Microparticles derived from human erythropoietin mRNA-transfected mesenchymal stem cells inhibit epithelial-to-mesenchymal transition and ameliorate renal interstitial fibrosis

Abstract

BackgroundRenal tubulointerstitial fibrosis (TIF) plays an important role in the progression of chronic kidney disease (CKD) and its pathogenesis involves epithelial-to-mesenchymal transition (EMT) upon renal injury. Recombinant human erythropoietin (rhEPO) has been shown to display novel cytoprotective effects, in part by inhibiting transforming growth factor (TGF)-beta 1-induced EMT. Here, we evaluated the inhibitory effects of microparticles (MPs) derived from human EPO gene-transfected kidney mesenchymal stem cells (hEPO-KMSCs) against TGF-beta 1-induced EMT in Madin-Darby canine kidney (MDCK) cells and against TIF in mouse kidneys with unilateral ureteral obstruction (UUO).MethodsEMT was induced in MDCK cells by treatment with TGF-beta 1 (5ng/mL) for 48h and then inhibited by co-treatment with rhEPO (100IU/mL), mock gene-transfected KMSC-derived MPs (MOCK-MPs), or hEPO-KMSC-derived MPs (hEPO-MPs) for a further 48h. UUO was induced in FVB/N mice, which were then treated with rhEPO (1000IU/kg, intraperitoneally, every other day for 1week), MOCK-MPs, or hEPO-MPs (80 mu g, intravenously). Alpha-smooth muscle actin (alpha -SMA), fibronectin, and E-cadherin expression were evaluated in MDCK cells and kidney tissues, and the extent of TIF in UUO kidneys was assessed by immunohistochemical staining.ResultsTGF-beta 1 treatment significantly increased alpha -SMA and fibronectin expression in MDCK cells and decreased that of E-cadherin, while co-treatment with rhEPO, MOCK-MPs, or hEPO-MPs markedly attenuated these changes. In addition, rhEPO and hEPO-MP treatment effectively decreased phosphorylated Smad2 and Smad3, as well as phosphorylated p38 mitogen-activated protein kinase (MAPK) expression, suggesting that rhEPO and rhEPO-MPs can inhibit TGF-beta 1-induced EMT via both Smad and non-Smad pathways. rhEPO and hEPO-MP treatment also significantly attenuated the extent of renal TIF after 1week of UUO compared to MOCK-MPs, with hEPO-MPs significantly reducing myofibroblast and F4/80+ macrophage infiltration as well as EMT marker expression in UUO renal tissues in a similar manner to rhEPO.ConclusionsOur results demonstrate that hEPO-MPs modulate TGF-beta 1-induced EMT in MDCK cells via the Smad2, Smad3, and p38 MAPK pathways and significantly attenuated renal TIF in UUO kidneys.