Proc. Natl. Acad. Sci. USA Vol. 87, pp. 7457-7461, October 1990 Cell Biology
Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells DENNIS BROWN, XIN LIANG ZHU, AND WILLIAM S. SLY Renal Unit, Massachusetts General Hospital and Department of Pathology, Harvard Medical School, Boston, MA 02114; and The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104
Contributed by William S. Sly, July 12, 1990
membranes by a phosphatidylinositol-glycan (PI-G) anchor
Rat carbonic anhydrase (CA) IV was purified ABSTRACT by affinity chromatography and used to produce a specific antiserum in rabbits for immunolocalization studies in rat kidney. CA IV was localized in apical plasma membranes of the proximal convoluted tubule and the thick ascending limb of Henle. Both of these segments are involved in bicarbonate reabsorption in the rat. Immunofluorescent staining of the brush border was faint in the S1 segment, greatest in the S2 segment, and absent from the S3 segment of the proximal tubule. CA IV was also -detected in the basolateral plasma membrane of proximal-tubule and thick-ascending-limb epithelial cells by immunofluorescence and immunoelectron mi croscopy. In the proximal tubule, an extracellular membrane CA had been previously suggested on the basis of electrophysiological studies. CA IV was not detected in intercalated cells of the collecting ducts. These cells contain, in contrast, abundant cytosolic CA H. Thus, the distribution of CA IV is quite distinct from that of CA H; it corresponds with the localization of an isoenzyme(s) that did not stain with antibodies against CA II but that was revealed by histochemical-staining procedures. We conclude that the apical CA IV is the luminal CA responsible for bicarbonate reabsorption in the proximal tubule and the thick ascending limb in the rat kidney. These studies also suggest that CA IV plays a role in bicarbonate transport across the basolateral plasma membrane in these two segments of the rat nephron.
(14).
That renal CA mediates bicarbonate reabsorption by the proximal tubule has been known for many years (15). Initially, CA II was thought to be the primary isozyme involved (16). However, micropuncture studies indicated that a luminal CA was responsible for most bicarbonate reabsorption in the isolated tubule (17). The observation that CA II-deficient patients had only a mild proximal component to their renal tubular acidosis and that they had a dramatic bicarbonaturic response to infused acetazolamide also suggested that a CA other than CA II plays a dominant role in bicarbonate reabsorption (18, 19). Kidney CAs have been localized both by enzyme histochemistry and biochemical assays, which do not readily distinguish different isozymes, as well as by immunocytochemistry with antibodies specific for the soluble isozymes (4, 20-24). In general, histochemical procedures give a more intense and widespread distribution of enzyme activity than is observed with antibodies to the soluble isozymes. Brushborder membranes and certain other membranes, in particular, appeared to contain an intense activity for an isozyme that was not reactive with antiserum to CA II (20). CA IV would be a good candidate to explain this CA activity, but until now, no immunological reagents have been available to test this possibility. In this study, we have raised an antibody against the 39-kDa CA IV from rat lung, which reacts with CA IV of the same molecular weight in rat kidney, to examine the distribution of CA IV in rat kidney. CA IV immunostaining was abundant in the brush border of certain segments of the proximal tubule, as well as in apical and basolateral plasma membranes of cells of the thick ascending limb of Henle, where CA II immunostaining was weak or absent. On the other hand, CA IV could not be detected in intercalated cells of the collecting ducts, which show intense staining for CA II (21, 22). Thus, the distribution of CA IV is quite distinct from that of CA II. It corresponds well with the activity previously shown by histochemical staining in nephron segments that are unstained or only poorly stained with anti-CA II antibodies.
Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of CO2. At least seven isozymes (CA I-CA VII) of CA have been identified in mammals; they differ in physical and kinetic properties, susceptibility to inhibitors, subcellular localization, and tissue-specific expression (1-3). In renal epithelial cells, CA II is the only soluble form of the enzyme (4), but a membraneassociated enzyme has been demonstrated in isolated brush borders and basolateral membranes of rat (5, 6) and canine (7, 8) kidney, as well as in plasma membranes from human kidney (9, 10). Wistrand and Knuuttila (11) recently reported the purification and characterization of a 35-kDa membraneassociated enzyme from human renal membranes and suggested it may be the human analog of the 52-kDa membraneassociated enzyme from bovine lung, which had been designated CA IV (12). We recently purified CA IV from human lung and kidney (13) and presented evidence that the 35-kDa membrane-associated CAs in these two tissues were identical and that the 52-kDa bovine CA IV is larger only because it contains five to six N-linked oligosaccharide chains not present on human CA IV. Furthermore, over half of the CA IV in both tissues was released from membranes by phosphatidylinositol-specific phospholipase C, suggesting that much of the CA IV in lung and kidney is attached to
MATERIALS AND METHODS Antibody Preparation. CA IV was purified to homogeneity from rat lung and used to raise antibody in rabbits exactly as described recently for CA IV from human lung (12). CA I and CA II cross-reacting species were removed from the antiserum by passing it over an affinity column containing rat erythrocyte CA I and CA II immobilized on Affi-Gel 10. An affinity-purified antibody was also prepared from this antiserum by adsorbing the anti-rat CA IV antibodies to immobilized human lung CA IV and eluting the cross-reaching
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. §1734 solely to indicate this fact.
Abbreviations: CA, carbonic anhydrase; PI-G, phosphatidylinositol
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antibodies retained on the human lung CA IV column with 2 M MgCl2/0.1 M glycine*HCl buffer, pH 2.5. No difference was found in the immunolocalization pattern using crude antiserum or the affinity-purified antibodies. Tissue Preparation. Adult Sprague-Dawley rats were anesthetized with Inactin (Byk-Guiden Pharmaceuticals), and their kidneys were perfused via the abdominal aorta, 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 (25) for 10 min. Kidneys were removed and immersed overnight in the same fixture at 49C. Immunocytochemistry. For light microscopy, sagittal slices of fixed kidneys were rinsed extensively in phosphatebuffered saline (PBS; 0.9% NaCl/10 mM sodium phosphate buffer, pH 7.4) and then immersed in 30% (wt/vol) sucrose in PBS for at least 1 hr. Tissue was then mounted on a cryostat support in a drop of mounting medium and frozen in liquid nitrogen. Five-micrometer sections were cut on a Reichert Frigocut cryotome and picked up on gelatin-coated glass slides. After initial incubation in PBS/1% bovine serum albumin, sections were incubated with a 1:500 dilution of anti-CA antiserum for 1 hr at room temperature. Sections were rinsed three times for 5 min each in PBS and then incubated with goat anti-rabbit IgG-fluorescein isothiocyanate (final concentration, 15 pug/ml) for 30 min. After being further rinsed, sections were mounted in glycerol/PBS, 1:1 (vol/vol) containing 4% (wt/vol) 1-propyl gallate to retard quenching of the fluorescence signal (26). Photographs were taken on a Nikon FXA photomicroscope with Kodak T-Max 400 film, pushed to 1600 ASA during development. For electron microscopy, tissue was treated as above except that cryostat sections were cut at a thickness of 20 /km. These sections were incubated overnight in 1:500 diluted antibody, rinsed for 1 hr in six changes of PBS, and then incubated for 2 hr in goat anti-rabbit IgG-biotin. After being rinsed for an additional 1 hr, the sections were incubated in avidin/biotinylated horseradish peroxidase complex (ABC reagent; Vector Laboratories) for 1 hr, rinsed, and exposed to diaminobenzidine at 1 mg/ml in 0.03% hydrogen peroxide/ 100 mM Tris-HCl buffer, pH 8.0. After 10 min, the reaction was stopped by washing with PBS, and sections were placed for 1 hr in 1% reduced osmium tetroxide in distilled water. Tissue slices were then dehydrated in ethanol and embedded in LX-112 resin. Thin sections were examined either un-
FIG. 1.
Distribution of CA IV in rat
kidney
cortex. Some prox-
imal-tubule profiles show a bright immunofluorescent staining of the brush border as well as a marked basolateral staining. Other proximal-tubule profiles show considerably weaker staining. (Bar = 50
AM.)
stained or stained with lead citrate and uranyl acetate and photographed in a Phillips CM10 electron microscope. Measurement. To quantify the fluorescence level in different cell types and in different regions of the epithelial cells, sections were examined in a Nikon FXA microscope by using a 40x oil-immersion objective. An attached Dage-MTI (Michigan City, IN) camera attached to an image intensifier (Videoscope, Dallas), a frame averager (Poynting, Oak Park, IL), and an SMI image-analysis system (Southern Micro Instruments, Atlanta) was used to digitize images and to analyze relative fluorescent intensities. Camera and intensifier gain were adjusted so that fluorescence intensity and output voltage were linear, and background signal was subtracted from the digitized image. By placing the cross-hair of the cursor on different areas of the same cell or on areas of different cell types, random pixel intensities were selected as a measure of fluorescence intensity. Images were saved on the hard disk of an IBM comripatible computer. Data represent the means ± SEM taken from 20 measurements for each cell type and each region. Fluorescence intensities were plotted in histogram
FIG. 2. (A) Part of a rat kidney cortex, stained for CA IV, showing the urinary pole of a glomerulus (G). The S1 segment of the proximal convoluted tubule in direct connection with the glomerular parietal epithelial cells is only weakly fluorescent (arrows). In contrast, some other segments of the proximal tubule are brightly stained. (Bar = 50 Atm.) (B) Part of a rat kidney cortex close to a medullary ray, showing a mosaic pattern of staining in some proximal-tubule segments (arrows). Proximal-tubule segments within the medullary ray (S3 segments) are negative. The pattern of staining is interpreted to represent short tubule segments in which positive cells characteristic of the S2 segment lie adjacent to negative cells characteristic of the S3 segment. Note the presence of positive thick ascending limbs in the medullary ray. (Bar = 50 Am.)
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form using the Cricket Graft program (Cricket Software, Malvern, PA) running on an Apple Macintosh II computer. RESULTS In the cortex, the most striking fluorescence was seen in the brush border of a population of proximal tubules. In these tubules, the cytoplasm showed a weak fluorescence, and the basolateral plasma membrane was also stained (Fig. 1). However, many proximal tubule profiles were unstained or only weakly fluorescent. Based on the observation that proximal tubules in continuity with glomeruli were only poorly stained (Fig. 2A), the brightly positive segments probably represent the S2 region of the proximal convoluted tubule; the initial S1 segment contains considerably less CA IV antigenicity. In the outer medulla (outer stripe), the straight proximal tubule (S3 segment) was unstained (Figs. 2B and 3). At the boundary between the S2 and S3 segments of the proximal tubule, brightly stained S2 cells were found adjacent to unstained S3 cells in the same tubular profile (Fig. 2B). A similar heterogeneous pattern of staining in this region of the proximal tubule has previously been reported using lectin-gold complexes to stain cell-surface glycoconjugates that are segment-specific in the proximal tubule (27).
Proc. Natl. Acad. Sci. USA 87 (1990)
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In addition to proximal tubules, cells of the thick ascending limb of Henle were stained (Figs. 2 and 3). Although apical plasma membranes appeared more brightly stained, basolateral membranes of these cells were also labeled (Fig. 3B). Both cortical and medullary thick limbs were stained, but the labeling appeared heavier in medullary segments. In both the inner and outer stripe of the outer medulla, collecting duct cells and thin limbs of Henle were unstained. In the inner medulla, all tubule segments were unstained. No fluorescence was detected in any nephron segment when the specific antiserum was replaced by the same concentration of preimmune serum. By electron microscopy, the enzyme could be clearly localized on both apical and basolateral plasma membranes in both proximal tubules (Fig. 4A) and thick ascending limbs (Fig. 4B). Staining was restricted to membranes and was not present in the cytosol. The apparent cytosolic fluorescence seen by immunofluorescence was, therefore, a result of the staining of closely apposed basolateral plasma membrane infoldings that characterize these renal epithelial cells. A quantitative measurement of fluorescence in the three segments of the proximal tubule confirmed that the brush border of the S2 segment showed a much greater level of staining
FIG. 3. (A) Corticomedullary boundary of a rat kidney. On left, S2 segments of the proximal tubule within the cortex are heavily stained for CA IV. On right, proximal S3 segments in the outer stripe of the outer medulla are negative. Thick ascending limbs (arrows) are positive, but collecting ducts (CD) are unstained. (Bar = 50 ,Lm.) (B) Higher magnification of part of the outer stripe of the outer medullar. The CA IVpositive tubules are thick ascending limbs of Henle (arrows). The apical membrane appears more brightly stained than the basolateral plasma membrane. The proximal S3 segments are unstained, and both intercalated and principal cells in collecting ducts (CD) are negative. (Bar = 30 gm.)
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Proc. Natl. Acad. Sci. USA 87 (1990)
I
f
B
FIG. 4. Basolateral plasma membrane staining (arrowheads) in the proximal-tubule S2 segment (A) and the thick ascending limb of Henle (B). Tissue was stained with the ABC reagent-horseradish peroxidase procedure for electron microscopy before embedding in LX 112. No staining of the cytosol could be detected, and mitochondrial membranes are unstained. (Bar = 1 ,gm.)
than the two other regions (Fig. 5). The analysis also showed that while the staining of the S3 segment was no greater than background levels seen in the presence of preimmune serum, the brush border staining in the S1 segment was significantly greater than background. The apparent cytosolic staining measured in this quantification was, based on electron microscopic results, a result of the staining of the closely apposed basolateral plasma membrane infoldings that characterize these epithelial cells.
DISCUSSION The intense immunostaining with antibodies to CA IV in the brush border of proximal tubules indicates that CA IV is the luminal CA proposed to play a dominant role in bicarbonate reabsorption by the proximal tubule (17). Although the S1 CA IV STAINING OF PROXIMAL TUBULES
a) c C a)
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.......... ..--..
BB - Brush border CY - Cytoplasm
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83
Proximal Tubule Region
FIG. 5. Quantitative measurement of fluorescence intensity in S1, S2, and S3 regions of the proximal tubule by digitized image analysis. Brush border (BB) and cytoplasm (CY) were measured in these three segments. The fluorescence intensities for brush border and cytoplasm in the S3 segment were not significantly different from background levels measured with preimmune serum as primary antibody (data not shown). Levels of brush-border fluorescence in the Si and S2 segments, as well as cytoplasm fluorescence in the S2 segment, were all significantly greater (Student's t test: P < 0.001) than background. Bars represent the means + SEM of 20 measurements in different cells from the same section.
segment of the proximal convoluted tubule showed a low level of staining, by far the highest concentration of CA IV reactivity was found in the S2 segment. Staining was not detectable in the S3 segment of the proximal tubule. This result agrees with physiological data from the rabbit proximal tubule, in which the S3 but not the S2 segment develops a luminal disequilibrium pH, indicating that an apical, membrane-associated CA is absent from the S3 segment (28). The apical plasma-membrane immunoreactivity detected in the thick ascending limb of Henle is striking and represents an important finding. There is no direct functional evidence indicating whether CA is located in the apical membrane of this segment (29), but histochemical studies suggested that it might be present in the rat (20). In the rat (30, 31), but not rabbit (32), both medullary and cortical thick ascending limbs actively absorb bicarbonate. Proton transport into the lumen of the thick ascending limb, as in the proximal tubule, is mediated primarily by Na+/H+ exchange, a mechanism with limited capacity to transport protons against a pH gradient (29). The rat thick ascending limb acidifies its lumen at a rate of at least 10 pmol/min per mm (30, 31), and a luminal CA is necessary to prevent the development of a pH disequilibrium that would limit proton transport. The clear immunostaining of basolateral plasma membranes in both proximal tubules and thick ascending limbs implies a function for CA IV in basolateral bicarbonate transport in these segments. Such a role has been suggested in the rat proximal S2 segment by physiological studies (33). Based on our present data, CA IV may also play a similar role in the thick ascending limb. In rat proximal tubules and thick ascending limbs, CA II, the major soluble isozyme of CA in the kidney, could barely be detected by immunocytochemistry (4, 21, 34). However, abundant CA activity was detected by enzyme cytochemistry in these same segments (20). Much of the dense reaction product that develops during the cytochemical incubation procedure appeared associated with epithelial-cell plasma membranes, indicating an important contribution of membrane-bound enzyme to total CA activity in these segments. The present study shows directly that CA IV is concentrated in the plasma membranes of these epithelial cells and probably is responsible for the bulk of the staining seen with the histochemical procedure of Hansson (35). In contrast, CA IV could not be detected in either intercalated or principal cells of collecting ducts or in thin limbs of Henle. The expression of CA IV in both apical and basolateral plasma membrane domains presents an interesting topological problem. Most enzymes involved in transepithelial transport are specifically targeted to only one pole of the cell. An
Proc. Natl. Acad. Sci. USA 87 (1990)
Cell Biology: Brown et al. exception to this rule is the proton-pumping ATPase in kidney collecting-duct intercalated cells; this enzyme can be targeted to both apical and/or basolateral plasma membranes in these specialized cells (36). Although the basis for this unusual sorting of proton pumps in intercalated cells is not known, a potential mechanism for apical vs. basolateral sorting of CA IV can be envisaged. In recent studies, it has been shown that over half of the CA IV in human kidney membranes can be released from membranes by treatment with phosphatidylinositol-specific phospholipase C (13). However, the fact that a significant fraction was not released implies that while much of the kidney CA IV is membraneattached by a PI-G anchor, some CA IV may be anchored by another mechanism, such as a transmembrane-spanning domain. Because PI-G-linked proteins have recently been proposed to move exclusively to the apical pole of epithelial cells (37), the apical CA IV may possibly represent the PI-G-linked fraction of this enzyme, whereas the fraction targeted to the basolateral plasma membrane may be the transmembrane form. We recently reported that CA IV could be identified enzymatically and immunologically in membranes from the urine of normal subjects and from CA II-deficient patients (38). The CA IV in these membranes is presumably derived from proximal-tubule brush borders and apical membranes of the thick ascending limb, or both. The CA IV in these urinary membranes was almost completely released by treatment with phosphatidylinositol-specific phospholipase C, suggesting that the CA IV in membranes found in the urine (and presumably, therefore, derived from apical plasma membranes) is anchored by a PI-G linkage. Establishing whether PI-G-linked and transmembrane forms of CA IV are actually both present in kidney and are differentially targeted to opposite poles of epithelial cells will require a detailed study on the effect of phospholipase C on CA IV release from distinct membrane regions. The authors gratefully acknowledge Drs. Seiji Sato, Abdul Waheed, and Mark Knepper for helpful suggestions and discussions. We are grateful to John Natale for excellent technical assistance and to Ms. Elizabeth Torno for editorial assistance. This work was supported by National Institutes of Health Grants DK38452 (D.B.) and GM34182, DK40163 (W.S.S.), and Clinical Research Grant 6-407 from the March of Dimes (W.S.S.). D.B. is an Established Investigator of the American Heart Association. 1. Hewett-Emmett, D., Hopkins, P. J., Tashian, R. E. & Szelusniak, J. (1984) Ann. N. Y. Acad. Sci. 429, 338-358. 2. Tashian, R. E. (1989) BioEssays 10, 186-192. 3. Fernley, R. T. (1988) Trends Biochem. Sci. 13, 356-359. 4. Spicer, S. S., Sens, M. A. & Tashian, R. T. (1982) J. Histochem. Cytochem. 30, 864-873. 5. Wistrand, P. J. & Kinne, R. (1977) Pflagers Arch. 370,121-126. 6. Eveloff, J., Swenson, E. R. & Maren, T. H. (1979) Biochem. Pharmacol. 28, 1434-1437.
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7. Vincent, S. H. & Silverman, D. H. (1980) Arch. Biochem. Biophys. 205, 51-56. 8. Sanyal, G., Pessah, N. I. & Maren, T. H. (1981) Biochim. Biophys. Acta 657, 128-137. 9. McKinley, D. N. & Whitney, P. L. (1976) Biochim. Biophys. Acta 445, 780-790. 10. Wistrand, P. J. (1979) Upsala J. Med. Sci. 26, 75 (abstr.). 11. Wistrand, P. J. & Knuuttila, K. (1989) Kidney Int. 35,851-859. 12. Whitney, P. L. & Briggle, T. V. (1979) J. Biol. Chem. 257, 12056-12059. 13. Zhu, X. L. & Sly, W. S. (1990) J. Biol. Chem. 265, 8795-8801. 14. Low, M. G., Stiernberg, J., Waneck, G. L., Flavell, R. A. & Kincaide, P. W. (1988) J. Immunol. Methods 113, 101-111. 15. Dobyan, D. C. & Bulger, R. E. (1982) Am. J. Physiol. 243, F311-F324. 16. Karlmark, B., Agerup, B. & Wistrand, P. J. (1979) Acta Physiol. Scand. 106, 145-150. 17. Lucci, M. S., Tinker, J. P., Weiner, I. M. & DuBose, T. D. (1983) Am. J. Physiol. 245, F443-F449. 18. Sly, W. S., Whyte, M. P., Krupin, T. & Sundaram, V. (1985) Pediatr. Res. 19, 1033-1036. 19. Sly, W. S. (1989) in The Metabolic Basis ofInherited Disease, eds. Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D. (McGraw-Hill, New York), pp. 2857-2868. 20. Lonnerholm, G. & Ridderstrale, Y. (1980) Kidney Int. 17, 162-174. 21. Brown, D., Roth, J., Kumpulainen, T. & Orci, L. (1983) Am. J. Physiol. 245, F110-F118. 22. Brown, D., Roth, J., Kumpulainen, T. & Orci, L. (1982) Histochemistry 75, 209-213. 23. Lonnerholm, G. & Wistrand, P. J. (1983) Pediatr. Res. 17,
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24. Lonnerholm, G. & Wistrand, P. J. (1984) Kidney Int. 25, 886-898. 25. McLean, I. W. & Nakane, P. F. (1974) J. Histochem. Cy-
tochem. 22, 1077-1083.
26. Giloh, H. & Sedat, S. W. (1982) Science 217, 1252-1255. 27. Brown, D., Kunz, A., Wohlwend, A., Vassalli, J.-D. & Orci,
L. (1983) C. R. Acad. Sci. Paris 297, 501-506.
28. Kurtz, I., Starr, R., Balaban, R. S., Garvin, J. S. & Knepper, M. A. (1986) J. Clin. Invest. 78, 989-996. 29. Knepper, M. A. & Burg, M. B. (1988) in Diseases of the Kidney, eds. Schrier, J. W. & Gottschalk, C. W. (Little, Brown, Boston), pp. 211-239. 30. Good, D. W., Knepper, M. A. & Burg, M. A. (1984) Am. J. Physiol. 247, F35-F44. 31. Good, D. W. (1985) Am. J. Physiol. 248, F821-F829. 32. lino, Y. & Burg, M. B. (1981) Jpn. J. Physiol. 31, 99-107. 33. Burckhardt, B. C., Sato, K. & Fromter, E. (1984) Pflugers Arch. 401, 34-42. 34. Spicer, S. S., Stoward, P. J. & Tashian, R. E. (1979) J. Histochem. Cytochem. 27, 820-831. 35. Hansson, H. P. (1967) Histochemie 11, 112-128. 36. Brown, D., Hirsch, S. & Gluck, S. (1988) Nature (London) 331, 622-624. 37. Lisanti, M. P., Caras, I. W., Dovitz, M. A. & RodriguezBoulan, E. (1989) J. Cell Biol. 109, 2145-2156. 38. Sato, S., Zhu, X. L. & Sly, W. S. (1990) Proc. Natl. Acad. Sci.
USA 87, 6073-6076.
Localization of membrane-associated carbonic anhydrase type IV in kidney epithelial cells DENNIS BROWN, XIN LIANG ZHU, AND WILLIAM S. SLY Renal Unit, Massachusetts General Hospital and Department of Pathology, Harvard Medical School, Boston, MA 02114; and The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO 63104
Contributed by William S. Sly, July 12, 1990
membranes by a phosphatidylinositol-glycan (PI-G) anchor
Rat carbonic anhydrase (CA) IV was purified ABSTRACT by affinity chromatography and used to produce a specific antiserum in rabbits for immunolocalization studies in rat kidney. CA IV was localized in apical plasma membranes of the proximal convoluted tubule and the thick ascending limb of Henle. Both of these segments are involved in bicarbonate reabsorption in the rat. Immunofluorescent staining of the brush border was faint in the S1 segment, greatest in the S2 segment, and absent from the S3 segment of the proximal tubule. CA IV was also -detected in the basolateral plasma membrane of proximal-tubule and thick-ascending-limb epithelial cells by immunofluorescence and immunoelectron mi croscopy. In the proximal tubule, an extracellular membrane CA had been previously suggested on the basis of electrophysiological studies. CA IV was not detected in intercalated cells of the collecting ducts. These cells contain, in contrast, abundant cytosolic CA H. Thus, the distribution of CA IV is quite distinct from that of CA H; it corresponds with the localization of an isoenzyme(s) that did not stain with antibodies against CA II but that was revealed by histochemical-staining procedures. We conclude that the apical CA IV is the luminal CA responsible for bicarbonate reabsorption in the proximal tubule and the thick ascending limb in the rat kidney. These studies also suggest that CA IV plays a role in bicarbonate transport across the basolateral plasma membrane in these two segments of the rat nephron.
(14).
That renal CA mediates bicarbonate reabsorption by the proximal tubule has been known for many years (15). Initially, CA II was thought to be the primary isozyme involved (16). However, micropuncture studies indicated that a luminal CA was responsible for most bicarbonate reabsorption in the isolated tubule (17). The observation that CA II-deficient patients had only a mild proximal component to their renal tubular acidosis and that they had a dramatic bicarbonaturic response to infused acetazolamide also suggested that a CA other than CA II plays a dominant role in bicarbonate reabsorption (18, 19). Kidney CAs have been localized both by enzyme histochemistry and biochemical assays, which do not readily distinguish different isozymes, as well as by immunocytochemistry with antibodies specific for the soluble isozymes (4, 20-24). In general, histochemical procedures give a more intense and widespread distribution of enzyme activity than is observed with antibodies to the soluble isozymes. Brushborder membranes and certain other membranes, in particular, appeared to contain an intense activity for an isozyme that was not reactive with antiserum to CA II (20). CA IV would be a good candidate to explain this CA activity, but until now, no immunological reagents have been available to test this possibility. In this study, we have raised an antibody against the 39-kDa CA IV from rat lung, which reacts with CA IV of the same molecular weight in rat kidney, to examine the distribution of CA IV in rat kidney. CA IV immunostaining was abundant in the brush border of certain segments of the proximal tubule, as well as in apical and basolateral plasma membranes of cells of the thick ascending limb of Henle, where CA II immunostaining was weak or absent. On the other hand, CA IV could not be detected in intercalated cells of the collecting ducts, which show intense staining for CA II (21, 22). Thus, the distribution of CA IV is quite distinct from that of CA II. It corresponds well with the activity previously shown by histochemical staining in nephron segments that are unstained or only poorly stained with anti-CA II antibodies.
Carbonic anhydrases (CAs) are zinc metalloenzymes that catalyze the reversible hydration of CO2. At least seven isozymes (CA I-CA VII) of CA have been identified in mammals; they differ in physical and kinetic properties, susceptibility to inhibitors, subcellular localization, and tissue-specific expression (1-3). In renal epithelial cells, CA II is the only soluble form of the enzyme (4), but a membraneassociated enzyme has been demonstrated in isolated brush borders and basolateral membranes of rat (5, 6) and canine (7, 8) kidney, as well as in plasma membranes from human kidney (9, 10). Wistrand and Knuuttila (11) recently reported the purification and characterization of a 35-kDa membraneassociated enzyme from human renal membranes and suggested it may be the human analog of the 52-kDa membraneassociated enzyme from bovine lung, which had been designated CA IV (12). We recently purified CA IV from human lung and kidney (13) and presented evidence that the 35-kDa membrane-associated CAs in these two tissues were identical and that the 52-kDa bovine CA IV is larger only because it contains five to six N-linked oligosaccharide chains not present on human CA IV. Furthermore, over half of the CA IV in both tissues was released from membranes by phosphatidylinositol-specific phospholipase C, suggesting that much of the CA IV in lung and kidney is attached to
MATERIALS AND METHODS Antibody Preparation. CA IV was purified to homogeneity from rat lung and used to raise antibody in rabbits exactly as described recently for CA IV from human lung (12). CA I and CA II cross-reacting species were removed from the antiserum by passing it over an affinity column containing rat erythrocyte CA I and CA II immobilized on Affi-Gel 10. An affinity-purified antibody was also prepared from this antiserum by adsorbing the anti-rat CA IV antibodies to immobilized human lung CA IV and eluting the cross-reaching
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. §1734 solely to indicate this fact.
Abbreviations: CA, carbonic anhydrase; PI-G, phosphatidylinositol
glycan. 7457
7458
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Proc. Natl. Acad. Sci. USA 87 (1990)
antibodies retained on the human lung CA IV column with 2 M MgCl2/0.1 M glycine*HCl buffer, pH 2.5. No difference was found in the immunolocalization pattern using crude antiserum or the affinity-purified antibodies. Tissue Preparation. Adult Sprague-Dawley rats were anesthetized with Inactin (Byk-Guiden Pharmaceuticals), and their kidneys were perfused via the abdominal aorta, 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 (25) for 10 min. Kidneys were removed and immersed overnight in the same fixture at 49C. Immunocytochemistry. For light microscopy, sagittal slices of fixed kidneys were rinsed extensively in phosphatebuffered saline (PBS; 0.9% NaCl/10 mM sodium phosphate buffer, pH 7.4) and then immersed in 30% (wt/vol) sucrose in PBS for at least 1 hr. Tissue was then mounted on a cryostat support in a drop of mounting medium and frozen in liquid nitrogen. Five-micrometer sections were cut on a Reichert Frigocut cryotome and picked up on gelatin-coated glass slides. After initial incubation in PBS/1% bovine serum albumin, sections were incubated with a 1:500 dilution of anti-CA antiserum for 1 hr at room temperature. Sections were rinsed three times for 5 min each in PBS and then incubated with goat anti-rabbit IgG-fluorescein isothiocyanate (final concentration, 15 pug/ml) for 30 min. After being further rinsed, sections were mounted in glycerol/PBS, 1:1 (vol/vol) containing 4% (wt/vol) 1-propyl gallate to retard quenching of the fluorescence signal (26). Photographs were taken on a Nikon FXA photomicroscope with Kodak T-Max 400 film, pushed to 1600 ASA during development. For electron microscopy, tissue was treated as above except that cryostat sections were cut at a thickness of 20 /km. These sections were incubated overnight in 1:500 diluted antibody, rinsed for 1 hr in six changes of PBS, and then incubated for 2 hr in goat anti-rabbit IgG-biotin. After being rinsed for an additional 1 hr, the sections were incubated in avidin/biotinylated horseradish peroxidase complex (ABC reagent; Vector Laboratories) for 1 hr, rinsed, and exposed to diaminobenzidine at 1 mg/ml in 0.03% hydrogen peroxide/ 100 mM Tris-HCl buffer, pH 8.0. After 10 min, the reaction was stopped by washing with PBS, and sections were placed for 1 hr in 1% reduced osmium tetroxide in distilled water. Tissue slices were then dehydrated in ethanol and embedded in LX-112 resin. Thin sections were examined either un-
FIG. 1.
Distribution of CA IV in rat
kidney
cortex. Some prox-
imal-tubule profiles show a bright immunofluorescent staining of the brush border as well as a marked basolateral staining. Other proximal-tubule profiles show considerably weaker staining. (Bar = 50
AM.)
stained or stained with lead citrate and uranyl acetate and photographed in a Phillips CM10 electron microscope. Measurement. To quantify the fluorescence level in different cell types and in different regions of the epithelial cells, sections were examined in a Nikon FXA microscope by using a 40x oil-immersion objective. An attached Dage-MTI (Michigan City, IN) camera attached to an image intensifier (Videoscope, Dallas), a frame averager (Poynting, Oak Park, IL), and an SMI image-analysis system (Southern Micro Instruments, Atlanta) was used to digitize images and to analyze relative fluorescent intensities. Camera and intensifier gain were adjusted so that fluorescence intensity and output voltage were linear, and background signal was subtracted from the digitized image. By placing the cross-hair of the cursor on different areas of the same cell or on areas of different cell types, random pixel intensities were selected as a measure of fluorescence intensity. Images were saved on the hard disk of an IBM comripatible computer. Data represent the means ± SEM taken from 20 measurements for each cell type and each region. Fluorescence intensities were plotted in histogram
FIG. 2. (A) Part of a rat kidney cortex, stained for CA IV, showing the urinary pole of a glomerulus (G). The S1 segment of the proximal convoluted tubule in direct connection with the glomerular parietal epithelial cells is only weakly fluorescent (arrows). In contrast, some other segments of the proximal tubule are brightly stained. (Bar = 50 Atm.) (B) Part of a rat kidney cortex close to a medullary ray, showing a mosaic pattern of staining in some proximal-tubule segments (arrows). Proximal-tubule segments within the medullary ray (S3 segments) are negative. The pattern of staining is interpreted to represent short tubule segments in which positive cells characteristic of the S2 segment lie adjacent to negative cells characteristic of the S3 segment. Note the presence of positive thick ascending limbs in the medullary ray. (Bar = 50 Am.)
Cell
Biology:
Brown et al.
form using the Cricket Graft program (Cricket Software, Malvern, PA) running on an Apple Macintosh II computer. RESULTS In the cortex, the most striking fluorescence was seen in the brush border of a population of proximal tubules. In these tubules, the cytoplasm showed a weak fluorescence, and the basolateral plasma membrane was also stained (Fig. 1). However, many proximal tubule profiles were unstained or only weakly fluorescent. Based on the observation that proximal tubules in continuity with glomeruli were only poorly stained (Fig. 2A), the brightly positive segments probably represent the S2 region of the proximal convoluted tubule; the initial S1 segment contains considerably less CA IV antigenicity. In the outer medulla (outer stripe), the straight proximal tubule (S3 segment) was unstained (Figs. 2B and 3). At the boundary between the S2 and S3 segments of the proximal tubule, brightly stained S2 cells were found adjacent to unstained S3 cells in the same tubular profile (Fig. 2B). A similar heterogeneous pattern of staining in this region of the proximal tubule has previously been reported using lectin-gold complexes to stain cell-surface glycoconjugates that are segment-specific in the proximal tubule (27).
Proc. Natl. Acad. Sci. USA 87 (1990)
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In addition to proximal tubules, cells of the thick ascending limb of Henle were stained (Figs. 2 and 3). Although apical plasma membranes appeared more brightly stained, basolateral membranes of these cells were also labeled (Fig. 3B). Both cortical and medullary thick limbs were stained, but the labeling appeared heavier in medullary segments. In both the inner and outer stripe of the outer medulla, collecting duct cells and thin limbs of Henle were unstained. In the inner medulla, all tubule segments were unstained. No fluorescence was detected in any nephron segment when the specific antiserum was replaced by the same concentration of preimmune serum. By electron microscopy, the enzyme could be clearly localized on both apical and basolateral plasma membranes in both proximal tubules (Fig. 4A) and thick ascending limbs (Fig. 4B). Staining was restricted to membranes and was not present in the cytosol. The apparent cytosolic fluorescence seen by immunofluorescence was, therefore, a result of the staining of closely apposed basolateral plasma membrane infoldings that characterize these renal epithelial cells. A quantitative measurement of fluorescence in the three segments of the proximal tubule confirmed that the brush border of the S2 segment showed a much greater level of staining
FIG. 3. (A) Corticomedullary boundary of a rat kidney. On left, S2 segments of the proximal tubule within the cortex are heavily stained for CA IV. On right, proximal S3 segments in the outer stripe of the outer medulla are negative. Thick ascending limbs (arrows) are positive, but collecting ducts (CD) are unstained. (Bar = 50 ,Lm.) (B) Higher magnification of part of the outer stripe of the outer medullar. The CA IVpositive tubules are thick ascending limbs of Henle (arrows). The apical membrane appears more brightly stained than the basolateral plasma membrane. The proximal S3 segments are unstained, and both intercalated and principal cells in collecting ducts (CD) are negative. (Bar = 30 gm.)
.
Cell
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A
Biology: Brown et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
I
f
B
FIG. 4. Basolateral plasma membrane staining (arrowheads) in the proximal-tubule S2 segment (A) and the thick ascending limb of Henle (B). Tissue was stained with the ABC reagent-horseradish peroxidase procedure for electron microscopy before embedding in LX 112. No staining of the cytosol could be detected, and mitochondrial membranes are unstained. (Bar = 1 ,gm.)
than the two other regions (Fig. 5). The analysis also showed that while the staining of the S3 segment was no greater than background levels seen in the presence of preimmune serum, the brush border staining in the S1 segment was significantly greater than background. The apparent cytosolic staining measured in this quantification was, based on electron microscopic results, a result of the staining of the closely apposed basolateral plasma membrane infoldings that characterize these epithelial cells.
DISCUSSION The intense immunostaining with antibodies to CA IV in the brush border of proximal tubules indicates that CA IV is the luminal CA proposed to play a dominant role in bicarbonate reabsorption by the proximal tubule (17). Although the S1 CA IV STAINING OF PROXIMAL TUBULES
a) c C a)
1
,1 c1
Ca U a1) 0
-
,,....,.......
So0
,.-: .
.......... ...
.......... ..--..
BB - Brush border CY - Cytoplasm
...
-...
....
_
..............
.... .
-. . . . . . . . .
... y-
CY
:'.,' ;:-S----3....'.''.
.........
cy
CY
83
Proximal Tubule Region
FIG. 5. Quantitative measurement of fluorescence intensity in S1, S2, and S3 regions of the proximal tubule by digitized image analysis. Brush border (BB) and cytoplasm (CY) were measured in these three segments. The fluorescence intensities for brush border and cytoplasm in the S3 segment were not significantly different from background levels measured with preimmune serum as primary antibody (data not shown). Levels of brush-border fluorescence in the Si and S2 segments, as well as cytoplasm fluorescence in the S2 segment, were all significantly greater (Student's t test: P < 0.001) than background. Bars represent the means + SEM of 20 measurements in different cells from the same section.
segment of the proximal convoluted tubule showed a low level of staining, by far the highest concentration of CA IV reactivity was found in the S2 segment. Staining was not detectable in the S3 segment of the proximal tubule. This result agrees with physiological data from the rabbit proximal tubule, in which the S3 but not the S2 segment develops a luminal disequilibrium pH, indicating that an apical, membrane-associated CA is absent from the S3 segment (28). The apical plasma-membrane immunoreactivity detected in the thick ascending limb of Henle is striking and represents an important finding. There is no direct functional evidence indicating whether CA is located in the apical membrane of this segment (29), but histochemical studies suggested that it might be present in the rat (20). In the rat (30, 31), but not rabbit (32), both medullary and cortical thick ascending limbs actively absorb bicarbonate. Proton transport into the lumen of the thick ascending limb, as in the proximal tubule, is mediated primarily by Na+/H+ exchange, a mechanism with limited capacity to transport protons against a pH gradient (29). The rat thick ascending limb acidifies its lumen at a rate of at least 10 pmol/min per mm (30, 31), and a luminal CA is necessary to prevent the development of a pH disequilibrium that would limit proton transport. The clear immunostaining of basolateral plasma membranes in both proximal tubules and thick ascending limbs implies a function for CA IV in basolateral bicarbonate transport in these segments. Such a role has been suggested in the rat proximal S2 segment by physiological studies (33). Based on our present data, CA IV may also play a similar role in the thick ascending limb. In rat proximal tubules and thick ascending limbs, CA II, the major soluble isozyme of CA in the kidney, could barely be detected by immunocytochemistry (4, 21, 34). However, abundant CA activity was detected by enzyme cytochemistry in these same segments (20). Much of the dense reaction product that develops during the cytochemical incubation procedure appeared associated with epithelial-cell plasma membranes, indicating an important contribution of membrane-bound enzyme to total CA activity in these segments. The present study shows directly that CA IV is concentrated in the plasma membranes of these epithelial cells and probably is responsible for the bulk of the staining seen with the histochemical procedure of Hansson (35). In contrast, CA IV could not be detected in either intercalated or principal cells of collecting ducts or in thin limbs of Henle. The expression of CA IV in both apical and basolateral plasma membrane domains presents an interesting topological problem. Most enzymes involved in transepithelial transport are specifically targeted to only one pole of the cell. An
Proc. Natl. Acad. Sci. USA 87 (1990)
Cell Biology: Brown et al. exception to this rule is the proton-pumping ATPase in kidney collecting-duct intercalated cells; this enzyme can be targeted to both apical and/or basolateral plasma membranes in these specialized cells (36). Although the basis for this unusual sorting of proton pumps in intercalated cells is not known, a potential mechanism for apical vs. basolateral sorting of CA IV can be envisaged. In recent studies, it has been shown that over half of the CA IV in human kidney membranes can be released from membranes by treatment with phosphatidylinositol-specific phospholipase C (13). However, the fact that a significant fraction was not released implies that while much of the kidney CA IV is membraneattached by a PI-G anchor, some CA IV may be anchored by another mechanism, such as a transmembrane-spanning domain. Because PI-G-linked proteins have recently been proposed to move exclusively to the apical pole of epithelial cells (37), the apical CA IV may possibly represent the PI-G-linked fraction of this enzyme, whereas the fraction targeted to the basolateral plasma membrane may be the transmembrane form. We recently reported that CA IV could be identified enzymatically and immunologically in membranes from the urine of normal subjects and from CA II-deficient patients (38). The CA IV in these membranes is presumably derived from proximal-tubule brush borders and apical membranes of the thick ascending limb, or both. The CA IV in these urinary membranes was almost completely released by treatment with phosphatidylinositol-specific phospholipase C, suggesting that the CA IV in membranes found in the urine (and presumably, therefore, derived from apical plasma membranes) is anchored by a PI-G linkage. Establishing whether PI-G-linked and transmembrane forms of CA IV are actually both present in kidney and are differentially targeted to opposite poles of epithelial cells will require a detailed study on the effect of phospholipase C on CA IV release from distinct membrane regions. The authors gratefully acknowledge Drs. Seiji Sato, Abdul Waheed, and Mark Knepper for helpful suggestions and discussions. We are grateful to John Natale for excellent technical assistance and to Ms. Elizabeth Torno for editorial assistance. This work was supported by National Institutes of Health Grants DK38452 (D.B.) and GM34182, DK40163 (W.S.S.), and Clinical Research Grant 6-407 from the March of Dimes (W.S.S.). D.B. is an Established Investigator of the American Heart Association. 1. Hewett-Emmett, D., Hopkins, P. J., Tashian, R. E. & Szelusniak, J. (1984) Ann. N. Y. Acad. Sci. 429, 338-358. 2. Tashian, R. E. (1989) BioEssays 10, 186-192. 3. Fernley, R. T. (1988) Trends Biochem. Sci. 13, 356-359. 4. Spicer, S. S., Sens, M. A. & Tashian, R. T. (1982) J. Histochem. Cytochem. 30, 864-873. 5. Wistrand, P. J. & Kinne, R. (1977) Pflagers Arch. 370,121-126. 6. Eveloff, J., Swenson, E. R. & Maren, T. H. (1979) Biochem. Pharmacol. 28, 1434-1437.
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7. Vincent, S. H. & Silverman, D. H. (1980) Arch. Biochem. Biophys. 205, 51-56. 8. Sanyal, G., Pessah, N. I. & Maren, T. H. (1981) Biochim. Biophys. Acta 657, 128-137. 9. McKinley, D. N. & Whitney, P. L. (1976) Biochim. Biophys. Acta 445, 780-790. 10. Wistrand, P. J. (1979) Upsala J. Med. Sci. 26, 75 (abstr.). 11. Wistrand, P. J. & Knuuttila, K. (1989) Kidney Int. 35,851-859. 12. Whitney, P. L. & Briggle, T. V. (1979) J. Biol. Chem. 257, 12056-12059. 13. Zhu, X. L. & Sly, W. S. (1990) J. Biol. Chem. 265, 8795-8801. 14. Low, M. G., Stiernberg, J., Waneck, G. L., Flavell, R. A. & Kincaide, P. W. (1988) J. Immunol. Methods 113, 101-111. 15. Dobyan, D. C. & Bulger, R. E. (1982) Am. J. Physiol. 243, F311-F324. 16. Karlmark, B., Agerup, B. & Wistrand, P. J. (1979) Acta Physiol. Scand. 106, 145-150. 17. Lucci, M. S., Tinker, J. P., Weiner, I. M. & DuBose, T. D. (1983) Am. J. Physiol. 245, F443-F449. 18. Sly, W. S., Whyte, M. P., Krupin, T. & Sundaram, V. (1985) Pediatr. Res. 19, 1033-1036. 19. Sly, W. S. (1989) in The Metabolic Basis ofInherited Disease, eds. Scriver, C. R., Beaudet, A. L., Sly, W. S. & Valle, D. (McGraw-Hill, New York), pp. 2857-2868. 20. Lonnerholm, G. & Ridderstrale, Y. (1980) Kidney Int. 17, 162-174. 21. Brown, D., Roth, J., Kumpulainen, T. & Orci, L. (1983) Am. J. Physiol. 245, F110-F118. 22. Brown, D., Roth, J., Kumpulainen, T. & Orci, L. (1982) Histochemistry 75, 209-213. 23. Lonnerholm, G. & Wistrand, P. J. (1983) Pediatr. Res. 17,
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