Urine concentrating mechanisms examined by molecular biology based techniques

本研究从三个方面阐明了肾脏尿浓缩功能的机制：1）肾脏尿浓缩相关转运蛋白的克隆及其结构与功能关系的研究。作为新的转运蛋白，我们克隆了AQP 3、AQP 6、ClC-K2、ClC-3和ClC-5。AQP 3是肾集合管的基底外侧型水通道，可透过甘油等小分子溶质。AQP 6与AQP 3同属一个亚家族，其功能特征与AQP 3相似。ClC-5具有强烈的外向整流特性，其mRNA分布于多种组织中。2）尿浓缩能力信号转导机制的研究。我们观察到脱水可上调AQP 2和AQP 3的mRNA表达。通过分离这两个基因的5 '端基因组序列，鉴定了这一调控机制。我们还发现，A-激酶在调节AQP 2中起着关键作用。ClC-K1和ClC-K2也在进行类似的研究。3）基础研究信息在临床医学中的应用。本课题的研究成果对临床有一定的指导意义。在许多水平衡紊乱的实验模型中检查了AQP 2的调节。尿中AQP 2的测定在水平衡紊乱的诊断中具有重要的临床意义。抗AQP抗体的免疫组化研究也鉴别了肾癌的起源。

In this research, we intended to clarify three aspects of the mechanisms underlying kidney urinary concentrating ability.1) Cloning of transport proteins related to kidney urinary concentrating ability and examinationof their structure and function relationships. As new trasporters, we have cloned AQP3, AQP6, ClC-K2, ClC-3, and ClC-5. AQP3 is the basolateral type water channel of kidney collecting duct and it permeates small solutes such as glycerol. AQP6 also belongs to the same subfaily as AQP3, and it functional characters are similar to AQP3. ClC-5 has an unique character of strong outward rectification and its mRNA distributes in many tissues.2) Idnetification of the signaling mechanisms controlling urinary concentrating ability. We observed the upregulation of mRNA of AQP2 and AQP3 by dehydration. The mechanisms for this regulation was identifyed by isolation of genomic sequences of the 5'region of the both genes. We also identified that A-kinse plays critical roles in the regulation of AQP2. Similar studies are under way for ClC-K1 and ClC-K2.3) Application of basic research information ot clinical medicine. Research results obtained in this project have many implication to clinical field. Regulation of AQP2 was examined in many experimental models of water balance disorders. Urinary measurement of AQP2 has been shown to be of clinical importance in diagnosis of water balance disorders. Also immunohistochemical study using antibodies against AQPs differentiated the origin of renal carcinoma.