RAPID COMMUNICATION
Immunoelectron microscopic localization of NBC3 sodium-bicarbonate cotransporter in rat kidney

Tae-Hwan Kwon1, Alexander Pushkin2, Natalia Abuladze2, Søren Nielsen1, and Ira Kurtz2

1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark; and 2 Division of Nephrology, University of California, Los Angeles, School of Medicine, Los Angeles, California 90095-1689


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we produced a rabbit peptide-derived polyclonal COOH-terminal antibody that selectively recognizes NBC3, to determine the cellular and subcellular localization of NBC3 in rat kidney, using immunocytochemistry and immunoelectron microscopy. Immunocytochemistry with cryostat sections and semithin cryosections revealed specific staining of intercalated cells (ICs) in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting ducts. In the connecting tubule and in the cortical and medullary collecting duct, the labeling was associated with both type A and type B ICs. In type A ICs, labeling was confined to the apical and subapical domains, whereas in type B ICs, basal domains were exclusively labeled. In contrast, collecting duct principal cells were consistently unlabeled, and this was confirmed using anti-aquaporin-2 antibodies, which labeled principal cells in parallel semithin cryosections. Glomeruli, proximal tubules, descending thin limbs, ascending thin limbs, thick ascending limbs, distal convoluted tubules, and vascular structures were unlabeled. For immunoelectron microscopy, tissue samples were freeze-substituted, and immunolabeling was performed on ultrathin Lowicryl HM20 sections. Immunoelectron microscopy demonstrated that NBC3 labeling was very abundant in the apical plasma membrane, in intracellular vesicles, and in tubulocisternal profiles in the subapical domains of type A ICs. In type B ICs, NBC3 was mainly present in the basolateral plasma membrane. Immunolabeling controls using peptide-absorbed antibody were consistently negative. In conclusion, NBC3 is highly abundant in the apical plasma membrane of type A ICs and in the basolateral plasma membrane of type B ICs. This suggests that NBC3 plays an important role in modulating bicarbonate transport in the connecting tubule and collecting duct.

acid-base; bicarbonate transport; intercalated cell; vacuolar proton-adenosinetriphosphatase


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY plays a critical role in acid-base homeostasis by the reabsorption of filtered bicarbonate and the generation of new bicarbonate. In the proximal tubule, HCO-3 reabsorption occurs via the transcellular coupling of the apical Na+/H+ exchanger (NHE3) with the basolateral Na(HCO3)n cotransporter, kNBC1, which plays a critical role in mediating electrogenic bicarbonate efflux (2, 5, 8, 21). Recently, kNBC1 has been localized by in situ hybridization to the proximal tubule in rabbit kidneys (2), and immunohistochemical analysis has revealed its presence in the basolateral plasma membrane of rat proximal tubule S1 and S2 segments (22), strongly suggesting that kNBC1 mediates basolateral proximal tubule bicarbonate efflux. In the distal nephron, acid secretion is primarily mediated by a vacuolar H+-ATPase and H+-K+-ATPase located in the apical plasma membranes of type A intercalated cells (4, 7, 13). Basolateral bicarbonate efflux is mediated by the anion exchanger AE1 in this cell type (3, 24). In type B intercalated cells, the vacuolar H+-ATPase is located on the basolateral membrane (3, 6). This cell type is thought to mediate collecting duct bicarbonate secretion via an apical anion exchanger that has not yet been identified.

In addition to kNBC1, which mediates electrogenic basolateral proximal tubule bicarbonate, other members of the NBC family have been identified and functionally characterized that could potentially play a role in mediating acid-base transport in the kidney. pNBC1 mediates electrogenic sodium bicarbonate cotransport in the pancreas and is expressed at low levels in the kidney, where its physiological function is currently unknown (1, 9). An NBC-like clone, NBC2, isolated from a human retina cDNA library has not been functionally characterized (11).

NBC3 has been recently cloned from human skeletal muscle and functionally characterized 1 (20). Unlike NBC1, NBC3 was demonstrated to be electroneutral, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) inhibitable, and DIDS insensitive. Preliminary experiments using RT-PCR had suggested that NBC3 was present in the kidney, and therefore to explore its distribution further, a peptide-derived rabbit antibody was produced. In the present study, we identified the localization of NBC3 in rat kidney and determined its cellular and subcellular localization using immunocytochemistry and immunoelectron microscopy.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Antibody against NBC3. A synthetic peptide corresponding to amino acids 1197-1214 of the COOH terminal of human NBC3 (20) was used to generate a polyclonal antibody. The purified peptide was coupled to keyhole limpet hemocyanin for immunization in rabbits. This region of NBC3 is nearly identical in human, rat, and rabbit, and the antibody shows cross-reactivity between species (see RESULTS section).

Transient expression in HEK-293 cells. NBC3 was subcloned from pCR-Script SK+ into pCDNA 3.1 vector (Invitrogen) using Not I and Xho I restriction sites. HEK-293 cells (American Type Culture Collection, Rockville, MD) were grown at 37°C, 5% CO2, in DMEM supplemented with 10% fetal bovine serum, 200 mg/l L-glutamine, and penicillin/streptomycin cocktail (Gemini Bio-Products, Calabasas, CA). At 12-16 h before transfection, a 90% confluent 10-cm polystyrene plate (Becton-Dickinson, Franklin Lakes, NJ) of cells was split 1:6 into 10 ml of medium and seeded onto a 10-cm plate. The cells were then transfected with purified plasmids (Qiagen Plasmid; Santa Clarita, CA) by using FuGene6 reagent (Boehringer Mannheim). FuGene6 transfections were carried out according to the manufacturer's instructions at 37°C, 5% CO2, for 6 h. The transfection medium was then removed by rinsing twice with 10 ml of 1× PBS and adding back 10 ml growth medium, and the cells were then incubated at 37°C, 5% CO2, overnight. The 10-cm plate of transfected cells was then trypsinized and transferred to a 24-well plate containing glass coverslips, and the cells were left at 37°C, 5% CO2, overnight. The 24-well plate of transfected cells was rinsed twice with 1× PBS, and incubated with 400 µl 3% paraformaldehyde for 15 min at room temperature. The paraformaldehyde was replaced with 400 µl methanol (-20°C) for 5 min. The cells were then rinsed twice with 1× PBS and processed for immunofluorescence. The primary antibody, NBC3-C1 (1/100 dilution) was applied for 1 h at 37°C. Following several washes in PBS, goat anti-rabbit IgG conjugated with Alexa 488 (1/500 dilution, Molecular Probes) was applied for 1 h at 37°C. The coverslips were rinsed in PBS and mounted in Cytoseal 60 (Stephens Scientific). A liquid-cooled PXL charge-coupled device camera (model CH1, Photometrics), coupled to a Nikon Microphot-FXA epifluorescence microscope, was used to capture and digitize the fluorescence images. The images were transferred to a Silicon Graphics Indy 5000 computer using ISEE 4.0 software (Inovision), and printed on a Kodak 8650 PS color printer.

Membrane fractionation for immunoblotting. The kidneys from normal Munich-Wistar rats were divided into cortex, outer stripe and inner stripe of the outer medulla, and inner medulla. These tissues were homogenized [0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 mM leupeptin and 1 mM phenylmethylsulfonyl fluoride (PMSF)] using an Ultra-Turrax T8 homogenizer (IKA Labortechnik; Staufen, Germany), at maximum speed for 30 s, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles (15). Gel samples (Laemmli sample buffer containing 2% SDS) were made of this pellet. Transfected and control HEK-293 cells were disrupted using a glass homogenizer in 0.1 M Tris-HCl buffer pH 7.5, containing 1 mM EDTA, 8.5 mM leupeptin, 1 mM PMSF, and 1% Triton X-100. The homogenate was spun at 18,000 g for 20 min. The supernatant was mixed with Laemmli sample buffer (3:1 vol/vol).

Electrophoresis and immunoblotting. Samples of membranes from rat kidney cortex, outer stripe and inner stripe of the outer medulla, and inner medulla were run on 6-16% gradient polyacrylamide minigels (Bio-Rad Mini Protean II). After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h, and incubated overnight at 4°C with immune serum diluted as 1:2,000. The labeling was visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P448, diluted 1:3,000; DAKO, Glostrup, Denmark) using enhanced chemiluminescence system (Amersham Pharmacia Biotech; Buckinghamshire, UK). Immunolabeling controls were performed using preabsorption of the immune serum with the immunizing peptide.

Deglycosylation. For N-glycosidase F (PNGase F) digestion, 100 µl membrane fraction from the inner stripe of the outer medulla was incubated at room temperature for 6, 12, or 24 h in the presence of 5 U of PNGase F obtained from Boehringer (Mannheim, Germany), respectively. Boiling the suspensions in Laemmli sample buffer stopped the enzymatic reactions, and samples were analyzed by immunoblotting.

Immunocytochemistry and immunoelectron microscopy. Kidneys from normal Munich-Wistar rats were fixed by retrograde perfusion via the aorta with periodate-lysine-paraformaldehyde (PLP: 0.01 M NaIO4, 0.075 M lysine, and 2% paraformaldehyde, in 0.0375 M Na2HPO4 buffer, pH 6.2). Tissue blocks prepared from cortex, outer and inner stripe of outer medulla, and inner medulla were cryoprotected with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid nitrogen (18). For preparation of cryostat sections, tissue was cryoprotected in 25% sucrose. Cryostat sections (10 µm) and semithin sections (0.8-1 µm, Reichert Ultracut S cryoultramicrotome) were incubated overnight at 4°C with immune serum (diluted 1:4,000), and labeling was visualized with HRP-conjugated secondary antibody (P448, 1:100; DAKO) (26). In double-label fluorescent studies, the vacuolar H+-ATPase was localized with a mouse monoclonal antibody, kindly supplied by Dr. S. Gluck (E11, diluted 1:2), which was mixed with the primary antibody against NBC3. The labeling was visualized using a rhodamine-conjugated swine anti-rabbit antibody (DAKO R156, diluted 1:40) mixed with a fluorescein-conjugated goat anti-mouse antibody (DAKO F479, diluted 1:20).

For immunoelectron microscopy, the frozen samples were freeze-substituted in a Reichert AFS freeze substitution unit (16, 17, 26). In brief, the samples were sequentially equilibrated over 3 days in methanol containing 0.5% uranyl acetate at temperatures gradually raised from -80°C to -70°C, then rinsed in pure methanol for 24 h while increasing the temperature from -70°C to -45°C, and infiltrated with Lowicryl HM20 and methanol 1:1, 2:1, and finally pure Lowicryl HM20 before ultraviolet polymerization for 2 days at -45°C and 2 days at 0°C. Immunolabeling was performed on ultrathin Lowicryl HM20 sections. Sections were pretreated with a saturated solutions of NaOH in absolute ethanol (2-3 s), rinsed, and preincubated for 10 min with 0.1% sodium borohydride and 50 mM glycine in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100. Sections were rinsed and incubated overnight at 4°C with immune serum diluted in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100 with 0.2% milk (diluted 1:4,000). After rinse, sections were incubated for 2 h at room temperature with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10, 1:50; BioCell Research Laboratories, Cardiff, UK). The sections were stained with uranyl acetate and lead citrate before examination in Philips CM100 or Philips 208 electron microscopes.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RT-PCR analysis of NBC3 in rat kidney. High-stringency Northern blotting analysis of human RNA was not sufficiently sensitive to detect NBC3 transcripts in whole kidney (20). To determine whether NBC3 is expressed in kidney, RT-PCR analysis of human and rat kidney total RNA (Clontech) was performed using specific primers for amplifying NBC3. The following primers were used: sense, 5'-CGAACAGGTCTGTCTGCCTC-3' (855-874); antisense, 5'-CTTTCAGCGCTGCTTCTAAG-3'(1133-1152); nested sense, 5'-CTTCTTCAAGAGCTGGAACCC-3'(931-951); and nested antisense, 5'-GCGGATGAATTACTACTGTG- GG-3'(1101-1122). As shown in Fig. 1, NBC3 mRNA is expressed in human and rat kidney. Controls were negative, and sequencing of the PCR product confirmed the presence of NBC3 in rat and human kidney (not shown). Since RT-PCR indicated the presence of NBC3 in the kidney, an antibody to NBC3 was produced to establish further the presence of NBC3 protein in the kidney and to establish the cellular and subcellular localization.


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Fig. 1.   RT-PCR amplification of human and rat NBC3. Total RNA from human and rat kidney (Clontech) was reversed transcribed using AMV reverse transcriptase. A specific region of NBC3 was amplified using the following primers mentioned in results (RT-PCR analysis of NBC3 in rat kidney). Lane 1, 1-kb ladder; lane 2, human kidney; lane 3, rat kidney.

Immunoblotting of NBC3 in rat kidney membranes. To establish the presence and to examine the cellular distribution of NBC3 protein in rat kidney, a rabbit antibody raised against a peptide corresponding to human NBC3 was produced. The antibody recognized the peptide on immunoblot (not shown) and specifically recognized an ~200-kDa band on immunoblots using membrane fractions from rat renal cortex, outer stripe of the outer medulla, inner stripe of the outer medulla, and inner medulla (Fig. 2A). In rat kidney membranes, the strongest signals were obtained in membranes from outer medulla (Fig. 2A), whereas only a very weak band was detected in membranes from kidney inner medulla (Fig. 2A). A band of the same size was also seen in immunoblots of membrane fractions from human kidney (not shown). The specificity of the labeling was confirmed using anti-NBC3 antibody preabsorbed with the immunizing peptide (Figs. 2B and 3C).


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Fig. 2.   Immunoblot analysis of NBC3. A: anti-NBC3 immunoblot of membranes from rat kidney cortex (COR), outer (OSOM) and inner stripe (ISOM) of the outer medulla, and inner medulla (IM). Anti-NBC3 specifically recognizes ~200-kDa band. B: immunoblot of anti-NBC3 preabsorbed with immunizing peptide. Band was not detected with anti-NBC3 preabsorbed with immunizing peptide. C: immunoblot analysis of N-glycosylation of NBC3. Anti-NBC-3 immunoblot of membranes from ISOM of rat kidney before (-) or after (+) digestion with N-glycosidase F (PNGase F). Treatment with PNGase F resulted in a shift in molecular size to ~160 kDa.

The size of the recognized protein is larger than the predicted size of ~136 kDa (20), suggesting that NBC3 is glycosylated or posttranslationally modified in other ways. Consistent with this, treatment of membranes from the inner stripe of the outer medulla of rat kidney with PNGase F for 6, 12, or 24 h resulted in a shift in molecular size to ~160 kDa (Fig. 2C).

Expression of NBC3 in HEK-293 cells. To further characterize the antibody, HEK-293 cells were transfected with NBC3. Immunoblotting revealed a band of similar size to that seen in rat kidney membranes (cf. Figs. 2A and 3C) and controls using peptide-absorbed antibody were negative (Fig. 3C). Immunofluorescence microscopy revealed that NBC3 labeling in transfected cells was predominantly associated with plasma membrane domains (Fig. 3A), whereas no labeling was observed in nontransfected cells (Fig. 3B).


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Fig. 3.   Immunocytochemical (A and B) and immunoblot (C) analysis of NBC3 expression in HEK-293 cells. A: transfected cells showing a predominant plasma membrane localization of NBC3. B: untransfected cells. C: lane 1, transfected cells; lane 2, untransfected cells

Localization of NBC3 in rat kidney using immunohistochemistry and immunocytochemistry. As demonstrated in Fig. 4, A-C, immunohistochemistry revealed prominent labeling of the connecting tubule and of cortical, outer medullary, and inner medullary collecting ducts in rat kidney. In contrast, glomeruli, proximal tubules, descending thin limbs, ascending thin limbs, thick ascending limbs, distal convoluted tubules, and vascular structures were consistently unlabeled, as also confirmed by immunocytochemistry and immunoelectron microscopy. Immunohistochemistry using cryostat sections of human kidney revealed extensive labeling of collecting duct intercalated cells (not shown) consistent with the labeling pattern in rat.


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Fig. 4.   Immunocytochemical analyses of cellular and subcellular localization of NBC3 in rat kidney using immunoperoxidase labeling of cryostat sections (A-G) and semithin cryosections (H and I). A-C: low magnification reveals that in cortex (A), inner stripe of outer medulla (B), and inner medulla (C), NBC3 labeling is only seen in connecting tubules and in intercalated cells of collecting ducts (arrows). D: in cortical collecting duct, labeling is associated with apical and subapical domains of type A intercalated (arrows), whereas in type B intercalated cells the labeling is mainly confined to basal domains (arrowheads). E: connecting tubule (CT) intercalated cells exhibits apical labeling. "DCT" indicates transition of distal convoluted tubule into the connecting tubule. F: abundant labeling is seen in both type A and type B (arrowhead) intercalated cells in cortical collecting duct (CCD). G: in inner stripe of outer medulla, labeling is exclusively associated with apical and subapical domains of type A intercalated cells (arrows). H and I: higher resolution immunocytochemistry in semithin sections of cortex shows apical and subapical labeling of type A intercalated cells (arrows) and basal labeling (arrowheads) in type B intercalated cells. G, glomerulus; PT, proximal tubule; T, thick ascending limb. Magnifications, ×170 (A-C) and ×680 (D-I).

Higher resolution immunocytochemistry of sections from cortex revealed that in the cortical collecting duct (Fig. 4D) and connecting tubule (Fig. 4E), NBC3 labeling was present in intercalated cells, whereas NBC3 was absent in the collecting duct principal cells. This distribution pattern was confirmed by immunocytochemistry using anti-aquaporin-2 which labeled principal cells in parallel semithin cryosections (not shown). Moreover, this labeling pattern was also confirmed by immunoelectron microscopy (see section below). NBC3 labeling was observed of both type A and in type B intercalated cells in the connecting tubule and cortical collecting duct. In type A intercalated cells, labeling was predominantly confined to the apical and subapical domains (arrows in Fig. 4, D, E, H, and I), whereas in type B intercalated cells only basal domains were labeled (arrowheads in Fig. 4, D, F, and H). Also, this labeling pattern was confirmed by immunoelectron microscopy (see section below). The intercalated cells of the outer stripe of the outer medulla (not shown) and the inner stripe of the outer medulla (Fig. 4G), which all have type A properties, exhibited abundant apical labeling (Fig. 4G).

Kidney inner medulla intercalated cells (initial segment), which are known to exhibit typical type A properties, also have extensive apical NBC3 immunolabeling (Fig. 4C). The presence of NBC3 in this nonabundant cell type in the inner medulla is consistent with the relatively weak but distinct NBC3 signal obtained by immunoblotting in membranes from kidney inner medulla compared with outer medulla (Fig. 2A).

Double immunofluorescence labeling of NBC3 and H+-ATPase revealed an almost complete overlap in the labeling pattern in the collecting duct (not shown), indicating that all intercalated cells express NBC3.

Immunoelectron microscopy of NBC3 in type A intercalated cells. Immunoelectron microscopy was conducted using immunogold labeling of sections prepared from kidney tissue embedded in Lowicryl HM20 by cryosubstitution. In sections from the inner stripe of the outer medulla (where only type A intercalated cells are present in rat kidney), very abundant immunogold labeling was observed in type A intercalated cells (Fig. 5), whereas no other cells exhibited immunogold labeling (not shown). The apical plasma membrane exhibited very strong NBC3 immunogold labeling with staining of both microplicae and plasma membrane areas between microplicae. In addition, strong labeling was observed of intracellular vesicles and of tubulocisternal profiles in the subapical domains of the cell (Fig. 5). Immunolabeling controls (Fig. 5, inset) using antibody preabsorbed with excess immunizing peptide produced no labeling. As shown in Fig. 6, there was some heterogeneity between type A intercalated cells with respect to the relative abundance of the NBC3 labeling of the apical plasma membrane and intracellular vesicles. In some cells, very abundant NBC3 labeling was observed in the apical plasma membrane (Fig. 6A), whereas in other cells less extensive apical plasma membrane labeling was observed (Fig. 6B). Intracellular vesicles and tubulocisternal profiles consistently exhibited strong labeling (Figs. 5 and 6). Immunoelectron microscopy of sections from kidney inner medulla revealed abundant NBC3 immunogold labeling of the apical plasma membrane and intracellular vesicles and tubulocisternal profiles in intercalated cells (not shown), thereby confirming the immunohistochemistry results.


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Fig. 5.   Immunoelectron microscopy in collecting duct type A intercalated cell from inner stripe of outer medulla. Abundant labeling is associated with apical plasma membrane domains and intracellular vesicles and tubulocisternal profiles in the subapical part of cell. Inset: immunolabeling control. Peptide-absorbed immune serum does not produce labeling in collecting duct cells in the inner stripe of the outer medulla. PC, principal cell; IC, intercalated cell; M, mitochondria. Magnification, ×44,700.



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Fig. 6.   Immunoelectron microscopy of collecting duct type A intercalated cells from inner stripe of outer medulla (A and B). A: very abundant labeling is seen in apical plasma membrane (arrows). B: very abundant labeling is seen of intracellular vesicles and tubulocisternal profiles in another type A intercalated cell (arrowheads). M, mitochondria. Magnification, ×44,400.

Immunoelectron microscopy of NBC3 in type B intercalated cells. As shown in Fig. 4, connecting tubule cells exhibited significant NBC3 labeling. Both type A and type B intercalated cells exhibited significant labeling with type A cells showing apical labeling (Fig. 4) and type B intercalated cells showing basolateral labeling (Fig. 4). Immunoelectron microscopy confirmed the labeling of type B intercalated cells. As shown in Fig. 7, the type B intercalated cells from the connecting tubule exhibited significant labeling of the basolateral plasma membrane (arrows, Fig. 7B) and intracellular vesicles in the basal part of the cell (not shown), whereas the apical plasma membrane was completely unlabeled (not shown). In type B cells in the cortical collecting duct, an identical labeling pattern was seen (Fig. 8). Extensive labeling was associated with the basolateral plasma membrane (Fig. 8, arrows), and some labeling was also associated with intracellular vesicles (Fig. 8, arrowheads).


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Fig. 7.   Immunoelectron microscopy of connecting tubule. A: survey view of section of a connecting tubule showing three different cell types; principal cell (PC), type B intercalated cell (IC-B), and connecting tubule cell (CT). Rectangle indicates the area presented at higher magnification in B. B: abundant labeling is seen in basolateral plasma membrane (arrows) of type B intercalated cell. BM, basement membrane; CT, connecting tubule cell; E, endothelium; IC-B, type B intercalated cell; M, mitochondria; N, nucleus; and PC, principal cell. Magnifications, ×5,500 (A) and ×42,200 (B).



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Fig. 8.   Immunoelectron microscopy of cortical collecting duct. A: survey view of section of a cortical collecting duct showing three different cell types: principal cell (PC), type A intercalated cell (IC-A), and type B intercalated cell (IC-B). Rectangles indicate the areas presented at higher magnification in B and C. B and C: abundant labeling is seen in basolateral plasma membrane (arrows) and intracellular vesicles (arrowheads) in the basal part of the type B intercalated cell. BM, basement membrane; M, mitochondria; and N, nucleus. Magnifications, ×5,200 (A) and ×43,700 (B and C).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of present study demonstrate that in rat kidney, NBC3 is specifically localized in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting duct. Moreover, high-resolution immunocytochemistry and immunoelectron microscopy demonstrates that both type A and type B intercalated cells in the connecting tubule and the collecting duct exhibit strong NBC3 immunolabeling, whereas principal cells are unlabeled. In type A intercalated cells, NBC3 labeling is confined to the apical plasma membrane and subapical intracellular vesicles and tubulocisternal profiles. In contrast, in type B intercalated cells, NBC3 is abundantly expressed in the basolateral plasma membrane and is absent in the apical plasma membrane domains. The abundance of NBC3 in both type A and type B intercalated cells strongly indicates that NBC3 may play an important role in bicarbonate transport in the connecting tubule and collecting duct.

Specificity of the antibody. The antibody was raised against a synthetic peptide corresponding to the COOH-terminal amino acids of human NBC3 (amino acids 1197-1214). This sequence is nearly identical in human, rabbit, and rat. Consistent with these findings, immunoblotting demonstrated that the antibody labeled NBC3 in membranes prepared from rat and human kidney (this study) as well as from rabbit kidney (20a). Furthermore, immunocytochemistry demonstrates that the antibody labeled intercalated cells both in rat and human kidneys, as recently shown in the rabbit kidney (20a), further confirming that the anti-human NBC3 antibody recognizes both rat and human NBC3. Immunoblotting revealed a ~200-kDa band, and PNGase F treatment reduced the molecular size consistent with a considerable glycosylation. Immunolabeling controls using preabsorbed antibody with the immunizing peptide ablated the labeling completely both in immunoblotting (Fig. 2B) and immunohistochemistry and immunoelectron microscopy (Fig. 5, inset). Thus the observations strongly support the view that the antibody specifically and selectively recognizes NBC3 in rat kidney.

Presence of NBC3 in intercalated cells. The abundant expression of NBC3 in the connecting tubule and collecting duct intercalated cells is consistent with the well-established role of these cells in acid-base transport and regulation of acid-base homeostasis. In the connecting tubule and cortical collecting duct, at least two types of intercalated cells are known to be involved functionally in this transport (3, 6, 13, 14, 23, 24), and both of these cell types express NBC3 (Figs. 4-8). The H+ -secreting type A intercalated cells possess a vacuolar H+-ATPase in the apical endocytic vesicles and a Cl-/HCO-3 exchanger (AE1) in the basolateral membrane for transporting HCO-3 to the interstitium (3, 24). The acid secretion has been demonstrated to be regulated at least in part by insertion and retrieval of the tubulovesicular structures that contain the H+-ATPase into and from the apical plasma membranes in response to subacute or chronic changes in acid-base status of the animal (13). In this process, the endosomal acidification mediated by a vacuolar H+-ATPase has been implicated to play a role in maintaining the endocytic activity (12, 19).

The HCO-3-secreting type B cells have these transport processes in the opposite membranes, i.e., the vacuolar H+-ATPase in the basolateral plasma membrane and a Cl-/HCO-3 exchanger (not yet identified) in the apical plasma membrane (3, 6). A sodium-coupled HCO-3 transporter has not previously been identified in collecting duct intercalated cells. The present finding of NBC3 in the apical part of the type A intercalated cells and in the basolateral part of type B intercalated cells strongly suggests that NBC3 may play a significant role in HCO-3 transport in these cells including a potential role for Na-HCO-3 transport in outer medullary collecting duct, although the exact physiological role needs to be established in both cell types. This requires additional studies, and presently one can only speculate on specific functions.

Functional studies have revealed that NBC3 is electroneutral, EIPA-inhibitable, and DIDS insensitive (20). Thus the direction of transport of HCO-3 appears to be dependent on the sodium and HCO-3 gradients across the specific cell in which it is expressed. It also remains unknown whether NBC3 plays a role in transepithelial Na+ reabsorption in the collecting duct.

Our finding that the localization of NBC3 resembles the subcellular localization of the vacuolar H+-ATPase in both type A and type B intercalated cells indicates that they may be functionally associated. Additional channels have also been identified in the same cells and in a similar subcellular distribution. Gunther et al. (10) demonstrated that ClC-5 chloride channel exhibited a similar although not completely overlapping staining pattern with the vacuolar H+-ATPase in type A intercalated cells (10), and recently it has been reported that aquaporin-6, a unique water channel protein, was found to be associated with intracellular vesicles and tubulocisternal profiles in intercalated cells also housing the vacuolar H+-ATPase (26). Aquaporin-6 conducts both water and Cl-, and this can be regulated (gated) by changes in pH (25). Thus NBC3, vacuolar H+-ATPase, ClC-5 chloride channel, and aquaporin-6 may play distinct roles in regulating vesicle acidification, modulating the activity of the vacuolar H+-ATPase, and regulating acid-base transport in intercalated cells.

The abundance of NBC3 in intracellular vesicles and in the apical plasma membrane of type A intercalated cells raises the possibility that NBC3 may be trafficked in a regulated fashion from intracellular vesicles to the apical plasma membrane in response to acute or chronic changes in acid-base balance. Conversely, NBC3 may be retrieved by endocytosis from the apical plasma membrane into the intracellular compartment. Further studies are necessary to establish this.

Conclusion. The results of present study demonstrate that NBC3 is specifically localized in the connecting tubule and in cortical, outer medullary, and initial inner medullary collecting duct, where it resides in the apical plasma membrane and subapical intracellular vesicles in type A intercalated cells and in the basolateral plasma membrane and vesicles in the basal part of type B intercalated cells. The abundance of NBC3 in both type A and type B intercalated cells strongly suggests an essential role for NBC3 in modulating bicarbonate transport in the connecting tubule and collecting duct.


    ACKNOWLEDGEMENTS

We thank Inger Merete Paulsen, Zhila Nikrozi, Mette Vistisen, and Gitte Christensen for expert technical assistance.


    FOOTNOTES

Support for this study was provided by the Karen Elise Jensen Foundation, Novo Nordic Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, and the University of Aarhus, EU Commission (TMR and Biotech programs), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-46976, the Iris and B. Gerald Cantor Foundation, the Max Factor Family Foundation, the Verna Harrah Foundation, the Richard and Hinda Rosenthal Foundation, and the Fredericka Taubitz Foundation. N. Abuladze is supported by a Training Grant J891002 from the National Kidney Foundation of Southern California.

1 Recently, H. Amlal, C. E. Burnham, and M. Soleimani (Am. J. Physiol. Renal Physiol. 276: F903-F913, 1999) reported an NBC-like partial clone, which was also called NBC-3. An ~4.4-kb transcript was highly expressed in brain and spinal cord.

Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).

Received 29 July 1999; accepted in final form 19 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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