Immunocytochemical and immunoelectron microscopic localization
of
-,
-, and
-ENaC in rat kidney
Henrik
Hager1,
Tae-Hwan
Kwon1,2,
Anna K.
Vinnikova3,
Shyama
Masilamani4,
Heddwen L.
Brooks4,
Jørgen
Frøkiaer5,
Mark A.
Knepper4, and
Søren
Nielsen1
1 Department of Cell Biology, Institute of Anatomy,
University of Aarhus, DK-8000 Aarhus C; 2 Department of
Physiology, School of Medicine, Dongguk University, Kyungju 780-714, Korea; 3 Research Service, McGuire Veterans Affairs Medical
Center, Richmond, Virginia 23249; 4 Laboratory of Kidney and
Electrolyte Metabolism, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland 20892; and
5 Department of Clinical Physiology, Aarhus University Hospital
and Institute of Experimental Clinical Research, DK-8200 Aarhus N,
Denmark
 |
ABSTRACT |
Epithelial sodium channel
(ENaC) subunit (
,
, and
) mRNA and protein have been localized
to the principal cells of the connecting tubule (CNT), cortical
collecting duct (CCD), and outer medullary collecting duct (OMCD) in
rat kidney. However, the subcellular localization of ENaC subunits in
the principal cells of these cells is undefined. The cellular and
subcellular localization of ENaC subunits in rat kidney was therefore
examined. Immunocytochemistry demonstrated the presence of all three
subunits in principal cells of the CNT, CCD, OMCD, and IMCD. In cortex
and outer medulla, confocal microscopy demonstrated a difference in the
subcellular localization of subunits.
-ENaC was localized mainly in
a zone in the apical domains, whereas
- and
-ENaC were found
throughout the cytoplasm. Immunoelectron microscopy confirmed the
presence of ENaC subunits in both the apical plasma membrane and
intracellular vesicles. In contrast to the labeling pattern seen in
cortex,
-ENaC labeling in IMCD cells was distributed throughout the
cytoplasm. In the urothelium covering pelvis, ureters, and bladder,
immunoperoxidase and confocal microscopy revealed differences the
presence of all ENaC subunits. As seen in CCD,
-ENaC was present in
a narrow zone near the apical plasma membrane, whereas
- and
-ENaC were dispersed throughout the cytoplasm. In conclusion, all
three subunits of ENaC are expressed throughout the collecting duct
(CD), including the IMCD as well as in the urothelium. The
intracellular vesicular pool in CD principal cells suggests ENaC
trafficking as a potential mechanism for the regulation of
Na+ reabsorption.
aldosterone; collecting duct; urothelium; epithelial sodium
channel; intracellular trafficking; sodium transport
 |
INTRODUCTION |
IN SEVERAL TIGHT
EPITHELIA, electrogenic entry of Na+ from the lumen
into the cells is mediated by the epithelial sodium channel (ENaC)
located in the apical plasma membrane. This represents the
rate-limiting step for Na+ absorption and is characterized
by inhibition with submicromolar concentrations of the diuretic
amiloride (11). On the basolateral side, the Na-K-ATPase
actively transports sodium out of the cell into the extracellular
interstitium and provides the driving force for Na+
absorption (11). ENaC, belonging to the degenerin/ENaC
gene superfamily, is composed of three homologous subunits,
-,
- and
-ENaC.
ENaC is known to be present in several organs, including kidney, where
ENaC subunits are known to be expressed in the connecting tubule (CNT),
cortical collecting duct, (CCD), and outer medullary (OMCD) collecting
duct (7, 21, 30). In these segments ENaC participates in
the fine regulation of Na+ reabsorption mediated by the
hormones controlling sodium and water balance, e.g., the
mineralocorticoid aldosterone and vasopressin (8, 12, 18,
21). This Na+ reabsorption via ENaC is electrogenic,
hence creating a lumen negative gradient that promotes the passive
chloride reabsorption via the paracellular pathway (6, 34)
as well as the secretion of potassium into the lumen (29).
The latter may also be enhanced by the increased activity of
Na-K-ATPase mediated by the mineralocorticoid as well as by the
aldosterone-sensitive K+ channels in the apical membranes
(37).
Perhaps owing to the high turnover number of ENaC, physiological rates
of sodium transport may occur in the collecting duct with relatively
few copies of the ENaC complex compared with, for example, the
collecting duct water channel aquaporin-2 (AQP2). The relatively low
abundance of ENaC in the collecting duct cells has added to the
challenge of discovering how its activity is regulated at the level of
ENaC protein because standard-quality antibodies may have difficulty in
demonstrating labeling above background levels. Recently, we have
developed a set of antibodies to the three ENaC subunits that appear to
offer an improvement in labeling efficiency in immunoblots and
immunocytochemistry (21). In this paper, we exploit these
antibodies in high-resolution light microscopic as well as electron
microscopic localizations of ENaC in the kidney to provide new
information regarding the cellular and subcellular localization of all
three subunits along the collecting duct system. Specifically in this
study, we have focused on the following four major objectives:
1) to define the localization of each of the three ENaC
subunits along the axis at the collecting duct, including evaluating
whether ENaC subunits are present in the inner medullary collecting
duct (IMCD); 2) to define the subcellular localization by
confocal microscopy to evaluate whether there is axial heterogeneity in
the subcellular distribution of ENaC subunits; 3) to
evaluate by confocal microscopy whether there are significant
differences in the subcellular distribution of each subunit within the
same cell; and 4) to define the subcellular localization of
ENaC subunits by immunoelectron microscopy to establish the hypothesis
that ENaC is present in both the apical plasma membrane and in the
intracellular vesicles. Its presence in vesicles would be compatible
with the view that ENaC regulation may involve regulated trafficking,
as seen with vasopressin regulation of AQP2. These objectives were
explored through the use of immunoblotting, immunocytochemistry, laser
confocal microscopy, and immunoelectron microscopy.
 |
METHODS |
Antibody against ENaC subunits, AQP2, and
H+-ATPase.
Antibodies raised in rabbits against synthetic peptides were used.
Affinity-purified polyclonal antibodies against
-,
-, and
-ENaC (LL766AP, LL558AP, and LL550AP, respectively) have been
described previously (21). An additional antiserum against
-ENaC was also raised. For this, a peptide from the cytoplasmic NH2 terminus of rat
-ENaC (4),
NH2-KGDKREEQGLGPEPSAPRQPC-COOH, corresponding to
amino acids 48-67 with an additional cysteine at the COOH
terminus, was synthesized, coupled to keyhole limpet hemocyanine, and
used for immunization of rabbits by using a standard immunization
protocol. Both immune serum and affinity-purified antibodies were used
for immunoblotting and immunocytochemistry. The immune serum was
diluted 1:3,000, whereas the affinity-purified antibodies were used at
a concentration of between 3 and 8 µg/ml. Preimmune serum from rabbit
L766, L558, and L550 were purified by affinity chromatography by using
protein A columns (Pierce, Rockford, IL) and used for immunoblotting controls.
Polyclonal rabbit antibodies against rat AQP2 (10) have
been well characterized previously (24, 25), and AQP2 is
known to be present at the principal cells of the CNT, CCD, OMCD, and IMCD in rat kidney (26). Monoclonal mouse antibodies
against H+-ATPase (31 kDa of H+-ATPase/E11,
kindly provided by Dr. S. Gluck) were also used.
Renal tissue preparation and immunoblotting.
The kidneys from normal Sprague-Dawley rats were divided into cortex,
outer medulla, and inner medulla. The inner medulla was further divided
into three zones: base, middle, and tip. These tissues were homogenized
in chilled, buffered isolation solution containing 250 mM sucrose, 10 mM triethanolamine (Calbiochem, La Jolla, CA), 1 µg/ml leupeptin
(Bachem, Torrance, CA), and 0.1 mg/ml phenylmethylsulfonyl fluoride (US
Biochemical, Toledo, OH), adjusted to pH 7.6, using a tissue
homogenizer (Omni 2000, Omni International, Warrenton, VA) fitted with
a 10-mm microsawtooth generator. Protein concentrations of the
homogenates were measured by a Pierce BCA Protein Assay Reagent kit
(Pierce). All samples were then diluted with isolation solution to a
protein concentration of between 1 and 3 mg/ml and solubilized at
60°C for 15 min in Laemmli sample buffer. Twenty micrograms of
protein from each sample were loaded onto individual lanes
electrophoresed on 12% polyacrylamide gels (precast, Bio-Rad,
Hercules, CA). The proteins were transferred from the gels
electrophoretically to pure nitrocellulose membranes (Bio-Rad). After a
30-min, 5% milk block, membranes were probed overnight at 4°C with
the appropriate affinity-purified polyclonal antibody (see above). For
probing blots, all antibodies were diluted into a solution containing
150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl
Tween 20, and 0.1 g/dl BSA (pH 7.5). The secondary antibody was goat
anti-rabbit IgG conjugated to horseradish peroxidase (Kirkegaard and
Perry Laboratories, Gaithersburg, MD) used at a concentration of 0.1 mg/ml. Sites of antibody-antigen reaction were visualized by using
luminol-based enhanced chemiluminescence (LumiGLO, Kirkegaard and Perry
Laboratories) before exposure to X-ray film. For differential
centrifugation, the homogenates were subjected to sequential
centrifugation at 4,000, 12,000, and 200,000 g as previously
described (20). The pellets were resuspended in isolation
buffer, solubilized in Laemmli sample buffer, and subjected to
electrophoresis for immunoblotting as described above.
Immunocytochemistry and immunoelectron microscopy.
Kidneys from normal Munich-Wistar rats (n = 5) were
fixed by retrograde perfusion via the aorta with 2% paraformaldehyde, in 0.1 M cacodylate buffer, pH 7.4. For semithin sections (0.8-1 µm), tissue blocks prepared from cortex, outer and inner stripes of
outer medulla, and inner medulla were cryoprotected with 2.3 M sucrose
containing 2% paraformaldehyde, mounted on holders, and rapidly frozen
in liquid nitrogen (27). For immunoperoxidase and
immunofluorescence microscopy, other kidney blocks containing all
kidney zones were dehydrated and embedded in paraffin. For light and
laser confocal microscopy, the paraffin-embedded tissues were cut into
2-µm sections on a rotary microtome (Micron). The staining was
carried out by using indirect immunofluorescence or indirect
immunoperoxidase. The sections were dewaxed and rehydrated. For
immunoperoxidase labeling, endogenous peroxidase was blocked by 0.5%
H2O2 in absolute methanol for 10 min at room
temperature. To reveal antigens, sections were put in 1 mM Tris
solution (pH 9.0) supplemented with 0.5 mM EGTA and heated by using a
microwave oven for 10 min. Nonspecific binding of Ig was prevented by
incubating the sections in 50 mM NH4Cl for 30 min, followed
by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2%
gelatin. Sections were incubated overnight at 4°C with primary
antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton
X-100. After rinsing with PBS supplemented with 0.1% BSA, 0.05%
saponin, and 0.2% gelatin for 3 × 10 min, the sections for laser
confocal microscopy were incubated in Alexa 488-conjugated goat
anti-rabbit antibody (Molecular Probes) diluted in PBS supplemented
with 0.1% BSA and 0.3% Triton X-100 for 60 min at room
temperature. For double labeling, Alexa 546-conjugated goat
anti-mouse antibody (Molecular Probes) was added as well. After rinsing
with PBS for 3 × 10 min, the sections were mounted in glycerol
supplemented with antifade reagent (N-propyl gallat). For
immunoperoxidase, the sections were washed (see above) followed
by incubation in horseradish peroxidase-conjugated goat anti-rabbit
Ig (DAKO P448) diluted in PBS supplemented with 0.1% BSA and
0.3% Triton X-100. The microscopy was carried out by using a Leica
DMRE light microscope and a Zeiss LSM510 laser confocal microscope.
Immunoelectron microscopy.
For immunoelectron microscopy, the frozen samples were
freeze-substituted in a Reichert AFS freeze substitution unit
(16, 27, 38). In brief, the samples were sequentially
equilibrated over 3 days in methanol containing 0.5% uranyl acetate at
temperatures gradually raised from
80 to
70°C, then rinsed in
pure methanol for 24 h while the temperature was increased from
70 to
45°C, and infiltrated with Lowicryl HM20 and methanol 1:1,
2:1 and, finally, pure Lowicryl HM20 before ultraviolet polymerization for 2 days at
45°C and 2 days at 0°C. Immunolabeling was
performed on ultrathin Lowicryl HM20 sections. Sections were pretreated with a saturated solution of NaOH in absolute ethanol (2-3 s), rinsed, and preincubated for 10 min with 0.1% sodium borohydride and
50 mM glycine in 0.05 M Tris (pH 7.4) supplemented with 0.1% Triton
X-100. Sections were rinsed and incubated overnight at 4°C with
antibodies diluted in 0.05 M Tris (pH 7.4) supplemented with 0.1%
Triton X-100 with 0.2% milk. After rinsing, sections were incubated
for 1 h at room temperature with goat anti-rabbit IgG conjugated
to 10-nm colloidal gold particles (1:50, GAR.EM10, BioCell Research
Laboratories, Cardiff, UK). The sections were stained with uranyl
acetate and lead citrate before examination in Philips CM100 or Philips
208 electron microscopes. Immunolabeling controls were performed by
using preabsorption of the immune serum with the peptide used for immunization.
 |
RESULTS |
Immunoblotting of
-,
-, and
-ENaC in rat kidney.
Immunoblotting of fractions prepared from rat kidney homogenates by
differential centrifugations (Fig. 1)
revealed that the affinity-purified antibodies directed to each of the
three ENaC subunits recognize a single band of apparent molecular mass
in the 83- to 90-kDa range in renal membranes but not in cytosol (200,000-g supernatant). Membrane fractionation revealed the
presence of all three ENaC subunits in the membrane fractions that were enriched for either plasma membranes or intracellular vesicles. The
bands were not present with purified IgG from preimmune serum from the
same rabbits.

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Fig. 1.
Immunoblotting of - (top), -
(middle), and -subunits (bottom) of the
epithelial sodium channel (ENaC) in rat kidney fractions enriched for
plasma membranes (17,000-g pellet), intracellular vesicles
(200,000-g pellet), or cytoplasmic proteins
(200,000-g supernatant). Twenty micrograms were loaded in
each lane. Left: labeling with affinity-purified antibodies.
Right: labeling with protein A-purified IgG prepared from
preimmune sera. Equal concentrations of IgG were used for antibody and
preimmune serum in each case.
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To examine the presence of ENaC subunits in the different zones of rat
kidney, immunoblots of homogenates from the rat whole kidney, cortex,
outer medulla, and three different zones of the inner medulla (base,
middle, and tip) were performed by using the same affinity-purified
rabbit anti-rat antibodies (Fig. 2, arrowheads). In all the investigated zones of the rat kidney including inner medulla, the antibodies labeled distinct bands, corresponding to
the three ENaC subunits (Fig. 2). This indicated that
-,
- and
-subunits of the ENaC are present in all kidney zones including inner medulla. The relative abundance of each subunit appears to
decrease toward the inner part of the inner medulla, however. The faint
bands in the immunoblots of
- and
-ENaC (Fig. 2, tip of inner
medulla) may represent modified
- and
-ENaC, e.g., ubiquinated or otherwise posttranslationally modified.

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Fig. 2.
Immunoblotting of - (top), -
(middle), and -ENaC (bottom) in rat kidney.
Immunoblots of membrane fractions from the rat kidney cortex (CTX),
outer medulla (OM), and 3 different zones of the inner medulla [base
(1), middle (2), and tip (3)] using
the affinity-purified rabbit anti-rat antibodies against -, -,
and -ENaC subunits, respectively. In all the investigated zones of
the rat kidney including inner medulla, each antibody labeled a
distinct band at ~83-90 kDa in membrane fractions.
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Localization of
-,
-, and
-ENaC in rat kidney by using
immunohistochemistry.
In both kidney cortex and outer medulla, immunoperoxidase
microscopy using paraffin-embedded rat kidney tissues
demonstrated that immunolabeling of the
-,
- and
-subunits of
ENaC was exclusively associated with principal cells in the CCD (Fig.
3, A-F and
D-F, arrows) as well as in the OMCD (not shown). In
contrast, intercalated cells exhibited no labeling of ENaC subunits in
the CCD (Fig. 3, D-F, arrowheads) as well as in the
OMCD (not shown). Importantly, the principal cells of the IMCD also
exhibited significant labeling of
-,
-, and
-subunits (Fig. 3,
G-L), with decreasing labeling along the axis. A faint
but distinct labeling of the ENaC subunits was found all the way to the
papillary tip (Fig. 3, G-L). Immunolabeling controls
with peptide-preabsorbed antibodies were negative in all zones of the
kidney (Fig. 4, A-F).

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Fig. 3.
Immunoperoxidase microscopy of -, -, and -ENaC subunits
using paraffin-embedded rat kidney tissues. A-C:
immunolabeling of the -, -, and -ENaC subunits are exclusively
associated with principal cells in the cortical collecting ducts (CCD),
respectively. G, glomerulus. D-F: high magnification
reveals that ENaC subunits are exclusively associated with principal
cells in the CCD (arrows), whereas intercalated cells exhibited no
labeling (arrowheads). G-I: in the inner medulla,
principal cells also exhibit a significant labeling of -, -, and
- ENaC, respectively. J-L: high magnification
reveals a distinct labeling of the ENaC subunits in the terminal
collecting duct [inner meduallry collecting duct (IMCD)] principal
cells. Magnification: ×220 (A-C, G-I);
×550 (D-F,
J-L).
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Fig. 4.
Immunolabeling controls with
peptide-preabsorbed antibodies show no labeling in cortex
(A-C), medulla (D-F), or the urothelium
covering the pelvis (G-I). Magnification ×250
(A-F); ×630 (G-I).
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Cellular and subcellular localization of
-,
-, and
-ENaC
subunits determined by single- and double-labeling confocal laser
microscopy.
To evaluate the cellular and subcellular localization of
-,
-, and
-ENaC subunits, single- and double-labeling confocal microscopy was undertaken, and the labeling pattern was compared with
that of AQP2 and H+-ATPase. AQP2 labeling was restricted to
principal cells (Fig. 5, A and
C), whereas H+-ATPase labeling was restricted to
intercalated cells of the collecting duct (Fig. 5,
B-C). Double labeling using antibodies against
-,
-, and
-ENaC subunits and H+-ATPase revealed that
(Fig. 5, D and F)-,
(Fig. 5, G and
I)-, and
-ENaC (Fig. 5, J and L)
were exclusively localized in the principal cells (arrows), consistent
with previous results. Importantly, confocal microscopy revealed that
in CNT (not shown) and CCD,
-ENaC labeling was exclusively
restricted to a narrow zone in the apical part of the principal cells
including the plasma membrane domains (Fig. 5, D and
F, arrows). Compared with the labeling of AQP2 (in
neighboring sections; see Fig. 5, A and C),
-ENaC labeling was considerably more restricted to the very apical
part of the principal cells. In contrast,
- and
-subunits were
observed to be distributed throughout the cytoplasm with no distinct
increased labeling in the apical domains of the principal cells [Fig.
5, G and I and J and L
(arrows), respectively].

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Fig. 5.
Colocalization of aquaporin-2
(AQP2; C), -ENaC (F), -ENaC (I),
and -ENaC (L) with H+-ATPase in the CCD.
A-C: AQP2 is present at the apical part of the
principal cells (A and C, arrows), whereas
H+-ATPase is present at the apical part of the type-A
intercalated cells (B and C, arrowheads). Thus
AQP2 is exclusively expressed in the principal cells (C,
arrows), whereas H+-ATPase is located at the intercalated
cells (C, arrowheads) in the same duct. D-L:
(D and F, arrows)-, (G and
I, arrows)-, and -ENaC (J and L,
arrows) are exclusively localized at the principal cells. In contrast,
H+-ATPase is located only at the intercalated cells
(E, H, and K, arrowheads). Confocal
microscopy also reveals that the -ENaC is mainly localized at the
apical portion of the principal cells (D and F,
arrows) in contrast to - and -ENaC (G and I
and J and L, respectively). Magnification: ×800
(A-L).
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To evaluate whether there was an axial heterogeneity in the subcellular
localization of ENaC subunits along the axis of the collecting duct,
confocal laser microscopy was performed in cortex (Fig.
6, A-C), outer medulla
(not shown), and inner medulla (Fig. 6, D-F). In
contrast to the labeling pattern seen in cortex (with
-ENaC labeling
restricted to the apical part of the principal cells),
-ENaC
labeling in IMCD principal cells was distributed more evenly in the
cytoplasm (cf. Fig. 6, A and D). In the outer medulla the labeling pattern of
-ENaC is more similar to the pattern
seen in CCD, although slightly less polarized (not shown). Labeling of
- and
-ENaC in the IMCD was similar to that seen in CCD (cf. Fig.
6, B-C and E-F).

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Fig. 6.
Confocal microscopy using
immunofluorescence labeling of ENaC subunits in sections from kidney
cortex (A-C) or from inner medulla
(D-F). A-C: a significant labeling of
-, -, and -ENaC subunits is exclusively associated with
principal cells of the collecting ducts (arrows), whereas intercalated
cells were not labeled. -ENaC is mainly localized at the apical
portion of the principal cells (A, arrows). Arrowheads,
basal portions of the principal cells. In contrast, labeling of -
and -subunit is observed as dispersed throughout the cytoplasm with
no distinct labeling at the apical part of the cells (B and
C, arrows, respectively). D-F: in the inner
medulla, principal cells also exhibit a significant labeling of -,
-, and -subunit, respectively. Magnification: ×1,000
(A-C); ×630 (D-F).
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Immunoelectron microscopy of the ENaC in collecting duct principal
cells.
Immunoelectron microscopy was performed by using immunogold labeling of
sections prepared from kidney tissue embedded in Lowicryl HM20 by
cryosubstitution. In sections from the kidney cortex, immunogold
labeling of the
-subunit of the ENaC was observed of principal cells
in the CCD [Fig. 7, PC (arrows)],
whereas intercalated cells exhibited no immunogold labeling (Fig. 7,
IC). In the principal cells, immunolabeling of the
-subunit was
predominantly associated with intracellular vesicles (Figs. 7 and
8, arrows), some of which are clearly
revealed in the section whereas others are more difficult to see
because of weak fixation and/or tangentional sectioning. Also, distinct
labeling of the apical plasma membrane was seen (Fig. 8, arrowheads).
This immunolabeling pattern was consistent with the confocal
microscopic observations, demonstrating mainly intracellular
labeling of the
-subunit (Figs. 5G and 6B).
Consistently, immunogold labeling of the
-subunit was also mainly
associated with intracellular vesicles (Fig.
9,
A-C), but some labeling was also seen
associated with the apical plasma membrane domains (Fig. 9A, arrowhead) in kidneys of rats with no sodium restriction
and no aldosterone treatment. Immunogold labeling of
-ENaC revealed weak labeling of intracellular vesicles and apical plasma membrane close to background levels (not shown). Thus immunoelectron microscopy demonstrates that ENaC subunits are present in both intracellular vesicles and the apical plasma membrane.

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Fig. 7.
Immunoelectron microscopy of the -ENaC subunit in the
CCD in ultrathin Lowicryl HM20 sections. Immunogold labeling of the
-ENaC subunit in principal cells (PC, arrows) is observed, whereas
intercalated cells (IC) exhibit no immunogold labeling. In the PC,
immunolabeling of the -subunit is predominantly associated with
intracellular vesicles (arrows). Magnification: ×63,000.
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Fig. 8.
Immunoelectron microscopy of the -ENaC subunit in the
CCD in ultrathin Lowicryl HM20 sections. Immunogold labeling of the
-ENaC subunit in principal cells is predominantly associated with
intracellular vesicles (arrows), whereas little immunogold labeling is
seen at the apical plasma membrane domain (arrowheads). Magnification:
×63,000.
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Fig. 9.
Immunoelectron microscopy of the -ENaC subunit
(A-C) in the principal cells of the CCD in ultrathin
Lowicryl HM20 sections. Immunogold labeling of the -ENaC subunit in
principal cells is predominantly associated with intracellular vesicles
(arrows), whereas little immunogold labeling is seen at the apical
plasma membrane domain (arrowheads). L, lumen; M, mitochondria.
Magnification: ×63,000.
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Localization of
-,
- and
-subunits of ENaC in urothelium.
A marked ENaC subunit labeling of the transitional epithelium
(urothelium) covering the renal pelvic wall was also found (Fig. 10). Consistent with the difference in
the subcellular localization in the CCD principal cells, both
immunoperoxidase (Fig. 10, A, C, and
E) and confocal microscopy (Fig. 10, B,
D, and F) revealed that
-ENaC was located in
and/or near the apical plasma membranes (Fig. 10, A and
B), whereas
(Fig. 10, C and D)-
and
-ENaC (Fig. 10, E and F) were located
throughout the cytoplasm in a diffuse pattern. The umbrella cells were
seen to be devoid of
-ENaC, whereas they expressed
- and
-ENaC. This distribution pattern was also seen in urothelium of the
urinary bladder (not shown). Immunolabeling controls were negative
(Fig. 4, G-I).

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Fig. 10.
Immunoperoxidase (A, C, and E)
and confocal microscopy (B, D, and F)
of ENaC subunits in the surface epithelium (urothelium) covering the
pelvis. -ENaC is located in and/or near the apical plasma membranes
(A and B), whereas (C
and D)- and -ENaC (E and F)
were located throughout the cytoplasm in a diffuse pattern.
Magnification ×630.
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DISCUSSION |
We have demonstrated that
-,
-, and
-ENaC are present not
only in the principal cells of the CNT, CCD, and OMCD but are also
abundantly expressed in principal cells of the IMCD, consistent with
previous physiological data from isolated perfused IMCD
(17). Thus all three subunits of ENaC are present along
the entire collecting duct. Moreover, we have demonstrated that the
subcellular localization of the three ENaC subunits is distinctly
different and also differs along the axis of the collecting duct. Laser
confocal microscopy demonstrated that
-ENaC is predominantly present
in the extreme apical domains of the principal cells in CCD and OMCD.
In contrast, labeling of
- and
-ENaC is mainly associated with
intracellular vesicles dispersed in the entire cytoplasm with no
increase in labeling in the apical plasma membrane domains. The
differences in the subcellular localization among
-,
- and
-ENaC subunits suggest that there are some differences in the
regulation of ENaC subunits. In contrast to the difference in the
subcellular localization of
-ENaC vs.
- and
-ENaC in CCD and
OMCD, there was virtually no difference in the subcellular localization
of all three subunits in IMCD, where most labeling was observed in
cytoplasmic areas. Thus there is an axial heterogeneity in the
subcellular localization of
-ENaC. Immunoelectron microscopy
demonstrated that ENaC subunits are present in both the apical plasma
membrane and intracellular vesicles, consistent with the possibility
that ENaC regulation may involve trafficking from vesicles to the
apical plasma membrane, analogous to vasopressin regulation of AQP2
trafficking. Finally, we demonstrated that all three ENaC subunits were
localized in the urothelium covering the proximal portions of the
urinary tract, thereby extending previous studies showing the presence
of ENaC in bladder urothelial cells (33). Again, there was
a distinct difference in the subcellular localization because
-ENaC
(in contrast to
- and
-ENaC) was localized in a highly polarized fashion, in a narrow region in the apical section of the cells. Moreover, in the superficial cell layer (the umbrella cells) only
- and
-ENaC were present, demonstrating that
-ENaC
may not be uniformly present in all cells expressing
- and
-ENaC.
ENaC localization in the renal collecting duct.
The present observation that
-,
-, and
-ENaC are expressed in
principal cells of the IMCD is consistent with physiological data
demonstrating amiloride-inhibited sodium conductance in the apical
membrane of isolated and perfused rat IMCD (17). One of
the early studies on ENaC expression in kidney (7)
reported that ENaC was not present in the IMCD and suggested that
another channel would be involved in sodium reabsorption at this site, whereas earlier immunocytochemical works demonstrated ENaC in rat IMCD
(3, 36). Our data showing the presence of all three subunits in IMCD strongly support the view that ENaC subunits are
involved in IMCD sodium reabsorption. The discrepancy between the two
studies is likely to be due to differences in the level of expression
along the collecting duct axis. As demonstrated in this study, there is
indeed an axial heterogeneity in ENaC expression levels because ENaC
immunolabeling is somewhat stronger in the CCD and OMCD compared with
IMCD. Thus the lack of identification of ENaC in IMCD in the previous
study may simply be a matter of detection level. In support of this
view, we optimized the labeling procedures in this study, which
resulted in a 3- to 10-fold increase in labeling efficiency (with
conventional methods, 3-10 times higher antibody concentration was
necessary to achieve labeling). Thus the presence of ENaC also in the
IMCD supports the view that ENaC is also involved in
amiloride-sensitive sodium reabsorption at this site.
Different subcellular localization of
- vs.
- and
-ENaC
subunits in collecting duct principal cells.
ENaC consists of at least three structurally related subunits (
-,
-, and
-ENaC), and all three subunits participate in channel
formation as the absence of any one results in a significant reduction
of sodium current expression in Xenopus laevis oocytes. We
demonstrated by immunoblotting, immunocytochemistry, confocal microscopy, and immunoelectron microscopy that ENaC subunits are present in both intracellular vesicles and the apical plasma membrane. Moreover, we have demonstrated that immunolabeling of
-ENaC is mainly present at the apical domains of the principal cells, whereas the labeling of
- and
-ENaC is associated with intracellular vesicles dispersed in the entire cytoplasm, with sparse labeling of the
apical plasma membranes. This may suggest that there are some
differences in the regulation of ENaC subunits. Previous studies have
also shown discrepancies with regard to the three subunits. Increased
plasma aldosterone levels, induced by either sodium restriction or
aldosterone infusion, were associated with a marked increase in the
abundance of
-ENaC protein in kidneys, but the abundance of the
-
and
-ENaC subunit proteins did not increase (21).
Consistent with this,
-ENaC appears to be the only subunit of the
three that has been reported to be transcriptionally regulated by
aldosterone (1, 9, 28, 35). Moreover, 7-day water
restriction of Sprague-Dawley rats resulted in significantly increased
abundances of
- and
-ENaC but no change in
-ENaC, whereas
chronic vasopressin infusion increased the abundance of the all three
ENaC subunit proteins (8). The implication of changes in
abundance of one or two subunits and not the other(s) in sodium
transport capacity is not known. Because the synthesis of the
-ENaC
subunit has been suggested to be a rate-limiting factor of the
multimeric ENaC complex (22), sodium transport could be
expected to be proportional to the abundance of the
-ENaC protein
levels. Further studies are needed to assess the impact of differential
regulation and differential subcellular localization of the ENaC
subunits on sodium transport. As discussed above,
-ENaC labeling in
the IMCD reveals enhanced vesicular labeling (in addition to labeling
of plasma membrane domains) compared with CCD and OMCD [interestingly,
this is exactly similar to the labeling pattern of AQP2 (see Fig. 5,
for example), which also shows this segmental difference]. The higher
molecular weight band in the immunoblot is likely to reflect important
issues of ENaC biology. The top band in the
immunoblot of
-ENaC (Fig. 2) may represent modified
-ENaC, i.e.,
ubiquinated or otherwise modified. Moreover, immunoblotting of
-ENaC
reveals a second, higher molecular weight band in the middle portion
and tip of the inner medulla (Fig. 2). The reason is unknown but is
likely to reflect chemical modification of the subunit.
Mechanisms of regulation of sodium reabsorption in collecting duct
by ENaC.
Renal regulation of sodium reabsorption or excretion is essential to
the regulation of extracellular fluid volume as well as to the control
of blood pressure. Sodium is reabsorbed in the different renal tubular
segments through several renal sodium transporters: in proximal tubule,
type 3 Na+/H+ exchanger (NHE3) and type II
Na+-Pi cotransporter (NaPi-2) are both
expressed apically (2, 23), whereas Na-K-ATPase is heavily
expressed in the basolateral membrane of renal tubule cells, and are
responsible for sodium reabsorption (32). The loop of
Henle generates a high osmolality in renal medulla by driving the
countercurrent multiplier, which is dependent on NaCl absorption by the
thick ascending limb (TAL). The apically expressed Na-K-2Cl
cotransporter (rat type 1 bumetanide-sensitive cotransporter) and NHE3,
in conjunction with basolaterally expressed Na-K-ATPase are mainly
responsible for sodium reabsorption by the TAL (15). In
the distal convoluted tubule, the thiazide-sensitive Na-Cl
cotransporter is involved in apical sodium reabsorption (14). As described above, regulation of sodium
reabsorption also occurs in the CNT and collecting duct, and ENaC plays
a critical role in this. The importance of ENaC in volume regulation
has been demonstrated in recent studies that have identified mutations in ENaC as the basis of the pathogenesis of Liddle's syndrome, a
disorder characterized by volume expansion and hypertension (13,
31) as well as type I pseudohypoaldosteronism, a disorder characterized by volume depletion and hypotension (5).
ENaC is regulated by the adrenal mineralocorticoid hormone vasopressin and insulin, which markedly increase the apical permeability of the
collecting duct to sodium.
In principle, there may be at least two mechanisms for regulation of
ENaC activity in the apical plasma membrane. One may involve regulation
of ENaC expression, and in addition regulated trafficking of ENaC
subunits may be involved, analogous to vasopressin regulation of AQP2
(i.e., short-term regulation of trafficking and long-term regulation of
expression). Masilamani et al. (21) recently demonstrated
by immunofluorescence microscopy that sodium restriction in rats was
associated with a redistribution of diffuse intracellular labeling of
ENaC subunits to the apical regions of the principal cells as well as a
marked increase in
-ENaC labeling (21). Similar
observations have been recently reported by Loffing et al.
(19). They suggested that ENaC protein may be regulated by
both intracellular trafficking to the apical plasma membrane domains
(short-term regulation) and by increasing protein abundance (long-term
regulation) in response to aldosterone or other stimuli for enhancing
sodium reabsorption in the distal nephron. Our study showing ENaC
subunits in both the apical plasma membrane and intracellular vesicles
is consistent with the possibility of regulated trafficking from
vesicles to the apical plasma membrane. Future studies will be aimed at
defining whether ENaC subunits undergo significant regulated
trafficking in response to aldosterone, vasopressin, or other hormones.
Localization of ENaC subunits in pelvic urothelial cells.
Our data demonstrate that all three subunits of ENaC are expressed in
urothelial cells covering the renal pelvis. This extends previous data
showing the presence of ENaC in bladder urothelium (33).
Two observations in particular are curious. First, there is a
pronounced difference in the subcellular distribution of
-ENaC vs.
- and
-ENaC. This is similar to what is seen in the CCD and OMCD,
which is that
-ENaC has a pronounced polarized distribution with
labeling of the plasma membrane domains and very little labeling within
the cell. In contrast, both
- and
-ENaC are distributed in a
dispersed vesicular pattern in the cytoplasm. Second, there is a
complete absence of
-ENaC immunolabeling in the outer cell layer
(the umbrella cells) whereas significant labeling of
- and
-ENaC
was observed. This raises the possibility that ENaC subunits may be
separately expressed and the potentiality that additional isoforms
exist to replace
-ENaC. The role of ENaC at this site and potential
regulation of ENaC in the urothelial cells will require additional analysis.
 |
ACKNOWLEDGEMENTS |
The authors thank Zhila Nikrozi, Inger Merete Paulsen, Helle
Høyer, Mette Vistisen, and Gitte Christensen for expert technical assistance.
 |
FOOTNOTES |
Support for this study was provided by the Karen Elise Jensen
Foundation, the Novo Nordic Foundation, the Danish Medical Research Council, the University of Aarhus Research Foundation, the University of Aarhus, Dongguk University, the Commission of the European Union
(EU-Biotech Program and EU-TMR Program), and the intramural budget of
the National Heart, Lung, and Blood Institute.
Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
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.
Received 11 August 2000; accepted in final form 9 February 2001.
 |
REFERENCES |
1.
Asher, C,
Wald H,
Rossier BC,
and
Garty H.
Aldosterone-induced increase in the abundance of Na+ channel subunits.
Am J Physiol Cell Physiol
271:
C605-C611,
1996[Abstract/Free Full Text].
2.
Biemesderfer, D,
Rutherford PA,
Nagy T,
Pizzonia JH,
Abu-Alfa AK,
and
Aronson PS.
Monoclonal antibodies for high-resolution localization of NHE3 in adult and neonatal rat kidney.
Am J Physiol Renal Physiol
273:
F289-F299,
1997[Abstract/Free Full Text].
3.
Brown, D,
Sorscher EJ,
Ausiello DA,
and
Benos DJ.
Immunocytochemical localization of Na+ channels in rat kidney medulla.
Am J Physiol Renal Fluid Electrolyte Physiol
256:
F366-F369,
1989[Abstract/Free Full Text].
4.
Canessa, CM,
Horisberger JD,
and
Rossier BC.
Epithelial sodium channel related to proteins involved in neurodegeneration.
Nature
361:
467-470,
1993[ISI][Medline].
5.
Chang, SS,
Grunder S,
Hanukoglu A,
Rosler A,
Mathew PM,
Hanukoglu I,
Schild L,
Lu Y,
Shimkets RA,
Nelson-Williams C,
Rossier BC,
and
Lifton RP.
Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.
Nat Genet
12:
248-253,
1996[ISI][Medline].
6.
Dietl, P,
Schwiebert E,
and
Stanton BA.
Cellular mechanisms of chloride transport in the cortical collecting duct.
Kidney Int Suppl
33:
S125-S130,
1991[Medline].
7.
Duc, C,
Farman N,
Canessa CM,
Bonvalet JP,
and
Rossier BC.
Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry.
J Cell Biol
127:
1907-1921,
1994[Abstract].
8.
Ecelbarger, CA,
Kim GH,
Terris J,
Masilamani S,
Mitchell C,
Reyes I,
Verbalis JG,
and
Knepper MA.
Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney.
Am J Physiol Renal Physiol
279:
F46-F53,
2000[Abstract/Free Full Text].
9.
Escoubet, B,
Coureau C,
Bonvalet JP,
and
Farman N.
Noncoordinate regulation of epithelial Na channel and Na pump subunit mRNAs in kidney and colon by aldosterone.
Am J Physiol Cell Physiol
272:
C1482-C1491,
1997[Abstract/Free Full Text].
10.
Fushimi, K,
Uchida S,
Hara Y,
Hirata Y,
Marumo F,
and
Sasaki S.
Cloning and expression of apical membrane water channel of rat kidney collecting tubule.
Nature
361:
549-552,
1993[ISI][Medline].
11.
Garty, H,
and
Palmer LG.
Epithelial sodium channels: function, structure, and regulation.
Physiol Rev
77:
359-396,
1997[Abstract/Free Full Text].
12.
Grunder, S,
and
Rossier BC.
A reappraisal of aldosterone effects on the kidney: new insights provided by epithelial sodium channel cloning.
Curr Opin Nephrol Hypertens
6:
35-39,
1997[ISI][Medline].
13.
Hansson, JH,
Nelson-Williams C,
Suzuki H,
Schild L,
Shimkets R,
Lu Y,
Canessa C,
Iwasaki T,
Rossier B,
and
Lifton RP.
Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle's syndrome.
Nat Genet
11:
76-82,
1995[ISI][Medline].
14.
Kim, GH,
Masilamani S,
Turner R,
Mitchell C,
Wade JB,
and
Knepper MA.
The thiazide-sensitive Na-Cl cotransporter is an aldosterone-induced protein.
Proc Natl Acad Sci USA
95:
14552-14557,
1998[Abstract/Free Full Text].
15.
Knepper, MA,
Kim GH,
Fernandez-Llama P,
and
Ecelbarger CA.
Regulation of thick ascending limb transport by vasopressin.
J Am Soc Nephrol
10:
628-634,
1999[Free Full Text].
16.
Kwon, TH,
Pushkin A,
Abuladze N,
Nielsen S,
and
Kurtz I.
Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney.
Am J Physiol Renal Physiol
278:
F327-F336,
2000[Abstract/Free Full Text].
17.
Light, DB,
McCann FV,
Keller TM,
and
Stanton BA.
Amiloride-sensitive cation channel in apical membrane of inner medullary collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
255:
F278-F286,
1988[Abstract/Free Full Text].
18.
Loffing, J,
Loffing-Cueni D,
Macher A,
Hebert SC,
Olson B,
Knepper MA,
Rossier BC,
and
Kaissling B.
Localization of epithelial sodium channel and aquaporin-2 in rabbit kidney cortex.
Am J Physiol Renal Physiol
278:
F530-F539,
2000[Abstract/Free Full Text].
19.
Loffing, J,
Pietri L,
Aregger F,
Bloch-Faure M,
Ziegler U,
Meneton P,
Rossier BC,
and
Kaissling B.
Differential subcellular localization of ENaC subunits in mouse kidney in response to high- and low-Na diets.
Am J Physiol Renal Physiol
279:
F252-F258,
2000[Abstract/Free Full Text].
20.
Marples, D,
Frøkiaer J,
Dorup J,
Knepper MA,
and
Nielsen S.
Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex.
J Clin Invest
97:
1960-1968,
1996[Abstract/Free Full Text].
21.
Masilamani, S,
Kim GH,
Mitchell C,
Wade JB,
and
Knepper MA.
Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney.
J Clin Invest
104:
R19-R23,
1999[Abstract/Free Full Text].
22.
May, A,
Puoti A,
Gaeggeler HP,
Horisberger JD,
and
Rossier BC.
Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel alpha subunit in A6 renal cells.
J Am Soc Nephrol
8:
1813-1822,
1997[Abstract].
23.
Murer, H,
and
Biber J.
Molecular mechanisms of renal apical Na/phosphate cotransport.
Annu Rev Physiol
58:
607-618,
1996[ISI][Medline].
24.
Nielsen, S,
Chou CL,
Marples D,
Christensen EI,
Kishore BK,
and
Knepper MA.
Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.
Proc Natl Acad Sci USA
92:
1013-1017,
1995[Abstract].
25.
Nielsen, S,
DiGiovanni SR,
Christensen EI,
Knepper MA,
and
Harris HW.
Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.
Proc Natl Acad Sci USA
90:
11663-11667,
1993[Abstract].
26.
Nielsen, S,
Kwon TH,
Christensen BM,
Promeneur D,
Frøkiaer J,
and
Marples D.
Physiology and pathophysiology of renal aquaporins.
J Am Soc Nephrol
10:
647-663,
1999[Abstract/Free Full Text].
27.
Nielsen, S,
Pallone T,
Smith BL,
Christensen EI,
Agre P,
and
Maunsbach AB.
Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1023-F1037,
1995[Abstract/Free Full Text].
28.
Ono, S,
Kusano E,
Muto S,
Ando Y,
and
Asano Y.
A low-Na+ diet enhances expression of mRNA for epithelial Na+ channel in rat renal inner medulla.
Pflügers Arch
434:
756-763,
1997[ISI][Medline].
29.
Sansom, SC,
and
O'Neil RG.
Mineralocorticoid regulation of apical cell membrane Na+ and K+ transport of the cortical collecting duct.
Am J Physiol Renal Fluid Electrolyte Physiol
248:
F858-F868,
1985[Abstract/Free Full Text].
30.
Schmitt, R,
Ellison DH,
Farman N,
Rossier BC,
Reilly RF,
Reeves WB,
Oberbaumer I,
Tapp R,
and
Bachmann S.
Developmental expression of sodium entry pathways in rat nephron.
Am J Physiol Renal Physiol
276:
F367-F381,
1999[Abstract/Free Full Text].
31.
Shimkets, RA,
Warnock DG,
Bositis CM,
Nelson-Williams C,
Hansson JH,
Schambelan M,
Gill JR, Jr,
Ulick S,
Milora RV,
and
Findling JW.
Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel.
Cell
79:
407-414,
1994[ISI][Medline].
32.
Skou, JC.
The Na,K-pump.
Methods Enzymol
156:
1-25,
1988[ISI][Medline].
33.
Smith, PR,
Mackler SA,
Weiser PC,
Brooker DR,
Ahn YJ,
Harte BJ,
McNulty KA,
and
Kleyman TR.
Expression and localization of epithelial sodium channel in mammalian urinary bladder.
Am J Physiol Renal Physiol
274:
F91-F96,
1998[Abstract/Free Full Text].
34.
Stokes, JB.
Sodium and potassium transport by the collecting duct.
Kidney Int
38:
679-686,
1990[ISI][Medline].
35.
Stokes, JB,
and
Sigmund RD.
Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity.
Am J Physiol Cell Physiol
274:
C1699-C1707,
1998[Abstract/Free Full Text].
36.
Tousson, A,
Alley CD,
Sorscher EJ,
Brinkley BR,
and
Benos DJ.
Immunochemical localization of amiloride-sensitive sodium channels in sodium-transporting epithelia.
J Cell Sci
93:
349-362,
1989[Abstract].
37.
Wang, WH,
Schwab A,
and
Giebisch G.
Regulation of small-conductance K+ channel in apical membrane of rat cortical collecting tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
259:
F494-F502,
1990[Abstract/Free Full Text].
38.
Yasui, M,
Kwon TH,
Knepper MA,
Nielsen S,
and
Agre P.
Aquaporin-6: an intracellular vesicle water channel protein in renal epithelia.
Proc Natl Acad Sci USA
96:
5808-5813,
1999[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 280(6):F1093-F1106