Proteomic Analysis of Renal Response to Acidosis

描述（由申请方提供）：代谢性酸中毒期间，近端小管内发生的肾脏产氨和脱氨显著增加，促进酸排泄并产生碳酸氢盐。这种基本的适应性反应部分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意义。差异凝胶电泳用于比较从对照大鼠和从酸中毒2小时或1，3和7天的大鼠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其纯度约为95%。用DeCyder软件对2维凝胶进行成像和定量，并使用MALDI/TOF/TOF质谱法来鉴定响应于酸中毒而增加或减少的蛋白质。除了谷氨酸脱氢酶（GA）、谷氨酸脱氢酶（GDH）和磷酸烯醇丙酮酸羧激酶（PEPCK）（响应于酸中毒而诱导的三种充分表征的蛋白质）之外，该方法鉴定了17种先前未识别的蛋白质，其增加1.5至5.6倍，以及16种蛋白质，其减少0.67至0.03倍。这些变化中的一些通过蛋白质印迹分析得到证实。时间研究确定的蛋白质，要么是完全诱导1天内类似PEPCK或表现出更渐进的动力学类似GA和GDH。编码后一种蛋白质的所有mRNA都含有与pH响应元件同源的AU序列，所述pH响应元件在酸中毒期间介导GA mRNA的稳定性增加。因此，选择性mRNA稳定化可能是蛋白质表达增加的主要机制。使用ICAT标记试剂的进一步分析鉴定了许多具有相似倍数差异的相同蛋白质。这种方法还确定了11种额外的蛋白质，在慢性酸中毒7天后增加超过1.5倍。本申请中提出的研究有三个具体目标：利用ICAT和iTRAQ标记技术和蛋白质印迹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来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化蛋白质组的变化;并进行生物信息学分析以鉴定调节近端小管内的稳态适应的调节元件和潜在的信号传导机制。酸中毒的发作需要肾脏酸排泄和碳酸氢盐产生的适当增加。拟议的蛋白质组学分析将大大提高知识的复杂性的肾脏适应，有助于调节酸碱平衡，并提供新的见解有关的机制，肾近端小管的感官轻微的pH值变化，并激活特定的信号通路，以介导这一基本的反应。由此产生的假设将刺激未来的生物化学和药理学研究，这将改善对各种形式酸中毒患者的治疗。

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.