title: NAD(P)H oxidase and uncoupled nitric oxide synthase are major sources of glomerular superoxide in rats with experimental diabetic nephropathy.
abstract: Increased production of reactive oxygen species (ROS) in diabetes may be a common pathway linking diverse pathogenic mechanisms of diabetic vascular complications, including nephropathy. Assessment of the oxidative stress production pathway is therefore important for the prediction and prevention of diabetic complications. However, ROS production mechanisms remain unclear in diabetic glomeruli. To identify the source and determine the mechanisms of ROS production in the diabetic kidney, diabetes was induced with streptozotocin in rats. After 6 wk, glomerular ROS production had increased in the streptozotocin rat kidney, as assessed by dihydroethidium-derived chemiluminescence. ROS production was increased by the addition of NADH or L-arginine and was partially reduced by the addition of diphenylene iodonium or N(G)-nitro-L-arginine methyl ester, identifying NAD(P)H oxidase and nitric oxide (NO) synthase (NOS) as ROS sources. The mRNA and protein expression of endothelial NOS (eNOS), as measured by real-time RT-PCR and Western blotting, increased significantly (mRNA level, 1.3-fold; protein level, 1.8-fold). However, the dimeric form of eNOS was decreased in diabetic glomeruli, as measured by low-temperature SDS-PAGE. Production of renal ROS and NO by uncoupled NOS was imaged by confocal laser microscopy after renal perfusion of 2',7'-dichlorofluorescein diacetate (a ROS marker) and diaminorhodamine-4M AM (a NO marker) with L-arginine. Accelerated ROS production and diminished bioavailable NO caused by NOS uncoupling were noted in the diabetic kidney. Administration of tetrahydrobiopterin (BH4), a cofactor for eNOS, reversed the decreased dimeric form of eNOS and glomerular NO production. Our results indicate that NAD(P)H oxidase and uncoupling of eNOS contribute to glomerular ROS production, mediated by the loss of BH4 availability. These mechanisms are potential key targets for therapeutic interventions.
authors.affiliation: Div. of Nephrology, Dept. of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. minosatou-npr@umin.ac.jp
authors.forename: Minoru
authors.initials: M
authors.lastname: Satoh
authors.forename: Sohachi
authors.initials: S
authors.lastname: Fujimoto
authors.forename: Yoshisuke
authors.initials: Y
authors.lastname: Haruna
authors.forename: Sayaka
authors.initials: S
authors.lastname: Arakawa
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Horike
authors.forename: Norio
authors.initials: N
authors.lastname: Komai
authors.forename: Tamaki
authors.initials: T
authors.lastname: Sasaki
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Tsujioka
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Makino
authors.forename: Naoki
authors.initials: N
authors.lastname: Kashihara
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives* / Q000494:pharmacology; D003928:Diabetic Nephropathies / Q000188:drug therapy / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D007678:Kidney Glomerulus / Q000201:enzymology*; D008297:Male; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013481:Superoxides / Q000378:metabolism; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000378:metabolism
chemical_list: D000975:Antioxidants; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D019255:NADPH Oxidases; C003402:sapropterin