Study The Function And Mechanism Of Tisp40 In Renal Tubulointerstitial Fibrosis

Chronic kidney disease(CKD)caused by various causes,has become a public health problem that seriously endangers human health.Acute kidney injury(AKI)accelerates the development of CKD,while patients with CKD have a higher probability of suffering from AKI.Ischemic reperfusion(I/R)injury is one of the main causes of AKI.Epithelial-mesenchymal transition(EMT)is a determinant of renal tubulointerstitial fibrosis,which represents a loss of epithelial markers,including E-cadherin,and an increase in mesenchymal markers,such as alpha smooth muscle actin(a-SMA).Under severe and repetitive stimulation,the de-differentiated tubular epithelial cells release profibrotic mediators that cause a transformation from tubulointerstitial cells,including fibroblasts and pericytes,to scar-producing cells,leading to the accumulation and deposition of extracellular matrix(ECM),such as fibronectin and collagen I.TGF-β plays a key role and activates downstream Smad signaling in mediating the process of renal tubulointerstitial fibrosis.Tisp40,a transcription factor of the CREB/CREM family,is associated with cell apoptosis and proliferation.In our recent study of renal(I/R)injuries,we found that Tisp40 significantly increased after I/R.Therefore,we speculate that there is a close interaction between Tisp40 and renal interstitial fibrosis,and Tisp40 may be involved in the genesis and development of renal interstitial fibrosis.In this study,we investigated the expression,function and mechanism of Tisp40 in I/R-induced renal interstitial fibrosis and TGF-β1-induced renal cellular fibrosis in vivo and in vitro.Our results showed that Tisp40 expression was significantly upregulated in I/R-induced renal interstitial fibrosis and TGF-P-induced renal cellular fibrosis.We also found that Tisp40 could regulate the phosphorylation of Smad2 and Smad3,and induce epithelial mesenchymal transition(EMT)and extracellular matrix(ECM)deposition through TGF-β/Smad signaling pathway,including the upregulated expression of alpha smooth muscle actin(a-SMA),fibronectin(FN)and collagen I,and the downregulated expression of E-cadherin.Our study also illustrated that Tisp40 did not participate in the regulation of phosphorylation of Smad2 and Smad3 without TGF-β stimulation.In summary,our results showed that Tisp40 is upregulated in renal interstitial fibrosis,and Tisp40 downregulation reduce the phosphorylation of Smad2 and Smad3 via TGF-β/Smad signaling,to inhibit the EMT and ECM deposition and alleviate renal interstitial fibrosis.Part 1 Study the relation between Tisp40 and renalinterstitial fibrosisObjective To investigate the expression change of Tisp40 in I/R-induced renal interstitial fibrosis and TGF-β-induced renal cellular fibrosis in mouse renal tubular epithelial cell line TCMK-1.To verify Tisp40-overexpresssion TCMK-1/Tisp40 cells transfected by lentivirus vector and screen the highest silence-efficiency one in 3 constructed siRNA-Tisp40.Methods In vivo,the model of renal fibrosis was induced by I/R in C57BL/6 mouse.The location and expression of Tisp40 in renal tissue were detected by immunohistochemistry,Western blot and RT-PCR.In vitro,TCMK-1 cells were treated by TGF-β3(10ng/ml)for 24h.The expression changes of Tisp40 protein in different doses of TGF-β1(0.1,1,lOng/ml)and stimulation time(4,8,12,24,48h)was detected by Western blot.Tisp40 mRNA and protein expression was delected by RT-PCR and Western blot in TCMK-1/wildtype(TCMK-1/wt)and TCMK-1/Tisp40 cells.Tisp40-overexpresssion TCMK-1/Tisp40 cells were also evaluated by immunofluorescence.The silence-efficiency of 3 constructed siRNA-Tisp40(siRNAl-Tisp40,siRNA2-Tisp40 and siRNA3-Tisp40)was screened by Western blot.Results In vivo,the expression of Tisp40 in the sham operation group(Sham)was slight,and the expression of Tisp40 mRNA and protein in the model group(I/R)increased significantly(p<0.05).Tisp40 mainly distributed in the renal tubules.In vitro,Tisp40 increased dose-dependently and time-dependently with the change of TGF-β1 doses(0.1,1,lOng/ml)and stimulation time(4,8,12,24,48h).The expression of Tisp40 was basically stable after the stimulation of TGF-β1(10ng/ml)for 24h.TCMK-1/wt cells expressed slight Tisp40 before the stimulation of TGF-p.the mRNA and protein expression of Tisp40 in TGF-β-induced TCMK-1/wt and TCMK-1/Tisp40 cells increased significantly(p<0.05).The expression of Tisp40 in TCMK-1/Tisp40 cells was higher than that of TCMK-1/wt cells(P<0.05)with or without TGF-β stimulation,indicating that Tisp40-overexpression cell line TCMK-1/Tisp40 was successfully constructed.All of the 3 constructed siRNA-Tisp40 silence Tisp40,and siRNA3-Tisp40 had the highest silence-efficiency.Conclusions Tisp40 was upregulated in I/R-induced renal interstitial fibrosis and TGF-β-induced renal cellular fibrosis,suggesting that there is a closely relation between Tisp40 and renal interstitial fibrosis.Part 2 Study the role of Tisp40 in Renal interstitial fibrosisObjective To investigate the role of Tisp40 in I/R-induced Renal interstitial fibrosis and TGF-p-induced renal cellular fibrosisMethods In vivo,the model of renal fibrosis was induced by I/R in wild-type(Tisp40+/+)and Tisp40 knockout(Tisp40-/-)C57BL/6 mice.Renal interstitial fibrosis was evaluated by Masson staining and hydroxyproline analysis.The mRNA and protein expression of Tisp40 was detected by RT-PCR and Western blot.The mRNA and protein expression of a-SMA,E-cadherin、fibronectin and collagen I was detected by immunohistochemistry,RT-PCR and Western blot.In vitro,the mRNA and protein expression of a-SMA,E-cadherin、fibronectin and collagen I in TCMK-1/wt,TCMK-1/Tisp40 and TCMK-1/vector was detected by RT-PCR and Western blot with or without TGF-β1 stimulation(10ng/ml)for 24h.The protein expression and location of fibronectin was also detected by immunofluorescence.In addition,we transfected siRNA3-Tisp40 with the highest silence efficiency into TCMK-1/wt cells via Lipofectamine RNA iMAX for 48h.The mRNA and protein expression of a-SMA,E-cadherin.fibronectin and collagen I was detected by RT-PCR and Western blot with or without TGF-β1 stimulation(10ng/ml)for 24h.The protein expression and location of fibronectin was also detected by immunofluorescence.Results In vivo,the mRNA and protein expression of Tisp40 was slight in wild-type Tisp40(+/+)and Tisp40 knockout(Tisp40-/-)C57BL/6 mice without I/R induction.After ischemia-reperfusion injury,the mRNA and protein expression of Tisp40 in wild-type mice significantly increased(p<0.05),while the mRNA and protein expression of Tisp40 stayed low level in Tisp40 knockout mice.Compared with I/R-induced wild-type mice,the mRNA and protein expression of a-SMA,fibronectin and collagen I in I/R-induced Tisp40 knockout mice obviously decreased(p<0.05),and the mRNA and protein expression of E-cadherin significantly increased(p<0.05).In vitro,compared with TGF-β-induced TCMK-1/wt and TCMK-1/vector cells,the mRNA and protein expression of a-SMA,fibronectin and collagen I in TGF-β-induced TCMK-1/Tisp40 cells obviously increased(p<0.05),and the mRNA and protein expression of E-cadherin significantly decreased(p<0.05)in TGF-β-induced TCMK-1/Tisp40 cells.Tisp40 overexpression had no effect on the mRNA and protein expression of a-SMA,E-cadherin,fibronectin and collagen I without TGF-βstimulation.Similarly,although siRNA-Tisp40 effectively silenced Tisp40 expression,it did not affect mRNA and protein expression of a-SMA,E-cadherin,fibronectin and collagen I without TGF-β1 stimulation.Compared with the control group(Scramble),the mRNA and protein expression of a-SMA,fibronectin and collagen I in siRNA group(siRNA-Tisp40)obviously decreased(p<0.05),and the mRNA and protein expression of E-cadherin significantly increased(p<0.05)in siRNA-Tisp40 treated TCMK-1 cells with TGF-β,stimulation.Conclusion Tisp40 downregulation alleviated renal interstitial fibrosis by inhibiting epithelial mesenchymal transition(EMT)and extracellular matrix(ECM)deposition.Tisp40 upregulation played an opposite role.Our results indicate that Tisp40 plays an important role in renal interstitial fibrosis.Tisp40 knockout protects kidney by inhibiting EMT and ECM deposition.Part 3 Study the molecular mechanism of Tisp40 in Renal interstitial fibrosisObjective To investigate the regulation role of Tisp40 in TGF-p/Smad and Snail signal pathways,and to demonstrate the specific molecular mechanism of Tisp40 in I/R-induced renal interstitial fibrosis and TGF-β-induced renal cellular fibrosis.Methods In vivo,the protein expression of Smad2,Smad3 and pSmad2/3 in I/R-induced wild-type mice was detected by Western blot,to verify the I/R-induced activation of TGF-β/Smad signaling.The model of renal fibrosis was induced by I/R in Tisp40+/+ and Tisp40-/-C57BL/6 mice.The mRNA and protein expression of TGF-β was detected by RT-PCR and Western blot.The protein expression of Smad2 and Smad3 was detected by Western blot.The protein expression of pSmad2/3 and Snail was detected by immunohistochemistry and Western blot.In vitro,the protein expression of Smad2,Smad3,pSmad2/3 and Snail in TCMK-1/wt,TCMK-1/Tisp40 and TCMK-1/vector was detected by Western blot with or without TGF-β1 stimulation(10ng/ml)for 1h.In addition,we transfected siRNA3-Tisp40 with the highest silence efficiency into TCMK-1/wt cells via Lipofectamine RNA iMAX for 48h.The protein expression of Smad2,Smad3,pSmad2/3 and Snail was detected by Western blot with or without TGF-β1 stimulation(10ng/ml)for 1h.Results In vivo,ischemia-reperfusion injury could activate TGF-β/Smad signaling.The protein expression of TGF-β,Smad2,Smad3,pSmad2/3 and Snail was slight in kidney of Tisp40+/+ and Tisp40-/-C57BL/6 mice without I/R induction.After ischemia-reperfusion injury,the protein expression of TGF-β,Smad2,Smad3,pSmad2/3 and Snail in Tisp40+/+and Tisp40-/-mice significantly increased(p<0.05).Compared with I/R-induced wild-type mice,the protein expression of TGF-P,Smad2 and Smad3 in I/R-induced Tisp40 knockout mice had no statistic difference(p>0.05),while the protein expression of pSmad2/3 and Snail significantly decreased(p<0.05).In vitro,the protein expression of Smad2,Smad3,pSmad2/3 and Snail in TCMK-1/wt,TCMK-1/Tisp40 and TCMK-1/vector cells stayed low level without TGF-P stimulation,while the protein expression of Smad2,Smad3,pSmad2/3 and Snail was upregulated with TGF-β stimulation.Compared with TGF-β-induced TCMK-1/wt and TCMK-1/vector cells,the protein expression of Smad2 and Smad3 in TGF-β-induced TCMK-1/Tisp40 cells had no statistic difference(p>0.05),while the protein expression of pSmad2/3 and Snail significantly increased(p<0.05)in TGF-β-induced TCMK-1/Tisp40 cells.Tisp40 overexpression had no effect on the protein expression of Smad2,Smad3,pSmad2/3 and Snail without TGF-β1 stimulation.It was likely that TGF-βwas no sufficient to activate fibrosis.Similarly,although siRNA-Tisp40 effectively silenced Tisp40 expression,it did not affect protein expression of Smad2,Smad3,pSmad2/3 and Snail without TGF-β1 stimulation.Compared with the control group(Scramble),the protein expression of Smad2 and Smad3 in siRNA group(siRNA-Tisp40)had no statistic difference(p>0.05),while the protein expression of pSmad2/3 and Snail significantly decreased(p<0.05)in siRNA-Tisp40 treated TCMK-1 cells with TGF-p stimulation.Conclusion Tisp40 regulated renal interstitial fibrosis via modulating the phosphorylation of Smad2 and Smad3 in TGF-β/Smad signaling.Tisp40 downregulation protected fibrotic kidney by inhibiting the phosphorylation of Smad2 and Smad3 via TGF-β/Smad signaling.Our results provide a new therapeutic target in the clinical treatment of renal interstitial fibrosis.