Priming prevent nephrotoxic acute renal failure through stimulation of antioxidant defense mechanism

ABSTRACT Introduction: Priming is the mechanism of protection induced by a previous exposition of a cell or organ to low or equal concentrations of a toxic substance. Objective: To analyze the mechanism of priming induced by the previous exposition to gentamicin in human proximal tubular cells and nephrotoxic acute renal failure (ARF). Methods: Wistar rats and tubular cells were exposed to gentamicin 2mM during 24h or 40 mg/kg during 3 days and after one rest week were exposed to the same concentration during 24h in cells and additional ten days in rats. The primed animals were compared to control rats receiving vehicle and GENTA animals treated with the gentamicin during the same period. Biochemical parameters were analyzed. The oxidative stress was analyzed by urinary hydroperoxides and carbonylated protein while antioxidant defense was studied by antioxidant activity of the plasma (FRAP), catalase, superoxide dismutase, heme-oxygenase 1 (HO-1) immunostaining and enzymatic activity in kidney. Necrosis, apoptosis, proliferation, endothelin 1 (ET-1) and HO-1 expression were studied in cells. Results: Priming of the animals inhibited the increase in creatinine, urea, sodium excretion and urinary protein induced by gentamicin. Bosentan, ET-1 receptor antagonist, and hemin, HO-1 inducer, potentiate the inhibition. The mechanism of protection was mediated by induction of the antioxidant enzymes HO-1, catalase and SOD activity and oxidative stress reduction. Priming inhibited cell death and induced proliferation through ET-1 production. Conclusion: Priming is a persistent and multifactorial mechanism, the stimulation of the antioxidant defense could mimics partially the priming process and prevent the ARF.

摘要

引言：预适应（priming）是指细胞或器官预先暴露于低浓度或等浓度有毒物质后所诱导产生的保护机制。
目的：分析预先暴露于庆大霉素（gentamicin）后人近端肾小管上皮细胞及肾毒性急性肾衰竭（acute renal failure, ARF）模型中，预适应的作用机制。
方法：将Wistar大鼠及人近端肾小管上皮细胞分别暴露于2mM庆大霉素环境中24小时，或按40mg/kg的剂量对大鼠连续给药3天；两组对象均经过1周洗脱期后，再以相同浓度分别对细胞继续暴露24小时、对大鼠继续给药10天。将预适应组大鼠与仅给予赋形剂的对照组大鼠，以及同期仅接受庆大霉素处理的庆大霉素组（GENTA组）大鼠进行对比分析。检测各项生化指标；通过尿液氢过氧化物与羰基化蛋白水平评估氧化应激状态；通过血浆铁还原抗氧化能力（ferric reducing ability of plasma, FRAP）、过氧化氢酶、超氧化物歧化酶的活性，以及肾脏组织血红素氧合酶1（heme-oxygenase 1, HO-1）的免疫组化染色及酶活性，评价抗氧化防御系统功能。在细胞层面检测细胞坏死、凋亡、增殖情况，以及内皮素1（endothelin 1, ET-1）与HO-1的表达水平。
结果：预适应可抑制庆大霉素诱导的肌酐、尿素氮、钠排泄量及尿蛋白水平升高。内皮素受体拮抗剂波生坦（bosentan）与HO-1诱导剂氯化血红素（hemin）可增强该保护效应。该保护机制通过诱导抗氧化酶HO-1、过氧化氢酶与超氧化物歧化酶的活性，降低氧化应激水平实现。预适应可抑制细胞死亡，并通过促进ET-1的生成诱导细胞增殖。
结论：预适应是一种持续且多因素的保护机制，激活抗氧化防御系统可部分模拟预适应过程，从而预防肾毒性急性肾衰竭的发生。