Arginine vasopressin (AVP) is essential for maintaining body fluid homeostasis. The antidiuretic effects of AVP are initialized by binding of AVP to the type-2 vasopressin receptor (V2R) in the kidney collecting duct (CD), resulting in the exocytic insertion of aquaporin-2 (AQP-2) water channels into the apical plasma membrane. In this study, we describe the generation and characterization of a polyclonal antibody targeted against the NH2 terminus of the rat V2R. HEK-293 cells overexpressing the rat, mouse, or human V2R showed strong intracellular immunolabeling. Additionally, immunostaining of M-1 kidney cells expressing a V2R-green fluorescent protein (GFP) fusion construct showed colocalization between GFP and antibody-specific V2R labeling. Immunoblots of rat kidney showed 43- and 47-kDa proteins in all zones that were both reduced to 34-kDa by N-glycosidase F. Protein solubilization with nonionic detergents or the use of homobifunctional cross-linkers demonstrated that the rat V2R exists as a protein complex in native kidney. Immunohistochemistry of rat and mouse kidney revealed abundant labeling of the CD. Double-labeling confocal immunofluorescence microscopy [using distal convoluted tubule/connecting tubule (CNT)-specific marker calbindin and CNT/CD-specific marker AQP-2] showed V2R labeling in both CD and CNT. There was a complete absence of labeling in vascular structures and other renal tubules, including the thick ascending limb (TAL), although RT-PCR of microdissected tubules showed expression of V2R mRNA in TAL. Confocal microscopy demonstrated that at the subcellular level, V2R labeling was predominantly intracellular in normal kidneys, although some staining was apparent in basolateral membrane domains. Confocal microscopy of isolated inner medullary collecting duct tubules showed that the V2R is expressed both intracellularly and in basolateral membrane domains.
- water balance
- G protein-coupled receptor
arginine vasopressin (AVP) is essential in the regulation of body fluid homeostasis. In response to small increases in plasma osmolality or a reduction in the effective circulating blood volume, AVP is released by the posterior pituitary gland and promotes water reabsorption in the kidney collecting duct, enhanced urinary concentration in the thick ascending limb (TAL), and vasoconstriction via four subtypes of receptors.
The type-2 vasopressin receptor (V2R) (2, 32), a member of the large seven-transmembrane class 1 G protein-coupled receptor family (21), mediates the antidiuretic effects of AVP. In the kidney collecting duct, AVP binds to the V2R, resulting in activation of adenylyl cyclase through V2R-coupled GTP-binding protein Gs. This increases intracellular cAMP levels and leads to activation of protein kinase A (PKA), intracellular Ca2+ oscillations (66), and activation of myosin light-chain kinase (8), as well as cAMP-dependent, PKA-independent activation of the Rap-GEF Epac (67). Through multiple mechanisms, V2R-mediated signaling leads to the exocytic insertion of aquaporin (AQP)-2-bearing vesicles into the apical plasma membrane (reviewed in Refs. 6, 40). In addition, long-term exposure to vasopressin leads to an increase in AQP-2 synthesis via increased gene transcription, increasing the total abundance of AQP-2 in the cells (13). Together, these mechanisms increase collecting duct water permeability.
A defective or enhanced ability of the kidney to respond to vasopressin results in a number of inherited or acquired water balance disorders (reviewed in Refs. 40, 64). One rare inherited disease, resulting from the failure of the renal tubules to respond to AVP, is nephrogenic diabetes insipidus (NDI). This is either X-linked, caused by mutations in the V2R gene (XNDI), or autosomal recessive, caused by mutations in the AQP2 gene (50). Acquired forms of NDI are very common and include drug-induced forms (e.g., lithium, antibiotics, and anticancer drugs), electrolyte disorders (hypokalemia, hypercalcemia), obstruction of the urinary tract (kidney stones, prostate hypertrophy), or acute and chronic renal failure (e.g., 14–16, 30, 31, 33, 34). Acquired water balance disorders can also result in severe water retention, such as is seen in conditions like congestive heart failure, liver cirrhosis, and syndrome of inappropriate vasopressin secretion (SIADH) (5, 17, 25, 41, 57).
The clinical importance of the V2R in water balance disorders is underlined by the current use of V2R antagonists in a clinical setting (18, 19, 49, 65). However, because of incomplete biological information about V2R regulation, features such as why agonist effects dramatically decline with time (limiting their use), and why vasopressin escape occurs in liver cirrhosis but not in congestive heart failure, are still poorly understood.
Despite the overwhelming evidence implicating the V2R in numerous physiological and pathophysiological processes, the progress in studying these disease states has been restricted, possibly because of difficulties in raising antibodies against the large seven-transmembrane receptor. Two previous studies have addressed V2R expression in rat kidney using peptide-targeted antibodies (42, 54); however, there are clear differences in V2R distribution between the two reports. In 1995, Nonoguchi and colleagues (42), using an antibody raised against the intracellular loop between the fifth and sixth transmembrane domain, saw V2R expression in some TAL segments and the collecting duct. In their study, V2R labeling was not only apparent in the basolateral membrane, but also the luminal membrane of the collecting ducts, especially in terminal inner medullary collecting duct (IMCD). In contrast, Sarmiento and colleagues (54), using an antibody directed against the second intracellular loop, saw V2R expression in all TALs, distal convoluted tubules (DCT), connecting tubules, and collecting ducts. V2R labeling was clearly apparent in the apical membrane, with some staining interpreted as being associated with the basolateral membrane. Furthermore, from the study of Sarmiento and colleagues, it is clear that immunolabeling of the DCT is of equal intensity to labeling of the collecting duct, which is in contrast to several other reports on V2R expression/function (7, 26, 35, 58).
Because of the previous conflicting reports, and as a first step for future studies addressing the in vivo regulation of the V2R in multiple disease states, the aim of this study was to develop a new peptide-targeted antibody against the V2R and determine the precise segmental and subcellular localization of the V2R in the kidney.
A 19-amino acid synthetic peptide, MLLVSTVSAVPGLFSPPSSC (COOH-terminal cysteine added for affinity purification), corresponding to amino acids 1–19 of the rat V2R (accession no. NP_062009), was produced by standard solid-phase, peptide-synthesis techniques, purified by high-performance liquid chromatography, and conjugated to maleimide-activated keyhole limpet hemocyanin via covalent linkage to the COOH-terminal cysteine. Two rabbits were immunized by using a combination of Freund's complete and incomplete adjuvants. The antisera obtained were affinity-purified by using a column on which 2 mg of the immunizing peptide was immobilized via covalent linkage to agarose beads (SulfoLink Kit; Pierce). After a series of experimental procedures, it was determined that the antiserum from rabbit no. 7251 produced the most reliable results, and thus was used for all subsequent experimental procedures (referred to as 7251AP). Immunoprecipitation experiments followed by protein identification by tandem mass spectroscopy confirmed specificity of the antibody (see Supplemental Materials, available with the online version of this article).
All animal protocols in this study were approved by the boards of the Institute of Anatomy and Institute of Clinical Medicine, University of Aarhus, according to the licenses for the use of experimental animals issued by the Danish Ministry of Justice.
Rat, mouse, and human V2R cDNA coding sequences were generated by standard RT-PCR methodologies and cloned into the pcDNA3.1 vector (Invitrogen) for expression in HEK-293 cells. Subsequently, the rat V2R coding region was reamplified and cloned into both the pAcGFP1-N1 (creating V2R-GFP) and the pAcGFP1-C1 (creating GFP-V2R) vectors (Clontech) by standard molecular methods. All constructs were fully sequenced for verification before use.
Transfection of HEK-293 and M-1 kidney cells.
Cells were cultured at 37°C, 5% CO2 in a humidified incubator in modified DMEM/F12 media (Invitrogen) containing 10% FCS (Invitrogen), 2 mM l-glutamine (Sigma), 100 U/ml penicillin, and 100 U/ml streptomycin. On reaching confluency, cells were routinely split into 12-well plates containing poly-lysine-coated glass coverslips. When cells were 80–90% confluent, they were transfected with 500 ng/well of cDNA constructs using Lipofectamine-2000 transfection reagent (Invitrogen) and the manufacturer's recommended protocol. For HEK-293 control transfections, cells were transfected with the vector pcDNA3.1 containing inverted V2R coding sequences. After 6 h, the cells were washed with PBS, returned to fresh culture medium, and analyzed after 48 h. Cells were fixed for 20 min in 4% paraformaldehyde in PBS at 4°C. After being washed several times with PBS, cells were permeabilized with 0.3% Triton X-100, 1% BSA in PBS for 30 min at room temperature, washed, and then incubated overnight at 4°C with 7251AP antibody. After several washes in PBS, specific staining was visualized using goat anti-rabbit IgG and Alexa Fluor 488 or 546 (Molecular Probes, Invitrogen), and nuclei were counterstained with TO-PRO-3 iodide (Molecular Probes, Invitrogen). Laser confocal microscopy was carried out with a Leica TCS-SP2 laser confocal microscope. For some samples, transfection was performed in six-well plates without coverslips, and gel samples were prepared (see Immunoblotting).
After removal, the right kidney was dissected into cortex (CTX), inner stripe of outer medulla (ISOM), and inner medulla (IM). The tissue was immediately homogenized in ice-cold isolation solution (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.4) containing the protease inhibitors leupeptin (8.5 μM) and phenymethylsulfonyl fluoride (1 mM) and the phosphatase inhibitors Na+-ortho-vanadate, NaF, and okadaic acid. The tissue was homogenized with an Ultra-Turrax T8 homogenizer, and the homogenate was centrifuged at 2,000 g for 15 min at 4°C. For subcellular fractionation experiments, homogenates underwent a series of differential centrifugation steps at 4°C: 1) 1,000 g for 10 min (pellet contains nonhomogenized tissue); 2) 4,000 g for 20 min (pellet contains nuclei, mitochondria, and some larger plasma membranes); 3) 17,000 g for 20 min (pellet enriched for plasma membranes); and 4) 200,000 g for 1 h (pellet enriched in intracellular vesicles, while supernatant contains cytosolic proteins). Total protein concentrations were measured (BCA kit; Pierce) in the supernatants, and the samples were solubilized in Laemmli sample buffer (5×; 7.5% SDS, 30% glycerol, 50 mM Tris pH 6.8, bromphenol blue, 25 mM dithiothreitol) at 65°C for 15 min. SDS-PAGE was performed on 12% polyacrylamide or 4–15% gradient polyacrylamide gels (Ready Gels, Bio-Rad). The proteins were transferred electrophoretically (Mini Trans-Blot Cell, Bio-Rad) to nitrocellulose membranes. Membranes were blocked for 1 h at room temperature with 5% nonfat dry milk and probed overnight at 4°C with NH2-terminal antibody V2R 7251AP. Membranes were washed and exposed to a horseradish peroxidase-conjugated secondary antibody (Dako P448, goat anti-rabbit IgG, 1:3,000) for 1 h at room temperature. After being washed, sites of antibody-antigen reaction were visualized using the enhanced chemiluminescence (ECL) Western Blotting Detection Reagents and ECL film (Amersham Pharmacia).
Deglycosylation of V2R protein in kidney homogenates.
Kidney CTX, ISOM, and IM total protein homogenates were prepared as described above. Twenty micrograms of homogenate was denatured by heating to 100°C for 10 min after addition of SDS (0.5% final concentration) and 0.1 M dithiothreitol. Samples were allowed to cool before addition of NP-40 detergent (1% final concentration) and 1,500 U PNGase F (New England Biolabs) or isolation solution (negative control). The mixture was incubated at 37°C for 60 min. The reaction was quenched by the addition of 5× Laemmli sample buffer (4:1, vol:vol). These samples were heated again to 65°C for 15 min before SDS-PAGE and immunoblotting.
Cross-linking of V2R-associated proteins.
Kidney CTX and ISOM total protein homogenates were prepared as described above. Before solubilization, 200 μl of protein sample was incubated with 10 mM of each of three commercially available (Pierce) cross-linkers: the membrane-permeable, homobifunctional N-hydroxysuccinade ester bis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone [BSOCOES; molecular weight (MW) = 436.36 g/mol; linker arm length, 13 Å]; and the membrane-permeable, homobifunctional imidoester cross-linkers dimethyladipimidate (DMA; MW = 245.15; linker arm length, 8.6 Å) and dimethylpimelimidate (DMP; MW 259.17; linker arm length, 9.2 Å). Control samples included total protein homogenates incubated with the solvent used to dissolve the corresponding cross-linker alone. All samples were incubated for 60 min at room temperature with bidirectional mixing. The reaction was quenched with 1 M Tris (pH 7.4), at a final concentration of 50 mM, and incubated for an additional 30 min at room temperature with bidirectional mixing. Laemmli sample buffer (4:1, vol:vol) was added, and the samples were heated to 65°C for 15 min before SDS-PAGE and immunoblotting.
The right kidney was clamped, removed, and processed for immunoblotting (see above). The left kidney was fixed by retrograde perfusion via the abdominal aorta. Kidneys were initially perfused with cold 0.01 M PBS (pH 7.4) for 15 s, followed by cold 3% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) for 3 min. The kidney was removed, and the midregion was sectioned into 2- to 3-mm transverse sections and immersion-fixed for an additional 1 h, followed by 3 × 10 min washes with 0.1 M cacodylate buffer, pH 7.4. The tissue was dehydrated in graded ethanol, incubated overnight in xylene, and embedded in paraffin, and 2-μm sections were cut on a rotary microtome (Leica Microsystems, Herlev, Denmark). Immunolabeling was performed as described previously in detail (39). The V2R 7251AP antibody was used at a final IgG concentration of 0.8 μg/ml. Labeling was detected using a horseradish peroxidase-conjugated secondary antibody (Dako P448, goat anti-rabbit IgG) and visualized with 0.05% 3,3′-diaminobenzidine tetrachloride (DAB, Kemen Tek, Copenhagen, Denmark). For dual-labeling studies with H+-ATPase and pendrin, single labeling was performed as described above. Sections were then incubated in 3.5% H2O2 in methanol to remove any remaining peroxidase from the first staining. Sections were incubated overnight at 4°C with H+-ATPase or pendrin antibody before being incubated with horseradish peroxidase-conjugated secondary antibody. For detection, Vector SG substrate (Vector Laboratories) was used as the chromagen to produce a gray-blue label, which is easily distinguished from the brown label produced by DAB in the first immunolabeling procedure for V2R. Light microscopy was carried out with a Leica DMRE (Leica Microsystems).
For confocal laser scanning microscopy studies, the following secondary fluorescent antibodies were used at a 1:1,000 dilution: goat anti-rabbit IgG, Alexa Fluor 488; goat anti-mouse IgG, Alexa Fluor 546; and goat anti-chicken IgG, Alexa Fluor 546 (Molecular Probes, Invitrogen). Laser confocal microscopy was carried out with a Leica TCS-SP2 laser confocal microscope.
IMCD tubule preparation and labeling.
IMCDs were purified from rat renal IMs as previously described (9). After low-speed centrifugation (70 g for 10 s) to sediment the heavier IMCD cells from the lighter non-IMCD cells, the tubules were attached to glass slides using Cell-Tak Cell adhesive (BD Biosciences). Tubules were fixed in 4% paraformaldehyde in PBS for 10 min at room temperature, washed in PBS, permeabilized with 0.3% Triton X-100, 1% BSA in PBS for 10 min at room temperature, washed, and then incubated overnight at 4°C with primary antibody. After several washes in PBS, specific staining was visualized using goat anti-rabbit IgG, Alexa Fluor 488 (Molecular Probes, Invitrogen), and nuclei were counterstained with TO-PRO-3 iodide (Molecular Probes, Invitrogen). Laser confocal microscopy was carried out with a Leica TCS-SP2 laser confocal microscope. For some tubules, the permeabilization step was omitted.
Recognition of V2R in transiently transfected kidney cells.
To establish the cellular and subcellular distribution of the V2R, a peptide-derived polyclonal antibody (raised in rabbits) against the amino terminus of rat V2R was produced and affinity purified. To assess the specificity of the new antibody (7251AP), HEK-293 cells were transiently transfected with constructs encoding either the rat, human, or mouse V2R and subsequently immunolabeled with the affinity purified antisera. As demonstrated in Fig. 1, HEK-293 cells expressing either the rat, human, or mouse V2R exhibited strong staining, which was predominantly associated with intracellular compartments. Nontransfected cells or cells transfected with a vector containing “inverted” rat V2R were not labeled, demonstrating specificity of the antibody for the V2R. Because of the punctate pattern of intracellular labeling, and to further confirm specificity of the antibody for the V2R alone, we transiently transfected M-1 kidney cells with constructs encoding rat V2R fused either at the NH2 terminus (GFP-V2R) or COOH terminus (V2R-GFP) to eGFP. Immunolabeling of these cells with 7251AP followed by confocal microscopy (Fig. 2A) showed that for the V2R-GFP construct there was colocalization between V2R antibody-specific labeling and GFP. GFP-V2R constructs failed to show any labeling, most likely because of the NH2-terminal GFP tag preventing antibody binding to the epitope. In addition, transfected cells that were subsequently immunoblotted and probed with either GFP or V2R antibodies showed protein bands of the sizes expected of the fusion constructs (Fig. 2B). Taken together, these results indicate that the 7251AP antiserum was specific for the V2R.1
Immunoblotting analysis of V2R in rat kidney.
To further characterize the anti-V2R antibody, immunoblotting was performed using whole protein homogenates from rat kidney IM and CTX. Figure 3A shows a representative immunoblot probed with the 7251AP antibody. In both IM and CTX, a protein “doublet” of approximately 43 and 47 kDa is apparent, consistent with the predicted molecular weight of the rat V2R plus glycosylation.2 To evaluate the specificity of the 7251AP antibody for V2R, preabsorption controls were carried out. Figure 3B shows an identical blot probed with 7251AP antibody previously incubated overnight with the immunizing peptide. All bands were ablated after preincubation with the immunizing peptide.
Glycosylation of V2R.
To test whether the protein doublet of 43 and 47 kDa is due to N-glycosylation of the V2R, protein homogenates from rat IM, ISOM, and CTX were incubated with PNGase F or vehicle. Figure 4 shows a representative immunoblot (PAGE performed on a 4–15% polyacrylamide gradient gel) probed with the 7251AP antibody. After PNGase F treatment, a single 34-kDa protein band was apparent, with the disappearance of the 47-kDa protein band. In addition, after long exposure, the appearance of further high-molecular-weight protein bands was apparent in the treated samples (see below). Similar results were found with an additional deglycosylation experiment performed with PNGase F from a different manufacturer (not shown). An identical blot probed with 7251AP antibody previously incubated overnight with the immunizing peptide resulted in disappearance of all bands (not shown). These results demonstrate that 1) V2R is N-glycosylated in vivo; and 2) the 43- and 47-kDa bands represent two states of V2R glycosylation, perhaps N-linked glycosylation at two distinct sites.
Subcellular distribution of V2R.
Serial centrifugation of IM and ISOM protein homogenates followed by immunoblotting was performed as a first step toward understanding the subcellular distribution of the V2R. This previously well-characterized technique relies on the relative different sizes/weights of different cellular compartments to separate them as follows: 4,000 g (nuclei, mitochondria, and some larger plasma membranes); 17,000 g (plasma membrane enriched); 200,000 g (intracellular vesicles) and 200,000 g supernatant (snt) (smaller vesicles and cytosolic proteins). The identity of each intracellular compartment was confirmed using antibodies against the Na-K-ATPase, which was most abundant in the membrane-associated fractions, and the AQP-2 water channel, which was most abundant in membrane-associated and intracellular vesicle-enriched fractions (Fig. 5). From these studies, we observe that the V2R is abundantly expressed in 43- and 47-kDa forms in the intracellular vesicle-enriched fractions. However, the V2R is much less abundant in 43- and 47-kDa forms in the membrane-associated fraction but expressed in a higher-molecular-weight form, possibly a dimer (see below).
Stabilization of V2R protein complexes by chemical cross-linking.
In several of our immunoblotting studies (especially with overexposure of films, not shown), and particularly in the membrane fraction from the subcellular fractionation studies described above, larger-molecular-weight moieties were observed. If the V2R is complexed either with itself [similar to other G protein-coupled receptors (48)] or with other proteins, homobifunctional cross-linking agents have the potential of stabilizing these complexes. To examine whether the native V2R is found in the rat kidney as high-molecular-weight complexes, we performed cross-linking studies in total protein homogenates from rat ISOM and CTX using three different homobifunctional cross-linking agents. Cross-linked samples were run on 4–15% polyacrylamide gradient gels, immunoblotted, and probed with the NH2-terminal antibody 7251AP (Fig. 6). In the absence of cross-linking, or with the cross-linking agents DMA or DMP, protein bands were observed at 47 kDa, which we propose is the monomeric form of the native V2R3 , and at ∼85 kDa. When cross-linking was performed with the membrane-permeable, homobifunctional N-hydroxysuccinade ester BSOCOES, several higher-molecular-weight complexes containing the V2R were stabilized. The approximate molecular masses of these complexes are 90 and 180 kDa, possibly representing the native V2R as a homodimer and homotetramer. From these results, we conclude that the rat V2R is present in the membrane as a high-molecular-weight complex.
Localization of V2R in normal rat kidney by immunohistochemistry.
To investigate the distribution of the V2R in normal rat kidney, we initially performed immunoperoxidase microscopy using paraffin-embedded kidney samples from normal rats and the 7251AP antibody. Figure 7 shows the localization of the V2R in different kidney zones (CTX, ISOM, and IM) in normal rats. In the CTX (Fig. 7A) and the ISOM (Fig. 7C), immunolabeling of the V2R was exclusively associated with collecting duct cells. In the IM, abundant labeling of the V2R was observed in all IMCD cells (Fig. 7E). Interestingly, no labeling was observed using the 7251AP antibody in segments identified as TALs. Labeling was predominantly intracellular in all regions, but some labeling was associated with the basolateral membrane domains (arrows). Immunolabeling controls with antibodies preabsorbed overnight with the immunizing peptide showed no labeling in any of the zones (not shown). Immunolocalization studies using cryosections that had not been exposed to organic solvents or the paraffin embedding process showed a similar pattern of labeling (not shown).
Localization of V2R in normal mouse kidney by immunohistochemistry.
To examine the distribution of the V2R in normal mouse kidney, we performed immunoperoxidase microscopy using paraffin-embedded mouse kidney and the 7251AP antibody. Figure 8 shows the localization of the V2R in the IM. At low magnification, abundant labeling is associated with all IMCD segments (Fig. 8A). Immunolabeling controls with antibody preabsorbed overnight with the immunizing peptide showed no labeling (Fig. 8A, inset). At higher magnification (Fig. 8B), it is clear that the labeling is associated with all IMCD cells, and there is no labeling of thin limbs of Henle's loop (tL). Labeling was predominantly intracellular, but some labeling was associated with the basolateral membrane domains (arrows). In addition, similarly to the rat, there was no labeling of TAL segments.
Cellular and subcellular localization of rat V2R determined by double-labeling confocal laser scanning microscopy.
To evaluate the cellular and subcellular localization of the rat V2R and to confirm the results of the immunoperoxidase microscopy, double-labeling confocal microscopy was undertaken using the 7251AP antibody. Figure 9 shows labeling of V2R (green) and the vasopressin-regulated water channel AQP2 (red) in normal rat kidney IM and CTX. In both the kidney regions, V2R labeling was predominantly intracellular (arrows, Fig. 9, A and D), whereas AQP-2 labeling was both intracellular and in the apical membrane domains (arrowheads, Fig. 9, B and E). All tubule segments that labeled for V2R were also positive for AQP-2 (Fig. 9, C and F) and thus were identified as either collecting ducts or connecting tubules (CNT, see below). In all kidney regions, very little colocalization of V2R and AQP2 was apparent in the apical plasma membrane (Fig. 9, C and F).
To determine the intracellular compartment that the majority of the V2R resides in, we performed colocalization studies with known markers for the endoplasmic reticulum, Golgi, lysosomes, and the peroxisome (not shown). There was little colocalization with any of the markers, suggesting that the majority of the V2R resides in a different location from the compartments examined.
Colocalization studies with AQP-2 revealed that the V2R is localized to either collecting ducts, CNT, or both. To distinguish between the CNT and collecting ducts, we performed double immunolabeling for V2R and calbindin. In the rat, calbindin is known to be abundantly expressed in cells of the DCT and CNT and is present in only a few collecting duct cells in the superficial cortex. Figure 10 shows labeling of V2R (Fig. 10A, green) and calbindin (Fig. 10B, red) in normal rat kidney CTX. V2R labeling was apparent in both calbindin-positive tubules (Fig. 10C), identified as CNT, and calbindin-negative tubules (not shown), which were identified as cortical collecting ducts (CCDs). In the CNT, V2R immunolabeling is found in the basolateral plasma membrane domains, with some intracellular labeling. Combined, these data indicate that, in the rat, the V2R is localized to both collecting ducts and the CNT throughout the kidney.
Rat V2R is expressed in collecting duct principal cells.
To confirm that in collecting ducts the V2R is only expressed in principal cells and not in intercalated cells, we performed double-labeling immunohistochemistry with the V2R 7251AP antibody and known markers for intercalated cells: the Cl−/HCO3− exchanger pendrin or the B1 subunit of the vacuolar H+-ATPase. It has previously been demonstrated that pendrin is localized to the apical plasma membrane domains of type B intercalated cells and non-A/non-B intercalated cells, whereas the B1 subunit of the vacuolar H+-ATPase is a marker for all types of intercalated cells (apical in type A, basolateral in type B). Figure 11 shows double labeling of a cortical region of the kidney from a normal rat with peroxidase-conjugated secondary antibodies. Pendrin (dark blue) is clearly localized to the apical pole of a subset of intercalated cells (Fig. 11A, arrowheads), whereas the V2R (brown) is both intracellular and in the basolateral membrane domain of pendrin negative cells (arrows). Additionally, the vacuolar H+-ATPase (dark blue) clearly labels all subtypes of intercalated cells (Fig. 11B, arrowheads), whereas the V2R (brown) is both intracellular and in the basolateral membrane domain of H+-ATPase-negative cells (arrows). We conclude from these data that, within the collecting duct, the rat V2R is expressed exclusively in principal cells.
Distribution of V2R in isolated rat IMCDs.
Surprisingly, in kidneys isolated from normal animals, we found that the majority of the V2R was localized inside the cell in some unknown intracellular compartment. To be certain that our antibody was capable of labeling membrane-associated V2R, we isolated IMCD tubules (after AVP washout), attached them to glass slides, and performed confocal laser scanning microscopy. Permeabilized IMCDs labeled for V2R (green) and nuclei (red) showed both intracellular and membrane associated V2R labeling (Fig. 12A). At higher magnification (Fig. 12A, inset, top right), the membrane labeling is much more apparent. An image acquired at a different level of cross section on an alternative IMCD tubule (Fig. 12B) clearly shows basolateral staining, similar to that of an IMCD tubule (Fig. 12C) labeled for the basolateral membrane-associated water channel AQP-4. IMCD tubules labeled for V2R (green) without permeabilization showed weak basolateral membrane labeling in some cells (Fig. 12D). In addition, in some cells, labeling appeared to be associated with the apical membrane domain, but this was not a common feature. For the majority of nonpermeabilized tubules, cell nuclei were not labeled with the cell-impermeable nuclei marker TOPRO-3 (not shown), indicating that the tubules were intact. Taken together, these results indicate that the 7251AP antibody is capable of labeling membrane-associated V2R.
V2R mRNA is expressed in both the IMCD and TAL.
Immunohistochemistry determined that V2R labeling with the 7251AP antibody was restricted to the kidney collecting ducts and the CNT. There was a complete absence of labeling in TAL segments, despite this tubule segment being vasopressin responsive. The apparent lack of labeling could be due to several factors; e.g., 1) the expression of the V2R in the TAL is below the detection limit for our antibody; 2) the V2R expressed in the TAL has an alternative NH2 terminus and thus would not be recognized by our NH2-terminal antibody; or 3) the effects of vasopressin in this segment are mediated by another type of receptor. Therefore, we performed RT-PCR on microdissected rat kidney IMCD or TAL nephron segments using primers specific for AQP-2, Na-K-2Cl cotransporter (NKCC2), and the NH2 terminus and COOH terminus of the V2R (Fig. 13). cDNA products representing AQP-2 are only detected in IMCD (Fig. 13A), whereas NKCC2 is only apparent in TAL (Fig. 13B), demonstrating a pure preparation of microdissected tubules. In contrast, V2R mRNA (corresponding to both the NH2 and the COOH terminus) is detected in both IMCD and TAL. Identical reactions performed in the absence of reverse transcriptase confirmed that the specific cDNA products did not result from genomic DNA contamination. In addition, we performed RT-PCR using the same primer sets and microdissected mouse DCTs. We did not see expression of the V2R in this tubule segment (data not shown).
In the kidney, the interaction between AVP and the V2R is a critical first step in maintaining body water homeostasis. In an oversimplified view, binding of AVP to the V2R results in activation of Gs, subsequent activation of adenylyl cyclase, and an increase in cAMP. Within the kidney collecting duct, this cascade of events results in the insertion of AQP-2 into the apical plasma membrane, facilitating water reabsorption (40). Despite the cDNA encoding the V2R being isolated nearly 15 years ago (2, 32), there is still a lack of studies of the receptor at the whole organism level. Thus, despite their being a wealth of data on the function/regulation of the receptor in vitro, little is known about the regulation of the receptor in vivo. The aim of this work was to generate and comprehensively characterize a novel polyclonal antibody directed against the extracellular NH2 terminus of the rat V2R (termed V2R 7251AP) and to determine the precise segmental and subcellular distribution of the receptor in kidney.
We employed single- and double-labeling immunoperoxidase microscopy combined with double-labeling confocal microscopy (using specific markers for individual nephron segments) to determine the precise segmental and subcellular distribution of the V2R in the kidney. Our results show that in the rat, our antibody localizes the V2R to the principal cells of the CNT, CCD, outer medullary collecting duct, and IMCD. In the mouse, V2R labeling is localized to the medullary collecting ducts; however, we cannot rule out V2R expression in the CNT, especially as the transition from the CNT to collecting duct is very gradual in the mouse. In agreement with our findings, RT-PCR on microdissected rat kidney tubules has shown that V2R mRNA is expressed predominantly in the collecting duct (26, 58), in situ hybridization of rat kidney has demonstrated V2R mRNA in renal arcades and collecting ducts (26, 44, 45), and in mouse and human, V2R mRNA has been detected in the medullary collecting ducts (7).
The major finding in our localization studies was that at a subcellular level in “normal” rats and mice, the V2R was predominantly expressed intracellularly, although some V2R labeling was apparent in the basolateral membrane domains. The results of our V2R immunolocalization studies are substantiated by our biochemical studies (see below). Two previous studies have addressed V2R expression in rat kidney using peptide-targeted antibodies (42, 54), and although there are similarities between our results and the previously published reports, there are also several differences. Using an antibody raised against the intracellular loop between the fifth and sixth transmembrane domain, Nonoguchi et al. (42) described that the V2R is localized to some TALs and to the basolateral and weakly, to the apical membrane of collecting ducts. Sarmiento et al. (54), using an antibody directed against the second intracellular loop of the V2R, localized the receptor to all TALs, DCTs, connecting tubules and collecting ducts, where staining is interpreted as being present on basolateral membranes and also on the luminal membranes of tubular cells. However, on close examination of the immunolabeling from these studies, we believe that the labeling is not considerably different from the labeling observed in the present study, with the majority of the labeling being observed intracellularly with some staining of basolateral membranes. One clear difference between the present study [and the study of Nonoguchi and colleagues (42)] and the study of Sarmiento et al. (54) is that we did not observe any labeling of DCT, whereas in the study of Sarmiento et al., strong immunolabeling of the DCT, which was predominantly apical, was of equal intensity to labeling of collecting duct and CNT segments. The physiological explanation for such strong V2R expression throughout this tubule segment is unclear.
If there really is a difference in V2R immunolocalization between the current study and the previous reports, what potentially is the cause? The majority of immunolabeling in our present study is from formaldehyde-fixed, paraffin embedded tissue sections [similar to the study of Sarmiento et al. (54)], whereas in the study of Nonoguchi et al. (42), it was from frozen tissue sections not exposed to organic solvents. However, when we repeated our studies using simple cryosections that are not exposed to solvents, we observed a similar subcellular distribution. Another possibility that one could speculate is that with our antibody, some epitope masking might occur in one location (plasma membrane) that prevents or restricts immunostaining. However, we clearly demonstrate that in isolated IMCD tubules, our antibody detects membrane-associated V2R; thus epitope masking is unlikely to be the reason for reduced membrane labeling in the tissue sections. Thus we believe that our immunolocalization of the V2R is a true reflection of where the majority of the V2R is situated under normal conditions. Whether this subcellular distribution changes under different physiological conditions, for example changes in circulating AVP levels, remains to be determined.
In our present study, we found that the V2R is expressed in the rat CNT. Until recently, it was generally believed that regulation of water reabsorption by the kidney occurred exclusively in the collecting duct. The basis for this thinking is based primarily on studies in rabbits showing that 1) the rabbit CNT possesses very low water permeability that is insensitive to vasopressin (22), and 2) microdissected rabbit CNTs possess very low vasopressin-sensitive adenylate cyclase activity (38). However, micropuncture studies by Gottschalk and colleagues showed that water can be reabsorbed proximal to the collecting duct (20), and in other studies, in contrast to the rabbit, in species with a high concentrating ability, e.g., the rat and mouse, vasopressin increases adenylate cyclase activity in the CNT (37). Furthermore, in the rat CNT both AQP-2 and the epithelial sodium channel, ENaC, have been shown to be both expressed and regulated by vasopressin (e.g., 10, 11, 55). Thus the expression of the V2R in the CNT, and thus the vasopressin-sensitive regulation of water and salt reabsorption by the CNT, is likely to be of physiological importance. In terms of water balance, the reabsorption of water in the CNT of the antidiuretic kidney will enhance maximal urinary osmolality and water conservation by allowing the water to be absorbed in the kidneys cortical labyrinth where blood flow is very rapid.
Unlike previous reports (42, 54), and despite extensive investigation, we did not find any labeling of the V2R in the TAL with our antibody, although we did determine that V2R mRNA is expressed in microdissected rat TAL segments. The medullary and cortical segments of the TAL play central roles in the regulation of water excretion (reviewed in Ref. 28). The medullary TAL actively reabsorbs NaCl, which drives the countercurrent multiplication process responsible for concentrating the urine. More than 30 years ago, studies by Morel and colleagues (23, 24, 35) localized adenylate cyclase activity to the rabbit, rat and mouse TAL and showed that the activity increases in response to vasopressin. Subsequent studies have demonstrated that vasopressin increases the intracellular level of cAMP in microdissected TAL segments (62, 63). The major effects of vasopressin on the TAL are thought to be mediated by the V2R. Indeed, the presence of the V2R in the TAL has been demonstrated by radiotracer binding studies (1) in microdissected rat TAL segments, by in situ hybridization (44, 45), by RT-PCR (58), and by immunohistochemistry (42, 54). The apparent lack of labeling in our studies could be due to several factors, but it is most likely that expression of the V2R in the TAL is below the detection limit of our antibody. Indeed, compared with CCD, in rabbit medullary TAL an ∼100-fold greater concentration of AVP is required to induce a 2-fold increase in adenylate cyclase activity (24), indicating that the V2R may be expressed at much lower levels. Furthermore, in addition to the V2R, several other receptors expressed in rodent TAL are coupled to adenylyl cyclase (36); thus the overall function of the TAL in the urinary concentrating process may be due to the regulatory actions of several hormones.
In biochemical studies, we determined that the V2R exists both as a low-molecular-weight doublet and as several high-molecular-weight complexes. In its low-molecular-weight form, the V2R exists as a minor protein of 43 kDa and a major protein band of 47 kDa. These molecular masses are consistent with sizes reported previously from kidney homogenates (42, 46, 47) or cells that express V2R fusion proteins (3, 4, 29, 43, 51, 56), and we propose that these represent the monomeric forms of the native V2R plus glycosylation. Indeed, treatment with N-glycosidase F reduced the native protein forms to a single protein band of 34 kDa, indicating that a single polypeptide chain is glycosylated to two different extents. We propose that the 47-kDa form represents the fully glycosylated native receptor, whereas the 43-kDa form is a partially processed high-mannose form (52). Since most integral membrane proteins targeted to the plasma membrane exhibit N-linked glycosylation, the finding that the V2R is glycosylated is not surprising. Indeed, the PNGase F-induced decrease in apparent mass (∼14 kDa) is consistent with the presence of two N-glycosylation sites in the rat V2R (32).
Serial centrifugation studies confirmed our immunolocalization studies and showed that the V2R is most abundant in intracellular vesicle-associated fractions. In the membrane-associated fractions, the weak V2R labeling is predominantly as high-molecular-weight complexes (these are much more apparent after overexposure of film). Furthermore, when inner medulla homogenates were subjected to homobifunctional cross-linkers before immunoblotting, numerous high-molecular-weight complexes were stabilized. Traditionally, G protein-coupled receptors have been thought to act as monomers, but emerging data have challenged this opinion. Indeed, many transmembrane receptors are known to dimerize as part of their normal function, e.g., receptor tyrosine kinases (60). Previous studies in cell culture have reported that the human V2R forms homo- and heterodimers (53, 61) or oligomeric complexes by domain swapping (56), and recently in an individual cell-free expression system the V2R has been shown to exist as a dimer (27). Our studies show for the first time that the native V2R protein exists as at least a dimer in vivo. In cell culture, dimerization of the V2R occurs early in the secretory pathway (68), although it has also been reported that formation of oligomers could be a consequence of overproduction of the protein in overexpression systems and not a required step for receptor function (53). In addition to forming homodimers, several heterodimer combinations have been reported to be formed between the V2R, the vasopressin V1a receptor (V1aR), and the oxytocin receptor (OTR), e.g., V1aR/V2R (59, 61) or V2R/OTR (12, 61). The V1aR/V2R interaction is thought to be of functional importance, as it results in substantial cointernalization of the two receptors (59). Our present studies do not establish whether the complex is simply a dimer of V2R or a heterooligomer.
In summary, we have developed and comprehensively characterized a novel polyclonal antibody against the V2R. This antibody will be an essential tool in understanding the regulation of the V2R in body water homeostasis and in studying dysregulation of the V2R in numerous physiological and pathophysiological processes.
R. A. Fenton is supported by a Marie Curie Intra-European Fellowship, the Carlsberg Foundation (Carlsbergfondet) and the Danish National Research Foundation (Danmarks Grundforskningsfond). L. Brønd is supported by The Danish Heart Association (Hjerteforeningen). The Water and Salt Research Center at the University of Aarhus is established and supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). Additional support for this study was provided by the WIRED program (Nordic Council and the Nordic Centre of Excellence Program in Molecular Medicine).
We are grateful to Peter Deen for advice regarding cell culture and for providing stably transfected MDCK cells. We thank Christian Westburg, Inger Merete Paulsen, Helle Høyer, and Zhila Nikrozi for expert technical assistance.
↵1 Furthermore, immunoprecipitation experiments followed by protein identification by tandem mass spectroscopy confirmed specificity of the antibody (see Supplemental Materials, available with the online version of this article).
↵2 The protein doublet of approximately 43 and 47 kDa is clearly visible in some rat protein samples, whereas in other samples only the predominant 47-kDa protein band is apparent.
↵3 Parallel blots (not shown) probed for rat UT-A1 (a urea transporter protein expressed in the collecting duct) showed, as previously determined, stabilization of a large-molecular-mass complex of ∼∼200 kDa with all three cross-linking agents, indicating that the chemical cross-linking agents were functional.
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