ARTICLE |
Correspondence to: Seppo Parkkila, Dept. of Anatomy and Cell Biology, Box 5000, FIN-90014 University of Oulu, Finland. E-mail: seppo.parkkila@oulu.fi
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Summary |
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Carbonic anhydrase isozyme XII (CA XII) is a novel membrane-associated protein with a potential role in von HippelLindau carcinogenesis. Although Northern blotting has revealed positive signal for CA XII in normal human kidney, this is the first study to demonstrate its cellular and subcellular localization along the human nephron and collecting duct. Immunohistochemistry with a polyclonal antibody (PAb) raised against truncated CA XII revealed distinct staining in the basolateral plasma membrane of the epithelial cells in the thick ascending limb of Henle and distal convoluted tubules, and in the principal cells of the collecting ducts. A weak basolateral signal was also detected in the epithelium of the proximal convoluted tubules. In addition to the normal kidney specimens, this immunohistochemical study included 31 renal tumors. CA XII showed moderate or strong plasma membrane-associated expression in most oncocytomas and clear-cell carcinomas. The segmental, cellular, and subcellular distribution of CA XII along the human nephron and collecting duct suggests that it may be one of the key enzymes involved in normal renal physiology, particularly in the regulation of water homeostasis. High expression of CA XII in some renal carcinomas may contribute to its role in von HippelLindau carcinogenesis.
(J Histochem Cytochem 48:16011608, 2000)
Key Words: aquaporin-2, cancer, carbonic anhydrase, carcinoma, immunohistochemistry, kidney, plasma membrane
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Introduction |
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THE HISTOLOGICAL LOCALIZATION of CA activity has been extensively studied in kidney (
Membrane-associated CA IV is expressed on the apical brush-border membrane of the rat proximal tubule cells (less on the basolateral membrane) and on the cells of the thick ascending limbs of Henle (
The reports on a novel membrane-associated isozyme, CA XII, have raised considerable interest because its mRNA is expressed in the normal human kidney and overexpressed in some malignant cells, including non-small-cell lung carcinoma cells and renal cancer cells (
Immunohistochemistry for CA XII has recently been reported in the human endometrium (
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Materials and Methods |
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Antibodies
The anti-human CA XII antiserum to the secretory form of human CA XII was raised in rabbits as described earlier for CA IV (
Immunocytochemistry
Samples of normal human kidney (n = 4) and renal neoplasms (n = 31) were obtained together with routine histopathological specimens taken during surgical operations for renal carcinoma. The renal tumor specimens included 16 clear-cell carcinomas (grade 1, n = 4; grade 2, n = 2; grade 3, n = 7; grade 4, n = 3), two chromophilic carcinomas, two chromophobic carcinomas, five oncocytomas, one angiomyolipoma, and five Wilms' tumors. The procedures had the approval of the ethics committee of Oulu University Hospital and the research was carried out according to the provisions of the Helsinki Declaration of 1975. Each tissue sample was divided into several small pieces about 5 mm thick. The specimens were fixed in Carnoy's fluid (absolute ethanol + chloroform + glacial acetic acid 6:3:1) for 6 hr at 4C or in 4% neutral-buffered formaldehyde for 2448 hr at room temperature (RT). The samples were then dehydrated, embedded in paraffin in a vacuum oven at 58C, and sections of 5 µm were placed on gelatin-coated microscope slides. The immunostaining of tissue sections was performed using the biotinstreptavidin complex method, employing the following steps: (a) pretreatment of the sections with undiluted cow colostral whey for 40 min and rinsing in PBS; (b) incubation for 1 hr with the anti-human CA XII serum or nonimmune rabbit serum diluted 1:100 or 1:500 in PBS containing 1% bovine serum albumin (BSA); (c) treatment with cow colostral whey for 40 min and rinsing in PBS; (d) incubation for 1 hr with biotinylated swine anti-rabbit IgG (Dakopatts; Glostrup, Denmark) diluted 1:300 in 1% BSAPBS; (e) incubation for 30 min with peroxidase-conjugated streptavidin (Dakopatts) diluted 1:500 in PBS; and (f) incubation for 2 min in DAB solution containing 9 mg 3,3'-diaminobenzidine tetrahydrochloride (Fluka; Buchs, Switzerland) in 15 ml PBS + 5 µl 30% H2O2. The sections were washed three times for 10 min in PBS after incubation Steps b, d, and e. Additional control experiments were performed using 50 µg CA XII protein/microscope slide to block the specific immunoreaction. All the incubations and washings were carried out at RT and the sections were finally mounted in Permount (Fisher Scientific; Fair Lawn, NJ). The stained sections were examined and photographed with a Leitz Aristoplan microscope (Wetzlar, Germany). The intensity of CA XII immunostaining in the renal tumor specimens was scored by two of the investigators (S. Parkkila and T.J. Karttunen) on a scale of 0 to 4 as follows: 0, no reaction; 1, occasional positive cells; 2, weak reaction; 3, moderate reaction; 4, strong reaction. The statistical analyses of intensity scores were performed using one-way analysis of variance.
To determine the cellular distribution of CA XII in the human kidney, the tissue sections were stained using a double immunofluorescence method and analyzed by confocal laser scanning microscopy. The steps in the double immunostaining were as follows: (a) pretreatment of the sections with 1% BSAPBS for 40 min; (b) incubation for 1 hr with guinea pig anti-human CA II serum diluted 1:50 or goat anti-human AQP2 IgG diluted 5 µg/100 µl, and rabbit anti-human CA XII serum diluted 1:50 in 1% BSAPBS; (c) washing three times for 5 min in PBS; (d) incubation for 1 hr with 1:50 diluted fluorescein isothiocyanate (FITC)-conjugated goat anti-guinea pig IgG (Sigma Chemical; St Louis, MO) or 1:10 diluted FITC-conjugated donkey anti-goat IgG (Santa Cruz), and 1:30 diluted tetramethylrhodamine isothiocyanate (TRITC)-conjugated swine anti-rabbit IgG (Dakopatts) in 1% BSAPBS; and (e) washing three times for 5 min in PBS. The immunofluorescent sections were examined with a confocal laser scanning microscope (Leitz CLSM; Leica Microsystems, Heidelberg, Germany).
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Results |
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Localization of CA XII in Human Kidney
Fig 1 shows immunoperoxidase staining of CA XII in the human kidney. In the cortex (Fig 1A), the strongest expression of CA XII was located in the epithelial cells of the distal convoluted tubules, thick ascending limb of Henle, and collecting ducts. The immunoreaction was most distinct in the basolateral plasma membrane of the epithelial cells. The epithelium of the proximal convoluted tubules showed only a weak reaction on the basolateral surfaces. In the medulla (Fig 1B), CA XII was located in selected cells in the epithelium of the collecting ducts, where the positive reaction was confined to the basolateral plasma membrane. The control immunostaining of the renal cortex using nonimmune rabbit serum remained negative (Fig 1C).
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Fig 2 provides an additional example of control staining for the specificity of the antibody for CA XII. Antibody-stained sections of renal cortex were compared with sections exposed to antibody in the presence of blocking CA XII protein. The positive staining seen in Fig 2A was blocked by the added antigen (Fig 2B).
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For a more detailed cellular distribution, CA II was used as a marker for the intercalated cells (
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Expression of CA XII in Renal Tumors
An outline of the CA XII immunoreactivities estimated in the renal tumors is presented in Fig 4. The mean staining intensity varied from moderate to strong in all tumor categories except angiomyolipoma and Wilms' tumor, the latter showing much weaker immunoreaction compared to other tumors (p<0.001). In clear-cell carcinomas, the immunoreaction showed a trend to correlate with the histological grade, being slightly weaker in well-differentiated carcinomas, although this difference did not reach a statistical significance because of the small number of grade 1 tumors. This finding is illustrated in Fig 5A5C, in which Fig 5A shows weak signal in grade 1 clear-cell carcinoma, while the positive reaction became stronger in grade 2 and 4 tumors (Fig 5B and Fig 5C, respectively). In all clear-cell carcinomas, the most prominent signal was localized to the plasma membrane of the malignant cells. Fig 5D shows a plasma membrane-associated immunoreaction for CA XII in oncocytoma. Fig 5E demonstrates that the Wilms' tumor specimens were mainly negative for CA XII.
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Discussion |
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The present observations provide the first opportunity to compare the expression of CA XII to the known distribution pattern of CA activity along the segments of the human nephron and collecting duct. Because CA XII expressed in mammalian cell lines shows distinct CA enzymatic activity (
The physiological role of basolateral CA in renal epithelial cells is unclear. In the proximal convoluted tubule, basolateral CA has been suggested to facilitate Na+/HCO3- co-transport by preventing the development of alkaline disequilibrium pH in the interstitium (
The present immunohistochemical study revealed that CA XII is expressed not only in the normal human kidney but also in most clear-cell carcinomas and oncocytomas. In contrast, the other membrane-associated isozyme, CA IX, shows high expression only in renal carcinomas and is not expressed in normal kidney (
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Acknowledgments |
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Supported by grants from the National Institutes of Health (DK40163 and GM34182) to WSS and from the Sigrid Juselius Foundation to SP.
Received for publication May 2, 2000; accepted July 10, 2000.
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