Proteomic Analysis of Renal Response to Acidosis

描述（由申请人提供）：代谢性酸中毒时，近端小管内发生的肾氨生成和糖异生明显增加，促进了酸的排泄并产生碳酸氢盐。这种基本的适应性反应在一定程度上是通过改变多种蛋白质的表达来维持的。初步的蛋白质组学分析确定了对这种反应进行彻底的蛋白质组学和生物信息学分析的可行性和重要性。采用差异凝胶电泳法比较对照大鼠和酸化2小时、1、3、7天大鼠近端小管中蛋白的表达。近端小管通过胶原酶消化和Percoll密度梯度离心分离，经western blot分析纯度约为95%。使用DeCyder软件对二维凝胶进行成像和定量，使用MALDI/TOF/TOF质谱法鉴定酸中毒反应中增加或减少的蛋白质。除了谷氨酰胺酶（GA）、谷氨酸脱氢酶（GDH）和磷酸烯醇丙酮酸羧激酶（PEPCK）这三种已被充分表征的在酸中毒反应中诱导的蛋白质外，该方法还鉴定了17种以前未被识别的蛋白质，它们的数量增加了1.5- 5.6倍，16种蛋白质的数量减少了1.5- 5.6倍。67倍和0.3倍。western blot分析证实了部分变化。时间研究发现，蛋白质要么在1天内完全诱导，类似于PEPCK，要么表现出更渐进的动力学，类似于GA和GDH。所有编码后一种蛋白的mRNA都含有一个au序列，该序列与酸中毒期间介导GA mRNA稳定性增加的ph反应元件同源。因此，选择性mRNA稳定可能是酸中毒反应中蛋白表达增加的主要机制。使用ICAT标记试剂进行进一步分析，鉴定出许多具有相似折叠差异的相同蛋白质。这种方法还发现了11种额外的蛋白质，这些蛋白质在慢性酸中毒7天后增加了1.5倍以上。本申请中提出的研究有三个具体目的：利用ICAT和iTRAQ标记技术和western blot分析来确认和扩展初始蛋白质组学数据；进行蛋白质组学分析，以检测和量化急性和慢性代谢性酸中毒期间膜蛋白和磷蛋白组的变化；并进行生物信息学分析，以确定介导近端小管内稳态适应的调节元件和潜在的信号机制。/相关性酸中毒的发生需要适当增加肾酸排泄和碳酸氢盐的产生。提出的蛋白质组学分析将极大地提高对肾脏适应的复杂性的认识，这些适应有助于调节酸碱平衡，并提供关于肾近端小管感知pH值轻微变化并激活特定信号通路以介导这一基本反应的机制的新见解。由此产生的假设将刺激未来的生化和药理学研究，以改善患有各种形式酸中毒的患者的治疗。

DESCRIPTION (provided by applicant): The pronounced increases in renal ammoniagenesis and gluconeogenesis that occur within the proximal tubule during metabolic acidosis facilitate the excretion of acid and generate bicarbonate. This essential adpative response is sustained, in part, by altered expression of multiple proteins. Preliminary proteomic analyses established the feasibility and the significance of performing a thorough proteomic and bioinformatic analysis of this response. Difference gel electrophoresis was used to compare protein expression in proximal tubules isolated from control rats and from rats that were made acidotic for 2 hours or for 1, 3 and 7 days. Proximal tubules were isolated by collagenase digestion and Percoll density gradient centrifugation and shown by western blot analysis to be approximately 95% pure. The 2-dimensional gels were imaged and quantified with DeCyder software and MALDI/TOF/TOF mass spectrometry was used to identify the proteins that are increased or reduced in response to acidosis. In addition to glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK), three well- characterized proteins that are induced in response to acidosis, this approach identified 17 previously unrecognized proteins that are increased between 1.5- to 5.6-fold and 16 proteins that are decreased between .67- and .03-fold. Some of these changes were confirmed by western blot analysis. Temporal studies identified proteins that either are fully induced within 1 d similar to PEPCK or exhibit more gradual kinetics similar to GA and GDH. All of the mRNAs that encode the latter proteins contain an AU-sequence that is homologous to the pH-response element that mediates the increased stability of GA mRNA during acidosis. Thus, selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis. Further analysis using the ICAT labeling reagent identified many of the same proteins with similar fold differences. This approach also identified 11 additional proteins that are increased more than 1.5-fold after 7 days of chronic acidosis. The research proposed in this application has three specific aims: to utilize ICAT and iTRAQ labeling techniques and western blot analysis to confirm and extend the initial proteomic data; to perform proteomic analyses to detect and quantify changes in membrane proteins and the phosphoproteome that occur during acute and chronic metabolic acidosis; and to conduct a bioinformatic analysis to identify the regulatory elements and potential signaling mechanisms that mediate the homeostatic adaptations within the proximal tubule. /Relevance The onset of acidosis requires the appropriate increase in renal acid excretion and bicarbonate production. The proposed proteomic analyses will greatly enhance knowledge of the complexity of the renal adaptations that contribute to the regulation of acid-base balance and provide novel insight regarding the mechanism by which the renal proximal tubule senses slight changes in pH and activates specific signaling pathways to mediate this essential response. The resulting hypotheses will stimulate future biochemical and pharmacological studies that will improve the treatment of patients who present with various forms of acidosis.