糖尿病肾病中PEDF通过ATGL导致尿蛋白增加的作用及其机制

Diabetic kidney disease (DKD) is the most frequent cause of chronic kidney failure in both developed and developing countries. The microalbuminuria stage is the first clinically detectable sign of glomerular damage and is important for the DKD early treatment. However, the regulation of urinary albumin excretion has not been defined. The plasma pigment epithelium-derived factor (PEDF), an endogenous anti-angiogenic molecule, is significantly increased in DKD, but its role is still unclear. Our preliminary work demonstrated that intravenous infusion of exogenous PEDF led to increased urinary albumin to creatinine ratio (ACR), and this effect was inversed in the adipose triglyceride lipase (ATGL) knockout mouse, which suggested that PEDF may induce proteinuria development through its receptor ATGL in DKD. In addition, our previous work have proved that PEDF could induce the actin rearrangement through RhoA/ROCK pathway. So it is speculated that PEDF/ATGL may induce podocyte injury through RhoA/ROCK mediated actin rearrangement, which induces the proteinuria development. In order to clarify the role of PEDF and its mechanism in DKD, an STZ-induced mouse model of insensate diabetic neuropathy and the podocyte were chosen, and PEDF monoclonal antibody (PEDF-mAb) or recombinant lentivirus vectors targeting PEDF receptors ATGL will be applied to inhibit the PEDF. The ACR, kidney functional magnetic resonance imaging (fMRI) , actin arrangement, cell apoptosis, et al will be detected. All the studies were hoped to prove the key role of PEDF/ATGL in the urinary albumin excretion, and may offer a therapeutic targets for managing proteinuria development in DKD.

糖尿病肾病已成为导致终末期肾病的首要原因。蛋白尿期是糖尿病肾病发生发展和治疗干预的重要阶段，但糖尿病时尿蛋白排泄调节机制尚未阐明。申请者前期研究和文献证据提示：血浆PEDF与足细胞膜上ATGL结合，激活胞内Rho/ROCK通路引起微丝重排，导致足突融合、足细胞与肾小球基底膜黏附力下降是糖尿病肾病中蛋白尿发生发展的关键分子机制和干预治疗环节。本课题拟构建糖尿病小鼠模型，结合ATGL基因敲除小鼠和足细胞系，利用功能磁共振、WB、病毒转染等研究方法，从动物和细胞水平，明确血浆PEDF在糖尿病肾病时的变化及其在蛋白尿发生发展中的作用，揭示PEDF增加尿蛋白排泄的作用靶点和环节，以及PEDF激活ATGL后导致足细胞损伤的分子机制，探讨抑制PEDF/ATGL对糖尿病肾病的保护作用。以期证实“PEDF/ATGL-Rho/ROCK-微丝重排-足细胞损伤”未知途径在糖尿病肾病蛋白尿调控中的作用和地位。

糖尿病肾病已成为导致终末期肾病的首要原因。蛋白尿期是糖尿病肾病发生发展和治疗干预的重要阶段，前期研究和文献证据提示：血浆PEDF与足细胞膜上ATGL结合，激活胞内Rho/ROCK通路引起微丝重排，导致足突融合、足细胞与肾小球基底膜黏附力下降是糖尿病肾病中蛋白尿发生发展的关键分子机制和干预治疗环节。本课题构建1型糖尿病和2型糖尿病小鼠模型，对正常C57小鼠及糖尿病小鼠分别注射PEDF蛋白后，正常小鼠和糖尿病小鼠均出现蛋白尿，血浆肌酐水平升高，光镜下肾小球体积缩小，肾小囊增大，电镜下足细胞足突融合、数量减少。实验明确在1型和2型糖尿病中，PEDF均通过作用于肾小球足细胞，诱导足突融合，破坏肾小球滤过屏障，增加尿蛋白排泄。在体外试验中通过对正常足细胞和高糖处理足细胞给予PEDF干预，观察到正常足细胞处理后，细胞连接受损，通过细胞间缝隙漏出的荧光物质明显增加，同时紧密连接蛋白ZO-1表达减少，足细胞标志物表达减少，微丝聚合增加，胞周出现张力丝结构。高糖处理后足细胞出现类似的改变，而PEDF处理会加重高糖对足细胞的损伤。进一步体外实验发现高糖处理或PEDF处理均可激活足细胞RhoA活性（RhoA蛋白表达无明显改变），并使ROCK1蛋白表达增加，RhoA抑制剂胞外酶C3可以有效抑制RhoA激活和ROCK1表达增加，明显缓解高糖或PEDF处理导致的足细胞细胞损伤。本研究证实PEDF通过RhoA/ROCK信号通路诱导足细胞微丝骨架重排导致肾小球滤过屏障受损，尿蛋白生成及排泄增加，参与糖尿病肾病的发病机制。