自噬在乙二醇诱导的大鼠肾内晶体形成中的调控作用

Role of autophagy in formation of intrarenal crystals induced by ethylene glycol in rats

目的:探讨自噬在乙二醇诱导的大鼠肾内晶体形成中的调控作用。方法:健康雄性SPF级SD大鼠40只,分为正常对照组、结石模型组、N-乙酰半胱氨酸(NAC)处理组(0.75%乙二醇+NAC)、雷帕霉素处理组(0.75%乙二醇+雷帕霉素)和氯喹处理组(0.75%乙二醇+氯喹),每组8只。干预4周后,应用Western blot和免疫组化检测各组肾脏组织中自噬关键蛋白LC3-II的表达水平;透射电镜观察各组肾组织中自噬泡的数量;应用试剂盒检测各组24 h尿液中总超氧化物歧化酶(T-SOD)和谷胱甘肽过氧化物酶(GSH-Px)的水平;全自动生化仪检测各组血清肌酐及尿素氮的水平;ELISA法检测各组24 h尿液中的中性粒细胞明胶酶相关脂质运载蛋白(NGAL)和肾损伤分子1(Kim-1)的水平。钙盐染色法观测各组肾脏晶体的沉积情况,评价肾组织的病理学变化。结果:与结石模型组相比,雷帕霉素处理组和氯喹处理组中LC3-II的表达水平和自噬泡数量均增加,而在NAC处理组中LC3-II的表达水平和自噬泡数量均减少(P<0.05)。与结石组相比,雷帕霉素处理组中T-SOD和GSH-Px水平降低,而氯喹处理组和NAC处理组中T-SOD和GSH-Px的活性增加(P<0.05)。与正常组相比,结石组中血清肌酐、尿素氮、NGAL和Kim-1的水平及肾脏内晶体沉积增加(P<0.05);在雷帕霉素处理组中肾脏损伤进一步加重,晶体沉积进一步增加,而在氯喹处理组和NAC处理组中肾脏损伤明显减轻,晶体的沉积明显减少。结论:乙二醇激活自噬后可以促进大鼠肾内晶体的形成。应用抗氧化剂和自噬抑制剂不仅可以降低肾脏内的自噬水平,还可以减轻肾脏损伤,减少晶体沉积,降低肾结石的形成率。

AIM: To investigate the role of autophagy in the formation of intrarenal crystals induced by ethy-lene glycol in rats. METHODS: Healthy male SPF SD rats(n=40) were divided into 5 groups(n=8): normal control group, stone model group, rapamycin treatment group(0.75% ethylene glycol + rapamycin), chloroquine treatment group(0.75% ethylene glycol + chloroquine) and N-acetylcysteine(NAC) treatment group(0.75% glycol + NAC). After 4 weeks of intervention, Western blot and immunohistochemistry were applied to detect the expression levels of autophagy protein LC3-II in the kidney tissues of each group. The number of autophagic vacuoles in each group was observed under transmission electron microscope. The level of total superoxide dismutase(T-SOD) in urine from each group was detected by T-SOD kit, while the level of glutathione peroxidase(GSH-Px) was measured by GSH-Px kit. The serum levels of creatinine and urea nitrogen in each group were examined by automatic biochemical analyzer. The levels of neutrophil gelatinase-associated lipocalin(NGAL) and kidney injury molecule 1(Kim-1) in 24 h urine of each group were measured by ELISA. Von Kossa staining was used to observe the deposition of renal crystals in each group for evaluating the pathological changes of renal tissues. RESULTS: Compared with model group, the expression level of autophagy protein LC3-II and the quantity of autophagic vacuoles in rapamycin treatment group and chloroquine treatment group were increased, while those in NAC treatment group were reduced(P<0.05). Compared with model group, the urine levels of T-SOD and GSH-Px were decreased in rapamycin treatment group, while those in chloroquine treatment group and NAC treatment group were increased(P<0.05). Compared with normal group, the serum levels of creatinine, urea nitrogen, NGAL and Kim-1, and the crystal deposition in the kidneys in model group were increased. The renal damage and crystal deposition were further increased in rapamycin treatment group, while those in chloroquine and NAC treatment groups were significantly reduced. CONCLUSION: Ethylene glycol-activated autophagy promotes the formation of intrarenal crystals in rats. After application of antioxidants and autophagy inhibitors, the level of autophagy in the kidney, the renal tissue damage, the crystal deposition and the formation rate of kidney stones are reduced.