Mechanism Of Podocyte Apoptosis Regulated By Cdc42/YAP/NFAT Signal Pathway

BackgroundPodocytes are highly differentiated cells with complex actin cytoskeletal architecture that play a key role in maintaining the integrity of glomerular filtration barrier.Therefore,loss of podocyte will surely disturb the function of glomerular.Apoptosis is one of the main reasons that causes loss of podocyte and sequentially induces proteinuria.Accumulating evidence show that podocyte apoptosis is one of the most important mechanisms in the pathogenesis of many kidney diseases,such as chronic kidney diseases,diabetic nephropathy,focal segmental glomerular sclerosis,et al.Thus,preventing podocyte apoptosis will be a promising therapeutic target for treating these kidney diseases.However,the concert mechanisms that cause podocyte apoptosis in these kidney diseases are still far from being fully understood.Rho family small GTPases RhoA,Racl,and Cdc42 are the three most extensively studied prototypes and are known powerful regulators of actin cytoskeletal dynamics,cell adhesive interactions,motility,or cell polarity.However,these Rho-GTPases also control other important cellular functions such as gene expression,proliferation and apoptosis.A large body of data implicates RhoA,Rac1,and Cdc42 in the pathogenesis of disease processes in the kidney including glomerular diseases.Recent studies also indicated that podocyte-specific deletion of Cdc42 in vivo,but not of RhoA or Rac1,leads to congenital nephrotic syndrome,podocyte foot process effacement and glomerulosclerosis.However,whether Cdc42 is involved in podocyte apoptosis remains unknown.Meanwhile,further studies will also be required to unravel the detailed downstream signaling cascades of Cdc42,for instance,Nwasp,in podocytes and the exact mechanisms leading to podocyte apoptosis and proteinuria.Yes-associated protein(YAP)is a major downstream cascade of the Hippo pathway which controls the expression of genes that promote cell proliferation and inhibits apoptosis.Under normal condition,dephosphorylated YAP localizes in the nucleus and functions as a transcriptional co-activator that mainly through interacting with TEA domain family member(TEAD)family transcriptional factors to induce target gene expression.YAP phosphorylation promotes its cytoplasmic sequestration and inactivation.Recent studies have indicated that YAP is an antiapoptotic molecule in podocyte,and podocyte-specific deletion of YAP leads to proteinuria kidney diseases.Moreover,loss of stress fiber caused by insufficient Cdc42 can result in reducing transcriptional activity and nuclear localization of YAP.Therefore,it is likely that Cdc42 deficiency may induce podocyte apoptosis by reducing nuclear YAP.The nuclear factor of activated T cells(NFAT),which are the substrate for calcineurin(CaN),represent a family of Ca2+ dependent transcription factors.Four isoforms,NFATI,NFAT2,NFAT3 and NFAT4,have been identified so far.NEAT is expressed in most immune system cells and plays a pivotal role in the transcription of cytokine genes and other genes critical for the immune response.Our previous study found that high glucose increased intercellular Ca2+ concentration,and subsequent induced the activation of calcineurin and NFAT2(increased NFAT2 nuclear accumulation).Activation of NFAT2 induced by high glucose was completely blocked by NFAT inhibitor 11R-VIVIT,and was also blocked by the calcinerin inhibitor CsA.In addition,the apoptosis inducing effect of high glucose was abolished by cotreatment with 11R-VIVIT.These data demonstrated that activation of NFAT2 could resulte in podocyte apoptosis.Previous studies also showed that inhibition of RhoGTPases caused by activation of calcineurin/NFAT could result in disruption of podocyte cytoskeleton and cell apoptosis.What's more,mice heart specific knock out Cdc42 could cause activation of NFAT and myocardial hypertrophy.Thus,these data indicated that loss of Cdc42 could activate NFAT thereby led to podocyte apoptosis.In addition,as mice heart specific knock out Cdc42 could cause activation of NFAT and myocardial hypertrophy,and ablation of Cdc42 decreased nuclear localization of YAP in mouse embryonic fibroblasts and other kidney cells,led to a reduction of YAP-dependent gene expression and caused a severe defect in nephrogenesis that strikingly phenocopies loss of YAP,which could function as a transcriptional co-activator to inhibit dendrin in the podocyte nuclear thereby inhibited cell apoptosis.Therefore,base on these previous studies,we assume that there may be a direct relationship between YAP and NFAT in the podocyte nuclear which is similar to YAP and dendrin,as they are both closely linked to cytoskeletal protein Cdc42 and podocyte apoptosis.Some previous studies demonstrated that HG?LPS?ADR could induce culture podocyte apoptosis,and murine models of LPS or ADR-induced proteinuria and podocytopathy were widely used.In addition,murine model of ADR-induced proteinuria and podocytopathy has been used as the model of FSGS as well.Thus,to explore the mechanism of podocyte apoptosis,we study podocyte apoptosis in HG,LPS or ADR treated podocyte in vitro,and db/db,LPS or ADR mice in vivo.In this study,to explore the mechanism of podocyte apoptosis,we study podocyte apoptosis in HG,LPS or ADR treated podocyte in vitro,and db/db,LPS or ADR mice in vivo.We found that Cdc42 expression and activity were decreased in injured podocytes in vitro and vivo,and loss of Cdc42 in vitro and vivo by siRNA and specific inhibitor ML-141 respectively caused podocyte apoptosis and proteinuria,accompanied with increased pro-apoptotic Bax and decreased anti-apoptotic Bcl-2 gene and protein expression.Thus,these results demonstrated that Loss of Cdc42 caused podocyte apoptosis.We also found that YAP expression were significantly decreased in HG,LPS or ADR injured podocytes in vitro and in vivo,and cell apoptosis rate was markedly increased in vitro cultured YAP knockdown podocytes.Thus,these results demonstrated that Loss of YAP could also cause podocyte apoptosis.Our results further demonstrated that insufficient Cdc42 or neuronal Wiskott-Aldrich syndrome protein(Nwasp),its downstream effector,could decrease the mRNA and protein expression of YAP,which had been regarded as an anti-apoptosis protein in podocyte.Moreover,our data indicated that loss of stress fibers caused by HG,LPS,ADR or Cdc42/Nwasp deficiency decreased YAP mRNA and protein expression and induced podocyte apoptosis.Therefore,these data indicated that Cdc42 could positively regulate YAP,maybe through stress fibers.Cell apoptosis induced by Cdc42/Nwasp/stress fiber deficiency was significantly inhibited in podocytes with over expressed active YAP.Therefore,YAP mediated Cdc42/Nwasp/stress fiber deficiency-induced podocyte apoptosis.Moreover,we found that NFAT was activated in Cdc42,Nwasp,stress fibers or YAP deficiency podocyte,YAP could bind to NFAT in podocyte nuclear and inhibit its nuclear protein expression,inhibition of NFAT could significantly abolish Cdc42/Nwasp/stress fibers/YAP deficiency-induced podocyte apoptosis.Therefore,Cdc42/Nwasp/stress fibers/YAP/NFAT signal pathway may potentially play an important role in regulating podocyte apoptosis.Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases.Methods1.Animal studiesAnimal care and experiments were performed in accordance with the ARRIVE guidelines,and were approved by the Ethics Committee for animal research of Guangdong General Hospital.6 male C57BL/KsJ db/db mice and 5 age-matched wild-type(BKS)mice and 36 male BALB/c mice aged 8 weeks were housed in the animal centre of Zhongshan School of Medicine,Sun Yat-sen University.Mice urinary albumin and creatinine were measured using mouse albumin-specific ELISA(Bethyl Laboratories,Inc,USA)and creatinine kits(Cayman Chemical,USA)respectively,according to the manufacturer's instructions.Proteinuria was expressed as the ratio of albumin to creatinine.Mice were anaesthetized(ketamine,70 mg/kg,i.p.)before killed and kidney tissue were collected for paraffin section and frozen section.Podocyte number counting was analyzed by immunohistochemical staining WT-1 in paraffin section.Podocyte apoptosis,Cdc42 and YAP protein expression were analyzed by immunofluorescent assay in frozen section.1.1.Diabetic mice modelThe C57BL/KsJ db/db mice were obese and known to develop type 2 diabetes,followed by diabetic kidney disease.For urine collection,individual mice were caged once every 2 weeks in a metabolic cage for 24 h from week 12 to week 20.Fasting blood glucose was measured in tail-vein blood,weekly,using a one touch ultra glucometer and Test Strip(Lifescan,Milpitas,CA,USA)after 6 h of fasting.Body weight was obtained every week.After 8 weeks of observation,mice were anaesthetized(ketamine;70 mg kg-1,i.p.)before killed and then kidney tissue were collected for further study.1.2.Murine models of LPS-induced proteinuria and podocytopathyFive male BALB/c mice were randomized to each of the following treatments:LPS-induced proteinuric mice were intraperitoneally injected with 200 ?g LPS(1 mg/ml in sterile LPS-free saline,serotype:E.coli 0111:B4,Sigma)in a total volume of 200 ?l;control mice were intraperitoneally injected with equal volumes of sterile LPS-free saline.Mice were followed for 24 h before they were killed and the kidneys resected for further investigation.Urine was collected before and 24 h after LPS injection.1.3.Murine models of ADR-induced proteinuria and podocytopathyFive male BALB/c mice were injected once with ADR(2 mg/ml in sterile ADR-free saline,Doxorubicin hydrochloride,Sigma)at a dose of 12mg/kg body weight via the tail vein on day 0.Five control BALB/c mice were injected with an equal volume of saline only.Urine of all mice was collected on day 0,4,7,14.1.4.Murine models of Cdc42 inhibitionFive male BALB/c mice were intraperitoneally injected with 100ul ML-141(selective inhibitor of Cdc42,1 mg/ml in 30%dimethylsulfoxide-saline,Tocris Bioscience,UK)daily for 3 weeks.Five blank control mice and five Dmso control mice were intraperitoneally injected with 100 ul saline and 30%dimethylsulfoxide-saline respectively everyday for 3 weeks.Urine of all mice was collected weekly before killed.2.Cell culture and treatment2.1.Podocyte cultureThe conditionally immortalized mouse podocyte cell line(MPC)was a kind gift from Dr.Jochen Reiser(Rush University Medical Center,Chicago,IL,USA),and cultured as described previously.Cells were cultured at 33?,5%C02 in RPMI-1640 medium(Gibco BRL,Gaithersburg,MD,USA)supplemented with 10%fetal bovine serum(FBS,Gibco BRL,USA)and recombinant IFN-y(growth permissive conditions;CYT-358,ProSpec,Tany Technogene Ltd,Israel)for proliferation.To induce differentiation,podocytes were reseeded and cultured at 370C,5%C02 in 100 cm2 culture dish coated with 12 mg/ml type-I collagen(BD Bioscience,Bedford,MA,USA)and in dulbecco's modified eagle medium(DMEM,with 5.3 mM glucose,Invitrogen,USA)supplemented with 5%FBS,deprived of IFN-y(growth restrictive conditions)for 10 to 14 days.2.2.Identification of differentiated podocyteWe compared the morphological difference between proliferated podocytes(33?,with 10%FBS and IFN-y)and differentiated podocytes(37?,with 5%FBS,and deprived of IFN-y)under microscopy.After differentiation,podocytes was confirmed by the identification of synaptopodin,a podocyte differentiation marker,under laser confocal microscopy.2.3.Podocyte treatment106 cells were cultured at 37?,5%C02 for 8 days before synchronized into quiescence by growing cells in serum-free RPMI-1640 medium for 24 h,and then treated with Mannitol(MA,add to 30mM glucose),high glucose(HG,add to 30mM glucose),LPS(100?g/ml),ADR(0.125?g/ml),NFAT inhibitor 11R-VIVIT(100nM)or siRNA(50nM)for 48 h.Each reaction was repeated in triplicates.3.Detection of podocyte apoptosis in vitro and in vivo3.1.Detection of podocyte apoptosis in vitroApoptotic cells in different groups(treated with HG?LPS?ADR?siRNA or plasmids)were determined using an Annexin V/PI apoptosis detection kit according to manufacturer's protocol.Briefly,podocytes were resuspended with binding buffer followed by incubation with 5 ml of Annexin V(conjugated with FITC)and 5 ml of PI in the dark for 10 min.Cell fluorescence was then analyzed using a Cell Lab QuantaTM SC Flow cytometer.Cells positive for Annexin V-FITC were considered as apoptosis.3.2.Detection of podocyte apoptosis in vivoApoptotic cell death in kidney frozen sections was detected by using the TUNEL kit(Roche Molecular Biochemicals,Mannheim,Germany)as described previously.Cells positive for both TUNEL and WT-1 are apoptotic podocytes.The quantifications of these data were expressed as the means of apoptotic podocytes from 20 randomly selected glomeruli from at least three samples.Photomicrographs were taken with laser confocal microscopy(LCSM,Zeiss KS 400,Postfach,Germany).4.Detection of active and total Cdc42 protein expression in podocytes4.1.Detection of active Cdc42 protein expressionActive Cdc42 was measured by G-LISA Cdc42 Activation Assay Biochem kit(colorimetric assay,Cytoskeleton,USA)following the manufacturer's instruction.And the signal was measured at 490 nm with a microplate reader(MRX,Dynatech Laboratories).Total Cdc42 protein expression of each group was measured by WB,as described below.Cell lysate from each group containing the same amount of protein was measured in both Cdc42 GTPase activation assay and WB.Results were expressed as fold activity of stimulated in relation to non-stimulated controls normalized to total Cdc42 protein content.4.2.Detection of total Cdc42 protein expressionAs for in vitro culture podocytes,an aliquot of cell lysates containing 30 ?g of protein was used for Western Blotting to detect the expression of total Cdc42 protein.As for in vivo podocytes,we use laser confocal microscopy to obtain the micrographs of dual-color fluorescence staining of kidney glomeruli from human and mice for Cdc42 and synaptopodin.The quantification of the total Cdc42 protein expression in podocytes(both positive for synaptopodin and Cdc42)was analyzed by Image-Pro Plus 6.0.5.Detection of the nuclear and cytoplasmic YAP protein expression in podocytesFor in vitro cultured podocytes,nuclear protein was extracted from cell lysates according to the nuclear and plasma protein extraction kit instructions,and then the nuclear YAP protein expression was detected by Western Blotting.In addition,we use immunofluorescence to analyze the karyoplasmic distribution of YAP protein in cultured podocytes.For in vivo podocytes,we use laser confocal microscopy to obtain the micrographs of dual-color fluorescence staining of kidney glomeruli from human and mice for YAP and WT-1.The quantification of the nuclear YAP protein expression in podocytes(both positive for YAP and WT-1)was analyzed by Image-Pro Plus 6.0.6.Transfection of siRNAs and plasmids6.1.Transfection of siRNAsThe siRNAs against Cdc42,Nwasp,YAP,and control were designed and synthesized by RIBOBIO CO.LTD.(Guangzhou,China).106 cells were cultured at 37?,5%CO2 for 8 days before synchronized into quiescence by growing cells in serum-free RPMI-1640 medium for 24 h,and then they were transfect into podocytes for 48 h using Lipofectamine 2000 reagent(Invitrogen,Carlsbad,CA)following the manufacturer's protocol.The sequences of siRNAs used in this study were as follows:siCdc42 5'GCAAGAGGAUUAUGACAGA dTdT 3',siNwasp 5'GGUGUC GCUUGUCUGGUUA dTdT3',siYAP 5'-CGAGAUGAGAGCACAGACA dTdT-3'.6.2.Transfection of plasmidsLentiviral packaging wild-type YAP or YAP S112A mutation plasmids(CS-Mm06093-Lv128,GeneCopoeia,China)were generated in HEK293T cells that were expanded and transfected using the Lenti-PacTM HIV Expression Packing Kit(HPK-LvTR-20,GeneCopoeia,China)according to the manufacturer's protocols,106 cells were cultured at 37?,5%C02 for 8 days before synchronized into quiescence by growing cells in serum-free RPMI-1640 medium for 24 h,and then podocytes were infected in 6-well plate(Corning,USA)with the lentivirus particles overnight before transfected with siRNAs.Forty eight hours after transfection,the cells under different conditions were collected.The effect of overexpression of active YAP in podocytes was identified by increasing nuclear YAP protein expression analyzed by Western Blotting.7.The co-immunoprecipitation test of nuclear YAP and NFAT proteins in podocytesNuclear protein was extracted from cell lysates according to the nuclear and plasma protein extraction kit instructions.The target proteins were bound to YAP antibody and NFAT antibody respectively and then were precipitated by ProteinA/G agarose beads before SDS-polyacrylamide gel electrophoresis.Accordingly,NFAT antibody and YAP antibody were used to incubate the PVDF membrane containing YAP and NFAT protein precipitated by ProteinA/G agarose beads.Finally,the combination of YAP and NFAT protein in podocytes nuclei was identified by analyzing the bands in the graphs.8.Statistical analysisAll values are expressed as mean ± standard deviation(SD).Statistical analysis was performed using the statistical package SPSS for Windows Ver.19.0(SPSS,Inc.,Chicago,IL,USA).All experimental observations were repeated more than three times.Statistical analysis of the data from multiple groups was analyzed by one-way ANOVA with LSD-test,Bonferonni-test,Student-Newman-Keuls-tests or Tamhane's-test.Data from two groups were compared by Student's t-test.P-values<0.05 were considered significant.Results1.Apoptosis was significantly increased in HG,LPS or ADR injured podocytes in vitro and vivo.There was a marked reduction in glomerular WT-1-positive cells in glomeruli of db/db mice,LPS mice or ADR mice relative to control mice.TUNEL staining demonstrated that podocyte apoptosis was obviously increased in db/db mice,LPS mice or ADR mice compared to control mice.Consisted with the results above,urinary albumin excretion(?g·mg-1 creatinine),the parameters of proteinuria and podocytopathy,was also remarkably increased in db/db mice,LPS mice or ADR mice as compared with that in control mice.In addition,cell apoptosis was significantly increased in HG,LPS or ADR injured podocytes in vitro.Consisted with the results above,the mRNA and protein expression of Bax,a well-recognized indicator of apoptosis were significantly increased in HG,LPS and ADR treated podocytes comparing to normal controls.However,Bcl-2,the indicator of anti-apoptosis was significantly decreased in HG,LPS and ADR treated podocytes.These data indicated that we had successfully created podocyte apoptosis models in vitro and in vivo.2.Cdc42 expression and activity were decreased in injured podocytes in vitro and vivoThe mRNA and total protein expression of Cdc42 were significantly decreased in injured podocyte induced by HG,LPS or ADR comparing to normal controls.Similarly,Cdc42 activity was also obviously decreased in HG or LPS or ADR treated podocytes.Immunofluorescent staining exhibited Cdc42 expression was decreased in glomerular podocyte of LPS or ADR-injected mice,comparing with vehicle control mice respectively.Consisted with the results from mice,in patients with DN or FSGS,the protein expression of Cdc42 was also markedly decreased in glomerular podocytes.3.Loss of Cdc42 caused podocyte apoptosis and proteinuriaApoptosis was markedly increased in vitro cultured Cdc42 knockdown podocytes compare to siCon.The mRNA and protein analysis of apoptosis signals Bax showed significantly increased expression in Cdc42 silenced podocytes,while apoptosis negative related gene and protein Bcl-2 was inversely declined.In addition,there was a marked reduction in glomerular WT-1-positive cells in glomeruli of mice treated with Cdc42-specific inhibitor ML-141,Furthermore,measurement of apoptosis cells by TUNEL staining showed that the number of apoptosis podocytes in glomerular was increased in mice treated with ML-141 comparing to the control mice.Urinary albumin excretion was also significantly increased in ML-141 treated mice as compared with control mice.4.Nwasp and YAP expression were decreased in injured podocytesThe results of real time-qPCR and western blot analysis indicated that the mRNA and protein expression of Nwasp were markedly decreased in podocytes treated with HG or LPS or ADR.Results from immunofluorescence and western blotting demonstrated that both nuclear and cytoplasmic YAP protein expression was obviously decreased in injured podocytes induced by HG or LPS or ADR.Similar results from PCR to analysis the mRNA expression of YAP were also obtained in HG or LPS or ADR treated podocytes.In addition,in patients with DN or FSGS,the nuclear protein expression of YAP was also markedly decreased in glomerular podocytes.5.Loss of Nwasp or YAP induced podocyte apoptosis in vitroCell apoptosis rate was markedly increased in vitro cultured Nwasp or YAP knockdown podocytes compared with siCon.Consisted with the results above,the mRNA and protein expression of Bax,a well recognized indicator of apoptosis were significantly increased in Nwasp or YAP knockdown podocytes comparing to siCon.However,Bcl-2,the indicator of anti-apoptosis was significantly decreased in Nwasp or YAP knockdown podocytes.6.Overexpression of active YAP alleviated podocyte apoptosis induced by HG,LPS or ADRConsisted with the results of mentioned above,cell apoptosis rate was markedly increased in vitro cultured podocytes treated with HG,LPS or ADR,respectively.However,after over expressed active YAP in vitro podocytes,cell apoptosis induced by HG,LPS or ADR was significantly alleviated.7.Loss of Cdc42/Nwasp decreased YAP expression in podocytesThe results of real time-qPCR and western blot analysis indicated that the mRNA and protein of Nwasp were markedly decreased in Cdc42 knockdown podocytes.Results from both immunofluorescence and western blotting demonstrated that both nuclear and cytoplasmic YAP protein expression was obviously decreased in Cdc42 or Nwasp knockdown podocytes.Similar results from real time-qPCR to analysis the mRNA expression of YAP were also obtained in Cdc42 or Nwasp knockdown podocytes.Furthermore,specifically inhibited Cdc42 expression with ML-141 in mice also decreased nuclear YAP protein expression.Thus,these data strongly indicated that Cdc42/Nwasp could positively regulate YAP expression.8.Stress fiber/YAP probably mediated Cdc42/Nwasp deficiency-induced podocyte apoptosisPhalloidin-stained stress fiber was decreased in vitro Cdc42 or Nwasp knowdown podocytes accompanied with increased podocyte apoptosis.This was consistent with above results that stress fiber was also decreased while podocyte apoptosis was increased in HG,LPS or ADR injured podocytes.Inhibited stress fiber formation with the specific inhibitor LatA resulted in increased podocyte apoptosis accompanied with decreased YAP mRNA and protein expression,but not changed the protein expression of Cdc42 and Nwasp.These data indicated that Cdc42/Nwasp could positively regulate YAP expression probably through stress fiber.We also investigated the apoptotic effect of overexpression of active YAP in Cdc42 or Nwasp knockdown podocytes or LatA treated podocytes.As expected,podocyte apoptosis induced by Cdc42 or Nwasp knockdown or LatA treatment was significantly abolished by over expressing active YAP.These data mentioned above indicated that Stress fiber/YAP probably mediated Cdc42/Nwasp deficiency induced podocyte apoptosis.9.NFAT mediated Cdc42/Nwasp/strsss fiber/YAP deficiency-induced podocyte apoptosisNFAT was significantly activated in Cdc42 siRNA.Nwasp siRNA.LatA or YAP siRNA treated podocytes.Moreover,YAP protein could bind to NFAT protein in the podocyte nuclear and inhibited its expression.Inhibition of NFAT significantly abolished podocyte apoptosis induced by Cdc42,Nwasp or YAP knockdown or LatA treatment.Thus,these data demonstrated that NFAT mediated Cdc42/Nwasp/strsss fiber/YAP deficiency induced podocyte apoptosisConclusions1.Loss of Cdc42 caused podocyte apoptosis and proteinuria.2.Cdc42 could positively regulate YAP expression probably through stress fiber.3.YAP mediated Cdc42/Nwasp/strsss fiber deficiency-induced podocyte apoptosis.Moreover,YAP protein could bind to NFAT protein in the podocyte nuclear and inhibited its expression,thereby to prevent podocyte apoptosis.4.NFAT mediated Cdc42/Nwasp/strsss fiber/YAP deficiency-induced podocyte apoptosis.Cdc42/Nwasp/stress fibers/YAP/NFAT signal pathway may potentially play an important role in regulating podocyte apoptosis.Maintaining necessary Cdc42 would be one potent way to prevent proteinuria kidney diseases.