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
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ABSTRACT |
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bicarbonate; transport; electrogenic
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 (13, 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 (4850), 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.
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METHODS |
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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 690718 (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 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).
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RESULTS |
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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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DISCUSSION |
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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 4.58), 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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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