Ho Cheng, Lee Pei-Hsien, Huang Wei-Jan, Hsu Yen-Chen, Lin Chun-Liang, Wang Jeng-Yi
Division of Endocrinology and Metabolism, Chang Gung Memorial Hospital, Chiayi, Taiwan.
Nephrology (Carlton). 2007 Aug;12(4):348-56. doi: 10.1111/j.1440-1797.2007.00809.x.
The formation of methylglyoxal (MGO), a highly reactive dicarbonyl compound, is accelerated under diabetic conditions. Although recent studies have suggested that apoptotic cell death is involved in diabetic nephropathy, the precise mechanism of MGO-induced renal fibrosis remains to be elucidated.
Rat kidney mesangial cells with or without pretreatment with inhibitors, including superoxide dismutase, catalase, L-NAME, diphenylene iodonium, rotenone, allopurinol, PD98059, SB203580 and SP600125 were cultured in medium containing 100 microM MGO. In the MGO-treated cell culture system, fibrosis-related signalling pathway was assessed by enzyme-linked immunosorbent assay, reverse transcription-polymerase chain reaction and western blotting.
Expression of fibronectin induced by MGO was highest after 48 h treatment. Superoxide production rapidly increased after 2 h and remained at a high level for 24 h. Scavenging O(2) (-) reversed transforming growth factor beta 1 (TGF-beta1) and fibronectin mRNA level. Pretreatment with diphenylene iodonium significantly suppressed MGO-induced superoxide, TGF-beta1 expression and fibronectin gene expression, indicating that NADPH oxidase is responsible for inducing superoxide formation and subsequently induced renal fibrosis. High MGO rapidly enhanced Ras activation in 1 h and progressively increased cytosolic p38 activation. Additionally, SB203580 pretreatment reduced MGO promotion of fibronectin gene activation suggesting that cytosolic p38 activation might affect MGO-induced renal mesangial fibrosis. Inhibiting Ras activity with manumycin A significantly reduced the promoting effect of MGO on superoxide synthesis, and fibronectin expression.
Induction of superxoide by Ras via p38 pathway activates fibrotic gene transcription of mesangial cells. Reduction of oxidative stress by scavenging superoxide may offer an alternative strategy for controlling MGO-induced renal fibrosis.
甲基乙二醛（MGO）是一种高反应性二羰基化合物，在糖尿病条件下其形成会加速。尽管最近的研究表明凋亡性细胞死亡与糖尿病肾病有关，但MGO诱导肾纤维化的确切机制仍有待阐明。
用超氧化物歧化酶、过氧化氢酶、L-硝基精氨酸甲酯、二亚苯基碘鎓、鱼藤酮、别嘌呤醇、PD98059、SB203580和SP600125等抑制剂预处理或未预处理的大鼠肾系膜细胞在含有100微摩尔MGO的培养基中培养。在MGO处理的细胞培养系统中，通过酶联免疫吸附测定、逆转录-聚合酶链反应和蛋白质印迹法评估纤维化相关信号通路。
MGO诱导的纤连蛋白表达在处理48小时后最高。超氧化物产生在2小时后迅速增加，并在24小时内保持在高水平。清除O(2) (-)可逆转转化生长因子β1（TGF-β1）和纤连蛋白mRNA水平。用二亚苯基碘鎓预处理可显著抑制MGO诱导的超氧化物、TGF-β1表达和纤连蛋白基因表达，表明NADPH氧化酶负责诱导超氧化物形成并随后诱导肾纤维化。高浓度MGO在1小时内迅速增强Ras激活，并逐渐增加细胞溶质p38激活。此外，SB203580预处理降低了MGO对纤连蛋白基因激活的促进作用，表明细胞溶质p38激活可能影响MGO诱导的肾系膜纤维化。用马马霉素A抑制Ras活性可显著降低MGO对超氧化物合成和纤连蛋白表达的促进作用。
Ras通过p38途径诱导超氧化物激活系膜细胞的纤维化基因转录。通过清除超氧化物降低氧化应激可能为控制MGO诱导的肾纤维化提供一种替代策略。
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