Isolation and characterization of rat kidney active urea transporter

描述(由申请人提供)：尿素在尿液浓缩机制中起着重要作用。在过去的十年里，在了解尿素在肾脏中的再吸收和再循环方面取得了重大进展，包括克隆了两个促进尿素转运蛋白基因UT-A和UT-B。然而，来自生理学研究的有力证据表明，哺乳动物肾脏中存在一种钠依赖的活性转运体，它可以向上移动某些肾单位节段的尿素。虽然活性尿素转运蛋白是在40多年前提出的，但还没有一个被表征。本项目的总体目标是通过两种独立的方法从肾脏内髓(IM)中克隆这种新的活性尿素转运蛋白：1)改进的双试剂法抑制消减杂交(TtSSH)；2)利用尿素吸收缺陷的酵母菌株进行表达筛选。在成功鉴定了新型尿素转运蛋白之后，我们将研究活性尿素转运蛋白的性质。我们希望我们在这里应用的两种方法将引导我们发现新的基因。我们相信，该项目的成功将填补尿素在肾脏中主动转运的分子证据的空白，并将扩大我们对尿液浓缩机制和水平衡的理解。 公共卫生相关性 强有力的生理证据表明，40多年来，哺乳动物肾脏存在活跃的尿素转运活性。本项目的总体目标是通过两种独立的方法从肾脏IM克隆新型活性尿素转运蛋白。该项目的成功将扩大我们对尿液浓缩机制和水稳态的理解。

DESCRIPTION (provided by applicant): Urea plays an important role in the urinary concentrating mechanisms. In the past decade, significant progress has been made in understanding urea reabsorption and recycling in kidney including the cloning of the two facilitated urea transporter genes, UT-A and UT-B. However, strong evidence from physiological studies demonstrated that the existence of a sodium- dependent active transporter in mammalian kidney, which can uphill move urea in some nephron segments. Though the active urea transporter was proposed over 40 years ago, none has yet been characterized. The overall objective of this project is to clone this novel active urea transporter from kidney inner medulla (IM) by using two independent approaches: 1) modified two-tester suppression subtraction hybridization (ttSSH) and 2) expression screening using a urea uptake defective yeast strain. After having successfully identified the novel urea transporter, we will investigate the nature of the active urea transporter. We hope the two approaches we apply here will lead us to discover the new genes. We believe the success of this project will fill in the gap with the molecular evidence how urea is actively transported in kidney and will extend our understanding on the urinary concentrating mechanism and water homeostasis. PUBLIC HEALTH RELEVANCE Strong physiological evidence demonstrated the existence of active urea transport activity in mammalian kidney over 40 years. The overall objective of this project is to clone the novel active urea transporter from kidney IM by using two independent approaches. The success of this project will extend our understanding on the urinary concentrating mechanism and water homeostasis.