肾小管上皮细胞通过Bim/NFAT通路介导足细胞骨架改变的分子机制研究

Diabetic kidney disease (DKD) is an important microvascular complications of diabetes that affects around 30% of diabetic patients and is the most common cause of end-stage renal disease(ESRD). Damage to podocytes, which is considered to be a possible early pathological marker for DKD, is important due to its role in causing albuminuria and glomerular damage. Functional podocytes rely on the cytoskeleton to stabilize the glomerular capillaries and participate in the barrier function of the glomerular filter via interactions with cell–cell junction and cell–matrix proteins. Dysregulation of the highly specialized podocyte actin cytoskeleton is closely associated with albuminuria. Most researchers agree that initial lesions in DKD affect the glomerular compartment; however, the progression of the disease shows the strongest correlation with the changes in the tubules. Our preliminary studies showed that apoptosis of proximal tubular epithelial cells (PTEC) occurs much earlier than that of glomerular in the development of DKD, and the increased activity of proapoptosis protein Bim (Bcl-interacting mediator) was closely related to the early lesions of PTEC. Recent study indicates that the cross-talk between PTEC and podocyte plays an important role in DKD. In our current study, co-culture system was used to examined the interaction between PTEC and podocytes and found that the podocyte skeleton was damaged when co-culture with Bim-transfected HK-2 cells. The exactly mechanism is remain unclear. The nuclear factor of activated T cells(NFAT), which are the substrate for calcineurin(CaN), represent a family of Ca2+ dependent transcription factors. Recent studies have also demonstrated that the activation of NFAT was involved in cytoskeleton injury and DKD. What’s more, our further studies showed that the activation of NFAT was closely related with Bim in PTEC. In addition, several studies clarified that NFAT transcription complexes assemble on DNA to activate genes primary dedicated to cell-cell inteactions in the nucleus. Based on our preliminary studies above, we propose the hypothesis that Bim/NFAT pathway in PTEC plays an important role in promoting the damage and rearrangement of podocyte skeleton through regulating specific effector molecule activation and release in DKD. To confirm the detail mechanisms, PTEC conditional Bim gene knockout mice were used in our further study to clear Bim’s function and its molecular mechanism in mediating PTEC-podocyte crosstalk in inducing DKD..The significance of the study is to investigate a new mechanism and potential target for the treatment of DKD. This study will help us to get a better understanding of Bim-mediated tubule-podocyte crosstalk in DKD.

既往糖尿病肾病（DKD）发病机制的研究多注重单一环节或细胞的调控，而忽略了细胞间相互作用。肾小球足细胞和肾小管近端上皮细胞（PTEC）是执行肾脏功能的两种主要细胞，二者如何相互作用导致DKD，尚未阐明。我们前期发现：早期DKD肾小管病变早于肾小球；且证实Bim是介导PTEC病变的关键。前期共培养发现PTEC中Bim升高可致足细胞骨架损伤。活化T细胞核因子(NFAT)负责调控细胞间信息传递，且参与了DKD及细胞骨架损伤。预实验发现PTEC中Bim可活化NFAT。故推测：肾小管PTEC中Bim激活NFAT，通过诱导特定分子分泌，介导肾小球足细胞骨架损伤，共同致DKD进展。本课题将用cre-loxp条件性敲除技术构建特异性PTEC Bim基因敲除小鼠，结合激光捕获显微切割等方法，突破既往研究局限性，聚焦“Bim/NFAT介导管球对话”导致DKD的新机制，为今后DKD的诊疗开辟新思路、新靶点。

糖尿病肾病（DKD）治疗的硬终点获益不佳，亟需替代方案。既往DKD研究多局限于单一细胞中，忽略了细胞间交流的作用。近端小管上皮细胞（PTECs）和足细胞是肾脏中肾小管和肾小球的代表性功能单位，我们前期研究发现，DKD中肾小管病变早于肾小球，Bim是介导PTEC病变的关键。本课题通过构建PTECs-足细胞共培养体系证实，早期的肾小管病变通过调控Bim-NFAT通路介导肾小球足细胞骨架损伤。进一步通过上述共培养体系结合 Sino human lncRNA array V3.0 芯片，对共培养后足细胞内 lncRNA 表达谱进行检测，发现LncRNA NONHSAT179542.1 是“管球交流”中触发足细胞骨架损伤介导其早期病变的关键始动分子，并且MICAL2是LncRNA NONHSAT179542.1介导足细胞损伤的潜在下游靶点。该课题同时采用cre-loxp条件性敲除技术构建PTECs条件性Bim敲除小鼠（Ggt1-Cre Bimfl/fl），通过构建糖尿病肾病小鼠模型，结合转录组测序、免疫浸润分析等方法对上述研究进行了进一步验证与深入，突破既往研究局限性，发现管球交流中，Bim-LncRNA NONHSAT179542.1-MICAL2轴介导DKD早期足细胞损伤导致DKD的新机制，为今后DKD的诊疗开辟新思路、新靶点。