Response of the mitochondrial proteome of rat renal proximal convoluted tubules to chronic metabolic acidosis - PubMed (original) (raw)

Response of the mitochondrial proteome of rat renal proximal convoluted tubules to chronic metabolic acidosis

Dana M Freund et al. Am J Physiol Renal Physiol. 2013.

Abstract

Metabolic acidosis is a common clinical condition that is caused by a decrease in blood pH and bicarbonate concentration. Increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis. A workflow was developed to characterize the mitochondrial proteome of the proximal convoluted tubule. Based upon the increase in specific activity of cytochrome c oxidase, the isolated mitochondria were enriched eightfold. Two-dimensional liquid chromatography coupled with mass spectrometry was utilized to compare mitochondrial-enriched samples from control and chronic acidotic rats. Proteomic analysis identified 901 proteins in the control and acidotic samples. Further analysis identified 37 peptides that contain an N-ε-acetyl-lysine; of these, 22 are novel sites. Spectral counting analysis revealed 33 proteins that are significantly altered in abundance in response to chronic metabolic acidosis. Western blot analysis was performed to validate the calculated changes in abundance. Thus the current study represents the first comprehensive analysis of the mitochondrial proteome of the rat renal proximal convoluted tubule and its response to metabolic acidosis.

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Figures

Fig. 1.

Fig. 1.

Enrichment of cytochrome c oxidase and peroxisomal biogenesis factor 3 in isolated mitochondrial samples. A: homogenates of proximal convoluted tubules (PCT) and the mitochondrial fractions (M) isolated from three control (Ctrl) and three 7-day chronic acidotic (CMA) rats were assayed for cytochrome c oxidase activity and protein concentration. Values are means ± SE of the fold-increase in specific activities of the triplicate acidotic PCT or M samples, normalized to the control PCT samples. B: corresponding increases in peroxisomal biogenesis factor 3 were quantified by Western blot analysis. Values are means ± the range of fold-increases measured in duplicate biological samples.

Fig. 2.

Fig. 2.

Proteomics workflow. PCT were isolated from 3 control and three 7-day CMA rats. The tubules were homogenized, and the mitochondria were isolated by differential and sucrose gradient centrifugations. Following tryptic digestion, duplicate injections of the resulting peptides were analyzed by 2-dimensional liquid chromatography (2D-LC) coupled directly with tandem mass spectrometry (MS/MS). Proteins were identified by searching the mass spectra vs. the Uniprot-KB Rat protein sequence database. Spectral counting was performed to determine significant changes in protein abundance.

Fig. 3.

Fig. 3.

Proteomic analysis. A: Venn diagram of total proteins identified in the mitochondria prepared from PCT isolated from control and 7-day CMA rats. A total of 901 proteins were identified; 878 were common in both samples, while 12 and 11 were unique to the control and acidotic samples, respectively. Pie charts are shown of the cellular locations (B) and cellular processes (C) as determined by gene ontology (GO) analysis of the identified proteins.

Fig. 4.

Fig. 4.

Western blot analysis of the temporal changes in lysine acetylation of total mitochondrial proteins during development of acidosis. A: mitochondrial samples were isolated from PCT of three control, three 1-day acidotic (MA), three 3-day MA, and three 7-day CMA rats. Western blot analysis was performed using an anti-_N_-ε-acetyl-lysine antibody. The bands were imaged and quantified with an Odyssey Infrared Imager. B: acetylation of mitochondrial proteins increased during the onset of acidosis. The combined intensities of each lane in A were normalized to the level of succinate dehydrogenase (SDH), which served as a loading control. Values are means ± SE of triplicate samples.

Fig. 5.

Fig. 5.

Western blot analysis of the temporal changes in abundance of proteins identified by spectral count analysis. A: separate mitochondrial samples were isolated from PCT of three control, three 1-day MA, three 3-day MA, and three 7-day CMA rats. Western blot analysis was performed using 8 specific antibodies to validate the changes in protein abundance calculated by comparing the spectral counts of control and 7-day CMA samples. MW are molecular weight standards. Each blot was normalized to a mitochondrial protein that served as a loading control. The bands were imaged and quantified with an Odyssey Infrared Imager. B: comparison of the fold-changes calculated from Western blot analyses (open bars) and spectral counts (filled bars). Values are means ± SE of the fold-increase in the triplicate 7-day acidotic samples normalized to the control samples. C and D: time course of the fold-changes in the 8 proteins during development of chronic acidosis derived from the Western blot analyses in A. Values are means ± SE of triplicate samples.

Fig. 6.

Fig. 6.

Effect of unilateral nephrectomy on lysine acetylation and protein abundance of mitochondrial proteins. A: Western blot analysis of homogenates of mitochondria isolated from PCT from 3 sham-operated and 3 uninephrectomized (UNX) rats was performed using an antibody specific for _N_-ε-acetyl-lysine. B: Western blot analysis was performed using antibodies that are specific for 8 of the proteins that were identified to increase significantly by spectral counting during 7-day CMA. Each blot was normalized to a mitochondrial protein that served as a loading control. MW are molecular weight standards. The bands were imaged and quantified with an Odyssey Infrared Imager. C: comparison of fold-changes in protein expression in UNX rats. Values are means ± SE of the intensities of the triplicate UNX samples normalized to the sham-operated control samples. The dashed line indicates no change in abundance.

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