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

有机阳离子转运体OCT2表达于肾近端小管基底外膜，被认为介导基本药物的个体间和个体内药代动力学变异。然而，表达调控的机制尚不清楚。在本研究中，我们使用缺失的构建体和突变的ares构建体，详细分析了睾酮介导的OCT2表达调控机制。以大鼠基因组DNA为模板和OCT1、OCT3特异性引物，采用PCR方法分离OCT1、OCT3的启动子区域。通过筛选大鼠基因组文库分离OCT2启动子区。睾酮刺激OCT2转录将每个启动子区域插入pGL3载体后，将其引入具有大鼠雄激素受体的培养猪肾源性肾上皮细胞LLC-PK1细胞。睾酮刺激OCT2启动子活性在1 nM及以上，而雄激素受体拮抗剂尼鲁胺抑制OCT2启动子活性。使用位于OCT2启动子区域的缺失构建体和突变ARE构建体分析了缺失构建体和AREsPromoter活性突变构建体的转录活性。综上所述，我们明确了两个不同的AREs， ARE-1和ARE-3在睾酮刺激OCT2转录活性中具有相关作用。

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.