解剖学报 ›› 2014, Vol. 45 ›› Issue (4): 555-560.doi: 10.3969/j.issn.0529-1356.2014.04.021

• 组织学胚胎学发育生物学 • 上一篇    下一篇

mTOR复合物在糖尿病肾病小鼠肾组织中的不同分布和表达

赵鸿1  冀倩倩2,3 李永霞2 段秋红4 姚丽君2*   

  1. 1. 华中科技大学附属同济医院创伤外科, 武汉 430022; 2. 华中科技大学附属协和医院肾内科, 武汉 430030; 3. 湖北省中山医院肾内科, 武汉 430033; 4. 华中科技大学同济医学院生物化学与分子生物学教研室, 武汉 430022
  • 收稿日期:2014-02-10 修回日期:2014-04-06 出版日期:2014-08-06 发布日期:2014-08-06
  • 通讯作者: 姚丽君 E-mail:drylj@hotmail.com
  • 基金资助:

    蛋白激酶C-α在小鼠尿浓缩功能中的作用研究;SGK3缺失致Ezrin泛素化在慢性肾病中的作用及机理研究

Different distribution and expression of mammalian target of rapamycin complex in the kidney of diabetic nephropathy mice

ZHAO Hong1 JI Qian-qian 2, 3 LI Yong-xia2 DUAN Qiu-hong4 YAO Li-jun 2*   

  1. 1. Department of Trauma Surgery,Tongji Hospita, Huazhong University of Science and Technology, Wuhan 430022, China;2. Department of Nephrology, Union Hospital, Huazhong University of Science and Technology, Wuhan 430030, China;3. Department of Nephrology, Zhongshan Hospital, Wuhan 430033, China; 4. Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
     
  • Received:2014-02-10 Revised:2014-04-06 Online:2014-08-06 Published:2014-08-06
  • Contact: YAO Li-jun E-mail:drylj@hotmail.com

摘要:

目的 探讨哺乳动物雷帕霉素靶分子复合物(mTORC)在糖尿病肾病(DN)小鼠肾组织中的分布、表达。方法 14只C57BL/6小鼠随机分成对照组和DN组,每组各7只。DN组小鼠予以链脲菌素腹腔注射建立小鼠DN模型,采用生化技术检测小鼠血、尿肌酐以及白蛋白水平,组织学染色检测肾脏病理变化,免疫荧光以及免疫印迹技术检测肾组织中mTOR、mTOR第2448位丝氨酸磷酸化修饰后mTOR(p-mTOR)、mTORC1(Raptor)、mTORC2(Rictor)以及mTOR信号通路下游的效应蛋白磷酸化S6K1(p-S6K1)的分布和表达。 结果 DN组小鼠血糖、尿白蛋白/肌酐比值明显增加(P<0.01),肾小球明显增大(P<0.05)。mTOR、Raptor以及Rictor在正常以及DN小鼠肾皮质和髓质中均有表达,主要表达在肾小球系膜区、毛细血管袢、皮质近曲小管以及外髓和内髓集合管上皮细胞中。其中正常小鼠内髓肾组织中未见p-S6K1表达,正常以及DN小鼠肾小球中未见p-mTOR表达。免疫印迹检测表明,DN小鼠肾组织中mTOR、p-mTOR、Raptor、Rictor以及p-S6K1均明显上升(P<0.05)。 结论 mTORC广泛分布于小鼠肾组织且参与DN的发生发展,但mTOR第2448位丝氨酸磷酸化并不直接参与高血糖介导的肾小球损伤。

关键词: 哺乳动物雷帕霉素靶分子, 糖尿病肾病, Rictor, Raptor, 免疫荧光, 免疫印迹法, 小鼠

Abstract:

Objective To investigate the different distribution and expression of mammalian target of rapamycin complex (mTORC) in the kidney of diabetic nephropathy (DN) mice. Methods Fourteen eight-week-old male C57BL/6 mice were assigned to 2 groups: the control group (n=7) and the streptozotocin (STZ)-induced DN group (n=7). Blood and urinary variables including glucose, albumin, creatinine and albumin/creatinine ratio were assessed 2 weeks after STZ injection. Hematoxylin-eosin staining was performed for renal pathological analyses. The distributions of mTOR, phosph-ser2448-mTOR(p-mTOR), mTORC1(Raptor), mTORC2(Rictor) and phosph-ser240/244-S6K1 (p-S6K1) were determined by immunofluorescence. The expression levels of mTOR, p-mTOR, mTORC1(Raptor), mTORC2(Rictor), S6K1 and p-S6K1 were detected by Western blotting. Results Two weeks after STZ injection, the diabetic mice developed albuminuria (P<0.01) and renal hypertrophy (P<0.05). The immunofluorescence positive staining for mTOR, Raptor, and Rictor was distributed in the epithelial cells of proximal tubules, glomerular mesangium and capillary loops as well as the medullary collecting ducts of the control mouse kidney. These positive signals increased in the DN mouse kidney (P<0.05). However, pS6K1 was not detected in the inner medulla of control mouse and p-mTOR was not found in the glomeruli of both control and DN mice. Conclusion mTORC is widely expessed in the mouse kidney and participates in the development of DN, whereas the 2448 serine phosphorylation of mTOR may be not implicated in the hyperglycemia mediated glomerular injury.

Key words: Mammaliam target of rapamycin, Diabetic nephropathy, Rictor, Raptor, Immunofluorescence, Western blotting, Mouse