A study of oxidant stress induced cellular injury in cultured mesangial cells

为建立急性肾功能衰竭的体外模型，用1 mM次黄嘌呤（HX）和50 u/ml黄嘌呤氧化酶（XO）联合作用于培养的大鼠内髓集合管细胞，产生氧化应激。氧化应激诱导1）增加细胞内钙离子浓度（Ca）i，并通过荧光指示剂碘化丙啶检测降低细胞活力。2)氧化应激诱导的细胞损伤通过去除细胞外钙、给予钙拮抗剂维拉帕米和细胞外代谢性酸中毒来预防。其次，在培养的系膜细胞中应用相同的操作。1 mM HX和5 u/ml XO联合诱导的氧化应激可引起细胞内钙离子浓度升高，并通过碘化丙啶强度检测细胞损伤。钙拮抗剂维拉帕米和细胞外代谢性酸中毒的管理，防止氧化应激诱导的细胞损伤。在本方案中，苏佩 关于我们 还应用了过氧化物歧化酶（SOD），然而，在本文所检测的任何浓度（100 u/ml至3000 u/ml）下，SOD均不提高细胞活力。加入过氧化氢酶（一种氢氧化物的清除剂）后，细胞活力明显提高到对照组水平。第三，同样的方法应用于离体兔近端直小管（PST）。采用体外离体管状微灌流法对PST进行灌流。1 mMHX不影响PST的水重吸收率（Jv），与0.5u/mlXO联合作用20-40 min可引起急性肾小管坏死常见的刷状缘膜脱离。在此条件下，Jv显著下降，40 min时Jv降至接近零.维拉帕米的应用可部分阻止HX和XO引起的形态学和功能变化，而基底侧代谢性酸中毒则完全阻止了这些变化。结果表明，次黄嘌呤和黄嘌呤氧化酶联合作用可引起体外培养的内髓集合管、系膜细胞和近端直小管的细胞损伤。钙拮抗剂部分阻止氧化应激引起的细胞损伤，细胞外代谢性酸中毒完全阻止细胞损伤。SOD单独给药不能防止氧化应激诱导的细胞损伤，然而，与过氧化氢酶的组合可以完全防止氧化应激诱导的细胞损伤。少

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 … More 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