Comparison of the AT 1 -receptor blockers candesartan, irbesartan and losartan for inhibiting renal microvascular constriction

Paper Keywords: isolated perfused hydronephrotic rat kidney, renal microvessels, AT1 antagonists, candesartan, irbesartan, losartan *Laboratory for P...
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Keywords: isolated perfused hydronephrotic rat kidney, renal microvessels, AT1 antagonists, candesartan, irbesartan, losartan *Laboratory for Physiology and † Department of Nephrology, Institute for Cardiovascular Research (ICaR-VU), Vrije Universiteit, Amsterdam, The Netherlands Correspondence to: Dr William F van Rodijnen Laboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), Vrije Universiteit, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands Tel: +31 20 4448380 Fax: +31 20 4448255 E-mail: w.van_rodijnen. [email protected] JRAAS 2001;2 (suppl 1):S204-S210

Journal of the ReninAngiotensinAldosterone System (Including other peptidergic systems)

March 2001 Volume 2 Supplement 1 S204

Comparison of the AT1-receptor blockers candesartan, irbesartan and losartan for inhibiting renal microvascular constriction William F van Rodijnen,* Ton A van Lambalgen*, Marco E van Teijlingen,* Geert-Jan Tangelder,* Piet M ter Wee†

Abstract Angiotensin II (Ang II) type 1 (AT1) receptor blockers differ in their affinity for the AT1-receptor, suggesting a dissimilar potency for inhibiting Ang II-induced vascular constriction. In the present study, we compared the effects of candesartan, irbesartan and losartan on the renal microvascular constriction to locally-formed Ang II, using isolated, perfused hydronephrotic rat kidneys. Addition of 1 nmol/L angiotensin I (Ang I, the precursor of Ang II) significantly reduced the diameters of interlobular arteries (ILAs; -47.6±2.6%), afferent arterioles (AAs; -43.6±2.3%) and efferent arterioles (EAs; -31.6±2.4%). Candesartan and irbesartan were more potent in antagonising the constriction to Ang I than losartan. By contrast, candesartan and irbesartan differed only slightly in potency. After a washing period of 60 minutes with drug-free medium, a second application of Ang I failed to induce vasoconstriction only in candesartan-treated kidneys. Pretreatment of hydronephrotic kidneys with candesartan, to further explore its antagonistic properties, shifted the dose-response curves of Ang II approximately 2 log units to the right without reducing the maximal Ang IIinduced constriction of ILAs, AAs or EAs. Additionally, dose-response curves of Ang II were similar after short (10 minutes) and prolonged (60 minutes) preincubation with candesartan. Our findings indicate that candesartan and irbesartan are more potent inhibitors of renal microvascular constriction to locallyformed Ang II than losartan. The inhibitory effect of candesartan is more prolonged, suggesting a slow dissociation from the AT1-receptor. Additionally, candesartan was found to block the Ang II-induced constriction of renal microvessels in a surmountable manner. Introduction The renin angiotensin-aldosterone system (RAAS) plays a pivotal role in the regulation of systemic blood pressure and local renal haemodynamics. One of its effector peptides, angiotensin II (Ang II), acts as a strong vasoconstrictor and stimulates sodium and fluid retention. Increased levels of Ang II are thought to be critically involved in the development and maintenance of hypertension and congestive heart failure. In these clinical settings, pathophysiological actions of Ang II are predominantly mediated via the Ang II type 1 (AT1)

receptor. Therefore, therapeutic efforts have focused on the development of drugs that specifically antagonise this receptor subtype. Currently, several non-peptide AT1-receptor blockers are available.1,2 Of these, losartan was the first to be approved for clinical use, followed by several others, including irbesartan and candesartan. To differentiate between AT1-receptor blockers, several in vitro studies have been performed to assess the affinity of these drugs for the AT1-receptor. Based on displacement of radiolabelled Ang II in membrane preparations from different tissues, candesartan was found to have the highest affinity for the AT1-receptor, followed by irbesartan and losartan.2-4 The affinity of candesartan, irbesartan and losartan for the AT1-receptor is reflected in their potency for inhibiting AT1-mediated actions. In Chinese hamster ovary cells expressing the transfected human AT1-receptor, the Ang II-induced inositol phosphate accumulation was inhibited with a potency rank order of candesartan > irbesartan > losartan.5 Although there is general consensus that Ang II-induced vasoconstriction is mediated by the AT1-receptor,few studies have directly compared the potency of AT1-receptor blockers for antagonising Ang II-induced constrictor responses. Using isolated rabbit aortic strips, Cazaubon et al.4 reported that irbesartan was 6–7 times more potent than losartan in inhibiting Ang II-mediated contraction. However, candesartan was not included in this study. Similarly, in the hydronephrotic rat kidney preparation, losartan inhibited the renal microvascular constriction in response to Ang II.6 However, the effects of candesartan and irbesartan on Ang II-induced constriction were not investigated. Thus, although evidence from radioligand displacement experiments suggest that candesartan, irbesartan and losartan differ in their potency for inhibiting AT1mediated responses, the potency rank order of these antagonists for blocking Ang II-induced constriction in vascular preparations is unclear. Recently, the duration of AT1-receptor antagonism was explored in isolated rat portal veins. In contrast to irbesartan and losartan, candesartan produced a long-lasting antagonism of the vascular response to Ang II, as demonstrated by maintained inhibition, despite extensive washing

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of the preparation.7 The long-lasting action of candesartan is attributed to its slow dissociation from the AT1-receptor.8 In isolated rabbit aortic rings3,7,9 and rat portal veins,7 candesartan caused a marked reduction of the maximal response to Ang II, suggesting insurmountable antagonism.Although still not completely clear, the slow dissociation rate of candesartan from the AT1-receptor is also thought to underlie this antagonistic behaviour. In contrast, pretreatment of rabbit aortas7,9 or rat portal veins7 with losartan or irbesartan produced a rightward shift of the dose-response curves of Ang II, without altering the maximal effect, suggesting surmountable antagonism. Local Ang II generation is likely to play an important role in the kidney,with respect to the regulation of glomerular haemodynamics and sodium and fluid retention.10 The ability of the kidney to generate substantial amounts of Ang II is demonstrated by the rapid conversion of angiotensin I (Ang I) to Ang II,which results in vasoconstriction of the renal microvasculature.11 In the present study, we compared the potency of candesartan, irbesartan and losartan in inhibiting renal microvascular constriction to locally-formed Ang II. We treated hydronephrotic rat kidneys with Ang I, followed by increasing concentrations of either candesartan, irbesartan or losartan. After performing doseresponse curves, kidneys were perfused with control tissue culture medium for 1 hour. Subsequently, Ang I was reapplied to study the duration of the inhibitory effect of candesartan,irbesartan and losartan in the kidney. Because a second constrictor response to Ang I was absent only in candesartan-treated kidneys, special attention was paid to the antagonistic properties of this particular AT1-receptor antagonist. Materials and methods Animals Experiments were performed using hydronephrotic kidneys from male Sprague-Dawley rats (Harlan, Zeist, The Netherlands). The rats were housed individually in macrolon cages and had access to pelleted food and water ad libitum.The Institutional Animal Care and Use Committee approved all procedures as described below.

Journal of the ReninAngiotensinAldosterone System (Including other peptidergic systems)

March 2001 Volume 2 Supplement 1

Chemicals The perfusion medium used for in vitro perfusion of hydronephrotic rat kidneys consisted of Dulbecco's modified Eagle medium (DMEM) supplemented with (in mmol/L) 23.8 bicarbonate, 5.5 D-glucose, 1 sodium pyruvate and 5.6 HEPES. Medium was equilibrated with 95% air / 5% CO2, resulting in a pO2 of approximately 150 mm Hg, a pCO2 of approximately 35 mmHg and a pH of 7.4. Temperature was maintained at 37°C. The AT1-receptor blockers, candesartan, irbesartan and losartan were kindly provided by AstraZeneca R&D (Mölndal, Sweden), Sanofi (Montpellier, France) and Merck (Rahway, NJ),

respectively. Stock solutions were prepared fresh on the day of an experiment. Candesartan was dissolved in a 1:4 mixture of 1 N Na2CO3 and physiological saline. Fifty percent dimethylformamide was used as solvent for irbesartan, while losartan was dissolved in distilled water. Angiotensin I (Ang I), angiotensin II (Ang II) and arginine vasopressin were dissolved in distilled water and the stock solutions were stored at –20°C until use. DMEM, D-glucose, dimethylformamide, Ang II, Ang I and arginine vasopressin were obtained from Sigma-Alldrich (Zwijndrecht, The Netherlands), bicarbonate was from Merck (Darmstadt, Germany) and pyruvate and HEPES were purchased from GIBCO-BRL (Breda,The Netherlands). Induction of unilateral hydronephrosis Rats were anaesthetised using Hypnorm® (fluanison (4 mg/kg) and fentanyl (0.126 mg/kg), i.m., Janssen Pharmaceutica, Beerse, Belgium) and diazepam (2 mg/kg, i.p., Centrafarm, Etten-Luer, The Netherlands). To induce hydronephrosis, the left ureter was exposed and subsequently tied off with a suture. Six to eight weeks after surgery, renal tubular tissue had undergone almost complete atrophy, allowing direct microscopic visualisation of the renal microvasculature.12,13 At that stage, the hydronephrotic kidney was excised for in vitro perfusion. In vitro perfusion of hydronephrotic kidneys The technique for isolation and in vitro perfusion of hydronephrotic rat kidneys has been described in detail by Loutzenhiser et al.14 Briefly, rats were anaesthetised using pentobarbital (50 mg/kg, i.p., Nembutal®, Sanofi Sante B.V., Maassluis, The Netherlands) and ketamine (25 mg/kg, i.m., Kombivet, Etten-Leur, The Netherlands). The hydronephrotic kidney was exposed through a wide abdominal incision and its renal artery was cannulated via the abdominal aorta. Under continuous perfusion with DMEM, this kidney was excised and connective tissue, fat and the renal capsule were removed. Subsequently, the kidney was moved to the stage of an inverted microscope (Axiovert 100, Zeiss, Weesp, The Netherlands), equipped with a thin glass viewing port on the bottom surface.12 A single-pass perfusion setup was used for in vitro perfusion.14 Briefly, preheated and oxygenated DMEM was pumped on demand from a large reservoir through a heat exchanger to a small reservoir, supplying the renal artery. The driving force for perfusion of the hydronephrotic kidney was provided by the inflow of gas (95% air / 5% CO2) into the small reservoir. During the experiment, renal perfusion pressure was measured at the level of the renal artery and kept constant at 80 mmHg by adjusting the pressure within the small pressurised reservoir, using a back-pressure regulator. A rolling pump (Masterflex, Chicago, IL) was used to pump the venous effluent away from the microscope chamber.

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Figure 1 Effects of candesartan, irbesartan and losartan on the vasoconstriction of interlobular arteries (ILAs), afferent arterioles (AAs) and efferent arterioles (EAs) in response to angiotensin I (1 nmol/L).The corresponding IC50 values are given in Table 1. * = this point and the preceding part of the dose-response curve were significantly different from the basal diameter (p

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