Ramipril prevents basal arterial constriction and enhanced myogenic tone in the femoral artery in mildly uraemic normotensive rats

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1 Clinical Science (1999) 97, (Printed in Great Britain) 233 Ramipril prevents basal arterial constriction and enhanced myogenic tone in the femoral artery in mildly uraemic normotensive rats Tessa SAVAGE*, Aisling C. MCMAHON*, Adrian MULLEN*, Rachel M. TRIBE and Magdi M. YAQOOB* *Anthony Raine Research Laboratories, St. Bartholomews Hospital, Dominion House, 59 Bartholomew Close, West Smithfield, London EC1A 7BE, U.K., and Fetal Health Research Group, St. Thomas Hospital, London SE1 7EH, U.K. A B S T R A C T Some aspects of vascular reactivity are altered in mild experimental uraemia, as shown by increased myogenic tone and a reduced lumen diameter in the femoral artery. This study was conducted to investigate the prevention of these uraemia-induced vascular abnormalities by the angiotensin-converting enzyme inhibitor (ACE-I) Ramipril. Ten male Wistar rats were rendered uraemic (U) by 5/6th nephrectomy, and 10 control (C) rats were concurrently sham-operated. After 4 weeks, both groups were given daily subcutaneous injections of 3 µg of Ramipril for a further 4 weeks. Tail-cuff systolic blood pressure was then recorded and the rat was killed. Isolated femoral arteries were mounted on a pressure myograph and pressurized at 40 mmhg for baseline measurements of the lumen internal diameter. Myogenic tone was then assessed over a range of intravascular pressures from 40 to 160 mmhg. Biochemically, serum urea and creatinine were significantly higher in the uraemic (U) group [urea: U, 23 3 mmol/l; C, 6 1 mmol/l (P 0 001); creatinine: U, mmol/l, C, mmol/l (P 0 01)]. Systolic blood pressure was the same in both groups (U, mmhg; C, mmhg). The mean baseline internal diameter was the same in both groups (U, µm; C, µm, not significant), as was mean myogenic tone (U, 4 7 1%; C,3 4 1%). In conclusion, there were no differences in baseline lumen diameter or myogenic tone in uraemic compared with control femoral arteries of rats treated with Ramipril, which suggests that Ramipril may prevent the development of elevated myogenic tone and decreased lumen diameter previously observed in this model of uraemia. These results suggest that these specific vascular abnormalities in uraemia may be mediated by renin or bradykinin, or by the direct action of angiotensin II on vascular smooth muscle. INTRODUCTION For over 20 years, cardiovascular disease has persisted as the leading cause of mortality, accounting for over 50% of deaths in patients with end-stage renal disease (ESRD), both in Europe [1] and in the U.S.A. [2]. An important prognostic risk factor is left ventricular hypertrophy, which is found in 60 70% of dialysis patients [3], and independently confers a mortality rate of 70% within 5 years in this population [4]. Changes in the structure and function of large arteries have been found to correlate with changes in left ventricular dimensions in ESRD patients [5], even in those without pre-existing cardiac or vascular disease [6]. Recently we have reported that, in the femoral artery of mildly uraemic rats, myogenic tone is increased and the basal arterial lumen diameter is Key words: femoral artery, myogenic tone, pressure myography, Ramipril, uraemia. Abbreviations: ACE-I, angiotensin-converting enzyme inhibitor; ESRD, end-stage renal disease; i.d., internal diameter; PSS, physiological salt solution; KPSS, potassium-substituted PSS; SHR, spontaneously hypertensive rat. Correspondence: Mrs T. Savage.

2 234 T. Savage and others reduced in the absence of hypertension before structural changes become apparent [7]. Enhanced vasoconstriction and concurrent loss of endothelium-dependent relaxation is regarded as an early marker of endothelial dysfunction in atherosclerosis, even before it becomes angiographically apparent [8,9]. Therefore it is possible that the reduced lumen diameter observed in uraemic rats may indicate endothelial dysfunction in this model of uraemia. In spontaneously hypertensive rats (SHRs), angiotensin-converting enzyme inhibitors (ACE-Is) both reverse and regress cardiac and vascular hypertrophy [10] and, in hypertensive patients, intervention with the ACE- I Ramipril was shown to induce regression of carotid intima-media thickness [11]. At sub-anti-hypertensive doses (0 01 mg day kg ), Ramipril has been shown to halt the progression of atherosclerosis, reduce the amount of surface atherosclerotic lesions present in the aorta and restore impaired endothelial function in cholesterol-fed rabbits [9,12]. Of particular interest was the observation that, in SHRs treated with the same dose, although hypertension and its associated vascular hypertrophy did develop, the response of the aorta to the vasoconstrictor noradrenaline (0 01 µm) was reduced, and relaxation in response to acetylcholine ( µm) was enhanced [13], suggesting that the observed changes were not related to structural alterations in the vascular wall. Given these findings, the aim of the present study was to investigate whether intervention with the ACE-I Ramipril might prevent the enhanced basal constriction previously observed in mildly uraemic rats [7]. We also aimed to assess the effects of Ramipril on myogenic tone. METHODS Animals Male Wistar rats ( g) were housed in the on-site Biological Services Unit individually in holding rooms at a constant temperature of 21 2 C and humidity of 40%. The 24 h day was fixed in a 12 h light 12 h dark cycle. All procedures had prior approval from the U.K. Home Office (project licence ) and were performed in accordance with the Animals Scientific Procedures Act Induction of uraemia The 5 6th nephrectomy animal model of uraemia was chosen, as it induces mild uraemia without the confounding factor of hypertension which is present in other uraemic models [14]. All animals underwent two surgical procedures in pairs 1 week apart. Anaesthesia was induced initially by an intramuscular injection into a hind leg of 0 18 ml of Hypnorm (fentanyl citrate mg ml and fluanisone 10 mg ml; Janssen-Cilag Ltd., Saunderton, High Wycombe, Bucks., U.K.), followed by 0 06 ml of Diazepem (Phoenix Pharmaceuticals Ltd., Gloucester, U.K.), which was given intraperitoneally. The rat was then shaved in the abdominal area for the first stage of surgery, which involved decapsulation and removal of two-thirds of the left kidney for the uraemic group (n 10) and decapsulation of the left kidney for the sham-operated control group (n 10). The following week, the rats were shaved over the area covering the right flank; the uraemic group underwent a total right nephrectomy and the control group was sham-operated as described above. For 8 weeks, the rats were pair-fed to control for appetite suppression associated with uraemia. Administration of Ramipril As Ramipril has been shown to exert effects on the vasculature independently of its anti-hypertensive properties [9,12], and since in previous experiments using the same model at 8 weeks of uraemia there was no evidence of hypertension [7], we elected to give a sub-antihypertensive dose of Ramipril. Beginning 4 weeks after the second stage of surgery, both groups were given Ramipril (3 µg day) subcutaneously into the scruff of the necks at the same time each day for the remaining 4 weeks before initiation of the experiment. This dose has been shown to have positive effects on the vasculature in SHRs [13]. Anaesthesia On each study day, one rat was studied. The rat was given 0 25 ml of Hypnorm into a rear thigh muscle, and a further 1 6 ml of diazepem was then given intraperitoneally to induce deep anaesthesia. This was tested by lifting the rat s head and checking that its neck was truly relaxed, and then sharply pinching its claw. If there was no physical reaction, it was assumed that the rat was sufficiently anaesthetized to proceed (usually 5 10 min later). As concurrent experiments were being performed by colleagues on cardiac myocytes, the rat was killed following the opening of the thoracic cavity and rapid removal of the whole heart. Systolic blood pressure This was measured on three consecutive days using the tail-cuff method (Programmed Electrosphygmomanometer PE-300; Nargo Bio-Systems, Houston, TX, U.S.A.) 1 week before the rat was killed. The rats were placed in restrainers with chocolate at the head end, which kept them still while systolic blood pressure was recorded on their tails held stable by the end of the restrainer. The mean of five readings on each day was recorded, but for analysis the mean from day 3 only was used, as the first 2 days were considered as a training period. Uraemic and control rats were handled identically with respect to the measurement of systolic blood pressure, to reduce the chances of systematic error.

3 Effects of Ramipril on myogenic tone in uraemia 235 Bloods Cardiac venepuncture was performed to obtain bloods for urea and electrolytes and a full blood count at the time that the rat was killed. Dissection of the femoral artery The femoral artery and surrounding muscle was excised from both legs immediately after the rat was killed, and pinned on to a silastic Petri dish containing cold physiological salt solution (PSS) and left to equilibrate for about 15 min. Two segments of the femoral artery (approx. 5 mm) were dissected under a microscope (Stemi SV6; Zeiss) using microscissors and fine forceps. Pressure myography The technique employed for studying the femoral artery was perfusion myography, which has been described extensively elsewhere [15]. Briefly, the vessel was mounted in the myograph chamber, composed of two opposing microglass cannulae (tips 70 µm), and secured at each end with two sutures. The vessel was then perfused with PSS both intraluminally and extraluminally. On either side of the chamber was a series of threeway taps with windkessels to dampen pulsatile flow and solid-state in-line pressure transducers. These proximal and distal pressure transducers monitored the respective pressures at each end of the vessel, and the mean intraluminal pressure was calculated and maintained by the pressure servo control pump. The internal diameter (i.d.) was measured continuously by the video dimension analyser, and the vessel was displayed on the video monitor which was connected to a charge-coupled device (CCD) camera positioned on the inverted microscope ( 10). Chemicals and solutions All other chemicals were purchased from Sigma Aldrich Co. (Poole, Dorset, U.K.), and all solutions were made up on the day of experimentation. PSS consisted of 5 litres of AnalaR water to which was added (mmol l): 119 NaCl, 4 7 KCl, 1 17 MgSO, 25 NaHCO, 1 18 NaH PO,0 026 EDTA and 5 5 glucose; PSS was kept refrigerated until use. To each 1 litre of PSS was added 2 5 M CaCl immediately before use. When calcium-free PSS was used, 0 38 g of the calcium chelater EGTA was added per litre of PSS. In potassiumsubstituted PSS (KPSS), equimolar KCl (mmol l) replaced NaCl. Experimental protocol At the start of the experiment, intraluminal perfusion of the vessel was stopped; the intraluminal pressure was raised to 40 mmhg and the vessel was left to equilibrate in PSS, which constantly perfused the vessel extraluminally at a rate of 19 ml min for 40 min. The PSS was maintained at 37 C and gassed continously with 95% O 5% CO to yield a ph of 7 4. The viability of the vessel was then tested by a standard procedure as described elsewhere [16], by direct extraluminal application to the perfusion chamber of both L-phenylephrine (1 µmol l) in KPSS, to ensure rapid vasoconstriction, and acetylcholine (1 µmol l) to check the function of the endothelium by subsequent vessel dilatation. Criteria for a suitable vessel was dilatation to 90% or more of its original diameter. If this did not occur, then the vessel was discarded and another vessel from the same rat was mounted and the procedure repeated. The intraluminal pressure was then raised in incremental steps of 20 mmhg up to 160 mmhg. At each step, the vessel i.d. was recorded after 10 min. The pressure was then reduced down to 40 mmhg and the vessel was left to equilibrate in Ca-free PSS for 40 min, and then passive diameters were obtained every 10 min at each intravascular pressure as above. Myogenic tone was calculated as follows: Myogenic tone at (Ca-free PSS i.d.) (PSS i.d.) each intravascular 100 Ca-free PSS i.d. pressure (%) Statistical analysis Data are expressed as means S.E.M. For each vessel, a summary score representing the average myogenic tone for each group was calculated. The means of these and the other parameters in the control and uraemic groups were compared using Student s t-test for unpaired data, as we could not be certain that the rats came from the same litter. Significance was assumed when P RESULTS General data Data were lost from one control rat due to two separate vessels leaking when pressurized. Therefore data were successfully collected from ten uraemic (U) rats and nine control (C) rats. The baseline characteristics are presented in Table 1. The body weight of the two groups of rats did Table 1 Baseline characteristics of Ramipril-treated control and uraemic rats Significance of differences: *P 0 01; **P Variable Control (n 9) Uraemic (n 10) Body weight (g) Serum creatinine (µmol/l) * Serum urea (mmol/l) ** Haemoglobin (g/dl) ** Systolic blood pressure (mmhg)

4 236 T. Savage and others Figure 1 i.d. of the femoral artery pressurized at 40 mmhg in uraemic (U; n 10) and control (C; n 9) rats Figure 2 Mean myogenic tone (at mmhg) in the femoral arteries of uraemic (U; n 10) and control (C; n 9) rats not differ significantly at the time of experiments, indicating that the uraemic group was not volumeexpanded. Serum urea was raised 3-fold and serum creatinine about 2-fold in the uraemic rats compared with controls, and haemoglobin was significantly lower in the uraemics, although still within the normal range for male Wistar rats (11 18 g dl) [17]. Systolic blood pressure was the same and normotensive in both groups (U, mmhg; C, mmhg), as was expected using the model of uraemia [14]. Pressure myography Following the 40 min equilibration period when the vessel was pressurized at 40 mmhg, the lumen i.d. was the same in both groups (U, µm, C, µm; not significant) (Figure 1). Moreover, passive diameters obtained in calcium-free PSS were also similar in the two groups (U, µm; C, µm). The mean myogenic tone for all intravascular pressures did not differ between the two groups (U, 4 7 1%; C, 3 4 1%) (Figure 2), and there was no significant difference at any single intraluminal pressure. DISCUSSION The principle findings of this study are that there were no differences in baseline lumen diameter or myogenic tone in the femoral arteries of uraemic compared with control rats treated with low-dose Ramipril. This differs from a previous study in which we observed a significantly reduced lumen diameter and elevated myogenic tone in vessels from untreated uraemic rats compared with those from control rats [7]. This suggests that Ramipril may prevent the development of these specific vascular abnormalities in this model of uraemia, and that the abnormalities might be mediated by renin or bradykinin, or by the direct action of angiotensin II on vascular smooth muscle. The use of ACE-Is has been shown to exert a protective renal effect by slowing down the progression of renal disease in both diabetic [18] and non-diabetic [19] populations, and clinical studies of patients with pre- ESRD have demonstrated a 19 20% regression in left ventricular mass index with a substantial improvement in diastolic left ventricle function after treatment with enalapril or captopril for 12 months [20]. As changes in large-artery function and structure have been associated with increased left ventricular mass in ESRD patients [5], it is possible that our results may be of potential clinical vascular benefit to patients before reaching ESRD. We deliberately elected to use Ramipril at a sub-antihypertensive dose, as hypertension is not usually a feature of this model of experimental uraemia [14], and this dose has been shown to reduce both cardiac and vascular hypertrophy in other experimental models [10]. Ramipril is an ACE-I which results in reduced angiotensin II formation, accumulation of bradykinins and even increased angiotensin-(1 7) formation. It may be that rendering angiotensin II production below normal with Ramipril contributed to the prevention of the elevation of myogenic tone. Several experimental studies have demonstrated that there are many beneficial cardiovascular effects of ACE-Is and the resulting specific AT -receptor antagonism. These include a reduction in neointima formation following carotid balloon denudation in rats [21 23], a decrease in blood pressure and vascular hypertrophy in SHRs [13,23,24], an improvement in endothelial function and cessation of atherosclerosis in rabbits [9], a decrease in the size of myocardial infarcts in rabbits, and regression of left ventricular hypertrophy in rats following aortic banding [12]. In our study, we cannot comment on the relative contributions of angiotensin II and bradykinin activities to account for our results, as Ramipril blocks all ACE activity. Thus further study is required to elucidate the precise mechanisms underlying our results. To summarize, there were no differences in baseline lumen diameter or myogenic tone in the femoral arteries of uraemic compared with control rats treated with Ramipril. This suggests that Ramipril may prevent the development of elevated myogenic tone and decreased lumen diameter observed previously in uraemic vessels compared with control vessels using this model of experimental uraemia. These observations suggest that the use of an ACE-I (Ramipril) in uraemic patients may confer a cardiovascular benefit as well as a renoprotective advantage.

5 Effects of Ramipril on myogenic tone in uraemia 237 ACKNOWLEDGMENTS This work is dedicated to the late Professor A. E. G. Raine. We thank J. M. Wilson for her technical assistance in administering the Ramipril. REFERENCES 1 Raine, A. E. G., Margreiter, R., Brunner, F. P. et al. (1992) Report on management of renal failure in Europe, XXII, Nephrol. Dial. Transplant. 7 (suppl. 2), USRDS 1997 Annual Data Report (1997) Am. J. Kidney Dis. 30 (suppl. 2) 3 Foley, R. N., Parfrey, P. S. and Harnett, J. D. (1992) Left ventricular hypertrophy in dialysis patients. Semin. Dial. 5, Silberberg, J. S., Barne, P. E., Prichard, S. S. and Sniderman, A. D. (1989) Impact of left ventricular hypertrophy on survival in end-stage renal disease. Kidney Int. 36, London, G. M., Guerin, A. P., Marchais, S. J. et al. (1996) Cardiac and arterial interactions in end-stage renal disease. Kidney Int. 50, Savage, T., Clarke, A. L., Giles, M., Tomson, C. R. V. and Raine, A. E. G. (1998) Calcified plaque is common in the carotid and femoral arteries of dialysis patients without clinical vascular disease. Nephrol. Dial. Transplant. 13, Savage, T., McMahon, A. C., Mullen, A. M. et al. (1998) Increased myogenic tone precedes structural changes in mild experimental uraemia in the absence of hypertension. Clin. Sci. 95, Sharpe, N. (1993) The effects of ACE inhibition on progression of atherosclerosis. J. Cardiovasc. Pharmacol. 22 (suppl. 9), S9 S12 9 Riezebos, J., Vleeming, W., Beems, R. B. et al. (1994) Comparison of the antiatherogenic effects of Isradipine and Ramipril in cholesterol-fed rabbits: I, effect on progression of atherosclerosis and endothelial dysfunction. J. Cardiovasc. Pharmacol. 23, Lundie, M. J., Friberg, P., Kline, R. L. and Adams, M. A. (1997) Long-term inhibition of the renin angiotensin system in genetic hypertension: analysis of the impact on blood pressure and cardiovascular structural changes. J. Hypertens. 15, Mayet, J., Stanton, A. V., Sinclair, A.-M. et al. (1995) The effects of antihypertensive therapy on carotid vascular structure in man. Cardiovasc. Res. 30, Linz, W., Wiemer, G. and Scholkens, B. (1993) Contribution of bradykinin to the cardiovascular effects of Ramipril. J. Cardiovasc. Pharmacol. 22 (suppl. 9), S1 S8 13 Gohlke, P., Lambert, V., Kuwer, I., Bartenbach, S., Schnelli, A. and Unger, T. (1993) Vascular remodelling in systemic hypertension. Am. J. Cardiol. 71, 2E 6E 14 Ibrahim, H. N. and Hostetter, T. H. (1998) The renin aldosterone axis in two models of reduced renal mass in the rat. J. Am. Soc. Nephrol. 9, Osol, G. and Cipolla, M. (1993) Interaction of myogenic and adrenergic mechanisms in isolated, pressurized uterine radial arteries from late-pregnant and non-pregnant rats. Am. J. Obstet. Gynecol. 168, Tribe, R. M., Thomas, C. R. and Poston, L. (1998) Flowinduced dilatation in isolated resistance arteries from control and streptozotocin-diabetic rats. Diabetologia 41, Wolfenson, S. and Lloyd, M. (1994) Handbook of Laboratory Animal Management and Welfare, Oxford University Press, Oxford 18 Hoeschler, D. and Bakris, G. (1994) Antihypertensive therapy and progression of renal disease. J. Cardiovasc. Pharmacol. 23 (suppl. 1), S34 S38 19 Giatras, I., Lau, J. and Levey, A. S. (1997) Effect of angiotensin-converting enzyme inhibitors on the progression of nondiabetic renal disease: a meta-analysis of randomized trials. Ann. Intern. Med. 127, Dyadyk, A. I., Bagriy, A. E., Lebed, I. A., Yarovaya, N. F., Schukina, E. V. and Taradin, G. G. (1997) ACE inhibitors captopril and enalaril induce regression of left ventricular hypertrophy in hypertensive patients with chronic renal failure. Nephrol. Dial. Transplant. 12, Kauffman, R. F., Bean, J. S., Zimmerman, K. M., Brown, R. F. and Steinberg, M. I. (1991) Losartan, a non-peptide angiotensin II (ang II) receptor antagonist, inhibits neointima formation following balloon injury to rat carotid arteries. Life Sci. 49, PL-223 PL Farhy, R., Peterson, E. and Scicli, A. G. (1997) Kinins and the events influenced by an angiotensin-converting enzyme inhibitor during neointima formation in the rat carotid artery. J. Hypertens. 15, Kim, S. and Iwao, H. (1997) Involvement of angiotensin II in cardiovascular and renal injury: effects of an AT - receptor antagonist on gene expression and the cellular phenotype. J. Hypertens. Suppl. 15, S3 S7 24 Hashimoto, Y., Kurosawa, Y., Minami, K., Fushimi, K. and Narita, H. (1998) A novel angiotensin II-receptor antagonist, 606A, induces regression of cardiac hypertrophy, augments endothelium-dependent relaxation and improves renal function in stroke-prone spontaneously hypertensive rats. Jpn. J. Pharmacol. 76, Received 1 February 1999; accepted 29 March 1999