Small interfering RNA-mediated functional silencing of vasopressin V2 receptors in the mouse kidney
Ali Hassan1,2,
Ying Tian3,
Wei Zheng1,2,
Hong Ji1,2,
Kathryn Sandberg1,2 and
Joseph G. Verbalis1,3
1 Center for the Study of Sex Differences in Health, Aging and Disease, Georgetown University Medical Center, Washington, District of Columbia
2 Department of Medicine, Division of Nephrology and Hypertension, Georgetown University Medical Center, Washington, District of Columbia
3 Department of Medicine, Division of Endocrinology and Metabolism, Georgetown University Medical Center, Washington, District of Columbia
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ABSTRACT
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The antidiuretic effects of arginine vasopressin (AVP) on the kidney are mediated by V2 subtype AVP receptors (V2R). To investigate the role of regulation of V2R in water and sodium homeostasis, we have developed a method for small interfering RNA (siRNA)-mediated inhibition of V2R expression in vivo. Three 21-nt siRNA sequences were chosen that specifically targeted the mouse V2R but shared no appreciable sequence homology to any other known mouse genes, including the vasopressin V1a and V1b receptors. Additionally, an siRNA sequence that shared no significant matches to any known mammalian gene sequences was chosen for use as a control. Chemically synthesized siRNA was complexed with the liposomal transfection reagent DOTAP. Each mouse (male C57BL/6) received 3.6 nmol (
50 µg) of either the control (nonsilencing) or one of the V2R-targeting siRNAs via intravenous injection. Forty-eight hours after injection membranes were prepared from the inner medulla of the kidneys, and V2R expression was measured by a radioligand binding assay and Western immunoblotting. Treatment with one of the V2R-targeting siRNAs (R2) caused a 39.7 ± 8.7% reduction in V2R-specific binding compared with the control (n = 11, P < 0.05) and a 37.0 ± 2.3% reduction in V2R protein expression as measured by Western immunoblotting (n = 4, P < 0.001). Additionally, real-time PCR revealed that R2 siRNA treatment induced a 68.8 ± 2.2% reduction in V2R mRNA. However, this siRNA treatment did not alter the animals basal urine concentrating capacity under unstimulated conditions. In subsequent experiments, treatment with R2 siRNA was found to significantly attenuate the antidiuretic effects the V2R-specific AVP agonist 1-desamino-[8-D-arginine]vasopressin (dDAVP). Mice were infused with dDAVP (0.25 ng/h) for 3 days to produce maximal antidiuresis and then were injected with either the R2 siRNA or the nonsilencing control. On day 2 after treatment, urine osmolality was significantly decreased from 3,455 ± 72 in control animals (n = 12) to 3,155 ± 129 mosmol/kgH2O in R2 siRNA-treated animals (n = 12) (P < 0.05); similarly, on day 2 24-h urine volume was significantly increased from 0.86 ± 0.07 ml/day to 1.11 ± 0.06 ml/day in R2 siRNA-treated animals (P < 0.05). In summary we have demonstrated that RNA interference methodology can be used successfully in vivo to significantly reduce functional expression of the V2R in the mouse kidney.
inner medulla; in vivo gene silencing; G protein-coupled receptor
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INTRODUCTION
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ARGININE VASOPRESSIN (AVP) is the major physiological regulator of renal water excretion. AVP acts in the kidney to increase the water permeability of the renal distal tubules and collecting ducts, thereby accelerating water reabsorption (40). The antidiuretic effects of vasopressin are mediated by vasopressin V2 receptors (V2Rs), adenylate cyclase-coupled members of the G protein-coupled receptor family that are present in the renal distal tubules and collecting ducts (3, 44). Vasopressin regulates the water permeability of renal collecting tubule in two ways. Short-term regulation is achieved by shuttling of aquaporin-2 (AQP2) water channels from intracellular vesicles into the apical plasma membrane (24). Long-term regulation occurs through increasing the abundance of AQP2 protein (19). The vasopressin V2R is structurally, genetically and pharmacologically distinct from the two other vasopressin receptors, the V1a receptor, expressed predominantly in the liver and vasculature (22), and the V1b receptor, expressed almost exclusively in the pituitary (5, 17, 37).
In some pathophysiological circumstances, the renal ability to concentrate urine in response to AVP is decreased. Often, this decrease in renal concentrating ability is associated with a concomitant reduction in vasopressin V2R expression. For example, the effects of 1-desamino-[8-D-arginine]vasopressin (dDAVP)-induced antidiuresis are mitigated by the "renal escape" phenomenon (7, 41). In the rat, the increased urine volume and decreased urine osmolality that indicate the beginning of the initiation of the escape process coincide with downregulation of kidney vasopressin V2R binding (39). Similarly, rats with chronic renal failure exhibit a marked decrease in vasopressin V2R density and the virtual absence of V2R mRNA without changes in other G protein-coupled receptors, suggesting that vasopressin resistance during chronic renal failure is also due, at least in part, to vasopressin V2R downregulation (38). Furthermore, lipopolysaccharide-induced endotoxemia results in decreases in both V2R mRNA and density in the kidney inner medulla, and this downregulation is also associated with decreased renal capacity to concentrate urine (11). Since renal AQP2 expression and distribution are mainly regulated by vasopressin V2R-mediated increases in cAMP, this temporal association between decreases in urine concentrating capacity and kidney vasopressin V2R expression suggests that alterations in V2R levels play an important role in regulating renal function; however, correlative studies cannot prove a causal relationship by virtue of this temporal association. Indeed, in some instances V2R expression is reduced without any apparent defect in urine-concentrating capacity. For example, dDAVP-treated rats are able to maintain maximal AQP2 expression and urine osmolalities without manifesting escape even though vasopressin V2R binding sites are significantly reduced (7, 39). Similarly, following 72 h water deprivation, the vasopressin V2R density (Bmax) in rat renal tubular epithelial basolateral cells is reduced by 38% without affecting the affinity (Kd) of the receptor (36). This is paradoxical, since V2R expression is reduced in a circumstance in which increased water reabsorption would be clearly advantageous. One potential explanation for these phenomena is that there is a relatively large population of "spare" vasopressin V2Rs (i.e., a receptor reserve) in the kidney and, as a result, V2R expression must be reduced below a certain threshold before there is any reduction in second messenger generation and thus urine concentrating capacity (36, 39). To further investigate the degree to which changes in vasopressin V2R expression contribute to alterations in renal function, we developed a method for reducing V2R expression in vivo.
RNA interference (RNAi) is a mechanism whereby small double-stranded RNA molecules interact with mRNA containing homologous sequences in a highly sequence-specific manner. This process is directed by endogenously expressed protein components known as an RNA-induced silencing complex (RISC), the actions of which ultimately result in the degradation of the mRNA (9). Because interaction of the siRNA with its target is highly sequence specific, typically only the target mRNA is affected and off-target effects are minimal (10). Although RNAi has been observed in a wide range of eukaryotes (31, 43), silencing of mammalian genes has only recently been demonstrated using this technique (4, 8). In mammalian cells, gene silencing can be induced by transfection of cells with 21-nt RNA duplexes or short interfering RNA (siRNA). Recently, a small number of studies have demonstrated that RNAi techniques can also be used to knock down gene expression in vivo (20) following transfection of siRNA via both cationic liposomal transfection (32, 33, 35) and hydrodynamic transfection of siRNA (16). Several studies have demonstrated in vivo gene silencing of transgenically expressed reporter genes [e.g., green fluorescent protein (GFP) or luciferase] (15, 16, 20), but to date no study has shown that siRNA silencing of G protein-coupled receptor expression in a mammalian system has functionally significant physiological effects.
In vivo silencing of the vasopressin V2R has posed a substantial challenge. First, the vasopressin V2R is expressed predominantly in the inner medulla of the kidney (44). Thus it was unclear whether siRNA could be delivered to this tissue with sufficiently high efficiency to achieve a significant reduction in V2R expression. Second, even if it were possible to reduce vasopressin V2R expression in vivo, it was unclear whether this reduction in expression would be of sufficient magnitude to result in a physiological effect. Therefore, the aim of this study was to determine whether siRNA-mediated RNA interference could be employed successfully in vivo to reduce vasopressin V2R expression in the mouse kidney, and if so, to determine whether this methodology could be used to investigate the role of V2R downregulation in regulating renal function.
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MATERIALS AND METHODS
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Small interfering RNA (siRNA) constructs.
Three different siRNA duplexes (R1, R2, and R3) targeting the mouse vasopressin V2R were selected using standard siRNA design rules (9). BLAST searching showed that these siRNA constructs did not match the V1a or V1b vasopressin receptor subtypes, nor did they have any significant homology to any other known mouse gene. Additionally, an siRNA construct that matched no known mammalian genes was selected for use as a nonsilencing control. The sequences of these four siRNA constructs are shown in Fig. 1. All siRNA duplexes were chemically synthesized by Qiagen (Carlsbad, CA; HPP grade).

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Fig. 1. Sequences of short interfering RNA (siRNA) constructs used in this study. In addition to a control (i.e., nonsilencing) siRNA construct, three different siRNA duplexes were designed that targeted the coding region of the mouse V2 vasopressin receptor (V2R) mRNA (GenBank accession no. NM_019404). These siRNA constructs targeted the following nucleotides within the vasopressin V2R mRNA sequence: R1, 260280; R2, 680700; R3, 12021222.
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siRNA transfections.
Mice (adult male C57BL/6, 1724 g, 59 wk; Taconic, Germantown, NY) were transfected with siRNA using a procedure similar to that described by Sorensen et al. (35). Mice were anesthetized by intraperitoneal injection of pentobarbital (60 mg/kg, Nembutal; Fisher Scientific, Pittsburgh, PA). Once anesthetized, the animals were volume loaded by intraperitoneal injection of 1.5 ml of isotonic saline to ensure adequate renal perfusion 30 min prior to siRNA infusion. No behavioral changes were associated with this intraperitoneal saline injection. Then, 3.6 nmol (equivalent to
50 µg) of siRNA was complexed with the liposomal transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP; Roche Diagnostics, Indianapolis, IN) in HEPES-buffered saline (pH 7.4). The final volume of the transfection mixture was 198 µl, and the siRNA/DOTAP charge ratio was 1:1. The siRNA-DOTAP solution was injected through a catheter (PE-10 gauge; Becton-Dickinson, Franklin Lakes, NJ) inserted into the jugular vein. Following infusion of the siRNA mixture over a period of
1 min, the jugular was sutured and the animals were allowed to recover. Animals were monitored continuously post injection. All animals were awake and walking 45 min after siRNA injection. No behavioral changes were apparent. Mortality was low (94% survival). All experiments were carried out under the supervision of and in accordance with the regulations of the Georgetown University Animal Care and Use Committee.
Osmotic minipump infusion of dDAVP.
Under light methoxyflurane anesthesia, mice were subcutaneously implanted with osmotic minipumps (model 1002; Alzet, Palo Alto, CA), which delivered 0.25 ng dDAVP (Ferring Pharmaceuticals, Suffern, NY) per hour. Pumps were implanted at midday 72 h prior to siRNA injection.
Preparation of kidney inner medullary tissue.
Forty-eight hours after siRNA infusion animals were killed by decapitation, the kidneys were quickly removed and rinsed in ice-cold PBS and were then sliced along the corticomedullary axis to separate the medulla from the cortex. The inner medullary region of the kidneys was dissected. The remaining portion of the inner medulla was homogenized in ice-cold buffer A (50 mM Tris·HCl, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 1.0 µg/ml leupeptin, 10 µg/ml aprotinin, and 0.04 U/ml antipain; pH 7.4) using a StedFast SL1200 homogenizer (Fisher Scientific) fitted with a Teflon pestle. The resulting homogenate was centrifuged at 25 g for 10 min at 4°C. The pellet was discarded, and the supernatant was recentrifuged in ice-cold buffer A at 15,000 g for 30 min at 4°C. The membrane preparations were gently vortexed and resuspended in ice-cold buffer A to a final concentration of 0.51.0 mg/ml. Membrane protein concentrations were determined by protein assay using BSA as the standard (Bio-Rad Laboratories, Richmond, CA).
Vasopressin V2R radioligand binding assay.
V2R binding was determined in inner medulla membrane preparations as previously described by Tian et al. (39) using the V2R antagonist, d(CH2)5 [D-Ile2,Ile4,Tyr-NH29]AVP, which has a tyrosine iodination site at the carboxyl terminal distant from the ligand receptor binding site (courtesy of Dr. Maurice Manning, Medical College of Ohio, Toledo, OH). This ligand was iodinated using a chloramine T procedure (14), and the monoiodinated V2R radioligand was purified by reverse-phase HPLC on a C18 column (Peptide Radioiodination Service, University of Mississippi).
Vasopressin V2R immunoblot.
V2R protein expression was determined by immunoblotting and chemiluminescent protein detection as previously described (6). In brief, inner medullary membrane preparations (7 µg) were mixed with 5x Laemmli sample buffer (4 vol of sample to 1 vol of Laemmli buffer) and heated to 60°C for 15 min to solubilize proteins. The proteins were then separated by SDS-PAGE using Bio-Rad precast 12.5% gels (Bio-Rad Laboratories, Hercules, CA). Following electrophoresis, proteins were transferred to nitrocellulose membranes at 0.6 A for 60 min in transfer buffer (50 mM NaPO4, 150 mM NaCl, 0.01% Tween-20). The vasopressin V2R antibody used is a rabbit anti-vasopressin V2R affinity-purified polyclonal antibody (AVPR-V2; Chemicon International, Temecula, CA), use of which has previously been described (25). The nitrocellulose membranes were incubated with the antibody (2.5 µg/ml) at 4°C overnight. Next, the membrane was incubated with the secondary antibody (1:10,000; Pierce Biotechnology, Rockford, IL) and chemiluminescence was carried out using LumiGlo reagents according to the manufacturers protocol (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Real-time PCR of vasopressin V2R mRNA.
Total RNA was extracted using the RNaqueous-4PCR silica-fiber binding RNA extraction kit (Ambion, Austin, TX). First-strand cDNA was prepared from total RNA (500 ng) using the iScript cDNA synthesis kit (Bio-Rad) with MMLV RNase H+ reverse transcriptase, oligo(dT), and random hexamers. Quantitation of V2R mRNA and 18S ribosomal RNA (for control) was performed by real-time PCR using an ABI Prism 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). The PCR reaction mixture for vasopressin V2R detection consisted of RNase-free water, TaqMan Universal PCR Master Mix (Applied Biosystems), and 300 nM specific primers and 10 µM probe [forward primer (663F) 5'-AGC TCT TCA TCT TTG CTC AAC GT-3'; reverse primer (755R) 5'-GTT CCG CTA GGT CCA CTG TAT-3'; probe (725T) 6FAM-CGA TTT GCA GAG CCA T-TAMRA], and cDNA samples. PCR reactions without reverse transcription were included to control for contamination by genomic DNA. Results obtained for vasopressin V2R mRNA levels were normalized to 18S ribosomal RNA.
Measurement of urine volume and osmolality.
Animals were housed in metabolic cages, and their urine was collected and stored at 20°C. Animals were acclimated to the cages for 3 days prior to siRNA infusion. Evaporation was prevented by adding 0.5 ml mineral oil to the collection tubes. Urine samples were collected daily at midday. Urine osmolality was measured using a vapor-pressure osmometer (model 5520; Wescor, Logan, UT).
Statistical analysis.
All results are expressed as means ± SE. Differences between treatment groups were analyzed statistically using Students t-test, or one-way ANOVA followed by Dunnetts test when more than two groups were compared. All analyses were performed using the statistical analysis software package, Prism 4.0 (GraphPad Software, San Diego, CA).
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RESULTS
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Effect of siRNA treatment on vasopressin V2R expression.
The relative efficacy of the three distinct siRNA duplexes were compared. As described above, mice were injected with 3.6 nmol (
50 µg) of each siRNA (Fig. 1), and 48 h later, V2R expression was assessed by radioligand binding assay (Fig. 2). The R2 siRNA duplex induced a marked reduction in V2R-specific binding in inner medullary membranes [39.7 ± 8.7% compared with the control siRNA, n = 11, P < 0.05 (Dunnetts test)]. Therefore, we selected the R2 siRNA duplex for use in all further experiments. Note that R3 siRNA appeared to be of approximately equivalent potency to R2; however, due to animal variability, the R3 siRNA effect did not reach statistical significance. The effect of R1 also did not reach statistical significance and showed a smaller trend toward reduced V2R expression compared with either R2 or R3.

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Fig. 2. Effect of V2R-targeting siRNA treatment on monoiodinated V2 radioligand (125I-V2RA) binding in the mouse inner medulla. Binding assays were carried out as described in MATERIALS AND METHODS. Bars are means ± SE of V2R-specific binding expressed as a percentage of the specific binding in mice treated with the control siRNA on the same day. The n values for each treatment were: control, n = 11; R1, n = 6; R2, n = 11; R3, n = 3. *P < 0.05.
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In addition to measuring the effect of R2 siRNA treatment on V2R binding, we also determined the effect of R2 on V2R protein expression by Western analysis. V2R protein expression was reduced by 37.0 ± 2.3% (n = 4, P < 0.001, t-test) following treatment with R2 siRNA compared with control siRNA treatment (Fig. 3, A and B).

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Fig. 3. Western Blot analysis of vasopressin V2R expression in the mouse inner medulla following R2 siRNA treatment. A: V2R immunoblot showing V2R immunoreactive bands following either control siRNA or R2 siRNA treatment. B: semiquantitative analysis of V2R bands. Data are means ± SE. ***P < 0.001.
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Effect of siRNA treatment on vasopressin V2R mRNA. Under the same treatment conditions described above, R2 siRNA treatment induced a 68.8 ± 1.3% (n = 3, P < 0.05, t-test) reduction in V2R mRNA, as determined by quantitative real-time PCR analysis (Fig. 4).

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Fig. 4. Effect of siRNA treatment on vasopressin V2R expression in the mouse inner medulla. V2R mRNA was quantitated by real-time PCR analysis as described in MATERIALS AND METHODS. Data are means ± SE. *P < 0.05.
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Effect of siRNA treatment on urine volume and osmolality under unstimulated conditions.
Under basal conditions we observed no significant difference in the animals urine-concentrating capacity following R2 siRNA treatment. Forty-eight hours after siRNA infusion both urine osmolality [control, 2,241 ± 238 mosmol/kgH2O (n = 4) vs. R2, 2,436 ± 117 mosmol/kgH2O (n = 4), not significant] and urine volume [control, 1.38 ± 0.09 ml/day (n = 4) vs. R2, 1.19 ± 0.24 ml/day (n = 4), not significant].
Effect of siRNA treatment on urine volume and osmolality during dDAVP treatment.
Because we observed no alteration in urine concentrating capacity under basal conditions, we investigated whether siRNA-mediated silencing of the V2R could alter the animals urine-concentrating capacity under circumstances in which the V2R system was maximally stimulated. To induce maximal antidiuresis, animals were treated with the V2R-specific vasopressin agonist, dDAVP, via a continuous osmotic minipump infusion at a rate of 0.25 ng/h for the entire duration of the experiment. Three days after implantation of the dDAVP minipumps, each animal was intravenously injected with either control or R2 siRNA as described above. Twenty-four-hour urines were collected throughout the experiment.
Urine osmolality was significantly reduced following R2 siRNA treatment compared with control siRNA (Fig. 5A). Urine osmolality was significantly decreased on both day 2 [3,155 ± 129 (n = 12) vs. 3,455 ± 72 mosmol/kgH2O (n = 12), P < 0.05, t-test] and day 3 [2,861 ± 150 (n = 12) vs. 3,373 ± 128 mosmol/kgH2O (n = 12), P < 0.05, t-test] following siRNA treatment. On day 2 following infusion, urine volume was significantly increased from 0.86 ± 0.07 ml/day (n = 12) in control animals to 1.11 ± 0.06 ml/day (n = 12) in R2 siRNA-treated animals (P < 0.05, t-test) (Fig. 5B). Following this initial increase in urine volume after R2 siRNA infusion, urine volumes in the R2 siRNA-treated animals were not significantly different from the controls.

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Fig. 5. A: R2 siRNA treatment reduces urine osmolality during dDAVP-induced antidiuresis. Animals were treated with 0.25 ng/h dDAVP throughout the experiment by osmotic minipump infusion as described in MATERIALS AND METHODS. Three days after the commencement of the dDAVP infusion, animals were injected with either control or R2 siRNA. Urine osmolality was significantly reduced on days 2 and 3 following R2 siRNA injection, compared with the control. Data are means ± SE. *P < 0.05. B: time course of changes in urine volume following R2 siRNA treatment during dDAVP-induced antidiuresis. Data are means ± SE. *P < 0.05. **P < 0.01.
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DISCUSSION
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These data demonstrate that vasopressin V2R expression in the inner medulla of the kidney can be reduced in vivo via siRNA-mediated RNA interference in the mouse. Furthermore, the reduction in V2R expression induced by this siRNA treatment was of significant magnitude to reduce the animals functional ability to respond to vasopressin stimulation, as evidenced by significantly reduced urine osmolality and increased urine volume during continuous dDAVP infusion. The R2 siRNA duplex potently reduced V2R-specific binding by
40% at 48 h after transfection. Since Scatchard analysis was not performed, because of limitations in murine inner medulla membrane quantity, the reduction in specific binding could reflect a reduction in ligand affinity (Kd) or a reduction in receptor number (Bmax). (Note that to complete one radioligand saturation isotherm for Scatchard analysis, at least 34 mice would be required to generate sufficient tissue.) However, the reduction in specific binding most likely reflects a reduction in Bmax, since the magnitude of the reduction in binding closely correlated with the magnitude of the reduction in protein expression by Western immunoblotting. Furthermore, siRNA silences genes by reducing mRNA expression and thus would not be expected to affect ligand affinities.
The siRNA delivery method used in this study was based on the intravenous siRNA transfection protocol previously devised by Sorensen et al. (35). Each animal was injected with 3.6 nmol of siRNA complexed with the cationic liposomal transfection reagent, DOTAP, to improve the ability of the siRNA to cross the cell membrane. This siRNA dose has previously been demonstrated to be effective in inducing siRNA-mediated RNAi of transgenically expressed luciferase (16) and GFP (35) in the mouse kidney via intravenous delivery (16, 35). One study reported siRNA knockdown of GFP expression in the kidney of transgenic GFP mice, demonstrating that the mouse kidney contains the intracellular machinery necessary for RNA interference (16). It should be noted, however, that in that study, GFP knockdown was measured in the whole kidney rather than just the inner medulla. Most V2Rs are expressed in the inner medulla, and this tissue has considerably more variable blood flow than the cortex (23). To counter this potential problem, we volume-loaded the animals with an intraperitoneal injection of isotonic saline to increase blood flow to the renal medulla. We chose to measure the effect of siRNA treatment after 48 h, since the half-life of the vasopressin receptor, similar to most G protein-coupled receptors, is relatively long; half-lives of 46 h have been reported for the V2R when expressed in eukaryotic transient transfection systems (18, 29).
Although evidence from in vitro and cell culture systems suggests that any mRNA can be silenced using RNA interference techniques (9), a large degree of variability in the efficacy of siRNA duplexes has been observed (27, 30). The reasons for the lack of efficacy of some siRNA sequences is only partially understood (27). Possible explanations include the sequence of the target within the mRNA (13), the secondary structure of the siRNA duplex (30), and the binding of interfering proteins to the mRNA (9). Although we were able to use established siRNA design guidelines to select sequences likely to be effective in order to maximize the probability of identifying a highly potent siRNA sequence, we designed three separate siRNA duplexes that targeted the vasopressin V2R at different positions within the coding region of the V2R mRNA sequence. We then screened these three siRNA sequences for their efficacy at reducing V2R binding in the mouse kidney inner medulla. A nonsilencing siRNA duplex was selected for use as a control since recent evidence has clearly demonstrated that mock transfection is not an appropriate control in siRNA experiments (1, 34). Although all three siRNAs showed a trend toward reducing V2R-specific binding, only the effect of R2 reached statistical significance. Although the R3 siRNA duplex appeared to be equipotent to R2, the effects of R3 did not reach statistical significance. As described above, we can only speculate as to the mechanistic reasons for the apparent differences in potency of the three siRNA sequences.
The level of reduction of vasopressin V2R mRNA (68%) that we observed following R2 siRNA treatment suggests that delivery of the siRNA to the mouse inner medulla was highly efficient. It is likely that delivery of the siRNA to other tissues using this technique was also efficient; however, because expression of the vasopressin V2R is confined almost exclusively to the kidney, it is difficult to assess delivery to other tissues.
Although we were able to markedly reduce expression of the vasopressin V2R protein by 40% in the inner medulla of mice using the R2 siRNA construct, it is interesting that the basal urine-concentrating capacity of the mice was not altered under these conditions: 48-h treatment with R2 siRNA did not significantly alter either urine osmolality or volume. This result was not surprising, since, as described above, evidence suggests that there is a large reserve of vasopressin V2Rs in the rodent kidney. Therefore, under these basal, unstimulated conditions, sufficient V2Rs likely remained to mediate the level of signal transduction required to maintain adequate AQP2 membrane insertion and normal urine concentration capacity.
We hypothesized that a functional effect of silencing of the vasopressin V2R would be most easily observed under conditions of maximally stimulated antidiuresis. Therefore, we treated the mice with the vasopressin V2R-specific agonist, dDAVP, and then infused either the control or the R2 siRNA. Under these conditions, we observed a transient but significant decrease in urine osmolality and increase in urine volume in R2 siRNA-treated animals. Urine osmolality was significantly reduced on days 2 and 3 and urine volume was significantly increased on day 2 following R2 siRNA infusion. The transient nature of these changes in urine volume and osmolality are not entirely surprising,, given the nature of siRNA molecules. While the double-stranded structure of siRNA may provide some protection against degradation by RNases, siRNA molecules are rapidly degraded in plasma (2) and somewhat more slowly in cells (12). Therefore, any knockdown in receptor expression induced by siRNA-mediated RNAi is bound to be transient, with levels of the V2R mRNA returning to normal as the receptor-targeting siRNA is degraded. Subsequently, V2R protein expression would be expected to return to control levels as new receptors are translated from newly synthesized mRNA. Longer-term reductions in vasopressin V2R expression might be achieved through sequential injections of the siRNA construct or alternatively via transfection with vectors expressing the R2 sequence as part of a short-hairpin RNA (shRNA; Ref. 26). A number of shRNA vector expression systems have been demonstrated to be effective in vivo including plasmids (21), adenoviruses (42), and lentiviruses (28). These vector expression systems are particularly attractive because the shRNA duplex is continuously expressed in situ, and thus even relatively poor delivery of the vector to the target tissue could potentially result in long-term silencing.
In addition to the transient nature of the functional changes in urine osmolality and volume, the magnitude of the changes produced were not large. This might indicate that the induced decreases in V2R expression were just at the threshold for impairing V2R-mediated signal transduction, but further studies using alternative dosing and/or transfection strategies to produce larger graded knockdowns of the V2R will be required to ascertain this. Nonetheless, these studies constitute a clear "proof of concept" that siRNA methodology is capable of addressing important questions regarding the relation between G protein-coupled receptor expression and ligand-stimulated signal transduction in genetically intact in vivo systems.
In summary, the data presented demonstrate that siRNA-mediated RNA interference can be used to significantly reduce kidney vasopressin V2R expression in vivo and that this reduction in receptor expression is of sufficient magnitude to reduce the ability of the mice to sustain maximal antidiuresis during vasopressin stimulation. The data provide evidence that alterations in the level of genetically normal vasopressin V2Rs in the kidney can modulate maximal urine concentrating capacity. This represents, to our knowledge, the first demonstration of the use of siRNA silencing of a G protein-coupled receptor in a mammalian system to produce functionally significant physiological effects.
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ACKNOWLEDGMENTS
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This work was supported by National Institutes of Health (NIH) Grant DK-38094 and a National Kidney Foundation (National Capital Area) grant to J. G. Verbalis and NIH Grant HL-57502 and a grant from the Center for Biological Modulators, Korea Research Institute of Chemical Technology (South Korea), to K. Sandberg. A. Hassan is the recipient of an American Heart Association Jocelyn Beard Moran postdoctoral fellowship.
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FOOTNOTES
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
Address for reprint requests and other correspondence: A. Hassan, Georgetown Univ. Medical Center, 4000 Reservoir Rd., Rm. 377, Bldg. D, Washington, DC 20057 (e-mail: amh56{at}georgetown.edu).
doi:10.1152/physiolgenomics.00147.2004.
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REFERENCES
|
---|
- Anonymous. Whither RNAi? Nat Cell Biol 5: 489490, 2003.[CrossRef][ISI][Medline]
- Bertrand JR, Pottier M, Vekris A, Opolon P, Maksimenko A, and Malvy C. Comparison of antisense oligonucleotides and siRNAs in cell culture and in vivo. Biochem Biophys Res Commun 296: 10001004, 2002.[CrossRef][ISI][Medline]
- Birnbaumer M. Vasopressin receptors. Trends Endocrinol Metab 11: 406410, 2000.[CrossRef][ISI][Medline]
- Caplen NJ, Parrish S, Imani F, Fire A, and Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 98: 97429747, 2001.[Abstract/Free Full Text]
- DeKeyzer N, Engelborghs Y, and Volckaert G. Cloning, expression and purification of a sarcoplasmic calcium-binding protein from the sandworm Nereis diversicolor via a fusion product with chloramphenicol acetyltransferase. Protein Eng 7: 125130, 1994.[ISI][Medline]
- Ecelbarger CA, Chou CL, Lee AJ, DiGiovanni SR, Verbalis JG, and Knepper MA. Escape from vasopressin-induced antidiuresis: role of vasopressin resistance of the collecting duct. Am J Physiol Renal Physiol 274: F1161F1166, 1998.[Abstract/Free Full Text]
- Ecelbarger CA, Nielsen S, Olson BR, Murase T, Baker EA, Knepper MA, and Verbalis JG. Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat. J Clin Invest 99: 18521863, 1997.[Abstract/Free Full Text]
- Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, and Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411: 494498, 2001.[CrossRef][ISI][Medline]
- Elbashir SM, Harborth J, Weber K, and Tuschl T. Analysis of gene function in somatic mammalian cells using small interfering RNAs. Methods 26: 199213, 2002.[CrossRef][ISI][Medline]
- Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, and Tuschl T. Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J 20: 68776888, 2001.[Abstract/Free Full Text]
- Grinevich V, Knepper MA, Verbalis J, Reyes I, and Aguilera G. Acute endotoxemia in rats induces down-regulation of V2 vasopressin receptors and aquaporin-2 content in the kidney medulla. Kidney Int 65: 5462, 2004.[CrossRef][ISI][Medline]
- Holen T, Amarzguioui M, Babaie E, and Prydz H. Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway. Nucleic Acids Res 31: 24012407, 2003.[Abstract/Free Full Text]
- Holen T, Amarzguioui M, Wiiger MT, Babaie E, and Prydz H. Positional effects of short interfering RNAs targeting the human coagulation trigger tissue factor. Nucleic Acids Res 30: 17571766, 2002.[Abstract/Free Full Text]
- Hunter WM and Greenwood FC. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature 194: 495496, 1962.[ISI][Medline]
- Kobayashi N, Matsui Y, Kawase A, Hirata K, Miyagishi M, Taira K, Nishikawa M, and Takakura Y. Vector-based in vivo RNA interference: dose- and time-dependent suppression of transgene expression. J Pharmacol Exp Ther 308: 688693, 2004.[Abstract/Free Full Text]
- Lewis DL, Hagstrom JE, Loomis AG, Wolff JA, and Herweijer H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet 32: 107108, 2002.[CrossRef][ISI][Medline]
- Lolait SJ, OCarroll AM, Mahan LC, Felder CC, Button DC, Young W, III, Mezey E, and Brownstein MJ. Extrapituitary expression of the rat V1b vasopressin receptor gene. Proc Natl Acad Sci USA 92: 67836787, 1995.[Abstract/Free Full Text]
- Martin NP, Lefkowitz RJ, and Shenoy SK. Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination. J Biol Chem 278: 4595445959, 2003.[Abstract/Free Full Text]
- Matsumura Y, Uchida S, Rai T, Sasaki S, and Marumo F. Transcriptional regulation of aquaporin-2 water channel gene by cAMP. J Am Soc Nephrol 8: 861867, 1997.[Abstract/Free Full Text]
- McCaffrey AP, Meuse L, Pham TT, Conklin DS, Hannon GJ, and Kay MA. RNA interference in adult mice. Nature 418: 3839, 2002.[CrossRef][ISI][Medline]
- McCaffrey AP, Nakai H, Pandey K, Huang Z, Salazar FH, Xu H, Wieland SF, Marion PL, and Kay MA. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol 21: 639644, 2003.[CrossRef][ISI][Medline]
- Morel A, OCarroll AM, Brownstein MJ, and Lolait SJ. Molecular cloning and expression of a rat V1a arginine vasopressin receptor. Nature 356: 523526, 1992.[CrossRef][ISI][Medline]
- Nakanishi K, Mattson DL, Gross V, Roman RJ, and Cowley AW Jr. Control of renal medullary blood flow by vasopressin V1 and V2 receptors. Am J Physiol Regul Integr Comp Physiol 269: R193R200, 1995.[Abstract/Free Full Text]
- Nielsen S, Chou CL, Marples D, Christensen EI, Kishore BK, and Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane. Proc Natl Acad Sci USA 92: 10131017, 1995.[Abstract/Free Full Text]
- Nonoguchi H, Owada A, Kobayashi N, Takayama M, Terada Y, Koike J, Ujiie K, Marumo F, Sakai T, and 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: 17681778, 1995.[ISI][Medline]
- Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 16: 948958, 2002.[Abstract/Free Full Text]
- Reynolds A, Leake D, Boese Q, Scaringe S, Marshall WS, and Khvorova A. Rational siRNA design for RNA interference. Nat Biotechnol 22: 326330, 2004.[CrossRef][ISI][Medline]
- Rubinson DA, Dillon CP, Kwiatkowski AV, Sievers C, Yang L, Kopinja J, Rooney DL, Ihrig MM, McManus MT, Gertler FB, Scott ML, and Van Parijs L. A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat Genet 33: 401406, 2003.[CrossRef][ISI][Medline]
- Sadeghi HM, Innamorati G, and Birnbaumer M. Maturation of receptor proteins in eukaryotic expression systems. J Recept Signal Transduct Res 17: 433445, 1997.[ISI][Medline]
- Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, and Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115: 199208, 2003.[CrossRef][ISI][Medline]
- Sharp PA. RNA interference2001. Genes Dev 15: 485490, 2001.[Free Full Text]
- Sioud M and Sorensen DR. Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun 312: 12201225, 2003.[CrossRef][ISI][Medline]
- Sioud M and Sorensen DR. Systemic delivery of synthetic siRNAs. Methods Mol Biol 252: 515522, 2004.[Medline]
- Sledz CA, Holko M, de Veer MJ, Silverman RH, and Williams BR. Activation of the interferon system by short-interfering RNAs. Nat Cell Biol 5: 834839, 2003.[CrossRef][ISI][Medline]
- Sorensen DR, Leirdal M, and Sioud M. Gene silencing by systemic delivery of synthetic siRNAs in adult mice. J Mol Biol 327: 761766, 2003.[CrossRef][ISI][Medline]
- Steiner M and Phillips MI. Renal tubular vasopressin receptors downregulated by dehydration. Am J Physiol Cell Physiol 254: C404C410, 1988.[Abstract/Free Full Text]
- Sugimoto T, Saito M, Mochizuki S, Watanabe Y, Hashimoto S, and Kawashima H. Molecular cloning and functional expression of a cDNA encoding the human V1b vasopressin receptor. J Biol Chem 269: 2708827092, 1994.[Abstract/Free Full Text]
- Teitelbaum I and McGuinness S. Vasopressin resistance in chronic renal failure. Evidence for the role of decreased V2 receptor mRNA. J Clin Invest 96: 378385, 1995.[ISI][Medline]
- Tian Y, Sandberg K, Murase T, Baker EA, Speth RC, and Verbalis JG. Vasopressin V2 receptor binding is down-regulated during renal escape from vasopressin-induced antidiuresis. Endocrinology 141: 307314, 2000.[Abstract/Free Full Text]
- Verbalis JG. Vasopressin V2 receptor antagonists. J Mol Endocrinol 29: 19, 2002.[Abstract/Free Full Text]
- Verbalis JG and Drutarosky MD. Adaptation to chronic hypoosmolality in rats. Kidney Int 34: 351360, 1988.[ISI][Medline]
- Xia H, Mao Q, Paulson HL, and Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 20: 10061010, 2002.[CrossRef][ISI][Medline]
- Zamore PD. RNA interference: listening to the sound of silence. Nat Struct Biol 8: 746750, 2001.[CrossRef][ISI][Medline]
- Zingg HH. Vasopressin and oxytocin receptors. Baillieres Clin Endocrinol Metab 10: 7596, 1996.[CrossRef][ISI][Medline]
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