title: Induction of reactive oxygen species from isolated rat glomeruli by protein kinase C activation and TNF-alpha stimulation, and effects of a phosphodiesterase inhibitor.
abstract: Diabetic nephropathy is a major complication of diabetes leading to end-stage renal disease, which requires hemodialysis. Although the mechanism by which it progresses is largely unknown, the role of hyperglycemia-derived oxidative stress has recently been the focus of attention as the cause of diabetic complications. Constituent cells of the renal glomeruli have the capacity to release reactive oxygen species (ROS) upon stimulation of NADPH oxidase activated by protein kinase C (PKC). Hyperglycemia and insulin resistance in the diabetic state are often associated with activation of PKC and tumor necrosis factor (TNF)-alpha, respectively. The aim of this study is to clarify the signaling pathway leading to ROS production by PKC and TNF-alpha in rat glomeruli. Isolated rat glomeruli were stimulated with phorbol 12-myristate 13-acetate (PMA) and TNF-alpha, and the amount of ROS was measured using a chemiluminescence method. Stimulation with PMA (10 ng/ml) generated ROS with a peak value of 136+/-1.2 cpm/mg protein (mean+/-SEM). The PKC inhibitor H-7, the NADPH oxidase inhibitor diphenylene iodonium and the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin inhibited PMA-induced ROS production by 100%, 100% and 80%, respectively. In addition, TNF-alpha stimulated ROS production (283+/-5.8/mg protein/20 min). The phosphodiesterase inhibitor cilostazol activates protein kinase A and is reported to improve albuminuria in diabetic rats. Cilostazol (100 microg/ml) inhibited PMA, and TNF-alpha-induced ROS production by 78+/-1.8, and 19+/-2.7%, respectively. The effects of cilostazol were not additive with wortmannin. Cilostazol arrests oxidative stress induced by PKC activation by inhibiting the PI-3 kinase-dependent pathway, and may thus prevent the development of diabetic nephropathy.
authors.affiliation: Department of Disease Control and Homeostasis, Kanazawa University Graduate School of Medical Science, 13-1 Takara-Machi, Kanazawa, Ishikawa, Japan.
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Koike
authors.forename: Toshinari
authors.initials: T
authors.lastname: Takamura
authors.forename: Shuich
authors.initials: S
authors.lastname: Kaneko
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000494:pharmacology; D003921:Diabetes Mellitus, Experimental / Q000201:enzymology*; D003928:Diabetic Nephropathies / Q000201:enzymology / Q000517:prevention & control; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006943:Hyperglycemia / Q000201:enzymology; D007333:Insulin Resistance; D007678:Kidney Glomerulus / Q000201:enzymology*; D008297:Male; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000187:drug effects*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D002273:Carcinogens; D004791:Enzyme Inhibitors; D017382:Reactive Oxygen Species; D014409:Tumor Necrosis Factor-alpha; D011493:Protein Kinase C; D010727:Phosphoric Diester Hydrolases; D013755:Tetradecanoylphorbol Acetate