CLC-5 Inactivation and Hypercalciuria in Dent's Disease

Dent 病中的 CLC-5 失活和高钙尿症

• 批准号: 6559378
• 负责人: STEVEN J SCHEINMAN
• 金额: $ 15.2万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6559378
• 关键词: 25 hydroxycholecalciferol; calcium; cell line; chloride channels; gene targeting; genetically modified animals; hydroxylation; hypercalciuria; intracellular transport; ion transport; laboratory mouse; membrane activity; nephrolithiasis; oxalates; phosphates; renal tubular transport; voltage gated channel

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.

说明(申请人提供)：高钙尿症是草酸钙结石最常见的危险因素，通常有遗传成分。第一个被鉴定出该基因的遗传性高钙尿综合征是X连锁肾结石(Dent病)，它是由编码电压门控氯通道C1C-5的CLCN5基因的失活突变引起的，该基因表达在肾近端小管细胞的心尖下内涵体中。然而，除了低分子量蛋白尿之外，我们不知道这如何解释该疾病的其他特征，如高钙尿，这是登特型S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多的1，25(OH)2维生素D。 我们的假设是，C1C-5在内体中的功能缺陷会导致膜和蛋白质运输的改变，从而扰乱细胞囊泡运输网络的完整性。这可以解释顶端依赖钠的运输过程的功能变化，如磷酸盐的重吸收。此外，由于25(OH)2D的1a-羟化发生在近端肾小管细胞中，1a-羟基酶的失调可能是膜转运改变的次要后果。我们将在培养的HK-2人近端小管细胞中解决这一假设，在该细胞中，我们使用核酶来减少C1C-5的表达。在初步研究中，我们已经证明了这些击倒细胞的膜运输和溶质运输的变化。利用这一模型，我们的具体目标是确定C1C-5表达减少对：(1)膜转运，(2)溶质转运(特别是钠依赖的磷酸盐转运)，以及(3)维生素D的1a-羟化的后果。我们期待这些先导性研究将对Dent病草酸钙结石的形成机制提供重要的见解，并为利用这一体外细胞培养模型进一步研究高钙尿症和肾功能衰竭的机制提供基础。