NDT Advance Access originally published online on February 22, 2006
Nephrology Dialysis Transplantation 2006 21(6):1504-1513; doi:10.1093/ndt/gfl017
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© The Author [2006]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org
Original Articles: Experimental Nephrology
Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-ß1 dependent and TGF-ß1 independent pathways
Department of Medicine, University of Hong Kong, Hong Kong, China
Correspondence and offprint requests to: Professor Tak Mao Chan and Dr Susan Yung, Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam Road, Hong Kong SAR, China. Email: dtmchan{at}hkucc.hku.hk
Background. TGF-ß1 bioactivation, consequent to the interaction of latent TGF-ß1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-ß1 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-ß1 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-ß1 (0–10 ng/ml) for 24 h. Cell proliferation was assessed by [3H]-thymidine incorporation. TGF-ß1 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-ß1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-ß1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-ß1 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-ß1. TGF-ß1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-ß1.
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-ß1 dependent and TGF-ß1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy.
Keywords: fibronectin; glucose; proximal renal tubular epithelial cells; TGF-ß1; thrombospondin-1
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