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
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ABSTRACT |
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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
-subunit was undetectable, and the
- and
-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
- and
-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
- and
-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
-subunit and its insertion into the apical
plasma membrane.
aldosterone; trafficking; immunohistochemistry; distal nephron; sodium transport
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INTRODUCTION |
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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 -,
-, and
-subunits (7). We found that in mice fed a
high-Na diet (associated with low plasma aldosterone levels) that
-ENaC was undetectable and any apical plasma membrane localization of
-ENaC and
-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 -subunit.
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MATERIAL AND METHODS |
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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 atImmunohistochemistry
Primary antibodies.
ENaC was detected with rabbit antisera directed against rat -ENaC
(dilution 1:500),
ENaC (dilution 1:1,000), or
-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 againstEvaluation 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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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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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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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 -subunit was undetectable (Fig.
3A). All ENaC-positive cells
revealed a bright immunostaining for the
- and
-subunits all over
the cytoplasm (Fig. 3, C and E).
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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 - and
-subunits (Fig.
3, D and F) was associated with distinct
immunostaining for the
-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
- and
-ENaC increased; the
axial heterogeneity of apical ENaC abundance found in mice on a low-Na diet is well evident when comparing
-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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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 - and
-ENaC
subunits, as shown in Fig. 5,
A-C, for the
-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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In summary, under a high-Na diet the -subunit was undetectable, and
the
- and
-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
-subunit coincided with a shift of the
- and
-ENaC subunits from the cytoplasm to the apical plasma membrane. In
deeper regions of the collecting system,
- and
-ENaC subunits were found exclusively at cytoplasmic sites and the
-subunit was undetectable.
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DISCUSSION |
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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 -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 -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
-ENaC, whereas
- and
-ENaC appear to be more or less
constitutively expressed. The increased abundance of
-ENaC is caused
by an enhanced transcriptional and translational synthesis but does not
seem to be due to a prolonged half-life of the
-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
-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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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