Carbonic anhydrase XII mRNA encodes a hydratase that is differentially expressed along the rabbit nephron

George J. Schwartz, Anne M. Kittelberger, Richard H. Watkins, and Michael A. O'Reilly

Department of Pediatrics, University of Rochester School of Medicine, Rochester, New York 14642


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Membrane-bound carbonic anhydrase (CA) facilitates acidification in the kidney. Although most hydratase activity is considered due to CA IV, some in the basolateral membranes could be attributed to CA XII. Indeed, CA IV is glycosylphosphatidylinositol anchored, connoting apical polarization, but CA IV immunoreactivity has been detected on basolateral membranes of proximal tubules. Herein, we determined whether CA XII mRNA was expressed in acidifying segments of the rabbit nephron. The open reading frame of CA XII was sequenced from a rabbit kidney cortex cDNA library; it was 83% identical to human CA XII and coded for a 355-amino acid single-pass transmembrane protein. Northern blot analysis revealed an abundant 4.5-kb message in kidney cortex, medulla, and colon. By in situ hybridization, CA XII mRNA was expressed by proximal convoluted and straight tubules, cortical and medullary collecting ducts, and papillary epithelium. By RT-PCR, CA XII mRNA was abundantly expressed in cortical and medullary collecting ducts and thick ascending limb of Henle's loop; it was also expressed in proximal convoluted and straight tubules but not in glomeruli or S3 segments. FLAG-CA XII of ~40 kDa expressed in Escherichia coli showed hydratase activity that was inhibited by 0.1 mM acetazolamide. Unlike CA IV, expressed CA XII activity was inhibited by 1% SDS, suggesting insufficient disulfide linkages to stabilize the molecule. Western blotting of expressed CA XII with two anti-rabbit CA IV peptide antibodies showed no cross-reactivity. Our findings indicate that CA XII may contribute to the membrane CA activity of proximal tubules and collecting ducts.

in situ hybridization; reverse transcriptase-polymerase chain reaction; expressed protein; proximal tubule; medullary collecting duct


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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CARBONIC ANHYDRASE (CA) CATALYZES the reversible hydration of CO2 and dehydration of carbonic acid. These functions would make this enzyme important in renal acid-base transport. Of the 14 isoenzymes of the CA family identified thus far, two of the major renal enzymes are cytosolic CA II, which accounts for 95% of activity, and CA IV, which accounts for a substantial portion of the membrane-associated CA activity (6, 21, 37, 40). CA IV is a glycosylphosphatidylinositol (GPI)-linked protein (36, 41), indicating that it is likely to be expressed primarily on the apical membrane (8, 26). However, we have recently shown functional CA activity on the basolateral membranes of proximal straight tubules (34), and the identity of this basolateral CA activity is unclear.

When the kidneys of CA II-deficient mice were examined for histochemical activity (27), intense staining associated with the apical and basolateral plasma membranes was detected in proximal convoluted tubules. The absence of cytosolic CA II allowed the clear detection of CA hydratase activity associated with the plasma membranes. In addition, immunocytochemical studies that used three different antibodies to CA IV (9, 29, 31) consistently revealed both apical and basolateral labeling. The appearance of a basolateral signal suggests that a non-GPI-linked isoform of CA IV or an isoform of CA that cross-reacts with the antibodies to CA IV is present.

Recently, CA XII was identified, cloned, and characterized in renal cell carcinoma and normal human kidney (35). The sequence predicted a single-pass transmembrane protein with an extracellular CA domain. Expression was also noted in normal human colon (14, 35) and in colorectal tumors (14). Recently, Parkkila et al. (24), by using an antibody against a secreted form of human CA XII (14), showed staining in the basolateral membranes of cells of the thick ascending limb and distal tubules and in principal cells of the collecting ducts of human kidneys. A weak basolateral signal was also noted in proximal convoluted tubules.

The finding of an additional membrane CA was particularly relevant to our recent identification of functional basolateral CA activity in the rabbit renal proximal straight tubule (34). Because much acid-base physiology has been derived from studies in isolated rabbit nephron segments and CA is a key enzyme in acid-base physiology, we cloned rabbit CA XII, localized its mRNA in the kidney and other organs, as well as in some specific regions of the kidney, and characterized the expressed protein. Our findings are consistent with the hypothesis that CA XII is at least partially responsible for some membrane hydratase activity in the proximal tubule and collecting ducts.


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Animals and preparation of tissue. New Zealand White rabbits (1.5-2.5 kg) were purchased from Hazleton-Dutchland Farms (Denver, PA) and fed standard laboratory chow with free access to tap water. Each rabbit was anesthetized with an intracardiac injection of pentobarbital sodium (100 mg/kg) after premedication with intramuscular xylazine (5 mg/kg) and ketamine (44 mg/kg). The kidneys were rapidly removed and cut into coronal slices of 1- to 2-mm thickness. Tissue was dissected from cortex, outer medulla, and inner medulla of fresh kidneys with a razor blade and scraped from the colonic mucosa; these samples were snap frozen in liquid nitrogen, as described previously (29). Small pieces of tissue from lung, spleen, and heart were also cut and snap frozen. For in situ hybridization, a kidney was perfused via the renal artery with PBS until it blanched, followed by fixation with neutral buffered formalin; 2% paraformaldehyde, 75 mM lysine, and 10 mM sodium periodate; or Prefer (Anatech, Battle Creek, MI).

Preparation of total RNA. Frozen tissue was homogenized in five to six short bursts with a Tissuemizer (Ultra-Turrax, Janke-Kunkel, Tekmar, Cincinnati, OH) by using a S25N probe at 24,000 rpm in an acid guanidinium thiocyanate-phenol chloroform solution (TriReagent, Molecular Research Center, Cincinnati, OH), and total RNA was obtained according to the protocol provided by the manufacturer. The RNA was resuspended in diethylpyrocarbonate-treated water and quantified spectrophotometrically by the absorbance at 260 nm. Purity was determined from the ratio of absorbances at 260 and 280 nm and by ethidium bromide staining after size fractionation on 1.5% agarose minigels (38).

RT-PCR for detecting CA XII in rabbit kidney cortex. From the 3'-end of human CA XII (35), we prepared a degenerate antisense primer that encoded the last seven amino acids: 5'-GTC GAC TCA NGC RTG NGC YTC NGT YTC-3'. We used an upstream sense primer encoding five amino acids that are conserved among the membrane CAs (rabbit CA IV, human CA IX, human CA XII, and mouse CA XIV) (22, 25, 35, 38): 5'-AAG CTT CAR YTN CAY YTN CAY TGG-3'. Restriction sites (XhoI and HindIII) were added to the primers to facilitate cloning in other vectors. The predicted size of the PCR product was ~750 bp. The rabbit CA XII probe was obtained by amplifying 250 ng of reverse-transcribed rabbit kidney cortex RNA at 42°C for 5 cycles followed by subsequent amplification at 52°C for 40 cycles. The band was gel purified with low-melting-point agarose for use as a probe and for sequencing after ligation into pCR 2.1 cloning vector (TA cloning kit, Invitrogen, San Diego, CA) (38).

Screening of a rabbit kidney cDNA library for CA XII gene expression and sequencing of positive controls. The 750-bp RT-PCR fragment of CA XII obtained from rabbit kidney cortex total RNA was used to screen a quarter of a rabbit kidney cortex cDNA library constructed in Lambda ZAP II vector (Stratagene, La Jolla, CA) and grown on XL1-Blue recA- Escherichia coli host strain. Titration of this library revealed 3 × 109 plaque-forming units/ml. Duralon-UV membranes (Stratagene) were oriented on 150-mm NZY plates and used to lift DNA from the plaques. Prehybridization and overnight hybridization were carried out in 0.5 M sodium phosphate-1 mM EDTA-7% SDS buffer (pH 7.2) at 55°C according to the manufacturer's instructions (Bio-Rad). The double-strand RT-PCR product was labeled with [32P]dCTP by random hexanucleotide extension (Roche Diagnostics, Indianapolis, IN), and unincorporated radioactivity was removed by separation over a G-50 column. Washes were performed at low stringency [first in 40 mM sodium-1 mM EDTA-5% SDS (pH 7.2) and then as above but in 1% SDS] at 55°C. Four rounds of screening yielded two positive clones, from which the inserts and the Bluescript phagemid (Stratagene) were excised in vivo from Lambda ZAP. The estimated sizes of the two clones were 1.9 and 1.4 kb, and these were sequenced by PCR (Prism DyeDeoxy Terminator, Perkin Elmer, Foster City, CA). Because the smaller clone appeared identical to the larger one but did not yield a full-length open reading frame, only the larger clone was sequenced with oligonucleotide primers designed every 400 bp along both strands of the sequence. The nucleotide sequence was assembled and aligned to the human sequence and RT-PCR fragment with MacVector (version 6.5, Accelrys, Princeton, NJ).

Northern blot analysis of total RNA from kidney and other organs. Northern blot analysis was performed as previously described (38). Briefly, 5-20 µg of total RNA were denatured in 3% formaldehyde, size fractionated on 1.5% agarose gels, transferred to nylon filters (Zeta-Probe, Bio-Rad, Hercules, CA), and hybridized with DNA probes labeled with 32P[dCTP] by random hexanucleotide extension (Roche Diagnostics). Each Northern blot was run once with one set of tissues or organs. The DNA probes were a 750-bp fragment of rabbit CA XII (see below), a 600-bp fragment of rabbit CA IV (38), and rat beta -actin (housekeeping gene) (23), which were labeled to a specific activity of 1.5 × 109 counts · min-1 · µg-1. Autoradiography was performed with Kodak XAR film at -80°C for 1-4 days.

In situ hybridization. After overnight fixation, the tissue was dehydrated in a series of graded alcohols, placed in xylene, and then embedded in paraffin following a standard protocol (1). Sections were cut to 5- to 8-µm thickness, placed on positively charged slides (Superfrost+, VWR Scientific, Piscataway, NJ), and stored at -80°C.

Plasmid DNA containing 1.9 kb of rabbit CA XII cDNA was linearized with restriction enzymes XhoI or XbaI to make antisense and sense templates, respectively, for cRNA probes according to the protocol of Auten et al. (2). The identity of these probes was confirmed by sequence analysis. The probes were labeled with alpha -[33P]UTP to a specific activity of ~1.5 × 109 counts · min-1 · µg-1. Alkaline hydrolysis reduced the probe length to ~200 bp (39).

Before hybridization, slides were deparaffinized by immersion in xylene and then dehydrated through a graded ethanol series followed by proteinase K (GIBCO-BRL) digestion for 30 min at 37°C. Slides were then equilibrated with 100 mM triethanolamine-HCl (pH 8) and treated with 0.25% acetic anhydride. The slides were then washed in 2× SSC, dehydrated, and dried. Prehybridization was at 53°C for 3 h; hybridization was performed for 16 h at 53°C by using a hybridization solution containing 30 ng · kb-1 · ml-1 of probe. The hybridization solution was 50% formamide, 300 mM NaCl, 10 mM Tris · HCl (pH 8), 1 mM EDTA, 1× Denhardt's reagent, 10% dextran sulfate, and 0.5 mg/ml yeast tRNA.

After hybridization, slides were rinsed and digested with RNase A. The stringent rinse was in 0.1× SSC at 69°C followed by a rinse in 0.1× SSC at room temperature. Then, the slides were dehydrated by passing through graded ethanol washes, dipped in 1:1 dilution of NTB-2 emulsion (Eastman Kodak, Rochester, NY) and exposed at 4°C for 17 days, developed as described (38, 39), and counterstained with hematoxylin and eosin. Sections from at least three different animals were examined.

RT-PCR detection of CA XII expression in isolated nephron segments. Tubule segments were isolated from kidney slices under a dissecting microscope in chilled PBS (containing calcium and magnesium) plus 10 mM vanadyl ribonucleoside complex (5 Prime 3 Prime, West Chester, PA) to inhibit RNA degradation, as previously described (33). Some kidney slices were treated at 37°C with 0.1% collagenase (type I, Sigma) for 20-35 min. From the cortex were dissected glomeruli (usually 4-5 glomeruli/tube), proximal convoluted tubules, proximal straight tubules, thick ascending limbs, and cortical collecting ducts. From the outer medulla were obtained S3 proximal tubules and outer medullary collecting ducts primarily from the inner stripe. Inner medullary collecting ducts (IMCDs) were dissected mainly from the initial mid-inner medulla. Segments were measured for length, rinsed twice in PBS, transferred in 2 µl to a 0.5-ml microcentrifuge tube containing 1.5 µl of 7% Triton X-100 plus 8 U ribonuclease inhibitor (RNAsin, Promega, Madison, WI), sonicated 5 min at room temperature, and frozen at -80°C. Most segments were 1-2 mm in length. Dissection of proximal tubules was generally completed within 1 h of the animal's death; distal segments could be successfully obtained for another 30-60 min.

For each run of five segments, we used a positive control of 1 ng total RNA from rabbit kidney cortex and a negative control of 0 ng RNA. Reverse transcription was performed for each sample for 1 h at 42°C in 20 µl final volume by using 250 ng of random hexamers, 1 µl from each of four 10 mM deoxynucleotide stocks, RT buffer, and 200 U Superscript II (Invitrogen) RNase H- RT according to the manufacturer's instructions. PCR was performed in a 50-µl reaction by using 2.5 U Platinum Taq DNA polymerase (Invitrogen). For CA XII, 8 µl of first-strand cDNA was amplified for 40 cycles by using 60 pmol of sense and antisense primers in 2 mM MgCl2 with an annealing temperature of 55°C. For CA II, an identifying gene (33) of proximal tubules and collecting ducts, 8 µl of first-strand cDNA was amplified for 40 cycles by using 60 pmol of sense and antisense primers in 1.5 mM MgCl2 with an annealing temperature of 52°C. For L32, a ribosomal protein used as a general housekeeping gene (12), 1 µl of first-strand cDNA was amplified for 35 cycles by using 50 pmol of sense and antisense primers in 1 mM MgCl2 with an annealing temperature of 55°C. Each PCR was completed with a final 7-min extension at 72°C and storage at 4°C.

The primers for CA XII were 5'-ACA TGT ACC TCC AGG GCC-3' (sense, bases 308-325) and 5'-TCA GGC ATG GGC CTC GGT CTC-3' (antisense, bases 1045-1066), and the expected size of the PCR product was 758 bp. The primers for CA II (33) were 5'-ATG TCC CAT CAC TGG GGG TAC-3' (sense, bases 1-21) and 5'-TGG CTC CTC AGG TTC CGC CTC CTT-3' (antisense, bases 718-738), and the expected size of the PCR product was 738 bp. The primers for L32 (12) were 5'-AAG AAG TTC ATC AGG CAC CAG T-3' (sense, bases 691-712) and 5'-GCA GCA TGT GCT TGG TTT TCT T-3' (antisense, bases 829-850), and the expected size of the PCR product was 159 bp.

Twenty microliters of each sample were size fractionated on 1.2% agarose minigels (1 for each gene), and the products were visualized by ultraviolet fluorescence after ethidium bromide staining. Each gel was photographed with identical settings. The products were scored as present, absent, or faintly positive. If L32 was faintly positive or negative, the tubule was discarded. Proximal tubule segments were considered satisfactory only if they expressed CA II mRNA (33). Segments that were faintly positive for CA XII were counted as positive. Segments (10-25) were microdissected from eight rabbits. Collagenase had no effect on the expression of CA XII (data not shown), and collagenase-treated segments were pooled with those obtained without collagenase, as previously reported (33).

Transient expression of rabbit CA XII in E. coli and in COS cells. The full-length open reading frame of rabbit CA XII (1,068 bp) was amplified by PCR from the large cDNA clone with restriction sites XhoI and HindIII engineered at each end. The PCR product was directionally subcloned in frame into an NH2-terminal pFLAG-cytomegalovirus-1 expression vector (Sigma) via HindIII/SalI digestions. After transformation into DH5alpha cells, colonies were selected in ampicillin and plasmid preparations were prepared. Some preparations were induced by 0.5 mM isopropyl thiogalactoside for 24 h. CA XII-FLAG was obtained from the membrane fraction and media and examined by Western blotting using M2 anti-FLAG monoclonal antibody (Sigma). In addition, the protein from 4 liters of media was concentrated to provide adequate amounts for determination of hydratase activity.

The full-length open reading frame of rabbit XII was directionally subcloned in frame into pcDNA3 (Invitrogen), which provides a high level of constitutive expression via its cytomegalovirus-enhancer promoter. The CA XII-pcDNA3 (335-670 µg) was transfected into COS-7 cells by using 4 µg lipofectamine (GIBCO-BRL) on 12-well tissue culture plates (20-mm diameter) according to the manufacturer's instructions. After 48 h, the cells were scraped off the well plates and membrane proteins were isolated in Sato's buffer (25 mM triethanolamine, pH 8.1, 59 mM Na2SO4, and 1 mM benzamidine chloride) plus the protease inhibitors 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 (29).

Stable expression of rabbit CA XII in cultured rat IMCD cells. To obtain larger amounts of material that were posttranslationally modified by a mammalian system, we expressed these CAs in immortalized rat IMCD cells. We selected immortalized rat IMCD cells to express CA XII, because they are acidifying epithelia in culture and reasonably represent the IMCD in situ (3, 17). The full-length open reading frame of rabbit XII was directionally subcloned in frame into pcDNA3 (Invitrogen). This vector has a neomycin resistance site to facilitate stable expression. In preliminary studies, we determined that 600 µg/ml of Geneticin (GIBCO-BRL) killed nontransfected IMCD cells within 2 wk. The IMCD cells were transfected with CA XII in pcDNA and grown in six-well plates in 600 µg/ml of Geneticin. Individual surviving colonies were obtained with cloning cylinders and eventually expanded into T-75 flasks. Once confluent, the cells were scraped off and homogenized, and membranes were obtained from the fraction that was centrifuged at 100,000 g for 1 h. The protein was quantified by the BCA assay (Pierce, Rockford, IL) with BSA as a standard.

A similar approach was used to stably express the full-length rabbit CA IV open reading frame in IMCD cells (30). CA IV-transfected cells expressed abundant CA activity and a protein of similar size to rabbit CA IV (29). These cells were used as CA IV-positive controls for the IMCD cells expressing CA XII.

CA hydratase activity. Crude membrane samples were solubilized in Sato's buffer (25 mM triethanolamine, pH 8.1, 59 mM Na2SO4, and 1 mM benzamidine chloride), which also contained protease inhibitors, as noted above (29). Hydratase activity in expressed rabbit CA XII was measured at 1-2°C as a colorimetric end point assay by using imidazole/Tris buffers, CO2 as a substrate, and p-nitrophenol as the pH indicator (4, 6). This miniature assay utilized 50 µl membrane sample plus water and 50 µl p-nitrophenol in buffer for a total volume of 100 µl. An enzyme unit (EU), normalized to 1 mg protein, is the amount of homogenate necessary to halve the reaction blank time and was corrected for miniaturization of Maren's micromethod (18) by dividing the present results by 10 (4). CA hydratase activity was computed from log (B/S)/log 2 (4) and normalized to 1 mg protein, wherein B is the time measured for the boiled inactivated enzyme or matched buffer devoid of enzyme and S is the time measured for the membrane sample. In practice, we added enough membrane protein such that the reaction time was approximately one-half of the blank.

Characterization of the expressed CA XII hydratase activity was performed by examining comparable amounts of membrane that were boiled (denatured) for 3 min, exposed for 20 min to the general CA inhibitor acetazolamide (0.01-500 µM) (4) or to 0.1-2% SDS; the latter inhibits CA II but not CA IV activity (6, 37, 41). The expressed CA IV hydratase activity was examined in a similar fashion to provide a comparison. All hydratase assays were performed in duplicate or triplicate.

Analysis and statistics. The intensity of the signals in the autoradiograms was analyzed by scanning densitometry (SigmaGel, Jandel, San Rafael, CA). To compensate for differences in quantity of total RNA on each lane of the membrane in the Northern blot analysis, each CA value was normalized to its respective value of beta -actin.

CA XII protein secondary structure analysis was obtained from algorithms contained in MacVector and Gene Runner (version 3.04, Hastings Software, Hastings, NY) programs.

In situ hybridization images were processed by using Spot software (version 3.0.4, Diagnostic Instruments, Sterling Heights, MI) and further optimized with Photoshop (version 6.0, Adobe Systems, San Jose, CA). The images were collected and presented in montage form by using Powerpoint (version 2000, Microsoft, Bellevue, WA).


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Cloning and sequencing of rabbit CA XII. Using a degenerate antisense primer situated at the end of the coding region plus an upstream sense primer encoding five amino acids that are conserved among the membrane CAs, we obtained a 720-bp RT-PCR product from rabbit kidney total RNA (Fig. 1). Sequence analysis of this product revealed 76% identity to human CA XII (35). This cDNA was then used to screen one-quarter of a rabbit kidney cortex cDNA library. Two clones were obtained after four rounds of screening, and each was sequenced from the ends by using primers derived from the Bluescript phagemid. Clone 3-2a1 spanned the entire open reading frame of CA XII, and it was sequenced in both directions by using a series of internal primers every 300-400 bases. The GenBank accession number is AF-263367. Comparison with the full-length open reading frame of human CA XII revealed 83% identity at the nucleotide level, but the rabbit sequence contained three additional nucleotides at the 3'-end for a total of 1,068 through the open reading frame. These three additional bases were confirmed by sequencing several RT-PCR products generated from the distal half of the molecule.


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Fig. 1.   Schematic diagram of rabbit carbonic anhydrase (CA) XII open reading frame (ORF) and poly A tail (AAA), the two cDNA clones obtained from the rabbit kidney cortex library, and the initial RT-PCR product obtained from rabbit kidney total RNA. The lengths and position of the molecules were drawn to scale.

The encoded amino acid sequence revealed 77% identity and an additional 4% similarity to human CA XII; the rabbit sequence predicted 355 amino acids in the translated polypeptide compared with 354 amino acids in the human sequence (Fig. 2). There was an addition of one amino acid (glycine) in the putative cytoplasmic region.


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Fig. 2.   Alignment of rabbit (Rab) and human (Hum) CA XII amino acid sequences. The dark-gray boxes indicate identical amino acids, whereas the light-gray boxes indicate similar amino acids. The identical amino acids are listed below the paired sequences, and a dot indicates a similar amino acid. An absence of gray indicates different amino acids. There is a one amino acid gap at 332 in human CA XII.

From Kyte-Doolittle hydropathy plots and von Heijne and Goldman, Engleman, and Steitz methods to identify transmembrane regions, we predicted that the rabbit CA XII protein contained an NH2-terminal signal sequence of 26 amino acids, followed by a CA domain, a putative transmembrane region (amino acids 294-327), and a terminating hydrophilic sequence of 28 amino acids, which corresponded to a cytoplasmic COOH terminus. Thus rabbit CA XII was predicted to be a single-pass transmembrane protein with a cytoplasmic tail. Compared with human CA XII, the predicted secondary sequences were similar in size and location. The COOH terminus contained putative sites for cAMP and casein kinase II phosphorylation.

Each of the histidines, which bind zinc and/or serve as proton shuttle agents for human CA XII, was encoded by the rabbit sequence (amino acids 61, 94, 119, 121, 133, 145, and 148). Comparison to each of the high-activity rabbit CAs (II and IV) showed conservation of these histidines, except for His61 (Fig. 3). One pair of cysteines was conserved in rabbit and human CA XII at amino acids 50 and 230, suggesting the possibility of intramolecular disulfide bonds, which could confer partial resistance to denaturation of hydratase activity by SDS. CA IV, which has two pairs of cysteines (38, 41), is resistant to denaturation by 0.2-10% SDS (6, 37). The location of these two cysteines in CA XII was reasonably well conserved compared with rabbit CA IV.


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Fig. 3.   Alignment of amino acid sequences of rabbit CA XII, IV, and II, beginning with 1 of CA XII and finishing with 355 of CA XII, which because of gaps numbers 363. The dark-gray boxes indicate identical amino acids, whereas the light-gray boxes indicate similar amino acids. Identical amino acids are listed below the matched sequences, and a dot indicates a similar amino acid. An absence of gray indicates different amino acids.

The extracellular portion of the molecule had three potential sites for N-asparagine glycosylation and four for myristolation, which would suggest an increase in molecular mass above the 39.6 kDa predicted from the amino acid sequence.

Expression of CA XII in rabbit kidney and other organs. Using the 720-bp RT-PCR product as a probe for CA XII, we examined the expression of this gene in the zones of the kidney and in a few other organs. Figure 4A shows the expression of CA XII mRNA in mouse kidney and in outer medulla, inner medulla, and cortex from rabbit kidney. CA XII was seen as a 4.5-kb single transcript that was most abundant in kidney cortex and less so in inner medulla and outer medulla. Figure 4B shows a Northern blot of cortex, colon, lung, spleen, and heart tissue. CA XII was well expressed by cortex and colon but not by lung, spleen, or heart. This was in contrast to CA IV, which was abundantly expressed in colon, lung, and kidney cortex and much less so in heart. beta -Actin was used to compensate for loading differences and was expressed in each of these tissues, although the molecular mass was smaller in heart. When these samples were expressed as CA XII/actin ratios, the cortex was 3.7, colon 2.0, lung <0.03, spleen <0.03, and heart <0.03. For CA IV/actin, the ratios were colon 3.4, cortex 2.0, lung 1.0, heart 0.2, and spleen <0.03.


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Fig. 4.   A: Northern blot of 20 µg of total RNA from mouse kidney (MK) and rabbit kidney outer medulla (OM), inner medulla (IM), and cortex (CTX). A single band was seen slightly smaller than the 28S marker (~4.5 kb), heaviest in the cortex. B: Northern blot of total RNA from rabbit tissues including cortex (Ctx; 7 µg), colon mucosa (Co; 10 µg), lung (Lu; 20 µg), spleen (Spl; 20 µg), and heart (Hrt; 20 µg) probed for CA XII and CA IV and then reprobed for actin. Expression of CA XII and CA IV was normalized to actin and expressed as a ratio.

In situ hybridization. CA XII was abundantly expressed in the inner cortex, as seen in the low-power darkfield views of the corticomedullary junction (Fig. 5A); proximal convoluted tubules (arrows) but not glomeruli expressed it. The signal was not abundant in the outer stripe of the outer medulla (Fig. 5B). In the inner stripe of the outer medulla and the inner medulla, CA XII was well expressed over medullary collecting ducts (Fig. 5, B and C).


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Fig. 5.   Darkfield views of CA XII in situ hybridization in rabbit kidney. Left: low-power views with antisense probe. Right: high-power views with sense probe; they can be compared with the views from Fig. 6. A: corticomedullary junction showing juxtamedullary glomeruli (G) and abundant signal over proximal convoluted tubules (arrows) surrounding these glomeruli; less signal was seen in the adjacent outer stripe of the outer medulla. B: view of outer medulla at junction of inner stripe (IS) and outer stripe (OS); signal is observed over tubules (arrows) in the inner stripe. C: inner medulla shows collecting ducts expressing CA XII (arrows). D: high-power sense view of midcortex showing glomerulus (G) in center surrounded by proximal tubules. E: sense view of inner medulla showing collecting ducts (arrows). Sense views did not show concentration of signal over any particular structures.

High-power views over the inner cortex (Fig. 5D) and inner medulla (Fig. 5E) with a sense probe did not reveal any specific increase in grain density over proximal tubules or decrease over a glomerulus in the cortex (Fig. 5D) or over the epithelia of IMCDs (Fig. 5E, arrows).

High-power views showing side-by-side hematoxylin/eosin and dark field images, by using an antisense probe, confirmed a specific CA XII signal over proximal convoluted tubules, but little was observed over glomeruli of the cortex (Fig. 6, A and B). The medullary rays showed an intermediate signal, and at high power this signal appeared to be primarily over proximal straight tubules and cortical collecting ducts (C in Fig. 6, C and D). This signal appeared to be less than that observed over neighboring proximal convoluted tubules. In the inner stripe of the outer medulla, there was a definite signal detected over the outer medullary collecting ducts (C in Fig. 6, E and F). In the initial inner medulla, abundant signal was observed over the epithelia of IMCDs (C in Fig. 6, G and H) and of collecting ducts in the terminal inner medulla (C in Fig. 6, I and J). In these last views, there appeared to be a specific signal noted over the papillary epithelium, a finding that was not seen by using a sense probe (not shown).


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Fig. 6.   High-power views of rabbit kidney with an antisense probe with hematoxylin and eosin views (left) and darkfield (right). A and B: cortex view with glomerulus (G), proximal convoluted tubules (P), and a collecting duct (C). Signal was seen over proximal tubules and collecting duct and nearly absent from the glomerulus. C and D: cortex view of medullary ray showing proximal straight tubules (S), cortical collecting duct (C), and proximal convoluted tubule (P). Signal appeared heaviest over the proximal convoluted tubules and less over proximal straight tubules, thick ascending limbs, and collecting duct. E and F: outer medulla view showing signal over outer medullary collecting ducts (C) from the inner stripe. G and H: inner medulla view showing inner medullary collecting ducts (IMCD; C) with abundant signal over their cells. I and J: papillary view showing terminal IMCDs (C) and papillary epithelium (Pa) expressing abundant CA XII mRNA.

Expression of CA XII mRNA by individual nephron segments. RT-PCR was performed on microdissected nephron segments (Table 1), and a representative run is shown in Fig. 7. Glomeruli did not express CA XII (an average of 5 glomeruli were pooled for each sample). Proximal convoluted and straight tubules generally expressed CA XII mRNA; 13 of 15 convoluted segments averaging 1.4 ± 0.1 mm and 15 of 20 straight segments averaging 1.5 ± 0.1 mm were positive. Only 1 of 11 medullary proximal straight tubules, S3 segments averaging 1.2 ± 0.1 mm, expressed CA XII mRNA. Thick ascending limbs of Henle's loop (averaging 1.6 ± 0.2 mm) expressed CA XII mRNA in more than one-half of the cases (14/24). Surprisingly, 20 of 24 of these segments expressed CA II as well. CA XII mRNA, as well as CA II, was readily detected in collecting ducts. Cortical collecting ducts (averaging 1.3 ± 0.1 mm) abundantly expressed CA XII (25/25) as well as CA II (25/25), whereas 7 of 9 outer medullary collecting ducts (averaging 1.2 ± 0.1 mm) and 12 of 15 IMCDs (averaging 1.2 ± 0.1 mm) expressed CA XII.

                              
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Table 1.   CA XII mRNA expression by isolated nephron segments



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Fig. 7.   Examination of microdissected tubules for expression of CA XII, CA II, and L32 by RT-PCR. The marker lane (M) shows a 100-bp ladder with heavy band for 600 bp. The signal for CA XII is seen at 758 bp, for CA II at 738 bp, and for L32 at 159 bp. Lane 1, 5 glomeruli (Glo); lane 2, thick ascending limb of Henle's loop (TAL); lane 3, cortical collecting duct (CD); lane 4, medullary proximal straight tubule (S3); lane 5, cortical proximal straight tubule (PST); lane 6, 1 ng of total RNA from rabbit cortex (positive control; 1); lane 7, 0 ng total RNA (negative control; 0). In this representative run, both cortical collecting duct and proximal straight tubule express CA XII, whereas thick ascending limb of Henle's loop, cortical collecting duct, medullary proximal straight tubule, and cortical proximal straight tubule express CA II; each segment expresses L32.

Expression of CA XII FLAG protein. To characterize the activity of CA XII and determine whether it reacted with our anti-CA IV antibodies, we expressed it by using a FLAG-tagged expression plasmid. CA XII-FLAG was obtained from the membrane fraction of E. coli and expressed as a ~40-kDa protein (arrow) as seen by Western blotting using M2 anti-FLAG antibody (Fig. 8). From the 355 amino acids encoded by the open reading frame, the predicted molecular mass would be 39.6 kDa, and the FLAG tag would add ~1 kDa to this sequence, totaling ~40.6 kDa, similar to what was observed in the Western blot assuming no posttranslational modification by the E coli. Adding 0.05 mM of the inducer isopropyl thiogalactoside for 24 h increased the expression of CA XII-FLAG (not shown).


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Fig. 8.   Western blot with M2 anti-FLAG antibody to detect CA XII-FLAG expressed by fractions of Escherichia coli. Left: CA XII was expressed at ~40 kDa in the membrane fraction (Memb) but not in the periplasmic (Peri) or secreted (Sec) fractions. Right: when the media was concentrated, secreted CA XII-FLAG was detected in 300 µg of protein (Sec).

The membrane protein was suspended in Sato's buffer plus 0.5% Triton X-100 and assayed for hydratase assay. The membranes yielded 1.5 ± 0.1 EU/mg of hydratase activity (Table 1), and this was 69% inhibited by 0.1 mM acetazolamide and 87% by 0.5 mM acetazolamide and completely inhibited by 1% SDS or boiling for 3 min. The protein from the media contained 1.3 ± 0.1 EU/mg activity, and this was fully inhibited by 0.1 µM acetazolamide or by boiling and 70% inhibited by 1% SDS.

Transient expression of CA XII in COS cells. COS-7 cells were transiently transfected with CA XII-pcDNA3, and 48 h later were scraped, homogenized, and centrifuged to yield membrane proteins. Fifteen micrograms of membrane proteins yielded 1 EU of hydratase activity (6.5 ± 0.3 EU/mg, Table 2). This activity was completely inhibited by 0.1 mM acetazolamide or by boiling. Membranes from nontransfected COS cells had no detectable hydratase activity.

                              
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Table 2.   Expression of CA hydratase activity

Stable expression of CA XII by cultured rat IMCDs. Membrane protein was obtained from rat IMCD cells stably expressing CA XII-pcDNA and assayed for hydratase activity; three bulk preparations of membranes from several colonies averaged 17.0 ± 1.5 EU/mg (Table 2). Seven individual colonies showed ~1 EU of hydratase activity in 45-220 µg of membrane protein with a mean of 10.1 ± 2.1 EU/mg. In contrast, membranes from IMCD cells transfected with the vector alone showed minimal activity (0.2 ± 0.1 EU/mg, Table 2).

One clone of IMCD cells (2A9) that expressed CA XII was examined in greater detail. The hydratase activity was 11.6 EU/mg. The expressed membrane hydratase activity was completely inhibited by 0.1 µM acetazolamide and by boiling for 3 min and nearly so by 1% SDS. An SDS titration showed 57% inhibition by 0.1% SDS, 82 by 0.5%, 98 by 1%, and 100 by 2% SDS. An acetazolamide titration showed complete inhibition at 1 and 0.1 µM acetazolamide and 48% inhibition at 0.01 µM.

For comparison, seven independent clones of IMCD cells stably expressing CA IV had a mean hydratase activity of 18.9 ± 9.4 EU/mg. One clone studied in depth (2c) showed no inhibition in SDS up to 2% concentration (Table 2). For another comparison, membranes from rabbit kidney cortex expressed ~13 EU/mg (6). This hydratase activity was stable in 1% SDS but was completely inhibited by 2 µM acetazolamide (6). The resistance to SDS in the membrane fraction is consistent with most of the hydratase activity being CA IV.

Expression of CA XII does not cross-react with anti-CA IV antibodies. Because anti-CA IV antibodies identify basolateral staining in the proximal tubule, we determined whether CA XII, which may be expressed basolaterally, was detectable by anti-CA IV antibodies. Membrane proteins were obtained from IMCD cells stably transfected with CA XII and from IMCD cells expressing full-length CA IV. The membrane protein was transferred to nitrocellulose and probed with each of our anti-CA IV peptide antibodies (38). Figure 9 shows that 20 µg of membrane protein from IMCD cells stably expressing CA XII (CA 12) were not detectable with either of the two anti-CA IV peptide antibodies, anti-KDNV (left) or anti-YDQR (right). In contrast, protein from IMCD cells expressing CA IV (CA 4) was readily detectable with either antibody and revealed a protein of 46-50 kDa, similar to what has been observed previously in rabbit kidney (29, 31).


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Fig. 9.   CA XII protein stably expressed by IMCD cells (CA12) was not detectable with anti-KDNV (left) or anti-YDQR (right) CA IV peptide antibodies. The positive control was IMCD cells stably expressing CA IV (CA 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

With the identification of several new isoforms of CA, it is necessary to identify each isoform in a specific organ, localize and characterize it, and determine its function. Because an extensive physiology of acid-base homeostasis has been derived from isolated perfused segments of the rabbit nephron, we have systematically investigated CA expression in the rabbit kidney (5, 6, 29, 32-34, 39). With respect to CA, we have previously localized CA II and IV in specific nephron segments of rabbit kidney (4, 6, 29, 31-33, 39). The present study shows that rabbit CA XII was quite similar to human CA XII (83% identity at the nucleotide level; 76% identical and 83% similar at the amino acid level). However, rabbit CA XII had an additional cytoplasmic amino acid for a total of 355. The protein was predicted to be a single-pass transmembrane structure. Each of the three zinc-binding histidines of the high-activity CAs was conserved in CA XII.

Rabbit CA XII mRNA was abundantly expressed in kidney cortex and less so in medulla. In nonrenal tissues, CA XII was expressed in colon but not in lung, spleen, and heart. The most abundant expression of CA XII mRNA was detected in kidney cortex and colonic mucosa; other sections of the intestine were not investigated. In humans (35), CA XII mRNA is present in kidney and colon but not in other tissues. Thus CA XII mRNA appears to display a similar pattern of tissue distribution in human and rabbit. By immunocytochemistry, CA XII protein is highly expressed in colon (14) but is absent from the small intestine; the staining pattern is confined to the basolateral membranes of the enterocytes.

In addition to examining rabbit CA XII mRNA in various organs, we have expressed rabbit CA XII protein in several cell systems. First, by using an NH2-terminal FLAG sequence, we expressed rabbit CA XII in E. coli, and its molecular mass was nearly identical to that predicted for the protein from the amino acid sequence (~40 kDa); moreover, substantial hydratase activity was detected. Then, by using a transient transfection of COS cells, we expressed hydratase activity in the membranes derived from these cells. As with most of the active CAs, the hydratase activity was inhibited by boiling and by acetazolamide, as shown by Tureci et al. (35), who characterized expression of the human isoform.

Using the same CA XII construct, we investigated IMCD cells, a cell line that expresses H+-ATPase (3) and CA II (28) but not CA IV (28). The ample expression of CA XII mRNA in the IMCD of the rabbit kidney suggested to us that an IMCD cell line would serve as a good model. Indeed, transfected CA XII was stably expressed at high levels in the membranes of several clones of IMCD cells in culture. As a control, membranes from this cell line that had been transfected with vector alone (Table 1) or were not transfected (not shown) expressed very small amounts of membrane hydratase activity, the identity of which is not yet known. Titration of the CA XII-transfected cells with acetazolamide revealed an I50 of ~5 × 10-8 M, similar to that for partially purified CA IV (19, 37). Titration with SDS revealed significant inhibition even at concentrations as low as 0.1% SDS. This study indicated that CA XII had little stability in SDS and therefore was unlikely to have disulfide linkages that would stabilize it in detergents, despite the presence of a pair of cysteines in the extracellular domain (20, 37).

Renal localization of CA XII mRNA. In situ hybridization confirmed the abundant expression in the cortex, with heavy expression by proximal convoluted tubules and less so over proximal straight tubules. Medullary rays, which also include cortical collecting ducts and cortical thick ascending limbs, also showed CA XII mRNA expression, but there was virtually no expression over glomeruli. The medulla showed labeling over outer medullary collecting ducts but more abundant signal over collecting ducts from the initial and terminal inner medulla. The proximal tubule in the kidney and the colon are both involved in salt, water, and bicarbonate reabsorption, so it is conceivable that the CA XII is present in the basolateral membrane to provide additional CO2 to facilitate the reabsorption of NaCl and water via the Na-3HCO3 cotransporter. Indeed, we have shown in a recent physiological study (34) that basolateral CA activity facilitates bicarbonate and fluid absorption in the isolated perfused rabbit proximal straight tubule.

Examination of CA XII expression in isolated nephron segments by RT-PCR showed excellent agreement with the in situ hybridization studies. There was abundant expression of CA XII mRNA in cortical, outer medullary, and IMCDs, as well as thick ascending limbs of Henle's loop. Expression was also noted in proximal convoluted and straight tubules but not in S3 segments or glomeruli.

A recent immunohistochemical study (24) of human kidney using a polyclonal antibody to a COOH-terminally truncated CA XII protein showed distinct labeling over basolateral membranes of the thick ascending limb of Henle's loop and distal convoluted tubule cells and principal cells of the cortical collecting ducts. A basolateral signal was seen over the proximal convoluted tubules, as would be anticipated from our data. In the medulla, a signal was noted over basolateral membranes of some cells in the collecting duct epithelium, but localization within the medulla (inner vs. outer) was not specified. Double labeling with CA II, a cytosolic marker of intercalated cells, revealed that the majority of CA XII-positive cells in collecting ducts showed no reaction for CA II, indicating that the cells expressing CA XII were likely to be principal cells rather than intercalated cells. This was confirmed by aquaporin-2 staining. In some distal tubule cells, there was double labeling for CA XII and CA II, but the specific identification of such cells was not performed; intercalated cells are not usually found in distal tubule (13, 16).

Although our mRNA studies cannot provide information on the polarity of CA XII expression, they clearly agreed for the most part with the immunohistochemical study of Parkkilla et al. (24) that showed expression along the distal nephron as well as in proximal convoluted and straight tubules. Nevertheless, there were some minor differences between the RT-PCR findings and the in situ hybridization studies. The more extensive expression in proximal tubules observed by the latter method may possibly be attributable to cross-hybridization of alkaline-hydrolyzed shortened probe containing a common CA domain, with expressed CA II mRNA. In addition, the thickness of the sections could result in some lack of specificity. We cannot provide other explanations at this time and suggest that additional antibody localization studies be performed in the future. Because the antibody used by Tureci and colleagues (35) was made in rabbits, it is unlikely to be useful for examining CA XII expression in rabbit kidney.

Another unexplained finding is the detection of CA II mRNA in thick ascending limbs. The rabbit thick ascending limb, in contrast to that in mice and rats, does not express CA II activity (10, 11, 15), and we previously found no CA II mRNA in isolated rabbit thick ascending limbs (33). Perhaps the use of a more potent Taq polymerase contributed to the more recent positive finding. The fact that this segment in the rabbit does not express CA II activity (10, 11) suggests that the expression of CA II mRNA by rabbit thick ascending limbs is not physiologically important.

The molecular identities of the apical and basolateral membrane CAs have been suggested to be CA IV (9, 29, 31) on the basis of immunocytochemical labeling. This immunocytochemical finding is in contrast to the expectation that CA IV is more likely to be preferentially apically expressed because of being GPI anchored (7). It is possible that CA IV is expressed basolaterally as well, perhaps as a non-GPI-linked single-pass membrane isoform, or a basolateral CA isoform may be cross-reacting with anti-CA IV antibodies. Either of these choices is presently feasible. However, when CA XII was expressed in E. coli or IMCD cells, cross-reacting products were not detected with either of our anti-CA IV peptide antibodies. Thus it is likely that our anti-CA IV antibodies are detecting an isoform on the basolateral membrane that is not CA XII.

Our studies show that CA XII mRNA is expressed in the rabbit kidney and predominately over proximal tubules, thick limbs, and collecting ducts. Although CA XII has been considered to be important in salt and water transport (24), our sequence analysis showed that each of the histidines expressed by high-activity CAs was preserved, and the expressed protein had substantial hydratase activity. These findings suggest that CA XII might be involved in CO2 fluxes (i.e., H+ transport) and therefore could be expressed by acidifying segments of the nephron, such as the proximal tubule and medullary collecting duct. The abundance of the mRNA and the localization to these acidifying nephron segments are compelling reasons to generate a specific CA XII antibody that is useful for studies in rabbit kidney.


    ACKNOWLEDGEMENTS

We appreciate the help of Dr. Lois Arend in reviewing the in situ hybridizations.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-50603 (G. J. Schwartz) and HL-58774 (M. A. O'Reilly). Additional support was provided by the Thomas H. Maren Foundation (G. J. Schwartz).

Address for reprint requests and other correspondence: G. J. Schwartz, Box 777, Univ. of Rochester Medical Ctr., 601 Elmwood Ave., Rochester, NY 14642 (E-mail: George_Schwartz{at}urmc.rochester.edu).

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

First published October 8, 2002;10.1152/ajprenal.00370.2001

Received 19 December 2001; accepted in final form 2 October 2002.


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