SRC INDUCES ENDOTHELIAL-TO-MESENCHYMAL TRANSITION AND RENAL ALLOGRAFT FIBROSIS VIA PROMOTING WNT/β-CATENIN PATHWAY

Endothelial-to-mesenchymal (EndMT) is recognized as an important source of myofibroblasts contributing to the pathogenesis of allograft interstitial fibrosis in our previous studies. Increased proto-oncogene tyrosine-protein kinase Src (Src) activity has been associated with several organs fibrosis including some renal diseases, but its role in allograft fibrosis of kidney transplant remains elusive. In the present study, we used immunohistochemistry (IHC) and immunofluorescence (IF) staining to detect the active form of Src (phopsho-Src Tyr416) of chronic renal allograft dysfunction (CAD) and healthy patients. Moreover, we cultured human umbilical vein endothelial cells (HUVEC) and human arterial endothelial cell (HAEC) which were treated with transforming growth factor-β1 (TGF-β1). Biomarkers of EndMT, as well as total Src and Src Tyr416, were examined in various doses or at various time points with or without PP1, a selective inhibitor of Src, using Western Blotting or qRT- PCR. Furthermore, knockdown or overexpressed Src plasmids in HUVEC and HAEC were constructed to confirm the exclusive effect of Src on the EndMT. Finally, rat renal transplant model was established and injected by PP1. Allograft tissues from human and rats were also collected and prepared for HE, Masson’s trichrome, IHC staining and western blotting assays. As a result, we demonstrate Src activity has been upregulated in glomerulus and vascular endothelium of rat renal transplant model and CAD patients. We found also TGF-β1 significant promoted development of EndMT and Src Tyr416 in a time-dependent and dose-dependent manner through raising β-catenin nuclear translocation. More importantly, PP1 alleviated the progression of EndMT and allograft interstitial fibrosis in vivo and in vitro by inhibiting WNT/β-catenin pathway. Thus, Src is an important mediator of allograft interstitial fibrosis, and suggest that Src will be a potential therapeutic target for prevention and treatment of renal allograft interstitial fibrosis.