ARTICLE |
Correspondence to: Sylvie Breton, Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129.
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Summary |
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In kidney epithelial cells, a variety of physiological processes are dependent on the active recycling of membrane proteins between intracellular vesicles and the cell surface. Although clathrin-mediated endocytosis occurs in several renal cell types, endocytosis can also occur by non-clathrin-coated vesicles, including pinocytotic structures known as caveolae that contain a novel coat protein, caveolin. Exo- and endocytosis of a vacuolar H+-ATPase in intercalated cells also occurs via specialized "coated" vesicles that do not contain clathrin. The aim of this study was to localize caveolin in the kidney and, in addition, to determine whether it could be a component of the H+-ATPase recycling process. Using an antibody against the - and ß-isoforms of caveolin-1, our immunocytochemical data show a marked heterogeneity in the cellular expression of this isoform of caveolin in kidney. In contrast, caveolin-3 was not detectable in renal epithelial cells. Caveolin-1 was abundant in endothelial cells and smooth muscle cells and was present in the parietal cells of Bowman's capsule. Distal tubule cells, connecting tubule cells, and collecting duct principal cells exhibited marked punctate basolateral staining, corresponding to the presence of caveolae detected by electron microscopy, whereas all intercalated cells were negative in both cortex and medulla. These data indicate that although caveolin-1 may participate in basolateral events in some kidney epithelial cell types, it does not appear to be involved in the regulated recycling of H+-ATPase in intercalated cells. Therefore, these cells recycle H+-ATPase by a mechanism that involves neither clathrin nor caveolin-1. (J Histochem Cytochem 46:205214, 1998)
Key Words: immunocytochemistry, caveolin, kidney, intercalated cell, endocytosis
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Introduction |
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Recycling OF MEMBRANE PROTEINS plays a key role in modulating the transport of fluid, electrolytes, and proteins in epithelial cells lining different segments of the urinary tubule (
However, endocytosis can also occur via other pathways, and considerable attention has been focused on caveolae as mediators of an alternative route of internalization that does not involve clathrin (
We previously showed that vigorous endocytosis of the vacuolar H+-ATPase and fluid-phase markers occurs in kidney collecting duct intercalated cells via a clathrin-independent mechanism (
The aim of the present study was to examine the distribution of caveolin in kidney epithelial cells and to determine whether it is located in those cell types in which clathrin-independent endocytosis is prevalent. Our results show that caveolin has a distinctive basolateral pattern of distribution in several epithelial cell types along the urinary tubule but that it is not detectable in intercalated cells where H+-ATPase-coated vesicles are predominantly involved in endocytosis.
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Materials and Methods |
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Antibodies
Two anti-caveolin monoclonal antibodies (MAbs) were used in this study. One recognizes only the -isoform of caveolin-1 (MAb 2234), and the other recognizes both the
- and ß-isoforms (MAb 2297). All of the positive results presented were obtained using the latter antibody, suggesting that the isoform detected in the kidney by immunocytochemistry was the ß-isoform. Antibodies against caveolin-3, found in muscle cells (
Western Blotting
To confirm specificity of the anti-caveolin antibodies, Western blots of kidney outer medulla were performed as previously described (
Animals
Male SpragueDawley rats were anesthetized and kidneys were fixed in vivo with PLP buffer containing 2% paraformaldehyde, 10 mM sodium periodate, 70 mM lysine (PLP), and 5% sucrose as previously described (
For preparation of 4-µm sections, tissues were cryoprotected in 30% sucrose before sectioning with a Reichert Frigocut microtome using disposable knives. For 1-µm sections, tissues were immersed in 2.3 M sucrose for at least 1 hr before freezing in liquid nitrogen and sectioning on glass knives in a Reichert FC4D ultracryomicrotome as previously described (
Immunostaining
Tissue sections were immunostained as previously described
(
Some sections were double stained to confirm the identity of the cells that were positive or negative for caveolin in the collecting duct. After application of the anti-caveolin antibody followed by secondary antibody coupled to CY3, rabbit polyclonal antibodies against either the AQP2 water channel (a principal cell marker, affinity-purified, diluted 1:4), AE1 (a marker of A-intercalated cells, diluted 1:1600), or the 56-kD subunit of the H+-ATPase (a marker of A- and B-intercalated cells, diluted 1:100) were applied for 2 hr, followed by a goat anti-rabbit IgG coupled to FITC, diluted 1:60 (Jackson Immunologicals).
Sections were photographed on a Nikon FXA microscope and some color images were captured using an Optronics 3-bit CCD color camera (Optronics Engineering; Goleta, CA), and IP Lab Spectrum (Scanalytics; Vienna, VA) acquisition and analysis software running on a Power PC 8500. Separate images for CY3 and FITC staining were obtained from double stained specimens, and the individual images were color-separated into their RGB components. The red (for CY3) and green (for FITC) components were merged and the composite images were imported as TIFF files into Adobe Photoshop 3.04 for size reduction and printing on a Tektronix Phaser 440 dye-sublimation color printer.
Endosome Labeling by FITCDextran Uptake
Some rats were anesthetized as above and injected with 1 ml of a 25 mg/ml solution of FITCdextran (10,000 kD; Molecular Probes, Eugene, OR) into the jugular vein, as previously described (
Electron Microscopy
Conventional Electron Microscopy.
Caveolae (pinocytotic invaginations) and other coated membrane vesicles were detected in renal epithelial cells by conventional electron microscopy in rat kidneys fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer.
Immunogold Electron Microscopy.
PLP-fixed kidneys were used for immunogold electron microscopy on ultrathin frozen sections as previously described (
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Results |
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Western Blotting
The anti-caveolin /ß antibody MAb 2297 detected in the kidney a single band at 21 kD, corresponding to the ß-isoform, whereas in the intestine a single band at a slightly higher molecular weight, probably representing the
-isoform, was observed. Both
- and ß-isoforms are present in the epididymis (Figure 1). The caveolin band in the inner stripe of the outer medulla was fainter than in intestine or epididymis, in which caveolin-containing smooth muscle cells are very abundant. These results demonstrate that, in the kidney, only a single band at the expected molecular weight of the ß-isoform of caveolin is recognized (
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Immunofluorescence Microscopy
Only the antibody against both - and ß-caveolin gave positive immunostaining in the kidney. In contrast,
-caveolin-1 and caveolin-3 antibodies did not label renal epithelial cells (not shown). These results indicate that the isoform localized in the kidney is probably ß-caveolin-1.
Cortex. Caveolin was most abundant at the basolateral pole of distal convoluted tubules and connecting tubules, where its pattern of staining suggested an association with the highly infolded basolateral plasma membrane of these cells (Figure 2 and Figure 3AC). The apical pole was unstained. In the connecting segment, basolateral staining was present in some cells, whereas others were unstained. In the collecting duct, a mosaic pattern of basolateral staining was also seen (Figure 3D). Identification of the negative cells as intercalated cells was confirmed by double staining (see below). In addition to tubule staining, the parietal epithelium of Bowman's capsule was positive and some cells in the glomerulus were labeled (Figure 2A and Figure 2B). Peritubule capillaries were unstained, but both smooth muscle cells and endothelial cells were brightly positive in small vessels, including afferent and efferent glomerular arterioles (Figure 2B). Proximal tubule cells from all segments (S1, S2, S3) showed no detectable staining (Figure 2A and Figure 3A), and cortical thick ascending limbs of Henle were negative.
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Double Staining.
In connecting segments and collecting ducts, all cells positive for caveolin were negative for both AE1 (Figure 3B) and the H+-ATPase 56-kD subunit (Figure 3C), but were apically stained with anti-AQP2 antibodies (Figure 3D). Caveolin-negative cells all contained the 56-kD H+-ATPase subunit (Figure 3C), identifying them as intercalated cells (
Outer Medulla: Outer Stripe and Inner Stripe. Collecting duct principal cells (AQP2-positive) showed an irregular, punctate basolateral staining for caveolin (Figure 4A and Figure 4B, inner stripe). Intercalated cells, identified by an absence of AQP2 staining and by the presence of basolateral AE1 and apical H+-ATPase (not shown), did not contain detectable caveolin. S3 proximal tubule segments in the outer stripe (not shown) and thick ascending limbs were also unstained (Figure 4A and Figure 4B). Endothelial cells of some vasa recta in the vascular bundles were brightly stained, whereas others were not fluorescent. Thin descending limbs of Henle inside the vascular bundles (descending thin limbs of short-looped nephrons) were stained (not shown).
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Inner Medulla/Papilla. Collecting duct principal cells, vasa recta, and some thin limbs of Henle were labeled. Intercalated cells comprise only 510% of the collecting duct epithelium in the initial portion of the inner medulla, and then disappear completely from collecting ducts in the middle and the tip of the papilla. In these regions, most of the epithelial cells (principal cells) were stained with both caveolin (Figure 4C) and AQP2 (Figure 4D). The papillary surface epithelium was unstained.
FITCDextran Uptake
As previously reported, FITCdextran injected into the jugular vein of rats is a fluid-phase marker of apical endocytosis in the kidney (
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Electron Microscopy
In many cell types, caveolae were associated with the basolateral plasma membrane. Figure 6A and Figure 6B show the basal region of a connecting tubule epithelial cell (Figure 6A) and a collecting duct principal cell (Figure 6B). The basal plasma membrane infoldings of the connecting tubule cell penetrate much more deeply into the cytoplasm, and caveolae are found at different levels along these membrane infoldings. In contrast, the basal infoldings of the principal cell are much shallower but also contain caveolae. The marked difference in geometry of these infoldings is responsible for the different width of the immunofluorescent band of caveolin staining seen in these cell types (compare Figure 3C and Figure 3D). Caveolae were not detected in the basolateral or apical plasma membranes of intercalated cells or proximal tubule epithelial cells, consistent with the absence of labeling with anti-caveolin antibodies. In contrast to epithelial cells, large numbers of caveolae were present in endothelial cells of the descending vasa recta (Figure 6C), arterioles, and the glomerular capillaries, as well as in smooth muscle cells and adipocytes.
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Morphologically recognizable caveolae were rarely seen in ultrathin frozen sections, probably because of the SDS treatment that was required to reveal caveolin antigenicity. Nevertheless, heavy immunogold labeling for caveolin was found in cells in which caveolae were abundant. As an example, an endothelial cell from a descending vasa recta is shown in Figure 6D. Many gold particles are located at the basolateral pole of this cell, precisely the site where caveolae are concentrated, as shown by conventional electron microscopy in Figure 6C. In epithelial cells, some small clusters of gold particles were found over the basal infoldings of connecting segment and collecting duct principal cells (not shown), but lack of membrane resolution made it impossible to attribute this staining to the presence of the single, isolated caveolae that occur in these cells.
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Discussion |
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Epithelial cells lining the urinary tubule carry out many specialized functions that, in several cases, involve the exo- and endocytotic recycling of membrane proteins (
Our present results show that the coat protein caveolin is not detectable in intercalated cells, whereas caveolin is abundant in some other kidney cell types. The distal convoluted tubule had the brightest staining, which was, as in principal cells, uniquely basolateral. Interestingly, a Ca2+-ATPase has been located in caveolae in many cell types and is abundant in the basolateral membrane of the distal tubule (
The role of caveolin in the basolateral membrane of principal cells is unknown, but a variety of proteins, including signal transduction proteins, have been found in caveolae (s and adenylate cyclase (
s and G
i2 on the basolateral membrane domain of vasopressin-sensitive principal cells (
It has been proposed that GPI-anchored membrane proteins are concentrated in caveolae in several cell types (
The absence of caveolin and clathrin from H+-ATPase-recycling vesicles in intercalated cells suggests that these vesicles represent a distinct class of coated vesicle. Whether the H+-ATPase subunits that form this coat have roles in vesicle trafficking in addition to (or in parallel with) their function as ion-transporting proton pumps in intercalated cells and related H+-secreting cells (-adaptin subunit also co-purifies with the proton pump under some conditions (
The presence of caveolin in defined membrane domains further supports the notion that different renal epithelial cells, and even different membrane domains in the same cell, have evolved specialized membrane trafficking pathways that are involved in the regulation and modulation of epithelial transport pathways in this complex tissue.
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Footnotes |
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1 Present address: Albert Einstein College of Medicine, Dept. Molecular Pharmacology, Bronx, NY.
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Acknowledgments |
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Supported by grants from the National Institutes of Health, DK 42956 (DB) and DK38452 (SB). ML was supported by an NIH FIRST award GM-50443, by a grant from the W. M. Keck Foundation to the Whitehead Fellows program, and by a grant from the Elsa U. Pardee Foundation. SB was partially supported by a grant from National Sciences and Engineering Research Council of Canada, by a Hoechst Marion Roussel fellowship from the National Kidney Foundation, and by a Claflin Distinguished Scholar Award from the Massachusetts General Hospital.
Received for publication April 28, 1997; accepted August 7, 1997.
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