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

描述（由申请人提供）：肾结石是一种常见的严重疾病。尿柠檬酸盐是最重要的钙结石抑制剂，通过保持钙溶解。肾脏中柠檬酸盐转运的调节尚未得到充分的研究，仍然知之甚少。单一转运蛋白NaDC 1被认为负责肠和肾柠檬酸盐重吸收。然而，我们最近发现了一种新的钙敏感性柠檬酸盐转运过程。本申请的总体目标是确定肾脏近端小管顶端柠檬酸盐转运的机制和调节，通过一种新的顶端钙敏感转运蛋白，以及NaDC 1和NaDC 3的相对作用。我们的中心假设是，新的柠檬酸盐重吸收过程的功能，以限制柠檬酸盐转运近端小管时，钙增加。阐明先前未鉴定的柠檬酸盐转运蛋白可能导致增加尿柠檬酸盐的新治疗。目的：1）明确新型钙敏感性柠檬酸盐转运体及其在近端小管中的作用。基于三种近端小管细胞系中的数据，我们的工作假设是，除了NaDC 1之外，近端小管的顶侧还存在一种新型的钙敏感性二羧酸转运蛋白。我们将在这些细胞系中使用NaDC 1的敲低来证明这一点。我们还将确定柠檬酸盐转运速率和剩余柠檬酸盐转运过程的细胞外钙敏感性 发现于从基因敲除小鼠解剖的近曲小管节段生长的近曲小管细胞中。 2)确定NaDC 3在近端小管重吸收柠檬酸盐中的作用。基于我们令人惊讶的初步数据，我们将确定NaDC 3是否在顶膜上表达并解释钙敏感性柠檬酸盐重吸收。我们将确定近端小管中NaDC 3的定位并确定顶端表面上的钙敏感性。我们还将确定基底外侧NaDC 3的作用。 3)确定NaDC 1在柠檬酸盐的肾重吸收和肠吸收中的作用。我们的工作假设是NaDC 1不是肾脏和肠道中柠檬酸盐转运的唯一机制。其他根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NaDC 1敲除小鼠，并在酸中毒和高钙尿症的基线和条件下比较-/-与+/+小鼠的肾脏柠檬酸盐重吸收。我们还将测定NaDC 1敲除小鼠的肠道柠檬酸盐吸收。 这些研究将建立新的范式，以了解柠檬酸盐在肾脏近端小管和肠道中的重吸收，以及这些范式如何在酸中毒和高钙尿症中受到影响。这些结果预计将产生重要影响，因为了解近端小管中柠檬酸盐转运的调节将大大改善预防钙结石的策略。

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.