自噬在镉致大鼠肾脏损伤中的作用

目的:探讨不同浓度氯化镉染毒对大鼠肾脏功能的损伤及自噬在肾脏损伤中的作用,初步阐明镉损伤毒作用机制。方法:将40只大鼠随机分为空白对照组、低剂量染毒组(0.2 mg·kg^-1 CdCl₂)、中剂量染毒组(0.4 mg·kg^-1 CdCl₂)和高剂量染毒组(0.8 mg·kg^-1 CdCl₂),每组10只。腹腔注射给予氯化镉溶液制备肾损伤模型,空白对照组大鼠给予等量生理盐水,观察大鼠一般状态和体质量。5周后,取大鼠肾脏组织和血液,检测肾功能相关指标肌酐、尿素氮和β2微球蛋白表达水平;原子吸收光谱法测定肾脏组织中镉水平;免疫组织化学法检测肾脏组织中自噬相关Beclin1蛋白的表达水平;Western blotting法检测肾脏组织中自噬相关蛋白LC3B的相对表达水平。结果:各染毒组大鼠明显精神萎靡,从染毒第3周开始,高剂量染毒组大鼠体质量增长减慢,显著低于空白对照组(P<0.05);至染毒结束,中、高剂量染毒组大鼠体质量均显著低于空白对照组(P<0.05)。中、高剂量染毒组大鼠血清肌肝、尿素氮和β2微球蛋白表达水平较空白对照组显著升高(P<0.05),且高剂量染毒组大鼠血清肌肝水平显著高于低剂量染毒组(P<0.05),中、高剂量染毒组大鼠尿素氮水平显著高于低剂量组(P<0.05)。原子吸收光谱法检测,各染毒组大鼠肾脏组织中蓄积大量金属镉,空白对照组仅见微量镉蓄积,且低、中和高剂量染毒组大鼠镉的蓄积量呈递增趋势。与低剂量染毒组比较,中、高剂量染毒组大鼠肾脏组织中镉水平显著升高(P<0.05);与中剂量染毒组比较,高剂量染毒组大鼠肾脏组织中镉水平显著升高(P<0.05)。免疫组织化学法检测,各染毒组大鼠肾脏组织中Beclin1蛋白表达水平显著高于空白对照组(P<0.05),且中、高剂量染毒组大鼠肾脏组织中Beclin1蛋白表达水平显著高于低剂量组(P<0.05),同时高剂量染毒组大鼠肾脏组织中Beclin1蛋白表达水平显著高于中剂量组(P<0.05)。Western blotting检测,各染毒组大鼠肾脏组织中LC3B蛋白相对表达水平显著高于空白对照组(P<0.05),且中、高剂量染毒组大鼠肾脏组织中LC3B蛋白相对表达水平显著高于低剂量组(P<0.05)。结论:在0.2~0.8 mg·kg^-1剂量范围内,随着氯化镉染毒剂量增加,镉对大鼠肾脏功能损伤逐渐加重,其机制可能与增加肾脏细胞自噬有关联。

Role of autophagy in kidney damage of rats caused by cadmium

Objective To explore the kidney damage in the rats caused by different concentrations of CdCl₂ and the role of autophagy in the kidney damage,and to clarify the mechanism of the kindney damage caused by CdCl₂.Methods 40 Wistar rats were randomly divided into blank control group,low dose group (0.2 mg·kg^-1 CdCl₂),middle dose group(0.4 mg·kg^-1 CdCl₂) and high dose group (0.8 mg·kg^-1 CdCl₂),and there were 10 rats in each group. The cadmium chloride solution was administrated by intraperitoneal injection to prepare the rat models of kidney damage. The same volume of normal saline was injected into the rats in blank control goup. The general status and body weights of all rats were observed. The kidney tissue and blood of the rats were obtained 5 weeks later. The renal function indexes including creatinine,blood urea nitrogen,and β2-microglobulin were detected;the levels of cadmium in the kidney tissue of the rats in various groups were detected by atomic absorption spectrometry;the relative expression level of LC3B and expression level of Beclin1 protein were detected by Western blotting and immunohistochemical methods,respectively.Results Obvious depression was seen in the rats in CdCl₂ groups,and the body weights of the rats in high dose group were significantly lower than those in blank control group from 3 weeks after exposure to cadmium (P<0.05);the body weights of the rats in middle dose group and high dose group were significantly lower than those in blank control group at the end of this experiment (P<0.05). The results of blood biochemical detection showed that the levels of creatinine,blood urea nitrogen, and β2-microglobulin of the rats in middle dose group and high dose group were significantly higher than those in blank control group (P<0.05),and the levels of creatinine of the rats in high dose group were significantly higher than those in low dose group (P<0.05);the levels of blood urea nitrogen of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05). The results of atomic absorption spectrometry showed that there were a small amount of cadmium accumulation of the rats in blank control group,but a large amount of metal cadmium accumulated in the kidney tissue were seen in low,middle and high dose groups and the levels of cadmium were increased with the increasing of the exposure dose;the levels of cadmium of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05);the levels of cadmium of the rats in high dose group were significantly higher than those in middle dose group (P<0.05).The immunohistochemistry results showed that the expression levels of Beclin1 protein in the kidney tissue of the rats in CdCl₂ groups were significantly higher than those in blank control group(P<0.05),and the expression levels of Beclin1 protein of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05);the expression levels of Beclin1 protein of the rats in high dose group were significantly higher than those in middle dose group (P<0.05).The Western blotting results showed that the relative expression levels of LC3B protein in the kidney tissue of the rats in CdCl₂ groups were significantly higher than those in blank control group (P<0.05),and the relative expression levels of LC3B protein in the kidney tissue of the rats in middle dose group and high dose group were significantly higher than those in low dose group (P<0.05).Conclusion At the range from 0.2 to 0.8 mg·kg^-1,the kidney damage gets more and more serous with the increasing of the exposure dose of CdCl₂,and autophagy may be one of the mechanisms.