Purinergic P2X₁ receptor, purinergic P2X₇ receptor, and angiotensin II type 1 receptor interactions in the regulation of renal afferent arterioles in angiotensin II-dependent hypertension

Abstract

In ANG II-dependent hypertension, ANG II activates ANG II type 1 receptors (AT₁Rs), elevating blood pressure and increasing renal afferent arteriolar resistance (AAR). The increased arterial pressure augments interstitial ATP concentrations activating purinergic P2X receptors (P2XRs) also increasing AAR. Interestingly, P2X₁R and P2X₇R inhibition reduces AAR to the normal range, raising the conundrum regarding the apparent disappearance of AT₁R influence. To evaluate the interactions between P2XRs and AT₁Rs in mediating the increased AAR elicited by chronic ANG II infusions, experiments using the isolated blood perfused juxtamedullary nephron preparation allowed visualization of afferent arteriolar diameters (AAD). Normotensive and ANG II-infused hypertensive rats showed AAD responses to increases in renal perfusion pressure from 100 to 140 mmHg by decreasing AAD by 26 ± 10% and 19 ± 4%. Superfusion with the inhibitor P2X₁Ri (NF4490; 1 μM) increased AAD. In normotensive kidneys, superfusion with ANG II (1 nM) decreased AAD by 16 ± 4% and decreased further by 19 ± 5% with an increase in renal perfusion pressure. Treatment with P2X₁Ri increased AAD by 30 ± 6% to values higher than those at 100 mmHg plus ANG II. In hypertensive kidneys, the inhibitor AT₁Ri (SML1394; 1 μM) increased AAD by 10 ± 7%. In contrast, treatment with P2X₁Ri increased AAD by 21 ± 14%; combination with P2X₁Ri plus P2X₇Ri (A438079; 1 μM) increased AAD further by 25 ± 8%. The results indicate that P2X₁R, P2X₇R, and AT₁R actions converge at receptor or postreceptor signaling pathways, but P2XR exerts a dominant influence abrogating the actions of AT₁Rs on AAR in ANG II-dependent hypertension.

INTRODUCTION

In ANG II-induced hypertension, ANG II activates ANG II type 1 receptors (AT₁Rs) inducing systemic hypertension and vasoconstriction (3, 50). Chronic activation of AT₁Rs elicits renal vasoconstriction and contributes to renal injury (16, 42). In particular, chronic ANG II infusions elicit sustained renal afferent arteriolar vasoconstriction as supported by the increased intrarenal levels of ANG II (46) and vasodilator responses to AT₁R blockade (47). However, purinergic P2X receptors (P2XRs) also exert substantial afferent arteriolar vasoconstriction in ANG II-induced hypertension, as evidenced by augmented levels of renal interstitial ATP (14) and renal microvascular responses to P2XR blockade (12).

Under normal and hypertensive conditions, there is a direct association between renal perfusion pressure (RPP) and renal interstitial ATP (14, 36, 37, 39) derived from sheer stress-induced release of ATP as well as release from macula densa cells stimulated by tubuloglomerular feedback signals (1). Further support is provided by a study that used an ATP-sensitive biosensor and reported that increases in arterial pressure increase cortical ATP levels (39). Studies using the juxtamedullary nephron preparation have demonstrated that interstitial ATP activates P2X₁Rs and elicits afferent arteriolar vasoconstriction contributing to the autoregulatory response to increases in perfusion pressure (20). Activation of P2XRs by increased concentrations of ATP has been implicated in the genesis and maintenance of salt-sensitive hypertension (14, 26), DOCA-salt-induced hypertension (27), and ANG II-induced hypertension (14). Interestingly, in a micropuncture study in ANG II-dependent hypertension, P2X₁R or P2X₇R blockade returned renal afferent arterial resistance to near-normal values even though blood pressure remained elevated, indicating the persistence of elevated systemic ANG II concentrations (12). These results provide basic evidence implicating a complex interaction between P2X₁R, P2X₇R, and AT₁R in regulating renal afferent arterial resistance and raise the conundrum of how P2XR and AT₁R can both be responsible for the increased afferent arteriolar vascular resistance existing in hypertensive conditions.

A possible explanation that resolves the conundrum is that P2XR and AT₁R share common receptor or postreceptor signaling mechanisms, which converge to elicit renal vasoconstriction in ANG II-induced hypertension. Accordingly, we posited that P2XR and AT₁R share pos-receptor signaling pathways but that P2XRs are dominant in the control of afferent arteriolar resistance. Because in in vivo studies AT₁R inhibition markedly reduces arterial pressure, which reduces interstitial ATP levels, the in vitro juxtamedullary nephron preparation was selected to allow maintenance of perfusion pressure at hypertensive levels as well as direct visualization of afferent arteriolar responses to AT₁R, P2X₁R, and P2X₇R inhibition either individually or combined.

MATERIALS AND METHODS

The experimental protocol was approved by Tulane University Institutional Animal Care and Use Committee. Sprague-Dawley male rats weighing 220–250 g (Charles River Laboratories) were housed in metabolic cages and maintained in a temperature-controlled room regulated on a 12:12-h light-dark cycle with free access to water and standard rat chow.

Induction of Hypertension

ANG II was infused into male Sprague-Dawley rats (220–250 g, Sigma, St. Louis, MO) via subcutaneous osmotic minipumps (Alzet model 2002, Durect, Cupertino, CA) implanted while the rats were under isoflurane anesthesia. A minipump delivered ANG II at a rate of 80 ng/min. Experiments were performed 13–14 days after the initiation of ANG II infusion. Hypertensive rats prepared in this manner have been shown to have increased renal interstitial ATP and ANG II tissue levels (14, 31, 46).

Measurement of Systolic Blood Pressure

Systolic blood pressure in awake rats was measured using tail-cuff plethysmography. In ANG II-infused rats, systolic blood pressure increased significantly throughout the duration of 13–14 days from 111 ± 5 to 183 ± 6 mmHg (n = 25). In normotensive rats, the range of systolic blood pressure was 101–127 mmHg (x̄ = 109 ± 7 mmHg, n = 15).

Juxtamedullary Nephron Preparation

Afferent arteriolar diameters (AAD) were assessed in vitro using the isolated blood-perfused juxtamedullary nephron technique combined with videomicroscopy, as previously described (11, 41). Each experiment used one male Sprague-Dawley rat (Charles River Laboratories) weighing 300–380 g serving as the kidney donor as well as the blood donor. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a cannula was inserted in the left carotid artery for blood collection. Donor blood was collected in a heparinized (500 units) syringe via the carotid arterial cannula and centrifuged to separate the plasma and cellular fractions. The buffy coat was removed and discarded. After sequential passage of the plasma through 5-µm and 0.22-µm filters (Gelman Sciences, Ann Arbor, MI), erythrocytes were added to achieve a hematocrit of 33%. This reconstituted blood was passed through a 5-µm nylon mesh and thereafter stirred continuously in a closed reservoir that was pressurized with a 95% O₂-5% CO₂ gas mixture. The right kidney was perfused through a cannula inserted in the superior mesenteric artery and advanced into the right renal artery. The perfusate was Tyrode solution (pH 7.4) containing 5.1% BSA and a mixture of l-amino acids. The kidney was excised and sectioned longitudinally, retaining the papilla intact with the perfused dorsal two-thirds of the organ. The papilla was reflected to expose the pelvic mucosa and tissue covering the inner cortical surface. Overlying tissue was removed to expose the tubules, glomeruli, and related vasculature of the juxtamedullary nephrons. The arterial supply of the exposed microvasculature was isolated by ligating the larger branches of the renal artery. After the dissection was completed, the Tyrode perfusate was replaced with the reconstituted blood. Perfusion pressure was monitored by a pressure catheter centered in the tip of the perfusion cannula. RPP was regulated by adjusting the rate of gas inflow into the blood reservoir and set at 100 mmHg. The inner cortical surface of the kidney was continuously superfused with warmed (37°C) Tyrode solution containing 1% BSA. The tissue was transilluminated on the fixed stage of a microscope (Nikon) equipped with a water immersion objective (40). Video images of the microvessels were transferred by a Newvicon camera (model NC-67M, Dage-MTI, Michigan City, IN) through an image enhancer (model MFJ-1452, MFJ Enterprises, Starkville, MS) to a video monitor (Conrac Display Systems, Covina, CA), and single afferent arterioles were visualized. The video signal was recorded on DVD for later analysis. AADs were measured at 5-min intervals using a calibrated digital image-shearing monitor (Instrumentation for Physiology and Medicine, San Diego, CA).

Experimental Protocols

A single afferent arteriole close to its glomerulus that showed robust blood flow was selected for study. After a 10-min equilibration period, an experimental protocol was initiated, consisting of three to five periods of a 5-min duration. For each treatment, doses of the inhibitor compounds were selected on the basis of pilot experiments, and the following doses were used in the study: 1) 1 µM AT₁ receptor inhibitor (SML1394, olmesartan, Sigma), 2) 1 µM P2X₁R inhibitor (NF449, Calbiochem), and 3) 1 µM P2X₇R inhibitor (A438079, Calbiochem).

Autoregulatory Responsiveness

Initially, the autoregulatory responses were assessed by measurement of AAD at a control RPP of 100 mmHg and following an acute increase in RPP to 140 mmHg. AADs were measured in kidneys from normotensive (n = 15) and hypertensive (n = 25) rats.

Experiments on Kidneys From Normotensive Rats

Effects of AT₁R, P2X₁R, and P2X₇R blockade on AAD at increased RPP of 140 mmHg.

AAD was measured at a RPP of 100 mmHg and at increased RPP to 140 mmHg, which was maintained. To evaluate the influence of AT₁R and P2XR, AAD was determined before and during treatment with each selective blocker by superfusion of solutions containing 1 µM SML1394 (n = 5) or 1 µM NF449 (n = 5) or 1 µM A438079 (n = 5).

Blockade of ATP action on afferent arterioles with P2X₁R and P2X₇R blockers.

Because ATP is the endogenous ligand for P2XRs, these experiments were designed to determine the efficacy of P2XR blockers in reversing the vasoconstrictor action of 5 µM ATP (Sigma). AAD was measured at a RPP of 100 mmHg before and during treatment with ATP alone and with superimposed P2X₁R blockade with 1 µM NF449 (n = 4) or P2X₇R blockade with 1 µM A438079 (n = 4) and then with combined 1 µM NF449 plus 1 µM A438079 in each group.

Effect of AT₁R and P2X₁R blockade on AAD during treatment with ANG II at a RPP of 100 mmHg followed by an increase in RPP to 140 mmHg.

The effect of ANG II on AAD was assessed using ANG II (1.0 nM) with RPP maintained at 100 mmHg (n = 7). After a 5-min control period, RPP was increased to 140 mmHg with continuous superfusion with ANG II, and AAD was determined. The P2X₁R blocker (NF449) was then superfused for 5 min followed by the addition of the AT₁R blocker (SML1394) to determine the residual effect of ANG II on AAD.

Experiments on Kidneys From Hypertensive Rats

Effect of AT₁R, P2X₁R, and P2X₇R blockade on afferent arterioles maintained at increased RPP in hypertensive rat kidneys.

These experiments were designed to determine the role of the AT₁R on afferent arterioles (n = 5) of chronic ANG II-infused hypertensive rats, which have increased renal interstitial ATP as well as elevated ANG II levels. AAD was measured at perfusion pressures of 100 and 140 mmHg. With RPP maintained at 140 mmHg, the tissue was superfused with 1 µM SML1394, and AAD was determined. The superfusate was changed to 1 µM NF449, and AAD was determined. In addition, AAD responses to combined treatment with NF449 plus A439079 were determined.

Effect of AT₁R, P2X₇R, and P2X₁R plus P2_X7R blockade on AAD at increased RPP.

These experiments were designed to evaluate the effects of AT₁R blockade on afferent arterioles (n = 5). While RPP was held at 140 mmHg, the superfusion solution containing 1 µM SML1394 was superfused, and the change in ADD was determined. Subsequently, the superfusate was changed to 1 µM A439079, and AAD was determined. Finally, arteriolar responses to combined treatment with NF449 plus A439079 were determined.

Effect of P2X₁R and AT₁R blockade on AAD at increased RPP.

This series was designed to determine the effects of P2X₁R blockade to vasodilate afferent arterioles at elevated RPP followed by additional AT₁R inhibitor (n = 5). RPP was increased from 100 to 140 mmHg; the arteriolar response to 1 µM NF449 was then determined. After 5 min, during the continued presence of NF449 effect, the superfusion solution with 1 µM SML1394 was added, and the additional change in AAD was determined.

Effect of P2X₇R and AT₁R blockade on AAD at increased RPP.

These experiments were performed to determine the effects of P2X₇R blockade to vasodilate afferent arterioles (n = 5). RPP was increased from 100 to 140 mmHg. The change in AAD following superfusion with 1 µM A438079 was determined. After 5 min with continued treatment with A438079 effect, 1 µM SML1394 was added, and AAD was determined.

Effects of P2X₁R plus P2X₇R and AT₁R blockade on AAD following increases in RPP to 140 mmHg.

These experiments were performed to determine the effects of combined treatment with P2X₁R plus P2X₇R blockade on AAD with superimposed AT₁R blockade (n = 5). Following the increase in RPP from 100 to 140 mmHg, the arteriolar response to 1 µM NF449 plus 1 µM A438079 was determined. After 5 min in the continued presence of NF449 plus A438079, the effect of superfusion with 1 µM SML1394 was determined.

Statistical Analysis

Data were evaluated using Prism software (Graph Pad, San Diego, CA). Results were evaluated using one-way ANOVA with repeated measures followed by the Newman-Keuls multiple-range test. P values of <0.05 were considered statistically significant. All values are expressed as means ± SD.

RESULTS

Systolic Blood Pressure Measured in Awake Rats During the Development of Hypertension

In ANG II-infused rats, systolic blood pressure increased progressively throughout the duration of 13–14 days from 111 ± 5 to 183 ± 6 mmHg (n = 25). In normotensive rats, the range of systolic blood pressure was 101–127 mmHg with an average of 109 ± 7 mmHg (n = 15).

Autoregulatory Capability in Kidneys From Normotensive and Hypertensive Rats

Changes in AAD were measured at the control RPP of 100 mmHg and in response to increases in RPP, which elicited pressure-mediated autoregulatory reductions in AAD. Under control conditions of 100 mmHg, AAD from normotensive rats averaged 12.9 ± 1.2 μm and decreased by 26 ± 10% (9.5 ± 1.6 μm, n = 15, P < 0.05) when RPP was increased to 140 mmHg. In kidneys from ANG II-infused hypertensive rats, the increase in RPP from 100 to 140 mmHg decreased AAD by 19 ± 4% (14.9 ± 0.1 vs. 12 ± 0.2 μm, n = 25, P < 0.05), suggesting reduced autoregulatory capability in AAD from hypertensive rats, as has been previously reported (19).

Experiments in Kidneys From Normotensive Rats

AAD responses to AT₁R, P2X₁R, and P2X₇R inhibition at increased RPP.

In separate experiments, we determined the effects of AT₁R, P2X₁R, and P2X₇R inhibition on AAD after pressure-mediated autoregulatory responses. In this series, increasing RPP from 100 to 140 mmHg decreased AAD from 12.9 ± 2 to 9.4 ± 1.6 μm. Treatment with AT₁R inhibitor alone did not alter AAD (9.4 ± 1.3 vs. 9 ± 1.1 μm, n = 5; Fig. 1A). Upon treatment with P2X₁R inhibitor, AAD returned to a value similar to that at a RPP of 100 mmHg (from 9.1 ± 2.1 to 13.2 ± 1.7 μm, n = 5, P < 0.05; Fig. 1B). Treatment with P2X₇R inhibitor did not vasodilate AAD as much (from 10 ± 1.4 to 10.8 ± 1.9 μm, n = 5, P < 0.05; Fig. 1C), indicating a minor role of P2X₇R in the augmented preglomerular resistance in normal kidneys, consistent with a study that showed a lower abundance of P2X₇R protein in kidneys from normotensive rats (12).

Fig. 1.

Responses of afferent arterioles to ANG II type 1 receptor (AT₁R), purinergic P2X₁ receptor (P2X₁R), and purinergic P2X₇ receptor (P2X₇R) inhibition under renal perfusion pressure (RPP) at 140 mmHg in kidneys from normotensive rats. A−C: afferent arteriolar responses to AT₁R inhibitor (AT₁Ri; A), P2X₁R inhibitor (P2X₁Ri; B), and P2X₇R inhibitor (P2X₇Ri; C). Data are expressed as means ± SD; n = 5 per group. *P < 0.05 vs. RPP at 100 mmHg; #P < 0.05 vs. RPP at 140 mmHg.

AAD responses to ATP followed by P2X₁R and P2X₇R blockade.

The effects of 5 µM ATP on AAD at control normotensive pressure showed a rapid initial vasoconstriction from a control of 14.5 ± 0.9 μm to 11.2 ± 0.7 μm (n = 8, P < 0.05). P2X₁R inhibition restored AAD from 10.9 ± 0.6 to 14.0 ± 0.8 μm (P < 0.05). Further treatment with combined P2X₁R plus P2X₇R inhibitors did not cause further vasodilation (Fig. 2A). In separate experiments, P2X₇R inhibitors did not vasodilate AAD significantly (12.2 ± 0.6 vs. 11.5 ± 0.7 μm). However, combined treatment with P2X₁R plus P2X₇R inhibitor returned AAD to values similar to control (14.8 ± 0.7 μm; Fig. 2B).

Fig. 2.

Afferent arteriolar responses to ATP alone followed by ATP plus inhibition of P2X₁ receptor (P2X₁R) or the combination of P2X₁R + P2X₇ receptor (P2X₇R) inhibition in kidneys from normotensive rats at baseline pressure. After steady-state diameters were established, ATP (5 µmol/L) was superfused over the blood vessels. A: afferent arteriolar responses to ATP followed by ATP plus P2X₁R inhibitor (P2X₁Ri) and ATP plus P2X₁Ri and P2X₇R inhibitor (P2X₇Ri). B: the order of the inhibitors was changed, and afferent arteriolar responses to ATP and ATP plus P2X₇Ri were measured followed by ATP under P2X₁Ri + P2X₇Ri (n = 4 per group). Data are expressed as means ± SD. *P < 0.05 vs. renal perfusion pressure (RPP) at 100 mmHg; #P < 0.05 vs. RPP at 100 mmHg + ATP; &P < 0.05 vs. RPP at 100 mmHg + ATP + P2X₇Ri.

Effects of P2X₁R and AT₁R inhibition in the presence of elevated ANG II and increased RPP.

To simulate conditions in kidneys from hypertensive rats, we tested the acute effects of P2X₁R inhibition in the presence of elevated ANG II and increased RPP. ANG II (1 nM) caused significant decreases in AAD by 16 ± 4% (11.9 ± 1.2 vs. 10.0 ± 1.4 μm); an increase in RPP caused further decreases in AAD by 19 ± 5% to 8.1 ± 0.7 μm (n = 7, P < 0.05). Treatment with P2X₁R inhibitor significantly increased AAD by 30 ± 6% to values greater than the value observed at 100 mmHg plus ANG II (10.5 ± 1.1 μm) and similar to the baseline value at 100 mmHg. In the presence of P2X₁R inhibition, further treatment with AT₁R inhibitor increased AAD only slightly more by 11 ± 7% (11.6 ± 1.1 μm), bringing it closer to the baseline value at 100 mmHg (11.9 ± 1.2 μm; Fig. 3).

Fig. 3.

Acute effect of ANG II infusion in afferent arterioles under high renal perfusion pressure (RPP) followed by P2X₁ receptor (P2X₁R) or ANG II type 1 receptor (AT₁R) inhibition in normotensive rats. The afferent arteriolar response to ANG II (1 nmol/L) superfusion of the vessel at 100 mmHg was followed by a step increase in RPP to reach 140 mmHg. Superfusion of the P2X₁R inhibitor (P2X₁Ri) was then followed by the AT₁R inhibitor (AT₁Ri; n = 7). Data are expressed as means ± SD. *P < 0.05 vs. RPP at 100 mmHg; #P < 0.05 vs. RPP at 100 mmHg + ANG II; &P < 0.05 vs. RPP at 140 mmHg + ANG II.

Experiments in Kidneys From Hypertensive Rats

Interactions between P2XR and AT₁R on AAD at elevated RPP.

ANG II-infused hypertensive rats have increased interstitial ATP and ANG II tissue levels, indicating that, under these conditions, it is possible that both P2XR and AT₁R regulate AAD. In this series, we determined the interactions between P2X₁R, P2X₇R, and AT₁R on AAD at the increased perfusion pressure. An increase in RPP from 100 to 140 mmHg significantly decreased AAD by 20 ± 5% (14.9 ± 0.9 vs. 11.9 ± 1.2 μm, P < 0.05). Treatment with P2X₁R inhibitor significantly increased AAD by 20 ± 14% (14.4 ± 0.5 μm, P < 0.05) to values similar to those observed at a RPP of 100 mmHg; AAD increased slightly to 15.3 ± 0.7 μm upon treatment with the AT₁R blocker (Fig. 4A). In separate experiments, initial treatment with the AT₁R inhibitor increased AAD by 8 ± 5% (13.2 ± 0.8 vs. 12.2 ± 0.6 μm, P < 0.05). Superimposed treatment with P2X₁R inhibitor significantly increased AAD by 12 ± 5% (14.8 ± 0.6 μm, P < 0.05) to values similar to those at a RPP of 100 mmHg (14.7 ± 0.6 μm). Further treatment with P2X₁R plus P2X₇R inhibitors elicited further increases in AAD to 15.3 ± 0.8 μm, which were similar to baseline values (Fig. 4B).

Fig. 4.

Effect of ANG II type 1 receptor (AT₁R) and P2X₁ (P2X₁R) and P2X₇ receptor (P2X₇R) blockade on afferent arteriolar diameter under high renal perfusion pressure (RPP) in kidneys from hypertensive rats. A: afferent arteriolar responses to increases in RPP from 100 to 140 mmHg were tested followed by P2X₁R inhibitor (P2X₁Ri) and then AT₁R inhibitor (AT₁Ri). B: the order of the inhibitors was reversed. First, afferent arteriolar responses to increases in RPP were followed by AT₁Ri and P2X₁R blockade (P2X₁Ri); then, the combination of P2X₁R plus P2X₇R inhibitor (P2X₁Ri) was tested. Data are expressed as means ± SD; n = 5 per group. *P < 0.05 vs. RPP at 100 mmHg; #P < 0.05 vs. RPP at 140 mmHg; &P < 0.05 vs. RPP at 140 mmHg + AT₁Ri.

Effects of AT₁R, P2X₇R, and P2X₁R plus P2X₇R blockade on AAD at elevated RPP.

In this series, increasing RPP from 100 to 140 mmHg decreased diameters by 22 ± 1% (14.4 ± 0.7 vs. 11.3 ± 0.6 μm, P < 0.05). Treatment with the P2X₇R inhibitor significantly increased AAD by 15 ± 3% (13.0 ± 0.6 μm, P < 0.05). AAD increased further by 10 ± 1% (14.3 ± 0.7 μm, P < 0.05) by treatment with the AT₁R inhibitor (Fig. 5A). In separate experiments, initial treatment with the AT₁R blocker increased AAD by 12 ± 8% (13.7 ± 0.7 vs. 12.2 ± 0.7 μm, P < 0.05). Further treatment with P2X₇R inhibitor significantly increased AAD by 7 ± 5% (14.7 ± 0.7 μm, P < 0.05) to a value similar to that at a RPP of 100 mmHg (15.1 ± 0.5 μm). Additional treatment with P2X₁R plus P2X₇R inhibitors fully restored AAD to 15.4 ± 0.8 μm (Fig. 5B).

Fig. 5.

Effects of P2X₇ receptor (P2X₇R) or ANG II type 1 receptor (AT₁R) inhibition alone and P2X₁ receptor (P2X₁R) plus P2X₇R blockade on the afferent arteriolar response to an increase in renal perfusion pressure (RPP) in hypertensive rats. A: afferent arteriolar responses to increases in RPP were followed by P2X₇R inhibitor (P2X₇Ri) and AT₁R inhibitor (AT₁Ri). B: afferent arteriolar responses to increases in RPP followed by AT₁Ri and P2X₇R inhibitor (P2X₇Ri) and the combination of P2X₁Ri plus P2X₇Ri. Data are expressed as means ± SD; n = 5 per group. *P < 0.05 vs. RPP at 100 mmHg; #P < 0.05 vs. RPP at 140 mmHg; &P < 0.05 vs. RPP at 140 mmHg + AT₁Ri; ^αP < 0.05 vs. RPP at 140 mmHg + P2X₇Ri.

Effect of P2X₁R plus P2X₇R and AT₁R blockade on AAD at elevated RPP.

In this series, increasing RPP from 100 to 140 mmHg significantly decreased diameters by 19 ± 5% (14.9 ± 0.7 vs. 12 ± 0.7 μm, P < 0.05). Treatment with P2X₁R plus P2X₇R inhibitors significantly increased AAD by 25 ± 8% (15 ± 0.7 μm, P < 0.05) to a value not significantly different from the value at a RPP of 100 mmHg. Further treatment with AT₁R inhibitor after treatment with the P2X₁R inhibitor plus P2X₇R inhibitor did not significantly modify AAD (15.4 ± 0.5 μm, not significant; Fig. 6).

Fig. 6.

Effect of P2X₇ receptor (P2X₇R), P2X₁ receptor (P2X₁R), and ANG II type 1 receptor (AT₁R) blockade on the afferent arteriolar response to increased renal perfusion pressure (RPP) in hypertensive rats. Afferent arteriolar responses to increases in RPP (n = 5) were followed by treatment with P2X₁R inhibitor (P2X₁Ri) plus P2X₇R inhibitor (P2X₇Ri) and after the addition of AT₁R inhibitor (AT₁Ri) alone. Data are expressed as means ± SD. *P < 0.05 vs. RPP at 100 mmHg; #P < 0.05 vs. RPP at 140 mmHg.

DISCUSSION

Under inappropriate stimulation of the renin-angiotensin system, the increased circulating and tissue ANG II concentrations acting on AT₁Rs increase systemic vascular resistance. The accompanying progressive increase in renal vascular resistance is due predominantly to the afferent and efferent arteriolar vasoconstriction mediated primarily by AT₁R activation due to the increased ANG II concentrations (34). During the early stages of ANG II-dependent hypertension, renal arterioles are highly responsive to angiotensin-converting enzyme inhibition and angiotensin receptor blockers, indicating the predominant influence of the augmented ANG II levels activating AT₁Rs (34). The increased renal vascular resistance is mediated by both the direct effect of AT₁R actions on renal vascular smooth muscle as well as augmentation of the sensitivity of the tubuloglomerular feedback mechanism (34). There is strong evidence based on cross-transplantation studies that ANG II causes hypertension through direct actions on kidney AT₁Rs (6). Also, ANG II-dependent responses of the renal circulation were almost abolished in AT₁R knockout mice (6). In particular, afferent arteriolar reactivity to ANG II is enhanced in early stages of angiotensin-dependent hypertension (17).

Furthermore, in response to increases in RPP, intrarenal paracrine factors, including autacoids of the purinergic system, become activated. Increases in arterial pressure result in progressive increases in interstitial levels of ATP, which acts on P2XRs and P2Y receptors (P2YRs). Functional evidence for P2XR and P2YRs to the overall regulation of renal afferent arterioles and autoregulatory behavior has been provided in several studies (18, 22, 38). P2Y₁R and P2Y₂R are also expressed in afferent arterioles, whereas P2Y₁Rs are expressed only in efferent arterioles (44). P2YR stimulation by low concentrations of selective agonists such as UTP or ATP-γ-S elicited vasodilation, but vasoconstriction occurred at higher concentrations (5, 9). However, the role of P2YRs in autoregulation and tubuloglomerular feedback responses remains uncertain (18, 22, 38). On the other hand, P2XRs, particularly P2X₁R, elicit marked afferent arteriole vasoconstriction potentially compounding the ANG II-mediated effects.

During acute and transient increases in arterial pressure, the increases in ATP released by endothelial cells in response to increases in shear stress and by macula densa cells in response to tubuloglomerular feedback mechanism-mediated signals provide the protective regulation of afferent arteriolar resistance to maintain glomerular pressure and glomerular filtration rate. The dialysis method has shown sustained increases in interstitial ATP concentrations at increased arterial pressure (25, 37). A more recent study has demonstrated increases in interstitial ATP using a real-time electrochemical detection method that provides continuous measurement of interstitial ATP (39). Thus, this mechanism provides a rapid critical autoregulatory protective function preventing the increases in blood pressure from disrupting the kidney’s homeostatic function or causing glomerular and microvascular injury by excessive intrarenal pressures.

Arterial pressure-induced increases in intrarenal ATP levels are sustained in ANG II-dependent hypertension and presumably continue to provide the protective function that is served in acute situations (14). Thus, in ANG II-dependent hypertension, two powerful vasoconstrictor systems are each extending their influence on the renal vasculature, but whether their interactions are additive, overlapping, or synergistic remains unclear. If additive, the kidney’s homeostatic function would be threatened by devastating excessive vasoconstriction, which does not occur. Yet, when treated with angiotensin receptor blockers, the renal vascular resistance decreases toward normal levels in association with decreases in arterial pressure, thus maintaining or even increasing renal blood flow and glomerular filtration rate (4). This response suggests that AT₁R activity is dominant, but the contribution of the decreased renal vascular resistance associated with the reductions in interstitial ATP caused by the decreases in arterial pressure is difficult to determine. In contrast, administration of P2XR inhibitors counteracts the activation of P2XRs in the kidney without causing associated changes in arterial pressure (12, 14). Indeed, P2XR blockade chronically or acutely administered to ANG II-induced hypertensive rats does not reduce the elevated arterial pressure but elicits profound changes in segmental renal vascular resistance (12). In a series of micropuncture studies, which allowed evaluation of glomerular dynamics in response to nonselective (13) or selective (12) P2XR inhibition, the results suggested a marked dominance of P2XRs on segmental vascular resistance. The combination of P2X₁R and P2X₇R completely normalized afferent arteriolar resistance in ANG II-infused hypertensive rats and increased single nephron plasma flow and filtration rate as well as glomerular pressure to values above normal because of the maintained arterial pressure. Under these conditions of maintained arterial pressure, it appeared that the P2XR effect was clearly dominant in regulating renal vascular resistance, thus raising the conundrum of how both angiotensin via AT₁R and ATP via P2XR could be exerting the dominant effect to regulate renal vascular resistance in hypertension. To address this conundrum, it was necessary to study the actions of blockade of AT₁R and P2XR, either alone or in combination, at hypertensive perfusion pressures, which could be maintained independent of the changes in renal vascular resistance. To achieve this requirement and to view directly the responses of the afferent arterioles, we used the in vitro blood perfused juxtamedullary nephron preparation. Importantly, the kidney preparation was perfused with its own blood so that kidneys from hypertensive rats were perfused with blood having the composition as affected by the 2 wk of ANG II perfusion.

We first evaluated the responses in kidneys from normotensive rats to determine the relative interactions between AT₁R and P2XR. There are no pure selective antagonists for P2XRs. However, there are some antagonists with higher selectivity for certain receptors, such as NF449 for P2X₁R and A438079 for P2X₇R. These antagonists have a higher affinity for their receptors (7, 28, 29, 35). In normotensive rats studied at normotensive pressures, ATP elicited a robust afferent constriction, which was blocked almost completely by the P2X₁R inhibitor, whereas there was no significant effect of P2X₇R inhibition. These results are consistent with data indicating that renal P2X₇R abundance is very low in normotensive rats (12). In response to an increase in RPP from 100 to 140 mmHg, kidneys from normotensive rats responded with a robust afferent arteriolar constriction that was mitigated primarily by the P2X₁R inhibitor without contribution of the P2X₇R inhibitor. Furthermore, AT₁R blockade did not modify AAD significantly. These data are consistent with previous findings that the vasoconstriction caused by α,β-methylene ATP, a potent P2X₁R agonist, was blocked by NF279 (20, 23). Additionally, P2X₁R knockout mice displayed an impaired pressure-mediated autoregulatory response (21). Blockade of AT₁Rs did not modify the response of AAD to the increase in RPP to 140 mmHg. These data are consistent with the findings that autoregulatory responses are not mediated by AT₁Rs (25) but are strongly dependent on P2X₁R activation, indicating that P2X₁R serves as an important mediator of vascular resistance responses to increases in RPP. They are also consistent with the localization of P2X₁R in the smooth muscle of afferent arterioles from normal rats (12, 44, 45). In contrast, no changes were observed with superimposed P2X₇R inhibition, which is consistent with the low abundance of P2X₇R in normal arterioles (31, 45).

To further study the interaction between AT₁R and P2XR in kidneys from control rats, we simulated “in vivo” conditions by superfusing ANG II over the tissues and clearly demonstrated ANG II-mediated constriction. The addition of ANG II at baseline RPP caused a 10% decrease in AAD. Even in the presence of ANG II, an increase in RPP to 140 mmHg caused a 19% further decrease in AAD. P2X₁R blockade returned AAD to a value between those seen at baseline RPP and with ANG II superfusion. With the addition of the AT₁R inhibitor, AAD returned to baseline values. These results indicate a predominant P2X₁R effect on the autoregulatory component along with a maintained influence of AT₁Rs in the regulation of AAD during combined elevation in arterial pressure and acute treatment with ANG II.

To evaluate transformation effects in chronic ANG II-dependent hypertension, afferent arteriolar responses in kidneys from ANG II-infused hypertensive rats demonstrated greater interactions between the effects of AT₁R and P2X₁R. Blockade of P2X₁R at high RPP mitigated the AT₁R-indued constriction in renal afferent arterioles. These findings are consistent with increased expression of P2X₁R in this model of hypertension (12).

In afferent arterioles from hypertensive rats, specific P2X₁R or P2X₇R blockade returned renal AAD to near-normal values, and only very slight effects of AT₁R blockade were observed. When the AT₁R blocker was used first, there was a small but distinctive increase in AAD, which indicates a small residual effect of AT₁R. Addition of the P2X₁R and P2X₇R inhibitors restored AAD to control levels. The ability of P2X₇R inhibitor to significantly augment AAD can be attributed to a higher expression of P2X₇R in afferent arterioles of hypertensive rats (12).

Collectively, these results provide functional evidence that renal P2X₁R and P2X₇R exert dominant roles in the regulation of AAD in ANG II-infused hypertension and abrogate the AT₁R influence on AAD. Understanding how these two receptors interact during the signaling process is crucial to understand the control of renal afferent arteriolar resistance under hypertension.

The intracellular mechanisms by which P2XR and AT₁R interact will need to be studied further. AT₁R is a G protein-coupled receptor, and P2XRs are ligand-gated ion channels. Both P2XR and AT₁R mediate vasoconstriction via an increase in cytosolic Ca²⁺, but the intracellular mechanisms are different. Stimulation of AT₁R activates Gα_q/11 protein, which induces the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) and production of inositol 1,4,5-trisphosphate and diacylglycerol by activation of phospholipase C-β. Inositol 1,4,5-trisphosphate then induces intracellular Ca²⁺ mobilization from the endoplasmic reticulum to the cytosol. In contrast, activation of ligand-gated ion channels causes a remarkable influx of Ca²⁺ from the extracellular compartment (8), which leads to further increases in Ca²⁺ release from intracellular stores. This concert of regulation of Ca²⁺ signaling pathways plays a key role in controlling G protein-couple receptor- and ligand-gated ion channel-mediated renal arteriolar vasoconstriction.

PIP₂, as a cofactor, may also be a critical regulatory factor for the P2X₁R and P2X₇R signaling pathway. It has been shown that the P2XR intracellular domain, containing a dual cluster of basic amino acids, enables PIP₂ binding (2), which strongly potentiates the activities of P2X₁R and P2X₇R. It has been suggested that protein-PIP₂ interaction induces a conformational change in the transmembrane segment (TM2) linking to the proximal COOH-terminal domain, which can influence channel opening activity. This evidence suggests that AT₁R shares an important signaling pathway with P2X₁R and P2X₇R involving PIP₂. Among the downstream components involved in the AT₁R signaling pathway, AT₁R is not limited to Gα_q/11 protein but also increases Rho kinase activity via G_l2/l3. During ANG II treatment, Rho kinase inhibition in ANG II-infused rats led to greater vasodilation compared with control rats (32, 49). These data suggest that ANG II-activated AT₁R-mediated activation of the Rho kinase pathway contributes to the increased afferent arteriolar resistance. Interestingly, the P2X₁ receptor agonist α,β-methylene ATP reduced AAD, but this response was eliminated during Rho kinase inhibition (24, 34). These data demonstrate that Rho kinase activation is an important part of the intracellular signaling mechanisms that connect P2XR activation with vasoconstriction. Thus, it is possible that Rho kinase contributes to AT₁R and P2XR signaling interactions (15).

Besides posttranslational interactions that mediate downstream signaling pathways, it is known that G protein-coupled receptors can interact directly with ligand-gated ion channels, leading to a functional and reciprocal modulation of each receptor type (30, 48). P2XRs are composed of three subunits, and the COOH-terminal tail of P2XR subunits interacts directly with the main intracellular loop of other membrane receptors. One noticeable feature of P2X₇R is that it has a long COOH-terminal tail (43), a structural factor that facilitates physical interaction between receptors. The new insights of receptor interaction showed that AT₁R can form complexes with other receptors (10, 13, 33, 40). However, a physical interaction between AT₁R and P2XR has not been reported, but it would be interesting to investigate the possible physical interactions between P2X₁R or P2X₇R and AT₁R in the control of renal afferent arteriolar resistance.

In conclusion, this work provides functional evidence that renal P2X₁R and P2X₇R exert dominant roles in the regulation of AAD in kidneys from ANG II-dependent hypertensive rats during elevation in arterial pressure and abrogate most of the AT₁R influence on AAD.

Understanding how these two receptors interact during the signaling process is key to unlocking how renal afferent arteriolar resistance is controlled during hypertension. Resolving the mechanisms will facilitate our ability to design therapeutic interventions that will prevent the decline of kidney function early in the progression of hypertension.

GRANTS

This work was supported by National Institute of General Medical Sciences Grant CoBRE P30-GM-103337 and by the Lavin-Bernick endowment at Tulane (to L. G. Navar).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.K., M.F., and L.G.N. conceived and designed research; S.K. performed experiments; S.K. analyzed data; S.K., W.S., M.F., and L.G.N. interpreted results of experiments; S.K. prepared figures; S.K., W.S., M.F., and L.G.N. drafted manuscript; S.K. and L.G.N. edited and revised manuscript; S.K. approved final version of manuscript.