Anthony K. Cook

Physiological role for P2X1 receptors in renal microvascular autoregulatory behavior

This study tests the hypothesis that P2X1 receptors mediate pressure-induced afferent arteriolar autoregulatory responses. Afferent arterioles from rats and P2X1 KO mice were examined using the juxtamedullary nephron technique. Arteriolar diameter was measured in response to step increases in renal perfusion pressure (RPP). Autoregulatory adjustments in diameter were measured before and during P2X receptor blockade with NF279 or A1 receptor blockade with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). Acute papillectomy or furosemide perfusion was performed to interrupt distal tubular fluid flow past the macula densa, thus minimizing tubuloglomerular feedback‐dependent influences on afferent arteriolar function. Under control conditions, arteriolar diameter decreased by 17% and 29% at RPP of 130 and 160 mmHg, respectively. Blockade of P2X1 receptors with NF279 blocked pressuremediated vasoconstriction, reflecting an attenuated autoregulatory response. The A1 receptor blocker DPCPX did not alter autoregulatory behavior or the response to ATP. Deletion of P2X1 receptors in KO mice significantly blunted autoregulatory responses induced by an increase in RPP, and this response was not further impaired by papillectomy or furosemide. WT control mice exhibited typical RPPdependent vasoconstriction that was significantly attenuated by papillectomy. These data provide compelling new evidence indicating that tubuloglomerular feedback signals are coupled to autoregulatory preglomerular vasoconstriction through ATP-mediated activation of P2X1 receptors.

ETA and ETB receptors differentially modulate afferent and efferent arteriolar responses to endothelin

The segment‐specific actions of endothelin peptides and agonists have not been thoroughly investigated in the renal microcirculation. The current studies were performed to assess the relative contribution of ETA and ETB receptors to the renal pre‐ and postglomerular arteriolar responses to ET‐1. Experiments determined the effect of selective ETA (A‐127722; 30 nM) and ETB (A‐192621; 30 nM) receptor blockade, on arteriolar responses to ET‐1 concentrations of 1 pM to 10 nM in rat kidneys using the isolated juxtamedullary nephron technique. Renal perfusion pressure was set at 110 mmHg. Baseline afferent arteriolar diameter was similar in all groups and averaged 17.8±0.6 μm (n=14). In control experiments (n=6), ET‐1 produced significant concentration‐dependent decreases in arteriolar diameter, with 10 nM ET‐1 decreasing diameter by 85±1%. Selective blockade of ETA receptors (n=6) prevented ET‐1‐mediated vasoconstriction, except at concentrations of 1 and 10 nM. Similarly, the vasoconstrictor profile was right shifted during selective ETB receptor blockade (n=4). Combined ETA and ETB receptor blockade (n=5) completely abolished afferent arteriolar diameter responses to ET‐1. ETB selective agonists (S6c and IRL‐1620) produced disparate responses. S6c produced a concentration‐dependent vasoconstriction of afferent arterioles. In contrast, S6c produced a concentration‐dependent dilation of efferent arterioles that could be blocked with an ETB receptor antagonist. IRL‐1620, another ETB agonist, was less effective at altering afferent or efferent diameter and produced a small reduction in pre‐ and postglomerular arteriolar diameter. These data demonstrate that both ETA and ETB receptors participate in ET‐1‐mediated vasoconstriction of afferent arterioles. ETB receptor stimulation provides a significant vasodilatory influence on the efferent arteriole. Furthermore, since selective ETA and ETB receptor antagonists abolished preglomerular vasoconstrictor responses at lower ET‐1 concentrations, these data support a possible interaction between ETA and ETB receptors in the control of afferent arteriolar diameter.

Afferent and Efferent Arteriolar Vasoconstriction to Angiotensin II and Norepinephrine Involves Release of Ca2+ From Intracellular Stores

Renal vascular responses to angiotensin II (Ang II) and norepinephrine (NE) are reported to involve both mobilization of calcium from intracellular stores and activation of calcium influx pathways. The present study was conducted to determine the contribution of calcium release from intracellular stores to afferent and efferent arteriolar responses to Ang II and NE. Experiments were performed in vitro using the blood-perfused, juxtamedullary nephron technique combined with videomicroscopy. The responses of afferent and efferent arterioles to Ang II and NE were determined before and after depletion of intracellular calcium pools with 1 mumol/L thapsigargin. Positive control responses were obtained with 55 mmol/L KCI. Ang II concentrations of 0.1, 1.0, and 10 nmol/L decreased afferent arteriolar diameter by 10 +/- 4%, 17 +/- 4%, and 29 +/- 6%, respectively (P < .05; n = 8). NE also decreased afferent diameter by 5 +/- 1%, 13 +/- 1%, and 57 +/- 9% at concentrations of 10, 100, and 1000 nmol/L, respectively (P < .05; n = 6). Thapsigargin treatment shifted the afferent arteriolar concentration-response curves for both Ang II and NE significantly to the right. Nevertheless, KCI evoked a pronounced vasoconstriction and decreased afferent diameter by 56 +/- 7% (P < .05; n = 6). Postglomerular responses to Ang II and NE were abolished by thapsigargin. During the control period, efferent diameter decreased by 3 +/- 1%, 7 +/- 2%, and 14 +/- 4% for the three Ang II concentrations and 3 +/- 1%, 5 +/- 1%, and 15 +/- 4% in response to the three NE concentrations, respectively. These responses were completely eliminated in the presence of thapsigargin, whereas KCI evoked an efferent arteriolar vasoconstriction of 57 +/- 9% (P < .05). These data demonstrate that agonist-induced calcium release from intracellular stores represents an essential component in the afferent and efferent arteriolar response to Ang II and NE. Furthermore, they suggest that efferent arteriolar responses to these agents may rely more heavily on calcium release from this store, whereas afferent responses may include activation of other pathways.

Chemokine receptor 2b inhibition provides renal protection in angiotensin II-salt hypertension

The present study was designed to determine whether chemokine receptor 2b (CCR2b) contributes to the development of renal injury in salt-sensitive angiotensin II (ANG) hypertension. Rats were infused with ANG and fed a high-salt diet (HS) for 14 days. Rats were divided into 4 groups: HS; HS administered the CCR2b antagonist, RS102895; Ang/HS hypertensive; and Ang/HS hypertensive administered RS102895. CCR2b inhibition slowed the progression of blood pressure elevation during the first week of ANG/HS hypertension; however, it did not alter blood pressure in the HS group. At 2 weeks, arterial pressure was not significantly different between ANG/HS and ANG/HS hypertensive rats administered RS102895. Renal cortical nuclear factor &kgr;B activity increased in ANG/HS hypertension compared with the HS group (0.11±0.006 versus 0.08±0.003 ng of activated nuclear factor &kgr;B per microgram of protein), and RS102895 treatment lowered nuclear factor &kgr;B activity in ANG/HS hypertension (0.08±0.005 ng of activated nuclear factor &kgr;B per microgram of protein). Renal tumor necrosis factor-&agr; and intercellular adhesion molecule-1 expression increased, and Cyp2c23 expression decreased in ANG/HS hypertension compared with the HS group, and CCR2b inhibition reduced tumor necrosis factor-&agr; and intercellular adhesion molecule-1 and increased Cyp2c23 expression. Histological immunostaining revealed increased renal monocyte and macrophage infiltration in ANG/HS hypertensive rats with decreased infiltration in rats receiving RS102895 treatment. Albuminuria and cortical collagen staining also increased in ANG/HS hypertensive rats, and RS102895 treatment lowered these effects. Afferent arteriolar autoregulatory responses to increasing renal perfusion pressure were blunted in ANG/HS hypertension, and RS102895 treatment improved this response. These data suggest that CCR2b inhibition protects the kidney in hypertension by reducing inflammation and delaying the progression of hypertension.

Effect of Epithelial Sodium Channel Blockade on the Myogenic Response of Rat Juxtamedullary Afferent Arterioles

The mechanotransduction mechanism underlying the myogenic response is poorly understood, but evidence implicates participation of epithelial sodium channel (ENaC)-like proteins. Therefore, the role of ENaC on the afferent arteriolar myogenic response was investigated in vitro using the blood-perfused juxtamedullary nephron technique. Papillectomy was used to isolate myogenic influences by eliminating tubuloglomerular feedback signals. Autoregulatory responses were assessed by manipulating perfusion pressure in 30-mm Hg steps. Under control conditions, arteriolar diameter increased by 15% from 13.0±1.3 to 14.7±1.2 &mgr;m (P<0.05) after reducing perfusion pressure from 100 to 70 mm Hg. Diameter decreased to 11.3±1.1 and 10.6±1.0 &mgr;m after increasing pressure to 130 and 160 mm Hg (88±1 and 81±2% of control diameter, P<0.05), respectively. Pressure-mediated autoregulatory responses were significantly inhibited by superfusion of 10 &mgr;mol/L amiloride (102±2, 97±4, and 94±3% of control diameter), or 10 &mgr;mol/L benzamil (106±5, 100±3, and 103±3% of control diameter), and when perfusing with blood containing 5 &mgr;mol/L amiloride (106±2, 97±4, and 97±4% of control diameter). Vasoconstrictor responses to 55 mmol/L KCl were preserved as diameters decreased by 67±4, 55±8, and 60±4% in afferent arterioles superfused with amiloride or benzamil, and perfused with amiloride, respectively. These responses were similar to responses obtained from control afferent arterioles (64±6%, P>0.05). Immunofluorescence revealed expression of the &agr;, &bgr;, and &ggr; subunits of ENaC in freshly isolated preglomerular microvascular smooth muscle cells. These results demonstrate that selective ENaC inhibitors attenuate afferent arteriolar myogenic responses and suggest that ENaC may function as mechanosensitive ion channels initiating pressure-dependent myogenic responses in rat juxtamedullary afferent arterioles.

Calcium Mobilization Contributes to Pressure-Mediated Afferent Arteriolar Vasoconstriction

Preglomerular responses to vasoactive agonists utilize calcium released from intracellular stores and activation of calcium influx pathways to elicit vasoconstriction. The current study was performed to determine the role of calcium release from intracellular stores on the afferent arteriolar response to increases in perfusion pressure. Experiments were performed, in vitro, using the blood perfused, juxtamedullary nephron technique combined with videomicroscopy. The response of afferent arterioles to 30 mm Hg increases in perfusion pressure was determined before and after depletion of intracellular calcium pools with a 10-minute preincubation with 1 micromol/L thapsigargin or 100 micromol/L cyclopiazonic acid. Afferent arteriolar diameter averaged 20.2+/-1.0 microm (n=19) at a control perfusion pressure of 100 mm Hg. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent caliber by 10.7+/-1.0% (P<.05 versus con) and by 24.7+/-1.6% (P<.05 versus diameter at 130 mm Hg); respectively. Thapsigargin significantly increased afferent diameter by 21+/-2% (n=6) at 100 mm Hg and prevented pressure-induced autoregulatory responses. Afferent diameter averaged 24.3+/-1.7, 24.5+/-1.8 and 24.3+/-1.8 microm at perfusion pressures of 100, 130 and 160 mm Hg; respectively. Cyclopiazonic acid treatment also inhibited autoregulatory behavior but did not alter resting vessel diameter. Afferent arteriolar diameter (n=6) averaged 21.4+/-1.9 microm at 100 mm Hg and 20.9+/-2.1 and 20.5+/-2.2 microm at 130 and 160 mm Hg; respectively. Additional studies were performed to assess the role of phospholipase C activity in pressure-mediated autoregulatory behavior of afferent arterioles. Step increases in perfusion pressure decreased afferent diameter by 10.7+/-3.8 and 21.7+/-4.1%; respectively. Administration of the phospholipase C inhibitor, U-73122, (5 micromoles/L) did not significantly alter baseline diameter but did attenuate the pressure-mediated vasoconstrictor response. Increasing perfusion pressure to 130 and 160 mm Hg reduced afferent diameter by only 6.5+/-1.5 and 10.0+/-2.0%; respectively. These data demonstrate that interruption of calcium mobilization with thapsigargin, cyclopiazonic acid, or phospholipase C inhibition markedly attenuates pressure-mediated afferent arteriolar vasoconstriction and suggests that autoregulatory adjustments in afferent arteriolar diameter involve calcium release from inositoltrisphosphate(IP3)-sensitive intracellular stores.

Impaired Ca2+ Signaling Attenuates P2X Receptor–Mediated Vasoconstriction of Afferent Arterioles in Angiotensin II Hypertension

This study tested the hypothesis that afferent arteriolar responses to purinoceptor activation are attenuated, and Ca2+ signaling mechanisms are responsible for the blunted preglomerular vascular reactivity in angiotensin II (Ang II) hypertension. Experiments determined the effects of ATP, the P2X1 agonist &bgr;,&ggr;–methylene ATP or the P2Y agonist UTP on arteriolar diameter using the juxtamedullary nephron technique and on renal myocyte intracellular Ca2+ concentration ([Ca2+]i) using single cell fluorescence microscopy. Six or 13 days of Ang II infusion significantly attenuated the vasoconstrictor responses to ATP and &bgr;,&ggr;–methylene ATP (P<0.05). During exposure to ATP (1, 10, and 100 &mgr;mol/L), afferent diameter declined by 17±2%, 29±3%, and 30±2% in normal control rats and 8±3%, 7±3%, and 22±3% in kidneys of Ang II–infused rats (13 days). Renal myocyte intracellular calcium responses to ATP or &bgr;,&ggr;–methylene ATP were also decreased in Ang II hypertensive rats. In myocytes of control rats, peak increases in [Ca2+]i averaged 107±21, 170±38, and 478±79 nmol/L at ATP concentrations of 1, 10, and 100 &mgr;mol/L, respectively. Ang II infusion for 13 days decreased the peak responses to ATP (1, 10, and 100 &mgr;mol/L) to 65±13, 102±20, and 367±73 nmol/L, respectively. The peak increases in [Ca2+]i in response to &bgr;,&ggr;–methylene ATP were also reduced in Ang II hypertensive rats. However, angiotensin hypertension did not change the UTP-mediated vasoconstrictor responses or the myocyte calcium responses to UTP. These results indicate that the impaired autoregulatory response observed in Ang II–dependent hypertension can be attributed to impairment of P2X1 receptor–mediated signal transduction.

Afferent arteriolar responsiveness to altered perfusion pressure in renal hypertension

The present study was performed to determine the role of afferent arterioles in the impaired autoregulatory response shown to occur in the contralateral kidney of Goldblatt hypertensive rats. The responsiveness of juxtamedullary afferent arterioles to alterations in perfusion pressure was studied in the nonclipped kidney of two-kidney, one clip hypertensive and sham-operated rats. Systolic pressure, 5–6 weeks after clipping, averaged 184±6 mm Hg in the hypertensive rats (n = 16) and 121 ± 3 mm Hg in the sham-operated control rats (n = 7). By using the in vitro blood-perfused juxtamedullary nephron technique, afferent arterioles were directly visualized, and their inside diameters were measured by videomicroscopic methods. In sham-operated kidneys perfused with blood from normotensive rats, afferent arteriolar diameter averaged 22.8 ±1.8 μm at a renal arterial perfusion pressure of 151±1 mm Hg and increased to 24.8±1.8 μm when perfusion pressure was reduced to 110±2 mm Hg. Conversely, in hypertensive kidneys perfused with blood from either hypertensive or normotensive rats, the afferent arterioles failed to vasodilate and actually exhibited a slight decrease in diameter from 24.6 ± 1.3 to 23.0 ± 2.3 μm in response to the same reduction in perfusion pressure. Vasodilator capability, however, could be demonstrated in response to verapamil and sodium nitroprusside, which increased afferent diameter in both the sham-operated and hypertensive groups of rats. Thus, unlike arterioles from normotensive rats, juxtamedullary afferent arterioles from two-kidney, one clip Goldblatt hypertensive rats fail to vasodilate after a reduction in perfusion pressure. This impaired autoregulatory ability might result from an intrinsic functional defect in the vasculature of the high pressure contralateral kidney.

Rho-kinase inhibition reduces pressure-mediated autoregulatory adjustments in afferent arteriolar diameter

Preglomerular resistance is regulated by calcium influx- and mobilization-dependent mechanisms; however, the role of Rho-kinase in calcium sensitization in the intact kidney has not been carefully examined. Experiments were performed to test the hypothesis that Rho-kinase inhibition blunts pressure-mediated afferent arteriolar autoregulatory behavior and vasoconstrictor responses evoked by angiotensin II and P2X1 receptor activation. Rat kidneys were studied in vitro using the blood-perfused juxtamedullary nephron technique. Autoregulatory behavior was assessed before and during Rho-kinase inhibition with Y-27632 (1.0 microM; n = 5). Control diameter averaged 14.3 +/- 0.8 microm and increased to 18.1 +/- 0.9 microm (P < 0.05) during Y-27632 treatment. In the continued presence of Y-27632, reducing perfusion pressure to 65 mmHg slightly increased diameter to 18.7 +/- 1.0 microm. Subsequent pressure increases to 130 and 160 mmHg yielded afferent arteriolar diameters of 17.5 +/- 0.8 and 16.6 +/- 0.6 microm (P < 0.05). This 11% decline in diameter is significantly smaller than the 40% decrease obtained in untreated kidneys. The inhibitory effects of Y-27632 on autoregulatory behavior were concentration dependent. Angiotensin II responses were blunted by Y-27632. Angiotensin II (1.0 nM) reduced afferent diameter by 17 +/- 1% in untreated arterioles and by 6 +/- 2% during exposure to Y-27632. The P2X1 receptor agonist, alpha, beta-methylene ATP, reduced afferent arteriolar diameter by 8 +/- 1% but this response was eliminated during exposure to Y-27632. Western blot analysis confirms expression of the Rho-kinase signaling pathway. Thus, Rho-kinase may be important in pressure-mediated autoregulatory adjustments in preglomerular resistance and responsiveness to angiotensin II and autoregulatory P2X1 receptor agonists.

P2X1 receptor-mediated vasoconstriction of afferent arterioles in angiotensin II-infused hypertensive rats fed a high-salt diet.

Experiments tested the hypothesis that P2 receptor reactivity is impaired in angiotensin (Ang) II hypertensive rats fed an 8%NaCl diet (Ang II+HS). Juxtamedullary afferent arteriolar autoregulatory behavior was determined over a pressure range of 65 to 200 mm Hg. Arteriolar responsiveness to P2X1 (&bgr;,&ggr;-methylene ATP) or P2Y2 receptor (uridine triphosphate) activation was determined in vitro. Systolic blood pressure averaged 126±3 and 225±4 mm Hg in control and Ang II+HS rats, respectively (P<0.05). In control kidneys, &bgr;,&ggr;-methylene ATP (10−8 to 10−4 mol/L) reduced arteriolar diameter by 8±3%, 13±5%, 19±5%, 22±6%, and 24±9%, respectively, whereas uridine triphosphate reduced diameter by 2±1%, 2±2%, 9±3%, 37±7%, and 58±7%. Autoregulation was markedly blunted in Ang II+HS kidneys, with arteriolar diameter remaining essentially unchanged when perfusion pressure increased to 200 mm Hg compared with a 40±2% decline in diameter observed in normal kidneys over the same pressure range (P<0.05). P2X1 receptor–mediated vasoconstriction was significantly attenuated in Ang II+HS kidneys. &bgr;,&ggr;-Methylene ATP reduced arteriolar diameter by 1±1%, 3±2%, 6±1%, 9±3%, and 7±1%, respectively (P<0.05), versus control rats. Similar patterns were noted when hypertensive perfusion pressures were used. Uridine triphosphate–mediated responses were unchanged in Ang II+HS rats compared with control, indicating preservation of P2Y2 receptor function. Ang II+HS blunted P2X1-mediated increases in intracellular Ca2+ concentration in preglomerular smooth muscle cells. Therefore, Ang II+HS rats exhibit attenuated afferent arteriolar responses to P2X1 receptor stimulation. These data support the hypothesis that P2X1 receptors are important for pressure-mediated autoregulatory responses. Impairment of P2X1 receptor function may explain the hypertension-induced decline in renal autoregulatory capability.