CONTROL OF RENAL GLUTAMINASE INDUCTION DURING ACIDOSIS

描述：(改编自申请人的摘要)：最近的研究来自 首席研究员的实验室表明，线粒体 大鼠肾脏内谷氨酰胺酶(GA)活性增加7-20倍 慢性代谢性酸中毒时的近端小管。这一适应 有助于肾脏氨生成的持续增加和 糖异生作用是部分补偿酸中毒所必需的。 GA活性的提高是GA基因稳定的结果。这 响应由嵌合构造的表达式建模，该嵌合构造包含 LLC-PK1-F+细胞中GA基因的3‘-非翻译区，一个pH反应区 猪肾近端小管样细胞系。删除分析具有 将功能元件映射到2446碱基内的340碱基序列 GA基因的3‘-非翻译区。此序列赋予增强的 当结合到一个 嵌合的mRNA.RNA凝胶位移分析表明，大鼠肾皮质 胞浆提取物含有一种具有高亲和力和 与该序列的特定结合。绑定的级别降低 从严重酸化的大鼠中提取的提取物中有显著的作用。这个 LLC-PK1-F+细胞表达四种GA mRNAs，其中只有一种是由 将细胞转移到酸性介质中(pH 6.9，10 mM HCO3-)。RNaseH 映射表明4GA mRNAs在长度和序列上不同 它们的3‘-非翻译区。这个系统将被用来追求 赤霉素A基因多种异构体的功能分析及其机制 PH响应性稳定。拟议研究的具体目标 是：1)克隆和鉴定GA基因的多种异构体；2)克隆和鉴定GA基因 确定GA基因组的组织和表达机制；3) 进一步定义pH反应不稳定元件，并克隆和 确定相关结合蛋白的特征；以及4)确定 赤霉素A基因的降解和稳定机制。建议的分析 旨在为潜在的药理学方法提供洞察， 在各种临床情况下可刺激肾脏氨化。 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.