Regulation of proximal tubule vacuolar H⁺-ATPase by PKA and AMP-activated protein kinase

Abstract

ϩ -ATPase (VATPase) mediates ATP-driven H ϩ transport across membranes. This pump is present at the apical membrane of kidney proximal tubule cells and intercalated cells. Defects in the V-ATPase and in proximal tubule function can cause renal tubular acidosis. We examined the role of protein kinase A (PKA) and AMP-activated protein kinase (AMPK) in the regulation of the V-ATPase in the proximal tubule as these two kinases coregulate the V-ATPase in the collecting duct. As the proximal tubule V-ATPases have different subunit compositions from other nephron segments, we postulated that V-ATPase regulation in the proximal tubule could differ from other kidney tubule segments. Immunofluorescence labeling of rat ex vivo kidney slices revealed that the V-ATPase was present in the proximal tubule both at the apical pole, colocalizing with the brush-border marker wheat germ agglutinin, and in the cytosol when slices were incubated in buffer alone. When slices were incubated with a cAMP analog and a phosphodiesterase inhibitor, the V-ATPase accumulated at the apical pole of S3 segment cells. These PKA activators also increased V-ATPase apical membrane expression as well as the rate of VATPase-dependent extracellular acidification in S3 cell monolayers relative to untreated cells. However, the AMPK activator AICAR decreased PKA-induced V-ATPase apical accumulation in proximal tubules of kidney slices and decreased V-ATPase activity in S3 cell monolayers. Our results suggest that in proximal tubule the V-ATPase subcellular localization and activity are acutely coregulated via PKA downstream of hormonal signals and via AMPK downstream of metabolic stress.AMPK; PKA; acid-base homeostasis; metabolic stress THE KIDNEY plays a vital role in acid-base homeostasis, by H ϩ secretion and/or reabsorption of filtered HCO 3 Ϫ , in a variety of nephron segments, including the proximal tubule (26, 34). The proximal tubule epithelium reabsorbs ϳ80% of the filtered HCO 3 Ϫ (21). Proximal tubular mechanisms to reabsorb luminal HCO 3Ϫ require H ϩ secretion into the lumen. Both apical Na ϩ /H ϩ exchangers (NHEs) and the vacuolar H ϩ -ATPase (V-ATPase) contribute to these processes (13,21,51,54,67). Apical membrane NHE3 contributes approximately two-thirds of the total H ϩ secretion, while approximately one-third is mediated by the V-ATPase (21). The V-ATPase is a protein complex that transports H ϩ across membranes by hydrolyzing ATP, and in the proximal tubule it has been identified by microscopy in endocytic vesicles, at the base of the brush border and also sometimes in microvilli (9). H ϩ transport by the V-ATPase is also important in apical endocytosis (19,42,63). Defects in V-ATPase and in proximal tubule function can cause proximal renal tubular acidosis (RTA) (29).Changes in V-ATPase subcellular localization are associated with the regulation of H ϩ secretion (8, 39, 56). However, the potential role of kinases in V-ATPase regulation in the proximal tubule has not yet been elucidated. This pump exhibits similar regulation in kidney A...

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“…Kidneys from C57BL/6 male mice were fixed in 4% PFA and embedded in Tissue-Tek optimal cutting temperature compound before staining 4 -5-m cryosections with goat anti-aquaporin 2 (C-17) (0.2 g/ml Santa Cruz Biotechnology, sc-9882) and rabbit anti-DHHC3 (at 3.3 g/ml; ab31837, Abcam, Cambridge, MA) antibodies, as previously described (67,68). Validation of the anti-DHHC 3 antibody is described below.…”

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Vacuolar ATPase as a potential therapeutic target and mediator of treatment resistance in cancer

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