DEVELOPMENT OF WATER DIURETICS THAT INHIBIT WATER CHANNEL

本研究的目的是明确肾脏集合管加压素水通道AQP2的分子结构和调控机制，并开发具有利尿功能的AQP2抑制剂。首先，我们利用定点突变和基因表达技术对首次克隆的AQP2进行了分子结构分析。我们能够定位水通道蛋白2分子中的水通道，并提出了水通道蛋白2的三维结构模型。在我们的模型中，AQP2的水通道由AQP2的五个亲水环中的两个组成，AQP2是一个由五个亲水环连接的六个跨膜片段的膜蛋白。其次，我们发现AQP2的细胞内运输参与了加压素对肾集合管顶端膜渗透性的调节。结果发现，加压素刺激后，储存在内体膜上的AQP2被转移到表面膜，胞质中的囊泡被胞吐到顶端膜，导致顶端膜的渗透水通透性增加。在这些调控步骤中，我们证明了丝氨酸256被蛋白激酶A磷酸化是cAMP依赖性调节AQP2胞吐所必需的。基于这些发现，我们通过随机筛选寻找潜在的水利尿剂，如结合在水孔附近并抑制水渗透性的药物，或作为抑制AQP2的加压素依赖性胞吐的药物。我们获得了几种抑制水渗透性的试剂。为了确定体内效应，将药剂静脉内或腹膜内给予大鼠，但由于药剂的毒性，无法确定利尿效应。将继续筛选，以寻找毒性较小的AQP2抑制剂。

The purposes of this research was to Identify molecular structure and regulatory mechanisms of vasopressin water channel AQP2 In kidney collecting duct, and to develop functional Inhibitors of AQP2 that will be of use as a water diuretics in clinical medicine.First, we examined molecular structure of AQP2, which was first cloned by the investigator, using molecular techniques of site-directed mutagenesis and gene expression. We were able to localize the aqueous pore in the AQP2 molecule and proposed a three-dimensional structure model of AQP2. In our model aqueous pore of AQP2 is composed of two of five hydrophilic loops of AQP2, which is a membrane protein with six transmembrane segments connected by five hydrophilic loops.Second, we found that intracellular trafficking of AQP2 is involved in the vasopressin regulation of osmotic water permeability of kidney collecting duct apical membrane. It was observed that, after vasopressin stimulation, AQP2 stored on endosomal membranes was transferred to the surface membrane with exocytosis of vesicles from cytoplasm to the apical membrane, resulting in the increase of osmotic water permeability of the apical membrane. In these regulatory steps, we proved that phosphorylation of serine 256 by protein kinase A is required for cAMP-dependent regulatory exocytosis of AQP2.Based on these findings, we looked for AQP2 inhibitors = potential water diuretics by random screening, as agents that bind near the aqueous pore and inhibit water permeability, or as agents that inhibit vasopressin-dependent exocytosis of AQP2. We obtained several agents that inhibited water permeability. To determine the effects in vivo, the agents were given to rats intravenously or intraperitoneally, but because of toxicity of the agents it was not able to determine diuretic effects. Screening will be continued to find AQP2 inhibitor with less toxicity.