The Role Of Rac1 Signaling In Diabetic Podocyte Injury

Background:Ras-related C3 botulinum toxin substrate 1(Rac1),a member of Rho family small GTPases,is a multi-functional molecule implicated in various cellular processes,such as cell adhesions,proliferation and cellular plasticity.Abnormal Rac1 activation is involved in reactive oxygen species(ROS)production and inflammatory responses,and is linked to the pathogenesis and development of diabetic nephropathy(DN).There are data implicating that podocytes might undergo epithelial-mesenchymal transition(EMT),a process during which epithelial cells are transformed to mesenchymal cells,under pathological conditions.And Racl is involved in EMT in cancer cells via potentiating two types of downstream effectors p21-activated kinase 1(PAK1)and mitogen activated protein kinases(MAPKs).P38 MAPK belongs to the latter and plays a critical role in inflammatory responses and regulation of cytoskeletal stability.We previously demonstrated that high glucose treatment could lead to EMT in HK-2 cells when p38 MAPK was found hyperactivated,which was also responsible for the development of diabetic kidney fibrosis;inhibition of p38 MAPK by using shRNAs markedly reversed these abnormalities.In the present study,we cultured immortalized mouse podocytes and generated podocyte-specific Rac1 RNAi transgenic(TG)mice;we investigated the role of signaling cascade of Rac1/PAK1/p38 MAPK in high glucose-induced EMT of podocytes and proteinuria under diabetic conditions;we also attached importance to the interactions between Rac1 signaling pathway and a pivotal transcriptional factor β-catenin;lastly and intriguingly,we revealed that podocyte-specific Racl deficiency was protective against diabetic podocyte damage and proteinuria,providing clear evidence for a early curative potentiality of targeting Racl in diabetic nephropathy.Part Ⅰ Rac1 in high glucose-induced podocyte epithelial-mesenchymal transitionObjective:1.To explore whether high glucose stimulates podocyte EMT.2.To determine whether EMT of podocytes leads to malfunction or monolayer disruption.3.To investigate the potential mechanisms through which Rac1 signaling pathway affects podocyte EMT.4.To investigate the impacts of blockade of Rac1 signaling pathway on podocyte EMT.Methods:1.Cell culture.Conditionally immortalized mouse podocytes were kindly provided by Professor Peter Mundel(Harvard Medical School)via Professor Jie Ding(Peking University).In brief,podocytes were cultured on type Ⅰ collagen in RPMI 1640 supplemented with 10%fetal bovine serum,100 U/ml penicillin,and 100mg/ml streptomycin under permissive conditions 33℃ plus 10 U/ml mouse recombinantγ-interferon.Cell differentiation was induced by maintaining podocytes at 37℃without γ-interferon under nonpermissive conditions for at least 14 days.2.Determination of podocyte EMT markers and function before and after high glucose(30mmol/l,HG)treatment:(1)alterations of podocyte morphology by inverted phase microscope;(2)assessment of podocyte makers p-cadherin and ZO-1,as well as mesenchymal markers FSP-1、α-SMA and desmin by western blot,real time PCR and immunofluorescence;(3)apoptotic rates by flow cytometry;(4)albumin permeability and monolayer funciton by transwell assay;(5)F-actin distribution by FITC-phalloidin staining and cell motility by transwell assay.3.Determination of signaling cascade of Rac1/PAK1/p38 MAPK:(1)construction,transfection and efficiency validation of Rac1 shRNAs ① positive transfection rates was determined by fluorescence microscope ②protein and mRNA levels of Racl were assessed by western blot and real time PCR;(2)preparation of inhibitors IPA-3 and SB203580 for PAK1 and p38 MAPK:the powder was first diluted in dimethyl sulfoxide(DMSO)as a stock liquid stored at-20℃.For in vitro use,the stock liquid was added into the culture media to a final concentration of 10μM;(3)Racl activation was measured by GST pull-down;(4)phosphorylation levels of PAK1 and p38 MAPK in podocytes under different conditions were detected by western blot.4.Impacts of blockade of Racl signaling on podoycte EMT and function:(1)EMT markers and podocyte functions were analyzed;(2)levels of transcriptional factors β-catenin and Snail were measured by western blot and immunofluorescence;(3)interaction between Racl cascade and p-catenin ① construction of Racl expression vectors including constitutive active-Racl(CA-Racl),wide type-Rac1(WT-Racl)and dominant negative-Rac1 Rac1(DN-Racl)② construction of P-catenin expression vectors(Flag-β-catenin)and an empty vector as the control ③cotransfection of expression vectors of Rac1 and β-catenin ④interplay between Rac1/PAK1 and β-catenin by co-immunoprecipitation(CO-IP)⑤interaction between p38 MAPK and β-catenin by in vito binding assay.Results:1.HG for 48h led to decreased expression of p-cadherin and ZO-1,whereas increased expression of mesenchymal hallmarks FSP-1,α-SMA and desmin(P<0.01);albumin permeability was significantly increased(P<0.01),simultaneously F-actin was re-assembly at the cortex of cells,together with increased cell motility(P<0.01).2.HG for 48h triggered Racl hyperactivation(P<0.01),as well as hyperphosphorylation of both PAK1 and p38 MAPK(P<0.01);moreover,β-catenin was found dephosphorylated at its N-terminus,and Snail was upregulated(P<0.05),when β-catenin showed marked nuclear accumulation.3.Rac1 shRNA significantly inhibited the expression of EMT markers(P<0.05),and reinstated podocyte dysfunction(P<0.05);also the aberrant activation of Rac1 or hyperphosphorylation of PAK1 and p38 MAPK were simultaneously rectified by Rac1 shRNA(P<0.05);the enhanced activities of transcriptional factors β-catenin and Snail,as well as β-catenin nuclear migration were partially prohibited by Racl shRNA(P<0.05);notably,the addition of IPA-3 or SB203580 showed limited impacts on Racl activation(P>0.05),and PAK1 hyperphosphorylation was not altered by SB203580(P>0.05).4.Racl directly phosphorylated β-catenin at its C-terminus,through which Racl resulted to less ubiquitylated β-catenin;p38 MAPK interacted β-catenin in a MEF2c phosphorylation-dependent way.Conclusion:1.HG stimulation resulted in podocyte EMT and malfunction.2.Racl was involved in podocyte EMT via potentiating two major downstream signals PAK1 and p38 MAPK.3.Aberrant activation of signaling cascade of Racl/PAKl/p38 MAPK under HG conditions promoted transcriptional factor β-catenin activity and nuclear accumulation,which affected EMT-related genes and proteins,thus driven the occurrence of podocyte EMT.4.Blokade of Racl/PAKl/p38 MAPK signaling pathway was protective against podocyte EMT.Part Ⅱ Generation and characterization of podocyte-specific Racl RNAi transgenic miceObjective:1.Generation and characterization of podocyte-specific Racl RNAi TG mice.Methods:1.Construction of podocyte-specific Racl shRNA vectors:(1)amplification of sequences of Nephrin promotor and mirRacl;(2)connecting pUp-Nephrin and pDown-SM30/mirRacl by digestion;(3)construction of pLV.EX2d.null-Nephrin>SM30/mirRacl expression vector by gateway technology.2.Establishment of podocyte-specific Rac1 RNAi TG mice:(1)FO TG mice ①the vector DNA was linearized and purified ②newborn mice were obtained by DNA microinjection into the germ cell ③F0 TG mice were obtained by genotyping;(2)Rac1 RNAi TG mice were bred and prepared by FO mice intercrossing with WT C57BL/6 mice,DNA extraction from the murine tail and genotyping.3.Interference efficiency validation of Rac1 RNAi TG mice:(1)Rac1 expression was detected in kidney sections by immunofluorescence doubled stained with synaptopodin in both wide type(WT)and TG mice;(2)Rac1 protein and mRNA levels were determined by western blot and real time PCR in primary podocytes from WT and TG mice.Results:1.Murine tail DNA PCR showed podocyte-specific TG mice were successfully genetically modified.2.Rac1 staining was reduced in Racl RNAi TG mice in comparison with WT littermates,without significant impacts on synaptopodin expression.3.Both protein and mRNA levels of Rac1 were decreased in primary podocytes from Racl RNAi TG mice than WT controls(P<0.05).Conclusion:1.Successful geneation of podocyte-specific Rac1 RNAi TG mice.Part Ⅲ Targeting Racl signaling is protective against diabetic podocyte injuryObjective:1.Generation of murine models of diabetes in WT and TG mice.2.To determine the beneficial effects of podocyte-specific Racl inhibition on diabetic podocyte injury.3.To investigate the protective role of targeting Rac1 in diabetic proteinuria and kidney impairment.Methods:1.Generation of murine models of diabetes:(1)by intraperitoneal injection with streptozotocin(STZ)(WT-STZ/TG-STZ),or citric buffer as controls(WT-control/TG-control);(2)standard:blood glucose constantly>16.7mmol.2.Metabolic data were determined during 4W-12W:weight,systolic blood pressure(SBP),blood glucose,24h urine total protein(24h UTP).3.Diabetic kidney lesion:(1)podocyte counts,average width of process,rates of process fusion and average thickness of the glomerular basement membrane(GBM)were observed by transmission electron microscope(TEM);(2)expression of slit diaphragm(SD)components p-cadherin and ZO-1,as well as fibroblastic markers FSP-1、α-SMA、desmin and matrix metalloproteinases(mmp9);(3)pathological lesion of the kidney were revealed by PAS staining;(4)apoptotic rates of podocytes were examined by WT-1 staning..4.GTP-Rac1 in primary podocytes was examined by GST pull-down assay;phosphorylation levels of PAK1 and p38 MAPK were measured by western blot.Results:1.Mild foot process effusion was revealed by TEM in WT-STZ mice at as early as 6W,when SD proteins p-cadherin and ZO-1 began to decrease and desmin was upregulated;promoted Racl activation,as well as enhanced PAK1 and p38 MAPK phosphorylation were observed in primary WT-STZ podocytes(P<0.01);at 8W,proteinuria occurred(P<0.01),together with increased expression of mmp9;at 12W,severe process effacement was revealed when proteinuria was exacerbated(P<0.01)and mmp9 was heavily stained,simultaneously,de novo of FSP-1 was exhibited by IHC.2.During the experimental period,WT-STZ mice weighed significantly less than WT-controls(P<0.01);whereas from 4W to 8W,TG-STZ weighed a little more than WT-STZ counterparts(P<0.05).3.During the experimental period,foot process effusion was attenuated in TG-STZ mice than WT-STZ mice;and SD proteins p-cadherin and ZO-1,as well as desmin were partially revesed;the expression of mmp9 and FSP-1 were also diminished.4.At 6W,phosphorylation levels of PAK1 and p38 MAPK were both decreased in TG-STZ primary podocytes than WT-STZ primary controls(P<0.05).5.During the experimental period,proteinuria was ameliorated in TG-STZ mice compared with WT-STZ littermates(P<0.05);concomitantly,it was found by PAS and Masson that the expansion of GBM and mesangial areas were reduced in TG-STZ mice.6.During the experimental period,there were no distinguishable differences between TG-STZ mice and WT-STZ littermate controls in either SBP or blood glucose,or podocyte counts(P>0.05).Conclusion:1.Diabetic proteinuria was secondary to podocyte damage.2.Targeting podocyte Racl was protective against diabetic podocyte injury via deactivating its downstream effectors PAK1 and p38 MAPK,reinstating podocyte SD protein expression and function,and reversing podocyte dedifferentiation.3.Podocyte-specific Rac1 deficiency was able to decrease proteinuria via maintaining podocyte integrity and to preserve the kidney via alleviating GBM and mesangial injury.