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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.sup.-/-]) 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.sup.-/-] 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.sup.-/-] 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. advanced glycation; advanced glycation end products; receptor of advanced glycation end products doi:10.1152/ajprenal.00591.2009

Signaling via the receptor of advanced glycation end products (RAGE)-though complex and not fully elucidated in the setting of diabetes-is considered a key injurious pathway in the development of diabetic nephropathy (DN). We report here that RAGE deletion resulted in increased expression of fibrotic markers (collagen I and IV, fibronectin) and the inflammatory marker MCP-1 in primary mouse mesangial cells (MCs) and in kidney cortex. RNA sequencing analysis in MCs from RAGE-/- and wild-type mice confirmed these observations. Nevertheless, despite these gene expression changes, decreased responsiveness to transforming growth factor-β was identified in RAGE-/- mice. Furthermore, RAGE deletion conferred a more proliferative phenotype in MCs and reduced susceptibility to staurosporine-induced apoptosis. RAGE restoration experiments in RAGE-/- MCs largely reversed these gene expression changes, resulting in reduced expression of fibrotic and inflammatory markers. This study highlights that protection against DN in RAGE knockout mice is likely to be due in part to the decreased responsiveness to growth factor stimulation and an antiapoptotic phenotype in MCs. Furthermore, it extends our understanding of the role of RAGE in the progression of DN, as RAGE seems to play a key role in modulating the sensitivity of the kidney to injurious stimuli such as prosclerotic cytokines. [ABSTRACT FROM AUTHOR]

AMP-activated protein kinase (AMPK) is a key metabolic enzyme, responding to low levels of energy by increasing energy production, namely via increased glucose uptake and fatty acid β-oxidation. Therefore, activators of AMPK are potential candidates for the therapy of type 2 diabetes mellitus (T2DM). We aimed to characterise a new mechanism of AMPK activation using two novel compounds (Bak01 and Bak02) in human primary skeletal muscle cell culture. Skeletal muscle cells were isolated from vastus lateralis biopsies from 5 healthy male controls and 5 males with T2DM matched for age, BMI and blood pressure (fasting glucose mmol/L: 5.1±0.4 and 8.3±2.3 respectively p=0.02). We demonstrate that two novel agents Bak01 and Bak02, stimulate glucose uptake and fat oxidation in primary human skeletal muscle cultures via direct activation of AMPK. Phosphorylation of skeletal muscle AMPK was increased in response to Bak01 and Bak02 after 5min incubations (202±45%, p=0.01 and 203±36%, p=0.004 respectively), which returned to basal after 30mins. Glucose uptake increased in both healthy (131±10%, p =0.03) and T2DM cells (178±12%, p<0.001). These responses were equivalent to that observed with a pharmacological dose (100nM) of insulin (197±15%, p<0.001) in T2DM cells. Bak02 induced glucose uptake only in T2DM cells (144±17%, p<0.01). The downstream substrate of AMPK, acetyl CoA carboxylase, which controls fatty acid oxidation, was also phosphorylated in response to both compounds (Bak01: 250±66% p=0.002 and Bak02: 255±70%, p=0.004, 30min incubation). Both Bak01 and Bak02 compounds increased fatty acid oxidation equivalently in T2DM cells (141±13%, p=0.02 and 150±9 %, p=0.02 respectively) but induced no response in healthy cells. Inhibition of AMPK using a dominant-negative adenovirus abolished the effect of Bak01 and Bak02 on glucose uptake and fatty acid oxidation. These data clearly demonstrate that both Bak01 and Bak02 activate AMPK to increase glucose uptake and fatty acid oxidation in human primary skeletal muscle cell culture. If validated in vivo this mechanism may provide a platform for design of therapeutic agents to address the metabolic syndrome, T2DM and obesity. [ABSTRACT FROM AUTHOR]

Aims: Defects in the activity of enzyme complexes of the mitochondrial respiratory chain are thought to be responsible for several disorders, including renal impairment. Gene mutations that result in complex I deficiency are the most common oxidative phosphorylation disorders in humans. To determine whether an abnormality in mitochondrial complex I per se is associated with development of renal disease, mice with a knockdown of the complex I gene, Ndufs6 were studied., Results: Ndufs6 mice had a partial renal cortical complex I deficiency; Ndufs6gt/gt, 32% activity and Ndufs6gt/+, 83% activity compared with wild-type mice. Both Ndufs6gt/+ and Ndufs6gt/gt mice exhibited hallmarks of renal disease, including albuminuria, urinary excretion of kidney injury molecule-1 (Kim-1), renal fibrosis, and changes in glomerular volume, with decreased capacity to generate mitochondrial ATP and superoxide from substrates oxidized via complex I. However, more advanced renal defects in Ndufs6gt/gt mice were observed in the context of a disruption in the inner mitochondrial electrochemical potential, 3-nitrotyrosine-modified mitochondrial proteins, increased urinary excretion of 15-isoprostane F2t, and up-regulation of antioxidant defence. Juvenile Ndufs6gt/gt mice also exhibited signs of early renal impairment with increased urinary Kim-1 excretion and elevated circulating cystatin C., Innovation: We have identified renal impairment in a mouse model of partial complex I deficiency, suggesting that even modest deficits in mitochondrial respiratory chain function may act as risk factors for chronic kidney disease., Conclusion: These studies identify for the first time that complex I deficiency as the result of interruption of Ndufs6 is an independent cause of renal impairment.

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-alpha 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-alpha 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-alpha 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-alpha is downstream of the AGE-receptor for AGE interaction in diabetic renal disease and may provide a novel therapeutic target for diabetic nephropathy.