Kei Fukami

RAGE-Induced Cytosolic ROS Promote Mitochondrial Superoxide Generation in Diabetes

Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodents each induced renal cytosolic oxidative stress, which led to mitochondrial permeability transition and deficiency of mitochondrial complex I. Because of a lack of glucose-derived NADH, which is the substrate for complex I, these changes did not lead to excess production of mitochondrial superoxide; however, when we performed these experiments in hyperglycemic conditions in vitro or in diabetic rats, we observed significant generation of mitochondrial superoxide at the level of complex I, fueled by a sustained supply of NADH. Pharmacologic inhibition of AGE-RAGE-induced mitochondrial permeability transition in vitro abrogated production of mitochondrial superoxide; we observed a similar effect in vivo after inhibiting cytosolic ROS production with apocynin or lowering AGEs with alagebrium. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in mice. Taken together, these studies suggest that AGE-RAGE-induced cytosolic ROS production facilitates mitochondrial superoxide production in hyperglycemic environments, providing further evidence of a role for the advanced glycation pathway in the development and progression of diabetic nephropathy.

Inhibition of NADPH Oxidase Prevents Advanced Glycation End Product–Mediated Damage in Diabetic Nephropathy Through a Protein Kinase C-α–Dependent Pathway

OBJECTIVE—Excessive production of reactive oxygen species (ROS) via NADPH oxidase has been implicated in the pathogenesis of diabetic nephropathy. Since NADPH oxidase activation is closely linked to other putative pathways, its interaction with changes in protein kinase C (PKC) and increased advanced glycation was examined. RESEARCH DESIGN AND METHODS—Streptozotocin-induced diabetic or nondiabetic Sprague Dawley rats were followed for 32 weeks, with groups randomized to no treatment or the NADPH oxidase assembly inhibitor apocynin (15 mg · kg−1 · day−1; weeks 16–32). Complementary in vitro studies were performed in which primary rat mesangial cells, in the presence and absence of advanced glycation end products (AGEs)-BSA, were treated with either apocynin or the PKC-α inhibitor Ro-32-0432. RESULTS—Apocynin attenuated diabetes-associated increases in albuminuria and glomerulosclerosis. Circulating, renal cytosolic, and skin collagen–associated AGE levels in diabetic rats were not reduced by apocynin. Diabetes-induced translocation of PKC, specifically PKC-α to renal membranes, was associated with increased NADPH-dependent superoxide production and elevated renal, serum, and urinary vascular endothelial growth factor (VEGF) concentrations. In both diabetic rodents and in AGE-treated mesangial cells, blockade of NADPH oxidase or PKC-α attenuated cytosolic superoxide and PKC activation and increased VEGF. Finally, renal extracellular matrix accumulation of fibronectin and collagen IV was decreased by apocynin. CONCLUSIONS—In the context of these and previous findings by our group, we conclude that activation of NADPH oxidase via phosphorylation of PKC-α is downstream of the AGE–receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.

Role of advanced glycation end products (AGEs) and oxidative stress in vascular complications in diabetes.

BACKGROUND A non-enzymatic reaction between reducing sugars and amino groups of proteins, lipids and nucleic acids contributes to the aging of macromolecules, whose process has been known to progress at an accelerated rate under hyperglycemic and/or oxidative stress conditions. Over a course of days to weeks, early glycation products undergo further reactions such as rearrangements and dehydration to become irreversibly cross-linked, fluorescent protein derivatives termed advanced glycation end products (AGEs). SCOPE OF REVIEW In this paper, we review the role of AGE-oxidative stress axis and its therapeutic interventions in vascular complications in diabetes. MAJOR CONCLUSIONS AGEs elicit oxidative stress generation and subsequently cause inflammatory and thrombogenic reactions in various types of cells via interaction with a receptor for AGEs (RAGE), thereby being involved in vascular complications in diabetes. In addition, mitochondrial superoxide generation has been shown to play an important role in the formation and accumulation of AGEs under diabetic conditions. Further, we have recently found that a pathophysiological crosstalk between AGE-RAGE axis and renin-angiotensin system (RAS) could contribute to the progression of vascular damage in diabetes. GENERAL SIGNIFICANCE These observations suggest that inhibition of AGE-RAGE-oxidative stress axis or blockade of its interaction with RAS is a novel therapeutic strategy for preventing vascular complications in diabetes.

Molecular mechanisms of diabetic nephropathy and its therapeutic intervention.

Diabetic nephropathy is a leading cause of end-stage renal failure, which could account for disabilities and high mortality rates in patients with diabetes. Diabetic nephropathy seems to occur as a result of an interaction between metabolic and hemodynamic factors, which activate common pathways that lead to renal damage. Recent large landmark clinical studies have shown that intensive glucose control reduces the risk of the development and progression of diabetic nephropathy, and the blockade renin-angiotensin system (RAS) is also an important target for both metabolic and hemodynamic derangements in diabetic nephropathy. However, diabetic nephropathy remains the leading cause of end-stage renal failure in developed countries. Therefore, to develop novel therapeutic strategies that specifically target diabetic nephropathy may be helpful for most patients with diabetes. High glucose, via various mechanisms such as increased production of oxidative stress and advanced glycation end products (AGEs), and activation of the RAS and protein kinase C (PKC), elicits vascular inflammation and alters gene expression of growth factors and cytokines, thereby it might be involved in the development and progression of diabetic nephropathy. This article summarizes the molecular mechanisms of diabetic nephropathy and the potential therapeutic interventions that may prevent this devastating disorder even in the presence of hyperglycemia, control of which is often difficult with current therapeutic options.

Agents that block advanced glycation end product (AGE)-RAGE (receptor for AGEs)-oxidative stress system: a novel therapeutic strategy for diabetic vascular complications

Background: Diabetic vascular complications are leading causes of acquired blindness, end-stage renal failure, a variety of neuropathies, and accelerated atherosclerosis, which together could account for disabilities and high mortality rates in patients with diabetes. Since there is accumulating evidence that the advanced glycation end product (AGE)–RAGE (receptor for AGEs)–oxidative stress axis is involved in diabetic vascular complications, inhibition of the AGE–RAGE system may be a promising target for therapeutic intervention in these devastating disorders. Objective: In this review, we discuss several types of agent that may be able to inhibit the AGE–RAGE–oxidative stress system, and their therapeutic implications in vascular complications in diabetes. Methods: We have analyzed currently available scientific literature in the field of AGE–RAGE to create a comprehensive review on novel therapeutic agents for vascular complications in diabetes. Results/conclusion: Inhibition of AGE formation, blockade of the AGE–RAGE interaction, and suppression of RAGE expression or its downstream pathways may be novel therapeutic strategies for the treatment of vascular complications in diabetes.

Role of AGEs in Diabetic Nephropathy

Diabetic nephropathy is the most common cause of end-stage renal disease in the world, and accounts for a significant increase in morbidity and mortality in patients with diabetes. Therapeutic options such as strict blood pressure and/or glycemic control are effective for preventing the development and progression of diabetic nephropathy, but the number of diabetic patients on hemodialysis is still increasing. Therefore, a novel therapeutic strategy that could halt the progression of diabetic nephropathy should be developed. Advanced glycation end products (AGEs) are heterogeneous cross-linked sugar-derived proteins which could accumulate in glomerular basement membrane, mesangial cells, endothelial cells, and podocytes in patients with diabetes and/or end-stage renal failure. AGEs are thought to be involved in the pathogenesis of diabetic nephropathy via multifactorial mechanisms such as oxidative stress generation and overproduction of various growth factors and cytokines. Further, recently, the cross-talk between AGEs and the renin-angiotensin system (RAS) has been proposed to participate in diabetic nephropathy. In addition, activation of the RAS elicits ROS generation and subsequently stimulates growth factor and cytokine production by kidney cells as well. These observations suggest that combination therapy with inhibitors of the RAS and blockers of the AGEs formation and/or their downstream pathway may be a novel therapeutic option for preventing diabetic nephropathy. In this paper, we review the role of AGEs and their receptor system in the pathogenesis of diabetic nephropathy. We further discuss here the cross-talk between AGEs and the RAS in the development and progression of diabetic nephropathy.

Dimethylarginine Dimethylaminohydrolase Prevents Progression of Renal Dysfunction by Inhibiting Loss of Peritubular Capillaries and Tubulointerstitial Fibrosis in a Rat Model of Chronic Kidney Disease

Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is mainly degraded by dimethylarginine dimethylaminohydrolase (DDAH). It was recently reported that reduced DDAH expression could contribute to ADMA accumulation and subsequent elevation of BP in an experimental model of chronic kidney disease (CKD). ADMA is a strong predictor of the progression of CKD as well. However, a role for the ADMA-DDAH in the pathogenesis of CKD remains to be elucidated. This study investigated the effects of DDAH-elicited ADMA lowering on renal function and pathology in a rat remnant kidney model. Four weeks after five-sixths subtotal nephrectomy (Nx), the rats were given tail-vein injections of recombinant adenovirus vector encoding DDAH-I (Adv-DDAH) or control vector expressing bacterial beta-galactosidase (Adv-LZ) or orally administered 20 mg/kg per d hydralazine (Hyz), which served as a BP control model. In comparison with Adv-LZ or Hyz administration, Adv-DDAH decreased plasma levels of ADMA and inhibited the deterioration of renal dysfunction. Plasma levels of ADMA were associated with decreased number of peritubular capillaries, increased tubulointerstitial fibrosis, and proteinuria levels in Nx rats. These changes were progressed in Adv-LZ-or Hyz-treated Nx rats, which were ameliorated by DDAH overexpression. In addition, semiquantitative reverse transcriptase-PCR and immunohistochemistry for TGF-beta revealed that Adv-DDAH inhibited upregulation of TGF-beta expression in Nx rats. These data suggest that ADMA may be involved in peritubular capillary loss and tubulointerstitial fibrosis, thereby contributing to the progression of CKD. Substitution of DDAH protein or enhancement of its activity may become a novel therapeutic strategy for the treatment of CKD.

Protective role of pigment epithelium-derived factor (PEDF) in early phase of experimental diabetic retinopathy.

Pigment epithelium‐derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that PEDF may protect against proliferative diabetic retinopathy. However, a role for PEDF in early diabetic retinopathy remains to be elucidated. We investigated here whether and how PEDF could prevent the development of diabetic retinopathy.

Role of AGEs-RAGE System in Cardiovascular Disease

Advanced glycation end products (AGEs) are a heterogenous group of molecules formed during a non-enzymatic reaction between proteins and sugar residues. Recently, AGEs and their receptor (receptor for AGEs; RAGE) play a central role in the pathogenesis of cardiovascular disease (CVD), which accounts for disability and high mortality rate in patients with diabetes. AGEs initiate diabetic micro- and macrovascular complications through the structural modification and functional alteration of the extracellular matrix proteins as well as intracellular signaling molecules. Engagement of RAGEs with AGEs elicits intracellular reactive oxygen species (ROS) generation and subsequently activates mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-κB) signaling, followed by production of several inflammatory and/or profibrotic factors such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1), thereby being involved in the progression of atherosclerosis. Administration of soluble form of RAGE (sRAGE) could work as a decoy receptor for AGEs and might inhibit the binding of AGEs to RAGE, preventing the development and progression of atherosclerosis in animal models. Furthermore, AGEs/high mobility group box-1 (HMGB-1)-RAGE interaction is involved in heart failure, abdominal aortic aneurysm (AAA) and vascular calcification as well. Thus, blockade of the AGEs/HMGB-1-RAGE system may be a promising therapeutic target for preventing diabetes- and/or age-related CVD. We review here the pathological role of the AGEs/HMGB-1-RAGE system in various types of CVD.

IL-1RI deficiency ameliorates early experimental renal interstitial fibrosis

BACKGROUND IL-1beta has the potential to promote progressive renal disease by effects on macrophage recruitment and activation or by effects mediated through tubular cell transforming growth factor (TGF)-beta production, previously demonstrated in vitro. METHODS The in vivo roles of endogenous IL-1beta and its type I receptor (IL-1RI) in renal fibrosis were studied using wild-type C57BL/6 mice, IL-1beta(-/-) and IL-1RI(-/-) mice with unilateral ureteric obstruction. RESULTS After 7 days, IL-1RI(-/-) mice (IL-1alpha and IL-1beta deficient) were protected from injury and collagen accumulation. IL-1beta(-/-) mice demonstrated some histological protection, but no reduction in alpha1(1) procollagen mRNA or biochemically measured collagen accumulation. Compared with obstructed kidneys from wild-type mice, TGF-beta1 mRNA was reduced in IL-1RI(-/-) mice (with trends to reduced TGF-beta2 and TGF-beta3). Expression of a downstream TGF-beta effector, connective tissue growth factor, was decreased in IL-1RI(-/-) mice. IL-1RI(-/-) mice exhibited less tubulointerstitial apoptosis compared with wild-type mice. Macrophage infiltration and adhesion molecule mRNA expression was unchanged in IL-1beta(-/-) or IL-1RI(-/-) mice. While TNF expression was similar to wild-type mice, IFN-gamma expression was reduced in both IL-1beta(-/-) and IL-1RI(-/-) mice. IL-1RI(-/-) mice at 14 days showed a catch-up in fibrosis compared with wild-type mice. CONCLUSION IL-1/IL-1RI interactions are profibrotic in renal fibrosis. IL-1RI(-/-) mice were more protected at an early stage, associated with changes in TGF-beta and downstream mediators of fibrosis, but independent of the presence of infiltrating macrophages.