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。该基因首次被发现的遗传性高钙血症综合征是x连锁肾结石（登特氏病），它是由编码电压门控氯通道C1C-5的CLCN5基因突变失活引起的，该基因在肾近端小管细胞的根尖下内体中表达。然而，除了低分子（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，这是邓特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小管产生过多的1,25(OH)2维生素D。

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.