Mechanism of Renal Injury by Active Oxygen Species Produced by Cytochrome P450

糖尿病、缺血/再灌流和某些药物如环孢素可导致肾功能衰竭。自由基被认为是肾脏损伤的重要因素，但其机制尚不清楚。细胞色素P450(P450)是一种产生活性氧的单加氧酶。据报道，P450的一种抑制剂可减轻缺血/再灌流引起的肾脏损伤。本研究旨在探讨P450产生的自由基在肾损伤中的作用。首先，通过电子自旋共振(ESR)鉴定了产生羟基自由基的P450亚型。产生羟基自由基活性最高的是CYP2B1和3A2，其次是CYP2E1和4A2。CYP2B1和3A2是苯巴比妥(PB)在大鼠肝脏中的诱导形式。酒精和糖尿病可在肝脏和肾脏中诱导细胞色素P450-2E1的表达。细胞色素P4A2是一种主要的肾脏P450，由环孢素A诱导。PB可引起大鼠肝脏脂质过氧化，使DNA损伤标记物8-羟基-2‘-脱氧鸟苷(8-OHdG)升高。另外，用抗细胞色素P450-2B1和8-羟色胺G抗体进行免疫组织化学研究，发现其染色面积与8-羟色胺抗体相同。此外，取肾来源的LLC-PK1细胞作为缺血再灌注模型。乳酸脱氢酶(LDH)测定细胞损伤。由NADPH、P450-还原酶和P450组成的电子传递系统抑制剂减少了LDH的释放，表明该抑制剂减轻了由活性氧物种引起的细胞损伤。这些结果表明，P450产生的活性氧物种损害了膜脂并到达了基因组DNA。P450在自由基致肾损伤中起着重要作用。

Diabetes, ischemia/reperfusion, and some drus such as cyclosporin induce renal failure. Free radicals are thought to be an important factor in renal injury but its mechanism is not clear. Cytochrome P450 (P450) which is a monooxygenase produces active oxygen species. It is reported that an inhibitor of P450 reduces renal injury caused by ischemia/reperfusion. In this study, a role of free radical produced by P450 in renal injury was investigated. First, isoforms of P450 which produced hydroxyl radicals were identified by electron spin resonance (ESR). CYP2B1 and 3A2 had the highest activity for hydroxyl radical production and CYP2E1 and 4A2 had the next highest activity. CYP2B1 and 3A2 are phenobarbital (PB)-inducible forms in rat liver. CYP2E1 is induced in live and kidney by alcohol and diabetes. CYP4A2 is a major renal P450 and induced by cyclosporin. Treatment of rats with PB induced lipid-peroxidation and increased 8-hydroxy-2'-deoxyguanosine (8-OHdG), DNA damage maker, in rat liver. In addition, immunochemical study with antibodies against CYP2B1 and 8-OHdG revealed that areas stained with CYP2B1 antibody were identical with those with 8-OHdG antibody. Furthermore, LLC-PK1 cells which derived from kidney was used for ischemia/reperfusion model. Cell damage was assayd by release of lactate dehydrogenase (LDH). An inhibitor for electron transfer system constituted with NADPH, P450-reductase, and P450 decreased the release of LDH,indicating that the inhibitor decreased the cell damage caused by active oxygen species.These findings suggested that active oxygen species produced by P450 damaged membrane lipids and reached genomic DNA.P450 has an important role in renal injury caused by free radicals.