Vicki Thallas-Bonke

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.

Connective Tissue Growth Factor Plays an Important Role in Advanced Glycation End Product–Induced Tubular Epithelial-to-Mesenchymal Transition: Implications for Diabetic Renal Disease

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

Lack of the Antioxidant Enzyme Glutathione Peroxidase-1 Accelerates Atherosclerosis in Diabetic Apolipoprotein E–Deficient Mice

Background— Recent clinical studies have suggested a major protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx1) in diabetes-associated atherosclerosis. We induced diabetes in mice deficient for both GPx1 and apolipoprotein E (ApoE) to determine whether this is merely an association or whether GPx1 has a direct effect on diabetes-associated atherosclerosis. Methods and Results— ApoE-deficient (ApoE−/−) and ApoE/GPx1 double-knockout (ApoE−/−GPx1−/−) mice were made diabetic with streptozotocin and aortic lesion formation, and atherogenic pathways were assessed after 10 and 20 weeks of diabetes. Aortic proinflammatory and profibrotic markers were determined by both quantitative reverse-transcription polymerase chain reaction analysis after 10 weeks of diabetes and immunohistochemical analysis after 10 and 20 weeks of diabetes. Sham-injected nondiabetic counterparts served as controls. Atherosclerotic lesions within the aortic sinus region, as well as arch, thoracic, and abdominal lesions, were significantly increased in diabetic ApoE−/−GPx1−/− aortas compared with diabetic ApoE−/− aortas. This increase was accompanied by increased macrophages, &agr;-smooth muscle actin, receptors for advanced glycation end products, and various proinflammatory (vascular cell adhesion molecule-1) and profibrotic (vascular endothelial growth factor and connective tissue growth factor) markers. Quantitative reverse-transcription polymerase chain reaction analysis showed increased expression of receptors for advanced glycation end products (RAGE), vascular cell adhesion molecule-1, vascular endothelial growth factor, and connective tissue growth factor. Nitrotyrosine levels were significantly increased in diabetic ApoE−/−GPx1−/− mouse aortas. These findings were observed despite upregulation of other antioxidants. Conclusions— Lack of functional GPx1 accelerates diabetes-associated atherosclerosis via upregulation of proinflammatory and profibrotic pathways in ApoE−/− mice. Our study provides evidence of a protective role for GPx1 and establishes GPx1 as an important antiatherogenic therapeutic target in patients with or at risk of diabetic macrovascular disease.

Modulation of Soluble Receptor for Advanced Glycation End Products by Angiotensin-Converting Enzyme-1 Inhibition in Diabetic Nephropathy

Recent studies have identified that first-line renoprotective agents that interrupt the renin-angiotensin system not only reduce BP but also can attenuate advanced glycation end product (AGE) accumulation. This study used in vitro, preclinical, and human approaches to explore the potential effects of these agents on the modulation of the receptor for AGE (RAGE). Bovine aortic endothelial cells that were exposed to the angiotensin-converting enzyme inhibitor (ACEi) ramiprilat in the presence of high glucose demonstrated a significant increase in soluble RAGE (sRAGE) secreted into the medium. In streptozotocin-induced diabetic rats, ramipril treatment (ACEi) at 3 mg/L for 24 wk reduced the accumulation of skin collagen-linked carboxymethyllysine and pentosidine, as well as circulating and renal AGE. Renal gene upregulation of total RAGE (all three splice variants) was observed in ACEi-treated animals. There was a specific increase in the gene expression of the splice variant C-truncated RAGE (sRAGE). There were also increases in sRAGE protein identified within renal cells with ACEi treatment, which showed AGE-binding ability. This was associated with decreases in renal full-length RAGE protein from ACEi-treated rats. Decreases in plasma soluble RAGE that were significantly increased by ACEi treatment were also identified in diabetic rats. Similarly, there was a significant increase in plasma sRAGE in patients who had type 1 diabetes and were treated with the ACEi perindopril. Complexes between sRAGE and carboxymethyllysine were identified in human and rodent diabetic plasma. It is postulated that ACE inhibition reduces the accumulation of AGE in diabetes partly by increasing the production and secretion of sRAGE into plasma.

Genetic Targeting or Pharmacologic Inhibition of NADPH Oxidase Nox4 Provides Renoprotection in Long-Term Diabetic Nephropathy

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(-/-) 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-κB in streptozotocin-induced diabetic ApoE(-/-) 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.

Advanced Glycation End Products Are Direct Modulators of β-Cell Function

OBJECTIVE Excess accumulation of advanced glycation end products (AGEs) contributes to aging and chronic diseases. We aimed to obtain evidence that exposure to AGEs plays a role in the development of type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS The effect of AGEs was examined on insulin secretion by MIN6N8 cells and mouse islets and in vivo in three separate rodent models: AGE-injected or high AGE–fed Sprague-Dawley rats and nonobese diabetic (NODLt) mice. Rodents were also treated with the AGE-lowering agent alagebrium. RESULTS β-Cells exposed to AGEs displayed acute glucose-stimulated insulin secretory defects, mitochondrial abnormalities including excess superoxide generation, a decline in ATP content, loss of MnSOD activity, reduced calcium flux, and increased glucose uptake, all of which were improved with alagebrium treatment or with MnSOD adenoviral overexpression. Isolated mouse islets exposed to AGEs had decreased glucose-stimulated insulin secretion, increased mitochondrial superoxide production, and depletion of ATP content, which were improved with alagebrium or with MnTBAP, an SOD mimetic. In rats, transient or chronic exposure to AGEs caused progressive insulin secretory defects, superoxide generation, and β-cell death, ameliorated with alagebrium. NODLt mice had increased circulating AGEs in association with an increase in islet mitochondrial superoxide generation, which was prevented by alagebrium, which also reduced the incidence of autoimmune diabetes. Finally, at-risk children who progressed to T1D had higher AGE concentrations than matched nonprogressors. CONCLUSIONS These findings demonstrate that AGEs directly cause insulin secretory defects, most likely by impairing mitochondrial function, which may contribute to the development of T1D.

Dedifferentiation of Immortalized Human Podocytes in Response to Transforming Growth Factor-β: A Model for Diabetic Podocytopathy

OBJECTIVE Diabetic nephropathy is associated with dedifferentiation of podocytes, losing the specialized features required for efficient glomerular function and acquiring a number of profibrotic, proinflammatory, and proliferative features. These result from tight junction and cytoskeletal rearrangement, augmented proliferation, and apoptosis. RESEARCH DESIGN AND METHODS Experiments were performed in conditionally immortalized human podocytes developed by transfection with the temperature-sensitive SV40-T gene. Cells were then cultured in the presence of transforming growth factor (TGF)-β1 or angiotensin II in the presence or absence of a selective inhibitor of the TGF-β type I receptor kinase, SB-431542. Gene and protein expression were then examined by real-time RT-PCR and immunofluorescence, and correlated with changes observed in vivo in experimental diabetes. RESULTS Treatment of cells with TGF-β1 resulted in dynamic changes in their morphology, starting with retraction and shortening of foot processes and finishing with the formation of broad and complex tight junctions between adjacent podocytes. This dedifferentiation was also associated with dose- and time-dependent reduction in the expression of glomerular epithelial markers (nephrin, p-cadherin, zonnula occludens-1) and increased expression of mesenchymal markers (α−smooth muscle actin, vimentin, nestin), matrix components (fibronectin, collagen I, and collagen IV α3), cellular proliferation, and apoptosis. The induction of diabetes in mice was also associated with similar changes in morphology, protein expression, and proliferation in glomerular podocytes. CONCLUSIONS In response to TGF-β and other TGF-dependent stimuli, mature podocytes undergo dedifferentiation that leads to effacement of foot processes, morphologic flattening, and increased formation of intercellular tight junctions. This simplification of their phenotype to a more embryonic form is also associated with reentry of mature podocytes into the cell cycle, which results in enhanced proliferation and apoptosis. These “pathoadaptive” changes are seen early in the diabetic glomerulus and ultimately contribute to albuminuria, glomerulosclerosis, and podocytopenia.

Disparate effects on renal and oxidative parameters following RAGE deletion, AGE accumulation inhibition, or dietary AGE control in experimental diabetic nephropathy

Advanced glycation end products (AGEs) and the receptor for AGEs (RAGE) generate ROS, and therefore this study evaluated the effects of RAGE deletion, decreasing AGE accumulation, or lowering dietary AGE content on oxidative parameters in diabetic nephropathy (DN). Control and diabetic male wild-type and RAGE-deficient (RAGE-/-) mice were fed high- or low-AGE diets, with two groups given the inhibitor of AGE accumulation, alagebrium chloride, and followed for 24 wk. Diabetic RAGE-/- mice were protected against albuminuria, hyperfiltration, glomerulosclerosis, decreased renal mitochondrial ATP production, and excess generation of both mitochondrial and cytosolic superoxide. Whereas glomerulosclerosis, tubulointerstitial expansion, and hyperfiltration were improved in diabetic mice treated with alagebrium, there was no effect on urinary albumin excretion. Both diabetic RAGE-/- and alagebrium-treated mice had an attenuation of renal RAGE expression and decreased renal and urinary AGE (carboxymethyllysine) levels. Low-AGE diets did not confer renoprotection, lower the AGE burden or renal RAGE expression, or improve cytosolic or mitochondrial superoxide generation. Renal uncoupling protein-2 gene expression and mitochondrial membrane potential were attenuated by all therapeutic interventions in diabetic mice. In the present study, diverse approaches to block the AGE-RAGE axis had disparate effects on DN, which has potential clinical implications for the way this axis should be targeted in humans.

Targeted reduction of advanced glycation improves renal function in obesity

Obesity is highly prevalent in Western populations and is considered a risk factor for the development of renal impairment. Interventions that reduce the tissue burden of advanced glycation end-products (AGEs) have shown promise in stemming the progression of chronic disease. Here we tested if treatments that lower tissue AGE burden in patients and mice would improve obesity-related renal dysfunction. Overweight and obese individuals (body mass index (BMI) 26-39 kg/m(2)) were recruited to a randomized, crossover clinical trial involving 2 weeks each on a low- and a high-AGE-containing diet. Renal function and an inflammatory profile (monocyte chemoattractant protein-1 (MCP-1) and macrophage migration inhibitory factor (MIF)) were improved following the low-AGE diet. Mechanisms of advanced glycation-related renal damage were investigated in a mouse model of obesity using the AGE-lowering pharmaceutical, alagebrium, and mice in which the receptor for AGE (RAGE) was deleted. Obesity, resulting from a diet high in both fat and AGE, caused renal impairment; however, treatment of the RAGE knockout mice with alagebrium improved urinary albumin excretion, creatinine clearance, the inflammatory profile, and renal oxidative stress. Alagebrium treatment, however, resulted in decreased weight gain and improved glycemic control compared with wild-type mice on a high-fat Western diet. Thus, targeted reduction of the advanced glycation pathway improved renal function in obesity.