Immunolocalization of the electrogenic Na+-HCOminus 3 cotransporter in mammalian and amphibian kidney

Bernhard M. Schmitt1, Daniel Biemesderfer2, Michael F. Romero1, Emile L. Boulpaep1, and Walter F. Boron1

Departments of 1 Cellular and Molecular Physiology and 2 Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut 06520

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Electrogenic cotransport of Na+ and HCO-3 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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

SODIUM-BICARBONATE COTRANSPORTERS are members of an emerging superfamily of Na+-coupled HCO-3 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+-HCO-3 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+-HCO-3 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+-HCO-3 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).

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 (DH5alpha ; 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.

In addition, we also generated a fusion protein containing amino acids 338-391 of rkNBC (MBP-NBC-3). These residues correspond to a region of rkNBC just NH2-terminal to the first putative transmembrane segment. A third fusion protein contained the alpha -fragment of beta -galactosidase (MBP-beta gal; transcribed from wild-type pMAL-c2). A fourth fusion protein (MBP-NHE3) contained the COOH-terminal 131 amino acids of rabbit NHE3 (6). We used MBP-NBC-3, MBP-beta gal, and MBP-NHE3 as control fusion proteins in the preabsorption experiments with anti-MBP-NBC-5 serum.

LARGE-SCALE EXPRESSION AND PURIFICATION. E. coli harboring pMAL-NBC-5 were grown at 37°C in 500 ml LB/Amp. When the culture medium had reached an optical density of 0.4-0.6 at 550 nm, expression of the fusion protein gene was initiated by adding isopropylthiogalactoside (IPTG; final concentration 0.3 mM) and allowed to proceed for 4 h. Cells were then pelleted by centrifugation (3,000 g for 15 min at 4°C) and resuspended in 25 ml "column buffer" [in mM: 50 Tris, 300 NaCl, 1 phenylmethylsulfonyl fluoride (PMSF), and 1 sodium EDTA, pH 7.4 with HCl]. The cell suspension was sonicated vigorously for 20 min on ice. The sonicate was cleared from insoluble matter by centrifugation (15,000 rpm for 30 min at 4°C, SS-34 rotor, Sorvall) and diluted with 4 vol of column buffer. For batch affinity purification, 10 ml of amylose matrix (NEB) was added to this cleared lysate and mixed by end-over-end rotation overnight at 4°C. The amylose beads were washed five times in 50 ml of column buffer before the MBP fusion protein was specifically eluted with 5 ml of 10 mM maltose in column buffer. Protein concentration in the eluate was determined with the BCA kit (Pierce, Rockford, IL), and the purity of the fusion protein was assessed by SDS-PAGE and staining with Coomassie brilliant blue G250.

Immunization with fusion protein MBP-NBC-5. Two guinea pigs and two rabbits were subjected to the following immunization protocol: day 0, 1 ml preimmune bleed; day 1, subcutaneous injection of purified MBP-NBC-5 in complete Freund's adjuvant (guinea pig, 20 µg; rabbit, 50 µg); day 28, boost with MBP-NBC-5 in incomplete Freund's adjuvant (guinea pig, 20 µg; rabbit, 50 µg); day 38, 2-ml test bleed 1; day 56, boost (as on day 28); day 66, 2-ml test bleed 2. Subsequently, animals were boosted and bled in 4-wk intervals. Antisera were stored at 4°C with 0.03% sodium azide to prevent microbial growth.

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+-HCO-3 Cotransporter

Preparation of renal microsomes. Whole kidneys of adult Sprague-Dawley rats (Camm, Wayne, NJ), New Zealand White rabbits (Covance, Denver, PA), and the salamander A. tigrinum (Charles Sullivan, Nashville, TN) were removed under anesthesia (rats, 100 mg/kg ip pentobarbital; rabbits, same dose iv; salamander, submersion in 0.2% tricaine methanesulfonate). The kidneys were placed in ice-cold homogenization buffer (HB, in mM: 250 sucrose, 20 HEPES, pH 7.4 with HCl, 100 NaCl, 2 sodium EDTA, 1 PMSF, 0.001 leupeptin, and 0.001 pepstatin) and homogenized using a Polytron. The homogenate was centrifuged (15 min at 3,000 rpm, SS-34 rotor, 4°C), and the pellet (P1), containing debris and nuclei, was discarded. The supernatant (S1) was recentrifuged (45 min at 15,000 rpm, SS-34 rotor, 4°C), and the supernatant (S2) was discarded. The resulting microsomal pellet (P2), containing plasma and organellar membranes, was resuspended in HB, assayed for protein content, and stored at -20°C.

Electrophoresis and transfer. Proteins from the microsome preparation were separated by denaturing SDS-PAGE under reducing conditions (100 mM dithiothreitol or 0.5% beta -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-beta 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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Abstract
Introduction
Methods
Results
Discussion
References

Generation of Polyclonal Antisera

Fusion proteins MBP-beta gal, MBP-NBC-3, and MBP-NBC-5 all expressed well in E. coli (Fig. 1) and were water soluble. Using SDS-PAGE, we found major bands at ~50 kDa for MBP-beta gal and MBP-NBC-3 and at ~55 kDa for MBP-NBC-5 (Fig. 1), in accord with the calculated Mr values of the fusion proteins.


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Fig. 1.   Expression of fusion proteins in Escherichia coli. Coomassie-stained SDS-polyacrylamide gel (12.5%) of a crude lysate of E. coli expressing maltose binding protein (MBP) fusion proteins: beta gal, MBP plus alpha -fragment of beta -galactosidase; NBC-3, MBP plus amino acids 338-391 of the rat kidney Na+-HCO-3 cotransporter (rkNBC); NBC-5, MBP plus amino acids 928-1035 of rkNBC. Size markers, spaced in 10-kDa intervals, were run in parallel; the arrow indicates 50 kDa. Molecular masses of the 3 fusion proteins, calculated from the deduced amino acid sequences, are 50.6, 48.6, and 54.6 kDa, respectively. The lower of the two major bands in MBP-NBC-5 probably represents a partially degraded fusion protein. Representative of three blots.

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-beta 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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Fig. 2.   Specificity of the anti-MBP-NBC-5 serum: expression of NBC in HEK-293 cells. Untransfected or wild-type HEK-293 cells (A) and HEK-293 cells transiently transfected with NBC from salamander Ambystoma tigrinum (aNBC) (B) were subjected to indirect immunofluorescence staining, using the guinea pig anti-MBP-NBC-5 serum. Top: immunofluorescence images. Bottom: light transmission images of the same 2 preparations from top. Only the cells transfected with aNBC show labeling. A and B are representative of two cover slips each.

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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Fig. 3.   Specificity of the anti-MBP-NBC-5 serum: expression of NBC in Xenopus oocytes. A: functional expression of AE2 and rkNBC in oocytes of X. laevis. We injected oocytes with cRNAs for the Cl-/HCO-3 exchanger isoform 2 (AE2, left records) or for the rat kidney Na+-HCO-3 cotransporter (rkNBC, right records). Eight days after injection, we monitored intracellular pH (pHi) and membrane potential (Vm) with microelectrodes. Responses to removal of bath Cl- ("0 Cl-") or bath Na+ ("0 Na+") confirmed functional expression of AE2 and NBC, respectively. B: immunoblotting of oocytes. Protein extracts from one water-injected control oocyte, as well as the two oocytes shown in A were immunoblotted with rabbit anti-MBP-NBC-5 serum. In the water-injected control oocyte (H2O), no significant cross-reactivity of the antiserum with any of the endogenous Xenopus oocyte proteins is apparent. Similarly, no labeling is observed in the oocyte expressing AE2 (the same oocyte as represented in the left records of A). Molecular mass calculated from the deduced amino acid sequence of AE2 is ~151 kDa. In the oocyte expressing rkNBC (the same oocyte represented in the right records of A), however, the anti-MBP-NBC-5 serum strongly labels a single band of ~130 kDa. We obtained similar blots with one additional water-injected oocyte, one additional AE2-injected oocyte, and two additional rkNBC-injected oocytes.

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 HCO-3, 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/HCO-3 (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+-HCO-3 cotransporter in both immunohistochemical and immunoblotting assays.

Immunoblotting of the Native Renal Na+-HCO-3 Cotransporter

Immunoblotting of salamander, rat, and rabbit kidney. To identify the NBC protein in amphibian and mammalian species, we performed immunoblotting experiments with rabbit anti-MBP-NBC-5 serum on microsomes from whole kidneys of salamander, rat, and rabbit. As shown in Fig. 4, in salamander kidney the antiserum detected a single band of ~160 kDa. This Mr is substantially greater than the 116 kDa predicted from the cDNA sequence of aNBC. In rat kidney (lane 2) and rabbit kidney (lane 3), the predominant bands were at a Mr of ~130 kDa. In rat kidney, the antiserum also routinely detected minor bands at ~100 kDa and ~85 kDa; the band below ~80 kDa was variable. In the rabbit kidney, the antisera also routinely detected the ~100 kDa band; the lower Mr bands were variable.


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Fig. 4.   Immunoblot of Na+-HCO-3 cotransporter proteins from salamander, rat, and rabbit kidney. Microsome preparations from kidneys of the salamander A. tigrinum, Sprague-Dawley rats, and New-Zealand White rabbits were separated by SDS-PAGE and immunoblotted with rabbit anti-MBP-NBC-5 serum. In the salamander kidney, a single band of ~160 kDa is labeled, whereas the calculated molecular mass (Mr) for aNBC is ~116 kDa. This lane is representative of 3 blots. In rat and rabbit kidney, the antiserum labeled major bands at ~130 kDa, compared with the computed Mr of ~116 kDa. These lanes are representative of more than 10 blots.

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-beta gal and MBP-NBC-3, we probed, respectively, with sera previously depleted of antibodies directed against either MBP-beta gal (by preincubating with an excess of MBP-beta 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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Fig. 5.   Specificity of anti-MBP-NBC-5 serum: immunoblot of rat kidney proteins after antibody preabsorption. Microsome preparations from rat kidney were separated by SDS-PAGE and immunoblotted with rabbit anti-MBP-NBC-5 serum. Serum not preabsorbed with an MBP fusion protein (control) strongly labeled a band of ~130 kDa, as well as two additional, weaker bands of ~100 kDa and ~85 kDa. Serum preabsorbed with either the MBP-beta gal or the MBP-NBC-3 fusion proteins shows very similar labeling patterns. In contrast, serum preabsorbed with the MBP-NBC-5 fusion protein shows no labeling whatsoever (fourth lane). Thus labeling was specifically due to antibodies recognizing epitopes within the NBC-5 portion of the fusion protein.

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+-HCO-3 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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Fig. 6.   Immunolocalization of NBC in rat kidney: cellular localization. Indirect immunofluorescence microscopy was performed on semithin (0.5 µm) cryosections with guinea pig anti-MBP-NBC-5 serum. Top: fluorescence image. Bottom: light transmission image (magnification, ×500). A: preimmune serum. No labeling is seen with serum from the guinea pig obtained prior to immunization. B: immune serum, preabsorbed with an unrelated MBP fusion protein, MBP-NHE. Strong labeling is seen in proximal tubules (PT); other nephrons visible in this section (bottom of B) are not labeled. In PT cells, labeling was consistently observed exclusively on the basolateral membrane and its numerous infoldings but was completely absent from the apical brush border membrane. C: immune serum, preabsorbed with MBP-NBC-5 fusion protein. Absence of immunoreactivity demonstrates the specificity of the labeling in B.

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+-HCO-3 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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Fig. 7.   Immunolocalization of NBC in rat kidney: overview (magnification, ×35). DAB/immunoperoxidase labeling was performed on a 5-µm thick cross-section with guinea pig anti-MBP-NBC-5 serum. Strong, distinct staining is seen in the cortex, localizing exclusively to proximal tubules. Apart from PT, no other nephron segments in the cortex are labeled. Furthermore, no staining is found in outer or inner medulla.

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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Fig. 8.   Immunolocalization of NBC in salamander kidney; overview (magnification, ×40). A: indirect immunofluorescence microscopy with guinea pig anti-MBP-NBC-5 serum on 5-µm thick cryosection of a kidney from the salamander A. tigrinum. B: schematic drawing of the same section; the localization of the various nephron segments within the salamander kidney is illustrated by a fictitious nephron projected onto the section plane. The nomenclature of the distal tubule is that of Planelles and Anagnostopoulos (26). As shown in A, strong labeling is seen lateral to the crescent-shaped zone that contains the glomeruli (shaded area in B). Labeled segments in this zone probably correspond to the initial portion of late distal tubules. No labeling is found medial to the glomeruli, which includes the "diluting segments" or "early distal tubules." On other sections (not shown), proximal tubules were stained weakly and variably. PT, proximal tubule; IS, intermediate segment; ED, early distal tubule; LD, late distal tubule; CD, collecting duct; G, glomerulus.

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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Fig. 9.   Immunolocalization of NBC in salamander kidney; cellular localization (magnification, ×525). Indirect immunofluorescence microscopy with guinea pig anti-MBP-NBC-5 serum on 0.5-µm thin cryosections. A: fluorescence microscopic image. B: transmission light microscopic image (phase contrast). Fluorescent labeling is restricted to the basolateral face of the kidney tubule cells, as in rat and rabbit. Labeled cells exhibit late distal tubule cell morphology.

In the thicker proximal tubules, where the electrogenic Na+-HCO-3 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.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Transepithelial HCO-3 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+-HCO-3 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 HCO-3 reabsorption. Moreover, work on intact tubules with microelectrodes and pH-sensitive dyes, as well as experiments on membrane vesicles, demonstrates a high rate of electrogenic Na+-HCO-3 cotransport in the basolateral membrane of the proximal tubule (overview in Ref. 9). The absence of NBC immunoreactivity in the thin limbs of Henle's loop and all segments distal of the macula densa also is in keeping with the functional data (33) and with our knowledge of the particular mechanisms of HCO-3 transport in these tubule segments (15).

Although we saw strong labeling of the S1 and S2 proximal tubules of rat and rabbit, segments with high levels of HCO-3 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 HCO-3.

The proximal tubule contributes much less to the reabsorption of filtered HCO-3 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 HCO-3 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/HCO-3. 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+-HCO-3 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-3 cotransporter in immunoblotting and immunohistochemical applications. The results of our immunolocalization study support the current models of HCO-3 reabsorption by the mammalian proximal tubule, as developed from previous functional studies. Our results on the salamander also support the conclusion that the late distal tubule is the main site for HCO-3 reabsorption in the amphibian kidney, with a smaller contribution being made by the proximal tubule.

    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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Methods
Results
Discussion
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