Regucalcin介导的细胞膜Ca2+-ATP酶调控在草酸钙结石形成中的作用及机制研究

The disorder of calcium homeostasis in the epithelia of renal tubules is an important role in calcium oxalate stone formation, however, the mechanism is still unknown. Regucalcin(RGN) is a new calcium-binding protein, which plays a pivotal role in maintaining the intracellular calcium homeostasis. Our former study indicated that RGN was down-regulated in epithelia of renal tubules after treated with COM crystal. In calcium oxalate stone rat model, RGN was also significantly down-regulated in renal tissue, especially in those tissue surrounded by the calcium oxalate crystals. These results disclosed that RGN was involved in the pathogenesis of cellular damage and calcium oxalate stone formation. It is already known that RGN could regulate the expression and activity of plasma membrane Ca2+ ATPases (PMCA), whereas Ca2+ ATPases (PMCA) has been involved in cell injury and regulate the level of intracellular calcium, which will induce the adhesion of calcium oxalate crystals to the epithelia of renal tubules, and latterly, the calcium oxalate stone will be formed. . According to our former results and reported literatures, we suspect that RGN was involved in process of calcium oxalate stone formation by regulating the expression and activity of PMCA. In this study we try to explain the possible effect and mechanism of RGN on the formation of calcium oxalate stone. First, the role of RGN on cellular damage and crystal retention induced by calcium oxalate crystals was examined. Furtherly, the rat model with the RGN silence or over-expression in the kidney was established, which was involved in the study to confirm the role of RGN in calcium oxalate stone formation. . Our study aims to providing an experimental basis for the pathogenesis of oxalate stone stone formation and a new intervention strategy for the prevention and treatment of calcium oxalate stone.

肾小管上皮细胞内钙稳态的破坏被认为在草酸钙结石形成过程中具有重要作用，但具体机制不明。Regucalcin（RGN）是新近发现的一种钙结合蛋白。我们前期研究发现：草酸钙晶体不但可以明显诱导肾小管上皮细胞RGN表达下调，而且草酸钙结石大鼠肾组织中RGN的表达也降低，尤其在晶体粘附处明显，提示RGN参与了草酸钙晶体的粘附和结石形成。由于RGN可以调控细胞膜Ca2+-ATPases（PMCA）的表达及活性，而PMCA可以通过调控细胞内钙的转运导致细胞损伤，进而影响晶体粘附。我们推测RGN可能通过调控PMCA破坏细胞内钙稳态，参与了晶体粘附和结石形成过程。本研究拟在检测RGN对草酸钙晶体诱导的细胞损伤和晶体粘附影响的基础上，通过构建肾脏沉默和过表达RGN大鼠，研究RGN在草酸钙结石形成中的作用及可能机制，为明确草酸钙结石发病机制提供新的实验依据，为草酸钙结石的预防提供新的干预靶点。

Regucalcin（RGN）是一种钙结合蛋白，我们的研究结果显示RGN在草酸钙结石模型的大鼠肾脏组织中表达降低。进一步细胞实验结果表明，RGN可通过调节肾小管上皮细胞的氧化应激水平降低肾小管上皮细胞对一水草酸钙（COM）晶体的粘附。进一步研究表明，抗雄药物Casodex可通过抑制雄激素受体（AR）表达增加肾小管上皮细胞RGN蛋白的表达，进而降低肾小管上皮细胞的氧化应激水平及对COM晶体的粘附。机制研究表明，AR可通过增加miRNA-203的表达降低肾小管上皮细胞RGN蛋白的表达，进而增加肾小管上皮细胞的氧化应激水平及对COM晶体的粘附。进一步机制研究表明，AR可通过直接结合到miRNA-203启动子区ARE增加miRNA-203的表达，而miRNA-203可通过直接结合到RGN mRNA 3'-UTR降低RGN蛋白的表达。在应用5%羟脯氨酸建立大鼠草酸钙结石模型的基础上的动物实验结果表明，Casodex可有效抑制大鼠肾脏AR蛋白的表达，并减少大鼠肾脏miRNA-203的表达，进而增加大鼠肾脏RGN蛋白的表达，最终抑制大鼠草酸钙结石的形成。本研究通过多个体外细胞系及体内动物模型证明，AR可通过增加miRNA-203的表达降低RGN蛋白的表达，进而增加肾小管上皮细胞的氧化应激水平，最终促进草酸钙结石的形成，而Casodex可通过靶向这条新发现的AR/miRNA-203/RGN/氧化应激信号通路抑制草酸钙结石的形成，为草酸钙结石的发病机制研究提供新的思路及实验基础，为草酸钙结石的临床防治提供潜在的治疗靶点及靶向药物。此外，在完成计划研究内容的基础上，又进一步拓展了本项目的研究工作：本项目组研究发现氯喹介导的细胞自噬抑制可通过减弱草酸诱导的肾小管上皮细胞氧化应激损伤，进而减少高草酸尿诱导的大鼠肾脏草酸钙晶体形成，而大蒜素可有效改善草酸诱导的肾小管上皮细胞缝隙连接损伤，进而减少高草酸尿诱导的大鼠肾脏草酸钙晶体形成，为草酸钙结石的临床防治提供新的思路及潜在的靶向药物。