Control of Renal Glutaminase Expression during Acidosis

描述(由申请人提供)：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可滴定酸排泄方面的遗传或后天缺陷引起。慢性酸中毒会导致儿童智力迟缓，成人会出现骨软化、肾钙化和尿石症。代谢性酸中毒时，肾脏近曲小管谷氨酰胺分解代谢代偿性增加。由此导致的铵离子和碳酸氢根离子合成的增加促进了酸的排泄，并部分恢复了酸碱平衡。这种基本的适应性反应是由基因表达的显著变化所维持的，这些变化主要通过转录后调节发生。这种反应的一个范例是谷氨酰胺酶(GA)的持续增加，这种增加是通过选择性稳定GA mRNA而发生的，并由两个作为pH反应元件(PhRE)的8-ntAU-序列介导。在正常的酸碱平衡状态下，GA mRNA的快速周转发生在快速去烯化之前，而pH响应的稳定与去烯化的速率和程度的降低有关。HUR和p40-AUF1与PhRE具有较高的亲和力和特异性。初步实验证实，酸中毒可激活大鼠肾小管上皮细胞内质网应激信号通路，促进培养肾细胞RNA应激颗粒的形成和核内Hur的释放。因此，在正常的酸碱平衡中，PhRE结合蛋白可能有助于死烯基酶的募集和随后GA mRNA的胞外溶解衰变。在代谢性酸中毒中，与应激颗粒的联合可能促进GA mRNA的重塑，并促进其与稳定因子如Hur或p40AUF1的联系。为了验证这些新的假设，提出了以下具体目标：识别酸中毒开始时激活的内质网应激信号通路，并利用显性的负向和siRNA构建物来确定其在GA mRNA稳定中的作用；通过进行过表达和siRNA敲除实验以及通过利用蛋白质/RNA下拉、免疫荧光和质谱分析来确定Hur和AUF1的胞浆异构体的潜在作用，并利用蛋白质/RNA下拉、免疫荧光和质谱分析来建立它们与GA mRNA、细胞定位和磷酸化位点的时间关联；以及使用一种新的方法来快速纯化和鉴定在细胞中完整形成的PhRE/蛋白质复合体。由此产生的数据将确定近端小管细胞感知pH值轻微变化并调节其蛋白质组对酸中毒反应的重塑的机制。它还将显著提高对调节mRNA稳定性变化的基本机制的理解，并调节肾脏的基本生理反应。 公共卫生相关性：代谢性酸中毒是一种常见的临床症状，可导致成人骨软化、肾钙素沉着症和尿石症，并导致儿童智力低下。在终末期肾脏疾病中，代谢性酸中毒的发展与外周胰岛素抵抗的增加相关，是另一个发病率危险因素。为了弥补酸中毒的发作，需要适当增加肾酸排泄和碳酸氢盐的产生。这一基本反应是由肾脏谷氨酰胺分解代谢的适应性增加启动的，这种代谢仅发生在近曲小管内，并在很大程度上由细胞特异性的谷氨酰胺酶mRNA和蛋白质水平的显著增加维持。拟议的实验将极大地提高人们对肾脏感知酸碱平衡变化并传递这一信息以促进谷氨酰胺酶mRNA选择性稳定的机制的认识。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。该系统也是一个重要的范例，以了解生理调节的信使核糖核酸周转有助于基本的适应性反应的机制。此外，由此产生的数据将有助于在这个系统中和一般情况下，对mRNA周转的机制和调节的基础知识。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出现慢性代谢性酸中毒的患者的治疗。

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.