Urine concentrating mechanisms examined by molecular biology based techniques

在本研究中，我们试图从三个方面阐明肾脏尿液浓缩能力的机制：1)与肾脏尿液浓缩能力相关的转运蛋白的克隆及其结构和功能关系的研究。作为新的转运子，我们已经克隆了AQP3、AQP6、ClC-K2、ClC-3和ClC-5。AQP3为肾集合管底侧型水通道，可渗透甘油等小分子溶质。AQP6与AQP3同属亚门，其功能特性与AQP3相似。ClC-5具有外向整流性强的独特特性，其mRNA分布于多种组织中。2)控制尿浓缩能力的信号机制的研究。我们观察了脱水对AQP2和AQP3基因表达的影响。通过分离这两个基因5‘端的基因组序列，确定了这种调控的机制。我们还发现A-Kinse在AQP2的调控中起着关键作用。CLC-K1和CLC-K2.3)临床医学基础研究信息的应用也在进行类似的研究。本课题的研究成果对临床具有重要的指导意义。AQP2的调节在许多水平衡失调的实验模型中得到了检验。尿AQP2测定在水平衡紊乱的诊断中具有重要的临床意义。此外，使用水通道蛋白抗体的免疫组织化学研究也区分了肾癌的来源。

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.