.1. FENESTRATEDENDOTHELIUM....................................................................................11 2.2. GLOMERULARBASEMENTMEMBRANE......................................................................13 2.3. PODOCYTES.................................................................................................................16 3. NEPHROTICSYNDROME.........................................................................................................19 3.1. 3.2. 3.3. 3.4. CLINICALMANIFESTATIONS........................................................................................20 3.1.1. Proteinuria.......................................................................................................20 3.1.2. Hypoalbuminemia...........................................................................................21 3.1.3. Edema..............................................................................................................21 3.1.4. Hyperlipidemia................................................................................................22 COMPLICATIONS.........................................................................................................23 3.2.1. Thrombosis......................................................................................................23 3.2.2. Acuterenalinsufficiency.................................................................................23 3.2.3. Endocrinalterations........................................................................................24 3.2.4. Infections.........................................................................................................24 ETIOLOGYOFNEPHROTICSYNDROME.......................................................................24 3.3.1. CongenitalNephroticsyndromeofFinnishType............................................24 3.3.2. Diffusemesangialsclerosis..............................................................................26 3.3.3. Minimalchangedisease..................................................................................26 3.3.4. Focalsegmentalglomerulosclerosis...............................................................27 3.3.5. Membranousnephropathy.............................................................................29 TREATMENT.................................................................................................................30 3.4.1. Generalmanagement......................................................................................30 3.4.2. TreatmentinCNS............................................................................................31 3.4.3. TreatmentinchildrenandFSGS/MCDadults.................................................31 3.4.3.1. Corticosteroidtherapy................................................................................31 3.4.3.2. Immunosuppressivetherapy......................................................................32 3.4.4. TreatmentinMN.............................................................................................32 4. PATHOGENESISOFIDIOPATHICNEPHROTICSYNDROME.....................................................34 4.1. IMMUNECOMPLEXESDEPOSITION:MN....................................................................35 4.2. TCELLDEPENDENTCIRCULATINGFACTOR:SSNSANDSDNS.....................................37 4.3. GENETICCAUSE:SRNS.................................................................................................38 5. GENETICSOFSRNS.................................................................................................................42 5.1. AutosomalrecessiveisolatedSRNSandFSGS.............................................................42 5.1.1. NPHS1gene.....................................................................................................43 5.1.2. NPHS2gene.....................................................................................................45 5.1.3. PLCE1gene......................................................................................................47 5.1.4. CD2APgene.....................................................................................................49 5.1.5. PTPROgene.....................................................................................................50 5.1.6. MYO1Egene....................................................................................................50 5.1.7. ARHGDIAgene.................................................................................................51 5.1.8. ADCK4gene.....................................................................................................51 5.1.9. TTC21Bgene....................................................................................................52 5.1.10. CRB2gene........................................................................................................54 5.1.11. NUP107gene...................................................................................................54 5.1.12. NUP93gene.....................................................................................................55 5.2. 5.3. GenesimplicatedinautosomaldominantisolatedSRNSandFSGS...........................56 5.2.1. WT1gene........................................................................................................56 5.2.2. ACTN4gene.....................................................................................................58 5.2.3. TRPC6gene......................................................................................................59 5.2.4. INF2gene.........................................................................................................61 5.2.5. LMX1Bgene.....................................................................................................63 5.2.6. ANLNgene.......................................................................................................63 5.2.7. PAX2gene........................................................................................................64 GenesimplicatedinsyndromicformsofSRNSandFSGS...........................................65 6. CLINICALUTILITYOFGENETICTESTING.................................................................................67 AIMS............................................................................................................................... .............71 RESULTS............................................................................................................................... ........75 STUDYI............................................................................................................................... ....77 HLAͲDQA1andPLA2R1PolymorphismsandRiskofIdiopathicMembranousNephropathy77 STUDYII............................................................................................................................... ...93 TargetednextͲgenerationsequencinginsteroidͲresistantnephroticsyndrome:mutationsin multipleglomerulargenesmayinfluencediseaseseverity...................................................93 STUDYIII............................................................................................................................... 113 ContributionofTTC21Bgenetoglomerularandcystickidneydiseases.............................113 STUDYIV............................................................................................................................... 123 ClinicalutilityofakidneyͲdiseasegenepanelforgeneticdiagnosisofcysticandglomerular inheritedkidneydiseases.....................................................................................................123 DISCUSSION............................................................................................................................... 151 IDIOPATHICNEPHROTICSYNDROME........................................................................................152 IDIOPATHICMEMBRANOUSNEPHROPATHY:THEIMPORTANCEOFGENETICS.......................152 THEKIDNEYͲDISEASEGENEPANEL:THEINTEGRATIVEAPPROACH..........................................155 SRNS/FSGS:FROMGENETICSTOGENOMICS............................................................................159 TTC21BGENE:CAUSATIVEANDMODIFYINGROLE...................................................................162 CONCLUSIONS........................................................................................................................... 165 BIBLIOGRAPHY........................................................................................................................... 169 ANNEXES.......................................................................................................................195 ABBREVIATIONS A,Ala Alanine AIIRA AngiotensinIIreceptorantagonists ACEi AngiotensinͲconvertingͲenzymeinhibitors AD Autosomaldominant ADAS Autosomaldominantalportsyndrome ADPKD Autosomaldominantpolycystickidneydisease ADTKD Autosomaldominanttubulointerstitialkidneydisease AFP AlphaͲfetoprotein ANP AtrialNatriureticPeptide AR Autosomalrecessive ARAS Autosomalrecessivealportsyndrome ARPKD Autosomalrecessivepolycystickidneydisease AS Alportsyndrome Ca 2+ Calciumion CAKUT Congenitalanomaliesofkidneyandurinarytract C,Cys Cysteine CD2AP CD2ͲAssociatedProtein CI Confidenceinterval CKD Chronickidneydisease CN Copynumber CNS Congenitalnephroticsyndrome CNF CongenitalnephroticsyndromeofFinnishtype CNV Copynumbervariant CoQ10 CoenzymeQ10 D,Asp Asparticacid DAD Diaphanousregulatorydomain DAG Diacylglycerol Deletion/insertion Indel DID Diaphanousinhibitorydomain DMS Diffusemesangialsclerosis DNA DeoxyriboNucleicAcid DSC Doublingofserumcreatinine E,Glu Glutamicacid 1 ELISA EnzymeͲlinkedimmunosorbentassay ESRD EndͲstagerenaldisease F,Phe Phenylalanine FCGR3 FcgammareceptorIII FH1,FH2 Forminhomologydomains1,2 FNIII FibronectintypeIII fs Frameshift FSGS Focalsegmentalglomerulosclerosis g,mg Gram,milligram G,Gly Glycine GBM Glomerularbasementmembrane GEC Glomerularendothelialcell GFB Glomerularfiltrationbarrier GFR Glomerularfiltrationrate h Hour h Heterozygous H Homozygous H,His Histidine HDL Highdensitylipoprotein HLAͲDQA1 HLAcomplexclassIIHLAͲDQɲͲchain1 IFA IndirectfluorescentͲantibodytechnique IFT139 Intraflagellartransportprotein139 Ig Immunoglobulin IKD Inheritedkidneydiseases IMN IdiopathicMN INF2 Invertedformin2 INS Idiopathicnephroticsyndrome IP3 Inositol1,4,5Ͳtriphosphate JATD Jeuneasphyxiatingthoracicdystrophy K,Lys Lysine kDa Kilodalton l,dl,ml Liter,deciliter,milliliter L,Leu Leucine LG Lamininglobular LMX1B LIMhomeodomainͲcontainingtranscriptionfactor1B LMͲ521 Lamininɲ5ɴ2ɶ1 2 LN LamininNͲterminal M,Met Methionine 2 m Metresquare MAF Minorallelefrequency MCD Minimalchangedisease mDias MouseDiaphanousͲrelatedformins MG Mutationgroup min Minutes MLPA MultiplexligationͲdependentprobeamplification Mmol Millimole MN Membranousnephropathy mo Months MOI Modeofinheritance MYO1E Myosin1E n Samplesize N,Asn Asparagine NC1 Noncollagenousdomain ND Nodata NEP Neutralendopeptidase NGS NextͲgenerationsequencing Nm Nanometre NPHP Nephronophthisis NPHPͲRC NephronophthisisͲrelatedciliopathies NS Nephroticsyndrome NSR NonͲspontaneousremission OR Oddsratio P PValue P,Pro Proline PCR Polymerasechainreaction PH Pleckstrinhomology PKD Polycystickidneydisease PLA2R1 MͲtypephospholipaseA2receptor PLCɸ1 PhospholipaseCepsilon1 PLC_X,PLC_Y Phospholipasecatalyticdomains Q,Gln Glutamine R,Arg Arginine 3 RA1,RA2 RasGTPbindingdomainfromguaninenucleotideexchangefactors RAAS ReninͲangiotensinͲaldosteronesystem RasGEF_CDC25 GuaninenucleotideexchangefactorforRasͲlikesmallGTPases Ref Reference rs RefSNP S,Ser Serine sd Syndrome SD Standarddeviation SDNS SteroidͲDependentNephroticSyndrome SNP Singlenucleotidepolymorphism SOD2 Superoxidedismutase2 SRNS SteroidͲResistantNephroticSyndrome SSNS SteroidͲSensitiveNephroticSyndrome suPAR Solubleurokinaseplasminogenactivatorreceptor T,Thr Threonine THSD7A ThrombospondintypeͲ1domainͲcontainingprotein7A TM Transmembrane TRP Tetratricopeptiderepeatdomains TS Tuberoussclerosis UCV Unclassifiedsequencevariants uPAR Urokinaseplasminogenactivatorreceptor V,Val Valine VEGF Vascularendothelialgrowthfactor vs Versus VS Variantscore WAGR Wilmstumor,aniridia,genitourinaryabnormalities,andmental retardation WT1 WilmsTumor1 XLAS XͲlinkedalportsyndrome Y,Tyr Tyrosine y,yr Years * Stopcodon 4 PRESENTATION Thisthesisisacontributiontotheknowledgeofthemolecularbasesofidiopathicnephrotic syndrome.IthasbeenperformedintheMolecularBiologylaboratoryofFundacióPuigvertin Barcelona.Thislaboratoryperformsgeneticdiagnosticofinheritedrenaldiseasessince2002 aswellasresearchonthemolecularbasesofthesediseases. Thefirstpartofthisthesispresentstheassociationofgeneticpolymorphismswiththeriskto developidiopathicmembranousnephropathyandwithitsclinicalcourse.Ourresultsshowed that risk alleles within HLAͲDQA1 and PLA2R1 genes are associated with a higher risk of idiopathicmembranousnephropathy.Inaddition,thecombinationoftheriskallelesforboth genesresultsinanincreasedriskofidiopathicmembranous nephropathy.Forthefirsttime, we presented evidence of the contribution of these polymorphisms to predict response to immunosuppressivetherapyanddeclineinrenalfunction. ThesecondpartofthesethesisfocusesonthestudyofthemolecularbasisofsteroidͲresistant nephrotic syndrome. Our results contribute to enhance the genotypic spectrum associated withthisdiseaseinseveralaspects:1)mutationsinanSRNS/FSGSgenetogetherwithCOL4A3 have been detected in patients with increased disease severity, 2) the homozygous p.P209L mutation in TTC21B gene is not the only causative mutation of FSGS, and 3) heterozygous deleterious TTC21B variants may aggravate the phenotype of patients with glomerular and cystic kidney inherited kidney diseases. In addition our results also improved the genetic diagnosisofinheritedkidneydiseases.WedevelopedakidneyͲdiseasegenepanelthatallowed amorecomprehensivecharacterizationofpatientsenablingthedifferentialdiagnosisofcystic andglomerularinheritedkidneydiseasesandtheidentificationofstructuralvariants,complex inheritancepatternsandmosaicmutations.Thisapproachiscurrentlyusedinroutinegenetic diagnostic of patients from all over Spain and other countries, providing a nice example of translationalresearch. This thesis consisted of six parts. The introduction section describes the generalities of the glomerularfiltrationbarrier,aswellastheclinicalfeaturesandmolecularbasesofidiopathic nephroticsyndrome.Theaimssectionreflectstheglobalandtheparticularaimsofthepresent thesis.TheResultssectioncontainsthefourstudiesperformedinthisprojectwithasummary of each one at the beginning. These results are discussed the Discussion section. Finally, the conclusionsderivedfromourresultsarepresentedintheConclusionsection. 5 6 INTRODUCTION 7 8 1. KIDNEYANATOMYANDPHYSIOLOGY Thekidney’smainfunctionistofilterwastematerialsoutofthebloodandpassthemout of the body as urine. The kidney’s microscopic functional units that filter blood to produce urine are nephrons. An adult human kidney is known to contain an average of one million nephrons.Basedontheirlocation,therearetwotypesofnephrons:corticalnephrons(85%), which are located deep in the renal cortex, and juxtamedullary nephrons (15%), which lie in therenalcortexclosetotherenalmedulla(Figure1). Nephrons are functionally divided into a filtration unit called the renal corpuscle and a reabsorptionunitnamedtherenaltubule: Ͳ TherenalcorpuscleiscomposedoftheglomerulusandBowman’scapsule,separated bytheurinaryspacealsonamedBowman’sspace. o The glomerulus is a network of tangled capillaries where the actual filtering takesplace.Itiscomposedofthreecelltypes:glomerularendothelialcellsthat form the intricately tortuous inner capillary tuft; podocytes or visceral epithelialcellsthattightlywraparoundtheexteriorofglomerularcapillaries; and mesangial cells that together with the mesangial matrix provide a structural reinforcement for the glomerular vasculature. Glomerular endothelial cells and podocytes share a common extracellular cell matrix knownastheglomerularbasementmembrane(GBM).Thesethreelayersform theglomerularfiltrationbarrier(GFB). o Bowman’scapsuleisacuplikeenclosurecomposedofparietalepithelialcells thatsurroundthetuftofcapillaries. Ͳ Therenaltubuleisalongconvolutedstructurethatemergesfromtheglomerulus.Its functionistoconcentrateurineandrecovernonͲwastesolutesfromtheprimaryurine. Itisdividedintothreepartsbasedontheirfunction:theproximalconvolutedtubule, the loop of Henle and the distal convoluted tubule, which empties its filtrate into collectingducts. 9 Figure e 1. Basic kidneyanatomy (adapted fro om: “KidneySStucture.” Bou undless Biolog gy. and Kurts etal., 2013)). T The renal arrtery enters the kidneyy through th he renal hilius and braanches into small arterioles that caarry the bloo od to the reenal cortex. In the renaal cortex, blo ood enters to t the glomeruli througgh the afferrent arteriole in the vaascular pole. Inside thee glomeruluss, the affereent arteriolee immediateely branchess into the ellaborated glomerular caapillary tuft. LowͲ molecularͲweighttplasmawassteproductssarefilteredacrosstheG GFBformingtheprimaryyurine hepassageo ofalbuminaandlargermacromolecules(>15kDaa)that intheeurinaryspaace,whileth are necessary n for maintainin ng normal ho omeostasis is restricted.. The filtrateed blood exitts the glomeruli througgh the efferrent arteriolles. The effferent arterioles descen nd into the renal ullaandsepaarateintoth heperitubulaarcapillariess.Theperitubularcapillaariessurroun ndthe medu proximalanddisttaltubules,aaswellastheeloopofHenle,wherettheyareknow wnasvasa.These capilllariesconverrgeintoveinsthatendin ntherenalve ein. T Theprimary dintheurin naryspaceflowstothep proximalcon nvolutedtub buleat urineformed theu urinarypoleo oftherenalccorpuscle.There,mucho ofthewaterrandnutrien ntsinitiallyfiltered into the primaryy urine are reabsorbed d. Then, the e filtrate enters the loo op of Henle e that desceendsdeepin ntothemedullaofthekkidney,make esahairpintturn,andretturnstotherenal corteex. In the loo op of Henle water and ions are reaabsorbed. Neext, the filtrate passes to t the distall convoluted d tubule an nd finally urrine from th he distal co onvoluted tu ubules of se everal nephronsenters thecollectin ngduct.Inth hisstructureswaterand ionsarestilllreabsorbed d.The oncentrated urine throu ugh the renaal medulla aand into the renal colleccting duct caarries the co 10 pelviss. In the ren nal pelvis, urine from many m collecting ducts co ombines and flows out of o the kidneeysandintottheureter. 2. THEGLOM T MERULARFITLRATIO ONBARRIER T The GFB liess between the t vasculatture and th he urinary space s being the only barrier b betw ween the blo oodstream and a the prim mary urine. This structu ure is a dyn namic and highly selectivefilterthatsieveson nthebasiso ofmolecular sizeandeleectricalcharggegeneratin ngthe primaaryurine. T driving component The c o the glomerular filtrattion is the glomerular of g p pressure, wh hich is highlyy controlled by the regulation of th he vascular resistance of o both afferent and effferent arterioleviadiffeerentmechanisms.Otherrfactorsdetterminingtheeglomerularrfiltrationarrethe oncottic pressure due to thee high conceentration off intracapillaary plasma p proteins, and the hydro ostaticpressure,exerted dbyafluidattrest(Leeuw wisetal.,201 10). T GFB is composed The c of three layeers: the fene estrated end dothelium, tthe GBM an nd the podo ocytes, visceeral epitheliaal cells with h interdigitaating foot processes p th hat form th he slit diaph hragm(Figurre2). Figure e 2. A) Schematic represeentation of a a glomerular capillary loop showing in nteraction be etween podoccytes, mesanggial cells and the endothelium. B) Enlarrged view of the glomerulaar filtration barrier: b fenesttrated endoth helium, glomerular basement membran ne and podoccyte foot processes with the t slit diaphragm(Leeuwisetal.,2010). 2 FENESTTRATEDEN 2.1. NDOTHELIU UM T The glomeru ular capillariies are heavily perforaated with trranscellular pores know wn as fenesstraeandareenotsurroun ndedbysmo oothmusclecells.Theseffenestraeare60Ͳ100nm mwide 11 and comprise c ap pproximatelyy 20% of the endotheliaal surface, making m glom merular capillaries efficieentportalsffortherapidpassageofh highvolumessoffluid.Basedontheffenestraesize e,one wouldexpectthaatalbumin(3 3.5nmofrad dius)couldpassthrough thefenestra ae(Levick&SSmaje helium has a a very low sieving s coeffficient of alb bumin 1987). However, the fenestrrated endoth umplaysanaactiverolein nrenalfiltration(Sarin20 010). indicaatingthattheglomerularendotheliu T Theglycocaly yxisanegativeͲchargedggelͲlikesurfaacestructureethatlinestheluminalfaaceof glomerularcapillaariesandtheefenestralsurfaces,andactsasasizzebarrierto albuminfiltration Q 20 002). It is composed d of proteo oglycans with their bound b (Rosttgaard & Qvortrup glyco osaminoglycaans, glycopro oteins and glycolipids. g Around A 90% of these glyycosaminogllycans areh heparinsulfateandhyalu uronanand theyactas amolecular scaffold.Plaasmaprotein nsare absorrbed within the glycocaalyx by bindiing multivale ent to glyco osaminoglycaans, thus cre eating stericchindrancettoproteinfiltration.Thissbarrierfunctionwillbeefurthermodifiedbythe efluid drag throughtheeglycocalyx whichallowsfordynam micequilibriumwithglycocalyxͲboundand freeccirculatingprroteins(Haraaldssonetall.,2008). T cellͲsurfaaceͲanchoreed glycocalyxx and the broad The b coat of o >200 nm thick formed by plasm ma proteins absorbed within w the glycocalyx constitute c the endotheelial surface layer (Hjalm marsson et al., 2004). This T layer fo orms the firsst barrier to o albumin paassage acrosss the glomerular filtrattion barrier and ensuress that the albumin is larrgely confineed to the cap pillary lumen(Figure3). Figure e 3. Schemattic representaation of the glomerular fiiltration barrier with a deetailed view of the glycoccalyxcomponents.ESL,end dothelialsurfaacelayer(adaptedfromHaaraldssonetall.,2008) 12 2.2. GLOMERULARBASEMENTMEMBRANE TheGBMisa250to400nmthinmeshworkofextracellularmatrixproteins.Mostofthe GBMissituatedbetweentheglomerularendothelialcellsandthepodocytesalthoughitisalso foundbetweenmesangialcellsandpodocytes. TheGBMprovidesstructuralsupportfortheglomerularcapillariesandharborsligandsfor receptors of the adjacent endothelial cells, podocytes, and mesangial cells (Yurchenco & Patton2009;Miner2005).Inaddition,itactsasasizeͲandchargeͲselectivefiltrationbarrier, impeding the passage of molecules negatively charged and molecules larger than approximately5nm(Deenetal.,2001). The GBM is composed of laminin, type IV collagen, heparan sulfate proteoglycan and nidogen (Figure 4). These components are synthesized by podocytes and glomerular endothelialcellsandsecretedtotheextracellularspace(StJohn&Abrahamson2001).During glomerulogenesis the separate podocyteͲderived and endotheliumͲderived basement membranes fuse to form the immature GBM. After glomerular maduration both podocytes and endothelial cells are equally important for maintaining its structure and function (Suh & Miner2013). Ͳ Lamininsarelargeheterotrimericglycoproteinscomposedofthreedifferentchains:ɲ, ɴandɶ.Thesechainsassembletoformatleast15ɲͲɴͲɶdistinctlaminintrimerswith cruciform, YͲshaped, or rodͲshaped structures. Each laminin trimer has the following parts: o A long arm formed by association of ɲ, ɴ and ɶ chains via coiled coil interactions and disulfide bonding. At the distal end, there is a large laminin globular(LG)domainformedexclusivelybytheCͲterminaloftheɲchainthat linklaminintrimerstothepodocytesandendothelialcellsviainteractionswith integrinsanddystroglycansreceptors. o ThreeshortarmscalledlamininNͲterminal(LN)domainscorrespondingtothe NͲterminalglobulardomainsofthethreechains,whichplaysanessentialrole inpolymerizationoftrimerstoformanetwork. The major laminin trimer found in the mature GBM is laminin ɲ5ɴ2ɶ1 (LMͲ521) (St John&Abrahamson2001).Oncelamininissecretedtotheextracellularspace,laminin trimersselfͲpolymerizebyinteractionsamongLNdomainsandformalamininnetwork 13 (Cheng et al., 1997). This polymerization step is a reversible and calciumͲdependent processthatrequiresaminimumconcentrationoflaminintrimerstoformaninitiation complex(Yurchenco&Cheng1993).Therefore,lamininappearstobeessentialforthe initial formation of GBM. Then, interactions of laminin with other basement membrane molecules (type IV collagen, nidogen, and sulfated proteoglycan) enable theassemblyofaGBM. Ͳ Type IV collagens represent half the total proteins of a mature GBM. There are 6 genetically distinct collagen IV ɲ chains (ɲ1Ͳɲ6) and they assemble to form three differentprotomers,eachcomposedofthreeɲchains:ɲ1ɲ1ɲ2,ɲ3ɲ4ɲ5andɲ5ɲ5ɲ6. Each collagen IV ɲ chain has three domains: the NͲterminal 7S domain, a central collagenousdomainandanoncollagenousdomain(NC1)attheCͲterminus. The central collagenous domain consists of many GlyͲXͲY amino acid triplet repeats (withXandYusuallybeinglysineorproline)thatallowtheassemblingofthechains intoatriplehelix.TherearealsomultipleinterruptionsoftheGlyͲXͲYrepeatsscattered throughout the collagenous domain that provide flexibility to the collagen IV protomers and therefore, to the collagen IV network. Both the 7S and the NC1 domainsareinvolvedinlinkingtrimerstoeachothertopromotecollagenIVnetwork formation. In addition, the NC1 domain is crucial for directing the composition of collagenheterodimers. MaturationoftheGBMinvolvesthetransitionoftheɲ1ɲ1ɲ2collagentotheɲ3ɲ4ɲ5 collagen protomers as the predominant collagen complex. This transition might be requiredtoaccommodatetheincreasedbloodpressureafterbirth,sinceɲ3ɲ4ɲ5type IVcollagenproducesamoreheavilycrossͲlinkedandmoreproteaseͲresistantnetwork comparedtotheɲ1ɲ1ɲ2typeIVcollagennetwork(Miner&Sanes1994). Ͳ NidogenͲ1 and nidogenͲ2 are two homologous glycoproteins containing three globularͲlike domains with two rodͲlike domains that separate the globularͲlike domains. NidogenͲ1 binds to both, laminin ɶ1 chain short arm and type IV collagen. NidogensaresupposedtoprovideextrastabilitytoGBMundersituationsofunusual stress,buttheyarenotrequiredfortheinitialformationoftheGBM. Ͳ Heparan sulfate proteoglycans have a protein core with covalently linked sulphated glycosaminoglycansidechains.Thesulphatedsidechainsgiveanegativechargetothe proteoglycan, however they do not seem to play critical roles in the GFB. The most 14 common heparan sulphate proteoglycan in mature GBM is agrin, although in glomerulogenesisthereappearstobecoͲdistributionofperlecanandagrin(Groffenet al.,1998).TheNͲterminaldomainofagrinbindsavidlytotheLMͲ541longarm,andits CͲterminal contains domains that can bind to cellͲsurface receptors such as dystroglycan and integrins. These properties of agrin suggest that it could be implicatedinmediatingchargeselectivitywithintheGFBandinlinkingtheGBMtothe adjacentcells.Inaddition,heparansulphatesidechainsareimportantforbindingand sequestering growth factors in some contexts, such as the passage of vascular endothelialgrowthfactor(VEGF)frompodocytestofenestratedendothelium. Figure4:ComponentsoftheglomerularbasementmembraneandthepodocyteͲglomerularbasement membraneinterface(Lennonetal.,2014). These components are distributed as a highly organized labyrinth of interconnected polygonal fibrils of varying thickness ranging from 4 to 10 nm. The fibrils are more densely 15 packeedwithinthe corewherre theyhaveeheterogene eousporesaaveraging10 0 nmin diam meter. The ɲ3ɲ4ɲ5 collagen is con ncentrated at the core e with the minor m ɲ1ɲ1 1ɲ2 closer to the endothelialside, whereastheelamininLM MͲ521andagrinarebiom modallydisttributed(Figu ure5) (Scott&Quaggin2015). Figure e5.Molecularorganization noftheglomeerularbaseme entmembran ne.ESL,endotthelialsurface elayer; FP,fo ootprocesses;GBM,glomerularbasementmembrane e;GEC,glomerularendotheelialcell;IR,in ntegrin recep ptor(Scott&Q Quaggin2015)). 2 PODOC 2.3. CYTES P Podocytesar rehighlyspeecialized,terminallydiffe erentiatedep pithelialcellssthatenwraapthe outerr aspect of the GFB. Th hey perform m highly speccialized funcctions, which h includes: a size barrieer to proteins, a chargee barrier to proteins, maintenance m of the capillary loop shape, s countteraction of the intraglo omerular preessure, synthesis and maintenance m of the GBM M, and produ uction and secretion of VEGF reequired forr glomerular endothelial cells inttegrity (Shan nkland2006)). M Mature podo ocytes consisst of morpho ologically an nd functionally different components: cell body,majorproccesses,secon ndaryprocesssesandfoo otprocesses..Thecellbo odyessentiallylies pace and it contains c the nucleus and d the cell machinery. m Frrom the cell body in thee urinary sp arise long major processes that t brancheed into seco ondary proceesses, which in turn spliit into foot processes.FFootprocessesbranchan ndinterdigittatewithneighboringfo ootprocessess,and mplex adjaccent foot processes are directly linkked by the glomerular slit diaphgraam. This com structture of the podocyte is achieved by a unique cytoskelettal architectture consistiing of 16 microtubuleͲrich major and secondary processes, and actinͲbased foot processes (Welsh & Saleem2012)(Figure6). Figure 6. A scanning electron micrograph of podocyte cells in a glomerulus shows the cell body (CB), major processes (MP), secondary processes (SP) and the finely interdigitating foot processes (Dennis KunkelMicroscopy,Inc.Welsh&Saleem2012) Footprocessescontainthreedistinctmembranecompartments:thebasalside,theapical side and the slit diaphgram. All three domains interact with the foot processes actin cytoskeleton, influencing signaling pathways and motility of the podocyte. The basal side anchorsthepodocytetotheGBMwithseveraltypesofintegrinsanddystroglycans(Kreidberg etal.,1996;Raatsetal.,2000).Theapicalsideisnegativelychargedduetothepresenceofthe anionicproteinspodocalyxin,podoplanin,andpodoendin(Kerjaschkietal.,1984;Matsuietal., 1999). This negative charge contributes to limit the passage of albumin (also negatively charged)andmaintainstheseparationamongadjacentpodocytes. The podocyte slit diaphgram is a unique cellͲcell contact with zipperͲlike structure and a constant width of 40 nm (Rodewald & Karnovsky 1974). The podocyte slit diaphgram representsaseparateclassofspecializedintercellularjunctionbecauseitintegratesstructural componentsofvariouscelljunctiontypes,includingtight,adhesion,gapandneuraljunctions (Reiser et al., 2000; Schnabel et al., 1990). Many of the molecular constituents of the slit diaphgrams have been identified, although their topological assembly into a functional complex is poorly understood. The ectodomains of several adhesion receptors in the slit diaphgram likely organize the bridge linking juxtaposed foot processes via a combination of homophilic and heterophilic receptorͲreceptor interactions. By virtue of their large ectodomains, nephrin and Fat1 are excellent candidates to associate in trans to connect opposingfootprocesses(Inoueetal.,2001;Khoshnoodietal.,2003)(Figure7). 17 Theslitdiaphgramcontainsuniqueproteinsthathavetofulfillatleastfourdifferenttasks: theyactasamacromolecularfilter,anchorthefiltertotheGBM,connecttheslitdiaphgramto theactincytoskeletonviaadaptorproteins,andarepartofasignalingcomplexthatintegrates andmediatesextracellularandintracellularsignalsregulatingtheplasticityoffoodprocesses (Grahammeretal.,2013). The first unique slit diaphgram protein identified was nephrin, a type I transmembrane protein with a cytoplasmatic CͲterminus, a transmembrane domain, a fibronectin type III domainandeightextracellularC2Ͳtypeimmunoglobulinsdomains.Nephrinmoleculesinteract intransconfigurationwitheachother,andprobablyoverlapinthemiddleofthegaptoforma dense midline, giving the slit diaphragm its zipperͲlike appearance. Therefore, nephrin is considered the core biomechanical component of the molecular sieve (Tryggvason 1999; Ruotsalainenetal.,1999). TheNephͲ1,NephͲ2andNephͲ3proteinssharecertainhomologywithnephrin,butlacka fibronectindomainandonlyhavefiveextracellularC2Ͳtypeimmunoglobulindomains(Sellinet al.,2003).Thesemoleculesareprobablytooshorttoparticipateinhomophilicinteractionsin transconfigurationacrossthe40nmgapoftheslitdiaphragm,butarelikelytointeractwith nephrininbothcisandtransconfigurations(Gerkeetal.,2003;Barlettaetal.,2003).Allthree NephproteinspossessaTHVmotifneartheirCͲterminalend,whichcanbindtoPDZdomains, therebyenablingrecruitmentofscaffoldingmolecules,suchasZOͲ1ortheaPKC/PARͲ3/PARͲ6 polaritycomplex.Neph1andnephrinseemtobeinvolvedinsignalingeventsfromtheurinary spacetothepodocytecytosol(Hartlebenetal.,2008). The third unique slit diaphragm protein identified to date is podocin, a member of stomatinfamilyofproteins.Podocinisanchoredtothemembraneviaitscentralpartandits NͲterminal and CͲterminal both face the cytoplasm. Podocin helps in the nephrin anchoring, seemstorecruitotherslitdiaphragmproteinstocholesterolͲrichmembranedomains,andto increasethesignalingpropertiesofthenephrinͲNeph1complex(Huber,Simons,etal.,2003; Gargetal.,2007). Thescaffold proteinsZOͲ1,CD2Ͳassociated protein and MAGIͲ2, aswellasCASKand the actinͲbinding proteins IQGAP1 and ɲͲactininͲ4, anchor nephrin (and potentially other slit diaphragm proteins) directly or indirectly to the actin cytoskeleton, which is crucial to both foot process spacing and stability (Huber, Hartleben, et al., 2003; Palmen 2002). These interactionsprobablystrengthenthebiomechanicalpropertiesoftheslitdiaphragmitself,and 18 might also enable it to relay signals to the actin cytoskeleton that, in turn, induce morphologicalchangestoprimaryandsecondaryfootprocesses(Grahammeretal.,2013). Figure7.Theglomerularslitdiaphragm.Anultrathinslitdiaphragmspansthefiltrationslitbetweenthe footprocesses,slightlyabovethebasementmembrane(Tryggvasonetal.,2006). 3. NEPHROTICSYNDROME Nephroticsyndrome(NS)ischaracterizedbymassiveproteinuria,hypoalbuminemia,and edema,althoughadditionalclinicalfeaturessuchashyperlipidemiaarealsousuallypresent.A recently published definition of NS considers that nephrotic range proteinuria of glomerular originandaserumalbuminconcentrationbelowthelowerlimitofnormalareessentialtoNS definition whereas edema, hyperlipidemia and lipiduria are commonly associated but not essential(Glassocketal.,2015). The central abnormality in all cases of NS is the development of massive proteinuria. Physiologically,thelivertriestocompensateforthisexcessivelosswithincreasedproteinand lipoprotein synthesis. NS develops when the loss of protein in urine exceeds the rate of albuminsynthesisintheliver,resultinginhypoalbuminemiaandedema. NS represents one of the most common diagnoses in pediatric nephrology. The annual incidence is estimated to be 2Ͳ7 per 100.000 children, with a prevalence of 16 per 100.000 children in Western countries (Giglio et al., 2015). There is a male preponderance among 19 young children, at a ratio of 2:1 to females, although this gender disparity disappears by adolescence,makingtheincidenceinadolescenceandadultsequalamongmalesandfemales. 3.1. CLINICALMANIFESTATIONS 3.1.1. Proteinuria Proteinuria is a condition in which urine contains an abnormal amount of protein. Nephrotic range proteinuria is defined as the proteinuria capable of producing NS and is consideredtobesuperiorto3.5g/24h/1.73m2inadultsor40mg/h/m2inchildren.However, theclinicalmanifestationsofNScanappearwithlowerlevelsofproteinuriaorbemissingin patientswithhigherlevelsofproteinuria.Forthisreason,itispreferredtoconsidernephrotic rangeproteinuria,theproteinuriaabletoproducehypoalbuminemia. The main proteins lost in NS have a molecular weight of 40Ͳ150 kDa, including albumin (the most abundant protein in plasma), IgG, transferrin, ceruloplasmin and ɲ1Ͳacid glycoprotein. There is also loss of small amounts of higher molecular weight proteins (200 kDa),likethesmallformsofhighdensitylipoprotein(HDL).However,proteinshigherthan200 kDasuchasIgM,macroglobulins,fibrinogen,XIIIfactor,fibronectinandlargelipoproteinsare notlostevenincaseofsevereproteinuria. The amount of proteinuria can be influenced by other factors different from the glomerular damage such as the reninͲangiotensin system activity, the hepatic capacity for albuminsynthesis,theproteindailyintakeandtheantihypertensivedrugadministration. Inhealthyconditions,theGFBavoidsthepassageofmoleculeswithasizesuperiorto70 kDatotheprimaryurine.Albuminhasamolecularweightof69kDaandanetchargeofͲ15 and is filtered through the GFB with a sieving coefficient of 0.0001. Interestingly, negatively chargedproteinshaveanincreasedclearancecomparedtopositivechargedonesduetothe negativechargesintheGBMheparansulfateproteoglycans. ProteinuriainNScanbeduetoastructuralalteration,disruptingthesizeselectivityofthe GFB and leading to massive nonselective proteinuria, or to an electrochemical alteration, affectingthechargeselectivityoftheGFBandpresentingwithselectivealbuminuria. 20 3.1.2. Hypoalbuminemia Hypoalbuminemia is a low level of albumin (inferior to 3 g/dl) in blood and it is a direct consequenceofproteinuria.Albuministhemostabundantplasmaproteinandrepresents70Ͳ 90%oftheproteinuriadetectedinNS.Thisfiltratedalbuminisinpartcatabolizedbytherenal tubule,whichincreasesitscatabolicrate.Tocompensatethislossofalbumininurine,theliver increasesthealbuminsynthesisevenina300%.Whentheproteinuriaandtherenalalbumin catabolism exceed its hepatic synthesis, hypoalbuminemia appears. The severity of the hypoalbuminemia is well correlated with the amount of proteinuria although other factors suchastheage,thenutritionalstateandthetypeofrenallesionalsoinfluence. In addition to the hypoalbuminemia, there is a decrease in the serum immunoglobulins, especially IgG, whereas high molecular weight immunoglobulins remain normal or even elevated. 3.1.3. Edema Edema is defined as an abnormal accumulation of fluid in the interstitial compartment. EdemasarethemainreasonfordiagnosisinchildrenwithNS.Theyaresoft,pittingandmainly localizedinsloperegionslikefootorsacrum,andinregionswithlowtissuepressuresuchas periorbital region. In advanced disease, patients may develop periorbital or genital edema, ascites,orpleuraleffusion. ItiswidelyacceptedthatNSpatientshaveanexcessoftotalbodysodiumandwater.Two major pathophysiological mechanisms have been proposed to explain the development of edemainNSbasedonthestatusoftheirintravascularvolume:theunderfillinghypothesisand theoverfillinghypothesis. Intheunderfillinghypothesis(Figure8A),thehypoalbuminemiacausesadecreaseinthe intravascular oncotic pressure. This in turn leads to fluid shift from intravascular to extravascular compartment and edema formation. The homeostatic response to the hypovolemia activates the renin angiotensin aldosterone system, the sympathetic nervous systemandthereleaseofantidiuretichormone.Gradually,theplasmavolumenormalizesasa resultoftheincreaseoftheextracellularspaceandtheedemaformation. In the overfill hypothesis (Figure 8B), there is an intrinsic defect that enhances tubular sodium and water reabsorption, independently of the hemodynamic situation. This causes 21 intravvascularvolu umeexpansiionandincreasedintravvascularhydrrostaticpresssure,which leads to ed dema formattion. Several mechanism ms to explain n this sodium and wateer retention in NS patientswithoveerfillingofth heintravascu ularcomparttmenthaveb beenpropossed.Suchpaatients wresistancettoAtrialNattriureticPep ptide(ANP), apolypeptid dethatisrelleasedinord derto show reducce the bloo od pressure by decreassing the am mount of waater, sodium m and fat in the circullatory system m. It has allso been suggested thaat intrarenal sodium rettention is due to activaationoftheepithelialsodiumchanneelinthecolle ectingduct. Figure e8.Pathophyysiologicalmeechanismsofeedemaformation.A)theunderfillinghyypothesisand B)the overfiillinghypotheesis(adaptedffromHernand doAvendaño2 2008). T Thefindingt hatmostNSSpatientshaavenormalo orelevatedintravascularrvolumesugggests that the overfilling hypotheesis might prevail p in most m cases. However, iin some children umecontractionisfound d.Ofnote,bo othhypothesesarenotm mutuallyexcclusive intravvascularvolu andtthevolumesstatusmayd dependonthestageof disease.Itisspossibleth hattheunderfilled state may be predominant in the acutee setting, in which massive proteinuria causes rapid a abrupt drop in plasm ma oncotic p pressure, wh hereas development of hypoalbuminemia and an overfilledstatemaybep predominant inthechron nicphasedu uringwhichp patientsmayyhave theo continuingsodium mretentionduetopersistentlowͲgradehypoalb buminemia. 3.1.4. Hyperlipide H emia H Hyperlipidem mia is an abn normally higgh concentraation of fats or lipids in blood. The most frequ uent lipid abnormality a is hypercholesterolemia appearin ng in 85% of patients. The 22 hypertriglyceridemiaislessfrequentandonlyappearswhentheserumalbuminisbelow1Ͳ2 g/dl.Lipiduria,theexcretionoffattycastsinurine,isalsocharacteristic. The pathogenesis of hyperlipidemia is multifactorial. Hypercholesterolemia seems to be the consequence of the decrease in the intravascular oncotic pressure that results in an increase in the hepatic synthesis of lipids and apolipoproteins. In addition, lipoproteins clearance is decreased due to the low level of tissue low density lipoproteins receptors. Hypertriglyceridemia is not due to the increase in triglycerides synthesis but a decrease in triglyceridescatabolism.Itiscorrelatedwiththealbuminclearancebutnotwiththedecrease intheintravascularoncoticpressure. The clinical significance of hyperlipidemia in NS is uncertain. Patients with NS and hyperlipidemiahaveanincreasedcardiovascularmortality,althoughthesepatientsalsohave othercardiovascularriskfactors.Inanimalmodels,hyperlipidemiaacceleratestheglomerular damage,promotingtheinflammatoryinfiltrateandrenalfibrosis. 3.2. COMPLICATIONS 3.2.1. Thrombosis RenalͲveinthrombosisandthromboemboliceventsingeneralconstituteoneofthemost severe complications in NS. Its incidence is estimated to be 5Ͳ60% of patients being more frequent at the beginning of the disease. In addition, only 10% of patients with renalͲvein thrombosispresentwithsymptoms:flankpain,grosshematuria,increasedrenalsize,andloss of renal function. In children, thromboses are more severe and in half of cases affect the arterialtree.Inadults,arterialthrombosisislesscommonthanvenousthrombosis,butitisa seriouscomplicationcausingimportantmorbidity. Severe hypoalbuminemia (less than 25 g/l), high proteinuria (more than 10 g/24h), high fibrinogenlevels,lowantithrombinIIIlevels(less than75%ofnormal),and hypovolemia are significantlyassociatedwithanexcessiveriskofthromboticcomplication. 3.2.2. Acuterenalinsufficiency Acute renal insufficiency appears in patients treated with diuretics with severe hypoalbuminemia.Itsmaincauseisintravascularvolumedepletion. 23 3.2.3. Endocrinalterations NSalterstheregulationofseveralendocrinesystemsduetotheurinarylossofhormones orduetothechangeintheirintravascularandextravasculardistribution. 3.2.4. Infections The risk of infections is increased in children. The most common and serious type of infectionisprimarybacterialperitonitis,whichisestimatedtohaveanincidenceof5%inNS children. Risk factors include low serum IgG levels due to urinary loss of IgG, abnormal T lymphocytefunction,anddecreasedlevelsoffactorsBandD,resultinginadecreasedability to opsonize encapsulated bacteria. In addition, the administration of steroids and immunosuppressivedrugsduringrelapsesfurtherincreasestheriskofinfection. 3.3. ETIOLOGYOFNEPHROTICSYNDROME NSmaybecausedbyavarietyofglomerularandsystemicdiseases.Approximately90%of thepediatriccasesareprimaryoridiopathicbutasmallproportionofthemaresecondaryto infectious agents and other glomerular and systemic diseases. Separation of these two categoriesisnoalwayseasybutitisimportanttodecidethetherapeuticapproach.Thisthesis isonlyfocusedontheidiopathicnephroticsyndrome(INS). INS can be associated with a variety of distinct clinic and pathological conditions largely dependentontheageatdiseaseonset.Theidentificationofthehistologicallesionmayhelpto elucidatethepathogenesisofthedisease. 3.3.1. CongenitalNephroticsyndromeofFinnishType Congenital nephrotic syndrome (CNS) appears within the first 3 months of life. CNS of Finnish type (CNF) represents the most severe form of NS. It was first described in Finland, whereithasanincidenceof1/8200birthsbutithasbeenreportedworldwide. CNFisanautosomalrecessive(AR)inheriteddisordercharacterizedbymassiveproteinuria even in utero, a large placenta, and characteristic microcystic dilatation of the proximal tubules.MutationsinNPHS1genearethemajorcauseofCNF,(explainedinthenextsection) (Kestiläetal.,1998). 24 AprenataldiagnosisofCNFissuspectedasearlyas16to18weeksofgestationifelevated ɲͲfetoproteinlevelsaredetectedinthematernalserumortheamnioticfluid,reflectingfetal proteinuria. The abnormally large placenta (placental/weight ratio over 0.25) constricts the fetus likely causing premature delivery of the fetus with postural deformities. The child is typically small for the gestational age, with widened cranial fontanelles, a small lowͲbridged nose, and edema in approximately 25% of cases. Proteinuria is detectable at birth and it is extremely high ranging from 1 to 6 g/day. Initially, proteinuria is highly selective but it becomespoorlyselectiveasthediseaseprogresses.Heavyandconstantproteinuriainevitably leadsfullͲblownNSsoonafterbirthwithlifeͲthreateningedema,malnutrition,failuretothrive, andsecondarycomplications.Microhematuriaorsignsoftubulardysfunction(aminoaciduria and glycosuria) may also be present. Renal function is usually normal at birth but it progressivelydeclinesreachingendͲstagerenaldisease(ESRD)withinthefirst2to3yearsof life.ThereisahighmortalitywithinthefirstyearduetothecomplicationsofNS,particularly infectionsandsepsis. Thehistologicallesionsaredetectedmorefrequentlyafter3monthsofage.Theearliest manifestation is focal cystic dilatation of proximal tubules (Figure 10), although it is not presentinallthecases,and/oranincreaseinmesangialcellularityandmatrix,producingslight glomerular enlargement. As disease progresses, the characteristic lesions of CNF appear consistingofradialdilatationsoftheproximaltubules,mesangialproliferationandglomerular lesions of focal segmental sclerosis. Immunofluorescence does not detect immune deposits although as the disease progresses, IgM and complement C3 can be found. Effacement of podocytes foot processes and disappearance of the slit diaphragm are seen in electronic microscopy. Figure 10. Histological lesions of congenital nephrotic syndrome of Finnish type at light microscopy. Hematoxylineosinstaining(Duicuetal.,2009). 25 3.3.2. Diffusemesangialsclerosis Diffuse mesangial sclerosis (DMS) accounts for approximately 10% of CNS cases and one thirdofinfantileNScases(from3to12months)(Gbadegesinetal.,2008). DMSischaracterizedbyonsetofNSinthefirstyearoflifeandrapidprogressiontoESRD, oftenwithin1to3months.Thisentityhasbeendescribedasanisolateddisorderoraspartof syndromeswithrenalandextrarenalabnormalitiessuchasDenysͲDrashorPiersonSyndrome. DMS is pathologically defined by mesangial expansion and sclerosis that evolves towards obliteration of the capillary lumen and contraction of the glomerular tuft (Figure 11). Immunofluorescenceshowsnoglomerularimmunedepositsalthough nonspecificstainingof the glomerular mesangium for IgM, C3, and C1q may be identified in some glomeruli. The majorfindinginelectronicmicroscopyisextensiveeffacementoffootprocesses. Figure 11. Histological lesions of diffuse mesangial sclerosis at light microscopy masson trichrome staining(fromD’Agatietal.,2005). 3.3.3. Minimalchangedisease Minimalchangedisease(MCD)isthemajorhistologicalfindinginINSchildrenaccounting for more than 80%. The most frequent age at onset is 2Ͳ6 years (median 2.5) and is more commoninmalesthanfemales(Bansal2014).Inadults,MCDisresponsibleforaround15Ͳ20% ofINScases,thereisahigherincidenceinelderlythanmiddleͲagedadultsandtheincidenceis equalbetweenbothgenders(Cameron1996;Korbetetal.,1988;Haasetal.,1997). MCD virtually always manifests as NS at the onset, thus, the predominant findings are heavy proteinuria, hypoalbuminemia, edema and elevated serum cholesterol. Proteinuria in MCDisusuallyhighlyselective,consistingpredominantlyofalbumin.MostchildrenwithMCD 26 do not have evidence of renal insufficiency, hematuria, or hypertension. This entity is highly steroidresponsivebutinsomecasesmayfollowaremittingandrelapsingcourse.Progression toESRDisnotpartofthenaturalhistoryofMCD. MCD is pathologically characterized by minimal or no glomerular alterations on light microscopy(Figure12).The“minimal”findingsincludepodocyteswellingandmildmesangial expansion. Immunofluorescence does not detect glomerular immune deposits although in a minority of cases, there may be weak mesangial positivity for immunoglobulin IgM, with or without C3. Electronic microscopy shows extensive effacement of foot processes in the absenceofotherabnormalitiesoftheperipheralcapillarywalls. Figure12.Histologicallesionsofminimalchangediseaseatlightmicroscopy.Massontrichromestaining (fromkidneypathologywebsite). 3.3.4. Focalsegmentalglomerulosclerosis Focal segmental glomerulosclerosis (FSGS) is one of the most frequent patterns of injury foundinINSaccountingforapproximately10%ofchildren,20Ͳ50%ofadolescentsandaround 20Ͳ30% of adults (Hogg et al., 2007; Cameron 1996; Korbet et al., 1988; Haas et al., 1997). FSGS lesions can occur as a result of many causes, including circulating factors, mutations in podocytegenesorbesecondarytodrugs,infectionsormaladaptiveresponsestonephronloss (Bose&Cattran2014). Ethnicity plays important roles in the incidence of FSGS being 3Ͳ7 times higher in young black men as compared with whites (Freedman et al., 2009). The reported annual incidence ratesforFSGSis5casespermillionpopulationinwhites,comparedwith24casespermillion population in African Americans. This increased incidence is partly explained by genetic risk factors in 2 important podocyte function proteins: nonmuscle myosin heavy chainͲ9 (MYH9) andapolipoproteinL1(APOL1).ThreepolymorphismsinMYH9genewerereportedtoconfer 27 riskforidiopathicFSGSandhypertensiveESRDamongblacksinARmodel(Koppetal.,2008). Further investigation revealed two variants, called G1 and G2, in the last exon of the neighboring gene APOL1 that had stronger association with FSGS and were under strong selectiononlyinAfrica(Genoveseetal.,2010). Proteinuria is a defining feature of FSGS, typically accompanied by hypoalbuminemia, hypercholesterolemia, and edema. Approximately 75Ͳ90% of children and 50Ͳ60% of adults with FSGS have NS at presentation (D’Agati et al., 2011). FSGS is associated with a poor response to corticosteroid treatment (only about 50% of patients respond to therapy). It is usually a progressive disorder with <5% spontaneous remission and a 50% ESRD rate over a periodof5Ͳ8yearsfromthetimeoftherenalbiopsyinpatientsthatareeitherunresponsive to treatment or not treated (Bose & Cattran 2014). Black race, increased degrees of proteinuria,andrenalinsufficiencyareassociatedwithaworseoutcome.Approximately40% of patients who undergo kidney transplantation develop recurrence of the disease in the allograft(D’Agatietal.,2011). FSGSispathologicallydefinedasglomerularsclerosisinvolvingasubsetofglomeruli(focal) andaportionoftheglomerulartuft(segmental)(Figure13).Becausejuxtamedullarynephrons are often affected first, adequate glomerular sampling is needed to identify the diagnostic lesions.Asthediseaseprogresses,amorediffuseandglobalpatternofsclerosisevolvesand tubularatrophyandinterstitialfibrosisdevelop.Immunofluorescencetypicallyrevealscoarse segmentalstainingforIgMandC3entrappedinareasofhyalinosis.Onelectronicmicroscopy, themajorfindingisextensiveeffacementofthefootprocesseswithoutotherabnormalitiesin theGBM.ThereareseveralFSGSvariantsbasedonthepathologicfindings,frombesttoworst prognosis:tip,FSGSnototherwisespecified,cellular,collapsing,andperihilar,beingthemost common FSGS not otherwise specified. Also, increased severity of interstitial fibrosis and tubularatrophyinbiopsyspecimensareassociatedwithworseoutcome. 28 Figure13:Histologicallesionsoffocalsegmentalglomerulosclerosisatlightmicroscopy.400x,masson trichromestaining(fromkidneypathologywebsite). 3.3.5. Membranousnephropathy Membranous nephropathy (MN) is one of the most common forms of NS in adults, accountingforabout20%ofcases(Glassock2010).Itcompriseslessthan2%ofallidiopathic NScasesinchildrenundertheageof5years,butitsincidenceincreasesprogressivelythrough adolescence and into adulthood with a peak incidence in individuals aged 30Ͳ50 years. The disease incidence is also influenced by sex being more common in males than females (sex ratio 2:1). This condition is idiopathic in approximately 70Ͳ80% of cases, whereas the remaining are secondary to a variety of autoimmune, infectious, neoplastic, and other systemicdiseases,orfollowingexposuretocertainmedications(Glassock2010). The predominant clinical presentation of patients with MN is NS, occurring in 60Ͳ80% of them.Theremainingpatientspresentwithsubnephroticproteinuria,oftenasymptomaticthat maybedetectedasanincidentalfindingduringaroutinemedicalexamination,andabout60% ofthemwillprogresstofullNS(Fervenzaetal.,2008;Hladunewichetal.,2009).Microscopic hematuriaoccursinupto50%ofpatientswithMN,butredbloodcellscastsandmacroscopic hematuria are rare (Wasserstein 1997). About 80% of patients with MN have normal blood pressure and glomerular filtration rate at presentation. Acute renal failure is uncommon (Ronco&Debiec2015). IMNnaturalhistoryfollowsthreemajorclinicalcourses:spontaneousremission,stablebut persistentproteinuria,orprogressiontoESRD.Spontaneousremissionoccursinapproximately onethirdofpatients,usuallywithinthefirsttwoyearsafterpresentation(Polancoetal.,2010; Polancoetal.,2012).Theothertwothirdsofpatientscanbedividedequallyintothosewith persistent proteinuria who will maintain normal renal function long term and those patients whowillprogresstorenalfailuredespiteimmunosuppressivetherapy(Glassock2003).MNis thesecondorthirdleadingcauseofESRDinpatientswithprimaryglomerulonephritisandthe glomerulopathy that recurs most frequently after kidney transplantation (in about 40% of cases)andthreatensgraftfunction(Ronco&Debiec2015). MNispathologicallydefinedasacapillarywallthickening,normalcellularity,positiveIgG and C3 staining along capillary walls on immunofluorescence, and accumulation of immune depositsontheouteraspectoftheGBMonelectronmicroscopycalledspikes(Figure14). 29 Figure e 14: Histolo ogical lesions of membran nous nephrop pathy. A) Hem matoxilin eossin staining at light micro oscopy: capillaary loops aree thickened and a prominent but cellulaarity is not iincreased. B) Silver staining at light microscopy: m t there are chaaracteristic “spikes” of gllomerular basement mem mbrane unedeposits.C C)Immunoflu uorescencemiicrograph:sub bepithelialdepositsofmain nlyIgG betweeentheimmu and complement c appear in a diffuse granu ular pattern in the glomeerular basement membrane. D) Electrron microscopy: electron dense immune depositss are seen scattered s witthin the thicckened glomeerularbasemeentmembranee(renalpatho ologywebsite). 3 TREATM 3.4. MENT IN NStreatmen ntrequiresaglobaltheraapeuticapprroachtocou unteractthe glomerular lesion butalsothederivvedsystemicccomplicatio ons.Generalmeasuresto otreatproteeinuriaande edema mostpatientss,whereasspecifictreatmentdependsontheaggeatonsetaand/or arecommoninm thehistologicalleesion. R Renalbiopsy mongthediffferent isconsidereedmandatorryinadultpaatientstodistinguisham renallesionsbecausethetreeatmentapproachdifferssamongtheedifferenten ntities.Inchildren r biopsy is only perfformed when the NS is steroid resisstant as most of patients are the renal respo ondent. 3.4.1. Generalma G anagement P Proteinuriais streatedwithreninͲangiotensinͲaldo osteronesysttem(RAAS)blockersinclluding both,,angiotensin nͲconvertingͲenzymeinh hibitors(ACEEi)andangio otensinIIrecceptorantagonists (AIIRA A) as they have a syn nergic effectt. These dru ugs avoid the formatio on and action of angio otensinII,ah hormonethaathasavaso oconstrictive eactionand increasesblo oodpressure e.The vasod dilationofth heglomerulaarefferentaarterioledescreasesthe intraglomerrularpressurreand thereeforedecreassesproteinleakage. 30 General measures to control edema include salt restriction, lying in supine position, and use of diuretics drugs. Moderate exercise, a low cholesterol diet, hydroximetilglutarilͲCoA reductase inhibitors, and weight lost in obese patients are recommended to reduce hiperlipidemia.Hygienic,dieteticandpharmacologicalmeasuresareneededtocounteractthe highmorbidityandmortalityassociatedwithNSanditscomplications. 3.4.2. TreatmentinCNS The goals of therapy during the first months are to control edema and possible uremia, prevent and treat complications such as infections and thromboses, and provide optimal nutritionsothatthechildgrowsanddevelopsasnormallyaspossible. CNS patients are resistant to corticosteroids and immunosuppressive therapy, therefore theonlycurativeapproachisrenaltransplantation.Bilateralnephrectomyanddialysismaybe necessarytoavoidthecomplicationsofNS. 3.4.3. TreatmentinchildrenandFSGS/MCDadults 3.4.3.1. Corticosteroidtherapy CorticosteroidsarethefirstͲlinetreatmentformostINS.ChildrenpresentingwithINSare treated empirically with corticosteroids because there is a high incidence of MCD, which is associated with a high rate of steroid responsive. A complete remission is considered if proteinuria < 100mg/m2/day or < 1+ on urine dipstick for 3 consecutive days, and urine protein/creatinine ratio < 20 mg/mmol. Partial remission consists of a proteinuria of 0.1Ͳ1 g/m2/day and absolute urine protein/creatinine ratio between 20 and 200 mg/mmol (Trautmannetal.,2015). Adults with FSGS or MCD as renal lesion are also initially treated with corticosteroids. A completeremissionisdefinedbyproteinuria<0.3g/dayandnormalizationofserumalbumin toatleast3.5g/dl.Partialremissionisconsideredifproteinuria0.3Ͳ3.5g/daywithareduction inproteinuriabyatleast50%fromthebaseline. Basedonpatients’responsetosteroidtherapy,INScanbedividedinto: 31 Ͳ SteroidͲSensitive Nephrotic Syndrome (SSNS): patients who achieve remission in response to corticosteroid treatment alone. Approximately 80Ͳ90% of these patients have one or more relapses, but they mostly continue to respond to corticosteroids throughout their subsequent course and the longͲterm prognosis, including the maintenanceofnormalkidneyfunction,isgood.Themostfrequentrenalhistological lesionofSSNSisMCD. Ͳ SteroidͲDependent Nephrotic Syndrome (SDNS): patients that initially respond to corticosteroidtreatmentbyachievingcompleteremissionbutdeveloparelapseeither while still receiving steroids at lower doses or within 2 weeks of discontinuation of treatmentfollowingasteroidtaper.SuchpatientstypicallyrequirecontinuedlowͲdose treatmentwithsteroidstopreventdevelopmentofrelapse. Ͳ SteroidͲResistant Nephrotic Syndrome (SRNS): Patients who fail to achieve remission after8weeks(children)or>4 months (adults)ofcorticosteroid treatment.Themost frequent renal histological lesions of SRNS are FSGS or DMS. Steroid resistant MCD adultsshouldbereͲevaluatedforothercausesofNS. Itisestimatedthatabout80%ofchildrenand60%ofadultswithINSrespondtosteroid therapy with complete resolution of proteinuria and edema. Among this steroid sensitive group, the clinical course is variable, with up to 60% having frequent relapses or becoming dependentonsteroidtherapytomaintaintheminremission.Theremaining20%ofchildren and 40% of adults with INS do not achieve a sustained remission after standardized corticosteroidtherapy(ArbeitsgemeinschaftfurPadiatrische1988;Antignac2002;Troyanovet al.,2005). 3.4.3.2. Immunosuppressivetherapy Immunosuppressive therapy is considered in patients with SRNS, frequent relapses or steroiddependency.Therearedifferentimmunosuppressivetherapiesavailableanddifferent therapeuticschemesdependingontheageatonsetand/orthehistologicallesion(reviewedin KDIGOguidelines(GlomerulonephritisWorkGroup2012)). 3.4.4. TreatmentinMN Spontaneous remission is a wellͲknown characteristic of MN occurring in approximately onethirdofpatients,butitmaybedelayedforaslongas18Ͳ24months(Polancoetal.,2010). 32 Thus,theinitialapproachinMNpatientsconsistsofa6Ͳmonthobservationalperiod,inwhich onlyantiproteinuricagentsaregiven.Patientswithpersistentproteinuriaandnodeclineafter 6 months are treated with immunosuppressors accoding to KDIGO guidelines (GlomerulonephritisWorkGroup2012). IMN patients presenting with nonͲnephrotic proteinuria typically only receive ACEi and AIIRAandtheyhaveaveryfavorablelongͲtermprognosis(Hladunewichetal.,2009;Ponticelli & Glassock 2014). However, a delay in immunosuppressive treatment in patients presenting withNSwouldexposethemtothecomplicationsofNS(vandenBrandetal.,2012).Inthese patients, controversy exists regarding the proper timing of immunosuppression and the best therapeuticregimen,duetothelowtherapeuticindexofimmunosuppressivedrugsandtheir side effects (Remuzzi et al., 2002; Perna et al., 2004; Glassock 2004; du BufͲVereijken et al., 2005).Anumberofcliniciansproposetorestrictimmunosuppressivetherapytopatientswith sustainedheavyproteinuriaorinthefaceofworseningrenalfunction(Cattran2005). Severalmarkersofdiseaseprogressionhelpclinicianstopredicttheclinicaloutcomeofthe disease. Predictors of spontaneous remission are baseline proteinuria less than 8 g/day, female sex, age younger than 50 years old and preserved renal function at presentation (Cattran2005).However,eveninpatientswithlargeproteinuria,spontaneousremissioncan occurinupto20Ͳ25%ofthem(Polancoetal.,2010). The Toronto Risk Score is calculated by a validated algorithm that correctly identifies patientsatriskofdiseaseprogressionwith85Ͳ90%accuracy.Thisscoreisbasedonthelevelof proteinuriaduringa6Ͳmonthperiodofmaximumproteinuria,creatinineclearanceatthestart of that period, and the change in creatinine clearance over the course of those 6 months. According to the score obtained, patients are grouped into lowͲ, middleͲ, and highͲrisk for progression and a different treatment approach is indicated (Figure 15) (Pei et al., 1992; Cattranetal.,1997).Themaindisadvantageofthismodelisthatanobservationalperiodofat least6months(ormoreiftheperiodofmaximumproteinuriadoesnotcorrespondwiththe onsetofthedisease)isneededtocalculatethescore,whichprolongspatientexposuretorisks associatedwithNS. 33 Figure e 15. A treaatment algorrithm that combines the e predictive factors and best evidencce for conseervativeandth henimmunossuppressivetreatment(Catttran2005). M Measuremen nt of the urinary excretion of ɴ2Ͳmicroglobulin and ɲ1Ͳmicrroglobulin in n spot urinee samples was w also sho own to accurately pred dict progresssive loss off kidney fun nction (Bran ntenetal.,20 005;Hofstraetal.,2008).VandenBrandetal.co omparedtheeprognosticvalue of the Toronto Risk R score with w the urinary lowͲmolecular weight proteins (ɴ2Ͳmicroglo obulin ɲ bulin) and showed s thatt both prognostic meth hods had higgh sensitivity and and ɲ1Ͳmicroglo specificity with no n significantt differencess between th hem. Howevver, urinary markers havve the ntagethatth heprognosisscanbeestaablishedwith hasinglemeeasurement (vandenBraandet advan al., 2012). 2 The clinical comp plexity of thee disease su uggests that a combinattion of prognostic markkerswouldbeethebestop ptionforpreedictionofcliinicaloutcom me. 4. PATHOGEN P NESISOFIDIOPATH HICNEPHR ROTICSYN NDROME IN NSisdueto thedevelop pmentofstructuraland functionaldefectsintheeGFB,resulttingin its in nability to restrict r urinaary loss of protein. Po odocyte foott process eeffacement is i the invariable feature observed not only in n NS patientts but also in all proteeinuric glomerular diseaases, and it can result from seveeral triggers including immune i cau uses and ge enetic abnormalities (Deegens et al., a 2008). Briefly, idiopaathic MN (IM MN) is conssidered an organͲ o withMCDorrFSGS specificautoimmunedisease.SSNSandSSDNSpatientts,generallypresentingw usedbyaTͲcelldependen ntcirculatinggfactor.Finaally,in onreenalbiospy,aareconsidereedtobecau CNSaandSRNSpaatients,thelaatterusuallyshowingFSG GSorDMSaashistologicaallesion,age enetic defecctisconsiderredtoberessponsibleforrthedisease. 34 4 IMMUN 4.1. NECOMPLLEXESDEPO OSITION:M MN M is an immunologically mediated disease chaaracterized by MN b the depo osition of immune comp plexes in thee subepithellial space, which w causess a thickenin ng of the GBM. The immune deposits consist of several components c , including IgG, I antigens, and the m membrane attack a comp plex. IgG4 iss usually th he most pro ominent IgG G subclass deposited d in n idiopathic IMN, altho ough variablee amounts of o IgG1 are also associaated with im mmune depo osits; by con ntrast, deposition of IgG G1, IgG2 and d IgG3 exceeeds depositio on of IgG4 in i secondaryy MN (Imai et al., 1997;Kurokietal.,2002;Ohttanietal.,20 004;Segawaetal.,2010)). nthepast10 0years,greaatprogressh hasbeenmaadeintheun nderstandinggofthemole ecular In patho omechanism ms of human n MN inspirred by studies of experimental mo odels. Studies on Heym mannnephrittisratsledtotheconceptthatapodocyteantiggen,megalin n,couldserveasa targeetofcirculatingantibodiesleadingto otheinsitu uformationo ofimmuneccomplexes(FFigure 16) (Heymann ( e al., 1959 et 9; Kerjaschki & Farquhar 1982). However, H m megalin is neither expreessedinhum manpodocyttesnordetecctedinthessubepithelialdepositsin patientswitthMN (Herrrmannetal.,2012). Figure e16.InͲsitufo ormationofim mmunedeposstis.Circulatin ngantibodiesbindtoanendogenouspod docyte antigeen(adaptedfrromRonco&Debiec2015). In n humans, a a number off podocyte antigens a havve been iden ntified (Figurre 16). Antib bodies that target podocyte antigeens accumu ulate as immune depo osits leadingg to comple ement onalimpairm mentoftheG GFBandprotteinuria(Ron nco&Debiecc2012). activaation,functio C Circulatingan ntibodiesagainstneutralendopeptid dase(NEP)w wereidentifiiedinachild dwith neonatal MN (D Debiec et all., 2002). NEP is a me embraneͲbou und enzymee that can digest biologically activve peptides (Turner et al., 2001). The mother was NEP deficient due to MEgene,wh hichcodifies foramembranemetallo oͲendopeptid dase.Shebe ecame mutaationsonMM alloim mmunized to o the paterrnally inherited NEP an ntigen expreessed in thee placenta during d pregn nancyandtrransplacentaallytransferrredNEPantiibodiestoth hechildcaussing MN. Thefact 35 thatrabbitsinjectedwiththeIgGfractionfromthemotheralsodevelopedMNandproteinuria wasanadditionalproofthatthediseasewasrelatedtocirculatingantiͲNEPantibodies(Debiec et al., 2002). In the five cases with alloimmune neonatal MN identified so far, the mothers were NEP deficient due to homozygous truncating deletion in exon 7 or compound heterozygoteforthismutationandasecondmutationinexon15oftheMMEgene(Debiecet al., 2004; Vivarelli et al., 2015). This finding provided the proof of concept that a podocyte antigen could be responsible for human MN, as is the case for megalin in rats, and laid the foundationfortheidentificationofpodocyteautoantigensinadultswithIMN. The identification of antibodies to the MͲtype phospholipase A2 receptor (PLA2R1) in about 70% of patients with IMN indicated that PLA2R1 is the major target antigen in this disease (Beck et al., 2009). Recently, Tomas et al. detected circulating antibodies to thrombospondintypeͲ1domainͲcontainingprotein7A(THSD7A)in8Ͳ14%ofpatientsnegative forantiͲPLA2R1,indentifyingTHSD7AasthesecondmajorautoantigeninIMN(Tomasetal., 2014). PLA2R1 and THSD7A proteins are detected in normal human podocytes, and both antigens colocalize with IgG4 in subepithelial deposits. Both proteins have quite similar structural and biochemical properties consisting of a large extracellular region with multiple and repeated disulfideͲbonded and NͲglycosylated domains. IgG4 antibodies against these antigenswerepresentinserafrompatientswithIMNbutnotintheserumofhealthycontrols, or patients with secondary MN. Furthermore, IgG eluted from biopsy samples reacted with recombinant PLA2R or THSD7A. Autoantibodies to both proteins recognize their target antigens only under nonͲreducting conditions. Interestingly, patients with IMN have an autoimmune response against either PLA2R or THSD7A, but not both. This finding suggests PLA2RͲassociated and THSD7AͲassociated MN are two separate entities (Beck et al., 2009; Tomasetal.,2014). Antibodiesagainstthecytosolicproteinssuperoxidedismutase2(SOD2),aldosereductase, andɲͲenolasewereidentifiedinboth,serumandglomeruliofpatientswithIMN(Prunottoet al., 2010; Bruschi et al., 2011). These proteins are not present or minimally expressed on normal podocyte membranes but are “neoͲexpressed” in glomeruli of patients with IMN. Mechanisms of translocation of these intracellular molecules have been proposed to explain the development of these autoantiboides. In vitro studies have shown that SOD2 can be inducedbyoxidativestresssuchasexposuretohydrogenperoxide.AntiͲɲͲenolaseantibodies arenotspecificforMNastheyhavebeendetectedinawiderangeofautoimmunediseases 36 without glomerular involvement and in healthy individuals. The pathogenic role of these antibodiesremainsuncertain. There are still 15Ͳ25% of IMN cases in which no target antigen has been identified indicating that there may be asͲyet unidentified antigens or that these patients have been wronglyclassifiedasidiopathicowingtoanundetectedsecondarycauseofthedisease(Tomas etal.,2014). IthaslongbeenknownthatIMNisassociatedwithcertainHLAclassIIimmuneresponse genes (Klouda et al., 1979; Le Petit et al., 1982; Vaughan et al., 1989; Ogahara et al., 1992; Chevrieretal.,1997).ThesingleͲnucleotidepolymorphism(SNP)rs35771982inPLA2R1gene wasassociatedwithIMNinKoreanandTaiwanesecohorts(Liuetal.,2010;Kimetal.,2011). GenomeͲwideassociationstudieshavereportedhighlysignificantassociationofanHLAͲDQA1 allele on chromosome 6p21 and a PLA2R1 allele on chromosome 2q24 with IMN in white Europeanpatients.Interestingly,carryingtheriskallelesofbothgeneshadanadditiveeffect, thus,forapersonwhoishomozygousforbothriskallelestheoddsratiofordevelopingIMNis close to 80, compared to individuals homozygous for the protective alleles. These findings suggest that genetic background plays a significant role in the predisposition of the primary MN(Stanescuetal.,2011). 4.2. TCELLDEPENDENTCIRCULATINGFACTOR:SSNSANDSDNS An underlying immune defect is supposed to cause SSNS, SDNS and the subset of SRNS thatrespondtoimmunosuppressiveagentsand/orrelapseafterkidneytransplantation.These patientsgenerallypresentwithMCDorFSGSonrenalbiopsy. In1954,Gentilietal.hypothesizedthatINSwascausedbyacirculatingfactor,basedon daringexperimentsinwhichtheyadministeredplasmafrominfantswithINStononͲnephrotic children and observed a minimal increase in proteinuria (Gentili et al., 1954). In 1974, ShalhoubsuggestedthatINSwasmediatedbyaTͲcellͲdependentcirculatingfactorthatwould affectglomerularpermeability(Shalhoub1974). Clinical evidence for the existence of a circulating permeability factor in idiopathic MCD wasbasedontheresponsivenessofmostformsofprimaryMCDtocorticosteroids,alkylating agents,calcineurininhibitors,andmycophenolatemofetil,allofwhichareknowninhibitorsof T lymphocyte function (Gbadegesin & Smoyer 2008). Regarding FSGS, the best evidence for 37 theexistenceofcirculatingpermeabilityfactorcamefromclinicalobservationsofproteinuria and FSGS recurrence after kidney transplantation (Hoyer et al., 1972). Additional evidence came from studies reporting remission of proteinuria by plasma exchange or immunoadsorption,especiallyifinstitutedearlyin thecourseof recurrentdisease(Dantal et al.,1994;Arteroetal.,1994;Deegensetal.,2004).Moreover,serumorplasmafrompatients withrecurrentFSGSinducedproteinuriainratsandincreasedalbuminpermeabilityinisolated glomeruli.Inthepastfourdecades,manyinvestigatorshavesearchedtheresponsiblefactors, thusfarwithlittlesuccess(Maasetal.,2014). In2011,solubleurokinaseplasminogenactivatorreceptor(suPAR)wasproposedtobethe permeabilityfactorcausingFSGS.suPARisa20Ͳ50kDproteinwiththreehomologousdomains produced by cleavage and release of membrane bound urokinase plasminogen activator receptor(uPAR)(Behrendtetal.,1990).Inculturedpodocytesandinanimalmodels,increased podocyte uPAR expression resulted in foot process effacement and actin cytoskeleton rearrangement due to the activation of integrin ɴ3, one of the main proteins anchoring podocytestotheGBM(Weietal.,2011). Inhumans,asignificantly increasedserumsuPAR levelswasfoundin patientswithFSGS comparedtohealthyanddiseasecontrolsbuttheserelationshipsdisappearedwhenadjusting forrenalfunction.Ofnote,theELISAtestusedinthesestudiesdidnotdiscernbetweenfullͲ length glycosylated suPAR from fragments. In addition, both shortͲterm and prolonged administration of recombinant suPAR in mice did induce neither albuminuria, nor foot processes effacement. Based on these findings, fullͲlength suPAR is not the cause of FSGS. However, the specific pathologic type of suPAR could be unglycosylated and/or suPAR fragments, likely explaining the negative results in ELISA experiments and with recombinant suPAR(Deegens&Wetzels2014;Reiseretal.,2014). 4.3. GENETICCAUSE:SRNS InheritedstructuraldefectsofGFBareresponsibleofaproportionofSRNSpatients.These patientsaremostlyresistanttoimmunosuppressiveagents,almostinvariablyprogresstoESRD anddonorelapseafterrenaltransplantation(Machuca,Benoit,etal.,2009). Mutationsinmorethan30geneshavebeenassociatedwithSRNStodate.Mostofthese mutationsareinpodocytegenesanddisruptpodocytefunctioneitherthroughslitdiaphragm disassembly, damaging cell architecture or metabolism, disturbing cellͲmatrix interactions, 38 and/orimpedingsignalingpathways.SyndromicformsofNSarecausedbyinheriteddefectsin structuresnonspecifictothekidney,suchasmitochondrialandlysosomalorganellesthatmay alsoleadtopodocytedysfunction.MutationsinafewgenesencodingGBMproteinshavealso beenassociatedwithNS(Figure17)(Vivante&Hildebrandt2016). Figure 17. Genetic causes of SRNS. Most of the mutated proteins reside in the podocyte (MalagaͲ Dieguez&Susztak2013). Inherited genetic defects in all these genes only explained approximately 30% of SRNS casesconsistingof70%offamilialand15Ͳ25%ofsporadicSRNScases(Santín,Bullich,etal., 2011;McCarthyetal.,2013).Thepercentageofpatientswithmutationsdecreasesastheage at onset increases (Figure 18). Mutation screening of the largest cohort of SRNS patients studiedsofar,consistingof2016patients(1783families),identifiedageneticdiseasecausein 29.5%ofcases.Specifically,mutationswereidentifiedin69.4%ofcongenitalcases(onsetfrom 0Ͳ3 months), 49.7% of infantile cases (from 4 to 12 months), 25.3% of young children cases (from 13 months to 6 years), 17.8% of older children cases (from 7 to 12 years), 10.8% of adolescents(from13Ͳ18years)and21.4%ofyoungadults(from19to25years).Inthisstudy the most frequent mutated gene was NPHS2 accounting for 49.8% of patients, followed by WT1 in 17.3% of patients and NPHS1 in 14.8% of patients. Most of the remaining genes identifiedareraremutationsinvolvingafewfamilies(Sadowskietal.,2014). 39 Figure e 18. Percentage of patieents with cau usative mutattion detected d in one of tthe 21ͲSRNS genes analyzzedinrelation ntoageofon nsetofprotein nuria(Sadowskietal.,2014 4). M Mutationscr eeningofth hemostfreq quently mutaatedgenesinlargecoho ortsrevealed dthat the distribution d of the caussative gene depends on n the age att onset, the familial/spo oradic status, and the mode m of inheritance. Baased on thesse criteria, our o group prroposed a ge enetic testin ng approach h in children n and adultts with SRN NS (Figure 19). Mutations in NPHSS1 are respo onsibleform mostoftheC CNFpatientssbutCNSis geneticallyh heterogeneo ousinnonͲFinnish populations.Mosstpatientsin nwhomthe diseasebegganinthefirrstweekoflifehadmutaations in the NPHS1 geene, whereaas mutationss in NPHS2 were the main m genetic cause of CNS C in m than 1 1 month afte er birth (Macchuca et al.,, 2010). The most patient in whom NS began more uentcauseofinfantilean ndchildhood donsetSRNSS(from13m monthsto12 years)areN NPHS2 frequ mutaationsalthou ughNPHS1m mutationsareealsofound d.WT1ͲrelateedSRNSism mostlydetectedin sporaadicpatientsswithanonssetrangingfrromcongeniitaltochildhood.Inadoleescenceand dadult onsettpatientsthep.R229Qvvariantincom mpoundhete erozygosityw withapatho ogenicmutattionin NPHSS2geneand INF2mutationsarethemostfrequentcauseso ofthediseasse(Santín,Bullich, etal.,,2011). M Mostoftheg genesinvolveedinpediatrricSRNSfollo owanARinh heritance,includingmutaations inNP PHS1,NPHS2 2,andPLCE1amongotheers.Theexce eptionisWTT1ͲrelatedSR RNSwhichfo ollows andaautosomaldo ominant(AD D)pattern.TheADformssofSRNS(IN NF2,TRPC6aamongotherrs)are generally charactterized by a milder diseease course with typically adolescen nt (from 13 to 18 o variable degrees of proteinu uria, and slo ow progressiion to yearss) or adult (>18 years) onset, ESRD D(Conlonet al.,1995;Raanaetal.,20 003).Sincem mostpatientsdonoexhiibitfullͲblow wnNS, theseeformsareu usuallyreferrredtoasADFSGS(Boyerretal.,2015 5). 40 Figure19.GeneticapproachinchildrenandadultswithSRNS(Santín,Bullich,etal.,2011). Of note, the renal histological lesion is not considered in our genetic testing algorithm because in most children renal biopsy is not performed. In addition, the renal histological lesionisnotspecificasmostcasespresentwithFSGSindependentlyofthemutatedgene.The exceptionisDMSlesion,whichissuggestiveofWT1orPLCE1mutations. The high genetic heterogeneity and phenotypic variability of SRNS hampers the genetic diagnosis of the disease. The advent of nextͲgeneration sequencing technologies has revolutionized the genetics field facilitating the identification of new genes associated with SRNSandprovidingthetoolstodealwiththehighgeneticheterogeneityofSRNS.However, there are still a large proportion of SRNS patients with no underlying genetic defect found; therefore,numerousfurthergenesareexpectedtobeidentifiedascausativeofSRNS. 41 5. GENETICSOFSRNS 5.1. AutosomalrecessiveisolatedSRNSandFSGS Table1.GenescausativeofautosomalrecessiveisolatedSRNSandFSGS. Gene Reference MOI Typicalageat diagnosisof proteinuria/NS Tyical histology Ageat ESRD Numberoffamilies (patients) published NPHS1 KestilaMetal.,1998 AR 0Ͳ10years CNF,MCD, FSGS 7months– 15years >270(>300) NPHS2 BouteNetal.,2000 AR 0Ͳ40years MCD,FSGS 2Ͳ50years >600(>850) PLCE1 HinkesBetal.,2006 AR 0Ͳ8years DMS,FSGS 5months– 12years 50(71) CD2AP LowikMMetal.,2007 ARͲ (AD) 10months FSGS 3years 1(1)* CUBN Ovuncetal.,2011 AR 4Ͳ5years ND ND 1(2) PTPRO OzaltinFetal.,2011 AR 5Ͳ14years MCD,FSGS 18years 2(5) MYO1E SannaͲCherchiSetal.,2011 AR 2monthsͲ9years FSGS 6Ͳ>15 years 10(14) CFH Sethietal.,2012 AR childhood FSGS ND 1(1) ARHGDIA GuptaIRetal.,2013 AR 2Ͳ3weeks DMS 3months 3(6) ADCK4 AshrafSetal.,2013 AR <1Ͳ21years FSGS 7Ͳ23years 20(41) TTC21B CongEHetal.,2014 AR 9Ͳ30years FSGS 12Ͳ35years 7(13) EMP2 Geeetal.,2014 AR 3years MCD ND 1(1)** CRB2 EbarasiLetal.,2015 AR 9monthsͲ6years FSGS NA 5(4) NUP107 Miyakeetal.,2015 AR 2Ͳ11years FSGS 4Ͳ12years 5(9) NUP93 Braunetal.,2016 AR 1Ͳ6years FSGS 1Ͳ11years 6(7) Abbreviations:AD,autosomaldominant;AR,autosomalrecessive,CNF,congenitalnephroticsyndrome ofFinnishtype;DMS,diffusemesangialsclerosis;ESRD,endͲstagerenaldisease;FSGS,focalsegmental glomerulosclerosis; MCD, minimal change disease; MOI, mode of inheritance; ND, no data; NS, nephroticsyndrome *onlytheARpatientisconsidered **Mutationsinthisgenein3families(4patients)withsteroidͲsensitiveNShavebeenreported. 42 5.1.1. NPHS1gen N e T TheNPHS1g enespans26kbonchro omosome19 9q13andcon ntains29exons(Kestila etal., 1994). NPHS1 encodes nep phrin, the major m structtural protein n of the GFB. Nephrin n is a transsmembrane adhesion protein of 1241 amino acids and approximateely 135 kDaa that belon ngs to the im mmunoglobu ulin superfam mily. This prrotein contains 8 C2Ͳtyp pe IgͲlike domains andaafibronectin ntypeIIIrep peatintheextracellular regions,asingletransmembranedo omain, and a a cytosolic CͲterminal C end. The extrracellular do omain of nep phrin forms homodimerrs and heterrodimers witth NEPH1. NephrinͲNEP N H1 interactio ons control nephrin sign naling, glomerular perm meability and d podocyte cell polarityy (Figure 20 0) (Garg et al., 2007; Liu et al., 2003; Hartlebenetal.,2 2008). Figure e20.Structurreandlocation nofthenephrinproteinintheslitdiaph hragm.FP,foo otprocesses;SSD,slit diaphragm(adapteedfromMachuca,Benoit,eetal.,2009). M Mutations in NPHS1 aree the major cause of CN NS includingg CNF, the m most severe form (Kestilä et al., 19 998). In Finland, two fo ounder mutaations accou unt for moree than 94% of all 8%ofpatien ntsandconsistsofatwo oͲbase mutaations.TheFinͲmajor(p.L41fs*91)acccountfor78 paird deletioninexxon2thatreesultsinatru uncatedprotteinofonly9 90aminoacids.TheFinͲminor (p.R1 1109*)accou untsfor16%ofpatientsaandisanonssensemutatiioninexon2 26thatresulttsina prem maturestopccodonleadingtoaproteiinof1108am minoacids.Thesetwomu utationsboth hlead totheabsenceofnephrinin thepodocyyteslitdiaphragm(Kestiläetal.,199 98).InnonͲFinnish or mutations are rare an nd most patients have private p populations, the FinͲmajor and FinͲmino nͲFinnishpatientsislow werthaninFinnish mutaations.Thefrrequencyof NPHS1mutaationsinnon 43 patients,accountingfor39%to58%ofcongenitalNScases(Hinkesetal.,2007;Heeringaetal., 2008;Schoebetal.,2010). To date, more than 250 different mutations have been described, including nonsense, missense, frameshift insertions/deletions, and splicing mutations spanning over the whole gene. In vitro functional assays have shown that most NPHS1 missense mutations lead to protein retention in the endoplasmic reticulum, possibly as a result of protein misfolding, resultingindefectiveintracellularnephrintraffickingouttothecellsurface(Liuetal.,2001). Interestingly, in vitro treatment with chemical chaperone rescued several of the missense mutantsfromtheendoplasmicreticulumtothecellsurface(Liuetal.,2004). The spectrum of renal disease related to NPHS1 mutations has broadened with the identification of patients with NPHS1 mutations and childhood and even adult onset SRNS (Philippeetal.,2008;Santín,GarcíaͲMaset,etal.,2009).Philippeetal.foundNPHS1mutations in 10 of 160 patients presenting with SRNS at a mean age of 3 years (from 6 months to 8 years), MCD or FSGS on renal biopsy, and ESRD at a mean age of 13.6 years (from 6 to 25 years).Santínetal.detectedNPHS1mutationsinapatientdiagnosedat27yearsofageand with unimpaired renal function after two years of followͲup (Santín, GarcíaͲMaset, et al., 2009). All these patients carried a severe mutation and one mild mutation in compound heterozygousstate.Mildmutationsexhibitednormaltraffickingtotheplasmamembraneand maintained the abilities to form nephrin homodimers and to heterodimerize with NEPH1, suggestingthatpartialstructuralandsignalingfunctionwaspreserved(Philippeetal.,2008). Patients carrying two mutations in the cytoplasmic domain were also associated with a mild phenotype(Machucaetal.,2010).Thehomozygousp.R1160*mutationhasbeenreportedto result in a mild CNF disease in about 50% of cases, mostly females. These patients have histologicalfindingsconsistentwithCNFandeithermildproteinuriaorcompleteremissionup to19yearsofage(Kozielletal.,2002).Asummaryofthemildmutationsreportedtodateis showninFigure21. 44 Figure e 21. NPHS1 mutations deetected in thee following sttudies: Liu et t al., 2001; Ph hilippe et al., 2008; Machuca et al., 20 010, and Sch hoeb et al., 2010. 2 Schemaatic representtation of the localization of the ns and protein functional domains. d Ig, IgͲlike domain; FNIII, fibro onectin mutattions with resspect to exon typeIIIdomain;TM M,transmemb branedomain(adaptedfrom mPhilippeetal.,2008). 5.1.2. NPHS2gen N e T NPHS2 gene The g spans approximateely 25 kb on n chromosom me 1q25Ͳq3 31 and contaains 8 exonss (Boute et al., 2000). NPHS2 N encod des podocin, a 42ͲkDa integral mem mbrane protein of 383aaminoacidstthatbelongsstothestom matinfamily. Podocinisalmostexclussivelyexpressedin thekkidneyattheepodocyteslitdiaphragm m.Itisprediictedtobeaanintegralm membraneprotein insertted in the membrane through a short s hydrophobic regio on resultingg in a hairpiinͲlike topollogy with in ntracellular NͲ N and CͲterrminal ends. It also con ntains a pro ohibitin hom mology domaain that allo ows podocin binding to cholesterol in plasma membrane. This protein n was show wn to accumulate in oligomeric form m in lipidͲraftt microdomaains and to recruit neph hrin in theseespecializedmicrodomainsoftheslittdiaphragmplasmamem mbrane(Hub ber,Simons,etal., 2003). By also interacting with w CD2AP and TRPC6 6, podocin represents r aan importan nt link weentheslitd diaphragmaandthepodo ocytecytoske eleton. betw T Theclassical phenotypeaassociatedw withNPHS2m mutationsis earlyͲchildhoodNS(neaarlyall beforre6yearsoffage),resisttanttocorticcosteroidsandimmunossuppressive agents,and rapid progrressiontoESSRD(beforettheendofth hefirstdecaadeoflife)w withlowrecu urrenceafterrrenal transsplantation.H HistologicalffindingsshowFSGSinap pproximatelyy80%ofcassesalthough hMCD arealsofound(B Bouteetal., 2000;Webeeretal.,200 04;Hinkesettal.,2008). NPHS2mutaations alsoaareafrequeentcauseof CNS(15Ͳ39% %),infantile SRNS(29Ͳ35.2%)and,ttoalesserextent, adulttonsetSRNS(16%)(Boucchirebetal., 2014;Sadow wskietal.,20 014). M Mutationsin NPHS2geneeaccountfo orapproximaately30Ͳ40% %offamilialccases(Karle etal., 2002;Webereta al.,2004;Berdelietal.,2 2007;Hinkessetal.,2007 7;Hinkeset al.,2008)an nd10Ͳ 45 30%ofsporadic(Caridietal.,2001;Caridietal.,2003;Berdelietal.,2007;Gbadegesinetal., 2008;Megremisetal.,2009;Jungraithmayretal.,2011). Todate,morethan110NPHS2pathogenicmutationshavebeenreported(LOVDmutation database). They are distributed throughout the entire gene and consist of all kinds of alterations, incuding missense, nonsense, frameshift, and splicing mutations. Missense mutations are the most frequent type of alteration accounting for 42% of the mutations (Bouchirebetal.,2014). NPHS2 mutations are frequently found in patients from Central Europe, North America, Turkey, the Middle East, North Africa, and South America. However, they are not a major causeofSRNSinAfrican AmericanandAsianpatients.Severalfounder mutationshavebeen identified:p.R138QinEurope,p.R138*intheIsraeliͲArabpopulations,p.V260EintheComoros Islands,andp.A284VinSouthAmerica.TheR138Qmutationisthemostprevalentmutationin European patients accounting for 32% and 44% of all affected NPHS2 alleles in two large European series (Weber et al., 2004; Hinkes et al., 2008). However, in Spanish population it was only found in 2 of 14 alleles, accounting for 14% of all NPHS2 affected alleles (Santín, TazónͲVega,etal.,2011).Thearginineresidueatposition138ishighlyconservedamongthe stomatinͲlikeproteinfamilymembersandiscrucialforpodocinfunction(Bouteetal.,2000). The protein resulting from the substitution of glutamine for arginine is retained in the endoplasmicreticulum,impairingtheintracellulartraffickingofpodocintocellmembraneand losingitsabilitytorecruitnephrininlipidrafts(Huber,Simons,etal.,2003). The nature of the mutations correlates with the age at onset of NS (Figure 22). Patients withframeshiftornonsensemutationsinhomozygousorcompoundheterozygousstatehave anearlierageatNSonsetthan those carrying two missensemutations (mean1.75years vs. 4.17 years) (Hinkes et al., 2008). Missense mutations leading to retention of the mutant proteinintheendoplasmicreticulum(suchasthep.R138Qorthep.R168H)resultinanearlier onsetofdiseasethanmutationsthatdonotdisruptthemutantproteintraffictotheplasma membrane(20.8±4vs.128.7±9months,respectively)(Rosellietal.,2004).Incongenitaland infantileSRNS,the94.1%ofpatientswithNPHS2mutationswerefoundtocarryatleastone truncating mutation or homozygous p.R138Q mutations, (Hinkes et al., 2007). Missense mutations like the p.G92C, p.V180M and p.R238S are associated with a milder phenotype, a laterageatonsetofNSandESRD.Thesemutationsallowthetargetingofthepodocinmutant tothecellmembrane,likelyresultinginapartialfunctionofthepodocinthatmayexplainthe laterageatonsetofNS(around10years)(Weberetal.,2004)andthelessseveredisease. 46 Figure e 22. The natture of NPHSS2 mutation determines d th he age at onset of SRNS. CNSͲFT, conggenital nephrrotic syndrom me of Finnish type; FSGS, focal segmen ntal glomerulo osclerosis; mo o, months; y,, years (adap ptedfromHild debrandt&Heeeringa2009). P Patients carrying the p.R R229Q and a a pathogenic mutation have been aassociated with w a signifficantly laterr onset of th he disease. These patie ents represen nt almost alll the adult onset casess with NPHSS2 mutations and they develop ESR RD at a meean age of 2 26.1 ± 18.9 years (Tsukkaguchi et al., a 2002). The T p.R229Q Q is the most frequentlly reported nonͲsynonyymous NPHSS2variantinCaucasians (Franceschinietal.,200 06).Ahigherfrequency ofp.R229Q allele wasffoundinSRN NS,particulaarlyinEurop peanorSoutthAmerican populationss,thanincontrols (Machuca,Humm mel,etal.,20 009).InSpanishpopulattion,p.R229Qwasfound din5.3%of SRNS o 1.98% of o controls (SSantín, TazónͲVega, et al., a 2011). In addition, in n vitro patients and in only m pro otein to nephrin, studies demonsttrated decreased binding of the p.R229Q mutant maybepathogenic(Tsukkaguchietal.,2002).Reecently,Tory etal. suggeestingthatthisvariantm demo onstratedthatthepatho ogenicityoftthep.R229Q Qvariantdep pendsontheetransͲassocciated mutaation. The p.R229Q variant lead to disease phe enotype onlly when it is associated d with certain specific CͲterminal C N NPHS2 mutaations, whicch exert a dominant d neegative effe ect on 29Q podocin n through altered a dimeeritzation and mislocaliization. In ccontrast, paatients p.R22 carryingthep.R2 229Qinhom mozygousstateareasymptomatic(To oryetal.,20 014).Interesstingly panish patien nts with latee onset SRN NS, the p.R229Q was freequently fou und in comp pound in Sp heterrozygosity with w the p.A A284V path hogenic muttation, whicch is found in a consserved haplo otype(Santín n,TazónͲVegga,etal.,201 11). 5.1.3. PLCE1gene P e T ThePLCE1ge enespans33 34.4kboncchromosome e10q23and contains34 4exons.Thissgene encod desthephosspholipaseC Cepsilon1(P PLCɸ1)prote ein,acytoplaasmicenzym methatbelon ngsto thep phospholipassefamilyof proteinsand dcatalyzesthehydrolysisofpolypho osphoinositid desto 47 generate the second messengers: inositol 1,4,5Ͳtriphosphate (IP3) and diacylglycerol (DAG). Theseproductsinitiatethecascadeofintracellularpathwaysofcellgrowthanddifferentiation. This protein has two isoforms: PLCɸ1 isoform A of 2302 amino acids (258 kDa), and PLCɸ1 isoform B of 1994 aminoacids (224 kDa). The PLCɸ1 protein contains the following putative protein domains: RasGEF_CDC25 (guanine nucleotide exchange factor for RasͲlike small GTPasesdomain),PHdomain(Pleckstrinhomologydomain),EFhand,phospholipasecatalytic domains(PLC_XandPLC_Y),C2motif(proteinkinaseCconservedregion2,subgroup2)and RA1 and RA2 domains (RasGTP binding domain from guanine nucleotide exchange factors) (Hinkesetal.,2006).PLCɸ1isexpressedinthepodocytes,neuraltissue,andskeletalmuscleof mouse embryos, as well as the skin, skeletal muscle and heart of adult mice (Tadano et al., 2005; Wu et al., 2003; Bai et al., 2004). PLCɸ1 has an important role during glomerular development,andrecruitsnephrintotheplasmamembraneviaIQGAP1. MutationsinPLCE1genewereidentifiedinpatientswithearlyͲonsetNSandDMSorFSGS onrenalhistology(Hinkesetal.,2006).StudiesinlargecohortsrevealedthatPLCE1mutations are the major cause of idiopathic DMS, accounting for approximately 28.6Ͳ33% of idiopathic DMSpatients(Gbadegesinetal.,2008).Mutationsinthisgenewerealsodetectedinabout8% of FSGS cases without NPHS2 mutations. Patients with PLCE1 mutations have an earlyͲonset NS, mean age of 22.8 ± 5.0 months, and lead to ESRD at a mean age of 39.1 ± 6.3 months (Boyeretal.,2010). Todate,morethan40differentmutationshavebeendescribed,predominantlytruncating mutationswithno clusteringarounda specificgeneregion.Intheinitialcohortreported, all the individuals with homozygous truncating mutations presented DMS as histological lesion whereastwopatientscarryinghomozygousnonͲtruncatingmutationspresentedFSGS(Hinkes et al., 2006). However, in a more extensive study of 139 patients with earlyͲonset SRNS, truncating and missense mutations led to similar renal presentation and evolution and were detectedinpatientswithboth,DMSandFSGShistologicallesions.Recently,anearlierageof onsethasbeenreportedforpatientscarryingsplicesitemutationscomparedwithCͲterminal truncatingmutations(afteraminoacidresidue1000)ormissensemutations(Sadowskietal., 2014).RenalprognosisisworstinpatientswithDMScomparedtothosewithFSGS:meanage atonsetofNS15.3±6.0monthsvs.30.5±7.7months,andmeanageatESRD20.9±5.8vs. 57.2±7.3months,respectively(Boyeretal.,2010). InindividualswithPLCE1mutations,thepresenceofDMSlesionswiththeappearanceof immatureglomeruli,togetherwiththereducednephrinandpodocinexpression,indicatesthat 48 PLCɸ1 is necessary for proper progression of glomerular development at the capillary loop stage.PLCɸ1mightinteractwithIQGAP1inthevicinityoffootprocessesandmightserveasan assemblyscaffoldfortheorganizationofamultimolecularcomplexinvolvedinmorphogenetic processesofglomerulardevelopmentatthecapillaryloopstage(Hinkesetal.,2006). Four families with transiently affected or asymptomatic family members carrying homozygoustruncatingmutationshavebeenreported(Gilbertetal.,2009;Boyeretal.,2010). This finding raised the possibility that modifier genes or environmental factors could play a role in the renal phenotypic variability observed in individuals carrying PLCE1 mutations, but additionalvariantsin19candidategenes,including16phospholipases,wereruledout(Boyer etal.,2010).Theeffectofmodifiersgenescouldalsoexplaintheabsenceofanobviousrenal phenotypeinthePlce1knockoutmousemodel(Hinkesetal.,2006). 5.1.4. CD2APgene CD2AP spans approximately 170 kb on chromosome 6p12.3 and contains 18 exons. This geneencodestheCD2ͲAssociatedProtein (CD2AP), aproteinof 639aminoacidsand80kDa. CD2APproteinwasoriginallyclonedasaninteractionpartnerofCD2,acelladhesionmolecule found on the surface of T lymphocytes and natural killer cells that is implicated in TͲcell adhesion to antigenͲpresenting cells. In the kidney CD2AP is expressed in proximal tubules, collecting ducts and podocytes, where it interacts with nephrin (Shih et al., 1999), podocin (Schwarzetal.,2001)andFͲactin(Lehtonenetal.,2002)suggestingitsroleinanchoringthe slitdiaphragmtotheactincytoskeleton. TheCd2apnullmousemodelwasfoundtopresentnotonlyanimpairedTͲcellfunctionin vitro but also CNS leading to ESRD by 6Ͳ7 weeks of age (Shih et al., 1999). In addition, mice with Cd2ap haploinsufficiency developed glomerular changes at 9 months of age and had increasedsusceptibilitytoglomerularinjurybynephrotoxicantibodiesorimmunecomplexes (Kimetal.,2003). To date, only one homozygous CD2AP mutation in an infantile form of NS (Lowik et al., 2007),twocompoundheterozygousCD2APmutationsinacongenitalonsetNS(AlͲHamedet al., 2013), and five heterozygous mutations in pediatric and adult FSGS cases have been described(Kimetal.,2003;Lowiketal.,2008;Giganteetal.,2009).Thehomozygouspatient had failure to thrive secondary to nephroticͲrange proteinuria and microscopic hematuria at theageof10months,FSGSonhistology,andprogressedtoESRDbytheageof3yearswithno 49 recurrence after renal transplantation. Both parents were heterozygous and asymptomatic, discarding a haploinsufficient phenotype in this family (Lowik et al., 2007). Studies in large internationalcohortsdidnotidentifyanymutationinCD2AP,suggestingthatthecontribution ofthisgenetoSRNSislimited(Sadowskietal.,2014). 5.1.5. PTPROgene PTPROgeneencodestheproteintyrosinephosphatasereceptortypeO.Thisproteinwas identifiedinrabbitasapodocyteͲspecifictransmembraneproteinlocalizedtotheapicalside offootprocesses.Thisreceptormightplayaroleinmaintainingfootprocessstructureand/or functionbyregulatingtyrosinephosphorylationofpodocyteproteins(Thomasetal.,1994). PTPROmutationswereidentifiedintwoTurkishconsanguineousfamilies(fiveindividuals) bygenomeͲwideanalysisfollowedbyhomozygositymapping.PatientswithPTPROmutations hadanonsetofSRNSbetween5and14yearsoldwithFSGSorMCDonhistologyandsevere footprocesseffacement.OnepatientreachedESRDattheageof18yearswhereastheothers hadpreservedrenalfunctionat7,11,12and15yearsold(Ozaltinetal.,2011). 5.1.6. MYO1Egene MYO1E encodes myosin 1E (MYO1E), a member of the ubiquitously expressed class I myosins. In podocytes MYO1E is associated with the plasma membrane and is implicated in podocyteactincytoskeletonorganizationandmotility(Nabetetal.,2009;Meleetal.,2011). Mutations in the MYO1E gene have been described in 5 families (7 individuals) with isolatedSRNS,accountingforapproximately6%ofpatients.However,allthefamiliesreported to date had some degree of consanguinity. Thus, MYO1E mutations do not appear to be a common cause of SRNS in familial or sporadic cases but to account for a number of consanguineousfamilies(Meleetal.,2011;AlͲHamedetal.,2013). AllthefamiliespresentedwithisolatedSRNSand,insomecases,microhematuriawasalso described. They had an age at onset ranging from 4 months to 9 years and developed ESRD between10and13yearsofage,althoughfourpatientspreservednormalrenalfunctionat11, 13 and 14 (n=2) years old. Renal biopsy showed FSGS in all the cases except one with MCD (Meleetal.,2011;SannaͲCherchietal.,2011;AlͲHamedetal.,2013).Affectedmemberswere resistant to treatment with corticosteroids but some of them responded to immunosuppressivetreatmentwithcyclosporine(Meleetal.,2011)(MeleCetal2011). 50 Myo1eͲknockoutmicehaveproteinuria,podocytefootprocesseseffacement,andchronic renal disease, mimicking the phenotype of individuals with MYO1E mutations (19 Mele). Functional studies in some of the mutations found revealed an abnormal subcellular localization of the protein or an impaired function due to the loss of important MYO1E domains(Mele). 5.1.7. ARHGDIAgene The ARHGDIA gene encodes the RhoͲGDP dissociation inhibitorͲalpha (RhoGDIɲ), which sequestersRhoͲGTPasesinaninactivestateinthecytosol.AhomozygousmutationinͲframe deletioninARHGDIAwasfoundintwosiblingsofconsanguineousPakistaniparentsbywholeͲ exome sequencing. Both siblings presented CNS with generalized edema, severe proteinuria andhypoalbuminemia.TherenalbiopsyperformedinoneofthemrevealedDMS.Onesibling reachedESRDat3monthswhereastheotherdied.ThemutationinthisfamilyledtoalossͲofͲ function of RhoGDIɲ likely disturbing the balance of RHOͲGTPases within podocytes, which wouldcausedearrangementoftheactincytoskeleton,podocyteinjury,andNS(Guptaetal., 2013). Gee et al. identified two more families with homozygous missense mutations in ARHGDIA gene by combining homozygosity mapping with wholeͲexome sequencing. These families presented with early onset SRNS (from 14 days to 2.4 years), DMS on renal biopsy (when performed), and rapidly progressed to ESRD (from 6 weeks to 3 years). Extrarenal featuressuchasintellectualdisability,sensorineuralhearingloss,seizuresorcorticalblindness werereportedinsomepatients.Themissensemutationsinthesefamiliesabrogateinteraction with RHO GTPases, increase active GTPͲbound RAC1 and CDC42, and result in a migratory phenotypicchangeinpodocytes(Geeetal.,2013). 5.1.8. ADCK4gene The ADCK4 gene encodes the AarF Domain Containing KinaseͲ4, a protein of 544 amino acidsandapproximately60kDa.Thisproteinlocalizestothemitochondriaandfootprocesses inpodocytesandissuggestedtobeinvolvedincoenzymeQ10(CoQ10)biosynthesis(Ashrafet al.,2013). Recessive mutations in ADCK4 have been identified in a total of 20 SRNS families (41 individuals), accounting for approximately 2% of SRNS screened. However, this prevalence might be overestimated by the fact that most families were consanguineous (Ashraf et al., 2013;Korkmazetal.,2015). 51 The disease first manifested in adolescence, typically with mild to moderate proteinuria and no or mild edema. Hematuria was present in 25% of patients at diagnosis. All patients showed FSGS on biopsy. Advanced CKD was present in almost half of patients at time of diagnosisandprogressiontoESRDoccurredexclusivelyintheseconddecadeoflife(Korkmaz etal.,2015). PatientswithADCK4mutationswerefoundtohavereducedcellularCoQ10content(Ashraf et al., 2013; Desbats et al., 2015). Hereditary defects of CoQ10 biosynthesis cause SRNS as a partofmultiorganinvolvementbutmayalsocontributetoisolatedSRNS.PatientswithADCK4 mutationsshowedlargelyrenalͲlimitedphenotype,withonlysevenpatientspresentingsigns and symptoms compatible with neurologic dysfunction (Ashraf et al., 2013; Korkmaz et al., 2015). 5.1.9. TTC21Bgene TheTTC21Bgenespans96.37Kbonchromosome2q24andcontains29exons.Thisgene encodestheretrogradeintraflagellartransportprotein139(IFT139),consistingof1216amino acids and 151 kDa. It contains multiple tetratricopeptide repeat domains (TRP) that bind specific peptide ligands and are thought to mediate protein–protein interactions (Brinker et al.,2002).IFT139isacomponentoftheintraflagellartransportͲAcomplexthatassociateswith themotorproteindynein2toregulateretrogradetraffickingintheprimarycilium(Taschneret al.,2012). TheprimaryciliumisamicrotubuleͲbasedantennaͲlikeorganellepresentonthesurfaceof mostcells.Itiscomposedoftheaxoneme(amicrotubuleͲbasedcorestructure)surroundedby aciliarymembranethatiscontinuouswiththeplasmamembrane.Theprimaryciliumplaysan important role in sensing flow changes and mediating signaling pathways involved in the establishment of cell polarity during development (Ishikawa & Marshall 2011). The intraflagellartransportinvolvesmovementoflargeproteincomplexes,includingIFTparticles consistingoftwodistinctcomplexes:AandB.Theanterogrademovement(fromcytoplasmto ciliary tip) is powered by kinesinͲ2 while the retrograde movement (from the ciliary tip to cytoplasm)ispoweredbydynein2(Figure24). 52 Figure23.Structureandorganizationoftheprimarycilium(Valenteetal.,2014). Mice carrying a homozygous null mutation in Ttc21b were embryonic lethal and their phenotypeoverlappedwiththeclinicalfeaturesofsevereciliopathiesinhumans(Tranetal., 2008). Mutations in TTC21B gene have been identified in nine individuals with different ciliopathies: Jeune Asphyxuating Thoracic Dystrophy (a chondrodysplasia that often leads to death in infancy); nephronophthisis with extraͲrenal manifestations; and isolated nephronophthisis(Davisetal.,2011;Ottoetal.,2011;McInerneyͲLeoetal.,2014).Ofnote,all patients with isolated nephronophthisis carried the homozygous p.P209L mutation. Unexpectedly,thissamemutationhasrecentlybeenidentifiedinsevenfamilieswithFSGSand tubulointerstitiallesions.Allthereportedpatientswiththep.P209Lmutationsharethesame haplotype, indicating a founder effect for this mutation in patients of Portuguese or North African origin. Therefore, TTC21B is the first ciliary gene involved in a glomerular disorder (HuynhCongetal.,2014). The clinical phenotype associated with TTC21B mutations is characterized by lateͲonset proteinuria (9Ͳ23 years), high blood pressure and ESRD at ages 15Ͳ32 years. Renal biopsies show FSGS together with characteristic alterations of nephronophthisis such as tubular basement membrane thickening, indicating the coexistence of primary glomerular and tubulointerstitialalterations(HuynhCongetal.,2014). TheIFT139proteinisexpressedatthebaseofprimaryciliumindistaltubulesbutalsoin podocytes.Fetalpodocyteshaveaprimaryciliumbutitislostinthemaduration,thenIFT139 expressionisenhancedandrelocalizedalongthemicrotubulenetwork.Functionalstudieson the effect of the p.P209L mutation in glomerulus suggested that it causes cytoskeleton 53 alterations that may destabilize podocyte architecture leading to FSGS (Huynh Cong et al., 2014). 5.1.10. CRB2gene TheCRB2geneencodestheputativetransmembraneCrumbshomolog2protein,aprotein required for podocyte foot process arboritzation, slit diaphragm formation, and proper nephrintrafficking(Ebarasietal.,2015). HomozygousorcompoundheterozygousmutationsinCRB2genehavebeenidentifiedby homozygositymappingandwholeexomesequencinginfourfamilieswithchildhoodͲonset(9 monthsͲ6 years) isolated SRNS and FSGS as histological lesions (Ebarasi et al., 2015). All affected children carried a missense mutation in at least one allele, in accordance with the embryoniclethalityofCrb2knockoutmice(Xiaoetal.,2011).Ofnote,CRB2mutationswere also detected in five fetuses and one infant with cerebral ventriculomegaly and echogenic kidneyswithhistopathologicalfindingsofcongenitalnephrosis(Slavotineketal.,2015). 5.1.11. NUP107gene The NUP107 gene encodes the essential scaffold protein of the nuclear pore complex NUP107.Thisproteinfacilitatestheefficienttransferofmacromoleculesbetweenthenucleus and cytoplasmina highlyselectivemannerandplayspivotalrolesin thenuclearframework andgeneexpression(StrambioͲDeͲCastilliaetal.,2010). RecessivemutationsinNUP107genehavebeenidentifiedbywholeͲexomesequencingin five SRNS families (nine individuals). In four of the five families, all the individuals showed earlyͲonsetSRNSmanifestingat2Ͳ3yearsofageandreachedESRDbefore10yearsofage.In theremainingfamily,thetwoaffectedindividualshadadiseaseonsetafter10yearsold,one ofthemreachedESRDat12yearsoldwhereastheotherhaspreservedrenalfunctionatthe current34yearsofage.Inalltheaffectedindividualsrenal biopsiesrevealedFSGSandthey wereresistanttocorticosteroidtherapybutpartiallyrespondenttoimmunosuppressivedrugs. None of the five patients that underwent renal transplantation experienced recurrence of SRNS(Miyakeetal.,2015). A total of four different mutations were found in the five families. Interestingly, the heterozygousp.D831Amutationwascommoninallthefivefamilies.Thismutationislikelyto be specific to East Asian because it has only been reported in Japanese population. 54 Furthermore, haplotype analysis confirmed that all the families shared the same haplotype confirming a common ancestor for this mutation. Mutations in NUP107 are likely to cause immatureand/orhypoplasticpodocytes,oratleastimpairedpodocytesthatareprogressively destroyedbyincreasedfiltrationpressureafterbirth(Miyakeetal.,2015). 5.1.12. NUP93gene Mutations in NUP93, encoding nuclear pore protein 93 likely involved in nuclear pore complexassembly,havebeenrecentlyidentifiedinsixfamilieswithSRNS(Braunetal.,2016). Phenotypically,affectedmembershadSRNSthatmanifestedbetween1and6yearsofage andcausedESRDbetweentheagesof1and11years.RenalbiopsyshowedFSGSorDMS.In addition,therewasarenaltubularphenotypeinvolvingtubulardilationwithproteincastsand interstitial cell infiltration. Electron microscopy showed partial podocyte foot process effacement. One patient showed partial response to corticosteroids, and two patients respondedtoimmunosuppressivetherapywithcyclosporineA(Braunetal.,2016). Five different mutations were detected in homozygous or compound heterozygous state including three missense mutations, one small deletion and one splicing mutation. The mutations p.G591V and p.Y629C apparently represent European and Turkish founder alleles, respectively.FunctionalstudiesrevealedthatNUP93mutationscauseddisruptednuclearpore complex assembly and abrogated interaction with SMAD4, thus identifying aberrant SMAD signalingasanewdiseasemechanismofSRNS(Braunetal.,2016). 55 5.2. GenesimplicatedinautosomaldominantisolatedSRNSandFSGS Table2.GenescausativeofautosomaldominantisolatedSRNSandFSGS. Gene Reference MOI Typicalageatdiagnosis ofproteinuria/NS Tyical histology Ageat ESRD Numberoffamilies (patients)published WT1 Coppesetal.,1992 AD 3Ͳ59years FSGS 10Ͳ69years 7(31) ACTN4 Kaplanetal.,2000 AD 3Ͳ54years FSGS 6Ͳ59years 12(>75) TRPC6 Reiseretal.,2005 AD 2Ͳ75years FSGS 0Ͳ20years afteronset >28(>62) INF2 Brownetal.,2010 AD 5Ͳ72years FSGS 13Ͳ70years >60(>221) LMX1B Boyeretal.,2013 AD 5Ͳ70years FSGS 28Ͳ70years 8(>28) AD ND FSGS 12Ͳ29years 1(3) ARHGAP24 Akileshetal.,2011 ANLN Gbadegesinetal., 2014 AD 9Ͳ69years FSGS 35Ͳ75years 2(12) PAX2 Baruaetal.,2014 AD 7Ͳ68years FSGS 30Ͳ58years 7(24) Abbreviations: AD, autosomal dominant; ESRD, endͲstage renal disease; FSGS, focal segmental glomerulosclerosis;MOI,modeofinheritance;ND,nodata;NS,nephroticsyndrome. 5.2.1. WT1gene The WT1 gene spans 47.86 kb on chromosome 11p13 and contains 10 exons. This gene encodes Wilms Tumor protein 1 (WT1), a zinc finger transcription factor of 449 amino acids and49kDathatcontainsanNͲterminaltransactivationdomain(exon1)andfourzincͲfingers at the CͲterminal (exons 7Ͳ10). WT1 encodes numerous isoforms, which are products of alternative translation start sites, alternative splicing, and RNA editing. Of particular interest areWT1(+KTS)andWT1(ͲKTS)variants,whichdifferbythepresenceofthreeaminoacids(KTS) betweenthezincfingersthreeandfour.Thepresenceofthisinsertinfluencesthemolecular biochemicalpropertiesoftheresultingprotein.WhileWT1(ͲKTS)bindDNAefficientlyandacts as a transcriptional activator, WT1(+KTS) seem to have higher affinity to RNA and splicing factors(Hammesetal.,2001). WT1proteinplaysacrucialroleinkidneyandgenitaltractdevelopment(PritchardͲJones etal.,1990;Rivera&Haber2005).Inthefetalkidney,WT1isabundantlyexpressedinareasof 56 active glomerulogenesis whereas in the adult kidney, WT1 expression persists in podocytes anditsintegrityisrequiredforproperGFBfunction(Morrisonetal.,2008). MutationsinWT1geneareassociatedwithawidespectrumofADsyndromesinchildren including Wilms tumor (Gessler et al., 1990), syndromic forms of glomerular disease and genitourinaryabnormalities:DenysͲDrash(Pelletieretal.,1991),FraiserSyndromes(Klamtet al., 1998) and WAGR syndrome (Wilms tumor, Aniridia, Genitourinary abnormalities, and mental Retardation)(Miller et al., 1964), and isolated SRNS (Jeanpierre et al., 1998; Schumacheretal.,1998;Itoetal.,1999;Denamuretal.,2000;Dharnidharkaetal.,2001;Itoet al., 2001). Interestingly, WT1Ͳassociated disorders may include SRNS either as an initial symptomorasalaterdevelopmentinapatientdiagnosedonthebasisofextrarenalfeatures. Thetypeandlocalizationofthemutationshasbeenclearlyassociatedwiththerenaland extrarenalphenotypes.Germlinealterations,usuallytruncatingmutations,locatedthroughout theentirecodingsequencepredisposetoWilmstumor.Heterozygousmissenseortruncating mutations in exons 8 and 9, mostly de novo, cause DenysͲDrash syndrome or isolated SRNS (Muchaetal.,2006).ThesemutationsaffecttheDNAͲandRNAͲbindingaffinityofthesecond and third zinc finger domains. Heterozygous mutations in the donor splice site of intron 9 resultinFraiserSyndrome.These mutationsleadtoareductionofthe+KTSisoformcausing the alteration of the normal ratio of +KTS/ͲKTS isoforms in the cell (Barbaux et al., 1997). Finally, large genomic rearrangement affecting chromosome 11p13 may disrupt WT1 among severalgenes,resultinginWAGRsyndrome. The renal phenotype associated with WT1 mutations is characterized by an earlyͲonset SRNSandrapidprogressiontoESRD.Inpatientswithmutationsinexons8and9affectingthe DNA binding capacity (classically associated with DenysͲDrash Syndrome) NS presents in the first months of life, may be preceded by isolated proteinuria, and is always resistant to corticosteroidtherapy.ProgressiontoESRDoccursmostoftenbefore4yearsofage,andno recurrence is observed after renal transplantation (Habib et al., 1985). Histological findings show DMS in most patients. In contrast, patients with KTS mutations, causing Fraiser syndrome, have a childhoodͲonset proteinuria usually between 2 and 6 years of age, almost invariably FSGS on biopsy, and slow progression leading to ESRD in adolescence or early adulthood. Therenalphenotypeofpatientscarryingotherexonicmutationsisdeterminedbythetype of mutation. Most subjects with truncating mutations develop Wilms tumor early in life 57 whereas SRNS occurs later, typically in the second decade after nephrectomy. Missense mutations affecting amino acids in nonͲDNA binding positions are characterized by an intermediate renal phenotype manifesting within the first 5 years of life, usually presenting withDMSonbiopsy,andprogressingtoESRDatapproximately10yearsold. Femalepatients(46,XX)carryingeithermissenseorspliceͲsiteWT1mutationscommonly do not exhibit genital anomalies, have normal puberty, and therefore present with isolated sporadicSRNS(Ruf,Schultheiss,etal.,2004;Muchaetal.,2006).Missensemutationsinexon8 or 9 (p.R458Q, S461F, or R471K) affecting the second and third zinc fingers have been identifiedinfamilieswithisolatedADFSGS,includingmaleswithoutgenitalabnormalitiesor developmentaltumorswhotransmittedthemutation(Coppesetal.,1992;Benettietal.,2010; Guaragnaetal.,2013;Zhuetal.,2013;Halletal.,2015). Recently, a detailed study compared 61 patients with WT1Ͳrelated SRNS with 700 SRNS patients negative for WT1 mutations and revealed several genotypeͲphenotype correlations. WT1 mutated patients presented more frequently with chronic kidney disease and hypertension at diagnosis and exhibit more rapid disease progression compared to WT1 negative patients. FSGS is the most prevalent histopathological finding in WT1 nephropathy butDMSislargelyspecificforWT1diseaseandispresentin34%ofDMScases(Lipskaetal., 2014). This finding goes against the results obtained by Gbadegesin et al. who identified mutationsinPLCE1in28.6%offamilieswithDMSwhereasmutationsinWT1wereonlyfound in8.5%ofthesefamilies(Gbadegesinetal.,2008).Extrarenalphenotypesarealmostexclusive toWT1andmostlycomprise:sexreversaland/orurogenitalabnormalities(52%),Wilmstumor (38%),andgonadoblastoma(5%)(Lipskaetal.,2014). 5.2.2. ACTN4gene AgenomeͲwidescanperformedina100ͲmemberkindredallowedMathisetal.tomapthe firstlocusofADFSGSonchromosome19q13(Mathisetal.,1992;Mathisetal.,1998).Linkage analysis including additional families helped to reduce the size of the region and led to the identificationofthreenonconservativemissensemutationsintheACTN4gene(Kaplanetal., 2000).ACTN4genecomprises21exonsandencodestheɲͲactininͲ4protein,aproteinof911 aminoacidsandabout105kDathatbelongstothespectringenesuperfamily.ɲͲactininͲ4isan ubiquitously expressed actinͲbinding protein, that interconnects actin filaments in podocyte foot processes (Ichimura et al., 2003). Its structure is a headͲtoͲtail antiparallel homodimer withthefollowingparts:twocalponinhomologousdomains(CH1,CH2)withactinbindingsites 58 intheNͲterminal,followedbyfourspectrinrepeatsandtwoEFhanddomainsthatcanbind Ca2+ionsintheCͲterminal(Baronetal.,1987;Imamuraetal.,1988;Leinweberetal.,1999). Affectedcasestypicallypresentwithproteinuriastartingintheadolescenceorlater,with FSGSlesionsonkidneyhistology,andslowlyprogresstoESRDinthefifthdecadeoflife.Also, heterozygousACTN4mutationshaveseldombeenreportedinchildhoodͲonsetNS(Kaplanet al., 2000; Weins 2005; Pollak et al., 2007; Choi et al., 2008). Disease incomplete penetrance andgermlinemosaicismhasbeendescribed(Weins2005;Choietal.,2008). MutationsintheACTN4genewerereportedtoaccountforapproximately2%ofADFSGS (Barua et al., 2013). However, recent studies in large cohorts did not identify any patient carryingACTN4mutations,suggestinganevenlowercontributionofmutationsinthisgeneto ADFSGS(Sadowskietal.,2014). Todate,16mutationshavebeenreported.Mostofthemaremissensemutationsinvolving residueswithinornexttotheactinbindingsiteofɲͲactininͲ4(Kaplanetal.,2000;Ichimuraet al.,2003;Weins2005;Baruaetal.,2013;Giglioetal.,2015).Someofthesemutationshave been shown to increase the affinity of ɲͲactininͲ4 to FͲactin and/or induce ɲͲactininͲ4 mislocalization and the formation of ɲͲactininͲ4 and FͲactin aggregates around the nucleus with subsequent impairment of podocyte spreading and motility (Kaplan et al., 2000; Weins 2005; Yao et al., 2004; Michaud 2003; Michaud et al., 2006). KnockͲin and knockout mouse modelsandinvitrodatasuggestbothgainͲofͲfunctionandlossͲofͲfunctionmechanisms(Yao etal.,2004). 5.2.3. TRPC6gene TRPC6 spans approximately 421 kb on chromosome 11q22 and contains 13 exons. This gene encodes the short transient receptor potential canonical 6 (TRPC6), a protein of 931 amino acids and around 106 KDa. TRPC channels are tetrameric proteins composed of six transmembraneͲspanningdomainsandNͲandCͲterminalfacingthecytosol(Venkatachalam& Montell 2007). The fifth and sixth transmembrane domains line the pore of the ion channel (Hofmannetal.,2002)(Figure26).TRPC6isareceptorͲoperatednonͲselectivecationchannel, whichallowsintracellularCa2+toflowintothecellinresponsetoactivationbyDAGsecondary tophospholipaseCmediatedsignalcomingfromstimulationofspecificcellsurfacereceptors (Hofmannetal.,1999;Montell2005).Inthekidney,TRPC6isexpressedinrenaltubulesandin glomeruli,withpredominanceinpodocytes.TRPC6coͲlocalizeswithpodocinandnephrinand 59 immu unoprecipitationstudies showedphyysicalinteractionbetweenTRPC6an ndthesepro oteins, indicaating that th he channel is integrated into the signaling s complex at th he slit diaphragm. Thereefore,TRPC6 6iscriticalin nadjustingth hecationicionfluxcloseetotheslitd diaphragm(R Reiser etal.,2005).Inaaddition, Mo olleretal. deemonstrated dthatTRPC6 6isfunction nallyconnecttedto Mölleretal.,2007). theactincytoskeletoninthepodocyte(M Figure e24.Structurreandlocationofthe TRPC C6proteinintheslitdiaphragm.FP,foootprocesses;SSD,slit diaphragm. Ank, ankyrin a repeaat; cc, coiledͲcoil; CIRB, caalmodulin and d IP3 recepto orͲbinding (ad dapted al.,2009;Dryeer&Reiser2010). fromMachucaeta L Linkage analyysis of a 399Ͳmember family f with FSGS identiffied a locuss on chromo osome 11q21Ͳq22thatliinkedtothedisease(Wiinnetal.,19 999).Thisreggioncontain nedseveralkknown genessaswellas multiplenovelandpred dictedgeness,whichwerresystematiccallyscreene edfor mutaationsbydireectsequencing.Afterexaaminationoff42genes,TTRPC6emerggedasacand didate onth hebasisofreeportsofdettectionofTR RPC6mRNAiinthekidneyy(Riccioeta al.,2002;Garcia& Schilling 1997). Sequencing S of the 13 exons of th his gene ideentified a m missense muttation 12Qinexon 2thatsegreegatedwithtthediseaseiinallavailab blefamilymeembersconssistent p.P11 with anautosomaldominanttpattern(W Winn2005).A Additionalwo orkwasrepo ortedbyReiseret dfivefamilieeswith mutationsinTR RPC6thatseggregatedwitth FSGS,alth hough al.whoidentified mpletepenettrancewaso observed(Reeiseretal.,2005). incom B Basedonthe esetwostudiesthephen notypeassocciatedwithTTRPC6mutattionsislateͲͲonset ADFSSGSandvariiablerateof progression ntoESRD(W Winn2005;Reeiseretal.,2 2005).Howe ever,a signifficant numb ber of sporradic and childhoodͲon c nset FSGS cases c have been identified. ChildhoodͲonset SRNS has been reporteed in nine paatients carryying TRPC6 m mutations (SSantín, e al., 2009; Heeringa et al., 2009; Buscher et al., a 2012; Gigante et al.,, 2011; Mir et al., Ars, et 2012).Renalbiop psytypicallyshowedFSG GSbutcollapssingglomeru ulopathy(Gigganteetal.,2011; opoulos et al., a 2011) and MCD have been repo orted (Gigan nte et al., 20 011; Mottl et e al., Liako 60 2013).Thishighphenotypicvariabilitycanalsooccurwithinmembersofthesamefamilyeven withmutationcarriersshowingeithernosignsormildsignsofthedisease,indicatingthatthe disease is not fully penetrant (Reiser et al., 2005; Santín, Ars, et al., 2009). Studies in large cohorts revealed that TRPC6 mutations account for approximately 8% of AD FSGS patients (Sadowskietal.,2014). Todate,19differentmutationshavebeenreportedin22familial/sporadicpatientsfrom differentethnicbackground.ThreemutationsarelocatedintheANK1,twointheANK2,onein ANK3, one in ANK4, and five additional NͲterminal mutations are located outside of the predictedANKdomains(Reiseretal.,2005;Winn2005;Santín,Ars,etal.,2009;Giganteetal., 2011;Heeringaetal.,2009;Buscheretal.,2012;Hofstraetal.,2013;Miretal.,2012).Seven other TRPC6 mutations were located to the CͲterminal cytoplasmic tail (Reiser et al., 2005; Santín,Ars,etal.,2009;Giganteetal.,2011;Zhuetal.,2009;Mottletal.,2013),fourofthem mapping to a predicted coiledͲcoil domain at the CͲterminal (Gudermann et al., 2004). No mutationshavebeenidentifiedinthetransmembrane,extracellularorporeͲformingregionsof thechannel. Several of these mutations are gainͲofͲfunction mutations leading to increased channel activity by increasing calcium current amplitudes or by delaying channel inactivation. The p.N143S, p.S270T and p.K874* mutations did not modify the calcium influx during the functional assays, suggesting that an abnormality other than increased current amplitude is the cause of disease in individuals with these mutations. The several possibilities include altered channel regulation (despite normal amplitude), altered interaction with other slit diaphragmproteinsandalteredproteinturnover(Reiseretal.,2005). 5.2.4. INF2gene TheINF2genespansapproximately33kbonchromosome14q32andcontains23exons. Thisgeneencodestheinvertedformin2(INF2),aproteinof1249aminoacidsandaround136 KDa that belongs to formin family. Formins are widely expressed proteins governing several dynamiceventsthatrequireremodelingoftheactincytoskeletonsuchascellpolarity,celland tissuemorphogenesis,andcytokinesis(Boyer,Benoit,et al.,2011).TheINF2protein hasthe uniqueabilitytoacceleratebothpolymerizationanddepolymerizationofactin(Chhabraetal., 2009). This protein contains the following domains, from NͲterminal to CͲterminal: the diaphanous inhibitory domain (DID), the formin homology domains FH1 and FH2, and the diaphanousregulatorydomain(DAD).TheFH2domaindirectlymediatesactinassembly,and 61 theFH1domainsacceleratefilamentelongation.InteractionbetweenDIDandDADblocksthe ability of FH2 to interact with actin, inhibiting actin depolymerization but not actin polymerization. This step is inhibited by the binding of Rho GTPases to DID impeding its interactionwithDAD(Boyer,Benoit,etal.,2011;Gbadegesinetal.,2012). MutationsinINF2genewereidentifiedasacauseofADFSGSbyBrownetal.(Brownetal., 2010).StudiesinlargecohortsdescribedthatINF2mutationsaccountforapproximately9%Ͳ 17%offamilialADFSGSand<1%ofsporadiccases(Boyer,Benoit,etal.,2011;Gbadegesinet al.,2012;Baruaetal.,2013).MutationsinthisgenearealsothemaincauseofCharcotͲMarieͲ ToothdiseasewithFSGS(Boyer,Nevo,etal.,2011). The clinical phenotype of individuals with INF2 mutations is characterized by moderate proteinuria, without overt NS, manifesting at early adolescence or adulthood that is progressiveandoftenledtoESRD.However,nephroticrangeproteinuriaandfullͲblownNSat onset have been described. Microscopic hematuria and hypertension at onset have been noted in some individuals. Most individuals have an onset of the disease in the second to fourth decade of life with onset of renal failure in the third decade (Barua et al., 2013) but significantvariabilityhasbeenreported.Consideringallthepatientspublishedtodate,ageat proteinuriaonsetrangesfrom5yearsto72yearsandageatESRDonsetfrom13to70(Brown etal.,2010;Boyer,Benoit,etal.,2011).IntraͲfamilialvariabilityintheclinicalexpressionofthe diseaseisalsowidewithclinicallyunaffectedmemberscarryingthesamemutationthattheir affected son or daughter, consistent with incomplete penetrance that is often seen in AD diseases(Leeetal.,2011;Boyer,Benoit,etal.,2011;Gbadegesinetal.,2012). All the mutations described to date (more than 40) are highly conserved residues within theDIDdomain.Mostofthemclusterinexon4and2,accountingforapproximately80%and 10%ofmutations,respectively.Mutationsinexons3and6havebeenreportedintwoandone families,respectively(Baruaetal.,2013;SanchezͲAresetal.,2013). Many of the mutations reported are recurrent with certain codons appearing to be hotspots, such as Arg218 or Arg214. Mutations in Arg218 have been described in eight unrelatedfamilies,sixofthemcarrythep.R218Qmutation(Brownetal.,2010;Boyer,Benoit, etal.,2011;Gbadegesinetal.,2012;Baruaetal.,2013)whereastheremaining2familiescarry thep.R218Wmutation(Baruaetal.,2013).MutationsinArg214havebeendescribedineight familiesfourofthemcarrythep.R214H(Brownetal.,2010;Gbadegesinetal.,2012)mutation while the remaining four families carry the p.R214C mutation (Boyer, Benoit, et al., 2011; 62 Gbadegesinetal.,2012;Baruaetal.,2013).Apossiblefoundereffectofthesemutationswas studied by haplotype analysis but no common ancestral diseaseͲassociated haplotype was found(Boyer,Benoit,etal.,2011). INF2 mutations have been suggested to cause defects in actinͲmediated podocyte structuralmaintenanceandrepair(Brownetal.,2010).Mostofthemutationsreportedhave beenshowntocausemislocalizationofINF2inpodocytecultureandsomeofthemalsoinhibit the interaction between INF2ͲDID and mDiasͲDAD (mouse DiaphanousͲrelated formins), suggesting that changes are functionally important for regulation of actin cytoskeleton (Gbadegesinetal.,2012). 5.2.5. LMX1Bgene The LMX1B gene encodes the LIM homeodomainͲcontaining transcription factor 1B (LMX1B) that is essential during development. Mutations in LMX1B cause NailͲPatella syndromeassociatingdysplasiaofthepatellae,nailsandelbows,iliachorns,glaucomaand,in somecases,FSGSwithspecificlesionscharacterizedbythepresenceoftypeIIIcollagenfibrils in the GBM by electron microscopy (Dreyer et al., 1998). However, LMX1B mutations have been identified in four families (14 individuals) with AD FSGS but no glomerular membrane anomalysuggestiveofnailͲpatellaͲlikerenaldiseasebyelectronmicroscopyandnoextrarenal features (Boyer et al., 2013; Edwards et al., 2015). These patients presented significant albuminuriabetween5and70yearsofage,andsomeofthemreachedESRDbetween26and 70yearsofage.Thethreedifferentmutationsdetected(p.R246Q,p.R246Pandp.R249Q)are locatedintheLMX1Bhomeodomain.Theaffectedresiduesarelikelytoplayanimportantrole in strengthening the interaction between the homeobox domain of LMX1B and DNA, by holdinginteractionswiththeDNA(Boyeretal.,2013). 5.2.6. ANLNgene The ANLN gene encodes for anillin, an FͲactin binding protein that is enriched in the cytoskeleton.HeterozygousmissensemutationsinANLNhaverecentlybeendescribedintwo familieswithADFSGSbygenomeͲwidelinkagestudyandwholeͲexomesequencing.Theageat proteinuria onset varied from 9 to 69 years and ESRD occurred between 35 and 75 years (Gbadegesinetal.,2014). 63 5.2.7. PAX2gene PAX2isatranscriptionfactor expressedfromthefourthweek ofgestationin thekidney andplayacriticalroleinkidneydevelopment(Nakanishi&Yoshikawa2003).Mutationsinthis gene have typically been associated with congenital abnormalities of the kidney and urinary tract (CAKUT) as part of a syndrome known as renal coloboma or papillorenal syndrome (Sanyanusinetal.,1995).Baruaetal.identifiedaPAX2mutationinafamilywithADFSGSby exomesequencing.SequencingofthePAX2geneinFSGSfamiliesrevealedaPAX2mutationin 7ofthe175familiesanalyzed(24patients),suggestingthatmutationsinPAX2mightaccount forapproximately4%offamilieswithFSGS(Baruaetal.,2014). The affected patients had various degrees of proteinuria diagnosed between 7 and 68 years old (mostly second to fourth decades) and FSGS on biopsy. ESRD occurred in nine patientsbetween30and58yearsold.KidneyultrasoundinpatientswithPAX2ͲrelatedFSGS either was normal or could reveal echoic or small kidneys, dilated renal pelvis, or calyceal diverticulum. Most patients did no exhibit any extrarenal symptom, but renal coloboma was diagnosedretrospectivelyinonefamily(Baruaetal.,2014). Interestingly, a patient with a PAX2 mutation together with the homozygous p.R229Q in theNPHS2genehasbeenreported.ThispatientpresentedwithsubͲnephroticproteinuriaat7 months. At 20 years old he presented nephroticͲrange proteinura, FSGS on renal biopsy and hisrenalfunctionprogressivelydeterioratedreachingESRDat33yearsofage.Norecurrence ofthediseasewasobservedafteroneyearofrenaltransplantation.Additionally,hepresented ocularabnormalitiesinaccordancewiththeocularabnormalitiesseeninrenalcoloboma(Kerti etal2013). 64 5.3. GenesimplicatedinsyndromicformsofSRNSandFSGS Gene Reference MOI Typicalageatdiagnosis ofproteinuria/NS Tyicalhistology AgeatESRD Syndrome:recurrentextrarenalinvolvement Nºfamilies(patients) published SRNSwithgenitourinaryfeatures WT1 Pelletieretal.,1991 AD 0–10years DMS 0–15years DennysͲDrashsd:Wilmstumorinbothgenders,genitalanomalies in46,XYpatients >170 WT1 Klamtetal.,1998 AD 7monthsto34years FSGS 5toш34years Fraisersd:Pseudohermaphroditism,gonadoblastomain46,XY patients >60 AD 9–76years 22Ͳ52 NailͲPatellasd:hypoplasticnails/patellae,iliachorns >35(>54) AR 2–12years FSGS 3.8–22years SchimkeImmunoͲosseousdysplasia:spondyloepiphysealdysplasia, TͲcelldeficiency,cerebralischemia,broadnasaltip, hyperpigmentedmacules >90 5–47years FSGS 13–51years MELAS:myopathy,encephalopathy,lacticacidosis,andstrokeͲlike episode,diabetes,anddeafness >30(>45) SRNSwithosseousdysplasia LMX1B Dreyeretal.,1998 SMARCAL1 Boerkoeletal.,2002 FSGS,NPSͲ nephropathy MitochondriopathieswithSRNS Matern MTTL1 Gotoetal.,1990 COQ2 Quinziietal.,2006 AR 0–30months FSGS 0toш5years COQ10deficiency:encephalomyopathy,hypotonia,seizures, lactateacidosis 10(13) PDSS2 Lopezetal.,2006 AR 0Ͳ23months ND Nodata COQ10deficiency:encephalomyopathy,hypotonia,seizures, lactateacidosis 3(3) COQ6 Heeringaetal.,2011 AR 2monthsto6.4years FSGS COQ10deficiency:sensorineuraldeafness,seizure 8(16) SCARB2 Balreiraetal.,2008 AR 9to>59years FSGS Progressivemyoclonicepilepsy,cerebralandcerebellaratrophy, peripheralneuropathy,hearingimpairment 18(24) al 5monthsto 9.3years 9to>59years SRNSwithcutaneousfeatures 65 Gene Reference MOI ITGB4 Kambhametal.,2000 AR LAMB3 Hataetal.,2005 ITGA3 CD151 Typicalageatdiagnosisof Nºfamilies(patients) Tyicalhistology AgeatESRD Syndrome:recurrentextrarenalinvolvement 2monthsto10years FSGS NA Junctionalepidermolysisbullosa,naildystrophy,recurrent hemorrhagiccystitis,desquamationoflaryngealmucosa 2(2) AR ч4months DMS Epidermolysisbullosa,naildystrophy 1(1) Nicolaouetal.,2012 AR 0Ͳ4years FSGS Interstitiallungdisease,epidermolysisbullosa 6(6) Karamaticetal.,2004 AR ND ND Pretibialepidermolysisbullosa,sensorineuraldeafness,lacrimal ductstenosis,naildystrophy 2(2) Intellectualdisability 3(6) proteinuria/NS NA(5 months*) 2weekstoш4 years Thickeningof GBM published SRNSwithintellectualdisability 6weeksto3 ARHGDIA Guptaetal.,2013 AR 0–2.4years DMS KANK4 Geeetal.,2015 AR 2months FSGS ND XPO5 Braunetal,,2016 AR 2years MCD ND Speechdevelopmentdelay 1(1) AR 0–6years DMS 0Ͳ21years Piersonsd:microcoria,abnormallens,VI,hypotonia,motordelay >60(>70) years Intellectualdisability,shortstature,facialdysmophism,atrialseptal defect,dilativecardiomyopathy,leukocytosis 2(2) SRNSwithotherfeatures LAMB2 HasselbacherKetal., 2006 WDR73 Colinetal.,2014 AR 5to>13years FSGS ш5years GallowayͲMowatsd:secondarymicrocephaly,intellectual deficiency,corticalandcerebellaratrophy,facialdysmorphy,optic atrophy 2(3) INF2 Boyeretal.,2011 AD 10–46years FSGS 12–47years Charcot–Marie–Toothneuropathy,deafness 27(44) NUP205 Braunetal.,2016 AR 2Ͳ3years FSGS 7years Bicuspidaorticvalve,aorticinsufficiency,aorticrootenlargement 1(2) Abbreviations: AD, autosomal dominant; AR, autosomal recessive, diffuse mesangial sclerosis; ESRD, endͲstage renal disease; FSGS, focal segmental glomerulosclerosis; MCD,minimalchangedisease;MOI,modeofinheritance;ND,nodata;NPS,NailͲpatellasyndrome;NS,nephroticsyndrome;sd,syndrome. 66 6. CLINICALUTILITYOFGENETICTESTING TheidentificationofthegeneticcauseofSRNShasseveralclinicalimplications: Ͳ Provides an unequivocal molecular diagnosis for the patients and their family membersandmaydiagnosepreviouslyunrecognizedaffectedfamilymembers. Ͳ Predicts resistance to corticosteroid and/or immunosuppressive therapy. Therefore, theunnecessaryextensionofcorticosteroidtreatmentaswellasthepotentialriskand sideeffectsassociatedwiththesetreatmentscanbeavoided(Weberetal.,2004;Ruf, Lichtenberger,etal.,2004;Buscheretal.,2010;Giglioetal.,2015). Ͳ A low risk of relapse after renal transplantation, hence, living donor renal transplantationisencouraged. Ͳ Geneticcounseling(prenatalorpreimplantationgeneticdiagnosis)forfamilyplanning can be offered to couples with known risk for severe NS, in whom the causative mutationshavebeenidentifiedinafirstaffectedchild. Ͳ Prognosisinformationdependingonwhichgeneismutatedandthetypeofmutations consideringthegenotypeͲphenotypecorrelationsexplainedintheprevioussection. Althoughtheseclinicalimplicationsaregenerallyapplicabletoallpatientswithagenetic cause of SRNS identified, specific particularities and/or exceptions have been described dependingonthemutatedgene. Most patients with NPHS1 mutations are resistant to corticosteroids and immunosuppressive treatment. However, five patients (three families) with compound heterozygous or homozygous missense mutations and partial remission have been reported (Kitamuraetal.,2007;Heeringaetal.,2008;Schoebetal.,2010).Twosiblingswithfrequent relapsingNSthatcarriedcompoundheterozygousp.C265Randp.V822MmutationsinNPHS1 gene achieved remission of proteinuria without immunosuppressive treatment. Functional studiesshowedthatthep.C265Rmutationwastrappedintheendoplasmicreticulumwhereas thep.V822Mmutationpartiallyreachedtheplasmamembrane,likelyexplainingtheirmilder disease(Kitamuraetal.,2007).Intheremainingthreepatients(twofamilies)partialremission duetosteroidtreatmentwasachieved(Heeringaetal.,2008;Schoebetal.,2010). PostͲtransplantation recurrence in NPHS1 mutated patients is very low. However, in a studyofpatientswithFinͲMajororFinͲminormutations(thatleadtotheabsenceofnephrin) recurrenceoccurredin25%ofpatientsatameantimeof12monthspostͲtransplantation.All 67 these patients were homozygous for the FinͲmajor mutation, which leads to an early stopͲ codon and total absence of nephrin in the native kidney. In almost half of them antibodies againstnephrinweredetected(Patrakkaetal.,2002).Similarly,KuusniemietalreportedpostͲ transplantrecurrenceratein30%ofpatientshomozygousfortheFinͲmajormutationand70% of them showed antiͲnephrin antibodies (Kuusniemi 2007). Two patients with mutations different from the FinͲmajor and postͲtransplant recurrence have also been reported (Srivastavaetal.,2006;Chaudhurietal.,2012).InoneofthemantiͲnephrinantibodieswere detected (Chaudhuri et al., 2012). Thus, recurrence in these patients was considered an immune process against the nephrin present in the graft and treatment with immunosuppressiveagentsledtoremissioninsomecases(Flynnetal.,1992). Duringpregnancy,elevatedAFPinthematernalserumandamnioticfluidisindicativeof CNS. However, elevated AFP is not a specific marker of CNS as it has also been observed in NPHS1 heterozygous fetal carriers and in a fetus with DennysͲDrash syndrome (Devriendt et al., 1996; Patrakka et al., 2002). Therefore, prenatal genetic diagnosis is required to confirm CNSbeforeconsideringmedicalterminationofpregnancy. NPHS2mutatedpatientsdonotgenerallyrespondtocorticosteroidorimmunosuppressive therapy,butapartialreductionofproteinuriahasbeenreportedwithcyclosporineAineight patients(Ruf,Lichtenberger,etal.,2004;Malinaetal.,2009;Machuca,Hummel,etal.,2009). However, complete remission in SRNS caused by NPHS2 mutations have not been reported (Ruf, Lichtenberger, et al., 2004; Buscher et al., 2010). PostͲtransplant disease recurrence is significantly low in patients with NPHS2 mutations than in patients with without mutations, 4.5% vs. 28.5%, respectively according to a recent study in a large international cohort (Trautmannetal.,2015).However,postͲtransplantdiseaserecurrencehasbeendescribedin 12patients(Carraroetal.,2002;Bertellietal.,2003;Billingetal.,2004;Weberetal.,2004; Hocker et al., 2006; BeckerͲCohen et al., 2007; Trautmann et al., 2015). The most common mutationsinthesepatientswasthep.R138*andthep.R138Q.Thereisnoevidenceofpodocin antibodies in these patients, thus the pathomechanism of disease recurrence in NPHS2 mutatedpatientsremainsunknownandisprobablymultifactorial(Holmberg&Jalanko2014). TheidentificationofPLCE1representsthefirstmolecularcauseofNSthatmightresponse totherapyinsomeindividuals. Two childrenrespondedtotreatmentwith corticosteroidsor cyclosporineAachievingacompleteremissionandremainedfreeofproteinuriaafterseveral yearsoffollowͲup(Hinkesetal.,2006).Basedonthesefindings,ithasbeenspeculatedthat the arrest of glomerular development through PLCE1 mutations may be reversible by 68 treatmentwithcorticoidsorcyclosporineAviainductionofaredundantmechanismsuchas the activity of another phospholipase C (Hinkes et al., 2006). Thus, corticosteroid and immunosuppressivetreatmentisadvisedinpatientswithPLCE1mutations.NopostͲtransplant recurrencehasbeendescribedtodate. PatientswithWT1mutationsweredescribed tobe unresponsivetotherapy. However,a partial response to immunosuppressive agents has been described in some patients (Gellermann et al., 2010; Buscher et al., 2010). Gellermann et al. reported a favorable response to a therapy consisting of cyclosporine A in combination with corticosteroids in 3 patients with FSGS associated with WT1 mutations (Gellermann et al., 2010). Büscher et al. reported 2 patients who achieved a partial response to cyclosporine A: one phenotypically femaleFraiserpatient(karyotypeXY)affectedbyaWT1spliceͲsitemutationsandonepatients (karyotypeXX)withisolatedFSGSaffectedbyaWT1missensemutation(Buscheretal.,2010). InpatientswithWT1mutations,thekaryotypeoffemalesshouldbeinvestigatedinorder to exclude the 46 XY karyotype with pseudohermaphroditism. These patients should also be screened for the development of for the development of Wilms Tumor or gonadoblastoma (Roodetal.,2012).Geneticcounselingisessentialinbothfemaleandmales,astheyhavea 50% risk to transmit the mutation. Although most WT1 mutations are de novo, parents of a child with a WT1 mutation should be advised of a low probability of recurrence due to the possibilityofgerminalmosaicism,whichhasanunknownlikelihood. PotentialtherapyisavailableforsomerarecausesofSRNS.Forinstance,identificationof mutations in a gene encoding enzymes of the CoQ10 biosynthesis (COQ2, COQ6, ADCK4 or PDSS2)maywarranteeexperimentaltreatmentwithCoQ10(Heeringaetal.,2011;Montiniet al., 2008) as partial response to treatment with coenzyme Q10 has been described in individuals with SRNS and mutations in COQ2 (Montini et al., 2008), COQ6 (Heeringa et al., 2011) and ADCK4 (Ashraf et al., 2013). Similarly, individuals with TRPC6 mutations may potentially be amenable to treatment with calcineurin inhibitors (Schlondorff et al., 2009), patients carrying CUBN mutations may be amenable to treatment with vitamin B12, and individuals with ARHGDIA mutations may theoretically be responsive to the eplerenone treatment(Geeetal.,2013). GenetictestinginSRNSshouldbestronglyadvisedinthosepatientswithalikelyagenetic causeofthedisease:1)inCNSpatients,2)inchildhoodͲonsetSRNSpatients,bothfamilialand sporadic,3)inadultonsetpatientswithafamilyhistoryofthedisease,and4)inpatientsfrom 69 consanguineous marriages. This advice is based on the relatively high prevalence of genetic causesinthesegroups. 70 AIMS 71 72 Theglobalaimofthepresentthesiswastoincreaseourknowledgeonthecontributionof genetics to the pathogenesis and clinical outcome of idiopathic membranous nephropathy (IMN)andSRNS/FSGS. Thefirstpartofthisthesiswasfocusedontheassociationofgeneticpolymorphisms:SNPS andCopynumbervariants(CNVs),withtherisktodevelopIMNanditsclinicalcourse.Specific aimsofthispartwere: Ͳ To validate the association of HLAͲDQA1 and PLA2R1 genes with the risk to develop IMNinSpanishpopulation. Ͳ To test the putative association of CNVs within FCGR3A and FCGR3B genes with susceptibilitytoIMNinSpanishpopulation. Ͳ Toassessthecontributionofthesegeneticfactorstopredicttheclinicaloutcomeand renalfunctiondecline. ThesecondpartofthisthesisaimedtoanalyzethegeneticcausesofSRNS/FSGSaswellas toidentifygeneticmodifiersofdiseaseseverity.Particularaimsofthispartwere: Ͳ ToimprovethegenetictestingofSRNS/FSGS. Ͳ To assess the putative influence of mutations in multiple glomerular genes on SRNS/FSGSphenotypevariability. Ͳ TostudythecausativeandmodifyingroleofTTC21Bgene Thefinalaimofthepresentthesiswastodevelopanefficientandcomprehensivegenetic diagnosis including all known genes causative of glomerular and cystic inherited kidney diseases. 73 74
