Vacuolar H+-ATPase apical accumulation in kidney intercalated cells is regulated by PKA and AMP-activated protein kinase

Abstract

The vacuolar H(+)-ATPase (V-ATPase) in type A kidney intercalated cells is a major contributor to acid excretion in the collecting duct. The mechanisms of V-ATPase-trafficking regulation in kidney intercalated cells have not been well-characterized. In developmentally related epididymal clear cells, we showed previously that PKA, acting downstream of soluble adenylyl cyclase (sAC), induces V-ATPase apical membrane accumulation. These PKA-mediated effects were inhibited by activators of the metabolic sensor AMP-activated kinase (AMPK) in clear cells. Here, we examined the regulation of V-ATPase subcellular localization in intercalated cells by PKA and AMPK in rat kidney tissue slices ex vivo. Immunofluorescence labeling of kidney slices revealed that the PKA activator N(6)-monobutyryl cAMP (6-MB-cAMP) induced V-ATPase apical membrane accumulation in collecting duct intercalated cells, whereas the V-ATPase had a more cytosolic distribution when incubated in Ringer buffer alone for 30 min. V-ATPase accumulated at the apical membrane in intercalated cells in kidney slices incubated in Ringer buffer for 75 min, an effect that was prevented by treatment with PKA inhibitor (mPKI). The V-ATPase distribution was cytosolic in intercalated cells treated with the carbonic anhydrase inhibitor acetazolamide or the sAC inhibitor KH7, effects that were overridden by 6-MB-cAMP. Preincubation of kidney slices with an AMPK activator blocked V-ATPase apical membrane accumulation induced by 6-MB-cAMP, suggesting that AMPK antagonizes cAMP/PKA effects on V-ATPase distribution. Taken together, our results suggest that in intercalated cells V-ATPase subcellular localization and therefore its activity may be coupled to acid-base status via PKA, and metabolic status via AMPK.

Fig. 1.

PKA agonists induce rapid apical accumulation of the V-ATPase in intercalated cells. Confocal images of V-ATPase E subunit immunofluorescence labeling in kidney slices incubated in Ringer buffer alone for 30 min display a cytoplasmic distribution for the V-ATPase (A; L indicates the position of the lumen of the collecting duct). Addition of 1 mM N⁶-monobutyryl cAMP (6-MB-cAMP) plus 0.5 mM 3-isobutyl-1-methylxanthine (IBMX) induced an apical/luminal accumulation of the V-ATPase (B). Regions of interest (ROIs) were outlined for each cell at apical and cytoplasmic regions for quantification using previously described methods (4, 6, 66), as illustrated in A and B, insets (arrows indicate the cells depicted in the insets, where the scale bar represents 2.5 μm). C: quantification of the mean (±SE) V-ATPase-associated mean pixel intensity (MPI) apical/cytoplasmic ratio [arbitrary units (A.U.)] was used as a measure of apical V-ATPase accumulation, which was greater in the presence of PKA agonists. Data were obtained from at least 3 separate experiments measuring a total of at least 30 cells per condition (*P < 0.05 vs. Ringer pH 7.4, 30 min). Scale bar = 10 μm.

Fig. 2.

Carbonic anhydrase and cAMP/PKA acutely regulate V-ATPase subcellular localization in kidney intercalated cells. Confocal images of immunofluorescence staining of fixed kidney slices using an antibody against the V-ATPase E subunit in collecting duct are shown. A: slices incubated in Ringer buffer for 75 min had V-ATPase accumulation at the apical membrane of intercalated cells. Unless otherwise stated, the apical/luminal membrane of the intercalated cells is facing upwards in the image. B: this accumulation was prevented by incubation with 100 μM acetazolamide, a carbonic anhydrase inhibitor, for the same period of time. C: additional presence of 6-MB-cAMP (100 μM + 0.5 mM IBMX) in the solution restored the V-ATPase to the apical membrane of intercalated cells in the presence of acetazolamide. D: graph summarizing the apical-to-cytoplasmic MPI of V-ATPase-associated fluorescence. Error bars are SE (*P < 0.05 with respect to control, Ringer pH 7.4, 75 min; **P < 0.05 with respect to Ringer + acetazolamide, 75 min). Scale bar = 10 μm.

Fig. 3.

Soluble adenylyl cyclase (sAC) inhibitor KH7 prevents the HCO₃⁻-mediated V-ATPase apical accumulation. Confocal images of V-ATPase E subunit immunofluorescence labeling in kidney slices incubated in Ringer buffer alone for 75 min (A) or in the presence of the sAC inhibitor KH7 (40 μM; B) or in the presence of KH7 with the addition of the PKA activator 6-MB-cAMP (1 mM) for the last 30 min (C). V-ATPase accumulation at the apical membrane of intercalated cells was prevented by KH7. Intercalated cells in tissue slices treated with the PKA activator 6-MB-cAMP had apical V-ATPase accumulation even in the presence of a sAC inhibitor. E: quantification of the mean (±SE) V-ATPase-associated MPI apical-to-cytoplasmic ratio under the different conditions (*P < 0.05 with respect to control, Ringer pH 7.4, 75 min; D). Scale bar = 10 μm.

Fig. 4.

PKA inhibitor mPKI prevents apical V-ATPase accumulation in kidney intercalated cells. Confocal images of V-ATPase E subunit immunofluorescence labeling in kidney slices incubated either in Ringer buffer for 75 min (A) or in the presence of the specific PKA inhibitor mPKI (10 μM; B). Quantification of the mean (±SE) V-ATPase-associated MPI apical-to-cytoplasmic ratio under the different conditions demonstrates that mPKI prevented apical membrane accumulation (*P < 0.05 vs. Ringer buffer control, 75 min; C). Scale bar = 10 μm.

Fig. 5.

AMP-activated kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) prevents V-ATPase apical accumulation at 75 min. Confocal images illustrating cellular distribution changes of the V-ATPase E subunit in collecting duct cells of kidney slices treated with Ringer buffer (A) vs. Ringer buffer + 2 mM AICAR (B). C: quantification of the mean (±SE) V-ATPase-associated MPI apical-to-cytoplasmic ratio under the different conditions confirms a significant cytoplasmic redistribution of the V-ATPase in the presence of AICAR. *P < 0.05 vs. Ringer pH 7.4, 75 min. Scale bar = 10 μm.

Fig. 6.

AMPK activator AICAR prevents PKA-mediated V-ATPase apical accumulation. Confocal images of kidney slices using an antibody against the V-ATPase E subunit in collecting duct cells. A: slices incubated in Ringer buffer for 30 min display a cytosolic V-ATPase distribution in intercalated cells. B: presence of AICAR (2 mM) in the buffer did not induce subcellular localization changes compared with the 30-min Ringer buffer control. C: presence of PKA activator 6-MB-cAMP (1 mM) during the 30-min incubation induced a redistribution of the V-ATPase to the apical membrane of intercalated cells. D: this PKA-mediated accumulation did not occur when AICAR was also present in the buffer. E: quantification of the mean (±SE) V-ATPase-associated MPI apical-to-cytoplasmic ratio under the different conditions reveals a significant inhibition by AICAR of PKA-mediated apical V-ATPase accumulation (*P < 0.05 vs. control, Ringer buffer pH 7.4, 30 min).