Acid-Base and Ammonia Transport in the Collecting Duct

描述(由申请人提供)： 肾脏的NH4+排泄对酸碱平衡至关重要，通常至少占每日尿酸净排泄量的三分之二。最近的研究发现了新的非红系Rh蛋白，它们与非哺乳动物物种的NH4+转运蛋白有关。正常的酸碱平衡对健康和防止慢性肾脏疾病的进展至关重要。这项建议的主要目的是确定肾Rh糖蛋白在NH3/NH4+转运和CO2转运方面的功能和结构特性，这也是酸碱平衡的关键。有待探索的性质包括它们对pH的临界敏感性；作为气体运输者的潜在作用；以及它们可能 起着NH4+和NH3转运体的双重作用。具体地说，我们将解决以下目标：1)Rh糖蛋白的运输特性：这个目标的主要假设是Rhbg和Rhcg是NH4+(可能还有NH3)的转运体。2)Rh糖蛋白的结构-功能特性：对关键残基进行定点突变和功能分析，以确定不同Rh糖蛋白转运NH3/NH4+的不同原因。3)Rh糖蛋白转运NH3和CO2：我们将确定Rh蛋白是转运NH3还是CO2。这一特性是新颖和独特的，因为很少有膜蛋白被确定为气体运输的媒介。我们的研究使用了多种技术(PHI研究、电压钳实验、定点突变)，这些技术应该清楚地确定Rh蛋白是作为NH3、CO2还是NH4+转运体，以及这种转运体的特征。这项工作依赖于体外实验，因为表征NH3/NH4+转运的固有困难。拟议的工作是实质性的，但我们的最终目标是了解这些过程在肾脏中的生理意义和调节。 公共卫生相关性： 酸碱紊乱在退伍军人中很常见，特别是在慢性肾脏疾病、糖尿病和高血压期间。本应用旨在了解与酸碱平衡有关的肾功能，以便对这些疾病进行适当的诊断和治疗。正在研究的详细方面是被称为Rh蛋白的蛋白质，它调节尿液中的氨排泄，这是肾脏维持酸碱平衡的主要机制。

DESCRIPTION (provided by applicant): Renal excretion of NH4+ is critical for acid-base homeostasis, normally accounting for at least two-thirds of daily net acid excretion in the urine. Recent studies have identified new non- erythroid Rh proteins that are related to the NH4+ transporters in non-mammalian species. Normal acid-base balance is essential for health and prevention of progression of chronic kidney disease. The main purpose of this proposal is to determine the functional and structural properties of renal Rh glycoproteins with respect to NH3/NH4+ transport and CO2 transport which is also critical for acid-base homeostasis. Among the properties to be explored are their critical sensitivity to pH; a potential role as transporters of gases and the possibility that they serve dual roles as NH4+ and NH3 transporters. Specifically we will address the following aims: 1) Transport characteristics of Rh glycoproteins: The main hypothesis of this aim is that Rhbg and Rhcg are transporters of NH4+ (and possibly NH3). 2) Structure-function characteristics of Rh glycoproteins: Site-directed mutagenesis of critical residues & functional assays will be conducted to determine why different Rh glycoproteins transport NH3/NH4+ differently. 3) Transport of NH3 and CO2 by Rh glycoproteins: We will determine if Rh proteins transport NH3 or CO2. This property is novel & unique in that very few membrane proteins have been identified as mediators of gas transport. Our studies employ a combination of techniques (pHi studies, voltage clamp experiments, site-directed mutagenesis) that should clearly establish whether Rh proteins act as NH3 or CO2 or NH4+ transporters and the characteristics of this transport. This work relies on in vitro experiments because of the difficulties inherent in characterizing NH3/NH4+ transport. The proposed work is substantial, but our ultimate goals are to understand the physiologic significance and regulation of these processes in the kidney. PUBLIC HEALTH RELEVANCE: Acid-base disorders are common in Veterans particularly during chronic kidney disease, diabetes, and hypertension. The present application aims to understand the kidney functions involved in acid-base homeostasis so that these disorders can be diagnosed and treated appropriately. The detailed aspects being studied are proteins called Rh proteins that regulate ammonia excretion in the urine, the primary mechanism that kidneys maintain acid-base balance.