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Inhibition of NADPH Oxidase Prevents Advanced Glycation End Product–Mediated Damage in Diabetic Nephropathy Through a Protein Kinase C-α–Dependent Pathway Vicki Thallas-Bonke 1 , Suzanne R. Thorpe 2 , Melinda T. Coughlan 1 , Kei Fukami 1 , Felicia Y.T. Yap 1 , Karly C. Sourris 1 , Sally A. Penfold 1 , Leon A. Bach 3 , Mark E. Cooper 1 3 and Josephine M. Forbes 1 3 1 Juvenile Diabetes Research Foundation (JDFR) Albert Einstein Centre for Diabetes Complications, Diabetes and Metabolism Division, Baker Medical Research Institute, Melbourne, Victoria, Australia 2 Department of Chemistry and Biochemistry, University of South Carolina, Columbia, South Carolina 3 Department of Medicine and Immunology, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia Address correspondence and reprint requests to Vicki Thallas-Bonke, JDRF Albert Einstein Centre for Diabetes Complications, Diabetes and Metabolism Division, Baker Medical Research Institute, P.O. Box 6492, St. Kilda Rd., Central, Melbourne, Victoria, Australia, 8008. E-mail: vicki.thallas{at}baker.edu.au Abstract 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. AGE, advanced glycation end product CML, carboxymethyllysine ELISA, enzyme-linked immunosorbent assay PKC, protein kinsase C RAGE, receptor for AGE ROS, reactive oxygen species VEGF, vascular endothelial growth factor Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 24 October 2007. DOI: 10.2337/db07-1119. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted October 18, 2007. Received August 8, 2007. DIABETES

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) 26–39kg/m2) 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.

The accumulation of advanced glycation end products is thought to be a key factor in the initiation and progression of diabetic nephropathy. Here we determined whether the size of the ligands for the receptor for advanced glycation end products (RAGEs) that were present in the serum of patients with type 2 diabetes modulates their pathogenic potential. Serum was collected from control subjects and patients with type 2 diabetes with varying degrees of renal disease (normo-, micro-, or macroalbuminuria). The titers of the RAGE ligands N-carboxymethyllysine (CML), S100A, S100B, and high-mobility group box 1 (HMGB1) were measured by enzyme-linked immunosorbent assay in serum as well as in pooled size-fractionated serum. We also measured cellular binding of serum fractions to mesangial cells transfected with RAGE and examined the downstream signaling pathways. Circulating CML was increased in patients with type 2 diabetes, whereas HMGB1 was decreased. S100A8, S100BA9, and soluble RAGE were unchanged. The high-molecular-weight (over 50kDa) serum fraction contained the greatest proportion of RAGE ligands, with all immunoreactivity and cellular binding observed only with serum fractions over 30kDa. High-molecular-weight serum from macroalbuminuric patients showed greater RAGE binding capacity, modulation of cell-surface RAGE expression, increased phospho-protein kinase C-α, and p65 nuclear factor κB DNA-binding activity, which were competitively inhibited by soluble RAGE or CML neutralizing antibodies. These data show that ligands that activate RAGE present in the circulation of patients with type 2 diabetes and nephropathy are predominantly of high molecular weight.

Aims The accumulation of advanced glycation end products is implicated in the development and progression of diabetic kidney disease. No study has examined whether stimulating advanced glycation clearance via receptor manipulation is reno‐protective in diabetes. Podocytes, which are early contributors to diabetic kidney disease and could be a target for reno‐protection. Materials and methods To examine the effects of increased podocyte oligosaccharyltransferase‐48 on kidney function, glomerular sclerosis, tubulointerstitial fibrosis and proteome (PXD011434), we generated a mouse with increased oligosaccharyltransferase‐48kDa subunit abundance in podocytes driven by the podocin promoter. Results Despite increased urinary clearance of advanced glycation end products, we observed a decline in renal function, significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane thickening and foot process effacement and tubulointerstitial fibrosis. Analysis of isolated glomeruli identified enrichment in proteins associated with collagen deposition, endoplasmic reticulum stress and oxidative stress. Ultra‐resolution microscopy of podocytes revealed denudation of foot processes where there was co‐localization of oligosaccharyltransferase‐48kDa subunit and advanced glycation end‐products. Conclusions These studies indicate that increased podocyte expression of oligosaccharyltransferase‐48 kDa subunit results in glomerular endoplasmic reticulum stress and a decline in kidney function. Here, we present studies where we have selectively over‐expressed the gene encoding for OST48 within podocytes, a cell type intimately involved in the pathology of DKD. In this novel study, despite facilitating renal clearance of AGEs, overexpression of podocyte OST48 resulted in a decline in kidney function and significant glomerular damage and dysfunction exacerbated by diabetes. We have also identified for the first time the proteomic profiles detailing the mechanisms driving the decline in GFR and provided ultra‐resolution imaging visualizing the selective binding of AGE‐OST48 within the podocytes.

In this Review, the authors examine how the development of diabetes mellitus has come full circle from initiation to complications and suggest that the development of diabetes mellitus and the progression to chronic complications both require the same mechanistic triggers. Glycaemic control, reduction of blood pressure using agents that block the renin–angiotensin system and control of dyslipidaemia are the major strategies used in the clinical management of patients with diabetes mellitus. Each of these approaches interrupts a number of pathological pathways, which directly contributes to the vascular complications of diabetes mellitus, including renal disease, blindness, neuropathy and cardiovascular disease. However, research published over the past few years has indicated that many of the pathological pathways important in the development of the vascular complications of diabetes mellitus are equally relevant to the initiation of diabetes mellitus itself. These pathways include insulin signalling, generation of cellular energy, post-translational modifications and redox imbalances. This Review will examine how the development of diabetes mellitus has come full circle from initiation to complications and suggests that the development of diabetes mellitus and the progression to chronic complications both require the same mechanistic triggers. Key Points The development of diabetes mellitus and its associated vascular complications share common pathogenic pathways Vascular damage is often the result of imbalances in glucose handling at many sites within the cardiovascular system Dyslipidaemia and abnormalities in cellular energetics are frequently seen in both the development of diabetes mellitus and its associated vascular complications Research programs should now investigate patterns of damage across the body in patients with diabetes mellitus Grouping and characterization of patterns in the initiation of diabetes mellitus to enable comparison with patterns in vascular complications of diabetes mellitus might result in superior therapies

The accumulation of advanced glycation end products (AGEs) have been implicated in the development and progression of diabetic kidney disease (DKD). There has been interest in investigating the potential of AGE clearance receptors, such as oligosaccharyltransferase-48 kDa subunit (OST48) to prevent the detrimental effects of excess AGE accumulation seen in the diabetic kidney. Here the objective of the study was to increase the expression of OST48 to examine if this slowed the development of DKD by facilitating the clearance of AGEs. Groups of 8-week-old heterozygous knock-in male mice (n = 9–12/group) over-expressing the gene encoding for OST48, dolichyl-diphosphooligosaccharide-protein glycosyltransferase ( DDOST +/−) and litter mate controls were randomised to either (i) no diabetes or (ii) diabetes induced via multiple low-dose streptozotocin and followed for 24 weeks. By the study end, global over expression of OST48 increased glomerular OST48. This facilitated greater renal excretion of AGEs but did not affect circulating or renal AGE concentrations. Diabetes resulted in kidney damage including lower glomerular filtration rate, albuminuria, glomerulosclerosis and tubulointerstitial fibrosis. In diabetic mice, tubulointerstitial fibrosis was further exacerbated by global increases in OST48. There was significantly insulin effectiveness, increased acute insulin secretion, fasting insulin concentrations and AUC insulin observed during glucose tolerance testing in diabetic mice with global elevations in OST48 when compared to diabetic wild-type littermates. Overall, this study suggested that despite facilitating urinary-renal AGE clearance, there were no benefits observed on kidney functional and structural parameters in diabetes afforded by globally increasing OST48 expression. However, the improvements in insulin secretion seen in diabetic mice with global over-expression of OST48 and their dissociation from effects on kidney function warrant future investigation.

Background/Aims: The formation of advanced glycation end products (AGEs) is accelerated in patients with diabetic nephropathy. The aim of this study was to ascertain if the urinary excretion of proteins modified by advanced glycation can be used as biomarkers for albuminuria in individuals with type 1 or type 2 diabetes. Methods: Community-based patients with type 1 (n = 68) or type 2 diabetes (n = 216) attending a diabetes clinic of a tertiary referral hospital were classified as having normoalbuminuria (Normo, albumin excretion rate (AER) <20 µg/min), microalbuminuria (Micro, AER 20–200 µg/min) or macroalbuminuria (Macro, AER ≧200 µg/min). Serum and urine AGE-modified proteins were measured. Results: In patients with both type 1 diabetes and type 2 diabetes, there was a clear association between the degree of albuminuria and urinary AGE-modified proteins (p < 0.0001). Exclusive to patients with type 1 diabetes, urinary excretion of the AGE carboxymethyllysine correlated with AER, whereas patients with type 2 diabetes and macroalbuminuria had an increase in urinary methylglyoxal, an AGE intermediate. These changes were independent of isotopic glomerular filtration rate levels. Serum concentrations of AGEs or soluble receptor for AGEs were not consistently associated with albuminuria in either type 1 or type 2 diabetes. Conclusions: Urinary excretion of proteins modified by AGEs may be useful biomarkers of albuminuria in individuals with type 1 and type 2 diabetes, warranting prospective investigation in larger diabetic cohorts.

The AGER gene encodes for a number of RAGE isoforms, with the membrane bound signal transduction and decoy circulating soluble RAGE being the best characterised. Here we demonstrate a novel nuclear isoform of RAGE in mice and human kidney cortex which by cell and size fractionation we determined to be approximately 37kda. This nuclear RAGE isoform is functional and binds to DNA sequences within the upstream 5' promoter region of its own gene, AGER. This binding was shown to be abrogated by mutating the DNA consensus binding sequences during electromobility shift assay (EMSA) and was independent of NFκ-B or AP-1 binding. Cotransfection of expression constructs encoding various RAGE isoforms along with AGER gene promoter reporter-plasmids identified that the most likely source of the nuclear isoform of RAGE was a cleavage product of the nt-RAGE isoform. In obese mice with impaired kidney function, there was increased binding of nuclear RAGE within the A. Region of ager gene promoter with corresponding increases in membrane bound RAGE in renal cells. These findings were reproduced in vitro using proximal tubule cells. Hence, we postulate that RAGE expression is in part, self-regulated by the binding of a nuclear RAGE isoform to the promoter of the AGER gene (encoding RAGE) in the kidney. We also suggest that this RAGE self-regulation is altered under pathological conditions and this may have implications for chronic kidney disease.

The accumulation of advanced glycation end products is implicated in the development and progression of diabetic kidney disease. No study has examined whether stimulating advanced glycation clearance via receptor manipulation is reno-protective in diabetes. Podocytes, which are early contributors to diabetic kidney disease and could be a target for reno-protection. To examine the effects of increased podocyte oligosaccharyltransferase-48 on kidney function, glomerular sclerosis, tubulointerstitial fibrosis and proteome (PXD011434), we generated a mouse with increased oligosaccharyltransferase-48kDa subunit abundance in podocytes driven by the podocin promoter. Despite increased urinary clearance of advanced glycation end products, we observed a decline in renal function, significant glomerular damage including glomerulosclerosis, collagen IV deposition, glomerular basement membrane thickening and foot process effacement and tubulointerstitial fibrosis. Analysis of isolated glomeruli identified enrichment in proteins associated with collagen deposition, endoplasmic reticulum stress and oxidative stress. Ultra-resolution microscopy of podocytes revealed denudation of foot processes where there was co-localization of oligosaccharyltransferase-48kDa subunit and advanced glycation end-products. These studies indicate that increased podocyte expression of oligosaccharyltransferase-48kDa subunit results in glomerular endoplasmic reticulum stress and a decline in kidney function.