Reactive oxygen species (ROS) generated by Nox NADPH oxidases may play a critical role in the pathogenesis of diabetic nephropathy (DN). The efficacy of the Nox1/Nox4 inhibitor GKT137831 on the manifestations of DN was studied in OVE26 mice, a model of type 1 diabetes. Starting at 4-5 mo of age, OVE26 mice were treated with GKT137831 at 10 or 40 mg/kg, once-a-day for 4 wk. At both doses, GKT137831 inhibited NADPH oxidase activity, superoxide generation, and hydrogen peroxide production in the renal cortex from diabetic mice without affecting Nox1 or Nox4 protein expression. The increased expression of fibronectin and type IV collagen was reduced in the renal cortex, including glomeruli, of diabetic mice treated with GKT137831. GKT137831 significantly reduced glomerular hypertrophy, mesangial matrix expansion, urinary albumin excretion, and podocyte loss in OVE26 mice. GKT137831 also attenuated macrophage infiltration in glomeruli and tubulointerstitium. Collectively, our data indicate that pharmacological inhibition of Nox1/4 affords broad renoprotection in mice with preexisting diabetes and established kidney disease. This study validates the relevance of targeting Nox4 and identifies GKT137831 as a promising compound for the treatment of DN in type 1 diabetes.

Mesangial matrix accumulation is an early feature of glomerular pathology in diabetes. Oxidative stress plays a critical role in hyperglycemia-induced glomerular injury. Here, we demonstrate that, in glomerular mesangial cells (MCs), endothelial nitric oxide synthase (eNOS) is uncoupled upon exposure to high glucose (HG), with enhanced generation of reactive oxygen species (ROS) and decreased production of nitric oxide. Peroxynitrite mediates the effects of HG on eNOS dysfunction. HG upregulates Nox4 protein, and inhibition of Nox4 abrogates the increase in ROS and peroxynitrite generation, as well as the eNOS uncoupling triggered by HG, demonstrating that Nox4 functions upstream from eNOS. Importantly, this pathway contributes to HGinduced MC fibronectin accumulation. Nox4-mediated eNOS dysfunction was confirmed in glomeruli of a rat model of type 1 diabetes. Sestrin 2-dependent AMP-activated protein kinase (AMPK) activation attenuates HG-induced MC fibronectin synthesis through blockade of Nox4-dependent ROS and peroxynitrite generation, with subsequent eNOS uncoupling. We also find that HG negatively regulates sestrin 2 and AMPK, thereby promoting Nox4-mediated eNOS dysfunction and increased fibronectin. These data identify a protective function for sestrin 2/AMPK and potential targets for intervention to prevent fibrotic injury in diabetes.T he pathological manifestations of early diabetes in the glomerular microvascular bed include glomerular mesangial cell hypertrophy associated with an increase in mesangial matrix accumulation (1, 2). These events precede the development of irreversible glomerulosclerosis (1, 2). Data from animal models of diabetes, as well as from cultured cells, indicate that hyperglycemia and high glucose (HG) increase extracellular matrix expansion in mesangial cells (MCs) (1, 2).Oxidative stress with increased generation of reactive oxygen species (ROS) has emerged as a critical pathogenic factor in the development of diabetic nephropathy (DN) (1-3). However, the protective effects of traditional antioxidants are very limited. Identifying sources of ROS should help in designing rational therapy to modulate oxidative stress. Although multiple pathways may result in ROS generation, numerous studies identified NADPH oxidases of the Nox family as major sources of ROS in various nonphagocytic/stromal cells, including most kidney cells (4-6). Evidence from studies in cultured cells suggests that the isoform Nox4 is required for the damaging effects of HG that contribute to microvascular complications of diabetes in the retina, the heart, or the kidney (7-13). We have previously reported that Nox4-dependent ROS generation mediates glomerular hypertrophy and mesangial matrix accumulation in early type 1 diabetes (13). In MCs, we showed that Nox4-derived ROS result in the increased fibronectin expression induced by HG (13,14). Nevertheless, the mechanisms that Nox4 utilizes to exert this biological effect remain unclear, and the upstream regulators or downstream effectors of the oxidase are n...

Background: Oxidative stress is critical for the fibrotic response of mesangial cells (MCs) to angiotensin II. Results: Nox4-and mitochondrial reactive oxygen species (ROS)-dependent endothelial nitric-oxide synthase (eNOS) uncoupling led to fibronectin accumulation in MCs stimulated by angiotensin II. Conclusion: The Nox4/mitochondrial ROS/eNOS pathway mediates angiotensin II-induced MC injury. Significance: Targeting Nox4 and mitochondrial ROS is a promising therapeutic approach.

Evidence for gadolinium-based contrast agent-(GBCA-) induced disease continues to mount. Risk factors for gadolinium-induced systemic fibrosis are entirely unexplored. Obesity-related renal injury is characterized by activation of glomerular mesangial cells and podocyte damage with alteration of lipid metabolism/lipid accumulation in both cell types resulting in matrix accumulation and eventual progression to glomerulosclerosis. We examined the consequences of GBCA treatment in the kidneys from mice with normal kidney function and the potential interplay between obesity and gadolinium exposure. We found that administration of GBCA (4 weeks) causes significant renal fibrosis and podocyte injury that are associated with metabolic disorders as evidenced by dyslipidemia. Metabolomic analysis demonstrated that renal lipid metabolism and metabolic markers of collagen turnover are significantly altered by gadolinium. GBCA stimulates myeloid-derived fibrocytes to the kidney. Obesity was induced by feeding a group of mice a high fat diet (HFD) for 22 weeks. Groups were sub-randomized to GBCA treatment versus none for 4 weeks before sacrifice. HFD-induced fibrosis and podocyte injury were worsened by GBCA. Similarly, HFD-mediated hyperlipidemia and lipid metabolites were exacerbated by gadolinium. This is the first evidence that GBCA causes significant metabolic disorders and kidney injury in mice without renal insufficiency and that the injurious actions of GBCA are amplified by obesity. The understanding of the functional interplay between gadolinium and obesity will allow the development of therapeutic interventions or the establishment of effective preventive measures to reduce gadolinium-and obesity-mediated renal pathologies.

High-glucose increases NADPH oxidase 4 (NOX4) expression, reactive oxygen species generation, and matrix protein synthesis by inhibiting AMP-activated protein kinase (AMPK) in renal cells. Because hydrogen sulfide (HS) inhibits high glucose-induced matrix protein increase by activating AMPK in renal cells, we examined whether HS inhibits high glucose-induced expression of NOX4 and matrix protein and whether HS and NO pathways are integrated. High glucose increased NOX4 expression and activity at 24 h in renal proximal tubular epithelial cells, which was inhibited by sodium hydrosulfide (NaHS), a source of HS. High glucose decreased AMPK phosphorylation and activity, which was restored by NaHS. Compound C, an AMPK inhibitor, prevented NaHS inhibition of high glucose-induced NOX4 expression. NaHS inhibition of high glucose-induced NOX4 expression was abrogated by (ω)-nitro-l-arginine methyl ester, an inhibitor of NOS. NaHS unexpectedly augmented the expression of inducible NOS (iNOS) but not endothelial NOS. iNOS siRNA and 1400W, a selective iNOS inhibitor, abolished the ameliorative effects of NaHS on high glucose-induced NOX4 expression, reactive oxygen species generation, and, matrix laminin expression. Thus, HS recruits iNOS to generate NO to inhibit high glucose-induced NOX4 expression, oxidative stress, and matrix protein accumulation in renal epithelial cells; the two gasotransmitters HS and NO and their interaction may serve as therapeutic targets in diabetic kidney disease.