Proteomic analysis of the effect of metabolic acidosis on the apical membrane of the renal proximal convoluted tubule
Date
2011
Authors
Walmsley, Scott J., author
Curthoys, Norman, advisor
Deluca, Jennifer, committee member
Dobos, Karen, committee member
Laybourne, Paul, committee member
Prenni, Jessica, committee member
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Abstract
Metabolic acidosis is a physiological disturbance which results in a decrease in blood and extracellular pH and HCO3-. The renal response to this disturbance is initiated in the proximal convoluted tubule (PCT) of the kidney. At the PCT, the brush border membrane facilitates solute reabsorbtion and excretion of acid during acidosis. However, the extent of the global remodeling of proteins at the brush border remains mostly unknown. Therefore a proteomic investigation of the remodeling of theseproteins during metabolic acidosis at the brush border was completed. First, using LTQ mass spectrometry and spectral counting, an enrichment method was tested that analyzed brush border membrane vesicles (BBMV) from cortex versus those which were derived from purified proximal convoluted tubules. From these results we detected and hypothesized that enzymes of glucose metabolism localized at the brush border would be altered in abundance during acidosis at the PCT brush border. Next, we performed a quantitative analysis of the temporal response to metabolic acidosis during 1-d, 3-d and 7-d acidosis using Q-TOF mass spectrometry and spectral counting. As expected, the results indicated a decrease of enzymes of glucose metabolism including Fructose-1,6-bisphosphatase 1 and Enolase A. Aldolase A was found to be transiently decreased during 1-d and 3-d acidosis. In addition, the Na+-glucose transporter 2 was found to be transiently increased during 1-d and 3-d acidosis. Finally, to confirm these abundance changes detected using spectral counting, an accurate mass and time tag method was developed. Using this method, we successfully developed an AMT database of the previously identified spectra. This database was used to match peptides detected using QTOF-LC-MS to the previously identified peptides. Peptide abundance by spectral counting was validated using the more accurate peak intensities and were generally in concordance with those abundance measurements using spectral counting. The developed model suggested a mechanism for internalization of these enzymes of glucose metabolism in support of glutamine metabolism, which is central to the cellular response to acidosis by the PCT.
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Subject
renal physiology
mass spectrometry
proteomics
metabolic acidosis