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*Department of Medicine, Division of Nephrology, and Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York.
Correspondence to Dr. Barbara J. Ballermann, Professor of Medicine, The Albert Einstein College of Medicine, 1300 Morris Park Avenue,, Bronx, New York 10461. Phone: 718-430-3252; Fax: 718-430-8963; E-mail: bjballer{at}aecom.yu.edu
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Introduction |
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Several lines of evidence have suggested that endothelial fenestrae form through fusion of caveolae. Caveolae are diaphragmed, cholesterol-rich vesicular plasma membrane invaginations with a lipid composition similar to that of lipid raft microdomains (3). They contain high levels of the caveolae-specific protein caveolin-1, which is required for their formation (4,5). Ultrastructural studies of cultured endothelial cells show that organelles resembling caveolae and containing caveolin-1 can undergo fusion to form transendothelial channels (6–8), a postulated stage in fenestrae formation. Like caveolae, most endothelial cell fenestrae in vivo contain bridging diaphragms (9,10), and their peristomal plasma membrane is cholesterol-rich, as in caveolae (11). Furthermore, the protein PV-1, initially isolated from the apical plasma membrane of pulmonary endothelium (12), is specifically associated with the bridging diaphragms of endothelial fenestrae and caveolae (9). In regard to this study, it is notable, however, that PV-1 was uniquely absent from glomerular endothelial cell fenestrae (9), possibly due to the lack of or a different molecular composition of fenestral diaphragms in these cells.
VEGF-mediated induction of endothelial fenestrae has also been linked to fusion of caveolin-1–containing vesicles (13). Typically, VEGF is highly expressed in epithelial cells neighboring fenestrated endothelia (14), and VEGF can rapidly induce fenestrae in capillaries in vivo (15). VEGF-mediated induction of fenestrae is accompanied by an increase in the number of fused caveolar vesicles (16), and such vesiculo-vacuolar organelles express caveolin-1 (13). Recently, Chen et al. (6) similarly found that caveolin-1–lined transendothelial channels can be induced by VEGF in cultured glomerular endothelial cells in vitro. However, Esser et al. (16), who observed caveolin-1 in fusing caveolae in vitro, noted that caveolin-1 is absent from fenestrae of cultured endothelial cells and fenestrae of choroid plexus endothelium in vivo.
Taken together, the data reported so far suggest that both caveolae and fenestrae are spanned by diaphragms and that the protein PV-1 is a component of caveolar diaphragms and diaphragms of some but not all fenestrae. Furthermore, fusion of caveolin-1–containing caveolae occurs in response to VEGF in vitro, and such fused caveolae have been reported to form transendothelial channels/fenestrae. However, fenestrae have not been shown to contain caveolin-1. If caveolae are indeed precursors of fenestrae, it would be expected that caveolin-1–deficient mice, which cannot form caveolae (4,5), would also exhibit defects in their fenestrae.
To determine whether glomerular endothelial fenestrae in vivo represent fused caveolae, we explored caveolin-1 localization in mouse and human renal glomeruli in situ and determined the ultrastructure of glomerular endothelial fenestrae in caveolin-1–deficient mice. We report here that caveolin-1 is expressed by glomerular endothelial cells but is excluded from the fenestrated region of these cells. Furthermore, although caveolae do not form in caveolin-1–deficient mice, glomerular endothelial cell fenestration is normal, implying that glomerular endothelial cell fenestrae in vivo do not form through fusion of caveolae.
Although the emphasis of the study was on the role of caveolin-1 in fenestrae formation, we also noted that caveolin-1 is highly expressed by podocytes. Caveolin-1 localizes, at least partially, to podocyte filtration slits and interacts with the podocyte slit diaphragm proteins nephrin and CD2AP, findings consistent with the lipid raft nature of the podocyte filtration slit plasma membrane.
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Materials and Methods |
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Human renal cortex represents discarded normal cortex removed at the time of tumor nephrectomy according to protocol FWA-00000140 approved by the Albert Einstein College of Medicine Committee on Clinical Investigations. Portions of the healthy pole of kidneys were fixed by immersion in 4% paraformaldehyde in PBS for 16 h following with 18% sucrose in PBS for 10 h. The samples were embedded in OCT (Tissue-tek; Sakura Finetech, Torrance, CA), and 3- or 4-µm sections were cut on a Leica CM 3050 S cryostat. Hematoxilin-eosin stained sections were examined by light microscopy and found to be normal.
Immunofluorescence
Rabbit polyclonal anti-caveolin-1 antibody (N-20; Santa Cruz Biotechnology, Santa Cruz, CA) or mouse monoclonal anti-caveolin-1 (C060; BD Transduction Laboratories, Lexington, KY) was used to detect caveolin-1 in mouse and human tissue sections. Rabbit polyclonal anti-nephrin antibody (a kind gift from Dr. Lawrence Holzman, University of Michigan, Ann Arbor, MI), rabbit anti-synaptopodin (17), and rabbit anti-podocin (18) were used to detect nephrin, synaptopodin, and podocin, respectively, in human and mouse tissue sections. Rhodamine-labeled Ulex Europaeus Agglutinin I (UEA; Vector Laboratories, Burlingame, CA) was used to label endothelial cells in human tissue sections. Sections were treated with 0.3% Triton X-100 in PBS for 10 min, washed 3 times for 5 min with PBS, and blocked with 2% FCS, 2% BSA, and 0.2% fish gelatin in PBS for 30 min at room temperature (RT). This was followed by incubation with anti-caveolin-1 antibody (1:200 in blocking solution) for 1 h at RT. The sections were again washed 3 times for 5 min with PBS, followed by incubation with Alexa 488–conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) at a concentration of 1 µg/ml for 30 min at room temperature. After washing, some of the sections were incubated with rhodamine-labeled UEA at a concentration of 10 µg/ml for 30 min at RT. For the nephrin/caveolin-1 colocalization study, human kidney sections were blocked as described above. Caveolin-1 monoclonal antibody was applied for 1 hr (1:100) at RT, the sections were washed and polyclonal anti-nephrin antibody (1:100) was applied for 1 hr. After washing, Cy3 anti-mouse IgG (1:150) and Alexa 488 anti-rabbit IgG (1:500) were applied for 45 min at RT. For experiments in caveolin-1–deficient mice, synaptopodin (1:100), podocin (1:100), nephrin (1:100), and caveolin-1 (1:200) antibodies were applied to frozen kidney sections of both wild-type and caveolin-1–deficient mice for 1 h. As secondary antibody, Alexa 488 anti-rabbit IgG (1:500) was applied for 45 min at RT. The blocking and washing steps were done as described above. The sections were mounted using ProLong Antifade kit (Molecular Probes). They were then evaluated by confocal microscopy using a Bio-Rad MR600 confocal fluorescence microscope.
Immunogold Electron Microscopy
Ultrathin (100 to 120 nm) frozen sections were prepared from perfusion-fixed mouse kidneys on a Leica Cryo Ultra Cut cryostat. Blocking was done with 2% FCS, 2% BSA, and 0.2% fish gelatin in PBS for 30 min at RT. Polyclonal rabbit anti-caveolin-1 IgG at a 1:200 dilution was applied for 1 h at RT followed by three 5-min washes with PBS and incubation with donkey anti-rabbit IgG coupled to 18 nm of colloidal gold (Jackson ImmunoResearch, West Grove, PA). After rinsing with PBS, the sections were postfixed with 2% glutaraldehyde and 0.5% tannic acid and counterstained with 2% osmium tetroxide. This was followed by staining with 1% uranyl acetate and then 0.003% lead citrate in 2% polyvinyl alcohol in dd H2O. The sections were air-dried in RT and then observed with a JEOL 1200EX transmission electron microscope.
Morphometry
The density of caveolin-1 immunogold labeling was determined for tissue sections from three separate mice processed independently. Digital photomicrographs (magnification, x15,000 to x30,000) were overlaid with a grid (5 x 5 mm). The area representing podocyte cell body, podocyte foot processes, endothelial cell body, fenestrated portion of endothelium, and background for each photomicrograph was determined by point-counting, using the intersects of the grid as evenly spaced points. Counts from each photomicrograph were converted to µm2 by correcting for the magnification. Gold particles associated with each structure were then quantified and expressed per µm2. Data represent the mean gold particle density/µm2 for each structural component from 4 to 10 photomicrographs per animal. Comparisons were made by the t test.
Transmission Electron Microscopy
Immersion-fixed mouse kidneys were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol, and embedded in LX112 resin (LADD Research Industries, Burlington, VT). Ultrathin sections were cut on a Reichert Ultracut E, stained with uranyl acetate followed by lead citrate, and viewed on a JEOL 1200EX transmission electron microscope at 80kV.
Protein Extraction and Immunoprecipitation
Glomeruli from adult C57 black mice were isolated as described in (17). They were pelleted by centrifugation for 5 min at 1000 x g at 4°C and resuspended in homogenization buffer (20 mM Tris, 0.1% NP-40, 150 mM NaCl, pH 7.5) supplemented with protease inhibitors as described (18). Glomeruli were then homogenized at 4°C with 15 strokes of a Dounce homogenizer. Insoluble material was pelleted at 14,000 x g for 10 min at 4°C and reprocessed once as described. The soluble material from both extractions was pooled and passed 20 times through a 27-gauge needle before use. For each immunoprecipitation, samples containing 0.5 mg protein were precleared with protein A-sepharose 4B (Sigma, St. Louis, MO) in 0.5 ml TNE (250 mM NaCl, 5 mM EDTA, 10 mM Tris, protease inhibitors, pH 7.4) for 1 hr at 4°C. The extracts were then incubated with 1 µg of anti-nephrin IgG (19), anti-CD2AP IgG (20), or anti-snRNP IgG (Progen, Heidelberg, Germany) for 2 h at 16°C in the presence of 0.4% SDS. Alternatively, the extracts were incubated with 2 µg of caveolin-1 IgG (N-20, Santa Cruz) or 1 µg of caveolin-1 IgG (2234, BD Transduction Laboratories) overnight at 4°C in the presence of 0.4% SDS. The SDS was included to disrupt lipid-protein interactions in this raft microdomain fraction. The nonspecific aggregates were pelleted at 14,000 x g for 5 min at 4°C. Protein A/G-sepharose 4B beads were then added to the supernatants and incubated for 90 to 180 min at 4°C. The beads were washed five times in TNE buffer and eluted by boiling at 95°C for 5 min in Laemmli sample buffer (21) containing 5% 
-Mercaptoethanol.
SDS-PAGE and Western Blotting
Samples containing equal protein concentrations were separated on 4 to 15% Tris-HCl gradient gels (Bio-Rad, Hercules, CA) in a standard manner. The resolved proteins were transferred to Immobilon-P membranes (Millipore, Bedford, MA). The blots were blocked in 5% nonfat dry milk in TBS-Tween for 1 h at room temperature followed by overnight incubation with rabbit polyclonal anti-caveolin-1 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) or monoclonal anti-eNOS (1:800; BD Transduction Laboratories, Lexington, KY) at 4°C. Alternatively, the blots were incubated with rabbit polyclonal anti-nephrin (1:2000) or rabbit polyclonal anti-CD2AP (1:2000). After washing in TBS-Tween (3 times for 10 min), horseradish peroxidase-conjugated donkey anti-rabbit IgG or donkey anti-mouse IgG at a concentration of 1:4000 was applied (in TBS-Tween) for 1 h. Bands were visualized using enhanced chemiluminescence (Renaissance; NEN Life Science Products, Boston, MA).
Blood Urea Nitrogen Assay
Blood was collected from the clipped tail of three female caveolin-1–deficient and three female littermate wild-type mice, all 3 mo of age. Serum was prepared by centrifugation (6000 x g; 6 min at RT). Three separate assays (BUN Infinity Reagent; Sigma, St Louis, MO) were run according to the manufacturer’s instructions. Standard concentrations of urea were used for reference.
Urinary Protein Excretion
Urine was collected from the same three female caveolin-1–deficient and three littermate wild-type mice. The urinary protein excretion was evaluated by electrophoresis of the urine samples on a 4 to 15% gradient SDS-PAGE gel followed by staining with Coomassie blue in a standard manner. Albumin (60 kD; 0.1 µg/µl) was included as a molecular size control.
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Glomerular Caveolin-1 Localization by Immunogold Electron Microscopy
To definitively localize caveolin-1 expression in glomerular capillary cells, immunogold labeling followed by transmission electron microscopy was performed. Caveolin-1 immunogold labeling was observed in glomerular endothelial cell bodies (EB) (Figure 2, I-B) but never in the fenestrated portion of the glomerular endothelium, (Figure 2, I-A through I-C). Caveolin-1 immunogold labeling was also observed in podocytes, both in the podocyte cell body and in foot processes (Figure 2, I-A through I-C). In foot processes, caveolin-1 was often, but not invariably, found at the filtration slit, (Figure 2, I-C). This is the first demonstration that caveolin-1 is highly expressed by renal glomerular podocytes.
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The caveolin-1 immunogold density for podoctyes is shown in Figure 2, II-B. The total area of podocyte foot processes and cell bodies counted was 16.25 and 31.74 µm2, respectively. Some but not all of the caveolin-1–linked gold particles were found at the podocyte filtration slit.
Glomerular Ultrastructure and Function in Caveolin-1–Deficient Mice
To obtain more conclusive evidence of whether caveolae are required glomerular endothelial cell fenestrae precursors, we examined the morphology of the glomerular endothelium in caveolin-1–deficient mice. These mice have previously been extensively characterized (4); they neither express caveolin-1, nor can they form caveolae. Transmission electron microscopy revealed that glomerular endothelial cell fenestrae are indistinguishable in density and size in the caveolin-1–deficient mice compared with wild-type mice (Figure 3, I-A and I-B, n = 3/group).
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Interaction of Caveolin-1 with Nephrin and CD2AP
We observed some caveolin-1 localization to podocyte filtration slits; therefore, we evaluated nephrin and CD2AP immunoprecipitates from whole mouse glomerular lysates for the presence of caveolin-1. The immunoprecipitation was done in the presence of SDS to preclude nonspecific associations of these proteins due to their location in lipid raft microdomains (18,23). Western blotting with caveolin-1 antibody of nephrin and CD2AP immunoprecipitates shows that caveolin-1 is co-immunoprecipitated from the lysates with both nephrin and CD2AP antibodies (Figure 4, I-A, lanes 2 and 4) but not with antibodies to the nuclear protein snRNP (Figure 4, I-A, lane 6). Also, the supernatants of the nephrin and CD2AP immunoprecipitates contained almost no detectable caveolin-1 (Figure 4, I-A, lanes 3 and 5), whereas caveolin-1 remained in the supernatant after immunoprecipitation with snRNP (Figure 4, I-A, lane 7). To control for possible nonspecific interaction of non-podocyte caveolin-1 with nephrin and CD2AP in the lysates, we determined whether nephrin and CD2AP immunoprecipitates contain eNOS, a protein known to interact with caveolin-1 in endothelial cells. As shown in Figure 4 (I-B, lanes 2 and 4), eNOS was not immunoprecipitated with anti-nephrin or anti-CD2AP antibodies but remained in the lysates. In addition, immunoprecipitates prepared with anti-caveolin-1 polyclonal or monoclonal antibodies were probed for the presence of nephrin and CD2AP. This experiment was conducted in a similar manner to the previous co-immunoprecipitations. Immunoprecipitates prepared with anti-caveolin-1 polyclonal antibodies and blotted for nephrin (Figure 4, II-A) or CD2AP (Figure 4, II-B) show co-immunoprecipitation of both proteins with caveolin-1 (Figure 4, II, lane 2 in A and B). Not shown, immunoprecipitates prepared with monclonal anti-caveolin-1 antibodies gave identical results. It therefore seems unlikely that the results in Figure 4, I-A, can be explained by nonspecific association of endothelial cell caveolin-1 with nephrin or CD2AP.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The view that endothelial fenestrae could represent fused caveolae came from the observations that VEGF, known to induce fenestrae in vivo (15,24), also stimulates fusion of caveolae in endothelial cells to form caveolin-1–containing vesiculo-vacuolar structures (13,16) and caveolin-1–lined transendothelial channels (6) in vitro. Furthermore, the protein PV-1 is found in both the bridging diaphragms of endothelial caveolae and in the fenestrae of most endothelia (9). However, Esser et al. (16) reported that caveolin-1 was not present in the fenestrated region of choroid plexus endothelial cells in vivo or in fenestrae of cultured endothelial cells. Our finding showing that caveolin-1 is excluded from glomerular fenestrae is consistent with the observation by Esser et al. (16) for choroid plexus. We also find that fenestrae ultrastructure is normal in caveolin-1–deficient mice. Caveolin-1 is essential for caveolae formation (4,5); therefore, glomerular fenestrae must be distinct from endothelial caveolae, and glomerular fenestrae formation must proceed through mechanisms other than caveolar fusion.
Our study does not rule out the possibility that fenestrae in glomerular endothelial cells differ from those in other fenestrated endothelia. In fact, studies with cationic markers have long suggested that the composition of endothelial fenestral diaphragms differ in different organ beds (25). Also, the observation that the caveolar diaphragm protein PV-1 localizes to peritubular fenestral diaphragms but not glomerular fenestrae is consistent with the view that the molecular composition of bridging diaphragms differs between renal peritubular and glomerular fenestrae or that glomerular endothelial fenestrae do not contain bridging diaphragms (9). Therefore, it is still possible that caveolar fusion could form fenestrae in non-glomerular endothelial cells.
Caveolin-1 immunoreactivity in glomeruli has previously been described and was thought to represent mainly endothelial cell expression (22). We found a similar pattern of caveolin-1 immunofluorescence as that reported by Breton et al. (22), (Figure 1, I-A and I-B) but noted that the endothelial-specific marker UEA co-localized only partially with caveolin-1 (Figure 1, II-C and II-F). We postulate that those regions labeled with UEA but not with caveolin-1 likely represent the fenestrated portion of the glomerular endothelial cells. The regions labeled by caveolin-1 antibodies but not by UEA could represent podocyte or mesangial cell labeling. Indeed, Tamai et al. (26) recently reported that caveolin-1 is present in mesangial cells. Still, the pattern of glomerular caveolin-1 immunofluorescence (Figure 1) does not suggest a predominant mesangial location. Using caveolin-1 immunogold electron microscopy, we find that caveolin-1 is expressed at high levels in glomerular podocytes. Caveolin-1 expression in podocytes has not previously been described.
Caveolin-1 immunogold localization in podocytes was often, but not exclusively, found at the podocyte filtration slits (Figure 2, I-C). The filtration slit proteins, including nephrin (19), CD2AP (27), and podocin (18), are associated with lipid rafts (23). These filtration slit proteins all are required to maintain the glomerular barrier to protein filtration, as deletion of nephrin, CD2AP, and podocin all result in congenital proteinuria and progressive focal and segmental glomerular sclerosis (20,28,29). Caveolae, like membrane regions at the filtration slit, also represent a type of lipid raft microdomain; therefore, we explored whether caveolin-1 can associate with some of the known slit diaphragm proteins. Immunoprecipitation of nephrin and CD2AP from glomerular lysates resulted in co-immunoprecipitation of caveolin-1, (Figure 4, I-A). By contrast, eNOS, known to be associated with caveolin-1 in endothelial cell caveolae (30), did not co-immunoprecipitate with nephrin or CD2AP (Figure 4, I-B). Hence, nonspecific association of endothelial caveolin-1 with nephrin or CD2AP in the glomerular lysates was ruled out. Immunoprecipitation of caveolin-1 from glomerular lysates resulted in co-immunoprecipitation of both nephrin and CD2AP as well, further validating the experiments described above (Figure 4, II-A and II-B). In addition, dual labeling with anti-nephrin and anti-caveolin-1 antibodies results in partial co-localization of the antibodies around the capillary loops of the glomeruli, further increasing the evidence for nephrin and caveolin-1 association (Figure 4, III-C). Nevertheless, podocyte foot process ultrastructure was normal in mice deficient in caveolin-1, no increase in urinary protein concentration was observed, and their BUN was normal (Figure 3). In addition, expression of the podocyte proteins, synaptopodin, podocin, and nephrin, did not differ between caveolin-1–deficient mice and their wild-type controls. It is therefore evident that caveolin-1 is not critical for the formation or function of podocyte slit diaphragms. The exact role of caveolin-1 in podocyte slit diaphragm function remains to be defined.
In conclusion, caveolin-1 is not found in glomerular endothelial cell fenestrae, nor is it necessary for the formation of normal fenestrae. Hence, the possibility that endothelial fenestrae represent fused caveolae has been ruled out, at least for glomerular endothelial cells. The interaction of caveolin-1 with nephrin and CD2AP in podocytes is consistent with the previous findings that the filtration slit plasma membrane represents a type of lipid raft microdomain.
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