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J. Biol. Chem., Vol. 276, Issue 50, 47087-47093, December 14, 2001

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Short Term Effect of Aldosterone on Na,K-ATPase Cell Surface Expression in Kidney Collecting Duct Cells^*

Vanessa Summa^§, David Mordasini^§¶, Frank Roger^¶, Marcelle Bens, Pierre-Yves Martin^¶, Alain Vandewalle, François Verrey^**, and Eric Féraille^¶

From the  Institute of Physiology, University of Zürich, Zürich, CH-8057 Switzerland, the ^¶ Division of Nephrology and Department of Pathology, University of Geneva, CH-1211 Geneva, Switzerland, and  INSERM, Unité 478, Faculté de Médecine Xavier-Bichat, 75870 Paris Cedex 18, France

Received for publication, July 27, 2001, and in revised form, October 4, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Aldosterone controls extracellular volume and blood pressure by regulating Na⁺ reabsorption, in particular by epithelia of the distal nephron. A main regulatory site of this transcellular transport is the epithelial sodium channel (ENaC) that mediates luminal Na⁺ influx. The Na,K-ATPase (Na⁺ pump) that coordinately extrudes Na⁺ across the basolateral membrane is known to be regulated by short term aldosterone as well. We now show that in the cortical collecting duct (CCD) from adrenalectomized rats, the increase in Na,K-ATPase activity (approximately 3-fold in 3 h), induced by a single aldosterone injection, can be fully accounted by the increase in Na,K-ATPase cell surface expression (+ 497 ± 35%). The short term aldosterone action was further investigated in cultured mouse collecting duct principal cells mpkCCD_cl4. Within 2 h, maximal Na,K-ATPase function assessed by Na⁺ pump current (I_p) measurements and Na,K-ATPase cell surface expression were increased by 20-50%. Aldosterone did not modify the Na⁺ dependence of the Na⁺ pumps and induced transcription- and translation-dependent actions on pump surface expression and current independently of ENaC-mediated Na⁺ influx. In summary, short term aldosterone directly increases the cell surface expression of pre-existing Na⁺ pumps in kidney CCD target cells. Thus, aldosterone controls Na⁺ reabsorption in the short term not only by regulating the apical cell surface expression of ENaC (Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001) Am. J. Physiol. 280, F675-F682) but also by coordinately acting on the basolateral cell surface expression of the Na,K-ATPase.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The mineralocorticoid hormone aldosterone plays a central role in the regulatory network that controls body sodium and volume homeostasis (1). It displays a potent Na⁺ retaining action at the level of tight epithelia, in particular in the aldosterone-sensitive distal nephron (ASDN),¹ a portion of the kidney tubule that extends from the second part of the distal convoluted tubule to the medullary collecting duct (2-4). The segment-specific cells of the ASDN express a transcellular pathway for Na⁺ reabsorption that is composed of the luminal epithelial Na⁺ channel (ENaC) and the basolateral Na,K-ATPase (Na⁺ pump). The activity of this Na⁺ reabsorptive pathway is controlled by aldosterone mostly via the mineralocorticoid receptor and to some extent also via the glucocorticoid receptor (5-8). To prevent these nuclear corticosteroid receptors from being activated by glucocorticoids, the ASDN cells express the 11-hydroxysteroid dehydrogenase type 2, which metabolizes glucocorticoids (9).

The transcription- and translation-dependent action of aldosterone on Na⁺ transport starts progressively after a lag period of ~30 min in adrenalectomized rats and amphibian model epithelia (10-12). This action has been schematically divided into an early regulatory phase (lasting 2-4 h after the lag period) and a late anabolic phase (12). The early, short term action of aldosterone is considered to be largely mediated by induced regulatory proteins, such as the serum- and glucocorticoid-inducible protein kinase, that are thought to act on the pre-existing Na⁺ transport machinery (13-15).

The action of aldosterone on Na⁺ transport at the level of ASDN cells is pleiotropic. For instance, aldosterone is known to act on both major Na⁺ transport proteins, apical ENaC, and the basolateral Na,K-ATPase (1, 16). As regards the mechanism by which ENaC is regulated, a recent study has shown that a single aldosterone injection given to adrenalectomized (ADX) rats induces the synthesis of new ENaC -subunits and, in the early ASDN, the translocation of , ,ENaC to the apical cell surface as well (4).

Concerning the effect of aldosterone on the Na,K-ATPase, measurements performed two decades ago on cortical collecting ducts (CCD) of adrenalectomized rats have shown that aldosterone restores within 1-3 h the Na,K-ATPase hydrolytic activity V_max and/or the number of ouabain binding sites (17-21). The nature of this effect was not known because the data could not distinguish between the possibilities that additional Na⁺ pump units are synthesized, that Na⁺ pumps are activated in situ, or that Na⁺ pumps are recruited/translocated from an intracellular pool to the cell surface.

The aim of the present study was to characterize the mechanism of the short term aldosterone effect on Na,K-ATPase activity in the mammalian ASDN. We first demonstrated in isolated CCDs of ADX rats that the regulatory effect of aldosterone on Na,K-ATPase activity could be accounted for by the regulation of the cell surface expression of the Na,K-ATPase. Because a qualitatively similar effect of aldosterone was detected in the mouse collecting duct principal cell line mpkCCD_cl4 (22), this cell line proved to be a useful in vitro model for further investigations.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Experimental Model and Isolated Rat Kidney Tubules-- Male Wistar rats (Charles River Laboratories, Saint Germain de l'Arbresle, France) weighting 120-130 g were adrenalectomized under isoflurane anesthesia and had then free access to a 0.9% NaCl solution for 1 week. A single dose of aldosterone (10 µg/kg) was infused intravenously in 0.3 ml of 0.9% NaCl, whereas controls received diluent. 3 h later, the rats were anesthetized with pentobarbital (5 mg/100 g of body weight, given intraperitoneally), the left kidney was infused with incubation solution, and single CCDs were isolated by microdissection in ice-cold oxygenated incubation solution (120 mM NaCl, 5 mM KCl, 4 mM NaHCO₃, 1 mM CaCl₂, 1 mM MgSO₄, 0.2 mM NaH₂PO₄, 0.15 mM Na₂HPO₄, 5 mM glucose, 10 mM lactate, 1 mM pyruvate, 4 mM essential and nonessential amino acids, 0.03 mM vitamins, 20 mM HEPES, 0.1% (w/v) bovine serum albumin, pH 7.45) containing aprotinin (10 millitrypsin-inhibitor units/ml) and leupeptin (20 mg/ml) as described (23). The length of tubular segments that served as reference for Na,K-ATPase activities and Western blot analysis was determined from the photographs of microdissected CCDs.

Cell Culture-- mpkCCD_cl4 cells (passages 18-40) were cultured as described in Ref. 22. For filter cultures, the cells were seeded at high density on Transwell filters (Costar) and kept for 6-8 days in standard medium. 24 h prior to the experiments, epithelia were placed into serum- and hormone-deprived medium. To obtain maximal effects that are mediated via the mineralocorticoid and the glucocorticoid receptors, aldosterone was used at 10⁶ M (5-8).

Measurement of Na,K-ATPase Activity-- The hydrolytic activity of the Na,K-ATPase was determined under V_max conditions by the release of P_i from [-³²P]ATP on isolated rat CCDs permeabilized by freeze/thawing, as previously described (24). This permeabilization procedure has been shown to reveal a pool of Na,K-ATPase that correlates with the cell surface expressed Na⁺ pumps (23). The Na,K-ATPase activity was measured on quadruplicate samples of four to six CCDs and taken as the difference between the mean ATPase activities in the absence (150 mM choline chloride, 2 mM ouabain) or presence of Na⁺ and K⁺ (100 mM NaCl and 5 mM KCl). The results are expressed as pmol ATP × mm¹ × h¹.

Measurement of the Ouabain-sensitive ⁸⁶Rb⁺ Uptake-- The cation transport activity of Na,K-ATPase was measured as ouabain-sensitive ⁸⁶Rb⁺ uptake under conditions of initial rate on mpkCCD_cl4 epithelia grown on 12-mm diameter Transwell filters. Incubations with aldosterone and/or drugs were for the indicated times in serum- and hormone-deprived medium (see above). The transport activity of Na,K-ATPase was determined, as previously described (23), in quadruplicate samples following a preincubation of 30 min in the presence or absence of 2 mM ouabain. Protein content was determined in parallel by using the BCA assay (Pierce). The ouabain-sensitive ⁸⁶Rb⁺ uptake was calculated as the difference between the mean values with or without 2 mM ouabain, and the results are expressed as pmol Rb⁺ × µg protein¹ × min¹.

Electrophysiology-- The equivalent short circuit current was calculated according to Ohm's law from the values of transepithelial potential difference and resistance measured with a Millicell (Millipore) device. Na⁺ pump current (I_p) was measured as previously described (25). Confluent mpkCCD_cl4 cells grown on Transwell filters (24-mm diameter) were transferred for 3 h to HEPES-buffered Dulbecco's modified Eagle's medium and kept under air atmosphere at 37 °C. The cell layers were then permeabilized apically to monovalent ions with 35 µg/ml amphotericin B for 15 min at 37 °C in Na⁺-free buffer containing 116 mM K⁺ gluconate, 1.8 mM CaCl₂, 1.6 mM MgCl₂, 0.8 mM KH₂PO₄, 2 mM D-glucose, 12 mM essential amino acids, 2 mM nonessential amino acids, 0.4 mM glutamine, 25 mM HEPES, 3 mM Ba(OH)₂ and adjusted to pH 7.4 with Tris. The filters were then transferred to a Ussing chamber, and Na⁺ was added to the desired final concentration by mixing Na⁺ buffer (Na+ replacement of K⁺) with the Na⁺-free buffer. Measurements were done under short circuit conditions before and after the addition of 1 mM ouabain to the basolateral compartment, and the I_p was calculated as the difference between the total and the ouabain-resistant current. For estimating V_max and the Na⁺ concentration required for half-maximal activation (K_0.5), the I_p max of the control epithelia was estimated for each experiment by fitting a sigmoidal curve with a nonlinear regression analysis routine (Prism, GraphPad) to the data, using a Hill coefficient fixed at 2.3 (26). After normalization with the I_p max of the controls, the data were pooled, and the mean Ks_0.5 was determined.

Measurement of Total and Cell Surface Expression of the Na,K-ATPase -Subunit-- The solubilization of proteins from 100 microdissected isolated CCDs (0.2-0.5-mm length) and Western blotting were performed as previously described (23). Proteins were blotted onto a polyvinylidine difluoride membrane (Immobilon-P, Millipore), incubated overnight with a polyclonal antibody (dilution, 1:2500) raised against the -subunit of Na,K-ATPase (27), and then incubated with a second anti-rabbit IgG antibody (dilution, 1:10000) coupled to horseradish peroxidase (Transduction Laboratories, Lexington, KY). Detection was performed by chemiluminescence using the Super Signal substrate method (Pierce).

Measurement of cell surface Na,K-ATPase was performed on isolated rat CCDs as described (23) using EZ-Link sulfossuccinimidobiotin (Sulfo NHS-S-S-Biotin; Pierce) for labeling of cell surface proteins. Biotinylated proteins were precipitated with streptavidin-agarose beads (Immunopure immobilized streptavidin; Pierce). In the present case, the beads were then washed three times with anti-protease-containing buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 20 µg/ml leupeptin, 10 millitrypsin-inhibitor units/ml aprotinin) and once with 10 mM Tris-HCl, pH 7.4. Solubilization of the precipitated proteins, SDS-PAGE, and Western blotting were as described above. The amounts of proteins loaded on each lane corresponded to the same initial length of isolated CCDs (± 5%). The chemiluminescence signals were quantified by densitometry and expressed in each experiment as percentages of the control. The results are expressed as the means ± S.E. from several animals.

For mpkCCD_cl4 epithelia grown on Transwell filters (12-mm diameter), a similar procedure was used for cell surface biotinylation, streptavidin-agarose precipitation, and Western blotting (23). The protein concentration of the lysates was measured with the BCA protein assay (Pierce) to load equal amounts of proteins on each lane for Western blotting and to submit equal amounts of proteins to streptavidin-agarose precipitation.

Statistics-- Statistical analysis of Na,K-ATPase activities, ⁸⁶Rb⁺ uptakes and I_p were done by unpaired Student t test or by analysis of variance for comparison of two or more than two groups, respectively. Statistical analysis of Na,K-ATPase -subunit immunoreactivity was done using the Mann-Whitney U test or the Kruskal-Wallis test for comparison of two or more than two groups, respectively. The results are expressed as the means ± S.E. from n independent experiments. Each experiment was performed with tubules from one animal or with cells from one passage. A p value less < 0.05 was considered significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Short Term Aldosterone Increases in Vivo Na,K-ATPase Hydrolytic Activity and Cell Surface Expression in Intact Rat Cortical Collecting Duct-- The effect of a single intravenous infusion of aldosterone on Na,K-ATPase activity and expression was investigated in CCDs of ADX rats. As reported earlier (17-19), short term aldosterone (3 h) increased the maximal hydrolytic activity of the Na,K-ATPase by ~200% (ADX control, 323 ± 80; ADX aldosterone, 940 ± 150 pmol ATP × mm¹ × h¹; n = 9; p < 0.01). We performed Western blotting to test whether or not this increase in enzyme activity could be explained by an increase in the cellular pool of the Na,K-ATPase. As shown in Fig. 1B, this did not appear to be the case because the -subunit pool was increased only by 63 ± 7% (n = 8), and the -subunit was increased similarly by 71 ± 21% (n = 8; data not shown). To test the hypothesis that it was mainly the cell surface pool of the Na,K-ATPase that was increased by the short aldosterone treatment, we performed cell surface biotinylation followed by precipitation with streptavidin-agarose beads and Western blotting. Fig. 1C shows that aldosterone increased the amounts of cell surface Na,K-ATPase -subunit by 497 ± 35% (n = 8). Thus, the increase in maximal Na,K-ATPase hydrolytic activity observed in CCDs of adrenalectomized rats 3 h after a single aldosterone injection appears to be entirely mediated by an increase in its cell surface expression.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Effect of aldosterone (single injection, 3 h) on Na,K-ATPase hydrolytic activity (Vmax), and on the Na,K-ATPase -subunit total and cell surface pools in CCDs of adrenalectomized rats. A, effect of aldosterone on Na,K-ATPase activity. The activity was determined in Vmax conditions on freeze/thawing permeabilized CCDs of control (Ctl) and aldosterone-injected (Ald) rats. The results, expressed as percentages of controls, are the means ± S.E. from nine separate experiments. **, p < 0.01. B, effect of aldosterone on the total Na,K-ATPase -subunit pool detected by Western blotting. Bands from a representative immunoblot are shown in the upper panel (two different samples for control and aldosterone (3 h)), and the bars in the lower panel represent the densitometric quantification values, expressed as percentages of control, from six independent experiments. *, p < 0.05. C, effect of aldosterone on the cell surface expression of the Na,K-ATPase -subunit. Cell surface proteins from microdissected CCDs were biotinylated, solubilized, and precipitated by streptavidin-agarose beads, and the Na,K-ATPase -subunit was detected by Western blotting. A representative immunoblot (two different samples for control and aldosterone (3 h)) is shown in the upper panel, and the bars in the lower panel represent the densitometric quantification values, expressed as percentages of control, from seven independent experiments. **, p < 0.01.

Short Term Aldosterone Increases in Vitro the Function of Na,K-ATPase in Cultured mpkCCD_cl4 Cells-- We used the ex vivo CCD model of cultured mpkCCD_cl4 cells grown on filter supports to characterize more extensively the mechanism underlying the short term effect of aldosterone. This cell line was derived from mouse CCD and was shown to express an aldosterone-inducible amiloride-sensitive electrogenic Na⁺ transport (22). As expected, 1 µM aldosterone increased the transepithelial short circuit current (I_sc) already within 1 h and further during the second hour of treatment (Fig. 2). The transport activity of the Na,K-ATPase (Na⁺ pump), measured as ⁸⁶Rb⁺ uptake, increased in response to aldosterone with a time course close to the I_sc (Fig. 2).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Time course of aldosterone action on the transepithelial current, ouabain-sensitive 86Rb+ uptake and pump current in mpkCCDcl4 cells. Confluent mpkCCDcl4 cells grown of polycarbonate filters were incubated in the absence or presence of 1 µM aldosterone. The transepithelial equivalent short circuit current (Isc) (triangles, n = 17), the ouabain-sensitive 86Rb+ uptake (circles, n = 6) and the Na+ pump current (Ip) (diamonds, n = 4-6) are expressed as percentages of control (means ± S.E.). All test values were significantly different from their control (p < 0.05).

Because the ⁸⁶Rb⁺ uptake assay does not discriminate between kinetic activation of the Na⁺ pump by intracellular Na⁺ concentration ([Na⁺]_i) versus regulatory changes in pump number or activity, ouabain-sensitive pump currents were measured. The apical membrane of the epithelia was selectively permeabilized to monovalent cations with amphotericin B, thus allowing the control of [Na⁺]_i. Under these experimental conditions, aldosterone increased the current carried by the Na⁺ pump by ~30%, following during the first 2 h a similar time course as the increase in transepithelial I_sc (Fig. 2). Na⁺ activation experiments were then performed to determine whether this regulatory effect of aldosterone on Na,K-ATPase function corresponded to a change in Na⁺ affinity or to an increase in the maximal transport capacity of the basolateral Na⁺ pumps. Fig. 3 shows that the apparent affinity for Na⁺ was not changed by aldosterone (K_0.5 control, 8.5 mM; K_0.5 aldosterone, 10.1 mM; n = 4; difference not significant). Similar to the observations made in rat CCDs (Fig. 1), these results suggested that the effect of aldosterone might correspond to an increase in the number of functional Na⁺ pumps at the basolateral membrane.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Aldosterone increases the Na+ pump current (Ip) without modifying the apparent Na+ affinity of the Na+ pumps in mpkCCDcl4 epithelia. Confluent mpkCCDcl4 cells grown on filters were incubated in the absence (empty circles) or in the presence (filled circles) of 1 µM aldosterone for 2 h. The Ip was measured at various Na+ concentrations after permeabilization of the apical membrane to monovalent ions with amphotericin B. The results, expressed as percentages of control Ip max are the means ± S.E. from four independent experiments. *, p < 0.05. Sigmoidal dose-response curves were fitted to the experimental points using a Hill coefficient fixed at 2.3 (26). Derived K0.5 values (8.5 and 10.1 mM, for control and aldosterone, respectively) were not significantly different.

In summary, the results shown in Figs. 2 and 3 confirmed that short term aldosterone increases the electrogenic transepithelial sodium transport in cultured mpkCCD_cl4 cells. Furthermore, they showed that this effect not only reflects an increase in apical Na⁺ influx via ENaC and a consecutive kinetic activation of the Na,K-ATPase but also reflects a regulatory change in the maximal Na,K-ATPase function.

Aldosterone Increases the Cell Surface Expression of the Na,K-ATPase -Subunit in Cultured mpkCCD_cl4- Cells-- To test whether the effect of aldosterone on the pump function of mpkCCD_cl4 cells was due to an increase in Na,K-ATPase cell surface expression, we performed cell surface biotinylations followed by streptavidin-agarose precipitation and Western blotting. Fig. 4A shows that 1 µM aldosterone time-dependently increased the cell surface amount of the Na,K-ATPase -subunit. This effect was statistically significant after 2 h and, up to that time point, quantitatively similar to the increase in I_p. The cell surface expression continued to increase at later time points and reached a maximum after 6 h (increase by 60 ± 5.2%; n = 6; p < 0.05), whereas the I_P remained approximately at the same level (compare Figs. 2 and 4A). Western blotting on total cell extracts showed that the total pool of Na,K-ATPase -subunit increased as well but later than the surface expression and to a lesser extent, reaching a maximum at 24 h (increase by 38 ± 8.5; n = 8; p < 0.05) (Fig. 4B).

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Time course of aldosterone action on whole cell and cell surface Na,K-ATPase  subunit in cultured mpkCCDcl4 cells. Confluent mpkCCDcl4 cells grown on filters were incubated in the absence (Ctl) or presence of 1 µM aldosterone. A, effect of aldosterone on cell surface expression of the Na,K-ATPase -subunit. After cell surface protein biotinylation and streptavidin-agarose precipitation, the surface-expressed Na,K-ATPase -subunit was detected by Western blotting. The bands from a representative immunoblot are shown (upper panel). The bars (lower panel) represent the densitometric quantification (percentages of control, means ± S.E.) from six independent experiments. *, p < 0.05. B, effect of aldosterone on the total Na,K-ATPase -subunit pool. The -subunit was detected by Western blotting. The bands from a representative immunoblot are shown (upper panel). The bars (lower panel) represent the densitometric quantification (percentage of control, means ± S.E.) from six independent experiments. *, p < 0.05.

The correlation of the early aldosterone-induced increase in cell surface Na,K-ATPase (2 h) (Fig. 4A) with the increase in I_p (Fig. 2) suggests that the latter effect on I_p might result from the increase in Na⁺ pump cell surface expression. Although less pronounced, these results are qualitatively similar to those obtained in intact rat CCDs (Fig. 1). Thus, mpkCCD_cl4 cells appear to be a suitable model to study the mechanism of collecting duct Na,K-ATPase regulation.

Aldosterone Stimulates Na,K-ATPase Activity by a Transcription-dependent Mechanism That Is Independent of the Apical Na⁺Entry-- Aldosterone stimulates the expression and activity of the ENaC and thus the apical influx of Na⁺ in mkpCCD_cl4 cells (22). Therefore, the increase in Na⁺ pump function that appears to be mediated by an increase in Na,K-ATPase cell surface expression could have been secondary to an increase in [Na⁺]_i. To test this possibility, we prevented the apical Na⁺ influx during the aldosterone treatment with the ENaC inhibitor amiloride. As shown by the ⁸⁶Rb⁺ uptake experiments (performed after washout of amiloride for 5 min), the cellular transport response to aldosterone remained unchanged (Fig. 5). Importantly, the regulation of the Na,K-ATPase measured by the I_p and the increase in Na,K-ATPase cell surface expression were maintained as well, indicating that the early effect of aldosterone at the level of the Na⁺ pump is not secondary to apical Na⁺ entry.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Amiloride does not prevent the stimulation of ouabain-sensitive 86Rb+ uptake, Ip, and Na,K-ATPase cell surface expression by aldosterone in cultured mpkCCDcl4 cells. Cells grown on filters were incubated in the absence or presence of 1 µM aldosterone and/or 1 µM amiloride for 2 or 3 h at 37 °C. The bars represent percentages of controls. A, effect of amiloride on the aldosterone-induced (2 h) stimulation of ouabain-sensitive 86Rb+ uptake. B, effect of amiloride on the aldosterone-induced (2 h) stimulation of Ip. C, effect of amiloride on the aldosterone-induced (3 h) increase in Na,K-ATPase cell surface expression. The results are the means ± S.E. from six (A), three (B), and seven (C) separate experiments. *, p < 0.05 versus control.

We next tested whether the effect of aldosterone on Na⁺ transport depends in cultured mpkCCD_cl4 cells on ongoing transcription and translation, as previously reported for intact rat CCDs (21) and amphibian epithelia (28). Actinomycin D (5 µM) or cycloheximide (20 µM) was added to prevent transcription or translation, respectively, during the aldosterone treatment. Both inhibitors blocked the effect of aldosterone on the ⁸⁶Rb⁺ uptake and also slightly decreased the base-line transport in untreated mpkCCD_cl4 cells (Fig. 6, A and C). This showed that the transepithelial Na⁺ transport and, in particular, its stimulation by aldosterone depend on ongoing transcription and translation in mpkCCD_cl4 cells as well. Similarly, the aldosterone-induced increase in Na,K-ATPase cell surface expression and activation of the Na⁺ pump function measured as I_p were also fully prevented by cycloheximide and actinomycin D, respectively (Fig. 6, B and D). Thus, the increase in basolateral Na⁺ pump function induced by aldosterone requires, similar to the stimulation of transepithelial Na⁺ transport, a functional transcription and translation machinery.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   Transcriptional and translational dependence of the effect of aldosterone on Na,K-ATPase surface expression and Ip in cultured mpkCCD cells. Cells grown on filters were incubated in the absence or presence of 1 µM aldosterone and/or 2 µM cycloheximide (A and B) or 5 µM actinomycin D (C and D) for 2 or 3 h at 37 °C. A, effect of cycloheximide on the aldosterone-induced (2 h) stimulation of ouabain-sensitive 86Rb+ uptake. B, effect of cycloheximide on the aldosterone-induced (3 h) increase in Na,K-ATPase cell surface expression. C, effect of actinomycin D on the aldosterone-induced stimulation of ouabain-sensitive 86Rb+ uptake. D, effect of actinomycin D on the aldosterone-induced stimulation of Ip. The results, expressed as percentages of control values, are the means ± S.E. from five (A and C), eight (B), and three (D) separate experiments. *, p < 0.05 versus control. p < 0.05 versus control.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Role of Na⁺ Pump Regulation by Short Term Aldosterone in CCD Cells-- The present study shows that short term aldosterone increases the maximal activity of the Na,K-ATPase in mammalian cortical collecting duct cells by increasing its cell surface expression. This finding seemingly opposes the current understanding that the single rate-limiting site of Na⁺ transport regulation by aldosterone is ENaC, at least in the short term.

Because the Na,K-ATPase activity (the actual pumping rate) strongly depends on [Na⁺]_i (positive cooperativity with Hill coefficient between 2 and 3) (16, 25, 29-31) and because [Na⁺]_i is generally below its half-maximal activation concentration (K_0.5) in CCD (30, 31), it has been suggested that the kinetic control of the pump function would suffice to match the basolateral efflux of Na⁺ to its luminal influx. However, a lack of Na,K-ATPase regulation would lead to fluctuations in [Na⁺]_i that would tend to blunt the transport response because the Na⁺ flux through ENaC activity strongly depends on [Na⁺]_i (32). For instance, the electrochemical driving force for Na⁺ influx via ENaC depends on [Na⁺]_i (33). This is particularly relevant in the collecting duct where luminal/urinary Na⁺ concentration can be quite low. Second, high [Na⁺]_i exerts a strong negative feedback regulation on ENaC function (33, 34). Furthermore, a coordinated regulation of the Na⁺ pumps might also be important in the collecting duct, because this part of the nephron expresses Na⁺ pumps with a high apparent affinity for Na⁺ (31). Thus, the Na⁺ pumps of the CCD have, at a given [Na⁺]_i, less kinetic reserve than Na⁺ pumps with a lower apparent affinity. Taken together, it is not surprising that the regulation of transepithelial Na⁺ transport by aldosterone relies, besides on the regulation of ENaC, also on the regulation of the Na⁺ pumps, as demonstrated in this study.

The extent by which aldosterone stimulates the maximal Na⁺ pump current (independent of [Na⁺]_i) in mpkCCD_cl4 epithelia (20-50% after 2 h) represents approximately half of the effect aldosterone exerts on the ouabain-sensitive ⁸⁶Rb⁺ uptake (40-100% after 2 h). Because this latter assay measures the actual pumping rate of the Na,K-ATPase and because the apparent Na⁺ affinity of the Na,K-ATPase is not altered by aldosterone (Fig. 3), this indicates that approximately half of the Na⁺ pump activation measured by ⁸⁶Rb⁺ uptake must result from a kinetic activation of Na⁺ pumps, presumably in response to an increase in [Na⁺]_i that is secondary to the increase in apical Na⁺ influx. Hence, it appears that in our experimental conditions the increase in transcellular Na⁺ transport observed in mpkCCD_cl4 cells is supported, at the basolateral membrane, in part by an increase in the number of active pumps and that the other part relies on the kinetic activation of the Na⁺ pumps.

Pathway Leading to Aldosterone-induced Na,K-ATPase Cell Surface Expression-- Blocking ENaC function during the aldosterone treatment with a low amiloride concentration did not prevent the aldosterone-induced increase in Na⁺ pump surface expression and Na⁺ pump current. Thus, these effects are not secondary to an increase in apical Na⁺ influx. They may rather be mediated by (an) aldosterone-induced regulatory protein(s) that directly trigger(s) the redistribution of Na,K-ATPase toward the basolateral membrane. It remains to be investigated whether aldosterone-induced serum and glucocorticoid-induced kinase 1, which appears to play a major role in the control of ENaC cell surface expression (4, 36), also plays such a role for the control of Na,K-ATPase cell surface expression.

Previous reports have suggested that aldosterone determines in rat CCD the existence of a pool of inactive Na⁺ pumps that can be recruited/activated in response to cAMP or cell volume increase (37, 38). The present study shows that aldosterone-deprived CCD cells (ADX rats) contain nonetheless a large pool of inactive Na⁺ pumps that can be recruited by aldosterone. Thus, one can speculate that CCD cells lack, in the absence of aldosterone, (a) gene product(s) other than the Na⁺ pump that is(are) required to allow physiological stimuli such as cAMP and volume increase to recruit/activate Na⁺ pumps.

Interestingly, the cAMP-induced recruitment/activation of Na⁺ pumps in CCDs has been recently shown to be mediated by the redistribution of Na⁺ pumps to the cell surface, similar to the short term effect of aldosterone described in the present study (23). Thus, it will be interesting to clarify the relationship between the mechanisms involved in the aldosterone- and the cAMP-induced Na⁺ pump translocation events.

The late effect of aldosterone that leads to an increase in the cellular Na⁺ pump pool of CCD (39) was also observed in mpkCCD_cl4 cells in the present study. This late increase in the Na⁺ pump pool is parallel to the late part of the increase in Na⁺ pump cell surface expression (later than 2 h after aldosterone addition) and can therefore fully account for it. Interestingly, the recently described effect of puromycin-induced nephrotic syndrome on Na,K-ATPase expression in the CCD resembles the late effect of aldosterone described here (40).

Unlike the Na,K-ATPase cell surface expression, the I_p did not increase between 2 and 6 h after aldosterone addition. We hypothesize that this lack of correlation might be the result of other regulatory effects that modulated the activity of pumps expressed at the cell surface (16). It is noteworthy to mention that, similarly, the increase in Na,K-ATPase cell surface expression measured in CCDs (~6-fold) was larger than the corresponding increase in Na,K-ATPase activity (3-fold), also suggesting the possibility of an in situ counterregulation.

Lack of Short Term Change in Apparent Na⁺ Affinity-- The short term effect of aldosterone on Na,K-ATPase V_max, i.e. on its cell surface expression, that we have observed in mammalian CCD and mpkCCD_cl4 cells, is clearly different from the effect previously observed in amphibian A6 cells. In this latter model, short term aldosterone appeared to increase the Na⁺ affinity of a subpopulation of Na⁺ pumps and had no measurable effect on the Na,K-ATPase cell surface expression (25, 41). However, recent reports on the expression of CHIF and its interaction with the Na,K-ATPase suggest that aldosterone may act in the long term on the apparent Na⁺ affinity of Na⁺ pumps in mammalian collecting duct as well. Indeed, CHIF was shown to be expressed in the medullary collecting ducts of rats, to be up-regulated in the kidney by low salt diet (high aldosterone), and to associate with coexpressed Na,K-ATPase in Xenopus oocytes, thereby increasing its apparent affinity for [Na⁺]_i (35, 42). Such a long term effect of aldosterone on the apparent intracellular Na⁺ affinity of Na⁺ pumps would increase the driving force for apical Na⁺ entry via ENaC. Thus, we can hypothesize that aldosterone increases the driving force for Na⁺ entry via synergistic mechanisms: 1) as shown in this study, by increasing the amount of cell surface expressed Na⁺ pumps, in the short term by a translocation mechanism and in the long term by increasing the Na⁺ pump cellular pool and 2) by increasing the apparent affinity of Na⁺ pumps for [Na⁺]_i. Some axial heterogeneity of this putative effect can be anticipated, because increasing the driving force for apical Na⁺ entry via ENaC is physiologically most relevant for distal portions of the ASDN where maximal Na⁺ retention leads to very low luminal Na⁺ concentrations.

In conclusion, aldosterone exerts, already in the short term, a pleiotropic action on the transcellular Na⁺ reabsorption machinery of CCD cells. Besides regulating the luminal influx of Na⁺ at the level of ENaC, it also controls the surface expression of Na⁺ pumps. This mechanism should prevent an excessive increase in [Na⁺]_i that otherwise would exert a negative feedback on the apical Na⁺ influx via ENaC. Thus, Na,K-ATPase regulation contributes to the short term control of transepithelial Na⁺ transport by aldosterone.

	ACKNOWLEDGEMENTS

We thank Martine Rousselot for technical assistance and Christian Gasser for artwork.

	FOOTNOTES

^* This work was supported by Swiss National Science Foundation Grants 31-59141.99 (to F. V.) and 31-50830.99 (to E. F.), by the Hartmann-Müller Stiftung in Zürich, and by the Carlos and Elsie de Reuter Foundation in Geneva.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^** To whom correspondence should be addressed: Inst. of Physiology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. Tel.: 41-1-635-5044; Fax: 41-1-635-6814; E-mail: verrey@physiol.unizh.ch.

Published, JBC Papers in Press, October 11, 2001, DOI 10.1074/jbc.M107165200

	ABBREVIATIONS

The abbreviations used are: ASDN, aldosterone-sensitive distal nephron; ENaC, epithelial sodium channel; CCD, cortical collecting duct; ADX, adrenalectomized.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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