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,,1
* Department of Medicine, University of California San Diego, San Diego, California, USA;
Veterans Affairs San Diego Healthcare System, San Diego California, San Diego, California, USA;
Department of Pharmacology, University of California San Diego, La Jolla, California, USA;
Department of Surgery, University of California San Diego, San Diego, California, USA; and
|| Department of Pediatric Nephrology, Hôpital Trousseau, Paris, France
1Correspondence: Departments of Medicine and Pharmacology, University of California San Diego and VA San Diego Healthcare System, 3350 La Jolla Village Dr. (9151), San Diego, CA, 92161 USA. E-mail: vvallon{at}ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-ENaC, greater medullary expression of the Na-K-2Cl-cotransporter NKCC2, and greater furosemide-sensitive Na+ reabsorption in association with greater renal medullary expression of aquaporin-2 and vasopressin-dependent renal cAMP formation and water reabsorption despite similar vasopressin levels compared with wild type. Of note, smaller increases in plasma aldosterone were required to adapt renal Na+ excretion to restricted intake in knockout mice, suggesting a facilitation in renal Na+ retention. The results thus identify a previously unrecognized role for P2Y2 receptors in blood pressure regulation that is linked to an inhibitory influence on renal Na+ and water reabsorption. Based on these findings in knockout mice, we propose that a blunting in P2Y2 receptor expression or activity is a new mechanism for salt-resistant arterial hypertension.—Rieg, T., Bundey, R. A., Chen, Y., Deschenes, G., Junger, W., Insel, P. A., Vallon, V. Mice lacking P2Y2 receptors have salt-resistant hypertension and facilitated renal Na+ and water reabsorption
Key Words: ATP • epithelial sodium channel ENaC • vasopressin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The investigation of genetic disorders affecting BP has led to the identification of genetic abnormalities associated with several rare forms of hypertension that affect renal salt reabsorption (2)
. Because such mutations appear to contribute to only a small extent to hypertension in the general population, greater understanding is needed regarding candidate genes and mechanisms that contribute to the regulation of renal salt and fluid transport and BP.
Extracellular nucleotides (e.g., ATP) regulate a broad range of physiological and pathophysiological processes through the activation of two classes of receptors, ligand-gated P2X and G protein-coupled P2Y receptors (3)
. Ion transport is one such physiological process that is regulated by nucleotide receptors. Various P2X and P2Y receptors are expressed in different regions of the kidney, including the vasculature and the glomerulus but also in the tubular epithelia and collecting duct system (4
5
6
7
8)
. Although accumulating evidence implicates P2 receptors in the regulation of renal epithelial transport (9)
, because of the lack of receptor subtype-specific antagonists, the in vivo role of such receptors is unclear.
The renal collecting duct is part of the aldosterone-sensitive distal nephron (ASDN), which expresses in its apical membrane the epithelial sodium channel ENaC, the latter being a primary target of the mineralocorticoid aldosterone and critically involved in the regulation of renal Na+ reabsorption and K+ excretion (10)
, as well as in genetic forms of arterial hypertension (2)
. In vitro application of ATP to the apical surface of mouse cortical collecting ducts reduces amiloride-sensitive short-circuit currents (11)
, indicating the inhibition of ENaC-mediated Na+ reabsorption by ATP. The pharmacological profile of these effects indicated that P2Y2 receptors might mediate these responses to ATP (11
, 12)
. In vivo evidence for an inhibitory effect of luminal ATP on Na+ reabsorption in the collecting duct has been provided in microperfusion experiments in rats (13)
. Subsequent experiments with more selective agonists, however, failed to identify the receptor responsible for this inhibitory effect. In addition to an effect on Na+ reabsorption, a variety of experimental approaches has provided evidence that local ATP release and P2Y2 receptor stimulation may inhibit water reabsorption in the collecting duct through the release of PGE2 and suppression of adenylyl cyclase activity (14
15
16
17
18)
.
The present studies aimed to identify the in vivo contribution of P2Y2 receptors to renal Na+, K+, and water transport and regulation of BP. To this end, we performed experiments in gene-targeted mice that lack P2Y2 receptors. Previous studies in these mice have identified important roles for the P2Y2 receptor in a variety of processes including neuronal growth (19)
, neutrophil chemotaxis (20)
, stimulation of K+ secretion in the colon (21)
, nucleotide-regulated Ca2+ signaling in lung fibroblasts and airway epithelial cells (22)
, and nucleotide-stimulated Cl– secretion in trachea and gallbladder (23)
, but neither BP nor renal function has been evaluated. We report here that such mice have salt-resistant hypertension in association with abnormalities in renal Na+ and fluid physiology.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Heterozygous P2Y2-deficient mice were back-crossed to C57BL/6J (Harlan Indianapolis, IN, USA) for a total of 10 generations. Heterozygous animals from the final backcross were bred to each other yielding P2Y2–/– mice from which a breeding colony was established. A breeding colony of C57BL/6J mice was established to provide wild-type mice. All animals were bred and raised under the same conditions at the University of California San Diego. Age-matched adult male mice were used for experiments.
Metabolic cage experiments in awake mice
Twety-four hour urine collections were performed in trained pairs of mice subjected to indicated diets for 7days (24
, 25)
. Diets applied included a standard diet (0.44%Na+; 0.97%K+), a high K+ diet (0.15%Na+; 5%K+), a low NaCl diet (0.02%Na+; 1%K+), and a high NaCl diet (0.85%NaCl in drinking water plus standard diet). Blood was taken by puncturing the retrobulbar plexus under brief isoflurane anesthesia.
Systolic arterial BP and heart rate in awake mice were determined by the tail-cuff method as described previously (26
, 27)
.
Diuretic and natriuretic responses in awake mice were determined over 2 h in response to intraperitoneal application of one of the following: furosemide (25 mg/kg); chlorothiazide (25 mg/kg); amiloride (2 mg/kg); or V2R antagonist SR121463 (1 mg/kg).
Clearance experiments under anesthesia
Experiments were performed in mice anesthetized with 100 mg/kg ip thiobutabarbital and 100 mg/kg im ketamine as described (28
, 29)
. The femoral artery was cannulated for direct blood pressure measurement. The jugular vein was cannulated for infusion (145 mM NaCl, 2.25 g/dl BSA; 500 µl/h/30g bw). For assessment of GFR and effective renal plasma flow, 3H-inulin (20 µCi/h/30 g bw) as well as 14C-labeled and unlabeled paraaminohippurate (PAH; 0.7 µCi/h/30g bw and 3.6 mg/h/30g bw) were added. Quantitative urine collections were performed using a bladder catheter.
Determination of plasma, urine and fecal concentrations
Na+ and K+ were determined by flame-emission photometry, aldosterone (Diagnostic System Laboratories, Webster, TX, USA) and renin concentration (GammaCoat, DiaSorin, Stillwater, MN, USA) by radioimmunoassay (30)
, PGE2 with a PGE2 EIA kit (Cayman Chemical, Ann Arbor, MI, USA) and urinary ATP by high-performance liquid chromatography (31)
.
Acute water loading was performed by oral gavage (3% of bw) and mice were then placed for 2 h in metabolic cages. Electolyte-free-water clearance {Cle-H2O = urinary flow rate ·[1-([Na+]urine+[K+]urine)/ [Na+]plasma)]} was calculated according to Rose (32)
where [Na+]urine, [K+]urine, and [Na+]plasma refer to the respective ion concentrations in urine or plasma, respectively.
Immunoblot analysis
Mice were sacrificed and kidneys either used whole or dissected into cortical and medullary sections prior to homogenization in buffer (250 mM sucrose, 10 mM triethanolamine). Immunoblotting was performed using the NuPage gel system (Invitrogen, Carlsbad, CA, USA). Equal lane loading (20 µg protein) was achieved using a Bio-Rad Protein assay (Bio-Rad, Richmond, CA, USA). Chemiluminescent detection was performed with ECL Advance (Amersham, Piscataway, NJ, USA). Antisera to NKCC2 and -ENaC were generous gifts from J. Loffing (University of Lausanne) and A. Doucet (Institut des Cordeliers), respectively. AQP2 antisera were from Santa Cruz Biotech (Santa Cruz, CA, USA) and caveolin-1 and GAPDH antisera from BD transduction labs (Franklin Lakes, NJ, USA). Densitometric analysis of immunoblots was performed using LabWorks 4.6 (UVP, Upland, CA, USA) and Prism 4.0 (GraphPad, San Diego, CA, USA) software.
IMCD preparation
IMCD were isolated using a modification of the method of Chou et al. (33)
. IMCD aliquots were incubated at 37°C for 10 min with 200 µM 3-isobutyl-1-methylxanthine (IBMX). In certain experiments, ATP
S (100 µM) was coapplied and desmopressin (1 pM-3 nM) was added and the incubation continued for 5 min. Reactions were terminated by addition of ice-cold 7.5% trichloroacetic acid and cAMP content of samples assessed by radioimmunoassay (34)
.
Statistics
Data are mean ± SE and were tested for significance using the Student’s t test. Only results with P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). P2Y2–/–, however, exhibited lower plasma concentrations of aldosterone, renin, and K+ as well as lower hematocrit values than WT (Fig. 1
A).
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WT and P2Y2–/– showed no significant differences in food and fluid intake or total fecal mass and urinary fluid excretion as determined in conscious mice in metabolic cages (Table 1)
. Moreover, no significant differences were observed in fecal or urinary excretion of Na+, K+ or Cl– (Table 1)
. The ratio of K+ to Na+ excretion was not significantly different in urine but was lower in feces of P2Y2–/– compared with WT (Fig. 1B
). The lower fecal ratio in P2Y2–/– was associated with, and thus may be the consequence of, lower plasma concentrations of aldosterone compared with WT (Fig. 1A
).
Basal arterial BP, GFR, and renal reabsorption
Renal clearance experiments were performed under inactin/ketamine anesthesia and arterial catheterization. P2Y2–/– showed a significantly greater systolic, diastolic and mean arterial BP than WT (Fig. 1C
). Heart rate was not different between P2Y2–/– and WT (487±22 vs. 480±18 beats/min, NS). Measurements of 3H-inulin and 14C-PAH clearances revealed no significant differences between P2Y2 –/– and WT in GFR and effective renal plasma (not shown) and blood flow, respectively (Fig. 1C
). These findings indicate that P2Y2–/– have arterial hypertension that is associated with intact renal autoregulation, as reflected by a greater renal vascular resistance (RVR) in the P2Y2–/– animals (Fig. 1C
). Despite the greater blood pressure in P2Y2–/–, absolute (not shown) and fractional urinary excretion of fluid, Na+, and K+ was not different between genotypes (Fig. 1C
), indicating altered renal function in P2Y2–/– mice receiving standard NaCl diet.
Arterial BP, heart rate and aldosterone in response to varying NaCl intake
Guyton proposed that hypertension is associated with impaired renal Na+ excretion and an abnormality of the renal function curve that describes the relationship between urinary output of Na+ and BP (1)
. We thus assessed these relationships in P2Y2–/– and WT mice by determining systolic arterial BP and heart rate by the tail-cuff method in trained conscious mice on standard NaCl diet and on either low or high NaCl diet for 7 days. Figure 2
A shows a shift to the right in the curve relating urinary output of Na+ and BP in P2Y2–/– compared with WT. In the range of Na+ intake/excretion tested, the slope of the curve was not different between genotypes, thus indicating a salt-resistant form of hypertension in P2Y2–/–. Conscious P2Y2–/– on standard NaCl diet had significantly lower heart rates than WT (Fig. 2A
). A role of the baroreceptor reflex in the lower heart rate was implicated by the finding that the differences in heart rate were absent under inactin/ketamine anesthesia (see above); barbiturate anesthesia has been shown to nearly abolish the heart rate response to aortic depressor nerve stimulation (35)
. Moreover, P2Y2–/– exhibited an inverse relationship between salt intake and heart rate (Fig. 2A
). These changes were associated with persistently lower plasma concentrations of aldosterone in P2Y2–/– irrespective of Na+ intake/excretion (Fig. 2A
). Notably, a smaller increase in plasma aldosterone concentrations in response to a low NaCl diet was sufficient to restrict renal Na+ excretion in P2Y2–/– compared to WT (
2- vs. 5-fold).
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Responses to dietary K+ loading
Plasma concentrations of K+ and aldosterone are key determinants of renal K+ excretion. P2Y2–/– showed evidence of facilitated renal K+ excretion, such that lower circulating K+ and aldosterone concentrations were sufficient to maintain renal K+ excretion (Fig. 1A
; Table 1
). Moreover, P2Y2–/– animals adapted urinary K+ excretion in response to high K+ intake for 7 days to similar values as WT and this occurred under conditions of similar plasma concentrations of aldosterone (Fig. 2B
) and K+ in P2Y2–/– and WT (5.3±0.3 vs. 4.7±0.1 mM, NS). Thus, facilitation of urinary K+ excretion and K+ balance by P2Y2 receptors dissipated under conditions that required higher rates of renal K+ excretion than occur on a standard diet.
Pharmacological blockade and renal expression of sodium transport proteins
To further identify the sites of facilitated or enhanced renal Na+ reabsorption in P2Y2–/– and assess the ambient contribution of specific transport mechanisms, we determined urinary Na+ excretion over a period of 2 h in conscious mice (receiving standard NaCl diet) in response to acute treatment with drugs that target specific Na+ transport mechanisms: furosemide [25 mg/kg ip; an inhibitor of Na+-2Cl–-K+-cotransporter NKCC2 in thick ascending limb (TAL)], chlorothiazide (25 mg/kg ip; an inhibitor of Na+-Cl–-cotransporter NCC in distal convoluted tubule), and amiloride (2 mg/kg ip; an inhibitor of ENaC in the ASDN; low doses of amiloride were used to prevent additional proximal tubular effects).
Chlorothiazide induced similar urinary Na+ excretions in both genotypes (Fig. 3
A). The response to amiloride was preserved in P2Y2–/– despite lower expression of -ENaC compared with WT, as determined by immunoblotting (Fig. 3B
). In comparison, both Na+ excretion in response to furosemide (Fig. 3A
) and the renal medullary expression of NKCC2 (Fig. 3C
) were greater in P2Y2–/– than WT, implying an enhanced NKCC2 activity in TAL of P2Y2–/–.
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Studies on basal renal water transport
Net urinary reabsorption and excretion of fluid, as well as urinary osmolality and electrolyte-free water clearance (Cle-H2O), were not different between P2Y2–/– and WT in basal metabolic cage or clearance experiments (see Fig. 1C
, Table 1
as well as Fig. 4
, the latter showing results from metabolic cage experiments in conscious mice). Whereas urinary excretion of PGE2 was not different (Fig. 4)
, the excretion of ATP and cAMP (Fig. 4)
and renal medullary expression of aquaporin-2 (Fig. 3C
) were greater in P2Y2–/– than WT despite similar 24h urinary excretion of vasopressin (Fig. 4)
, which is a surrogate for mean daily plasma vasopressin concentrations (36)
. These findings indicate that lack of P2Y2 receptors potentially increases adenylyl cyclase activity and aquaporin-2 expression without inducing net effects on urinary flow rate or osmolality (see Discussion for further explanation).
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To more directly assess signaling and transport mechanisms in the aquaporin-2-expressing segments, we determined the acute response to the vasopressin V2 receptor (V2R) antagonist SR121463 (1 mg/kg ip) (37)
. In WT animals SR121463 lowered urine cAMP (P<0.05) and osmolality and increased urinary flow rate and Cle-H20 (Fig. 4)
. The results for cAMP are consistent with previous studies showing that a portion of cAMP generated in the collecting duct is released from the cells (38)
and that vasopressin activation contributes to urinary cAMP (39)
. These changes in WT were associated with an increase in urinary PGE2 but a reduction in urinary ATP (Fig. 4)
, implying a tonic inhibition of PGE2 formation but stimulation of ATP release by V2R activation. In P2Y2–/–, SR121463 elicited a significantly greater diuresis and Cle-H2O associated with lower urinary osmolality compared with WT (Fig. 4)
, indicating greater basal reabsorption of fluid in the collecting duct of P2Y2–/– (consistent with greater urinary cAMP and aquaporin-2 expression in these animals).
SR121463 induced similar increases in urinary PGE2 in P2Y2–/– and WT and abolished the differences observed in basal urinary excretion of both ATP and cAMP (Fig. 4)
. Experiments in freshly isolated inner medullary collecting ducts (IMCD) revealed that cAMP formation induced by the V2R agonist, desmopressin, was inhibited by the stable P2 receptor agonist ATPS in WT but not P2Y2–/– (Fig. 5
). Together these results indicate that in the absence of P2Y2 receptors, vasopressin acting via the V2R has enhanced effects on cAMP formation (in the presence of a P2Y2 agonist) and aquaporin-2-mediated water reabsorption.
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Responses to acute water loading
Water loading (3% of body weight by oral gavage) was performed followed by quantitative urine collection over 2 h. In WT, this maneuver induced similar changes to those observed in response to V2R blockade in urine flow rate, urine osmolality and Cle-H20 (Fig. 4)
. Likewise, water loading led to decreased urinary cAMP and increased PGE2 (Fig. 4)
and these changes were associated with a reduction in urinary vasopressin excretion. In contrast to V2R blockade, however, water loading enhanced urinary ATP excretion (P<0.05).
In P2Y2–/–, the water load-induced increase in Cle-H2O was similar to that in WT, even though urinary vasopressin and cAMP excretion were significantly higher in P2Y2–/–. Moreover, this was associated with greater urinary excretion of both PGE2 and ATP in P2Y2–/–. The greater increase in urinary PGE2 in P2Y2–/– compared to WT may have caused greater urinary Na+ excretion (data not shown), as a mechanism to explain greater urine osmolality despite greater urinary flow rates in response to water loading (Fig. 4)
. Thus, in the absence of P2Y2 receptors, a lesser suppression of vasopressin and cAMP is sufficient to induce the same increase in free water excretion as in normal mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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41
42)
and humans (43)
. Thus, hypertension in P2Y2–/– may be salt-resistant because the baroreceptor response to variations in salt intake is intact.
Guyton proposed that all types of hypertension have impaired renal Na+ excretion and that the rise in BP stabilizes Na+ homeostasis by increasing renal Na+ excretion via pressure natriuresis (1)
. Notably, smaller increases in plasma aldosterone were required to adapt renal Na+ excretion to restricted intake in knockout mice, suggesting a facilitation in renal Na+ retention. Previous in vitro studies proposed a role for P2Y2 receptors in the suppression of ENaC-mediated Na+ reabsorption (11)
. Moreover, in the A6 cell line, the P2Y receptor agonist ATPS reduces the open probability of apical ENaC via activation of phospholipase C (44)
. Based on experiments in Xenopus oozytes and M1 collecting duct cells, Kunzelmann and coworkers (45)
proposed the following mechanism for the regulation of ENaC by P2Y2 receptors: under resting conditions the inner leaflet of the lipid bilayer contains a high concentration of phosphatidylinositol-bisphosphate (PIP2), the latter binding the N terminus of ß-ENaC, thereby maintaining ENaC channel activity. Stimulation of P2Y2 receptors activates phospholipase C, which hydrolyzes and lowers the concentration of PIP2 with resultant decreases in PIP2 binding to the N terminus of ß-ENaC and in ENaC activity by lowering the open probability. We found that despite lower ENaC expression, and thus potentially lower ENaC channel number in the apical membrane (46
, 47)
, amiloride-sensitive Na+ reabsorption was not significantly affected in P2Y2–/–. These in vivo findings would be consistent with the concept that lack of P2Y2 receptor activation results in greater ENaC open probability; compensatory decreases in circulating aldosterone concentrations and ENaC expression may serve to normalize net ENaC activity.
The current studies revealed greater furosemide-induced natriuresis and greater medullary NKCC2 expression in P2Y2–/– compared with WT. Salt reabsorption via NKCC2 in the water-impermeable thick ascending limb is an important determinant of the renal diluting capacity. In this regard, the combined finding of greater urinary flow rates in P2Y2–/– vs. WT in response to V2R blockade but similar natriuretic responses to amiloride provided indirect evidence for a greater delivery of a more hypotonic fluid to the collecting duct in knockout mice, which would be consistent with a greater diluting activity in the thick ascending limb. The mechanism(s) by which P2Y2 receptors may regulate NKCC2 remain(s) to be determined. Up-regulation of medullary NKCC2 has been proposed to contribute to hypertension in spontaneously (salt-sensitive) hypertensive rats (48
, 49)
, "programmed hypertension" induced by low-protein diet during pregnancy (50)
and the early phase of hypertension in the Milan strain of rats (51
, 52)
. Whether dysfunction of P2Y2 receptors contributes to those forms of hypertension will require further study.
Previous in vitro studies have implied that extracellular ATP acts via P2Y2 receptors to inhibit apical membrane small-conductance K+ channels of the cortical collecting duct (53)
, which may explain the facilitated renal K+ excretion in P2Y2–/– under standard K+ diet. Another potential factor in those animals is a greater flow rate of early distal tubules (as deduced from experiments with V2R blockade), which may enhance K+ secretion. Of note, the facilitation of urinary K+ excretion in P2Y2–/– dissipated in response to a high K+ diet. Thus, conditions requiring high rates of renal K+ excretion blunt the inhibition of such excretion by P2Y2 receptors.
The present studies provide evidence that V2R stimulation triggers P2Y2 receptor-mediated inhibition of cAMP formation. Thus, P2Y2 receptor activation can feedback inhibit cAMP-dependent effects of vasopressin and as a consequence, P2Y2–/– mice have greater expression of aquaporin-2 and greater reabsorption of fluid in the collecting duct compared with WT. Cell swelling induces ATP release (54)
and thus the greater fluid reabsorption may relate to greater basal urinary excretions of ATP in P2Y2–/–. Urinary ATP excretion was similar in the genotypes during inhibition of water transport by V2R blockade but modestly greater under basal conditions and much greater in response to acute water loading in P2Y2–/– compared with WT. We speculate that a lack of P2Y2 receptor-mediated feedback inhibition of water transport and activation of regulatory volume decrease results in greater cell volumes and ATP release. This idea is consistent with an autocrine/paracrine signaling role of ATP release and P2Y receptor activation, as suggested by studies of regulatory volume decrease in hepatocytes (55)
and biliary epithelial cells (56)
.
Although one view is that ATP released from epithelial cells in a number of different organs has autocrine and/or paracrine actions, many of these cells are also supplied by sympathetic nerves, which release ATP as a cotransmitter (57)
. Thus, basolateral P2Y2 receptors in the collecting duct may also be stimulated by ATP released from sympathetic nerves. ATP detected in urine is less likely to result from the latter source but from renal epithelia and perhaps from the lower urinary tract, where the release of ATP could be stimulated by distension or hypotonicity. Notably, water loading and V2R blockade induced similar increases in urinary flow rates and decreases in urinary tonicity in WT and thus potentially exposed the urothelial cells of the lower urinary tract to similar distension and hypotonicity but the observed effects on urinary ATP excretion were opposite in response to these two maneuvers.
Local formation of PGE2 can inhibit water, Na+ and urea uptake in collecting ducts (58
59
60)
. In both genotypes, V2R blockade and acute water loading increased urinary PGE2. Greater urinary PGE2 increases in response to water loading in P2Y2–/–, however, were unexpected, based on evidence that P2Y2 receptors mediate ATP-induced PGE2 formation and release in collecting ducts (15
, 17)
. The current findings imply that P2 receptors other than P2Y2 contribute to ATP-induced cellular PGE2 release and/or the lack of P2Y2 receptors facilitates cell swelling, which triggers enhanced release of PGE2 by other mechanisms.
Integrating the current findings, we propose that greater reabsorption of water in the collecting duct and potentially of Na+ via NKCC2 and/or via ENaC initially induce an isotonic hyperreabsorption in P2Y2–/–. Suppression of aldosterone and the induced pressure natriuresis, however, stabilize Na+ and fluid homeostasis (Fig. 6
). Impaired renal Na+ reabsorption can also induce secondary increases in peripheral resistance (1)
. ATP-evoked relaxation of the murine aorta is mainly mediated by P2Y2 receptors (61)
; however, the latter studies of P2Y2–/– mice did not include assessment of small arteries that are the primary determinants of peripheral resistance. Future experiments will need to assess if lack of vascular P2Y2 receptors contributes to increased BP in such mice.
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In summary, the current results with P2Y2–/– mice provide novel evidence for an in vivo role of P2Y2 receptors in the regulation of both BP and the renal reabsorption of Na+ and fluid. The results warrant further studies to explore effects of P2Y2 receptor agonism on renal Na+ and fluid transport and BP as well as the potential role of genetic polymorphisms of P2Y2 receptors (62)
in the pathophysiology of essential hypertension.
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ACKNOWLEDGMENTS |
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-ENaC were generous gifts from J. Loffing (University of Lausanne, Switzerland) and A. Doucet (Institut des Cordeliers, France), respectively. SR121463 was kindly provided by C. Serradeil-Le Gal (Sanofi-Aventis, Toulouse). Received for publication April 13, 2007. Accepted for publication May 17, 2007.
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, this induces greater deliveries of Na+ and fluid out of the proximal tubule 
. Greater Na+ reabsorption via NKCC2 in water impermeable TAL normalizes net Na+ delivery but causes the delivery of greater amounts of more hypotonic fluid to the early distal convoluted tubule 
. When the vasopressin system and thus the distal water permeability is suppressed, the latter facilitates free-water clearance. Under basal conditions, however, the excess water is reabsorbed via increased AQP2 activity, such that net urinary Na+ and fluid excretion is normal in P2Y2–/– 
.