Mechanism Of RTECs Transdifferentiation Induced By AAⅠ

Objective: To investigate the role of AAⅠon mouse renal tubular epithelial cells (RTECs) in vitro, and whether TGF-β1/Smads signal transduction is involved in AAⅠinduced RTECs-myofibroblast transdifferentiation, and to find out the target gene of AAⅠ. It would provide help to reveal the mechanism of renal interstitial fibrosis induced by AAⅠ.Methods: Mouse primary RTECs were cultured and identificated by morphology observation. After RTECs treated with or without AAⅠ, immunofluorescence staining, RT-PCR and ELISA were used to analyze cytokeratin 18 (CK18), vimentin,α-SMA and TGF-β1/Smads signal moleculars expression or distribution. AAⅠin culture medium was added to RTECs simultaneously with, or without the addition of SB-431542, immunofluorescence staining was used to observe changes of cells morphology. RTECs were transfected with Smad7-luc reporter plasmid vector, luciferase activities of RTECs treated with or without AAⅠwere measured by the Dual-Luciferase? reporter assay system. CDS sequence of mouse Smad7 was amplified by RT-PCR, conjugated to vector, transformed to E.coli. Plasmids were extracted and sequencing.Results: Mouse RTECs could be successfully cultured by mechanical separation and collageanseⅠdigestion; cells after passage 4 lost epithelial characteristics. High doses of AAⅠcaused death of RTECs; as time increased, 50ng/ml of AAⅠstimulated RTECs to transdifferentiate into myofibroblast. As showed by immunofluorescence staining, CK18 exprssion was down-regulated, vimentin andα-SMA expression was up-regulated in RTECs treated with AAⅠ, while SB-431542 could block these changes. AAⅠstimulated TGF-β1 mRNA expression and protein secretion; AAⅠinduced Smad2 mRNA expression, while significantly inhibited Smad7 expression in dose- and time- 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. Mouse primary culture is first successfully cultured in this study in China. 2. RTECs are confirmed to be target cells of AAⅠ. 3. TGF-β1/Smads signal transduction is involved in EMT induced by AAⅠ, SB-431542 can block the EMT process induced by AAⅠ. 4. Smad7 is one of the target genes in RTECs of AAⅠ.