Sestrin 2 and AMPK Connect Hyperglycemia to Nox4-Dependent Endothelial Nitric Oxide Synthase Uncoupling and Matrix Protein Expression

Abstract

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...

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