ARTICLE |
Correspondence to: Kari Kaunisto, Dept. of Anatomy and Cell Biology, Box 5000, FIN-90014 University of Oulu, Finland. E-mail: Kari.Kaunisto@oulu.fi
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Summary |
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Membrane-associated carbonic anhydrase (CA) has a crucial role in renal HCO3- absorption. CA activity has been localized to both luminal and basolateral membranes of the tubule epithelial cells. CA XII is a transmembrane isoenzyme that has been demonstrated in the basolateral plasma membrane of human renal, intestinal, and reproductive epithelia. The present study was designed to demonstrate the distribution of CA XII expression in the rodent kidney. A new polyclonal antibody to recombinant mouse CA XII was used in both Western blotting and immunohistochemistry. Western blotting analysis revealed a 4045-kD polypeptide in CA XII-expressing CHO cells and isolated membranes of mouse and rat kidney. Immunofluorescence staining localized CA XII in the basolateral plasma membranes of S1 and S2 proximal tubule segments. Abundant basolateral staining of CA XII was seen in a subpopulation of cells in both cortical and medullary collecting ducts. Double immunofluorescence staining identified these cells as H+-secreting type A intercalated cells. The localization of CA XII in the peritubular space of proximal tubules suggests that it may play a role in renal HCO3- absorption, whereas the function of CA XII in the type A intercalated cells needs further investigation.
(J Histochem Cytochem 51:12171224, 2003)
Key Words: HCO3- absorption, proximal tubule, collecting duct
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Introduction |
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CARBONIC ANHYDRASE (CA) plays an important role in the control of acidbase balance by facilitating urinary acidification. Up to 5% of renal CA activity is membrane-associated, while the remaining 95% corresponds to cytoplasmic CA II (
Membrane-associated renal CA activity has earlier been attributed to CA IV, which is expressed in proximal tubules and the thick ascending limb of rat nephrons (
CA XII was originally identified as a tumor-associated gene expressed in renal and lung cancer cells (
Marked species differences in CA distribution along the nephron have been reported (
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Materials and Methods |
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Antibodies
Polyclonal rabbit antiserum to mouse CA XII was used in Western blotting and IHC. This antibody was produced as follows. Chinese hamster ovary (CHO) cells were transfected with a gene construct containing a truncated secretory form of mouse CA XII according to earlier procedures (
In double-labeling studies, rat anti-mouse CA XIV (
Animals and Tissue Preparation
Adult male SpragueDawley rats and Balb/c mice were sacrificed by CO2 asphyxiation followed by decapitation. For Western blotting, kidneys were removed and rapidly frozen in liquid nitrogen. For IHC, kidneys were perfused in situ through the abdominal aorta with 3% paraformaldehyde in PBS, removed, and cut into coronal slices. The slices were further immersion-fixed for 23 hr at room temperature, cryoprotected overnight in 20% sucrosePBS and rapidly frozen in liquid nitrogen-chilled isopentane.
Western Blotting
CHO cells expressing truncated mouse CA XII or full-length mouse CA XIV cDNA (
Cell lysates or renal membrane proteins were subjected to standard SDS-PAGE under reducing conditions. After electrophoresis, the polypeptides were transferred to Immobilon-P membranes (Millipore; Bedford, MA). The membranes were immunostained using rabbit anti-mouse CA XII, anti-mouse CA XIV, or preimmune serum as primary antibodies and peroxidase-conjugated goat or donkey anti-rabbit IgG as secondary antibody, as described previously (
Immunohistochemistry
Sections were cut at 5 µm using a Microm Cryo-Star microtome (Microm; Walldorf, Germany), dried onto Superfrost Plus microscope slides (Menzel; Braunschweig, Germany), and incubated with PBS containing 20% cow colostral whey for 30 min. The sections were then incubated for 2 hr (mice) or overnight (rats) with polyclonal rabbit anti-CA XII or preimmune serum, diluted 1:1001:300 in 1% BSAPBS, washed three times for 5 min in PBS, and incubated for 1 hr with Alexa 488- or 568-coupled goat anti-rabbit IgG, diluted 1:300 in PBS. After four 5-min washes in PBS, slides were mounted in Immu-mount (Shandon; Pittsburgh, PA).
For double immunofluorescence staining, the rabbit anti-CA XII was detected with Alexa 592-coupled donkey anti-rabbit IgG (1:200) and the goat antibodies against calbindin D28K (1:100), AQP2 (diluted 5 µg/100 µl), or TammHorsfall glycoprotein (1:100) with Alexa 488-coupled donkey anti-goat IgG (1:200). In the double-labeling experiments, the rabbit anti-mouse CA XII antibodies were detected with Alexa 568-coupled goat anti-rabbit IgG (1:300), rat anti-mouse CA XIV (1:20) with Alexa 488-coupled goat anti-rat IgG (1:300), and mouse monoclonal anti-human AE1 (1:10) with Alexa 488-coupled goat anti-mouse IgG (1:300). The sections were first examined with a conventional epifluorescence microscope (Nikon; Tokyo, Japan) and the final images were obtained with a confocal laser scanning microscope (Zeiss; Göttingen, Germany).
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Results |
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Antibody Characterization and CA XII Expression in the Kidney
The specificity of rabbit anti-mouse CA XII antiserum was tested by SDS-PAGE and Western blotting of lysates from CHO cells transfected with cDNAs for truncated mouse CA XII or full-length mouse CA XIV. Polypeptides of 4244 kD for truncated mouse CA XII and 5055 kD for full-length mouse CA XIV reacted with their respective antisera (Fig 1). There was no crossreaction between mouse CA XII and XIV antisera. No reaction was seen with preimmune serum.
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Western blotting analysis revealed a diffuse 4045 kD polypeptide band in both mouse and rat kidney membrane samples (Fig 2). In addition, anti-CA XII antibody recognized a faint unidentified band of 65 kD in both species. When specific antiserum was replaced by preimmune serum, no signal was detected. The observed size of the CA XII-specific band is in agreement with the size of the glycosylated human CA XII (
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IHC Localization of CA XII in the Kidney
The distribution of CA XII along the rodent nephron and its subcellular localization were studied using indirect immunofluorescence. Low-magnification images of mouse kidney cortex demonstrate CA XII immunostaining in proximal tubules and cortical collecting ducts (Fig 3A). In the medulla of mouse kidney, strong immunostaining was seen in tubules, which represented medullary collecting ducts (Fig 3C). CA XII-positive cells were seen in the cortical and outer medullary collecting ducts but not in the inner medulla (not shown). In proximal tubules, CA XII immunostaining was intense in the S1 segment and decreased towards the S2 segment (Fig 3A and Fig 3B), whereas S3 proximal tubules were negative (not shown). The labeling was restricted to the basolateral plasma membrane, while the luminal brush border membrane was negative (Fig 3A and Fig 3B). In the cortical (Fig 3A) and medullary collecting ducts (Fig 3C), the labeling was present in the basolateral plasma membrane of an epithelial cell subpopulation. In rat kidney, the staining pattern was similar, although the signal was weaker in proximal tubules where it was clearly visible after longer incubations with the primary antibody (Fig 3D). Collecting ducts in the rat kidney cortex and outer medulla showed strong basolateral immunostaining in a subpopulation of epithelial cells (Fig 3E and Fig 3F). The control immunostaining with preimmune serum remained negative (Fig 3G).
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We have recently reported that another membrane-associated CA, CA XIV, was localized in the S1 and S2 proximal tubule segments and in the initial segment of the thin descending limb mouse and rat kidney (
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Discussion |
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Membrane-associated CA activity plays an important role in renal acidification even though it comprises only 5% of total renal CA activity (
CA activity has been identified in basolateral membranes, in addition to renal luminal membranes, by biochemical (
In the present study we used polyclonal antibodies and IHC to investigate the distribution and cellular localization of CA XII in mouse and rat nephron. Basolateral localization of CA XII in the proximal tubule indicates that at least three isoenzymes, CA IV, XIV, and XII, may contribute to CA activity in that membrane domain. The importance of basolateral CA in proximal tubule to HCO3- and fluid absorption has been clearly defined by
We found intense immunostaining in the basolateral membrane of intercalated cells in cortical and outer medullary collecting ducts. The finding of basolateral CA in intercalated cells is in agreement with earlier histochemical studies on mouse kidney (
The localization of mouse and rat renal CA XII has similarities but also some differences compared to the human enzyme (
In summary, we have studied the localization of CA XII in the rodent kidney using a new antibody raised against the recombinant mouse enzyme. CA XII was localized to the basolateral plasma membrane of proximal tubules and type A intercalated cells of the collecting ducts. Therefore, CA XII is one of the three CA isoenzymes responsible for the intercellular CA activity needed for efficient HCO3- and fluid absorption in the proximal tubule. The possible role of CA XII in the basolateral membrane of type A intercalated cells needs further studies using selective inhibition of that activity.
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Acknowledgments |
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Supported by a grant from the Sigrid Juselius Foundation to S.P. and by grants DK53405, DK40163, and GM34182 from the National Institutes of Health to W.S.S.
We thank Dr Michael Jennings for providing the AE1 antibody and Dr Maritta Pietilä for helping in confocal microscopy. The technical assistance of Ms Sirpa Kellokumpu and Ms Lissu Hukkanen is gratefully acknowledged.
Received for publication November 26, 2002; accepted May 8, 2003.
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