循环线粒体DNA对足细胞的损伤作用及其机制

Mitochondria derived from bacteria in evolution, and retain certain features of bacteria, including unmethylation of CpG motif in their DNA (mtDNA). A recent study by Zhang Q et al (Nature, 2010) has shown that mtDNA can also act on Toll-like receptor 9 (TLR9)，thereby activating neutrophils and causing tissue injuries. Moreover, studies have also shown that proteinuric patients of trauma, burning and pre-eclampsia have an elevated serum mtDNA level, and that TLR9 is upregulated in podocytes in some glomerular diseases studied. We therefore hypothesize that circulating mtDNA could directly induce podocyte injury, leading to proteinuria and glomerular disease. Our most recent experiments have demonstrated that mtDNA injection into mice causes massive proteinuria and foot process effacement of podocytes, thus supporting our hypothesis. In the present grant application, we propose 1) in vitro study to determine if mtDNA can cause injury of cultured podocytes through TLR9, NFkB or p38 pathways, 2) in vivo study with mice to investigate the mechanism underlying mtDNA-induced proteinuria by determining if mtDNA induces proteinuria by directly injuring podocytes via TLR9, NFkB or p38 pathways, or by systemic inflammatory response that is potentially induced by mtDNA, and 3) examination of patients of various glomerular diseases to determine if circulating mtDNA levels are elevated, and if TLR9, NFkB and p38 are upregulated or activated. These studies are expected to reveal a novel mechanism underlying podocyte and glomerular injury, thereby providing a new therapeutic approach that targets circulating mtDNA or TLR9.

线粒体在进化上起源于细菌，故其DNA（mtDNA）中的CpG为非甲基化。近来研究表明，mtDNA能像细菌DNA一样，作用于Toll样受体9（TLR9），活化中性粒细胞并造成组织损伤（Zhang,Nature,2010）；伴有蛋白尿的创伤，烧伤及子痫病人的血清mtDNA升高；以及TLR9在足细胞损伤中表达上调。据此，我们推测循环中的mtDNA能直接损伤足细胞并导致蛋白尿。我们把mtDNA处理小鼠，造成了大量蛋白尿及足细胞足突融合，从而支持该假说。本课题拟：1）用细胞实验确定mtDNA能否经TLR9/NFkB/p38通路而损伤足细胞，2）用动物实验确定mtDNA是否经TLR9/NFkB/p38通路造成足细胞损伤和蛋白尿，并评估系统炎症反应的作用，3）检测各类肾小球病患血清mtDNA水平，以及足细胞TLR9/NFkB/p38表达和活化。本研究有望揭示足细胞及肾小球损伤新机制，为其防治提供新途径。

肾脏病是一种常见病，在我国患者总数超过1亿。足细胞是肾小球滤过屏障的关键组成，其损伤可启动肾小球病变，导致肾小球硬化和肾功能丧失。因此，弄清足细胞的损伤机制是肾小球疾病防治的关键环节之一。本课题根据本领域的最新进展以及前期研究，提出线粒体DNA或DAMPs对足细胞的损伤作用，并提出其可能的机制是通过TLR9及其下游的p38和NFkB信号，造成足细胞凋亡。本研究目的在于确定mtDNA对足细胞的损伤作用，并阐明其损伤作用的机制。我们首先利用制备的线粒体DAMPs，包括线粒体DNA，对大鼠和小鼠进行了尾静脉注射，确定循环mtDAMPs或DNA对足细胞的损伤效应。通过收集和测定尿液中的蛋白水平，评估线粒体DAMPs和mtDNA对足细胞的影响，发现线粒体DAMPs能诱导蛋白尿，证明线粒体DAMPs或DNA对足细胞的损伤作用，但蛋白尿水平较低且不能持续，提示需要长期的mtDAMPs或mtDNA的处理才可能诱导足细胞损伤。我们还对mtDNA对足细胞的损伤作用及其机制进行了研究，在嘌呤霉素氨基核苷（PAN）损伤模型中发现PAN促进细胞内源mtDNA向内体-溶酶体的转移并显著上调TLR9，并激活MAPK38和NFkB信号，造成足细胞凋亡。用siRNA将TLR9敲低则能消除PAN效应，从而证实mtDNA是通过TLR9诱导足细胞凋亡。我们进一步敲低溶酶体特异的DNase2，以增加溶酶体mtDNA累积，发现加重凋亡；反之，过表达DNase2促进溶酶体内mtDNA降解则发现凋亡增加。我们还在大鼠和FSGS病人肾标本进行了验证研究。总之，我们阐明了足细胞损伤的一个新的机制，对足细胞病的预防和治疗提供了新的靶点和途径。此外，本课题的部分资金还用于了新课题探索，包括对肾小球细胞进行单细胞RNA-seq分析从而鉴定了大批足细胞和系膜细胞的必需基因；还对miR-30在足细胞作用机制进行了研究。已发表标注基金SCI论文7篇，包括J Clin Invest，Kidney Int， Sci Rep， Plos One等。该课题研究过程中，有三名研究生的参与，并获得很好的科研培训。