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Am J Physiol Renal Physiol 293: F1166-F1177, 2007. First published July 11, 2007; doi:10.1152/ajprenal.00196.2007
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Vasopressin V2 receptor expression along rat, mouse, and human renal epithelia with focus on TAL

K. Mutig,1 A. Paliege,1 T. Kahl,1 T. Jöns,1 W. Müller-Esterl,2 and S. Bachmann1

1Department of Anatomy, Charité Universitätsmedizin, Berlin; and 2Institute of Biochemistry II, University of Frankfurt Medical School, Frankfurt, Germany

Submitted 24 April 2007 ; accepted in final form 27 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In renal epithelia, vasopressin influences salt and water transport, chiefly via vasopressin V2 receptors (V2Rs) linked to adenylyl cyclase. A combination of vasopressin-induced effects along several distinct portions of the nephron and collecting duct system may help balance the net effects of antidiuresis in cortex and medulla. Previous studies of the intrarenal distribution of V2Rs have been inconclusive with respect to segment- and cell-type-related V2R expression. Our study therefore aimed to present a high-resolution analysis of V2R mRNA expression in rat, mouse, and human kidney epithelia, supplemented with immunohistochemical data. Cell types of the renal tubule were identified histochemically using specific markers. Pronounced V2R signal in thick ascending limb (TAL) was corroborated functionally; phosphorylation of Na+-K+-2Cl– cotransporter type 2 (NKCC2) was established in cultured TAL cells from rabbit and in rats with diabetes insipidus that were treated with the V2R agonist desmopressin. We found solid expression of V2R mRNA in medullary TAL (MTAL), macula densa, connecting tubule, and cortical and medullary collecting duct and weaker expression in cortical TAL and distal convoluted tubule in all three species. Additional V2R immunostaining of kidneys and rabbit TAL cells confirmed our findings. In agreement with strong V2R expression in MTAL, kidneys from rats with diabetes insipidus and cultured TAL cells revealed sharp, selective increases in NKCC2 phosphorylation upon desmopressin treatment. Macula densa cells constitutively showed strong NKCC2 phosphorylation. Results suggest comparably significant effects of vasopressin-induced V2R signaling in MTAL and in connecting tubule/collecting duct principal cells across the three species. Strong V2R expression in macula densa may be related to tubulovascular signal transfer.

antidiuretic hormone; thick ascending limb; kidney; sodium-potassium-chloride cotransporter type 2; bumetanide-sensitive cotransporter type 1

ANTIDIURETIC HORMONE [arginine vasopressin (AVP)] serves to regulate body fluid osmolality, blood volume, blood pressure, and vascular tone. In the kidney, two major receptor subtypes [V1a and V2 receptors (V1aR and V2R)] have been characterized (22, 24). V1aRs are principally localized in the renal vasculature and glomeruli and mediate the vasopressor effect of AVP (2, 32). The tubular antidiuretic effect of AVP is mediated by the V2R and adenylyl cyclase-dependent cAMP signaling (13, 22). Since the early work of Morel (25), sites of V2R-mediated AVP action along the nephron have been defined at different levels, such as transport (13, 15, 17), ligand binding (2, 35, 44), receptor mRNA expression (9, 32, 47), and immunohistochemical localization (30, 39). The collecting duct (CD) is generally considered the principal target for the antidiuretic action of AVP, but available data suggest a role for AVP also in the distal tubule (4, 38, 39). At both sites, AVP serves to control the renal concentrating mechanism. In the thick ascending limb (TAL), AVP stimulates transepithelial NaCl transport via increased abundance and phosphorylation of Na+-K+-2Cl– cotransporter type 2 (NKCC2) (10, 11, 14, 15). In the CD, the main action of AVP is promotion of water reabsorption via the aquaporin type 2 (AQP-2) water channel (13, 17, 28). Clinically, defective V2R function is associated with nephrogenic diabetes insipidus (DI) (22). Vasopressin agonists and antagonists have been characterized in animal models and studied for use in clinical medicine (10, 11, 43).

Regarding the prominent functional role of V2R-mediated effects of AVP in the kidney, an exact mapping to the renal nephron segments and cell types is of major importance. Previous work has presented information on V2R distribution across several mammalian species, but results have in part been inconclusive, and information on cell-type-specific localization of V2R biosynthesis is scarce (9, 30, 32, 39, 47). These studies showed solid V2R expression in the CD, with some reference to regional differences in intensity believed to reflect AVP-induced effects on the stimulation/insertion of AQP-2 and urea transporter(s) (4, 28, 36). Localization of V2R was further reported in rat kidney TAL (10, 39) and macula densa (32); binding of oxytocin, which is structurally related to AVP, and expression of oxytocin receptor in macula densa as well (33, 45) support a role for AVP and, possibly, oxytocin in the regulation of glomerular filtration rate (37). A recent study analyzing V2R distribution in mouse and human kidney confirmed V2R expression in CD, but not in TAL (9).

Interest in genetically engineered mice, as well as recent data on the functional significance of AVP-related signaling in the distal tubule, has stimulated us to study in more detail the distribution of the V2R in mouse nephron segments with use of nonradioactive in situ hybridization and antibody staining combined with immunostaining by segment- and cell-type-specific markers, and we have analyzed the rat kidney in parallel. Since there is further awareness of the potency to clinically manipulate AVP-related effects in volume disorders and edema, we have extended our approach to the study of the human kidney.

Our results have clearly shown dominant expression of the V2R in medullary TAL (MTAL), macula densa, and medullary CD (MCD), intermediate expression in connecting tubule (CNT) and cortical CD (CCD), and low expression in cortical TAL (CTAL) and distal convoluted tubule (DCT) in all three species. To illustrate the functional significance of V2R signaling in TAL, we have studied short-term effects of the V2R agonist desmopressin (dDAVP) in cultured TAL cells and in Brattleboro rats with central DI by evaluating the intensity and distribution of NKCC2 phosphorylation in the response.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, tissues, and cells. Adult male Wistar rats (n = 3), Brattleboro rats with DI (Harlan; n = 20), and C57/Bl6 mice (n = 3) were bred in the local animal facility (Charité Berlin) and kept on standard diet and tap water. Animals were anesthetized by an injection of pentobarbital sodium (0.06 mg/g body wt ip). The abdominal cavity was opened, and kidneys were perfused retrogradely through the abdominal aorta using 3% paraformaldehyde (41). Human kidney samples (n = 3) were derived from tumor nephrectomy; tissues from the healthy parts of the kidneys were immersion fixed in paraformaldehyde. The local council on animal care approved the protocols (permission number G006-2/05 Land Berlin); the standards correspond to the requirements of the American Physiological Society. The human kidneys were from tumor nephrectomy material obtained after written consent of the respective patients from Charité hospital. Tissues were prepared for standard cryostat and paraffin sectioning. Series of 5-µm-thick consecutive sections were cut from rat, mouse, and human kidney samples for in situ hybridization and immunohistochemistry. For study of V2R-mediated changes in expression and phosphorylation status of NKCC2, DI rats were randomly divided into three groups: 1) control, vehicle (intraperitoneal saline)-treated animals (n = 8), 2) animals treated with desmopressin [1-desamino-8-D-Arg vasopressin (dDAVP); 1 ng/g body wt ip] for 30 min (n = 8), and 3) animals treated with vehicle and then subjected to 4 h (from 4 to 8 PM) of water deprivation (n = 4). At the end of the experiments, four DI rats, each from groups 1, 2, and 3, were perfusion fixed for morphological analysis. The remaining DI rats of groups 1 and 2 were killed by an overdose of pentobarbital sodium (Nembutal), the kidneys were removed, and pieces of outer medulla and cortex were dissected. Simian virus 40-transformed rabbit TAL (rbTAL) cells obtained from rabbit kidney medulla were cultured as described elsewhere (48). Cells were incubated with vehicle or dDAVP (1 x 10–7 mol/l; Sigma) in FCS-free culture medium for 30 min. Cells were then harvested and homogenized in sucrose buffer.

In situ hybridization. In situ hybridization was performed as described elsewhere (42). Briefly, a 584-bp PCR product was amplified from rat kidney cDNA using specific primers for V2R: 5' CAg CAg CCA ggA ggA ACT AC 3' (forward) and 5' gTg CCA CAA ACA CCA TCA Ag 3' (reverse). The amplified fragment was cloned into the pCR4-TOPO vector (Invitrogen) and verified by DNA sequencing. The digoxigenin (DIG)-11-UTP-labeled antisense riboprobe was synthesized by in vitro transcription (DIG RNA labeling kit Sp6/T7, Roche). Since sequence homology was high across species (97% for mouse and 85% for human V2R), the same probe was used for all species. For in situ hybridization, dewaxed paraffin sections were treated with proteinase K, hybridized for 18 h at 40°C with hybridization mix (2.5 ng mRNA/µl), washed, and incubated with sheep anti-DIG alkaline phosphatase-conjugated antibody (DAKO). Signal was generated using 4-nitro blue tetrazolium chloride. For control, a 592-bp nonsense probe was applied in parallel with the antisense probe. Sections were rinsed with PBS, coverslips were applied using PBS-glycerol, and sections were viewed by bright-field microscopy.

Immunohistochemistry. For identification of V2 mRNA-expressing tubular segments, rabbit polyclonal antibody against V2R was applied (39). For further cell-type-specific identification of the in situ signal, segment-specific antibodies were applied using double labeling or separate labeling of consecutive sections (3, 6, 7). The following polyclonal antibodies were used: guinea pig antibody against the NH2-terminal 85 amino acids of NKCC2 (2.1 antibody) (41) and sheep anti-Tamm-Horsfall protein (THP) antibody (BioTrend) for TAL; rabbit anti-Na+-Cl– cotransporter (NCC) antibody (provided by D. H. Ellison, Portland, OR) for DCT; sheep anti-11beta-hydroxysteroid dehydrogenase (11beta-HSD2) antibody (Chemicon) for late distal convoluted tubule (DCT2), CNT, and CCD (7); goat anti-AQP-2 antibody (Santa Cruz Biotechnology) for CNT, CCD, and MCD principal cells; and rabbit anti-vacuolar H+-ATPase antibody (Santa Cruz Biotechnology) or rabbit anti-anion exchanger 1 (AE1) (16) antibody for intercalated cells. For separate immunohistochemical labeling, sections were dewaxed and boiled in citrate buffer (pH 6.0) for 5 min. Sections were blocked with 5% milk powder in PBS for 1 h and incubated with primary antibody diluted in 5% milk powder for 1 h at room temperature and then incubated overnight at 4°C. For multiple staining, antibodies were sequentially applied, with a wash step between applications. Next, appropriate secondary fluorescent Cy3- or Cy2-conjugated antibodies (DIANOVA) were applied over a 2-h period. For bright-field V2R detection, an LSAB-2 System-AP enhancer kit (DakoCytomation) was used. Sections were washed, coverslips were applied with PBS-glycerol, and sections were viewed in a Leica DMRB microscope equipped with a SPOT 32 camera and MetaView 3.6a software (Diagnostic Instruments, Universal Imaging).

Evaluation of phosphorylated NKCC2. For analysis of NKCC2 phosphorylation, rabbit antiserum against phosphorylated NKCC2 (2 NH2-terminal threonines; see Ref. 11 for peptide sequence) was produced and affinity purified using selection with phosphorylated and nonphosphorylated peptides (Pineda, Berlin, Germany). The affinity-purified antibody against phosphorylated NKCC2 was termed pT2 antibody. Preabsorption tests were performed immunohistochemically using the phosphorylated, as well as the nonphosphorylated, peptides (10-fold excess). The size of phosphorylated NKCC2 as recognized by pT2 antibody was confirmed by Western blot on rat kidney extracts. Specificity was further confirmed by colocalization with 2.1 antibody. Paraffin sections of human kidney were incubated with pT2 antibody and then horseradish peroxidase (HRP) for detection. For double staining, incubation of frozen sections with pT2 antibody was followed by application of 2.1 antibody. HRP- and fluorescent Cy3-coupled secondary antibodies were used for detection.

Western blot. Nuclei were removed by homogenization of excised rat kidney zones and rbTAL cells in sucrose-triethanolamine buffer and centrifugation at 1,000 g for 15 min at 4°C. The supernatants were separated by PAGE [50 µg protein/lane, as determined by a bicinchoninic acid protein assay reagent kit (Pierce); 8–10% gel]. After electrophoretic transfer to PVDF membrane, equity in protein loading and blotting was verified by membrane staining using 0.1% Ponceau red staining. After they were blocked in 5% milk, PVDF membranes were incubated with mouse monoclonal antibody against the COOH-terminal 310 amino acids of NKCC2 (T4 antibody; Developmental Studies Hybridoma Bank, University of Iowa) or pT2 or 2.1 primary antibodies for 1 h at room temperature, incubated overnight at 4°C, and then exposed to HRP-conjugated secondary antibodies for 2 h at room temperature. Immunoreactive bands were detected by chemiluminescence and exposed to X-ray films, and the signals were scanned and densitometrically evaluated. Monoclonal mouse anti-beta-actin antibody (Sigma) was used to normalize all data for expression of the housekeeping gene beta-actin.

Real-time PCR. Samples of the outer medulla and cortex from DI rats were homogenized, and total RNA was prepared using the RNeasy total RNA kit (Qiagen). Genomic DNA was digested by DNase, and cDNA was synthesized by reverse transcription of 5 µg of total RNA (cDNA synthesis kit, Invitrogen). Specific TaqMan gene expression assays for NKCC2 and GAPDH were generated by Applied Biosystems. Amplification was performed using the real-time PCR TaqMan Fast 7500 (Applied Biosystems). Samples were incubated at 94°C for 10 min, and then 45 cycles of 95°C (3 s) and 60°C (30 s) were run. Threshold cycle (Ct) values were set in the linear phase of exponential amplification. The difference between values obtained for NKCC2 and the housekeeping gene GAPDH ({Delta}Ct) was calculated and compared between treatments and kidney zones.

Analysis of data. Western blot and real-time PCR results were evaluated using routine parametric descriptive statistics. Groups were compared with two-way ANOVA and t-tests; Bonferroni's correction was used as appropriate. P < 0.05 was accepted as significant. Values are means ± SD.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
We have localized V2R mRNA using DIG-labeled antisense probes hybridized to rat, mouse, and human kidney. For additional information, we have used specific antibody to V2R. Double-staining techniques or serial sectioning was applied for cell type and segment identification. Results were largely similar among species. Overviews of coronal kidney sections present a clear predominance of V2R mRNA expression intensity within the inner stripe of the outer medulla, intermediate strength in the inner medulla, and weaker signals in the outer stripe and the cortical segments. The dominant outer medullary signal was due to the parallel, strong V2R expression in MTAL and MCD (Fig. 1). V2R immunoreactive signal essentially paralleled these patterns but revealed less axial heterogeneity in the distal tubule segments.


Figure 1
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  		Fig. 1. Zonal and segmental distribution of baseline vasopressin V2 receptor (V2R) mRNA expression in rat, mouse, and human renal epithelia. Left: schematic representation of V2R mRNA distribution (see Figs. 2–7). Right: overview scale in situ hybridization images across species. Approximate intensity levels of V2R mRNA expression are represented by different colors. Kidney zones are as follows: cortex (C), outer medulla (OM) with outer stripe (OS) and inner stripe (IS), and inner medulla (IM). Tubule segments are as follows: medullary and cortical thick ascending limb (MTAL and CTAL), macula densa (MD), distal convoluted tubule (DCT), connecting tubule (CNT), and cortical and medullary collecting duct (CCD and MCD). a–c: Nonsense probe hybridization control images at outer-to-inner medullary transition. d–l: Representative images of V2R mRNA expression in renal cortex (d–f), at outer-to-inner medullary transition (g–i; note strong labeling of MTALs), and in inner medulla (j–l). Arrows point to corresponding kidney zones. Original magnification x200.

 
Cortical vasculature, interstitium, glomeruli, and proximal nephron. Generally, no significant V2R mRNA expression was encountered in cortical and medullary vasculature, interstitium, glomeruli, proximal tubules, or thin limbs of the loop of Henle of rat and mouse kidneys. In human kidney, the findings were similar, except for positive signals occasionally found in glomerular parietal epithelium and in the glomerular tuft; the latter, however, was irregular and could not be clearly assigned to a cell type.

Hypothalamic V2R mRNA signal. In situ hybridization of rat brain sections was performed for additional confirmation of the specificity of the applied V2R riboprobe. Hybridization with the antisense riboprobe produced a specific signal in hypothalamic paraventricular nucleus, as previously characterized functionally (50), whereas application of the nonsense probe did not produce a signal (Fig. 2, g and h).


Figure 2
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  		Fig. 2. V2R mRNA distribution in rat kidney. a and b: Colocalization of V2R mRNA in MTAL (*) identified by Na+-K+-2Cl– type 2 cotransporter (NKCC2) immunostaining [antibody against NH2-terminal 85 amino acids of NKCC2 (2.1)]. V2R mRNA-positive MCD (#) are negative for NKCC2. c and d: V2R mRNA expressing MCD are labeled by aquaporin type 2 (AQP-2) immunostaining, whereas MTAL are negative for AQP-2. Dashed lines, outer-to-inner medullary transitions. e and f: Juxtaglomerular thick ascending limb (TAL) expressing V2R mRNA in macula densa (between flanking lines); parallel staining for Tamm-Horsfall protein (THP) in TAL spares macula densa. Glomeruli (G) are shown by dashed lines. g and h: Labeling of rat brain sections detects V2R expression in neurons of hypothalamic paraventricular nucleus (arrows in g), whereas the nonsense probe does not produce a signal (h). III, 3rd ventricle. Original magnification x400.

 
TAL. The TAL, identified by concomitant staining with antibody against NKCC2 or THP, displayed significant V2R mRNA expression in the medullary part. The intensity of the inner stripe MTAL signal was strongest (Fig. 1, g–i; Fig. 2, a and b; Fig. 3, a and b; and Fig. 4, a and b), whereas the intensity of the outer stripe MTAL signal was intermediate. In the cortical part, expression was weak, except for the macula densa cells, which showed strong V2R mRNA expression; cells were identified by the absence of THP (Fig. 2, e and f; and Fig. 4, e and f) or by their specific topography (Fig. 3, e and f). Specific antibody to V2R showed significant staining of MTAL and CTAL segments across species as well (Figs. 3, g and h; and Fig. 4, g and h). Staining was stronger at the luminal than at the basolateral cell aspect and, particularly, in mice showed somewhat less pronounced axial heterogeneity than the mRNA signal.


Figure 3
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  		Fig. 3. V2R distribution in mouse kidney. a and b: Colocalization of V2R mRNA in MTAL (*) identified by NKCC2 immunostaining (2.1 antibody). V2R mRNA-positive MCD (#) are negative for NKCC2. c and d: V2R mRNA expressing MCD are labeled by AQP-2 immunostaining, whereas MTAL are negative for AQP-2. Dashed lines, outer-to-inner medullary transitions. e and f: juxtaglomerular TAL expressing V2R mRNA in macula densa (between flanking lines) concomitantly immunolabeled for NKCC2; glomeruli (G) are shown by dashed lines. g and h: Immunohistochemical localization of V2R in medulla (g) and cortex (h) showing TAL (*) and collecting duct (CD) profiles (#). Original magnification x400.

 

Figure 4
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  		Fig. 4. V2R distribution in human kidney. a and b: Colocalization of V2R mRNA in MTAL (*) identified by NKCC2 immunostaining (2.1 antibody). V2R mRNA-positive MCD (#) are negative for NKCC2. c and d: MCD are labeled by AQP-2 immunostaining, whereas MTAL are negative for AQP-2. Arrows point to intercalated cells, which show strong V2R but no AQP-2 signals. e and f: Juxtaglomerular TAL with V2R mRNA expressed in macula densa (between flanking lines) concomitantly immunolabeled for Tamm-Horsfall protein (THP) in CTAL, but not in macula densa; glomerulus (G) is shown by dashed lines. g and h: Immunohistochemical localization of V2R in medulla (g) and cortex (h) showing TAL (*;) and CD profiles (#). Arrow points to an intercalated cell (g). Original magnification x400.

 
DCT, CNT, and CCD. In DCT, identified by concomitant NCC immunostaining, there was significant V2R mRNA expression that was moderately stronger than in the preceding CTAL (Fig. 5, a–i; and Fig. 6, a and b). In the CNT and CCD immunostained by AQP-2, V2R mRNA signals were of intermediate intensity (Fig. 1). CNT segments were identified by their initial transition from DCT2 immunostained for NCC and 11beta-HSD2 or for complementary expression of NCC and AQP-2 (Fig. 5, a–i; and Fig. 6, c and d). The majority of cortical intercalated cells of CNT and CCD, as identified by parallel H+-ATPase immunostaining, were V2R mRNA negative; those with cytosolic/basolateral H+-ATPase staining, likely to correspond to the B phenotype, were generally unstained, whereas those with luminal staining, corresponding to the A phenotype, in part expressed the receptor (Fig. 7, a–d).


Figure 5
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  		Fig. 5. V2R mRNA distribution in distal convolutions and collecting ducts of rat (a–f) and mouse cortices (g–i). Multiple labeling in consecutive serial sections demonstrates low- to medium-intensity V2R mRNA levels in DCTs (1) identified by Na+-Cl– cotransporter (NCC), 11beta-hydroxysteroid dehydrogenase (11HSD2), and lack of AQP-2. CNT (2), the onset of which is defined by mutually exclusive NCC vs. AQP-2 signals or by partial overlap of NCC and 11HSD2, representing DCT-to-CNT transitions (horizontal bars), and NCC-negative AQP-2-positive CCD portions (3) showed stronger signals. Original magnification x400.

 

Figure 6
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  		Fig. 6. V2R mRNA distribution in distal convolutions and collecting ducts of human cortex. a and b: V2R mRNA signal in NCC-positive DCTs. c and d: V2R mRNA signal in AQP-2-positive CNT/CCD. Dashed lines, positive nephron segments. G, glomeruli. Arrows in a and c show occasional V2R mRNA signal in glomerular cells. Original magnification x400.

 

Figure 7
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  		Fig. 7. V2R expression in medullary and cortical intercalated cells across species. Intercalated cells are identified on consecutive or double-labeled sections immunostained for H+-ATPase, anion exchanger type 1 (AE1), or AQP-2. Cortical and medullary type A intercalated cells (arrows) are labeled by apical H+-ATPase or basolateral AE1 antibodies, whereas type B cells (arrowheads) show cytosolic/basolateral expression of H+-ATPase. Some cortical type A cells express V2R mRNA; cortical type B cells lack V2R mRNA (a–d). Medullary intercalated cells express V2R mRNA throughout in rat (e–h), show parallel V2R-immunoreactive, AQP-2-negative signal in mouse (i and j), and express V2R mRNA in human kidney (k and l). Original magnification x400.

 
Medullary vasculature, interstitium, thin limbs, and MCD. Medullary vasculature, interstitium, and thin limbs were devoid of V2R signal. In the MCD, identified by AQP-2 immunostaining, V2R mRNA was markedly expressed in all three species, with the strongest label in human kidney (Fig. 2, c and d; Fig. 3, c and d; and Fig. 4, c and d). Medullary type A intercalated cells, identified by basolateral AE1 and apical H+-ATPase immunoreactivity and lack of AQP-2 signal, strongly expressed V2R mRNA throughout (Fig. 4, c and d; and Fig. 7, e–l). V2R expression in MCD and CCD was further confirmed by immunohistochemistry, which revealed a strong signal in MCD and a weaker signal in CCD (Fig. 3g; and Fig. 4, g and h). The strength of the TAL and CD signals was similar. Medullary intercalated cells, as identified by their morphology, demonstrated strong immunoreactive V2R signal throughout (Fig. 4g).

Effects of AVP on TAL transporter. To verify the significant V2R expression in TAL, we studied AVP-dependent activation of the major TAL ion transporter NKCC2 in AVP-deficient DI rats that had been supplemented with dDAVP for 30 min or had been subjected to 4 h of water deprivation. Distribution and abundance of NKCC2, as well as phosphorylated NKCC2, were evaluated using 2.1 and pT2 antibodies, respectively.

In vehicle-treated DI rats, pT2 signal was not detectable in the medulla, whereas scattered pT2-positive TAL segments were observed in the cortex (Fig. 8, a, d, and g; Fig. 9, b and f). Macula densa cells showed selectively stronger expression of phosphorylated NKCC2 than adjacent TAL cells (Fig. 9, i and j). In low-power magnification, stimulation of DI rats with dDAVP resulted in drastic increases of pT2 signal intensities in CTAL and MTAL compared with vehicle treatment (Fig. 8, a, b, d, e, g, and h), whereas water deprivation for 4 h did not significantly affect NKCC2 phosphorylation (Fig. 8, c, f, and i). Detailed double-staining analysis revealed moderate increases in 2.1 signals, as opposed to more marked increases in pT2 signals, in single CTAL and MTAL profiles, respectively, upon dDAVP treatment (Fig. 9, a–h). pT2 signals on dDAVP-stimulated DI rat kidneys were completely abolished by preincubation with phosphorylated peptide used for immunization during the preabsorption test, whereas application of the nonphosphorylated peptide did not affect the reaction (Fig. 9, k and l); pT2 band specificity was also demonstrated by Western blot (Fig. 9m). These changes were confirmed by Western blot. Cortical and outer medullary kidney samples of vehicle- or dDAVP-treated DI rats were analyzed. dDAVP treatment resulted in augmented 2.1 signals (+79 ± 11% in cortex and +85 ± 11% in outer medulla, P < 0.05; Fig. 10, a, b, g, and h), as well as increased pT2 signals (+86 ± 5% in cortex and +198 ± 18% in outer medulla, P < 0.005; Fig. 10, c, d, g, and h). Data have been normalized for beta-actin expression. Quantification of the NKCC2 mRNA in cortical and outer medullary samples did not reveal significant differences between treatments (Fig. 10, i and j). These results were extended to the study of cultured, immortalized rbTAL cells superfused with vehicle or dDAVP. rbTAL cells express V2R (unpublished RT-PCR data and Western blot; Fig. 11a). Exposure to dDAVP for 30 min resulted in augmented T4 signals (+154 ± 32%, P < 0.05), as well as increased pT2 signals (+151 ± 37%, P < 0.05; Fig. 11b). Data have been normalized for beta-actin expression.


Figure 8
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  		Fig. 8. Comparative overviews of experimentally altered NKCC2 phosphorylation in kidneys of Brattleboro rats with diabetes insipidus (DI) using affinity-purified antibody against phosphorylated NKCC2 (pT2 antibody). Zonal differences between groups are shown in vehicle-treated rats (a, d, and g), rats treated for 30 min with 1-desamino-8-D-Arg-vasopressin (dDAVP; b, e, and h), and rats deprived of water for 4 h (c, f, and i). Dashed lines, outer-to-inner medullary transitions. Original magnification x200.

 

Figure 9
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  		Fig. 9. Representative TAL profiles from vehicle- and dDAVP-treated DI rat kidneys. a–j: Image pairs after 2.1 (NKCC2) and pT2 (phosphorylated NKCC2) immunostaining. *, Double-stained profiles. a–d, Renal cortex; e–h, medulla; i and j, macula densa. Note mild increases in 2.1 signals as opposed to more pronounced increases in pT2 signals after dDAVP stimulation, indicating enhanced NKCC2 phosphorylation. Macula densa at steady state shows selectively high phosphorylated NKCC2 signal; glomerulus (G). Preabsorption of pT2 antibody with use of 10-fold excess of phosphorylated peptide abolished signal (k). Nonphosphorylated peptide had no influence on pT2 staining on TAL profiles (l). m: Single pT2 band on whole lane, Western blot, with size marker indicated. Original magnifications x400 (a–j) and x200 (k and l).

 

Figure 10
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  		Fig. 10. Western blot and real-time PCR analyses of cortical (left) and outer medullary (right) tissues from vehicle- and dDAVP-treated DI rats. a–d: Representative blots revealing bands for 2.1 (a and b) and pT2 (c and d) antibody staining at 160 kDa. e and f: Loading controls (beta-actin-immunoreactive bands). g and h: respective densitometric evaluations, with vehicle groups set at 100%. Note disproportionately enhanced signal for phosphorylated NKCC2 in medulla of the dDAVP group (h). i and j: Real-time PCR showing cycle threshold difference ({Delta}Ct) between values obtained for NKCC2 and housekeeping gene GAPDH. *P < 0.01 vs. vehicle. §P < 0.01, pT2 vs. 2.1 in dDAVP group.

 

Figure 11
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  		Fig. 11. Western blot analysis showing baseline immunoreactive V2R levels (a) and vehicle vs. dDAVP treatment in TAL cells from rabbit (rbTAL, b). a: 3 representative bands from independent rbTAL lysates show V2R at ~50 kDa. b: representative blots revealing bands for antibody against COOH-terminal 310 amino acids of NKCC2 (T4) and pT2 antibody staining at 160 kDa, as well as loading controls (beta-actin) at 42 kDa (top) and respective densitometric evaluations of T4 and pT2 immunoreactivity, with the vehicle groups set at 100% (bottom). *P < 0.05 vs. vehicle.

 
Application of pT2 antibody to human kidney resulted in substantial CTAL (Fig. 12a) but weak MTAL staining (data not shown). This finding may be related to the functional and endocrine status of the patient's kidney during surgery, possibly preventing medullary activation of NKCC2. By contrast, similar to rodents, human macula densa cells showed a prominent, selective expression of phosphorylated NKCC2 exceeding the level in adjacent CTAL cells (Fig. 12b).


Figure 12
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  		Fig. 12. Localization of phosphorylated NKCC2 in human kidney showing pT2-immunoreactive cortical TAL profiles (a) and selectively enhanced labeling of macula densa cells (between flanking lines; b). Glomeruli (G) are shown by dashed lines. Original magnification x400.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study shows the distribution of V2R mRNA expression along the nephron in rat, mouse, and human kidneys, complemented with an immunohistochemical approach for detection of the immunoreactive protein. With respect to local AVP signaling in the loop of Henle, V2R-dependent phosphorylation of NKCC2, reflecting surface expression of the transporter, was studied in TAL. Expression of V2R was established along the major distal epithelia. Medullary and cortical TAL, macula densa and post-macula segment, distal convolutions, CCD and MCD epithelia, and a subset of type A intercalated cells of the three species expressed V2R mRNA to distinct degrees of intensity, but with no major interspecies differences. Localization of V2R mRNA to these epithelia likely reflected sites of receptor biosynthesis, since parallel immunohistochemistry from this and previous work has provided comparable results, and functional studies have, in part, indicated regional heterogeneity in the renal response to AVP (10, 30, 39, 40). The topographical results have corroborated and extended earlier results obtained in rats by means of labeled ligand binding (20, 35), immunohistochemistry (30, 39), or in situ hybridization (32). We have extended the information on overall V2R mRNA distribution and its cell-type assignment by analyzing mouse and human kidneys.

The major finding of the present study is the localization of significant V2R mRNA synthesis to the TAL in the three species, revealing axial heterogeneity with solid expression in the medullary TAL and weak signal along the cortical TAL. This result has previously been obtained in rats at the mRNA (22, 32) and immunoreactive protein (30, 39) levels, although without clear assignment to the particular portions. A heterogeneity in V2R mRNA signal intensity between long- and short-looped medullary TAL profiles, as reported earlier in rats (30), was not evident from our results; rather, staining was equally strong in all profiles in the three species. Extending molecular localization of V2R to the mouse and human kidney, we have corroborated the significance of a marked medullary presence of the receptor in TAL across representative mammalian species. Concentration of the V2R immunoreactive signal at the luminal aspect of TAL across species reflects earlier data from the rat (39) but is at variance with previous demonstration of V2R signal located predominantly at the basolateral, and less so at the luminal, aspects (30). This difference may be antibody based; however, since, in TAL basolateral membrane, folding is extensive whereas the luminal plasma membrane has little surface amplification, the number of receptors per cell side, reflected by the distinct immunoreactivities, may still be comparable. If we consider the apparent presence of V2R on both sides of TAL, strong luminal vs. weak basolateral staining may therefore reflect similar receptor densities on each cell pole. Whether this is associated with antagonistic AVP effects of the two locations, as described earlier for the MCD (30), is not clear from the present data.

Our results of a strong V2R signal in TAL is at variance with another study reporting no V2R mRNA in the TAL of mice and humans (9). The reason for this discrepancy may be technical, since less-resolving radio-labeled in situ hybridization was used in that study, and it was stated that a diffuse but intensive V2R mRNA labeling was prevailing in medulla. Distal epithelia may therefore have escaped identification. In terms of specificity and resolution, our findings of rat hypothalamic paraventricular neurons showing distinct V2R staining have demonstrated the viability of our tools.

Functionally, there is solid evidence of a role for V2R signaling via cAMP accumulation in medullary ion transport by the TAL, promoting the "single effect" of the countercurrent multiplication as part of the urinary concentrating mechanism (38). In cortical TAL, AVP may further activate Mg2+ and Ca2+ transport, yet, probably via V1R (29, 49). The zonal distribution of V1R/V1aR, however, remains obscure (12, 46), and others explicitly found no V1aR transcripts in cortical TAL (9).

Effects of AVP and selective V2R agonists and antagonists on TAL have been studied with respect to the gene products involved in transepithelial NaCl transport. Significant changes in the expression or phosphorylation state of key Na+ transporters have been reported (10, 11, 18). In Brattleboro DI rats lacking circulating vasopressin, a decreased steady-state expression of the key ion transporters in TAL was shown to be restored after long-term application of the V2R agonist dDAVP (10). Also, in the short term, dDAVP exerts significant effects in TAL, driving phosphorylation and apical trafficking of the major TAL transporter NKCC2 in mouse kidney (11, 31). Although this function has earlier been estimated to be relevant only in rodents with their specifically high concentrating ability by some (for review see Ref. 4), the high V2R expression in the human kidney suggests an analogous, relevant function of AVP in medullary TAL as well. In addition, a parallel, AVP-independent mechanism was postulated on the basis of NKCC2 stimulation in DI rats after short-term water deprivation (23).

To functionally corroborate our data on V2R expression in TAL, we have studied the short-term induction of NKCC2 by AVP in DI rats regionally, determining the abundance of NKCC2 mRNA, whole protein, and NH2-terminal threonine phosphorylation. Data were supplemented by the study of our cultured rbTAL cell line, which expresses V2R and NKCC2. Western blot V2R signal in rbTAL cells showed an expected, dominant band at ~50 kDa (39); the NKCC2 band generated with the T4 antibody also corresponded to the expected size of 160 kDa (18). In DI rats, there were clear increases in phosphorylated NKCC2 signal along the entire length of the TAL, with a sharper rise in its medullary portion in DI rats. Corresponding changes were found in the rbTAL cells. In part, our observations thus mirror earlier data on NKCC2 trafficking in mice (11). However, at variance with the mouse data, simultaneous increases in the absolute amounts of NKCC2, but not NKCC2 mRNA, were found, possibly reflecting AVP-dependent changes in NKCC2 protein stability or posttranscriptional activation. By contrast, our study failed to reveal an increase of phosphorylated NKCC2 upon water deprivation in the DI rat, which underlines the unique relevance of AVP in TAL transepithelial transport stimulation.

We furthermore found consistent, selectively strong V2R expression in macula densa cells in all three species. Previously, radiolabeled in situ signal was reported in rat kidney macula densa (32), and possible roles of vasopressin V1 and oxytocin/oxytocin receptor signaling in macula densa function were highlighted (2, 33). In parallel with V2R expression, we found high-level baseline signals of phosphorylated NKCC2 in macula densa across species, suggesting that AVP may influence tubuloglomerular signaling via modulation of NKCC2-dependent transpepithelial transport. This observation may be related to AVP-dependent acute changes in glomerular filtration rate in rat kidney (4, 37). However, the detection of selectively high phosphorylated NKCC2 immunoreactivity in DI rat macula densa underscores AVP-independent, constitutive activation of the cotransporter at this site.

Our results of V2R expression in DCT, CNT, and CCD confirm previous findings in rat (30, 39) and extend the information on the cell-type-specific detail in the three species. Functionally, the presence of V2R in DCT, at least in its terminal portion (i.e., DCT2) (3), agrees with published data on increased NCC biosynthesis rate upon long-term dDAVP application in rats (10). The presence of mineralocorticoid receptors in DCT, CNT, and CCD and the presence of 11beta-HSD2 in DCT2, CNT, and CCD, along with functional data, have underlined local steroid sensitivity of NaCl transport (for review see Refs. 3 and 21). The parallel expression of V2R and the epithelial Na+ channel (ENaC), the major distal Na+ transporter, further emphasizes the manifold hormonal influences on the distal convolutions and collecting system. Functionally, Na+ transport and ENaC abundance have been shown to increase under cAMP-dependent vasopressin/V2R stimulation (8, 10, 26). A link to the colocalized V1R expression (39, 46) and its potentially antagonistic role may be relevant with respect to the cellular control of luminal vs. basolateral transport (for review see Ref. 4).

The major function of vasopressin relates to the CD, with the hormone acting at physiological concentrations of 10–12 M to exert its antidiuretic actions; the V2R-dependent effect is coupled to adenylyl cyclase, inducing PKA-mediated phosphorylation of AQP-2 and its translocation to the apical membrane of CD principal cells (for review see Ref. 27). Our findings of moderate cortical and stronger medullary V2R mRNA expression levels across species agree with earlier histochemical (9, 30, 35, 39) and functional (4, 27) data. Less is known about intercalated cells with respect to AVP-induced signaling. This study has clearly defined that a subset of cortical type A intercalated cells and all medullary type A intercalated cells were expressing V2R mRNA. Type A intercalated cells may modulate net acid excretion and bicarbonate reabsorption. Acute and chronic effects of vasopressin on acid-base transport have been partially characterized (1, 5, 34). Type A intercalated cells are dominant in the renal medulla (19). At this site, they have been shown to express a newly identified basolateral Cl–/HCO3– exchanger upon dDAVP treatment in DI rats (34), suggesting a possible functional link to V2R signaling. Type B intercalated cells, which constitute a significant fraction in the CNT and CCD in rat and mouse kidney, were devoid of V2R mRNA. However, long-term dDAVP application in DI rats had marked effects on acid-base transporters also in the type B intercalated cells, possibly owing to the regulatory requirements induced by local solute concentrations (1).

In summary, a comparative, cell-type-oriented analysis of V2R distribution in rat, mouse, and human kidney has been presented at high resolution. Similar expression patterns have been found across these species. Dominant V2R expression was observed in medullary TAL and MCD, consistent with the prominent medullary effects of AVP on its major target products, AQP-2 and NKCC2. Functional analysis of AVP-dependent phosphorylation of NKCC2 in the different portions of TAL and cultured rbTAL cells has confirmed the role of V2R expression in this segment. Our study provides new insights into the organization of renal volume handling.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This project was supported by Deutsche Forschungsgemeinschaft Grant FOR 667.


   ACKNOWLEDGMENTS
 
We thank F. Grams for expert technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. Bachmann, Institut für Vegetative Anatomie, Charité-Universitätsmedizin Berlin, Campus Charité-Mitte, Philippstr. 12, D-10115 Berlin, Germany (e-mail: sbachm{at}charite.de)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amlal H, Sheriff S, Faroqui S, Ma L, Barone S, Petrovic S, Soleimani M. Regulation of acid-base transporters by vasopressin in the kidney collecting duct of Brattleboro rat. Am J Nephrol 26: 194–205, 2006.[CrossRef][Web of Science][Medline]
  2. Arpin-Bott MP, Kaissling B, Waltisperger E, Rabhi M, Saussine P, Freund-Mercier MJ, Stoeckel ME. Historadioautographic localization of oxytocin and V1a vasopressin binding sites in the kidney of developing and adult rabbit, mouse and merione and of adult human. Exp Nephrol 10: 196–208, 2002.[CrossRef][Web of Science][Medline]
  3. Bachmann S, Bostanjoglo M, Schmitt R, Ellison DH. Sodium transport-related proteins in the mammalian distal nephron—distribution, ontogeny and functional aspects. Anat Embryol (Berl) 200: 447–468, 1999.[CrossRef][Medline]
  4. Bankir L. Antidiuretic action of vasopressin: quantitative aspects and interaction between V1a and V2 receptor-mediated effects. Cardiovasc Res 51: 372–390, 2001.[Abstract/Free Full Text]
  5. Bichara M, Mercier O, Houillier P, Paillard M, Leviel F. Effects of antidiuretic hormone on urinary acidification and on tubular handling of bicarbonate in the rat. J Clin Invest 80: 621–630, 1987.[Web of Science][Medline]
  6. Biner HL, Arpin-Bott MP, Loffing J, Wang X, Knepper M, Hebert SC, Kaissling B. Human cortical distal nephron: distribution of electrolyte and water transport pathways. J Am Soc Nephrol 13: 836–847, 2002.[Abstract/Free Full Text]
  7. Bostanjoglo M, Reeves WB, Reilly RF, Velazquez H, Robertson N, Litwack G, Morsing P, Dørup J, Bachmann S, Ellison DH. 11beta-Hydroxysteroid dehydrogenase, mineralocorticoid receptor, and thiazide-sensitive Na-Cl cotransporter expression by distal tubules. J Am Soc Nephrol 9: 347–358, 1998.[CrossRef]
  8. Butterworth MB, Edinger RS, Johnson JP, Frizzell RA. Acute ENaC stimulation by cAMP in a kidney cell line is mediated by exocytic insertion from a recycling channel pool. J Gen Physiol 125: 81–101, 2005.[Web of Science][Medline]
  9. Carmosino M, Brooks HL, Cai Q, Davis LS, Opalenik S, Hao C, Breyer MD. Axial heterogeneity of vasopressin receptor subtypes along the human and mouse collecting duct. Am J Physiol Renal Physiol 292: F351–F360, 2007.[Abstract/Free Full Text]
  10. Ecelbarger CA, Kim GH, Wade JB, Knepper MA. Regulation of the abundance of renal sodium transporters and channels by vasopressin. Exp Neurol 171: 227–234, 2001.[CrossRef][Web of Science][Medline]
  11. Gimenez I, Forbush B. Short-term stimulation of the renal Na-K-Cl cotransporter (NKCC2) by vasopressin involves phosphorylation and membrane translocation of the protein. J Biol Chem 278: 26946–26951, 2003.[Abstract/Free Full Text]
  12. Gonzalez CB, Figueroa CD, Reyes CE, Caorsi CE, Troncoso S, Menzel D. Immunolocalization of V1 vasopressin receptors in the rat kidney using anti-receptor antibodies. Kidney Int 52: 1206–1215, 1997.[Web of Science][Medline]
  13. Grantham JJ, Burg MB. Effect of vasopressin and cAMP on permeability of isolated collecting tubules. Am J Physiol 211: 255–259, 1966.[Free Full Text]
  14. Hebert SC, Culpepper RM, Andreoli TE. NaCl transport in mouse medullary thick ascending limbs. I. Functional nephron heterogeneity and ADH-stimulated NaCl cotransport. Am J Physiol Renal Fluid Electrolyte Physiol 241: F412–F431, 1981.[Abstract/Free Full Text]
  15. Hebert SC, Andreoli TE. Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle. II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl– absorption. J Membr Biol 80: 221–233, 1984.[CrossRef][Web of Science][Medline]
  16. Huber S, Asan E, Jöns T, Kerscher C, Püschel B, Drenckhahn D. Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis. Am J Physiol Renal Physiol 277: F841–F849, 1999.[Abstract/Free Full Text]
  17. Inoue T, Nonoguchi H, Tomita K. Physiological effects of vasopressin and atrial natriuretic peptide in the collecting duct. Cardiovasc Res 51: 470–480, 2001.[Abstract/Free Full Text]
  18. Kim GH, Ecelbarger CA, Mitchell C, Packer RK, Wade JB, Knepper MA. Vasopressin increases Na-K-2Cl cotransporter expression in thick ascending limb of Henle's loop. Am J Physiol Renal Physiol 276: F96–F103, 1999.[Abstract/Free Full Text]
  19. Kim J, Kim YH, Cha JH, Tisher CC, Madsen KM. Intercalated cell subtypes in connecting tubule and cortical collecting duct of rat and mouse. J Am Soc Nephrol 10: 1–12, 1999.[Abstract/Free Full Text]
  20. Kirk KL, Buku A, Eggena P. Cell specificity of vasopressin binding in renal collecting duct: computer-enhanced imaging of a fluorescent hormone analog. Proc Natl Acad Sci USA 84: 6000–6004, 1987.[Abstract/Free Full Text]
  21. Loffing J, Kaissling B. Sodium and calcium transport pathways along the mammalian distal nephron: from rabbit to human. Am J Physiol Renal Physiol 284: F628–F643, 2003.[Abstract/Free Full Text]
  22. Lolait SJ, O'Carroll AM, McBride OW, Konig M, Morel A, Brownstein MJ. Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature 357: 336–339, 1992.[CrossRef][Medline]
  23. Michimata M, Mizukami K, Suzuki M, Kazama I, Nakamura Y, Suzuki K, Yanagisawa T, Imai Y, Sasaki S, Matsubara M. Vasopressin-independent renal urinary concentration: increased rBSC1 and enhanced countercurrent multiplication. Kidney Int 64: 933–938, 2003.[CrossRef][Web of Science][Medline]
  24. Morel A, O'Carroll AM, Brownstein MJ, Lolait SJ. Molecular cloning and expression of a rat V1a arginine vasopressin receptor. Nature 356: 523–526, 1992.[CrossRef][Medline]
  25. Morel F. Sites of hormone action in the mammalian nephron. Am J Physiol Renal Fluid Electrolyte Physiol 240: F159–F164, 1981.[Abstract/Free Full Text]
  26. Nicco C, Wittner M, DiStefano A, Jounier S, Bankir L, Bouby N. Chronic exposure to vasopressin upregulates ENaC and sodium transport in the rat renal collecting duct and lung. Hypertension 38: 1143–1149, 2001.[Abstract/Free Full Text]
  27. Nielsen S, Kwon TH, Christensen BM, Promeneur D, Frokiær J, Marples D. Physiology and pathophysiology of renal aquaporins. J Am Soc Nephrol 10: 647–663, 1999.[Abstract/Free Full Text]
  28. Nishimoto G, Zelenina M, Li D, Yasui M, Aperia A, Nielsen S, Nairn AC. Arginine vasopressin stimulates phosphorylation of aquaporin-2 in rat renal tissue. Am J Physiol Renal Physiol 276: F254–F259, 1999.[Abstract/Free Full Text]
  29. Nitschke R, Frobe U, Greger R. Antidiuretic hormone acts via V1 receptors on intracellular calcium in the isolated perfused rabbit cortical thick ascending limb. Pflügers Arch 417: 622–632, 1991.[CrossRef][Web of Science][Medline]
  30. Nonoguchi H, Owada A, Kobayashi N, Takayama M, Terada Y, Koike J, Ujiie K, Marumo F, Sakai T, Tomita K. Immunohistochemical localization of V2 vasopressin receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts. J Clin Invest 96: 1768–1778, 1995.[Web of Science][Medline]
  31. Ortiz PA. cAMP increases surface expression of NKCC2 in rat thick ascending limbs: role of VAMP. Am J Physiol Renal Physiol 290: F608–F616, 2006.[Abstract/Free Full Text]
  32. Ostrowski NL, Young WS 3rd, Knepper MA, Lolait SJ. Expression of vasopressin V1a and V2 receptor messenger ribonucleic acid in the liver and kidney of embryonic, developing, and adult rats. Endocrinology 133: 1849–1859, 1993.[Abstract/Free Full Text]
  33. Ostrowski NL, Lolait SJ. Oxytocin receptor gene expression in female rat kidney. The effect of estrogen. Adv Exp Med Biol 395: 329–340, 1995.[Medline]
  34. Petrovic S, Amlal H, Sun X, Karet F, Barone S, Soleimani M. Vasopressin induces expression of the Cl–/HCO3– exchanger SLC26A7 in kidney medullary collecting ducts of Brattleboro rats. Am J Physiol Renal Physiol 290: F1194–F1201, 2006.[Abstract/Free Full Text]
  35. Phillips PA, Abrahams JM, Kelly JM, Mooser V, Trinder D, Johnston CI. Localization of vasopressin binding sites in rat tissues using specific V1 and V2 selective ligands. Endocrinology 126: 1478–1484, 1990.[Abstract/Free Full Text]
  36. Promeneur D, Kwon TH, Frokiær J, Knepper MA, Nielsen S. Vasopressin V2-receptor-dependent regulation of AQP2 expression in Brattleboro rats. Am J Physiol Renal Physiol 279: F370–F382, 2000.[Abstract/Free Full Text]
  37. Roald AB, Tenstad O, Aukland K. The effect of AVP-V2 receptor stimulation on local GFR in the rat kidney. Acta Physiol Scand 168: 351–359, 2000.[CrossRef][Web of Science][Medline]
  38. Sands JM, Kokko JP. Current concepts of the countercurrent multiplication system. Kidney Int Suppl 57: S93–S99, 1996.[Medline]
  39. Sarmiento JM, Ehrenfeld P, Anazco CC, Reyes CE, Troncoso S, Figueroa CD, Müller-Esterl W, Gonzalez CB. Differential distribution of the vasopressin V2 receptor along the rat nephron during renal ontogeny and maturation. Kidney Int 68: 487–496, 2005.[CrossRef][Web of Science][Medline]
  40. Sauter D, Fernandes S, Goncalves-Mendes N, Boulkroun S, Bankir L, Loffing J, Bouby N. Long-term effects of vasopressin on the subcellular localization of ENaC in the renal collecting system. Kidney Int 69: 1024–1032, 2006.[CrossRef][Web of Science][Medline]
  41. Schmitt R, Ellison DH, Klussmann E, Bachmann S. Renal expression of sodium transporters and aquaporin-2 in hypothyroid rats. Am J Physiol Renal Physiol 284: F1097–F1104, 2003.[Abstract/Free Full Text]
  42. Schmitt R, Kahl T, Mutig K, Bachmann S. Selectively reduced expression of thick ascending limb Tamm-Horsfall protein in hypothyroid kidneys. Histochem Cell Biol 121: 319–327, 2004.[CrossRef][Web of Science][Medline]
  43. Serradeil-Le Gal C, Wagnon J, Valette G, Garcia G, Pascal M, Maffrand JP, Le Fur G. Nonpeptide vasopressin receptor antagonists: development of selective and orally active V1a, V2 and V1b receptor ligands. Prog Brain Res 139: 197–210, 2002.[Web of Science][Medline]
  44. Stoeckel ME, Freund-Mercier MJ, Palacios JM, Richard P, Porte A. Autoradiographic localization of binding sites for oxytocin and vasopressin in the rat kidney. J Endocrinol 113: 179–182, 1987.[Abstract/Free Full Text]
  45. Stoeckel ME, Freund-Mercier MJ. Autoradiographic demonstration of oxytocin-binding sites in the macula densa. Am J Physiol Renal Fluid Electrolyte Physiol 257: F310–F314, 1989.[Abstract/Free Full Text]
  46. Tashima Y, Kohda Y, Nonoguchi H, Ikebe M, Machida K, Star RA, Tomita K. Intranephron localization and regulation of the V1a vasopressin receptor during chronic metabolic acidosis and dehydration in rats. Pflügers Arch 442: 652–661, 2001.[CrossRef][Web of Science][Medline]
  47. Terada Y, Tomita K, Nonoguchi H, Yang T, Marumo F. Different localization and regulation of two types of vasopressin receptor messenger RNA in microdissected rat nephron segments using reverse transcription polymerase chain reaction. J Clin Invest 92: 2339–2345, 1993.[Web of Science][Medline]
  48. Welker P, Geist B, Frühauf JH, Salanova M, Groneberg DA, Krause E, Bachmann S. Role of lipid rafts in membrane delivery of renal epithelial Na+,K+-ATPase, thick ascending limb. Am J Physiol Regul Integr Comp Physiol 293: R1328–R1337, 2007.
  49. Wittner M, di Stefano A, Wangemann P, Nitschke R, Greger R, Bailly C, Amiel C, Roinel N, de Rouffignac C. Differential effects of ADH on sodium, chloride, potassium, calcium and magnesium transport in cortical and medullary thick ascending limbs of mouse nephron. Pflügers Arch 412: 516–523, 1988.[CrossRef][Web of Science][Medline]
  50. Yang J, Chen JM, Song CY, Liu WY, Wang G, Wang CH, Lin BC. Through the central V2, not V1 receptors influencing the endogenous opiate peptide system, arginine vasopressin, not oxytocin in the hypothalamic paraventricular nucleus involves in the antinociception in the rat. Brain Res 1069: 127–138, 2006.[CrossRef][Web of Science][Medline]



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