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

有机阳离子转运蛋白OCT 2表达于肾近端小管的基底外侧膜，被认为介导了基础药物药代动力学的个体间和个体内变异性。然而，表达调控的机制知之甚少。在本研究中，我们详细分析了睾丸激素介导的OCT 2表达调控机制，使用缺失的构建体和具有突变ARE的构建体。OCT 1、2和3启动子区的克隆以大鼠基因组DNA为模板，使用OCT 1和OCT 3的特异性引物，通过PCR分离OCT 1和OCT 3的启动子区。通过筛选大鼠基因组文库，分离出OCT 2的启动子区.将每个启动子区域插入pGL 3载体后，将其与大鼠雄激素受体一起导入LLC-PK 1细胞（培养的猪肾上皮细胞）中。OCT 2的启动子活性被1 nM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.使用缺失的构建体和具有突变的ARE的构建体分析转录活性使用缺失的构建体和具有位于OCT 2的启动子区域中的突变的ARE的构建体分析启动子活性。因此，ARE-1和ARE-3在睾酮刺激OCT 2转录活性中具有重要作用。

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.