Control of Renal Glutaminase Expression during Acidosis

描述（由申请方提供）：代谢性酸中毒是一种明显的酸碱平衡紊乱，由代谢、肾脏处理碳酸氢盐和排泄可滴定酸的遗传或获得性缺陷引起。慢性酸中毒可引起儿童智力迟钝，成人骨软化症、肾钙质沉着症和尿石症。在代谢性酸中毒期间，近曲小管中发生肾谷氨酰胺催化剂的代偿性增加。由此产生的铵和碳酸氢根离子合成的增加促进了酸的排泄并部分恢复酸碱平衡。这种基本的适应性反应是通过主要通过转录后调节发生的基因表达的显著变化来维持的。这种反应的范例是通过选择性稳定GA mRNA而发生的转氨酶（GA）的持续增加，并且由作为pH反应元件（pHRE）起作用的两个8-nt AU序列介导。GA mRNA在正常酸碱平衡期间发生的快速周转之前是快速去腺苷化的，而pH响应稳定化与去腺苷化的速率和程度降低相关。HuR和p40-AUF 1以高亲和力和特异性结合pHRE。初步实验证实，酸中毒的发作激活大鼠肾近端小管中的ER应激信号通路，并促进RNA应激颗粒的形成和培养的肾细胞中HuR从细胞核中的释放。因此，在正常酸碱平衡期间，pHRE结合蛋白可促进去腺苷酸酶的募集和随后GA mRNA的核酸外切衰变。在代谢性酸中毒期间，与应激颗粒的关联可能促进GA mRNA的重塑，并促进其与稳定因子如HuR或p40 AUF 1的关联。为了验证这些新的假设，提出了以下具体目标：鉴定响应于酸中毒发作而激活的ER应激信号通路，并利用显性负性和siRNA构建体来确定其在GA mRNA稳定中的作用;通过进行过表达和siRNA敲低实验以及利用蛋白质/RNA pull-技术来表征HuR和AUF 1的胞质异构体的潜在作用。下，免疫荧光和质谱分析，以建立其与GA mRNA，细胞定位和磷酸化位点的时间关联;并采用一种新的协议，以快速纯化和鉴定pHRE/蛋白质复合物，在完整的细胞中形成。由此产生的数据将定义近端小管细胞感知pH值轻微变化并介导其蛋白质组重塑以响应酸中毒的机制。它还将显著增强对调节mRNA稳定性变化和介导肾脏基本生理反应的基本机制的理解。 公共卫生相关性：代谢性酸中毒是一种常见的临床疾病，在成人中可导致骨软化症、肾钙质沉着症和尿石症，在儿童中可导致智力迟钝。在终末期肾病期间，代谢性酸中毒的发生与外周胰岛素抵抗增加相关，并且是额外的发病风险因素。为了补偿酸中毒的发作，需要适当增加肾脏的酸排泄和碳酸氢盐的产生。这种基本反应由仅在近曲小管内发生的谷氨酰胺的肾catalysts的适应性增加引发，并且在很大程度上由谷氨酰胺酶mRNA和蛋白质水平的细胞特异性和显著增加维持。所提出的实验将极大地增强对肾脏感知酸碱平衡变化的机制的认识，并将此信息传递给肾脏，以促进转氨酶mRNA的选择性稳定。最近的蛋白质组学分析表明，在酸中毒过程中，近端小管中多个基因的表达是通过相同的mRNA稳定机制来调节的。该系统也是理解mRNA周转的生理调节有助于基本适应性反应的机制的重要范例。此外，由此产生的数据将有助于mRNA周转的机制和调节的基础知识，无论是在这个系统中，并在一般情况下。这些知识将构成生化和药理学研究的基础，这些研究可能会改善慢性代谢性酸中毒患者的治疗。

DESCRIPTION (provided by applicant): Metabolic acidosis is a pronounced disturbance in acid-base balance that is caused by genetic or acquired defects in metabolism, in renal handling of bicarbonate, and in the excretion of titratable acid. Chronic acidosis causes mental retardation in children and osteomalacia, nephrocalcinosis and urolithiasis in adults. During metabolic acidosis, a compensatory increase in renal glutamine catabolism occurs in the proximal convoluted tubule. The resulting increases in ammonium and bicarbonate ion synthesis facilitate the excretion of acid and partially restore acid-base balance. This essential adaptive response is sustained by pronounced changes in gene expression that occur primarily through post-transcriptional regulation. A paradigm for this response is the sustained increase in glutaminase (GA) that occurs through selective stabilization of GA mRNA and is mediated by two 8-nt AU-sequences that function as a pH-response element (pHRE). The rapid turnover of GA mRNA that occurs during normal acid base-balance is preceded by a rapid deadenylation, while the pH-responsive stabilization is associated with a decreased rate and extent of deadenylation. HuR and p40-AUF1 bind to the pHRE with high affinity and specificity. Preliminary experiments establish that onset of acidosis activates an ER-stress signaling pathway in rat renal proximal tubules and promotes the formation of RNA stress granules and the release of HuR from the nucleus in cultured kidney cells. Therefore, during normal acid-base balance, a pHRE binding protein may facilitate the recruitment of a deadenylase and the subsequent exonucleolytic decay of GA mRNA. During metabolic acidosis, association with stress granules may promote remodeling of the GA mRNA and facilitate its association with stabilizing factors such as HuR or p40AUF1. To test these novel hypotheses, the following specific aims are proposed: to identify the ER-stress signaling pathway that is activated in response to onset of acidosis and to utilize dominant negative and siRNA constructs to establish its role in stabilization of GA mRNA; to characterize the potential roles of HuR and the cytosolic isoforms of AUF1 by performing over expression and siRNA knockdown experiments and by utilizing protein/RNA pull-down, immunofluorescence, and mass spectroscopic analysis to establish their temporal association with GA mRNA, cellular localization, and sites of phosphorylation; and to employ a novel protocol to rapidly purify and identify pHRE/protein complexes that are formed in intact cells. The resulting data will define the mechanism by which the proximal tubule cell senses slight changes in pH and mediates the remodeling of its proteome in response to acidosis. It will also significantly enhance understanding of the fundamental mechanisms that regulate changes in mRNA stability and mediate an essential physiological response of the kidney. PUBLIC HEALTH RELEVANCE: Metabolic acidosis is a common clinical condition that contributes to osteomalacia, nephrocalcinosis and urolithiasis in adults and causes mental retardation in children. During end stage renal disease, the development of metabolic acidosis correlates with increased peripheral insulin resistance and is an additional morbidity risk factor. To compensate for the onset of acidosis requires an appropriate increase in renal acid excretion and bicarbonate production. This essential response is initiated by an adaptive increase in renal catabolism of glutamine that occurs solely within the proximal convoluted tubule and is sustained, in large part, by the cell-specific and pronounced increase in the level of glutaminase mRNA and protein. The proposed experiments will greatly enhance knowledge of the mechanism by which the kidney senses changes in acid-base balance and transmits this information to promote selective stabilization of glutaminase mRNA. Recent proteomic analysis indicates that expression of multiple genes in the proximal tubule during acidosis is regulated through the same mechanism of mRNA stabilization. This system is also an important paradigm for understanding the mechanism by which the physiological regulation of mRNA turnover contributes to an essential adaptive response. In addition, the resulting data will contribute fundamental knowledge of the mechanism and regulation of mRNA turnover, both in this system and in general. This knowledge will constitute the basis for biochemical and pharmacological studies that may improve the treatment of patients who present with chronic forms of metabolic acidosis.