肾小管上皮细胞表型转化在Randall钙斑形成机制中的作用

Urolithiasis is a quite common disease in urology, which is seriously harmful for human life. And it is always the research concentration in urology field. In recent years, many scholars both at home and abroad through directly observing the Randall plague in renal papillae area by percutaneous nephroscope and analyzing its elements, have found that almost all the patients with idiopathic calcium oxalate stone have Randall plague. So that, Randall plague is regarded as the place where idiopathic calcium oxalate stone begins. Years of studies have identified phenotype transformations of renal tubular epithelial cells can be carried out. In our study of Genetic Hypercalciuric Stone-forming (GHS) rats, in renal, the expression of some factors（Cbfa1, OPN, ALP, sox9, Collagen protein II）which were regarded by researchers as the special markers of osteoblast-like cells and chondrocyte-like cells and closely related with bone formation were detected. But the same markers were not found in control rats. And our research result of immunohistochemistry ( Collagen protein II ) of human kidneys also confirmed that the renal tubular epithelial cells could change to become the chondrocyte-like cells.Bone morphogenetic proteins（BMPs）is a protein family of important bone anabolic factors, which are closely related with ectopic calcification. The research in the vascular calcification has shown that: BMP2 signal pathway can regulate the phenotype transformations of vascular smooth muscle cells（VSMCs）into osteoblast-like cells or chondrocyte-like cells. Meanwhile, we found that the expressions of BMP2, MSX2, cbfα1 and Osterix in both mRNA and protein levels in GHS rats were significantly higher than that in control rats. So it is shown that BMP2 signal way was closely related with the formation of idiopathic calcium oxalate calculi. So we emphasis that BMP2 signal pathway, activated in the condition of hypoxicischemic stress、high calcium urine or inflammation, can regulate the phenotype transformations of renal tubular epithelial cells of Henle loops and cause the formation of Randall plague. This study is proposed by constructing epithelial cell lines tracer GHS rats model to research the effect of phenotype transformations on the formation of Randall plague and the role of BMP2 signal pathway on cellular phenotype transformations, perfecting the regulation mechanism of BMP2 signale pathway in stone formation and providing the theoretical base of therapy and prevention of renal idiopathic calcium oxalate stone.

最新研究表明肾脏Randall钙斑是位于肾乳头下间质的异位钙化，是尿石形成的基础和起始环节。本小组在对GHS大鼠和特发性草酸钙结石患者的研究中，发现了肾小管上皮细胞向成骨样细胞和软骨样细胞表型转化的证据，并发现BMP2信号通路与特发性草酸钙结石的形成密切相关，且该通路可调控细胞进行表型转化。基于以上研究，我们认为：在特定微环境的刺激作用下，细胞内BMP2信号通路被激活，进而调控肾Henle袢细段小管上皮细胞向成骨样细胞和软骨样细胞进行表型转化，并最终导致细段小管基底膜钙化和Randall钙斑形成。本研究拟通过构建上皮细胞系示踪GHS大鼠模型，追踪肾Henle袢细段小管上皮细胞表型转化过程，研究表型转化在Randall钙斑形成中的作用；并通过在活体和细胞两个不同水平抑制BMP2信号通路，研究其对细胞表型转化的影响，完善BMP2信号通路在结石形成中的调控机制，为尿石症基础理论研究提供新的思路。

研究表明肾脏Randall 钙斑是位于肾乳头下间质的异位钙化，是尿石形成的基础和起始环节。本课题小组在对遗传高钙尿结石形成（GHS）大鼠和特发性草酸钙结石患者的研究中，发现了肾小管上皮细胞向成骨样细胞和软骨样细胞表型转化的证据，并发现BMP2 信号通路与特发性草酸钙结石的形成密切相关，且该通路可调控细胞进行表型转化。基于以上研究，我们认为：在特定微环境的刺激作用下，细胞内相关信号通路被激活，进而调控肾Henle袢细段小管上皮细胞向成骨样细胞或软骨样细胞进行表型转化，并最终导致基质钙化和Randall 钙斑形成。本研究利用GHS大鼠作为特发性高钙尿症的动物模型，通过体内体外实验证明了肾结石形成中可能存在潜在的上皮间充质转分化（EMT）及成骨软骨分化机制。同时验证了IH病人肾小管上皮细胞也存在相似的转分化过程。并通过检测GHS大鼠及IH病人中血、尿中TGF-β1含量推测TGF-β1及其下游信号Wnt11信号通路与钙离子共同促进肾小管上皮细胞向成骨软骨分化。而阻断PRECs细胞Wnt11信号以及L型钙离子通道后，PRECs细胞EMT以及分化过程均被明显逆转，E-cadherin表达上调，而Snail1，Col2A1，OPN，Sox9和Runx2表达均减弱。同时通过外源性TGF-β1/钙离子对NRK单独或联合作用发现，TGF-β1可显著增加NRK间充质表型，而钙离子单独作用并不显著，联合作用可以显著促进细胞EMT进程以及成骨软骨分化。同时在研究计划之外，通过基因芯片技术及生物信息学分析成功鉴定了GHS大鼠肾脏内差异表达的miRNA及mRNA,并构建了GHS大鼠肾脏miRNA-mRNA调控网络和信号通路，为特发性高钙尿结石病肾脏基因水平发生的改变提供了新思路。