TRPV5和TRPV6介导的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 which were regarded by researchers as the special markers of osteoblast-like cells and chondrocyte-like cells were detected. We also found that high levels of VD3/VDR through the activation of the BMP2 pathway, regulated the phenotype transformations of renal tubular epithelial cells and cause the formation of Randall plague. After the treatment of high dose of VD3, expression of the transient receptor potential vanilloid member 5 which control the oncentration of calcium ion in and out cells were decreased.So we emphasis that phenotypic transformation of renal tubular epithelial cells, induced by BMP2 signal pathway which was activated in the condition of high levels of VD3/VDR might decrease the expressions of TRPV5 and /or TRPV6 and inhibit the migration of calcium ion, break the balance of calcium ion in and out cells in kidney and cause the formation of calcium plaque. This study was proposed by the GHS rat model, confirmed the migration of calcium ion in and out cells on the formation of Randall plaque in vivo and vitro, respectively. Meanwhile, the phenotypic transformation of renal tubular epithelial cells, induced by BMP2 signal pathway which was activated in the condition of high levels of VD3/VDR might regulate the expressions of TRPV5 and /or TRPV6.

最新研究表明，肾Randall钙斑是尿石形成的基础和起始环节，但其形成机制仍不清楚。本人前期研究已证实，VD3/VDR通过BMP2信号通路调控肾小管上皮细胞表型转化在肾结石形成中发挥重要作用。进一步研究显示：高浓度钙离子可激活BMP2信号通路；VD3/VDR可抑制细胞膜上调控细胞内外钙离子浓度的钙通道蛋白TRPV5的表达，而细胞膜上主要的钙通道蛋白有TRPV5和TRPV6。因此，我们推测：VD3/VDR通过抑制细胞膜钙通道蛋白（TRPV5和/或TRPV6）的表达，干扰细胞外钙离子的内流，使细胞外钙离子浓度升高，而高浓度钙离子可激活BMP2信号通路，使肾小管上皮细胞发生表型转化，导致肾Randall钙斑的形成。本研究拟通过上调、下调VD3/VDR，分别从体内和体外两个方面，证实VD3/VDR通过钙通道调控肾小管上皮细胞表型转化的机制。

本人前期研究已证实，VD3/VDR通过BMP2信号通路调控肾小管上皮细胞表型转化在肾结石形成中发挥重要作用。但VD3/VDR激活BMP2信号通路的机制并不清楚。在本研究中，我们检测了处理前后TRPV5、TRPV6，BMP2, Runx2和Osterix以及软骨样细胞标记因子（sox9，Collagen protein II）、成骨样细胞标记因子（OPN、OCN）、上皮细胞标记因子（Keratin、E-cadherin）的表达，并采用Yang Hsueh的方法检测细胞内钙盐沉积变化。结果显示：与正常细胞组比较，VDR-siRNA组的BMP2、Runx2、Osterix、collagenproteinII、SOX9、OPN、OCN表达降低，Keratin、E-cadherin表达增加，VD3处理组中BMP2、Runx2、Osterix、collagenproteinII、SOX9、OPN、OCN表达增加，Keratin、E-cadherin表达降低；VD3+CuCl2组较VD3组TRPV5、TRPV6，BMP2, Runx2、Osterix以及软骨样细胞标记因子（sox9，Collagen protein II）、成骨样细胞标记因子（OPN）表达均有所下降。我们还采用免疫荧光染色的方法观察上皮细胞表型转化过程，其结果显示：正常细胞组中，E-cadherin、OPN及sox9均有表达，与正常细胞组相比，VDR-siRNA转染组中，E-cadherin表达增加，OPN及sox9表达减少，VD3处理组中OPN及sox9表达明显增加，E-cadherin未见表达，VDR-siRNA转染的VD3处理组中OPN及sox9表达增加，E-cadherin表达减少。以上结果表明，在高水平VD3/VDR的条件下，诱导细胞膜上的钙离子转运通道的表达，使细胞内的钙离子浓度升高，激活BMP2信号通路各分子，使上皮细胞可能向成骨样或软骨样细胞进行分化，导致结石的形成。