1The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C; 2Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark; 3Department of Medicine, Harvard Medical School, Molecular and Vascular Medicine Unit and Renal Unit, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215; and 4Emory University School of Medicine, Renal Division, Atlanta, Georgia 30322
Submitted 21 November 2003 ; accepted in final form 26 January 2004
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ABSTRACT |
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immunohistochemistry; collecting duct; nephron segments
AE2 is thought to be constitutively expressed in almost all mammalian cells and has accordingly been detected by RT-PCR in all rat kidney nephron segments (5). Functionally, AE2 exhibits unique properties relative to AE1. First, AE2 exchange activity increases with both increasing intracellular pH and extracellular pH (23, 24). Second, the anion exchange activity of AE2 is increased by hypertonic extracellular conditions. In Xenopus laevis oocytes, hypertonicity increases AE2-mediated Cl/HCO3 exchange, which acts in tandem with increased Na+/H+ exchange to generate a regulatory volume increase (14). Third, AE2-mediated anion exchange is regulated by extracellular ammonium (13).
In addition to its functions related to cellular pH and volume regulation, AE2 may play a role in transepithelial bicarbonate transport and urinary acidification in rat kidney. Immunohistochemical studies showed AE2 immunoreactivity in varying amounts in basolateral plasma membrane domains of epithelial cells in all rat kidney nephron segments distal to the proximal tubule, with the strongest labeling in the medullary thick ascending limb (mTAL), inner medullary collecting duct (IMCD), and macula densa (2). Functional studies confirmed the presence of AE2 in the mTAL (10). AE2 mRNA has been shown to be present in rat proximal tubules by RT-PCR. However, by immunohistochemistry, AE2 protein expression is low in this segment (2). Thus AE2 likely plays a minor role in bicarbonate absorption in the proximal tubule.
In contrast to the strongly labeled basolateral regions of macula densa cells, AE2 is weakly detected in the cortical TAL (cTAL) and weakly detected or absent in distal convoluted tubule, connecting tubule, and the cortical portion of the CD (2). As the CD extends into the medulla, immunolabeling increases gradually and is most intense in the most terminal portion of the IMCD (tIMCD) (2). Across the apical membrane of the rat terminal IMCD, secretion of chloride and absorption of bicarbonate (28) occur in tandem with Cl uptake and HCO3 efflux across the basolateral membrane (electroneutral Na+-independent Cl/HCO3 exchange) (22). Because the rat tIMCD does not express AE1 (1), and because AE2 is highly expressed in rat tIMCD, AE2 might be the gene product that mediates this electroneutral Cl/HCO3 exchange (2, 25).
Three splice variants of AE2 (AE2a, b, and c), which differ in tissue and cellular distribution, have been described in the rat (29). By Northern blot analysis, mRNA of all isoforms has been detected in the stomach, whereas only AE2a and AE2b mRNA have been detected in the kidney (29). However, AE2c mRNA has been detected in the kidney by RT-PCR (2). In rabbit stomach, AE2 protein expression as well as DIDS-sensitive Cl/HCO3 exchange are more than 10-fold higher in parietal cells than in mucous cells. Moreover, Northern blot analysis has shown that more AE2a than AE2b mRNA is present in mucous cells, whereas AE2b mRNA is the dominant isoform in parietal cells (21). The cell type-specific expression of each AE2 isoform may reflect the distinct functional regulation of Cl/anion exchange in these gastric cell types. The comparative distribution of AE2a and AE2b protein in rat kidney remains unreported.
The purpose of this study was to describe the segmental and cellular distribution of the AE2a and AE2b polypeptides in the rat kidney and to determine whether their levels are regulated independently in response to chronic NH4Cl loading. This was achieved by immunoblotting, immunohistochemistry, and immunoelectron microscopy using antibodies specific to the unique NH2-terminal AE2a and to an amino acid (aa) sequence common to AE2a and b but different from AE2c.
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MATERIALS AND METHODS |
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AE2a (-222).
Polyclonal antibodies were raised in rabbits against a synthetic peptide CSSAPRRPASGADSLHTPEPES that corresponds to aa222 of the rat and mouse AE2a isoform. This amino acid sequence is absent from AE2c, and only the latter five aminoacids are present in the amino acid sequences of rat AE2b and mouse AE2b1.
Anti-AE2a/b (-101117).
Polyclonal antibodies were raised in rabbits against a synthetic peptide CPGRKPRRRPGASPTGET corresponding to aa101117 of the rat and mouse AE2a isoform and aa88104 in the rat and mouse AE2b sequences. This peptide sequence is not found in rat AE2c. Affinity purification was performed by binding antibodies from hyperimmune serum to the peptide immunosorbent, eluting with citrate-phosphate, pH 2.2, and neutralizing the eluate with Tris base. Sodium azide was added at 0.1% as an antimicrobial. Antibody specificity was tested on cells overexpressing recombinant mouse AE2a, AE2b, and AE2c.
Anti-AE2a, b, c ( 12241237,
-COOH-term).
An antibody raised against aa12241237 of the AE2 sequence (
-COOH-term) previously described (2) detects a shared COOH terminus of all AE2 isoforms and was used to test total AE2 expression in transfected cells.
Immunoblotting of cell lysates of ECr-293 cells expressing mouse AE2a, AE2b1, and AE2c1. EcR-293 cells (Clontech) were grown in DME plus 10% FCS with penicillin, streptomycin, and Zeocin and transiently transfected (SuperFect, Qiagen) with mouse AE2a, AE2b1, and AE2c1, or no vector. The following day, cells were exposed to 5 mM ponasterone. Forty-eight hours later, cells were scraped, extracted with 1% Triton X-100, 75 mM NaCl, 10 mM Na phosphate, and 10 mM Tris·HCl, pH 7.4. Cell protein was quantified by BCA assay (Pierce) before extraction. Proteins were separated by 520% SDS-PAGE, transferred to nitrocellulose, and blots were developed with the indicated antipeptide antibodies. Peroxidase-conjugated anti-Ig was from Jackson ImmunoResearch; enhanced chemiluminescence detection reagents were from Pierce.
Immunofluorescence analysis of EcR-293 cells expressing mouse AE2a, AE2b1, and AE2c1. EcR-293 cells grown on plastic dishes were transiently transfected with AE2a cDNA. After 20 h, cells were trypsinized and replated on polylysine-coated multichamber slides. After a 5-h recovery period, cells were induced with 5 mM ponasterone. EcR-293 cells plated on polylysine-coated multichamber microscope slides, with stable expression of AE2b1 or AE2c1 cDNAs, were similarly induced with 5 mM ponasterone. After 24 h of induction, cells were rinsed, fixed in 3% paraformaldehyde for 30 min at room temperature, quenched with 50 mM glycine in PBS, washed with PBS, incubated with 1% SDS for 15 min for epitope unmasking, and subjected to immunostaining. Primary antibody was applied for 2 h at room temperature, slides were washed, and then secondary antibodies were applied for 45 min. Slides were visualized with a Bio-Rad MRC-1024 confocal immunofluorescence microscope.
Animals. Male Munich-Wistar rats (250300 g) from Møllegaard Breeding Centre were kept on a standard rodent diet (Altromin 1320, Lage, Germany) until use. Non-Swiss albino mice were purchased from Harlan (Ardmore, TX). The use of animals in the experiments in the study complied with institutional and National Institutes of Health standards.
NH4Cl loading of rats with fixed water and food intake. Rats were assigned randomly to either the control or treated group. Before the treatment, the rats were kept in metabolic cages for 3 days to record baseline values of urine output and pH. Each morning rats were given a fixed amount of ground rat chow (0.068 g/g body wt) mixed with water (0.168 g/g body wt). The experimental group (n = 12) was given 0.033 mmol NH4Cl/g body wt in the food for 7 days, whereas the control group (n = 12) consumed the same amount of food but without NH4Cl. The NH4Cl loading induced a marked drop in urinary pH (5.76 vs. 7.94), but the groups did not differ in plasma acid-base status or change in body weight indicating that the animals were in steady state with respect to acid intake and excretion (11).
HCl loading of rats with fixed water and food intake. For comparison with the NH4Cl loading, a control experiment using HCl loading was performed. The experiment followed the same protocol as the NH4Cl-loading experiment, except that HCl were used instead of NH4Cl. The experimental group was given 0.033 mmol HCl/g body wt in the food for 7 days, whereas the control group consumed the same amount of food but without HCl. Similarly to NH4Cl loading, HCl loading induced a marked drop in urine pH compared with the control group (6.29 vs. 8.25, n = 14), and no difference was observed in plasma pH [7.33 (n = 9) vs. 7.36 (n = 11)].
Rat kidney membrane fractionation and immunoblotting. Kidneys from normal rats and six rats from the NH4Cl-loaded group were used. The kidneys were divided into three zones: cortex/outer stripe of the outer medulla (Ctx/OS), inner stripe of the outer medulla (ISOM), and inner medulla (IM). The tissue was homogenized in 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin and 1 mM phenylmethylsulfonyl fluoride using an ultra-Turrax T8 homogenizer (IKA Labortechnik) at a maximum speed (25,000 rpm) for 30 s, and the homogenates were centrifuged at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatants were solubilized in Laemmli sample buffer containing 3% SDS (final concentration). SDS-PAGE electrophoresis was performed of 30 µg protein in each lane of 9% polyacrylamide gels (Bio-Rad Mini Protean II) and run for 10 min at 100 V followed by 40 min at 200 V. After transfer by electroelution (0.025 M Tris, 0.19 M glycine, and 20% methanol, pH 8.3, 100 V, 1 h) 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 anti-AE2 antibodies. The labeling was visualized with horseradish peroxidase (HRP)-conjugated secondary antibodies (P448, DAKO, Glostrup, Denmark, diluted 1:3,000) using the enhanced chemiluminescence system (Amersham International). The chemiluminescence was recorded on film (Amersham), which was subsequently scanned using a flatbed scanner. Two-dimensional rolling ball background subtraction and the Gel-plot2 macro in Scion Image (Windows version of National Institutes of Health Image) were used for densitometric analysis.
Mouse kidney membrane fractionation and immunoblotting. Six-week-old non-Swiss albino mice were anesthetized with 100% O2 at 1 l/min with 4% isofluorane before death. Preparation of kidney lysates and immunoblotting were performed as reported previously (9). Kidneys were place in ice-cold solution containing 250 mM sucrose, 10 mM triethanolamine, 1 µg/ml leupeptin (Sigma), and 0.1 mg/ml PMSF (US Biochemical, Toledo, OH), pH 7.6. Kidney tissue was dissected to separate cortex, outer medulla, and IM. Tissue samples from each of these regions were homogenized with a tissue homogenizer (Omni/Tech Quest, Warrenton, VA) at 15,000 rpm on ice for 15 s. The homogenization step was repeated twice. Protein content was measured using the method of Lowry et al. (19). Membranes were solubilized for 15 min at 60°C in Laemmli sample buffer. SDS-PAGE was performed on minigels of 8% polyacrylamide. Each lane was loaded with 25 µg protein. The proteins were transferred from the gels electrophoretically onto nitrocellulose membranes. After being blocked with 5 g/dl nonfat dry milk, membranes were probed with the anti-AE2a and anti-AEa/b antibodies at 1:10,000 and 1:50,000 dilutions, respectively. Donkey anti-rabbit IgG conjugated to HRP at a 1:5,000 dilution (31458, Pierce, Rockford, IL) was used as a secondary antibody. Sites of antibody-antigen reaction were visualized using luminal-based enhanced chemiluminescence (KPL; Kirkegaard and Perry, Gaithersburg, MD) before exposure of X-ray film (X-OMAT AR; Eastman Kodak, Rochester, NY).
Immunohistochemistry of rat kidney sections. Kidneys from normal rats, 12 NH4Cl-loaded rats, and 12 control rats were fixed by retrograde perfusion via the aorta with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and postfixed for 2 h in similar fixative. Kidneys from 9 HCl-loaded and 9 control rats were fixed similarly, except that 3% paraformaldehyde and 1-h postfixation were applied. Kidney slices containing all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissues were cut at 2-µm thickness on a rotary microtome (Leica). The sections were dewaxed in xylene and rehydrated through 99% ethanol to 96% ethanol. After 30-min incubation in 0.3% H2O2 in methanol to block endogenous peroxidase activity, rehydration was completed through 96 and 70% ethanol. To reveal antigens, sections were placed in 10 mM Tris buffer (pH 9.0) supplemented with 0.5 mM EGTA (Titriplex VI, Merck) and heated in a microwave oven for 10 min. Nonspecific binding of Ig was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with AE2 antibodies diluted in 10 mM PBS (pH 7.4) containing 0.1% Triton X-100 and 0.1% BSA. For light microscopy, sections were incubated with HRP-linked goat anti-rabbit secondary antibodies (P448, DAKO), labeling visualized by DAB technique and the sections counterstained using Mayers hematoxylin.
Immunoelectron microscopy.
For immunoelectron microscopy, the kidneys were perfused through the abdominal aorta with 0.01 M periodate-0.075 M lysine-2% paraformaldehyde in 37 mM phosphate buffer (pH 6.2) for 5 min and fixed additionally by immersion in the same fixative for 2 h at room temperature and then overnight at 4°C. Sections of tissue were cut transversely through the entire kidney on a Vibratome at a thickness of 50 µm and washed with 50 mM NH4Cl in PBS three times for 15 min followed by incubation for 3 h in PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin. The tissue sections were then incubated overnight at 4°C in PBS containing 1% BSA and AE2a/b (-101117) antibody (1:1,000). After several washes with PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin, the sections were incubated for 2 h in peroxidase-conjugated goat anti-rabbit IgG (P448, DAKO), diluted 1:100 in PBS containing 1% BSA. The sections were then rinsed, first in PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin and subsequently in 0.01 M PBS buffer, pH 7.4. For the detection of HRP, the sections were incubated in 0.1% DAB in 0.01 M PBS buffer for 5 min, after which H2O2 was added to a final concentration of 0.01% and the incubation was continued for 10 min. After three washes in 0.01 M PBS buffer, tissue slices were fixed in 1% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h at 4°C and washed three times for 10 min with 0.1 M sodium cacodylate buffer. This was followed by postfixation in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4°C. After being rinsed in 0.1 M sodium cacodylate buffer, the tissue was dehydrated in a graded series of alcohol and propylene oxide and embedded in TAAB resin. Ultrathin sections were stained with lead citrate and photographed with a Philips CM100 transmission electron microscope.
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RESULTS |
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Immunoelectron microscopic localization of AE2 in IM. To determine the subcellular localization of AE2, electron microscopy using a preembedding HRP-visualized immunolabeling technique was employed (15). Antigen-antibody interactions are detected as a dark precipitate. AE2a/b immunolabeling was detected in the basolateral plasma membrane in IMCD cells in the inner third of the IM (Fig. 5).
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DISCUSSION |
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Immunoblotting of rat kidney samples confirmed the distribution of the AE2 isoforms observed by immunohistochemistry. In both the rat and mouse, greater AE2a expression was detected in the ISOM than in the IM. At first glance, results of immunoblots appear to differ from studies using immunohistochemistry, which suggest that AE2a expression is greater in IMCD than mTAL. However, basolateral membrane proteins of the mTAL comprise a particularly large fraction of the total proteins in ISOM tissue homogenates due to the deep basolateral invaginations of the mTAL cells (26). In the mTAL of the ISOM, lateral invaginations are numerous and often extend two-thirds or more of the distance from the base of the cell to the luminal border (26). Moreover, the mTAL represents more than 70% of the total protein in the outer medulla (4), whereas the tIMCD represents only 2030% of the total protein in the IM (16). Taken together, basolateral plasma membrane proteins of the mTAL comprise a much larger fraction of the total protein content in ISOM than the fraction of IM protein from basolateral membranes of the tIMCD. Thus, if equal amounts of total protein from the outer and IM were loaded on a gel, greater basolateral membrane protein would be loaded per lane from the mTAL than from the tIMCD. The same bias may not be present by immunohistochemistry.
Alternatively, the apparent discrepancy between immunoblotting and immunohistochemistry with respect to relative intensities of immunoreactivity using the AE2a antibody may occur because the AE2a-specific epitope, localized at the NH2 terminal of AE2a, is less accessible in the mTAL than in the tIMCD in fixed tissue, perhaps due to interactions with other proteins.
Increased cTAL immunolabeling in NH4Cl-loaded rats is likely due to upregulation of AE2b.
By both immunoblotting and immunohistochemistry, AE2a/b immunoreactivity was greatly increased in the kidney cortex from NH4Cl-loaded rats relative to controls. No change in AE2a expression was detected in kidney from rats ingesting NH4Cl. Increased AE2 expression was noted by immunohistochemistry as intensified labeling within cTAL. Because AE2a immunoreactivity did not change with NH4Cl ingestion, we conclude that the increased labeling found with the AE2a/b antibody (-101117) is largely due to increased abundance of the AE2b isoform in cTAL following NH4Cl loading. Similar changes in AE2a/b immunolabeling were seen following 7 days of HCl loading, supporting the conclusion that the changes in AE2b expression in cTAL are involved in the renal compensation of a sustained acid load.
AE2a is widely expressed and is thought to serve general cellular functions such as regulation of intracellular pH and cell volume. However, the tissue distribution of rat AE2b mRNA, present in liver, kidney, stomach, and intestine, appears to be more limited (29). The present study demonstrates that immunolabeling of AE2a/b, but not AE2a, is increased in rat kidney cTAL following oral NH4Cl loading. Our data therefore suggest that AE2a and AE2b polypeptide levels are regulated independently. Rat AE2b mRNA is transcribed from a promoter located in intron 2 of the AE2 gene. A number of consensus binding sites for transcription factors have been identified upstream of the initiation site of rat AE2b (29). Although little is known about the signals that may induce AE2b polypeptide expression in rat cells, the human AE2b2 promoter can be regulated by HNF1 (20).
Functional role of AE2 in TAL and CD. Rat cTAL absorbs bicarbonate (3). Therefore, AE2 expressed in cTAL might participate in the process of bicarbonate absorption and urinary acidification in this segment, particularly following NH4Cl ingestion. Increased absorption of bicarbonate in the cTAL would help correct the metabolic acidosis that follows NH4Cl ingestion. Alternatively, increased AE2 expression might modulate Cl excretion following NH4Cl ingestion. Chloride channels expressed on the basolateral plasma membrane differ between the cTAL and mTAL (30). Thus the mechanism of Cl absorption and its regulation may differ between the cTAL and mTAL. Whether upregulation of AE2 in cTAL helps maintain acid-base or chloride balance (or both) following NH4Cl ingestion remains to be determined.
After ingestion of NH4Cl, net acid secretion increases in rat IMCD, both in vivo and in vitro (28). As described above, strong AE2a immunoreactivity was seen in tIMCD, and AE2a may be the only AE2 isoform present in IMCD. The results of this study do not allow any conclusions regarding the presence of AE2b in IMCD. Expression of AE2 protein in IM was not seen to change with NH4Cl loading. Thus if AE2 contributes to the increased absorption of HCO3 observed following NH4Cl ingestion, either AE2-mediated transport is not rate limiting or AE2 activity increases in this treatment model through a mechanism that does not involve changes in protein expression. NH4Cl loading is associated with a number of adaptive changes in the kidney, such as an increase in the interstitial concentration of NH4+ in the IM (12). In heterologous expression systems, AE2 is activated by increases in extracellular NH4+ concentration. Thus activation of AE2 by NH4+ might increase AE2-mediated HCO3 efflux across the basolateral membrane of the tIMCD in vivo, following NH4Cl ingestion, in the absence of changes in AE2 protein expression.
In conclusion, in the chronic NH4Cl-loading model of metabolic acidosis, AE2 protein expression is upregulated in the cortex, likely through increased abundance of AE2b polypeptide(s) in the cTAL. In the IM, a region in which the AE2a polypeptide is relatively more abundant, no change in AE2 protein expression was observed. Whether the observed upregulation of AE2 contributes to the increase in net acid secretion observed in this treatment model remains to be determined.
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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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REFERENCES |
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