Effects of angiotensin II, arginine vasopressin and tromboxane A2 in renal vascular bed: role of rho-kinase
Alessandro Cavarape1,
Johannes Bauer2,
Ettore Bartoli3,
Karlhans Endlich4 and
Niranjan Parekh5
1 Department of Experimental and Clinical Pathology and Medicine, Chair of Internal Medicine, University of Udine, Udine, 2 Department of Medical Science, Chair of Internal Medicine, University of Piemonte Orientale A. Avogadro, Novara, Italy, 3 Department of Neonatology, 4 Department of Cell Biology and Anatomy and 5 Department of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany
Correspondence and offprint requests to: Alessandro Cavarape, MD, Department of Experimental and Clinical Pathology and Medicine (DPMSC), Chair of Internal Medicine, Piazza S. Maria della Misericordia, 1, I-33100 Udine, Italy. Email: alessandro.cavarape{at}dpmsc.uniud.it
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Abstract
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Background. Angiotensin II (Ang II), arginine vasopressin (AVP) and tromboxane A2 (TxA2) are dissimilar vasoconstrictors involved in regulating renal circulation. Whereas Ang II is primarily a physiological modulator, AVP and TxA2 play important roles under pathological conditions. Previously, we have shown variable importance of intracellular Ca2+ and protein kinase C for their mode of action (Ang II > AVP >U-46619), but the cell signalling via rho-associated kinase (ROK) is a common pathway. The aim of this study was to determine their sites of action in the renal vascular bed and the corresponding role of ROK at the microvascular level.
Methods. Glomerular blood flow (GBF) and luminal diameter of different vessels (1070 µm) were measured in the split hydronephrotic kidney of anaesthetized rats. The tissue bath concentration of Ang II, AVP or the TxA2 agonist U-46619 was adjusted to reduce GBF by
50%. The measurements were repeated after adding a sub-maximal dose of the ROK inhibitor Y-27632 into the bath.
Results. Ang II constricted all vessels significantly, the constriction being least in the proximal segment of the arcuate artery (
70 µm). Significant constrictions due to AVP were found only in interlobular and arcuate arteries (2070 µm), but not in the afferent and efferent arterioles. U-46619 constricted only the arcuate artery (
50 µm). Y-27632 (104 M) dilated all vessels significantly and increased GBF by 65%. Thereafter, effects of all agonists were severely attenuated. Control reductions in GBF could be obtained at higher concentrations of AVP (10-fold) and U-46619 (5-fold) and a lesser GBF reduction with Ang II (100-fold) without changes in the respective patterns of vascular constriction.
Conclusions. Our data indicate that the agonists, in the order Ang II, AVP and TxA2, constrict larger vessels within the renal vascular tree via activation of ROK. Therefore, ROK inhibitors may provide a therapeutic tool to antagonize pathological vasospasm of conduit vessels, which are resistant to other vasodilators.
Keywords: glomerular blood flow; renal haemodynamics; split hydronephrotic kidney
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Introduction
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Renal vascular resistance is modulated in health and disease by a variety of endocrine and paracrine hormones [1]. Agonists bind to their specific receptors along the renal vascular tree to change the corresponding vessel diameters. In the kidney, effects of vasoactive agents can be assessed from changes in total renal resistance by measuring renal blood flow and systemic blood pressure. Measurement of glomerular pressure further permits differentiation between the pre- and postglomerular resistance. Though cumbersome, casting of renal vascular tree after in vivo fixation is used to analyse the pattern of diameter changes of all vessels [2]. Different renal vessels including vasa recta can be visualized in the in vitro perfused juxtamedullary nephron preparation [1,3]. However, most of the studies using this preparation investigated effects of different agonists only on afferent and efferent arterioles [1]. In the model of split hydronephrotic kidney, with complete tubular atrophy, all vessels from the arcuate artery to the efferent arteriole (1070 µm), are accessible to intravital microscopic measurements [4]. A number of studies using this model [5] showed substantial differences in the pattern of sites of action for different agents. It was remarkable that agonists considered to be involved in physiological regulation of renal circulation, such as angiotensin II (Ang II) and norepinephrine, constricted predominantly small vessels, whereas those involved under pathological/inflammatory conditions (serotonin, leukotrienes) constricted preferentially large preglomerular vessels.
The cell signalling pathways activated by agonists regulate vascular tone ultimately by increasing the phosphorylation status of myosin light chain (MLC) in smooth muscle cells. The major cell signalling pathways involved in this process were known to be mobilization of intracellular Ca2+ via IP3, Ca2+ influx through L-type channels, activation of one or more isoforms of protein kinase C (PKC), and more recently, activation of rho-associated kinase (ROK) [6,7]. Whereas mobilization and influx of Ca2+ accelerate phosphorylation of MLC by Ca2+-calmodulin dependent MLC kinase, PKC and ROK inhibit MLC phosphatase (Ca2+ sensitization). A number of studies indicate that the role of Ca2+ mobilization, Ca2+ influx and PKC can be highly variable for effects of different vasoconstrictor agonists [8], whereas ROK seems to be pivotal for all agonists [8,9]. In a previous study on mesenteric circulation [10] we found that the vasoconstrictor effect of Ang II was highly dependent on Ca2+ mobilization, Ca2+ influx and PKC activation in an additive manner, whereas that of a thromboxaneA2 agonist, U-46619, was independent of these pathways. Arginine vasopressin (AVP) took an intermediate position between Ang II and U-46619 (N. Parekh, unpublished results).
The aim of our study was to find out in the hydronephrotic kidney of the rat if the pattern of renal vascular constriction due to agonists correlated in some manner with the cell signalling pathways, and to investigate the role of ROK at a microvascular level. For this purpose, we measured effects of topically administered Ang II, AVP and U-46619 on vascular diameter and glomerular blood flow (GBF) under control conditions and after inhibiting ROK by Y-27632.
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Materials and methods
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Preparation of the hydronephrotic kidney
The study was performed on female Wistar rats (230280 g) in accordance with national animal protection guidelines. The technique and experimental procedures have been described previously in detail [4,11]. Briefly, unilateral hydronephrosis was induced by ligating the left ureter through a flank incision during pentobarbital sodium anaesthesia (Nembutal, 60 mg/kg i.p.; Ceva, Bad Segeberg, Germany). Two to three months later, the final experiments were performed under thiobutabarbital anesthesia (Inactin, 100 mg/kg i.p.; Byk Gulden, Konstanz, Germany). Rats were placed on a thermostat table to keep body temperature at 37°C. The trachea was intubated for free breathing, the left jugular vein cannulated for replacing fluid loss (3.5 ml/h), and the left femoral artery for recording systemic blood pressure. The left hydronephrotic kidney was exposed through a flank incision and split along the great curvature with a thermal cautery. The dorsal half of the split kidney was sutured to a semicircular-shaped wire frame and suspended above a transparent port into a tissue bath. The entry of the renal hilus into the chamber was sealed with silicon grease, the bath was filled with 30 ml of an isotonic, isocolloidal solution (Haemaccel, Behringwerke AG, Marburg, Germany) and maintained at 37°C. In this preparation with intact blood supply and innervation, renal vessels are accessible to transillumination microscopy. Experiments were started after a stabilization period of 60 min.
Measurements of vessel diameters and GBF
The animal table was mounted on the stage of a microscope with a closed-circuit video system. A set of vessels in series with a glomerulus were selected for measuring diameters. The vessels consisted of two segments each (proximal and distal) of arcuate artery, interlobular artery and afferent arteriole and one segment of efferent arteriole (
100 µm downstream of the glomerulus). The luminal vessel diameters were measured from a calibrated monitor. To determine GBF, the velocity of red blood cells was measured in the efferent arteriole of the selected glomerulus by using a velocity tracking correlator (Model 102B; IPM Inc., San Diego, CA, USA). In order to obtain the GBF, the measured red cell velocity was multiplied by the luminal cross-section of the efferent arteriole and corrected for the Fahreus-Lindqvist effect [11].
Experimental procedure
The experiments were designed to investigate patterns of vasoconstriction induced by equipotent doses (GBF, 50%) of Ang II, AVP and U-46619 (Sigma) under control conditions and after inhibition of ROK by Y-27632 (a gift from Welfide Corp.). All agents were administered via tissue bath. Each measurement consisted of a set of either three or four periods (vehicle, agonist and washout with vehicle or the combination: vehicle, Y-27632, Y-27632 + agonist, and washout with Y-27632). Two to four measurements were done per animal. Preliminary experiments served to determine tissue bath concentrations of agonists required to reduce GBF by
50% (before and after Y-27632), the concentration of Y-27632 to produce sub-maximal rise in GBF, and the time needed to obtain full effects of different agents. Concentrations are given in Results, and time cspans were: Ang II, <5 min; AVP, <10 min; U-46619 and Y-27632 <20 min. Therefore, periods for the experimental protocol were fixed at 20 min for U-46619 and Y-27632 and their washouts and 10 min for others.
Statistical analysis
The data are presented as mean ± SEM. Changes in vascular diameters and GBF are expressed as percentage changes from the preceding control values. Statistical comparisons were done by t-tests for multiple comparisons after analysis of variance (Bonferroni). Values of P < 0.05 were considered statistically significant.
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Results
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Table 1 gives the absolute control values for different vessel diameters, GBF and systemic blood pressure before the interventions done in this study, whereas the Figures 14 describe the relative changes in these parameters due to corresponding interventions. Neither the agonists nor Y-27632 administered to the tissue bath affected the blood pressure.
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Table 1. Control values (mean ± SEM) of luminal vessel diameters, GBF and blood pressure prior to the indicated interventions (agonists or Y-27632) in the first four columns and after administration of Y-27632 (without agonists) in the last six columns
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Fig. 1. Effects of Ang II, AVP and U-46619 on vessel diameters and GBF. Vessels are proximal (P) and distal (D) segments of arcuate artery (Ar), interlobular artery (IL), afferent arteriole (Af) and efferent arteriole (Eff). The number of experiments are given in Table 1. *P < 0.05 vs control.
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Fig. 4. Effects of 3 x 109 M AVP under control conditions and that of 3 x 109 and 3 x 108 M AVP after 104 M Y-27632.
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Fig. 3. Effects of 108 M Ang II under control conditions and that of 108 and 106 M Ang II after 104 M Y-27632.
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Fig. 5. Effects of 2 x 106 M U-46619 under control conditions and that of 2 x 106 and 105 M U-46619 after 104 M Y-27632.
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As shown in Figure 1, concentrations of 108 M Ang II, 3 x 109 M AVP and 2 x 106 M U-46619 were equipotent and reduced GBF by
50%. Corresponding patterns of changes in vessel diameters, however, were different. In accordance with earlier findings [5], Ang II constricted all pre- and postglomerular vessels, the constriction being the least for the largest vessel segment (proximal arcuate artery). On the other hand, AVP constricted predominantly larger vessels (interlobular and arcuate artery, >20 µm) and U-46619 only the largest vessel (arcuate artery, >45 µm). Though the changes in GBF and hence, in renal vascular resistance were similar for all agonists, the vasoconstriction induced by U-46619 was lesser and in fewer of the investigated segments than for AVP. This discrepancy indicates substantial constriction of larger upstream vessels by U-46619. These vessels are located in thick tissue near the hilus of the hydronephrotic kidney, and are barely accessible to transillumination microscopic measurements.
At a concentration of 104 M, Y-27632 increased GBF by 64 ± 7% (Figure 2) and dilated all vessels, tendentially more larger vessels. After administration of Y-27632, Ang II had no effect and AVP and U-46619 caused minor reductions in GBF (3 ± 1 and 6 ± 2%, respectively). Thereafter, concentrations of agonists were increased in order to study vasoconstrictor effects of agonists in the presence of Y-27632. With Ang II it was not possible to reduce GBF to a level comparable to the control value despite a 100-fold higher concentration (Figure 3), also constriction of vessels was attenuated. The reduction in GBF was significantly less than that under control conditions (20 ± 4 vs 45 ± 4%), and the effect could not be surmounted by higher concentrations. Reductions in GBF in the range of the corresponding control values could be attained for AVP at a 10-fold higher concentration (Figure 4) and for U-46619 at a 5-fold higher concentration (Figure 5). Y-27632 did not cause discernible changes in the pattern of vasoconstriction by agonists.
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Discussion
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The present data indicate differences in patterns of vasoconstriction induced by Ang II, AVP and U-46619 along the renal vascular tree. Major constriction by Ang II was located in small resistance vessels (arterioles) considered to be involved in physiological regulation of renal blood flow. On the other hand, constriction due to U-46619 resided in large conduit vessels responsible for pathological vasospasm. AVP influenced diameters of intermediate arteries. Furthermore, our data confirm that activation of ROK is pivotal for the action of all exogenous agonists as well as for the basal tonus.
The constrictor effect of Ang II on pre- and postglomerular vessels, particularly the afferent and efferent arterioles, has been studied extensively [1]. In the hydronephrotic kidney, the role of Ang II has been repeatedly determined by using Ang II, converting enzyme inhibitor, receptor antagonist and the precursor Ang I [5]. All these studies show that the largest constriction occurs in distal interlobular artery and proximal afferent arteriole (1525 µm). A similar pattern of vasoconstriction is also reported for norepinephrine, neuropeptide Y and adenosine A1 agonist [5].
Harrison-Bernard and Carmines [3] measured effects of increasing doses of AVP on diameters of different vessels in the blood perfused juxtamedullary nephron preparation. In accordance with our data, they found major vasoconstriction in the arcuate and interlobular arteries, but in contrast to our data from cortical nephrons, they observed also a small but significant effect on proximal afferent arteriole. AVP (V1a) receptors responsible for vasoconstriction have been shown to be localized in the interlobular and large arteries and in the afferent and efferent arterioles of the juxtamedullary nephrons [12]. In contrast to our data, evidence for AVP-induced arteriole constriction has been shown for the isolated perfused rabbit afferent arteriole [13] and for the afferent and efferent arterioles of the isolated perfused rat hydronephrotic kidney [14]. The pattern of vasoconstriction observed for AVP in the present study resembles that reported for endothelin [5].
Vasoconstriction induced by the TxA2 agonist U-46619 was confined to the arcuate artery and presumably to the upstream interlobar artery. This pattern of vasoconstriction is known to be induced by topical administration of serotonin [2], leukotrienes (D4 and E4) [5], by intravenous infusion of cyclosporin A [5,11], and by complement activation with cobra venom factor [5]. In the isolated perfused hydronephrotic kidney [15], however, intrarenal administration of a TxA2 (U-44069) has been shown to constrict both afferent and efferent arterioles, whereas other vessels were not examined. Therefore, it cannot be ruled out that depending upon the experimental design and the dose of the agonist, also microvessels are susceptible to TxA2 or AVP.
In this study, ROK was inhibited by Y-27632 at a bath concentration of 104 M, which is much higher than the reported value in the micromolar range [9]. It should be pointed out that due to diffusion limitations, an equivalent concentration of any agent administered via the tissue bath to the hydronephrotic kidney is more than an order of magnitude higher than the corresponding plasma concentration. In the normal kidney ROK is completely inhibited by adjusting plasma concentration to
5 x 106 M. Also a 50% reduction in blood flow is attained at an Ang II concentration of 2 x 109 vs 108 M in this study [8].
The present study confirms the ubiquitous role of ROK for agonist-induced vasoconstriction at the microvascular level in vivo in the hydronephrotic kidney. The effects of agonists were distinctly attenuated after inhibition of ROK by Y-27632, and at least five times higher doses were needed to obtain comparable changes in vessel diameters and GBF. Similar results were also obtained in normal kidneys by measuring changes in renal blood flow [8]. In that study, norepinephrine, Ang II, AVP and U-46619 were infused into the renal artery and ROK was inhibited by co-infusion of Y-27632 or of a structurally different compound HA-1077. With the introduction of Y-27632 as a cell permeable ROK inhibitor [9], the central role of ROK was demonstrated in smooth muscle preparation using histamine, serotonin, acetylcholine, endothelin, phenylephrine, Ang II and U-46619 [9,16]. In vivo studies with Y-27632 have concentrated on its effects on pathological vasoconstriction. Orally administered Y-27632 has been shown to reduce systemic blood pressure in different models of hypertensive rats more effectively than in normotensive ones [9]. The basilar artery of spontaneously hypertensive rats with enhanced myogenic tone has been reported to relax more in response to topically administered Y-27632 than that of normotensive animals [17]. The vasospasm of canine basilar artery induced by subarachnoid haemorrhage could be reversed by topical application of Y-27632 [18], and hypercontractility of spastic segments of porcine coronary artery could be reversed by intracoronary injection of Y-27632 [19].
Inhibition of ROK increased GBF by 65% and dilated all pre- and postglomerular vessels (Figure 2), presumably by antagonizing endogenous vasoconstrictors. The dilator effect was largest in the first three upstream vessel segments. In an earlier study [5], L-type Ca2+ channels were blocked by nitrendipine, which produced a different pattern of vasodilation. Nitrendipine dilated only preglomerular vessels, the effect being largest in the fourth and fifth segments (distal interlobular artery and proximal afferent arteriole), and GBF was not measured. It is interesting to note that all vasodilators tested in the hydronephrotic kidney also had their maximal effects on these small preglomerular vessels (prostacyclin, artrial natriuretc peptide, parathyroid hormone, dopamine, acetylcholine, sodium nitroprusside) [5]. In normal kidneys, in which large vessels contribute lesser to total resistance than in the hydronephrotic kidney [5], the rise in renal blood flow induced by intrarenal infusion of Y-27632 was lesser (25 vs 64% in the hydronephrotic kidney), but greater than that induced by nifedipine (14%) [8]. Taken together, the data suggest that ROK is more important for MLC phosphorylation than Ca2+, particularly in the large conduit vessels.
Endogenous agonists responsible for constriction of large vessels in the hydronephrotic kidney are not well known. In this model, Ang II and endothelin are involved in basal tone [5], endothelin having a greater effect on larger vessels. Renal TxA2 synthesis is known to increase in obstructive uropathy and during the early induction phase of hydronephrosis [20]. Preliminary experiments with TxA2 receptor antagonist BM 13505 (Daltroban), however, did not indicate an elevated level of TxA2 in our model of end-stage hydronephrosis. Similar results have also been reported for isolated perfused hydronephrotic kidney [14].
In conclusion, we have demonstrated that in contrast to Ang II, which constricts predominantly resistance vessels, AVP and TxA2 agonist constrict conduit vessels. Furthermore, it could be shown that the effects of all agonists could be substantially attenuated by inhibition of ROK. Therefore, ROK could be regarded as a therapeutic target for the treatment of diseases associated with constriction of conduit vessels.
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Acknowledgments
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Y-27632 was a gift from the Welfide Corporation. We thank Rudolf Dussel for expert technical assistance. This study was supported by grants from the Italian Society of Nephrology, ItalianGerman co-operation grant (Vigoni CRUI-DAAD Program), the German Research Foundation (Graduiertenkolleg: Experimentelle Nieren- und Kreislaufforschung).
Conflict of interest statement. None declared.
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Received for publication: 20.11.02
Accepted in revised form: 9. 4.03