REGULATION OF CITRATE TRANSPORT

尿柠檬酸盐是已知的钙性肾结石最重要的内源性抑制剂之一。尿中柠檬酸盐的排泄主要是由其在近端小管的部分重吸收决定的。以前，研究柠檬酸盐转运的一个主要障碍是缺乏合适的细胞培养模型。然而，我们现在已经明确地确定了一种研究近端小管细胞系柠檬酸盐重吸收的方法（见初步数据）。此外，最近从近端小管中克隆了一个根尖二羧酸转运蛋白，这使得人们对柠檬酸盐转运的调控有了新的认识。这与我们的新的培养模型将允许我们检查几个关键问题在分子调控柠檬酸盐运输。具体目的是表征在近端小管柠檬酸盐吸收的急性和慢性调节。柠檬酸盐转运的急性调节pH和二价阳离子，特别是将解决。虽然之前的各种研究已经解决了pH对柠檬酸盐运输的影响，但我们提出的研究将能够同时直接解决细胞内pH（用荧光探针测量）和细胞外pH的作用。关于二价阳离子，我们的初步数据显示细胞外钙和镁对柠檬酸盐运输的显着影响。我们的研究结果表明，在临床范围内的腔内钙和镁浓度的变化可能是确定尿柠檬酸盐排泄的重要因素。拟议的研究也将解决柠檬酸盐重吸收的慢性调节。我们的细胞培养方法将允许我们直接检查pH，低钾血症和饥饿的慢性调节机制。我们假设柠檬酸盐重吸收的变化是由柠檬酸转运体的丰度和/或细胞分布的变化引起的。在拟议的研究中，我们将能够测量这些刺激对极化细胞中柠檬酸转运的直接体外影响，以及对mRNA和蛋白质水平以及二羧酸转运体的细胞分布的直接体外影响。

Urinary citrate is one of the most important known endogenous inhibitors of calcium nephrolithiasis. Urinary citrate excretion is primarily determined by its fractional reabsorption in the proximal tubule. Previously, a major impediment to studies of citrate transport has been the lack of a suitable cell culture model. However, we have now clearly identified a method to study citrate reabsorption in a proximal tubule cell line (see preliminary data). In addition, the recent cloning of an apical dicarboxylate transporter from the proximal tubule by Pajor allows new insight into the regulation of citrate transport. This with our new culture model will allow us to examine several critical issues in the molecular regulation of citrate transport. The specific aims are to characterize both acute and chronic regulation of citrate absorption in the proximal tubule. Acute regulation of citrate transport by pH and divalent cations, in particular will be addressed. Although a variety of previous studies have addressed the influence of pH on citrate transport, our proposed studies will be able to directly address simultaneously the role of intracellular pH (to be measured with fluorescent probes) as well as extracellular pH. In regards to divalent cations, our preliminary data demonstrate dramatic effects of extracellular calcium and magnesium on citrate transport. Our findings suggest that changes in luminal calcium and magnesium concentrations within the ranges seen clinically may be important in determining urinary citrate excretion. The proposed studies will also address chronic regulation of citrate reabsorption. Our cell culture method will allow us to directly examine the mechanism of chronic regulation by pH, hypokalemia, and starvation. We hypothesize that changes in citrate reabsorption with each of these result from changes in the abundance and/or cellular distribution of the citrate transporter. In the proposed studies we will be able to measure the direct in vitro effects of these stimuli on citrate transport in polarized cells, and on mRNA and protein levels and cellular distribution of the dicarboxylate transporter.