Membrane rafts-redox signalling pathway contributes to renal fibrosis via modulation of the renal tubular epithelial-mesenchymal transition.

KEY POINTS: Membrane rafts (MRs)-redox signalling pathway is activated in response to transforming growth factor-β1 (TGF-β1) stimulation in renal tubular cells. This pathway contributes to TGF-1β-induced epithelial-mesenchymal transition (EMT) in renal tubular cells. The the MRs-redox signalling pathway is activated in renal tubular cells isolated from angiotensin II (AngII)-induced hypertensive rats. Inhibition of this pathway attenuated renal inflammation and fibrosis in AngII-induced hypertension. ABSTRACT: The membrane rafts (MRs)-redox pathway is characterized by NADPH oxidase subunit clustering and activation through lysosome fusion, V-type proton ATPase subunit E2 (encoded by the Atp6v1e2 gene) translocation and sphingomyelin phosphodiesterase 1 (SMPD1, encoded by the SMPD1 gene) activation. In the present study, we hypothesized that the MRs-redox-derived reactive oxygen species (ROS) are involved in renal inflammation and fibrosis by promoting renal tubular epithelial-mesenchymal transition (EMT). Results show that transforming growth factor-β1 (TGF-β1) acutely induced MR formation and ROS production in NRK-52E cells, a rat renal tubular cell line. In addition, transfection of Atp6v1e2 small hairpin RNAs (shRNA) and SMPD1 shRNA attenuated TGF-β1-induced changes in EMT markers, including E-cadherin, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) in NRK-52E cells. Moreover, Erk1/2 activation may be a downstream regulator of the MRs-redox-derived ROS, because both shRNAs significantly inhibited TGF-β1-induced Erk1/2 phosphorylation. Further in vivo study shows that the renal tubular the MRs-redox signalling pathway was activated in angiotensin II (AngII)-induced hypertension, as indicated by the increased NADPH oxidase subunit Nox4 fraction in the MR domain, SMPD1 activation and increased ROS content in isolated renal tubular cells. Finally, renal transfection of Atp6v1e2 shRNA and SMPD1 shRNA significantly prevented renal fibrosis and inflammation, as indicated by the decrease of α-SMA, fibronectin, collagen I, monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and tumour necrosis factor-α (TNF-α) in kidneys from AngII-infused rats. It was concluded that the the MRs-redox signalling pathway is involved in TGF-β1-induced renal tubular EMT and renal inflammation/fibrosis in AngII-induced hypertension.