1 Department of Respiratory Diseases, Aarhus University Hospital, 2 The Water and Salt Research Center, and 3 Institute of Human Genetics, University of Aarhus, DK-8000 Aarhus, Denmark; and 4 Department of Neurobiology and Anatomy, University of Texas Medical School, Houston, Texas 77030
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ABSTRACT |
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The vasopressin-induced trafficking of aquaporin-2 (AQP2) water channels in kidney collecting duct is likely mediated by vesicle-targeting proteins (N-ethylmaleimide-sensitive factor attachment protein receptors). Hrs-2 is an ATPase believed to have a modulatory role in regulated exocytosis. To examine whether Hrs-2 is expressed in rat kidney, we carried out RT-PCR combined with DNA sequence analysis and Northern blotting using a digoxigenin-labeled Hrs-2 RNA probe. RT-PCR and Northern blotting revealed that Hrs-2 mRNA is localized in all zones of rat kidney. The presence of Hrs-2 protein in rat kidney was confirmed by immunoblotting, revealing a 115-kDa protein in kidney and brain membrane fractions corresponding to the expected molecular size of Hrs-2. Immunostaining and confocal laser scanning microscopy of LLC-PK1 cells (a porcine proximal tubule cell line) transfected with Hrs-2 DNA confirmed the specificity of the antibody and revealed that Hrs-2 is mainly localized in intracellular compartments, including cathepsin D-containing lysosomal/endosomal compartments. The cellular and subcellular localization of Hrs-2 in rat kidney was examined by immunocytochemistry and confocal laser scanning microscopy. Hrs-2 immunoreactivity was observed in collecting duct principal cells, and weaker labeling was detected in other nephron segments. The labeling was predominantly present in intracellular vesicles, but labeling was also observed in the apical plasma membrane domains of some cells. Colabeling with AQP2 revealed colocalization in vesicles and apical plasma membrane domains, suggesting a role for Hrs-2 in regulated AQP2 trafficking.
vesicle-targeting receptors; N-ethylmaleimide-sensitive factor attachment protein receptors; collecting duct; aquaporin-2
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INTRODUCTION |
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TOTAL BODY WATER BALANCE CRITICALLY DEPENDS on the regulation of renal water excretion by the peptide hormone vasopressin (the antidiuretic hormone), which acts on the kidney collecting duct principal cells to regulate collecting duct water reabsorption (12, 20). Aquaporin-2 (AQP2) is the predominant vasopressin-regulated water channel that is important for regulation of the water permeability of the apical plasma membrane of collecting duct principal cells for tight regulation of body water balance (11, 20, 22). The action of vasopressin increases the osmotic water permeability of the renal collecting duct by triggering exocytosis of AQP2-bearing vesicles to the apical plasma membrane to increase the density of AQP2 in the apical plasma membrane (15, 17, 22, 26, 31).
The molecular mechanisms responsible for the vasopressin-stimulated docking and fusion of AQP2-containing intracellular vesicles with the apical plasma membrane are poorly understood. However, recent studies suggest that the regulated trafficking in epithelia may involve the same basic principles as described in the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) hypothesis (5, 25, 27, 28, 32). This general model has arisen largely on the basis of studies of the molecular mechanisms by which synaptic vesicles dock and fuse with plasma membrane, and it is believed to underlie most membrane transport processes in virtually all cells (1, 5, 25, 27, 28, 32). The SNARE hypothesis predicts that a vesicle-targeting membrane protein (v-SNARE) binds to cognate proteins on the target membrane (t-SNARE) to form a complex, which is recognized and dissociated by the cystolic NSF and SNAPs. The v-SNARE vesicle-associated membrane protein (VAMP) has been identified in kidney collecting duct principal cells (7, 9, 21) as well as in AQP2 vesicles (21). In addition, two families of t-SNAREs, the syntaxins and the homologs of SNAP-25 (synaptosomal-associated protein of 25 kDa), have been identified. Among the t-SNAREs, syntaxin-4 has been shown to be present in collecting duct principal cells and is localized to the apical plasma membrane (13). Also, SNAP-23, the SNAP-25 isoform, is expressed in the collecting duct principal cells (8). Although only indicative, the localization of these proteins in domains where AQP2 is present indicates a role of SNARE vesicle-targeting receptors in vasopressin regulation of AQP2 trafficking.
Recently, a SNAP-25-associated protein (Hrs-2) was isolated from the brain (3). Hrs-2 is believed to have a modulatory function in the secretory process by regulating the assembly of fusogenic SNARE complexes (29). Hrs-2 is also associated with the SNAP-25 isoform SNAP-23 (30). Because SNAP-23 has been identified in the rat kidney collecting duct, we hypothesize that Hrs-2 may be present in kidney as well. The purposes of the present study were 1) to investigate whether Hrs-2 mRNA and protein are present in the kidney and, if this were the case, 2) to establish its cellular and subcellular distribution. This was achieved by using RT-PCR, Northern blotting, immunoblotting, cell transfection, immunocytochemistry, and confocal laser scanning microscopy.
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METHODS |
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Experimental animals. Studies were performed in adult Munich-Wistar rats (Møllegard Breeding Centre). The rats were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water.
Purification of total RNA.
Total RNA from tissues from three zones of kidney (cortex, inner stripe
of the outer medulla, and inner medulla) and cerebellum was extracted
by the acid guanidinium thiocyanate-phenol-chloroform method with
modifications (4, 24). One frozen tissue was homogenized
(Ultra-Turrax T8, IKA Labortechnik) for 30 s with 1 ml solution D
(741 mg/ml guanidinium thiocyanate, 39 mM Na-citrate, pH 7.0, 0.78%
sarcosyl, 7 µl/ml -mercaptoethanol), 100 µl 2 M sodium acetate
(pH 4.0), and 1 ml water-saturated phenol. Subsequently, 0.15 volume
CHCl3-isoamylalcohol (49:1) was added to the
homogenate. The final suspension was cooled on ice for 30 min, and
samples were then centrifuged at 4,000 g for 30 min at
4°C. The aqueous phase containing the RNA was mixed with 1 vol
isopropanol and left at
20°C for 15 min followed by centrifugation
at 4,000 g for 30 min at 4°C. The resulting RNA pellet was
washed in 70% ethanol and finally dissolved in 50 µl TE-buffer and frozen.
Northern blot analysis. Northern blot analysis (4) was performed by using a digoxigenin-labeled Hrs-2 RNA probe. This probe was made by in vitro transcription (MAXIscript In Vitro Transcription kit, Ambien), where a pBluescript vector containing the entire Hrs-2 cDNA was used as a template. Total RNA was separated on a 1% agarose or 2 or 6% formaldehyde gel followed by blotting on a nylon membrane filter (Hybond-N, Amersham Life Science, Buckinghamshire, UK). To reveal the size of Hrs-2 mRNA, a digoxigenin-labeled RNA molecular weight marker (Boehringer Mannheim) was used. Prehybridization was performed at 55°C for 30 min in 5× standard sodium citrate (SSC), 50% formamide, 0.1% sarcosyl, 0.02% SDS, and 2% blocking solution (blocking reagent in maleic acid, Boehringer Mannheim). After prehybridization, blots were hybridized with the digoxigenin-labeled RNA probe at 55°C for 24 h. After hybridization, blots were washed in 2× SSC, 0.1% SDS at room temperature for 2 × 5 min followed by washing in 0.1 × SSC, 0.1% SDS at 68°C for 30 min. Blots were equilibrated for 1 min in maleic acid containing 0.3% Tween 20 and blocked for 30 min. After incubation for 30 min with anti-digoxigenin-AP conjugate, blots were washed for 30 min in maleic acid containing 0.3% Tween 20 and equilibrated for 5 min in 0.1 M Tris · HCL, 0.1 M NaCl. The bands were visualized by using a chemiluminescent substrate, CSPD (Boehringer Mannheim).
RT-PCR experiments. The starting material was total RNA extracted from rat cerebellum, inner medulla, inner stripe of outer medulla, and cortex. The sequence-specific primers were designed from the published rat Hrs-2 cDNA sequence (3) (GenBank accession no. U87863). The following primers were chosen: a sense primer (bp 1719-1740; 5'- CTGGGGGTGTACTCTACCAGC-3') and an antisense primer (bp 2635-2654; 5'- CACACAGTTCACATCGGAC-3'). Base pair numbering is relative to the ATG codon, believed to be the site of translation initiation. These two primers amplify a 935-bp PCR product.
cDNA was synthesized from RNA-samples containing 2 µg RNA by using Expand RT (Boehringer Mannheim) together with dNTP (1 mM of each dNTP, Amersham Pharmacia Biotech, Piscataway, NJ) as described by the manufacturer. RNA in water was diluted (by tapping) with 20 pmol of the antisense primer to a volume of 11 µl. The reaction mixture was incubated at 65°C for 5 min to allow the primer to anneal to the mRNA. cDNA was then synthesized at 42°C for 1 h. The PCR was performed in a total volume of 50 µl with 20 pmol of each primer, 200 µM of each dNTP, 1× reaction buffer (Qiagen, Valencia, CA), 2.5 U of HotStar Taq DNA polymerase (Qiagen), and 2 µl of cDNA. Thus the quantity of cDNA amplified corresponded to 0.2 µg of RNA in the RT-PCR. Temperature cycling conditions consisted of 15 min at 95°C (to activate HotStar Taq DNA polymerase) followed by 30 cycles for 30 s at 94°C (denaturation), 30 s at 55°C (annealing), and 2 min at 72°C (extension), with a final extension for 7 min at 72°C. Several control experiments were performed: 1) RNA was directly amplified, without RT, ensuring that products were not the result of amplification of genomic DNA; 2) negative controls for the RT-step (without added RNA) and the PCR (without added DNA) were performed to control for contamination of the reagents for these reactions. For analysis, 2 µl of the RT-PCR products were size-fractionated by electrophoresis through a 2% agarose gel (containing 0.5 µg/ml ethidium bromide) in 1× Tris-acetate-EDTA buffer. After electrophoresis, the product bands were photographed under ultraviolet light (EagleEye II, Stratagene). The product was analyzed on a 1% low-melting agaose gel, and the band corresponding to the 935-bp product band was excised and purified by using the Wizard PCR preps DNA purification system (Promega, Madison, WI). DNA sequencing was done with the ThermoSequenase kit (Amersham Life Science, Cleveland, OH) on a 381 Sequenator (Amersham Life Science). Sequence analysis was done using the blast program from NCBI.Transfection of Hrs-2 in LLC-PK1 cells.
LLC-PK1 cells (a porcine proximal tubule cell line kindly
provided by Dr. Rikke Nielsen, Dept. of Cell Biology, Inst. of Anatomy, Univ. of Aarhus) were grown in 75-cm2 flasks (Nunc,
Roskilde, Denmark) or 10-cm2 slideflasks (Nunc) at 37°C
and 5% (vol/vol) CO2 in DMEM (In Vitro, Copenhagen,
Denmark) containing 10% (vol/vol) FCS (GIBCO-BRL). The pcDNA3/myc
vector (Invitrogen, Groningen, The Netherlands) harboring the
full-length rat Hrs-2 cDNA was used to transfect the
LLC-PK1 cells utilizing FuGENE 6 transfection reagent
(Roche Diagnostic). As a negative control for Hrs-2 expression, the
vector without the Hrs-2 cDNA insert was used. The cells were fixed
42 h after transfection with freshly prepared 4% (wt/vol)
paraformaldehyde (Merck) for 5 min at 4°C, followed by wash in PBS.
The cells were then permeabilized with 70% (vol/vol) ethanol for 20 min at 20°C.
Immunostaining and localization of Hrs-2 in LLC-PK1 cells by confocal laser scanning microscopy. A two-layer immunostaining procedure was used with three washing steps in PBS between incubations. Fixed cells were incubated for 60 min at room temperature with anti-Hrs-2 monoclonal antibody (2, 3, 30), 9E10 anti-Myc monoclonal antibody against the c-Myc tag (RDI, Flanders, NJ), or anti-cathepsin D polyclonal antibody (DAKO, Glostrup, Denmark). Subsequently the cells were incubated for 60 min with fluorescein-conjugated goat anti-mouse secondary antibodies or with fluorescein-conjugated goat anti-rabbit secondary antibodies (diluted 1:1,000, Molecular Probes). The microscopy was carried out by using a Leica TCS 4D confocal laser scanning microscope.
Membrane fractionation for immunoblotting. Tissues from two zones of kidney (inner stripe of the outer medulla and inner medulla) and those from the cerebellum were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 µM leupeptin, 1 mM phenylmethylsulfonyl fluoride) by using an ultra-turrax T8 homogenizer (IKA Labortechnik), at maximum speed for 10 s, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular membranes (14, 15). From the resultant pellets, gel samples (Laemmli sample buffer containing 2% SDS) were made.
Electrophoresis and immunoblotting. The gel samples were run on 12% polyacrylamide minigels (Bio-Rad Mini Protean II). The gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% skim-milk in 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5 (PBS-T) for 1 h and incubated with anti-Hrs2 monoclonal antibody (2, 3, 30). The labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (P447, DAKO, diluted 1:3,000) by using the enhanced chemiluminescence system (Amersham International).
Immunoperoxidase and confocal laser scanning microscopy of brain and kidney cells. Kidneys and brains from normal Munich-Wistar rats (n = 3) were fixed by retrograde perfusion via the aorta or cardiac perfusion, respectively. The fixative contained 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4. For semithin sections (0.8-1 µm), tissue blocks prepared from brain, cortex, outer and inner stripe of outer medulla, and inner medulla were cryoprotected with 2.3 M sucrose, mounted on holders, and rapidly frozen in liquid nitrogen (23). For immunoperoxidase, immunofluorescence, and confocal laser scanning microscopy, other kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. For light and confocal laser scanning microscopy, the paraffin-embedded tissues were sectioned at 2 µm on a microtome (Leica). The staining was carried out using indirect immunofluorescence or indirect immunoperoxidase (16). The sections were dewaxed and rehydrated. For immunoperoxidase labeling, endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 10 min at room temperature. To reveal antigens, sections were incubated in 1 mM Tris solution (pH 9.0), supplemented with 0.5 mM EGTA, and heated using a microwave oven for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with anti-Hrs-2 antibody (2, 3, 30) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After a rinse with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 30 min, the sections for confocal laser microscopy were incubated in Alexa 546-conjugated goat anti-mouse antibody (Molecular Probes) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100 for 60 min at room temperature. After a rinse with PBS for 30 min, the sections were mounted in glycerol supplemented with antifade reagent (N-propyl-gallat). For immunoperoxidase, the sections were washed (see above) followed by incubation in horseradish peroxidase-conjugated goat anti-mouse immunoglobulin (DAKO P447) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton-X-100. For double immunolabeling, sections were incubated simultaneously with monoclonal anti-Hrs-2 and with either polyclonal rabbit anti-rat AQP2 antibody or with polyclonal rabbit anti-rat cathepsin D antibody. The microscopy was carried out using a Leica DMRE light microscope and a Zeiss LSM510 laser confocal microscope.
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RESULTS |
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Hrs-2 mRNA expression in rat kidney detected by RT-PCR.
Recently, SNAP-23 was identified in rat kidney (8), and
because Hrs-2 is associated with SNAP-25 (3), RT-PCR was
carried out using Hrs-2-specific primers to investigate whether Hrs-2 is also present in the kidney (Fig.
1A). RT-PCR amplifications were performed on total RNA extracted from rat kidney zones (cortex, inner stripe of the outer medulla, and inner medulla). Expression of
Hrs-2 mRNA was studied using a single pair of primers flanking a 935-bp
DNA fragment. As shown in Fig. 1A, Hrs-2 mRNA was detected in rat kidney. As control, experiments were also carried out using total RNA extracted from the rat cerebellum (Fig. 1A), where
Hrs-2 has previously been identified (3). Amplification of
the RNA directly with PCR without RT yielded no detectable product on the ethidium bromide-stained agarose gels, confirming that the RNA was
not contaminated with cDNA or genomic DNA. Furthermore, all RT-PCR
experiments included "blank" controls from which RNA was omitted to
control for DNA contamination. The 935-bp PCR product obtained from the
RT-PCR amplification of rat inner medulla RNA was sequenced and
compared with the reported sequence for rat Hrs-2 (3). The
determined sequence matched the reported sequence with 100% agreement,
confirming the identity of the 935-bp PCR product to the published
Hrs-2 cDNA sequence.
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Hrs-2 mRNA expression in rat kidney detected by Northern blot analysis. Because RT-PCR experiments revealed that Hrs-2 mRNA was detected in rat kidney, Northern blot analysis was performed with total RNA isolated from rat kidney inner medulla, inner stripe of outer medulla, and cortex and a Hrs-2 RNA probe hybridizing to Hrs-2 mRNA. As a positive control, total RNA isolated from rat cerebellum was also used. As seen in Fig. 1B, the Hrs-2 RNA probe labels a single band in all the examined parts of rat kidney, which has a size equivalent to the size of the band observed in rat cerebellum (corresponding to ~2.8 kb).
Analysis of the subcellular localization of transfected Hrs-2
protein in LLC-PK1 cells.
To confirm antibody specificity and to further establish the
subcellular distribution of Hrs-2 protein in kidney cells,
immunostaining experiments were performed using the LLC-PK1
cell line transfected with full-length rat Hrs-2 cDNA, which was COOH
terminally fused to the c-myc sequence of the mammalian expression
vector pcDNA3/myc. After transfection, the chimera was overexpressed in
LLC-PK1 cells. The intracellular distribution of the fusion
protein was analyzed using in situ immunostaining and confocal laser
scanning microscopy. The anti-Hrs-2 labeling was mainly found to be
associated with intracellular compartments (Fig.
2A), but cells also
occasionally (<10% of the cells) exhibited labeling of the plasma
membrane in addition to the intracellular labeling (data not shown).
The specificity of the labeling pattern was confirmed by the use of cells transfected with the vector containing no Hrs-2 insert. No
positive signals were observed in these cells. This specific labeling
pattern was further supported with the use of anti-Myc antibody showing
a similar labeling pattern (data not shown). Thus these experiments
further confirmed the specificity of the antibody and demonstrate that
Hrs-2 is mainly an intracellular protein, although occasionally it is
also present in the plasma membrane.
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Hrs-2 protein is expressed in rat kidney. On the basis of the finding that Hrs-2 is expressed in rat kidney at the mRNA level, we carried out further studies using a previously characterized anti-Hrs-2 antibody (2, 3, 30) to determine whether Hrs-2 protein is also detectable in rat kidney. Figure 2B shows an immunoblot using samples containing plasma membranes and intracellular membranes from rat kidney zones (inner medulla and inner stripe of the outer medulla) and cerebellum. Hrs-2 protein is detected in all three samples, demonstrating the presence of Hrs-2 protein in the kidney. The presence of Hrs-2 in samples containing plasma membranes and intracellular membranes is consistent with previous evidence that Hrs-2 is at least partly membrane associated.
Immunocytochemical localization of Hrs-2 protein in kidney.
To further confirm the specificity of the Hrs-2 antibody,
immunolabeling of sections from rat brain was performed. Corresponding to previous reports, Hrs-2 immunosignals were detected in neuronal elements in different regions of the central nervous system
(30). In the cerebellum, immunosignals were present in the
molecular, Purkinje cellular, and granular layers. Purkinje cell bodies
and apical dendrites ramifying into the molecular layer were strongly labeled (Fig. 3A). In the
cerebral cortex, cell bodies and the initial portion of the apical
dendrites of the cortical pyramidal cells were labeled in all cortical
layers (Fig. 3B). Pyramidal cells in the CA1 and CA3 areas
of the hippocampus were also immunopositive (Fig. 3C),
whereas tracts of the corpus callosum remained unlabeled (Fig.
3D).
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Localization of Hrs-2 and cathepsin D proteins in
collecting duct using confocal laser scanning microscopy.
To test whether some of the intracellular compartments, in which Hrs-2
was localized, are late endosomes or lysosomes, the localization of
Hrs-2 was compared with that of cathepsin D [an endosomal/lysosomal
marker (6)] using double-labeling confocal laser scanning
microscopy. In LLC-PK1 cells transfected with
the mammalian expression vector pcDNA3/myc harboring the rat Hrs-2 cDNA
sequence (Fig. 4,
A-C), Hrs-2 occasionally colocalizes with cathepsin D, although most Hrs-2 labeling was confined to cathepsin D-negative structures. Consistent with this, immunolabeling of Hrs-2
and cathepsin D in kidney sections revealed some degree of
colocalization (Fig. 4, D-F) in cortical
collecting duct cells. Occasionally, there was a high degree of
separation in some collecting duct cells (Fig. 4,
G-I).
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Localization of Hrs-2 and AQP2 proteins in collecting duct using
confocal laser scanning microscopy.
The distribution of Hrs-2 in different segments of rat kidney
collecting duct was also determined by immunofluorescence and confocal
laser scanning microscopy. Figure
5A shows labeling of cortical
collecting duct. Hrs-2 labeling is associated with principal cells of
the collecting duct (arrows and arrowheads), whereas intercalated cells
exhibit little or no labeling (*). Hrs-2 immunolabeling is mainly
associated with intracellular vesicles (arrowheads), but occasionally
(minor fraction of the collecting duct principal cells) Hrs-2 labeling
was also associated with the apical plasma membrane domains (arrows).
The same labeling pattern was also observed in outer medullary
collecting duct (Fig. 6A) and
in inner medullary collecting duct (Fig. 6D).
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DISCUSSION |
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Regulation of AQP2 trafficking by vasopressin is central to the overall process that precisely regulates body water balance. Hence, an understanding of the molecular mechanisms involved in regulation of AQP2 trafficking is crucial to understanding the physiology and pathophysiology of water balance and water balance disorders. AQP2 is stored in the membranes of intracellular vesicles in the collecting duct principal cells (18). On stimulation by vasopressin, these AQP2-bearing vesicles fuse with the apical plasma membrane, thus increasing the water permeability of the apical plasma membrane and, concomitantly, of the epithelium as a whole (17).. Docking of these vesicles with the apical plasma membrane has been proposed to be mediated by binding of v-SNAREs with t-SNAREs (7, 13, 21). This process has been thought to be essential for synaptic vesicle exocytosis from neurons, where at least one v-SNARE (VAMP-2) forms a complex with at least two t-SNAREs, syntaxin and SNAP-25 (28). The likelihood that such a complex may be involved in AQP2 vesicle targeting to the apical plasma membrane of collecting duct principal cells drew support from the observations that the v-SNARE VAMP-2 and synaptotagmin VIII have been identified in the collecting duct principal cells, including in AQP2-bearing vesicles (10, 21). In addition, syntaxin-4 and the SNAP-25 homolog SNAP-23 have been found to be expressed in collecting duct principal cells (8, 13).
Hrs-2 is an ATPase that binds to SNAP-25 in a calcium-regulated manner (3). The binding of Hrs-2 to the SNAP-25-syntaxin complex inhibits the binding of VAMP. Thus Hrs-2 is suggested to play a negative role in the fusion-reaction between the vesicles and the plasma membrane. Because Hrs-2 also binds to SNAP-23 (30), in the present study we sought to determine whether Hrs-2 is expressed in the renal collecting duct. Hrs-2 mRNA was detected in the collecting duct by both RT-PCR and Northern-blotting, whereas Hrs-2 protein was detected by immunoblotting. Immunostaining of LLC-PK1 cells transfected with Hrs-2 demonstrated that Hrs-2 is not associated with the plasma membrane. Immunocytochemistry and confocal laser scanning microscopy revealed Hrs-2 immunolabeling mainly in vesicles in principal cells of the collecting duct and the virtual absence of Hrs-2 in intercalated cells. Occasionally, some cells also showed labeling of the apical plasma membrane domains. Colabeling of Hrs-2 and AQP2 strongly suggests colocalization on both vesicle and the apical plasma membrane.
Presence of Hrs-2 in kidney collecting duct principal cells. The expression of Hrs-2 at the mRNA level in kidney collecting duct was demonstrated by RT-PCR (Fig. 1A) and Northern blotting (Fig. 1B) using total RNA extracted from kidney homogenates. Direct sequencing of the RT-PCR product confirmed that the amplified product was derived from Hrs-2 mRNA. Hrs-2 mRNA is present in inner medulla, inner stripe of the outer medulla, and cortex, providing the evidence that Hrs-2 is broadly distributed in the kidney. As a positive control, total RNA from cerebellum was used, because Hrs-2 has been detected in the brain (3). The presence of Hrs-2 protein in kidney collecting duct was demonstrated by immunoblotting. A 115-kDa Hrs-2 band was seen on immunoblots using membrane fractions from inner medulla and inner stripe of the outer medulla (Fig. 2B), corresponding to the expected molecular size of Hrs-2. The fact that Hrs-2 is present in rat kidney membrane fractions containing mainly plasma membranes and intracellular membranes indicates that Hrs-2 is at least partly associated with these membranes. Two control experiments were performed to ensure the specificity of the anti-Hrs-2 antibody (2, 3, 30). Hrs-2-overexpression in the LLC-PK1 cell line followed by immunostaining using either anti-c-Myc or anti-Hrs-2 antibodies resulted in the same labeling pattern revealing labeling of intracellular compartments (Fig. 2A), which is consistent with previous observations (30). Moreover, immunocytochemical localization of Hrs-2 protein in brain (Fig. 3, A-D) demonstrated that Hrs-2 is present in neuronal elements in different regions of the central nervous system, as previously reported (30).
Colocalization of Hrs-2 and AQP2 and of Hrs-2 and cathepsin D. The distribution of Hrs-2 in kidney was determined by immunoperoxidase microscopy (Fig. 3, E-F). Hrs-2 is present in cortex, inner stripe of the outer medulla, outer stripe of the outer medulla, and inner medulla. The Hrs-2 labeling is mainly localized to intracellular compartments of the collecting duct. Immunofluorescence confocal laser scanning microscopy was used to determine the Hrs-2 distribution in kidney and to determine whether Hrs-2 colocalizes with AQP2 and with cathepsin D (Figs. 4-7). The Hrs-2 labeling pattern in collecting duct from cortex, inner medulla, and outer medulla shows mainly labeling of principal cells, with almost no labeling of intercalated cells. The labeling is seen in both intracellular vesicles and near or in the apical plasma membrane. It should be emphasized that only a minority of collecting duct principal cells exhibited plasma membrane labeling, consistent with the results obtained from transfection of LLC-PK1 cells with Hrs-2 also revealing predominant intracellular labeling but with occasional cells exhibiting plasma membrane labeling. It should also be noted that there is a difference in the subcellular localization pattern in the collecting duct principal cells in the three different kidney zones examined. Hrs-2 labeling in the inner medulla is, to a greater extent, localized in the apical membrane domains compared with the labeling in cortical and outer medullary collecting duct. Although some labeling was also found associated with proximal tubule cells, most other nephron segments or vascular structures exhibited less immunostaining, suggesting that in the kidney Hrs-2 is most abundant in collecting duct principal cells.
In this study we have demonstrated the presence of Hrs-2 in the principal cells of the collecting duct, a protein implicated in regulation of the formation of the SNAP-25-syntaxin-VAMP complex. Moreover, we have established that to some extent there is colocalization of Hrs-2 and AQP2 in intracellular vesicles and the apical plasma membrane. This supports the previous studies suggesting that SNARE proteins may be involved in AQP2 trafficking. Thus far syntaxin-4 has been identified in the apical plasma membrane of collecting duct principal cells (13), whereas VAMP-2 is present in substantial amounts in AQP2-containing vesicles (21). In addition, SNAP-23, the SNAP-25 homolog, has been detected in both intracellular vesicles and the apical plasma membrane (8). Thus evidence has been obtained that collecting duct principal cells express all three components (VAMP-2, syntaxin-4, and SNAP-23) of a putative SNARE complex analogous to that demonstrated in the central nervous system. The presence of syntaxin-4 and SNAP-23 in the apical plasma membrane and of VAMP-2 in AQP2-containing vesicles indicates that the SNAP-23-syntaxin-4-VAMP-2 complex may be responsible for the selective targeting of AQP2 to the apical plasma membrane. The results of the present studies on Hrs-2 and previous studies on SNAP-23 indicate that Hrs-2 codistributes with SNAP-23 in that both are localized on the apical plasma membrane and the intracellular vesicles. This is in accordance with recent observations that Hrs-2 interacts with SNAP-23. Overall, the present data, combined with previous studies on SNAP-23, VAMP-2, and syntaxin-4, strongly suggest that Hrs-2 may be in a position to regulate the trafficking and/or insertion of AQP2-containing vesicles with the apical plasma membrane. To further analyze which compartments are labeled with anti-Hrs-2 antibody, we performed double immunofluorescence labeling of Hrs-2 and the endosomal/lysosomal marker cathepsin D. In both Hrs-2-transfected cells and in kidney collecting duct cells, there was some degree of colocalization, indicating that Hrs-2 is associated, in part, with endsomal/lysosomal compartments. This is consistent with the presence of Hrs-2 in relatively large cytoplasmic structures observed by single-labeling techniques. Its presence in lysosomes/endosomes indicates that Hrs-2 may be involved, in part, in membrane trafficking associated with endocytosis. Thus, taken together, our results suggest that Hrs-2 may have a role in the trafficking and/or degradation of AQP2. Although there is some degree of colocalization of Hrs-2 and AQP2, the observations in some collecting duct principal cells show almost a complete separation of Hrs-2 and AQP2. This suggests that Hrs-2 is likely to play a role in a particular step of the regulated trafficking of AQP2 but not in all steps. The different steps that may or may not be regulated involve 1) bringing AQP2 from the Golgi into a compartment that can be recruited for regulated trafficking of AQP2-bearing vesicles, for example; 2) trafficking of AQP2-bearing vesicles along microtubles to the apical pole of the cells; 3) docking and fusion of AQP2-bearing vesicles with the apical plasma membrane; 4) endocytic retrival of AQP2 from the apical plasma membrane maybe also involving a lateral migration of AQP2 in the apical plasma membrane to coated pits for endocytosis, for example; and 5) trafficking of AQP2 into a compartment that allows recycling of AQP2 in response to a second stimulation (e.g., via vasopressin-cAMP-protein kinase A). It is not yet clear at which step Hrs-2 might be acting to regulate AQP2 trafficking. In summary, we have demonstrated the presence of Hrs-2 in principal cells of the renal collecting duct, the site of vasopressin-regulated water transport in the kidney. Hrs-2 is predominantly present in intracellular vesicles/endosomes/lysosmes, but some Hrs-2 could also be detected in the apical plasma membrane domains. In addition, Hrs-2 colocalizes with AQP2 to a significant extent. However, additional studies are crucial to provide direct evidence for the specific regulatory role of Hrs-2 as well as the structural SNARE proteins (syntaxin-4, VAMP-2, SNAP-23) in regulated membrane trafficking and, hence, regulated trafficking of channels and transporters in collecting duct principal cells. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Inger Merete Paulsen and Trine Møller for expert technical assistance.
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FOOTNOTES |
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Support for this study was provided by the Karen Elise Jensen Foundation, Novo Nordisk Foundation, Danish Medical Research Council, the European Commission Training, Mobility, and Research Program, and the Biotech Program of the 4th Framework Program and Key Action 3.1.2 of the 5th Framework Program (S. Nielsen), the Danish Allergy Research Center (A. Shukla, H. J. Hoffmann, and R. Dahl), and the Mallinckrodt Foundation (A. J. Bean). The Water and Salt Research Center at the University of Aarhus was established by support from Danmarks Grundforskningsfond.
Address for reprint requests and other correspondence: S. Nielsen, The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
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.
Received 9 January 2001; accepted in final form 26 April 2001.
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