Journal Article

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

Josephine M. Forbes, Felicia Y.T. Yap, Suzanne R. Thorpe, Karly C. Sourris, Vicki Thallas-Bonke, Sally A. Penfold, Melinda T. Coughlan, Leon A. Bach, Kei Fukami, Mark E. Cooper

2008

Overview

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

• Publisher: American Diabetes Association
• Subject: Acetophenones - therapeutic use / Animals / Associated diseases and complications / Biological and medical sciences / Development and progression / Diabetes Mellitus, Experimental - enzymology / Diabetes Mellitus, Experimental - pathology / Diabetes. Impaired glucose tolerance / Diabetic nephropathies / Diabetic Nephropathies - enzymology / Diabetic Nephropathies - pathology / Endocrine pancreas. Apud cells (diseases) / Endocrinopathies / Enzyme Inhibitors - therapeutic use / Enzyme-Linked Immunosorbent Assay / Etiopathogenesis. Screening. Investigations. Target tissue resistance / Glomerular Mesangium - drug effects / Glomerular Mesangium - enzymology / Glomerular Mesangium - pathology / Glycation End Products, Advanced - antagonists & inhibitors / Glycosylation / Kidneys / Lysine - analogs & derivatives / Lysine - analysis / Male / Medical sciences / NADP (Coenzyme) / NADPH Oxidases - antagonists & inhibitors / Nephrology. Urinary tract diseases / Nicotinamide adenine dinucleotide phosphate / Physiological aspects / Protein Kinase C-alpha - metabolism / Protein kinases / Rats / Rats, Sprague-Dawley / Receptor for Advanced Glycation End Products / Receptors, Immunologic - drug effects / Receptors, Immunologic - physiology / Superoxides - metabolism / Urinary system involvement in other diseases. Miscellaneous