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 而细胞质中的磷酸烯醇式丙酮酸羧激酶(PEPCK)是 大鼠肾脏近曲小管内增加对 代谢性酸中毒。这种适应对于维持增长是必要的。 代偿性慢性肾功能衰竭时肾脏的氨异生和糖异生 酸中毒。酶活性的增加是由于 由于各自mRNAs水平的增加而导致的合成。这个 PEPCK的诱导是在转录水平上调节的。这个 启动和协调肾脏相关变化的机制 基因表达尚不清楚。已分离出大鼠PEPCK的全部基因 并进行了测序。许多PEPCK启动子元件和相关的 参与肝脏调节的转录因子 PEPCK基因已被鉴定和鉴定。此外，LLC-PKF+ 细胞--一种已建立的肾近端小管糖异生生线 上皮细胞显示PEPCK mRNA水平的适应性变化 对酸性介质中生长的响应与观察到的结果非常相似 活着。因此，这个系统非常适合用来描述 肾脏内特定细胞感知pH值变化的机制 和/或HCO3浓度，并将该信息转导到改变基因 表情。这一机制也可以调节协调适应 在维持谷氨酰胺的器官间代谢中 肾脏氨生成增加。建议的具体目标 研究正在确定PEPCK基因的pH响应机制 在LLC-PK-F+细胞中的表达，以映射顺式调节 参与这一反应的元素，利用转基因小鼠 确定已确定的监管要素是否对 对酸中毒的活体反应，以开发鉴定和定量的分析方法 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 洞察潜在的药物方法，可能会刺激 在引起代谢的各种临床条件下的氨生成 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.