钙敏感受体多态性影响江西赣南地区含钙肾结石形成的分子机制研究

Gannan, located at the south of Jiangxi province, is one of the most popular area with urolithiasis in China. Calcium nephrolithiasis (CN) is the most common form of urinary stone. Recently, the genetic factors in the formation of urinary stone are increasingly spectacular.In pre-experiment,a few investigations demonstrated that some polymorphisms in calcium-sensing receptor (CASR) gene were associated with the risk of idiopathic hypercalciuria and CN, and exerted influence on urinary Ca level. However, the exact mechanism is not clear. These studies also indicated that the most of the allels of the CASR gene associated with urolithiasis are located at promotor regoin or have the linkage disequilibrium with it. Moreover, the interacting proteins with CASR and protein kinase C-mediated phosphorylation of CASR are the main factors affecting the activation of CASR. We supposed that the polymorphisms of CASR are associated with the formation of CN through the genetic transcription regulation and the post-translational modification of CASR. In order to validate the hypothesis, we will go on detecting the other polymorphisms in CASR gene, and finding the genotypes and haplotypes associated with the risk and the phenotypes of CN. The molecular mechanism for the effect of CASR polymorphisms on the formation of CN will be investigated through the special haplotypes affecting the genetic transcription regulation and the post-translational modification of CASR. The investigation will provide a new leads to the genetic treatment of CN.

江西省赣南地区为我国泌尿系结石的高发区之一。含钙肾结石(CN)最常见。本项目组前期研究结果和文献资料表明，钙敏感受体(CASR)基因的几个多态性位点与特发性高钙尿症和CN发病风险相关。而且影响尿中Ca2+浓度。但其机制尚不清楚。现有研究的共同特点是，这些与泌尿系结石相关的CASR基因多态性位点多位于启动子区或与启动子区多态性位点存连锁不平衡性。另外，CASR相互作用蛋白和蛋白激酶C介导的CASR磷酸化是影响CASR活性的主要因素。我们推测，CASR基因多态性位点可能通过CASR基因转录调节和CASR蛋白翻译后修饰参与CN形成。为验证这一假说，本项目将继续检测CASR其它SNPs，分析基因型和单体型与CN发病风险及表型的相关性，并从特定单体型影响CASR基因转录调控和蛋白质翻译后修饰入手，探讨CASR单体型影响CN形成的分子机制，为CN的基因治疗提供新的线索和思路。

资料表明，钙敏感受体(CaSR)基因的几个多态性位点与特发性高钙尿症和CN发病风险相关。但其机制尚不清楚。本项目将继续检测CaSR其它单核苷酸多态性（SNP），分析其多态性与CN的相关性及分子机制。.本项目选择含钙肾结石（CN）患者220例和健康人群250人。选择CaSR基因中频数≥0.05，或文献报道与肾结石相关的11个SNPs位点：rs9740、rs1501899、rs3749207、rs4678174、rs6776158、rs7652589、rs1042636、rs1801725、rs1801726、rs7632399、rs9883981，采用TaqMan-MGB 探针法进行检测。结果显示，CN组中rs1501899位点的A等位基因和GG基因型频率高于对照组。单体型AATCAGAGCAA、AGCTGAGGCGG与CN的形成呈正相关，而单体型AGCTAGGGCAA与CN的形成呈负相关。.采用荧光定量RT-PCR检测CaSR mRNA表达，CN组肾组织中CaSR mRNA平均相对含量低于对照组。采用Western blot法检测受体活性修饰蛋白（RAMP）、小凹蛋白（Caveolin）和CaSR蛋白表达。CN组RAMP、Caveolin平均相对含量高于对照组, CN组CaSR平均相对含量低于对照组。.CaSR重组慢病毒载体转染人肾小管上皮细胞HK-2，分别采用Western-blot和RT-PCR法检测RAMP、丝蛋白、Caveolin、CASR、PKC的表达。结果显示，RAMP、丝蛋白、CaSR、PKC mRNA和蛋白表达均增高，而Caveolin mRNA和蛋白表达均降低。这表明，RAMP、丝蛋白、PKC、Caveolin与CaSR表达存在相关性，可能参与了CaSR蛋白的活性修饰。 .将CaSR基因进行rs6776158位点点突变，并构建荧光载体；成功构建SOX17载体，将两种载体转染人肾小管上皮细胞HK-2。结果显示：CaSR与SOX17共转染组荧光素酶活性显著增高。表明CaSR启动子区rs6776158位点点突变后不能与SOX17转录因子结合，其活性降低。.总之，CaSR基因多态性与CN显著相关，其作用可能通过影响CASR基因转录调控和蛋白质修饰实现。本项目研究结果为泌尿系结石成因和机制研究以及泌尿系结石的防治提供了新的线索和思路。.