链脲佐菌素糖尿病肾病大鼠模型肾小球损伤的机制

Mechanism of glomerular damage in rat model of STZ diabetic nephropathy

目的:探讨糖尿病肾病大鼠模型肾小球细胞损伤中自噬与凋亡的关系。方法:采用结扎左肾门血管及输尿管并注射链脲佐菌素(STZ)50mg/kg诱导高血糖症。6周后测定血糖、24h尿蛋白定量、血清尿素氮(BUN)、肌酐(Cr),摘取肾脏10%中性福尔马林固定,进行HE、PAS、Masson、Gordon-Sweet染色,并进行Beclin1、LC3-Ⅱ、PI3K、AKT、bcl-2、Bax、PTEN、凋亡(Tunel)染色观察。结果:模型组血糖、24h尿蛋白量、BUN、Cr均较正常对照组明显增高(P<0.01)。形态学观察显示,模型组肾小球体积增大,肾小管局灶性萎缩,肾小管细胞坏死,基底膜增厚,胶原纤维增多,肾组织内网状支架破坏,网状纤维减少。免疫组化结果显示LC3-Ⅱ和Beclin1在正常对照组肾小球均未见表达(-),在模型组均有表达,LC3-Ⅱ(+),Beclin1(+++);PI3K在正常对照组仅有少量表达(±),在模型组为强阳性表达(+++),AKT在正常对照组表达为阴性(-),在模型组表达显著增加(++)。PTEN在正常对照组为强阳性表达(+++),在模型组表达下降(+);凋亡细胞Tunel染色在正常对照组与模型组均呈阴性表达(-)。结论:糖尿病肾病中自噬通路被激活,而凋亡通路被抑制,提示糖尿病肾病中自噬通路的激活可能是肾小球细胞损伤的因素。

Objective： To investigate the relationship between autophagy and apoptosis in renal cell damage of diabetic nephropathy model. Methods： Ligation of the left renal hilar vessels and ureter and injection of streptozotocin （50mg/kg） were used to induce hyperglycemia. Six weeks later, blood glucose, 24-hour urinary protein, blood urea nitrogen （BUN） and Cratinine （Cr） were measured. Kidney was picked and fixed with 10% neutral formalin, then was staining with HE, PAS, Masson, Gordon- Sweet. And Beclinl, LC3- Ⅱ, PI3K, AKT, bcl-2, Bax, PTEN was detected with immunohistochemistry, and apoptosis with Tune/staining. Results： Blood glucose, 24h urine protein, BUN and Cr in the model group were higher than those in the normal control group （P〈0.01）. Renal pathological morphology showed that compared with the normal control group, the model showed glomerular mesangial expansion, basement membrane thickening and collagen fibers increasing and reticular fibers decreasing. Immunohistochemistry showed that there were no LC3- Ⅱ and Beclinl expression （-） in the control group, high expression in the model group [LC3-Ⅱ （＋）, Beclinl （＋＋＋）], and there was little PI3K expression （±） in normal control group and strong expression in the model group （＋＋＋）, AKT was negative （-） in the normal control group and high expression （＋＋） in the model group. PTEN was strongly positive （＋＋＋） in normal control group and decreased （＋） in the model group. Apoptotic ceils were negative in both normal control group and model group with tunel staining. Conclusion： Diabetic nephropathy autophagy pathway was activated, and the apoptosis pathway was inhibited, suggesting that autophagy was a major factor in diabetic nephropathy.