Proteomic Analysis of Renal Response to Acidosis

肾脏对酸中毒反应的蛋白质组学分析

• 批准号: 7423935
• 负责人: NORMAN P. CURTHOYS
• 金额: $ 18.01万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-15 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7423935
• 关键词: Acid-Base Equilibrium; Acidosis; Acids; Acute; Apical; Bicarbonate Ion; Bicarbonates; Biochemical; Bioinformatics; Biological; Calmodulin; Chronic; Colorado; Computer software; Core Facility; Data; Density Gradient Centrifugation; Digestion; Electrolytes; Excretory function; Exhibits; Future; Gel; Gene Expression; Gluconeogenesis; Glutamate Dehydrogenase; Glutaminase; Glutamine; Hour; Hypertrophy; Image; Individual; Kidney; Kinetics; Knowledge; Label; Laboratories; Left; Mass Spectrum Analysis; Mediating; Membrane Proteins; Messenger RNA; Metabolic acidosis; Metabolism; Mitochondria; Molecular; Numbers; Patients; Percoll; Phosphoenolpyruvate Carboxylase; Plasma; Production; Proteins; Proteomics; Proximal Kidney Tubules; Rattus; Reagent; Regulation; Regulatory Element; Research; Research Personnel; Response Elements; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Techniques; United States National Institutes of Health; Universities; Western Blotting; Work; ammonium bicarbonate; basolateral membrane; collagenase; day; gel electrophoresis; improved; insight; novel; protein expression; research study; response; symporter; system N protein 1; two-dimensional

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.

描述（由申请人提供）：代谢性酸中毒期间近端肾小管内发生的肾脏产氨和糖异生的显着增加促进酸的排泄并产生碳酸氢盐。这种重要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该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意义。使用差异凝胶电泳来比较从对照大鼠和酸中毒2小时或1、3和7天的大鼠中分离的近端小管中的蛋白质表达。通过胶原酶消化和 Percoll 密度梯度离心分离近端小管，并通过蛋白质印迹分析显示纯度约为 95%。使用 DeCyder 软件对二维凝胶进行成像和定量，并使用 MALDI/TOF/TOF 质谱法来识别因酸中毒而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) 这三种因酸中毒而诱导的特征明确的蛋白质之外，该方法还鉴定了 17 种以前未识别的蛋白质，这些蛋白质在 1.5- 至 5.6- 之间增加。倍和 16 种蛋白质减少了 0.67 至 0.03 倍。其中一些变化通过蛋白质印迹分析得到证实。时间研究发现蛋白质要么在 1 天内完全诱导，类似于 PEPCK，要么表现出类似于 GA 和 GDH 的更渐进的动力学。编码后一种蛋白质的所有 mRNA 均含有与 pH 响应元件同源的 AU 序列，该元件在酸中毒期间介导 GA mRNA 稳定性的增加。因此，选择性 mRNA 稳定可能是响应酸中毒而增加蛋白质表达的主要机制。使用 ICAT 标记试剂进行进一步分析，鉴定出许多具有相似倍数差异的相同蛋白质。这种方法还鉴定出了另外 11 种蛋白质，这些蛋白质在慢性酸中毒 7 天后增加了 1.5 倍以上。本申请提出的研究有三个具体目标：利用ICAT和iTRAQ标记技术以及蛋白质印迹分析来确认和扩展初始蛋白质组数据；进行蛋白质组分析，以检测和量化急性和慢性代谢性酸中毒期间发生的膜蛋白和磷酸蛋白质组的变化；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信号机制。 /相关性酸中毒的发生需要适当增加肾脏酸排泄和碳酸氢盐的产生。所提出的蛋白质组学分析将极大地增强对有助于酸碱平衡调节的肾脏适应复杂性的认识，并提供关于肾近端小管感知 pH 轻微变化并激活特定信号通路介导的机制的新见解。这一重要的回应。由此产生的假设将刺激未来的生化和药理学研究，从而改善对各种形式酸中毒患者的治疗。