Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule

Scott J. Walmsley, Dana M. Freund, Norman P. Curthoys

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH 4 + and HCO 3 - ions. Many of these adaptations involve proteins associated with the apical membrane. To quantify the temporal changes in these proteins, proteomic profiling was performed using brush-border membrane vesicles isolated from proximal convoluted tubules (BBMV PCT) that were purified from normal and acidotic rats. This preparation is essentially free of contaminating apical membranes from other renal cortical cells. The analysis identified 298 proteins, 26% of which contained one or more transmembrane domains. Spectral counts were used to assess changes in protein abundance. The onset of acidosis produced a twofold, but transient, increase in the Na +-dependent glucose transporter and a more gradual, but sustained, increase (3-fold) in the Na +-dependent lactate transporter. These changes were associated with the loss of glycolytic and gluconeogenic enzymes that are contained in the BBMV PCT isolated from normal rats. In addition, the levels of γ-glutamyltranspeptidase increased twofold, while transporters that participate in the uptake of neutral amino acids, including glutamine, were decreased. These changes could facilitate the deamidation of glutamine within the tubular lumen. Finally, pronounced increases were also observed in the levels of DAB2 (3-fold) and myosin 9 (7-fold), proteins that may participate in endocytosis of apical membrane proteins. Western blot analysis and accurate mass and time analyses were used to validate the spectral counting.

Original languageEnglish (US)
Pages (from-to)F1465-F1477
JournalAmerican Journal of Physiology - Renal Physiology
Volume302
Issue number11
DOIs
StatePublished - Jun 1 2012

Keywords

  • AMT analysis
  • Brush-border membranes
  • Mass spectrometry
  • Metabolic acidosis
  • Spectral counting

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