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. 2010 Mar;298(3):F643-54.
doi: 10.1152/ajprenal.00584.2009. Epub 2010 Jan 6.

cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells

Affiliations

cAMP stimulates apical V-ATPase accumulation, microvillar elongation, and proton extrusion in kidney collecting duct A-intercalated cells

Teodor G Păunescu et al. Am J Physiol Renal Physiol. 2010 Mar.

Abstract

Kidney proton-secreting A-intercalated cells (A-IC) respond to systemic acidosis by accumulating the vacuolar ATPase (V-ATPase) in their apical membrane and by increasing the length and number of apical microvilli. We show here that the cell-permeant cAMP analog CPT-cAMP, infused in vivo, results in an almost twofold increase in apical V-ATPase accumulation in AE1-positive A-IC within 15 min and that these cells develop an extensive array of apical microvilli compared with controls. In contrast, no significant change in V-ATPase distribution could be detected by immunocytochemistry in B-intercalated cells at the acute time point examined. To show a direct effect of cAMP on A-IC, we prepared cell suspensions from the medulla of transgenic mice expressing EGFP in IC (driven by the B1-subunit promoter of the V-ATPase) and exposed them to cAMP analogs in vitro. Three-dimensional reconstructions of confocal images revealed that cAMP induced a time-dependent growth of apical microvilli, starting within minutes after addition. This effect was blocked by the PKA inhibitor myristoylated PKI. These morphological changes were paralleled by increased cAMP-mediated proton extrusion (pHi recovery) by A-IC in outer medullary collecting ducts measured using the ratiometric probe BCECF. These results, and our prior data showing that the bicarbonate-stimulated soluble adenylyl cyclase (sAC) is highly expressed in kidney intercalated cells, support the idea that cAMP generated either by sAC, or by activation of other signaling pathways, is part of the signal transduction mechanism involved in acid-base sensing and V-ATPase membrane trafficking in kidney intercalated cells.

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Figures

Fig. 1.
Fig. 1.
Immunofluorescence labeling for the A subunit of the V-ATPase in control (Con; A) and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP)-treated (cAMP; B) rat outer medullary collecting duct (OMCD) intercalated cells (IC) imaged by conventional widefield microscopy on 5-μm kidney cryosections. The apical membranes of all A-IC exhibit bright V-ATPase staining (green), but in the cAMP-treated rat (B), extensively stained microvilli are readily detectable (arrows; see also inset). In contrast, the apical membranes of IC from the control rat (A) have a much smoother appearance. Bar = 5 μm. Inset bar = 2.5 μm.
Fig. 2.
Fig. 2.
Confocal microscopy showing A-subunit V-ATPase immunostaining in the OMCD of control and cAMP-treated rat kidney. In IC from control animals (A), the V-ATPase (green) localizes throughout the region between the apical membrane and the cell nucleus identified by 4,6-diamidino-2-phenylindole staining (blue), and few apical microvilli are seen. Following CPT-cAMP treatment, the apical membrane staining is brighter and exhibits clearly visible microvilli, whereas the subapical staining above the nucleus appears depleted (B). Bars = 5 μm.
Fig. 3.
Fig. 3.
Mean pixel intensity of apical B1 V-ATPase-associated immunofluorescence in 3 control (black bars) and 3 cAMP-treated rats (gray bars) is shown as means ± SE. The dark gray bar represents the average of the control group, 1,433 ± 123 (n = 3). The average mean pixel intensity (MPI) of all 124 cells quantified from the 3 control animals was 1,477. The light gray bar represents the average of the cAMP-treated group, 2,364 ± 219 (n = 3), representing a 65% increase over controls. The corresponding average MPI of all 77 A-ICs quantified from cAMP-treated rats was 2,415.
Fig. 4.
Fig. 4.
Immunogold electron microscopy on LR White resin-embedded rat kidney showing distribution of the V-ATPase A subunit in the apical domain of A-type IC from OMCD. The A-subunit of the V-ATPase is labeled with 10-nm gold particles. The V-ATPase is mainly associated with the subapical cytoplasm and vesicles in control animals (A) and is expressed at high levels on the extensive apical membrane microvilli that develop in cAMP-treated rats (B). Bars = 0.5 μm.
Fig. 5.
Fig. 5.
Quantification of gold particle density and apical membrane length in LR White resin-embedded control and cAMP-treated rat collecting duct A-type IC. The cAMP treatment induces a 2-fold increase in the number of gold particles located to the apical membrane and microvilli, from 70 ± 12 to 130 ± 18 (means ± SE; A). The apical membrane length normalized to the cell width increases after cAMP treatment from 3.8 ± 0.3 to 6.0 ± 0.7 (B). Gold particle density per unit length of membrane also increased from 3.1 ± 0.4 to 4.7 ± 0.2 particles/μm (C). All increases are statistically significant, as indicated by the respective P values. The quantification includes n = 31 OMCD A-IC from 3 control rats and n = 29 cells from 3 cAMP-treated animals.
Fig. 6.
Fig. 6.
Comparison of gold particle labeling intensity (particles per μm length of membrane) of the apical plasma membrane (solid bars) vs. the membranes of subapical vesicles (hatched bars) in A-IC from outer medullary collecting ducts of control, nonstimulated rat kidneys. The labeling intensity of the vesicles was significantly greater (P < 0.002 for each tissue) than that of the apical surface under nonstimulated conditions in all tissues examined. The quantification compared the labeling intensity of a total of 121 vesicles from 14 different OMCD IC from 3 animals with the control values for membrane labeling that are also presented in Fig. 5.
Fig. 7.
Fig. 7.
Quantification of the percentage of B-IC showing various patterns of V-ATPase staining before and after cAMP treatment in vivo. The staining categories were based on those previously used to define V-ATPase localization in IC (6, 53). BL, basolateral. No visually detectable or statistically significant change in the numbers of B-IC with the different staining patterns was detectable in any of the cAMP-treated animals compared with controls. (P = 0.88 by 2-way ANOVA).
Fig. 8.
Fig. 8.
Immunogold labeling of the V-ATPase A subunit in the basolateral region of B-IC from cortical collecting ducts of control (A) and cAMP-treated (B) LR White resin-embedded rat kidneys. The basolateral labeling intensity was not significantly different in control tissues compared with those exposed to cAMP. Quantitative data supporting this observation are provided in results. Bar = 0.5 μm.
Fig. 9.
Fig. 9.
Spinning disk confocal microscopy showing representative examples of microvillar development induced by CPT-cAMP (A and B) and 8-bromo (Br)-cAMP (C and D) treatment of IC that were isolated from the medulla of transgenic mice that express EGFP driven by the promoter of the V-ATPase B1 subunit. A and C show the cells before cAMP analog addition, and B and D show the same cells after 40-min exposure to the respective cAMP analog with new apical microvilli now clearly visible (arrows). E and F show a cell that was first exposed to the PKA inhibitor myristoylated (m) PKI and then for 40 min to CPT-cAMP in the continued presence of mPKI. No microvillar growth was detectable in the presence of mPKI. Bar = 5 μm
Fig. 10.
Fig. 10.
Spinning disc confocal microscopy showing an example of a dramatic morphological change induced by CPT-cAMP treatment that was seen in some of the IC isolated from transgenic mice expressing EGFP driven by the promoter of the V-ATPase B1 subunit. This cell initially had no visible microvilli (A) but progressively developed an array of extensive microvilli-like projections at the “apical” pole after 12 (B), 24 (C), 36 (D), 48 (E), and 60 min (F) of exposure to 1 mM CPT-cAMP. Bar = 10 μm.
Fig. 11.
Fig. 11.
Immunogold electron microscopy (LR White resin-embedded rat kidney) showing an extreme example of extensive growth of V-ATPase (A subunit)-containing apical microvilli at the apical domain of an OMCD A-IC from a rat infused for 15 min with both cAMP and theophylline. Bar = 0.5 μm.
Fig. 12.
Fig. 12.
V-ATPase activity measured as intracellular pH (pHi) recovery rate in OMCD A-IC from control and cAMP-treated mice, shown here as means ± SE. The rate of pHi recovery increased significantly (**P = 0.0036) after 15-min exposure to 300 μM 8-Br-cAMP (n = 110 cAMP-treated cells from 3 mice).

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References

    1. Al-Awqati Q. Terminal differentiation of intercalated cells: the role of hensin. Annu Rev Physiol 65: 567–583, 2003 - PubMed
    1. Alper SL. Genetic diseases of acid-base transporters. Annu Rev Physiol 64: 899–923, 2002 - PubMed
    1. Alper SL, Natale J, Gluck S, Lodish HF, Brown D. Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase. Proc Natl Acad Sci USA 86: 5429–5433, 1989 - PMC - PubMed
    1. Bagnis C, Marshansky V, Breton S, Brown D. Remodeling the cellular profile of collecting ducts by chronic carbonic anhydrase inhibition. Am J Physiol Renal Physiol 280: F437–F448, 2001 - PubMed
    1. Bastani B, Haragsim L. Immunocytochemistry of renal H-ATPase. Miner Electrolyte Metab 22: 382–395, 1996 - PubMed

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