Angiotensin II stimulates α3(IV) collagen production in mouse podocytes via TGF-β and VEGF signalling: implications for diabetic glomerulopathy

Abstract

Background. The podocyte is bathed in an angiotensin II (AngII)-rich ultrafiltrate, but the impact of AngII on podocyte pathobiology is not well known. Because podocytes play a direct role in the glomerular basement membrane (GBM) thickening of diabetes, the α3(IV) collagen chain was examined. Podocyte expression of α3(IV) collagen may involve the transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF) systems. Methods. Cultured mouse podocytes were treated with various doses of AngII for selected periods of time, with or without inhibitors of TGF-β and VEGF signalling, SB-431542 and SU5416, respectively. TGF-β1 and VEGF were assayed by enzyme-linked immunosorbent assay (ELISA); α3(IV) collagen, TGF-β type II receptor and phospho-Smad2 were assayed by immunoblotting. Results. AngII ≥10⁻¹⁰ M was found to stimulate the production of α3(IV) collagen significantly in as short a time as 3 h. The expression of α3(IV) collagen was influenced by the TGF-β system, but AngII did not increase the podocyte's production of TGF-β1 ligand; rather, it increased the expression of the TGF-β type II receptor and activated the TGF-β signalling system through Smad2. Despite the TGF-β receptor upregulation, synergy between AngII and TGF-β1 to boost α3(IV) collagen production was not observed. However, blockade of TGF-β signalling with SB-431542 prevented AngII from stimulating α3(IV) collagen production. Podocyte expression of α3(IV) collagen was also increased by the autocrine activity of VEGF. Podocytes were stimulated to secrete VEGF by 10⁻¹⁰ M or higher AngII after 48 h. Blockade of the endogenous VEGF activity by SU5416 prevented AngII-stimulated α3(IV) collagen production. Conclusions. AngII stimulates the podocyte to produce α3(IV) collagen protein via mechanisms involving TGF-β and VEGF signalling. Alterations in α3(IV) collagen production may contribute to GBM thickening and perhaps proteinuria in diabetes.