Mechanisms of podocyte injury in diabetes: role of cytochrome P450 and NADPH oxidases.

We investigated the role of cytochrome P450 of the 4A family (CYP4A), its metabolites, and NADPH oxidases both in reactive oxygen species (ROS) production and apoptosis of podocytes exposed to high glucose and in OVE26 mice, a model of type 1 diabetes. Apoptosis, albuminuria, ROS generation, NADPH superoxide generation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo. Exposure of mouse podocytes to high glucose resulted in apoptosis, with approximately one-third of the cells being apoptotic by 72 h. High-glucose treatment increased ROS generation and was associated with sequential upregulation of CYP4A and an increase in 20-hydroxyeicosatetraenoic acid (20-HETE) and Nox oxidases. This is consistent with the observation of delayed induction of NADPH oxidase activity by high glucose. The effects of high glucose on NADPH oxidase activity, Nox proteins and mRNA expression, and apoptosis were blocked by N-hydroxy-N′-(4-butyl-2-methylphenol) formamidine (HET0016), an inhibitor of CYP4A, and were mimicked by 20-HETE. CYP4A and Nox oxidase expression was upregulated in glomeruli of type 1 diabetic OVE26 mice. Treatment of OVE26 mice with HET0016 decreased NADPH oxidase activity and Nox1 and Nox4 protein expression and ameliorated apoptosis and albuminuria. Generation of ROS by CYP4A monooxygenases, 20-HETE, and Nox oxidases is involved in podocyte apoptosis in vitro and in vivo. Inhibition of selected cytochrome P450 isoforms prevented podocyte apoptosis and reduced proteinuria in diabetes.

我们研究了4A家族细胞色素P450（CYP4A）、其代谢产物以及NADPH氧化酶在暴露于高糖的足细胞的活性氧（ROS）产生和凋亡以及1型糖尿病模型OVE26小鼠中的作用。 在体外和体内测量了凋亡、蛋白尿、ROS产生、NADPH超氧化物产生、CYP4A和Nox蛋白表达以及mRNA水平。 将小鼠足细胞暴露于高糖环境会导致细胞凋亡，到72小时时约有三分之一的细胞发生凋亡。高糖处理增加了ROS的产生，并与CYP4A的依次上调以及20 - 羟基二十碳四烯酸（20 - HETE）和Nox氧化酶的增加相关。这与观察到的高糖对NADPH氧化酶活性的延迟诱导是一致的。高糖对NADPH氧化酶活性、Nox蛋白和mRNA表达以及凋亡的影响可被CYP4A抑制剂N - 羟基 - N′ -（4 - 丁基 - 2 - 甲基苯酚）甲脒（HET0016）阻断，并可被20 - HETE模拟。在1型糖尿病OVE26小鼠的肾小球中，CYP4A和Nox氧化酶表达上调。用HET0016治疗OVE26小鼠可降低NADPH氧化酶活性以及Nox1和Nox4蛋白表达，并改善细胞凋亡和蛋白尿情况。 CYP4A单加氧酶、20 - HETE和Nox氧化酶产生的ROS参与了体外和体内足细胞的凋亡。抑制特定的细胞色素P450同工酶可防止足细胞凋亡并减少糖尿病中的蛋白尿。