Symmetric dimethylarginine alters endothelial nitric oxide activity in glomerular endothelial cells.

Circulating symmetric dimethylarginine (SDMA) is increased in patients with chronic kidney disease. SDMA is considered an inert metabolite, but because it can transported into cells, we studied the effect of SDMA on glomerular endothelial cells. SDMA suppressed VEGF-induced endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide production, but not VEGFR2 activation and signaling leading to eNOS activation. SDMA caused eNOS uncoupling and increased superoxide anion production in response to VEGF. All these effects were blocked by preventing cellular uptake of SDMA with a molar excess of arginine. These data show that SDMA interferes with nitric oxide production by uncoupling eNOS and leads to oxidative stress in glomerular endothelial cells. In conclusion, our data show that SDMA is not an inert metabolite and that it could contribute to oxidative stress in the renal endothelium.

Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes.

The present study was performed to investigate the underlying mechanism, particularly the roles of reactive oxygen species (ROS) and protein kinase C (PKC), in the diabetes-induced canonical transient receptor potential 6 (TRPC6) downregulation. We found that high glucose (HG) significantly reduced TRPC6 protein expression in cultured mesangial cells (MCs). TRPC6 protein was also significantly reduced in the glomeruli but not in the heart or aorta isolated from streptozotocin-induced diabetic rats. In the cultured MCs, H(2)O(2) suppressed TRPC6 protein expression in a dose- and time-dependent manner, which emulated the HG effect. Catalase as well as superoxide dismutase were able to prevent the inhibitory effect of HG on TRPC6. The antioxidant effect observed in cultured cells was also observed in diabetic rats treated with tempol for 2 wk, which exhibited a preservation of TRPC6 in the glomeruli. Specific knockdown of Nox4, a component of NADPH oxidase, increased TRPC6 protein expression. Furthermore, the PKC activator phorbol 12-myristate 13-acetate (PMA), but not its analog 4α-phorbol 12, 13-didecanoate (4α-PDD), suppressed TRPC6 expression, and this PMA effect was not affected by catalase. Moreover, Gö6976, but not LY333531, attenuated the negative effect of HG on TRPC6 expression. Gö6976 also inhibited H(2)O(2) effect on TRPC6. Furthermore, either knockdown of TRPC6 or HG treatment significantly decreased ANG II-stimulated MC contraction, and the HG-impaired MC contraction was rescued by overexpression of TRPC6. These results suggest that hyperglycemia in diabetes downregulated TRPC6 protein expression in MCs through a NADPH oxidase Nox4-ROS-PKC pathway, proving a mechanism for impaired MC contraction in diabetes.

NAD(P)H oxidase mediates TGF-beta1-induced activation of kidney myofibroblasts.

TGF-beta1 expression closely associates with activation and conversion of fibroblasts to a myofibroblast phenotype and synthesis of an alternatively spliced cellular fibronectin variant, Fn-ED-A. Reactive oxygen species (ROS), such as superoxide, which is a product of NAD(P)H oxidase, also promote the transition of fibroblasts to myofibroblasts, but whether these two pathways are interrelated is unknown. Here, we examined a role for NAD(P)H oxidase-derived ROS in TGF-beta1-induced activation of rat kidney fibroblasts and expression of alpha-smooth muscle actin (alpha-SMA) and Fn-ED-A. In vitro, TGF-beta1 stimulated formation of abundant stress fibers and increased expression of both alpha-SMA and Fn-ED-A. In addition, TGF-beta1 increased both the activity of NADPH oxidase and expression of Nox2 and Nox4, homologs of the NAD(P)H oxidase family, indicating that this growth factor induces production of ROS. Small interfering RNA targeted against Nox4 markedly inhibited TGF-beta1-induced stimulation of NADPH oxidase activity and reduced alpha-SMA and Fn-ED-A expression. Inhibition of TGF-beta1 receptor 1 blocked Smad3 phosphorylation; reduced TGF-beta1-enhanced NADPH oxidase activity; and decreased expression of Nox4, alpha-SMA, and Fn-ED-A. Diphenyleneiodonium, an inhibitor of flavin-containing enzymes such as the Nox oxidases, had no effect on TGF-beta1-induced Smad3 but reduced both alpha-SMA and Fn-ED-A protein expression. The Smad3 inhibitor SIS3 reduced NADPH oxidase activity, Nox4 expression, and blocked alpha-SMA and Fn-ED-A, indicating that stimulation of myofibroblast activation by ROS is downstream of Smad3. In addition, TGF-beta1 stimulated phosphorylation of extracellular signal-regulated kinase (ERK1/2), and this was inhibited by blocking TGF-beta1 receptor 1, Smad3, or the Nox oxidases; ERK1/2 activation increased alpha-SMA and Fn-ED-A. Taken together, these results suggest that TGF-beta1-induced conversion of fibroblasts to a myofibroblast phenotype involves a signaling cascade through Smad3, NAD(P)H oxidase, and ERK1/2.

Nox4 NAD(P)H oxidase mediates Src-dependent tyrosine phosphorylation of PDK-1 in response to angiotensin II: role in mesangial cell hypertrophy and fibronectin expression.

Activation of glomerular mesangial cells (MCs) by angiotensin II (Ang II) leads to hypertrophy and extracellular matrix accumulation. Here, we demonstrate that, in MCs, Ang II induces an increase in PDK-1 (3-phosphoinositide-dependent protein kinase-1) kinase activity that required its phosphorylation on tyrosine 9 and 373/376. Introduction into the cells of PDK-1, mutated on these tyrosine residues or kinase-inactive, attenuates Ang II-induced hypertrophy and fibronectin accumulation. Ang II-mediated PDK-1 activation and tyrosine phosphorylation (total and on residues 9 and 373/376) are inhibited in cells transfected with small interfering RNA for Src, indicating that Src is upstream of PDK-1. In cells expressing oxidation-resistant Src mutant C487A, Ang II-induced hypertrophy and fibronectin expression are prevented, suggesting that the pathway is redox-sensitive. Ang II also up-regulates Nox4 protein, and siNox4 abrogates the Ang II-induced increase in intracellular reactive oxygen species (ROS) generation. Small interfering RNA for Nox4 also inhibits Ang II-induced activation of Src and PDK-1 tyrosine phosphorylation (total and on residues 9 and 373/376), demonstrating that Nox4 functions upstream of Src and PDK-1. Importantly, inhibition of Nox4, Src, or PDK-1 prevents the stimulatory effect of Ang II on fibronectin accumulation and cell hypertrophy. This work provides the first evidence that Nox4-derived ROS are responsible for Ang II-induced PDK-1 tyrosine phosphorylation and activation through stimulation of Src. Importantly, this pathway contributes to Ang II-induced MC hypertrophy and fibronectin accumulation. These data shed light on molecular processes underlying the oxidative signaling cascade engaged by Ang II and identify potential targets for intervention to prevent renal hypertrophy and fibrosis.

Arachidonic acid-dependent activation of a p22(phox)-based NAD(P)H oxidase mediates angiotensin II-induced mesangial cell protein synthesis and fibronectin expression via Akt/PKB.

Angiotensin II (Ang II) induces protein synthesis and hypertrophy through arachidonic acid (AA)- and redoxdependent activation of the serine-threonine kinase Akt/PKB in mesangial cells (MCs). The role of NAD(P)H oxidase component p22( phox ) was explored in this signaling pathway and in Ang II-induced expression of the extracellular matrix protein fibronectin. Ang II causes activation of Akt/PKB and induces fibronectin protein expression, effects abrogated by phospholipase A(2) inhibition and mimicked by AA. Ang II and AAalso elicited an increase in fibronectin expression that was reduced with a dominant negative mutant of Akt/PKB. Exposure of the cells to hydrogen peroxide stimulates Akt/PKB activity and fibronectin synthesis. The antioxidant N-acetylcysteine abolished Ang II- and AA-induced Akt/PKB activation and fibronectin expression. Western blot analysis revealed high levels of p22( phox ) in MCs. Antisense (AS) but not sense oligonucleotides for p22( phox ) prevented ROS generation in response to Ang II and AA. AS p22( phox ) inhibited Ang II- or AA-induced Akt/PKB as well as protein synthesis and fibronectin expression. These data provide the first evidence, in MCs, of activation by AAof a p22( phox )-based NAD(P)H oxidase and subsequent generation of ROS. Moreover, this pathway mediates the effect of Ang II on Akt/PKB-induced protein synthesis and fibronectin expression.