Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets

Johannes Loffing1, Laurence Pietri2, Fintan Aregger1, May Bloch-Faure2, Urs Ziegler1, Pierre Meneton2, Bernard C. Rossier3, and Brigitte Kaissling1

1 Institute of Anatomy, University of Zurich, CH-8057 Zurich, Switzerland; 2 Institut National de la Santé et de la Recherche Médicale U367, F-75005 Paris, France; and 3 Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous electrophysiological experiments on renal cortical collecting ducts indicated that dietary sodium intake and variations in aldosterone plasma levels regulate the abundance of functional epithelial Na channels (ENaC) in the apical plasma membrane. In mouse kidney we investigated by immunohistochemistry whether feeding for 3 wk a diet with high (3% Na) and low (0.05% Na) Na content influences the distribution pattern of ENaC. In mice of all experimental groups, ENaC was apparent in cells from the late portion of the distal convoluted tubule (DCT2) down to the medullary collecting duct (CD). In mice on a high-Na diet (plasma aldosterone: 40.8 ± 2.0 ng/dl), the alpha -subunit was undetectable, and the beta - and gamma -ENaC were detected in the cytoplasm, but not in the apical plasma membrane of the cells. In contrast, in mice on a low-Na diet (plasma aldosterone: 93.6 ± 9.3 ng/dl) all three ENaC subunits were displayed in the subapical cytoplasm and in the apical membrane of DCT2, connecting tubule (CNT), and, although less prominent, in cortical CD cells. Apical plasma membrane immunostaining progressively decreased along the cortical CD, simultaneously with increasing cytoplasmic staining for beta - and gamma -ENaC. Thus our data on mice adapted to moderately low and high Na intake suggest that regulation of ENaC function in vivo involves shifts of beta - and gamma -subunits from the cytoplasm to the apical plasma membrane and vice versa, respectively. The insertion of these subunits into the apical plasma membrane coincides with upregulation of the alpha -subunit and its insertion into the apical plasma membrane.

aldosterone; trafficking; immunohistochemistry; distal nephron; sodium transport


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

REGULATION OF EPITHELIAL SODIUM reabsorption is crucial for the maintenance of whole body Na and fluid homeostasis. The rate-limiting step in epithelial Na reabsorption is Na entry across specific transport proteins in the apical plasma membrane. In the mammalian renal distal nephron and collecting duct, the major Na entry pathway is the recently cloned amiloride-sensitive Na channel (ENaC) (reviewed in Refs. 17, 20, 36). The activity of this channel is positively correlated with plasma levels of aldosterone (29). The latter are usually inversely correlated with dietary Na intake (2, 15, 29).

Although the pathophysiology of ENaC begins to be elucidated, the physiological regulation of ENaC in vivo is poorly understood (40). Theoretically, epithelial Na reabsorption via ENaC may be controlled either by changes of the conductance of single channels, by changes of the gating kinetics of functional channels present in the plasma membrane, or by changes in the number of channels in the plasma membrane. Electrophysiological experiments on different aldosterone-responsive epithelia indicate an effect of aldosterone on channel gating (24) and on the number of functional ENaC channels in the apical plasma membrane (3, 5, 18, 30). However, these methods are unable to disclose whether an increase in epithelial apical Na conductance relies on activation of previously silent channels in the plasma membrane or on insertion of additional either preexisting or newly synthesized channels into the plasma membrane.

Therefore, we investigated by immunohistochemistry in kidneys of mice the effect of prolonged differential sodium intake on abundance and subcellular distribution of ENaC. The mouse was selected because in this species genetically engineered animal models can be generated, allowing one to study the pathophysiological consequences of ENaC mutations causing human diseases. For instance, loss-of-function mutation of ENaC can lead to pseudohypoaldosteronism type I (33) mimicking the human phenotype. Conversely, a gain-of-function mutation (Liddle mutation) can be introduced into the mouse genome to generate a mouse model with a salt-sensitive hypertensive phenotype (34).

For our study, we choose a protocol that induced rather modest changes of plasma aldosterone levels, which might fall in the range of physiological variations for that hormone. Thus the observed effects can be expected to be physiologically relevant.

ENaC is a heteromultimeric channel, composed of three homologous alpha -, beta -, and gamma -subunits (7). We found that in mice fed a high-Na diet (associated with low plasma aldosterone levels) that alpha -ENaC was undetectable and any apical plasma membrane localization of beta -ENaC and gamma -ENaC was lacking. Yet, under a low-Na diet (associated with moderately increased plasma aldosterone levels) all three ENaC subunits were prominent in the apical plasma membrane, whereas the cytoplasmic ENaC abundance appeared to be decreased.

Our data provide evidence that in the mammalian distal nephron, chronically altered Na intake, associated with only modest variations of aldosterone plasma levels, regulates ENaC abundance in the apical cell membrane, most probably by shifts from the cytoplasm to the apical plasma membrane and vice versa. These shifts go along with changes in the abundance of the alpha -subunit.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Experimental Protocol

We studied kidneys of 24 male C57BL/6 mice (body weights 24 to 30 g; Iffa Credo, Arbresle, France). The mice were distributed into three groups (groups 1-3) of eight animals each. The mice of group 1 received a standard diet, containing 0.24% Na. Mice of groups 2 and 3 were fed for 3 wk diets with low (0.05%; group 2) and high (3.0%; group 3) Na contents (UAR, Epinay-S/Orge, France). All animals had free access to tap water.

Mice were housed individually in metabolic cages to record 24 h urinary volume and Na excretion. Urinary Na concentration was measured with an indirect potentiometer (Beckman model E2A). Plasma levels of aldosterone were measured by radioimmunoassay (kit from Sanofi Diagnostics Pasteur, France) from blood (100 µl) drawn from the retro-orbital venous plexus under light anesthesia with a combination of ketamine (Narketan 10, 80 mg/kg body wt; Chassot, Belp, Switzerland) and xylazine (Rompun, 33 mg/kg body wt; Bayer, Leverkusen, Germany).

Tissue Fixation and Processing

The kidneys of anesthetized (see above) mice were fixed by intravascular perfusion through the abdominal aorta according to standard procedures in our laboratory (10). The fixative consisted of 3% paraformaldehyde and 0.05% picric acid. It was dissolved in a 3:2 mixture of 0.1 M cacodylate buffer (pH 7.4, adjusted to 300 mosmol/kgH2O with sucrose) and 10% hydroxyethyl starch in saline (HAES-steril; Fresenius, Stans, Switzerland). After 5 min of fixation, the kidneys were rinsed by perfusion for 5 min with the 0.1 M cacodylate buffer. Thin tissue slices (1-2 mm thick) were mounted on small cork disks, frozen in liquid propane, and stored at -80°C until use. Sections (3-5 µm thick) were cut in a cryostat and placed on chrome-alum gelatin-coated glass slides.

Immunohistochemistry

Primary antibodies. ENaC was detected with rabbit antisera directed against rat alpha -ENaC (dilution 1:500), beta -ENaC (dilution 1:1,000), or gamma -ENaC (dilution 1:20,000). The specificity of these antisera had been described previously (12). The thiazide-sensitive NaCl cotransporter (NCC) was detected with a previously characterized rabbit antiserum (32), in a dilution of 1:8,000. Intercalated cells were labeled by monoclonal antibodies (dilution 1:4) against bovine H-ATPase (19). Labeling of the apical plasma membrane of tubular cells was made with a 1:500 dilution of biotin-conjugated Lycopersicon esculentum agglutinin (LEA; Sigma).

Incubations. The cryostat sections were stored in PBS until use and then preincubated for 10 min with 10% normal goat serum in PBS. Incubations with the primary antibodies, diluted in PBS/1% BSA, took place overnight in a humidified chamber at 4°C. After repeated rinsing in PBS, binding sites of the primary antibodies were detected with Cy3-conjugated donkey-anti-rabbit IgG (Jackson Immuno Research Laboratories, West Grove, PA) or with FITC-conjugated goat-anti-mouse IgG (Jackson Immuno Research Laboratories). Binding sites of LEA were visualized with a 1:100 dilution of FITC-conjugated streptavidin (Bioscience Products, Emmenbruecke, Switzerland).

For simultaneous labeling of different antigens (ENaC subunits and thiazide-sensitive NaCl cotransporter, NCC) with primary antisera, raised in one single species (rabbits), we applied the double-labeling technique, described by Hunyady et al. (21), using the tyramide-signal amplification kit (TSA-Direct; NEN, Boston, MA). Briefly, nonspecific binding sites were blocked with the TSA blocking buffer for 30 min. Afterward, sections were incubated with highly diluted antibodies against beta -ENaC (dilution 10-5) or gamma -ENaC (dilution 10-7) overnight at 4°C. After repeated washings with PBS/0.05% Tween 20, sections were incubated with a 1:100 dilution of a biotin-conjugated donkey-anti-rabbit IgG for 45 min followed by incubation with a 1:100 dilution of streptavidin horseradish peroxidase (HRP) for 30 min. Thereafter, FITC-conjugated tyramides were applied for 10 min in a 1:75 dilution in TSA amplification diluent. The HRP-mediated precipitation of the FITC conjugates disclosed the binding sites of the ENaC antibodies. Subsequently, the sections were incubated with rabbit anti-NCC, diluted 1:8,000. Binding sites of the latter antiserum was revealed by Cy3-conjugated donkey-anti-rabbit IgG, diluted 1:1,000. Incubations were made at room temperature.

Finally, the sections were rinsed with PBS, and coverslips were mounted, using DAKO-Glycergel (Dakopatts), to which 2.5% 1,4-diazabicyclo[2,2,2]octane (DABCO; Sigma, St. Louis, MO) was added as a fading retardant.

For control of unspecific binding of the antibodies, we performed control incubations with the respective preimmune sera or by omitting the primary antibody. All control experiments were negative. Applications of the rabbit antisera, directed against different defined antigens, were additional internal controls.

Evaluation of immunofluorescence. The sections were studied by epifluorescence (Polyvar microscope; Reichert Jung, Vienna, Austria). Images were acquired with a VISICAM CCD camera (Visitron, Puchheim, Germany) and processed by Image-Pro Plus v3.0 software (Media Cybernetics, Silver Spring, MD). Confocal laser-scanning microscopy was performed with a confocal unit (model TLS SP; Leica, Heidelberg, Germany) using the 488 nm and 568 nm laser lines of a argon/krypton laser for excitation of FITC and Cy3, respectively. For detection the emission settings of the spectrophotometer were set to 515-540 nm for FITC and 575-650 nm for Cy3. Images of FITC- and Cy3-related fluorescence were acquired sequentially to exclude crosstalk between the two channels. Optical deconvolution of serial confocal images was done with Hygens v2.1 imaging software (Bitplane, Zurich, Switzerland). For printing, images were imported into Adobe Photoshop v4.0.

Statistics

Data are given as means ± SE. Differences between means were compared by unpaired Student's t-test. Differences were considered to be statistically significant if P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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Effect of Diets on Urinary Sodium Excretion and Plasma Levels of Aldosterone

During the first 8 days after the switch from the control to the high-Na diet, 24 h urinary volume and Na excretion steadily increased before reaching a new steady state. Mice fed a low-salt diet had significantly lower 24 h urinary fluid and Na excretion then the high-salt-treated mice (Fig. 1). The lower Na excretion was observed from the first day on after the switch in the diet.


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Fig. 1.   Urinary volume and Na excretion in mice fed high- (HS) or low-Na diets (LS); 24 h diuresis (top) and natriuresis (bottom) were recorded during the first 2 wk (n = 8 animals/group).

Plasma levels of aldosterone were measured on day 21 after the diet switch, i.e., the day of death. In mice fed for 3 wk the high-salt diet, the plasma levels of aldosterone were 40.8 ± 2.0 ng/dl (1.13 ± 0.06 nM); in mice fed for 3 wk the low-salt diet, they were 93.6 ± 9.3 ng/dl (2.59 ± 0.26 nM).

Distribution of ENaC in Kidneys of Mice on Standard Diets

Our immunohistochemical data on the segmental distribution of ENaC agree with previous in situ hybridization and immunohistochemistry studies in rats (12, 37). In mice on standard diets (0.24% Na content) ENaC was displayed by the segment-specific cells in the second half of the distal convoluted tubule (DCT2), in the connecting tubule (CNT), in the cortical collecting duct (CCD), and in the medullary collecting duct (MCD). The ENaC-negative cells, interspersed among the ENaC-positive cells, were identified as intercalated cells (IC cells) on account of their strong binding to antibodies against the proton-ATPase (19) (Fig. 2, D and E).


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Fig. 2.   Distributions of NCC (A), gamma -ENaC (B), and beta -ENaC (C and D), and H-ATPase (E) in kidneys of mice on a standard Na-intake: cryostat sections. P, proximal tubule; a, cortical radial artery; v, cortical radial vein; CN, connecting tubule; D, DCT1; asterisk, DCT2. Double labeling for NCC (A) and gamma -ENaC (B). A: NCC related immunofluorescence is highly abundant in the luminal plasma membrane of DCT1 and progressively decreases along the DCT2 towards the connecting tubule. B: gamma -ENaC is undetectable in DCT1 and is colocalized with NCC in DCT2, which display weak cytoplasmic and distinct apical ENaC labeling (arrows). C: faint apical staining for beta -ENaC is visible in cells along the DCT2 (arrows) and at the transition to the connecting tubule; cytoplasmic staining becomes prominent in further downstream portions in the CNT and in the collecting duct (CD). D and E: double labeling of a collecting duct with beta -ENaC (D) and H-ATPase (E); beta -ENaC is found in the cytoplasm of the segment-specific cells (arrowheads) and is absent in intercalated cells (arrows), displaying high levels of H-ATPase. Bars in C and E = 40 µm.

The DCT2 cells coexpressed ENaC together with the DCT-specific thiazide-sensitive NaCl cotransporter (NCC). Immunostaining for the NCC gradually decreased in urinary flow direction, whereas the ENaC-related immunofluorescence progressively increased toward the CNT (Fig. 2, A-C)

Only in the most upstream ENaC-positive cells in the DCT2 and at the beginning of the CNT, weak ENaC-related immunostaining was observed in the apical plasma membrane (Fig. 2, B and C). Further downstream, ENaC immunostaining was distributed in a fine-granular manner throughout the cytoplasm, and the apical plasma membrane was consistently unstained (Fig. 2D).

Effect of Dietary Sodium Intake on ENaC Localization

The segmental distribution of ENaC in mice on a Na-rich or Na-poor diet was the same as in mice on a control diet. However, the subcellular distribution of the ENaC subunits differed markedly among the experimental groups. No differences were observed in the staining pattern for the NCC.

Na-rich diet (group 2). In these mice, any apical plasma membrane localization of ENaC subunits was absent. The alpha -subunit was undetectable (Fig. 3A). All ENaC-positive cells revealed a bright immunostaining for the beta - and gamma -subunits all over the cytoplasm (Fig. 3, C and E).


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Fig. 3.   ENaC subunits in connecting tubules of mice kept for 3 wk on a high-Na diet (A, C, and E) or on a low-Na diet (B, D, and F): cryostat sections. Under a high-Na diet, alpha -ENaC (A) is undetectable; beta - (C) and gamma -ENaC (E) are evenly distributed throughout the cytoplasm. Under a low-Na diet alpha -ENaC (B), beta -ENaC (D), and gamma -ENaC (F) are accumulated in the apical cell pole. Bars in B and F = 20 µm.

Na-poor diet (group 3). In contrast to the previous group the ENaC-positive cells revealed distinct and prominent immunostaining in the apical cell pole. Remarkably, the strong apical expression of beta - and gamma -subunits (Fig. 3, D and F) was associated with distinct immunostaining for the alpha -ENaC subunit (Fig. 3B) in the apical cell pole. For all three subunits, the apical staining was the most conspicuous in the CNT and progressively decreased in urinary flow direction. Inversely to the gradual decrease in the apical plasma membrane, the cytoplasmic abundance of beta - and gamma -ENaC increased; the axial heterogeneity of apical ENaC abundance found in mice on a low-Na diet is well evident when comparing beta -ENaC-related immunostaining in CNTs and CCDs from mice on a high-Na (Fig. 4A) and a low-Na diet (Fig. 4B).


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Fig. 4.   Detection of beta -ENaC in the renal cortex of mice kept for 3 wk on a high-Na diet (A) or on a low-Na diet (B): cryostat sections. P, proximal tubule; CD, cortical collecting duct (CCD); CN, CNT. A: after high-Na diet, beta -ENaC is found exclusively at intracellular sites in the CNT as well as in the CCD; in both portions of the collecting system, immunostaining shows a similar intensity. B: after a low-Na diet, beta -ENaC is present in the apical cell pole in both segments, apical staining is more prominent in the CNT than in the CCD; cytoplasmic immunostaining is almost completely absent in the CNT. Bar = 50 µm.

Apical Plasma Membrane Localization Of ENaC

To determine whether ENaC-subunits were inserted in the apical cell membrane or situated in an apical compartment distinct from the plasma membrane, we performed double labeling experiments with a biotin-conjugated membrane-binding lectin (LEA) and the ENaC antibodies. These preparations were studied by high-resolution confocal microscopy, and the serial confocal images were submitted to optical deconvolution.

In all experimental groups, the lectin bound to the apical plasma membrane of all cells in the DCT, CNT and CCD, and thick ascending limb. In most of these cells also the basolateral plasma membranes were more or less strongly stained. Endothelial plasma membranes were also labeled (not shown).

In mice kept on a high-Na diet, the distinct lectin labeling of the apical plasma membrane did not colocalize with beta - and gamma -ENaC subunits, as shown in Fig. 5, A-C, for the gamma -ENaC subunit. The latter was seen in a granular pattern diffusely distributed throughout the cytoplasm. In contrast, in mice kept on a low-Na diet, the lectin- and ENaC-related fluorescences were clearly colocalized in the apical plasma membrane of DCT2 and CNT cells (Fig. 5, D-F). In a few cells of the latter segment and many CD cells, ENaC staining was well present in the subapical cytoplasm but did not colocalize with the lectin in the apical plasma membrane.


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Fig. 5.   Confocal images of single CNT cells from mice, kept for 3 wk on a high-Na diet (A-C) and on a low-Na diet (D-F): double labeling with Lycopersicon esculentum agglutinin (LEA; green fluorescence) and for gamma -ENaC subunit (red fluorescence). The nuclei of the cells are not hit; under high-Na and low-Na diets, LEA-related immunofluorescence (green) is visible in the apical (arrowheads) and sometimes basolateral plasma membranes; after 3 wk of a high-Na diet, gamma -ENaC (B) is exclusively found in intracellular compartments and does not colocalize (C) with LEA binding; after 3 wk of a low-Na diet, gamma -ENaC (E) is shifted to the apical cytoplasm and colocalizes (yellow) with LEA in the apical plasma membrane (F). Bar = 2 µm.

In summary, under a high-Na diet the alpha -subunit was undetectable, and the beta - and gamma -ENaC subunits were seen exclusively in cytoplasmic domains. Axial differences in subcellular ENaC distribution were not discernible. Under a low-Na diet, pronounced axial differences in the cellular localization of ENaC subunits were apparent. In the most upstream ENaC-positive tubular portions, all three subunits were displayed in the apical plasma membrane. The immunohistochemical presence of the alpha -subunit coincided with a shift of the beta - and gamma -ENaC subunits from the cytoplasm to the apical plasma membrane. In deeper regions of the collecting system, beta - and gamma -ENaC subunits were found exclusively at cytoplasmic sites and the alpha -subunit was undetectable.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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In the present study we demonstrate by immunohistochemistry that the intracellular distribution of ENaC is affected by the dietary Na intake. Our data suggest that in vivo the control of ENaC-mediated Na reabsorption comprises, first, translocation of subunits between the cytoplasmic compartments and the apical plasma membrane and, second, regulation of the abundance of the alpha -subunit. Similar observations on rats have recently been reported by Masilamani et al. (26). In our study on mice, the changes in subcellular distribution of ENaC subunits occurred under comparably small alterations in Na intake, which elicited only moderate differences in plasma levels of aldosterone. In addition to changes in subcellular distribution, we demonstrate along the distal nephron pronounced axial heterogeneity in the apical abundance of ENaC subunits under normal diet as well as under low-Na diet.

Na intake, plasma aldosterone levels, and the number of functional Na channels in the apical plasma membrane are interrelated. For instance, in CCDs of rats (30), as well as in the A6 cell line, derived from Xenopus kidney cells (3, 5, 18), exogenous aldosterone administration augmented the number of apically expressed functional Na channels. In electrophysiological studies on isolated CCDs from rats on a standard laboratory salt diet (low plasma aldosterone levels) amiloride-sensitive Na transport and conductance were not measurable (14, 15, 29, 35, 39), whereas in CCDs from rats on a low-Na diet (high plasma aldosterone levels), amiloride-sensitive Na transport and currents were high (14, 15, 29). These functional data agree with our immunohistochemical observations on the subcellular distribution of ENaC subunits in the distal nephron of mice.

It has been proposed that in the kidney the synthesis of alpha -ENaC subunits might be a limiting factor for ENaC function (27). Several experiments on Xenopus kidney A6 cells (27), rabbit (11) and mouse (4) CCD cells in vitro, or on rat kidneys (2, 26, 28, 38) in vivo indicated that aldosterone or a low-Na diet primarily affects the synthesis of alpha -ENaC, whereas beta - and gamma -ENaC appear to be more or less constitutively expressed. The increased abundance of alpha -ENaC is caused by an enhanced transcriptional and translational synthesis but does not seem to be due to a prolonged half-life of the alpha -ENaC protein (27). Electrophysiological (7) and immunochemical experiments (6, 13) on oocytes injected with cRNAs coding for the individual subunits of ENaC established that the assembly of ENaC subunits to functioning channels and their translocation from intracellular pools to the plasma membrane critically depend on a sufficient abundance of alpha -ENaC. Our immunohistochemical observations in mice and a recent study in rats (26) suggest that the same mechanisms might be also pertinent for ENaC regulation in vivo.

Whether the changes in subcellular ENaC distribution, described in our study, are induced by the elevated aldosterone levels, accompanying the low Na intake, cannot be derived from our data. A dissociation of plasma aldosterone levels and ENaC activity can be deduced from experimental data by Pacha et al. (29) on Na currents in isolated collecting ducts obtained from rats on a low-Na diet; after feeding rats with a low-salt diet (about 10 times lower than the Na content of the low-Na diet used in our study), the Na channel activity evolved much faster than the increases in plasma aldosterone levels. Moreover, after salt repletion, the channel activity and aldosterone levels decreased, but the latter rebounded, whereas the channel activity stayed low (29). Also our observations on pronounced axial heterogeneity of the subcellular distribution of ENaC subunits along the distal nephron in mice point to the possibility that in addition to aldosterone plasma levels, other factors might interfere in the regulation of ENaC function. Among others, the progressive changes in tubular fluid composition might contribute to the regulation of ENaC. A regulatory role of altered intra- and extracellular ion concentrations (Na+, Ca2+, H+) on ENaC function (reviewed in Ref. 17) has been found in many in vitro (e.g., Refs. 8, 22, and 23) and in vivo (e.g., Refs. 16 and 31) experiments. For instance, it has been suggested that increasing intracellular Na concentrations downregulates ENaC function, a mechanism well known as "feed-back inhibition" of epithelial sodium channels (16). Recent data indicate that this mechanism might be related to increased endocytotic retrieval of functional channels from the plasma membrane (23).

The axial heterogeneity along the nephron in subcellular ENaC distribution reported in this study on mice and in a recent study on rabbit kidneys (25) is consistent with previous data on heterogeneity of Na transport rates along the distal nephron. In microperfused rat tubules, net Na reabsorption is well detectable in the "late" distal tubule (corresponding approximately to the DCT2 and CNT) (9) but not in the downstream tubular portion of the CCDs (39). Similarly, in isolated rabbit tubules, Na transport in CNTs had been found to be several times higher than in CCDs (1). The functional and immunohistochemical data on axial heterogeneity in the distal nephron suggest that depending on the functional demands for Na transport more or fewer collecting duct portions may be "recruited" for this function.

In conclusion, our data provide evidence that the activity of ENaC in the apical plasma membrane of mammalian distal nephron epithelia is controlled by regulation of ENaC cell surface expression, involving modulation of the abundance of the alpha -subunit and shifts of channel subunits between the cytoplasm and the apical plasma membrane. These mechanisms apparently allow efficient adaptation to chronic changes of functional demands. Our data on the segmental and subcellular distribution of ENaC in mice might constitute a basis for further studies on models of genetically modified ENaC and its functional consequences.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of L. Kläusli. We thank Dr. S. Gluck for supplying the monoclonal antibody against H-ATPase and Dr. S. Hebert for supplying the polyclonal antibody against NCC.


    FOOTNOTES

The studies were supported by the Swiss National Science Foundation Grant 31-47742.96 (to B. Kaissling) and by the French Institut National de la Santé et de la Recherche Médicale (to P. Meneton).

Part of the work has been presented at the "News in Aldosterone Action" Forefront Meeting of the International Society of Nephrology in Paris, August 15-18, 1999.

Address for reprint requests and other correspondence: B. Kaissling, Anatomisches Institut der Universität Zürich, Winterhurerstrasse 190, CH-8057 Zürich, Switzerland (E-mail: bkaissl{at}anatom.unizh.ch).

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. §1734 solely to indicate this fact.

Received 27 September 1999; accepted in final form 27 March 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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