MECHANISM OF PH-RESPONSE IN RENAL PEPCK GENE EXPRESSION

肾PEPCK基因表达的PH反应机制

• 批准号: 2143181
• 负责人: NORMAN P. CURTHOYS
• 金额: $ 16.83万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-04-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143181
• 关键词: acidity /alkalinity; enzyme biosynthesis; enzyme induction /repression; enzyme mechanism; epithelium; gene expression; genetic mapping; genetic regulatory element; genetically modified animals; gluconeogenesis; laboratory mouse; messenger RNA; molecular cloning; molecular genetics; phosphoenolpyruvate carboxylase; renal tubule acidosis; tissue /cell culture; transcription factor

The levels of the mitochondrial glutaminase and glutamate dehydrogenase and of the cytoplasmic phosphoenolpyruvate carboxykinase (PEPCK) are increased within the rat renal proximal convoluted tubule in response to metabolic acidosis. This adaptation is necessary to sustain increased renal ammoniagenesis and gluconeogenesis during a compensated chronic acidosis. The increased enzyme activities result from increased synthesis due to an increased level of the respective mRNAs. The induction of PEPCK is regulated at the level of transcription. The mechanism which initiates and coordinates the associated changes in renal gene expression are unknown. The entire rat PEPCK gene has been isolated and sequenced. Many of the PEPCK promoter elements and the associated transcription factors that participate in the regulation of the liver PEPCK gene have been identified and characterized. Furthermore, LLC-PKF+ cells, an established gluconeogenic line of renal proximal tubular epithelial cells, exhibit adaptive changes in PEPCK mRNA levels in response to growth in acidic medium that closely mimic those observed in vivo. Thus, this system is extremely well suited to characterize the mechanism by which specific cells within the kidney sense changes in pH and/or HCO3 concentration and transduce this information to alter gene expression. This mechanism may also regulate the coordinate adaptations in the interorgan metabolism of glutamine that are required to sustain increased renal ammoniagenesis. The specific aims of the proposed research are to determine the mechanism of the pH-response in PEPCK gene expression that occurs in LLC-PK-F+ cells, to map the cis-regulatory elements that participate in this response, to utilize transgenic mice to determine if the identified regulatory elements are essential for the in vivo response to acidosis, to develop assays to identify and quantitate the associated trans-acting factors, and to clone the participating transacting factors. The results of the proposed studies should provide insight into potential pharmacologic approaches that may stimulate ammoniagenesis in various clinical conditions which cause a metabolic acidosis.

线粒体谷氨酰胺酶和谷氨酸脱氢酶的水平 和细胞质磷酸烯醇丙酮酸羧激酶 (PEPCK) 的 大鼠肾近曲小管内响应增加 代谢性酸中毒。 这种适应对于维持增长是必要的 慢性代偿期肾氨生成和糖异生 酸中毒。 酶活性的增加是由于 由于各自 mRNA 水平的增加而导致合成。 这 PEPCK 的诱导在转录水平上受到调节。 这 启动和协调肾脏相关变化的机制 基因表达未知。 完整的大鼠PEPCK基因已被分离 并测序。 许多 PEPCK 启动子元件和相关的 参与肝脏调节的转录因子 PEPCK 基因已被鉴定和表征。 此外，LLC-PKF+ 细胞，肾近端小管已建立的糖异生细胞系 上皮细胞中，PEPCK mRNA 水平表现出适应性变化 对酸性介质中生长的反应与在中观察到的反应非常相似 体内。 因此，该系统非常适合表征 肾脏内特定细胞感知 pH 值变化的机制 和/或 HCO3 浓度并转导此信息以改变基因 表达。 该机制还可以调节坐标适应 在谷氨酰胺的器官间代谢中，维持 肾氨生成增加。 拟议的具体目标 研究目的是确定PEPCK基因中pH反应的机制 LLC-PK-F+ 细胞中发生的表达，以绘制顺式调控图谱 参与这种反应的元件，利用转基因小鼠 确定所确定的监管要素对于内部是否至关重要 对酸中毒的体内反应，开发检测方法来识别和定量 相关的反式作用因子，并克隆参与的 交易因素。 拟议研究的结果应提供 深入了解可能刺激的潜在药理学方法 在各种临床情况下产生氨，导致代谢 酸中毒。