PDF(Pubmed)
Abstract：
UNASSIGNED: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration.
UNASSIGNED: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA-protein interactions.
UNASSIGNED: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays.
UNASSIGNED: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.
UNASSIGNED: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA-protein interactions.
UNASSIGNED: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays.
UNASSIGNED: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.
摘要：
■G蛋白偶联胆汁酸受体（TGR5），第一个胆汁酸的G蛋白偶联受体，与胆汁酸相互作用后能够激活多种细胞内信号通路。TGR5在多种生理过程中发挥重要作用,被认为是治疗多种代谢性疾病的潜在靶点,包括2型糖尿病。有证据表明，TGR5基因缺失导致基础尿量增加，这表明它可能在肾脏水和盐的再吸收中起关键作用。本研究旨在阐明TGR5活化对尿液浓度的影响及其机制。
用TGR5激动剂(LCA和INT-777)处理小鼠3天。收集小鼠的24小时尿液并分析尿液生化参数。实时荧光定量PCR检测mRNA的表达,蛋白质印迹法检测蛋白表达。进行免疫组织化学和免疫荧光以检查蛋白质的细胞位置。培养的原代髓样收集管细胞用H89（PKA抑制剂）预处理1小时，随后12小时治疗LCA和INT-777。荧光素酶报告基因检测用于检测CREB对AQPs基因转录的影响。凝胶电泳迁移率变化测定用于分析DNA-蛋白质相互作用。
■用TGR5激动剂LCA和INT-777处理小鼠可显著减少尿量和增加尿渗透压,同时伴有AQP2和AQP3蛋白表达和膜转位的明显增加。在培养的原代髓样收集管细胞中，LCA和INT-777以cAMP/PKA依赖性方式剂量依赖性地上调AQP2和AQP3表达。机械上,AQP2和AQP3基因启动子都含有一个推定的CREB结合位点，如通过基因启动子驱动的荧光素酶和凝胶移位测定所评估的,其可以被CREB结合和激活。
■集体，我们的研究结果表明，TGR5的激活可以通过上调肾集合管中AQP2和AQP3的表达来促进尿液浓度。TGR5可能是治疗尿液浓度缺陷患者的有吸引力的靶标。
用TGR5激动剂(LCA和INT-777)处理小鼠3天。收集小鼠的24小时尿液并分析尿液生化参数。实时荧光定量PCR检测mRNA的表达,蛋白质印迹法检测蛋白表达。进行免疫组织化学和免疫荧光以检查蛋白质的细胞位置。培养的原代髓样收集管细胞用H89（PKA抑制剂）预处理1小时，随后12小时治疗LCA和INT-777。荧光素酶报告基因检测用于检测CREB对AQPs基因转录的影响。凝胶电泳迁移率变化测定用于分析DNA-蛋白质相互作用。
■用TGR5激动剂LCA和INT-777处理小鼠可显著减少尿量和增加尿渗透压,同时伴有AQP2和AQP3蛋白表达和膜转位的明显增加。在培养的原代髓样收集管细胞中，LCA和INT-777以cAMP/PKA依赖性方式剂量依赖性地上调AQP2和AQP3表达。机械上,AQP2和AQP3基因启动子都含有一个推定的CREB结合位点，如通过基因启动子驱动的荧光素酶和凝胶移位测定所评估的,其可以被CREB结合和激活。
■集体，我们的研究结果表明，TGR5的激活可以通过上调肾集合管中AQP2和AQP3的表达来促进尿液浓度。TGR5可能是治疗尿液浓度缺陷患者的有吸引力的靶标。
