Immunocytochemical and immunoelectron microscopic localization of alpha -, beta -, and gamma -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


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Epithelial sodium channel (ENaC) subunit (alpha , beta , and gamma ) 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. alpha -ENaC was localized mainly in a zone in the apical domains, whereas beta - and gamma -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, alpha -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, alpha -ENaC was present in a narrow zone near the apical plasma membrane, whereas beta - and gamma -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


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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, alpha -, beta - and gamma -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.


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Antibody against ENaC subunits, AQP2, and H+-ATPase. Antibodies raised in rabbits against synthetic peptides were used. Affinity-purified polyclonal antibodies against alpha -, beta -, and gamma -ENaC (LL766AP, LL558AP, and LL550AP, respectively) have been described previously (21). An additional antiserum against alpha -ENaC was also raised. For this, a peptide from the cytoplasmic NH2 terminus of rat alpha -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.


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Immunoblotting of alpha -, beta -, and gamma -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 alpha - (top), beta - (middle), and gamma -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.

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 alpha -, beta - and gamma -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 beta - and gamma -ENaC (Fig. 2, tip of inner medulla) may represent modified beta - and gamma -ENaC, e.g., ubiquinated or otherwise posttranslationally modified.


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Fig. 2.   Immunoblotting of alpha - (top), beta - (middle), and gamma -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 alpha -, beta -, and gamma -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.

Localization of alpha -, beta -, and gamma -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 alpha -, beta - and gamma -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 alpha -, beta -, and gamma -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 alpha -, beta -, and gamma -ENaC subunits using paraffin-embedded rat kidney tissues. A-C: immunolabeling of the alpha -, beta -, and gamma -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 alpha -, beta -, and gamma - 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).

Cellular and subcellular localization of alpha -, beta -, and gamma -ENaC subunits determined by single- and double-labeling confocal laser microscopy. To evaluate the cellular and subcellular localization of alpha -, beta -, and gamma -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 alpha -, beta -, and gamma -ENaC subunits and H+-ATPase revealed that alpha  (Fig. 5, D and F)-, beta  (Fig. 5, G and I)-, and gamma -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, alpha -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), alpha -ENaC labeling was considerably more restricted to the very apical part of the principal cells. In contrast, beta - and gamma -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), alpha -ENaC (F), beta -ENaC (I), and gamma -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: alpha  (D and F, arrows)-, beta  (G and I, arrows)-, and gamma -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 alpha -ENaC is mainly localized at the apical portion of the principal cells (D and F, arrows) in contrast to beta - and gamma -ENaC (G and I and J and L, respectively). Magnification: ×800 (A-L).

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 alpha -ENaC labeling restricted to the apical part of the principal cells), alpha -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 alpha -ENaC is more similar to the pattern seen in CCD, although slightly less polarized (not shown). Labeling of beta - and gamma -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 alpha -, beta -, and gamma -ENaC subunits is exclusively associated with principal cells of the collecting ducts (arrows), whereas intercalated cells were not labeled. alpha -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 beta - and gamma -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 alpha -, beta -, and gamma -subunit, respectively. Magnification: ×1,000 (A-C); ×630 (D-F).

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 beta -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 beta -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 beta -subunit (Figs. 5G and 6B). Consistently, immunogold labeling of the gamma -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 alpha -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 beta -ENaC subunit in the CCD in ultrathin Lowicryl HM20 sections. Immunogold labeling of the beta -ENaC subunit in principal cells (PC, arrows) is observed, whereas intercalated cells (IC) exhibit no immunogold labeling. In the PC, immunolabeling of the beta -subunit is predominantly associated with intracellular vesicles (arrows). Magnification: ×63,000.



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Fig. 8.   Immunoelectron microscopy of the beta -ENaC subunit in the CCD in ultrathin Lowicryl HM20 sections. Immunogold labeling of the beta -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 gamma -ENaC subunit (A-C) in the principal cells of the CCD in ultrathin Lowicryl HM20 sections. Immunogold labeling of the gamma -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.

Localization of alpha -, beta - and gamma -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 alpha -ENaC was located in and/or near the apical plasma membranes (Fig. 10, A and B), whereas beta  (Fig. 10, C and D)- and gamma -ENaC (Fig. 10, E and F) were located throughout the cytoplasm in a diffuse pattern. The umbrella cells were seen to be devoid of alpha -ENaC, whereas they expressed beta - and gamma -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. alpha -ENaC is located in and/or near the apical plasma membranes (A and B), whereas beta  (C and D)- and gamma -ENaC (E and F) were located throughout the cytoplasm in a diffuse pattern. Magnification ×630.


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We have demonstrated that alpha -, beta -, and gamma -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 alpha -ENaC is predominantly present in the extreme apical domains of the principal cells in CCD and OMCD. In contrast, labeling of beta - and gamma -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 alpha -, beta - and gamma -ENaC subunits suggest that there are some differences in the regulation of ENaC subunits. In contrast to the difference in the subcellular localization of alpha -ENaC vs. beta - and gamma -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 alpha -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 alpha -ENaC (in contrast to beta - and gamma -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 beta - and gamma -ENaC were present, demonstrating that alpha -ENaC may not be uniformly present in all cells expressing beta - and gamma -ENaC.

ENaC localization in the renal collecting duct. The present observation that alpha -, beta -, and gamma -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 alpha - vs. beta - and gamma -ENaC subunits in collecting duct principal cells. ENaC consists of at least three structurally related subunits (alpha -, beta -, and gamma -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 alpha -ENaC is mainly present at the apical domains of the principal cells, whereas the labeling of beta - and gamma -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 alpha -ENaC protein in kidneys, but the abundance of the beta - and gamma -ENaC subunit proteins did not increase (21). Consistent with this, alpha -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 beta - and gamma -ENaC but no change in alpha -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 alpha -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 alpha -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, alpha -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 beta -ENaC (Fig. 2) may represent modified beta -ENaC, i.e., ubiquinated or otherwise modified. Moreover, immunoblotting of gamma -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 alpha -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 alpha -ENaC vs. beta - and gamma -ENaC. This is similar to what is seen in the CCD and OMCD, which is that alpha -ENaC has a pronounced polarized distribution with labeling of the plasma membrane domains and very little labeling within the cell. In contrast, both beta - and gamma -ENaC are distributed in a dispersed vesicular pattern in the cytoplasm. Second, there is a complete absence of alpha -ENaC immunolabeling in the outer cell layer (the umbrella cells) whereas significant labeling of beta - and gamma -ENaC was observed. This raises the possibility that ENaC subunits may be separately expressed and the potentiality that additional isoforms exist to replace alpha -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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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