二甲双胍靶向TR4对肾结石形成的影响及分子机制研究

Renal calculus is one of the most common disease in urinary tract system, its recurrence rate is very high. Therefore, the mechanism study of kidney stone formation and how to prevent kidney stone is the current research focus. Our preliminary studies found that knocking down TR4 or using metformin as the TR4 inhibitor can alter the polarization and aggregation of macrophages and affect the ROS level of renal epithelial cells, result in the reduction number of calcium oxalate crystals adhered to renal tubular epithelial cells. In animal experiments, we found that metformin can reduce the number of CaOx crystals in mice and rats' kidney. According to these results, we hypothesize that metformin can reduce kidney stone formation via inhibiting TR4. In our project, we will use TR4 knockout mice, kidney tissue specific TR4 knockout mice and rat model to further verify the role of metformin /TR4/ROS in the formation of kidney stones. Meanwhile, we will utilize the oxidative stress chip and ChIP to explore the detail mechanism how TR4 regulate ROS. We will compare TR4 level in tissue of normal kidney with that of kidney with stones. At last we will investigate the prevalence of kidney stones in patients who were administrated with or without metformin in large scale. This project aims to reveal the new mechanism of renal calculi formation at cell, animal and clinic levels, and provide new ideas and theoretical basis for the prevention and treatment of renal calculi.

肾结石为泌尿系统常见疾病，且复发率高，肾结石的形成机制及防治是目前研究的热点。睾丸孤核受体4（TR4）在氧化应激、脂质代谢等生理过程中发挥重要作用。我们前期研究发现：调低TR4或应用TR4抑制剂二甲双胍可以降低巨噬细胞的极化和聚集，影响肾小管上皮细胞氧化应激水平，降低草酸钙结晶粘附。动物实验发现二甲双胍可以降低小鼠和大鼠肾草酸钙结晶数目。因此，我们推测二甲双胍可以通过TR4抑制肾结石形成。本研究拟运用TR4基因敲除小鼠、肾脏组织特异性TR4基因敲除小鼠和大鼠模型验证二甲双胍/TR4/ROS在肾结石形成中的作用；利用细胞因子氧化应激相关芯片、ChIP等探究TR4影响ROS的具体分子机制；比较正常肾组织与肾结石患者肾组织中TR4表达水平的差异；针对较大范围人群调查服用二甲双胍对肾结石患病率的影响。本课题将从细胞、动物、临床多个层次研究肾结石形成的新机制，为防治肾结石提供新的思路及理论依据。

本课题已按计划完成研究目标，首先我们在细胞层面验证二甲双胍可以影响TR4从而影响肾结石/结晶形成，进一步机制探索证明，肾小管上皮细胞中TR4通过转录水平调控MCP-1表达从而影响巨噬细胞聚集，而巨噬细胞聚集则会进一步影响肾小管上皮细胞氧化应激状态，从而影响肾结石/结晶形成。其次动物实验中应用大鼠及小鼠动物结石模型证明二甲双胍可以通过TR4转录调控MCP-1影响肾结石/结晶形成，后续采集小鼠结石模型样本进行转录组测序，筛选影响结石形成的相关基因及通路。最后我们收集2012-2017临床样本进行数据统计，结果发现服用二甲双胍的患者发生肾结石/结晶概率较低。目前相关研究成果共发表SCI论文6篇，其中影响因子10分以上4篇。项目共培养博士研究生2名，硕士研究生2名。