Glomerular matrix accumulation is a hallmark of diabetic renal disease. Serine/threonine kinase PKC-β1 mediates glucose-induced Akt S473 phosphorylation, RhoA activation, and transforming growth factor (TGF)-β1 upregulation and finally leads to matrix upregulation in mesangial cells (MCs). It has been reported that glucose-induced PKC-β1 activation is dependent on caveolin-1 and the presence of intact caveolae in MCs; however, whether activated PKC-β1 regulates caveolin-1 expression and phosphorylation are unknown. Here, we showed that, although the caveolin-1 protein level had no significant change, the PKC-β-specific inhibitor LY-333531 blocked caveolin-1 Y14 phosphorylation in high glucose (HG)-treated MCs and in the renal cortex of diabetic rats. The Src-specific inhibitor SU-6656 prevented the HG-induced association between PKC-β1 and caveolin-1 and PKC-β1 membrane translocation, whereas PKC-β1 small interfering RNA failed to block Src activation, indicating that Src kinase is upstream of PKC-β1 activation. Although LY-333531 blocked PKC-β1 membrane translocation, it had no effect on the PKC-β1/caveolin-1 association, suggesting that PKC-β1 activation requires the interaction of caveolin-1 and PKC-β1. PKC-β1-mediated Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation in response to HG were prevented by SU-6656 and nonphosphorylatable mutant caveolin-1 Y14A. In conclusion, Src activation by HG mediates the PKC-β1/caveolin-1 association and PKC-β1 activation, which assists in caveolin-1 Y14 phosphorylation by Src kinase. The downstream effects, including Akt S473 phosphorylation, RhoA activation, and fibronectin upregulation, require caveolin-1 Y14 phosphorylation. Caveolin-1 is thus an important mediator of the profibrogenic process in diabetic renal disease.
The use of antidiabetic drugs represents a major burden on the health care system. The high proportions of patients requiring antihypertensive drugs and lipid lowering drugs further increase drug expenditure. Most of these treatments have been shown to improve clinical outcomes and quality of life, if used appropriately. The impacts of these long-term medications on health care financing require careful evaluation to assess their cost-effectiveness.
Aim: RhoA/ROCK signaling plays an important role in diabetic nephropathy, and ROCK inhibitor fasudil exerts nephroprotection in experimental diabetic nephropathy. In this study we investigated the molecular mechanisms underlying the protective actions of fasudil in a rat model of diabetic nephropathy. Methods: Streptozotocin (STZ)-induced diabetic rats, to which fasudil or a positive control drug enalapril were orally administered for 8 months. Metabolic parameters and blood pressure were assessed during the treatments. After the rats were euthanized, kidney samples were collected for histological and molecular biological studies. VEGF, VEGFR1, VEGFR2 and fibronectin expression, and Src and caveolin-1 phosphorylation in the kidneys were assessed using RT-PCR, Western blot and immunohistochemistry assays. The association between VEGFR2 and caveolin-1 was analyzed with immunoprecipitation. Results: Chronic administration of fasudil (30 and 100 mg·kg -1 ·d -1 ) or enalapril (10 mg/kg, bid) significantly attenuated the glomerular sclerosis and albuminuria in the diabetic rats. Furthermore, fasudil treatment prevented the upregulation of VEGF, VEGFR1, VEGFR2 and fibronectin, and the increased association between VEGFR2 and caveolin-1 in the renal cortices, and partially blocked Src activation and caveolin-1 phosphorylation on tyrosine 14 in the kidneys, whereas enalapril treatment had no effects on the VEGFR2/ Src/caveolin-1 signaling pathway. Conclusion: Fasudil exerts protective actions in STZ-induced diabetic nephropathy by blocking the VEGFR2/Src/caveolin-1 signaling pathway and fibronectin upregulation. Thus, VEGFR2 may be a potential therapeutic target for the treatment of diabetic nephropathy.
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