1 Departments of Pediatrics and Medicine and 2 Division of Pediatric Nephrology, University of Rochester School of Medicine, Rochester 14642; and 3 Department of Medicine, College of Physicians and Surgeons of Columbia University, New York, New York 10032
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ABSTRACT |
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Carbonic anhydrase (CA) IV is a
membrane-bound enzyme that catalyzes the dehydration of carbonic acid
to CO2 and water. Using peptides from each end of the
deduced rabbit CA IV amino acid sequence, we generated a goat
anti-rabbit CA IV antibody, which was used for immunoblotting and
immunohistochemical analysis. CA IV was expressed in a variety of
organs including spleen, heart, lung, skeletal muscle, colon, and
kidney. Rabbit kidney CA IV had two N-glycosylation sites and
was sialated, the apparent molecular mass increasing by at least 11 to
~45 kDa in the cortex. Medullary CA IV was much more heavily
glycosylated than CA IV from cortex or any other organ, such
modifications increasing the molecular mass by at least 20 kDa. CA IV
was expressed on the apical and basolateral membranes of proximal
tubules with expression levels on the order of S2 > S1 > S3 = 0. Because CA IV is believed to be anchored to the apical membrane by
glycosylphosphatidylinositol, the presence of basolateral CA IV
suggests an alternative mechanism. CA IV was localized on the apical
membranes of outer medullary collecting duct cells of the inner stripe
and inner medullary collecting duct cells, as well as on
-intercalated cells. However, CA IV was not expressed by
-intercalated cells, glomeruli, distal tubule, or Henle's loop
cells. Thus CA IV was expressed by H+-secreting cells of
the rabbit kidney, suggesting an important role for CA IV in urinary acidification.
organs; intercalated cells; proximal tubule; medullary collecting duct; immunohistochemistry; Western blot
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INTRODUCTION |
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CARBONIC ANHYDRASE (CA) is an enzyme that is critical to acid-base homeostasis. Up to 5% of CA activity is membrane bound and corresponds to CA IV, with the remainder being primarily CA II (6, 33, 49, 65). CA IV catalyzes the dehydration of intraluminal carbonic acid that results from the secretion of protons into the lumen (12, 31). In CA II-deficient patients and mice, inhibition of CA activity (presumably CA IV) diminishes renal acid excretion, indicating a major role for CA IV in urinary acidification (3, 49).
The expression and functional activity of CA IV in different species have been extensively investigated; however, there have been major inconsistencies in the findings. It is well known that there are species differences in CA expression (12). Immunofluorescence studies of rat kidney using an affinity-purified antibody raised against the 39-kDa CA IV from rat lung (9) localized CA IV to apical and basolateral membranes of proximal tubules (S2 >> S1) and thick ascending limbs. No label was detected in intercalated cells or in cells of outer medullary (OMCD) and inner medullary collecting ducts (IMCD). In human kidney, a polyclonal antibody detected CA IV protein in some apical borders of cortical (CCD) and medullary collecting duct cells and weakly in the basolateral regions of proximal convoluted tubules (28). Surprisingly, no staining was found in the brush borders of the same tubules.
We recently studied the postnatal development of CA IV expression in rabbit kidney using an affinity-purified antibody raised against the 46- to 50-kDa CA IV from rabbit lung (46). Although this antibody detected the expression of CA IV in the proximal tubules, it failed to show expression of CA IV in the medullary collecting ducts of mature rabbits. This was inconsistent with the abundant expression of CA IV mRNA in mature OMCD and IMCD (53) and in inner medulla (63).
Membrane-bound CA IV activity has been detected biochemically in the
brush-border and basolateral membranes of proximal tubules (33, 41,
64). Histochemical studies also show CA activity in the apical
membranes of intercalated cells (28, 40). Functional studies have
identified luminal CA activity in rat proximal convoluted tubules (29),
along the inner stripe of rabbit OMCD (OMCDi) (51, 54), and
in the initial segment of rat IMCD (IMCDi) (61). Inhibition
of luminal CA IV eliminates nearly all net
HCO3 reabsorption in proximal tubule
(29) and OMCDi (54).
In view of the inconsistencies localizing renal CA IV (9, 28), it has been impossible to clearly associate CA IV with H+-secreting cells in the kidney. Hence, we prepared a new antibody to rabbit CA IV using two synthetic peptide antigens derived from either end of the deduced amino acid sequence (63). This antibody successfully detected CA IV by both immunoblotting and immunohistochemistry procedures. In the present work we were able to utilize this antibody to localize CA IV in various organs of the rabbit, characterize some basic biochemistry of renal CA IV, and determine the expression pattern of CA IV in the adult rabbit kidney.
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METHODS |
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Animals
New Zealand White rabbits were purchased from Hazleton-Dutchland Farms (Denver, PA). Adult females (1.5-2.5 kg) and pregnant dams were fed standard laboratory chow (Purina Mills, Richmond, IN) and allowed free access to tap water.Each rabbit was anesthetized by using an intraperitoneal injection of pentobarbital (100 mg) after sedation with intramuscular xylazine (5 mg/kg) and ketamine (44 mg/kg). The kidneys were rapidly removed and cut into coronal slices of 1-2 mm thickness. Cortex and inner medulla were separated from the slices. Tissue was coded and snap frozen.
Generation of Polyclonal Antiserum
From opposite ends of the deduced 308-amino acid sequence of rabbit CA IV (63), two peptides were synthesized (Fig. 1). The NH2-terminal amino acids, numbered 73-88 (YDQREARLVENNGHSV), provided a putative extracellular peptide downstream from the signal sequence that is removed from the mature protein (52). The COOH-terminal amino acids, numbered 263-278 (KDNVRPLQRLGDRSVF), provided a peptide that is likely to be upstream from the putative glycosylphosphatidylinositol (GPI) linkage (36, 52). Neither peptide was near one of the two deduced N-glycosylation sites of rabbit CA IV (63). These peptides were synthesized, coupled at their NH2 termini to ovalbumin, and injected into the same goat, using a proprietary immunization protocol (Quality Controlled Biochemicals, Hopkinton, MA). Crude antisera were tested by Western blotting of kidney membrane proteins and by immunohistochemistry (see below) until definitive identification of CA IV was established. The peptides were also used to affinity purify a portion of the antibody for immunohistochemistry and immunofluorescence. Aliquots of the peptides were retained for competition studies. The results described in this manuscript were obtained by using antibody that was affinity purified with the NH2-terminal peptide.
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Preparation of Kidney and Organ Membrane Proteins
Membrane proteins were prepared from 40-200 mg of each dissected zone of frozen kidney tissue by homogenizing on ice for three 30-s bursts using a Tissuemizer (Ultra-Turrax, Janke-Kunkel, Tekmar, Cincinnati, OH) with an S25N 10-G probe at 24,000 rpm in 7 ml Tris-SO4 buffer (25 mM Tris-SO4, 0.9% NaCl), pH 7.5. This buffer also contained protease inhibitors including 1 mM EDTA, 1 mM iodoacetate, 0.1 mg/ml 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride (Pefablock, Boehringer-Mannheim, Indianapolis, IN), 0.1 mg/ml 1,10-phenanthroline, 2 µg/ml pepstatin A, 5 µg/ml chymostatin, 10 µg/ml leupeptin, and 10 µg/ml aprotinin (7). Samples were stored on ice for 30 min, filtered through cheesecloth, and centrifuged at 1,000 g for 10 min at 4°C. The supernatant was centrifuged at 110,000 g for 60 min at 4°C. The pellet was washed twice and then solubilized in Sato's buffer (25 mM triethanolamine, pH 8.1, 59 mM Na2SO4, 1 mM benzamidine chloride) (42) containing 5% SDS, 0.1% saponin, and the same protease inhibitors as used in the homogenization buffer. Solubilization was performed by breaking up the membrane pellet with a pipette tip, agitating at room temperature, and passing the material 10 times through a series of decreasing needle sizes (smallest 21 gauge). The material was centrifuged at 12,000 rpm for 30 min at 15°C, and the supernatant was comprised of solubilized membrane proteins.Protein concentration was measured by using bicinchoninic acid (micro BCA protein assay, Pierce Biotec, Rockford, IL), with BSA as a standard. Twenty to fifty micrograms of membrane protein were generally size fractionated on reducing SDS-PAGE through a 10% separating and 4% stacking polyacrylamide gel.
Other organs were removed, frozen, and handled as described above. Samples of spleen, heart, lung, skeletal muscle, eye, liver, and colon were obtained. The lumen of the colon was perfused with PBS before homogenization.
Immunoblot Analysis
Fractionated proteins were transferred to nitrocellulose membranes by using a transblot electrophoretic transfer cell (Bio-Rad, Hercules, CA). After transfer each membrane was blocked overnight at 4°C in Tris-buffered Tween 20 (TBS-T)-5% milk-5% BSA and probed with a dilution of 1:200 crude goat anti-rabbit CA IV serum or 1:100 affinity-purified goat anti-rabbit CA IV for 2 h at room temperature. Then the filter was probed for an additional 2 h at room temperature with 1:4,000 horse anti-goat antibody conjugated to horseradish peroxidase (Cappel 55391, Organon, Durham, NC) that had been preabsorbed with 1% horse serum, 1% normal rabbit serum, 5% BSA, and 5% milk in TBS-T. After each exposure to antibody, the filter was washed for a total of 50 min in TBS-T. In some experiments the CA IV antibody was preabsorbed with 2 µg of each of the immunizing peptides used to generate the antibody. Signals were visualized by using enhanced chemiluminescence (Amersham, Arlington Heights, IL) and Kodak XAR (Rochester, NY) or Hyperfilm (Amersham). Molecular masses were estimated from the scanned films by using SigmaGel software (Jandel, San Rafael, CA).Analysis of CA Activity in Kidney Homogenates
Membranes from kidney cortex were obtained as described above, except that the kidney was initially perfused with cold PBS until it blanched. After ultracentrifugation of the homogenate, the pellet (membrane fraction) was resolubilized in Sato's buffer with 5% SDS plus 0.1% saponin. The solubilized material was quantified for protein.The end-point assay of CA hydratase activity in imidazole buffer at 4°C was a modification of that of Maren (4, 5, 30). Para-nitrophenol was used as a color pH indicator to determine when the CO2 gas had acidified the solution. One enzyme unit (EU) was defined as the amount of purified enzyme necessary to halve the control or uncatalyzed reaction time; this value was divided by 10 to correct for miniaturization of the assay (4, 6).
Deglycosylation with Peptide-N-Glycosidase F
One-tenth EU of CA IV in membrane homogenate was denatured by boiling for 3 min in 1% mercaptoethanol and placed on ice. The deglycosylation was carried out in buffer (containing 45 mM EDTA, 33% Triton X-100, 45 mM sodium phosphate, pH 7.4, and a protease inhibitor cocktail) plus 20 mU peptide-N-glycosidase F for 5 and 30 min at 37°C. Control incubations substituted water for the peptide-N-glycosidase F. The reaction was stopped by boiling for 3 min. Samples were fractionated on SDS-PAGE and examined for CA IV by immunoblotting.Purification of CA IV
Membranes from rabbit kidney were partially purified for CA IV by affinity chromatography by using the CA inhibitor para-(amino-methyl) benzene sulfonamide (pAMBS) coupled to 4% beaded agarose (Sigma Chemical St. Louis, MO), as previously described (6, 46, 66). Briefly, kidney membranes, obtained as described above, were added to sulfonilamide coupled to agarose beads (PAMBS-agarose, Sigma Chemical) and incubated with rocking for 60 min at 4°C. The beads were washed and CA IV eluted with 0.1 M sodium acetate, pH 5.0, 0.5 M sodium perchlorate, and 0.1% Brij. Fractions were collected and sorted by optical density at 280 nm. They were pooled and dialyzed overnight against 25 mM Tris-SO4, pH 7.5, 1 mM benzamidine chloride, 1 mM dithiothreitol, and 0.1% Brij.Affinity-purified rabbit CA IV yielded 3.6 EU of hydratase activity (6) from 14.4 g of kidney tissue. The enzyme activity was resistant to 0.2% SDS, which is a characteristic feature of the CA IV isoenzyme (6, 66) but was fully inhibited by 1 µM acetazolamide.
Neuraminidase Digestion of Purified CA IV
One-tenth EU of CA IV purified from whole kidney membranes was denatured withImmunohistochemistry
Kidneys were perfusion fixed in periodate-lysine-paraformaldehyde (PLP) or a nonformaldehyde-based fixative (Prefer, Anatech, Battle Creek, MI). Tissue was then cut into 1- to 2-mm slices perpendicular to the long axis. These pieces were allowed to fix in the same fixative at 4°C overnight. After being rinsed three times in 70% ethanol, the sections were embedded in paraffin and 4-µm sections were placed on charged slides (Superfrost +, VWR Scientific, Piscataway, NJ). After deparaffinization and hydration, endogenous peroxidase was quenched with 0.3% H2O2, and cells were permeabilized with 0.3% Triton X-100. Block was accomplished with 10% horse serum. The goat anti-rabbit affinity-purified CA IV antibody was applied at 1:125 dilution in 5% horse serum overnight at 4°C followed by biotin horse anti-goat secondary (Vector, Burlingame, CA), followed by avidin/biotinylated horseradish peroxidase (Vectastain Elite ABC kit), according to the instructions of the manufacturer. The substrate diaminobenzidine tetrahydrochloride was applied for 10 min to develop a brown color. We did not counterstain the sections with hematoxylin because of our concern for resolving faint staining, especially in the outer medulla. For confocal microscopy, sections were labeled with tertiary antibodies coupled to Texas red or fluorescein. Sections were obtained from ~5-10 different animals, and these were examined for each nephron segment.Double labeling was accomplished by using monoclonal antibody (MAb) B63
to the apical surface of -intercalated cells (14) (provided by Dr.
G. Fejes-Toth); MAb IVF12 to the band 3-like Cl
/HCO
3 exchanger
(AE1) on the basolateral membrane of
-intercalated cells (24)
(provided by Dr. M. Jennings); guinea pig polyclonal antibody against
the rabbit renal Na+/Ca2+ antiporter (38),
which labels primarily the basolateral membrane of majority cells of
the connecting tubule (provided by Dr. R. Reilly); antibody to
Tamm-Horsfall protein (Sigma Chemical), which identifies the apical
membranes of thick ascending limb cells. Avidin-biotin blocking was
used between the first and second labeling reactions, as specified by
the manufacturer (Vector). In each double label study the second
biotinylated tertiary antibody was coupled to alkaline phosphatase and
reacted with a red substrate (Vector). Because endogenous alkaline
phosphatase was not inhibited, this reaction also helped identify
proximal tubules by the red staining of the brush borders.
Antigen Retrieval
To maximize the observed signal on paraffin-embedded sections, we used various methods of antigen retrieval, including treatment with 1% SDS (1, 8), 0.1% saponin permeabilization followed by 0.05% saponin in all washes and exposures, and microwave treatment × 5 min in deionized water, in 1% zinc sulfate or in 1 mM EDTA (pH 8.0) (48). In addition, we made use of signal amplification techniques (62) by using avidin-biotin complexed to either horseradish peroxidase or alkaline phosphatase (Vectastain Elite ABC kit and ABC-AP kit; Vector Laboratories; see above). These efforts to improve immunostaining did not affect the pattern of proximal tubular staining observed with our affinity-purified anti-CA IV antibody (see RESULTS).Imaging of Sections
Sections were coverslipped with Refrax mounting medium (Anatech) and examined under an Olympus bright-field microscope; 35-mm slides were photographed with a Nikon F2 camera body attached to a ×2.5 camera port by using Elite 400-ASA film. Images from slides were acquired by using a UMax 1200 color scanner and Adobe Photoshop 4.0 software. Fluorescent sections were examined by using an Axiovert 100 laser scanning confocal microscope (model LSM 410; Carl Zeiss, Jena, Germany) (56). Excitation was accomplished with an argon-krypton laser producing lines at 488 or 568 nm. Images of the two different fluorochromes were collected at 1-µm-thickness optical sections by using Zeiss LSM-PC software. Confocal and bright-field images were subsequently processed by using Adobe Photoshop and Microsoft Powerpoint 97 software. ![]() |
RESULTS |
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Expression of CA IV Protein in Rabbit Kidney and Other Organs
Western blot analysis revealed that CA IV was expressed in rabbit kidney cortex as a single product with an approximate molecular mass of 45 kDa (Fig. 2). The signal was eliminated by preabsorption of either the crude antibody (not shown) or the affinity-purified antibody (see Fig. 2, right lanes) with 2 µg of immunizing peptides, indicating the specificity of the CA IV antibody. Similar competition was observed for kidney inner medullary CA IV (not shown).
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Figure 3 shows the expression of CA IV in a
variety of rabbit organs, which were chosen because of their expression
of CA IV in other species (except for spleen) (9, 10, 15, 16, 18, 21,
59, 60, 66). CA IV appeared most abundant in kidney cortex, kidney
medulla, lung, and heart (Fig. 3A), whereas less but detectable
expression was noted in spleen, skeletal muscle, eye, and colon (Fig.
3B). The approximate molecular mass of CA IV was ~45 kDa in
most of these samples. Surprisingly, the CA IV band from the inner
medulla had a molecular mass ranging from 47 to 60 kDa, appearing more
diffuse than the bands in the cortex and other organs, suggesting
substantially more posttranslational modification in medullary CA IV.
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N-Glycosylation and Sialation of Rabbit CA IV
To determine whether rabbit CA IV contained N-linked oligosaccharide (66), because two sites are predicted from the nucleotide sequence (52, 63), we treated 0.1 EU of CA IV from kidney membranes with 20 mU peptide-N-glycosidase F for 5 and 30 min at 37°C before size fractionating and immunoblotting. The cortex showed a rather definitive signal of 45 kDa, whereas the medulla was more diffuse at 47-60 kDa before digestion. Two deglycosylation products were observed in both cortical and medullary membranes after the treatment with peptide-N-glycosidase F, suggesting the presence of two N-glycosylation sites (Fig. 4). These findings confirm what had been observed by using a different antibody (46) and thereby validate our new antibody.
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The first deglycosylation resulted in a product of ~40 kDa in the cortex and 43 kDa in the inner medulla. The second resulted in a product of 34-35 kDa for both cortex and medulla, similar to the size predicted previously from the nucleotide sequence (52, 63). There appeared to be more glycosylation per site in the inner medulla and hence a larger molecular mass of the fully processed protein (up to ~60 kDa). Compared with the inner medulla and cortex, the outer medulla showed an intermediate amount and size of glycosylated protein (not shown). On the basis of the molecular mass of the mature proteins, the oligosaccharide chains at the two glycosylation sites could have added as much as 11 kDa to the cortical and 25 kDa to the inner medullary CA IV protein.
To determine whether there were sialic acid residues on the mature
protein, we affinity purified 0.1 EU of CA IV by sulfanilamide chromatography, with digestion overnight by using 13.5 mU of
neuraminidase (Fig. 5). Neuraminidase
treatment resulted in a ~1.2-kDa decrease in molecular mass,
indicating that there could be as many as four sialic acid residues
(molecular mass ~300 Da each) posttranslationally linked to rabbit
CA IV.
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Immunohistochemical Localization of CA IV in Rabbit Kidney
The subcellular localization in the kidney was examined in fixed paraffin sections by immunohistochemistry. In general, the most abundant expression of CA IV was detected after fixation with PLP or Prefer. Neutral buffered Formalin and 4% paraformaldehyde markedly reduced the CA IV labeling and were not further examined. Treatment of the PLP- and Prefer-fixed sections with 0.02% saponin followed by microwaving for 1 min in water consistently revealed optimal staining at the light and fluorescent microscopic level.Cortex.
Figure 6A shows a low-power
diaminobenzidine tetrahydrochloride-labeled picture of the kidney
cortex, showing signal over proximal straight tubules in the medullary
rays and more convoluted proximal tubules in the juxtamedullary
nephrons. These segments are probably S2 proximal tubules (25). The
glomeruli were consistently unlabeled. Proximal convoluted tubules
appeared variably stained at higher power, but proximal straight
tubules labeled much more heavily (Fig. 6B). Both apical and
basolateral membranes of the proximal tubules were labeled. The
expression in the cells of the cortical S2 proximal straight tubule
(Fig. 6C) appeared to be heavier at the apical than the
basolateral membrane. By confocal microscopy, a 1-µm section of
proximal straight tubule clearly showed labeling of both apical and
basolateral membranes (Fig. 6D) and again showed heavier label
in the apical membrane.
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Medulla.
There was no staining of the S3 proximal tubule (not shown). There was
modest apical staining of some OMCD cells in the outer stripe and inner
stripe, with slightly heavier apical labeling deep into the inner
stripe (Fig. 7A). In the inner
stripe, all or most of the cells in the OMCD appeared modestly labeled.
Heavy staining was noted in nearly all cells of the initial IMCD (Fig. 7B). Higher power showed that many, but probably not all,
OMCDi cells were labeled on the apical membrane and in an
apical-vesicular or cytosolic pattern (Fig. 7C, arrows).
IMCDi cells were more heavily labeled than
OMCDi cells (Fig. 7D, arrows). Confocal microscopy of 1-µm sections of IMCDi cells revealed predominantly
apical labeling (Fig. 7E, white arrows).
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Double Labeling of CA IV-Positive Cells in Kidney
B63 (-intercalated cells).
Previous studies have shown that monoclonal antibody B63 labels
HCO
3- secreting
-intercalated
cells in 1:1 agreement with apical peanut agglutinin (14). Cells
labeled with B63 (Fig. 8A, red,
arrows) in the CCD and connecting segments did not express the brown CA
IV label. Other cells within these segments expressed CA IV (brown,
arrowheads) but not B63 (red). By confocal microscopy, we confirmed
that
-intercalated cells (Fig. 8B, green, white arrow) did
not express CA IV and the minority cells that expressed CA IV (red,
white arrowhead) did not stain with B63.
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IVF 12 (-intercalated cells).
In addition to labeling AE1 (band 3) of red blood cells, IVF 12 identifies the kidney AE1 in
-intercalated cells (24, 45). In the
cortex (Fig. 8D), CCD contained band 3 positive cells (red basolateral label), which also labeled on their apical membranes for CA
IV (brown, arrows). Nearly every band 3 positive intercalated cell was
positive for CA IV in CCD, as well as in connecting segments (not
shown). This was confirmed by confocal microscopy, which showed
occasional cells in the CCD expressing apical CA IV (Fig. 8E,
green) and basolateral AE1 (red).
Anti-Na+/Ca2+
exchanger (connecting tubule cells).
This antibody labels the basolateral membranes of connecting tubule
cells in rabbit kidney cortex (38). These connecting tubule cells were
negative for CA IV (Fig. 8G). A minority cell type was negative
for the Na+/Ca2+ exchanger but expressed apical
CA IV (arrows). These were probably -intercalated cells, which are
known to populate the connecting segment (57). A much less intense
staining pattern for the Na+/Ca2+ exchanger was
also observed in some CCD cells, presumably principal cells, which were
also negative for CA IV (not shown).
Thick ascending limb cells. Thick ascending limb cells expressing Tamm-Horsfall protein did not express CA IV (not shown).
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DISCUSSION |
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CA IV is a membrane-bound enzyme that facilitates the
dehydration of carbonic acid and is therefore believed to play a key role in renal acidification. CA IV comprises ~5% of renal CA, with
the remaining 95% being cytosolic CA II (6, 33, 64, 65). During the
process of H+ secretion into the luminal fluid, CA IV
catalyzes the dehydration of carbonic acid generated from the titration
of filtered HCO3
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To investigate the cellular localization of CA IV in the rabbit kidney, we have used peptides from each end of the predicted amino acid sequence of the mature protein (63) to generate a goat anti-rabbit CA IV antibody. We confirmed the prediction from the nucleotide sequence that there were two N-glycosylation sites, using timed peptide-N-glycosidase digestions. In addition, neuraminidase treatment showed that the partially purified rabbit CA IV had several, perhaps four, sialic acid residues covalently attached. These studies indicate that mature rabbit CA IV contains complex oligosaccharides, probably added to the protein during processing in the Golgi apparatus. Because sialic acid bears a negative charge, it is likely that membrane-bound CA IV is negatively charged in the tubule lumens of rabbit kidney. These novel findings are in contrast to the human form of CA IV that contains no N-linked oligosaccharide chains or sialic acid residues (66) and to the rat form that has only one N-linked oligosaccharide chain (59).
In rabbit, CA IV was rather ubiquitous, appearing in lung, heart, skeletal muscle, colon, spleen, and eye, as well as kidney. Our experiments demonstrate the expression of CA IV with the molecular mass of ~45 kDa in all of these organs, except in the renal inner medulla, where the molecular mass was larger but less precise. We have also detected this heavier molecular mass form of CA IV in rabbit renal inner medulla by using a different antibody to CA IV (46). Because Northern analyses showed no difference in size of mRNAs coding for CA IV between cortex and inner medulla (63), we conclude that this heavier form results from a more extensive posttranslational modification of CA IV. Further investigations of the functional significance of this different glycosylation pattern are needed.
We consistently observed the expression of CA IV in regions of the tubule that show functional evidence for this enzyme. From CA inhibitor studies there is evidence for functional luminal CA in proximal tubule (26, 29), OMCDi (51, 54), and IMCDi (61). In the presence of CA inhibitor, each of these segments is known to secrete protons and generate an acid disequilibrium pH. To date, only our CA IV antibody has localized CA IV in each of these nephron segments.
There is also functional electrophysiological evidence for basolateral
CA in the proximal tubule, which indicates a role for this enzyme in
passive rheogenic HCO3 transfer across the peritubular membrane (2, 11). In addition,
HCO
3 transport across proximal tubule
basolateral membrane vesicles is inhibited by acetazolamide (20, 50).
These findings of membrane-bound CA activity are consistent with our
immunocytochemical finding of basolateral CA IV in proximal tubules.
The basolateral membrane is also the site where the
Na+-3HCO
3 cotransporter
moves HCO
3 at high rates. This
cotransporter probably moves one
CO2
3 plus one
HCO
3 along with one Na+
ion (34). Because the CO2
3
equilibrium concentration in physiological solutions is only 20-80
µM, >99% of the CO2
3 added
must be converted to HCO
3, according
to
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A previous study in rat kidneys, using an antibody generated from
purified rat CA IV (9), also showed proximal tubule labeling of CA IV
in both apical as well as basolateral membranes, with the heaviest
staining in the S2 segment, less in S1, and none in S3. Our findings in
rabbit kidney are comparable (Table 1), with the dual-membrane localization being confirmed by confocal microscopy. An explanation for more abundant expression in S2 compared
with S1 segments may be that a larger pH gradient is generated in the
S2 segment, thereby requiring more membrane-bound CA IV for catalyzing
the resulting carbonic acid. Although S3 proximal tubule segments can
generate a luminal disequilibrium pH through H+ secretion,
they do not appear to absorb much net
HCO3 or express functional luminal CA
IV (26), and this negative finding was confirmed by our
immunohistochemical studies.
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In human kidney a polyclonal antibody to CA IV (28) did not detect brush-border staining of proximal tubules and showed weak staining of the basolateral membranes. Other positive label was detected only in some collecting duct cells.
In the rat there was also staining of the thick ascending limb, a
segment which, in this species, absorbs
HCO3 (19). We found no labeling in the
rabbit thick ascending limb, consistent with previous histochemical
activity studies (13) and in keeping with this segment's not absorbing
HCO
3 in the rabbit (22).
No CA IV labeling was detected in cortical intercalated cells or in
medullary collecting duct cells of the rat kidney (9). We consistently
found modest but reproducible labeling of intercalated cells in the
OMCDo and in OMCDi cells. There was heavy
labeling of IMCDi cells and a subpopulation of cells in the
CCD. Confocal microscopy confirmed the apical and apical-vesicular
labeling of IMCD and intercalated cells. Double-labeling studies
indicated the latter to be -intercalated cells because of their
basolateral band 3 staining. Thus in the rabbit, each of these
proton-secreting collecting duct cells expressed CA IV protein on the
apical membrane.
These observations of CA IV protein in rabbit kidney agree well with
our previous study of CA IV mRNA in microdissected nephron segments
(53). CA IV mRNA (see Table 1) was detected in S1 and S2 proximal
tubules, and in three medullary collecting duct segments
(OMCDo, OMCDi, IMCDi). Consistent
with our observations of several immunohistochemical preparations, the
most intense mRNA was observed in IMCDi. Neither CA IV mRNA
nor protein was detected in glomeruli, S3 segments, thick ascending
limb segments, connecting segments, or distal tubules. It should be
noted that no CA IV RT-PCR signal had originally been detected in CCD
(53); however, in the present study CA IV protein was observed over -intercalated cells. This discrepancy might be due to the limited representation of
-intercalated cells in microdissected CCD.
-Intercalated cells only constitute ~20% of CCD intercalated cells (45, 47) or ~5% of total CCD cells, so that it could have been
difficult to reliably detect the CA IV signal by RT-PCR BY using
1-2 mm of tubule (25-50
-intercalated cells) as a template (53). Indeed, more recent examinations using more efficient RT-PCR
methodology have shown detectable CA IV mRNA in this segment (G. J. Schwartz, unpublished observations).
Our results support the findings of Ridderstrale et al. (39) showing CA activity in the membranes of cells in the rabbit CCD and medullary collecting duct. However, this study used a histochemical technique that does not distinguish between CA II and CA IV activities.
CA IV is believed to be a GPI-anchored protein (36) and, accordingly, would be expected to be solely expressed apically (27, 37). In addition, N-glycosylation is an apical sorting signal (43). Evidence for basolateral GPI-anchored proteins has only been obtained in a Fischer rat thyroid epithelial cell line (67), and the mechanism for the "mutant" behavior of this cell line has not been delineated. To our knowledge, there is no other organ or tissue that expresses a GPI-anchored protein on the basolateral membrane. Thus the finding of apical and basolateral CA IV in proximal tubules suggests a novel possibility that the basolateral form of CA IV could be GPI anchored. Alternatively, a non-GPI-linked form could be expressed basolaterally. Further studies are needed to distinguish between these alternatives.
In summary, we have generated an anti-CA IV antibody that works
effectively for both immunohistochemistry and immunoblotting. Biochemically, rabbit CA IV is heavily N-glycosylated, more so in the medulla than in the cortex, and this posttranslational modification adds 11-25 kDa to the molecular mass of the mature protein. It contains several sialic acid residues, which are probably added during processing in the Golgi apparatus and likely to cause CA
IV to be negatively charged in the tubule lumen. CA IV was found to be
more ubiquitous than previously realized, being expressed in several
organs including lung, heart, spleen, skeletal muscle, eye, and colon.
In the kidney CA IV was localized to the predominant H+-secreting epithelial cells, which include the S1
(convoluted) and S2 (convoluted and cortical straight) proximal tubules
(23), -intercalated cells of the CCD and connecting segment, and
medullary collecting duct cells (17, 23, 44). The association between the presence of luminal CA IV and rates of net
HCO
3 absorption indicates a critical
role for CA IV in renal acidification along the proximal tubule and
collecting duct. Interestingly, CA IV was also detected along the
basolateral membrane of proximal tubules, where high rates of
HCO
3 flux move via the
Na+-3HCO
3 cotransporter.
Inhibition of basolateral CA IV could lead to an alkaline
disequilibrium pH due to the accumulation of
CO2
3. These are the first studies
to localize CA IV in each of the net H+-secreting sites of
the rabbit kidney, thereby confirming previous functional studies and
the importance of CA IV in renal acidification. These studies are also
novel in showing for the first time the abundance and apical polarity
of CA IV in the medullary collecting duct.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Q. Al-Awqati for helping us to examine kidney sections by confocal microscopy. We are grateful to Drs. G. Fejes-Toth, M. Jennings, and R. Reilly for providing antibodies to counterlabel kidney sections.
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FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50603 (G. J. Schwartz).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. J. Schwartz, Div. of Pediatric Nephrology, Box 777, Univ. of Rochester, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: George_Schwartz{at}URMC.rochester.edu).
Received 10 August 1999; accepted in final form 16 December 1999.
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