Vasopressin regulates body water balance by controlling water permeability in kidney connecting tubules (CNT) and collecting ducts. In the CNT cells and the principal cells of the collecting duct, vasopressin binds to the V2 type of vasopressin receptor (V2R) located in the basolateral membrane. Vasopressin binding to V2R activates adenylyl cyclase via a stimulatory G protein Gs, resulting in increased intracellular cAMP concentration (1). Elevation of intracellular cAMP induces a rapid (within minutes) insertion of aquaporin-2 water channels into the apical membrane, thereby promoting the osmotically driven water reabsorption from the tubular lumen into the blood (2). In humans, loss-of-function mutations in V2R or aquaporin-2 genes leads to nephrogenic diabetes insipidus, a disease that is characterized by the inability of the kidney to concentrate urine (reviewed in reference ). The recently identified gain-of-function mutations in the V2R gene cause a nephrogenic syndrome that is characterized by hyponatremia and undetectable vasopressin levels (4).
Vasopressin is also involved in the long-term genomic regulation of renal water handling (reviewed in reference ). A number of vasopressin-dependent genes have been identified in various experimental settings and using various high-throughput techniques (6–8). Using gene expression profiling based on serial analysis of gene expression, we recently characterized the vasopressin-dependent gene network of the principal cell of the mouse cortical collecting duct (mpkCCDcl4 cells) that was stimulated for 4 h with vasopressin (8). One of the identified vasopressin-induced transcripts, namely the regulator of G protein signaling 2 (RGS2), belongs to the family of RGS proteins that control both the strength and the duration of signaling through G protein–coupled receptors (GPCR). In vitro, RGS2 has been shown as a direct inhibitor of at least four types of adenylyl cyclases (ACII, ACIII, ACV, and ACVI) as well as several GPCR. RGS2 has also been shown to act as a GTPase activating protein for G protein Gq, thereby inhibiting Gq signaling (reviewed in reference ). In vivo, mice that are deficient in RGS2 exhibit a strong hypertensive phenotype as a result of abnormally prolonged signaling by Gq-coupled vasoconstrictor receptors (10).
This study was undertaken to assess the role of RGS2 in water handling by the kidney. We hypothesized that stimulation of RGS2 expression by vasopressin could interfere with V2R or other GPCR signaling pathways and, thereby, influence water reabsorption in the nephron. Here we demonstrate that RGS2−/− mice exhibit an enhanced V2R signaling and increased renal responsiveness to vasopressin. Thus, RGS2 is likely involved in the negative feedback regulation of vasopressin effect in the kidney. This study provides the first evidence for the role of RGS2 in renal function.
Materials and Methods
Northern Blot Analysis
Northern blots were prepared with 2 μg of poly(A) RNA that was extracted from mpkCCDcl4 cells that were grown on filters. Cell culture conditions for mpkCCDcl4 cells have been previously described (8).
Microdissection of different parts of the nephron was performed from collagenase-treated kidneys as described previously (11).
Qualitative Reverse Transcriptase–PCR Analysis
Experiments were performed on male C57BL/6 mice (8 to 12 wk old) that were maintained on a standard diet with free access to water. RNA aliquots that originated from 1 mm of microdissected tubule were used in reverse transcriptase–PCR (RT-PCR; Titan One Tube RT-PCR System, Roche, Rotkreuz, Switzerland). The sense and antisense primers that were designed to amplify a 449-bp fragment of RGS2 mRNA were 5′-GGCAACGGCCCCAAGGTCGAGG-3′ and 5′-AAGCAGCCACTTGTAGCCTCTTG-3′, respectively. The sense and antisense primers that were designed to amplify a 444-bp fragment of V2R mRNA were 5′-CAGGAGGAGCTACTGGATGA-3′ and 5′-CAGGCTGAGAAGGAGTGAGA-3′, respectively. RT-PCR conditions were identical for both pairs of primers. The RT step was performed for 30 min at 50°C. The PCR conditions were as follows: 95°C for 30 s, 56°C for 30 s, and 68°C for 45 s (35 PCR cycles). The amplification products were run on a 2.5% agarose gel and stained by ethidium bromide. To check the specificity of the RGS2 primers, we cloned and sequenced the RT-PCR amplification product that originated from the CCD RNA samples. Six independent clones were sequenced. All six clones contained the identical 449-bp insert that corresponded to the amplified part of the RGS2 cDNA sequence.
Quantitative Real-Time PCR Analysis
Experiments were performed on 6-wk-old male C57BL/6 mice. In the water-restricted group, mice were deprived of water for 23 h. In the water-loaded group, mice were fed for 23 h with 17 ml of a water-loaded gelled diet that consisted of 12.8 ml of water and the same amount of nutrients and electrolytes as the water-restricted diet (4.5 g). The gelled diet was prepared as described previously (12). A total of 100 mm of both cortical thick ascending limb (CTAL) and CCD was microdissected from each mouse. Total RNA was extracted and reverse-transcribed using Superscript II reverse transcriptase (Invitrogen, Basel, Switzerland). Real-time quantitative PCR analysis was performed using the TaqMan system (Applied Biosystems, Foster City, CA). All reactions were performed in triplicate with the amount of cDNA corresponding to 1 mm of microdissected tubule. All primer sets that were used in this study were from Applied Biosystems: TaqMan Gene Expression Assay RGS2 Mm00501385_m1 (for RGS2), mouse ACTB (β-actin) Endogenous Control (for β-actin), and TaqMan Gene Expression Assay Hprt1 Mm00446968_m1 (for hypoxanthine guanine phosphoribosyl transferase I).
A colony of RGS2−/− mice (C57BL/6 background) was established from breeding pairs of RGS2 heterozygous mice originally described by Oliveira-Dos-Santos et al. (13).
cAMP Accumulation Experiments
Experiments were performed on wild-type or RGS2−/− male mice (8 to 12 wk old) that were maintained on a standard diet with free access to water. cAMP was measured in single pieces of CTAL or CCD (0.5 to 1.0 mm of tubular length) in the presence of 1 mM 3-isobutyl-1-methylxanthine, as described previously (14). Briefly, the microdissected nephron segments were kept on ice until analysis. After a preincubation step (10 min at 30°C), each sample was incubated for 4 min at 35°C in the presence of the agonist to be tested. Perkin Elmer cAMP RIA Kit (Waltham, MA) was used for measurement of cAMP levels. The amount of cAMP was calculated in femtomoles per millimeter of tubule length per 4-min incubation time at 35°C (fmol/mm per 4 min).
Metabolic Cage Experiments
Mice were housed in individual metabolic cages (Tecniplast, Buguggiate, Italy). All experiments were performed after 3 d of adaptation. Urine and plasma osmolarity as well as ionic composition were analyzed in the Laboratoire Central de Chimie Clinique, Centre Hospitalier Universitaire Vaudoise (CHUV) University Hospital (Lausanne, Switzerland).
Regulation of RGS2 Expression by Vasopressin in mpkCCDcl4 cells
The time-course and dosage-response effects of vasopressin on RGS2 mRNA expression were studied by Northern hybridization on RNA that were prepared from mpkCCDcl4 cells. As shown in Figure 1A, RGS2 mRNA levels were already increased at 30 min of vasopressin stimulation, reached a maximum at 1 h, and rapidly declined after 4 h. The dosage-response analysis (Figure 1B) revealed that RGS2 mRNA expression could be stimulated within the low physiologic range of hormone concentration (10−12 to 10−11 M). As shown in Figure 2, RGS2 expression was also upregulated by vasopressin at the protein level. Collectively, these results characterize RGS2 as a rapidly but transiently upregulated vasopressin-induced gene.
RGS2 mRNA Expression along the Nephron
To map the distribution of RGS2 along the mouse nephron, we performed RT-PCR analysis of RGS2 mRNA expression on RNA that was extracted from the microdissected nephron segments. Mice that were used in these experiments were allowed ad libitum access to food and water. As shown in Figure 3A, RGS2 mRNA is strongly expressed in the medullary thick ascending limb, CTAL, distal convoluted tubule (DCT), CNT, CCD, and outer medullary collecting duct. The RGS2 mRNA was barely detectable in the proximal convoluted tubule, proximal straight tubule, an thin descending and ascending limbs. This distribution was very similar to that of V2R mRNA expression (Figure 3B). It should be noted that although in our experiments the DCT was positive for V2R mRNA expression, the sensitivity of this nephron segment to vasopressin in the mouse is debated. In the rat, the V2R is expressed only in the late DCT (15). It is interesting that the V2R RT-PCR amplification products in the medullary thick ascending limb and CTAL as well as in the total kidney extract contained an additional band of a higher size. The upstream and downstream primers for V2R mRNA amplification were selected in the coding sequence of V2R cDNA, suggesting the possible existence of a thick ascending limb–specific V2R isoform in the mouse. The absence of cross-contamination between microdissected samples was validated by RT-PCR amplification of several additional mRNA species that are characteristic to the different parts of the nephron (see Supplementary Figure 1). Collectively, these results demonstrate that RGS2 expression in the kidney is restricted to the vasopressin-sensitive parts of the nephron.
In Vivo Regulation of RGS2 mRNA Expression
Water restriction is a highly potent stimulus for vasopressin secretion, whereas water loading efficiently suppresses plasma hormone concentration. To determine whether RGS2 expression correlates with circulating vasopressin levels, we performed real-time quantitative PCR analysis of RGS2 mRNA expression in CTAL and CCD that were microdissected either from water-loaded or from water-restricted mice. In the water-restricted group, mice were deprived of water for 23 h before kidney perfusion and microdissection. In the water-loaded group, mice were fed for 23 h with water-saturated gelled diet (see the Materials and Methods section). RGS2 mRNA expression levels were quantified using two independent internal standards, namely β-actin and hypoxanthine guanine phosphoribosyl transferase I. As shown in Figure 4, the RGS2 mRNA expression was significantly higher in both CTAL and CCD that were microdissected from water-restricted mice using both internal standards.
Quantitatively, the increase in RGS2 mRNA expression was higher in the CTAL compared with the CCD. It should be noted, however, that this quantification was performed at a single time point (23-h water restriction compared with 23-h water loading). It is possible that RGS2 mRNA, which is strongly but transiently induced by vasopressin in in vitro systems, does not follow the same accumulation/degradation kinetics between the CTAL and the CCD in vivo.
Agonist-Stimulated cAMP Levels in Wild-Type and RGS2−/− Mice
To assess directly the role of RGS2 in V2R signaling, we measured the cAMP levels in CCD that were microdissected from wild-type and RGS2−/− mice. All measurements of cAMP were performed in the presence of 1 mM 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor. The cAMP levels were measured in unstimulated CCD (basal cAMP levels) and in CCD that were treated with 10−9 or 10−8 M vasopressin for 4 min at 35°C. Basal cAMP levels were not different between wild-type and RGS2−/− mice (data not shown). Vasopressin-stimulated cAMP levels were higher in CCD that were microdissected from RGS2−/− mice at both hormone concentrations (Figure 5A).
It was previously demonstrated that mouse CCD cells express at least two Gq-coupled GPCR, namely endothelin-1 (ET-1) receptor B and α(1)-adrenergic receptor (12,16). Because RGS2 has also been shown to interfere with Gq-mediated signaling pathways, we measured the effects of ET-1 and phenylephrine on cAMP accumulation that was induced by vasopressin. As shown in Figure 5B, both ET-1 and phenylephrine provoked a significant inhibition of vasopressin-stimulated cAMP levels. However, there was no significant difference in the inhibitory effects between wild-type and RGS2−/− mice.
The increased cAMP levels in CCD that were microdissected from the RGS2−/− mice together with the rapid but transient upregulation of RGS2 expression by vasopressin suggest that vasopressin-stimulated RGS2 expression could be involved in the mean-term (30 min to several hours) negative feedback regulation of V2R signaling. The exact molecular targets of RGS2 in vasopressin-sensitive parts of the nephron remain unknown. However, the V2R itself or adenylyl cyclases ACIII, ACV, and ACVI, which are abundantly expressed in the principal cell of the collecting duct, are likely candidates for future studies (7,18).
Renal Function in RGS2−/− Mice
From our cAMP experiments, we hypothesized that RGS2−/− mice should exhibit an increased functional responsiveness to vasopressin in the collecting ducts. To test this hypothesis, we assessed renal water handling of RGS2−/− mice in two different experimental settings. In the first set of experiments, RGS2−/− mice or their wild-type littermates were housed individually in metabolic cages and allowed free access to food and water. In these baseline conditions, there was no difference in body weight; urine volume; urine and plasma osmolality; plasma Na concentration; and urine Na, K, Cl, PO4, Ca, and Mg excretion (Table 1). In addition, we did not find any significant difference in expression of several vasopressin-sensitive transporters (aquaporin-2, β and γ subunits of the amiloride-sensitive epithelial sodium channel, and α1 and β1 subunits of the Na, K-ATPase), as tested by Western blot analysis that was performed on microdissected nephron segments (Figure 6).
The second experimental setting examined acute differences in water handling between RGS2−/− and wild-type mice. In this protocol, we measured urine output in mice that were exposed to 23 h of water restriction followed by acute water loading (2 ml of intraperitoneal water injection). Urine was collected hourly during the first 5 h followed by water loading. The last fraction was collected between 5 and 7.5 h after water loading. As shown in Figure 7A, RGS2−/− mice excreted less urine in the beginning of the collection period than their wild-type littermates and more urine at the end of the collection period. Importantly, the total urine volume at the end of the collection period was not different between RGS2−/− and wild-type mice (0.57 ± 0.06 ml [n = 15] versus 0.72 ± 0.07 ml [n = 11], respectively). To check whether the observed difference is due to the difference in V2R signaling, we performed a similar water-loading experiment in which we added SR121463B, a V2R-specific antagonist, to the injected water. As shown in Figure 7B, in the presence of SR121463B, there was no difference in urine excretion between RGS2−/− and wild-type mice. Comparison of urine flow in the presence or absence of SR121463B also demonstrated in our experimental settings (23-h water deprivation followed by a 2 ml of intraperitoneal water injection with or without SR121463B) that the circulating vasopressin levels remain significant, at least in the beginning of the collection period. Indeed, during the 1- to 2- and 2- to 3-h collection periods, the mice that were administered an injection only of water excreted three to four times less urine than mice that were administered an injection of water complemented with SR1212463B. These results clearly indicate that the vasopressin/V2R-mediated signaling pathway is responsible for the observed difference in water excretion between the wild-type and RGS2−/− mice.
Collectively, our findings are compatible with a negative regulatory role of RGS2 in V2R signaling. Thus, RGS2−/− mice represent the first mouse model to exhibit a gain-of-function phenotype in water handling by the kidney. In humans, two gain-of-function missense mutations in the V2R gene were recently shown to cause a nephrogenic disorder that is characterized by hyponatremia, serum hyposmolarity, and suppressed vasopressin secretion. As proposed by Feldman et al. (4), these mutations result in constitutive V2R activation in the absence of antagonist, thereby leading to dysregulation of receptor function. This disease was labeled as the nephrogenic syndrome of inappropriate diuresis (4) or as the pseudo-SIADH (19) to emphasize that it represents a subtype of a relatively common syndrome of inappropriate antidiuretic hormone secretion (SIADH). In general, the pseudo-SIADH and SIADH have identical clinical features with the exception of high and low vasopressin levels, respectively. As discussed by Feldman et al. (4), cases of SIADH with low or undetectable vasopressin levels represent as much as 10 to 20% of affected patients. Our study indicates that in addition to V2R-activating mutations, the inactivating mutations of the RGS2 gene should be considered in these patients. The pseudo-SIADH was studied only in a very limited number of patients (two children of <3 mo of age). In addition to the species difference, it is difficult to compare the pseudo-SIADH clinical features with the phenotype of the RGS2−/− mice. However, the gain of function in the V2R signaling pathway and the inability to excrete a free water load (at least transiently) remain common features between patients with pseudo-SIADH and the RGS2−/− mouse model.
This work was supported by the Swiss National Fund for Scientific Research, grant 3100A0–105592/1 (D.F.).
Some of the data were presented in abstract form at the annual meeting of the American Society of Nephrology; November 14 through 19, 2006; San Diego, CA.
We thank Dr. Claudine Serradeil-Le Gal (Sanofi Synthélabo, Toulose, France) for the generous gift of SR121463B.
Published online ahead of print. Publication date available at www.jasn.org.
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