Notch 信号调控小管上皮细胞去分化在肾脏缺血耐受中的作用

Dedifferentiation is a protective and adaptive phenomenon. In order to survive from the injury, dedifferentiated cell has enhanced viability and proliferation potential. Notch signaling is important for maintaining the undifferentiated cell state. Our previous study showed that 20-min ischemia pretreatment protected the kidney from 40-min ischemia/reperfusion injury occurring four days later. However, the protective mechanism is unclear. We found hypoxia microenvironment, following ischemia pretreatment, can activate Notch signaling and promote the dedifferentiation of epithelial cells in proximal tubules. Thus, we speculate that tubular cell dedifferentiation is mediated by Notch survival signaling,and cell dedifferentiation in turn enhances ischemia tolerance in the kidney. In this study, Western blot, immunohistochemistry, confocal laser, and cell transfection technology will be used. In vivo and in vitro experiments, following ischemia or hypoxia pretreatment, we will observe the dynamic change in renal tubular epithelial cell phenotype, and further demonstrate its relationship with Notch signaling activation and renal ischemia tolerance. In the next step, through overexpressing of Notch intracellular domain and inhibiting Notch with γ-secretase inhibitor, the impact of Notch activation on cell dedifferentiation, energy metabolism, cell proliferation, and as well as on renal structure and function will be elucidated. This project study may be helpful to reveal the protective mechanism of delayed preconditioning, and provide experimental basis for the prevention and treatment of ischemia-associated injury in the kidney.

去分化是有机体长期进化过程中获得的一种保护性和适应性现象，细胞去分化后具有更强的存活力和增殖修复潜能，而Notch是调控细胞分化方向的重要信号通路。我们前期研究发现：预缺血（20 min）能诱导肾脏耐受4天后的第二次长时间缺血（40 min）,但机制尚不清楚。预实验显示：预处理营造的适度低氧微环境能激活Notch信号和促进肾小管上皮细胞去分化。推测：Notch信号调控的小管上皮细胞去分化可能是延迟性缺血预适应诱导肾脏缺血耐受的重要机制。研究采用免疫印迹、激光共聚焦、脂质体细胞转染等技术，结合体内和体外实验，1）观察预缺血后肾小管上皮细胞表型和生物学特性的动态变化，分析其与Notch信号活化和肾脏缺血耐受的关系；2）研究Notch信号抑制或过表达对细胞分化状态、能量代谢、增殖及肾脏结构和功能的影响。期望通过本项目研究，揭示肾脏延迟性缺血预适应的关键保护机制，为缺血性肾损伤的防治提供实验依据。

研究背景：延迟性缺血预适应（DIPC）能提高肾小管上皮细胞的缺血耐受力，是强有力防治ARF的内源性措施。结合国内外同行的研究报道与我们的研究基础，推测：Notch信号通路调控的小管上皮细胞去分化可能是DIPC肾脏保护作用的关键机制。有望通过本项目研究，揭示肾脏延迟性缺血预适应的关键保护机制，为缺血性肾损伤的防治提供实验依据。.材料和方法：我们依照研究计划，首先建立了经典的体外HK-2细胞缺氧（CoCl2化学缺氧）模型、体内急性缺血性肾损伤模型以及肾脏晚期缺血预适应的大鼠模型，观察并验证急性缺血/缺氧条件下，HK-2细胞（体外研究）和大鼠肾组织（体内研究）中肾小管上皮细胞的去分化、增殖、凋亡和坏死，并分析Notch信号通路的表达变化。免疫组织化学法观察波形蛋白（Vimentin）、细胞增殖核抗原（PCNA）、Ki67的表达；Western blot检测Notch1、Notch2、Dll1、Hes1蛋白表达。在此基础上，用γ-分泌酶抑制剂DAPT阻断Notch信号，进一步观察Notch信号通路在小管上皮细胞去分化/增殖修复中的作用，干预Notch信号通路对肾脏缺血/缺氧性损伤和缺血耐受效果的影响。.结果：1）DIPC 能够减少肾小管上皮细胞凋亡和坏死，促进其去分化、增殖修复；2）短时间预缺血能诱导Notch2/ Dll1/ Hes1信号通路高表达；3）γ-分泌酶抑制剂DAPT阻断Notch信号后部分削弱了IPC的肾脏保护作用，肾小管上皮细胞的凋亡、坏死数增加，而细胞去分化、增殖率减少；4）另外，DAPT阻断I/R组的Notch信号后，肾小管上皮细胞缺血性损伤进一步加重，肾功能和肾小管间质损伤恢复延迟；5）体外实验阻断Notch信号，能抑制体外培养的肾小管上皮细胞系HK-2增殖，这种抑制作用在一定范围内具有剂量依赖性；阻断Notch信号会导致肾小管上皮细胞细胞缺氧性损伤进一步加重。.结论：本项目研究证实缺血预处理诱导易损区肾小管上皮细胞去分化，这些去分化的细胞一方面提高了自身低氧耐受力，另一方面可增殖分化以替代周围损伤或死亡的细胞，保持了肾小管结构的完整性，从而在整体肾脏的缺血耐受中发挥了重要作用；Notch是调控小管上皮细胞去分化、增殖修复的重要内源性信号通路，Notch信号通路激活可能是DIPC诱导肾脏缺血耐受的重要机制。