MECHANISM OF PH-RESPONSE IN RENAL PEPCK GENE EXPRESSION

线粒体谷氨酰胺酶和谷氨酸脱氢酶水平 和细胞质磷酸烯醇式丙酮酸羧激酶（PEPCK）是 在大鼠肾近曲小管内增加， 代谢性酸中毒 这种适应是必要的，以维持增加 代偿性慢性肾功能不全时的肾生氨和肾再生 酸中毒 酶活性的增加是由于 由于相应mRNA水平的增加，这些mRNA的合成增加。 的 PEPCK的诱导在转录水平上受到调节。 的 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基因已被分离出来 和序列测定 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。 LLC-PKF+ 细胞，一个建立的肾近端肾小管上皮细胞系， 上皮细胞，表现出PEPCK mRNA水平的适应性变化， 对酸性介质中生长的反应，与在 vivo. 因此，该系统非常适合表征 肾脏内特定细胞感知pH变化的机制 和/或HCO 3浓度，并将此信息用于改变基因 表情 这一机制也可能调节协调适应 在谷氨酰胺的器官间代谢中 肾氨生成增加。 建议的具体目标 研究旨在确定PEPCK基因的pH响应机制 LLC-PK-F+细胞中发生的表达，以绘制顺式调节蛋白。 参与这种反应的元素，利用转基因小鼠， 确定所确定的监管要素是否是必要的， 体内对酸中毒的反应，以开发鉴定和定量 相关的反式作用因子，并克隆参与 交易因素 拟议研究的结果应提供 深入了解潜在的药理学方法， 在各种临床条件下的氨生成，其引起代谢性 酸中毒

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.