Proteomic analysis of the adaptive response of rat renal proximal tubules to metabolic acidosis

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were similar to 95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression. The mass-to-charge ratios and the amino acid sequences of the recovered tryptic peptides were determined by MALDI-TOF/TOF mass spectrometry and the proteins were identified using Mascot software. This analysis confirmed the well-characterized adaptive responses in glutaminase (GA), glutamate dehydrogenase (GDH), and phosphoenolpyruvate carboxykinase (PEPCK). This approach also identified 17 previously unrecognized proteins that are increased with ratios of 1.5 to 5.6 and 16 proteins that are decreased with ratios of 0.67 to 0.03 when tubules from 7-day acidotic vs. control rats were compared. Some of these changes were confirmed by Western blot analysis. Temporal studies identified proteins that were induced either with rapid kinetics similar to PEPCK or with more gradual profiles 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 found in GA mRNA. Thus selective mRNA stabilization may be a predominant mechanism by which protein expression is increased in response to acidosis.