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. ? ? ?
