A New Target for Kidney Stone Prevention: Calcium-Sensitive Transport of Citrate

描述(申请人提供)：肾结石是一种常见病和重病。尿柠檬酸是钙结石最重要的抑制物，通过保持钙的溶解。对肾脏中柠檬酸转运的调节还没有得到充分的研究&仍然知之甚少。一个单一的转运体NaDc1被认为负责肠道和肾脏柠檬酸的重吸收。然而，我们最近发现了一种新的钙敏感的柠檬酸转运过程。这项应用的总体目标是确定通过一种新的尖端钙敏感转运蛋白的肾脏近端小管尖端柠檬酸转运的机制和调节，以及NaDC1和NaDC3的相对作用。我们的中心假设是，当钙含量增加时，新的柠檬酸重吸收过程可以限制柠檬酸在近端小管中的运输。阐明以前未知的柠檬酸转运体可能会导致新的治疗方法来增加尿柠檬酸。目的是：1)确定新的钙敏感的柠檬酸转运蛋白及其在近端小管中的作用。我们的工作假设基于三个近端小管细胞系的数据，即除了NaDC1外，近端小管顶端还存在一种新的钙敏感的二羧酸转运体。我们将在这些细胞系中使用NaDC1的敲除来证明这一点。此外，我们还将确定柠檬酸转运速率，即剩余柠檬酸转运过程的细胞外钙敏感性。 在从基因敲除小鼠解剖的近端小管片段中生长的近端小管细胞中发现。2)确定NaDC3在近端小管柠檬酸重吸收中的作用。基于我们令人惊讶的初步数据，我们将确定NaDC3是否表达在顶膜上，这是钙敏感的柠檬酸重吸收的原因。我们将确定NaDC3在近端小管中的定位，并测定根尖表面的钙敏感性。我们还将确定基底外侧NaDC3的作用。3)确定NaDC1在柠檬酸的肾重吸收和肠吸收中的作用。我们的工作假设是，NaDC1不是柠檬酸在肾脏和肠道中转运的唯一机制。其他心尖机制在酸中毒和高钙尿症等情况下可能都很重要。我们将使用NaDC1基因敲除小鼠，比较在基线和酸中毒和高钙尿条件下-/-到+/+小鼠的肾脏柠檬酸重吸收。我们还将测定NaDC1基因敲除小鼠的肠道柠檬酸吸收。这些研究将建立新的范式，以了解柠檬酸在肾脏近端小管和肠道中的重吸收，以及这些范式在酸中毒和高钙尿中是如何受到影响的。这些结果有望产生重要影响，因为了解近端小管柠檬酸盐运输的调节将极大地改进预防钙结石的策略。

DESCRIPTION (provided by applicant): Kidney stones are a common & serious illness. Urinary citrate is the most important inhibitor of calcium stones by keeping calcium soluble. The regulation of citrate transport in the kidney has received inadequate investigation & remains poorly understood. A single transporter NaDC1 has been assumed to be responsible for intestinal & renal citrate reabsorption. However we have recently identified a novel calcium-sensitive citrate transport process. The overall objective of this application is to determine the mechanisms & regulation of kidney proximal tubule apical citrate transport via a novel apical calcium-sensitive transporter, & the relative roles of NaDC1 & NaDC3. Our central hypothesis is that the novel citrate reabsorptive process functions to limit citrate transport in the proximal tubule when calcium is increased. Elucidation of previously unidentified citrate transporters can potentially lead to new treatments to increase urinary citrate. Aims are: 1) Define the novel calcium-sensitive citrate transporter & its role in the proximal tubule. Our working hypothesis, based on data in three proximal tubule cell lines, is that in addition to NaDC1, a novel calcium- sensitive dicarboxylate transporter is present on the apical side of the proximal tubule. We will use knockdown of NaDC1 in these cell lines to demonstrate this. Also we will determine citrate transport rates & the extracellular calcium-sensitivity for the remaining citrate transport process found in proximal tubule cells grown from dissected proximal tubule segments from knockout mice. 2) Determine the role of NaDC3 in proximal tubule reabsorption of citrate. Based on our surprising preliminary data, we will determine if NaDC3 is expressed on the apical membrane & accounts for calcium- sensitive citrate reabsorption. We will determine the localization of NaDC3 in the proximal tubule & determine the calcium sensitivity on the apical surface. We will also determine the role of basolateral NaDC3. 3) Determine the role of NaDC1 in renal reabsorption & intestinal absorption of citrate. Our working hypothesis is that NaDC1 is not the exclusive mechanism of citrate transport in the kidney & intestine. Other apical mechanisms may both be important in conditions such as acidosis & hypercalciuria. We will use NaDC1 knockout mice & compare renal citrate reabsorption in the -/- to +/+ mice both under baseline & conditions of acidosis & hypercalciuria. We will also determine intestinal citrate absorption in NaDC1 knockout mice.. These studies will establish new paradigms in understanding citrate reabsorption in both the kidney proximal tubule & the intestine, & how these paradigms are impacted in acidosis & hypercalciuria. These outcomes are expected to have an important impact since understanding the regulation of citrate transport in the proximal tubule will dramatically improve strategies in the prevention of calcium stones.