Temporal changes in the cytosolic proteome of the proximal convoluted tubule during the onset of metabolic acidosis
Date
2013
Authors
Schauer, Kevin Lee, author
Curthoys, Norman P., advisor
Prenni, Jessica E., committee member
Florant, Gregory L., committee member
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Abstract
A decrease in blood pH coupled with a decrease in blood bicarbonate concentration is a relatively common pathological condition that is referred to as metabolic acidosis. The proximal convoluted tubule cells in the kidney respond to this condition by increasing the extraction of plasma glutamine, and up-regulating ammoniagenesis and gluconeogenesis. These processes produce bicarbonate ions that are transported to the blood to help restore acid-base homeostasis. A few cytosolic proteins such as phosphoenolpyruvate carboxykinase have previously been identified to play a role in the renal response to metabolic acidosis, but further analysis is needed to better characterize the response of the entire proteome. Therefore, proximal convoluted tubule cells were isolated from rat kidney cortex tissue at various times after onset of acidosis and fractionated to separate the soluble cytosolic proteins from the remainder of the cellular components. The cytosolic proteins were analyzed using two-dimensional liquid chromatography tandem mass spectrometry to identify the constituent proteins. Spectral counting along with average MS/MS total ion current was used to quantify temporal changes in relative protein abundance. In all, 461 proteins were confidently identified in the samples, of which 24 exhibited statistically significant changes in abundance. To validate the technique, several of the abundance changes observed by spectral counting were confirmed via western blotting. Data from the cytosolic fractions were then combined with previous proteomic data and bioinformatics analysis was performed to better characterize the overall changes that occur in the proximal convoluted tubule during the onset of metabolic acidosis.
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Subject
metabolic acidosis
spectral counting
proximal convoluted tubule
proteomics