MECHANISM OF PH-RESPONSE IN RENAL PEPCK GENE EXPRESSION

肾PEPCK基因表达的PH反应机制

• 批准号: 3245130
• 负责人: NORMAN P. CURTHOYS
• 金额: $ 15.61万
• 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 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245130
• 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化酶（PEPCK）的 大鼠肾脏近端曲折小管的增加 代谢性酸中毒。 这种适应是维持增加的必要条件 补偿慢性期间的肾脏氨和糖异生 酸中毒。 增加的酶活性来自增加 由于各自的mRNA水平增加而导致的合成。 这 PEPCK的诱导在转录水平下受到调节。 这 启动和协调肾脏相关变化的机制 基因表达未知。 整个大鼠PEPCK基因已被分离 并测序。 许多PEPCK启动子元素和相关的元素 参与肝脏调节的转录因子 PEPCK基因已被鉴定并表征。 此外，LLC-PKF+ 细胞，已建立的肾近端葡萄糖生成系 上皮细胞，表现出PEPCK mRNA水平的自适应变化 对酸性培养基中观察到的酸性培养基的生长的反应 体内。 因此，该系统非常适合表征 肾脏感中特定细胞在pH中的变化的机制 和/或HCO3浓度，并将此信息转传用于改变基因 表达。 该机制还可能调节坐标适应 在维持谷氨酰胺的组织间代谢中 增加肾脏氨（氨化）。 提议的具体目的 研究是为了确定PEPCK基因中pH响应的机理 在LLC-PK-F+细胞中发生的表达，以绘制顺式调节 参与此响应的元素，将转基因小鼠用于 确定确定的调节元素是否对IN至关重要 体内对酸中毒的反应，开发测定以识别和定量 相关的跨性别因素，并克隆参与 交易因子。 拟议研究的结果应提供 深入了解可能刺激的潜在药理方法 在各种临床条件下的氨基化作用，导致代谢 酸中毒。