Carbonic anhydrase IV is expressed in H+-secreting cells of rabbit kidney

George J. Schwartz1,2, Ann M. Kittelberger1, Darlene A. Barnhart1, and Soundarapandian Vijayakumar3

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -intercalated cells. However, CA IV was not expressed by beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 HCO-3 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Deduced amino acid sequence of rabbit carbonic anhydrase (CA) IV. Peptide sequences chosen for generation of antibody are in bold and underlined. NH2-terminal sequence is downstream from first 18 amino acids, which serve as a probable signal sequence that is cleaved during maturation of enzyme (35). COOH-terminal sequence is upstream from serine (amino acid 280), which is believed to serve as an anchor for glycosylphosphatidylinositol (GPI) linkage to membrane (36).

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 with beta -mercaptoethanol by boiling for 3 min and then incubated overnight at room temperature with 13.5 mU of Vibrio cholerae neuraminidase (Boehringer-Mannheim) in buffer containing 50 mM sodium acetate, pH 5.09, 4 mM CaCl2, 0.1 mg/ml BSA, and 0.25 mg/ml Pefabloc. The sample was boiled for 3 min in 2× Laemmli loading buffer before immunoblot analysis.

Immunohistochemistry

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 beta -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 alpha -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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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 2.   Immunoblot analysis of CA IV in 20 µg of membranes from rabbit kidney cortex. Two left lanes: single product of CA IV (~45 kDa) in 2 different preparations (C1, C2) of kidney cortex probed with affinity-purified CA IV antibody. Two right lanes: this signal was successfully eliminated by competition with 2 µg of each of immunizing peptides.

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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Fig. 3.   A: immunoblot analysis of CA IV in 20 µg of membranes from organs of a rabbit. Expression of a ~45-kDa product was detectable in spleen (Spl), heart (Ht), lung (Lg), skeletal muscle (Sk), colon (Col), renal inner medulla (IM), and renal cortex (Ctx); most abundant expression was seen in lung and kidney. No signal was observed in eye or liver. Inner medullary CA IV was substantially larger, more diffuse and probably more posttranslationally modified than CA IV from other organs and zones of kidney. B: CA IV immunoblot analysis of 50-µg samples from skeletal muscle, eye, liver, and colon, showing strong signal at ~45 kDa in colon and skeletal muscle, with less seen in eye and no definite signal in liver.

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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Fig. 4.   Endoglycosidase digestion (20 mU of peptide-N-glycosidase F) of 0.1 CA EU (~50 µg) of membrane proteins from cortex and medulla reacted at 37°C for 0 (control), 5, and 30 min. Control inner medulla CA IV product appeared to be more diffuse and of larger molecular mass than that of cortex. Complete digestion product was 34-35 kDa from both cortex and medulla, and there was an intermediate-size product observed after 5 min of digestion. Glycosylsated medullary products appeared larger than those from cortex.

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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Fig. 5.   Neuraminidase (Neura) digestion of purified rabbit kidney CA IV. Digestion of 0.1 EU of partially purified kidney CA IV overnight at room temperature with 13.5 mU of neuraminidase reduced mass of CA IV by ~1.2 kDa. Purified kidney CA IV resembled that from cortex, rather than that from medulla, due to huge abundance of cortex in a whole kidney preparation.

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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Fig. 6.   CA IV immunohistochemistry of kidney cortex. A: low-power (×12.5) view shows brown staining [diaminobenzidine tetrahydrochloride (DAB) reaction] of proximal straight tubules (PST) in medullary rays and of convoluted proximal tubules in juxtamedullary nephrons, presumably S2 segments (S2). No expression was detected in glomeruli (G). B: higher power (×50) shows variable staining of proximal tubules [compare proximal convoluted tubules (PCT)], with PST appearing heaviest labeled at both apical and basolateral membranes; glomeruli failed to label. C: higher power view (×250) shows apical (Ap) and basolateral (BL) labeling of PST, with apical labeling being heavier. D: confocal fluorescent microscopic view (×250) of PST cross sections showing CA IV labeling (white arrows) at both apical and basolateral membranes; again, CA IV appeared heavier in apical region. E: histochemical visualization (×50) of medullary ray showing labeling of PST segments and apical labeling of 2 intercalated cells (arrows) in a collecting duct; another on opposite wall is not pointed out. F: confocal fluorescent microscopic view (×250) of connecting segment showing apical and apical-vesicular labeling of CA IV in presumed intercalated cells (white arrows).

In some medullary rays (Fig. 6E), it was evident that a few cells in an otherwise negatively stained CCD were labeled at the apical membrane; these were probably intercalated cells (arrows). Similar apical and apical-vesicular labeling of probable intercalated cells was also observed in connecting segments, as seen on the 1-µm confocal microscopy section (Fig. 6F, arrows). Double-labeling studies were used to positively identify the localization of CA IV in intercalated cells and are discussed below.

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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Fig. 7.   CA IV immunohistochemistry of kidney medulla. A: low-power view (×12.5) of inner stripe of outer medulla and into inner medulla shows modest staining of outer medullary collecting ducts (OMCD), with heavier staining noted in initial inner medullary collecting ducts (IMCD). B: low-power view (×12.5) of inner medulla shows intense staining of IMCD cells. C: high-power view (×250) shows apical and vesicular labeling of some cells (arrows) of OMCD in inner stripe. D: high-power view (×250) of initial IMCD shows intense apical and apical-vesicular labeling of nearly every cell (arrows). E: confocal fluorescent microscopic view (×250) of IMCDs confirms apical labeling of nearly all cells (white arrows).

Double Labeling of CA IV-Positive Cells in Kidney

B63 (beta -intercalated cells). Previous studies have shown that monoclonal antibody B63 labels HCO-3- secreting beta -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 beta -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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Fig. 8.   Double labeling of CA IV-positive cells. A: histochemistry (×100) using red label (alkaline phosphatase, arrows) to identify beta -intercalated cells with antibody B63 and brown label (diaminobenzidine, arrowheads) to identify CA IV-positive cells. In this CCD there is no double labeling of intercalated cells. B: confocal fluorescent microscopy (×250) confirms that beta -intercalated cells (labeled green, white arrow) did not label with CA IV (red, white arrowhead). Note red apical and basolateral CA IV labeling of adjacent proximal straight tubule. C: histochemistry (×50) using alkaline phosphatase substrate to label lumens of S3 proximal tubules red (S3) and diaminobenzidine to label OMCD intercalated cells near end of outer stripe (arrows). D: cortical collecting duct cells (arrows) labeled apically for CA IV (brown cap) and basolaterally for band 3 (AE1, red), indicating that these are alpha -intercalated cells. Adjacent proximal straight tubules are brown-labeled apically and basolaterally for CA IV. E: confocal fluorescent microscopy (×250) confirms double labeling of alpha -intercalated cells (white arrows) with apical CA IV (green) and basolateral band 3 (red). F: histochemistry (×100) in outer medulla near end of outer stripe showing OMCD cells (arrows) with apical CA IV (light brown) and basolateral band 3 (red). Note apical red staining of S3 proximal tubules (S3) that contain apical alkaline phosphatase. G: histochemistry (×100) of cortical connecting segment showing connecting tubule cells with basolateral labeling of Na+/Ca2+ antiporter (red) and intercalated cells with apical CA IV labeling (brown, arrows). Connecting tubule cells did not express CA IV.

We utilized the residual alkaline phosphatase of the S3 proximal tubules as a marker to distinguish the outer from inner stripes of the outer medulla (55, 58). This low-power view (Fig. 8C) showed the alkaline phosphatase-positive S3 proximal tubules (which were negative for CA IV) and the weakly staining CA IV-expressing cells of the OMCD of the outer stripe (OMCDo; arrow).

IVF 12 (alpha -intercalated cells). In addition to labeling AE1 (band 3) of red blood cells, IVF 12 identifies the kidney AE1 in alpha -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).

Similar double staining was observed in OMCD cells from the inner and outer stripes (Fig. 8F, arrows). Band 3 was usually basolateral in the OMCD (32, 45), whereas CA IV label was observed primarily in an apical and a vesicular pattern but was less intense than in cortical alpha -intercalated cells.

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 alpha -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).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 HCO-3
H<SUP>+</SUP> + HCO<SUP>−</SUP><SUB>3</SUB> ⇄ H<SUB>2</SUB>CO<SUB>3</SUB> <AR><R><C>CA IV</C></R><R><C> ⇄</C></R></AR> H<SUB>2</SUB>O + CO<SUB>2</SUB>
Thus one would expect that CA IV would be expressed by renal epithelial cells that secrete protons, namely proximal tubule and collecting duct cells in the rabbit. Evidence supporting the critical role of CA IV in urinary acidification comes from the experiment of nature observed in two CA II-deficient patients who were given the CA inhibitor acetazolamide (49). The patients showed a prompt rise in urinary pH and HCO-3 excretion, comparable to the increment observed in two control subjects. In mice that are deficient in CA II through a N-ethyl-N-nitrosourea-induced null mutation, baseline urinary pH was higher than in control or healthy littermates. More importantly, the CA inhibitor metazolamide increased urinary HCO-3 excretion and inhibited titratable acid excretion comparably in each of these groups (3). Thus when CA II is absent, inhibition of residual CA (presumably CA IV) markedly reduces urinary acidification.

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 HCO-3 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
CO<SUP>2−</SUP><SUB>3</SUB> + H<SUP>+</SUP> ⇄ HCO<SUP>−</SUP><SUB>3</SUB>
However, because the H+ concentration is ~0.04 µM, the local pH will immediately rise and the reaction will reach equilibrium. Further consumption of CO2-3 can only continue when additional H+ ions are generated from H2O and CO2, presumably via CA IV catalysis. Perhaps the CA IV localized at this site serves to facilitate the generation of additional H+ ions, which prevent local CO2-3 gradients from building up in the intercellular space. Interestingly, in beta -intercalated cells, which secrete HCO-3 at high rates via an apical Cl-/HCO-3 exchanger, there is no immunocytochemical evidence for CA IV on either membrane (see Fig. 8A).

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 HCO-3 or express functional luminal CA IV (26), and this negative finding was confirmed by our immunohistochemical studies.

                              
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Table 1.   CA IV expression in nephron segments

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 HCO-3 (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 alpha -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 alpha -intercalated cells. This discrepancy might be due to the limited representation of alpha -intercalated cells in microdissected CCD. alpha -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 alpha -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), alpha -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.


    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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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