CONTROL OF RENAL GLUTAMINASE INDUCTION DURING ACIDOSIS

描述：（改编自申请人摘要）：最近的研究， 主要研究者实验室已经表明， 转氨酶（GA）活性在大鼠肾脏中增加7至20倍。 慢性代谢性酸中毒时的近端小管。 这种适应 有助于肾氨生成的持续增加， 这是部分补偿酸中毒所必需的。 GA活性的增加是由于GA mRNA的稳定。 这 通过表达含有所述抗体的嵌合构建体来模拟应答。 LLC-PK 1-F+细胞中GA mRNA的3 '-非翻译区， 猪肾近端小管样细胞系。 删除分析 将功能元件映射到2446个碱基内的340个碱基序列 GA mRNA的3 '-非翻译区。 这个序列赋予了一个增强的 责任和pH响应稳定时，纳入一个 嵌合mRNA。 RNA凝胶位移分析证实大鼠肾皮质 胞质提取物含有表现出高亲和力的蛋白质， 与该序列的特异性结合。 结合水平降低 在从急性酸中毒大鼠制备的提取物中显著。 的 LLC-PK 1-F+细胞表达四种GA mRNA，其中只有一种是由 将细胞转移至酸性培养基（pH 6.9，10 mM HCO 3-）中。 rna酶H 作图表明，4GA mRNA的长度和序列不同， 3 '-非翻译区 该系统将被用于追踪 GA mRNA多种异构体的功能分析及其作用机制 pH响应稳定性。 拟议研究的具体目标 是：1）克隆和表征GA mRNA的多种亚型; 2） 确定GA基因组的组织和表达机制; 3） 进一步确定pH响应性不稳定元件并克隆， 表征相关的结合蛋白;以及4）确定 GA mRNA的降解和稳定机制。 建议的分析 旨在深入了解潜在的药理学方法， 其可在各种临床条件下刺激肾氨生成 导致代谢性酸中毒

DESCRIPTION: (Adapted from the applicant's abstract): Recent studies from the principal investigator's laboratory have shown that mitochondrial glutaminase (GA) activity is increased 7- to 20-fold within the rat renal proximal tubule during chronic metabolic acidosis. This adaptation contributes to the sustained increase in renal ammoniagenesis and gluconeogenesis which are essential to partially compensate the acidosis. The increased GA activity results from stabilization of the GA mRNA. This response is modeled by the expression of a chimeric construct containing the 3'-nontranslated region of the GA mRNA in LLC-PK1-F+ cells, a pH-responsive line of porcine renal proximal tubule-like cells. Deletion analysis has mapped the functional element to a 340-base sequence within the 2446-base 3'-nontranslated region of the GA mRNA. This sequence confers an enhanced liability and a pH-responsive stabilization when incorporated into a chimeric mRNA. RNA gel-shift analysis established that rat renal cortical cytosolic extracts contains a protein which exhibits high affinity and specific binding to this sequence. The level of binding is decreased significantly in extracts prepared from rats made acutely acidotic. The LLC-PK1-F+ cells express four GA mRNAs, only one of which is induced by transfer of the cells to acidic medium (pH 6.9, 10 mM HCO3-). RNase H mapping indicates that the 4GA mRNAs differ in the length and sequence of their 3'-nontranslated regions. This system will be used to pursue the functional analysis of the multiple isoforms of GA mRNA and the mechanism of the pH-responsive stabilization. The specific aims of the proposed research are: 1) to clone and characterize the multiple isoforms of GA mRNA; 2) to define the organization and mechanism of expression of the GA genome; 3) to further define the pH-responsive instability elements and clone and characterize the associated binding proteins; and 4) to determine the mechanism of GA mRNA degradation and stabilization. The proposed analysis is designed to provide insight into potential pharmacological approaches, which may stimulate renal ammoniagenesis in various clinical conditions leading to metabolic acidosis.