Cdc42介导的丝状伪足形成在糖尿病肾病足细胞损伤中的作用及机制

The chaos of cell cytoskeleton and polarity in podocyte participate in diabetic nephropathy (DN). Cdc42 and its induced filopodia play important roles in differentiation and polarity maintenance in most cell types, but unknown in podocyte. Our previous study showed that the overexpression and (or) activation of Cdc42 promotes epithelial differentiation and planar polarity, as well as protects against the cell body collapse after high glucose injury, and reduces the foot process fusion and proteinuria. In this program, three-dimensional cell culture in vitro and glomeruli in vivo were detected. The overexpression, site directed mutagenesis, interference knockdown, fluorescence real-time co-localization, and fluorescence resonance energy transfer will be used to evaluate the Cdc42 and filopodia growth in DN. The AAV2/CRISPR carried sgRNA and donor sequence targeted Q61 loci in podocyte will be induced in vivo and in vitro to explore the mechanism of protection against diabetic injury.

足细胞细胞骨架的紊乱、细胞极性的减弱与糖尿病肾病的发生发展密切相关。Cdc42及其介导的丝状伪足参与细胞分化和极性维持，但在足细胞中作用未明。课题组前期发现Cdc42过表达将促进足细胞上皮分化，增加细胞平面极性，高水平激活可对抗高糖损伤后胞体塌陷、糖尿病肾中足突融合脱落及蛋白尿，且与丝状伪足活动相关，推测Cdc42及其介导的丝状伪足可通过细胞骨架重构，修补胞间紧密连接和（或）锚定细胞基质连接三方面机制起保护作用。本项目拟以离体三维培养细胞和在体肾小球为主要观测对象，结合过表达、点突变、干扰敲除等分子手段及荧光实时共定位、能量共振迁移等成像方法，探讨Cdc42介导丝状伪足在糖尿病肾病进程中的发生、发展规律和调控方式；并利用腺相关病毒介导CRISPR/Cas9对足细胞Cdc42的Q61位点编码基因进行离体和在体水平编辑，探索潜在保护机制，从而为糖尿病肾病治疗提供新的潜在治疗靶点。

课题组前期发现Cdc42在糖尿病肾病相关足细胞分化和极性维持中具有重要作用，因此在本项目中通过基因编辑工具构建Cdc42、Cdc42L61和Cdc42N17过表达足细胞系并进行三维培养，阐述Cdc42的不同激活状态对足细胞在高糖状态下的呈现不同程度的损伤反应，究其机制与其在高糖状态下对Tau5通过AMPK实现199S位点，而非404S位点磷酸化上调相关。在构建小鼠db/db糖尿病模型鼠中，通过条件性激活方式对Cdc42进行敲入表达，可观察到足细胞在Cdc42L61过表达状态下，Tau5磷酸化显著增强，并推进了糖尿病肾病进程。进一步的，小管细胞在三维培养状态下对于高糖的应激反应进行了描述和探讨：相较于缺糖缺氧和TGF-beta处理，高糖处理赋予了肾小管细胞更高的细胞应变性和极化改变能力，通过单细胞测序我们获得了一系列稳态细胞和逃逸细胞间的差异表达基因，其中也包含Cdc42的显著变化，可用于后续研究糖尿病肾病中肾小管损伤的相关机制阐述，并为糖尿病肾病的防治提供相关可行性靶点。