CLC-5 Inactivation and Hypercalciuria in Dent's Disease

描述（由申请人提供）：高钙尿症是草酸钙结石病最常见的危险因素，通常具有遗传成分。第一个遗传性高尿钙综合征的基因被确定为X连锁肾结石（登特氏病），这是由失活突变的CLCN 5基因编码的电压门控氯离子通道C1 C-5，表达在近端肾小管细胞的近端内体。 然而，除了低分子量（LMW）蛋白尿，我们不知道这如何解释疾病的其他特征，如高钙尿，这是登特氏病患者草酸钙结石形成的主要危险因素，也不知道为什么近端肾小管产生过量的1，25（OH）2维生素D。 我们的假设是，C1 C-5在核内体中的功能缺陷导致改变的膜和蛋白质运输，扰乱细胞囊泡运输网络的完整性。这可以解释顶端钠依赖性运输过程，如磷酸盐的重吸收功能的改变。此外，由于25（OH）2 D的1a-羟基化发生在近端肾小管细胞中，因此1a-羟化酶的失调可能是膜运输改变的次要后果。我们将在培养的HK-2人近端肾小管细胞中解决这一假设，在该细胞中，我们使用了核酶来减少C1 C-5的表达。 在初步研究中，我们已经证明了这些敲低细胞中膜运输和溶质转运的变化。使用该模型，我们的具体目标是确定C1 C-5表达减少对以下方面的影响：（1）膜运输，（2）溶质转运（特别是Na依赖性磷酸盐转运）和（3）维生素D的1a-羟基化。我们希望这些试点研究将提供显着的见解草酸钙结石形成的机制，在登特氏病，并提供基础，进一步研究使用这种体外细胞培养模型，以解决高钙尿症和肾功能衰竭的机制。

DESCRIPTION (provided by applicant): Hypercalciuria is the most common risk factor for calcium oxalate stone disease and often has an hereditary component. The first inherited hypercalciuric syndrome for which the gene was identified was X-linked nephrolithiasis (Dent's disease), which is caused by inactivating mutations in the CLCN5 gene encoding the voltage-gated chloride channel C1C-5, expressed in subapical endosomes of renal proximal tubular cells. However, other than low molecular-weight (LMW) proteinuria, we do not know how this explains other features of the disease such as hypercalciuria, which is the major risk factor for calcium oxalate stone formation in patients with Dent' s disease, nor why proximal tubular production of 1,25(OH)2 vitamin D is excessive. Our hypothesis is that defective function of C1C-5 in endosomes leads to altered membrane and protein trafficking that perturbs the integrity of the cellular vesicular transport network. This could explain altered function of apical Na-dependent transport processes such as reabsorption of phosphate. Further, since 1a-hydroxylation of 25(OH)2 D occurs in proximal tubular cells, it is possible that dysregulation of the 1a-hydroxylase is a secondary consequence of altered membrane trafficking. We will address this hypothesis in cultured HK-2 human proximal tubular cells in which we have used a ribozyme to reduce C1C-5 expression. In preliminary studies we have demonstrated changes in membrane trafficking and solute transport in these knockdown cells. Using this model our specific aims are to determine the consequences of reduced C1C-5 expression on: (1) membrane trafficking, (2) solute transport (particularly Na-dependent phosphate transport), and (3) 1a-hydroxylation of vitamin D. We expect that these pilot studies will offer significant insights into the mechanisms of calcium oxalate stone formation in Dent's disease and provide the foundation for further studies using this in vitro cell culture model to address mechanisms of hypercalciuria and renal failure.