Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756
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ABSTRACT |
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Previous studies indicate
that the colonic H-K-ATPase mRNA is expressed as the distal nephron.
However, the exact intrarenal localization of the colonic H-K-ATPase
protein is still unclear. The goal of the present study was to
determine the cellular and subcellular localization of the colonic
H-K-ATPase protein in the rabbit kidney. We used three monoclonal
antibodies (MAbs) directed against different epitopes of the rabbit
colonic H-K-ATPase -subunit (HK
2) to localize
HK
2 protein by immunofluorescence labeling of kidney
sections and laser-scanning confocal microscopy. The specificity of the
MAbs was confirmed by reaction with a single ~100-kDa band on Western
blots of distal colon. Specific immunohistochemical reaction with the
apical membrane of surface epithelial cells was observed with all three
MAbs on distal colon sections. In rabbit kidney, immunofluorescence was
detected only on the apical membrane of connecting tubule cells.
Immunofluorescence was not detected in the cortical-, outer-, and
inner-medullary collecting ducts. Furthermore, costaining with
principal- and intercalated cell-specific MAbs and a MAb against the
thick ascending limb suggests that these cell types express
HK
2 protein at levels that are below the detection limit
with this method. We conclude that in the rabbit kidney, under normal
dietary conditions, the HK
2 protein is expressed in the
apical membrane of connecting tubule cells.
potassium transport; confocal microscopy; rabbit kidney
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INTRODUCTION |
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THE KIDNEY PLAYS A
CRITICAL role in maintaining potassium homeostasis and acid-base
balance. The fine regulation of both K+ and H+
excretion takes place in the collecting duct system. A number of
functional studies indicate that a family of H-K-ATPases participates in both K+ and H+ transport in the distal
nephron (for review see Refs. 31 and 33). At
least two genes encoding H-K-ATPase -subunits seem to be expressed
in rat kidney: the gastric isoform (or HK
1)
(27) and the colonic (or HK
2) H-K-ATPases
(8, 17). Additional diversity in the H-K-ATPase family is
generated by alternative splicing. Recently, two
NH2-terminal splice variants of the HK
2 mRNA
were cloned from both the rat (17) and the rabbit
(6) kidney, designated HK
2a and
HK
2b (rat) and HK
2a and
HK
2c (rabbit).
HK1 mRNA has been localized to the connecting segment
and the entire collecting duct of rats (2). Results from
several laboratories, including ours, demonstrated that
HK
2 mRNA is expressed in cells of the distal nephron. In
situ hybridization studies with rats on normal diets showed strong
labeling in the connecting tubule (CNT) and cortical collecting duct
(CCD) but only very low levels of expression in the outer medullary
collecting duct (OMCD) (1). Our previous studies
demonstrated that the HK
2 mRNA is present in the CNT,
CCD, and OMCD of rabbits (13, 14) and that the expression
in CNT plus CCD cells is increased after in vivo base loading of the
animals (14). Several studies have demonstrated that the
expression of HK
2 mRNA is increased in the rat medulla
by chronic hypokalemia (1, 16, 25).
Information on the intrarenal localization of the HK2
protein is scanty. Only one immunohistochemical study has been
published thus far by Sangan and co-workers (25). These
authors reported that a polyclonal antibody against the rat colonic
H-K-ATPase
-subunit labeled the apical membrane of principal cells
in the rat OMCD, whereas no specific labeling was observed in the CNT. Localization of HK
2 to OMCD principal cells
was an unexpected finding because previous functional studies indicated
that H-K-ATPase activity resides primarily in intercalated cells
(28-30, 33). This finding also conflicts with
previous in situ hybridization data showing intense signals in the CNT
and CCD (1) and a recent report that shows that the
HK
2 protein is present in the renal cortex in the rabbit
(6). The above immunohistochemical study was done in rat
kidney, and the intrarenal and subcellular localization of the
HK
2 protein in other species remains unknown.
Despite clear evidence that HK2 is expressed in the
kidney, its exact function is still unclear, in part because no
isoform-specific inhibitors against the various H-K-ATPases are
available. A recent study with HK
2-deficient mice
indicates that during K+ deprivation this transporter plays
a critical role in the colon to maintain K+ homeostasis
(20). On the basis of its presumed function (i.e., K+ reabsorption and H+ secretion), one would
expect the colonic H-K-ATPase to be present in the apical membrane of
OMCD cells, because this nephron segment exhibits net K+
reabsorption and acidification and this is the nephron segment in which
dietary K+ seems to regulate HK
2 mRNA
expression (1). Indeed, Codina and co-workers
(7) showed that HK
2 protein, which was
undetectable in the kidney in control rats, becomes detectable on
immunoblots from the medulla (but not the cortex) of rats subjected to
chronic hypokalemia.
In this study, we examined the cellular and subcellular localization of
the HK2 protein in rabbit kidney, under conditions of
normal K+ homeostasis. We used three new monoclonal
antibodies (MAbs) against different epitopes on the rabbit
HK
2 protein, in combination with laser-scanning confocal
microscopy. Our results demonstrate that the HK
2 resides
in the apical membrane of connecting tubule cells in the rabbit kidney.
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MATERIALS AND METHODS |
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Generation of antibodies against the rabbit HK2.
MAbs were generated against a fusion protein containing the
NH2-terminal region of the rabbit HK
2a
(amino acids 1-99). The rabbit HK
2a cDNA
(14) was used as a template to amplify the corresponding
region using a sense primer (5'-TCC GAA TTC ACA TGC GCC AGA GAA AGC
TGG-3') and an antisense primer (5'-CAA ACA GAC CCC AGA GAT CAT CTA GGT
ACC CAA-3'), which add KpnI and EcoRI restriction
sites. After digestion with these enzymes, the PCR product was ligated
into the pPROEx-1 vector (GIBCO-BRL), which contains an
NH2-terminal polyhistidine tag and a tobacco etch virus
(TEV) protease cleavage site. Direct sequencing was performed to verify
that the insert is in the correct reading frame and is identical to the
appropriate region of the rabbit HK
2 cDNA (13,
14). Production of the fusion protein was induced with isopropyl-
-D-thiogalactopyranoside in Escherichia coli
DH5
. The fusion protein was purified on Ni-NTA resin according to
the manufacturer's instructions (GIBCO-BRL). The purified fusion
protein was then cleaved with recombinant TEV protease (GIBCO-BRL) and passed over a Ni-NTA column again to remove the polyhistidine tag.
Balb/c female mice were immunized with 100 µg of the cleaved fusion
protein in complete Freund's adjuvant (ip) once and then twice with 50 µg of fusion protein in incomplete Freund's adjuvant at 2-wk
intervals. Blood was obtained from the tail for the determination of
antibody titer by enzyme-linked immunosorbent assay (ELISA) using the
purified fusion protein as antigen. MAbs were generated using the
spleen of the mouse with the highest serum titer, as described
previously (9, 11, 12). Hybridomas, which were positive in
ELISA, were tested for Western blotting and immunohistochemical staining (see below).
Immunoblotting. Tissue homogenates were prepared from rabbit distal colon, kidney cortex, medulla, stomach, and heart by snap freezing the tissues after dissection and homogenization in 1% SDS-containing solubilization buffer (12). Rabbit CCD cells were isolated by immunodissection (9, 11) and lysed in solubilization buffer. Homogenates of E. coli expressing the fusion protein were obtained by sonication and centrifuged at 10,000 g. Protein concentrations were determined by the bicinchoninic acid method (Pierce), and then dithiothreitol was added to a final concentration of 1 mM. Ten micrograms of protein per lane were electrophoresed on 12.5% SDS-polyacrylamide gel with a 4% stacking gel and transferred to polyvinylidene difluoride Immobilon membranes. The membranes were blocked in 5% nonfat milk in 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween-20 (TBST) for 1 h and probed with the mouse MAbs (undiluted culture supernatant) at room temperature for 1 h. Membranes were washed with TBST four times and then incubated with a 1:20,000-fold dilution of alkaline phosphatase-conjugated rabbit anti-mouse IgG at room temperature for 1 h. After membranes were washed again with TBST, antibody binding was localized by the enhanced chemiluminescence method (Amersham).
Immunoprecipitation was performed by covalently linking MAb 0121 to an affinity support using a Seize X protein A immunoprecipitation kit (Pierce) after the manufacturer's instructions.Immunohistochemistry and confocal microscopy.
For these experiments, five male New Zealand rabbits were used. The
rabbits were anesthetized and the kidneys were perfused with
periodate-lysine-paraformaldehyde (PLP) fixative (19), kept in PLP for an additional 2 h at room temperature, and finally embedded in paraffin. Distal colon was fixed similarly.
Four-micrometer-thick sections were cut and deparaffinized, followed by
quenching of endogenous peroxidase activity by incubation with 3%
H2O2. Immunohistochemistry was performed using
culture supernatants of hybridomas followed by 1:100 dilution of a
horseradish peroxidase-conjugated anti-mouse IgG (Zymed). Colonic
H-K-ATPase immunoreactivity was visualized using the tyramide signal
amplification system (TSA-Indirect, NEN; Ref. 3) and Cy-5-conjugated
streptavidin (1:500). The following antibodies were used as segment and
cell-specific markers: MAb DT17, which reacts with principal cells in
the CCD (11); MAb F13/483, which reacts with CNT cells and
principal cells in the CCD; ST.12, which reacts with rabbit CNT and CCD
(9, 11); and peanut lectin agglutinin (PNA), a marker of
-intercalated cells in the rabbit (18).
-Intercalated cells were labeled with either a MAb against H-ATPase
(34) or a MAb against the basolateral HCO3/Cl
exchanger, band 3 (26). As a CNT marker, we
used a MAb against the Ca/Na exchanger (23). In dual-label immunohistochemistry, the second antibody was either directly labeled
with FITC or Texas red (MAbs DT.17, F13/483, ST.12) or visualized using
Alexa-568 or Texas red-conjugated species-specific antibodies in
1:2,000-1:4,000 dilution (for antibodies against H-ATPase and the
basolateral HCO3/Cl exchanger, band 3). In the latter experiments, either isotype-specific fluorochrome-labeled secondary antibody was used, or in the case of directly labeled MAbs,
binding to the secondary antibody was prevented by blocking the
sections with 10% mouse serum before the second mouse antibody was
applied. Appropriate controls with the second antibody alone were
performed in each case. Sections were mounted in Vectashield (Vector
Laboratories). Fluorescence images were captured on a PXL-cooled
charge-coupled device camera (Photometrics) attached to an Olympus IMT2
microscope equipped with an epifluorescence attachment and standard
FITC, Cy-5, and Texas red filter sets. Fluorescence laser scanning
confocal microscopy was performed on a Bio-Rad MRC-1024 confocal system
as described (22).
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RESULTS |
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Generation and characterization of MAbs.
MAbs were generated against the rabbit HK2 by
immunization of mice with a fusion protein encompassing amino acids
1-99 in the rabbit HK
2a (13). By using
ELISA, we identified several hybridoma clones that produced antibodies
reacting with the fusion protein. MAbs were tested on Western blots of
lysates of bacteria expressing the fusion protein and lysates of rabbit
distal colon. Three of the antibodies, designated 121, 132, and 233, recognized a major protein band with the appropriate size (14,700 kDa)
in lysates of E. coli expressing the recombinant
rabbit HK
2 (Fig. 1A, lanes
2-4). Omission of the primary antibody or substitution with
an irrelevant antibody that did not react with the HK
2
antigen in ELISA (MAb 11) yielded no detectable signal, as shown in
Fig. 1A, lane 1. The faint smaller band
recognized by MAb 121 (Fig. 1A, lane 2) most
likely corresponds to a degradation product or an early termination
product. This band was not observed with the other two MAbs. MAbs 121, 132, and 233 reacted with a single band with an apparent molecular mass
of ~100 kDa on immunoblots originating from the distal colon (Fig.
1B, lanes 2-4). We made numerous,
unsuccessful attempts to detect HK
2 on blots prepared with total kidney, cortex, or medulla. It is important to note that
these blots did not reveal any nonspecific reaction of the MAbs either
(not shown). To compensate for the apparent low abundance of the
HK
2 protein in the kidney, we thus enriched for
HK
2 protein by immunoprecipitation. Subsequent
immunoblotting of the protein precipitated with MAb 121 yielded a
strong band around 100 kDa in the distal colon (Fig. 1C,
lane 1), and a weaker but still readily detectable band in
the renal cortex (lane 2). However, even with prior
immunoprecipitation, we failed to detect immunoreactivity in the
medulla (lane 3).
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Immunohistochemistry.
All three MAbs reacted strongly with the apical membrane of surface
epithelial cells in the distal colon (Fig.
2). This localization is very similar to
that observed in the rat colon (25).
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DISCUSSION |
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Although expression of H-K-ATPases in the kidney is well
established, the exact cellular localization and function mediated by
the individual H-K-ATPase isoforms are still unclear. The focus of the
present study is the colonic H-K-ATPase, which, according to a recent
study with knockout mice, plays a role in K conservation in the colon
during K deprivation (20). Results of mRNA localization studies indicate that HK2 is expressed in the CNT
(1, 13), CCD (2, 13, 14), OMCD, and inner
medullary collecting duct (IMCD) (2).
The major finding of this study is that the HK2 protein
is present in the apical membrane of connecting tubule cells in rabbit kidney. This conclusion is based on results obtained with MAbs that
seem to be directed against different epitopes on the rabbit HK
2 subunit. The specificity of these antibodies was
confirmed by immunoblotting (Fig. 1) and specific
immunohistochemical reaction on distal colon sections (Fig. 2).
The observation that in the rabbit kidney HK2 is
expressed in the apical membrane of CNT cells was unexpected for
several reasons. First, functional studies indicated that H-K-ATPase
activity is present in intercalated cells (28, 30, 33),
where it was assumed to participate in luminal acidification and
K+ reabsorption. However, in the rabbit kidney, we did not
observe immunoreactivity in intercalated cells that were clearly
labeled with various cell-specific markers (Figs. 3-6). On the
other hand, our data that connecting tubular cells express
HK
2 protein are in agreement with earlier in situ
hybridization studies in rats showing strong reactivity in CNT
(1). However, in the same report (1), mRNA
signals were observed in the medulla, whereas we failed to detect
HK
2 protein anywhere else than in a subpopulation of
cells within the CNT. Our data also differ from those obtained with a
polyclonal antibody generated against the NH2-terminal region of the rat colonic H-K-ATPase (25). In that study,
no staining was observed in the CNT or any other cortical tubule of
rats on a normal K+ diet, whereas principal cells in the
OMCD were labeled (25). Similarly, a recent report showed
that immunoblots from rats subjected to chronic hypokalemia reacted
with an anti-HK
2 antibody, whereas the cortex remained
negative (7).
There are several possible explanations for the apparent discrepancies between our data and those cited above. First, the diverging results from the studies by Ahn and coworkers (1) might be related to the different methods applied (detection of mRNA in their study vs. immunohistochemistry in ours). The discrepancies between earlier reports (1, 7, 25) and our findings might also be related to species differences. The rat and the rabbit have very different natural diets and metabolism, with respect to both K+ intake and acid excretion. Because the relative contribution of H-K-ATPases to K+ and acid-base homeostasis might vary, depending on the localization, it would seem logical that the intrarenal or cellular localization of this enzyme varies in species with different needs, to conserve K+ or secrete acid.
A further possibility is that the antibodies used in this study vs.
those used by Sangan et al. (25) recognize different splice variants of HK2 (17). The splicing
pattern is different between rat (17) and rabbit
(6). Although the antibodies used in this study were
directed against a region that is common to the two splice variants of
HK
2 in the rabbit, HK
2a and
HK
2c (6), it is not clear that the anti-rat
antibodies are also directed against shared epitopes.
It is also possible that the HK2 antigens are not
equally accessible to our MAbs in all nephron segments. If this were
the case, one would expect that antigen-unmasking techniques would reveal immunoreactivity at additional sites. However, using an antigen-retrieval method (5) we failed to reveal
additional immunoreactivity (data not shown). Finally, although with
the above described immunohistochemical techniques we could not detect any labeling in the CCD, OMCD, and IMCD of rabbit kidneys, these data
do not exclude the possibility that HK
2 is expressed in these segments at lower levels.
What might be the function of an H-K-ATPase located in the apical
membrane of connecting tubule cells? A recent study with knockout mice
indicates that, under normal dietary conditions, the elimination of
this isoform does not result in major consequences in either K or
acid-base balance. However, if these mice are K deprived, they lose
excessive amounts of K, suggesting that the main function of the
colonic H-K-ATPase is K conservation (20). The CNT and CCD
exhibit net K secretion, whereas an apical H-K-ATPase is expected to
reabsorb K. One possibility is that an H-K-ATPase in the apical
membrane of CNT cells might participate in K+ recycling,
thereby decreasing the obligatory K+ loss associated with
Na+ reabsorption by the kidney. Such an idea is compatible
with the observations of Wang and coworkers (32) that
HK2 mRNA expression is upregulated by Na depletion in
the renal cortex. H-K-ATPase expressed in the apical membrane of
K+-secreting cells might also exert a local "paracrine"
effect by acidifying the luminal microenvironment. Because an acidic
luminal pH inhibits K+ secretion (4), an
H-K-ATPase, besides recycling secreted K, may also limit K secretion
itself. Such a mechanism might be particularly beneficial under
conditions of K deprivation.
Another possible role of the colonic H-K-ATPase is to mediate enhanced
secretion of NH
In summary, our immunohistochemical data obtained with MAbs directed
against different epitopes on the rabbit colonic H-K-ATPase indicate
that, in the rabbit kidney under normal dietary conditions, the
HK2 protein is expressed in the apical membrane of
connecting tubule cells. The function of the colonic H-K-ATPase in this
nephron segment remains to be identified.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-39523, DK-55845, and DK-41841.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. Fejes-Tóth, Dept. of Physiology, Dartmouth Medical School, Lebanon, NH 03756 (E-mail: geza.fejes-toth{at}dartmouth.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 2 August 2000; accepted in final form 17 April 2001.
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