Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-β1 dependent and TGF-β1 independent pathways

Abstract

Background. TGF-β1 bioactivation, consequent to the interaction of latent TGF-β1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-β1 bioactivation and matrix synthesis in human proximal renal tubular epithelial cells under the influence of high glucose concentrations. This study thus investigates the role of TSP-1 in mediating elevated glucose-induction of TGF-β1 bioactivation and fibronectin (FN) synthesis in human proximal tubular epithelial cells. Methods. Human proximal renal tubular epithelial cells (HK-2 cells) were incubated with 5, 10, 20 or 30 mM D-glucose for up to 3 weeks either in the presence or absence of TSP-1 blocking peptide. In separate studies HK-2 cells were incubated with exogenous TSP-1 (0-10 ng/ml) or TGF-β1 (0-10 ng/ml) for 24 h. Cell proliferation was assessed by [ 3H]-thymidine incorporation. TGF-β1 transcript, secretion and bioactivity were investigated by quantitative real-time PCR, ELISA and the MLEC bioassay respectively. TSP-1 and FN synthesis were assessed by quantitative real-time PCR, ELISAs and Western blot analysis. Results. Elevated glucose concentrations increased TSP-1 synthesis, which was associated with reduced cell proliferation, increased TGF-β1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-β1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-β1 in HK-2 cells to initiate FN accumulation. Of interest is our observation that TSP-1 also increased matrix synthesis through a mechanism independent of TGF-β1. TGF-β1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-β1. Conclusions. TSP-1 plays an important role in the induction of matrix synthesis by high glucose concentrations in human proximal renal tubular epithelial cells, through TGF-β1 dependent and TGF-β1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy. © 2006 Oxford University Press.