Distribution and oligomeric association of splice forms of
Na+-K+-ATPase regulatory
-subunit in rat
kidney
Elena
Arystarkhova,
Randall K.
Wetzel, and
Kathleen J.
Sweadner
Laboratory of Membrane Biology, Neuroscience Center,
Massachusetts General Hospital, Charlestown, Massachusetts 02129
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ABSTRACT |
Renal Na+-K+-ATPase is associated
with the
-subunit (FXYD2), a single-span membrane protein that
modifies ATPase properties. There are two splice variants with
different amino termini,
a and
b. Both
were found in the inner stripe of the outer medulla in the thick
ascending limb. Coimmunoprecipitation with each other and the
-subunit indicated that they were associated in macromolecular complexes. Association was controlled by ligands that affect
Na+-K+-ATPase conformation. In the cortex, the
proportion of the
b-subunit was markedly lower, and the
a-subunit predominated in isolated proximal tubule
cells. By immunofluorescence, the
b-subunit was detected
in the superficial cortex only in the distal convoluted tubule and
connecting tubule, which are rich in
Na+-K+-ATPase but comprise a minor fraction of
cortex mass. In the outer stripe of the outer medulla and for a short
distance in the deep cortex, the thick ascending limb predominantly
expressed the
b-subunit. Because different mechanisms
maintain and regulate Na+ homeostasis in different nephron
segments, the splice forms of the
-subunit may have evolved to
control the renal Na+ pump through pump properties, gene
expression, or both.
sodium pump; nephron; immunofluorescence; confocal microscopy
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INTRODUCTION |
THE KEY ENZYME
that maintains a low intracellular concentration of Na+
inside the cell by generation of an electrochemical gradient across the
cell membrane is Na+-K+-ATPase. The enzyme has
two necessary subunits,
and
, but in the kidney it is tightly
associated with a small, 7.4-kDa protein, the
-subunit (25,
42). The
-subunit is a type I, single-span membrane protein
with the amino terminus exposed outside the cell (11, 59).
Expression of the
-subunit at the mRNA or protein level is almost
exclusively in the kidney (42, 59), although our recent
analysis of the expressed sequence tag database revealed other tissues,
such as placenta and mammary gland, as potential sources
(56). On the basis of sequence homology, the
-subunit belongs to a family of small (7.5- to 19-kDa) single-span membrane proteins, the FXYD family. The family also includes phospholemman (46), mammary tumor antigen-8 (MAT-8) (44),
channel-inducing factor (CHIF) (7), RIC (i.e., related to
ion channel) (26), and two new gene products (FXYD6 and
FXYD7) (56). Most of the homology is within the
transmembrane region and the extracellular PFXYD motif, which is
characteristic of the family. Outside the core motif, the
-subunit
sequence is unique.
In the kidney, the
-subunit occurs as two splice variants,
a and
b (34, 35, 55, 56).
The mRNA splice affects protein sequence only at the amino terminus.
These splice forms were found in independent cDNA libraries derived
from multiple organ types, predominantly from fluid- and
solute-transporting tissues, and the sequences of the homologs from
human, mouse, and rat were obtained (55). We also cloned
and sequenced
b-subunit cDNA from the rat (GenBank
AF233060). Recently, the genomic organization of the human FXYD2 gene
encoding the
-subunit of Na+-K+-ATPase was
described (41, 57). Two different promoter regions were
identified, thus providing a potential structural basis for differential regulation of expression of the splice variants of the
-subunit.
It is established that the
-subunit is not required for catalytic
activity (27, 51), but it has been implicated to play a
modulatory role in the function of the Na+ pump.
Experiments with different expression systems revealed that the
-subunit could alter
Na+-K+- ATPase affinity for
extracellular K+ in a voltage-dependent manner when
expressed in oocytes (10, 11), increase affinity for
ATP (48, 59, 60), or reduce apparent affinity for
Na+ and/or K+ (5, 10, 48). The
modulation of apparent Na+ affinity by the
-subunit
appeared to be abolished by a posttranslational modification of the
protein that occurred in cell culture (5). Interestingly,
the mechanism of the effect on apparent Na+ affinity was
ascribed to an increase in K+ antagonism of cytoplasmic
Na+ activation (48), similar to
tissue-specific kinetic differences observed earlier (45,
61).
With antibodies to the
-subunit, we recently showed that the
-subunit is not expressed everywhere in the kidney but instead has a
very specific distribution to certain nephron segments: the
-subunit
colocalized with the
1-subunit in all nephron segments except the cortical thick ascending limb (CTAL) and cortical collecting ducts (CCD) (5, 62). Here, evidence is presented that
splice variants of the
-subunit are differentially distributed in
renal segments. Where coexpressed, the alternative splice forms
appeared to be able to associate with the same macromolecular
Na+-K+-ATPase complex.
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MATERIALS AND METHODS |
Antibodies.
Splice-specific antibodies RNGA (rat amino terminus of the
a-subunit) and RNGB (rat amino terminus of the
b-subunit) were raised at Quality Control Biochemicals
(Hopkinton, MA) against the peptides Ac-TELSANHGGC and MDRWYLGGC,
corresponding to the amino-terminal sequences of
a- and
b-subunits, respectively. The peptides were conjugated
to tetanus toxoid as a carrier through the specially added cysteine
residues and injected into rabbits (two for each peptide). The
conjugate was chosen specifically to boost the immune response, since
neither peptide was predicted to be highly immunogenic. The specificity
of the sera was tested by ELISA with BSA-coupled peptides on solid
phase. The titers of crude sera against the peptides were >20,000
(RNGA) and >60,000 (RNGB) for the rabbits with better titer.
The RNGA antiserum was affinity purified against the immunizing peptide
on a SulfoLink column (Pierce, Rockford, IL). For some
immunofluorescence experiments, we employed an anti-TELSANHC
affinity-purified antibody kindly provided by S. J. D. Karlish (Weizmann Institute). Another antipeptide antibody (RCT-G1),
developed previously (5), was employed to detect the COOH
terminus of the
-subunit, which is identical in both splice
variants. Monoclonal anti-
-subunit antibody McG-11H was isolated
from a BALB/c mouse injected with the synthetic peptide CGGSKKHRQVNEDEL (corresponding to
the COOH-terminal 15 amino acids of the rat
-subunit) bound to
keyhole limpet hemocyanin. Monoclonal antibody McK1 (19)
was used to detect the
1-subunit of
Na+-K+-ATPase. Antipeptide antibody KETYY (gift
of J. Kyte, University of California, San Diego), specific for the COOH
terminus of the
-subunit of Na+-K+-ATPase
(9), was used in ELISA experiments. Monoclonal antibody VG4 (gift of N. Modyanov, Medical College of Ohio) against
Na+-K+-ATPase
-subunit was used for
immunoprecipitation (4).
Membrane preparation and characterization.
Isolation of membranes (microsomal fraction) containing
Na+-K+- ATPase from rat kidney outer medulla
or cortex by differential centrifugation was performed as described by
Jørgensen (31). Purified enzyme used in Fig.
1 was prepared by SDS extraction (31). Cortical tubule cells were prepared as described
previously (52). Briefly, two rats were euthanized, and
the kidneys were perfused with 300 ml of PBS. Freshly isolated renal
cortex was minced on ice to a pastelike consistency in 15 ml of DMEM in
an atmosphere of 95% O2-5% CO2 and incubated
with 6 mg/ml collagenase (Worthington; 269 U/mg) for 60 min at 37°C
and then chilled on ice. A cell suspension was obtained by pouring the
paste through graded sieves (180, 75, 63, and 53 µm). The cells were
washed four times by centrifugation at 100 g for 4 min
at 4°C. The last pellet was resuspended in DMEM. Electrophoresis was
carried out in SDS-N-[2-(hydroxymethyl)ethyl]glycine
(Tricine) gels (50) with 12.5% acrylamide. Proteins were
transferred to nitrocellulose, incubated with antibodies, and detected
with chemiluminescence using luminol reagents (Pierce). Blots were
scanned with a laser densitometer (Molecular Dynamics).

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Fig. 1.
Different electrophoretic mobilities of a-
and b-subunits on SDS gels. A: rat kidney
outer medulla microsomes (5 µg) were dissolved in electrophoresis
sample buffer, loaded in a wide lane, run on an
SDS-N-[2-(hydroxymethyl)ethyl]glycine (Tricine) gel, and
transferred to nitrocellulose. The blot was cut vertically in the
middle of the lane, and lane 1 was stained with RCT-G1
antibody (the shared COOH terminus of the -subunit splice forms) at
1:5,000 and lane 2 with the RNGB antibody
( b-subunit specific) at 1:5,000. The faster-migrating
band was identified as the b-subunit. B:
purified rat kidney outer medulla enzyme (25 µg) was similarly
resolved in a wide lane of a gel. The blot was cut into 3 pieces and
stained as follows: RCT-G1 at 1:10,000 (lane 3), Karlish
a-subunit antibody at 1:75 (lane 4), and RNGB
at 1:10,000 (lane 5). The slower-migrating band was
identified as the a-subunit.
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Immunoassay.
Antibody binding was measured using an ELISA. Titers were measured on
preparations of crude rat renal membranes from medulla or cortex
adsorbed to Nunc MaxiSorp Immunoplates. PBS containing 0.5% BSA and
0.1% Tween 20 was used for washing and dilution. The secondary
antibody was biotinylated goat anti-rabbit IgG, and color was developed
with streptavidin-conjugated horseradish peroxidase and
o-phenylenediamine (Bethesda Research Laboratories).
Immunoprecipitation.
Membranes from rat kidney outer medulla (1 mg/ml) were solubilized with
n-dodecyl octaethylene glycol monoether detergent (C12E8, 3 mg/ml; Calbiochem) for 10 min at room
temperature in a buffer containing 25 mM imidazole, pH 7.3, and 1 mM
EDTA. The extract was diluted with two volumes of detergent-free
buffer, insoluble material was precipitated by centrifugation for 30 min at 20,000 g (4°C), and the pellet was checked for
residual Na+-K+-ATPase. The supernatant was
incubated overnight with primary antibodies (RNGA, RNGB, or VG4) at
4°C. The immune complexes were collected after 2 h of incubation
with secondary goat anti-rabbit or goat anti-mouse IgG antibodies
covalently bound to magnetic beads (Bio-Mag, PerSeptive Diagnostics,
Cambridge, MA). The samples were washed four or five times in
solubilization buffer containing 0.05% C12E8.
Final precipitates were resuspended in electrophoresis sample buffer
containing 62.5 mM Tris-Cl, pH 6.8, 2% SDS, 1%
-mercaptoethanol, and 10% glycerol and heated for 15 min at 65°C before they were loaded on SDS-Tricine gels. Alternatively, rat kidney membranes were
solubilized with 1 or 0.3%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in a buffer containing 20 mM HEPES, pH 7.3, and 100 mM NaCl.
The rest of the procedure was the same as that described above. To test
the effect of Na+ pump ligands on the immunoprecipitation
of the
-subunit splice variants, membranes from rat kidney outer
medulla were first resuspended in a buffer containing 20 mM HEPES, pH
7.3, supplemented with 1) 100 mM NaCl, 2) 20 mM
KCl, 3) 100 mM NaCl, 5 mM MgCl2, and 5 mM ATP,
or 4) 5 mM MgCl2 and 5 mM Pi, with
or without 1 mM ouabain. Solubilization with
C12E8 was carried out as described above, and
the buffer composition was the same for all subsequent steps, except
detergent concentration was reduced from 0.1 to 0.05% during the washes.
Immunofluorescence.
Immunocytochemistry was performed as described elsewhere in more detail
(5, 62). Rats were given food and water ad libitum. Cryostat sections of periodate-lysine-paraformaldehyde-fixed rat kidney, sometimes treated with SDS for antigen retrieval, were concurrently probed with a mixture of mouse monoclonal anti-
-subunit COOH terminus antibody (McG-11H, 1:50) and either rabbit polyclonal anti-
b-subunit (RNGB, 1:500) or Karlish
anti-
a-subunit (1:100) antibody. The sections were
subsequently incubated with a mixture of Cy3-conjugated goat anti-mouse
IgG (1:300; Accurate, Westbury, NY) and FITC-conjugated goat
anti-rabbit IgG (1:200; Jackson ImmunoResearch, West Grove,
PA). Slides were examined, and images were collected on a Nikon TE300
fluorescence microscope equipped with a Bio-Rad MRC 1024 scanning laser
confocal system (version 3.2).
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RESULTS |
Mobility of
a- and
b-subunits on SDS
gels.
To assess distribution of the
-subunit splice variants in the
kidney, splice-specific antibodies were developed. The difference between the splices is restricted to the short amino-terminal segment
[8 amino acids in the
a-subunit are replaced with 6 residues in the
b-subunit (55)], and mass
spectroscopy indicates that the initiation methionine is removed from
the
a-subunit (35; Arystarkhova E and Sweadner KJ,
unpublished observations). Thus the following sequences were chosen as
the antigens: TELSANHGGC (
a-subunit) and MDRWYLGGC
(
b-subunit).
The specificity of the antibodies for the
-subunit of
Na+-K+-ATPase was first tested by Western blot
analysis. Figure 1A demonstrates immunostaining of rat
kidney microsomes. Unfortunately, RNGA
(
a-subunit-specific antibody) failed to recognize the
SDS-denatured protein, although it was active in other assays. RNGB
clearly recognized the lower band in the
-subunit doublet, implying
that the
b-subunit had a faster electrophoretic mobility
on SDS gels than the
a-subunit, despite the calculated
molecular masses (7,126 for the
a-subunit without
methionine and 7,238 for the
b-subunit, with the
assumption of no chemical modification of the primary structure).
Specific staining of the
a-subunit with the Karlish
a-subunit antibody is shown in Fig.
1B. This antibody, like RNGA, was also weak on blots, and to detect stain it was necessary to load a large amount of
purified enzyme, instead of microsomes and to use the antibody at a
high concentration. Heavy gel loading precluded good resolution of the
doublet; nonetheless, the
a-subunit migrated more slowly than the
b-subunit. The relative gel mobilities are in
agreement with results of Küster et al. (35), who
also measured mass values (with carboxyamidomethyl cysteine) of 7,184.0 for the
a-subunit (no methionine) and 7,337.9 for the
b-subunit (N-acetylated), and also with in vitro
synthesis experiments of Beguin et al. (10). This suggests
that factors other than peptide molecular mass (such as net charge or
labile posttranslational modification) influence electrophoretic
mobilities of the
-subunit splice variants on SDS gels.
Posttranslational modification of the
-subunit, detected in
transfectants or by in vitro translation with microsomes, has been
reported to generate doublets (5, 10, 42, 60). However, in
our hands and in the report of Beguin et al. (10), these
migrate closer together than
a- and
b-subunits, and no one has reported resolving four such
bands in preparations from the kidney. The
a-subunit
from renal cortex migrated more slowly than the
a-subunit from the medulla (see below), and so much is
yet to be determined about the factors that influence gel migration.
Immunoprecipitation of a
Na+-K+-ATPase
macromolecular complex.
Both antibodies were effective in immunoprecipitation and
coprecipitated the Na+-K+- ATPase
-subunit, validating the specificity of RNGA as well as RNGB.
(Coprecipitation with the
-subunit was not investigated, because it
migrates too close to immunoglobulin heavy chain on Tricine gels, but
it would be expected to be present on the basis of the literature.)
Surprisingly, evidence was found that the RNGA and RNGB antibodies
could precipitate each other's antigen as well. Rat renal medulla
microsomes were first solubilized with the nonionic detergent
C12E8, and then RNGA or RNGB serum was applied.
Immune complexes were collected and resolved on SDS gels, and the blots
were stained with the antibody against the shared COOH terminus,
RCT-G1. As shown in Fig. 2A,
addition of RNGA or RNGB antibody resulted in precipitation of a
complex containing
1-,
a-, and
b-subunits of Na+-K+-ATPase. The
ratio of one splice variant to another was similar in the starting
material and the final precipitates as judged by densitometry analysis.
The reaction was specific, since none of the subunits was precipitated
with preimmune serum (control lanes in Fig. 2A). Additional
bands in the top portion of the gel represent heavy and light chains of
immunoglobulins, since these bands were also seen in the control
samples containing no rat kidney microsomes (not shown). Figure
2B shows a similar experiment, but precipitation was with
RNGA compared with precipitation with an
-subunit-specific antibody,
and for final detection RNGB and RCT-G1 antibodies were used. Detection
of the
b-subunit in the RNGA precipitate strongly
suggests that the
-subunit splice variants in medullary membranes
associate in the same complex. Similar results were obtained with
low-salt buffers (Fig. 2A) and NaCl-containing buffers (Fig.
2B), suggesting that ionic strength was not a major factor.

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Fig. 2.
Coimmunoprecipitation of -subunit splice variants.
A: rat kidney outer medulla microsomes were treated with
n-dodecyl octaethylene glycol monoether
(C12E8) detergent in buffer A
containing 25 mM imidazole and 1 mM EDTA, pH 7.3. Immunoprecipitation
was carried out with splice-specific antibodies RNGA
(IP- a) or RNGB (IP- b) or with preimmune
sera (pre- a and pre- b). The blot was cut
horizontally in the middle, and the lower portion was stained with
RCT-G1 antibody ( a + b) and the
upper portion with McK1 ( 1); only the relevant portions
of the gel are shown. B: rat kidney outer medulla microsomes
were solubilized with C12E8 in buffer
B containing 20 mM HEPES, pH 7.3, and 100 mM NaCl and precipitated
with RNGA or the monoclonal antibody VG4 ( -subunit specific).
Identical blots were stained with McK1 ( 1) and RCT-G1
( a + b) or McK1 ( 1)
and RNGB ( b). The pellet represents unsolubilized
material, which was minimal. C: rat kidney outer medulla
microsomes were solubilized with 1 or 0.3% (wt/vol)
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in
buffer B and precipitated with RNGA or RNGB antibody. Blots
were stained with McK1 ( 1) and RCT-G1
( a + b). Results illustrate
coprecipitation of a- with b-subunit in
several conditions, notably low salt and NaCl.
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A similar composition of complexes was seen after solubilization with a
charged detergent, CHAPS. A proportional amount of the
1-subunit and both splice variants of the
-subunit
were recovered with RNGA or RNGB antibodies (Fig. 2C). If
the coprecipitation of subunits were due to incomplete dissociation of
supermolecular assemblies (such as complexes with cytoskeletal
components) by the detergent, one might expect it to be enhanced by
reducing the concentration of detergent. Reducing the CHAPS
concentration from 1 to 0.3%, from above to below its critical micelle
concentration, however, just reduced the solubilization of the enzyme
and the efficiency of immunoprecipitation, while the ratio of one
subunit to another in the immunoprecipitates was not changed. Thus the data suggest a strong interaction between the
1- (or
1-) and
-subunits within the ATPase complex in rat
kidney outer medulla membranes. The results do not rule out association
within one functional unit (i.e., 

2), but it is
more likely that it requires more complex multimeric assembly, for
example, a dimer or tetramer of the
-subunits with associated
-
and
-subunits, such as the (

)2- or
(

)4-subunit.
Further evidence that
-subunit association with the other
Na+-K+-ATPase subunits is specific is that the
stability of the complex proved to be sensitive to the enzyme ligands
present during the immunoprecipitation.
Na+-K+-ATPase goes through a series of
conformation changes during its turnover: in simplified form, E1
E1-P
E2-P
E2
E1, in which P represents phosphate
(transferred from ATP) covalently bound to an aspartate in the active
site. The best precipitation by RNGA and RNGB antibodies was obtained
in buffers with NaCl (Fig. 3, A and
C). This condition favors the E1 conformation. Buffers with
KCl, which favors E2, showed greatly reduced precipitation at the same
ionic strength. In tests of enzyme stability in the conditions used for
precipitation, we found that, in Na+, 45% of the activity
was retained after 30 min and 36% after 3 h. In K+,
75% was retained after 30 min and 59% after 3 h. This should eliminate the hypothesis that precipitation is reduced in
K+ because the enzyme is less stable. Greater stability in
K+ has been reported elsewhere (24, 32).

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Fig. 3.
Ligand-dependent association of -subunit with
Na+-K+-ATPase. Rat kidney outer medulla
microsomes were solubilized with C12E8 in 20 mM
HEPES, pH 7.3, supplemented with 100 mM NaCl (A), 20 mM KCl
(B), 100 mM NaCl, 5 mM MgCl2, and 5 mM ATP
(C), or 5 mM MgCl2, 5 mM Pi, and 1 mM ouabain (D) and precipitated with RNGA
(IP- a) or RNGB (IP- b) antibody.
"Pellet" is shown to demonstrate efficiency of solubilization. As
in Fig. 2, the blot was cut so that - and -subunits could be
stained separately. HC, heavy chain; LC, light chain. Results
illustrate coprecipitation of -subunit in conditions that favor
Na+-K+-ATPase E1 conformation and lability of
the complex in conditions that favor the E2 or E2-P conformation.
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The E2 conformation can also be obtained in the presence of NaCl by
including ATP and Mg2+, which causes the enzyme to be
phosphorylated and adopt the E2-P conformation without greatly changing
the precipitation conditions. This caused a substantial reduction in
precipitation and an apparent loss of coprecipitation of the
a-subunit with the
b-subunit, as judged
by gel mobility (Fig. 3C). Another way to arrive at the E2-P
conformation, the so-called "backdoor" way, is to incubate the
enzyme with its product, Pi, in the presence of
Mg2+ and ouabain and in the absence of Na+ or
K+. In this case, while precipitation of the
-subunits
alone was reasonable, the coprecipitation of
- with
-subunit
antibodies appeared to be reduced, as expected if the complex is more
labile. Coprecipitation of
b- with the
a-subunit antibody was again reduced compared
with that in NaCl (Fig. 3D), as it was in Na+,
Mg2+, and ATP. As shown in Fig. 2A, low ionic
strength alone did not cause the same effect. As discussed below, these
conformation-specific effects on complex stability are evidence that
the
-subunits are functionally integrated into
Na+-K+-ATPase and that the coprecipitation of
the two splice forms was not an artifact or the result of
-
aggregation.
Table 1 shows an analysis of the
immunoprecipitation results. The amount of precipitated protein
obtained was approximated by measuring the density of stain on X-ray
film, and interexperiment variation in stain intensities was corrected
by comparing the ratios of
- to
-subunit stain in the precipitate
with that in the starting material. This should not be regarded as true
quantification, because it does not take into account the lower overall
precipitation seen in E2 conditions. We also, of course, do not assume
that equal stain intensity with two different antibodies means equal yield of antigen. The derived average ratios show that ratios of
-
to
-subunit stain in precipitates with anti-
a- and
anti-
b-subunit antibodies in Na+ were
similar (~0.7) and that ratios of
- to
-subunit were larger by
4- to 15-fold in all E2 conditions. This indicates less recovery of
- than of
-subunit, consistent with dissociation of
the complex in E2. The yield of precipitate with the RNGA antibody in
Na+, Mg2+, and ATP was too low to quantify.
Expression of
-subunit splice variants in the kidney.
The first indication of differential distribution of the
-subunit
splice variants in the kidney was obtained by ELISA. Because activity
of Na+-K+-ATPase varies significantly along the
nephron, ELISA with membrane preparations from the medulla and cortex
were first normalized for the amount of
1-subunit with
anti-KETYY antibody as the reference. This antibody recognizes the COOH
terminus of the
-subunit (9). As shown in Fig.
4A, binding of KETYY appeared
similar when the amount of cortical membranes in each well was almost
doubled compared with medullary membranes. Comparable results were
found for RNGA binding to adjusted amounts of cortical and medullary
membranes, a difference of ~1.6-fold (Fig. 4B).
Unexpectedly, in these conditions, RNGB binding was ~3.5 times weaker
to cortical than to medullary membranes (3- to 4-fold in different
experiments; Fig. 4C). This implies that expression of the
b-subunit should be much lower in cortical segments of
the rat nephron than in the medulla, if it is assumed that all the
-subunit was in a complex with the
-subunit. Alternatively,
although less likely, accessibility of the RNGB epitope may be more
masked in one type of membrane than in the other, or the epitope could
be structurally modified.

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Fig. 4.
ELISA-detected difference in proportions of
a- and b-subunits. Microsomes from
medulla (1 µg/well) or cortex (2 µg/well) were adsorbed to Nunc
immunoplates and then incubated with different dilutions of KETYY (the
COOH terminus of the -subunit, A), RNGA
( a-subunit, B), and RNGB
( b-subunit, C) antibody. Data represent the
difference between total and nonspecific binding, which was evaluated
on BSA-coated plates. There was much less b-subunit
immunoreactivity in the cortex than in the medulla. OD450,
optical density at 450 nm.
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To evaluate whether expression of the
b-subunit in rat
cortex was indeed lower than in the medulla, Western blot analysis was
performed on isolated membrane fractions. Although only RNGB antibody
proved to be suitably sensitive for Western blots, combined use of RNGB
and the RCT-G1 antibody against the COOH terminus of the
-subunit
allowed us to monitor the expression pattern of splice variants of the
-subunit in the kidney. Figure
5A shows representative blots
stained with McK1 (
1-subunit), RCT-G1, and RNGB
antibodies. Staining for the
b-subunit was significantly weaker in the cortex than in the medulla, similar to our ELISA observation. Although we did observe some batch-to-batch variations, we
always saw much less
b-subunit expressed in the cortex.
Figure 5B represents a summary of the densitometric analysis
of four independent experiments. Identical blots were stained with McK1 and either RCT-G1 or RNGB antibodies and then scanned. To normalize the
data to the amount of
-subunit loaded, the ratio was taken between
either
(i.e.,
a +
b) or
b and the
1 staining, and the data were
expressed in arbitrary units. There was some (20%) decrease in the
ratio of
- to
1-subunit staining in the cortex compared with the medulla and a significant reduction (~70-80%) in the ratio of
b- to
1-subunit staining
in the cortex compared with the medulla. This indicates that
1) some cortical segments must express
without
and
2) some must express
a without (or with much
less)
b. The former conclusion is in agreement with our
previous findings that the
-subunit is not detectable in some
cortical segments, whereas expression of the
1-subunit
is ubiquitous along the nephron (5, 62). A similar
decrease in the level of
b-subunit expression in
cortical vs. medullary segments was observed in membrane preparations
from mouse kidney (not shown).

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Fig. 5.
Expression of the b-subunit was
significantly lower in the cortex than in the medulla. A:
microsomes from rat kidney outer medulla or cortex were separated on
SDS-Tricine gels and transferred to nitrocellulose, and blots were
stained with McK1 ( 1), RCT-G1 ( a + b), and RNGB ( b) antibody. B:
densitometric analysis. Blots were scanned, and the ratio of the
" "- and/or b-subunit to the
1-subunit stain in cortical (C) and medullary (M)
segments is expressed in arbitrary units. Data represent a summary from
4 independent experiments. The proportion of b-subunits
was significantly lower (P < 0.00005 by Student's
t-test) than " "-subunits in the cortex.
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Selective expression of the
a-subunit in proximal
tubules.
Because the great majority of renal cortex volume is represented by
proximal tubules, next we isolated individual cells from the cortex by
procedures commonly used to prepare proximal tubule cells (PTC) and
checked them for expression of
-subunit splice variants. Cortical
tissue (the very superficial cortex) was dissected and dissociated with
collagenase, and the cell suspension was gradually poured through
molecular sieves with different opening diameters. Expression of
-
and/or
b-subunits was analyzed on blots. As shown in
Fig. 6, a substantial amount of
-subunit with low mobility was detected in PTC, similar to rat
kidney outer medulla membranes. At the same time, expression of the
b-subunit was not detected in these samples. The
-subunit band in PTC appeared fuzzy, and it had a slightly slower
electrophoretic mobility on SDS gels than the
-subunit in control
samples; the appearance of the cortical sample in Fig. 5A
was similar. This may indicate a structural peculiarity of the
a-subunit in cortical membranes, such as
posttranslational modification, compared with medullary segments of the
nephron. Objectively, the
b-subunit could be present in
sieve-isolated PTC of rat nephron but with a modified amino-terminal
sequence (e.g., with oxidized methionine and/or tryptophan residues)
that would have made it undetectable by the RNGB antibody.
Consequently, to interpret the results correctly, the distribution of
-subunit splice variants was next evaluated on kidney sections using
confocal immunofluorescence microscopy.

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Fig. 6.
Selective expression of the a-subunit in
proximal tubule cells. Microsomes from rat kidney outer medulla and
proximal tubule cells were separated on SDS-Tricine gels and
transferred to nitrocellulose, and blots were stained with McK1
( 1; A), RCT-G1 ( a + b; B), and RNGB ( b;
C) antibodies. The b-subunit was undetectable
in isolated proximal tubule cells; the a-subunit was
found in proportion to the -subunit.
|
|
Immunolocalization of splice variants of the
-subunit in the
kidney.
Figures 7 and
8 show low-magnification images of the
rat kidney in which distribution of
-,
a-, or
b-subunit stain can be seen throughout the four major
layers (cortex, outer and inner stripes of the outer medulla, and inner
medulla). The boundary between the cortex and the outer stripe is the
point at which glomeruli (faint dark holes) can no longer be seen, and
the boundary between the outer stripe and the inner stripe is the point
at which proximal straight tubules (PST) terminate and the packing of
medullary thick ascending limbs (MTAL) becomes much more dense. The
boundary between the inner stripe of the outer medulla and the inner
medulla is very obvious because of the sharp drop in stain (for any
Na+-K+-ATPase subunit). The red stain shows the
monoclonal anti-
-subunit COOH terminus antibody McG-H11, and the
green stain shows the rabbit anti-TELSANHC or RNGB. Total
-subunit
stain [as described in more detail and shown at higher magnification
elsewhere (62)] was brightest in the MTAL in the inner
and outer stripes and in the distal convoluted tubule (DCT) and
connecting tubule (CNT) in the cortex. This
-subunit antibody also
stained the proximal convoluted tubule (PCT) and PST in proportion to
-subunit stain of these structures, which is much lighter than
-subunit stain of the thick ascending limb (TAL), DCT, or CNT. Our
laboratory also previously reported finding no
-subunit stain in the
CTAL (5), cortical collecting duct (CCD), or outer
medullary collecting duct (62).

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Fig. 7.
Localization of the
a-subunit in rat kidney by double-label confocal
immunomicroscopy with antibodies against the -subunit COOH terminus
( -subunit) and against the a-subunit. Layers of the
kidney are designated as follows: cortex (C), outer stripe of the outer
medulla (OS), inner stripe of the outer medulla (IS), and inner medulla
(IM). The odd structure to the right of the inner medulla is a fold in
the section. Red segments in the superficial cortex are distal
convoluted tubule and connecting tubule; in the outer stripe and base
of the cortex they are the medullary thick ascending limb (and the
initial portion of the cortical thick ascending limb). Segments that
appear red in the combined image did not stain well for the
a-subunit. Stain for the a-subunit was
fairly uniform in the cortex, with the exception of the blank glomeruli
and unstained cortical thick ascending limb. Brightly stained segments
in the inner stripe are the medullary thick ascending limb. Scale bar,
100 µm.
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|

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Fig. 8.
Localization of the
b-subunit in rat kidney. With this combination of
antibodies, the red segments (fainter stain) are the proximal
convoluted tubule in the cortex and the proximal straight tubule in the
outer stripe. Brightly stained distal convoluted tubule and connecting
tubule in the cortex and thick ascending limb in outer and inner
stripes of the medulla expressed the b-subunit
abundantly. Light b-subunit stain can also be seen in
the thick ascending limb in the lowest portions of the cortex, but not
in the superficial cortex. Scale bar, 100 µm.
|
|
The low-power images show the uniformity of the differences in splice
form distribution. Figure 7 compares stain for the
a-subunit with total
-subunit stain. Although it is
true that
a-subunit stain was much stronger in the inner
stripe than elsewhere, it was proportional to the amount of
-subunit
there (62). Stain for the
a-subunit was
light but fairly uniform in the cortex, apparently absent from
glomeruli (round black holes), but present clearly in the majority of
tubules. We have shown that the segments in the cortex that were
stained brightly red by the
-subunit COOH terminus antibody are DCT
and CNT (62), and
a-subunit stain of these
was reduced (perhaps absent) compared with the rest of the stain. That
the rest of the stain was in the PCT was supported by the light level
of stain (like
-subunit stain, not shown) and by the large numbers
of stained tubules. We have also seen
a-subunit stain of
initial segments of the PCT emerging from glomeruli (not shown).
Interestingly, a similar segregation of red and green stain was
observed in the outer stripe of the outer medulla. The PST were stained
lightly for the
a-subunit, while the MTAL segments that
ascend between them were predominantly red, not green or yellow,
suggesting little expression of the
a-subunit.
Figure 8 is the complementary image in which stain for
b- and
-subunits is compared. Stain for the
b-subunit was prominent in the DCT and CNT of the cortex
and in the MTAL of the outer and inner stripes of the outer medulla.
The
b-subunit stain was undetectable in the PCT. The
distinction between the
b- and all-
-subunit stain is
shown at higher magnification for the cortex in Fig. 9. In the combined image, the
colocalization of the all-
- and
b-subunit stain in
the DCT and CNT is apparent as yellow, while the PCT are red, which
indicates that they contain the
-subunit, but not the
b-subunit, and, therefore, should have the
a-subunit. The CNT can be tentatively identified by the
presence of unstained cells (intercalated cells). Elsewhere, we used
specific DCT, CNT, and CCD markers to discriminate among these distal
cortical segments with the all-
-subunit antibody, and so it is used
as the reference antibody here (62).

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Fig. 9.
Apparent absence of the
b-subunit from the proximal tubule. Experiment is
similar to that in Fig. 8, but at higher magnification in the
superficial cortex. In the combined image, the red stain by the
-subunit COOH terminus antibody in the proximal convoluted tubule
(PCT) is clear, confined to the basolateral membrane. Stain for
the b-subunit (green; yellow in the combined image) was
undetectable in the PCT. DCT, distal convoluted tubule; G, glomerulus.
Scale bar, 100 µm.
|
|
The TAL showed a gradient of differences in the expression of
a- and
b-subunits. In the MTAL in the
inner stripe of the outer medulla, both were abundant. In the outer
stripe, stain for the
a-subunit was greatly reduced
relative to stain for the
b-subunit. These
b-subunit-stained segments penetrated slightly into the
cortex but soon lost any detectable stain. No
-subunit stain was
seen with any of the antibodies in the CTAL in the superficial cortex.
We observed light stain for
a- and
b-subunits in two other structures (data not shown).
Macula densa cells stain prominently for the
-subunit on the basal
surface facing the juxtaglomerular apparatus (48, 62).
Here we observed stain of the macula densa by
a- and
b-subunit antibodies. Stain for the
b-subunit seemed qualitatively more prominent, but this
may be due to a higher background with the
a-subunit
antibody. In the deepest regions of the inner medulla (papilla),
Na+-K+-ATPase
-subunit stain was higher than
in the more superficial region visible in Figs. 7 and 8. We observed
stain for
a- and
b-subunits there as well.
In summary, there were notable differences in
a- and
b-subunit distribution. In the cortex, the
a-subunit predominated in the PCT and the
b-subunit in the DCT and CNT. This pattern was also
obtained in the outer stripe of the outer medulla, where PST expressed
the
a-subunit, while this portion of the MTAL expressed predominantly the
b-subunit. In contrast, both
-subunit splice variants were expressed abundantly in the portion of
the MTAL in the inner stripe of the outer medulla. The TAL in the
superficial cortex (CTAL) were not detectably labeled by
a- or
b-subunit antibodies, just as they
were not labeled by the all-
-subunit antibody. Table
2 summarizes the distributions.
 |
DISCUSSION |
Segment-specific differences in
-subunit distribution.
In the kidney, different segments of the nephron carry out different
specific tasks and are subject to complex regulation. It is well
established that Na+-K+-ATPase distribution and
activity vary markedly along the nephron (17, 18, 33, 40).
Along with unequal representation of Na+-K+-ATPase along the nephron, functional
differences in intrinsic properties of the pump have been reported,
including segment-specific differences in the affinity of
Na+-K+-ATPase for Na+ (8, 13,
21, 23). Furthermore, there is also much evidence for
segment-specific regulation of Na+-K+- ATPase
activity (22, 49). The molecular basis of these
differences has been highly controversial, and little consensus has
emerged on the subject. Although there was general agreement that
1- and
1-subunits predominated in the
renal medulla, the cortex was less well studied, and it was at one time
hypothesized that other isoforms were expressed in more distal
segments. However, no one has reported segment-specific localization of
any isoforms of Na+-K+-ATPase other than
1- and
1-subunits at the mRNA or protein level. We and others have reported that expression of the
-subunit reduced the apparent affinity for Na+ in transfected cells
(3, 5) and in Xenopus oocytes (10) or increased K+ antagonism of Na+ binding
(48), and there were effects on apparent K+
affinity (5) and ATP affinity (59, 60) as
well. The presence or absence of the
-subunit appears to correlate
roughly with the differences in apparent affinities for Na+
in successive segments of the nephron. Localization of the
-subunit and its variants provides a new framework for considering apparent segment-specific differences.
We previously described the distribution of
-subunit stain in the
rat kidney (62). Using immunofluorescence with the RCT-G1 antibody, which recognizes an epitope shared by the splice variants, and an antibody against the
1-subunit of
Na+-K+-ATPase, we demonstrated simultaneous
expression of
- and
-subunits in many tubule segments. The
-subunit stain was brightest in the MTAL and DCT, moderate in CNT,
low in PCT, and undetectable in glomeruli and collecting ducts.
However, the
1-subunit, but not the
-subunit, was
found in the CTAL and in some segments of the CCD. Here we extended
these studies by examining localization of
-subunit splice variants
in medullary and cortical segments with splice-specific antibodies.
The major outcome is that we found colocalization of
a-
and
b-subunits in medullary tubules, whereas their
distribution in the cortex appeared to be strongly biased to different
segments. As in our previous work, we observed light stain for the
-subunit in the PCT that colocalized with the
1-subunit. This form of the
-subunit appeared to be
a. The bright
-subunit stain in the DCT and CNT was
apparently predominantly
b. No staining with RNGA or
RNGB antibody was detected in the upper CTAL or CCD, whereas the
1-subunit was found there. The gradient of
a- and
b-subunit expression in the TAL is
intriguing in view of this segment's physiological role as the
diluting segment. If absence of the
-subunit results in higher ion
affinities, this correlates with the presence of lower interstitial ion
concentrations as the tubule ascends into the cortex.
While this report was in preparation, Pu et al. (48)
reported on immunocytochemical localization of
-subunit splice
variants in the rat kidney. Although much of their staining was similar to ours, there were some differences in results or interpretation. They
concluded that both splice variants of the
-subunit were expressed
in proximal tubules at low levels. Here, we did not detect
b-subunit in the proximal tubule in kidney sections or in isolated PTC. Whether this discrepancy originates in technical details (e.g., affinity or purity of the antibodies or fixation method)
or reflects potential differences in physiological status of the
animals used in the studies remains to be clarified; completely different fixation protocols were used. Pu et al. also detected the
b-subunit in the CTAL, but we detected it only close to
the MTAL of the adjacent outer stripe of the outer medulla, and all the
rest of our cortical
-subunit stain was in Tamm-Horsfall-negative segments. Pu et al. detected only the
a-subunit in the
macula densa, while we have detected both
a- and
b-subunits there. Finally, they reported
b-subunit expression in the CCD on the basis of finding
it in segments containing aquaporin 2, a distal tubule marker. We
hypothesize, however, that this apparent discrepancy is due only to the
difficulty of distinguishing the CNT and CCD without the use of
specific markers. In rodents, the CNT has been shown to express
aquaporin 2 (14), although this is not true of rabbits
(37). In our studies with the
-subunit COOH terminus antibody, the brightest cortical stain colocalized with the
thiazide-sensitive carrier (a DCT marker) or with calbindin (which
stains CNT brightly), while thin-celled, weakly calbindin-stained CCD
did not show detectable
-subunit (62). All these
interpretations are, of course, subject to the general caveat that
experimental conditions determine the limit of sensitivity of immunofluorescence.
CHIF is a homolog of the
-subunit that is expressed chiefly in the
kidney and distal colon; its gene name is FXYD4. It has recently been
demonstrated that CHIF expression is confined to the collecting duct
(in the cortex and outer and inner medulla) (54). This
distribution is complementary to that of the
-subunit, in that we
did not detect the
-subunit in the collecting duct until deep in the
papilla. Very recent evidence indicates that CHIF increases
Na+-K+-ATPase apparent affinity for
Na+, the opposite of the effect of the
-subunit
(10).
Oligomeric assembly of
Na+-K+-ATPase.
Coexpression of
a- and
b-subunits in a
majority of the MTAL in the inner stripe of the outer medulla was
unambiguous. Coimmunoprecipitation of
a- and
b-subunits along with the
-subunit from medullary membranes raises the issue of the multimeric architectural organization of the Na+-K+-ATPase complex in those segments.
Many reports support the idea that
Na+-K+-ATPase exists as an oligomer of two to
four
-subunits, recently reviewed by Taniguchi et al.
(58). If this is so, in the MTAL a random association of
a- and
b-subunits with dimers or
tetramers of the
-subunit would result in
coimmunoprecipitation. Coprecipitation of
a and
b could in principle reflect
-
association but was more likely random assembly of the
-subunits with dimers or
tetramers of 
protomers, because the
-subunit was also
coprecipitated. This is consistent with a 1:1 ratio of
- to
-subunit (
a +
b) (35) or, at a lower limit, 1:0.5 (36), as
observed in trypsin-digested purified renal medullary
Na+-K+-ATPase.
Ligand effects on the ability of anti-
-subunit antibodies to
precipitate Na+-K+-ATPase complexes support the
specificity of the coprecipitation. It seems unlikely that
-subunit
aggregation into
-subunit-only oligomers (the only other way to
explain coprecipitation) would be affected by specific conditions that
are known to affect
-subunit conformation. The interpretation of
ligand effects relative to the literature on
Na+-K+-ATPase oligomer association has some
interesting features, however. First, C12E8 can
be used to solubilize the Na+-K+-ATPase as
protomers of 
(and presumably
) (12, 28). These protomers soon oligomerize, however, either as diprotomers in equilibrium or as higher aggregates with time and various conditions (12), including ionic strength (15, 16) and
the presence of lipid and ATP (43). Studies with
membrane-associated enzyme indicated that formation of E2-P promotes
formation of
-
- and
-
-subunit links with several
bifunctional cross-linking reagents or copper-phenanthroline (6,
47). The above literature suggests a closer association between
-
- and
-
-subunits in E2 than in E1 conformations. Here,
precipitation by anti-
-subunit antibodies was antagonized by
formation of E2-P. If we consider what is likely to be happening on the
molecular level, however, there may be no contradiction. First, the
stability of
-subunit interaction with the rest of the complex may
differ from that of
-
-,
-
-, or even
-
-subunit
associations, inasmuch as the
-subunit may lose part of a favorable
binding contact when the other subunits are rearranged relative to one
another. Ligand effects on cross-linking are more pertinent to the
orientation of reactive groups than to complex stability per se. There
are good examples of subtle ligand effects on the accessibility of
sites near the membrane that support the importance of conformational
changes for Na+-K+-ATPase intersubunit
organization and function (30, 38, 39, 53).
Implications for a physiological role for the
-subunit.
We observed previously that expression of the
a-subunit
in stable renal cell transfectants can reduce
Na+-K+-ATPase apparent Na+ affinity
to a level similar to that observed in kidney membranes (5). The kinetic differences we observed were seen in
partially purified enzyme, which indicates a very stable functional
modification of enzyme properties. Qualitatively similar findings were
reported by Pu et al. (48), who additionally concluded
that the reduction in apparent Na+ affinity could be best
interpreted as an increase in K+ competition for
Na+ at the Na+ binding site. Selective
expression of splice variants in cortical segments of the nephron
provides another potential layer for
-subunit-mediated regulation of
Na+-K+-ATPase.
The present data do not address whether and how
a-
and
b-subunits differently regulate
Na+-K+-ATPase properties. With mammalian cell
transfectants, we have observed that expression of
a- or
b-subunits reduces apparent affinities for Na+ and/or K+ but that
posttranslational modifications, revealed by shifts in gel mobility,
complicate the functional effects (3, 5, 63). Others have
reported equivalent functional effects of
a- or
b-subunits in transfectants on K+ antagonism
of Na+ activation (and on affinity for ATP) in the presence
of mixed levels of potential posttranslational modifications detected
by gel mobility (48, 60) and equivalent effects of
unmodified
a- and
b-subunits in
Xenopus oocyte pump currents (10). Much remains
to be learned about whether and where
-subunit posttranslational modifications occur in the kidney, since they have not yet been detected by mass spectroscopy of purified protein (35). It
is clear that expression of unmodified
-subunit of either splice variant reduces the manifest affinity for Na+ in in vitro
conditions that resemble physiological conditions. Even if both
-subunit splice variants had the same functional effect most of the
time, the two FXYD2 gene alternative transcripts with their different
5'-untranslated regions could be subject to different transcriptional
or translational expression control.
In this work, we observed exclusive expression of the
a-subunit in PCT, where >70% of filtered
Na+ is reabsorbed. The process is under complex control of
many hormones and neurotransmitters, including
parathyroid hormone, dopamine, epinephrine and
norepinephrine, angiotensin II, insulin, and glucocorticoids (22). Although apparent affinity of
Na+-K+-ATPase for Na+ is lowest in
PCT among analyzed microdissected nephron segments, it can be altered
in response to some physiological stimuli. At low concentrations, the
stimulatory effect of angiotensin II on Na+-K+-ATPase results at least partially from
increased apparent affinity for Na+ (2).
Dopamine, the major negative regulator of Na+ reabsorption,
not only inhibits Na+-K+-ATPase in proximal
tubules, but it also alters apparent affinity of the enzyme for cations
(decreases apparent affinity for K+ and increases that for
Na+) (1, 29). In proximal tubules, insulin
induces an increase in the Na+ sensitivity without altering
the maximal reaction velocity (21). Similarly, stimulation
of Na+-K+-ATPase activity by protein kinase C
in the proximal tubule occurs through enhancement of the affinity for
Na+ (20). Thus apparent segment-specific
regulation suggests the presence of different isoforms of
Na+-K+-ATPase (which is not the case) or a
specific modification of the intrinsic properties of the pump in the
proximal tubules, such as, hypothetically, a posttranslational
modification of the
a-subunit. Conversely, the
-subunit may be inactivated by suppression of gene expression. In
both cases, the net effect would be antinatriuretic. Whether any of
these mechanisms is indeed involved in regulation of Na+
readsorption in proximal tubules requires further investigation.
 |
ACKNOWLEDGEMENTS |
We thank Drs. S. J. D. Karlish, N. Modyanov, and J. Kyte for their
generosity with antibodies, Drs. D. Brown and M. S. Feschenko for
useful discussions, and N. K. Asinovski for technical assistance.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant HL-36271 to K. J. Sweadner.
Address for reprint requests and other correspondence:
K. J. Sweadner, 149-6118, Massachusetts General Hospital, 149 13th St., Charlestown, MA 02129 (E-mail:
sweadner{at}helix.mgh.harvard.edu).
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.
10.1152/ajprenal.00146.2001
Received 9 May 2001; accepted in final form 3 October 2001.
 |
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