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中华肾病研究电子杂志 ›› 2017, Vol. 06 ›› Issue (03) : 120 -126. doi: 10.3877/cma.j.issn.2095-3216.2017.03.006

所属专题: 文献

论著

自噬和p53凋亡刺激蛋白在大鼠急性肾损伤模型中的表达及早期诊断价值
李青霖1, 王小丹1,(), 杜婧1, 李玉茹2, 傅博3, 吕杨3   
  1. 1. 100853 北京,解放军总医院南楼保健科
    2. 解放军总医院南楼检验科
    3. 肾脏疾病国家重点实验室
  • 收稿日期:2017-01-21 出版日期:2017-06-28
  • 通信作者: 王小丹
  • 基金资助:
    国家自然科学基金(81370452)

Early diagnosis value of autophagy and expression of apoptosis-stimulating proteins of p53 in acute kidney injury model of rats

Qinglin Li1, Xiaodan Wang1,(), Jing Du1, Yuru Li2, Bo Fu3, Yang Lyu3   

  1. 1. Division of Health Care, Clinical Department of South Building
    2. Division of Laboratory Test, Clinical Department of South Building
    3. State Key Laboratory of Kidney Diseases; General Hospital of PLA, Beijing 100853, China
  • Received:2017-01-21 Published:2017-06-28
  • Corresponding author: Xiaodan Wang
  • About author:
    Corresponding author: Wang Xiaodan, Email:
引用本文:

李青霖, 王小丹, 杜婧, 李玉茹, 傅博, 吕杨. 自噬和p53凋亡刺激蛋白在大鼠急性肾损伤模型中的表达及早期诊断价值[J]. 中华肾病研究电子杂志, 2017, 06(03): 120-126.

Qinglin Li, Xiaodan Wang, Jing Du, Yuru Li, Bo Fu, Yang Lyu. Early diagnosis value of autophagy and expression of apoptosis-stimulating proteins of p53 in acute kidney injury model of rats[J]. Chinese Journal of Kidney Disease Investigation(Electronic Edition), 2017, 06(03): 120-126.

目的

观察自噬相关蛋白和p53凋亡刺激蛋白( ASPPs)在肾损伤早期的表达变化,探讨自噬相关蛋白和ASPPs是否可能成为急性肾损伤(AKI)早期损伤生物标志物。

方法

建立顺铂AKI大鼠模型,将46只雄性SD青年大鼠随机分为假手术组(Sham),顺铂模型组;模型组大鼠一次性腹腔注射顺铂5 mg/kg,Sham组相同途径注射等容量生理盐水;在给药1、3、7、10、14 d时检测大鼠血清肌酐(Scr)、尿素氮(BUN)和胱抑素C (cystatin C,Cys C)水平;光镜观察大鼠肾脏病理变化;透射电镜观察大鼠肾小管上皮细胞超微结构变化及自噬体的情况;免疫印迹法检测肾脏组织LC3、Atg5-Atg12、Beclin 1、LAMP-2、p62、p53及iASPP和ASPP1表达情况。所有数据采用SPSS 17.0统计软件分析。

结果

青年大鼠暴露于顺铂1 d时,与Sham组比较,顺铂模型组大鼠Scr、BUN、胱抑素C显著升高(P<0.05);光镜检查发现肾小管损害明显加重;电镜结果显示,顺铂模型组大鼠平均自噬体数量明显增多;顺铂诱导后1 d开始,肾脏组织iASPP蛋白表达明显减少(P<0.05),Atg5-Atg12、Beclin 1、LAMP-2、p62、p53及ASPP1蛋白表达明显升高(P<0.05)。肾脏组织LC3表达从3 d时明显降低(P<0.05)。

结论

自噬和ASPPs在AKI发生早期即可出现并参与了AKI的发生发展,在Scr开始升高前,反应性自噬已经启动。自噬相关蛋白和ASPPs有望成为AKI更早期的损伤标志物,可能是AKI早期干预的新靶点,但仍需更深入的研究。

Objective

To observe the expression changes of autophagy-related proteins and apoptosis-stimulating proteins of p53 (ASPPs) in early kidney injury, in order to investigate whether autophagy-related proteins and ASPPs can be biomarkers of early acute kidney injury (AKI) induced by cisplatin in Sprague-Dawley (SD) rats.

Methods

A total of 46 eight-week-old male SD rats were randomly divided into sham group and cisplatin model group. The rats in the model group were injected intraperitoneally with cisplatin 5mg/kg once, while rats in the sham group were treated in the same way with normal saline of the same volume. Serum creatinine (Scr), blood urea nitrogen (BUN), and cystatin C (Cys C) were measured at 1, 3, 7, 10 and 14 days after the administration. The kidneys pathological changes were observed under light microscopy. Ultrastructural changes and autophagy of rat renal tubular epithelial cells were observed under transmission electron microscopy. Expressions of LC3, Atg5-Atg12, Beclin 1, LAMP-2, p62, p53, iASPP, and ASPP1 were detected with Western blotting.

Results

At 1 day after the administration, levels of Scr, BUN, and Cys C in the cisplatin group were significantly higher than those in the sham group (P<0.05), and the renal tubular injury was significantly increased under light microscopy. The results of electron microscopy showed that the average number of autophagy was significantly increased in the cisplatin model group. From 1 day after the administration, the expression of iASPP protein in kidneys tissues was significantly decreased (P<0.05), while the expressions of Atg5-Atg12, Beclin1, LAMP-1, p62, p53, and ASPP1 proteins in the cisplatin model group was significantly higher than those in the sham group (P<0.05). The expression of LC3 in renal tissues was significantly decreased from 3 days after the administration (P<0.05).

Conclusions

Autophagy and ASPPs occurred in early AKI, and were involved in the development and progression of AKI. Reactive autophagy had been initiated before Scr began to increase. Although autophagy-related proteins and ASPPs are expected to be earlier markers of AKI, which may provide new targets for early intervention in AKI, further studies are still needed.

图1 顺铂注射后不同时间各组大鼠血清肌酐、尿素氮和胱抑素C的变化
图2 顺铂注射后不同时间各组大鼠肾组织病理图片(PAS×400)
图3 顺铂注射后不同时间各组大鼠肾小管上皮细胞自噬体,箭头所示自噬体(电镜×50 000)
图4 顺铂注射不同时间各组大鼠平均出现的自噬体
图5 顺铂注射后不同时间各组大鼠肾组织自噬相关蛋白表达情况及定量分析
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