自噬与阿霉素肾病大鼠肾组织损伤的关系

The relationship between autophagy and renal tissue injury in adriamycin nephropathy rats

目的 观察阿霉素肾病（adriamycin nephropathy）大鼠不同病理时期自噬体的形成及自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ及Beclin-1的表达及分布情况,探讨自噬与肾组织损伤、肾脏疾病进展的关系.方法60只健康雄性SD大鼠被随机分成对照组和模型组,各30只,模型组按6.5 mg/kg一次性尾静脉注射阿霉素,对照组给予一次性尾静脉注射等量生理盐水.动态监测2、4、6、8和10周各组大鼠24h尿蛋白量、血清白蛋白（Alb）、血清总胆固醇（TC）、血肌酐（Scr）及尿素氮（BUN）的变化,光镜和透射电镜分别观察肾脏组织病理学变化以及足细胞结构和自噬体的形成,免疫荧光染色检测LC3-Ⅰ、LC3-Ⅱ及Beclin-1在肾组织的定位及分布变化,Western印迹法检测自噬相关蛋白LC3-Ⅰ、LC3-Ⅱ及Beclin-1的表达.结果 模型组第2周时24 h尿蛋白量明显升高,Alb下降,第4周时TC已明显升高,与对照组比较差异有统计学意义（P＜0.01）;模型组第4周时Scr及BUN呈轻度升高,第8周后肾功能恶化,呈进行性加重,与对照组比较差异有统计学意义（P＜0.01）;光镜下肾脏组织病理改变由微小病变（MCD）逐渐发展至局灶节段性肾小球硬化（FSGS）;透射电镜下早期出现系膜细胞增生,溶酶体等细胞器形态及分布异常,足突增宽及弥漫性融合,晚期出现足突消失及核固缩.透射电镜及免疫荧光可观察到对照组大鼠肾组织自噬较弱,而在模型组,随着肾病病情进展,自噬明显增强,维持在高水平;Western印迹法检测到模型组大鼠在肾病病情进展过程中,LC3-Ⅰ、LC3-Ⅱ及Beclin-1蛋白表达增强,与对照组比较差异有统计学意义（P＜0.05）.结论 自噬参与肾脏组织损伤及蛋白尿的产生;自噬可能与肾脏疾病的进展密切相关.

Objective To observe the formation of autophagosome,the expression and distribution of autophagy-related protein LC3-Ⅰ,LC3-Ⅱ and Beclin-1 in adriamycin nephropathy rats at different pathological periods,to explore the relationship between autophagy and renal tissue injury,the occurrence of proteinuria,the progression of renal disease.Methods Sixty normal male SD rats were randomly divided into control group （n=30） and model group （n=30）,the rats in model group were injected with adriamycin（6.5 mg/kg） via tail-vein for one time,while the rats in control group were injected with saline.Urine protein quantitation of 24 hour,the levels of serum albumin and total cholesterol were measured serially at the 2,4,6,8,10 weeks.The changes of kidney tissue pathology were detected after HE,PAS and Masson staining by light microscope.The formation of autophagy were detected by transmission electron microscopy,the localization and distribution of LC3-Ⅰ,LC3-Ⅱ and Beclin-1 were detected by indirect immunofluorescence staining in kidney tissue,the autophagy-related proteins LC3-Ⅰ,LC3-Ⅱ and Beclin-1 expression was detected by Western blotting.Results In model group,urinary protein began to increase at the first two weeks,serum albumin decreased at the same time,and total cholesterol increased in the four weeks.There was a statistically significant difference compared with the control group （P 〈 0.01）.The Scr and BUN were increased slightly at the four weeks in model group,and showed the deterioration of renal function after the eight weeks.There was a statistically significant difference compared with the control group （P 〈 0.01）.Mesangial cell proliferation,mitochondrial swelling and foot process broadening and integration appeared early in the model group,while foot process disappearing and nuclear pyknosis were observed in the late by transmission electron microscope;Renal pathology gradually changed from mesangial proliferation to focal segmental glomerulosclerosis （FSGS） by light microscope.A low expression of autophagy was detected in renal tissue of control group rats by transmission electron microscopy and immunofluorescence microscope;in model group,with the progression of disease,the autophagy was significantly enhanced and maintained at a high level.With the progression of disease,the autophagyrelated proteins LC3-Ⅰ,LC3-Ⅱ and Beclin-1 was significantly enhanced in the model group than the control group （P〈0.05）.Conclusion Autophagy is involved in renal tissue injury and the occurrence of proteinuria,closely related to the progression of renal disease.