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Am J Physiol Renal Physiol 291: F1033-F1044, 2006. First published June 6, 2006; doi:10.1152/ajprenal.00086.2006
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Locations, abundances, and possible functions of FXYD ion transport regulators in rat renal medulla

Kaarina Pihakaski-Maunsbach,1,2 Henrik Vorum,3 Bent Honoré,3 Shigeki Tokonabe,1,2 Jørgen Frøkiær,1,4 Haim Garty,5 Steven J. D. Karlish,5 and Arvid B. Maunsbach1,2

1The Water and Salt Research Center, 2Department of Cell Biology, Institute of Anatomy, 3Institute of Medical Biochemistry, and 4Department of Clinical Physiology, University of Aarhus, Aarhus, Denmark; and 5Department of Biological Chemistry, Weizmann Institute of Science, Rehovoth, Israel

Submitted 14 March 2006 ; accepted in final form 31 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The {gamma}-subunit of Na-K-ATPase (FXYD2) and corticosteroid hormone-induced factor (CHIF; FXYD4) are considered pump regulators in kidney tubules. The aim of this study was to expand the information about their locations in the kidney medulla and to evaluate their importance for electrolyte excretion in an animal model. The cellular and subcellular locations and abundances of {gamma} and CHIF in the medulla of control and sodium-depleted rats were analyzed by immunofluorescence and immunoelectron microscopy and semiquantitative Western blotting. The results showed that antibodies against the {gamma}-subunit COOH terminus and splice variant {gamma}a, but not splice variant {gamma}b, labeled intercalated cells, but not principal cells, in the initial part of the inner medullary collecting duct (IMCD1). In subsequent segments (IMCD2 and IMCD3), all principal cells exhibited distinct basolateral labeling for both the {gamma}-subunit COOH terminus, splice variant {gamma}a, and CHIF. Splice variant {gamma}b was abundant in the inner stripe of the outer medulla but absent in the inner medulla (IM). Double labeling by high-resolution immunoelectron microscopy showed close structural association between CHIF and the Na-K-ATPase {alpha}1-subunit in basolateral membranes. The present observations provide new information about the cellular and subcellular locations of {gamma} and CHIF in the renal medulla and show a new {gamma} variant in the IM. Extensive NaCl depletion did not induce significant changes in the locations or abundances of the {gamma}-subunit COOH terminus and CHIF in different kidney zones. We conclude that the unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during NaCl depletion.

Na-K-ATPase {gamma}-subunit; CHIF; kidney medulla

NA-K-ATPASE CONSISTS, IN MOST tissues, of a catalytic {alpha}- and a glycosylated beta-subunit, but in the kidney a {gamma}-subunit is also associated with the pump (16, 28). The latter belongs to the FXYD protein family, which contains small single-span membrane proteins characterized by a short invariant motif, FXYD, at the beginning of the signature sequence, in or close to the membrane span (35). The {gamma}-subunit, FXYD2, is expressed in the kidney as two splice variants, {gamma}a and {gamma}b, differing at their extracellular NH2 termini, whereas their intracellular COOH termini are identical (23). Corticosteroid hormone-induced factor (CHIF; FXYD4) also belongs to the FXYD family and can associate with the {alpha}-subunit of Na-K-ATPase (3, 18). Both CHIF protein and mRNA have been localized in the medullary collecting duct of the kidney (11, 33).

Much evidence, summarized in Refs. 6, 14, 17, 34, 37, suggests that FXYD proteins may participate in the regulation of the renal Na-K-ATPase. Both the {gamma}-subunit and CHIF appear to modulate Na-K-ATPase functions, but in opposite ways. Thus in different expression systems, {gamma} raises the apparent affinity for ATP (38), modulates the K+ activation of Na-K-ATPase, and reduces the affinity for cytoplasmic Na+ ions (1, 4, 38), whereas CHIF increases the affinity of the pump for cellular sodium (5, 18). Analyses of structure-function relationships of the {gamma}-subunit and CHIF demonstrate that transmembrane segments determine the opposite effects of CHIF and {gamma} on the sodium affinity of the pump (24).

The distribution of the {gamma}-subunit in rat kidneys has been studied by immunofluorescence microscopy (2, 31, 42) but the reported locations of the {gamma}-subunit and its splice variants have been somewhat at variance. Thus {gamma} was not observed in the inner (IMCD) or outer medullary collecting duct (15, 31), or lacking in the upper IMCDs but was weakly present in the deeper IMCDs (2). Recently, we preliminarily demonstrated that splice variants {gamma}a and {gamma}b have different distribution patterns in IMCDs (30). However, the precise location patterns of {gamma} and its splice variants in the renal medulla, and the relationship between CHIF and {gamma}, remain to be determined. For this reason, we have now extended the analysis of CHIF and {gamma} locations by confocal immunofluorescence and immunoelectron microscopy and determined to what extent they are colocalized. Second, we have analyzed the effects of extensive NaCl depletion on {gamma} and CHIF abundances, using Western blotting and confocal immunofluorescence microscopy. Several previous studies, cited above, have reported properties of the FXYD proteins in isolated cells or cell-free systems, but our aim was to determine what changes, if any, occur in vivo in rat kidneys during well-defined stress conditions. The results show that sodium depletion does not induce significant changes in the abundances of {gamma} and CHIF in different kidney zones. The unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during prolonged NaCl depletion.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male Munich-Wistar rats, 7–10 wk old and weighing 175–200 g, were obtained from the Møllegaard Breeding Center (Ejby, Denmark). After acclimatization, the rats were kept in separate metabolic cages for 10 days and individually ration-fed artificial hydrated diets containing defined amounts of all electrolytes, nutrients, trace elements, and water. No additional water was provided.

Protocol 1. Eleven control rats received a gelled food mixture that provided 17 g dry wt·day–1·100 g body wt–1 of an artificial diet (Altromin C-1036), 15 ml·day–1·100 g body wt–1 of water, and 2.0 meq·day–1·100 g body wt–1 of NaCl.

Protocol 2. Eleven rats were individually ration-fed the same diet as in protocol 1, except that no NaCl was added. The daily NaCl intake in this group was 0.02 meq·day–1·100 g body wt–1.

Experimental Procedures

At the time of death, blood was drawn from the inferior caval vein, collected in heparinized tubes, and plasma was obtained after centrifugation. Osmolalities of plasma and urine were measured with an Advanced Wide Range Osmometer 3W2 (Advanced Instruments). The plasma and urinary concentrations of creatinine and the plasma concentrations of sodium and potassium were determined (Kodak Ektachem 700XRC). The concentrations of urinary sodium and potassium were determined by standard flame photometry (Eppendorf FCM6341). Plasma and urine concentrations of chloride were determined (ABL 615, Radiometer, Copenhagen, Denmark). Plasma aldosterone concentrations were determined using a commercially available radioimmunoassay kit (Coat-A-Count, Diagnostic Products, Los Angeles, CA).

For surgical procedures, the rats were anesthetized by inhalation of halothane (Halocarbon, River Edge, NJ) and kept anesthetized until killed. The study was approved by the Danish National Committee on Animal Research Ethics.

The left kidney was first removed from the anesthetized rat for Western blotting. The right kidney was then perfusion fixed via the abdominal aorta with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer. After perfusion fixation, the tissue was postfixed in the same fixative for 2 h, rinsed in buffer, and prepared for immunocytochemistry (27).

Antibodies

Na-K-ATPase {alpha}1-antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). Na-K-ATPase anti-{gamma}-subunit COOH-terminal antibodies were raised in rabbits against KHRQVNEDEL, corresponding to the last 10 COOH-terminal residues. Anti-{gamma}a- and anti-{gamma}b-antibodies were raised in rabbits against the synthetic NH2-terminal residues TELSANHC ({gamma}a) or MDRWYLC ({gamma}b) and as previously described (23). Anti-CHIF antibodies were raised in rabbits against the synthetic peptide CRRNHTPSSLPE, corresponding to the COOH-terminal part of the protein (33). Monoclonal anti-peptide antibody to the 31-kDa bovine kidney vacuolar H+-ATPase subunit (19) was used to label intercalated cells.

Immunocytochemistry

For confocal microscopy, sagittal tissue blocks including all kidney zones were embedded in paraffin. Sections were cut cold on a Reichert Ultracut S (Leica, Vienna, Austria) microtome at 2-µm thickness. The sections were deparaffinized, and antigen retrieval was carried out by warming the sections to the boiling point in Tris-EGTA (TEG) buffer at pH 9. They were then preincubated in PBS containing 1% BSA or 0.1% skim milk powder and 50 mM glycine. The sections were incubated for 1 h at room temperature with monoclonal antibodies against the {gamma}-subunit of Na-K-ATPase, with polyclonal rabbit antibodies against the {gamma}-subunit COOH terminus and the NH2-terminal splice variants {gamma}a and {gamma}b, or against CHIF or H+-ATPase. For confocal microscopy, primary antibodies were detected with goat anti-rabbit IgG labeled with Alexa Fluor 488 or with goat anti-mouse IgG labeled with Alexa Fluor 546. For double labeling, the sections were incubated with mixed primary monoclonal and polyclonal antibodies, which were detected with a mixture of the above secondary antibodies. The sections were analyzed with a Leica TCS SL confocal laser-scanning microscope (Leica, Mannheim, Germany). Subsegments of the IMCD were defined as previously reported (36, 41): IMCD1, proximal 25%; IMCD2, middle 50%; and IMCD3, distal 25%, corresponding to the papillary tip.

For immunoperoxidase microscopy, the deparaffinized sections were treated with 0.5% H2O2 in absolute methanol for 10 min at room temperature to block endogenous peroxidase. Nonspecific binding of IgG was prevented by incubating the sections in 50 mM NH4Cl for 30 min, followed by blocking for 30 min in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C or 1 h at room temperature with primary antibodies diluted in PBS added with 0.1% BSA and 0.3% Triton X-100. After being rinsed, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (P448, DAKO, Glostrup, Denmark). Following peroxidase staining, the sections were analyzed with a Leica DMRE light microscope. For immunoelectron microscopy, small tissue blocks were trimmed from the perfusion-fixed kidneys from all kidney zones, cryoprotected with 2.3 M sucrose, mounted on holders, and frozen in liquid nitrogen. Immunoelectron microscopy was performed on either thin (80 nm) cryosections prepared from the frozen tissue with a Leica Reichert Ultracut S cryoultramicrotome (Leica) or on tissue that was cryosubstituted in a Reichert AFS freeze-substitution unit and embedded in Lowicryl HM20 as previously described (27). Ultrathin (50 nm) Lowicryl sections were prepared with a Leica Reichert Ultracut S ultramicrotome at room temperature. For immunoelectron microscopy, the ultrathin cryosections or Lowicryl sections were first preincubated in PBS containing 50 mM glycine and 1% BSA or 0.1% skim milk powder. The sections were then incubated with the different antibodies diluted 1:100–1:400 in the preincubation buffer for 1 h at room temperature. The primary antibodies were visualized using goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM1O, BioCell Research Laboratories, Cardiff, UK) diluted 1:50 in PBS with 0.1% skim milk powder and 5 mg/ml polyethylene glycol. Double immunogold labeling of the Na-K-ATPase {alpha}1-subunit and CHIF was performed by mixing the primary antibodies and detecting them with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles for CHIF, and goat anti-mouse IgG conjugated to 5-nm colloidal gold particles for the Na-K-ATPase {alpha}1-subunit. The Lowicryl sections were stained with saturated uranyl acetate and the ultrathin cryosections with 0.3% uranyl acetate in 1.8% methylcellulose for 10 min before examination with a FEI Morgagni 268D transmission electron microscope. The following immunolabeling controls were used for both confocal and electron microscopy: 1) substitution of the primary antibody with nonimmune rabbit IgG; 2) absorption of the antibodies with purified {gamma} peptides (~50-fold molar excess); and 3) incubation without the use of a primary antibody. All controls showed absence of labeling.

Immunoblotting

The tissue samples for Western blotting in protocols 1 and 2 were dissected from the cortex, inner stripe of outer medulla (ISOM), and inner medulla (IM). The samples were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) by using an Ultra-Turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany) at maximum speed for 30 s. The homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet enriched in plasma membranes. The pellets were dissolved in Laemmli sample buffer containing 2% SDS, and their protein contents were estimated using a Bio-Rad Protein Assay (Hercules, CA) based on the method of Bradford (7). Ten micrograms of prepared sample were loaded in each well and run on 4–20% Tris-glycine or 10–20% Tris-tricine polyacrylamide minigels (NOVEX, San Diego, CA) under reducing conditions. After transfer by electroelution to nitrocellulose membranes, the blots were blocked with 5% skim milk powder in PBS-Tris (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated for 2 h at 4°C with Na-K-ATPase anti-{alpha}1-, anti-{gamma}-subunit COOH terminus, anti-{gamma}a-, anti-{gamma}b-, and anti-CHIF antibodies, all diluted 1:100. The labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P217, P260, diluted 1:5,000, DAKO) using the enhanced chemiluminescence system (Amersham International, Littel Chalfont, UK).

Quantification and Statistics

For densitometry of immunoblots, imaging Kodak films were scanned in the transmissive mode on a GS-710 Calibrated Imaging Densitometer from Bio-Rad using the Quantity One software package, designating a value (band area x density) to each band proportional to the amount of protein. The mean ± SE of each type of protein band in the sodium-depleted animals (n = 11) was calculated from the individual values from each animal and then compared with the mean values of the corresponding bands from the control animals (n = 11) using Student’s t-test.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Localization of FXYD2 in Renal Medulla

The overall distribution of FXYD2, the {gamma}-subunit of Na-K-ATPase, in rat kidney medulla was revealed by confocal immunofluorescence and immunoperoxidase microscopy (Fig. 1, A–F). The expression of {gamma} in the outer medulla was intense in the medullary thick ascending limb of Henle (mTAL) with the three {gamma}-antibodies applied in this study, but their distribution varied strikingly between the different subsegments of the collecting ducts in the inner medulla (IMCD1–3), as summarized in Table 1.


Figure 1
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  		Fig. 1. Localization of the {gamma}-subunit of Na-K-ATPase in kidney medulla of control rats by immunofluorescence and immunohistochemistry in the inner stripe of the outer medulla (ISOM; A and D), initial portion of inner medulla (IM1; A and D), middle portion (IM2; B and E), and tip of the papilla (IM3; C and F). Antibodies against the COOH terminus of the {gamma}-subunit were visualized with fluorescent Alexa 488-conjugated secondary antibodies (A–C), and antibodies against splice variant {gamma}a were visualized with fluorescence (D) and peroxidase-conjugated secondary antibody (E and F). In the ISOM, the thick ascending limbs of Henle are strongly labeled with both {gamma}-antibodies (A and D). In IM1, the same antibodies label only intercalated cells (IC) in the initial portion of the collecting duct (IMCD1; arrowheads in A and D). The {gamma}-subunit is also labeled in the ascending thin limbs of Henle, some of which are observed in direct continuity with medullary thick ascending limbs (asterisks in A and D). In IM2, all collecting duct (CD) cells, which in this portion are exclusively principal cells (PC), show distinct basolateral labeling for the {gamma}-subunit (B and E), and immunolabeling with anti-{gamma}a is comparable to fluorescent immunolabeling with the anti-{gamma}-subunit COOH terminus. Also in IM3, all cells in CD (open triangles in C and F) are labeled, but less intensively than in IM2. Cells of thin limb of Henle (asterisks), on the other hand, show stronger expression with both antibodies in IM3 than in IM2. Bars = 50 µm.

 

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  		Table 1. Expression of the {gamma}-subunit, FXYD2, its splice variants {gamma}a and {gamma}b, and CHIF, FXYD4, in inner stripe of outer medulla and inner medulla of kidney in control rats in different nephron segments

 
The {gamma}-subunit as detected with rabbit anti-{gamma}-subunit COOH-terminal antibodies was not observed in principal cells in IMCD1 (Figs. 1A), but in contrast to several previous studies was strongly expressed in all principal cells in IMCD2 (Fig. 1B), as well as in the tall IMCD3 cells in the papillary tip (Fig. 1C). Intercalated cells in IMCD1, located adjacent to unlabeled principal cells (Fig. 1A) and identified by their apical H+-ATPase, showed strong basolateral labeling with antibodies against the COOH terminus (Fig. 2A) and colocalization with the Na-K-ATPase {alpha}1-subunit (Fig. 2B). Weak labeling for the {gamma}-subunit COOH terminus was additionally observed in intercalated cells of outer medullary collecting ducts (not shown). All cells in IMCD2 (Fig. 2C, yellow fluorescence) and IMCD3 (not shown) revealed colocalization of {alpha}1 and {gamma}. The exact stoichiometric ratio between the {alpha}1-subunit and {gamma} along the medullary collecting duct is not known but is clearly segment specific. Thus {gamma} was expressed in all IMCD2 (Fig. 1, B and E) and IMCD3 (Fig. 1, C and F) cells, but not in IMCD1 (Fig. 1, A and D) cells, except for intercalated cells (Fig. 2A), whereas the {alpha}1-subunit was uniformly expressed along the IMCD1–2 (Fig. 2, B and C) and IMCD3 (not shown).


Figure 2
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  		Fig. 2. Double immunolabeling of {gamma} (COOH terminus) with H+-ATPase and with the {alpha}1-subunit of Na-K-ATPase in IMCD1 (A and B) and in IMCD2 (C) of control rat kidney by confocal laser-scanning microscopy. ICs exhibit basolateral labeling for {gamma} (green) and apical labeling for H+-ATPase (red), whereas PC are unlabeled (A). Colocalization (yellow) of {alpha}1 (red)- and {gamma}-subunits (green) is only present in IC (B). Colocalization (yellow) of {alpha}1- and {gamma}-subunits is observed in all IMCD2 cells (C). Bar = 20 µm.

 
The immunofluorescence observations were extended to the subcellular level by immunoelectron microscopy. Thus in IMCD1 the gold-conjugated rabbit anti-{gamma}-subunit COOH-terminal antibody labeled exclusively the greatly amplified basolateral cell membrane of intercalated cells (Fig. 3, A and B), whereas the apical cell membrane was unlabeled, as were adjacent principal cells (Fig. 3, C and D). In IMCD2 and IMCD3, the anti-{gamma}-subunit COOH terminus labeled the basolateral cell membrane of all cells (Fig. 4, A and B). Labeling was stronger along the lateral membrane up to the junctional complex than on the basal part of the basolateral membrane adjacent to the basal lamina (Figs. 3B and 4A). Antibodies against the {gamma}-subunit COOH terminus labeled in the IM the basolateral membrane of the cells in the thin loops of Henle (Fig. 4C), which were identified through their direct continuity with mTALs (Fig. 1A) and characteristic ultrastructure (22).


Figure 3
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  		Fig. 3. Immunoelectron microscopic localization of the {gamma}-subunit of Na-K-ATPase with anti-COOH-terminal antibody in IMCD1 of control rat kidney. A: low-magnification electron micrograph showing IC with luminal microprojections adjacent to a PC. Open arrows point to the lateral border between the cells. Immunogold labeling is associated with the basolateral membrane of the IC. N, nucleus. B: higher magnification from the basal portion of IC with immunolabeling of the basolateral membrane (arrows). There is more label on the lateral than on the basal part of the basolateral membrane adjacent to the basal lamina (star). C: lateral portion of IC and PC. The IC membrane is labeled (arrows), whereas the PC is unlabeled. D: basolateral portions of 2 PC showing unlabeled basal cell membranes. Bars = 1 µm (A); 0.2 µm (B–D).

 

Figure 4
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  		Fig. 4. Immunogold electron microscopic localization of the {gamma}-subunit of Na-K-ATPase with anti-{gamma}-COOH-terminal (A–C) and anti-{gamma}a (D-E)-antibodies in thin cryosections of control rat inner medulla (IM). Both lateral and basal portions of the cell membrane of principal cells in IMCD2 (A) and IM3 (B) are labeled (arrows, A and C). Basolateral membrane of cells in thin loop of Henle is labeled, but the apical membrane is unlabeled (C). Arrows indicate junctional complexes. D: immunolocalization of {gamma}a in basolateral membrane of medullary thick ascending limb. E: immunolocalization of {gamma}a in basolateral membrane of IMCD2 cell. Some of the gold particles are marked by arrows. Basal lamina is indicated with a star. Bars = 0.2 µm.

 
Splice variant {gamma}a in the inner medulla was distributed as the {gamma}-subunit detected with antibody against the {gamma}-subunit COOH terminus (Fig. 1, D–F). Thus {gamma}a was expressed in intercalated cells of IMCD1 and in principal cells in IMCD2–3. Labeling for splice variant {gamma}a was also observed in intercalated cells of outer medullary collecting ducts (not shown). At the electron microscopical level, splice variant {gamma}a was distinctly associated with the basolateral membrane of mTAL (Fig. 4D) as well as IMCD2 (Fig. 4E) and IMCD3 cells (not shown).

Splice variant {gamma}b was not detected with specific {gamma}b-antibodies in cells of the inner medulla but was strongly expressed in mTAL in the ISOM (Fig. 5).


Figure 5
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  		Fig. 5. Single (A, B, F, and G) or double (C–E) labeling for FXYD proteins in the outer medulla and IM. Immunolocalization of {gamma}b splice variant of Na-K-ATPase at the border between the IM and outer medulla of a control (A) and a sodium-depleted (B) rat. Immunolabeling for {gamma}b is lacking in both the IMCD of control (A) and sodium-depleted rats (B) but is strong in medullary thick ascending limb in the ISOM. Immunofluorescence in the medullary thick ascending limb is more intense in the sodium-depleted rat than in control rat. Bars = 100 µm (A and B). In IM1, PC, but not IC, show basolateral label for corticosteroid hormone-induced factor (CHIF; green), whereas IC express apical H+-ATPase (red, C). D: colocalization (yellow) of {alpha}1-subunit (red) and CHIF occurs in PC in IM1, but only {alpha}1 is present in IC. E: CHIF and {alpha}1-subunit are colocalized (yellow) in all cells of IMCD2. Consecutive 2-µm sections labeled for CHIF (F) and COOH terminus of {gamma} (G) in IM2 are shown. The same cells are labeled for both CHIF and {gamma} (compare cells marked with asterisks in F and G). Bars = 20 µm (C–G).

 
Localization of FXYD4 in Renal Medulla

FXYD4, CHIF, was present in the collecting ducts of outer and inner medulla, including the papillary tip, as previously documented (33), but was absent in other tubular segments. It was expressed in all IMCD1 cells, except intercalated cells identified by anti-H+-ATPase (Fig. 5, C and D) and in all IMCD2 (Fig. 5, E and F) and IMCD3 cells (not shown). Importantly, serial 2-µm-thin sections, labeled alternatively with antibodies against CHIF and the {gamma}-subunit COOH terminus, clearly demonstrated that both antibodies labeled the basolateral domain of the same cells in IMCD2 (compare star-labeled cells in Fig. 5, F and G). CHIF also showed colocalization with the Na-K-ATPase {alpha}1-subunit in collecting duct cells (Fig. 5E) in the inner medulla except intercalated cells in IMCD1 (Fig. 5D).

By electron microscopy, CHIF was labeled in the basolateral membrane of all cells in the IMCD except intercalated cells (Fig. 6A). Also, here there was stronger labeling of the lateral than of the basal part of the basolateral membrane (Fig. 6A). High-resolution double immunolabeling for the {alpha}1-subunit and CHIF, using 5- and 10-nm colloidal gold particles, respectively, often showed a center-to-center distance of ~25 nm between two particles (Fig. 6B). This suggests that these two proteins are located very close in the membrane. Whether the proteins are in direct contact would require presently unavailable information about the steric interaction between the primary and secondary antibodies.


Figure 6
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  		Fig. 6. Immunogold electron microscopic localization of CHIF in thin cryosections of control rat IM demonstrating distinct labeling in the lateral and basal part of the basolateral membrane of PC in IM2. A: basal lamina is marked with a star. B: double immunolabeling for CHIF (10-nm gold) and {alpha}1-subunit of Na-K-ATPase (5-nm gold). In some places (within small circles), gold particles of different sizes are ~25 nm apart (24 ± 4 nm; n = 9). Bars = 0.2 µm.

 
Effects of NaCl Depletion on FXYD Proteins

Differences between sodium-depleted and corresponding control rats were observed by immunofluorescence only with respect to the expression of {gamma}b in the ISOM. Thus there was a stronger fluorescence for {gamma}b in mTAL in NaCl-depleted rats than in controls (Fig. 3, A and B). Both intercalated and principal cells in IMCD1, like cells in the loop of Henle, were unstained for {gamma}b in both conditions. No immunocytochemical differences were observed in the expression of CHIF in the renal medulla in response to sodium depletion (not shown). Furthermore, we did not observe changes in the cellular composition of the collecting duct, as reported following lithium-induced nephrogenic diabetes insipidus (12). Functional data for NaCl-depleted and control rats are shown in Table 2. In NaCl-depleted rats, plasma aldosterone levels were significantly increased, consistent with activation of the renin-angiotensin system. This was associated with significantly decreased values of urinary sodium and chloride concentrations, as well as urine osmolality compared with control animals. In contrast, body weight, plasma sodium, plasma potassium, plasma creatinine, and plasma osmolality did not show significant changes following sodium depletion.


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  		Table 2. Functional data for control and sodium chloride-depleted rats

 
Quantitation of FXYD Proteins in Control and NaCl Depletion

Anti-{gamma}-subunit COOH terminus, anti-{gamma}a-, anti-{gamma}b-, and anti-CHIF antibodies were applied on Western blots from the different zones of the control kidney. Anti-{gamma}-subunit COOH terminus applied to tissue from the ISOM recognized a doublet, a higher and a lower molecular mass band (Fig. 7, lane A). The higher molecular mass band corresponded to the band labeled with anti-{gamma}a-antibodies (Fig. 7, lane B), and the lower molecular mass band corresponded to the band labeled by anti-{gamma}b-antibodies (Fig. 7, lane C). Mixing anti-{gamma}a- and anti-{gamma}b-antibodies also gave two bands in the ISOM (Fig. 7, lane D), and regardless in which combination the antibodies were used, there was no indication of additional bands besides the two observed in Fig. 7, A–D. When the same anti-{gamma}-antibodies were reacted with blots from the IM, the anti-{gamma}-subunit COOH-terminal antibody revealed a doublet (Fig. 7, lane E). The higher molecular mass band corresponded to {gamma}a as seen from the reaction with the {gamma}a-antibody (Fig. 7, lane F). The molecular mass of the lower band is similar to {gamma}b. However, it cannot be the same {gamma}b because this band did not react with anti-{gamma}b-antibodies (Fig. 7, lane G). Thus the {gamma}b-antibody exclusively recognized the {gamma}b-variant in the ISOM (lane C), whereas the {gamma}-subunit COOH-terminal antibody recognized two proteins both in the IM and the ISOM: {gamma}a and {gamma}b in the ISOM and {gamma}a and an unknown {gamma}b-like protein in the IM.


Figure 7
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  		Fig. 7. Western blots showing expression of the {gamma}-subunit of Na-K-ATPase in the ISOM and the IM of control rat kidney using antibodies against the {gamma}-subunit COOH terminus and splice variants {gamma}a and {gamma}b. Anti-{gamma}-COOH-terminal antibodies recognize 2 bands in the ISOM, a higher molecular mass and a lower molecular mass band (lane A). The higher molecular mass band corresponds to {gamma}a (lane B), whereas the lower molecular mass band corresponds to {gamma}b (lane C). Mixing of anti-{gamma}a- and anti-{gamma}b-antibodies also gives 2 bands (lane D). The anti-{gamma}-COOH-terminal antibody also reveals 2 bands in IM membrane samples, a higher molecular mass band (being {gamma}a, lane F), and a lower molecular mass band with a similar molecular mass as {gamma}b observed in blots from the ISOM (lane E). However, the specific anti-{gamma}b-antibody does not recognize any bands in blots from the IM (lane G).

 
The quantitative zonal levels of the {gamma}-subunit COOH terminus, {gamma}b, and CHIF in control and NaCl-depleted rats are shown in Fig. 8. In protocol 2 (sodium depletion), there was a significant increase (29%) in {gamma}b expression in the ISOM, complete absence in the IM, and no significant changes in cortex. Both the {gamma}-subunit COOH terminus and CHIF showed unchanged levels in all zones of control and sodium chloride depleted animals.


Figure 8
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  		Fig. 8. Quantitative zonal expressions in the IM, ISOM, and cortex of the {gamma}-subunit COOH terminus, splice variant {gamma}b, and CHIF in 11 control (open bars) and 11 NaCl-depleted (filled bars) rats (protocol 1 vs. protocol 2). Samples containing equal amounts of protein from the IM, ISOM, or cortex were run in parallel in the same blot. The {gamma}-subunit COOH terminus was expressed at similar levels in control and depleted rats in all three zones. Splice variant {gamma}b was not detected in the IM with the specific anti-{gamma}b-antibody. However, in the ISOM of NaCl-depleted rat kidney {gamma}b is expressed significantly more than in control rats. No difference is observed in the cortex. CHIF is expressed at similar levels in the IM, ISOM, and in the cortex in control and depleted rats. Differences between NaCl-depleted and control rats are significant in the ISOM for {gamma}b (P < 0.05).

 

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The present study demonstrates that FXYD2, the {gamma}-subunit of Na-K-ATPase, and its splice variants {gamma}a and {gamma}b have a more complex distribution in the renal medulla than previously demonstrated and addresses the question of to what extent, if any, {gamma} and CHIF abundances are regulated during extensive NaCl deprivation. The reason {gamma} was previously largely overlooked in the IM may be due to its lower abundance in the IM compared with the ISOM. Furthermore, we have used different preparation procedures, including optimized perfusion fixation, antigen retrieval, and electron microscopic methods. The results show that anti-{gamma}-subunit COOH-terminal antibodies label a different protein than {gamma}b in the IM. Whereas antibodies against {gamma}b stained mTAL cells intensively in the ISOM, and gave one single band in the ISOM blots, they did not label any cells in the IM and were not detected in IM blots. Anti-{gamma}-subunit COOH-terminal antibodies, on the contrary, revealed two bands in immunoblots of the IM, one with a mass comparable to {gamma}a and one comparable to {gamma}b. The latter band cannot represent the same protein that reacted with the specific anti-{gamma}b antibodies in the ISOM, because {gamma}b was seen neither in blots (Fig. 7G) nor by immunofluorescence in the IM (Fig. 5). The identity of this lower molecular mass protein in the IM is presently not known. It possesses the same COOH terminus as {gamma}b in the ISOM, because it is recognized by the anti-{gamma}-subunit COOH-terminal antibody (Fig. 7E) but apparently has a different NH2 terminus than {gamma}b in the IM. It is possible that it represents a cell-specific posttranslational modification or cleavage product of {gamma}a or {gamma}b that is not recognized by anti-{gamma}a- or anti-{gamma}b-antibodies. Thus our work suggests the presence of at least three different {gamma} proteins in rat kidney medulla.

Extensive NaCl depletion for 10 days did not cause significant changes in abundances of {gamma}, as determined with {gamma}-subunit COOH-terminal antibodies, or of CHIF (Fig. 8). Previous studies have shown unchanged abundance of the Na-K-ATPase {alpha}1-subunit in whole kidney homogenates following a very similar prolonged sodium depletion (26). Although extensive NaCl depletion in these experiments did not induce any significant changes in {gamma}-subunit COOH-terminal abundance, there were large changes in urine composition, including a 25-fold decrease in sodium (Table 2). This observation may be compared with the situation in {gamma} knockout mice, where {gamma} was completely absent, but urine composition was nevertheless not different from controls, although the sodium affinity of the isolated enzyme showed a slight increase (21). These two lines of in vivo observations indicate that {gamma} may be a less strong determinant for urine electrolyte composition than suggested by the well-documented in vitro effects on Na-K-ATPase quoted in the introduction to this study. Instead, urine electrolyte composition may primarily be regulated by several other sodium transport systems, such as the NaCl cotransporter and the epithelial sodium channel, that exhibit significant changes during NaCl depletion (26).

Despite unchanged levels of the {gamma}-subunit COOH terminus, an increase in {gamma}b was observed in the ISOM. A possible explanation is that the different varieties of {gamma}-subunits are regulated differently during sodium depletion by the activated renin-angiotensin system. Thus the lower band detected in the IM with the {gamma}-subunit COOH-terminal antibodies, but not with {gamma}b-antibodies, may also be present in the ISOM. If these proteins are regulated in opposite ways, the {gamma}-subunit COOH terminus will appear unchanged (Fig. 8). To what extent the change in {gamma}b abundance in the ISOM is related to changes in sodium pump activity in mTAL remains to be established. It has previously been demonstrated in mice that hydration, with a fivefold decreased urine osmolality, reduced the abundance of Na-K-ATPase {alpha}1-subunit in the IM (10). In the present study, the rats were not diuretic (Table 2), and there was only a 26% decrease in urine osmolality. No changes in abundances of {gamma} or CHIF were observed in the cortex, suggesting that these proteins are not regulated in the cortex in response to NaCl depletion. Aldosterone has been shown to increase the CHIF mRNA level in the colon (40), and following a high-K+, low-sodium diet, CHIF protein can also be upregulated in the kidney (33). This was not observed in the present quantitative analysis, possibly because the diet contained slightly less potassium and the rats were ration-fed with a constant salt and water intake. In CHIF knockout mice, no abnormalities of salt and water balance were observed under resting conditions (20). Immunoprecipitation experiments have shown that different {gamma} and CHIF complexes may exist in IMCD, such as {alpha}/beta/{gamma}a or {alpha}/beta/CHIF, but not {alpha}/beta/{gamma}/CHIF (18). Our double immunolabeling, serial section staining, and Western blot analyses also suggest that {gamma} and CHIF in the IM may be associated with Na-K-ATPase in several ways. The presence of {alpha}1 and {gamma}, and the lack of CHIF, in the basolateral membrane of intercalated cells in IMCD1 thus suggests that {alpha}/beta/{gamma}a complexes are present in intercalated cells, whereas principal cells in IMCD1 may have only {alpha}/beta or {alpha}/beta/CHIF. The presence of {alpha}/beta/CHIF complexes is supported by high-resolution immunoelectron microscopy showing close association between immunogold labels for {alpha} and for CHIF in the basolateral membrane (Fig. 6B).

The distributions of {gamma} and its splice variants and CHIF raise the question as to their functional roles in the medulla. In vitro, the functional effects of CHIF and {gamma} are well established and the {gamma}-subunit and CHIF appear to modulate Na-K-ATPase in opposite ways as quoted above. Serial sections demonstrate that both CHIF and {gamma} are present in the same principal cells in IMCD2 (Fig. 5, F and G), although not necessarily in the same molecular complex as shown by immunoprecipitation (18). The presence of both these modulators in the same IMCD2 cells in theory suggests that they may influence the Na-K-ATPase pump in opposite ways, depending on their relative abundances. In the absence of significant changes in their abundances, it is possible that there are regulatory mechanisms, yet unknown, that determine whether the {alpha}/beta complex binds {gamma} or CHIF and thereby modifies the pump.

Intercalated cells are morphologically similar in outer medullary collecting ducts and IMCD (13, 22, 25). In the IM, all intercalated cells are type A cells (for a review, see Ref. 39) with apical vacuolar H+-ATPase (8) and the basolateral anion exchanger AE-1. They also show strong expression of the {gamma}-subunit COOH terminus and splice variant {gamma}a, in contrast to adjacent principal cells in IMCD1 (Fig. 1, A and D, Fig. 2, A and B, Fig. 3, B–D, Fig. 5, C and D). This suggests a different functional role for {gamma} in intercalated cells than in principal cells in IM1, because the expressions of the {alpha}-subunit in these two cell types in IM1 are comparable (32). Furthermore, the observations specify that the splice variant is {gamma}a and not {gamma}b, thus suggesting that the splice variants may also have different functions in intercalated cells. The basolateral membrane of the IMCD3 cells in the terminal IMCD is strongly labeled with {gamma}-subunit COOH-terminal and {gamma}a-antibodies (Fig. 1, C and F, Fig. 4B). Interestingly, immortalized mouse IMCD3 cells cultured under isotonic conditions do not contain any {gamma}, but express {gamma} when challenged osmotically to 550 mosmol/kgH2O (9). The upregulated {gamma} becomes expressed in the basolateral cell membrane of the cultured cells (29). In the present experiments, {gamma} was still present in IMCD3 cells following sodium depletion, which may relate to the fact that the urine osmolality remained above 750 mosmol/kgH2O (Table 2).

In summary, we have extended the information on the location of the {gamma}-subunit and CHIF in rat renal medulla, in particular as related to IMCD cell types. Furthermore, we have demonstrated that {gamma} and CHIF can be located in the same cells in IMCD2 and IMCD3 and observed physical CHIF-{alpha}-subunit interaction in the basolateral cell membrane. Importantly, prolonged sodium depletion did not induce significant changes in the abundances of the {gamma}-subunit COOH terminus and CHIF in different kidney zones, as determined by semiquantitative immunoblotting and confocal immunofluorescence microscopy. The unchanged levels of these two FXYD proteins suggest that they are not primary determinants for urine electrolyte composition during prolonged NaCl depletion.


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This study was supported by the Water and Salt Research Center, established and supported by the Danish National Research Foundation (Grundforskningsfonden), the Danish Medical Research Council, the Karen Elise Jensen Foundation, and the Israel Science Foundation. H. Garty and S. J. D. Karlish are the incumbents of the Hella and Derrick Kleeman and William Smithburg Chairs of Biochemistry, respectively.


   ACKNOWLEDGMENTS
 
We thank Else-Merete Løcke, Karen Thomsen, Gitte Kall, Kirsten Peterslund, and Albert Meyer for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. B. Maunsbach, The Water and Salt Research Ctr, Institute of Anatomy Univ. of Aarhus, DK-8000 Aarhus, Denmark (e-mail: Maunsbach{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.


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