MAD2B介导的细胞周期再进入在FSGS足细胞损伤中的作用及其机制

Podocyte injury and loss are characteristic features of focal segmental glomerulosclerosis. It has been well documented that cell cycle re-entry of post-mitotic podocytes is involved in the development of podocyte injury and loss. However, the mechanism of cell cycle re-entry and its pathogenic role in FSGS has not been fully clarified. Mitotic arrest deficient-like 2 (MAD2B) is an important molecule involved in the regulation of cell cycle. Our preliminary data showed that the expression of MAD2B was elevated significantly in the podocyte area of FSGS mice, which was negatively correlated with the number of podocytes. In addition, inhibition of MAD2B in cultured podocytes prevented cell cycle re-entry and subsequent cell injury, suggesting that MAD2B plays a vital role in inducing cell cycle re-entry of podocytes. According to bioinformatic analysis, we found that MAD2B might be phosphorylated by ATM (Ataxia-Telangiectasia Mutated) kinase, which is necessary for its degradation. On the basis of these previous experiments, we plan to investigate the significance of MAD2B-mediated cell cycle re-entry of podocytes in the pathogenesis of FSGS and the role of ATM in regulating MAD2B phosphorylation, by using cultured podocytes and conditional knockout/transgenic mice. Through these studies, we expect to deepen the current understanding of the pathogenesis of FSGS and provide evidence for it early intervention.

足细胞损伤和缺失是原发性局灶节段性肾小球硬化（FSGS）的主要病理特征。研究表明，细胞周期再进入是导致FSGS进程中足细胞损伤和缺失的早期细胞事件，然而其发生机制尚未明确。有丝分裂阻滞缺陷蛋白---MAD2B是调控细胞周期的重要分子。我们的前期研究发现，FSGS小鼠模型足细胞MAD2B表达升高，且其表达强度与足细胞数目呈负相关；而体外敲低MAD2B可抑制细胞周期再进入和足细胞损伤，提示MAD2B是介导足细胞细胞周期再进入的关键分子。进一步生物信息学分析和预实验结果表明，MAD2B的磷酸化及其泛素化降解可能受ATM调控。在上述前期研究基础上，本项目拟采用体外细胞模型、足细胞特异性基因敲除和转基因小鼠等为研究对象，深入探讨MAD2B介导的细胞周期再进入在FSGS进程中的病理生理作用及ATM对MAD2B的调控机制，期望深化对FSGS发病机理的认识，并为其早期防治提供新的理论依据。

足细胞损伤和丢失是局灶节段性肾小球硬化（Focal segmental glomerulosclerosis, FSGS）的特征性病变，也是导致FSGS持续进展的病理生理基础。研究表明，细胞周期再进入是导致FSGS进程中足细胞损伤和丢失的早期事件，然而其始动机制尚未明确。有丝分裂阻滞缺陷蛋白MAD2B（Mitotic arrest deficient-like 2）是调控细胞周期蛋白表达的重要分子，但其在FSGS相关的细胞周期及有丝分裂异常中的作用和具体机制尚无报道。本项目采用FSGS患者肾活检标本、条件性基因敲除小鼠、阿霉素肾病小鼠模型和条件永生化人足细胞为研究对象，研究足细胞重新进入细胞周期在FSGS进程中的病理生理意义并探讨MAD2B在足细胞的细胞周期再进入过程中的关键作用和上游调控机制。本项目主要研究结果如下：（1）MAD2B在FSGS患者肾小球、ADR诱导的小鼠FSGS模型及足细胞损伤模型中的表达水平均明显上调，且在FSGS患者及小鼠模型中均可发现足细胞重新进入细胞周期，提示MAD2B可能参与FSGS疾病进程中的足细胞细胞周期再进入及足细胞损伤；（2）敲低MAD2B表达可减轻足细胞损伤及细胞周期再进入，表明MAD2B可通过介导细胞周期再进入参与FSGS疾病进程；（3）ATM激酶是MAD2B调控足细胞细胞周期的上游分子，抑制ATM的活化可减轻ADR诱导的MAD2B上调、足细胞损伤及细胞周期再进入；（4）从机制方面，ATM激酶通过磷酸化MAD2B调控其泛素化修饰，减少MAD2B在细胞内降解，导致MAD2B的蓄积，从而参与对足细胞细胞周期的调控。这些研究结果从细胞周期调节异常的角度，深化了人们对足细胞损伤机制的认识，明确了ATM-MAD2B信号通路在FSGS足细胞损伤中的关键作用，为FSGS的早期防治提供新的理论基础。

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