The kidney is arguably the most important target of microvascular damage in diabetes. A substantial proportion of individuals with diabetes will develop kidney disease owing to their disease and/or other co-morbidity, including hypertension and ageing-related nephron loss. The presence and severity of chronic kidney disease (CKD) identify individuals who are at increased risk of adverse health outcomes and premature mortality. Consequently, preventing and managing CKD in patients with diabetes is now a key aim of their overall management. Intensive management of patients with diabetes includes controlling blood glucose levels and blood pressure as well as blockade of the renin-angiotensin-aldosterone system; these approaches will reduce the incidence of diabetic kidney disease and slow its progression. Indeed, the major decline in the incidence of diabetic kidney disease (DKD) over the past 30 years and improved patient prognosis are largely attributable to improved diabetes care. However, there remains an unmet need for innovative treatment strategies to prevent, arrest, treat and reverse DKD. In this Primer, we summarize what is now known about the molecular pathogenesis of CKD in patients with diabetes and the key pathways and targets implicated in its progression. In addition, we discuss the current evidence for the prevention and management of DKD as well as the many controversies. Finally, we explore the opportunities to develop new interventions through urgently needed investment in dedicated and focused research. For an illustrated summary of this Primer, visit: http://go.nature.com/NKHDzg.

Intrarenal oxidative stress plays a critical role in the initiation and progression of diabetic kidney disease (DKD). Enhanced oxidative stress results from overproduction of reactive oxygen species (ROS) in the context of concomitant, insufficient antioxidant pathways. Renal ROS production in diabetes is predominantly mediated by various NADPH oxidases (NOXs), but a defective antioxidant system as well as mitochondrial dysfunction may also contribute. Recent Advances: Effective agents targeting the source of ROS generation hold the promise to rescue the kidney from oxidative damage and prevent subsequent progression of DKD. Critical Issues and Future Directions: In the present review, we summarize and critically analyze molecular and cellular mechanisms that have been demonstrated to be involved in NOX-induced renal injury in diabetes, with particular focus on the role of increased glomerular injury, the development of albuminuria, and tubulointerstitial fibrosis, as well as mitochondrial dysfunction. Furthermore, novel agents targeting NOX isoforms are discussed. Antioxid. Redox Signal. 25, 657-684.

The identification of NOX4 as a major source of oxidative stress in stroke and demonstration of dramatic protection after stroke in mice by NOX4 deletion or NOX inhibition, opens up new avenues for treatment.

Synthesis and deposition of extracellular matrix (ECM) within the glomerulus and interstitium characterizes renal fibrosis, but the mechanisms underlying this process are incompletely understood. The profibrotic cytokine TGF-b1 modulates the expression of certain microRNAs (miRNAs), suggesting that miRNAs may have a role in the pathogenesis of renal fibrosis. Here, we exposed proximal tubular cells, primary mesangial cells, and podocytes to TGF-b1 to examine its effect on miRNAs and subsequent collagen synthesis. TGF-b1 reduced expression of the miR-29a/b/c/family, which targets collagen gene expression, and increased expression of ECM proteins. In both resting and TGF-b1-treated cells, ectopic expression of miR-29 repressed the expression of collagens I and IV at both the mRNA and protein levels by targeting the 39untranslated region of these genes. Furthermore, we observed low levels of miR-29 in three models of renal fibrosis representing early and advanced stages of disease. Administration of the Rho-associated kinase inhibitor fasudil prevented renal fibrosis and restored expression of miR-29. Taken together, these data suggest that TGF-b1 inhibits expression of the miR-29 family, thereby promoting expression of ECM components. Pharmacologic modulation of these miRNAs may have therapeutic potential for progressive renal fibrosis.

Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)-forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and Nox4). Here we show that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE 2/2 mice. Deletion of Nox4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-kB in streptozotocin-induced diabetic ApoE 2/2 mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify Nox4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.

Background-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. Methods and Results-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. Conclusions-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.

OBJECTIVE-Activation of the receptor for advanced glycation end products (RAGE) in diabetic vasculature is considered to be a key mediator of atherogenesis. This study examines the effects of deletion of RAGE on the development of atherosclerosis in the diabetic apoE Ϫ/Ϫ model of accelerated atherosclerosis. RESEARCH DESIGN AND METHODS-ApoEϪ/Ϫ and RAGE Ϫ/Ϫ / apoE Ϫ/Ϫ double knockout mice were rendered diabetic with streptozotocin and followed for 20 weeks, at which time plaque accumulation was assessed by en face analysis. RESULTS-Although diabetic apoEϪ/Ϫ mice showed increased plaque accumulation (14.9 Ϯ 1.7%), diabetic RAGE Ϫ/Ϫ /apoE Ϫ/Ϫ mice had significantly reduced atherosclerotic plaque area (4.9 Ϯ 0.4%) to levels not significantly different from control apoE Ϫ/Ϫ mice (4.3 Ϯ 0.4%). These beneficial effects on the vasculature were associated with attenuation of leukocyte recruitment; decreased expression of proinflammatory mediators, including the nuclear factor-B subunit p65, VCAM-1, and MCP-1; and reduced oxidative stress, as reflected by staining for nitrotyrosine and reduced expression of various NADPH oxidase subunits, gp91phox, p47phox, and rac-1. Both RAGE and RAGE ligands, including S100A8/A9, high mobility group box 1 (HMGB1), and the advanced glycation end product (AGE) carboxymethyllysine were increased in plaques from diabetic apoE Ϫ/Ϫ mice. Furthermore, the accumulation of AGEs and other ligands to RAGE was reduced in diabetic RAGECONCLUSIONS-This study provides evidence for RAGE playing a central role in the development of accelerated atherosclerosis associated with diabetes. These findings emphasize the potential utility of strategies targeting RAGE activation in the prevention and treatment of diabetic macrovascular complications. Diabetes 57:2461-2469, 2008 T he receptor for advanced glycation end products (RAGE) is a multiligand cell surface molecule belonging to the immunoglobulin superfamily (1). It is expressed as full-length, N-truncated, and C-truncated isoforms, generated in humans by alternative splicing (2). Activation of the full-length RAGE receptor has been implicated in a range of chronic diseases, including various diabetic complications and atherosclerosis (1). In particular, studies in RAGE Ϫ/Ϫ mice that carry the dominant-negative form of the receptor (2-6) and in RAGE-overexpressing mice (7) have confirmed an important role of RAGE activation in the development of diabetic nephropathy, neuropathy, and impaired angiogenesis. RAGE activation has also been implicated in the acceleration of atherosclerotic lesion formation as well as in the maintenance of proinflammatory and prothrombotic mechanisms, characteristic of diabetes-accelerated atherosclerosis (8,9). RAGE also represents an important mediator of oxidative stress in diabetes. Activation of RAGE in vitro leads to increased NADPH oxidase expression, mitochondrial oxidase activity, and downregulation of endogenous antioxidant activity (10,11). RAGE Ϫ/Ϫ mice have a suppression of neointimal proliferation after externally...

OBJECTIVEProgressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.RESEARCH DESIGN AND METHODSRat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.RESULTSBoth TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β–dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3′ untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.CONCLUSIONSmiR-200a and miR-141 significantly impact on the development and progression of TGF-β–dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.