PATHOPHYSIOLOGY OF HYPERCALCIURIA

本课题的目的是研究高蛋白摄入所致的病理生理基础。 高钙尿症。上皮性钙通道(ECaC1)存在于远端的顶膜中 肾脏的曲管(DCT)在维持整体钙稳态中起着重要作用。我们的初步结果表明，与高蛋白摄入量相关的高钙尿症至少部分是由于酸抑制了ECaC1介导的DCT中的钙重吸收。在特定的目标1中，我们将研究ECaC1酸抑制的分子机制。ECaC1酸度调节的“pH感应器”候选基因将被定点突变 诱变。野生型和突变型通道的活性将通过全细胞膜片钳记录进行检测。膜磷脂，磷脂酰肌醇，4，5-二磷酸(PIP2) 最近出现了一种独特的通道功能调节器。在特定的目标2中，我们将检验PIP2对ECaC1通道的调节强调了以下机制的假设 前列腺素E_2加重高蛋白摄入所致的高钙尿症。在具体目标3中，我们将证实管腔酸化抑制远端曲小管(DCT)对钙的重吸收。用大鼠DCT进行体内微灌流，观察管腔pH值对钙重吸收的影响。这些研究将有助于了解高钙尿症的机制，不仅在高蛋白质摄入量的情况下，而且在慢性代谢性酸中毒的情况下也是如此。

The goal of this project is investigate pathophysiological basis of high protein intake-induced hypercalciuria. Epithelial Ca 2+ channels (ECaC1) are present in the apical membrane of the distal convoluted tubule (DCT) of kidney and play an important role in the maintenance of overall calcium homeostasis. Our preliminary results indicate that hypercalciuria associated with a high protein intake is, at least partly, caused by acid inhibition of ECaC1-mediated Ca 2+ reabsorption in DCT. In Specific Aim 1, we will examine the molecular mechanism of acid inhibition of ECaC1. Likely candidates of "pH sensor" for acid regulation of ECaC1 will be mutated by site-directed mutagenesis. The activity of wild type and mutant channels will be examined by whole-cell patch-clamp recording. Membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) has recently emerged as a unique regulator of channel function. In Specific Aim 2, we will examine the hypothesis that PIP2 regulation of ECaC1 channel underlines the mechanism by which prostaglandin E2 worsens the high protein intake-induced hypercalciuria. In Specific Aim 3, we will confirm that luminal acidification inhibits Ca 2+ reabsorption in the distal convoluted tubules (DCT). In vivo microperfusion of rat DCT will be performed to examine the effect of luminal pH on Ca 2+ reabsorption. These studies will help understand the mechanism of hypercalciuria, not only during high dietary protein intake but also under conditions of chronic metabolic acidosis.