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.

有机阳离子转运蛋白 OCT2 在肾近曲小管基底外侧膜中表达，被认为介导碱性药物药代动力学的个体间和个体内变异性。然而，表达调控机制却鲜为人知。在本研究中，我们使用缺失的构建体和具有突变ARE的构建体详细分析了睾酮介导的OCT2表达调节机制。1。 OCT1、2和3的启动子区域的克隆使用大鼠基因组DNA作为模板以及OCT1和OCT3的特异性引物通过PCR分离OCT1和OCT3的启动子区域。通过筛选大鼠基因组文库分离得到OCT2启动子区。 2．睾酮刺激OCT2转录将各启动子区域插入pGL3载体后，将其导入LLC-PK1细胞中，该细胞是培养的猪肾来源的肾上皮细胞，具有大鼠雄激素受体。 OCT2启动子活性受1nM及更高浓度的睾酮刺激，并被雄激素受体拮抗剂尼鲁米特抑制。3．使用删除的构建体和具有突变ARE的构建体分析转录活性使用删除的构建体和位于OCT2启动子区域的突变ARE的构建体分析启动子活性。结论是，两种不同的 ARE，ARE-1 和 ARE-3，被阐明在睾酮刺激 OCT2 转录活性中具有相关作用。