Rac1信号通路在糖尿病肾病足细胞转分化中的作用及机制

Diabetic nephropathy is characterized by a progressive increase in proteinuria that is closely related to epithelial-mesenchymal transition (EMT)of podocytes. Rac1 plays an important role in podocytes injury of diabetic nephropathy, but whether Rac1 and its downstream factors PAK1 and p38MAPK participate in podocytes EMT during diabetic nephropathy and proteinuria remains elusive. This study focused on EMT of podocytes that was indicated to play a central role in mechanism of proteinuria in diabetic nephropathy and renal fibrosis. Rac1 signaling pathyway was investigated through knocking down Rac1 gene by small hairpin RNA (shRNA). We further established purified podocyte-specific Rac1 RNA interfering (RNAi) transgenic mouse model that could be stably inherited. Activation of Rac1 signaling pathway and its interactions with transcriptional factors Snail or β-catenin in podocyte EMT were investigated on protein and gene levels respectively; and potential mechanisms of diabetic nephropathy were discussed. This will provide us a novel intervention target for prevention and clinical treatment of diabetic nephropathy.

蛋白尿是糖尿病肾病早期最突出的临床表现，而足细胞发生上皮-间充质细胞转分化与蛋白尿的发生密切相关。Rac1在糖尿病肾病足细胞损伤中起重要作用，而Rac1及其下游信号分子PAK1、p38MAPK是否介导糖尿病肾病足细胞的转分化，参与糖尿病肾病蛋白尿的发生，目前尚无报道。本研究以糖尿病肾病蛋白尿形成和肾脏纤维化的中心环节——足细胞转分化为切入点，以Rac1信号通路作为研究靶点，通过Rac1 shRNA高效、特异性干扰足细胞Rac1基因表达；并进一步构建稳定足细胞特异性的Rac1 RNAi转基因小鼠，在基因和蛋白水平上深入研究Rac1通路激活及Snail、β-catenin表达在足细胞转分化中的作用，深入探讨糖尿病肾病蛋白尿的发生机制，这将为临床早期预防和治疗糖尿病肾病提供新的思路。

Ras相关的C3肉毒素底物1（Ras-related C3 botulinum toxin substrate1，Rac1）是Rho家族小G蛋白成员之一，在细胞粘附、增殖以及细胞骨架调节等生理活动中发挥重要作用。我们以条件永生化小鼠足细胞系及构建足细胞特异性Rac1 RNAi转基因小鼠作为研究对象，证实高糖培养48h后足细胞标志性蛋白p-cadherin和ZO-1表达减少而间充质样蛋白FSP-1、α-SMA及desmin出现重新表达，伴Transwell小室白蛋白流入量显著增加，同时F-actin骨架蛋白出现边集现象；高糖刺激导致Rac1蛋白活化，以及PAK1、p38 MAPK磷酸化水平增高，引起β-catenin的N端去磷酸化、Snail表达增加，以及β-catenin细胞核迁移；Rac1 shRNA显著抑制了足细胞EMT， PAK1、p38 MAPK磷酸化水平明显降低， β-catenin、Snail活性明显降低，β-catenin核迁移抑制；Rac1对β-catenin的C端丝氨酸位点具有直接磷酸化修饰作用，导致β-catenin泛素化水平降低；p38 MAPK通过磷酸化下游MEF2c来结合β-catenin。通过构建Rac1 RNAi转基因小鼠发现，WT-STZ小鼠6W时足细胞出现足突轻度融合，SD蛋白p-cadherin和ZO-1表达降低，desmin表达增加；电镜显示，各观察时间点TG-STZ组均较WT-STZ组足突融合减轻，免疫荧光显示p-cadherin及ZO-1表达水平回升，desmin，mmp9与FSP-1明显减少；各时间点TG-STZ组均较WT-STZ组蛋白尿显著减少，同时PAS示肾小球基底膜与系膜区病变程度降低。.综上所述，足细胞特异性Rac1通路抑制可以通过失活下游激酶PAK1/p38 MAPK改善糖尿病足细胞损伤，具体机制可能包括以下五方面：（1）保存与恢复足细胞裂孔膜结构与功能；（2）调控足细胞骨架并改善足突融合；（3）逆转足细胞去分化；（4）减轻早期GBM增厚以及系膜区基质扩大；（5）减少蛋白漏出，而不影响血压及血糖代谢。