自噬对肾大部切除大鼠肾小管上皮细胞程序性坏死的影响

目的:探讨自噬是否参与肾大部切除(SNx)大鼠肾小管上皮细胞的过度死亡,及其与程序性坏死的关系。方法:48只雄性SD大鼠随机分为control组(6只)和SNx组(42只),分别行假手术和SNx。将24只SNx大鼠分为0、4、8和12周组;其余SNx大鼠分为SNx+vehicle组、SNx+necrostatin-1(Nec-1)组和SNx+3-甲基腺嘌呤(3-MA)组,每组6只。检测0、4、8和12周组大鼠肾组织RIP1、RIP3、LC3和beclin-1的mRNA和蛋白表达水平;用Nec-1和3-MA干预SNx大鼠,Western blot法检测LC3-Ⅰ、LC3-Ⅱ和beclin-1的蛋白水平,透射电镜和TUNEL染色判定Nec-1和3-MA对SNx大鼠肾小管上皮细胞死亡的影响,并观察Nec-1和3-MA对SNx大鼠肾组织的病理变化、活性氧簇(ROS)、血尿素氮(BUN)和血肌酐(SCr)含量的影响。结果:SNx术后8周大鼠肾组织RIP1、RIP3、LC3和beclin-1的mRNA和蛋白水平达最高值(P0.01);Nec-1和3-MA干预SNx大鼠的LC3-Ⅱ/Ⅰ和beclin-1蛋白水平、发生程序性坏死的肾小管上皮细胞及TUNEL阳性细胞数量均显著降低(P0.01)。另外,Nec-1减低SNx大鼠肾组织的ROS含量,但3-MA无此作用。结论:自噬参与了SNx大鼠肾小管上皮细胞过度死亡;抑制自噬可减轻SNx大鼠肾小管上皮细胞程序性坏死及其肾损伤。

Effect of autophagy on necroptosis of renal tubular epithelial cells in subtotal nephrectomy rats

AIM: To explore whether autophagy is involved in the excessive death of renal tubular epithelial cells in subtotal nephrectomy(SNx) rats and the relationship between autophagy and necroptosis in the kidney of SNx rats. METHODS: Male Sprague-Dawley rats were randomly assigned to control group(n=6) and SNx group(n=42). The rats in SNx group were subjected to SNx. Sham surgery was performed in the rats in control group. The rats in SNx group were divided into subgroups at 0, 4, 8 and 12 weeks(n=6) and the other rats in SNx group were divided into SNx+vehicle group, SNx+necrostatin-1(Nec-1) group and SNx+3-methyladenine(3-MA) group. The expression of RIP1, RIP3, LC3 and beclin-1 at mRNA and protein levels was measured at 0, 4, 8 and 12 weeks by qPCR and immunohistochemistry. The effects of Nec-1 or 3-MA on the protein expression of LC3-I, LC3-II and beclin-1, and production of reactive oxygen species(ROS) in the rat kidney were determined by Western blot and DCFH-DA staining. The death of renal tubular epithelial cells in the SNx rats was observed by TUNEL staining and electron microscopy. Finally, the effects of Nec-1 and 3-MA on blood urea nitrogen(BUN), serum creatinine(SCr) and the pathological changes of the renal tissues were analyzed. RESULTS: The highest mRNA and protein levels of RIP1, RIP3, LC3 and beclin-1 appeared at the 8th week after SNx(P<0.01). Compared with the rats in SNx+vehicle group, the protein over-expression of LC3-II/I and beclin-1, renal tubular epithelial cells with typical morphological features of necroptotic cell death and TUNEL-positive renal tubular cells were decreased in the SNx rats treated with Nec-1 and 3-MA(P<0.01), but 3-MA did not reduce the increased concentration of ROS. In addition, treatment with Nec-1 and 3-MA obviously reduced BUN, SCr(P<0.05), glomerulosclerosis index and tubulointerstitial injury score(P<0.01). CONCLUSION: Autophagy participates in the excessive death of renal tubular epithelial cells in SNx rats. Inhibition of autograph prevents necroptotic cell death of renal tubular cells, and alleviates chronic renal injury in SNx rats.