Robo4/ARF6信号通路在糖尿病肾病肾小球血管内皮高通透性中的作用及机理

Endothelial/glomerular hyperpermeability plays a key role in the pathogenesis of microalbuminuria of diabetic nephropathy. The biochemical basis of glomerular hyperpermeability, however, has yet to be fully elucidated. It is known that Robo4 exclusively expresses in endothelial cells and functions as endothelial stabilization and vascular integrity by inhibition ARF6 activity. It is unknown if Robo4 and ARF6 participate in diabetic nephropathy pathogenesis. Our preliminary study showed that deletion of Robo4 significantly increased urinary albumin excretion and enhanced severity of glomerulosclrosis,which suggesting Robo4 may be involved in glomerular vascular stability. This project will study the role of Robo4 and its downstream target ARF6 in glomerular hyperpermeability induced by high glucose and in the initiation and development of diabetic nephropathy,and the molecular mechanism underlying the maintenance of vascular stability by activating Robo4 and blocking Arf6 activity also will be examined in vivo and in vitro study.More importantly, the concept of treatment of diabetic nephropathy targeting endothelial stabilization and integrity will be proposed and tested.

肾小球血管内皮高通透性是糖尿病微蛋白尿发病的重要机制之一,但肾小球血管通透性的调控机制仍然缺乏充分了解。Robo4特异表达于内皮细胞，通过抑制ARF6的活化，具有稳定血管通透性和完整性的功能。我们的前期实验发现Robo4功能缺失的糖尿病小鼠与野生鼠相比,呈现更显著蛋白尿及糖尿病肾病病理病变，提示Robo4参与了肾小球内皮通透性稳定。本项目在此基础上建立Robo4基因敲除糖尿病小鼠和肾小球内皮细胞培养模型，观察Robo4/ARF6信号通路分子与内皮通透性、内皮损伤、VEGF、eNOS/NO和氧化应激的内在联系，阐明Robo4和ARF6在高糖条件下对内皮屏障的稳定和瓦解作用机制。通过给予小分子化合物SecinH3定向抑制ARF6和Slit2蛋白定向激活Robo4，分别观察它们对糖尿病肾病内皮高通透性的治疗作用。本实验将为今后针对Robo4信号通路异常,筛选治疗糖尿病肾病的药物提供理论依据。

糖尿病肾病是终末期肾病的最主要病因，其发病机制及其防治策略研究具有重要的社会意义和经济意义。肾小球血管内皮高通透性和病理性血管新生是糖尿病微蛋白尿发病的重要原因之一,但其调控机制仍然不清楚。本研究分别采用人早期糖尿病肾病和ROBO4基因敲除小鼠模型、体外高糖培养系膜、内皮细胞模型，利用激光共聚焦、real-time PCR，Western Blot、基因转染技术，观察Slit2/ROBO1-ROBO4信号与肾小球高通透性、病理性血管新生的关系，阐明了 Robo4 信号通路稳定内皮屏障功能的作用机制和高糖环境下内皮血管新生的机制。结果主要包括：①ROB04/ARF6信号通路参与了糖尿病肾病（DN）患者肾小球内皮细胞（HRGEC）的病理损伤和尿蛋白的进展。②高糖诱导HRGEC高通透性与ROBO4-ARF6负调控信号通路缺失有关，表现为低表达ROBO4不足以抑制ARF6高活化。过表达Robo4定向抑制ARF6活性可抑制高糖诱导的RGEC高通透性。③高糖诱导人HRGEC高通透性与ARNO–ARF6 正调控信号通路高活化有关，表现为高表达ARNO促进ARF6活化。SecinH3 定向抑制 ARNO可抑制高糖诱导的HRGEC高通透性。④高糖环境诱发的人肾小球内皮血管新生与Slit2/Robo1/ PI3K/Akt/VEGF信号通路活化有关。本研究研究结果将为系列糖尿病血管病变的防治提供新的思路和更有价值的治疗靶点。