Evaluation and estimation of renal drug elimination based on promoter analyses of organic cation transporters

基于有机阳离子转运蛋白启动子分析的肾脏药物消除评估和估计

基本信息

批准号：
17590119
负责人：
OKUDA Masahiro
金额：
$ 2.3万
依托单位：
Mie University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590119/
关键词：
organic cation transporter renal proximal tubules OCT2 promoter gender differences testosterone transcriptional regulation basic drugs アンドロゲン受容体有機カチオントランスポータ LLC-PK1 nilutamide rOCT2 androgen response elements

项目摘要

Organic cation transporter OCT2, expressed in the basolateral membranes of renal proximal tubules, is considered to mediates inter-and intra-individual variability of pharmacokinetics of basic drugs. However, mechanism of expressional regulation is scarcely known. In the present study, we analyzed the mechanism of testosterone-mediated regulation of OCT2 expression in detail, using deleted constructs and constructs with mutated AREs.1. Cloning of promoter regions of OCT1, 2, and 3Promoter regions of OCT1 and OCT3 were isolated by PCR using rat genomic DNA as a template and specific primers for OCT1 and OCT3. Promoter region of OCT2 was isolated by screening rat genomic library.2. Stimulation of transcription of OCT2 by testosteroneAfter insertion of each promoter region into pGL3 vector, it was introduced into LLC-PK1 cells, cultured renal epithelial cells derived from pig kidney, with rat androgen receptor. Promoter activity of OCT2 was stimulated by testosterone at 1 nM and higher, and was inhibited by nilutamide, an antagonist of androgen receptor.3. Analyses of transcription activity using deleted constructs and constructs with mutated AREsPromoter activity was analyzed using deleted constructs and constructs with mutated ARE located in the promoter region of OCT2. As the conclusion, two distinct AREs, ARE-1 and ARE-3, were clarified to have relevant roles in the stimulation of transcription activity of OCT2 by testosterone.

在肾近端小管的基底外侧膜中表达的有机阳离子转运蛋白OCT2被认为可以介导碱性药物的药代动力学的个体间和个体内变异性。但是，表达调控的机制几乎不知道。在本研究中，我们使用已删除的构建体和具有突变的ARES的构造分析了睾丸激素介导的OCT2表达调节的机理。1。使用大鼠基因组DNA作为Oct1和Oct3的模板和特定引物，通过PCR分离Oct1和Oct3的Oct1，2和3迅速范围区域的启动子区域的克隆。通过筛选大鼠基因组文库分离Oct2的启动子区域2。通过将每个启动子区域插入PGL3载体刺激OCT2的转录，将其引入LLC-PK1细胞中，源自大鼠雄激素受体的源自猪肾的培养的肾上皮细胞。 Oct2的启动子活性在1 nm及更高时刺激了睾丸激素，并被雄激素受体的拮抗剂Nilutamide抑制。3。使用已删除的Arespromoter活性的已删除构建体和构造的转录活性分析，使用已删除的构造和突变的构造位于OCT2的启动子区域中。得出的结论是，阐明了两个不同的ARE-1和AR-3，在睾丸激素刺激OCT2的转录活性中具有相关的作用。