This study was conducted to examine the hypothesis that P2 purinoceptors contribute to pressure-induced autoregulatory adjustments of afferent arteriolar caliber. Experiments were performed in vitro using the blood-perfused juxtamedullary nephron technique. Afferent arteriolar diameter averaged 19.2 +/- 0.6 microns (n = 51) at control perfusion pressure of 100 mmHg and decreased when perfusion pressure was increased. Desensitization of P2 purinoceptors abolished the alpha, beta-methylene ATP-mediated afferent vasoconstriction and prevented pressure-dependent autoregulatory adjustments in afferent diameter. P2-purinoceptor saturation significantly decreased afferent caliber and attenuated pressure-induced autoregulatory responses. To block P2 receptors, afferent arterioles were treated with the P2-purinoceptor antagonists, pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid or suramin. P2-receptor blockade prevented the afferent arteriolar vasoconstriction evoked by increasing perfusion pressure from 100 to 130 and 160 mmHg. These data demonstrate that inhibition of P2 purinoceptor-dependent responses through receptor desensitization, receptor saturation, or purinoceptor blockade impairs normal autoregulatory behavior in rat juxtamedullary afferent arterioles. The results are consistent with the hypothesis that P2 purinoceptors participate in mediating autoregulatory adjustments in afferent arteriolar diameter.

1 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 ET A and ET B receptors to the renal pre-and postglomerular arteriolar responses to ET-1. 2 Experiments determined the effect of selective ET A (A-127722; 30 nM) and ET B (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. 3 Baseline afferent arteriolar diameter was similar in all groups and averaged 17.870.6 mm (n ¼ 14). In control experiments (n ¼ 6), ET-1 produced significant concentration-dependent decreases in arteriolar diameter, with 10 nM ET-1 decreasing diameter by 8571%. 4 Selective blockade of ET A receptors (n ¼ 6) prevented ET-1-mediated vasoconstriction, except at concentrations of 1 and 10 nM. Similarly, the vasoconstrictor profile was right shifted during selective ET B receptor blockade (n ¼ 4). Combined ET A and ET B receptor blockade (n ¼ 5) completely abolished afferent arteriolar diameter responses to ET-1. 5 ET B 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 ET B receptor antagonist. IRL-1620, another ET B agonist, was less effective at altering afferent or efferent diameter and produced a small reduction in pre-and postglomerular arteriolar diameter. 6 These data demonstrate that both ET A and ET B receptors participate in ET-1-mediated vasoconstriction of afferent arterioles. ET B receptor stimulation provides a significant vasodilatory influence on the efferent arteriole. Furthermore, since selective ET A and ET B receptor antagonists abolished preglomerular vasoconstrictor responses at lower ET-1 concentrations, these data support a possible interaction between ET A and ET B receptors in the control of afferent arteriolar diameter.

Studies were performed to determine the responsiveness of rat juxtamedullary afferent arterioles to receptor-selective P2-purinoceptor agonists. Experiments were performed in vitro using the blood perfused juxtamedullary nephron technique, combined with videomicroscopy. Renal perfusion pressure was set at 110 mmHg and held constant. Basal afferent arteriolar diameter averaged 22.0 ± 0.6 μm ( n = 69). Stimulation with 0.1, 1.0, 10, and 100 μM ATP ( n = 10) elicited a concentration-dependent vasoconstriction averaging 8 ± 2, 17 ± 2, 21 ± 4, and 23 ± 5%, respectively. A nearly identical afferent arteriolar vasoconstriction was observed in response to the P2X-selective agonist β,γ-methylene ATP ( n = 10); however, another P2X agonist, α,β-methylene ATP, evoked marked receptor desensitization ( n = 10). Vessel diameter decreased by ∼7 ± 2, 16 ± 2, 23 ± 3, and 22 ± 3%, respectively, over the same concentration range. The P2Y-selective agonist, 2-methylthio-ATP, evoked only a modest vasoconstriction, whereas UTP and adenosine 5′- O-(3-thiotriphosphate) (ATPγS) reduced afferent diameter markedly at concentrations >1.0 μM. Afferent arteriolar diameter decreased by 5 ± 4, 31 ± 8, and 72 ± 8% during UTP administration ( n = 7) at concentrations of 1.0, 10, and 100 μM, respectively. Similarly, ATPγS ( n = 6) decreased afferent diameter by 16 ± 2, 58 ± 8, and 98 ± 3%, respectively, over the same concentration range. Nitric oxide synthesis inhibition with N ω-nitro-l-arginine did not significantly alter the afferent arteriolar response to ATP but did potentiate ATP-mediated arcuate artery vasoconstriction. The following data suggest the presence of multiple P2 receptors on juxtamedullary afferent arterioles and are consistent with classification of those receptors as members of the P2X- and P2Y2 (P2U)-receptor subtypes.

Abstract-This study tested the hypothesis that afferent arteriolar responses to purinoceptor activation are attenuated, and Ca 2ϩ signaling mechanisms are responsible for the blunted preglomerular vascular reactivity in angiotensin II (Ang II) hypertension. Experiments determined the effects of ATP, the P2X 1 agonist ␤,␥-methylene ATP or the P2Y agonist UTP on arteriolar diameter using the juxtamedullary nephron technique and on renal myocyte intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) using single cell fluorescence microscopy. Six or 13 days of Ang II infusion significantly attenuated the vasoconstrictor responses to ATP and ␤,␥-methylene ATP (PϽ0.05). During exposure to ATP (1, 10, and 100 mol/L), afferent diameter declined by 17Ϯ2%, 29Ϯ3%, and 30Ϯ2% in normal control rats and 8Ϯ3%, 7Ϯ3%, and 22Ϯ3% in kidneys of Ang II-infused rats (13 days). Renal myocyte intracellular calcium responses to ATP or ␤,␥-methylene ATP were also decreased in Ang II hypertensive rats. In myocytes of control rats, peak increases in [Ca 2ϩ ] i averaged 107Ϯ21, 170Ϯ38, and 478Ϯ79 nmol/L at ATP concentrations of 1, 10, and 100 mol/L, respectively. Ang II infusion for 13 days decreased the peak responses to ATP (1, 10, and 100 mol/L) to 65Ϯ13, 102Ϯ20, and 367Ϯ73 nmol/L, respectively. The peak increases in [Ca 2ϩ ] i in response to ␤,␥-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 P2X 1 receptor-mediated signal transduction. (Hypertension. 2005;46:562-568.)

Impaired autoregulation in chronic kidney disease can result in elevation of glomerular capillary pressure and progressive glomerular damage; however, the factors linking chronic glomerular disorders to impaired autoregulation have not been identified. We tested the hypothesis that the cytokine most closely associated with progressive glomerular disease, transforming growth factor (TGF)-beta, may also attenuate autoregulation. Kidneys from normal rats were prepared for videomicroscopy, using the blood-perfused juxtamedullary nephron technique. Autoregulatory responses were measured under control conditions and during superfusion with TGF-beta1 (10 ng/ml). Control afferent arteriolar diameter averaged 18.4 +/- 1 microm and significantly decreased to 16.3 +/- 0.9 and 13.2 +/- 0.8 microm at perfusion pressures of 130 and 160 mmHg, respectively. In the presence of TGF-beta1, autoregulatory responses were completely blocked. In similar experiments performed using PDGF-BB (10 ng/ml) and HGF (25 ng/ml), the normal autoregulatory response was not affected. In vitro studies, using isolated preglomerular vascular smooth muscle cells, revealed that exposure to TGF-beta1 stimulated a rapid increase in reactive oxygen species (ROS) that was inhibited by NADPH oxidase inhibitors. In situ studies, with dihydroethidium staining, revealed a marked increase in renal vessel ROS production on exposure to TGF-beta1. Pretreatment of the juxtamedullary afferent arterioles with tempol, a ROS scavenger, or with apocynin, a NADPH oxidase inhibitor, prevented the impaired autoregulation induced by TGF-beta1. These data reveal a novel hemodynamic pathway by which TGF-beta could lead to progressive glomerular injury by impairing normal renal microvascular function.

The dynamic activity of afferent arteriolar diameter (AAD) and blood flow (AABF) responses to a rapid step increase in renal arterial pressure (100-148 mmHg) was examined in the kidneys of normal Sprague-Dawley rats (n = 11) before [tubuloglomerular feedback (TGF)-intact] and after interruption of distal tubular flow (TGF-independent). Utilizing the in vitro blood-perfused juxtamedullary nephron preparation, fluctuations in AAD and erythrocyte velocity were sampled by using analog-to-digital computerized conversion, video microscopy, image shearing, and fast-frame, slow-frame techniques. These assessments enabled dynamic characterization of the autonomous actions and collective interactions between the myogenic and TGF mechanisms at the level of the afferent arteriole. The TGF-intact and TGF-independent systems exhibited common initial (0-24 vs. 0-13 s, respectively) response slope kinetics (-0.53 vs. -0.47% DeltaAAD/s; respectively) yet different maximum vasoconstrictive magnitude (-11.28 +/- 0.1 vs. -7. 02 +/- 0.9% DeltaAAD; P < 0.05, respectively). The initial AABF responses similarly exhibited similar kinetics but differing magnitudes. In contrast, during the sustained pressure input (13-97 s), the maximum vasoconstrictor magnitude (-7.02 +/- 0.9% DeltaAAD) and kinetics (-0.01% DeltaAAD/s) of the TGF-independent system were markedly blunted whereas the TGF-intact system exhibited continued vasoconstriction with slower kinetics (-0.20% DeltaAAD/s) until a steady-state plateau was reached (-25.9 +/- 0.4% DeltaAAD). Thus the TGF mechanism plays a role in both direct mediation of vasoconstriction and in modulation of the myogenic response.

. Renal segmental microvascular responses to ANG II in AT1A receptor null mice. Am J Physiol Renal Physiol 284: F538-F545, 2003. First published November 12, 2002 10.1152/ajprenal.00340. 2002The relative contributions of AT 1A and AT1B receptors to afferent arteriolar autoregulatory capability and afferent and efferent arteriolar responses to ANG II are not known. Experiments were conducted in kidneys from wildtype (WT) and AT 1AϪ/Ϫ mice utilizing the in vitro bloodperfused juxtamedullary nephron technique. Direct measurements of afferent (AAD) and efferent arteriolar diameters (EAD) were assessed at a renal arterial pressure of 100 mmHg. AAD averaged 14.8 Ϯ 0.8 m for WT and 14.9 Ϯ 0.8 m for AT1AϪ/Ϫ mice. AAD significantly decreased by 7 Ϯ 1, 16 Ϯ 1, and 26 Ϯ 2% for WT mice and by 11 Ϯ 1, 20 Ϯ 2, and 30 Ϯ 3% for AT1AϪ/Ϫ mice (120, 140, 160 mmHg). AAD autoregulatory capability was not affected by the absence of AT1A receptors. AAD responses to 10 nM ANG II were significantly blunted for AT1AϪ/Ϫ mice compared with WT (Ϫ22 Ϯ 2 vs. Ϫ37 Ϯ 5%). ANG II (0.1-10 nM) failed to elicit any change in EAD for AT1AϪ/Ϫ mice. AAD and EAD reductions in ANG II were blocked by 1 M candesartan. We conclude that afferent arteriole vasoconstrictor responses to ANG II are mediated by AT1A and AT1B receptors, whereas efferent arteriolar vasoconstrictor responses to ANG II are mediated by only AT1A receptors in the mouse kidney. afferent arteriole; efferent arteriole; juxtamedullary nephron; candesartan; autoregulation THERE ARE AT LEAST TWO MAJOR angiotensin receptors: AT 1 and AT 2 . The AT 1 receptor is thought to mediate most of the actions of ANG II on renal hemodynamic and tubular function, including afferent and efferent arteriolar vasoconstriction (3, 9, 31), modulation of tubuloglomerular feedback sensitivity (16), sodium and fluid reabsorption (18), and growth and differentiation (29). Two subtypes of the AT 1 receptors, designated AT 1A and AT 1B , have been identified in the rat (7,12,13,23) and mouse (24). The AT 1A receptor is thought to be the predominant renal form. Terada et al. (28) reported localization of the AT 1 receptor mRNA in microdissected renal vascular segments (glomeruli, vasa recta bundle, and arcuate arteries) of the kidney by RT-PCR methods. Further studies identified AT 1A and AT 1B mRNAs in the same renal vascular structures, as well as the afferent arteriole (2, 7). Additionally, the AT 1 receptor protein has been localized to the entire rat renal vasculature using immunohistochemical techniques and antibodies that recognize specifically the AT 1A receptor (30) or both the AT 1A and AT 1B receptor subtypes (10,17,21,30). The mRNA and protein expression profiles of the AT 1 receptor subtypes have not been determined for the efferent arteriole. Furthermore, the contribution of the AT 1A and AT 1B receptors to the afferent and efferent arteriolar responses to ANG II have not been investigated.The AT 1A and AT 1B receptors are pharmacologically indistinguishable from each other, and so it has not been pos...

Abstract-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. Key Words: myogenic response Ⅲ epithelial sodium channel Ⅲ amiloride Ⅲ benzamil Ⅲ juxtamedullary nephrons Ⅲ autoregulatory response R enal autoregulatory behavior maintains a relatively constant renal blood flow despite changes in renal arterial pressure, a vital renal function for preventing hypertensioninduced renal injury. [1][2][3] In kidneys, autoregulation is accomplished through the combined influences of the myogenic and tubuloglomerular feedback (TGF) mechanisms. 4 The TGF response is a process by which the macula densa senses changes in distal tubule NaCl delivery and, in turn, modulates release of paracrine signals that alter afferent arteriolar tone. 5 The myogenic response is an intrinsic property of preglomerular arteries and afferent arterioles. Myogenic responses are characterized by vasoconstriction after an increase in transmural pressure or vasorelaxation after a decrease in transmural pressure. 6 The myogenic response is inherent to vascular smooth muscle and independent of endothelium. 7 Myogenic behavior is also observed in vascular elements of many other organs such as coronary, cerebral, and mesenteric arteries and cremaster arterioles and reflects an important mechanism for establishing ambient vascular tone and maintaining a relatively constant regional blood flow and capillary hydrostatic pressure. [7][8][9] The cellular mechanisms by which an increase in arterial pressure triggers the myogenic response have been investigated intensively. It is well established that increasing local transmural pressure leads to membrane depolarization of vascular smooth muscle cells, activation of voltage-gated L-type calcium channels, and vasoconstriction. 10 -13 However, the mechanisms by which the mechanical stimuli lead to activation of myogenic signaling cascades remain poorly understood.Currently several hypotheses have been proposed for linking mechanical stimuli to the cellular events producing a myogenic response. These include involvement of stretchactivated cation channels, perturbation of the actin cytoskeleton, specialized membrane domains, or extracellular matrix proteins such as integrins. 8,9,14 -16 The role of stretch-activated cation channels in the myogenic response was indirectly demonstrated in isolated perfused hydronephrotic rat kidneys lacking TGF responses. 17 In this model, pressure-mediated