Thrombospondin-1 induced by high glucose concentration mediates fibronectin synthesis in proximal renal tubular epithelial cells through both TGF-beta1 dependent and TGF-beta1 independent pathways

Abstract

Diabetic nephropathy (DN) is a major cause of endstage renal disease. Much of the research on DN to date has focused on glomerular abnormalities, and yet the progression of renal failure correlates with tubulointerstitial damage. We investigated the mechanisms of increased fibronectin and TGF-β1 synthesis in human proximal renal tubular epithelial cells (PTEC) upon exposure to high glucose concentrations. PTEC were pre-conditioned with physiological (5mM) or elevated (10mM, 20mM and 30mM) D-glucose concentrations for up to 3 weeks, with mannitol as the hexose control. Elevated glucose concentrations increased secreted and cell-associated thrombospondin-1 (TSP-1) in a time- and dose-dependent manner (p<0.05 compared to 5mM D-glucose), which preceded TGF-β1 bioactivation and the induction of fibronectin synthesis. To investigate the role of TSP-1 in matrix synthesis by PTEC, cells were stimulated for 24 h with varying concentrations of exogenous TSP-1 (0-10 ng/ml). Maximum induction of TGF-β1 mRNA, secretion and bioactivation was observed with 0.1ng/ml TSP-1. TSP-1 did not modulate fibronectin gene expression, but induced its secretion and extracellular deposition in a dose-dependent manner (p<0.05 compared to control). TGF-β neutralizing antibody (0-10 μg/ml) by itself did not alter constitutive fibronectin synthesis in PTEC, but co-incubation of cells with TGF-β1 neutralizing antibody and TSP-1 resulted in a 25.7±2.7% inhibition of fibronectin deposition. The inhibition remained incomplete despite adding higher concentrations of TGF-β1 neutralizing antibody. TGF-β1 in turn modulated TSP-1 synthesis. Our results showed that induction of TSP-1 by a high glucose environment mediates the increased fibronectin secretion by PTEC. Only part of the downstream mechanisms are TGF-β1 dependent, and autocrine interactions may exist between TSP-1 and TGF-β1.