Departments of 1 Cellular and Molecular Physiology and 2 Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Electrogenic
cotransport of Na+ and
HCO3 is a crucial element of
HCO
3 reabsorption in the renal
proximal tubule (PT). An electrogenic
Na+-HCO
3
cotransporter (NBC) has recently been cloned from salamander and rat
kidney. In the present study, we generated polyclonal antibodies (pAbs)
to NBC and used them to characterize NBC on the protein level by
immunochemical methods. We generated pAbs in guinea pigs and rabbits by
immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928-1035) of rat kidney NBC (rkNBC). By
indirect immunofluorescence microscopy, the pAbs strongly labeled
HEK-293 cells transiently expressing NBC, but not in untransfected
cells. By immunoblotting, the pAbs recognized a ~130-kDa band in
Xenopus laevis oocytes expressing
rkNBC, but not in control oocytes injected with water or cRNA for the
Cl
/HCO
3
exchanger AE2. In immunoblotting experiments on renal microsomes, the
pAbs specifically labeled a major band at ~130 kDa in both rat and
rabbit, as well as a single ~160-kDa band in salamander kidney. By
indirect immunofluorescence microscopy on 0.5-µm cryosections of rat
and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP),
the pAbs produced a strong and exclusively basolateral staining of the
PT. In the salamander kidney, the pAbs labeled only weakly the
basolateral membrane of the PT. In contrast, we observed strong
basolateral labeling in the late distal tubule, but not in the early
distal tubule. The specificity of the pAbs for both immunoblotting and
immunohistochemistry was confirmed in antibody preabsorption
experiments using either the fusion protein used for immunization or
similarly prepared control fusion proteins. In summary, we have
developed antibodies specific for NBC, determined the apparent
molecular weights of rat, rabbit, and salamander kidney NBC proteins,
and described the localization of NBC within the kidney of these
mammalian and amphibian species.
fluid and electrolytes; renal bicarbonate reabsorption; acid-base; polyclonal antibody; immunofluorescence; immunoblotting; rat; rabbit; salamander
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INTRODUCTION |
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SODIUM-BICARBONATE COTRANSPORTERS are members of an
emerging superfamily of
Na+-coupled
HCO3 transporters (29) and are
widespread throughout the animal kingdom, including invertebrates (12), birds (19), amphibia (8), and mammals (18).
Na+-HCO
3
cotransporters are present in a wide variety of epithelial and
nonepithelial tissues and cell types (for review, see Ref. 7). Based on
physiological experiments,
Na+-HCO
3
cotransporters seem to exist with
Na+:HCO
3
coupling ratios of 1:3, 1:2, and 1:1 (for review, see Ref. 7).
Na+-HCO
3
cotransport with a
Na+:HCO
3
stoichiometry of 1:3 normally moves
Na+ and
HCO
3 out of cells, as in the renal
proximal tubule (34). On the other hand, cotransport with a
stoichiometry of 1:2 or 1:1 can move
Na+ and
HCO
3 into cells, such as glial cells,
and often is an important mechanism of acid extrusion (for review, see
Ref. 7).
Na+-HCO3
cotransport is of special importance in the kidney, where it plays a
double role at the intersection of the two fundamental homeostatic
systems that control extracellular fluid (ECF) volume on the one hand
and systemic pH on the other. First,
HCO
3 reabsorption in the proximal
tubule promotes salt and water reabsorption (22, 23, 31), whereas ECF
volume expansion inhibits HCO
3
reabsorption (4, 20, 27). Second,
Na+-HCO
3
cotransport is of paramount importance for systemic pH homeostasis.
Reclamation of HCO
3 from the
glomerular filtrate, mainly in the proximal tubule, wards off the
urinary loss of HCO
3. The last step of
HCO
3 reclamation, the transfer of
HCO
3 from the cytoplasm across the
basolateral membrane back into the ECF, occurs almost exclusively via
the electrogenic
Na+-HCO
3
cotransporter (2, 34).
Since the initial description of the
Na+-HCO3
cotransporter by Boron and Boulpaep in 1983 (8), several investigators
have gained valuable insights into the physiology of
Na+-HCO
3
cotransporters using measurements of transport activity in native
tissues, cells, or membrane preparations (overview in Ref. 3). However,
neither antibodies nor polynucleotide probes have been available. Only
very recently have attempts to clone a
Na+-HCO
3
cotransporter (NBC) succeeded: the cDNA encoding a
Na+-HCO
3
cotransporter from the kidney of the salamander Ambystoma tigrinum (aNBC) was isolated
by expression cloning in Xenopus
laevis oocytes (29). Subsequently, cDNAs from rat
(rkNBC, Ref. 28) and human kidney (hNBC, Ref. 11) were obtained by exploiting the homology with aNBC or the
Cl
/HCO
3
exchangers, respectively. The cloning of these
Na+-HCO
3
cotransporters makes it possible to generate molecular probes.
In the present study, we describe the generation of polyclonal antisera
and report the first immunoblotting and immunolocalization data on
Na+-HCO3
cotransporters. We found that the
Na+-HCO
3
cotransporter proteins in the rat and rabbit kidneys each have an
apparent molecular mass
(Mr)
of ~130 kDa by SDS-PAGE. Because the predicted
Mr of NBC is 116 kDa, these results are consistent with, at most, modest
posttranslational modification. In contrast, aNBC has an apparent
Mr of ~160 kDa, suggestive of extensive posttranslational modification. By
immunofluorescence microscopy, we found that NBC is strongly expressed
in the basolateral membrane of the proximal tubule of rat and rabbit
and in the basolateral membrane of a distinct portion of the late
distal tubule of the salamander.
Portions of the present work have been published in abstract form (13, 14).
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METHODS |
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Generation of Polyclonal Antisera
Preparation of immunogen.
CLONING. We used standard procedures to prepare a fusion
protein (MBP-NBC-5) of maltose binding protein ("MBP") and the
COOH-terminal 108 amino acids (i.e., residues 928-1035) of rat
kidney NBC (rkNBC; GenBank accession no. AF004017) (5). Briefly, the
portion of the rkNBC cDNA sequence coding for the COOH terminus was
amplified by PCR using modified primers that added an
EcoR I restriction site on the
5' end, and a stop codon plus an
Xba I restriction site on the 3'
end (sense primer: 5'-Cg gAA TTC gCg ATT ATT TTT CCA gTC ATg
ATC-3'; antisense primer: 5'-Cg TCT AgA TCA gCA TgA TgT gTg
gCg TTC AAg g-3'). The purified PCR product was digested with
EcoR I and
Xba I, repurified, and ligated into
the MBP expression vector pMAL-c2 (NEB, Beverly, MA) that had been
linearized with EcoR
I/Xba I. The resulting construct
(pMAL-NBC-5) was transfected into Escherichia
coli (DH5; Life Technologies, Gaithersburg, MD) and propagated in Luria-Bertani medium containing 100 µg/ml ampicillin (LB/Amp). Bidirectional sequencing of the purified plasmid confirmed the absence of frame shifts or mutations.
Characterization of Antisera by Heterologous Expression
Expression in HEK-293 cells. For transient expression in HEK-293 cells, the original aNBC cDNA clone, including 5'- and 3'-untranslated regions (28), was ligated into the Not I site of pSV-SPORT-1 plasmid (Life Technologies). Near-confluent HEK-293, grown on coverslips, were transfected using the DEAE-dextran method (5) and grown for 48 h. Untreated cells, mock-transfected cells, and cells transfected with only the "empty" vector were used as controls. We studied expression of aNBC by indirect immunofluorescence microscopy. Briefly, cells were fixed in 3% paraformaldehyde/PBS, permeabilized in 0.3% Triton X-100/PBS, blocked in 20% goat serum/PBS, and incubated for 1 h at room temperature with the respective sera diluted 1:100 in 20% goat serum/PBS. After washing the sample three times in PBS, we incubated it for 1 h with the secondary antibody [anti-guinea pig IgG, heavy and light chain, F(ab')2, conjugated to fluorescein isothiocyanate; Zymed Laboratories], diluted 1:2,000 in 20% goat serum/PBS. After three 30-min washes in PBS, the coverslips were mounted on glass slides in Aqua-mount (Learner) and examined on a Zeiss Axiophot fluorescence microscope. For micrographs, 100 ASA black-and-white film (T-Max 100, Kodak) was used.Expression in oocytes of X. laevis.
cRNAs encoding rkNBC and the murine anion-exchanger isoform AE2 were
transcribed from pTLN-2 (28) or pBluescript (36), respectively, and
injected into stage V-VI oocytes of X. laevis. Expression of the respective ion transporters
was assessed 8 days later using microelectrodes to monitor membrane
potential (Vm)
and intracellular pH (pHi) changes in response to removal of external
Na+ and
Cl (28). Subsequently,
Triton X-100 extracts of individual oocytes were prepared as described
elsewhere (17), except that methionine was omitted from the extraction
buffer. The extracted proteins were separated by SDS-PAGE and
immunoblotted as described below.
Immunoblotting of the Native Renal
Na+-HCO3
Cotransporter
Electrophoresis and transfer. Proteins
from the microsome preparation were separated by denaturing SDS-PAGE
under reducing conditions (100 mM dithiothreitol or 0.5%
-mercaptoethanol) in a discontinuous system. If not indicated
otherwise, then 1-mm-thick 7.5% gels were used, prepared from a
premixed monomer stock [total monomer concentration T = 30%
(wt/vol), concentration of cross-linker C = 2.5% (wt/vol) (16);
Analytical Biochemicals, Natick, MA]. For the stacking gels, we
used a lower concentration of acrylamide together with an increased
concentration of cross-linker (T = 2.5%, C = 25%),
rendering them more rigid and less sticky than conventional stacking
gels (T = 3.5%, C = 2.5%). For running buffers, either a Tris-glycine
buffer [375 mM Tris, 0.1% (wt/vol) SDS, pH adjusted to 8.8 with
glycine-HCl] or a Tris-borate buffer (375 Tris, 0.1% SDS, pH
adjusted to 9.1 with boric acid) was used. Following electrophoretic
separation, proteins were transferred overnight at 0.5-1.0
mA/cm2 in a semi-dry blotting
apparatus (Bio-Rad Laboratories, Richmond, CA) onto polyvinylidene
difluoride (PVDF) membranes (Immobilon-P; Millipore, Bedford, MA) using
the discontinuous Tris-glycine buffer system described by the
manufacturer (Millipore).
Proteins on the membranes were stained with Coomassie blue G250. The membranes were photocopied and subjected to the immunodetection protocol. All estimates of Mr were obtained by comparison to unstained standards, spaced in regular 10-kDa intervals (GIBCO), and run in the same gel. Prestained Mr standards (BioRad) consistently yielded lower and more scattered Mr estimates.
Immunodetection. For immunodetection, membranes were blocked for 30 min at ~22°C in Blotto, which consists of 5% (wt/vol) Carnation nonfat dry milk (Nestlé Food, Glendale, CA) and 0.1% Tween-20, in PBS (in g/l: 8 NaCl, 1.44 Na2HPO4, 0.24 KH2PO4, and 0.2 KCl, pH 7.4). Subsequently, membranes were incubated with the antisera at the indicated dilutions in Blotto for 1-2 h at ~22°C, or overnight at 4°C, followed by three 10 min-washes in Blotto. These washes were followed by a 1-h incubation with the secondary antibody (horseradish-peroxidase-labeled, affinity-purified, species-specific goat anti-IgG/whole molecule antibodies; Sigma, St. Louis, MO; diluted 1:2,000 to 1:10,000 in Blotto), three 10 min-washes in Blotto, and one 10 min-wash in PBS. Bound horseradish-peroxidase label was detected by chemiluminescence according to the manufacturer's protocol (SuperSignal substrate; Pierce, Rockford, IL) and documented on Kodak XOMAT AR film.
Antibody preabsorption experiments.
Primary antibodies in Blotto were preabsorbed at ~22°C for 1 h
with 10 µg/ml of one of the fusion proteins: MBP-gal, MBP-NBC-3,
MBP-NHE3, or MBP-NBC-5. This preabsorption was followed by the standard
immunodetection protocol.
Immunolocalization of NBC in Rat, Rabbit, and Salamander Kidney
Tissue preparation for immunohistochemistry. SALAMANDER. Female specimens of the aquatic phase A. tigrinum, kept at 4°C, were anesthetized by submersion in 0.2% tricaine methanesulfonate. The abdomen was opened via two paramedian incisions and one transverse suprapubic incision. The kidneys were exposed and perfused for 15 min via the venous portal circulation with cold, amphibian NaCl Ringer buffered with 10 mM HEPES, pH 7.5. The perfusion solution was then switched to a periodate-lysine-paraformaldehyde fixative (PLP; in mM, 8 NaIO4, 60 L-lysine, 30 Na2HPO4, 4% paraformaldehyde, in PBS of 200 mosmol/kgH2O, pH 7.4). The kidneys were then removed, postfixed for 4-6 h in the same fixative, washed in PBS, and stored in 0.5% paraformaldehyde in PBS at 4°C.
RAT AND RABBIT. Adult New Zealand White rabbits and Sprague-Dawley rats were anesthetized with pentobarbital sodium, and the kidneys were perfusion-fixed by first inserting a cannula into the descending aorta distal to the renal arteries. The kidneys were then perfused retrograde with PBS, pH 7.4 at 37°C, to remove blood, followed by PLP fixative (in mM, 10 NaIO4, 75 L-lysine, 2% paraformaldehyde in PBS of 300 mosmol/kgH2O, pH 7.4). For cryostat sections, kidneys were cut in half on a midsagittal plane and postfixed in PLP for 4-6 h. The fixed tissue was then cryoprotected overnight in a 30% solution of sucrose in PBS. Five-micrometer cryosections were cut on a Reichert cryostat, and mounted on gelatin-coated slides. To obtain semithin (0.5 µm) cryosections, blocks of tissue (2- to 4-mm cubes) from fixed kidneys were cut sequentially from cortex, medulla, and papilla. Thus representative tissue from all zones of the kidney was selected, and care was taken to maintain their original orientation. Tissue blocks were postfixed in PLP for an additional 4-6 h at room temperature, cryoprotected by a 1-h incubation in 2.3 M sucrose in phosphate buffer (pH 7.2) with 50% polyvinylpyrrolidone, mounted on aluminum nails, and frozen in liquid nitrogen for storage. Cutting of semithin cryosections was carried out on a Reichert Ultracut E ultramicrotome fitted with an FC-4E cryoattachment, then sections were mounted on gelatin-coated slides.Indirect Immunofluorescence Microscopy
Indirect immunofluorescence microscopy was performed on either 5-µm cryosections or on 0.5-µm cryosections as described previously (6). Briefly, tissue sections were washed sequentially in PBS, then in 50 mM NH4Cl in PBS, and in blocking buffer (1% bovine serum albumin in PBS). These washes were followed by a 1-h incubation with the primary antiserum, diluted 1:50 in 50% goat serum in PBS. After a PBS wash, sections were incubated for 1 h with the secondary antibody [anti-guinea pig IgG, heavy and light chain, F(ab')2, conjugated to fluorescein isothiocyanate; Zymed], diluted 1:100 in 50% goat serum in PBS. Subsequently, slides were washed in PBS and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Micrographs were taken with a Zeiss Axiophot microscope using either Tri-X (ASA 400) or T-Max (ASA 100) films. ![]() |
RESULTS |
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Generation of Polyclonal Antisera
Fusion proteins MBP-
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The guinea pigs and rabbits immunized with MBP-NBC-5 (see
METHODS) yielded antisera that
reacted strongly with MBP-NBC-5 (not shown). However, these antisera
generated from MBP-NBC-5 also reacted with MBP-NBC-3 and with
MBP-gal (not shown), indicating that the MBP portion of the fusion
protein was itself a strong immunogen. Because MBP is found only in
bacteria, we did not expect this anti-MBP immunoreactivity in the
antisera to compromise the specificity in our study on vertebrates.
Another concern was the possibility of contamination of the immunogen
by bacterial components, which are known to elicit strong immune
responses. The experiments that follow were designed to test for these
two types of cross-reactivity.
Characterization of Antisera by Heterologous Expression
To test the specificity of our antisera for immunocytochemistry and immunoblotting applications, we expressed NBC heterologously in either HEK-293 cells or Xenopus oocytes.Indirect immunofluorescence microscopy in HEK-293 cells expressing aNBC. We performed indirect immunofluorescence microscopy on HEK-293 cells using the guinea pig anti-MBP-NBC-5 serum. Untreated HEK-293 cells, mock-transfected cells, and cells transfected with the "empty" vector showed only weak background fluorescence. On the other hand, virtually all cells transfected with aNBC in pSV-SPORT-1 exhibited intense fluorescence throughout the plasma membrane and cytoplasm (Fig. 2). The cytoplasmic staining probably reflects accumulation of NBC in the endoplasmic reticulum and Golgi complex, which is often observed upon overexpression. The test of antibody specificity, however, does not depend on the subcellular localization of NBC.
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Our findings confirm that the antiserum raised against the COOH terminus of rkNBC easily recognizes the COOH terminus of aNBC, which is ~88% identical with rkNBC at the amino acid level. The antisera do not significantly cross-react with any other components of these mammalian cells.
Immunoblots of oocytes expressing rkNBC. We next performed immunoblot experiments on Xenopus oocytes expressing rkNBC. For controls, we used oocytes injected with either water or cRNA encoding AE2. We chose AE2 as a negative control, because, among all proteins whose primary structure is known, the anion exchangers have the highest degree of homology to the NBCs. In several oocytes, we used electrophysiological techniques to confirm expression of AE2 or rkNBC. On several others, we performed the immunoblots using either rabbit anti-MBP-NBC-5 serum or rabbit anti-MBP-NBC-3 serum. However, in a single oocyte expressing AE2 and in another expressing rkNBC (Fig. 3), we sequentially performed both the electrophysiological characterization and the immunoblot.
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Microelectrode measurements of
Vm and
pHi in oocytes injected with cRNA
encoding AE2 or rkNBC showed high activity of the respective
transporters (Fig. 3A), indicating
strong expression of functional protein. Upon exposure to 5%
CO2/33 mM
HCO3, pH 7.5, the oocyte injected with
AE2 cRNA responded with an initial acidification, due to
CO2 influx, followed by a slow
recovery of pHi (Fig.
3A, left).
Cl
removal from the bath
solution substantially increased the rate of alkalinization, whereas
returning the Cl
produced
an acidification. These effects were repeatable. In fact, the
pHi decrease elicited by returning
Cl
was more pronounced
after the second zero-Cl
pulse, consistent with the stimulation of
Cl
/HCO
3
exchange at high pHi (10).
Consistent with the presence of an electroneutral
Cl
/HCO
3
exchanger is the observation that changing the extracellular
Cl
concentration had no
specific effect on
Vm.
The oocyte injected with rkNBC-cRNA instantly hyperpolarized by ~80
mV upon addition of
CO2/HCO3
(Fig. 3A, right). The hyperpolarization is caused
by the inward, electrogenic flux of
Na+ and
HCO
3 mediated by NBC. Upon removal of
extracellular Na+, the oocytes
depolarized by ~90 mV. We attribute this depolarization to the
outward, electrogenic movement of
Na+ and
HCO
3. This effect of removing
Na+, which is the hallmark of
electrogenic
Na+-HCO
3
cotransporters (8), was fully reversible and repeatable. Similar
results have been obtained previously in oocytes expressing either
salamander or rat kidney NBC (28, 29). Introducing
CO2/HCO
3
caused a rapid pHi decrease,
followed by a slower recovery. As expected, lowering the extracellular
Na+ concentration reversibly
lowered the rate of alkalinization. The
pHi changes produced by
electrogenic
Na+-HCO
3
cotransport are relatively slow (29), reflecting the small
surface-to-volume ratio of oocytes.
After completion of the microelectrode measurements (Fig. 3A), we prepared Triton X-100 extracts of the same two oocytes. These extracts contained most of the membrane proteins but very little of the abundant yolk proteins that would otherwise interfere with immunodetection. We separated these proteins by SDS-PAGE, transferred them to a PVDF membrane, and probed them with rabbit anti-MBP-NBC-5 serum. The antiserum strongly recognized a single band of ~130 kDa in the extracts from the oocyte expressing rkNBC (Fig. 3B). The predicted Mr of NBC from rat kidney is 116 kDa (28). The antiserum did not react with any proteins from the oocytes injected with either water or AE2 cRNA (Fig. 3B). We obtained identical results on other oocytes injected with either water, AE2 cRNA, or rkNBC cRNA, but not having been subjected to electrophysiological measurements. In these latter experiments, the results were the same with both rabbit anti-MBP-NBC-3 serum and rabbit anti-MBP-NBC-5 serum.
Together, the above data show that the antisera raised to the NBC
fusion proteins are specific for the electrogenic
Na+-HCO3
cotransporter in both immunohistochemical and immunoblotting assays.
Immunoblotting of the Native Renal
Na+-HCO3
Cotransporter
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Using guinea pig anti-MBP-NBC-5 or guinea pig anti-MBP-NBC-3, we similarly obtained predominant bands at ~160 kDa for salamander and at ~130 kDa for rat and rabbit.
Antibody preabsorption experiments on rat
kidney. The specificity of the labeling observed in the
kidneys of salamander, rat, and rabbit was tested in further
immunoblotting experiments. Figure 5 shows
a representative experiment on rat kidney proteins. In the control
lane, probed with native rabbit anti-MBP-NBC-5 serum, we saw the same
banding pattern as in Fig. 4, i.e., a major band at ~130
kDa and two minor bands at ~100 and ~85 kDa. In the lanes labeled
MBP-gal and MBP-NBC-3, we probed, respectively, with sera previously
depleted of antibodies directed against either MBP-
gal (by
preincubating with an excess of MBP-
gal) or MBP-NBC-3 (by
preincubating with an excess of MBP-NBC-3). Although the antisera used
in lanes 2 and
3 were thus depleted of antibodies
directed against MBP per se, the banding patterns were
undistinguishable from the one produced by the undepleted serum in
lane 1.
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In the lane labeled MBP-NBC-5, we probed with anti-MBP-NBC-5 serum that
had been depleted of antibodies directed against MBP-NBC-5 (by
preincubating with an excess of MBP-NBC-5). We observed no bands
whatsoever. These results demonstrate that all three bands observed in
lanes 1-3 were due to labeling by
antibodies specifically directed against the NBC-5 portion of rkNBC.
There is no evidence for cross-reactivity of antibodies directed
against either MBP per se or against bacterial contaminants. Because a
~130-kDa band was also observed in
Xenopus oocytes heterologously
expressing rkNBC (Fig. 3B), it is
likely that the ~130-kDa protein truly corresponds to the rat kidney
Na+-HCO3
cotransporter protein. The nature of the two minor bands at ~100 and
~85 kDa is not clear. However, the antibody depletion experiments
demonstrate that proteins in these two bands have NBC-specific
epitopes. The two bands may represent proteolytic fragments of the
~130-kDa protein or different NBC isoforms. We observed identical
bands of ~100 and ~85 kDa with anti-MBP-NBC-3 serum, but not with
preimmune serum. The bands persisted with higher concentrations of up
to seven different protease inhibitors or when we carried out the
tissue homogenization directly in sample-loading buffer or strong
denaturants, such as 7 M urea or 4.5 M guanidinum isothiocyanate.
Using an antibody preabsorption approach similar to that outlined for Fig. 5, we also demonstrated that the labeling (see Fig. 4) of salamander and rabbit kidney with rabbit anti-MBP-NBC-5 is specific. Furthermore, we also showed that guinea pig anti-MBP-NBC-5 specifically labels kidney membrane preparations from rabbit. For all antisera, preimmune bleeds, obtained from rabbits and guinea pigs prior to the first injection of immunogen, did not detect any of the bands shown in Figs. 4 and 5.
Immunolocalization of NBC in Rat, Rabbit, and Salamander Kidney
To determine the cellular and subcellular location of the NBC protein in the kidneys of rat, rabbit, and salamander, we performed immunofluorescence staining of semithin (0.5 µm) sections, and immunoperoxidase staining of standard (5 µm) PLP-fixed cryosections. We used guinea pig anti-MBP-NBC-5 serum in both cases.Antibody preabsorption experiments. For each tissue, we separately assessed the specificity of the antiserum, using procedures analogous to those described above for the immunoblots. Figure 6 shows the results of such an experiment on 0.5-µm-thick sections of rat kidney, with the fluorescence images on the top and the transmission images on the bottom. Using preimmune serum, we observed no staining (Fig. 6A), a finding that rules out the presence of antibodies against cytoskeleton components that are sometimes spontaneously found in rabbit serum. Using immune serum that had been preabsorbed to MBP-NHE3 (i.e., a fusion protein devoid of NBC-specific sequences), we observed a strong fluorescence signal in proximal tubule cells (Fig. 6B). The staining pattern is typical of the basolateral membrane. Finally, preabsorbing the immune serum with MBP-NBC-5 (i.e., the fusion protein used for immunization) completely abolished the fluorescence labeling (Fig. 6C). We observed similar specificity of the antiserum in comparable immunofluorescence experiments on rabbit and salamander.
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Immunolocalization of NBC in rat and rabbit
kidney. The experiments described in the previous
section establish that the anti-MBP-NBC-5 serum, in combination with
the immunofluorescence protocol, detects the
Na+-HCO3
cotransporter specifically and at high spatial resolution. We next used
this protocol to determine the distribution of NBC along rat and rabbit
nephrons, systematically examining multiple semithin (0.5 µm) and
standard (5 µm) sections with guinea pig anti-MBP-NBC-5 serum. Figure
7 is a low-magnification overview of a
5-µm-thick coronal section of a rat kidney, stained with guinea pig
anti-MBP-NBC-5 serum using an immunoperoxidase technique. The staining
is confined to the superficial and midcortical regions of the cortex
and is absent from the medulla. In higher powered views of 5- and
0.5-µm sections, the staining localized exclusively to proximal
tubules. NBC immunoreactivity was consistently absent in all other
cortical structures, including the thick ascending limb, cortical
collecting duct, glomerulus, vasculature, and interstitium.
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We observed significant heterogeneity of labeling for NBC along the rat proximal tubule, with staining being greater in the early portions than toward the end of this segment. The anti-MBP-NBC-5 serum consistently and heavily stained S1 segments, identified by their continuity with glomeruli. In contrast, the S3 segments, as determined by locating the proximal tubule/thin limb of Henle transition, were never stained (data not shown). Because of the difficulty in identifying the boundaries of S2 segments, we were not able to accurately determine at what point along the proximal tubule the staining for NBC fell below our detection limit.
Because all labeling was confined to proximal tubule segments, we can correlate the staining pattern directly with the known topography of the S1, S2, and S3 segments within the kidney. The strongest staining was within the cortical labyrinth (Fig. 7) and can be positively assigned to the S1 and/or the early S2 segments. We detected no labeling within the medullary rays and in the outer stripe of the outer medulla. These latter observations rule out substantial amounts of NBC-immunoreactive material in the proximal straight tubule, i.e., the late S2 and the S3 segments. Thus the indirect evidence for axial heterogeneity of NBC, based on topography within the kidney, was in good agreement with the more direct morphological evidence presented in the preceding paragraph.
The rabbit kidney exhibited the same staining pattern that we observed in the rat kidney, using the guinea pig anti-MBP-NBC-5.
Immunolocalization of NBC in salamander kidney. We next determined the distribution of NBC in the kidney of the salamander A. tigrinum, again examining multiple semithin (0.5 µm) and standard (5 µm) sections with guinea pig anti-MBP-NBC-5 serum. The overview of the salamander kidney shown in Fig. 8A reveals intensely stained tubules in a crescent-shaped zone. In all cases, the crescent-shaped zone was separated from the lateral surface of the kidney by ~2 mm of unlabeled tissue (see Fig. 8B). In Fig. 8A, the crescent-shaped zone overlaps the more medial glomerular zone. In other sections (not shown), the crescent-shaped zone was even more lateral and thus separated also from the glomeruli by a region of unlabeled segments. However, in no case were these intensely stained tubules seen medial to the glomeruli. By their position within the kidney, these intensely stained tubule segments can be identified as "late distal tubules," as defined by Planelles and Anagnostopoulos (26). These intensely stained tubules probably even conform to the narrower definition of the "late distal tubule" by Yucha and Stoner (35).
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In a higher magnification (Fig. 9), the cells within these intensely stained tubules lack a brush border and have a distinct distal tubule morphology. In addition, the staining pattern in the salamander kidney shows that NBC localizes exclusively to the basolateral membrane. The labeling follows the infoldings of the basolateral membrane, which are numerous and pronounced in the salamander distal tubule.
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In the thicker proximal tubules, where the electrogenic
Na+-HCO3
cotransporter was first identified (8), we detected only weak staining.
We saw no staining in any structures other than proximal and "late
distal" tubules. Thus, like in the mammal, NBC is expressed strictly
basolaterally in the salamander kidney. However, unlike in mammals, NBC
is most abundant in the distal tubule.
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DISCUSSION |
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Transepithelial HCO3 reabsorption in
the renal tubule (as in other epithelia) is a complex process that requires several transporters, as well as various carbonic anhydrases (15). The transport events and reactions that constitute
HCO
3 reabsorption are highly
coordinated, making it possible for the tubule cell to up- and
downregulate HCO
3 reabsorption without
catastrophic consequences for pHi.
With the exception of the electrogenic
Na+-HCO3
cotransporter, antibodies and/or nucleotide probes have been available for studying the other major players known to participate in
HCO
3 reabsorption (e.g.,
Na+/H+
exchanger, H+-ATPases, carbonic
anhydrase isoforms II and IV, and
Cl
/HCO
3
exchanger). Consequently, these other proteins could be studied in
great detail. Meanwhile, the
Na+-HCO
3
cotransporter could not be investigated by any methods other than
functional assays. The cloning of the renal
Na+-HCO
3
cotransporter has now made it possible to generate molecular probes
that could be instrumental in obtaining a more complete understanding
of proximal tubule HCO
3 handling. The
aim of the present work was to generate the first antibodies to NBC, to
establish methods to detect
Na+-HCO
3
cotransporters with these antibodies on blots and in tissue sections,
and to collect some basic information about the cotransporter protein
and its distribution within the kidney.
We have generated polyclonal antisera, and demonstrated by several approaches their suitability as specific probes for renal NBCs, both in immunoblotting and in immunohistochemistry applications. We found that the renal NBCs from rat and rabbit kidney exhibit very similar characteristics: 1) they react equally well with antisera raised against the carboxy terminus of the rkNBC, 2) they both have an apparent Mr of ~130 kDa on denaturing polyacrylamide gels, and 3) they are both abundantly expressed on the basolateral membranes of the S1 and S2 segments of the renal proximal tubule. In contrast, the NBC from salamander kidney exhibited a higher apparent Mr of ~160 kDa, consistent with posttranslational modification. Unlike rat and rabbit NBC, the salamander cotransporter is strongly expressed in the distal tubule, but only weakly expressed in the proximal tubule. Like rat and rabbit NBC, the salamander cotransporter in the kidney is strictly basolateral and confined to the tubules.
Rabbits immunized with the COOH-terminal portion of the rat NBC (MBP-NBC-5) generated antibodies that specifically interacted with both rat and rabbit NBC. Two conclusions can be drawn from this finding. First, rabbit kidney NBC, which has not yet been cloned, must be very similar to rkNBC, at least at the COOH terminus. Given the high similarity of NBC from such distant species as salamander and rat (i.e., ~86% amino acid identity; Ref. 29), this similarity between rabbit and rat NBC probably holds for the whole protein.
Second, the successful generation of NBC-specific antisera in rabbits immunized with MBP-NBC-5 suggests, but does not prove, that the COOH terminus of NBC faces the cytoplasm. This tentative conclusion is consistent with the predictions derived from the hydropathy plot. One might have expected that an exofacial localization of these 108 amino acids in the endogenous rabbit NBC would have induced immune tolerance with respect to these epitopes, so that no antibodies could have been raised against the fusion protein.
Immunolocalization of NBC in Rat and Rabbit Kidney
The strong expression of the NBC protein in the basolateral membrane of the proximal tubule, as detected by our NBC-specific antibodies, fully matches the strong functional expression of the cotransporter. The proximal tubule is the site of high rates of HCOAlthough we saw strong labeling of the S1 and S2 proximal tubules of
rat and rabbit, segments with high levels of
HCO3 reabsorption, we saw no NBC
immunoreactivity in either the late S3 or the thick ascending limb of
the loop of Henle, segments believed to have
substantially lower levels of HCO
3 reabsorption (21). Because the inherent limitations of antibody methods, the negative findings in our immunolocalization study should
be interpreted with caution. Thus it is impossible to rule out
completely the possibility that NBC was expressed in these segments but
not detected, for instance, as a consequence of either very low
expression levels, masking of epitopes (e.g., by fixatives or binding
to other cellular constituents), expression of immunologically different isoforms, or dependence of antibody performance on particular assay conditions.
Two recent in situ hybridization studies examined the localization of NBC mRNA expression in the kidney (1, 28). In rat kidney (28), NBC mRNA was observed in the terminal, straight portion of the S2 segment, but was not detected in the S1 or S3 segments or other nephron segments. In rabbit kidney (1), highest levels of NBC mRNA were found in the S1, with intermediate levels in S2 and very low levels in S3. No other nephron segments were labeled. In this study (1), the authors used a cRNA probe corresponding to a portion of hNBC that is 85% identical to one of the two rkNBC probes used in the other study (28).
Immunolocalization of NBC in Salamander Kidney
Within the nephron of the salamander A. tigrinum, NBC immunoreactivity was pronounced in the late distal tubule, but less strong in the proximal tubule. In both segments, the labeling was confined to the basolateral membranes. This distribution along the nephron differs markedly from the pattern seen in the two mammalian species studied, rat and rabbit. Although many parallels exist between amphibian and mammalian kidneys, substantial differences exist as well, some of which pertain to the renal handling of HCOThe proximal tubule contributes much less to the reabsorption of
filtered HCO3 in amphibians (24, 35) than in mammals (~40% vs. ~80%). Most of the
HCO
3 exiting the amphibian proximal
tubule is reabsorbed by the distal tubule, with minor variable
contributions from collecting tubules and urinary bladder (35). As
early as 1937, Montgomery and Pierce (24) showed that acidification of
the luminal fluid occurs only along a short portion of the distal
tubule of Necturus maculosus and
Rana pipiens. This portion is about
halfway between the intermediate segment (i.e., the junction between
the proximal and distal tubules) and the end of the distal tubule
(i.e., junction with ureter) and extends over only ~20% of the
length of this tubule segment (i.e., ~1-2 mm). In 1986, Yucha
and Stoner (35) systematically measured
HCO
3 reabsorption along the nephron in
isolated, perfused tubule segments of A. maculatum and A. tigrinum. They observed high rates of
HCO
3 reabsorption in the late distal
tubule and rates about one-third as high in the proximal tubule.
However, rates were not significantly different from zero in the early
and mid-distal segments, which corresponds functionally to the diluting
portion of the nephron.
Acidification of luminal fluid also occurs in the late distal tubule of
Amphiuma, as shown by Stanton and
colleagues in 1987 (30). Two different cell types contribute to this
acidification. In one of these (i.e., type I cells), basolateral
HCO3 exit is coupled to
Na+ and inhibited by SITS,
consistent with electrogenic
Na+-HCO
3
cotransport (30). In N. maculosus,
Planelles and Anagnostopoulos (26) provided strong evidence for a
basolateral, electrogenic
Na+-HCO
3
cotransporter in the late distal tubule, based on microelectrode
measurements of
Vm,
pHi, intracellular Na+ activity, and intracellular
Cl
activity.
In the early distal tubule of R. esculenta, also known as the diluting segment,
Oberleithner and coworkers (25) observed luminal acidification in the
nominal absence of
CO2/HCO3. In fused cells derived from this diluting segment, Wang and colleagues (32) found evidence for an electrogenic
Na+-HCO
3 cotransporter.
In Ambystoma, electrogenic
Na+-HCO3
cotransport activity is present in the proximal tubule, where the
cotransporter was originally identified (8), but has not been assessed
in the distal tubule. Our immunolocalization data in
Ambystoma show strong immunoreactivity
in the late distal tubule, where rates of
HCO
3 reabsorption are high (35), and
lesser immunoreactivity in the proximal tubule, where rates of
HCO
3 reabsorption are more modest
(35).
Given the relative abundance of NBC in the distal tubule of the salamander kidney, it is very possible that the cDNA isolated in the original expression cloning on NBC (29) actually was of distal tubule rather that proximal tubule origin.
Conclusion
We have developed antisera that specifically recognize the renal electrogenic Na+-HCO ![]() |
ACKNOWLEDGEMENTS |
---|
We thank Sue Ann Mentone for expert technical assistance, Dr. Seth Alper for the AE2 clone, and Dr. P. Isenring for help with the expression of NBC in HEK cells. B. M. Schmitt was supported by a Forschungsstipendium from the Deutsche Forschungsgemeinschaft. M. F. Romero was supported by a National Institute of Diabetes and Digestive and Kidney Diseases Research Service Award DK-09342 and by a grant from the American Heart Association. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-30344.
![]() |
FOOTNOTES |
---|
Present address of M. F. Romero: Dept. of Physiology and Biophysics, Case Western Reserve Univ., Cleveland, OH 44106-4970.
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. §1734 solely to indicate this fact.
Address for reprint requests: B. M. Schmitt, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., New Haven, CT 06520.
Received 17 April 1998; accepted in final form 11 September 1998.
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