Detection of ClC-3 and ClC-5 in epididymal epithelium: immunofluorescence and RT-PCR after LCM

Corinne Isnard-Bagnis1,*, Nicolas Da Silva1,*, Valérie Beaulieu1, Alan S. L. Yu3, Dennis Brown1,2, and Sylvie Breton1,2

1 Renal Unit and Program in Membrane Biology, Massachusetts General Hospital, Charlestown 02129; 2 Department of Medicine, Harvard Medical School, Boston 02215; and 3 Renal Division, Brigham and Women's Hospital, Boston, Massachusetts 02115


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Epithelial cells of the epididymis and vas deferens establish an optimum luminal environment in which spermatozoa mature and are stored. This is achieved by active transepithelial transport of various ions including Cl- and H+. We investigated the localization of three closely related members of the ClC family, ClC-3, ClC-4, and ClC-5, in the epididymis and vas deferens. RT-PCR using mRNA isolated by laser capture microdissection (LCM)-detected ClC-3 and ClC-5 transcripts but did not detect any ClC-4-specific transcript. Western blot and immunofluorescence analysis demonstrated that ClC-3 and ClC-5 proteins are present in all regions of the epididymis and in the vas deferens. ClC-5 is expressed exclusively in H+-ATPase-rich cells (narrow and clear cells). Confocal microscopy showed that ClC-5 partially colocalizes with the H+-ATPase in the subapical pole of clear cells. ClC-3 is strongly expressed in the apical membrane of principal cells of the caput epididymidis and the vas deferens and is less abundant in principal cells of the body and cauda epididymidis. These findings are consistent with a potential role for ClC-3 in transepithelial chloride transport by principal cells and for ClC-5 in the acidification of H+-ATPase-containing vesicles in narrow and clear cells. ClC-5 might facilitate endosome trafficking in the epididymis, as has been proposed in the kidney.

chloride channels, vacuolar H+-ATPase, principal cells, clear cells; laser capture microdissection


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CHLORIDE CHANNELS of the ClC family are voltage-gated channels of various conductances. They are involved in a wide variety of functions that include the regulation of electrical excitability, transepithelial transport, cell volume regulation, and acidification of intracellular organelles (25, 38, 54, 56). The ClC chloride channel family is composed of nine members encoded by genes that have been highly conserved throughout evolution. ClCs show a differential tissue distribution and function in both plasma membranes and intracellular organelles. ClC-3, ClC-4, and ClC-5 channels form a common branch of the ClC family. They are closely related and share at least 80% identity (25, 54, 56). ClC-3 and ClC-4 are expressed in several tissues including brain (3, 27, 51) and kidney (41), whereas ClC-5 is predominantly expressed in the kidney (21, 37, 41) but is also present in the intestine (53). A recent study demonstrated the presence of ClC-4 in the brush-border membrane (BBM) of enterocytes, where it colocalizes with the cystic fibrosis transmembrane conductance regulator (CFTR) (40).

In the kidney, ClC-5 is present below the BBM of proximal tubule cells, in the medullary thick ascending limb of Henle, and in collecting duct intercalated cells (16, 21, 37, 41, 47). ClC-5 colocalizes with the vacuolar H+-ATPase (21, 47). It is present in endosomes derived from the renal outer medulla (37) and is proposed to provide the anion conductance required for endosomal acidification. Mutations in ClC-5 cause Dent's disease, which is characterized by a variety of renal disorders including low-molecular-weight proteinuria (18, 35, 39). Recent models of mice lacking ClC-5 have shown a marked defect in proximal tubular endocytosis (44, 55), demonstrating the importance of ClC-5 in the endosomal pathway in the kidney.

The physiological role of ClC-3 is controversial and is currently under investigation. Though this channel was initially proposed to be responsible for regulatory volume decrease (reviewed in Ref. 38), subsequent evidence from several laboratories did not support this notion (reviewed in Ref. 56). In addition, a recent study revealed no difference in swelling-activated currents in hepatocytes and pancreatic acinar cells isolated from ClC-3 knockout mice compared with wild-type mice (51). ClC-3 was reported to be localized predominantly in intracellular structures (56). Stobrawa et al. (51) recently showed that ClC-3 is present in endosomal and lysosomal fractions from liver membranes. ClC-3 was also shown to be important for the acidification of synaptic vesicles (51). In the kidney, ClC-3 mRNA was detected in type B intercalated cells and was absent from type A intercalated cells (41).

Active transepithelial transport of water and various solutes and ions, including chloride, potassium, and sodium, also occurs in parts of the male reproductive tract. Epithelial cells of the epididymis and vas deferens establish the luminal environment in which spermatozoa mature and are stored (23, 24, 26, 46). Functional studies have shown that active chloride reabsorption occurs in the proximal regions of the epididymis concomitantly with sodium reabsorption (33). The luminal concentration of chloride further decreases as the fluid transits through the caput, corpus, and cauda epididymidis (33). Chloride channels, including CFTR, have been described in the epididymis (31, 52). Electrogenic chloride secretion mediated by a cAMP-activated, diphenylamine-2-carboxylic acid-sensitive chloride conductance and a calcium-activated chloride channel has been reported in primary cultures of rat and mouse epididymal cells (29, 30). Whereas chloride transport has been characterized in primary cultures of cells derived from the epididymis, very little is known about the pathways of chloride transport in the intact tissue.

In addition to chloride transport, acid/base transport occurs in the epididymis and vas deferens. Several lines of evidence have revealed net proton secretion and bicarbonate reabsorption in these organs. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration and pH are lower in the lumen of the epididymal tubules compared with blood (15, 32, 33). This acidic and low-bicarbonate environment helps to maintain sperm in an immotile but viable state while they mature and are stored in the epididymis (1, 4). Work from our laboratory has shown that narrow cells in the initial segments and intermediate zone and clear cells in the caput, corpus, and cauda epididymidis express high levels of the vacuolar H+-ATPase on their apical membrane and subapical vesicles (6-9, 13). We have also shown that the majority of net proton secretion is inhibited by bafilomycin in isolated vas deferens (6-8).

The purpose of the present study was to determine whether ClC-3, ClC-4, and ClC-5 are expressed in the epididymis and vas deferens, where they might contribute to transepithelial and/or endosomal chloride transport in these tissues. We used complementary approaches to study the expression of these chloride channels: RT-PCR on epithelial cells isolated by laser capture microdissection (LCM), and immunolocalization and Western blotting using antibodies that recognize ClC-3, ClC-4, and ClC-5.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Tissue preparation and LCM. Rat and mouse cauda epididymidis were dissected, placed in cryomolds, embedded in Tissue-Tek OCT medium, immediately frozen in dry ice, and stored at -80°C. Tissue was sectioned at 5 µm on a Reichert-Jung 2800 Frigocut cryostat (Spencer Scientific, Derry, NH). Sections were mounted on uncoated glass slides (Fisher Scientific, Pittsburgh, PA) and stored at -80°C. Before use, sections were fixed in 70% ethanol and stained with 0.1% toluidine blue O, followed by dehydration steps in 70, 95, and 100% ethanol. Slides were dipped in xylene twice for 5 min each time and then completely air-dried. LCM was performed immediately. Epithelial cells were recovered by using the PixCell II LCM system (Arcturus Engineering, Mountain View, CA). After microdissection (spot size 7.5 µm, pulse duration 1.5 ms, power 50 mW), LCM caps were snapped onto a 0.5-ml microcentrifuge tube containing guanidine isothiocyanate buffer for RNA extraction. Epithelial cell samples from at least three rat and three mouse epididymis were collected.

Total RNA extraction and DNase treatment. Total RNA was isolated from rat brain and epididymis samples using the RNAwiz reagent (Ambion, Austin, TX). Total RNA from laser capture-microdissected samples was isolated using the Micro RNA isolation kit (Stratagene, La Jolla, CA). Genomic DNA contamination was removed by incubation with 10 units of DNase I (MessageClean; GenHunter, Nashville, TN) for 30 min at 37°C. DNA-free RNA was then reextracted, resuspended in nuclease-free water, and stored at -80°C.

RNA amplification. RNA (10 µl) was amplified by T7-based amplification as described previously (36) with some modifications. Briefly, first-strand synthesis was performed at 42°C in a 20-µl reaction solution containing 0.5 µg of T7 oligo(dT) primer, 1× first-strand buffer, 10 mM DTT, 1.0 mM each dNTP, 40 units of RNase inhibitor (Promega, Madison, WI), and 200 units of SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA). Second-strand synthesis was performed in 150 µl with 40 units of DNA polymerase I, 2 units of Escherichia coli RNase H, and 10 units of E. coli DNA ligase in 1× second-strand buffer for 2 h at 16°C. The double-stranded cDNA was polished with T4 DNA polymerase and then purified with the QIAquick PCR kit (Qiagen, Valencia, CA). cDNA was transcribed in 20 µl for 3 h at 42°C with 2 µl of AmpliScribe T7 enzyme preparation (Epicentre, Madison, WI), 7.5 mM ATP, CTP, GTP, and UTP, and 10 mM DTT in 1× AmpliScribe T7 buffer. After an incubation for 15 min at 37°C with 1 unit of RNase-free DNase I, the amplified antisense RNA (aRNA) was purified with the RNeasy Mini kit (Qiagen). For the second round of amplification, first-strand synthesis was performed with 50 ng of random hexamers for 1 h at 37°C. T7 oligo(dT) primer (1 µl) was added, and the second-strand synthesis was performed as described above, except that E. coli DNA ligase was omitted. The double-stranded cDNA was purified and transcribed as above. After this second round of amplification, aRNA was quantified by measuring the absorbance at 260 nm. A third round of amplification was performed when necessary.

Reverse transcription and PCR. Total RNA or amplified aRNA were reverse-transcribed for 1 h at 42°C in a final volume of 50 µl with 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 5.0 mM MgCl2, 1.0 mM each dNTP, 1 U/µl RNase inhibitor, 2.5 µM random hexamers, and 2.5 U/µl MuLV reverse transcriptase (Applied Biosystems, Foster City, CA). Reverse transcription products were used as templates for PCR. Oligonucleotide primer pairs were designed to amplify a short sequence in the 3'-end of the mouse ClC-3 (GenBank accession no. NM_007711), mouse ClC-4 (NM_011334), mouse ClC-5 (NM_016691), and rat ClC-5 (D50497) cDNAs. Primers were synthesized by Sigma-Genosys (The Woodlands, TX). The sequences are shown in Table 1.

                              
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Table 1.   Sequence of the primers used for PCR

Reaction mixtures consisted of a 20-µl final volume containing 2 µl of template, 1.25 units of AmpliTaq Gold DNA polymerase (Applied Biosystems), 50 mM KCl, 10 mM Tris · HCl (pH 8.3), 2.0 mM MgCl2, 1.0 mM each dNTP, and 0.5 µM forward and reverse oligonucleotide primers. PCR was performed in a Flexigene thermal cycler (Techne, Princeton, NJ) with the following parameters: 8 min at 95°C to activate the polymerase, followed by 30-40 cycles of melting for 1 min at 95°C, annealing for 30 s at 60°C, and extension for 45 s at 72°C, and a final extension for 10 min at 72°C. The PCR products were analyzed by electrophoresis on a 2.5% agarose gel or a 4% MetaPhor high-resolution gel containing GelStar stain (BioWhittaker Molecular Applications, Rockland, ME). Negative controls were performed by omitting cDNA template from the PCR amplification. The identity of PCR products was confirmed by direct sequencing (Molecular Biology DNA Sequencing Core Facility, Massachusetts General Hospital).

Antibodies. The ClC antibodies used in the present study have been characterized previously (37). The C1 antiserum was generated by rabbit immunization with a fusion protein corresponding to the 108-amino acid polypeptide from the carboxy terminus of rat ClC-5. This fusion protein contains sequences that are common to ClC-3, ClC-4, and ClC-5 and is referred to as ClC-3,4,5 in the present study. Serum from the rabbit with the highest titer of specific antibody was affinity-purified. This antibody recognizes ClC-3, ClC-4, and ClC-5. The ClC-5 isoform-specific C2 antibody was prepared by taking the previously generated affinity-purified C1 polyclonal antiserum and immunoadsorbing it against fusion proteins containing the homologous ClC-3 and ClC-4 polypeptides. In the present study, we used a slightly different immunofluorescence procedure to stain epididymis sections from that published previously with these antibodies (37). To determine whether the antibodies retained their ability to recognize ClC-3, ClC-4, and ClC-5 using our procedure, we first examined their immunoreactivity in kidney sections. The pattern of staining using the C1 antibody was identical to that previously reported. However, using the C2 antibody, we obtained a strong staining in collecting duct intercalated cells, in addition to the proximal tubule and thick ascending limb staining that was published previously with this antibody (Fig. 1). This staining pattern is consistent with the patterns published by other groups using monoclonal and polyclonal antibodies (16, 21, 37, 41, 47).


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Fig. 1.   Immunofluorescence localization of ClC-5 in kidney cortex. Cryostat sections of rat kidney cortex were stained for ClC-5, using the C2 antibody (A), and for the vacuolar H+- ATPase (B). In addition to the localization of ClC-5 in proximal tubules (PT) previously published with the C2 antibody, intercalated cell staining is also observed. ClC-5 is located in the apical pole of intercalated cells, where it colocalizes with H+-ATPase (arrows). Bars = 15 µm.

To identify narrow and clear cells in the epididymis, we used an affinity-purified chicken antibody against the 14 carboxy-terminal amino acids of the 31-kDa subunit (E subunit) of the vacuolar H+-ATPase. This antibody has been described previously (5, 10).

Preparation of tissue cryosections. Adult male Sprague-Dawley rats were anesthetized with Nembutal (65 mg/kg ip) and perfused through the left ventricle with phosphate-buffered saline (PBS; 0.09% NaCl in 10 mM phosphate buffer, pH 7.4), followed by PLP fixative (4% paraformaldehyde, 10 mM sodium periodate, 10 mM lysine, and 5% sucrose in 0.1 mM sodium phosphate), as described previously (8, 13). Before PLP perfusion, one epididymis was removed together with its vas deferens and frozen in liquid nitrogen for Western blot analysis. The remaining organs were harvested after PLP perfusion, further fixed by immersion overnight in PLP at 4°C, washed three times in PBS, and stored until use in the same buffer containing 0.02% sodium azide. Fixed epididymides were divided into two parts (caput + proximal corpus and cauda + distal corpus). These epididymis regions and vas deferens were equilibrated in 30% sucrose overnight at 4°C. They were then embedded in Tissue-Tek (Miles, Elkhart, IN) before being frozen in liquid nitrogen and sectioned with a Reichert-Jung 2800 Frigocut cryostat. Sections (3 µm) were picked up onto Superfrost Plus charged glass slides (Fisher Scientific).

Immunofluorescence. Fixed sections were hydrated in PBS for 10 min and treated for 5 min with SDS (1% in PBS), an antigen retrieval technique that we described previously (14). The sections were washed three times with PBS for 5 min each time and then blocked in a solution of 1% BSA/PBS/sodium azide for 15 min before application of the primary antibody (diluted as detailed below) for 1.5 h at room temperature. Sections were washed twice for 5 min in high-salt PBS (PBS containing 2.7% NaCl to reduce background staining) and once for 5 min in normal PBS. Secondary antibody was applied for 1 h at room temperature, followed by washes as described above. For double staining, the two primary antibodies were applied at the same time with the appropriate dilutions, and the two secondary antibodies were also applied simultaneously. Primary antibodies used were the antibody against the 31-kDa subunit of the vacuolar H+- ATPase at a 1:20 dilution, the C1 antibody at a 1:25 dilution, or the C2 antibody at a 1:4 dilution. Secondary antibodies used were either donkey anti-chicken antibody coupled to fluorescein isothiocyanate (7.5 µg/ml; Jackson ImmunoResearch, West Grove, PA) or goat anti-rabbit IgG coupled to indocarbocyanine (Cy3; 2 µg/ml; Jackson ImmunoResearch). Slides were mounted in a 2:1 mixture of Vectashield (Vector Laboratories, Burlingame, CA) mounting medium and 1.5 M Tris solution (pH 8.9). Black-and-white photographs were taken on a Nikon FXA epifluorescence photomicroscope (Garden City, NY) using Kodak (Eastman Kodak, Rochester, NY) TMAX 400 film push-processed to 1600 ASA. For double-stained sections, separate images were taken for each wavelength by using a Hamamatsu Orca camera attached to a Nikon Eclipse 800 microscope and then pseudocolored and merged in Adobe Photoshop. The digitized images were printed on an Epson Stylus 600 inkjet printer.

To evaluate the specificity of the antibodies, we preincubated C1 and C2 antibodies for 1 h at room temperature in the presence of an excess of peptide. ClC-3 and ClC-3,4,5 peptides were used at a concentration of 90 µg/ml. When the results of peptide inhibition were to be compared, photos were taken at fixed exposure times and printed under identical conditions.

Confocal microscopy. For the purpose of studying the distribution of chloride channels and H+-ATPase in intracellular vesicles dispersed after microtubule disruption, some rats were treated with colchicine (0.5 mg · ml-1 · 100 g body wt-1 for 13 h). The epididymides were then perfused and fixed in vivo, as described in Preparation of tissue cryosections. Double-labeled sections from control and colchicine-treated animals were examined and image files acquired with a Zeiss Axioplan microscope equipped with a Radiance 2000 confocal laser scanning system (Bio-Rad, Cambridge, MA). Digitized images were stored on a Dell PowerEdge 2300 computer and printed on a Tektronix Phaser 440 dye sublimation color printer.

Immunoblotting. Rats were perfused through the left ventricle with PBS (pH 7.4) at 37°C. Tissue samples were removed, cut into smaller pieces with a razor blade onto an iced surface, transferred to 2 ml of ice-cold lysis buffer containing protease inhibitors (Mini-Complete; Boehringer-Mannheim, Indianapolis, IN), and homogenized using a small Potter and a 27-gauge needle. Lysis buffer consisted of 15 mM NaCl, 10 mM Tris (pH 7.4), 1% Triton X-100, 0.5% Igepal, 1 mM EDTA, 1 mM EGTA (pH 7.4), 200 µM PMSF, and one-quarter tablet of Complete protease inhibitor cocktail per 10 ml of buffer solution.

An enriched preparation of BBM was obtained from the epididymides of two rats, using the Mg2+ precipitation technique, as we have previously described (42, 43). Briefly, the tissue was homogenized in 30 ml of a buffer containing 250 mM sucrose, 18 mM Tris-HEPES, 1 mM EDTA, and Complete protease inhibitor, pH 7.4, using a PRO 200 homogenizer (20 strokes in a glass/Teflon Potter). The homogenate was incubated with 10 mM MgCl2 on ice for 20 min, followed by centrifugation at 7,700 g for 15 min. The supernatant was further centrifuged at 20,000 g for 15 min. The pellet was resuspended in a buffer containing 150 mM KCl, 5 mM Tris-HEPES, and Complete protease inhibitor, pH 7.4, by being passed through a 25-gauge, <FR><NU>5</NU><DE>8</DE></FR>-in.-long needle and was centrifuged at 1,900 g for 15 min. The supernatant was centrifuged at 30,900 g for 15 min. The final BBM-enriched pellet was resuspended in 100 µl of resuspension buffer by being passed through a needle and syringe as above. The protein concentrations of the initial homogenate and the final BBM preparation were measured using the BCA assay (Pierce, Rockford, IL).

Samples were loaded at 20 µg of protein per lane. Samples were heated to 70°C for 10 min in Laemmli sample buffer containing 2.5% beta -mercaptoethanol and were subjected to electrophoresis using a Tris-glycine-SDS running buffer (Boston BioProducts, Ashland, MA) on 4-20% polyacrylamide gels (BioWhittaker Molecular Applications). Proteins were then transferred by using a Bio-Rad (Richmond, CA) semi-dry transfer cell onto Immun-Blot polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked in blotting buffer (3% BSA in 137 mM NaCl, 25 mM Tris, 2.7 mM KCl, and 0.3% Tween 20, pH 7.4) for 4 h at 20°C and were incubated overnight at 4°C with the primary antibodies. The C1 and C2 antibodies were used at a concentration of 7.6 and 3 µg/ml, respectively. Control experiments were performed after the C1 antibody had been preincubated with the ClC-3,4,5 fusion polypeptide (40 µg/ml) or the ClC-3-specific polypeptide (40 µg/ml). Negative controls were also performed after the C2 antibody had been preincubated with the ClC-3,4,5 polypeptide at a concentration of 15 µg/ml. Donkey anti-rabbit IgG conjugated to horseradish peroxidase (Jackson ImmunoResearch) was applied to the membranes for 1 h at room temperature. Proteins were detected by using the Renaissance Western blot chemiluminescence reagent (NEN, Boston, MA) with Kodak X-Omat blue XB-1 films.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ClC mRNA expression in epididymis and brain. Rat epididymis and brain were dissected, and total RNA was extracted and treated with DNase I to remove DNA contamination. RNA samples were used as templates for RT-PCR. Analysis of the PCR products revealed specific products for ClC-3, ClC-4, and ClC-5 in the brain (Fig. 2). However, no product was observed for ClC-4 in the epididymis, indicating that ClC-4 mRNA is not expressed in this tissue.


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Fig. 2.   ClC mRNA expression in epididymis and brain. Total RNA was extracted from rat brain and epididymis. RT-PCR was carried out for ClC-3, ClC-4, and ClC-5 as described in MATERIALS AND METHODS. Specific products were amplified in the brain sample for ClC-3, ClC-4, and ClC-5 (112, 84, and 140 bp, respectively). In the epididymis, only ClC-3 and ClC-5 were successfully amplified, indicating that ClC-4 mRNA is not expressed at detectable levels in this tissue. bp, 100-bp DNA ladder.

Detection of ClC-3 and ClC-5 mRNAs in epithelial cells of the epididymis. We then examined whether ClC-3 and ClC-5 mRNAs were present in epithelial cells of the epididymis. Rat and mouse epithelial cells were microdissected by LCM, total RNA was isolated and linearly amplified using a T7 RNA polymerase-based method, and the generated amplified aRNA samples were used as a template for RT-PCR. As shown in Fig. 3, ClC-3 and ClC-5 transcripts were present in three independent rat and mouse samples. After amplification, nucleotide sequence analysis demonstrated that the 112- and 127-bp PCR products correspond to nucleotides 2628-2739 (mouse samples) and 2751-2862 (rat samples) of ClC-3 mRNA and to nucleotides 2393-2519 (mouse samples) and 2373-2499 (rat samples) of ClC-5 mRNA. These results provide direct evidence that epithelial cells from the epididymis express ClC-3 and ClC-5 transcripts. To determine whether ClC-3 and ClC-5 proteins were present in the epididymis and vas deferens, we performed immunocytochemistry.


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Fig. 3.   Detection of ClC-3 and ClC-5 mRNAs in epithelial cells of the epididymis. Laser capture microdissection (LCM) was used to harvest three samples (1, 2, 3) of epithelial cells from mouse (A) and rat (B) cauda epididymidis. RNA was isolated from LCM samples, amplified, and subjected to RT-PCR analysis with primer sets designed to amplify a short sequence in the 3'-end of ClC-3 (left) and ClC-5 cDNA (right). A RT-PCR product corresponding to the predicted size of the cDNA segment to be amplified (112 bp for ClC-3 and 127 bp for ClC-5) was detected in all samples. The identity of PCR products for ClC-3 and ClC-5 was confirmed by direct sequencing. bp, 20-bp DNA ladder.

ClC-3 and ClC-5 immunostaining in the proximal epididymis. Using the C1 antibody, which recognizes ClC-3, ClC-4, and ClC-5, we detected a widespread distribution of immunofluorescence staining in the epididymis. Because no ClC-4 mRNA was detected in the epididymis, the staining obtained with the C1 antibody is most probably due to the presence of ClC-3 and ClC-5 proteins. Figure 4 shows the different regions of the proximal epididymis at low magnification to allow a better visualization of the complex anatomy of this organ. The C1 antibody stained the apical membrane of epithelial cells of the efferent ducts (Fig. 4, A and E), proximal initial segment (Fig. 4, B and E), middle initial segment (Fig. 4, C and E), distal initial segment (Fig. 4E), intermediate zone (Fig. 4E), and caput epididymidis (Fig. 4, D and E). The staining was completely abolished in the presence of the ClC-3,4,5 polypeptide (data not shown).


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Fig. 4.   Immunofluorescence localization of ClC-3 and ClC-5 in cryostat sections of the proximal regions of the epididymis. Epithelial cells of ductuli efferentes (A), proximal initial segment (B), middle initial segment (C), and caput epididymidis (D) show an intense apical membrane staining with the C1 antibody, which recognizes ClC-3 and ClC-5. The image shown in E is a montage of 20 individual images taken with a ×4 objective. DE, ductuli efferentes; PIS, proximal initial segment; MIS, middle initial segment; DIS, distal initial segment; IZ, intermediate zone. Bars = 15 µm (A-D) or 250 µm (E).

The higher resolution black-and-white image shown in Fig. 5 indicates that the C1 antibody produced a strong staining in the apical membrane of epithelial cells of the proximal initial segment. In this part of the epididymis, narrow cells represent <3% of the total population of epithelial cells (2), so the majority of epithelial cells shown in Fig. 5 are principal cells. An intracellular staining compatible with the Golgi apparatus was also visible in these cells. No staining was observed in this segment, using the C2 antibody (not shown).


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Fig. 5.   Immunocytochemical localization of ClC-3 and ClC-5 in cryostat section of the proximal initial segment. This higher resolution black-and-white image shows that chloride channels are highly expressed in the apical stereocilia of the principal cells (C1 antibody). A weaker juxtanuclear staining compatible with Golgi staining is also shown. Bar = 15 µm.

Figure 6 shows two consecutive 3-µm sections from the proximal caput epididymidis stained using the C1 (Fig. 6A) and C2 antibodies (Fig. 6C). Whereas all epithelial cells exhibited a strong apical staining with the C1 antibody, only occasional cells were stained with the C2 antibody. Double staining for the H+-ATPase showed that both principal cells (negative for H+-ATPase) and clear cells (positive for H+-ATPase) are labeled with the C1 antibody (Fig. 6B) and that the C2 antibody, which preferentially labels ClC-5, produced an immunoreaction in clear cells exclusively (Fig. 6D). ClC-5 is predominantly located in the apical pole of clear cells, where H+-ATPase is also present.


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Fig. 6.   Double immunofluorescence staining showing chloride channels (red) and vacuolar H+-ATPase (green) in the caput epididymidis. Two consecutive 3-µm sections were double-stained using the C1 (A) and the H+-ATPase antibody (B) or the C2 (C) and the H+-ATPase antibody (D), respectively. A: bright apical staining of all epithelial cells was obtained with the C1 antiserum. Clear cells, identified by their positive reaction with the H+-ATPase antibody (B) also show a more diffuse intracellular staining (arrows in A and B). C: staining obtained with the C2 antiserum, which is enriched in anti-ClC-5 IgG, is restricted to clear cells (arrows in C and D), whereas no staining was detected in the apical pole of principal cells. Bars = 20 µm.

ClC-3 and ClC-5 immunostaining in the cauda epididymidis. In the cauda epididymidis, a pattern of staining similar to that seen in the caput epididymidis was observed using the C1 (Fig. 7A) and C2 antibodies (Fig. 7D). Double labeling for the H+-ATPase was performed to identify the clear cells (Fig. 7, B and E). Whereas the C1 antibody strongly labeled the apical membrane of principal cells (negative for H+-ATPase), a more diffuse staining was also observed in clear cells (positive for H+-ATPase; Fig. 7, A-C, arrows). With the C2 antibody, the principal cell staining was greatly diminished and the clear cell staining was predominant (Fig. 7, D-F, arrows). In the corpus epididymidis, a weaker staining was seen, but the pattern of staining observed with both the C1 and C2 antibodies was comparable to that seen in the cauda epididymidis (not shown). Toward the distal part of the cauda epididymidis, principal cells became negative with the C1 antibody (Fig. 8), and the immunoreactivity was almost completely restricted to clear cells. In contrast, in the proximal portion of the vas deferens (Fig. 8) all cells were strongly labeled with this antibody.


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Fig. 7.   Double immunofluorescence staining showing chloride channels (red) and H+-ATPase (green) in the cauda epididymidis. A and B: double staining using the C1 and the H+- ATPase antibody, respectively. C: superimposition of A and B. A bright apical membrane staining is observed in principal cells with the C1 antibody, whereas clear cells, positive for H+-ATPase (arrows in A-C) show a more diffuse labeling. D and E: double staining using the C2 and the H+-ATPase antibody, respectively. F: merged image. The C2 antibody (enriched in ClC-5 IgG) labels preferentially clear cells (positive for H+-ATPase; arrows in D-F) and produces a diffuse staining. A faint residual staining is also observed in the apical membrane of principal cells, which is probably due to a low amount of anti-ClC-3 IgG in the C2 antiserum (see control experiment in Fig. 12).



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Fig. 8.   Immunocytochemical localization of the chloride channels ClC-3 and ClC-5 in the distal part of the cauda epididymidis. Toward the distal end of the cauda epididymidis, which is adjacent to the proximal portion of the vas deferens, the C1 antibody labels clear cells exclusively, indicating that principal cells do not express ClC-3 in this region. The staining observed in clear cells indicates ClC-5 immunoreactivity (see control experiments in Figs. 12 and 13). Principal cells from the vas deferens (VD) show an intense staining in their apical membrane. Bar = 40 µm.

ClC-3 and ClC-5 immunostaining in the vas deferens. The proximal vas deferens was double-labeled for the H+-ATPase and the ClC chloride channels (Fig. 9). Similarly to most regions of the epididymis, all epithelial cells were stained with the C1 antibody (Fig. 9, A-C), and only H+-ATPase-positive cells, identified as clear cells, were stained with the anti-ClC-5-enriched C2 antibody (Fig. 9, D-F).


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Fig. 9.   Double immunofluorescence labeling for chloride channels (red) and the vacuolar H+-ATPase (green) in the vas deferens. A and B: one vas deferens section double-stained using the C1 and the H+-ATPase antibody, respectively. C: superimposition of A and B. An apical staining was observed in principal cells and clear cells (arrows in B and C) with the C1 antibody. D and E: double staining using the C2 and the H+-ATPase antibody, respectively. F: merged image. The C2 antibody (enriched in ClC-5 IgG) labels preferentially clear cells (positive for H+-ATPase; arrows in D---F). Bar = 10 µm.

Localization of ClC-5 by confocal microscopy in the epididymis. Confocal microscopy of an epididymis section from a control rat revealed that ClC-5 (Fig. 10A) is present in intracellular structures located in the apical pole and throughout the cytoplasm of clear cells, whereas the H+-ATPase is concentrated in the apical pole (Fig. 10B). The merged image shown in Fig. 10C indicates an apparent partial colocalization of ClC-5 with H+-ATPase in the apical region of these cells. However, because of the spatial resolution of light microscopy, it is impossible to conclude whether these two proteins actually colocalize in the apical membrane or in the same population of apical vesicles. Therefore, we induced a redistribution of these vesicles by in vivo treatment with colchicine, a microtubule-disrupting agent, as we have previously reported (7). As shown in Fig. 10E, microtubule disruption induced a marked redistribution of H+-ATPase-containing vesicles throughout the cytoplasm. A more moderate effect was seen for ClC-5 (Fig. 10D). The merged image (Fig. 10F) shows a mixture of distinct H+-ATPase-rich (green) and ClC-5-rich (red) vesicles, with only partial overlap in the fluorescence pattern (yellow).


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Fig. 10.   Confocal microscopy showing double immunofluorescence labeling for ClC-5 (red) and H+-ATPase (green) in the cauda epididymidis of control and colchicine-treated animals. A and B: a control epididymis section stained using the C2 (A) and H+-ATPase (B) antibody, respectively. ClC-5 is present in intracellular structures that are located in the apical pole and throughout the cytoplasm (A). H+-ATPase (B) is concentrated in the apical pole of clear cells, but a diffuse cytosolic staining is also detectable. The merged image (C) shows a partial colocalization of ClC-5 with H+-ATPase in the apical pole of the cells (yellow), but some cytoplasmic vesicles contain only ClC-5. D and E: an epididymis section from a colchicine-treated rat stained using the C2 (D) and H+-ATPase (E) antibody, respectively. After colchicine treatment, both ClC-5 and H+-ATPase are redistributed throughout the cytoplasm. The merged image in F shows a mixture of intracellular structures that contain exclusively ClC-5 (red vesicles) or H+-ATPase (green vesicles) and shows that only a subpopulation of intracellular vesicles contains both proteins (yellow vesicles). Bar = 10 µm.

ClC-3 and ClC-5 immunoblotting in the epididymis. Figure 11A shows immunoblots of total homogenates using the C1 (left) and C2 antibodies (right). Both antibodies revealed a predominant band at around 85 kDa. No staining was detected after preincubation of the C2 antibody with the ClC-3,4,5 polypeptide (Fig. 11A, right panel, right lane).


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Fig. 11.   Western blot of epididymis samples using the C1 and C2 antibodies. A: immunoblots of total homogenates with the C1 (left) and C2 (right) antibodies. A predominant band at around 85 kDa was detected with both antibodies. The band detected with the C2 antibody was completely abolished in the presence of an excess of ClC-3,4,5 polypeptide (right panel, right lane). B: immunoblots of total homogenates (H) and brush-border membranes (BBM) using the C1 antibody alone (left) or after preincubation of the C1 antibody with an excess of ClC-3-specific polypeptide (middle) or ClC-3,4,5 polypeptide (right). The 85-kDa band detected with the C1 antibody is significantly stronger in BBM compared with total homogenate. In BBM, an additional, weaker band slightly above 100 kDa was also detected. In the presence of the ClC-3 polypeptide, the 85-kDa band was partially inhibited in the homogenate and was completely inhibited in the BBM. The weaker band detected at around 100 kDa was not inhibited and probably represents a nonspecific band. The ClC-3,4,5 polypeptide completely abolished the 85-kDa band in both homogenate and BBM preparations.

To further identify the chloride channel isoforms that are detected with the C1 antibody, we performed competition experiments using the ClC-3-specific polypeptide and the ClC-3,4,5 polypeptide in total homogenate as well as in BBM. We observed a marked increase in the 85-kDa band intensity in BBM compared with homogenate (Fig. 11B, left). Because the predicted molecular masses of ClC-3 (84.4), ClC-4 (83.7), and ClC-5 (83.1) are very similar and are not likely to be distinguishable on a standard size Western blot, the 85-kDa band probably represents a combination of ClC-3 and ClC-5 proteins. The 85-kDa band detected in BBM was completely inhibited in the presence of the ClC-3 polypeptide (Fig. 11B, middle). In total homogenate, the 85-kDa band is partially inhibited in the presence of the ClC-3 polypeptide, indicating the presence of detectable amounts of ClC-5 in this preparation. This was confirmed by the complete inhibition of the signal in both homogenate and BBM in the presence of the ClC-3,4,5 polypeptide (Fig. 11B, right).

Immunocytochemical competition experiments with the ClC-3 and ClC-3,4,5 polypeptides. Specificity of the C1 antibody was further determined by immunofluorescence after the antibody had been preincubated in the presence of the ClC-3 polypeptide (Fig. 12). Figure 12A shows the immunoreactivity produced by the antibody alone, and Fig. 12B shows staining after preadsorption of the antibody with the ClC-3 polypeptide. The principal cell staining was completely abolished in the presence of ClC-3 polypeptide, whereas the clear cell staining was still present. These results indicate that ClC-3 is expressed mainly in principal cells, whereas ClC-5 is enriched in clear cells. These data do not exclude, however, the possibility that clear cells express ClC-3 in addition to ClC-5.


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Fig. 12.   Competition experiments using the C1 antibody and the ClC-3-specific fusion polypeptide in the cauda epididymidis. We further determined the specificity of the C1 antibody by immunofluorescence. Immunostaining was performed on cryostat sections of the cauda epididymidis under control conditions (C1 antibody alone; A) and after preincubation of the antibody with the ClC-3 polypeptide (B). The C1 antibody alone labels the apical membrane of principal cells (arrow, A) and intracellular structures of clear cells (arrowhead, A). After preincubation with the ClC-3 fusion protein, only the clear cell staining remained (arrowhead, B) and principal cell staining was abolished (arrow, B). These results indicate that ClC-3 is expressed mainly in principal cells, whereas ClC-5 is expressed in clear cells. Bar = 5 µm.

Specificity of the C2 antibody was also assessed after preincubation with the ClC-3,4,5 polypeptide (Fig. 13). Cauda epididymidis sections were double-stained using the H+-ATPase antibody (Fig. 13, A and C) and the C2 antibody, either alone (Fig. 13B) or after preincubation with the fusion protein (Fig. 13D). As shown above, the C2 antibody stained H+-ATPase-rich clear cells exclusively. This immunofluorescence staining was completely abolished in the presence of the ClC-3,4,5 polypeptide. These results confirm that clear cells express ClC-5.


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Fig. 13.   Competition experiments using the C2 antibody and ClC-3,4,5 polypeptide in the cauda epididymidis. Double staining for H+-ATPase (A and C) and ClC-5 using the C2 antibody (B and D) was performed on cryostat sections of the cauda epididymidis. The C2 antibody was applied alone (B) or after preincubation in the presence of the ClC-3,4,5 polypeptide (D). The C2 antibody labels clear cells exclusively (B), as indicated by their positive staining for H+-ATPase (A). The staining was completely abolished by the ClC-3,4,5 polypeptide (D). These results indicate that clear cells express ClC-5. Bar = 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present study, we provide direct evidence that mouse and rat epididymal epithelial cells, isolated by LCM, express ClC-3 and ClC-5 mRNA transcripts and do not contain ClC-4 mRNA transcripts. Using immunocytochemistry and confocal microscopy, we localized ClC-3 and ClC-5 proteins in the epithelium of the rat epididymis and vas deferens. The staining obtained with the C1 antibody represents both ClC-3 and ClC-5, whereas that obtained with the C2 antibody is more specific for ClC-5. These two chloride channel isoforms are expressed in the apical domain of epithelial cells, and the level of expression varies in different regions of the epididymis. Principal cells express ClC-3 in their apical membrane and are devoid of ClC-5, whereas clear cells express ClC-5 in intracellular vesicles. Determining whether clear cells also express ClC-3 will require the use of antibodies that are specific for this isoform, without cross-reactivity with ClC-5.

The present study shows that ClC-5 is expressed exclusively in H+-ATPase-rich cells of the epididymis and vas deferens. ClC-5 is present in intracellular structures that are mainly located in the apical pole and partially colocalizes with the H+-ATPase. We have previously shown that the bulk of proton secretion in the isolated vas deferens is achieved by H+-ATPase-rich cells (8) and is not dependent on the presence of extracellular chloride (6), indicating that concomitant chloride transport is not essential for transepithelial proton flux in this tissue. The high expression of ClC-5 in H+-ATPase-rich cells might therefore indicate its potential role in endosomal acidification.

An intracellular staining pattern for ClC-5 has also been observed in kidney epithelial cells that are enriched in H+-ATPase, such as proximal tubule cells and intercalated cells (21, 37, 41, 47). These cells have a high rate of endocytosis (11, 12, 28), and impairment of the endosomal acidification machinery has been proposed to reduce this function (20, 45). For example, inhibition of H+-ATPase following cadmium intoxication and concomitant inhibition of endosomal acidification was shown to markedly inhibit the reabsorptive capacity of the proximal tubule (22). In addition, ClC-5 mutations cause Dent's disease, which is also partially characterized by impairment in proximal tubule reabsorption, leading to low-molecular-weight proteinuria (18, 35). Because ClC-5 is present in kidney endosomes, it was proposed to provide the electrical shunt necessary for endosomal proton influx (21, 37, 39, 41). Direct evidence for a role of ClC-5 in endocytosis was recently demonstrated in two independent ClC-5-deficient mouse models, in which there was a marked defect in the rate of apical endocytosis in proximal tubules (44, 55). More recently, a novel fluorescent chloride indicator was used to demonstrate that endosomal acidification is accompanied by a significant accumulation of chloride in the endosomes (50).

In the epididymis and vas deferens, H+-ATPase-rich cells have a high endocytotic activity (7), and the intracellular localization of ClC-5 shown in the present study is compatible with a potential role in endosomal acidification. However, ClC-5 is located not only in the apical pole, where the H+-ATPase is expressed, but in many cells, it is also present throughout the cytoplasm. Therefore, it appears that ClC-5 does not completely colocalize with the H+-ATPase in epididymal and vas deferens cells. This only partial colocalization was also confirmed in the epididymis of rats that were treated with the microtubule-disrupting agent colchicine. We have previously shown that colchicine induces a marked redistribution of H+-ATPase-containing vesicles throughout the cytoplasm (7). Under these conditions, a better visualization of each vesicle is achieved at the resolution provided at the light microscopic level. In these tissues, a clear mixture of ClC-5-rich vesicles, H+-ATPase-rich vesicles, and vesicles that contained both proteins was observed. ClC-5 might be involved in the acidification of a subpopulation of H+-ATPase-containing vesicles, while another unidentified conductance is involved in the acidification of the remaining vesicles. Alternatively, the absence of ClC-5 channels in a subpopulation of H+-ATPase-containing vesicles might be an important factor in keeping these vesicles in a less acidified state. In addition, colchicine might have induced some changes in the composition of the vesicle population and might have induced an apparent mislocalization of ClC-5 and H+-ATPase into separate vesicle populations. Clearly, further experiments are needed to address these questions.

In contrast to ClC-5, ClC-3 is highly expressed in the apical BBM of most epididymal principal cells and does not appear to be present in intracellular structures. We have previously shown that the C1 antibody also labels the apical membrane of kidney proximal tubule cells (37). Previous studies regarding the physiological function of ClC-3 have produced conflicting results (38, 51, 56). Whereas early reports indicated that ClC-3 is responsible for plasma membrane chloride transport during cell volume regulation, more recent studies have shown that ClC-3 is present in endosomes and that it facilitates the acidification of synaptic vesicles in a manner similar to ClC-5 in endosomes (51). In addition, ClC-3 mRNA was observed in type B intercalated cells, and ClC-3 was postulated to be the chloride channel that may serve to counteract the ion imbalance caused by the secretion of bicarbonate via the apical Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (41). ClC-3 was recently reported to be present at the apical surface of a subpopulation of Caco-2 cells (40) and in the canalicular membrane of rat hepatocytes (49). In the present study, the apical membrane localization of ClC-3 might indicate that this channel is involved in transepithelial chloride transport in the epididymis and vas deferens. The level of expression of this channel varied in different epididymal regions, being highest in the initial segments, intermediate zone, and caput epididymidis and weakest in the corpus epididymidis. Interestingly, no staining was observed in principal cells from the portion of the cauda epididymidis that is adjacent to the vas deferens, indicating that chloride transport might be reduced in these regions. Chloride reabsorption occurs in the epididymis, and the chloride concentration progressively decreases as the luminal fluid transits through the epididymis (33). The bulk of chloride reabsorption occurs in the proximal regions of the epididymis including the initial segments, intermediate zone, and caput epididymidis, where luminal chloride concentration falls from 118 mM in the seminiferous tubules to 31 mM in the caput epididymidis (33). The high expression of ClC-3 correlates with the high rate of chloride reabsorption in these regions. Between the corpus and the cauda epididymidis, chloride concentration remains constant at around 24 mM (33), which also correlates with the low expression or absence of ClC-3 in these regions. In the vas deferens, where ClC-3 is highly expressed, a further decrease in chloride concentration is achieved (33). These results indicate that ClC-3 might be involved in net transepithelial chloride transport in the epididymis and vas deferens.

Both ClC-3 and ClC-5 are voltage dependent, and when these channels are expressed in the plasma membrane of Xenopus oocytes or CHO-K1 cells they result in a strong outwardly directed current (17, 19, 34, 48). The properties of these channels, in situ, are not yet characterized. However, assuming that they possess the same strong outwardly rectifying characteristics in their native tissue and membrane domains, these channels would not be expected to carry significant currents at physiological transmembrane potentials (reviewed in Ref. 56). Clearly, more experiments are required to reconcile the electrical properties of both of these channels with their proposed physiological roles.

In summary, our results show that ClC-5 is expressed exclusively in H+-ATPase-rich cells (narrow and clear cells) of the epididymis and vas deferens, where it might be an important (although partial) player in the acidification of endosomes. ClC-3 is present in the apical plasma membrane of principal cells and does not appear to be located in intracellular vesicles. ClC-3 is likely to mediate transepithelial chloride transport because its expression correlates with the regions of the epididymis and vas deferens that possess a high rate of chloride reabsorption.


    ACKNOWLEDGEMENTS

We thank Dennis Sgroi for invaluable assistance with the laser capture microdissection and molecular biology studies.


    FOOTNOTES

* C. Isnard-Bagnis and N. Da Silva contributed equally to this work.

This work was supported by National Institutes of Health Grants HD-40793 (to S. Breton) and DK-38452 (to S. Breton and D. Brown). A. S. L. Yu was supported by an American Heart Association, New England Affiliate, Grant-In-Aid. C. Isnard-Bagnis was partially supported by research fellowships from the Institut National de la Santé et de la Recherche Médicale, the Foundation Arthur Sachs (as part of the Fulbright Program), l'Assistance Publique, and the Association des Femmes Diplomées des Universités. The microscopy core facility used in this work was partially supported by a center grant for the study of inflammatory bowel disease (DK-43351) and a Boston Area Diabetes Endocrinology Research Center Grant (DK-57521).

Address for reprint requests and other correspondence: S. Breton, Massachusetts General Hospital East, Renal Unit, 149 13th St., Charlestown, MA 02129 (E-mail address: sbreton{at}receptor.mgh.harvard.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.

10.1152/ajpcell.00374.2001

Received 6 August 2001; accepted in final form 3 September 2002.


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ABSTRACT
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RESULTS
DISCUSSION
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Am J Physiol Cell Physiol 284(1):C220-C232
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