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Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase

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Proc. Nati. Acad. Sci. USA Vol. 86, pp , July 1989 Cell Biology Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar
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Proc. Nati. Acad. Sci. USA Vol. 86, pp , July 1989 Cell Biology Subtypes of intercalated cells in rat kidney collecting duct defined by antibodies against erythroid band 3 and renal vacuolar H+-ATPase (kidney band 3/bicarbonate transport/anion antiporter/proton pump/acid/base balance) S. L. ALPER*t, J. NATALEt, S. GLUCK, H. F. LODISH*, AND D. BROWN: twhitehead Institute, Cambridge, MA 02142; *Renal Unit and Division of Molecular Medicine, Beth Israel Hospital, Boston, MA 02115; trenal Unit, Massachusetts General Hospital, Charlestown, MA 02129; Renal Division, Jewish Hospital, and Department of Cell Biology, Washington University, Saint Louis, MO 63110; and IDepartment of Biology, Massachusetts Institute of Technology, Cambridge, MA Contributed by H. F. Lodish, April 27, 1989 ABSTRACT The cellular distributions of the kidney form of the erythrocyte band 3 chloride/bicarbonate exchanger and the kidney vacuolar H.transporting ATPase were examined in rat kidney collecting duct by immunocytochemical stining of adjacent semithin sections. Polyclonal anti-peptide antibodies directed against two regions of murine erythroid band 3 gave a pattern of basolateral labeling similar to that seen with antibodies directed against the entire protein. In the medullary collecting duct almost all intercalated cells expressed basolateral membrane band 3 and displayed apical membrane H+- ATPase. In the cortical collecting duct and the connecting segment, band 3 labeling was restricted to a subpopulation of intercalated cells. In the cortical collecting duct 46% of intercalated cells had apical H+-ATPase and basolateral band 3. Cells that had either basolateral or diffuse cytoplasmic staining for H+-ATPase were all band 3-negative and accounted for 53% of the intercalated cells. In addition, occasional intercalated cells with apical H+-ATPase appeared to lack basolateral band 3. These results demonstrate the coexpression of H+- ATPase and band 3 in opposite plasma membrane domains of a subpopulation of intercalated cells that are probably the acid-excreting (type A) cells. All other intercalated cells lacked immunoreactive band 3 and probably include the bicarbonateexcreting (type B) cells. The mammalian kidney achieves systemic acid/base balance by reabsorption offiltered bicarbonate in the proximal tubule (1, 2) and by reabsorption (3-5) or excretion of bicarbonate (6-8) in the collecting duct. The balance between net absorption and net excretion of bicarbonate in the collecting duct is subject to regulation by the acid/base status and mineralocorticoid status of the animal (6-8). The mitochondria-rich intercalated cells of the collecting duct are specialized for proton and bicarbonate transport and have been subtyped based on their morphological characteristics. By analogy with the turtle urinary bladder, in which a and 8 intercalated cells, respectively, are believed to excrete protons and bicarbonate into the urine (9), so-called A and B intercalated cells have been similarly defined in studies of isolated perfused rat and rabbit collecting ducts (5-8). Immunocytochemical characterization ofintercalated cells has revealed subtypes that either exhibit or lack immunoreactive band 3 protein in their basolateral membranes (10-14). The transport of bicarbonate across the basolateral plasma membrane of type A intercalated cells appears to be carried out by products of alternative transcripts of the erythroid band 3 gene (15-17). The kidney band 3 protein encoded by The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C solely to indicate this fact. the principal renal transcript lacks the 79 N-terminal residues of erythrocyte band 3 (17, 18). More recently, intercalated cell subtypes with either an apical, a basolateral, or a diffuse distribution of immunoreactive vacuolar H+-ATPase have been described (19, 20). Clarification of the relationship between the subtypes of intercalated cells defined immunochemically and those defined physiologically is important for an understanding of the dietary and hormonal regulation of urinary excretion of acid and base. In this study we employed anti-peptide antibodies against the erythroid anion exchanger band 3 and the renal vacuolar H -ATPase to characterize further intercalated-cell heterogeneity in the rat kidney. We demonstrate the colocalization of apical plasma membrane H+-ATPase and basolateral plasma membrane kidney band 3 in almost all intercalated cells of rat medullary collecting duct and in a subtype of intercalated cells of rat cortical collecting duct; these are presumably the acid-excreting A cells. In contrast, at least two definable subtypes of intercalated cells in the cortical collecting duct and connecting segment appear to lack kidney band 3. These include the base-excreting type B cells and a different, novel subtype of intercalated cell. MATERIALS AND METHODS Materials. 251I-labeled protein A was purchased from Amersham. Nitrocellulose membranes were from Schleicher & Schuell. LX-112 embedding resin was from Ladd Research Industries (Burlington, VT). Fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG was obtained from Kirkegaard and Perry Laboratories (Gaithersburg, MD). Keyhole limpet hemocyanin was from Calbiochem. Gelvatol was from Polysciences. n-propyl gallate was from Sigma. Sephadex G-25 was from Pharmacia. Succinimidyl 4- (N-maleimidomethyl)cyclohexane-1-carboxylate and m- maleimidobenzoic N-hydroxysuccinimide ester were obtained from Pierce. Affi-Gel 15 was from Bio-Rad. Antiserum to purified murine erythrocyte band 3 has been described (21). Polyclonal antiserum to the cytoplasmic (22) C-terminal dodecapeptide of murine band 3 was a gift of R. Kopito (23, 24). Polyclonal antisera to the bovine renal medullary vacuolar H -ATPase and to individual subunit polypeptides of the H+-ATPase were prepared as described (25). Preparation of Anti-Band 3 Anti-Peptide Antibodies. Synthetic peptides comprising amino acids and of murine erythroid band 3 (26), and each containing a C-terminal cysteine, were prepared by James Burton (University Hospital, Boston) by solid-phase synthesis using HCl/dioxane methodology. The crude peptides were dis- Abbreviations: B3RP; band 3-related protein; FITC, fluorescein isothiocyanate. 5429 5430 Cell Biology: Alper et al. solved in 10o acetic acid and purified on a Sephadex G-25 column (200-ml bed volume). Limit acid hydrolysates of the purified peptides were subjected to amino acid analysis by Paul Matsudaira (Whitehead Institute, Cambridge, MA) to confirm their identities. Each peptide (3-5 mg) was crosslinked to keyhole limpet hemocyanin with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the coupled peptide was mixed with complete Freund's adjuvant. New Zealand White rabbits were immunized by subcutaneous injection and were given booster injections of coupled peptide in incomplete Freund's adjuvant after 1 month and at 3-week intervals thereafter. Sera were tested by peptide ELISA and subsequently by Western immunoblotting (23, 27) with detection by 125I-labeled protein A autoradiography. Sera were affinitypurified either by adsorption to nitrocellulose strips (28) containing murine erythroid band 3 or by affinity chromatography over Affi-Gel 15 coupled to a peptide-albumin conjugate prepared by crosslinking with m-maleimidobenzoic N-hydroxysuccinimide ester. Preparation and Analysis of Erytbrocyte Membranes. CD-1 mice were bled from the tail vessels into phosphate-buffered saline (PBS: 0.9% NaCl/10 mm sodium phosphate, ph 7.4) containing heparin. After removal of the buffy coat, ghosts were prepared by lysis at 4 C in 50 volumes of 5 mm sodium phosphate, ph 8.0/1 mm phenylmethylsulfonyl fluoride. Protein was assayed by the bicinchoninic acid (BCA) method (29). SDS/PAGE was performed by the method of Laemmli (30). Tissue Preparation. Adult Sprague-Dawley rats were anesthetized with Inactin (Byk-Guiden Pharmazeutika), and their kidneys were perfused via the left ventricle, first with Hanks' balanced salt solution for 1-2 min and then with a fixative containing 2% paraformaldehyde, 10 mm sodium periodate, and 75 mm lysine (31) for 10 min. Kidneys were removed and immersed overnight in the same fixative at 4 C. Pieces of kidney from various kidney regions were separated and embedded in LX-112 resin without postfixation in osmium. Immunocytochemistry. Semithin (1,m) sections were cut from tissue embedded in LX-112 and were treated for 2 min with a mixture of 2 g of KOH, 5 ml of propylene oxide and 10 ml of methanol to remove the resin (32). Sections were incubated for 10 min in PBS containing 1% bovine serum albumin to reduce nonspecific background staining and then for 2 hr at room temperature with a 20-pl drop of the indicated kda Proc. Natl. Acad. Sci. USA 86 (1989) dilution of antiserum. After three 5-min rinses in PBS, sections were incubated for 1 hr with FITC-coupled goat anti-rabbit IgG (diluted 1:30) for detection of antigenic sites by immunofluorescence microscopy. Sections were rinsed three times for 5 min in PBS and mounted in glycerol/pbs (1:1, vol/vol) containing 4% n-propyl gallate to retard quenching of the fluorescence (33). Photographs of sections were taken on Kodak Tri-X pan film with an Olympus BHS 68 - it FIG. 1. Autoradiograph of Western blot of mouse erythrocyte ghost proteins probed with antibodies to band 3, followed by 125I-labeled protein A. Lanes: 1, preimmune a177 (diluted 1:50); 2, peptide column-purified a177 (1:50); 3, preimmune a178 (1:50); 4, peptide column-purified a178 (1:50); 5, preimmune ab3 (1:200); 6, ab3 serum (1:200); 7, act serum (1:200). FIG. 2. Consecutive semithin sections of LX-112-embedded rat kidney showing a collecting duct from the inner stripe of the outer medulla. The sections were incubated with anti-band 3 antibody ab3 (a), a178 (b), or act (c) followed by FITC-labeled goat anti-rabbit second antibody. The same intercalated cells can be found in a-c, and all three antibodies gave an identical pattern of basolateral staining in these cells. (Bars = 20 j.m.) Cell Biology: Alper et al. FIG. 3. Adjacent semithin sections of LX-112-embedded rat kidney showing a collecting duct from the inner stripe of the outer medulla. Incubation with affinity-purified antibody to the 31-kDa subunit of the H+-ATPase gave an exclusively apical staining of the intercalated cells in this segment of the urinary tubule (a). In contrast, incubation with an antibody against band 3 (a178) resulted in staining of the basolateral plasma membranes of the same intercalated cells (b). Principal cells were unstained with either antibody. (Bars = 20,um.) photomicroscope equipped for epifluorescence (Marcon Instrument, Norwood, MA). For the quantitation in Table 1, some sections from each animal were double-stained with anti-band 3 antibody and FITC-labeled goat anti-rabbit IgG in a first step, followed by anti-h+-atpase antibody and Texas red-labeled goat antirabbit IgG in a second step. Despite the fact that both primary antibodies were raised in the same species, kidney band 3 staining could be easily distinguished from H+-ATPase staining by using the appropriate filter combinations. The different labeling patterns were directly quantified under the microscope. Proc. Natl. Acad. Sci. USA 86 (1989) 5431 RESULTS Antibody Specificity. We used four antibodies raised against three synthetic peptides corresponding to defined regions of murine band 3: a178, against residues , in the middle of the N-terminal cytoplasmic domain; a176 and a177, against residues , an externally disposed region of the transmembrane domain; and act, against the cytoplasmic C-terminal dodecapeptide. The Western blot of erythrocyte ghost membrane proteins (Fig. 1) indicated that all three anti-peptide antisera specifically detected band 3, as did the antiserum directed against the intact protein (ab3). The result was the same when nitrocellulose afflinity-purified a178, a176 (data not shown), and act (23) were used. The different anti-band 3 antisera recognized epitopes of identical distribution in the kidney. In serial semithin sections of rat kidney medulla, the ab3, a178, and act antisera stained the basolateral surface of the same intercalated cells (Fig. 2). However, no signal was detected in kidney sections with antibodies a176 or a177, whether on embedded or frozen tissue sections (data not shown). These data support published evidence (10-14, 24) that the immunoreactive band 3 of the intercalated cell basolateral membrane, as well as the functional anion exchanger of purified medullary collecting duct cells (34), is highly similar or identical to erythroid band 3 throughout most of its length. We refer to this immunoreactive band 3 as kidney band 3 (17, 18). Colocalization of Kidney Band 3 and H+-ATPase. Adjacent semithin sections of rat kidney outer medulla (Fig. 3) and cortex (Figs. 4 and 5) were incubated with antibodies to H+-ATPase and to band 3. Intercalated cells were defined by their darker, more granular appearance in phase-contrast microscopy and by their characteristic staining patterns with antibody to H+-ATPase (19, 20). Table 1 summarizes the immunologic characteristics of 1375 intercalated cells examined in double-stained sections of kidney from three ad libitum-fed animals. Similar results were obtained from examination of 4675 additional total tubular cells in semithin sections (from three additional animals) stained with single antibodies. In the inner stripe of the outer medulla all intercalated cells were of type A, characterized by apical H+-ATPase and basolateral kidney band 3. Fig. 3 shows adjacent sections of medullary collecting duct of the inner FIG. 4. Adjacent semithin sections of LX-112-embedded rat kidney showing a connecting tubule from the cortex. Immunolabeling with affinity-purified antibodies against the 31-kDa subunit of the H+-ATPase proton pump (a) resulted in a heavy apical staining in some intercalated cells (arrows), while other intercalated cells showed a predominantly basolateral staining, with some diffuse staining of the cytoplasm (arrowheads). When the same cells were stained with anti-band 3 antibody a178 (b), the cell showing apical proton pumps had basolateral immunoreactive band 3 sites (arrows), whereas no band 3 staining was detected in the intercalated cells with basolateral proton pumps (arrowheads). Note that a delicate staining ofproton pumps was also detected in the apical plasma membrane ofother connecting tubule epithelial cells, and that proton pumps were also present at the base of the brush border in the proximal tubule (upper left). The bright spots in the cytoplasm of proximal tubules in both a and b represent autofluorescent lysosomes. (Bars = 7,um.) 5432 Cell Biology: Alper et al. Proc. Natl. Acad. Sci. USA 86 (1989) FIG. 5. Adjacent semithin sections of LX-112-embedded rat kidney cortex showing a cortical collecting tubule immunolabeled with affinity-purified antibody to the 31-kDa subunit of the H+-ATPase (a) or anti-band 3 antibody act (b). In addition to cells showing apical proton pumps (a) and basolateral band 3 immunoreactive sites (b), some intercalated cells had apical proton pumps (arrows in a) but no detectable band 3 on their basolateral plasma membrane (arrowheads in b). These cells were rare in our normal rats, comprising 1% of the total intercalated cell population. As described for Fig. 4, a band of proton pump antigenicity was located at the base of the brush border in proximal tubules (upper left), but the bright spots in the same tubules represent autofluorescent lysosomes. (Bars = 12 gm.) stripe, exhibiting six such type A intercalated cells. The neighboring principal cells were not stained with either antibody. In the outer stripe of the outer medulla (figure not shown) 88% of the intercalated cells were of type A. The remaining 12% were type B, characterized by a basolateral and/or diffuse distribution of H+-ATPase and an apparent absence of kidney band 3. These type B intercalated cells were all located in the juxtacortical region of the outer stripe. In the cortex, type A intercalated cells comprise 46%, while type B intercalated cells represented almost all of the remaining 54%. Fig. 4 shows a cross-section of a cortical connecting segment that displays a type A (arrows) and a type B intercalated cell (arrowheads) in close proximity. The same mix of intercalated cell types is present in the cortical collecting duct (data not shown). Fig. 5 shows two examples of the third variety of intercalated cell (arrows), infrequently found in the cortical collecting duct and connecting segment. The cells display apical H+-ATPase (arrows) but no detectable basolateral band 3 (arrowheads) and comprise 1% of the intercalated cells of the cortex. Kidney band 3 is absent from all non-intercalated cells that display H+-ATPase (20). Evident examples are in Fig. 4, in which proximal tubule cells show H+-ATPase at the base of the brush border, while connecting segment cells show a thin rim of H+-ATPase at their apical membrane. Table 1. Intercalated cell types defined by immunocytochemistry No. of cells (%) Location Total Type A Type B Novel Cortex (45.9) 295 (53.2) 5 (0.9) Medulla Outer stripe (88.0) 30 (12.0) 0 Inner stripe (100) 0 0 Immunocytochemical staining patterns of 1375 intercalated cells were examined in semithin LX-122 sections of rat kidney from three ad libitum-fed animals. Kidney band 3 was detected with the act serum. H+-ATPase was detected in the same double-stained section with antibody to the 56-kDa subunit. Cortex included both cortical collecting duct and connecting segment. The outer stripe and inner stripe of the outer medulla were defined by gross anatomical landmarks. Total intercalated cells were defined by their characteristic staining with H+-ATPase antibody (19) and represented 43.6% of total tubular cells. All percentage values (in parentheses) had SEM of 1.5% or less for three animals. DISCUSSION Vectorial excretion of acid or base by specialized epithelial cells requires several biochemical components: (i) intracellular carbonic anhydrase is necessary to generate H+ and HCOj; (ii) an acid-extruding mechanism must function on only one side of the cell; and (iii) a base-extruding mechanism must be present only on the opposite plasma membrane. These individual components of the acid/base excretory system have been examined previously in the mammalian collecting duct by immunocytochemistry. In separate studies, carbonic anhydrase (35-37), immunoreactive band 3 (10-14, 24), and the vacuolar H+-ATPase (19, 20) were detected in intercalated cells. Carbonic anhydrase was present in the intercalated cell cytoplasm (35-37) and, at least in some rabbit cells (37), apparently associated with the plasma membrane. Immunoreactive band 3 was localized to the basolateral membrane of only some intercalated cells (11, 12, 14). Band 3-negative intercalated cells in the rabbit cortical collecting duct were characterized by peanut lectin-labeling of their apical membranes (11). H+-ATPase was found in apical or in basolateral membranes of individual rat intercalated cells and was present diffusely throughout the cytoplasm of some cells (19, 20). By examination ofadjacent semithin sections of rat kidney, we have united the separate classifications of intercalated cells by their patterns of expression of H+-ATPase and of kidney band 3. We have found that 99% of intercalated cells with apical H+-ATPase also express kidney band 3 and restrict it to their basolateral membranes. These cells comprise all the intercalated cells in the inner stripe of the rat outer medullary collecting duct, 88% of the intercalated cells in the outer stripe, and 46% of the intercalated cells in the cortical collecting duct. We believe that t
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