Elevated glucose induction of thrombospondin-1 up-regulates fibronectin synthesis in proximal renal tubular epithelial cells through TGF-beta1 dependent and TGF-beta1 independent pathways.
BACKGROUND
TGF-beta1 bioactivation, consequent to the interaction of latent TGF-beta1 with thrombospondin-1 (TSP-1), correlates with matrix accumulation in mesangial cells. Tubulointerstitial damage predicts poor renal survival. There is little data on TGF-beta1 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-beta1 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-beta1 (0-10 ng/ml) for 24 h. Cell proliferation was assessed by [(3)H]-thymidine incorporation. TGF-beta1 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-beta1 bioactivity, and stimulation of FN synthesis. The inclusion of TSP-1 blocking peptide to cells stimulated with elevated glucose concentration abrogated activation of TGF-beta1 and induction of FN secretion. Exogenous TSP-1 increased bioactive TGF-beta1 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-beta1. TGF-beta1 in turn modulated TSP-1 synthesis, indicative of an autocrine loop between TSP-1 and TGF-beta1.


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-beta1 dependent and TGF-beta1 independent pathways. Pharmacological intervention targeting increased TSP-1 expression may interrupt the pathogenesis of diabetic nephropathy.
Department of Medicine, Queen Mary Hospital, University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.
Susan
S
Yung
Candice Y Y
CY
Lee
Qing
Q
Zhang
Shuk Kei
SK
Lau
Ryan C W
RC
Tsang
Tak Mao
TM
Chan
D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000378:metabolism*; D005353:Fibronectins / Q000096:biosynthesis*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000378:metabolism; D015398:Signal Transduction; D019700:Thrombospondin 1 / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
D005353:Fibronectins; D019700:Thrombospondin 1; D053773:Transforming Growth Factor beta1; D005947:Glucose