Mechanism Of RTECs Apoptosis And Renal Interstitial Fibrosis In Mouse Induced By AA Ⅰ

Objective:1.To investigate the effect of AAⅠin inducing apoptosis of the RTECs in mouse.2.Mouse model of AAN was used to assess the patterns of renal injury and TGF-β1/Smads signaling pathway during the evolution of tubulointerstitial damage and to relate them to the development of fibrosis,and to find out the target gene of AAⅠ.Methods:1.Mouse primary RTECs were cultured and identificated by morphology observation.After RTECs treated with or without AAⅠ,DNA ladder,Flow cytometry analysis(FCAS) the apoptosis of the RTECs.2.Mice were given AAⅠat different doses consecutively by gavage,pathological section, immunohistochemistry and RT-PCR were used to analyze cytokeratin 18(CK18),vimentin,a-SMA and TGF-β1/Smads signal moleculars expression or distribution.CDS sequence of mouse Smad7 was amplified by RT-PCR,conjugated to vector,transformed to E.coli. Plasmids were extracted and sequencing.Results:1.Mouse RTECs could be successfully cultured,with the increased of AAⅠ,cells lost epithelial characteristics.After 24h,20-80μg/mL of AAⅠstimulated RTECs,cells apparence late apoptosis.2.As showed by immunohistochemistry and RT-PCR,CK18 exprssion was down-regulated, vimentin andα-SMA expression was up-regulated in RTECs treated with AAⅠ,AAⅠstimulated TGF-β1 mRNA expression and protein secretion;AAⅠinduced Smad2 mRNA expression,while significantly inhibited Smad7 expression in dose-dependence manner.Several nucleotides and amino acids were changed compared to control of CDS sequence in mouse Smad7 gene,but the sites in one group cells were different from other groups after treated with AAⅠ.Conclusions:1.The apoptosis of mouse origin RTECs could be induced by AAⅠ,the degree of apoptosis is along with the increasing of AAⅠaction time and density.2.Mouse model of AAN was established successfully in this study,and RTECs are confirmed to be target cells of AAⅠ.3. TGF-β1/Smads signal transduction is involved in EMT induced by AAⅠ.