DEVELOPMENT OF WATER DIURETICS THAT INHIBIT WATER CHANNEL

本研究的目的是明确肾脏集合管中加压素水通道AQP2的分子结构和调控机制，并开发AQP2的功能抑制剂，将其作为利尿剂用于临床医学。首先，我们利用定点突变和基因表达的分子技术对该基因的分子结构进行了研究。我们能够定位AQP2分子中的水孔，并提出了AQP2的三维结构模型。在我们的模型中，AQP2的水孔是由AQP2的五个亲水环中的两个组成的，AQP2是一个膜蛋白，由五个亲水环连接六个跨膜段。第二，我们发现AQP2的细胞内转运参与了血管加压素对肾集合管顶膜渗透水通透性的调节。观察到血管加压素刺激后，储存在内膜上的AQP2转移到表膜，使囊泡从细胞质排出到顶膜，导致顶膜渗透水通透性增加。在这些调控步骤中，我们证明了蛋白激酶A对丝氨酸256的磷酸化是依赖cAMP的AQP2调节性胞吐所必需的。基于这些发现，我们通过随机筛选寻找AQP2抑制剂=潜在的水利尿剂，作为结合在房水孔附近并抑制水通透性的药物，或者作为抑制AQP2的血管加压素依赖的胞吐的药物。我们得到了几种抑制透水性的药剂。为了确定体内的效果，这些药物分别通过静脉或腹腔给药，但由于这些药物的毒性，无法确定利尿效果。将继续筛选，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.