Regulation of Aquaporin-2 Expression by the α2-Adrenoceptor Agonist Clonidine in the Rat1
- Departments of 1Internal Medicine (A.J., B.P.) and2Pharmacology and Therapeutics (B.P., D.D.S.), 3Faculty of Medicine, St. Boniface Research Centre (A.J., L.C.), University of Manitoba, Winnipeg, Manitoba, Canada
Abstract
Aquaporin-2 (AQP-2), the major water channel responsible for water balance, has been shown to be regulated by the binding of vasopressin to V2 vasopressin receptors in the medullary collecting duct. α2-Adrenoceptor agonists such as clonidine have been associated with an increase in free water clearance that was secondary to an inhibition of the ability of vasopressin to increase cAMP levels in the collecting ducts. This investigation focused on the possibility that this increase in free water clearance following administration of an α2-adrenoceptor agonist was associated with a reduction in medullary AQP-2 expression. In the anesthetized rat, clonidine increased urine flow rate (32 ± 5 versus 137 ± 16 μl/min, p < .05) and free water clearance (−58 ± 6 versus 3 ± 8 μl/min,p < .05) compared with the group receiving the saline vehicle infusion. The increase in free water clearance with clonidine administration was associated with a reduction in whole kidney AQP-2 mRNA levels (282 ± 25 versus 216 ± 11A units, p < .05). This decrease in water reabsorption was associated with a redistribution of AQP-2 away from the luminal membrane of the medullary collecting duct to the cytosol. These effects were not secondary to changes in serum vasopressin levels, as these were similar in the vehicle control and clonidine groups (59 ± 5 pg/ml versus 64 ± 7 pg/ml,p = NS). The rapid redistribution of AQP-2 and the reduction in AQP-2 mRNA following clonidine administration are consistent with the hypothesis that the α2 adrenoceptor regulates water excretion at least in part by effects on AQP-2.
The regulation of water excretion has implications for a number of clinical situations. The interaction of vasopressin and α2-adrenoceptors has been well documented. In isolated collecting tubules, vasopressin increased cAMP accumulation (Chabardes et al., 1984; Umemura et al., 1985) and water permeability (Krothapalli and Suki, 1984). This vasopressin-induced increase in water permeability, as well as the increase in cAMP, was found to be attenuated in the presence of an α2-adrenoceptor agonist in both isolated tubular segments (Chabardes et al., 1984; Krothapalli and Suki, 1984; Umemura et al., 1985) and isolated perfused rat kidney (Smyth et al., 1985). In vivo studies have confirmed the ability of α2-adrenoceptor stimulation to increase free water clearance secondary to the inhibition of the renal actions of vasopressin (Strandhoy et al., 1982; Gellai and Ruffolo, 1987;Blandford and Smyth, 1990).
More recently, the effects of vasopressin on water permeability in the kidney have been found to be mediated through the regulation of aquaporin-2 (AQP-2) water channels (Deen et al., 1994; Knepper, 1997). cAMP has been found to be a second messenger in AQP-2 gene transcription and translocation into the luminal membrane after stimulation of V2 receptors by vasopressin (Hozawa et al., 1996; Deen et al., 1997; Matsumura et al., 1997). As α2-adrenoceptor stimulation has been shown to decrease the ability of vasopressin to increase cAMP (Chabardes et al., 1984; Umemura et al., 1985), we determined the effects of clonidine, an α2-adrenoceptor agonist, on AQP-2 expression and localization. The present study reports on the ability of α2-adrenoceptor stimulation to decrease the level of whole kidney AQP-2 mRNA levels as early as 1 h after administration. In addition, AQP-2 redistributes into the cytosol away from the luminal membrane of medullary collecting duct cells. This effect of clonidine failed to alter plasma vasopressin levels, suggesting an effect at the level of the nephron. These findings may have implications for the potential treatment of disorders associated with impaired regulation of water balance.
Materials and Methods
Animal Preparation and Functional Studies.
Male Sprague-Dawley rats (200–225 g) had their right kidneys removed under ether anesthesia 7 to 10 days before the day of the experiment, as previously described (Blandford and Smyth, 1990). On the day of the experiment, the animals were anesthetized with 50 mg/kg pentobarbitone (BDH Chemicals Ltd., Poole, England) and placed on a thermostatically controlled heating blanket that maintained body temperature at 37.5°C. The left jugular vein was cannulated (PE-50) for the infusion of saline (97 μl/min). The carotid artery was cannulated with PE-160 tubing for the measurement of blood pressure with a Statham pressure transducer (model P23Dc; Farmingdale, NY) connected to a Grass polygraph model V (Astro-Med, Inc., Grass Div., West Warwick, RI). The left kidney was exposed by a flank incision, and the ureter was cannulated (PE-10). A 31-gauge needle was advanced through the aorta into the renal artery and secured with glue for the infusion of the clonidine directly into the kidney.
The preparation was allowed to stabilize for 45 min, after which a 30-min control urine collection was obtained. Immediately following the first urine collection, the intrarenal infusion (3.4 μl/min) of vehicle (saline) or clonidine (3 nmol/kg/min) was begun and maintained for the duration of the experiment. During this collection two additional urine collections of 30 min each were obtained. At the end of the experiment, a blood sample was obtained for measurement of vasopressin, and the kidney was removed for the studies described below.
Vasopressin Assay.
Cervical blood was collected in prechilled tubes containing EDTA (1 mg/ml of blood) and aprotinin (500 kallikrein inhibitor units/ml of blood). Plasma was separated by centrifugation at 1600g for 15 min at 0°C. Plasma vasopressin concentrations were determined with a kit (Peninsula Laboratories, Inc., Belmont, CA) according to the manufacturer’s instructions.
RNA Extraction and Northern Hybridization.
RNA isolation was performed by the method of Chomczynski and Sacchi (1987). Following extraction, total RNA was dissolved in sterile water, and the concentration was determined by absorbency readings at 260 nm. Twenty micrograms per lane were loaded onto a 1% agarose gel containing 20 mM 3-(N-morpholino)propanesulfonic acid, 1 mM EDTA, 5 mM sodium acetate (pH 7.0), and 2.2 M formaldehyde. After separation by electrophoresis, RNA was transferred to nylon membranes (Duralon UV; Stratagene, La Jolla, CA) and fixed by UV cross-linking (Stratalinker; Stratagene). Prehybridization was performed at 60°C for 4 h in 5× standard saline citrate (SSC), 5× Denhardt’s reagent, 50 mM Tris-hydrochloride (pH 7.5), 0.1% sodium pyrophosphate, 0.2% SDS, 200 μg/ml denatured salmon testes DNA, and 100 μg/ml yeast tRNA.
cDNA probe for rat AQP-2 (985-base pair fragment corresponding to major coding region of rat AQP-2) was generously provided by Dr. K. Fushimi (Fushimi et al., 1993). This probe was labeled with32P by the random priming method (Promega Corp., Madison, WI.). After hybridization at 42°C for 16 to 18 h in buffer containing 50% deionized formamide, 5× SSC, 1× Denhardt’s reagent, 50 mM Tris-hydrochloride, pH 7.5, 0.1% sodium pyrophosphate, 1% SDS, 100 μg/ml salmon testes DNA, and 100 μg/ml yeast tRNA and washing in 2× SSC and 0.1% SDS, membranes were subjected to autoradiography (Kodak XAR-5 film), stripped, and reprobed for glyceraldehyde-3-phosphate dehydrogenase. The signal for hybridized AQP-2 and GAPDH mRNA was quantified with the National Institutes of Health Image Program from a scan of the radiograph. All values are reported in absorbance (A) factored for variations in loading of total RNA as determined by the density of GAPDH.
Western Analysis.
Total protein was isolated from medullary tissue with Trizol (Life Technologies, Inc.) according to the manufacturer’s instructions. Tissue was homogenized in Trizol reagent (1 ml/100 g) with a tissue homogenizer. After separation of RNA and DNA, proteins were precipitated from the phenol-ethanol supernatant with isopropanol. The precipitated protein was pelleted by centrifugation, washed three times with 0.3 M guanidine hydrochloride in 95% ethanol, and given a final wash in ethanol. The protein pellet was vacuum dried and resuspended in 1% SDS in preparation for electrophoresis.
Protein samples were electrophoresed on a denaturing, 10% polyacrylamide gel, transferred onto a 0.45 μ polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA), and subjected to Western blotting as follows. After blocking with 5% nonfat dry milk in Tris-buffered saline with Tween 20 at room temperature for 1 h, the membrane was washed with three changes of Tris-buffered saline before incubation with anti-AQP-2 antibody (1:500 dilution, kindly provided by P. Yves-Martin and R.W. Schrier, University of Colorado) for 1 h (Xu et al., 1997). Following another three washes with Tris-buffered saline with Tween 20, the membrane was incubated with horseradish peroxidase-conjugated, donkey, anti-rabbit IgG (1:1000 dilution, Amersham Pharmacia Biotech, Piscataway, NJ) at room temperature for 1 h. Antibody-bound proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech). Equality of protein loading was confirmed by ponceau S staining.
Immunofluorescence.
On removal of kidneys, medullary tissue was dissected away from the renal cortex, embedded in optimal cutting temperature medium, and immediately immersed in liquid nitrogen and stored at −70°C until sectioning. Embedded medullary tissue was sectioned into 5-μm sections on a cryostat (Cryo-Cut; American Optical Scientific Instruments, Buffalo, NY) and placed on silanized slides. Sections were permeabilized by incubating with 0.1% Triton X-100 in phosphate-buffered saline. After blocking with universal blocking agent (DAKO Corp., Carpinteria, CA), sections were incubated with anti-AQP-2 antibody for 1 h at 37°C. Specific labeling was visualized by incubation for 1 h at 37°C with fluorescein-conjugated, goat, anti-rabbit antibody (1:100 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and photographed on an Olympus BH-2 fluorescence microscope (Olympus Optical Corp. Ltd., Tokyo, Japan) at a magnification of 300× using Kodak Ektrachrome 400 print film.
Statistical Analysis.
All results are expressed as mean ± S.E. The unpaired Student’s t test with the Bonferroni correction for multiple comparisons was used for the statistical comparisons between groups, and results were considered significant at the p < .05 level.
Results
Renal Function.
The values obtained during the first urine collection were similar in both groups (data not shown). For presentation, data from the third collection period have been shown (Table 1). Intrarenal infusion of clonidine at 3 nmol/kg/min failed to significantly alter blood pressure, heart rate, and creatinine clearance. Clonidine at this infusion rate increased urine flow rate and sodium excretion. This increase in urine flow rate was secondary to an increase in free water clearance and osmolar clearance. The plasma vasopressin levels were similar following the saline vehicle infusion and the clonidine infusion (59 ± 5 versus 64 ± 7 pg/ml; NS;n = 6).
Effects of clonidine on hemodynamics and renal function
AQP-2 Gene Expression, Protein Level and Distribution.
Compared with the group receiving the saline vehicle infusion, there was a reduction in whole kidney AQP-2 mRNA following only 1 h of clonidine administration (Fig. 1; 282 ± 25 versus 216 ± 11 A units;p < .05, n = 4). This suggested that the expression of this water channel was rapidly regulated. At this early time point, this difference in mRNA levels was not reflected by a decrease in AQP-2 protein as detected by Western blot analysis (Fig. 2).
Representative lanes from hybridization of 30 μg of total RNA with cDNA probe for rat AQP-2 and GAPDH. Clonidine-infused rats (lanes 1, 3, 5, and 7) and saline-infused rats (lanes 2, 4, 6, and 8).
Western blot analysis of total protein probed with anti-AQP-2 antibody in kidneys from saline-infused (lanes 1, 3, 5, and 7) and clonidine-infused (lanes 2, 4, 6, and 8) rats. When factored for variations in loading, as determined by ponceau S staining, there was no significant difference between groups. The 29-kD band represents AQP-2 and the 36- to 45-kD band represents AQP-2 in glycated form.
Localization of AQP-2 in Medullary Tissues.
Representative immunoflouresence profiles of saline- or clonidine-treated rats are shown in Figs. 3, A and B, respectively. AQP-2 shifted away from the luminal membrane to the cytoplasm in inner medullary collecting duct cells. This redistribution would lessen the ability of this region to abstract water from the glomerular ultrafiltrate and likely accounts for the increase in free water clearance that was seen with α2-adrenoceptor agonist administration.
Representative immunofluoresence profiles for the localization of AQP-2 in medullary tissue from saline- (A) and clonidine-treated (B) rats. Relocation of aquaporin-2 from the luminal membrane to the cytoplasm of inner medullary collecting duct cells in clonidine-treated rats.
Discussion
The ability of vasopressin to enhance water transport by renal epithelial cells has been previously shown to be inhibited by stimulation of α2-adrenoceptors (Krothapalli and Suki, 1984). In vivo studies by our laboratory and others (Strandhoy et al., 1982; Gellai and Ruffolo, 1987; Blandford and Smyth, 1990) have documented an increase in free water clearance at doses of α2-adrenoceptor agonists that do not alter plasma vasopressin levels (Leander et al., 1985). The importance of vasopressin in the response to α2-adrenoceptor agonists has been previously documented by the failure of α2-adrenoceptor agonists to increase free water clearance in Brattleboro rats (Gellai, 1990) and in normal rats pretreated with a specific V2 vasopressin receptor antagonist (Blandford and Smyth, 1990). In this study, the intrarenal administration of the α2-adrenoceptor agonist clonidine increased free water clearance. This occurred despite lack of significant differences in plasma vasopressin levels, systemic blood pressure, or glomerular filtration rate, as measured by creatinine clearance. Micropuncture studies have suggested that α2-adrenoceptor stimulation increases water and sodium excretion in the collecting tubule (Stanton et al., 1987). Vasopressin alters water excretion at this site (Krothapalli and Suki, 1984). This site is consistent with an interference with the effects of vasopressin on AQP-2 expression on the luminal surface of collecting duct cells. AQP-2 has recently been cloned and localized to the principal cells in the medullary collecting duct (Fushimi et al., 1993). It has been shown that expression of AQP-2 is increased in animal models of congestive heart failure (Nielsen et al., 1997) and decreased in rats during lithium administration (Marples et al., 1995), low protein diet (Sands et al., 1996), bilateral ureteric obstruction (Frokiaer et al., 1996), purine aminonucleoside nephrosis (Apostol et al., 1997), and hypokalemia (Marples et al., 1996).
The mechanism by which α2-adrenoceptor stimulation altered AQP-2 expression in the rat was most conceivably secondary to changes in cAMP levels. It has been well documented that in the rat, α2-adrenoceptor stimulation decreases vasopressin-mediated increases in cAMP levels (Chabardes et al., 1984; Krothapalli and Suki, 1984; Umemura et al., 1985; Pettinger et al., 1987). However, this effect of α2-adrenoceptor stimulation has not been observed in dogs or humans (Brooks et al., 1991; Edwards et al., 1992). cAMP has been documented as the second messenger that participates in the trafficking of AQP-2 to the luminal membrane (Deen et al., 1997).
Recently, it has been shown that the promoter region of AQP-2 contains a cAMP response element (Yasui et al., 1997). Therefore, the observed decrease in whole kidney mRNA for AQP-2 in clonidine-treated rats may well be related to decreased cAMP levels in principal cells of the medullary collecting duct. However, a stabilization rather than an increase in the rate of transcription of the AQP-2 gene may also explain these findings. The levels of AQP-2 mRNA appear to be acutely regulated to parallel the cellular location of this water channel and reflect a tight regulation of water-handling requirements as suggested by the decrease in AQP-2 message as early as 1 h after stimulation with clonidine. The lack of a decrease in AQP-2 protein at this early time point is not surprising, because it is likely that sufficient time had not passed for the change in mRNA to be translated into a change in protein abundance. In other studies, 24 h after a stimulus for AQP-2 expression, a parallel increase in AQP-2 protein levels has been observed. This decrease in AQP-2 mRNA in the present study occurred despite similar levels of plasma vasopressin with and without clonidine administration. Hence the ability of clonidine to influence AQP-2 shuttling and mRNA expression was unlikely to be secondary to a change in vasopressin binding to the V2vasopressin receptor.
In summary, the present study has demonstrated for the first time the ability of α2-adrenoceptor agonists to alter the expression and translocation of AQP-2 in the rat kidney. This alteration in AQP-2 expression occurred independently of changes in vasopressin activity and may represent an alternative modality for the treatment of disorders associated with impaired regulation of water balance.
Acknowledgments
We are indebted to Marilyn Vandel and Dianne Kropp for expert technical assistance.
Footnotes
-
Send reprint requests to: Dr. Asad Junaid, Department of Internal Medicine, Faculty of Medicine, BG007 St. Boniface Hospital, Winnipeg R2H 2A6, Manitoba. E-mail:junaid{at}cc.umanitoba.ca
-
↵1 Supported by funding from the Medical Research Council of Canada (to D.D.S.) and Health Sciences Center Foundation of Winnipeg (to A.J.).
- Abbreviations:
- aquaporin-2
- AQP-2
-
- Received February 19, 1999.
- Accepted July 25, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
