新疆地区维吾尔族、汉族klotho基因单核苷酸多态性与特发性草酸钙肾结石相关性研究

Most clinically idiopathic calcium oxalate kidney stones has no significant genetic background and systemic diseases. Klotho is important for the regulation of calcium and phosphorus role has been confirmed, but the researchs of kidney calcification are still the exception. Researchs has found that the abscene of Klotho impair the reabsorbtion of calcium ions mediated by TRPV5,and present as hypercalcemia and hypercalciuria, this process needs the participation of klotho. Studies suggest that something may impair the reabsorption of calcium mediated by klotho in collaboration with TRPV5 in Kidney collecting tubule and Distal convoluted renal tubule. Our project will apply Immunohistochemistry, Western Blot and Real Time-PCR technology,and make a depth study of the mechanism of klotho in calcification in Kidney stone formation at animal and molecular level. Other studies also found that the klotho gene polymorphism was associated with the risk of Kidney stone formation in Europen, American and Japanese population.High Resolution Dissolved Curves will be used in our project to study the correlation of klotho gene mutation in Uighur and Han population and idiopathic calcium oxalate Kidney stone disease,and mechanism of calcification in Kidney stone formation caused by its variation.Our project will illustrate mechanism of the Kidney stone formation and provide new ideas for its prevention strategy and provides new experimental data and theoretical basis.

临床上大多数特发性草酸钙肾结石无明显遗传背景和相关系统性疾病。Klotho对钙磷调节的重要作用已被确认，但在肾脏钙化及结石方面研究还不多见。有研究结果发现klotho缺失影响TRPV5通道对钙离子的重吸收，呈现出高血钙及高尿钙表现。提示由klotho协同TRPV5介导的钙在肾远曲小管/集合管的重吸收发生障碍。而Klotho能够激活TRPV5使其发挥正常功能。本课题将应用免疫组化、Western Blot及Real Time-PCR等技术，在动物和分子水平深入研究Klotho在肾脏钙化结石形成中的机制。另有研究发现Klotho基因的多态性与欧美人群肾结石的形成风险相关。本研究拟利用高分辨溶解曲线（HRM）检测等技术研究维吾尔族与汉族人群中Klotho基因多态性与特发性草酸钙肾结石疾病的相关性及其对肾脏钙化结石形成的作用机制。为阐明肾结石形成机制提供新的思路，为其防治策略提供新的实验数据和

本研究首先探讨Klotho蛋白对草酸钙肾结石大鼠肾小管上皮细胞氧化应激的影响及其机制，其次分别纳入维吾尔族及汉族特发性草酸钙肾结石患者各400余例和维吾尔族及汉族健康体检者各400例。通过病例对照研究的方法对新疆草酸钙肾结石患者，应用SnaPshot方法检测外周血DNA Klotho基因SNPs，进行基因分型，了解新疆地区维吾尔族、汉族病例组与正常组之间基因多态性的差异。共分析新疆维吾尔族、汉族人群Klotho基因六个位点rs1207568、rs3752472、rs564481、rs648202、rs145682430、rs650439。得出以下结论：（1）各位点与新疆地区汉族、维吾尔族特发性草酸钙肾结石存在基因多态性差异。（2）rs1207568、rs564481位点两等位基因在汉族正常组与维吾尔族正常组中分布有显著差异。（3）rs564481、rs3752472、rs145682430及rs650439汉族、维吾尔族病例组中分布有显著差异。（4）汉族人群rs3753472C等位基因、rs650439A等位基因是特发性草酸钙肾结石的发病保护因素之一。维吾尔族rs1207568C等位基因是特发性草酸钙肾结石发病风险因素之一。（5）Klotho mRNA的低表达致使肾小管上皮细胞氧化损伤,促使草酸钙晶体黏附成石。Klotho mRNA的低表达可能和Klotho的抗氧化损伤信号通路有关。