DNA结合蛋白ZBP1在草酸钙结晶诱导肾小管上皮细胞焦亡中的作用和机制研究

Pyroptosis has been identified as a kind of programmed cell death over the past few years and was found to be induced by calcium oxalate crystals in the renal tubular epithelial cells in the latest study. The injury of mitochondria is an important characteristic ultrastructure change of renal tubular epithelial cells in calcium oxalate stone formers demonstrated by our previous study. Mitochondrial injury often occurred accompanied by cytosolic DNA accumulation and pyroptosis activation, however, the involvement of DNA binding protein ZBP1, as a crucial cytosolic DNA sensor and centric regulator of pyroptosis, in the kidney stone formation has never been reported so far. In our preliminary experiment, mRNA and protein levels of ZBP1 were both upregulated in the mice kidney tissues with deposition of calcium oxalate crystals. Moreover, key markers of pyroptosis, like NLRP3, Caspase-1 and so on, were also upregulated, meanwhile, knockdown of ZBP1 in the renal tubular epithelial cells resulted in alleviation of pyroptosis caused by calcium oxalate crystals. According to these findings, we speculate that ZBP1 plays a key role in the pyroptosis of renal tubular epithelial cells induced by calcium oxalate crystals and promotes stone formation. In this study, we will investigate further that following the stimulation of calcium oxalate crystals, what happens to the distribution of cytosolic mitochondrial DNA (mtDNA) and whether it binds to ZBP1 specifically. We will also elucidate the molecular mechanisms by which calcium oxalate crystals upregulate ZBP1 and through which ZBP1 conducts pyroptosis in the renal tubular epithelial cells. The aim of this research is to discover new mechanism of calcium oxalate stone formation and provide candidate drug targets for prevention of stone formation and recurrence.

焦亡是近年来发现的细胞程序性死亡，最新研究显示草酸钙结晶诱导肾小管上皮细胞焦亡。我们前期发现，线粒体损伤是草酸钙结石患者肾小管上皮细胞超微结构变化重要特征。线粒体损伤可致胞质DNA聚积并激活细胞焦亡，DNA结合蛋白ZBP1是重要的DNA感受器和调节焦亡的中心分子，其是否参与肾结石发病尚无报道。我们预实验示，草酸钙结晶小鼠肾组织ZBP1的mRNA和蛋白表达上调，焦亡关键分子NLRP3、Caspase-1等表达升高；敲减ZBP1则可减少草酸钙结晶诱导肾小管上皮细胞焦亡。据此我们推测，ZBP1在草酸钙结晶诱导的肾小管上皮细胞焦亡中发挥重要作用并促进结石形成。本项目拟继续探索草酸钙结晶刺激条件下，线粒体DNA分布变化，是否与ZBP1特异性结合；阐明草酸钙结晶上调ZBP1和ZBP1调控肾小管上皮细胞焦亡的机制。本项目旨在发现草酸钙结石形成新机制，为预防结石形成和复发提供候选药物靶点。

焦亡是近年来发现的细胞程序性死亡方式，其在肾结石发病中的作用研究较少。线粒体损伤可致胞质DNA聚积并激活细胞焦亡，我们既往研究发现线粒体损伤是草酸钙结石患者肾小管上皮细胞超微结构变化重要特征。DNA结合蛋白ZBP1是重要的DNA感受器和调节焦亡的中心分子，我们对草酸钙肾结晶小鼠肾组织进行基因芯片分析及验证发现ZBP1的mRNA和蛋白表达上调，焦亡标志物NLRP3、Caspase-1、GSDMD表达升高，ELISA法检测小鼠血清提示体内炎症指标IL-1β和IL-18升高。使用一水草酸钙结晶（COM）刺激人肾小管上皮细胞系（HK2），流式细胞检测显示细胞死亡水平增加，qPCR检测提示HK2细胞内ZBP1、RIPK3、NLRP3、Caspase-1、GSDMD的mRNA表达水平表达明显上调；ELISA法检测细胞培养基中炎症指标IL-1β和IL-18升高。进一步利用Zbp1-siRNA干扰HK2细胞ZBP1表达，草酸钙结晶诱导的细胞死亡水平下降，细胞内NLRP3、Caspase-1、GSDMD分子mRNA表达水平较非干扰组降低，细胞培养基中IL-1β和IL-18水平下降。我们利用信号通路抑制剂与细胞共培育筛选参与ZBP1上调的信号通路，发现P38/MAPK通路抑制剂显著抑制了COM诱导的ZBP1的表达。免疫共沉淀实验发现COM促进了ZBP1与RIPK3的直接结合，细胞免疫荧光实验发现ZBP1与RIPK3在HK2胞质中存在共定位现象。最后，我们收集草酸钙结石患者和非肾脏疾病患者肾组织，免疫组化提示草酸钙结石患者肾组织ZBP1、IL-1β、Caspase-1、NLRP3蛋白表达水平升高，免疫荧光检测提示草酸钙结石患者肾组织ZBP1和焦亡关键分子Caspase-1表达增加，两者存在共定位现象。以上结果提示草酸钙结晶通过P38/MAPK通路上调ZBP1，ZBP1通过与RIPK3互作并活化下游信号通路，促进肾小管上皮细胞炎症反应和焦亡，并可能成为未来药物治疗靶点。本项目首次发现了DNA感受器ZBP1通过介导肾小管上皮细胞焦亡在草酸钙结石发病中的作用，阐明了ZBP1上下游信号通路，拓展了当前对于草酸钙结石发病机制的认识，为肾结石预防和治疗提供了新的思路。