Expression and localization of rat NBC4c in liver and renal uroepithelium

Natalia Abuladze, Alexander Pushkin, Sergei Tatishchev, Debra Newman, Pakan Sassani, and Ira Kurtz

Division of Nephrology, David Geffen School of Medicine, University of California, Los Angeles, California 90095-1689

Submitted 31 December 2003 ; accepted in final form 11 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies provided functional evidence for electrogenic Na+-HCO3 cotransport in hepatocytes and in intrahepatic bile duct cholangiocytes. The molecular identity of the transporters mediating electrogenic sodium-bicarbonate cotransport in the liver is currently unknown. Of the known electrogenic Na+-HCO3 cotransporters (NBC1 and NBC4), we previously showed that NBC4 mRNA is highly expressed in the liver. In the present study, we performed RT-PCR, immunoblotting, and immunohistochemistry to characterize the expression pattern of NBC4 in rat liver and kidney. For immunodetection, a polyclonal antibody against rat NBC4 was generated and affinity purified. Of the known human NBC4 variants, only the rat NBC4c ortholog was detected by RT-PCR in rat liver, and the molecular mass of the NBC4c protein was ~145 kDa. NBC4c protein was expressed in hepatocytes and in the cholangiocytes lining the intrahepatic bile ducts. In hepatocytes, NBC4c was localized to the basolateral plasma membrane, whereas intrahepatic cholangiocytes stained apically. The NBC1 electrogenic sodium cotransporter variants kNBC1 and pNBC1 were not detected by immunoblotting and immunohistochemistry in rat liver. The pattern of localization of NBC4c in the liver suggests that the cotransporter plays a role in mediating Na+-HCO3 cotransport in hepatocytes and intrahepatic cholangiocytes. Unlike the liver, the rat kidney expressed electrogenic sodium-bicarbonate cotransporter proteins kNBC1 and NBC4c. In kidney, NBC4c also had a molecular mass of ~145 kDa and was immunolocalized to uroepithelial cells lining the renal pelvis, where the cotransporter may play an important role in protecting the renal parenchyma from alterations in urine pH.

bicarbonate; transport; electrogenic


HEPATOCYTES TRANSPORT WATER AND SOLUTES across the basolateral (sinusoidal) and apical (canalicular) membranes in a vectorial manner (44). Hepatocyte canalicular secretion accounts for 30–60% of spontaneous basal bile flow (44). In a mechanism distinct from bile acid transport, hepatocytes secrete HCO3 across the canalicular membrane (14). Active transcellular HCO3 secretion into bile is thought to be mediated by basolateral electrogenic Na+-HCO3 cotransport coupled to canalicular Cl/HCO3 exchange mediated by AE2 (8, 9, 20, 2225, 27, 41). Exposure of the liver to HCO3 increases hepatic oxygen consumption and Na+-K+-ATPase activity (21). It is estimated that ~50% of Na+-K+-ATPase function is coupled to the recycling of Na+ that has entered the cells via basolateral electrogenic Na+-HCO3 cotransport, thereby maintaining the intracellular Na+ concentration (21). In addition to playing an important role in transcellular HCO3 secretion and regulation of the intracellular Na+ concentration, basolateral electrogenic Na+-HCO3 cotransport and to a lesser extent basolateral Na+/H+ exchange also play an important role in regulating intracellular pH (pHi) in response to ongoing cytoplasmic reactions that generate protons (protein catabolism) and consume HCO3 (urea cycle) (20). The efficiency of apical and basolateral H+/base transport is enhanced by plasma membrane carbonic anhydrase XIV (46).

In the liver, in addition to hepatocytes, intrahepatic cholangiocytes lining bile ducts also play an important role in hepatic bile formation and biliary HCO3 secretion (13, 30, 42). Cholangiocytes lining the biliary epithelium both secrete and absorb various substances that modify the bile secreted by hepatocytes. Ductal secretion in response to secretin increases bile flow by 10% in rats and by 30% in humans (4, 37, 64). In response to hormones such as secretin, acetylcholine, VIP, and bombesin, cholangiocyte HCO3 secretion is increased (30). The increase in transepithelial HCO3 secretion is mediated by an elevation of cytosolic cAMP levels, which is thought to activate apical CFTR Cl channels and subsequent apical Cl/HCO3 exchange (6, 7, 18, 19, 25, 41, 58, 59). In pigs, cholangiocyte HCO3 secretion is also dependent on cytosolic carbonic anhydrase II (15). Whether additional HCO3 transport processes are present on the apical membrane of cholangiocytes is currently unknown. Less is known about the mechanisms of cholangiocyte basolateral HCO3 transport. On the basis of data obtained from isolated cultured rat cholangiocytes, it was hypothesized that HCO3 influx is mediated by electrogenic Na+-HCO3 cotransport and Na+/H+ exchange (6, 60). The cells in these studies were nonpolarized, precluding membrane localization of the transport processes. Further experiments in human and porcine cholangiocytes failed to detect functional Na+-HCO3 cotransport (59, 66). In addition to secreting HCO3, cholangiocytes are thought to have transport processes capable of mediating fluid absorption (30, 42). Recently, the Na+/H+ exchanger NHE3 has been localized to the apical membrane of rat cholangiocytes by immunohistochemistry, where it may play an important role in fluid absorption from the bile duct lumen (43).

Although electrogenic Na+-HCO3 cotransport plays an essential role in hepatocyte HCO3 secretion and bile formation, the specific proteins mediating this process have not been identified. Of the members of the HCO3 transporter superfamily, NBC1 and NBC4 are the only Na+-HCO3 cotransporters known to be electrogenic (35). NBC1 cotransporters are widely expressed in various tissues, including kidney, pancreas, eye, duodenum, colon, heart, salivary gland, brain, prostate, epididymis, and thyroid (1–3, 10, 11, 28, 29, 39, 40, 45, 52, 54, 62, 65). However, NBC1 mRNA is very weakly expressed in liver (1), suggesting that another gene product likely mediates electrogenic Na+-HCO3 cotransport in hepatocytes and/or cholangiocytes.

NBC4c, encoded by the human SLC4A5 gene (48–50), was first shown to function as an electrogenic Na+-HCO3 cotransporter in mammalian epithelial cells (53) and was subsequently confirmed in Xenopus oocytes (67). NBC4 transcripts are most highly expressed in liver and to a lesser extent in kidney, brain, heart, pancreas, testis, and muscle (48, 53). The finding that NBC4 mRNA is highly expressed in liver suggests that the cotransporter is a candidate protein mediating the electrogenic flux of Na+-HCO3 in hepatocytes and/or cholangiocytes. In the present study, we addressed the question as to whether NBC4 is the major electrogenic Na+-HCO3 cotransporter in hepatocytes. Studies of native intrahepatic cholangiocyte HCO3 transport processes have not been performed to date. Therefore, an additional goal of this study was to identify the electrogenic Na+-HCO3 cotransporter in native intrahepatic bile duct cholangiocytes. Unlike the liver, in kidney the major electrogenic Na+-HCO3 cotransporter is kNBC1, which is responsible for mediating basolateral Na+-HCO3 efflux in the renal proximal tubule (2, 16, 51, 54). The finding that NBC4 mRNA is also expressed in kidney suggested that in addition to kNBC1, this transporter plays a previously uncharacterized role in renal Na+-HCO3 transport. Therefore, additional studies were done to determine the pattern of expression of NBC4 in the kidney.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cloning and sequence analysis of rat NBC4c. Total RNA from rat liver obtained from male 3-mo-old Sprague-Dawley rats was isolated using the RNAeasy Mini kit (Qiagen, Valencia, CA). After DNase treatment, the mRNA was reverse transcribed using avian myeloblastosis virus (AMV) reverse transcriptase (Roche Applied Science, Indianapolis, IN). Primers based on the sequence of human NBC4c (GenBank accession no. AAK97072) and murine expressed sequence tags were used to amplify the coding region of rat NBC4c. The nucleotide sequence of the coding region was confirmed using 5'- and 3'-rapid amplification of cDNA ends. The sequence of rat NBC4c (Fig. 1) was determined using an ABI 310 sequencer (PerkinElmer, Foster City, CA). All experiments were performed in accordance with the guidelines established by the Institutional Animal Research Committee of the University of California.



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Fig. 1. Alignment of rat NBC4c (GenBank accession no. AY496959) with human NBC4c (GenBank accession no. AAK97072).

 
RT-PCR amplification and sequence analysis. Total RNA from fresh tissues obtained from male 3-mo-old Sprague-Dawley rats was isolated using the RNAeasy Mini kit (Qiagen). After DNase treatment, the mRNA was reverse transcribed using AMV reverse transcriptase. Rat NBC4-specific primers that encompass the variable splicing region of NBC4 were used to identify which NBC4 splice variants were expressed in rat liver and kidney. The following NBC4 primers were used to amplify NBC4 cDNA from rat liver and kidney: sense, 5'-GGCTACCATCTGGACCTGTTCTGGGT-3' (2676–2701), and antisense, 5'-CAGTGGATACCGTTTTGGGGATC-3' (3369–3391). The expected size of the rat NBC4c PCR product was 716 bp, whereas the predicted size of the PCR products for the rat orthologs of NBC4a, NBC4b, and NBC4d splice variants were 764, 790, and 473 bp, respectively. To amplify rat kNBC1 (GenBank accession no. AF004017) and rat pNBC1 (GenBank accession no. NM_053424), specific sense primers for each variant and a common antisense primer were used: kNBC1-sense, 5'-CACTGAAAATGTGGAAGGGAAG-3' (29–50); pNBC1-sense, 5'-CATGTGTGTGACGAAGAAGAAGTAGAAG-3' (99–125); and common antisense, 5'-GACCGAAGGTTGGATTTCTTG-3' (539–559 in rat kNBC1 and 701–721 in rat pNBC1). The predicted sizes of the rat kNBC1 and pNBC1 PCR products were 528 and 652 bp, respectively. PCR was performed with the following parameters: 30 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and elongation at 68°C for 1 min. Negative controls included omission of the cDNA. GAPDH primers were used to validate the cDNA in each reaction. PCR products were separated by 2% agarose gel electrophoresis, cut from the gel, and purified with Suprec filters (Takara, New York, NY). All PCR products were excised and sequenced using an ABI 310 sequencer (PerkinElmer).

Transient expression of rat NBC4c in HEK-293 cells. Rat NBC4c was subcloned from pCR-Script SK+ into pCDNA 3.1 vector (Invitrogen, Carlsbad, CA). Human embryonic kidney (HEK)-293 cells (American Type Culture Collection, Manassas, VA) 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). Twenty-four hours before transfection, a 90% confluent 10-cm polystyrene plate (Becton Dickinson, Franklin Lakes, NJ) of cells was split 1:4 onto a 10-cm polystyrene plate with 12 ml of medium that was then immediately divided into a six-well plate (2 ml/well) containing fibronectin-coated coverslips (Discovery Labware, Bedford, MA). Twenty-four hours later, the six-well plate was transfected with purified plasmids (1 µg/µl; Qiagen, Santa Clarita, CA) using the standard calcium phosphate method. The transfection medium was removed after ~16 h and replaced with fresh media. After 5 h, the coverslips were rinsed twice with 1x PBS and then incubated with 1 ml of 4% paraformaldehyde for 2 min followed by 1 ml of methanol (~20°C) for 2 min. The cells were then rinsed twice with 1x PBS and processed for examination by immunofluorescence microscopy. The NBC4-specific antibody NBC4-r3 was applied at 1:100 dilution in PBS for 1 h at room temperature. After several washes in PBS, goat anti-rabbit IgG conjugated with Cy3 (1:500 dilution; Jackson ImmunoResearch, West Grove, PA) was applied for 1 h at room temperature. The slides were rinsed in PBS and mounted in Crystal/Mount (Biomeda, Foster City, CA). 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.

Generation/characterization of a polyclonal antibody to rat NBC4 and immunoblot analysis of rat tissues. A polyclonal antibody to rat NBC4 (NBC4-r3) was raised in rabbits against a synthetic peptide derived from the protein corresponding to amino acids 690–718 (LVPNTNMSVYTPLNLTALD) in rat NBC4c (GenBank accession no. AY496959) located in the second predicted extracellular loop. The antibody was affinity purified using Sepharose 4B columns with covalently attached NBC4 peptide. Liver, kidney cortex, renal pelvis, and pancreas obtained from 3-mo-old male Sprague-Dawley rats were disrupted in a glass homogenizer in 50 ml of 50 mM Tris·HCl buffer containing 1% Triton X-100 and the following protease inhibitors: 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 µg/ml pepstatin, 1 µg/ml leupeptin, and 1 µg/ml aprotinin (Roche Applied Science). After incubation for 45 min, the homogenate was centrifuged at 18,000 g for 20 min at 4°C. Reducing SDS-PAGE was performed using 7.5% polyacrylamide ready gels obtained from Bio-Rad (Hercules, CA). Proteins separated by SDS-PAGE were electrotransferred onto polyvinylidene difluoride membrane (Amersham Biosciences, Piscataway, NJ). Nonspecific binding was blocked by incubation for 1 h in TBS (20 mM Tris·HCl, pH 7.5, and 140 mM NaCl) containing 5% dry milk and 0.05% Tween 20 (Bio-Rad). The NBC4-r3-specific antibody was used at a dilution of 1:1,000. Previously well-characterized kNBC1- and pNBC1-specific antibodies (11) were used at a dilution of 1:2,000. Secondary horseradish peroxidase-conjugated species-specific antibodies (Jackson ImmunoResearch) were used at a dilution 1:20,000. Bands were visualized using an enhanced chemiluminescence (ECL) kit and ECL hyperfilm (Amersham Biosciences).

Immunohistochemistry. In 3-mo-old male Sprague-Dawley rats under isofluorane anesthesia, the aorta was perfused retrogradely with 4% paraformaldehyde, pH 7.4. The tissues were removed and cryoprotected overnight in 25% sucrose and then frozen immediately for sectioning. The NBC4-specific antibody NBC4-r3 was applied at 1:100 dilution in PBS for 1 h at room temperature to 5-µm cryostat sections attached to Probe On Plus glass slides (Fisher, Los Angeles, CA). After several washes in PBS, goat anti-rabbit IgG conjugated with Cy3 (1:500 dilution; Jackson ImmunoResearch) was applied for 1 h at room temperature. The slides were rinsed in PBS and mounted in Crystal/Mount (Biomeda). For double-labeling experiments, rat liver hepatocytes were labeled with NBC4-r3 as described and a monoclonal antibody against the canalicular isoform of the multidrug resistance-associated protein Mrp2 (47) (1:100, clone M2III-6; Alexis Biochemicals, San Diego, CA). Anti-Mrp2 was detected using goat anti-mouse IgG conjugated with Cy2 (1:500 dilution; Jackson ImmunoResearch) applied for 1 h at room temperature. In separate experiments, rat intrahepatic bile duct cholangiocytes were double labeled with NBC4-r3 and a monoclonal antibody against the {alpha}1-subunit of the Na+-K+-ATPase (1:100, clone 6H; Upstate Biotechnology, Lake Placid, NY). The Na+-K+-ATPase antibody was detected using goat anti-mouse IgG conjugated with Cy2 (1:500 dilution; Jackson ImmunoResearch) applied for 1 h at room temperature. In separate studies, sections of rat liver were stained with well-characterized kNBC1- and pNBC1-specific antibodies (11) applied at 1:100 dilution in PBS for 1 h at room temperature to 5-µm cryostat sections attached to Probe On Plus glass slides (Fisher). After several washes in PBS, goat anti-rabbit IgG conjugated with Cy3 (1:500 dilution, Jackson ImmunoResearch) was applied for 1 h at room temperature. The slides were rinsed in PBS and mounted in Crystal/Mount (Biomeda). As controls, in addition to liver, rat kidney sections were stained with the kNBC1-specific antibody, and rat pancreas sections were stained with the pNBC1-specific antibody using an identical protocol and Hoechst dye (5 µM in PBS) nuclear stain. The confocal images were captured with a Leica TCS SP inverted confocal microscope (Leica, Wetzlar, Germany) coupled to an argon-krypton laser (model 643; Melles Griot, Irvine, CA), and a two-photon system (Millenia 532-nm diode laser pumping a Tsunami Ti:sapphire laser; Spectra Physics, Mountain View, CA).


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The alignment of the amino acid sequence of the rat NBC4c (GenBank accession no. AY496959) with human NBC4c (GenBank accession no. AAK97072) is shown in Fig. 1. The rat ortholog of NBC4c shares 89% sequence homology with human NBC4c and also functions as an electrogenic Na+-HCO3 cotransporter (unpublished results; see also Ref. 53). Hydrophobicity analysis indicates that NBC4c has 10 transmembrane segments with cytoplasmic NH2 and COOH termini as recently demonstrated for NBC1 (63). The RT-PCR analysis of NBC4 expression in rat liver is shown in Fig. 2. Sequence analysis of excised PCR products revealed that only NBC4c was detected in mRNA samples from rat liver. Other splice variants previously detected in human heart and testes (48–50) were not found under any conditions. Rat testes expressed both NBC4c and NBC4d splice variants. The results indicate that only NBC4c is expressed in rat liver. Unlike NBC4c mRNA, kNBC1 mRNA was not detectable, and the pNBC1 transcript was only very weakly expressed in rat liver.



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Fig. 2. A: identification of NBC4 splice variants in rat liver (lanes 3–5), kidney pelvis (lanes 6–8), and testis (lanes 9–11). Rat NBC4-specific primers (lanes 3, 4, 6, 7, 9, and 10) that encompass the variable splicing region of NBC4 were used to identify the NBC4 splice variants in rat liver, kidney pelvis, and testis. A single PCR product (716 bp) corresponding to NBC4c was detected in rat liver (lane 3) and kidney pelvis (lane 6). In rat testis (lane 9), a major 716-bp PCR product and a minor 473-bp product corresponding to NBC4d were detected. Sequencing of the 716-bp PCR products from rat liver and kidney pelvis confirmed that these tissues expressed NBC4c. In addition, sequencing analysis confirmed that the major 716-bp PCR product in rat testis was NBC4c and that the minor PCR product was NBC4d. NBC4a (764 bp) and NBC4b (790 bp) were not detected. Experiments were performed with (lanes 3, 6, and 9) and without (lanes 4, 7, and 10) reverse transcriptase. A 1-kb ladder (0.075–12 kb, lane 1; Invitrogen), a 100-bp ladder (100–1,000 bp, lane 2; Invitrogen), and GAPDH (lanes 5, 8, and 11) are shown. Markers refer to 100-bp ladder. B: determination of potential NBC1 transcripts in rat liver. Rat liver (lanes 3–8), rat kidney (lanes 9–11), and rat pancreas (lanes 12–14) results are shown. kNBC1 primers (lanes 3, 4, 9, and 10) and pNBC1 primers (lanes 6, 7, 12, and 13) are also shown. Experiments were performed with (lanes 3, 6, 9, and 12) and without (lanes 4, 7, 10, and 13) reverse transcriptase. A 1-kb ladder (lane 1), a 100-bp ladder (lane 2), and GAPDH (lanes 5, 8, 11, and 14) are shown. Markers refer to 100-bp ladder.

 
Further experiments were done to characterize the size of the NBC4c protein and to determine the expression pattern in liver. The results of immunoblotting experiments are shown in Fig. 3. The NBC4-r3 antibody detected an ~145-kDa protein in rat liver. Preincubating the NBC4-r3 antibody with the specific immunizing peptide completely blocked this band. Previously characterized antibodies to the kidney (kNBC1) and pancreas (pNBC1) NBC1 variants (11) that label rat kidney and pancreas, respectively (Fig. 3), did not label rat liver (Fig. 3). Therefore, of the known electrogenic Na+-HCO3 cotransporter proteins, NBC4c is highly expressed in rat liver.



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Fig. 3. Characterization of rat NBC4c-specific polyclonal antibody NBC4-r3 in rat tissues. A: immunohistochemistry of human embryonic kidney (HEK)-293 cells expressing rat NBC4c. B: control untransfected HEK-293 cells. C: immunoblotting of HEK-293 cells expressing rat NBC4c (lane 1) and control untransfected HEK-293 cells (lane 2) probed with NBC4-r3 antibody. Detection of rat NBC4c in rat liver (lanes 3 and 4) and kidney pelvis (lanes 5 and 6) Triton X-100 extracts. NBC4-r3 antibody (lanes 3 and 5) and NBC4-r3 antibody preincubated with (lanes 4 and 6) the immunizing peptide (10 µg/ml) were used. For detection of potential NBC1 variants in rat liver, Triton X-100 extracts from rat liver were probed with kNBC1-specific (lane 7) and pNBC1-specific (lane 8) antibodies. As controls, Triton X-100 extracts of rat kidney (lanes 9 and 10) and pancreas (lanes 11 and 12) were probed with kNBC1-specific antibody (lane 9), kNBC1-specific antibody preincubated with the immunizing peptide (10 µg/ml, lane 10), pNBC1-specific antibody (lane 11), and pNBC1-specific antibody preincubated with the immunizing peptide (10 µg/ml; lane 12). Loading values were 10 µg (lanes 1 and 2), 12 µg (lanes 3 and 4), 95 µg (lanes 5 and 6), 100 µg (lanes 7 and 8), 8 µg (lanes 9 and 10), and 6 µg (lanes 11 and 12).

 
To further characterize the NBC4-r3 antibody, HEK-293 cells were transfected with rat NBC4c. Immunoblotting revealed a band of similar size to that seen in rat liver (Fig. 3). The specificity of the labeling was confirmed using the NBC4-r3 antibody preabsorbed with the immunizing peptide (Fig. 3). Immunofluorescence microscopy revealed the NBC4-r3 antibody-labeled transfected cells (Fig. 3), whereas no labeling was detected in untransfected cells (Fig. 3).

Sections of rat liver were stained with the NBC4-r3 antibody to determine the localization of the cotransporter. As shown in Figs. 4 and 5, NBC4c is highly expressed in rat hepatocytes. The cotransporter was localized predominantly to the basolateral sinusoidal membrane. All hepatocytes were stained similarly, although sometimes those cells near the portal triads stained more intensely, demonstrating zonal variation within a hepatic lobule. The sinusoidal cells lining the sinusoidal spaces were not stained. In addition, the endothelial cells lining the central vein, the portal vein, and the hepatic artery were devoid of immunostaining. In addition to labeling hepatocytes, the NBC4-r3 antibody strongly labeled bile duct intrahepatic cholangiocytes in the portal triads (Figs. 4 and 5). Most cholangiocytes in the intrahepatic bile ducts were stained apically. The localization of the other known electrogenic Na+-HCO3 cotransporter NBC1 proteins in the liver was examined using previously characterized antibodies (11). As shown in Fig. 5, antibodies against kNBC1 and pNBC1 did not label any cells in the liver, confirming the immunoblotting results (Fig. 3). The lack of detectable pNBC1 protein in rat liver suggests either that pNBC1 protein is expressed at a level that could not be detected by immunoblotting and immunohistochemistry or that pNBC1 transcripts, which are only weakly expressed in liver, are translationally silent (17). Figure 5 shows labeling of renal proximal tubules with the kNBC1 antibody and staining of pancreatic ducts with the anti-pNBC1 antibody.



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Fig. 4. Immunolocalization of NBC4c in rat liver using the NBC4-r3 antibody. A: immunofluorescence image of NBC4c localization in rat liver hepatocytes (H) and intrahepatic bile ducts (BD). The portal and hepatic veins are unlabeled. NBC4c strongly labeled the basolateral membrane of hepatocytes throughout the liver parenchyma. In intrahepatic bile ducts, many cholangiocytes were stained apically. Bar, 50 µm. B: immunolocalization of NBC4c in the liver showing basolateral membrane labeling of hepatocytes, whereas the central vein (CV) is unlabeled. Bar, 100 µm. C: lack of staining of the rat liver using the NBC4-r3 antibody incubated with the specific immunizing peptide (10 µg/ml). PV, portal vein. Bar, 100 µm. D: corresponding Nomarski image. Bar, 100 µm.

 


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Fig. 5. A: double-labeling immunofluorescence image of rat liver hepatocytes showing the basolateral (sinusoidal) localization of NBC4c (red; arrows) compared with the canalicular isoform of the multidrug resistance-associated protein Mrp2 (green; arrowheads). Bar, 50 µm. B: double-labeling immunofluorescence image of an intrahepatic bile duct showing the basolateral localization of the Na+-K+-ATPase (green; arrowheads) compared with the apical localization of NBC4c (red; arrows). Bar, 14 µm. C: lack of expression of kNBC1 in the rat liver. Bar, 50 µm. D: immunofluorescence image showing kNBC1 staining of kidney proximal tubules. Bar, 100 µm. E: lack of expression of pNBC1 in rat liver. Bar, 50 µm. F: immunofluorescence image showing pNBC1 staining of pancreatic ducts (red) and acinar cell nuclei (blue; Hoechst dye). Bar, 4 µm.

 
In addition to the liver, we previously detected NBC4 mRNA in kidney (48). Because the major electrogenic Na+-HCO3 cotransporter in the kidney is kNBC1, which is responsible for HCO3 reabsorption across the basolateral membrane of the proximal tubule, we performed additional studies to evaluate which NBC4 variant is present in the kidney, as well as its cellular localization. As shown in Fig. 2, the rat kidney pelvis only expresses the NBC4c mRNA, as does the liver. The results of immunoblotting experiments are shown in Fig. 3. The NBC4-r3 antibody detected an ~145-kDa protein in rat kidney pelvis. Preincubating the NBC4-r3 antibody with the specific immunizing peptide completely blocked this band. The immunolocalization of NBC4c in the rat kidney was determined using the NBC4-r3 antibody. The parenchyma of the cortex, outer medulla, and inner medulla were devoid of NBC4 staining, and the NBC4-r3 antibody labeled the pelvic uroepithelium (Fig. 6). Figure 6 depicts the immunolocalization of NBC4c in uroepithelial cells comprising a simple squamous epithelium lining the outer pelvic wall of the fornix adjacent to the inner stripe of the outer medulla as well as cells lining the inner pelvic wall at the upper portion of the inner medulla. In addition to the fornix, uroepithelial cells lining the upper, middle, and lower portions (tip) of the inner medulla expressed NBC4c. The staining pattern was predominantly apical, although in some cells there was no specific membrane localization of the cotransporter. Figure 6 shows the immunolocalization of NBC4c in the uroepithelium lining the outer pelvic wall. Simple squamous uroepithelial cells lining the outer medulla (inner stripe) peripelvic column (OMIS), low cuboidal cells lining the outer medulla (outer stripe) peripelvic column (OMOS), transitional uroepithelial cells lining the cortex, and transitional uroepithelial cells lining the hilum of the renal pelvis stained positively for NBC4c. Cells lining the OMIS and OMOS had predominant apical staining, whereas in some uroepithelial cells lining the cortex and hilum, the staining was more diffuse.



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Fig. 6. Immunolocalization of NBC4c in rat kidney using NBC4-r3 antibody. A: immunofluorescence image of labeled uroepithelial cells lining the fornix region of the pelvic uroepithelium. Bar, 100 µm. B: lack of staining of the pelvic uroepithelium using NBC4-r3 antibody incubated with specific immunizing peptide (10 µg/ml). Bar, 100 µm. C and D: higher magnification immunofluorescence images of uroepithelial cells lining the inner medulla. Bar, 50 µm. Middle portion (C) and tip (D) of inner medulla are shown. E–H: immunolocalization of NBC4c in the uroepithelium lining the outer pelvic wall. Bar, 50 µm. E: simple squamous uroepithelial cells lining the outer medulla (inner stripe) peripelvic column (OMIS). F: low cuboidal uroepithelial cells lining the outer medulla (outer stripe) peripelvic column (OMOS). G: transitional uroepithelial cells lining the cortex (C). H: transitional uroepithelial cells lining the hilum (H) of the renal pelvis.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous experiments in cultured hepatocytes demonstrated the importance of electrogenic Na+-HCO3 cotransport in mediating transcellular HCO3 secretion (6, 18, 20, 21). Using a combination of RT-PCR, immunoblotting, and immunohistochemical analysis, we have demonstrated for the first time specific expression of the electrogenic Na+-HCO3 cotransporter NBC4c in the basolateral membrane of hepatocytes. These findings, together with the lack of NBC1 expression in these cells, suggest that NBC4c mediates basolateral HCO3 influx in hepatocytes and thereby contributes to biliary HCO3 secretion. Furthermore, given the documented importance of basolateral electrogenic Na+-HCO3 cotransport in hepatocyte pHi regulation, in this regard, NBC4c likely plays an important additional role in ensuring that hepatocyte pHi remains within normal limits despite ongoing metabolic reactions that generate protons.

Electrogenic Na+-HCO3 cotransport was first demonstrated in rat cholangiocytes in studies of nonpolarized cultured cells (7, 60). Studies in pig cholangiocytes have instead provided evidence for a vacuolar H+-ATPase that might be responsible for net HCO3 flux (66), whereas in polarized cultured human cholangiocytes, HCO3 flux is mediated by Na+-dependent Cl/HCO3 exchange (59). Species and technique-related differences can potentially account for these variable findings. Our results suggest that NBC4c mediates electrogenic Na+-HCO3 cotransport in intrahepatic cholangiocytes. The finding that NBC4c was expressed apically in many cholangiocytes was unexpected. Fluid secretion in cholangiocytes is mediated by an apical CFTR homolog coupled to Cl/HCO3 exchange (6, 7, 18, 19, 25, 41, 58, 59). More recently, NHE3 has been immunolocalized to the apical membrane of rat cholangiocytes, where it may play a role in fluid absorption (43). In contrast, in the rat NRC-1 cholangiocyte cell line, NHE2 and not NHE3 mRNA could be detected (58). The apical localization of NBC4c in many cholangiocytes suggests a potential role for the cotransporter in luminal fluid secretion and/or absorption. Although previous studies documented the presence of electrogenic Na+-HCO3 cotransport in nonpolarized cultured rat bile duct cells grown on glass coverslips, the specific membrane localization of this transport process in polarized cells was not determined. Furthermore, given the potential heterogeneity of cholangiocyte cell subtypes (5) and the effect of culturing cells in vitro on the targeting of membrane transporters, it is necessary to study the mechanisms of single cell cholangiocyte ion transport in vivo. Furthermore, additional experiments are required to determine the potential functional requirement for NBC4 in modulating intrahepatic bile duct fluid transport and in pHi regulation.

Our results indicate that of the known NBC4 variants, rat liver and kidney uroepithelium express NBC4c only. In contrast to the liver, where NBC4c was expressed in cells previously shown to have electrogenic Na+-HCO3 cotransport, in the kidney the cotransporter was immunolocalized to epithelial cells lining the renal pelvis that were not known previously to have Na+-HCO3 cotransport function. These cells that cover the renal pelvis constitute a thin barrier separating the urine from the outer and inner medullary renal parenchyma and a portion of the cortex (31, 36, 57). NBC4c is the first Na+-HCO3 cotransporter identified in these cells. In addition to NBC4c, pelvic uroepithelial cells express basolateral AE2 (61). These findings are in keeping with the fact that uroepithelial cells are exposed to a wide range of urinary pH values (pH 4.5–8), necessitating potent pH regulatory transport processes. The anatomical relationship between the pelvic uroepithelium and the underlying renal parenchyma is also of interest, given the possibility of the transport of water, Na+, and urea between the urine and the outer and inner medulla (12, 56). Uroepithelial cells may contribute to active transepithelial Na+ transport, as suggested by Schütz and Schnermann (55) using an isolated human renal pelvis preparation. However, the exact transport processes mediating transepithelial Na+ transport in the pelvic uroepithelium are currently not known. In addition to its role in pHi regulation, future studies must determine the potential contribution of NBC4c to uroepithelial Na+ transport.

In addition to NBC4c, uroepithelial cells have an amiloride-sensitive Na+ channel (ENaC), which appears to be involved in afferent renal nerve activation in response to changes in renal pelvic pressure (34). Changes in renal pelvic urinary Na+ concentration alter afferent renal nerve activity under basal conditions and in response to increased pelvic pressure (34). Although the majority of afferent renal mechanosensory nerves are located in the smooth muscle layer immediately beneath the transitional cells, a few fibers extend into the uroepithelial cell layer (68). It has been suggested that changes in intracellular Na+ concentration in uroepithelial cells may facilitate depolarization of adjacent sensory nerve endings during pelvic wall stretch (33). A similar mechanism has been reported in the tongue, where altered cellular Na+ flux in non-taste cells augments the depolarization of taste cells (38). ENaC channel activity and neuronal activity are known to be pH sensitive (32). The importance of pHi in modulating both Na+ channel activity and neurosensory responses underscores an additional potentially important role for NBC4c in preventing large changes in the pH of the microenvironment of mechanosensory nerve fibers in the pelvic wall.

In summary, this study shows for the first time that NBC4c is the electrogenic Na+-HCO3 cotransporter in rat hepatocytes and intrahepatic cholangiocytes. NBC1 cotransporters are not expressed in rat liver, indicating an important role for NBC4c in mediating Na+-HCO3 cotransport in these cells. In contrast, both NBC1 and NBC4c are expressed in rat kidney and are localized to the proximal tubule and the pelvic uroepithelium, respectively. Whether tissue-specific differences in membrane targeting or regulation of ion flux and/or stoichiometry might account for the specific requirement for NBC4c in liver and pelvic uroepithelium requires further study.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-63125, DK-58563, and DK-07789, American Heart Association (Western Affiliate) Grant 0365022Y, the Max Factor Family Foundation, the Richard and Hinda Rosenthal Foundation, the Frederika Taubitz Foundation, and the National Kidney Foundation of Southern California Grant J891002.


    ACKNOWLEDGMENTS
 
This work was presented in part at the annual meetings of the American Society of Nephrology, November 2001 and November 2003.


    FOOTNOTES
 

Address for reprint requests and other correspondence: I. Kurtz, Division of Nephrology, Univ. of California, Los Angeles, 10833 Le Conte Ave., Rm. 7-155 Factor Bldg., Los Angeles, CA 90095-1689 (E-mail: IKurtz{at}mednet.ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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