REGULATION OF THE RENAL SALT AND WATER TRANSPORTERS

Carolyn Ecelbarger博士受过营养学家和肾脏生理学家的双重培训，她在弥合这两个领域之间的差距方面处于独特的地位。她的长期职业目标包括研究营养因素如何影响肾功能，特别是这与肾脏盐和水转运蛋白的表达和调节的关系。指导研究生涯奖（KO1）将为拟议的学员，Ecelbarger博士提供机会，在乔治城大学Joseph Verbalis博士（拟议导师）的指导和专业知识下，扩大她在生理学方面的培训。在美国国立卫生研究院Mark Knepper博士的实验室进行博士后培训期间，她的研究主要集中在了解尿浓缩机制中重要的蛋白质，包括肾水通道蛋白和Na- K-2Cl共转运蛋白。埃尔巴格博士与韦尔巴利斯博士的实验室合作完成了一个重点项目，涉及评估水通道蛋白表达在抗利尿激素逃逸的生理现象中的作用。患有这种疾病的患者最初会保留水分并出现低钠血症。然而，如果持续补水，最终它们开始排泄大量相当稀释的尿液，尽管血管加压素的循环水平很高。这种“抗利尿激素逃逸”的过程几十年来一直困扰着肾脏生理学家，尽管人们对其中的机制知之甚少。在大鼠模型中，Ecelbarger等人能够显示水通道蛋白-2蛋白的显著下调，水通道蛋白-2蛋白是水稳态的关键蛋白。自然尿潴留是抗利尿素逃逸的另一个生理组成部分，对其了解甚少。因此，该建议的具体目的总结如下：(1)评估在抗利尿素的抗利尿作用的生理“逃逸”过程中钠转运体的肾脏表达和细胞分布的变化；(2)研究抗利尿激素逃逸和慢性抗利尿激素暴露模型中水通道蛋白-2表达调控的信号事件；(3)评估长期肠外营养（TPN）对肾盐和水转运蛋白表达和调控的影响。主要的假设是，直接调节几种关键的肾小管盐和水转运体的表达和细胞分布是维持体内整体盐和水稳态的手段，尽管存在生理扰动，如血液加压素水平的变化。分别为每一具体目标简要概述了研究建议；(1)通过免疫印迹和免疫组织化学评价肾小管关键钠转运体表达和调控抗利尿素逃逸的时间过程；(2)利用多种生化和免疫学方法来评估v2受体/腺苷酸环化酶信号级联中的重要调控事件，并确定它们与水通道蛋白-2丰度的关系；(3)评估全肠外营养对TPN大鼠模型肾功能的影响、肾脏血流动力学指标以及盐转运蛋白和水转运蛋白的表达和调控。

Dr. Carolyn Ecelbarger, Ph.D. who has trained as both a nutritionist and a renal physiologist is in a unique position to bridge the gap between these fields. Her long-term career goals include investigating how nutritional factors affects renal function and especially how this relates to expression and regulation of salt and water transporters of the kidney. The mentored Research Career Award (KO1) will provide the proposed mentee, Dr. Ecelbarger, with the opportunity to broaden her training in physiology, under the guidance and expertise of Dr. Joseph Verbalis (proposed mentor), at Georgetown University. During her post-doctoral training at the National Institutes of Health in Dr. Mark Knepper's laboratory, her research focussed mainly on understanding those proteins which are important in the urinary concentrating mechanism, including the renal aquaporins and the Na- K-2Cl cotransporter. A key project of Dr. Ecelbarger's, done in collaboration with Dr. Verbalis' laboratory, involved assessing the role of aquaporin expression in the physiological phenomenon of vasopressin escape. Patients with this disorder initially retain water and become hyponatremic. However, if water loading continues, eventually they begin to excrete larger volumes of fairly dilute urine, despite the high circulating levels of vasopressin. This process of "vasopressin escape" has puzzled renal physiologists for decades, although little is actually understood about the mechanisms involved. In a rat model, Ecelbarger et al. were able to show dramatic down-regulation of aquaporin-2 protein, a critical protein in water homeostasis. Naturesis is another physiologic component of vasopressin escape of which little is understood. Thus, the specific aims of the proposal are summarized as follows: (1) to evaluate changes in renal expression and cellular distribution of sodium transporters during physiologic "escape" from the antidiuretic action of vasopressin; (2) to investigate signaling events involved in regulation of aquaporin-2 expression in models of vasopressin escape and chronic vasopressin exposure and (3) to assess the impact of long-term parenteral nutrition (TPN) on the expression and regulation of renal salt and water transporters. The main hypothesis is that direct regulation of expression and cellular distribution of several critical kidney tubule salt and water transporters is the means by which overall salt and water homeostasis is maintained in the body, despite physiological perturbations such as changes in blood vasopressin levels. The study proposals are briefly outlined for each specific aim, respectively; (1) to evaluate the time course of vasopressin escape with regard to expression and regulation of critical sodium transporters along the kidney tubule by immunoblotting and immunohistochemistry; (2) to utilize a variety of biochemical and immunolological approaches in order to assess important regulatory events in the V2-receptor/adenylyl-cyclase signaling cascade and determine how they relate to aquaporin-2 abundance; (3) to assess the effects of total parenteral nutrition on renal function, measures of renal hemodynamics, and expression and regulation of salt and water transporters in a rat model of TPN.