古谷 裕章 自治医科大学, 医学部, 助手 (40240981)
TABEI Kaoru Faculty of Medicine, Jichi Medical School Lecturer, 医学部, 講師 (90155234)
KUSANO Eiji Faculty of Medicine, Jichi Medical School Associate Professor, 医学部, 助教授 (50102249)
KURODA Yutaka Faculty of Medicine, Jichi Medical School Lecturer, 医学部, 助手 (70240984)
|Budget Amount *help
¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1995 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1994 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1993 : ¥1,000,000 (Direct Cost : ¥1,000,000)
To aim to produce a in vitro model of acute renal failure, oxidant stress was produced by the combination of 1 mM Hypoxanthine (HX) and 50u/ml Xanthine oxidase (XO) in cultured rat inner medullary collecting duct cells. Oxidant stress induced 1) increased the intracellular calcium ion concentration (Ca) i, and decreased cellular viability examined by a fluorescent indicator, propidium iodide. 2) oxidant stress induced cellular injury was prevented by a removal of extracellular calcium, administration of calcium antagonist, verapamil, and extracellular metabolic acidosis.
Secondary, the same maneuvers were applied in cultured mesangial cells. Oxidant stress, induced by the combination of 1 mM HX and 5 u/ml XO,induced the increase of intracellular calcium, and cellular injury, detected by the intensity of propidium iodide. Oxidant stress induced cellular injury was prevented by an administration of calcium antagonist, verapamil, and extracellular metabolic acidosis. In this protocol, supe
roxide dismutase (SOD) was also applied, however, SOD did not improve cellular viability, at any concentration examined here, 100 u/ml to 3000u/ml. The addition of catalase, an scavenger of hydroxide, to SOD markedly improved cellular viability to the level of control.
Thirdly, the same maneuvers were applied in isolated rabbit proximal straight tubules (PST). PST was perfused by in vitro isolated tubular microperfusion. 1 mM HX did not affect the water reabsorption rate (Jv) in PST.The combination of HX and 0.5 u/ml XO induced brush border membrane detachment in 20-40 min, which is usually seen in acute tubular necrosis. In this condition, Jv decreased markedly, and at 40 min.Jv went down to near zero. The application of verapamil partially prevented the morphological changes and functional changes induced by HX and XO.Basolateral metabolic acidosis completely prevented these changes. However, SOD did not afffect the oxidant stress induced cellular damage in isolated PST in vitro.
These data suggested that the combination of Hypoxanthine and xanthine oxidase induced cellular damage, in cultured inner medullary collecting duct, cultured mesangial cells and isolated proximal straight tubules in vitro. Calcium antagonist partially prevents oxidant stress induced cellular damage, and extracellular metabolic acidosis prevents cellular damage completely. The administration of SOD alone did not prevent oxidant stress induced cellular injury, however, the combination with catalase prevent it completely. Less