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.

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. The serine/threonine kinase Akt mediates glucose-induced upregulation of collagen I in mesangial cells through transactivation of the EGF receptor (EGFR). In addition, in renal tubular cells, glucose-induced secretion of TGF-␤ requires phosphoinositide-3-OH kinase, suggesting a possible role for Akt in the modulation of TGF-␤ expression, but the mechanisms of Akt activation and its involvement in TGF-␤ regulation are unknown. Here, in primary mesangial cells, high glucose induced AktS473 phosphorylation, which correlates with its activation, in a protein kinase C ␤ (PKC-␤)-dependent manner. Glucose led to PKC-␤1 membrane translocation and association with Akt, and PKC-␤1 immunoprecipitated from glucose-treated cells phosphorylated recombinant Akt on S473. PKC is known to mediate glucose-induced TGF-␤1 upregulation through the transcription factor AP-1; here, inhibitors of phosphoinositide-3-OH kinase, PKC-␤ and Akt, and dominant-negative Akt all prevented glucose-induced activation of AP-1 and upregulation of TGF-␤1. Finally, pharmacologic and dominant negative inhibition of EGFR blocked glucose-induced activation of PKC-␤1, phosphorylation of AktS473, activation of AP-1, and upregulation of TGF-␤1. In vivo, the PKC-␤ inhibitor ruboxistaurin prevented Akt activation in the renal cortex of diabetic rats. In conclusion, PKC-␤1 is an Akt S473 kinase in glucose-treated mesangial cells, and TGF-␤1 transcriptional upregulation requires EGFR/PKC-␤1/Akt signaling. New therapeutic approaches for diabetic nephropathy may result from targeting components of this pathway, particularly the initial EGFR transactivation. 20: 554 -566, 200920: 554 -566, . doi: 10.1681 The kidney is an important site of diabetic microvascular complications, and hyperglycemia is central to glomerular matrix accumulation. Although strict glucose control and inhibition of the reninangiotensin system are effective in delaying the development of nephropathy, disease progression often occurs. The development of new treatment approaches is thus an important goal. J Am Soc NephrolWe have shown that collagen I induction by high glucose (HG) requires activation of the serine/threonine kinase Akt, and this depends on transactivation of the EGF receptor (EGFR). 1 Akt activation requires membrane translocation and phosphorylation on two sites, S473 and T308. 2 Phosphoinositide-3-OH kinase (PI3K) is an upstream mediator of Akt activation, generating phosphorylated lipid second messengers that recruit proteins with pleckstrin homology domains such as Akt to the membrane. 3 At the membrane, phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates Akt at T308. 3 Several S473 kinases, however, have

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Glomerular sclerosis of diverse etiologies is characterized by mesangial matrix accumulation, with transforming growth factor-beta (TGFbeta) an important pathogenic factor. The GTPase RhoA mediates TGFbeta-induced matrix accumulation in some settings. Here we study the role of the membrane microdomain caveolae in TGFbeta-induced RhoA activation and fibronectin upregulation in mesangial cells (MC). In primary rat MC, TGFbeta1 time dependently increased RhoA and downstream Rho kinase activation. Rho pathway inhibition blocked TGFbeta1-induced upregulation of fibronectin transcript and protein. TGFbeta1-induced RhoA activation was prevented by disrupting caveolae with cholesterol depletion and rescued by cholesterol repletion. Compared with wild types, RhoA/Rho kinase activation was absent in MC lacking caveolae. Reexpression of caveolin-1 (and caveolae) restored these responses. Phosphorylation of caveolin-1 on Y14, effected by Src kinases, has been implicated in signaling responses. Overexpression of nonphosphorylatable caveolin-1 Y14A prevented TGFbeta1-induced RhoA activation. TGFbeta1 also activated Src, and its inhibition blocked RhoA activation. Furthermore, TGFbeta1 led to association of RhoA and caveolin-1. This was prevented by Src or TGFbeta receptor I inhibition, and by caveolin-1 Y14A overexpression. Last, fibronectin upregulation by TGFbeta1 was blocked by Src inhibition, not seen in caveolin-1 knockout MC, and restored by caveolin-1 reexpression in the latter. TGFbeta1-induced collagen I accumulation also required caveolae. TGFbeta1-mediated Smad2/3 activation, however, did not require caveolae. We conclude that RhoA/Rho kinase mediates TGFbeta-induced fibronectin upregulation. This requires caveolae and caveolin-1 interaction with RhoA. Interference with caveolin/caveolae or RhoA signaling thus represents a potential target for the treatment of fibrotic renal disease.

Increased intraglomerular pressure is an important hemodynamic determinant of glomerulosclerosis and can be modeled in vitro by exposing mesangial cells to cyclic mechanical strain. A previous study showed that RhoA mediates strain-induced production of fibronectin; herein is investigated the role of caveolae in RhoA activation. Cyclodextrin and filipin, agents that disrupt caveolae, abrogated strain-induced RhoA activation in mesangial cells. Caveolin-1 (cav-1), the defining protein of caveolae, was Y14 phosphorylated by strain, and this was inhibited by PP1, showing Src dependence. Strain also induced c-SrcY416 phosphorylation and hence activation. Strain increased RhoA association with cav-1, which was blocked by PP1. Cyclodextrin and filipin inhibited the strain-induced RhoA/cav-1 association, indicating dependence on caveolar structural integrity. Restoration of caveolae by coincubation of cyclodextrin with cholesterol rescued both RhoA activation and RhoA/cav-1 association in response to strain. Sucrose gradient detected a significant portion of RhoA in caveolae, with Src located exclusively in these domains. Finally, in cells that were infected with retrovirus that encodes the nonphosphorylatable cav-1 Y14A, RhoA/cav-1 association, RhoA activation, and fibronectin secretion in response to strain were abrogated. It is concluded that strain-induced RhoA activation depends on the integrity of caveolae and on physical association of cav-1 and RhoA. The phosphorylation of cav-1 at Y14 by Src kinases is required for this to occur. These studies define a novel function for cav-1 and caveolae as positive effectors of RhoA activation. Targeting caveolae thus may provide a new therapeutic option for glomerular sclerosis that is associated with elevated intraglomerular pressure.