Immunocytochemical localization of Na-K-ATPase
- and
-subunits in rat kidney
Randall K.
Wetzel and
Kathleen J.
Sweadner
Laboratory of Membrane Biology, Neuroscience Center,
Massachusetts General Hospital, Charlestown, Massachusetts 02129
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ABSTRACT |
The
-subunit of the Na-K-ATPase is a single-span
membrane protein that alters the kinetic properties of the enzyme. It
is expressed in the kidney, but our initial observations indicated that
it is not present in all nephron segments (Arystarkhova E, Wetzel RK,
Asinovski NK, and Sweadner KJ. J Biol Chem 274:
33183-33185, 1999). Here we used triple-label confocal
immunofluorescence microscopy in rat kidney with antibodies to
Na-K-ATPase
1- and
-subunits and nephron segment-specific
markers. Na-K-ATPase
1-subunit stain was low but unambiguous in
proximal segments, moderate in macula densa, connecting tubules, and
cortical collecting ducts, high in thick ascending limb and distal
convoluted tubules, and nearly undetectable in glomeruli, descending
and ascending thin limb, and medullary collecting ducts. The
-subunit colocalized at staining levels similar to
1-subunit in
basolateral membranes in all segments except cortical thick ascending
limb and cortical collecting ducts, which had
1-subunit but no
detectable
-subunit stain. Selective
-subunit expression may
contribute to the variations in Na-K-ATPase properties in different
renal segments.
immunofluorescence; confocal microscopy; nephron; colocalization; sodium pump
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INTRODUCTION |
THE RENAL CONTROL OF
NA+ and K+ balance is complex and entails
ensembles of apical and basolateral transporters that play specialized roles in different segments of the nephron (23, 36). One
of the most physiologically important transporters is the Na-K-ATPase, which is crucial for the absorptive, secretory, and concentrating capacity of the kidney (14). In the nephron, the
Na-K-ATPase has been localized on the basolateral surface of most
tubules and is directly responsible for sodium reabsorption and for
maintaining ion gradients that are used in the redistribution of water,
other ions, and solutes. The Na-K-ATPase is composed of two subunits,
(112 kDa) and
(32 kDa plus glycosylation). There are four known
-subunit isoforms (
1,
2,
3, and
4) and three known
-subunit isoforms (
1,
2, and
3) (for review, see Ref.
8), but thus far,
1- and
1-subunits are the only
isoforms generally accepted to be expressed as proteins in adult kidney
tubules (8, 16) or detected by quantitative PCR
(25). In addition, the Na-K-ATPase has a third
nonobligatory subunit,
, that is expressed predominantly in the
kidney (35). The
-subunit belongs to the FXYD gene
family of small single-span membrane proteins that function as ion
transport regulators or channels (43). Expression of the
-subunit was shown to alter the voltage sensitivity and interaction with extracellular K+ and Na+ of Na-K-ATPase in
Xenopus oocytes (7), and transfection and coexpression of
-subunit with
- and
-subunits in a rat kidney cell line stably decreased the apparent Na+ and
K+ affinity of the Na-K-ATPase measured in vitro
(4) and increased the affinity for ATP (reviewed in Ref.
46; Ref. 48). Recently, the difference in
apparent Na+ affinity has been ascribed to an increase in
K+ competition for Na+ activation of the pump
(39).
The luminal, interstitial, and intracellular concentrations of
Na+, K+, and other ions vary along the length
of the nephron, creating microenvironments that may require adjustment
of pump functional properties. Because the renal Na-K-ATPase operates
well below its maximal velocity and close to its Michaelis-Menten
constant, small changes in Na-K-ATPase apparent affinity for
Na+ or K+ can have major consequences for
epithelial transport (21). There are segment-specific
differences in Na+ apparent affinity (6, 10, 20,
22) that cannot be explained by the intrinsic properties of the
enzyme's known
1- and
1-subunit isoforms.
The distribution of Na-K-ATPase
-subunit in the kidney has been
examined extensively in previous studies, using both
immunohistochemistry (27, 30, 37, 41) and Western blots
(31, 50). In addition, Na-K-ATPase mRNA has been localized
in the nephron using in situ hybridization (11, 17) and by
segment-specific quantification of mRNA (25, 50). Although
there are few minor differences, these studies all indicate that the
highest expression levels of Na-K-ATPase are in the medullary and
cortical thick ascending limb (mTAL and cTAL, respectively) and distal
convoluted tubule (DCT). There are lower levels in the proximal
convoluted tubule (PCT) and cortical collecting duct (CCD), and very
low levels of expression in glomeruli, descending or ascending thin
limb of Henle (DTL and ATL, respectively), and outer and inner
medullary collecting duct (OMCD and IMCD, respectively). These data
correlate with studies that have examined the amount of Na-K-ATPase
hydrolytic activity in isolated nephron segments (14, 28),
indicating that the highest activity is in the TAL and DCT, moderate
activity is in the PCT and CCD, and very low activity is in the
proximal straight tubule (PST), DTL, and ATL.
The above studies examined either whole Na-K-ATPase or the
-
or
-subunits. The distribution of
-subunit is less well
studied. Mercer et al. (35) localized
- and
-subunits in the sheep kidney cortex using immunofluorescence and
found the strongest stain in DCT, connecting tubule (CNT), and
principal cells of the collecting duct and the weaker stain in proximal
segments. Furthermore, they noted that
- and
-subunits were
always colocalized and were either present or absent together. Hayward
et al. (25) identified
-subunit transcripts in PCT and
PST. Initial experiments with our anti-
-subunit antibodies, however,
indicated that some nephron segments appeared to express
- and
-subunits, but not
-subunits (4). Therefore,
we have used confocal immunofluorescence microscopy to examine the
expression of
in rat kidney sections double or triple stained with
antibodies to Na-K-ATPase
1-,
1-, and
-subunits,
combined with antibodies to known nephron segment-specific markers to
specifically identify the
-subunit-expressing segments.
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MATERIALS AND METHODS |
Antibodies.
Table 1 lists the antibodies used.
Monoclonal anti-
-subunit antibody McG-11H is an IgG 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.
Polyclonal anti-
-subunit antibody RCT-G1 was isolated from a rabbit
injected with the same peptide and has been previously described
(4). The specificities of antibodies McK1
(anti-
1-subunits) and Ball757 (anti-
1-subunits) have also been previously described (3, 5). Antibodies directed
against known markers of specific nephron segments were also used.
Anti-aquaporin-1 (AQP1) specifically labels PCT, PST, and DTL
(51), anti-rTSC labels DCT (38),
anti-Tamm-Horsfall labels the TAL (42), anti-neuronal nitric oxide synthase (nNOS) labels macula densa cells
(49), and anti-calbindin brightly labels CNT and lightly
labels DCT (45).
Immunocytochemistry.
Adult male CD rats were anesthetized just to the point of cessation of
respiration with ether and then immediately perfused with 150 ml of PBS
(0.01 M sodium phosphate, 0.15 M NaCl, pH 7.2) followed by perfusion
with 300 ml of 2% paraformaldehyde in periodate-lysine buffer (PLP
fixative) (32). The kidneys were removed, bisected, and
postfixed by immersion in fresh PLP for an additional 2 h with
gentle agitation at room temperature. They were rinsed in several
changes of PBS for several hours at room temperature and then
immersed in 30% sucrose in PBS overnight at 4°C. They were embedded
in TBS tissue-freezing medium (Triangle Biomedical Sciences, Durham,
NC) in aluminum boats, frozen on liquid nitrogen, and stored at
20°C. Cryostat sections (8-10 µm) were picked up on ProbeOn
Plus positively charged microscope slides (Fisher Scientific, Pittsburgh, PA) and stored at
20°C until use. Immediately before staining, slides were brought to room temperature and a PAP pen (Kiyota
International, Elk Grove, IL) was used to draw a hydrophobic ring
around the sections.
For double immunofluorescence with mouse and rabbit antibodies, slides
were rinsed in PBS for 10 min and then laid flat in a dark moist box
for all subsequent incubations. The sections were covered (~50 µl
per section) with 1% SDS in PBS for 5 min. This SDS treatment has been
shown to enhance immunostaining to Na-K-ATPase and other antigens in
the kidney (9). The slides were rinsed thoroughly in PBS
(3 × 10 min) and then similarly incubated with 1% normal goat
serum and 0.5% NEN blocking reagent (New England Nuclear, Boston, MA)
in PBS with 0.3% Triton X-100 (PBSt) for 1 h at room temperature.
This blocking solution was removed with an aspirator, and a mixture of
one mouse antibody and one rabbit antibody at the appropriate dilution
(see Table 1) in PBSt was immediately applied to the sections and
incubated overnight at 4°C. The slides were rinsed in PBS for 10 min, high-salt PBS (450 mM NaCl in 100 mM phosphate buffer, pH 7.2) for
5 min, and then normal PBS two more times for 10 min each. They were then incubated in 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) in PBSt for 2 h. Finally, they were rinsed in normal and high-salt PBS and coverslipped in Vectashield fluorescence mounting medium (Vector Laboratories, Burlingame, CA).
For triple-label immunofluorescence involving two different mouse
antibodies and a rabbit antibody, tyramide signal amplification (TSA)
was used for one antibody. Slides were rinsed in PBS for 10 min,
immersed in 0.25% H2O2 in PBS for 60 min to
quench endogenous peroxidase activity, rinsed in PBS for 5 min, and
incubated with SDS and blocking solutions as described above. The
sections were then incubated with the anti-
1-mouse monoclonal
antibody McK1 at a dilution of 1:750 in PBSt. This high dilution of
McK1 was detectable only with tyramide amplification and not with
directly conjugated fluorescent secondary antibodies. The slides were
rinsed in normal and high-salt PBS as described above, incubated in
biotinylated horse anti-mouse IgG (1:500; Vector, Burlingame, CA) in
PBSt for 2 h, rinsed in normal and high-salt PBS, incubated in
streptavidin-horseradish peroxidase (1:100; NEN) in PBSt, rinsed in
normal and high-salt PBS, and then incubated in Cy3-tyramide reagent
(1:100) in NEN amplification diluent (NEN) for 5-7 min. The slides
were rinsed in several changes of PBS for 30 min and then incubated
with a second mouse antibody and a rabbit antibody in PBSt
overnight at 4°C. They were then rinsed in normal and high-salt PBS
and incubated in Cy5-conjugated goat anti-mouse IgG (1:300) and
FITC-conjugated goat anti-rabbit IgG (1:200; Jackson) in PBSt for
2 h. The slides were then rinsed and coverslipped as above. It
should be noted that when tyramide amplification is used with high
dilutions of McK1, stain is not as uniform and crisp as usual, and
stain of segments like PCT that have less Na-K-ATPase is harder to
detect than usual. Figures 1, 2, 11, and 12 show conventional staining with McK1, which clearly reveals the light stain seen in proximal segments.

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Fig. 1.
Confocal images of kidney cortex double labeled with 1- and
-subunit antibodies. The 2 bottom images are of 1-
(red, Cy3) and -subunits (green, FITC) individually, whereas the
top image shows the 2 colors merged. 1- and -Subunits
were colocalized (yellow in the merged image) in dimly labeled
presumptive proximal convoluted tubule (PCT; asterisk) and in brightly
labeled presumptive distal convoluted tubule (DCT; thick arrow),
whereas glomeruli (G) were unlabeled. Some vertically oriented
presumptive cortical thick ascending limb (cTAL) contained bright
1-subunit stain but little or no -subunit stain (thin arrow).
Similarly, presumptive cortical collecting duct (CCD) had 1-subunit
stain but no -subunit stain (arrowhead). Scale bar, 50 µm.
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Fig. 2.
Kidney double labeled with 1- (a, d, g, j, m),
-subunit (b, e, h, k, n) antibodies, and 2-color merged
images (c, f, i, l, o) at the level of the cortex
(a-c), cortex/medulla border
(d-f), medulla outer stripe (OS)/inner stripe
(IS) border (g-i), medulla inner stripe
(j-l), and outer medulla/inner medulla (OM/IM)
border (m-o). 1- and -subunits colocalized in all
levels of the kidney except in vertically oriented tubules in the
cortex that contained bright 1- but no -subunit stain (red in the
merged image). Tubules in the OM contained bright 1- and -subunit
stain, whereas tubules in the IM contained little or no stain. Scale
bar, 50 µm.
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For triple-label immunofluorescence involving the goat
anti-Tamm-Horsfall antibody, the slides were rinsed, incubated in SDS and blocking solutions, and then incubated with McK1 (mouse), RCT-G1
(rabbit) and anti-Tamm-Horsfall (goat) primary antibodies in PBSt as
described above for regular double-label immunofluorescence. However,
because unbound anti-goat secondary antibody would bind to the goat
anti-mouse and goat anti-rabbit secondary antibodies, sections were
incubated first in Cy3-conjugated donkey anti-goat IgG (1:1,000;
Jackson) in PBSt for 2 h, rinsed thoroughly to remove any unbound
secondary antibodies, then incubated in the Cy5-conjugated goat
anti-mouse and FITC-conjugated goat anti-rabbit antibodies in PBSt.
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 |
Stain for the Na-K-ATPase
1-subunit in different nephron
segments was consistent with previous reports. Figure
1 illustrates a section from the rat
renal cortex double labeled with mouse monoclonal
1- and rabbit
polyclonal
-subunit antibodies, showing each antibody separately and
in a combined image. Glomeruli were unstained. Yellow in the combined
image shows locations of coexpression that can be seen in both lightly
labeled presumptive PCT (asterisk) and brightly labeled presumptive DCT
(thick arrow). Expression of
1- without
-subunits was very
obvious in the heavily stained straight tubules, which from their
shape, location, and cell morphology are likely to be cTAL (thin
arrow); further evidence is shown below. Closer inspection also shows
1- without
-subunits in thin-celled segments with a patchy
distribution of stain (arrowhead). As will also be shown below, the
stained cells are the principal cells of the CCD.
Figure 2 shows representative sections
from all levels of the kidney.
1- (red) and
-subunits (green)
were colocalized (yellow) on the basolateral surfaces of the most
prominently stained tubules throughout the kidney. Figure 2,
a-c, shows the same images as in Fig. 1 but reduced to
scale. This is superficial cortex, marked by the presence of three
glomeruli. Figure 2, d-f, shows the transition to the
outer stripe of the outer medulla, where DCT, cTAL, and proximal
tubules coexist. The lightly stained proximal tubules all had
-subunits, whereas the brightly stained tubules sometimes did and
sometimes did not. Figure 2, g-i, shows the transition from outer stripe to inner stripe, and j-l show inner
stripe, where mTAL stain predominates. At this location,
-subunits
always colocalized with
1-subunits. Figure 2, m-o,
shows the transition from outer medulla to inner medulla, where there
was little stain detected for either subunit.
Stain for the
1-subunit coincided with that for
1-subunit, as
shown in Fig. 3. This is a triple-label
experiment in which the
1-subunit antibody was diluted to the point
that tyramide amplification was the only way to detect it. This method
resulted in spotty stain appearance in regions of low antigen
concentration (red), but it allowed two antibodies from the same
species (mouse in this case) to be used on the same section. The
other mouse antibody (anti-
-subunit) was stained by conventional
directly conjugated secondary antibody (blue).
1-Subunit was stained
by a rabbit antibody, and in the combined image it can be seen that
1-,
1-, and
-subunit stain coincided (pink) in both lightly stained regions (presumptive PCT) and heavily stained regions (DCT). In
presumptive cTAL, only
1- and
1-subunits were seen (orange-yellow).

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Fig. 3.
Kidney cortex triple labeled with Na-K-ATPase 1- [red, tyramide
signal amplification (TSA)-Cy3], 1- (green, FITC), and -subunit
(blue, Cy5) antibodies. 1- and 1-subunits were always
colocalized, and were usually colocalized with -subunits. However,
the TAL contained 1- and 1-subunit stain, but not -subunit
(yellow in the merged image). Scale bar, 50 µm.
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To identify specific tubules within the nephron, sections were triple
labeled with
1-,
-subunits, and a segment-specific antibody
(Figs. 4-10). Because most of the marker antibodies were generated
in either mouse or rabbit, we used tyramide amplification to stain for
1-subunits using a dilution of McK1 that was not detectable with
directly conjugated secondary antibodies and then stained for the
marker and
-subunits using either mouse marker antibody with the
rabbit polyclonal anti-
-subunit, RCT-G1, or rabbit marker antibodies
with the mouse monoclonal anti-
-subunit, McG-11H. Elimination of any
one of the three primary antibodies eliminated all stain for that
specific antibody without affecting the stain from the other two (not
shown). One antibody (anti-Tamm-Horsfall) was generated in goat, and
therefore we used conventional triple-label immunofluorescence with
three directly conjugated secondary antibodies.

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Fig. 4.
Aquaporin-1 (AQP1), a marker of proximal tubule. Kidney cortex was
triple labeled with 1- (red, TSA-Cy3), AQP1 (green, FITC), and
-subunit (blue, Cy5) antibodies. Proximal segments containing AQP1
stain were very lightly labeled with 1- and -subunits.
Conversely, distal segments that were not labeled by the AQP1 antibody
were brightly stained by 1- and -subunits. Scale bar, 50 µm.
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Fig. 5.
Kidney OM triple labeled with 1 (red, TSA-Cy3)-, AQP1 (green,
FITC), and -subunit (blue, Cy5) antibodies. Proximal segments
containing AQP1 stain were very lightly labeled with 1- and
-subunits, which were always colocalized. Distal segments,
presumably mTAL, were brightly labeled by 1- and -subunit
antibodies but were devoid of AQP1 stain. The dashed line indicates the
border between the OS and the IS of the OM. Scale bar, 50 µm.
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Fig. 6.
Kidney medulla triple labeled with 1 (red, TSA-Cy3)-, AQP1
(green, FITC), and -subunit (blue, Cy5) antibodies. Proximal
segments in the OM and IM containing AQP1 stain showed little or no
1- or -subunit stain. Ascending segments were brightly
stained with 1- and -subunit antibodies in the OM but were very
lightly stained in the IM. The dashed line indicates the border between
the IS of the OM and IM. Scale bar, 50 µm.
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Fig. 7.
Tamm-Horsfall antigen, a marker of TAL. Kidney was triple labeled
with Tamm-Horsfall (red, Cy3), (green, FITC)-, and 1-subunit
(blue, Cy5) antibodies. Tamm-Horsfall stain was bright in TAL and much
lighter in DCT. Although the DCT was brightly stained by both 1- and
-subunit antibodies, the cTAL was only stained by 1- and not by
-subunits. Scale bar, 50 µm.
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Fig. 8.
Neuronal nitric oxide synthase (nNOS), a marker of macula densa.
High-magnification image of kidney cortex triple labeled with 1
(red, TSA-Cy3)-, nNOS (green, FITC), and -subunit (blue, Cy5)
antibodies. The cells of the macula densa contained bright cytoplasmic
nNOS stain, as well as 1- and -subunit stain on their basolateral
surface. The adjacent portion of the cTAL was brightly stained by the
1-subunit antibody, but not by -subunit. Scale bar, 25 µm.
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Fig. 9.
Thiazide-sensitive Na+-Cl cotransporter
(rTSC), a marker of DCT. Kidney cortex was triple labeled with 1
(red, TSA-Cy3)-, rTSC (green, FITC), and -subunit (blue, Cy5)
antibodies. DCT that were apically labeled with the rTSC antibody were
also basolaterally labeled with 1- and -subunits. Presumptive
cTAL contained only 1-subunit stain (arrow), whereas presumptive
connecting tubule (CNT) contained patchy 1- and -subunit stain
but not rTSC (arrowhead). Scale bar, 50 µm.
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Fig. 10.
Calbindin, a marker of CNT. Kidney cortex was triple labeled with
1 (red, TSA-Cy3)-, -subunit (green, FITC), and calbindin (blue,
Cy5) antibodies. Bright calbindin stain was seen in CNT, whereas
lighter stain was seen in DCT and collecting ducts. DCT containing
light calbindin stain were also brightly stained by 1- and
-subunits. CNT were brightly labeled by 1- and -subunit
antibodies. Calbindin-stained CCD were lightly labeled by 1-subunit,
but contained little or no -subunit stain. A macula densa can also
be seen in this image (arrowhead). Scale bar, 50 µm.
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To identify proximal and descending segments, we used an AQP1 antibody.
AQP1 stain was localized at the apical surface in PCT, PST, and DTL.
Figures 4, 5, and 6 show AQP1-stained segments at three different
levels in the kidney. The most superficial cortex is shown in Fig.
4, where the AQP1-stained PCT (green) all
showed basolateral stain for
(blue)- as well as
1-subunits (red). The DCT were brightly stained for
1- and
-subunits (bright purple in the combined image) but unstained for AQP1. Figure
5 shows the boundary between outer
medullary outer stripe (OS) and inner stripe (IS). The brightly
AQP1-stained segments (PST and short-loop DTL, depending on diameter)
had barely detectable stain for
1- or
-subunits. The bright
purple
1- and
-subunit stain was presumably in mTAL, judging from
the absence of AQP1. Figure 6 shows the
boundary between outer medulla and the inner medulla. Bright AQP1 stain
of DTL was uniform across the boundary, and contained little detectable
stain for either
1- or
-subunits, and the purple stain for
1-
and
-subunits in mTAL stopped abruptly at the boundary.
To identify the TAL, we used an antibody against Tamm-Horsfall antigen
that is known to brightly label both the mTAL and cTAL and to lightly
label the DCT (42). Figure 7
shows that cTAL that contained bright Tamm-Horsfall and
1-subunit
stain were devoid of
-subunit stain and were purple in the combined
image. In contrast, DCT, identified by light Tamm-Horsfall stain,
contained bright
1- and
-subunit stain and were aqua in the
combined image.
The cells of the macula densa, located in the juxtaglomerular apparatus
in the cTAL, are known to express nNOS (49). Using an
antibody against nNOS, we were able to clearly distinguish between
cTAL, which contain macula densa, and mTAL, which do not, and also to
clearly identify the transition from cTAL to DCT, which occurs
immediately after the cTAL passes the juxtaglomerular apparatus
(not illustrated). A juxtaglomerular apparatus is seen in
Fig. 8. The cytoplasm of the cells of the
macula densa was stained with nNOS antibody, and the cells had light to
moderate
1- and
-subunit stain on their basolateral surface.
However, the remaining portion of the adjacent cTAL contained only
1-subunit stain and not
-subunit stain. In the combined image,
the macula densa is largely green, whereas the surrounding cTAL is red,
and adjacent PCT are light purple.
The thiazide-sensitive Na+-Cl
cotransporter
(rTSC) antibody is a known marker of DCT (38). The apical
surface of the DCT was clearly labeled with anti-rTSC, whereas the
basolateral surface was very brightly stained with both
1- and
-subunit antibodies (Fig. 9). In the
combined image, these are the tubules with blue-purple basolateral
surfaces and green apical stain. Two other kinds of brightly stained
tubules can be seen: those with
1- but no
-subunits or rTSC,
which are presumably cTAL (arrow), and those with
1- and
-subunit
stain but only faint rTSC stain (arrowhead). The patchy
1- and
-subunit stain of these last tubules suggests connecting or
collecting tubules with intercalated cells, and these are identified
more specifically in the next figure.
The calbindin antibody is known to brightly stain CNT and also to
lightly stain late DCT and collecting duct principal cells (45). Many features can be seen in Fig.
10. cTAL, stained only for
1-subunits, is seen as long vertical tubules (red). DCT was stained
with only
1- and
-subunit antibodies (yellow). The CNT was
brightly stained by the calbindin antibody and by both
1- and
-subunit antibodies (purple to white in the three-color image). This
stain was patchy, consistent with Na-K-ATPase stain in principal cells.
The CCD was lightly but clearly stained by the calbindin antibody, and
the principal cells were stained for
1- but not
-subunits.
Interestingly, a macula densa with prominent
-subunit stain is also
visible in this figure (arrowhead), with bright
1-subunit stain in
the adjacent cTAL cells.
The collecting duct was stained with anti-V-type ATPase antibody
(H+-ATPase), which stains intercalated cells from the
cortex through the IMCD (1). For these experiments,
conventional double labeling was used to enhance our ability to detect
1-subunit at low levels, because tyramide amplification at very high
antibody dilutions gives less uniform stain of
1-subunits. Figure
11 shows superficial cortex
H+-ATPase-stained intercalated cells in CCD alternating
with
1-stained principal cells (arrowheads). In the
bottom panels, it can be seen that such
1-stained cells
do not have stain for
-subunits (red in the combined image), in
contrast to DCT (solid yellow) and presumptive CNT (patchy yellow).
Figure 1 also shows a presumptive CCD with patchy stain of
1- but
not
-subunits running vertically next to presumptive cTAL.

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Fig. 11.
H+-ATPase as a marker of intercalated cells.
Kidney cortex was double labeled with either 1-subunit (red, Cy3)
and H+-ATPase (green, FITC) antibodies (top) or
1 (red, Cy3)- and -subunit (green, FITC) antibodies
(bottom). H+-ATPase was seen in intercalated
cells and 1-subunit stain was seen in principal cells of the CCD
(arrowheads, top), but the CCD was not stained by the
-subunit antibody (arrowheads, bottom). Scale bar, 100 µm.
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Figure 12 shows the boundary between
outer and inner medulla. Here, H+-ATPase stain of medullary
collecting duct continued unbroken across the boundary, whereas
1-
and
-subunit stain of mTAL stopped abruptly. Under the conditions
used, stain for either
1- or
-subunits was undetectable in OMCD
or IMCD.

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Fig. 12.
Kidney medulla double labeled with either 1 (red,
Cy3)-subunit and H+-ATPase (green, FITC) antibodies
(top) or 1 (red, Cy3)- and -subunit (green, FITC)
antibodies (bottom). H+-ATPase was seen in
intercalated cells of the medulary collecting ducts (MCD) (arrowheads,
top). No 1- or -subunit stain was seen in the MCD
(arrowheads, bottom). Scale bar, 100 µm.
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DISCUSSION |
In this study, Na-K-ATPase
1-subunit expression was highest in
the TAL and DCT, intermediate in the CNT and CCD, and low in the PCT.
The
-subunit was colocalized with the
1-subunit and was stained
at levels similar to
1-subunit in all tubules except cTAL and CCD,
which had no detectable
-subunits. Figure 13 diagrams the results. Stain for
Na-K-ATPase has been detected in the medullary collecting duct by
others with more sensitive detection (27, 40, 41). For
present purposes, we can only say that the levels of expression must be
very low.

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|
Fig. 13.
Summary diagram of the Na-K-ATPase 1- and
-subunit expression patterns along the length of the nephron.
The thickness of each line represents the apparent level of expression.
The distributions of the various markers are also shown. Macula densa,
which consists of a small patch of cells in the cortical thick
ascending limb at the juxtaglomerular apparatus at the glomerulus, is
indicated on the diagram. These cells were positive for -subunits at
the basolateral surface. Drawn in the style of Piepenhagen et al.
(37).
|
|
Mercer et al. (35) previously examined the expression of
- and
-subunits in the sheep kidney cortex using
immunofluorescence. The strongest stain was seen in DCT, CNT, and
principal cells of CCD, and the weaker stain was seen in proximal
segments. Furthermore, they noted that
- and
-subunits were
always colocalized and were either present or absent together. This
contradicts our results, and the reason for the discrepancy is not
known. Although it is possible that there is a species difference
between rat and sheep, preliminary studies in our lab have shown that
the pattern of
-subunit expression in mouse kidney is similar to
that in rat kidney. It is possible that cTAL was simply not examined in
the sheep study. Hayward et al. (25) examined the levels
of
- and
-subunit mRNA in isolated proximal segments of the rat
kidney. Similar amounts of
- and
-subunit transcripts were found
in the PCT and in the PST, but other more distal segments were not examined.
While this paper was under review, Pu et al. (39) also
reported
-subunit localization in the rat kidney, with an antibody that detects both splice forms like ours, and with splice-specific antibodies. Their results differ from ours in two important respects, in that they reported finding
-subunit expression in cTAL and in
CCD. We hypothesize that this is due mainly to differences in the
identification of nephron segments. In their Fig. 7A, for example, a
-subunit stained segment is labeled cTAL, which according to our data may be more likely to be a piece of DCT cut in cross section. Next to it, under the "g" is a segment that we would guess
is authentic cTAL: unstained for
-subunit except for the surface
facing the glomerulus, which appears to be a macula densa. They further
concluded that
-subunit was present in cTAL on the basis of
colocalization with Tamm-Horsfall antigen; clear colocalization of
b-subunit with Tamm-Horsfall antigen can be seen in Fig.
8H, but we have seen such stain in TAL only in the deepest
cortex and in the OS of the outer medulla. Pu et al. (39)
identified
-subunit stain in CCD on the basis of colocalization with
AQP2, but in rodents AQP2 is also found in CNT (13), where we observed
. On the whole, though, the results are similar. Minor discrepancies could also be due to the fact that different fixation protocols were used.
Functional implications.
It is interesting to note that
1-,
1-, and
-subunits were
coexpressed throughout the nephron except in the cTAL and CCD. In fact,
although the expression of
1-,
1-, and
-subunits is very high
in the DCT, the adjacent cTAL is seemingly devoid of
-subunits
despite equally high-
1- and -
1-subunit expression. We have
previously shown that transfection and coexpression of
-subunit with
- and
-subunits in a rat kidney cell line reduced the
Na-K-ATPase apparent affinity for sodium and potassium in vitro (4). Therefore, one would expect that Na-K-ATPase in segments that do not express
-subunits would have a higher ion affinity than those that do. The Doucet and Feraille laboratories have
examined ion affinities in isolated segments from the rabbit and rat
nephron. Their results indicate that Na-K-ATPase affinity for sodium is
higher in the CCD than in the PCT, mTAL, or cTAL (6, 10, 19,
22) and that affinity in cTAL is higher than in PCT
(6). Affinity for potassium is similar in PCT, mTAL, and
CCD (15). The higher sodium affinity in the cTAL and CCD than in more proximal segments is consistent with the hypothesis that
segments that don't express
-subunits have higher Na+
affinity than those that do.
If
-subunit expression decreases Na-K-ATPase apparent affinity for
Na+ in vivo as it does in vitro (4), then one
would expect Na-K-ATPase in the cTAL to have a higher affinity for
Na+. As much as 30% of the filtered load of
Na+ is reabsorbed in the TAL, yet this portion of the
nephron is impermeable to water (24). Consequently, the
concentration of Na+ in the lumen decreases along the
length of the TAL such that the lowest concentration in the entire
nephron is in the cTAL. It is possible, therefore, that the conditions
in the cTAL require Na-K-ATPase pumps with higher Na+
affinity (no
-subunit). The lower affinity expected (but
not yet measured in isolated segments) in the
-subunit-expressing DCT and CNT may reflect the primary role of these segments to make
adjustments to luminal content. We speculate that if
-subunit expression can be regulated, it is here and in the collecting duct that
changes will be seen.
Expression of
-subunit in a human embryonic kidney cell line has
also been shown to increase Na-K-ATPase affinity for ATP in vitro
(39, 47, 48). This raises an intriguing apparent contradiction. The concentration of ATP in the cTAL has been reported to be 25-45% lower than in the mTAL or DCT (44). If
-subunit expression increases Na-K-ATPase affinity for ATP in vivo
as this in vitro experiment suggests, then the Na-K-ATPase in the cTAL may be less active because of its lower affinity for ATP.
Splice variants of
-subunit.
The renal Na-K-ATPase
-subunit has recently been shown to exist as
two variants, the
a- and
b-subunits, with
different NH2-terminal sequences (29, 43).
Because we have data indicating that expression of these variants may
result in similar Na+ but different K+
affinities (2), it is important to examine the
distribution of these two
-subunit variants in the nephron. The
monoclonal and polyclonal anti-
-subunit antibodies used here are
both directed against a portion of the COOH-terminus of
-subunit
that is identical in the splice variants. We have obtained data
indicating coexpression of
a- and
b-subunits in mTAL in the IS, but preferential
expression of
a-subunit in PCT and PST and preferential
expression of
b-subunit in DCT, CNT, and in mTAL in the
OS, again differ in part from the results reported by others
(39).
Hypomagnesemia.
Hypomagnesemia is a condition of magnesium wasting that occurs
when the kidney is unable to reabsorb sufficient
Mg2+ from the renal ultrafiltrate (for review, see
Ref. 12). This disease is characterized by low serum
Mg2+ levels, but different forms of hypomagnesemia involve
shifts in other serum and urine electrolytes. Normally, the bulk of
Mg2+ reabsorption (65-75%) in the kidney takes place
via a passive or paracellular pathway in the cTAL. Significant amounts
of Mg2+ (5-10%) are also reabsorbed via an active
transcellular pathway in the DCT. Not surprisingly, the different forms
of hypomagnesemia have been linked to mutations in genes that encode
ion channels and tight junction proteins in the cTAL and DCT
(34). Recently, Meij et al. (33) have
identified a mutation in the Na-K-ATPase
-subunit gene that is
responsible for the disorder known as isolated dominant renal
hypomagnesemia. This dominant negative mutation is caused by a single
residue substitution in the transmembrane domain of
-subunit (Gly to
Arg) that prevents proper membrane insertion or routing of
-subunit
(33). Because we have shown in the present study that
-subunit is not expressed in cTAL, it is possible that this
-subunit mutation is affecting active Mg2+ reabsorption
in the DCT by preventing insertion of
-
-
-subunit pumps and
possibly of other membrane proteins, by accumulating misfolded protein
in the endoplasmic reticulum. That the mutation does not have a more
obvious effect on Na+ excretion may be due to the presence
of one good copy of the gene and the existence of a threshold for
pathology that is reached only in the cells with the very highest
-subunit expression levels, as seen here, in DCT. It is notable that
-subunit is also implicated in preimplantation blastocoel formation
in mice (26), and yet humans with the mutation are clearly
viable. A knockout of the
-subunit gene would be more informative.
In conclusion, expression of Na-K-ATPase with and without the
regulatory
-subunit has been described in identified renal segments.
The segments without
-subunit, the cTAL, and the CCD, are those that
have been shown to have the Na-K-ATPase with the highest endogenous
affinity for Na+. Further work on the physiological role of
-subunit would be timely.
 |
ACKNOWLEDGEMENTS |
We thank W. J. Ball, Jr., S. Nielsen, S. C. Hebert, and D. Brown for their generosity with antibody markers. We also thank D. Brown and E. Arystarkhova for helpful discussions.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
Grant 5R01- 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 (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.
Received 16 February 2001; accepted in final form 14 May 2001.
 |
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