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-β requires phosphoinositide-3-OH kinase, suggesting a possible role for Akt in the modulation of TGF-β expression, but the mechanisms of Akt activation and its involvement in TGF-β regulation are unknown. Here, in primary mesangial cells, high glucose induced AktS473 phosphorylation, which correlates with its activation, in a protein kinase C β (PKC-β)-dependent manner. Glucose led to PKC-β1 membrane translocation and association with Akt, and PKC-β1 immunoprecipitated from glucose-treated cells phosphorylated recombinant Akt on S473. PKC is known to mediate glucose-induced TGF-β1 upregulation through the transcription factor AP-1; here, inhibitors of phosphoinositide-3-OH kinase, PKC-β and Akt, and dominant-negative Akt all prevented glucose-induced activation of AP-1 and upregulation of TGF-β1. Finally, pharmacologic and dominant negative inhibition of EGFR blocked glucose-induced activation of PKC-β1, phosphorylation of AktS473, activation of AP-1, and upregulation of TGF-β1. In vivo, the PKC-β inhibitor ruboxistaurin prevented Akt activation in the renal cortex of diabetic rats. In conclusion, PKC-β1 is an Akt S473 kinase in glucose-treated mesangial cells, and TGF-β1 transcriptional upregulation requires EGFR/PKC-β1/Akt signaling. New therapeutic approaches for diabetic nephropathy may result from targeting components of this pathway, particularly the initial EGFR transactivation.

We previously showed that ADAM17 mediates high glucose-induced matrix production by kidney mesangial cells. ADAM17 expression is increased in diabetic kidneys, suggesting that its upregulation may augment HG profibrotic responses. We thus studied the effects of HG on ADAM17 gene regulation. Primary rat mesangial cells were treated with high glucose (30mM) or mannitol as osmotic control. High glucose dose-dependently increased ADAM17 promoter activity, transcript and protein levels. This correlated with augmented ADAM17 activity after 24h vs 1h of high glucose. We tested involvement of transcription factors shown in other settings to regulate ADAM17 transcription. Promoter activation was not affected by NF-κB or Sp1 inhibitors, but was blocked by HIF-1α inhibition or downregulation. This also prevented ADAM17 transcript and protein increases. HIF-1α activation by high glucose was shown by its increased nuclear translocation and activation of the HIF-reponsive HRE-luciferase reporter construct. Assessment of ADAM17 promoter deletion constructs coupled with mutation analysis and ChIP studies identified HIF-1α binding to its consensus element at -607 as critical for the high glucose response. Finally, inhibitors of EGFR and downstream PI3K/Akt, or ADAM17 itself, prevented high glucose-induced HIF-1α activation and ADAM17 upregulation. Thus, high glucose induces ADAM17 transcriptional upregulation in mesangial cells, which is associated with augmentation of its activity. This is mediated by HIF-1α and requires EGFR/ADAM17 signaling, demonstrating the potentiation by ADAM17 of its own upregulation. ADAM17 inhibition thus provides a potential novel therapeutic strategy for the treatment of diabetic nephropathy.

Glomerular sclerosis of diverse etiologies is characterized by mesangial matrix accumulation, with transforming growth factor-β (TGFβ) an important pathogenic factor. The GTPase RhoA mediates TGFβ-induced matrix accumulation in some settings. Here we study the role of the membrane microdomain caveolae in TGFβ-induced RhoA activation and fibronectin upregulation in mesangial cells (MC). In primary rat MC, TGFβ1 time dependently increased RhoA and downstream Rho kinase activation. Rho pathway inhibition blocked TGFβ1-induced upregulation of fibronectin transcript and protein. TGFβ1-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 TGFβ1-induced RhoA activation. TGFβ1 also activated Src, and its inhibition blocked RhoA activation. Furthermore, TGFβ1 led to association of RhoA and caveolin-1. This was prevented by Src or TGFβ receptor I inhibition, and by caveolin-1 Y14A overexpression. Last, fibronectin upregulation by TGFβ1 was blocked by Src inhibition, not seen in caveolin-1 knockout MC, and restored by caveolin-1 reexpression in the latter. TGFβ1-induced collagen I accumulation also required caveolae. TGFβ1-mediated Smad2/3 activation, however, did not require caveolae. We conclude that RhoA/Rho kinase mediates TGFβ-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.

: Glomerular capillary hypertension is an important determinant of glomerulosclerosis in rats with subtotal renal ablation. Dietary supplementation with L-arginine increases renal nitric oxide (NO) production and limits glomerular injury in this model, and early benefits are seen without altered glomerular capillary pressure. In an in vitro model of hemodynamically mediated signaling, the authors have reported that subjecting MC to cyclic stretch/relaxation activates the mitogen-activated protein kinase p42/44 (Erk) cascade and that NO and cyclic GMP abrogate stretch-induced Erk activation by inducing actin cytoskeletal disassembly. The actin cytoskeleton is regulated by the Rho family of GTPases, including RhoA; therefore, the authors examined the role of RhoA in stretch-induced Erk activation and as an NO target. In primary rat MC subjected to cyclic mechanical strain, RhoA activity was maximally increased (2.4-fold) after 1 min of stretch, and Erk activation temporally followed. The Rho-kinase inhibitor Y-27632 attenuated Erk activation in a dose-dependent manner and prevented stretch-induced actin stress fiber formation. The NO donors S-nitroso-N-acetylpenicillamine and cGMP both inhibited stretch-induced RhoA and Erk activation and stress fiber formation. Infection of MC with the RhoA mutant RhoA-Ala188, which is resistant to NO-dependent phosphorylation, abrogated the effects of NO and cGMP on stretch-induced Erk activation and stress fiber formation. The authors conclude that the early activation of RhoA is essential for stretch-induced actin stress fiber formation and Erk activation in MC, events which are prevented by NO and cGMP through their action on RhoA. Inhibition of RhoA may thus be a new approach to the prevention of hemodynamically mediated glomerular injury. E-mail: krepinj@mcmaster.ca

The accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress, is caused by a wide range of physiologic and pathologic conditions. Cells respond to ER stress by activating a series of integrative stress pathways termed the unfolded protein response (UPR). This either may be adaptive and promote cell survival, or if the ER stress is chronic or excessive, may lead to cell death. The role of ER stress in the pathophysiology of both acute and chronic kidney diseases has been gaining increasing interest. This review highlights the current knowledge of ER stress in renal disease, with emphasis on more recent advances. Potential therapeutic options targeting ER stress are discussed. Antioxid. Redox Signal. 11, 2341–2352.