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Am J Physiol Renal Physiol 294: F638-F644, 2008. First published January 16, 2008; doi:10.1152/ajprenal.00344.2007
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Vasopressin regulation of inner medullary collecting ducts and compensatory changes in mice lacking adenosine A1 receptors

Timo Rieg,1,3,5 Kanishka Pothula,2 Jana Schroth,3 Joseph Satriano,1,3 Hartmut Osswald,5 Jürgen Schnermann,4 Paul A. Insel,1,2 Richard A. Bundey,2 and Volker Vallon1,2,3

Departments of 1Medicine and 2Pharmacology, University of California, 3Veterans Affairs San Diego Healthcare System, San Diego, California; 4National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and 5Institute of Pharmacology and Toxicology, Medical Faculty of Eberhard Karls University, Tübingen, Germany

Submitted 23 July 2007 ; accepted in final form 14 January 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Activation of adenosine A1 receptors (A1R) can inhibit arginine vasopressin (AVP)-induced cAMP formation in isolated cortical and medullary collecting ducts. To assess the in vivo consequences of the absence of A1R, we performed experiments in mice lacking A1R (A1R–/–). We assessed the effects of the vasopressin V2 receptor (V2R) agonist 1-desamino-8-D-arginine vasopressin (dDAVP) on cAMP formation in isolated inner medullary collecting ducts (IMCD) and on water excretion in conscious water-loaded mice. dDAVP-induced cAMP formation in isolated IMCD was significantly greater (~2-fold) in A1R–/– compared with wild-type mice (WT) and, in contrast to WT, was not inhibited by the A1R agonist N6-cyclohexyladenosine. A1R–/– and WT had similar basal urinary excretion of vasopressin, expression of aquaporin-2 protein in renal cortex and medulla, and acute increases in urinary flow rate and electrolyte-free water clearance in response to the V2R antagonist SR121463 or acute water loading; the latter increased inner medullary A1R expression in WT. Dose dependence of dDAVP-induced antidiuresis after acute water loading was not different between the genotypes. However, A1R–/– had greater inner medullary expression of cyclooxygenase-1 under basal conditions and of the P2Y2 and EP3 receptor in response to water loading compared with WT mice. Thus vasopressin-induced cAMP formation is enhanced in isolated IMCD of mice lacking A1R, but the adenosine-A1R/V2R interaction demonstrated in vitro is likely compensated in vivo by multiple mechanisms, a number of which can be "uncovered" by water loading.

arginine vasopressin; aquaporin-2; cyclooxygenase

ADENOSINE IS AN IMPORTANT regulator of kidney function and involved in the regulation of glomerular filtration rate (GFR), medullary blood flow, and renin release (for a review, see Ref. 48). Adenosine is a mediator of tubuloglomerular feedback (TGF) (29, 43, 45) and exerts differential effects on transport processes in the tubular and collecting duct (CD) system. Whereas adenosine stimulates reabsorption in the cortical proximal tubule (25), it induces transport inhibition in the medullary thick ascending limb (mTAL) (3, 49). These different effects may relate to a proposed role of adenosine in the metabolic control of kidney function, which helps to preserve the function and integrity of the hypoxic renal medulla (48). Moreover, interactions between adenosine and the antidiuretic hormone arginine vasopressin (AVP) have been demonstrated in a variety of species and different experimental models (1, 7, 52, 53).

AVP is a primary regulator of water reabsorption in the CD system and critically involved in the regulation of water balance and plasma osmolality. AVP acts via the Gs protein-coupled vasopressin V2 receptor (V2R) to stimulate adenylyl cyclase and thus the synthesis of cAMP. Increases in cAMP levels activate protein kinase A (PKA), which phosphorylates the water channel aquaporin-2 (AQP2), with subsequent delivery of the channel into the apical plasma membrane. cAMP and PKA have further actions in the regulation of AQP2 expression: PKA-mediated phosphorylation of a cAMP-response element binding protein (CREB protein) promotes its binding to DNA and increases the transcription of the AQP2 gene (27). By contrast, A1R are Gi protein-coupled receptors that inhibit cAMP formation (9). Activation of A1R can decrease AVP-induced cAMP formation and thus potentially decrease AVP efficiency and water reabsorption. In rat isolated, perfused inner medullary collecting ducts (IMCD), the selective A1R agonist N6-cyclohexyladenosine (CHA) antagonizes AVP actions, resulting in reduced AVP-induced cAMP formation and decreased osmotic water permeability (7). In cultured cells derived from the cortical CD of rabbits, the inhibitory effect of A1R agonists on AVP-stimulated cAMP formation is sensitive to pertussis toxin, indicating the involvement of Gi proteins (52). A follow-up study using the same experimental model showed that adenosine, applied from either the basolateral or the apical side, inhibits the ability of AVP to stimulate cAMP formation (53).

In situ hybridization, binding studies, and RT-PCR have localized A1R to all nephron segments (for a review, see Ref. 48). The studies in rats and mice revealed a strong corticomedullary gradient, with the highest density of A1R in TAL, CD, and, in particular, IMCD (40, 50, 51, 54), consistent with a prominent role of A1R in the regulation of CD function. The source of adenosine acting on A1R in the CD is not entirely clear but could involve breakdown of extracellular ATP (48) and/or the extracellular cAMP-adenosine pathway, as postulated by Jackson et al. (19). The latter pathway is initiated by the efflux of cAMP from cells following activation of adenylyl cyclase with conversion of cAMP to adenosine by the sequential actions of ecto-phosphodiesterase and ecto-5'-nucleotidase. Adenosine then binds to A1R to inhibit adenylyl cyclase and cAMP formation. Such a mechanism for feedback inhibition has been proposed for the stimulation by cAMP of water transport in the CD and renin secretion in the juxtaglomerular apparatus (7, 17, 18, 52, 53).

Water transport in the CD is also regulated by other local factors, including prostaglandins (PG), e.g., PGE2, derived from cyclooxygenase-1 and -2 (COX-1, -2), the rate-limiting enzymes for PG production (4). The medulla has the greatest capacity for PG synthesis and the most abundant PGE2 receptor is the EP3 receptor subtype (20), which is a Gi protein-coupled receptor that inhibits cAMP formation (4). The CD is also the predominant site for endothelin-1 (ET-1) production, and recent studies in CD-specific knockout mice of ET-1 provided evidence for a reduced ability to excrete an acute water load associated with heightened sensitivity to the cAMP-elevating effects of AVP, indicating that ET-1 has natriuretic and diuretic properties (13). In rat (24) and rabbit (23) isolated IMCD, ET-1-dependent PGE2 production has been shown. However, CD-specific knockout of ET-1 increased urinary PGE2 (14), indicating that other factors also contribute to the regulation of PGE2 formation in IMCD. Nitric oxide (NO) and its second messenger, cGMP, have been demonstrated to inhibit AVP-stimulated osmotic water permeability (12, 28). Moreover, NO can activate COX-1 and COX-2 by a mechanism independent of cGMP (39). Finally, local release of ATP and activation of P2Y2 receptors have been implicated in the regulation of water transport in the CD (21, 31, 46). Thus many autocrine and paracrine systems contribute to the local regulation of water transport in the CD and may be able to compensate when one is defective or lacking.

In the present experiments, we used gene-targeted mice lacking A1R (A1R–/–) and their wild-type littermates (WT) to assess the in vivo role of A1R on water transport. In particular, we assessed the consequences of the absence of A1R on the ability to excrete an acute water load and on vasopressin-regulated water transport by examining the effects of the V2R agonist 1-desamino-8-D-arginine vasopressin (dDAVP) on cAMP formation in isolated IMCD and on water excretion in conscious water-loaded mice. We also assessed the ambient vasopressin-dependent regulation of water reabsorption in mice lacking A1R and the effects of acute water loading on inner medullary mRNA expression of A1R. In addition, we determined inner medullary mRNA expression of ET-1, COX-1, and COX-2, the EP3 receptor, the P2Y2 receptor, and endothelial NO synthase (eNOS) under basal conditions and in response to acute water loading. The present results demonstrate defects in IMCD in vitro that accompany the absence of A1R in the A1R–/– mice and indicate ways in which such mice appear to compensate in vivo for the absence of A1R.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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All animal experimentation was conducted in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and was approved by the local Institutional Animal Care and Use Committee. Mice were housed in a 12:12-h light-dark cycle in standard rodent cages with free access to standard rodent chow (Harlan Teklad 7001, Madison, WI) and tap water. A1R–/– and WT littermates were from a subcolony of the original strain generated by Sun et al. (43) that is maintained at the San Diego VA Medical Center. Mice were generated by heterozygous crossing; their genetic background is a mix of 129Sv/J and C57BL/6.

Studies in isolated IMCD. IMCD were isolated from A1R–/– and WT mice using a modification of the method of Chou et al. (6). To prevent degradation of cAMP formed during incubation with dDAVP, IMCD aliquots were incubated at 37°C for 10 min with 1 µM 4-[3-(cyclopentyloxy)-4-methoxy-phenyl]-2-pyrrolidinone (Rolipram, A. G. Scientific, San Diego, CA), a phosphodiesterase inhibitor that is not an adenosine receptor antagonist (41). In certain experiments, CHA (1 µM, Sigma-Aldrich, St. Louis, MO) and/or dDAVP (1 pM-3 nM, final concentrations) was added, and the incubation was continued for 5 min. Reactions were terminated by addition of ice-cold 7.5% trichloroacetic acid, and the cAMP content of samples was assessed by radioimmunoassay (30).

Basal urine analysis. Spontaneously voided urine was collected from male WT and A1R–/– mice for the determination and correlation of AVP and osmolality. In addition, mice were placed for 24 h in metabolic cages (Tecniplast, Hohenpeissenberg, Germany) to assess total basal fluid excretion. To ensure quantitative urine collection, metabolic cages were siliconized and urine was collected under water-saturated oil (47). After the urine collections, the mice were anesthetized with isoflurane and blood was drawn from the retrobulbar plexus for determination of hematocrit, plasma Na+ or K+, and osmolality. Urine and plasma osmolality were measured by vapor pressure (Vapro, Wescor, Salt Lake City, UT). Concentrations of AVP and creatinine were measured using a commercial assay for AVP (IBL, Hamburg, Germany) and creatinine (Thermo Fisher Scientific, Waltham, MA), respectively. Plasma aldosterone was measured by radioimmunoassay (Diagnostic Systems Laboratories, Webster, TX). Urinary PGE2 was assayed by a PGE2 metabolite EIA kit (Cayman Chemical, Ann Arbor, MI).

Effect of V2R blockade and acute oral water loading in WT and A1R–/– mice. One set of WT and A1R–/– mice was subjected to the acute application of the V2R antagonist SR121463 (1 mg/kg body wt ip, 3.3 µl/g body wt) (31, 34). Acute water loading was performed by oral gavage (10 mM glucose solution, 3% of body weight). Mice were immediately placed in metabolic cages for quantitative urine collections over 3 h (for SR121463) and 2 h (for oral water loading), respectively, without access to food or water, as previously described (31, 33, 55).

Electrolyte-free water clearance {Cle-H2O = urinary flow rate·(1 – (([Na+]urine + [K+]urine)/[Na+]plasma))} was calculated according to Rose (36), where [Na+]urine, [K+]urine, and [Na+]plasma refer to the respective ion concentrations in urine or plasma, respectively. Concentration of Na+ and K+ in urine and plasma were measured using a flame photometer (ELEX 6361, Eppendorf, Hamburg, Germany).

Effect of dDAVP in water-loaded WT and A1R–/– mice. dDAVP experiments were carried out in WT and A1R–/– mice given free access to standard rodent chow and 5% glucose (Sigma-Aldrich) solution as drinking water for 7 days to suppress endogenous levels of AVP (15). Subsequently, the mice were randomized to acute water loading (10 mM glucose solution, 3% of body weight) given by oral gavage (31), immediately followed by intraperitoneal injection of vehicle or the V2R agonist dDAVP (Sigma-Aldrich). The mice were then placed in metabolic cages for quantitative urine collections over 2 h without access to chow or water as described (31). After 5–7 days of recovery, this procedure was repeated: each mouse was injected with vehicle (100 µl/30 g body wt sterile water) or dDAVP at the following doses: 0.0004, 0.004, 0.04, and 0.4 µg/kg body wt.

Immunoblot analysis. Mice were euthanized, and the kidneys were dissected into cortical and medullary sections before homogenization in buffer containing protease inhibitor cocktail (250 mM sucrose, 10 mM triethanolamine, Sigma-Aldrich and Roche Applied Science, Indianapolis, IN, respectively) using a tissue homogenizer (Tissuemizer, Tekmar, Cincinnati, OH), as described previously (47). Homogenates were centrifuged at 100,000 g for 40 min to obtain a membrane pellet. The pellet was resuspended in homogenization buffer, and equal lane loading (15 µg protein) was achieved using a Bio-Rad DC Protein assay (Richmond, CA). Samples were resolved on 4–12% NuPAGE gels in MOPS buffer (Invitrogen, Carlsbad, CA). Gel proteins were transferred to nitrocellulose membranes and immunoblotted with AQP2 (dilution 1:500, Santa Cruz Biotechnology, Santa Cruz, CA) or β-actin for a loading control (dilution 1:5,000, Sigma-Aldrich). Chemiluminescent detection was performed with ECL Plus (Amersham, Piscataway, NJ). Densitometric analysis was performed by ImageJ Software (National Institutes of Health, Bethesda, MD).

Quantitative PCR experiments. Water loading (10 mM glucose solution, 3% of body weight) given by oral gavage was performed as described above, and both kidneys from each mouse were removed after 2 h under deep xylazine/ketamine anesthesia (34). For determination of basal gene expression, mice were sham treated by oral gavage without application of fluid, and both kidneys were removed after 2 h. Total RNA from WT and A1R–/– mice inner medullas was isolated and homogenized using QIAshredder (Qiagen, Valencia, CA) according to the manufacturer's instructions. The extracted RNA was further purified using RNeasy minicolumns with DNase I treatment (Qiagen) to remove traces of genomic DNA present in the samples. cDNA was made by reverse transcribing total RNA using oligo(dT) priming and SuperScript II (Invitrogen, respectively) according to the manufacturer's instructions. cDNA samples were treated with RNase H (2 U/µg cDNA, 20 min at 37°C) before use in real-time PCR experiments. These were performed using a qPCR SYBR Green master-mix (Eurogentec, San Diego, CA) in a DNA Engine Opticon 2 (Bio-Rad, Hercules, CA). Template concentration was 10 ng reverse-transcribed RNA/25-µl reaction and was used in conjunction with primer pairs (forward/reverse) specific for the following murine genes: ET-1, 5'-AAC TCA GGG CCC AAA GTA CC-3'/ 5'-ACG AAA AGA TGC CTT GAT GC-3'; P2Y2, 5'-CGT GCT CTA CTT CGT CAC CA-3'/5'-GAC CTC CTG TGG TCC CAT AA-3'; A1R, 5'-CAT TGG GCC ACA GAC CTA CT-3'/5'-CAA GGG AGA GAA TCC AGC AG-3'; COX-1, 5'-CAC TGG TGG ATG CCT TCT CT-3'/5'-CCG TAC AGC TCC TCC AAC-3'; COX-2, 5'-TCC TCC TGG AAC ATG GAC TC-3'/5'-TGC AGC CAT TTC CTT CTC TC-3'; EP3, 5'-GGT CGC CGC TAT TGA TAA TG-3'/5'-TTG TTC ATC ATC TGG CAG AAC T-3'; eNOS, 5'-GGG AAA GCT GCA GGT ATT TG-3'/5'-CTG TGA TGG CTG AAC GAA GA-3'; and GAPDH, 5'-AAG GGC TCA TGA CCA CAG TC-3'/5'-CAT ACT TGG CAG GTT TCT CCA-3'. Primers were designed using Primer 3 software (37) and manufactured by Bioneer (Alameda, CA). Melting curve analysis and gel electrophoresis of PCR products verified that a single product of the expected size was generated with each primer set. Data analysis used the {Delta}{Delta}Ct method, i.e., cycle thresholds (Ct), were normalized to GAPDH expression and compared with WT or basal expression.

Statistical analysis. The data are expressed as means ± SE. An unpaired Student's t-test was performed, as appropriate, to analyze for statistical differences between groups with P < 0.05 considered statistically significant.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Experiments in isolated IMCD. Basal cAMP formation in isolated IMCD was reduced by ~50% in A1R–/– compared with WT mice. Addition of the A1R agonist CHA (1 µM) reduced basal, as well as forskolin-induced, cAMP formation in WT, but was without effect in A1R–/– mice (Fig. 1A). dDAVP-induced increases in cAMP formation were about twofold greater (P < 0.05) in A1R–/– compared with WT and, in contrast to WT mice, were not inhibited by CHA (Fig. 1B). Thus A1R–/– mice show an absence of CHA-mediated inhibition of cAMP formation in IMCD cells as well as an enhanced response to dDAVP, presumably because of the loss of tonic inhibition of this response that occurs in WT mice.


Figure 1
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  		Fig. 1. In vitro studies in freshly isolated inner medullary collecting ducts (IMCD) of adenosine A1 receptor knockout mice (A1R–/–) and wild-type littermates (WT). A: effect of A1R agonist N6-cyclohexyladenosine (CHA) on control and forskolin-stimulated cAMP formation in WT and A1R–/– mice. B: concentration-response relationship between cAMP formation and vasopressin V2 receptor activation by 1-desamino-8-D-arginine vasopressin (dDAVP) in absolute terms (left) and in the absence (vehicle) or presence of CHA (1 µM) relative to control values (middle and right, respectively); n = 4 mice/group. *P < 0.05 vs. WT.

 
Experiments in conscious animals and whole kidneys. Basal parameters of WT and A1R–/– mice are shown in Table 1. Body weight, hematocrit, plasma osmolality, and aldosterone concentrations were similar between genotypes. In addition, both genotypes had similar urinary osmolalities, vasopressin levels (corrected for urinary creatinine concentration) (Fig. 2A), and total levels of AQP2 protein expression in kidney cortex and medulla (Fig. 2B). Neither the expression of the glycosylated (35–45 kDa) nor the nonglycosylated (29 kDa) AQP2 protein was different between genotypes in cortex or medulla (not shown). Basal 24-h urinary flow rates were modestly, but significantly, greater in A1R–/– compared with WT mice, consistent with the loss of basal antidiuretic tone of A1R activation in the kidney. Application of the V2R antagonist SR121463 (1 mg/kg ip) increased urinary flow rates and Cle-H2O to the same extent in WT and A1R–/– mice (Fig. 2C), indicating that the ambient V2R-dependent water reabsorption is similar between genotypes and not enhanced in A1R–/– mice. Acute water loading showed that urinary flow rate and Cle-H2O were not different between genotypes (Fig. 2C) and, confirming previous results (42), most of the applied fluid was excreted within 2 h (WT: 93 ± 5%, A1R–/–: 90 ± 5%, not significant). The water load-induced maximal diuresis as well as the dDAVP-induced, dose-dependent antidiuresis under these conditions were similar in A1R–/– and WT mice (Fig. 2D).


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  		Table 1. Basal parameters in adenosine A1 receptor wild-type and knockout mice

 

Figure 2
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  		Fig. 2. In vivo experiments of water transport in A1R–/– and WT mice. A: arginine vasopressin-to-creatinine ratio vs. osmolality from spontaneous voided urine. B: Western blots for renal expression of aquaporin-2 (AQP2; 15 µg membrane protein/lane; n = 4/group) and corresponding densitometry. AQP2 expression was normalized to β-actin as a loading control. C: basal urinary flow rate and electrolyte free water clearance (Cle-H2O) were measured in 24-h metabolic cage experiments. The response to vasopressin V2 receptor (V2R-I) blockade (SR 121463; 1 mg/kg ip) or acute water loading (3% of body weight by oral gavage) was determined in acute metabolic cage experiments (3 and 2 h, respectively). D: effect of selective V2R activation by dDAVP on urinary flow rate in water-loaded mice (see METHODS for further details). For all experiments, except Western blots, n = 9–12 mice/group. *P < 0.05 vs. WT.

 
Quantitative PCR in basal conditions and after acute water loading. Real-time PCR experiments revealed that, under basal conditions, in addition to the lack of A1R, the A1R–/– mice have significantly increased inner medullary COX-1 expression compared with their WT littermates (Fig. 3A). Two hours after acute water loading (Fig. 3B), WT mice displayed a significant increase in inner medullary mRNA expression of A1R as well as of ET-1 and COX-2, whereas A1R–/– mice show a significantly greater mRNA expression of P2Y2 and EP3 receptors and reduction in COX-2 compared with WT. Consistent with this lower expression of COX-2 mRNA, water loading resulted in a significantly lower urinary PGE2 excretion in A1R–/– compared with WT mice (51 ± 16 vs. 152 ± 18 ng·min–1·g body wt–1, P < 0.05). The higher EP3 receptor expression in A1R–/– mice is consistent with, and may be a consequence of, lower COX-2 expression that results in lower formation (and excretion) of PGE2, an agonist at the EP3 receptors. By contrast, inner medullary eNOS expression was not different between genotypes and not altered by water loading.


Figure 3
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  		Fig. 3. Inner medullary expression of mRNA of key regulators of renal water transport under basal conditions or at 2 h after acute water loading (3% of body weight by oral gavage) in WT and A1R–/– littermates. A: differences between inner medullary gene expression in WT and A1R–/– mice under basal conditions. Statistical comparisons were made between mRNA expression levels of WT and A1R–/– mice; n = 3 mice/group. *P < 0.05. B: effect of acute water loading on inner medullary gene expression in WT and A1R–/– mice. ET-1, endothelin-1; P2Y2R, P2Y2 receptor; A1R, adenosine A1 receptor; COX-1 and COX-2, cyclooxygenase-1 and -2; prostaglandin E2 receptor subtype EP3, EP3R; eNOS, endothelial NO synthase. Statistical comparisons were made for effects of water loading vs. basal conditions in WT (#P < 0.05) or for different responses in gene expression to water loading in A1R–/– vs. WT mice (*P < 0.05; n = 3 mice/group).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The current in vitro experiments in freshly isolated IMCD demonstrate that a loss of A1R alters cAMP formation. In vivo experiments using either V2R blockade, V2R activation, or acute water loading in WT and A1R–/– mice, however, were unable to unmask a significant role of A1R in vasopressin control of renal water reabsorption but uncovered several compensatory changes, possibly indicating that a higher COX-1 expression under basal conditions and a higher P2Y2 and EP3 receptor expression after water loading compensate for the loss of the A1R.

AVP increases cAMP formation in the CD (31), and adenosine or adenosine analogs, via activation of A1R, can inhibit AVP-induced cAMP formation in the CD system (1, 7, 52, 53). Our in vitro results confirm this concept in mice lacking A1R and also support the idea that the action of forskolin on cAMP generation is modulated by both the activation of stimulatory heterotrimeric G proteins (16) and by inhibitory heterotrimeric G proteins. In accordance with this conclusion, the stimulatory effects of AVP and forskolin on cAMP formation are additive in rat MCD (26, 38), and N6-(L-2-phenylisopropyl)adenosine (PIA), an A1R selective agonist, inhibits forskolin-stimulated cAMP accumulation in rat hippocampal slices (10).

The higher urinary flow rate observed in A1R–/– mice compared with WT mice under basal conditions confirms previous studies that show modestly higher urinary flow rates and fluid intake in A1R–/– vs. WT mice (5, 32). This result argues against a dominant antidiuretic influence in the absence of A1R, which could result if AVP effects were unopposed. We observed no evidence for the latter, however, in A1R–/– mice: normal urinary levels of AVP were associated with normal urine and plasma osmolality and plasma aldosterone concentration. Moreover, V2R blockade or V2R activation did not uncover significant differences with regard to renal water transport between A1R–/– and WT mice. If adenosine were to activate A1R to persistently inhibit AVP-induced renal water reabsorption in vivo, then A1R–/– mice would be expected to have a higher urinary flow rate after V2R blockade, a compensatory downregulation of plasma AVP, and/or a leftward shift of the antidiuresis response curve to dDAVP application as well perhaps a greater renal AQP2 expression, but none of these effects were observed in mice lacking A1R. By contrast, a mouse strain with a high constitutive cAMP-phosphodiesterase activity shows polyuria and has low intracellular cAMP levels associated with lower renal AQP2 protein and mRNA expression levels (11). Furthermore, ET-1 is an autocrine inhibitor of AVP activity in the CD, and as a consequence, IMCD suspensions of CD-specific ET-1 knockout mice have enhanced AVP- and forskolin-stimulated cAMP accumulation and plasma AVP levels are reduced (13). Together, the present experiments indicate that in the absence of A1R, the lack of an inhibitory effect of adenosine that is observed in vitro is offset in vivo by other mechanisms. The in vitro experiments, however, did reveal some unexpected results: basal cAMP formation was lower in isolated IMCD of mice lacking A1R compared with WT. If A1R in the IMCD provide a physiological braking mechanism for cAMP formation, then one would expect basal cAMP formation to be increased rather than reduced in the absence of the receptors. The findings in the A1R–/– mice thus suggest that activation of compensatory mechanisms tonically lower cAMP formation and overcome the lack of A1R. A possible compensatory mechanism may be that under basal conditions A1R–/– mice have higher COX-1 expression, resulting in higher levels of PGE2 and perhaps other COX-generated arachidonic acid metabolites, which, by acting on prostaglandin receptors (e.g., EP3 receptors), reduce cAMP formation (4).

Acute water loading activates local renal mechanisms that inhibit AVP-induced water permeability and thus accelerate the excretion of free water to stabilize extracellular tonicity and cell volume. We observed that inner medullary A1R mRNA expression increased in WT mice in response to acute water loading, a result consistent with an autocrine/paracrine signaling role of adenosine and the A1R. The mechanisms that contribute to the upregulation of A1R under these conditions remain to be determined. The ability to enhance free water excretion in response to acute water loading, however, appeared unaltered in mice lacking A1R, indicating the activation of compensating mechanisms. In WT mice, acute water loading also upregulated inner medullary mRNA expression of both ET-1 and COX-2. The findings for ET-1 confirm previous studies in WT mice in which water loading was associated with increased CD ET-1 mRNA expression whereas CD ET-1 knockout mice had a reduced ability to excrete an acute water load (13). To our knowledge, this is the first study to show that acute water loading upregulates the inner medullary expression of COX-2. This higher COX-2 mRNA expression may relate to findings in healthy women who increase urinary PGE2 excretion for the first 3 h after water loading (35) as well as studies in healthy volunteers showing that the increase in free water clearance in response to water loading is attenuated to the same extent by nonselective COX inhibition as well as selective blockade of COX-2 (2). A compensatory upregulation of inner medullary mRNA expression of ET-1 or COX-2, however, did not occur in A1R–/– mice. Data shown here indicate that the upregulation of COX-2 in response to acute water loading is blunted in the absence of A1R. In contrast, the expression of the EP3 receptor is significantly higher in A1R–/– mice and thus may have compensated in response to water loading. Notably, experiments in EP3–/– mice have indicated that PGE2 modulates basal urine osmolality through the EP3 receptor; on the other hand, the regulation of urine volume and osmolality under basal conditions and in response to various physiological stimuli appears normal in EP3–/– mice (although acute water loading had not been tested), indicating potential compensation by other mechanisms (8).

Another potential compensatory mechanism is the release of ATP and activation of P2Y2 receptors, which inhibit cAMP formation and thus oppose effects of AVP in the CD (21, 44). In contrast to the present findings in mice lacking A1R, P2Y2–/– mice show enhanced renal water reabsorption in vivo by a V2R/cAMP-dependent mechanism and loss of inhibition of AVP-induced cAMP formation by the agonist ATP{gamma}S in isolated IMCD (31). The latter studies provided indirect evidence for the concept that water loading-induced cell swelling of CD triggers ATP release, which, by activation of P2Y2 receptors, inhibits water transport and stabilizes cell volume in the CD (31, 46). Notably, however, AVP-induced cAMP formation in isolated IMCD was not different between WT and P2Y2–/– mice (in the absence of ATP{gamma}S), whereas that response was potentiated in A1R–/– mice in the present experiments. Thus, with regard to AVP-induced cAMP formation and antidiuresis, A1R–/– mice have a phenotype in isolated IMCD, but not in vivo, whereas P2Y2–/– mice show a phenotype in vivo, but not in isolated IMCD. Explanations for these different findings remain unclear. In experiments in isolated IMCD, application of the V2R agonist is expected to trigger cAMP formation, perhaps followed by extracellular adenosine formation (18), activation of A1R, and inhibition of adenylyl cyclase activity, an effect that is blunted in mice lacking A1R. In the absence of osmotic gradients, however, the in vitro application of the V2R agonist may not alter cell volume and thus may not generate a stimulus for ATP release. In vivo, lesser compensation in the absence of P2Y2 receptors than of A1R may indicate that the feedback inhibition via cell volume-regulated ATP release and activation of P2Y2 receptors is a more potent system than the extracellular formation of adenosine (from cellular release of cAMP and extracellular breakdown of ATP) and the subsequent activation of A1R. Moreover, P2Y2 receptor mRNA expression levels were significantly increased in A1R–/– but not WT mice after acute water loading, indicating that P2Y2 receptor upregulation is likely part of a compensatory mechanism in A1R–/– mice. Previous studies in rats revealed that chronic water loading (48 or 96 h) resulted in a marked increase in inner medullary P2Y2 mRNA expression in conjunction with increased protein abundance (22).

In summary, using A1R knockout mice and a selective A1R agonist, the current experiments provide evidence for an inhibitory influence of A1R activation on cAMP formation in response to AVP stimulation of isolated IMCD. The studies also show an inhibitory effect of A1R activation in IMCD on the stimulation of cAMP formation by forskolin. The in vivo phenotype of A1R–/– mice suggests that the adenosine-A1R/V2R interaction demonstrated in vitro is likely compensated by increased COX-1 expression under basal conditions and by perhaps multiple mechanisms, including greater P2Y2 and EP3 receptor expression during water loading.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants GM-66232, DK-56248, and DK-28602, the Department of Veterans Affairs, and Deutsche Forschungsgemeinschaft (RI 1535/3-1 and 3-2).


   ACKNOWLEDGMENTS
 
SR121463 was kindly provided by Claudine Serradeil-Le Gal, Sanofi-Synthelabo Recherche. We thank Hannah Steigele for excellent technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Vallon, Depts. of Medicine and Pharmacology, Div. of Nephrology and Hypertension, Univ. of California San Diego and VA San Diego Healthcare System, 3350 La Jolla Village Dr. (9151), San Diego, CA 92161 (e-mail: vvallon{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
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 DISCUSSION
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