The Effects And Mechanisms Of Tisp40 On Renal Ischemia-reperfusion Injury

Part 1 The effects of Tisp40 on renal ischemia-reperfusion induced renal tubular epithelial cell apoptosisObjective: To observe the expression of Tisp40 protein after renal ischemiareperfusion(IR)injury,and explore the effects and mechanisms of Tisp40 on renal dysfunctions,morphology abnormalities and renal tubular epithelial cell apoptosis induced by renal IR.Methods: Firstly,wild-type mice were subjected with or without renal IR.The protein level of Tisp40 was detected by Western blot assay and IHC staining.Secondly,Tisp40 knockout mice and wild-type mice were subjected with or without renal IR.Mice renal function was evaluated by the serum levels of creatinine and urea nitrogen.The degree of renal tubular damage was detected by PAS staining.The level of cell apoptosis was detected by TUNEL staining and the IHC staining of Cleaved Caspase-3 protein.The protein levels of CHOP,Bax,Bcl-2,and Cleaved Caspase-3 were detected by Western blot assay.Results: Western blot assay and IHC staining showed that the expression of Tisp40 protein was increased in wild-type mice after renal IR,and Tisp40 protein was mainly expressed in renal tubular epithelial cells.Renal function tests showed that the serum levels of creatinine and urea nitrogen were decreased in Tisp40 knockout mice than in wild-type mice after renal IR.PAS staining showed that the renal injury score was decreased in Tisp40 knockout mice than in wild-type mice after renal IR.TUNEL staining and the IHC staining of Cleaved Caspase-3 protein showed that the number of apoptotic renal tubular epithelial cells was decreased in Tisp40 knockout mice compared with wild-type mice after renal IR.Western blot assay showed that the protein levels of CHOP,Bax,and Cleaved Caspase-3 were decreased and the expression of Bcl-2 protein was increased in Tisp40 knockout mice than in wild-type mice after renal IR.Conclusion: The expression of Tisp40 protein was increased after renal IR.Tisp40 deficiency can attenuate renal dysfunctions,morphology abnormalities,renal tubular epithelial cell apoptosis,and inhibit CHOP-mediated apoptosis in renal IR injury.Part 2 The effects of Tisp40 on hypoxia-reoxygenation induced apoptosis of renal tubular epithelial cells(HK-2)Objective: To observe the expression of Tisp40 protein after hypoxia-reoxygenation(HR)in renal tubular epithelial cells(HK-2),and explore the effects and mechanisms of Tisp40 on HR induced renal tubular epithelial cell apoptosis.Methods: Firstly,renal tubular epithelial cells(HK-2)were subjected with or without HR.The expression of Tisp40 protein was detected by Western blot assay and immunocytochemical staining.Secondly,we constructed renal tubular epithelial cell lines with or without Tisp40 gene overexpression,and subjected with or without HR.Flow cytometry and Hoechst staining were used to detect the apoptosis of renal tubular epithelial cells.The protein levels of CHOP,Bax,Bcl-2,and Cleaved Caspase-3 were detected by Western blot assay.Results: Western blot analysis showed that the expression of Tisp40 protein was increased in renal tubular epithelial cells after HR.Also,immunocytochemical staining showed that the expression of Tisp40 protein was significantly increased in the cytoplasm and nucleus of renal tubular epithelial cells after HR.Flow cytometry and Hoechst staining revealed that the apoptosis level of tubular epithelial cells was increased in of Tisp40 gene overexpression group than in vector group after HR.Western blot assay showed that the protein levels of CHOP,Bax,and Cleaved Caspase-3 were increased,and the expression of Bcl-2 protein was decreased in Tisp40 gene overexpression group than in vector group after HR.Conclusion: The expression of Tisp40 protein was increased in renal tubular epithelial cells(HK-2)after HR.Tisp40 gene overexpression aggravates HR induced apoptosis of renal tubular epithelial cells,and promotes CHOP-mediated cell apoptosis pathway.Part 3 The effects of Tisp40 on renal tubular epithelial cellproliferation in renal ischemia-reperfusion injuryObjective: To explore the effect of Tisp40 on renal tubular epithelial cell proliferation in renal IR injury.Methods: Tisp40 knockout mice and wild-type mice were subjected with or without renal IR.The expression of PCNA protein was detected by immunofluorescence staining.The expression of p-Erk1/2 protein was detected by IHC staining.Results: Immunofluorescence staining showed that the expression PCNA protein was increased in the nucleus of renal tubular epithelial cells in wild-type mice after renal IR.Moreover,the expression of PCNA protein was further increased in Tisp40knockout mice than in wild-type mice after renal IR.Also,IHC staining showed that the expression p-Erk1/2 protein was increased in renal tubular epithelial cells in wildtype mice after renal IR.Moreover,the protein level of p-Erk1/2 was further increased in Tisp40 knockout mice than in wild-type mice after renal IR.Conclusion: Tisp40 deficiency promotes renal tubular epithelial cell proliferation in renal IR injury.Part 4 The effects of Tisp40 on renal tubular-interstitialinflammatory response induced by renal ischemia-reperfusionObjective: To explore the effects of Tisp40 on renal tubular-interstitial inflammatory cell infiltration,inflammatory factor production,and the activation of NF-?B signaling after renal IR.Methods: Tisp40 knockout mice and wild-type mice were subjected with or without renal IR.The infiltration of inflammatory cells was observed by H&E staining.The infiltration levels of neutrophils(MPO-positive),macrophages(CD68-positive),and inflammatory factors(TNF-? and MCP-1)were detected by IHC staining.The protein levels of I?B?/NF-?B signaling were measured by Western blot assay.Results: H&E staining showed that many inflammatory cells infiltrated in renal tissue in wild-type mice after renal IR.However,the infiltration of inflammatory cells was reduced in Tisp40 knockout mice than in wild-type mice after renal IR.IHC staining showed that the protein levels of MPO,CD68,TNF-? and MCP-1 were decreased in Tisp40 knockout mice compared with wild-type mice after renal IR.Moreover,Western blot results showed that the protein levels of p-I?B? and p-P65 were increased in the renal of wild-type mice after IR.However,the protein levels of pI?B? and p-P65 were decreased in Tisp40 knockout mice than in wild-type mice after renal IR.Conclusion: Tisp40 deficiency limits neutrophil and macrophage infiltration,inflammatory factor production,and the activation of NF-?B signaling induced by renal IR.