Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Charlestown 02129; and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
Submitted 12 February 2003 ; accepted in final form 12 February 2004
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ABSTRACT |
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vacuolar H+-ATPase B subunit; intercalated cells; clear cells; urogenital tract; immunofluorescence
V-ATPases in collecting duct (CD) IC are critical for the regulation of body acid-base balance (13, 21), whereas those in the male reproductive tract maintain an acidic luminal pH at which spermatozoa mature and are stored (1, 24). Osteoclast V-ATPases play a vital role in bone reabsorption (5, 26), and V-ATPases in the inner ear are involved in maintaining the high K+ level of the endolymph that is essential for hearing (34). A variety of cell types in lower vertebrates and invertebrates also use plasma membrane V-ATPase to energize other ion transport processes (37). Furthermore, cell types in addition to IC express plasma membrane V-ATPase in the kidney; these include epithelial cells of the thick ascending limb of Henle (TAL), the distal convoluted tubule (DCT), the connecting segment (CS), and the proximal tubule (PT) (15, 26, 29). Thus the V-ATPase has a fairly widespread expression on plasma membranes, where it plays distinct but well-defined physiological roles in addition to its more ubiquitous function in the acidification of intracellular compartments.
The V-ATPase is a complex enzyme that is composed of many distinct subunits (13, 20, 28, 30). The holoenzyme can be divided into two parts or sectors. One is composed of transmembrane subunits (the V0 sector), whereas the other forms a large cytosolic domain (the V1 sector) that is anchored to the plasma membrane via its interaction with the V0 sector. Considerable effort has been expended to understand the role of the individual subunits within the enzyme, as well as the role of different isoforms of some subunits that have been identified in different cells and tissues. One of these, the B subunit, is a component of the catalytic V1 domain, the peripheral complex that contains the ATP hydrolysis site of the enzyme. First cloned as a 57-kDa subunit from Neurospora crassa (6) and Arabidopsis (27), the B subunit is known to be expressed in two highly homologous 56-kDa isoforms, B1 and B2, in animal tissues. B1, initially described as a "kidney" isoform, is expressed at high levels in certain epithelial cells that are specialized for regulated proton transport, including the V-ATPase-rich cells of the kidney CD (29) and epididymis (9). This isoform was also found in some tissues outside the urogenital tract, including the eye (36), inner ear (34), lung, and placenta (31). On the other hand, B2, originally referred to as the "brain" isoform, is expressed in most tissues and is considered to be ubiquitous (29, 31). However, in contrast to other specialized proton-secreting cells, osteoclasts express high levels of B2 on their bone-reabsorbing surface. The polarization of the V-ATPase B2 isoform to the ruffled membrane presumably indicates its involvement in regulating proton secretion and bone reabsorption by osteoclasts (26).
The relative distribution of the B1 and B2 isoforms in proton-secreting urogenital epithelial cells has not been examined in depth. In particular, it is unclear whether both isoforms are coexpressed in IC and epididymal clear cells and whether they have similar or distinct intracellular localizations. It has been proposed that the particular isoform composition of the V-ATPase within a cell or tissue might be an important factor in determining the intracellular location and/or function of the V-ATPase (22, 35). Furthermore, recent studies on a knockout mouse that lacks the B1 isoform of the V-ATPase have shown not only that the mouse is viable but also that it does not develop detectable systemic acidosis when allowed unrestricted access to food and water (19). Furthermore, the mice appear to be fertile (K. E. Finberg, Dept. of Pathology, Massachusetts General Hospital, personal communication). This suggests that normal V-ATPase function might be maintained by the presence of the B2 isoform in proton-secreting cells of the urogenital tract.
To investigate whether the 56-kDa B2 subunit of the V-ATPase could potentially play a role in the regulation of proton transport in kidney and epididymis epithelial cells, we raised isoform-specific affinity-purified rabbit polyclonal antibodies and used them to study the distribution of the two B subunit isoforms in these organs. We found the 56-kDa B2 subunit of the V-ATPase to be expressed not only in the kidney proximal convoluted tubule and TAL, but also in the DCT, CS, and both A- and B-type IC of the CD in both species. The B2 isoform was also detected in all proton-secreting cells of the epididymis in both rat and mouse. Under baseline conditions, the B2 isoform was found predominantly on intracellular vesicles, but under some conditions, such as chronic carbonic anhydrase inhibition, it was also expressed on the apical plasma membrane of A-IC cells in the kidney. These data indicate that the B2 isoform can be integrated into the V-ATPase holoenzyme in specialized proton-secreting cells. It could, therefore, play a role not only in the acidification of intracellular organelles but also in transepithelial proton secretion and the maintenance of acid-base homeostasis.
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MATERIALS AND METHODS |
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For the peptide competition assay, the peptides against which the B1 and B2 antibodies were raised were dissolved in phosphate-buffered saline (PBS) containing 0.02% (wt/vol) sodium azide. Each antibody was preincubated in the presence of a 10-fold (wt/wt) excess of the respective immunizing peptide for 1 h at room temperature before the immunofluorescence staining protocol described in Immunofluorescence and confocal microscopy. Protein concentration in the peptide and antibody samples was determined using a SmartSpec 3000 spectrophotometer (Bio-Rad Laboratories, Hercules, CA).
For all anti-V-ATPase antibodies described, the immunizing peptides were synthesized by the Massachusetts General Hospital Peptide/Protein Core Facility and the respective antibodies were raised commercially (Cocalico Biologicals, Reamstown, PA). Each antibody was affinity purified from whole serum with the respective immunizing peptide by using the SulfoLink kit (Pierce, Rockford, IL) according to the manufacturer's instructions.
A monoclonal antibody against the 28-kDa calcium-binding protein calbindin (mouse IgG1 isotype; Sigma-Aldrich, St. Louis, MO) was used at a final concentration of 14 µg/ml as a kidney connecting segment marker.
The following affinity-purified secondary antibodies were used: indocarbocyanine (Cy3)-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) at a final concentration of 1.5 µg/ml, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories) at 25 µg/ml, FITC-conjugated donkey anti-chicken IgY (Jackson ImmunoResearch Laboratories) at 25 µg/ml, and FITC-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) at 8.3 µg/ml.
Tissue preparation. Adult male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) and adult mice (C57BL6 x CBA F1 strain; Jackson Laboratory, Bar Harbor, ME) were anesthetized with pentobarbital sodium (Nembutal, 65 mg/kg body wt ip; Abbot Laboratories, North Chicago, IL) and perfused through the left cardiac ventricle with PBS (0.9% NaCl in 10 mM phosphate buffer, pH 7.4) followed by paraformaldehyde-lysine-periodate fixative (PLP: 4% paraformaldehyde, 75 mM lysine-HCl, 10 mM sodium periodate, and 0.15 M sucrose in 37.5 mM sodium phosphate). Both kidneys were removed and sliced, and both epididymides were harvested from each animal. The organs were further fixed by immersion in PLP for 4 h at room temperature and overnight at 4°C, extensively rinsed in PBS, and stored at 4°C in PBS containing 0.02% sodium azide until use. Where appropriate, one epididymis and one kidney were harvested and frozen in liquid nitrogen for Western blotting (see Protein extraction and Western blotting).
Immunofluorescence and confocal microscopy. Tissues prepared as described in Tissue preparation were cryoprotected in PBS containing 0.9 M sucrose overnight at 4°C and then embedded in Tissue-Tek OCT compound 4583 (Sakura Finetek USA, Torrance, CA), mounted on a cutting block, and frozen at 27°C. Sections (4 µm) were cut on a Reichert-Jung 2800 Frigocut cryostat (Leica Microsystems, Bannockburn, IL), collected onto Superfrost Plus precleaned, charged microscope slides (Fisher Scientific, Pittsburgh, PA), air-dried, and stored at 4°C until use.
Sections were rehydrated in PBS for 10 min and treated with 1% (wt/vol) SDS for 4 min for retrieval of antigenic sites, as previously described (18). Sections were subsequently washed three times for 5 min in PBS and incubated for 10 min in 1% (wt/vol) bovine serum albumin (BSA) in PBS with 0.02% sodium azide to prevent nonspecific staining, followed by a 90-min incubation in the primary antibody at room temperature. After three 5-min PBS washes, the secondary antibody was applied for 1 h at room temperature, and the slides were then rinsed again in PBS three times for 5 min. Slides were mounted in a 1:1 mixture of Vectashield medium (Vector Laboratories, Burlingame, CA) and 1.5 M Tris solution (pH 8.9). For dual staining with antibodies raised in different species, the primary antibodies were applied sequentially at the appropriate concentrations as described in Antibodies, with each primary antibody being followed by the corresponding secondary antibody.
Digital images were acquired by using a Nikon Eclipse 800 epifluorescence microscope (Nikon Instruments, Melville, NY) with an Orca 100 charge-coupled device camera (Hamamatsu, Bridgewater, NJ). Images were then analyzed by using IPLab version 3.2.4 scientific image processing software (Scanalytics, Fairfax, VA) and were imported into and printed from Adobe Photoshop version 6.0 image-editing software (Adobe Systems, San Jose, CA).
For confocal laser scanning microscopy, tissue sections were prepared as described. Confocal imaging was performed on a Radiance 2000 confocal microscopy system (Bio-Rad Laboratories) using LaserSharp 2000 version 4.1 software, and images were edited as described above.
Immunogold electron microscopy. Small pieces of rat kidney medulla prepared as described were cryoprotected in PBS containing 2.3 M sucrose. Ultrathin cryosections were cut on a Leica EM FCS at 80°C and collected onto Formvar-coated nickel grids. Sections were blocked on drops of 1% BSA in PBS for 10 min at room temperature and then incubated on drops of primary anti-B2 V-ATPase antibody or DAKO diluent alone (DAKO, Carpinteria, CA) for 2 h at room temperature. After being rinsed on drops of PBS, the grids were incubated on drops of goat anti-rabbit IgG secondary antibody coupled to 10-nm gold particles (Ted Pella, Redding, CA) for 1 h at room temperature. After being rinsed on drops of distilled water, the grids were stained on drops of uranyl acetate-tylose mixture for 10 min on ice and then collected on loops and allowed to dry. Sections were examined in a Philips CM 10 transmission electron microscope at 80 kV.
Protein extraction and Western blotting. Kidney and epididymis tissues from rats and mice were cut into smaller pieces and disrupted with a Tenbroeck tissue grinder in 3 ml of homogenization buffer [320 mM sucrose, 10 mM HEPES-KOH, pH 7.2, 1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, and Complete protease inhibitors from Roche Applied Science (Indianapolis, IN)]. Homogenates were centrifuged for 10 min at 1,000 g at 4°C. Triton X-100 was added to the supernatant to a final concentration of 1%. After a second homogenization and centrifugation for 30 min at 16,000 g at 4°C, the supernatant was collected and the protein concentration was determined with the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) using albumin as standard. Protein extracts were aliquoted and stored at 80°C. Protein (2030 µg) was diluted in Laemmli reducing sample buffer, boiled for 5 min, and loaded onto Tris-glycine polyacrylamide 420% gradient gels (Cambrex, Rockland, ME). After SDS-PAGE separation, proteins were transferred onto an Immun-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories). Membranes were blocked in Tris-buffered saline (TBS) with 5% nonfat dry milk and then incubated overnight at 4°C with the primary antibody diluted in TBS with 2.5% milk. For competition experiments, the primary antibody was incubated for 1 h with a 10-fold excess of the corresponding peptide before the overnight incubation with the membrane. After four washes in TBS with 0.1% Tween 20 (TBST), membranes were incubated with a donkey anti-rabbit IgG antibody conjugated to horseradish peroxidase for 1 h at room temperature. After four further washes, antibody binding was detected with the Western Lightning chemiluminescence reagent (Perkin Elmer Life Sciences, Boston, MA).
Immunoprecipitation. Epididymis homogenate (8001,000 µg) was incubated overnight at 4°C with a chicken antibody raised against the E subunit of the V-ATPase (see Antibodies) in a buffer containing 20 mM HEPES-KOH, pH 7.2, 100 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and protease inhibitors. Anti-chicken IgY (50 µl) immobilized on agarose beads (Promega, Madison, WI) was added. After another hour of incubation at 4°C, proteins bound to the beads were recovered by centrifugation for 30 s at 500 g. Beads were washed four times in the immunoprecipitation buffer containing 0.5% Triton X-100, resuspended in 30 µl of 2x Laemmli reducing sample buffer, and boiled for 5 min. After a brief centrifugation, supernatants were loaded on a polyacrylamide gel and analyzed as described Protein extraction and Western blotting.
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RESULTS |
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To further characterize V-ATPase expression in the CS and DCT, we performed a dual immunostaining experiment by using antibodies against the B2 and E subunits of the V-ATPase. In both CS (Fig. 5, AC) and DCT (Fig. 5, DF), most cells stained for both subunits (B2, red, and E, green). DCT cells expressed E V-ATPase localized mostly apically and subapically (Fig. 5E), where it colocalized with B2 (Fig. 5D). Unlike DCT cells, rat kidney CS cells exhibited a more diverse, mosaic-like pattern of staining for both B2 (Fig. 5A) and E subunits (Fig. 5B). Whereas most B-IC with distinct basolateral E subunit staining in the CS showed weak basolateral B2 staining, as in the CD (see below), cells with a more intense basolateral B2 staining were sometimes encountered (Fig. 5, AC, insets). In contrast, apical B2 staining was detectable in most cells in the CS.
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Detection of V-ATPase B1 and B2 subunits in the epididymis by indirect immunofluorescence. Cryostat sections of PLP-fixed rat cauda epididymis were stained using antibodies raised against the B1 and B2 subunits of the V-ATPase. Double labeling for B2 and E subunits of the V-ATPase in rat cauda epididymis confirmed that B2 expression is strongest in the previously described (810) subunit B1- and E-positive clear cells (Fig. 10).
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DISCUSSION |
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In the CD, the B2 isoform was found predominantly in the cytoplasm under baseline conditions. Some A-IC, however, showed a tight apical localization of B2, consistent with plasma membrane staining, as previously described for the B1 isoform (4, 29, 33). In these cells from control animals, B2 was colocalized apically with the 31-kDa E subunit. This apical membrane expression was confirmed by immunogold electron microscopy. The extent of this apical localization was greatly enhanced in rats treated with acetazolamide, which we have previously shown to "activate" A-IC by increasing apical V-ATPase insertion (2). Apical insertion of the B1 isoform is also greatly increased by acetazolamide treatment (2). We conclude that though the V-ATPase holoenzyme containing the B2 isoform is mainly found in the cytoplasm, where it plays a role in the acidification of intracellular organelles, it could also contribute to apical proton secretion in renal epithelia under some conditions. A proton-secretory role for the B2-containing V-ATPase has already been shown in osteoclasts, which do not express the B1 isoform (26).
In contrast to the strong apical membrane localization of B2 in some A-IC, basolateral plasma membrane expression of B2 in B-IC of the cortical CD was weak or absent, even in those cells in which the E subunit of the V-ATPase stained the basolateral pole of the cells more strongly than other cellular domains. However, in CS, distinct basolateral coexpression of the B2 and E subunits was found in a few cells. Furthermore, after acetazolamide treatment of rats, some B-IC in CD had a weak but distinct basolateral B2 staining. These results imply that the basolateral targeting and/or retention of the B2 subunit in B-IC may be less efficient than that of the B1 subunit. Interestingly, we previously reported that NHE-RF1 (Na+/H+ exchanger regulatory factor), a member of the PDZ family of proteins, was expressed in B-IC but not A-IC (11) and proposed that NHE-RF1 might be involved in the bipolar targeting of the B1 V-ATPase subunit in B-IC. However, NHE-RF1 is probably not required for apical V-ATPase targeting, given that it was not detectable in A-IC, in which the plasma membrane V-ATPase is uniquely apical. The weak B2 staining of basolateral membranes of B-IC might, therefore, be related to the absence of the COOH-terminal PDZ-binding sequence from this isoform. In contrast, the COOH terminus of the B1 isoform contains a well-defined DTAL motif that allows it to interact with NHE-RF1 (11), which may permit basolateral accumulation of B1. It is also likely that the level of expression of the B2 subunit in any given IC might also determine our ability to detect low levels of basolateral staining. As previously shown for the E subunit (2), acetazolamide treatment might also upregulate B2 subunit expression, allowing its detection on basolateral membranes under these conditions.
In the epididymis, B2 isoform expression was readily detected in proton-secreting clear cells of rat and mouse tissues. The clear cells are, thus, similar to renal IC with respect to B1 and B2 coexpression in cells specialized for proton secretion. However, convincing colocalization of B2 with the 31-kDa E subunit in the extensive apical microvilli of these cells was not found under control conditions or after acetazolamide treatment of rats. Instead, B2 was always concentrated in a population of subapical vesicles in clear cells in both rat and mouse epididymis. Unlike the B1 subunit, B2 did not colocalize extensively with the E subunit in the apical plasma membrane microvilli. It is conceivable that the B2 isoform might not play the same role in transmembrane proton transport in epididymal clear cells as in kidney IC. Alternatively, it is possible that the acetazolamide treatment to which the animals were subjected in this report could fully activate renal IC but not epididymal clear cells. This is supported by the fact that localization of the B1 isoform was not modified in epididymal clear cells after acetazolamide treatment (data not shown). Further studies on activation of proton secretion in the epididymis are necessary to address these possibilities.
The idea that the B2 isoform can serve as a possible backup or alternative mechanism for the active role played by the B1 isoform in proton secretion is suggested by recent studies on knockout mice lacking the B1 isoform of the 56-kDa subunit. These animals are viable and do not develop detectable systemic acidosis when allowed unrestricted access to food and water. Their urine has, however, a higher pH than that of normal mice (3, 19). These findings suggest that the V-ATPase necessary for distal acidification by IC is at least partially functional, despite the lack of the B1 subunit. Furthermore, male knockout mice lacking the B1 isoform appear to be fertile (K. E. Finberg, personal communication), implying that luminal acidification in the epididymis, which is necessary for sperm maturation and storage, is not critically impaired by the lack of the B1 V-ATPase subunit. Direct examination of the V-ATPase subunit composition of IC and clear cells from these knockout mice is needed to provide more insight into these issues.
The cellular role of V-ATPase holoenzymes with distinct subunit compositions is coming under closer scrutiny with the discovery of increasing numbers of subunits that have different cell- and tissue-specific isoforms. It has been proposed that various V-ATPase subunit aggregations are responsible for differential intracellular localization and different targeting mechanisms and functions of the proton pump (22, 35). Each V1 sector of the V-ATPase contains three B subunits. However, it is not known whether a single V-ATPase holoenzyme complex contains only one type of B subunit isoform or whether "hybrid" complexes containing both B1 and B2 subunits are present in those cells in which the two isoforms are coexpressed. Coimmunoprecipitations with other V-ATPase subunits have not yet offered a definitive answer to this question. Subunits a4, c, d1, E2, and G1 were shown to coprecipitate with both B isoforms, whereas C2-b, d2, and G3 coimmunoprecipitate with B1 alone, and a1, a2, a3, and C1 coprecipitate with B2 alone (35). It is clear, therefore, that B1 and B2 subunits can occur in unique and separate V-ATPase complexes, but whether B1/B2 interaction occurs in yet other manifestations of the holoenzyme has not been specifically addressed.
Besides the A- and B-IC of the CD, the 56-kDa B2 subunit of the V-ATPase is also expressed in the kidney in the proximal convoluted tubule, TAL, DCT, and CS in both rat and mouse. In the TAL, a difference in B isoform expression was seen between the IS of the outer medulla and the rest of this tubule segment. IS TAL cells express only the B2 isoform, whereas both B1 and B2 are clearly detectable in the OS and the cortical TAL regions. A striking apical colocalization of B1 and B2 was also found in distal tubule and CS epithelial cells. The functional relevance of these patterns of B subunit expression remains to be determined.
In summary, our present data show that the B2 subunit of the V-ATPase is expressed in most, if not all, proton-secreting cells in the epithelium lining the nephron and the CD, and it is also present in proton-secreting clear cells of the epididymis. This subunit has a mainly cytoplasmic distribution in most cells under baseline conditions, but apical plasma membrane localization can be observed in a few kidney IC in the renal medulla. This membrane staining is increased in CD IC after chronic acetazolamide treatment, which results in an increased "activity" of these cells. These data indicate that in addition to its role in the acidification of intracellular organelles, the V-ATPase holoenzyme containing the B2 subunit is also poised to play a role in transepithelial proton secretion under some conditions.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* T. G. Punescu and N. Da Silva contributed equally to this work.
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