ABSTRACT
Objective To investigate the effect of complement C3a on mouse podocytes phenotype transformation.Methods Purified C3a recombinant protein was used to stimulate mature mouse podocytes.The expression of the mature podocyte markers synaptopodin,podocin,nephrin,CD2-associated protein (CD2AP) and the mesenchymal cell markers fibroblast specific protein 1 (FSP-1),α-smooth muscle actin (α-SMA) were detected by RT-PCR,Western blotting,immunochemistry and immunofluorescence,respectively.Some podocytes were transfected with integrin-linked kinase (ILK) siRNA before the administration of C3a,the expression of nephrin and α-SMA were accessed by Western blotting,and the expression of Snail and α-actinin 4 were accessed by Western blotting and immunochemical method.The migration ability of podocytes was observed by scratch test.Results Immunocytochemistry and immunofluorescence analysis showed that synaptopodin,podocin,nephrin,CD2AP were highly expressed by mature mouse podocytes.The expression of these podocyte markers could be markedly inhibited after 24 h of C3a (0.1 μmol/L) treatment,and accompanied by the induction of mesenchymal markers FSP-1 and α-SMA.Compared with control group,the mRNA levels of synaptopodin,podocin,CD2AP and nephrin were significantly repressed by the administration of C3a in a dose-dependent manner,whereas the transcription of FSP-1 and α-SMA were remarkably up-regulated by C3a treatment (P ＜ 0.05,respectively).Western blotting analysis also confirmed the decrease of synaptopodin,podocin,nephrin and CD2AP protein and the increase of FSP-1 and α-SMA protein were closely depend on the C3a concentration (P ＜ 0.05,respectively).To further assess the downstream of C3a,some podocytes were transfected with ILK siRNA before the administration of C3a.Compared with C3a group,the protein levels of nephrin and α-SMA were significantly changed by the administration of ILK siRNA (P ＜ 0.05,respectively).The expression of α-actinin 4 and Snail induced by C3a were inhibited by ILK knockdown (P ＜ 0.05,respectively),accompanied by a decline of cell migration potency.Conclusion Complement fragment C3a can induce transformation of mouse podocytes to mesenehymal cells,and ILK signaling pathway is involved in this cell type transformation.