INTRODUCTION

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THE ABILITY OF MAMMALIAN organisms to conserve water depends on the release of arginine vasopressin (AVP), and the actions of this peptide on the vasopressin V₂ receptors (V₂R) depend on the basolateral membranes of the collecting ducts. This is accomplished by V₂R-stimulated insertion of aquaporin-2 (AQP-2) water channels (12, 21, 22, 28) into the apical membrane of the collecting duct epithelium. Paradoxically, it has been demonstrated in vitro that pharmacological amounts of AVP lead to the downregulation of the V₂R through multiple mechanisms (3, 4, 7, 14, 18, 26). It has also been reported using semiquantitative RT-PCR that prolonged water restriction for up to 72 h decreases the levels of V₂R mRNA in isolated collecting ducts (32). Additionally, Firsov et al. (9) demonstrated that stimulation of the V₂R using a highly selective V₂R agonist, 1-desamino-8-D-arginine vasopressin (DDAVP), resulted in a reduction in the V₂R mRNA in isolated renal tubules, which included thick ascending limbs of Henle and collecting ducts. Yet the downregulation of the V₂R, particularly within the renal medulla, would appear to be counterproductive during water restriction, and it remains unknown whether the reduction of the V₂R mRNA is associated with a parallel reduction in the V₂R protein.

The present study was, therefore, designed to determine whether specific regional differences of the V₂R mRNA and/or protein levels occur between the renal cortex and medulla during 24 and 48 h of water restriction. A highly sensitive competitive RT-PCR assay was developed to quantitate the changes in the steady-state level of the V₂R mRNA, and a polyclonal antibody specific to the V₂R protein was generated to determine alterations in the V₂R protein by Western blot analysis.

	METHODS

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Experimental animals. Male Sprague-Dawley rats (360-420 g) from Harlan (Madison, WI) were used in the protein and RNA isolation. All animals were fed a standard pellet diet (Purina Mills, St. Louis, MO) and ad libitum water to drink until the beginning of the water restriction protocol. All protocols were approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

Tissue isolation. Kidneys from control ad libitum water, 24 h, and 48 h water-restricted rats were collected, and the RNA and protein were isolated. The left kidney was used for the RNA extraction, and the right kidney was used for protein isolation. The kidneys were coronally cut, which allowed for the visualization and removal of the three distinct regions of the kidney: the cortex and outer and inner medulla.

RNA isolation. RNA was isolated from the renal cortex and outer and inner medulla using TRIzol reagent (GIBCO-BRL; Life Technologies, Gaithersburg, MD). The extracted RNA was resuspended in diethyl pyrocarbonate (DEPC)-treated water (50 µl for the outer and inner medulla and 100 µl for the cortex). The RNA was quantified using a Beckman ultraviolet spectrophotometer at 260 nm, and the resuspended RNA was then frozen at 80°C.

Preparation of oligonucleotide primers. All nucleotide primers were purchased from Operon Technologies (Alameda, CA). Oligonucleotide primers were chosen from the published full-length cDNA sequences of rat V₂R (19). The oligonucleotides used for amplification correspond to the following sequences: primer A, 5' ATG GTG GGC ATG TAT GCC TCC TCC TAC ATG 3' (position 399-427 bp); primer B, 5' AGT GTC ATC CTC ACG GTC TTG GCC A 3' (position 835-859 bp); and primer C, 5' GAC ATA GGC ACG AAG GCC CCA 3' (position 638-659 bp).

Construction of mutant V₂R. The initial V₂R PCR product was amplified with primers A and B and cloned into the pCRII vector (Invitrogen, Carlsbad, CA). The DNA insert was excised from the vector by EcoR I digestion and gel purified (Qiagen, Chatsworth, CA). The purified EcoR I fragment was digested with Dde I to delete a 29-bp fragment (bp 548-578), and the digestion products (149 and 282 bp) were isolated and gel purified. The DNA fragments were ligated for 1 h at 16°C with T4 DNA ligase (Pharmacia, Piscataway, NJ). The ligation product was amplified with primers A and B, and the PCR products with the expected size were cloned into the pCR2.1 vector (Invitrogen) and then subcloned into the pSP64 poly(A) vector (Promega, Madison, WI). Transformants with the correct size were isolated and sequenced to ensure the removal of the 29 bp in the vasopressin V₂ receptor deletion mutant.

In vitro transcription of mutant V₂R RNA. One microgram of BamH I-digested plasmid was transcribed with 10 U SP6 RNA polymerase (Promega). The transcription solution consisted of (in mM) 40 Tris · HCl, pH 7.9, 6 MgCl₂, 10 dithiothreitol, 2 spermidine, 0.5 rNTP mix, and 40 U RNAsin. The transcription reaction was incubated at 37°C for 60 min, and then 5 U RQ1 RNase-free DNase was added to digest the plasmid for 30 min at 37°C. The transcribed RNA was then re-extracted with 100 µl TRIzol reagent, as described earlier. The RNA pellet was resuspended with DEPC-treated water, and the RNA concentration was determined by ultraviolet spectrophotometry. The deletion mutant transcript was aliquoted and frozen at 80°C.

Competitive RT-PCR. Initial experiments were performed to determine the amounts of total RNA needed from each of the kidney regions to allow for the RT-PCR amplification to occur within the same range of deletion mutant transcript. The amount of total RNA necessary for RT was 1 ng for the inner medulla, 3 ng for the outer medulla, and 5-10 ng for the cortex in combination with different amounts of mutant V₂R transcript (ranging from 4 to 280 zmol), using 10 pmol of primer C. All RNA samples (i.e., the control group and the corresponding experimental groups) from each kidney region were run simultaneously in each assay to minimize potential interassay variability. The RNA was incubated at 65°C for 10 min and then immediately placed on ice. The RT-PCR was performed as previously described (25). All PCR reactions were performed in the same tube as the RT reaction and were adjusted to a final volume of 50 µl. The PCR reaction conditions were optimal using 1.0 mM MgCl₂, 50 pmol of each primer (A and C), and 2.5 U AmpliTaq polymerase (Perkin-Elmer Cetus, Norwalk, CT). The reaction mixture was first denatured at 96°C for 5 min and then at 80°C while AmpliTaq polymerase (2.5 U) was added to each of the reaction tubes. The reactions were cycled for 32 cycles at 94, 66, and 72°C for 1 min at each step. Samples were then incubated for an additional 7 min at 72°C for final extension. Negative controls used in this experiments were PCR amplification of sterile water, mutant V₂R RNA transcript, and tissue RNA without RT.

Competitive RT-PCR product analysis. Ten-microliter aliquots of the PCR products were electrophoresed on a 2% agarose gel (75% regular agarose, 25% low-melting agarose). After electrophoresis and ethidium bromide staining, the gel was scanned with a FluorImager (Molecular Dynamics, Sunnyvale, CA). The bands were quantitated using ImageQuant software and then subsequently plotted to determine the equivalence point (i.e., the point at which the number of known mutant V₂R transcripts is equal to the number of unknown V₂R mRNA molecules in a given amount of total RNA). Competitive RT-PCR assays were run in triplicate for each RNA sample to obtain an average calculated value for the equivalence point.

Plasma membrane-enriched fraction protein isolation. The kidneys from control ad libitum water-restricted and 24 and 48 h water-restricted rats were coronally sectioned, and the three renal regions (renal cortex and outer and inner medulla) were isolated. The renal tissue was homogenized in (in mM) 5 K₂HPO₄, 5 KH₂PO₄, 250 sucrose, pH 7.7, 0.1 EDTA, 0.1 phenylmethylsulfonyl fluoride, 2 µg/µl leupeptin, and 5 µg/µl pepstatin. The centrifugation protocol was a slight modification of Ecelbarger et al. (8), in which the homogenate was initially centrifuged at 1,000 g to remove any incompletely homogenized membrane fragments and nuclei. The supernatant was then centrifuged at 16,000 g for 20 min. The pellet, which contains predominantly plasma membrane protein, was then resuspended in homogenization buffer. The protein concentrations were determined at 595 nm using the Coomassie method (Pierce, Rockford, Il).

Western blot analysis. Sample buffer [2% SDS, 100 mM Tris · HCl, pH 6.8, 5% -mercaptoethanol, 12% (vol/vol) glycerol, and 0.02% (wt/vol) bromophenol blue] was added to the protein sample and then incubated at 100°C for 5 min. Aliquots of 4 µg of inner medullary, 16 µg of outer medullary, and 16 µg of cortical membrane protein were loaded onto each lane and size separated by electrophoresis through a 12% SDS-PAGE gel. The proteins were transferred onto a nitrocellulose membrane (Bio-Rad, Hercules, CA), which was then blocked overnight at 4°C with 12% nonfat dried milk in blotting solution. The following day, the membranes were incubated for 1 h with the primary antibody (1:1,500) at room temperature. To determine the specificity of the V₂R antibody, membranes were incubated with the V₂R antibody preadsorbed with the competing immunogenic peptide and the preimmune serum. The membranes were subsequently incubated for 1 h with secondary antibody (goat anti-rabbit IgG; 1:1,500, Bio-Rad) at room temperature. The membranes were placed in a chemiluminescent solution (WesternView; Transduction Laboratories, Lexington, KY) to enable visualization of the protein bands on film. The bands were quantitated by densitometry (Molecular Dynamics). Each of the membranes was stripped and reprobed with -actin (1:2,500) to ensure equal loading of proteins in each lane.

Statistical analysis. The significance of differences between groups was tested by one-way ANOVA with the use of SigmaStat software. If a probability value of P  0.05 was obtained, the Tukey test was then used for comparison of each individual group with the appropriate control.

	RESULTS

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Construction of deletion mutant V₂R transcript. The use of an exogenous internal RNA standard enabled precise quantitation of tissue V₂R mRNA in the present study. A deletion mutant RNA transcript was made in these studies that contained the same nucleotide sequence as the wild-type mRNA, except for a small nucleotide deletion of 29 bp. As shown in Fig. 1, the initial RT-PCR product of 461 bp was produced using primers A and B, and subsequent endonuclease digestion with Dde I removed the 29 bp (Fig. 1, hatched box) from the wild-type V₂R sequence. The deletion mutant for the V₂R was clearly distinguishable from the wild-type (tissue) V₂R by restriction enzyme digestion, and final validation on the removal of the 29-bp Dde I fragment was achieved by PCR sequencing (data not shown).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Diagrammatic representation of deletion mutant construct, in which native 461-bp vasopressin V2 receptor (V2R) PCR product generated with primers A and B was digested with Dde I (as indicated by arrows). Hatched box was the deleted Dde I DNA fragment (29 bp). Subsequent RT-PCR assays utilized primers A and C for amplification of tissue and mutant V2R.

Validation of the efficiency and reproducibility of competitive RT-PCR assay. As shown in Fig. 2, it was first established that the amplification of the deletion mutant RNA transcript and the tissue (wild type) RNA were similar, a prerequisite for accurate quantitation. Figure 2A is a representative experiment (from 5 independent experiments) of an ethidium bromide-stained gel, in which 1 ng of inner medullary total RNA (lanes 2-6) and 160 zmol of deletion mutant transcript (lanes 7-11) were RT-PCR amplified for 30-35 cycles in one-cycle increments. Figure 2B is a graphical representation of the band intensity of Fig. 2A, which demonstrates that the slope of the line for tissue V₂R mRNA () was parallel to the V₂R mutant (). These experiments showed that the efficiencies of RT-PCR amplification for the mutant and the tissue products were equivalent, a critical requirement for the quantification of mRNA using RT-PCR.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   A: effect of PCR product formation of the wild-type (lanes 2-6) and deletion mutant (lanes 7-11), as a result of PCR cycle number from 30 to 35 cycles. B: densitometric analysis of PCR product band intensity in A. Five independent experiments were performed to determine the efficiencies of the wild-type and mutant V2R.

To determine both the range of total RNA that could be measured by the assay and the accuracy of the competitive RT-PCR assay, three different amounts of inner medullary total RNA (1, 5, and 25 ng) were used (Fig. 3). To determine the equivalence point (i.e., the point at which the number of mutant V₂R transcripts equals the number of tissue mRNA), seven different amounts of mutant RNA transcript were added to a constant amount of tissue total RNA (either 1, 5, or 25 ng). Figure 3A demonstrates an ethidium bromide-stained gel of a competitive RT-PCR assay, using 1 ng of inner medullary total RNA with competing amounts of mutant V₂R RNA ranging from 4 to 400 zmol (lanes 2-8). Negative control reactions were performed in each assay, which included the PCR amplification of tissue RNA (lane 9) and mutant RNA (lane 10) without RT and PCR amplification of sterile water (lane 11). Figure 3B demonstrates the densitometric analysis of the gel shown in Fig. 3A, the results of which were used to determine the amount of tissue mRNA at the equivalence point. The majority of the graphical analyses for the quantitative RT-PCR assay had r > 0.9, and any analysis where r < 0.8 was discarded.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   A: ethidium bromide-stained agarose gel (2%) of an RT-PCR assay, in which a constant amount of inner medullary total RNA (1 ng) was competed with 7 different amounts of mutant RNA (4-400 zmol, zmol = 1021 mol; lanes 2-8). Lane 1, molecular weight marker (100-bp ladder), and the negative controls are shown in lane 9 (PCR amplification of tissue RNA without RT), lane 10 (PCR amplification of mutant RNA without RT), and lane 11 (PCR amplification of sterile water). B: representation of A, in which bands have been densitometrically analyzed, which allowed for the calculation of the tissue content of the V2R mRNA (in zmol). C: standard curve for the quantitative RT-PCR derived from 3 different amounts (1, 5, and 25 ng) of inner medullary total RNA, and value from A is included in this curve. Five separate RT-PCR experiments were performed at each amount of inner medullary total RNA.

Five independent experiments were conducted using 1, 5, and 25 ng of inner medullary total RNA, and the equivalence points were determined as shown in Fig. 3C. The calculated tissue RNA contents of the V₂R were 486 ± 46.0 zmol, using 25 ng of total RNA; 112 ± 13.3 zmol, using 5 ng of total RNA; and 24.0 ± 3.9 zmol, using 1 ng of total RNA. This shows that, for from 25 to 5 ng of total RNA, there was a 4.4-fold difference and that, for from 5 to 1 ng, there was a 4.7-fold difference. The competitive RT-PCR developed for the V₂R is therefore quite proportional throughout the range from 1 to 25 ng of total RNA from the inner medulla and enables small changes in V₂R mRNA to be determined in minute nanogram amounts of tissue RNA.

Competitive RT-PCR analysis for the cortex and outer and inner medulla. As illustrated in Fig. 4, the steady-state levels of cortical V₂R mRNA averaged 8.37 ± 1.06 zmol/ng total RNA (means ± SE) in control rats (n = 3) with ad libitum water. After 24 h of water restriction, the steady-state levels decreased significantly to 3.49 ± 0.39 zmol/ng total RNA (P < 0.05), which remained decreased, averaging 4.12 ± 0.23 zmol/ng total RNA (P < 0.05) after 48 h of water restriction. In the outer medulla, the V₂R mRNA levels averaged 44.5 ± 7.57 zmol/ng total RNA, which decreased to 27.9 ± 3.51 zmol/ng total RNA after 24 h of water restriction. After 48 h of water restriction, the V₂R mRNA of the outer medulla decreased further to an average of 15.5 ± 3.54 zmol/ng total RNA (P < 0.05). In the inner medulla, the V₂R mRNA levels averaged 95.8 ± 4.75 zmol/ng total RNA during control ad libitum water conditions, a value significantly higher than that found in the cortex and outer medulla under the same conditions (P < 0.001). During the 24-h water restriction period, the steady-state levels of the V₂R mRNA decreased to 60.3 ± 15.2 zmol/ng total RNA and continued to fall significantly to 28.7 ± 3.19 zmol/ng total RNA (P < 0.05) after 48 h of water restriction.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Tissue content for the V2R mRNA (in zmol, 1021 mol/ng total RNA) were determined in the cortex and outer and inner medulla under 3 different conditions: ad libitum water, 24-, and 48-h water restriction. * P < 0.05, experimental groups were significantly different from their respective control groups.  P < 0.001, significant difference between different regions of the kidney compared with the renal cortex under ad libitum access to water.

Specificity of the V₂R antibody. We have previously demonstrated the binding specificity of the V₂R antibody, which was produced in our laboratory (25). In the present study, we used inner medullary plasma membrane-enriched protein (16,000 g pellet) to illustrate the specificity of the V₂R antibody. Figure 5 demonstrates that neither the primary V₂R antibody preadsorbed with the competing antigenic peptide (preadsorption) nor the preimmune serum produced an immunogenic signal for the V₂R protein. The predominant band was found at ~40 kDa, which is the expected size of the V₂R. There was also an occasional band seen at ~80 kDa, which is presumably a receptor dimer of the V₂R, which has been previously observed for the human V₂R by Sadeghi et al. (27).

View larger version (94K): [in this window] [in a new window]   Fig. 5.   Western blots of V2R, using plasma membrane-enriched fraction (16,000 g pellet) from the inner medulla of Sprague-Dawley rats (4 µg/lane). Specificity of the V2R antibody was demonstrated by preincubation of the V2R antibody with the competing antigenic peptide (Preabs) and with preimmune serum (Preimm, 1:1,500). Expected size of the V2R is 40.5 kDa.

Western blot analysis of the V₂R protein. Western blot analyses were performed on the plasma membrane-enriched fractions (16,000 g pellet) from the cortex (Fig. 6) and outer (Fig. 7) and inner medulla (Fig. 8). Figure 6A illustrates the changes of the V₂R in the plasma membrane-enriched fraction from the cortex of rats after 24 (top, n = 6) and 48 (bottom, n = 6) h of water restriction compared with control rats with ad libitum water (control, n = 6). Figure 6B summarizes the densitometric measurements of the 40.5-kDa band, which is the expected size of the V₂R. The V₂R decreased significantly after 24 h of water restriction (means ± SE, 54.3 ± 7.0% of control) and remained decreased through 48 h of water restriction (58.0 ± 12.7%).

View larger version (41K): [in this window] [in a new window]   Fig. 6.   A: Western blots of the V2R of the plasma membrane-enriched fraction (16,000 g pellet) from the renal cortex of Sprague-Dawley rats under ad libitum water and 24 (top membrane) and 48 (bottom membrane) of water restriction (n = 6 rats for each group). Each lane was loaded with 16 µg of protein. B: densitometry data. Ad libitum control rats are expressed at 100%, and the 24- and 48-h water restriction rats are expressed as a percentage of the ad libitum control rat values (* P < 0.05).

View larger version (43K): [in this window] [in a new window]   Fig. 7.   A: Western blots of the V2R of the plasma membrane-enriched fraction (16,000 g pellet) from renal outer medulla of Sprague-Dawley rats under ad libitum water and 24-h and 48-h water restriction (n = 6 rats for each group). Each lane was loaded with 16 µg of protein. B: densitometry data. Ad libitum control rats are expressed at 100%, and the 24- and 48-h water restriction rats are expressed as a percentage of the ad libitum control rat values (* P < 0.05).

View larger version (45K): [in this window] [in a new window]   Fig. 8.   A: Western blots of the V2R of the plasma membrane-enriched fraction (16,000 g pellet) from renal inner medulla cortex of Sprague-Dawley rats under ad libitum water and 24- and 48-h water restriction (n = 6 rats in each group). Each lane was loaded with 5 µg of protein. B: densitometry data. Ad libitum control rats are expressed at 100%, and the 24- and 48-h water restriction rats are expressed as a percentage of the ad libitum control rat values.

Interestingly, within the renal medulla, the results of our analysis suggest a differential regulation of the V₂R within the plasma membrane. In the outer medulla, the plasma membrane-enriched fraction, as shown in Fig. 7A and densitometrically analyzed in Fig. 7B, shows that there is no change from control after 24 h of water restriction (n = 6, 5.0 ± 2.9% of control), but, after 48 h of water restriction, there is a significant decrease of 62 ± 5.2%. Within the inner medulla (Fig. 8), there was no significant alteration of the V₂R protein at either 24 or 48 h of water restriction, in contrast to that observed in the cortex and outer medulla.

There was no difference in -actin in the control ad libitum water compared with the 24- or 48-h water restriction rats (data not shown), confirming that there was equal loading of the protein samples in the gel.

	DISCUSSION

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Detection of tissue mRNA has traditionally been performed with Northern blot analyses or RNase protection assays. However, one major drawback of these techniques is their lack of sensitivity, and so this necessitates the pooling of tissue samples. To circumvent this problem, quantitation of mRNA by RT-PCR has recently been developed (11, 29). The majority of mRNA measurements by RT-PCR have been made using semiquantitative methods, in which the mRNA of interest is compared with an internal housekeeping gene, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or -actin. However, because of the intrinsic variability of the RT-PCR assay, particularly between different mRNAs, precise quantification of the tissue mRNA requires the utilization of a known exogenous RNA transcript. The development of an exogenous RNA transcript for use as an internal standard enables one to fully control the potential sources of variability that can arise in the measurement of mRNA, particularly during the RT reaction (11, 29). This provides a powerful RT-PCR-based approach for quantifying tissue mRNA levels with exquisite sensitivity for the determination of mRNA levels within small, unpooled tissue samples. The mutant RNA transcript, which we developed, possessed the same nucleotide sequence as the wild-type mRNA, minus the small nucleotide deletion (29 bp in our case). Importantly, this mutant RNA transcript exhibited nearly identical amplification efficiency as the tissue mRNA (Fig. 2), which enabled this transcript to be used as a valid internal standard to quantify unknown amounts of wild-type V₂R mRNA and to monitor and control for variations in efficiencies of both the RT and PCR reactions.

Vasopressin receptor-mediated response to water restriction. The hydraulic conductivity of the medullary collecting duct epithelial cells has long been known to be dependent on V₂R stimulation by AVP. Recent studies in our laboratory have also found that optimization of urine concentration is dependent on AVP-mediated reductions in medullary vasa recta blood flow, which minimizes the "washout" of the interstitial solute gradient required for water reabsorption. Specifically, 48 h of water restriction resulted in an elevation in plasma AVP levels from 3 to 20.5 pg/ml and an associated 30% reduction in vasa recta blood flow to the inner medulla as measured by laser-Doppler flowmetry in unanesthetized Sprague-Dawley rats (10).

The V₂R has been largely localized to extravascular structures, in particular, the medullary thick ascending limbs of Henle and the collecting ducts (9, 24, 25, 32). The enhanced hydraulic conductivity and urea reabsorption stimulated by AVP results from V₂R-mediated insertion of AQP-2 water channels and activation of urea UT1 transporters in the apical membranes of the collecting duct epithelium. Furthermore, stimulation of the V₂R in the medullary thick ascending limbs of Henle activates the Na⁺-K⁺-2 Cl cotransporter (2, 30), which increases NaCl reabsorption. This increased activity enhances the generation of the medullary sodium osmotic gradient and, together with urea, determines the corticopapillary interstitial solute gradient necessary for urine concentration.

Evidence for AVP-mediated V₂R regulation. Although there is a wealth of functional information related to the various actions of AVP on distinct tubular and vascular structures in the kidney, the physiological mechanisms that regulate the expression of the vasopressin receptors, in particular, the V₂R, are not clearly understood. The goal of the present study was to characterize the regulation of these receptors at the level of mRNA and protein within the cortex and outer and inner medulla of the rat kidney during the first 2 days of water restriction. In vitro studies suggest that elevation in AVP would result in the downregulation of the V₂R (3, 4, 7, 14, 18, 26). These studies demonstrate, however, that receptor downregulation is a multifactorial phenomenon that can involve receptor occupancy, receptor-mediated endocytosis, decrease in the receptor mRNA and/or protein levels, phosphorylation of the receptor, alterations in the affinity of the receptor to the agonist, or an uncoupling of the hormone receptor to its second messenger system. Several in vitro experiments have shown that cells transfected with the V₂R mRNA could be desensitized when exposed to high concentrations of AVP (3, 14). Scatchard plot analysis indicated that AVP altered the V₂R from a high-affinity receptor to a lower-affinity receptor (3). Conflicting results at this time make it unclear whether the desensitization is related to receptor phosphorylation (14, 26).

Recent studies (9, 32) have attempted to understand the in vivo effects on V₂R of chronic stimulation, using RT-PCR on RNA obtained from microdissected thick ascending limbs of Henle and collecting ducts. Terada et al. (32) reported a decrease of V₂R mRNA in medullary collecting ducts during 72 h of water restriction. However, the changes in the mRNA were calculated using a semiquantitative method, in which the V₂R band intensity was compared with that of GAPDH. Firsov et al. (9), however, developed a quantitative RT-PCR assay and found that intramuscular injections of a highly selective V₂R agonist, DDAVP, administered daily for 3 days, led to a decrease in the steady-state levels of the V₂R mRNA in isolated renal tubules, which included the medullary thick ascending limbs and collecting ducts. This suggested that chronic stimulation of the V₂R was responsible for the decrease in the V₂R mRNA by influencing either the transcription of the gene or the stability of the mRNA. The signaling pathways for such changes remains to be established.

Regional time-dependent downregulation of the V₂R mRNA and protein. The results of the present study demonstrate a global decrease in the renal V₂R mRNA during 48 h of water restriction. Because of the functional importance of the V₂R during water restriction, it was important in our study to determine whether the V₂R protein decreased concomitantly. This indeed was the case, but the reduction in protein levels appeared to decrease after the changes in the V₂R mRNA, and interestingly, in the inner medulla, the proteins were not significantly altered, even after 2 days of water restriction. Regional changes in V₂R protein expression with water restriction have not previously been studied. Steiner and Phillips (31) studied tissue from whole rat kidney and showed that water restriction for 72 h led to a 38% decrease in the B_max of AVP but did not affect the receptor affinity to AVP as found under in vitro conditions (3, 14).

We can only speculate about the mechanisms responsible for the regional time-dependent differences between V₂R mRNA and protein expression found in our study. The steady-state levels of the mRNA were ~12-fold higher in the inner medulla, compared with the cortex in the euvolemic rats. The observed decrease of cortical V₂R protein levels observed after 24 h of water restriction was correlated with a significant decrease in the steady-state levels of the V₂R mRNA. However, at 48 h, reduced V₂R mRNA in the renal inner medulla occurred in the absence of reduced V₂R protein. This could be explained by the observation that even though the V₂R mRNA was reduced, the levels still remained higher than those observed in the euvolemic state in the renal cortex, so protein translation may have continued throughout the 48 h period of water restriction. Alternatively, it is possible that elevations of extracellular osmolality within the renal medulla could enhance the stability of the protein within the epithelial membranes. Several investigators have found that hypertonicity leads to an enhancement in the adenylyl cyclase activity of vasopressin-sensitive cells (1, 16, 20), and it has been observed that hyperosmolality can inhibit the movement of clathrin into the coated pits within the plasma membrane (5, 13) and thereby decrease the occurrence of receptor-mediated endocytosis. These or other yet unknown mechanisms could explain why the V₂R within the plasma membrane of the inner medulla was not altered during the 48 h of water restriction.

Physiological significance for differential V₂R downregulation. The differential downregulation of the V₂R within the distinct regions of the kidney (i.e., cortex and outer and inner medulla) could contribute importantly to the optimization of urine concentration and water conservation. The delayed reduction of the V₂R in the inner medulla would prolong the conservation of free water during intermittent periods of water restriction. Studies by Lankford et al. (17) indicate that the water permeability of isolated perfused inner medullary collecting ducts is actually enhanced in 24 h water-restricted rats independent of AVP. In contrast, urea transport remained AVP dependent, which was attributed to a distinct post-second messenger pathway (6, 15, 23). Together with the present data, this would suggest that the maintenance of the V₂R within the plasma membrane of the inner medulla provides optimization of urea transport through the collecting duct epithelium to enhance the osmotic drive for water reabsorption during water restriction.

In conclusion, this study has found that water restriction leads to a differential, time-dependent downregulation of the V₂R mRNA and protein in the rat kidney. The stability of the plasma membrane V₂R protein within the inner medulla may allow the mammalian organism to optimize urine concentration and ultimately minimize water loss during prolonged water restriction.