Isolation and characterization of rat kidney active urea transporter

描述（由申请人提供）：尿素在尿浓缩机制中起重要作用。在过去的十年中，人们对尿素在肾脏中的重吸收和再循环的理解取得了重大进展，包括克隆了两个促进性尿素转运基因UT-A和UT-B。然而，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，它可以在某些肾单元段上坡运输尿素。虽然活性尿素转运蛋白早在40多年前就被提出，但尚未被表征。本项目的总体目标是通过两种独立的方法：1)改良的双测试抑制减法杂交（ttSSH）和2)尿素摄取缺陷酵母菌的表达筛选，从肾内髓（IM）中克隆出这种新的活性尿素转运蛋白。在成功鉴定新的尿素转运蛋白后，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采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运输的分子证据的空白，并将扩大我们对尿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.