RhoA/Rho-Kinase Contribute to the Pathogenesis of Diabetic Renal Disease

Abstract

OBJECTIVE—Accumulation of glomerular matrix proteins is central to the pathogenesis of diabetic nephropathy, with resident mesangial cells (MCs) known to upregulate matrix protein synthesis in response to high glucose. Because activation of the GTPase RhoA has been implicated in matrix upregulation, we studied its role in induction of the matrix protein fibronectin in diabetic MCs and in vivo in diabetic nephropathy. RESEARCH DESIGN AND METHODS—Glucose (30 mmol/l)-induced RhoA/Rho-kinase, AP-1 activation, and fibronectin upregulation were assessed by immunoblotting, luciferase, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, real-time PCR, Northern blots, and immunofluorescence. Streptozotocin-induced diabetic rats were treated with the ρ-kinase inhibitor fasudil, which was compared with enalapril, and functional and pathologic parameters were assessed. RESULTS—Glucose led to RhoA and downstream Rho-kinase activation. Mannitol was without effect. Activity of the transcription factor AP-1, increased in diabetic MCs and kidneys, is important in the profibrotic effects of glucose, and this was dependent on Rho-kinase signaling. Upregulation of fibronectin by glucose, shown to be mediated by activator protein-1 (AP-1), was prevented by Rho-kinase inhibition. RhoA siRNA and dominant-negative RhoA also markedly attenuated fibronectin upregulation by high glucose. Applicability of these findings were tested in vivo. Fasudil prevented glomerular fibronectin upregulation, glomerular sclerosis, and proteinuria in diabetic rats, with effectiveness similar to enalapril. CONCLUSIONS—High glucose activates RhoA/Rho-kinase in MCs, leading to downstream AP-1 activation and fibronectin induction. Inhibition of this pathway in vivo prevents the pathologic changes of diabetic nephropathy, supporting a potential role for inhibitors of RhoA/Rho in the treatment of diabetic renal disease.

Blocking VEGF/Caveolin-1 signaling contributes to renal protection of fasudil in streptozotocin-induced diabetic rats

“…We identified a previously unrecognized renoprotective mechanism for the The diabetic rats developed kidney hypertrophy, mild albuminuria, and progressive glomerulosclerosis. Chronic treatment with middle-dose (30 mg/kg) and high-dose (100 mg/kg) fasudil ameliorated the kidney injuries with no effect on blood pressure and glucose, which is consistent with previous studies [10,12] . Although there was a trend towards decreased blood pressure in the middle-dose fasudil group, this trend was not significant.…”

“…Recent studies have demonstrated that treatment with fasudil, a ROCK inhibitor, prevents the development of experimental diabetic nephropathy by inhibiting the signaling pathways involving angiotensin II, TGF-β, and ECM accumulation [9,10,27] . However, whether fasudil blocks VEGF signaling has not been addressed yet.…”

“…The present study demonstrated that fasudil inhibited VEGF upregulation in diabetic rat renal cortex. Similarly Peng et al [10] demonstrated that the Rho inhibitor Y-27632 prevented high glucose-induced upregulation of VEGF transcription in mesangial cells. These results suggest that VEGF upregulation is probably dependent on RhoA/ROCK signaling.…”

“…RhoA/ROCK signaling regulates numerous cell functions, including vascular contraction, actin cytoskeleton reorganization, cellular morphology, motility, adhesion and proliferation [7,8] . Recent studies have indicated that RhoA/ROCK signaling is an important contributor to diabetic nephropathy [9,10] . Fasudil, a relatively specific ROCK inhibitor, has nephroprotective actions during experimental diabetic nephropathy [11][12][13] .…”

“…Increased VEGF involves glomerular hypertrophy, proteinuria and ECM production [16][17][18] . Peng et al demonstrated that the upregulation of VEGF transcription in mesangial cells induced by glucose was prevented by the Rho inhibitor Y-27632 indicating that VEGF upregulation is possibly dependent on RhoA/ROCK signaling [10] . There are three known VEGF receptors: VEGFR1/fmsrelated tyrosine kinase 1 (Flt1), VEGFR2/kinase insert domain receptor (KDR), and Neuropilin-1 [19] .…”