Altered V-ATPase expression in renal intercalated cells isolated from B1 subunit-deficient mice by fluorescence-activated cell sorting

• Published in: American journal of physiology. Renal physiology 2013-03, Vol.304 (5), p.F522-F532
• Main Authors: Vedovelli, Luca, Rothermel, John T, Finberg, Karin E, Wagner, Carsten A, Azroyan, Anie, Hill, Eric, Breton, Sylvie, Brown, Dennis, Paunescu, Teodor G
• Format: Article
• Language: English
• Subjects: Adenosine triphosphatase; Animals; Cell Membrane - metabolism; Cells; Epithelial Cells - cytology; Epithelial Cells - metabolism; Flow Cytometry; Kidney - cytology; Kidney - metabolism; Kidney diseases; Male; Membranes; Mice; Mice, Inbred C57BL; Mice, Knockout; Protein Subunits - genetics; Protein Subunits - metabolism; Proteins; Rodents; Up-Regulation; Vacuolar Proton-Translocating ATPases - genetics; Vacuolar Proton-Translocating ATPases - metabolism
• ISSN: 1931-857X; 1522-1466
• DOI: 10.1152/ajprenal.00394.2012

Unlike human patients with mutations in the 56-kDa B1 subunit isoform of the vacuolar proton-pumping ATPase (V-ATPase), B1-deficient mice (Atp6v1b1(-/-)) do not develop metabolic acidosis under baseline conditions. This is due to the insertion of V-ATPases containing the alternative B2 subunit isoform into the apical membrane of renal medullary collecting duct intercalated cells (ICs). We previously reported that quantitative Western blots (WBs) from whole kidneys showed similar B2 protein levels in Atp6v1b1(-/-) and wild-type mice (Păunescu TG, Russo LM, Da Silva N, Kovacikova J, Mohebbi N, Van Hoek AN, McKee M, Wagner CA, Breton S, Brown D. Am J Physiol Renal Physiol 293: F1915-F1926, 2007). However, WBs from renal medulla (including outer and inner medulla) membrane and cytosol fractions reveal a decrease in the levels of the ubiquitous V-ATPase E1 subunit. To compare V-ATPase expression specifically in ICs from wild-type and Atp6v1b1(-/-) mice, we crossed mice in which EGFP expression is driven by the B1 subunit promoter (EGFP-B1(+/+) mice) with Atp6v1b1(-/-) mice to generate novel EGFP-B1(-/-) mice. We isolated pure IC populations by fluorescence-assisted cell sorting from EGFP-B1(+/+) and EGFP-B1(-/-) mice to compare their V-ATPase subunit protein levels. We report that V-ATPase A, E1, and H subunits are all significantly downregulated in EGFP-B1(-/-) mice, while the B2 protein level is considerably increased in these animals. We conclude that under baseline conditions B2 upregulation compensates for the lack of B1 and is sufficient to maintain basal acid-base homeostasis, even when other V-ATPase subunits are downregulated.