Autophagy caused by oxidative stress promotes TGF-β1-induced epithelial-to-mesenchymal transition in human peritoneal mesothelial cells

Abstract

Abstract Epithelial-to-mesenchymal transition (EMT) is an important cause of peritoneal fibrosis. However, pathophysiological mechanism of EMT, specifically its relationship with autophagy, is still unknown. This study aimed to evaluate the role of autophagy in TGF-β1-induced EMT in human peritoneal mesothelial cells (HPMCs). Primary cultured HPMCs were treated with TGF-β1 (2 and 5 ng/mL) and changes in the autophagy-related markers and association between autophagy and EMT were evaluated. Furthermore, we identified changes in EMT- and autophagy-related signaling pathways after autophagy and NOX4 inhibition. TGF-β1 increased the generation of NOX4 and reactive oxygen species (ROS) in HPMCs, which resulted in mitochondrial damage. Moreover, treatment with GKT137831 (20 µM), a NOX1/4 inhibitor, reduced ROS in the mitochondria of HPMC cells and reduced TGF-β1-induced mitochondrial damage. Additionally, the indirect inhibition of autophagy by GKT137831 (20 µM) downregulated TGF-β1-induced EMT, whereas direct inhibition of autophagy using 3-methyladenine (2 mM) or ATG5 gene silencing decreased the TGF-β1-induced EMT in HPMCs. The Smad2/3 pathways, autophagy-related PI3K Class III and AKT pathways, and MAPK signaling pathways, such as ERK and P38, were involved in the TGF-β1-induced EMT. Moreover, autophagy and NOX4 inhibition suppressed the activation of these signaling pathways. Inhibition of autophagy, either by direct inhibition of autophagy or indirectly by reducing mitochondrial damage through upstream NOX4 inhibition, resulted in reduced EMT in the HPMCs. Autophagy could serve as a therapeutic target to prevent peritoneal fibrosis in patients undergoing peritoneal dialysis.