Oxidative Stress and Renal Fibrosis: Recent Insights for the Development of Novel Therapeutic Strategies

In diabetes mellitus, vascular complications such as atherosclerosis are a major cause of death. The key underlying pathomechanisms are unclear. However, hyperglycemic oxidative stress derived from NADPH oxidase (Nox), the only known dedicated enzyme to generate reactive oxygen species appears to play a role. Here we identify the Nox1 isoform as playing a key and pharmacologically targetable role in the accelerated development of diabetic atherosclerosis. Human aortic endothelial cells exposed to hyperglycemic conditions showed increased expression of Nox1, oxidative stress, and proinflammatory markers in a Nox1-siRNA reversible manner. Similarly, the specific Nox inhibitor, GKT137831, prevented oxidative stress in response to hyperglycemia in human aortic endothelial cells. To examine these observations in vivo, we investigated the role of Nox1 on plaque development in apolipoprotein E-deficient mice 10 weeks after induction of diabetes mellitus. Deletion of Nox1, but not Nox4, had a profound antiatherosclerotic effect correlating with reduced reactive oxygen species formation, attenuation of chemokine expression, vascular adhesion of leukocytes, macrophage infiltration, and reduced expression of proinflammatory and profibrotic markers. Similarly, treatment of diabetic apolipoprotein E-deficient mice with GKT137831 attenuated atherosclerosis development. These studies identify a major pathological role for Nox1 and suggest that Nox1-dependent oxidative stress is a promising target for diabetic vasculopathies, including atherosclerosis.

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.

The overall production of nitric oxide (NO) is decreased in chronic kidney disease (CKD) which contributes to cardiovascular events and further progression of kidney damage. There are many likely causes of NO deficiency in CKD and the areas surveyed in this review are: 1. Limitations on substrate (l-Arginine) availability, probably due to impaired renal l-Arginine biosynthesis, decreased transport of l-Arginine into endothelial cells and possible competition between NOS and competing metabolic pathways, such as arginase. 2. Increased circulating levels of endogenous NO synthase (NOS) inhibitors, in particular asymmetric dimethylarginine (ADMA). Increased methylation of proteins and their subsequent breakdown to release free ADMA may contribute but the major culprit is probably reduced ADMA catabolism by the enzymes dimethylarginine dimethylaminohydrolases. 3. Reduced renal cortex abundance of the neuronal NOS (nNOS)alpha protein correlates with injury while increasing nNOSbeta abundance may provide a compensatory, protective response. Interventions that can restore NO production by targeting these various pathways are likely to reduce the cardiovascular complications of CKD as well as slowing the rate of progression.