Critical Role Of Serum Response Factor In Epithelial-mesenchymal Transition Of Diabetic Nephropathy

Background and ObjectiveThe incidence of diabetic nephropathy(DN)in diabetes mellitus(DM)patients is approximately 20%～40%,and DN is the most severe complication and chief cause of death in DM.With the rapid increase in the number of DM patients,the incidence of DN rises year by year.It has become the first reason leading to end-stage renal disease(ESRD)in some countries.The pathogenesis of DN is so complicated that the exact mechanism is not known yet.Many factors act not only alone but also together to cause and to aggravate DN.Further study of the pathogenesis of DN is needed to seek an efficient treatment of DN.Podocytes play a fundamental role in glomerular filtration and its charge barrier can effectively prevent the leakage of protein.In DN,podocyte EMT leads to the fusion and effacement of foot process.Eventually,podocyte separated from the glomerular basement membrane(GBM)to the renal capsule.Podocyte EMT is considered as the beginning process of podocyte injury,which can enhance the motility of podocytes and lead to proteinuria and glomerular fibrosis.In the process of EMT,epithelial markers(synaptopodin,ZO-1,P-cadherin)are downregulated along with upregulation of mesenchymal markers(collagen-1,fibronectin,FSP-1 and a-SMA).Although DN was traditionally considered as a primarily glomerular disease,it is now widely accepted that the rate of deterioration of renal function correlates best with the degree of renal tubulointerstitial fibrosis.Renal tubular epithelial cells(TECs)epithelial-mesenchymal transition(EMT)is considered as the beginning process of tubulointerstitial fibrosis.In the process of EMT,epithelial markers(E-cadherin,ZO-1)are downregulated along with upregulation of mesenchymal markers(fibronectin,collagen-1,a-SMA,FSP-1).Snail,Zeb and HLH family are important transcriptional repressors of E-cadherin and other about EMT.SRF is a highly conserved DNA binding protein of MADS-box transcription factor family and a main switch for the transcription of cytoskeletal and contractile genes in almost all cells across various species.SRF induces mesoderm cells migration and plays an important role in the extension of embryonic body axis,which suggests that SRF is critical for embryonic development.SRF can be activated through two different ways according to the sensitivity of target genes to the Ras-extracellular regulated kinase(ERK)-ternary complex factors(TCFs)or Rho-actin-myocardin related transcription factors(MRTFs).SRF has been shown to have an essential role in tumor progression,especially in the EMT and metastasis of epithelial tumor cells,such as hepatocellular carcinoma,gastric cancer and prostate cancer.In transgenic mice,high SRF expression can cause myocyte hypertrophy,interstitial fibrosis and muscle fiber damage.And SRF knockout of coronary arterial smooth muscle cells(SMCs)could reduce the migration and proliferation ability of SMCs.In addition,SRF plays a key role in TGF-β induced lung myofibroblast differentiation,and using PKA to reduce SRF expression and activity can inhibit this process.It is reported by Wang HM that SRF provokes EMT of TECs in unilateral uretereal obstruction(UUO)model and accelerates the high glucose-induced EMT in human peritoneal mesothelial cells,suggesting a close connection between upregulation of SRF and DN.However,how SRF regulates EMT of DN remains largely unknown.According to the information above,our hypotheses are:SRF is upregulated and translocates from cytoplasm to nuclei in the kidney of DN rats,which results in EMT of podocytes and TECs,renal fibrosis and proteinuria through Snail signaling pathway;CCG-1423(a small molecule inhibitor of SRF)can relieve EMT of podocytes and TECs and improve renal fibrosis and proteinuria in DN rats.To verify our hypotheses,we used Type 1 diabetic rats model,mouse conditionally immortalized podocyte cell line(mpc5)and human proximal renal tubular epithelial cells(HK-2 cells)to explore the role and mechanism of SRF in glomerular fibrosis and renal tubulointerstitial fibrosis.Furthermore,the therapeutic potential of this signaling was also investigated.MethodsTo evaluate the role and mechanism of SRF in glomerular fibrosis,mpc5 was pretreated with CCG-1423(1μM or 2μM)for 1h followed by high glucose treatment for 72h;To illustrate whether SRF provokes podocyte EMT,we used plasmids containing full-length complementary DNA(cDNA)sequence of mice SRF and transwell chamber migration assay to determine the migration and motility of podocytes.In vivo,rats obtained a single intraperitoneal injection of 60 mg/kg STZ.CCG-1423 was administered by daily intraperitoneal injection at doses of 0.01 and 0.02 mg/kg body weight for 8 weeks since the day after STZ injection.At the end of the study,rats were weighed and housed in metabolic cages to collect 24-h urine.Blood samples of rats were collected followed by systemic perfusion.Kidneys were quickly removed,decapsulated and weighed.Western blot and Quantitative reverse transcription polymerase chain reaction(QPCR)were used to measure the expression of SRF,pSRF,ZO-1,collagen-1,a-SMA and FSP-1 in mpc5 and renal cortex of rats.Immunofluorescence staining was used to determine the expression and localization of SRF in podocytes.Serum glucose,serum creatinine(Scr),blood urea nitrogen(BUN)and 24-h urinary protein(24-h UP)were measured by the Department of Pathology;The expression of SRF,cadherin,fibronectin in glomeruli of rats was detected through immunohistochemical staining.Periodic acid-Schiff(PAS)staining and Masson staining were used to demonstrate glomerular fibrosis.To evaluate the role and mechanism of SRF in renal tubulointerstitial fibrosis,HK-2 cells were pretreated with CCG-1423(1μM or 2μM)for 1h followed by high glucose treatment for 72h;To illustrate whether SRF provokes EMT of HK-2 cells,we used plasmids containing full-length complementary DNA(cDNA)sequence of human SRF and transwell chamber migration assay to determine the migration and motility of HK-2 cells.In vivo,rats obtained a single intraperitoneal injection of 60 mg/kg STZ.CCG-1423 was administered by daily intraperitoneal injection at doses of 0.01 and 0.02 mg/kg body weight for 8 weeks since the day after STZ injection.At the end of the study,rats were weighed and housed in metabolic cages to collect 24-h urine.Blood samples of rats were collected followed by systemic perfusion.Kidneys were quickly removed,decapsulated and weighed.Western blot and QPCR were used to measure the expression of SRF,pSRF,ZO-1,collagen-1 a-SMA and FSP-1 in HK-2 cells and renal medulla of rats.Immunofluorescence staining was used to determine the expression and localization of SRF in HK-2 cells.Serum glucose,serum creatinine(Scr),blood urea nitrogen(BUN)and 24-h urinary protein(24-h UP)were measured by the Department of Pathology;The expression of SRF,cadherin,fibronectin in renal medulla of rats was detected through immunohistochemical staining.PAS staining and Masson staining were used to demonstrate renal tubulointerstitial fibrosis.Result1.The role of SRF in podocyte EMT of DN1.1 High glucose mediated podocyte EMT and SRF upregulation Substantial increase in protein and mRNA level of SRF and pSRF was observed at 72 h after high glucose treatment.High glucose suppressed epithelial synaptopodin and ZO-1 expression,and induced collagen-1,FN,FSP-1 and a-SMA expression as shown by western blot or QPCR analysis.SRF transferred from podocyte cytoplasm to nuclei obviously and the quantity of SRF was also increased after high glucose treatment,according to immunofluorescence staining.1.2 SRF overexpression mediated podocyte EMT and barrier dysfunctionOverexpression of exogenous SRF reduced epithelial synaptopodin expression,and increased collagen-1,FN,FSP-1,α-SMA and Snail expression in podocytes,as shown by western blot and QPCR.Transwell chamber migration assay showed that overexpression of SRF significantly upregulated the migration of podocytes across the pores of transwell filters.Albumin filtration assay showed that albumin easily diffused across the monolayer of podocytes transfected with SRF cDNA,in contrast to that transfected with empty pcDNA 3.1 vectors.1.3 Inhibition of SRF preserved podocyte phenotypes in vitroCCG-1423 selectively suppressed the expression of pSRF and SRF in podocytes after high glucose stimulation for 72 h.Besides,simultaneous treatment of podocytes with CCG-1423 also significantly abolished the reduction of synaptopodin expression and the induction of collagen-1,FN,FSP-1,a-SMA and Snail expression.Immunofluorescence staining suggested that CCG-1423 not only inhibited SRF expression,but also reduced the transfer of SRF from cytoplasm to nucleus in podocytes treated with high glucose.1.4 SRF was upregulated and activated in renal cortex of diabetic ratsWestern blot analysis,quantitative RT-PCR and immunohistochemical staining were carried out using renal cortex tissues from control group or DM group.SRF was significantly upregulated in DM group compared to control group in a time-dependent manner in vivo.1.5 Biochemical analysis of ratsThe DN model was proved by biochemical test results,which showed that serum glucose,scr,BUN,24-h UP and KW/BW were increased in the DM group,while serum albumin was decreased.However,compared to DM group,only serum albumin was improved after CCG-1423 treatment.1.6 Inhibition of SRF ameliorated glomerular fibrosis and proteinuria in vivoCCG-1423 significantly reduced 24-h UP by about 50%,compared with the vehicle control.Besides,inhibition of SRF with CCG-1423 also significantly abrogated the reduction of synaptopodin expression and the induction of SRF,collagen-1.α-SMA,FSP-1 expression in renal cortex tissues,according to western blot analysis.Masson and PAS staining demonstrated that renal fibrosis was present in DM group,and after 8 weeks of treatment with CCG-1423,renal fibrosis and glomerulosclerosis index were dramatically ameliorated compared with vehicle controls.In addition,CCG-1423 significantly preserved glomerular P-cadherin expression and suppressed a-SMA and FN expression in DN rats according to immunohistochemical staining.2.The role of SRF in EMT of TECs in DN2.1 High glucose induced EMT and SRF upregulation in HK-2 cellsSubstantial increase in protein and mRNA level of SRF and pSRF was observed at 72 h after high glucose treatment.High glucose suppressed epithelial E-cadherin and ZO-1 expression,and induced collagen-1,FN,FSP-1 and a-SMA expression as shown by western blot or QPCR analysis.SRF transferred from cytoplasm to nuclei obviously and the quantity of SRF was also increased in HK-2 cells after high glucose treatment,according to immunofluorescence staining.2.2 SRF overexpression mediated EMT and migration of HK-2 cellsOverexpression of exogenous SRF reduced epithelial E-cadherin expression,and increased collagen-1,FN,FSP-1,a-SMA and Snail expression in HK-2 cells,as shown by western blot and QPCR.Transwell chamber migration assay showed that overexpression of SRF significantly upregulated the migration of HK-2 cells across the pores of transwell filters.2.3 Inhibition of SRF preserved phenotypes of HK-2 cells in vitroCCG-1423 selectively suppressed the expression of pSRF and SRF in HK-2 after high glucose stimulation for 72 h.Besides,simultaneous treatment of HK-2 cells with CCG-1423 also significantly abolished the reduction of E-cadherin expression and the induction of collagen-1,FN,FSP-1,a-SMA and Snail expression.Immunofluorescence staining suggested that CCG-1423 not only inhibited SRF expression,but also reduced the transfer of SRF from cytoplasm to nucleus in HK-2 cells treated with high glucose.2.4 Biochemical analysis of ratsThe DN model was proved by biochemical test,which showed that serum glucose,Scr,BUN,24-h UP and KW/BW were increased in the DM group,while serum albumin was decreased.2.5 SRF was upregulated and activated in renal medulla of diabetic ratsWestern blot analysis,quantitative RT-PCR and immunohistochemical staining were carried out using renal medulla tissues from control group or DM group.SRF was significantly upregulated in DM group compared to control group in a time-dependent manner in vivo.2.6 Inhibition of SRF improved renal tubulointerstitial fibrosis and albuminuria in vivoInhibition of SRF with CCG-1423 significantly abrogated the reduction of E-cadherin expression and the induction of SRF,snail,collagen-1,a-SMA and FSP-1 expression in renal medulla tissues,according to western blot analysis.PAS staining demonstrated that renal tubulointerstitial fibrosis was present in DM group,and after 8 weeks treatment with CCG-1423,renal tubulointerstitial fibrosis was dramatically ameliorated compared with vehicle controls.In addition,CCG-1423 significantly preserved renal tubular E-cadherin expression and suppressed Snail,FN and FSP-1 expression in DN rats according to immunohistochemical staining.More importantly,CCG-1423 at 0.02 mg/kg BW dramatically decreased 24-h urinary albumin excretion(UAE)by about 36.4%,compared with the vehicle control.Conclusion1.SRF is activated and upregulated and translocates from cytoplasm to nuclei in the kidney of DN rats,which results in EMT of podocytes and TECs,renal fibrosis and proteinuria through Snail signaling pathway.2.Inhibition of SRF with CCG-1423 can ameliorate EMT and the loss of podocytes and TECs,resulting in improved renal glomerular and tubular function of DN.3.Inhibition of SRF by CCG-1423 can attenuate renal glomerular and tubulointerstitial fibrosis with improved proteinuria and serum albumin,which can delay the progress of DN.That may be an attractive therapeutic strategy for DN.