Effects of B7-1 on the cytoskeleton rearrangement in podocytes induced by angiotensin Ⅱ / 中华肾脏病杂志

ABSTRACT

Objective To observe the effect of costimulatory molecule B7-1 on cytoskeleton rearrangement in mouse podocytes induced by angiotensin Ⅱ (Ang Ⅱ),and to study the underlying molecular mechanism of B7-1 in the pathological changes of podocytes.Methods All cultivation of conditionally immortalized mouse podocytes (MPC) in vitro were divided into the following groupsnormal control group,CTLA-4 group,Ang Ⅱ group (10-6 mmol/L 12 h,24 h;10-8 mmol/L 12 h,24 h) and CTLA-4 with Ang Ⅱ group.Transfect B7-1 RNA interference fragment (siRNA) to the mature podocytes,and then restimulated by Ang Ⅱ (10-6 mmol/L 12 h),the change of podocyte cytoskeleton after Ang Ⅱ stimulation were observed.The expression of B7-1 in each group was assayed by flow cytometry and Western blotting.The nephrin and p-nephrin protein levels in the four groups were also analyzed by Western blotting.At the same time,the podocyte cytoskeleton distribution as indicated by F-actin was observed by fluorescence microscopy.Results Flow cytometry and Western blotting showed that B7-1 was not expressed in the normal control group.Ang Ⅱ showed a concentration and time dependent induction of B7-1 expression in mouse podocytes (P ＜ 0.05).Western blotting indicated that Ang Ⅱ induced B7-1 protein expression (P ＜ 0.05).Expression of nephrin and p-nephrin was significantly down-regulated by Ang Ⅱ (P ＜ 0.05).Compared with the normal control group,the expression of podocyte protein nephrin and p-nephrin in Ang Ⅱ stimulation group was significantly reduced (P ＜ 0.05).Using FITC phalloidin fluorescence staining showed that CTLA-4+Ang Ⅱ stimulation group cytoskeleton rearrangement was improved significantly and F-actin recombinant score (mCFS) decreased compared with Ang Ⅱ group (P ＜ 0.05),suggesting that Ang Ⅱ led to the disorder of the podocytes cytoskeleton and the destruction of the cytoskeleton of podocytes by Ang Ⅱ could be improved after B7-1 blocking.Compared with the Ang Ⅱ stimulation,transfection of B7-1 siRNA + Ang Ⅱ stimulation group improved F-actin cytoskeletal rearrangement,and mCFS also decreased significantly (P ＜ 0.05),suggesting that transfection of B7-1 siRNA might improve the damage of Ang Ⅱ on podocytes cytoskeleton.Conclusions B7-1 participates in the process of cytoskeleton reconstruction and plays an important role in the pathological changes of podocytes.