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

糖尿病、缺血/再灌注以及环孢素等药物可引起肾功能衰竭。自由基被认为是肾损伤的重要因素，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抑制剂可减轻缺血/再灌注引起的肾损伤。本研究探讨了P450自由基在肾损伤中的作用。首先，利用电子自旋共振（ESR）鉴定了产生羟基自由基的P450同工异构体。CYP2B1和3A2产生羟基自由基的活性最高，其次是CYP2E1和4A2。CYP2B1和3A2是大鼠肝脏中苯巴比妥（PB）诱导的形式。酒精和糖尿病在活体和肾脏中诱导CYP2E1。CYP4A2是主要的肾P450，由环孢素诱导。PB处理大鼠肝脏中脂质过氧化和DNA损伤制造者8-羟基-2'-脱氧鸟苷（8-OHdG）升高。此外，用CYP2B1抗体和8-OHdG抗体进行免疫化学研究发现，CYP2B1抗体染色区域与8-OHdG抗体染色区域相同。采用肾源性LLC-PK1细胞建立缺血再灌注模型。通过乳酸脱氢酶（LDH）的释放测定细胞损伤程度。由NADPH、P450-还原酶和P450组成的电子传递系统抑制剂可减少LDH的释放，表明该抑制剂可减轻活性氧对细胞的损伤。这些发现表明P450产生的活性氧破坏了膜脂并到达基因组DNA。P450在自由基引起的肾损伤中起重要作用。

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.