1 Program in Membrane Biology and Renal Unit, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114; and 2 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a key role in docking and fusion of intracellular transport vesicles and may regulate apical and basolateral membrane protein delivery in epithelial cells. In a previous study, syntaxin 3 (a target SNARE) protein was detectable in the kidney only in intercalated cells. We now report a more widespread distribution of syntaxin 3 in a variety of renal epithelial cells after antigen retrieval. Sections of rat kidney were treated with SDS and incubated with antisyntaxin 3 antibodies. Strong basolateral membrane staining was seen in descending and ascending thin limbs of Henle, thick ascending limbs of Henle, the macula densa, distal and connecting tubules, and all cells of the collecting duct including A- and B-intercalated cells. The papillary surface epithelium and the transitional epithelium of the ureter were also stained, but proximal tubules were negative. Western blotting revealed a strong signal at 37 kDa in all regions, and the antigen was restricted to membrane fractions. SDS treatment was not necessary to reveal syntaxin 3 in intercalated cells. These data show that syntaxin 3 might be involved in basolateral trafficking pathways in most renal epithelial cell types. The exclusive basolateral location of syntaxin 3 in situ, however, contrasts with the apical location of this SNARE protein in some kidney epithelial cells in culture.
soluble N-ethylmaleimide-sensitive factor attachment protein receptor; urinary tubule; basolateral membrane
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INTRODUCTION |
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THE GENERATION AND MAINTENANCE of distinct membrane domains in all cell types depend on the fidelity of vesicle trafficking and fusion processes. Over the past few years, it has become clear that fusion between donor and target membranes is regulated by a complex machinery that involves the interaction of membrane-associated and soluble proteins. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of transmembrane proteins that are found on target (t-SNAREs) and vesicle membranes, and it is believed that specific interactions between cognate SNAREs represent an important step in vesicle docking and fusion (17, 18). More recent data have resulted in modifications of the original SNARE hypothesis, and the precise way in which SNAREs and other accessory proteins interact before and during vesicle-target membrane fusion is the subject of intense investigation (14, 21, 22). The series of protein-protein interactions envisaged in the SNARE hypothesis provides an elegant way in which the distinct protein and lipid fingerprint of functionally distinct membranes can be tightly controlled and modulated.
The syntaxins represent a major class of t-SNAREs (1, 4). Their intracellular distribution has been examined in different epithelial cells to determine whether they might play some role in generating the polarized epithelial cell phenotype. The function of the urinary tubule, for example, depends on the apical and basolateral expression of a multitude of different transport systems, all of which must be correctly targeted to ensure the vectorial movement of fluid, ions, and other molecules across the lumen-to-blood epithelial interface (8, 9). In Madin-Darby canine kidney (MDCK) cells, derived from dog kidney, transfected syntaxin 3 was restricted to the apical plasma membrane and syntaxin 4 to the basolateral membrane, while syntaxin 2 was present on both membrane domains (11). In contrast, studies on endogenous syntaxins in renal epithelia in situ revealed syntaxin 4 on the apical plasma membrane of principal cells and syntaxin 3 on the basolateral membrane of intercalated cells (13). However, this report also showed that syntaxin 3 mRNA was expressed in a cellular pattern that was more widespread than detectable protein (13). In the present study, therefore, the location of syntaxin 3 protein in the kidney was reexamined using an antigen retrieval technique that has been previously described (7). The results show that, although no apical localization of syntaxin 3 became apparent after antigen retrieval, basolateral syntaxin 3 was detectable in many more segments of the urinary tubule than previously reported. These data indicate that syntaxin 3 may have a more ubiquitous role in basolateral trafficking in the kidney than was originally envisaged.
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MATERIALS AND METHODS |
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Polyclonal Antibodies
The production and characterization of polyclonal antibodies against syntaxin 3 have been described previously (13). Briefly, a 23-amino acid peptide corresponding to the NH2 terminus of syntaxin 3 (KDRLEQLKAKQLTQDDDTDEVEC) was coupled to keyhole limpet hemocyanin and used to immunize rabbits by a standard protocol. The antisera from two rabbits were affinity purified with the immunizing peptide using the SulfoLink system (Pierce, Rockford, IL). Preimmune serum was purified on a protein A affinity column (Pierce) and was used for control incubations. Some incubations were also performed using the affinity-purified antibody that had been preabsorbed with the immunizing peptide.Preparation of Membrane Pellets From Rat Kidneys
Membranes from the cortex, outer medulla, and inner medulla were prepared from rat kidneys, as previously described (13). Adult Sprague-Dawley rats were killed by decapitation, and tissues were homogenized in ice-cold homogenization solution (250 mM sucrose and 10 mM triethanolamine) with protease inhibitors. Plasma membrane-rich pellets were obtained by centrifugation, and the 17,000-g pellet was used for Western blotting. For the inner medulla, the 17,000-g supernatant was further centrifuged at 200,000 g to obtain a pellet of low-density microsomes, as previously described (13).Electrophoresis and Western Blotting
Membranes were solubilized at 60°C for 15 min in Laemmli buffer, and proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 12% minigels. After transfer of the proteins, nitrocellulose membranes were blocked with blotting buffer (150 mM NaCl, 50 mM NaH2PO4, and 0.05% Tween 20, pH 7.5) that contained 5% nonfat dried milk and were incubated with affinity-purified antisyntaxin 3 antibody (0.5 µg/ml). Donkey anti-rabbit IgG coupled to horseradish peroxidase was then applied at a concentration of 0.16 µg/ml. Staining was revealed by enhanced chemiluminescence (LumiGlo, Kirkegaard and Perry Laboratories, Gaithersburg, MD).Experimental Animals and Tissue Fixation
Experiments were conducted using mature (300-350 g) male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA). Rats were anesthetized using a pentobarbital sodium (50 mg/ml) injection of 0.15 ml/100 g body wt. The kidneys were fixed via left ventricle cardiac perfusion with 150 ml of Hanks' balanced salt solution followed by paraformaldehyde-lysine-periodate (PLP) fixation. The original PLP recipe (15) was modified to increase the paraformaldehyde concentration from 2% to 4%. The kidneys were removed after 5 min of perfusion in PLP and placed in PLP fixative at room temperature for a further 4-6 h or overnight at 4°C. Tissue was washed three times in PBS and kept at 4°C in PBS containing 0.02% sodium azide before use.Antigen Retrieval and Immunofluorescence
PLP-fixed tissues were cryoprotected in a solution of 30% sucrose in PBS forFor immunostaining, sections were hydrated for 5 min in PBS and pretreated for 5 min with 1% SDS in PBS, an antigen retrieval technique that we have previously described (7). Slides were washed three times in PBS for 5 min each time and then preincubated in 1% BSA in PBS-0.02% sodium azide for 15 min. Sections were incubated in primary antisyntaxin antiserum (5 µg/ml) for 90 min at room temperature, washed twice for 5 min in high-salt PBS (2.7% NaCl) to reduce nonspecific staining, and washed once in normal PBS. Sections were then incubated for 1 h with secondary antibody, goat anti-rabbit IgG coupled to FITC (Jackson Immunologicals, West Grove, PA), and they were again washed as described above. Double labeling was performed by subsequent incubation of some sections with a chicken anti-H+-ATPase antibody and donkey anti-chicken IgG conjugated to Cy3 (Jackson Immunologicals). The chicken antibody was raised against the COOH terminus of the 31-kDa H+-ATPase subunit (E subunit), and its use has been described previously (3). The slides were mounted in Vectashield (Vector Labs, Burlingame, CA) diluted 1:1 with Tris buffer, pH 8.5, and examined using a Nikon E800 epifluorescence microscope. Syntaxin 3 staining was photographed using TMAX 400 film push-processed to 1600 ASA. Double-stained sections were digitally imaged using a Hamamatsu Orca charge-coupled device camera and IPLab Spectrum software (Scanalytic, Vienna, VA). Final pseudocolored images were imported into and printed from Adobe Photoshop.
Control incubations were performed using preimmune IgG or antibodies that had been preabsorbed with the syntaxin 3 immunizing peptide before the first incubation step. All control incubations were negative, illustrating the specificity of the staining patterns reported here.
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RESULTS |
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Western Blotting of Kidney Membranes
Syntaxin 3 was detected by Western blotting in all kidney regions (Fig. 1A). The recognized band was at 37 kDa. In contrast to previous data (13), an additional weaker 67-kDa band was not detected by the antisyntaxin antibody in the present series of experiments. In whole membranes, the strongest expression was observed in the kidney cortex and outer medulla. A weaker band was present in membranes from the inner medulla. When inner medullary membranes were fractionated further, the 37-kDa band was detectable only in the "plasma membrane-enriched" fraction, and not in the 200,000-g pellet, which is composed of smaller vesicles and light endosomes (Fig. 1B).
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Immunofluorescence Staining of Kidney Sections
Cortex.
In the kidney cortex, staining was bright in cortical collecting
ducts, connecting segments, and cortical thick ascending limbs (Fig.
2). No specific staining was detectable
in proximal convoluted tubules or in the glomerulus. Staining in
collecting ducts and connecting segments was basolateral, and all cell
types were stained. In the cortical thick ascending limb, staining was also basolateral. Double staining with antibodies against
H+-ATPase showed that the basolateral plasma membranes of
A- and B-intercalated cells were stained for syntaxin 3. Staining was stronger in B-intercalated cells than in cortical A-intercalated cells
(Fig. 3), and syntaxin 3 staining of
B-cells partially overlapped the basolateral H+-ATPase
staining pattern.
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Outer medulla.
In the outer medulla, collecting ducts, thick ascending limbs of Henle,
and thin limbs of Henle were brightly positive (Figs. 4 and
5A). In all cases, the
staining was basolateral. In the case of thin limbs, the resolution was
insufficient to determine the polarity of the staining. All collecting
duct cells were stained with an approximately equal intensity. Stained
thin descending limbs were identified by finding regions of transition
between unstained proximal tubule S3 segments (Fig. 4A) and
the descending thin limb. Stained thin ascending limbs were identified
at regions of transition with the thick ascending limb (Fig.
4B). In addition, the thin ascending limb staining had a
punctate appearance because of the highly interdigitated nature of the
epithelium in this tubule region.
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Inner medulla.
In the outer third of the inner medulla, the basolateral
membranes of all collecting duct epithelial cells were brightly stained (Fig. 5B). Thin limbs of Henle were weakly positive. For
comparative purposes, adjacent sections were stained without SDS
pretreatment. As described previously, the only cells showing positive
staining under these conditions were intercalated cells (Fig.
5C). However, the intercalated cell staining was somewhat
weaker in the absence of SDS. In the middle third and the tip of the
papilla, bright basolateral staining of principal cells was observed
(Fig. 6A). Principal cells in
these regions were weakly stained, even in the absence of SDS
pretreatment (Fig. 6B). In addition, papillary surface
epithelial cells also showed a marked basolateral staining for syntaxin
3, as did the cells of the transitional epithelium of the ureter (Fig.
6A).
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DISCUSSION |
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A previous study reported that the t-SNARE syntaxin 3 was
detectable by immunocytochemistry only in A-intercalated cells in the
kidney (13). This led to the suggestion that syntaxin 3 was somehow involved in the targeting of basolateral membrane proteins,
including the Cl/HCO
Our present results show that the renal distribution of the syntaxin 3 protein is much more widespread than was originally proposed. Virtually all cells and tubule segments examined showed strong basolateral expression of syntaxin 3. A notable exception was the proximal tubule, which correlates with the previously reported absence of syntaxin 3 message in this tubule segment (13). Thus basolateral syntaxin 3 expression appears to be a common feature of many different epithelial cells in the kidney, and its role in basolateral targeting processes is not restricted to A-intercalated cells. Indeed, A- and B-intercalated cells were basolaterally stained for syntaxin 3, and the brightest staining was in the B-intercalated cells. In their previous study, Mandon et al. (13) did not examine syntaxin 3 in the cortex and, therefore, did not describe B-intercalated cell staining, since these cells are present mainly in cortical collecting ducts. The positive staining after SDS treatment is probably due to the unmasking of cryptic antigenic epitopes that are at least partially unavailable for antibody binding under conventional incubation conditions. SDS could simply result in protein unfolding (i.e., change in the secondary or tertiary structure of a protein), or it could dissociate multiprotein complexes that hinder antibody binding. In cells where the fluorescence was strongest, some staining was detectable in the absence of SDS, e.g., in intercalated cells, as previously described (13). However, even in these cells, the staining intensity was increased after SDS treatment. Although the increased staining of syntaxin 3 in the present study is largely attributed to the use of an antigen retrieval technique, some other methodological differences could also have contributed to the different results. For example, Mandon et al. used paraformaldehyde alone as a fixative, whereas we used PLP in our present study. There may also be differences in sensitivity between peroxidase- and fluorescence-based immunodetection procedures.
Syntaxins belong to a family of proteins that are an integral part of the machinery that controls membrane fusion in all cell types (1, 4). Docking and fusion of transport vesicles with their correct target membrane are strictly regulated by a complex series of protein-protein interactions that together ensure that membrane proteins are delivered to the appropriate membrane domain. So-called SNARE proteins, including the syntaxins, are present on donor and acceptor membranes, and their interaction with other members of the fusion machinery is now beginning to be understood (17, 18, 20, 21). It was originally believed that different SNARE proteins would be expressed only on specific membrane domains and that they would interact with a cognate SNARE protein present on the fusion partner in each case. However, more recent data have indicated that the process is considerably more complex and that the distinction between t-SNAREs, such as syntaxins, and vesicle SNAREs, such as cellubrevin and synaptobrevin/VAMP, is not as clear cut as originally envisaged (19, 23). Both types of SNARE proteins can be located on the same membrane domain (21). Nevertheless, certain SNAREs do have a limited cellular distribution on particular membrane domains (1), which may be important for apical and basolateral polarization in epithelial cells.
In the kidney, we have now found that syntaxin 3 is an exclusively basolateral protein, implying that its role is indeed in regulating processes that occur at the basolateral membrane of these cells. Syntaxin 4, on the other hand, was found on the apical membrane of collecting duct principal cells, where it was proposed to have a role in apical aquaporin 2 (AQP2) targeting in the kidney (12). In contrast, evidence from MDCK cells clearly locates syntaxin 3 on the apical plasma membrane and syntaxin 4 on the basolateral membrane (11), precisely the opposite distribution to that found in the kidney in situ. The data in MDCK cells were obtained from transfected cells overexpressing the various syntaxin isoforms, and this must be borne in mind when these results are interpreted. However, the authors discuss this issue extensively in their report and concluded that the observed polarity of syntaxin expression probably does not result from an overexpression artifact.
With regard to the function of these syntaxin isoforms, it has been proposed that syntaxin 4 is involved in apical AQP2 targeting in the kidney (12). However, MDCK cells transfected with AQP2 also show apical AQP2 expression (10), despite the fact that the apical SNARE is syntaxin 3 in these cells (11). In addition, recent data from our laboratory confirm previous reports that AQP2 can be expressed basolaterally in inner medullary principal cells in the kidney in situ (5, 16), where syntaxin 3 is expressed. Thus assigning specific sorting properties to the syntaxin family of SNARE proteins is not a simple task and is likely to be dependent on the cell type under examination. From the present and previous data, it must be concluded that syntaxin 3 and syntaxin 4 are localized such that either might contribute to the polarized membrane insertion of AQP2. Functional studies will ultimately be required to dissect the complex series of events that lead to polarized insertion of membrane proteins in renal epithelial cells.
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ACKNOWLEDGEMENTS |
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We thank Valerie Beaulieu for technical help.
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FOOTNOTES |
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These studies were supported by National Institutes of Health grant DK-42956 to D. Brown and S. Breton. Part of the funding was from the Intramural Budget of the National Heart, Lung, and Blood Institute (project no. Z01-HL-01282-KE to M. A. Knepper).
Address for reprint requests and other correspondence: D. Brown, Program in Membrane Biology/Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129 (E-mail: brown{at}receptor.mgh.harvard.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00128.2001
Received 19 April 2001; accepted in final form 3 October 2001.
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