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

有机阳离子转运体OCT2表达于肾近端小管的基侧膜，被认为介导了基础药物药代动力学的个体间和个体内的变异性。然而，其表达调控机制目前尚不清楚。在本研究中，我们使用缺失的构建体和突变的AREs1构建体，详细分析了睾酮调节OCT2表达的机制。以大鼠基因组DNA为模板，以OCT1和Oct3的特异性引物为模板，利用聚合酶链式反应技术克隆OCT1、OCT1和OCT3基因的启动子区域。通过对大鼠基因组文库的筛选，获得OCT2基因的启动子区域。睾酮对OCT2转录的刺激作用将各启动子插入pGL3载体后，与大鼠雄激素受体一起导入猪肾上皮细胞LLC-PK1细胞。雄激素受体拮抗剂尼鲁胺对OCT2启动子活性有抑制作用。用缺失的构建体和具有突变的AREsPromoter活性的构建体分析转录活性，使用删除的构建体和突变的构建体位于OCT2的启动子区。结果表明，两个不同的ARE，ARE-1和ARE-3，在睾酮刺激OCT2转录活性中起着相关的作用。

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.