H/HCO3 transport in the collecting duct

说明(申请人提供)：氨代谢对正常健康至关重要。肾脏不适当的氨代谢会导致代谢性酸中毒，肝脏会导致氨性脑病。在中枢神经系统中，细胞外氨增加会改变神经功能，并可能导致脑病。因此，了解氨代谢的细胞和分子机制，包括氨的运输，是很重要的。最近的研究发现了一个新的氨离子(NH4+)特异性转运蛋白家族。这些蛋白质最早是在酵母和植物中发现的，同系物在自然界中普遍存在。在酵母、植物和细菌等模型系统中，这些是内在的膜蛋白，介导高亲和力的、特定于铵的运输，其表达受到生理调节。其中两个蛋白RhBG和RhCG在肾脏的连接段和集合管表达，并呈极化表达。这些观察结果使我们推测，RhBG和RhCG是完整的膜，生理调节的氨离子转运体，在肾氨代谢中发挥关键作用。该项目的长期目标是明确RhBG和RhCG在哺乳动物肾脏生理学中的作用。为此，本提案的具体目的是：(1)确定小鼠肾脏RhBG和RhCG的表达和囊泡转运的调节，以响应与肾脏氨代谢改变相关的特定临床条件；(2)确定细胞外氨刺激RhBG和RhCG介导的离子转运变化的机制；以及(3)确定RhBG和RhCG的特定离子转运特征。我们将利用肾脏氨代谢改变、代谢性酸碱中毒和低钾血症的体内动物模型，确定RhBG和RhCG表达的调节和细胞定位，培养的集合管细胞系mIMCD-3用于体外研究RhBG和RhCG介导的转运调节的细胞机制，以及确定RhBG和RhCG特异性离子转运特性的异源表达系统。

DESCRIPTION (provided by applicant): Ammonia metabolism is critical for normal health. Inappropriate ammonia metabolism in the kidney leads to metabolic acidosis and in the liver leads to ammonia encephalopathy. In the central nervous system increased extracellular ammonia alters neuronal function and can lead to encephalopathy. Accordingly, understanding the cellular and molecular mechanisms of ammonia metabolism, which includes ammonia transport, is important. Recent studies have identified a novel family of ammonium ion (NH4+)-specific transporters. These proteins were first identified in yeast and in plants, and homologues are present throughout nature. In model systems, such as yeast, plants and bacteria, these are intrinsic membrane proteins that mediate high-affinity, ammonium-specific transport and whose expression is physiologically regulated. Two of these proteins, RhBG and RhCG are expressed in the connecting segment and the collecting duct of the kidney, and exhibit polarized expression. These observations lead us to postulate that RhBG and RhCG are integral membrane, physiologically-regulated ammonium-ion transporters that play critical roles in renal ammonia metabolism. The broad, long-term objectives of this project are to define the roles of RhBG and RhCG in mammalian renal physiology. To do so, the Specific Aims of the current proposal are to: (1) Define the regulation of mouse renal RhBG and RhCG expression and vesicular trafficking in response to specific clinical conditions associated with altered renal ammonia metabolism; (2) Determine the mechanism of extracellular ammonia-stimulated changes in RhBG- and RhCG-mediated ion transport; and, (3) identify the specific ion-transport characteristics of RhBG and RhCG. We will utilize in vivo animal models of altered renal ammonia metabolism, metabolic acidosis and alkalosis and hypokalemia, to define the regulation of RhBG and RhCG expression and cellular localization, a cultured collecting duct cell line, mIMCD-3, for in vitro studies examining the cellular mechanisms underlying regulation of RhBG and RhCG-mediated transport, and heterologous expression systems in which to define the specific ion transport characteristics of RhBG and RhCG.