Regulation of Renal Bicarbonate and Chloride Absorption

描述(申请人提供)：慢性代谢性酸中毒引起一系列的体内平衡反应，旨在使血液pH值恢复到正常值。在肾近端小管，这种反应包括氢离子分泌、氨合成和柠檬酸吸收增加。该实验室的研究已经检验了这些适应发生的分子基础，重点是对顶膜Na/H逆向转运体NHE3的调节。在最后一次资助期间，我们发现内皮素、Pyk2、c-Src、ERK和焦点黏附蛋白发挥了重要作用。建议的研究继续我们的重点是近端小管感知细胞内pH下降并传递信号从而最终增加NHE3活性的机制。研究分为四个目的。在目标1中，研究将利用微阵列来识别新的酸调节和内皮素调节基因。在《目标2》中，我们将重点关注夜班。微阵列的初步结果表明，夜转素基因是急性酸中毒中最受调控的基因。夜曲蛋白是一种昼夜节律基因。研究将探讨昼夜节律和肾脏酸化之间的关系，以及夜猫素在近端小管酸性信号中的作用。在目标3中，我们将研究内皮素B受体。以往的研究表明，内皮素B受体可以激活NHE3，并在酸激活NHE3的过程中发挥关键作用。内皮素B受体的两个结构域被发现是关键，即C末端的尾部和第二细胞内环(ICL2)。研究将利用酵母双杂交系统来识别与ICL2结合的蛋白质。利用酵母双杂交系统，我们已经证明了醛缩酶B与C-末端结构域结合，现在将研究它在调节NHE3中的作用。最后，将进行研究，以确定内皮素B受体在酸信号中所起的具体作用。最后一个目标是研究PYK2。Pyk2在酸中毒时被激活，我们已经证明它直接受环境pH的调节，这表明它可以作为pH传感器。研究将确定负责其pH敏感性的特定结构域/氨基酸，并确定Pyk2在酸信号级联中所起的作用。

DESCRIPTION (provided by applicant): Chronic metabolic acidosis induces a series of homeostatic responses that are designed to return blood pH to normal values. In the renal proximal tubule, this response includes increases in hydrogen ion secretion, ammonia synthesis and citrate absorption. Studies from this laboratory have examined the molecular basis by which these adaptations occur, focusing on regulation of the apical membrane Na/H antiporter, NHE3. During the last grant period, we showed that significant roles are played by the endothelins, Pyk2, c-Src, ERK, and focal adhesion proteins. The proposed studies continue our focus on the mechanisms by which the proximal tubule senses a decrease in intracellular pH and relays the signals so as to eventually increase NHE3 activity. Studies are divided into four aims. In aim 1, studies will utilize microarrays to identify novel acid-regulated and endothelin-regulated genes. In aim 2 we will focus on nocturnin. Preliminary results with microarrays have identified nocturnin as the most highly regulated gene in acute acidosis. Nocturnin is a circadian rhythm gene. Studies will examine the relationship between circadian rhythm and renal acidification and the role of nocturnin in acid signaling in the proximal tubule. In aim 3 we will study the endothelin B receptor. Previous studies have demonstrated that the endothelin B receptor can activate NHE3 and that it plays a key role in activation of NHE3 by acid. Two domains within the endothelin B receptor have been found to be key, the C-terminal tail and the second intracellular loop (ICL2). Studies will utilize the yeast two-hybid system to identify proteins that bind to ICL2. Using the yeast two-hybrid system, we have shown that Aldolase B binds to the C-terminal domain, and will now study its role in regulating NHE3. Lastly, studies will be performed to determine the specific role played by the endothelin B receptor in acid signaling. The last aim will study Pyk2. Pyk2 is activated in acidosis and we have shown that it is directly regulated by ambient pH, indicating that it can serve as a pH sensor. Studies will determine the specific domains/amino acids responsible for its pH sensitivity, and define the role that Pyk2 plays in the acid signaling cascade.