霉酚酸酯抑制大鼠肾脏缺血再灌注损伤诱导的肾小管上皮细胞凋亡

Mycophenolate mofetil inhibits renal ischemia/reperfusion injury-induced tubular cell apoptosis

目的研究霉酚酸酯(MMF)对大鼠肾脏缺血再灌注(I/R)损伤诱导的肾小管上皮细胞凋亡的保护作用及机制。方法Wistar大鼠随机分为假手术组、I/R组、I/R+MMF组。双侧肾动脉夹闭30 min再灌注18 h制作动物模型。观察肾功能及肾脏病理改变。以原位末端标记(TUNEL)及DNA电泳方法检测肾小管上皮细胞凋亡情况。RT—PCR法测定肾组织肿瘤坏死因子α(TNF-α)mRNA表达。Westein印迹法测定肾组织半胱氨酸天冬氨酸蛋白酶(caspase)3、8蛋白表达。结果肾脏缺血30 min再灌注18 h后,大鼠肾功能明显减退,肾脏病理改变明显,大量肾小管上皮细胞凋亡,肾组织中TNF-αmRNA表达及caspase-3、-8蛋白表达显著增加。MMF能显著改善肾功能和肾脏组织病理学改变,减轻肾小管上皮细胞凋亡,显著下调TNF-αmRNA表达及caspase-3、-8蛋白表达。结论MMF能抑制肾脏I/R损伤诱导的肾小管上皮细胞凋亡,保护肾功能,其作用至少部分是通过减少肾组织局部TNF-α含量,从而影响了caspase依赖的外源性凋亡途径实现的。

Objective To investigate the protective effect of mycophenolate mofetil (MMF) on tubular cell apoptosis in a rat model of renal ischemia/reperfusion(I/R) injury. Methods A total of 24 male Wistar rats were randomly divided into three groups (sham-operated group, I/R group and I/R+ MMF group). Rats except the sham were subjected to 30 min of renal ischemia by clamping both renal arteries with non-traumatic vascular clamps. The animals were pretreated with vehicle or MMF (20 mg·kg-1·d-1) by gavage beginning 2 d before ischemia and maintained during the entire study. Rats were sacrificed at 18 h of reperfusion. Blood sample and both kidneys were harvested for further analysis. Renal function following I/R was determined by measuring serum creatinine (Scr). The changes of renal morphology were observed by periodic acid-Schiff (PAS) staining. Tubular cell apoptosis was confirmed by DNA laddering and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect mRNA expression of renal TNF-α. Protein expression of renal caspase-3,-8 was analyzed by Western blot. Results Scr levels were significantly increased after renal I/R injury. Kidneys of I/R injury model rats displayed significant pathologic changes. Renal I/R injury induced a significant increase of apoptotic tubular cells, DNA fragmentation accompanied by increased mRNA expression of TNF-α and protein expression of caspase-3, -8. MMF significantly improved renal function and attenuated renal histopathologic changes compared to rats with I/R injury. Apoptotic tubular cells decreased by pretreatment with MMF. MMF significantly inhibited the up-regulation of TNF-α mRNA expression and caspase-3,-8 protein expression as well. Conclusions Pretreatment with MMF decreases tubular cell apoptosis, which maybe, at least partly, due to the weakened death receptor (extrinsic) pathway of apoptosis resulting from decreased TNF-α content.