Evaluation of a rapid pressor response test in healthy cats Amanda Erickson Coleman, DVM; Chad W. Schmiedt, DVM; Tiffany L. Jenkins, BS; Emily D. Garber, BS; Lisa R. Reno, BS; Scott A. Brown VMD, PhD

Objective—To evaluate angiotensin I and angiotensin II rapid pressor response tests in healthy cats. Animals—6 purpose-bred sexually intact male cats. Procedures—Telemetric blood pressure (BP) implants were placed in all cats. After 2 weeks, cats were anesthetized for challenge with exogenous angiotensin I or angiotensin II. Continuous direct arterial BP was recorded during and immediately after IV administration of boluses of angiotensin I or angiotensin II at increasing doses. Blood pressure responses were evaluated for change in systolic BP (SBP), change in diastolic BP (DBP), and rate of increase of SBP by 4 observers. Results—Following IV angiotensin I and angiotensin II administration, transient, dosedependent increases in BP (mean ± SEM change in SBP, 25.7 ± 5.2 and 45.0 ± 9.1; change in DBP, 23.4 ± 4.7 mm Hg and 36.4 ± 7.8 mm Hg; for 100 ng of angiotensin I/kg and angiotensin II/kg, respectively) and rate of increase of SBP were detected. At angiotensin I and II doses < 2.0 ng/kg, minimal responses were detected, with greater responses at doses ranging from 20 to 1,000 ng/kg. A significant effect of observer was not found. No adverse effects were observed. Conclusions and Clinical Relevance—The rapid pressor response test elicited dosedependent, transient increases in SBP and DBP. The test has potential as a means of objectively evaluating the efficacy of various modifiers of the renin-angiotensin-aldosterone system in cats. Ranges of response values are provided for reference in future studies. (Am J Vet Res 2013;74:1392–1399)

S

ystemic arterial hypertension is a well-recognized cause of morbidity in cats, particularly those of advanced age.1–4 In contrast to human patients, in whom essential (primary) hypertension is common, affected cats typically develop this disorder in association with other conditions, with CKD and hyperthyroidism implicated most frequently.2,4 The harmful effects of chronic hypertension have been well described and include injury to the heart, kidneys, eyes, and CNS.1,5–8 In patients with CKD, development of hypertension is complex and multifactorial. In human patients, hypertension associated with CKD is traditionally ascribed to impaired renal salt handling, excessive activation of the RAAS, sympathetic nervous system overactivity, and endothelial dysfunction.9 The role of RAAS activation in hypertensive cats with CKD has been examined in both naturally occurring disease and in exReceived February 20, 2013. Accepted July 10, 2013. From the Departments of Small Animal Medicine and Surgery (Coleman, Schmiedt, Jenkins, Garber, Reno, Brown) and Physiology and Pharmacology (Brown), College of Veterinary Medicine, University of Georgia, Athens, GA 30602. Supported in part by the Veterinary Medical Experiment Station of the College of Veterinary Medicine, University of Georgia. The authors thank Drs. Daniel C. Loper and Deborah Keys for technical assistance. Address correspondence to Dr. Coleman ([email protected]). 1392

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ACE ACE-i ARB BP CKD DBP ∆DBP ∆PP ∆SBP ∆SBPrate HR RAAS RPRT SBP SBPmax Tmax

ABBREVIATIONS

Angiotensin-converting enzyme Angiotensin-converting enzyme inhibitors Angiotensin II receptor blocker Blood pressure Chronic kidney disease Diastolic blood pressure Maximum change in diastolic blood pressure Change in pulse pressure between baseline and time at peak pressor response Maximum change in systolic blood pressure Rate of systolic blood pressure increase during pressor response Heart rate Renin-angiotensin-aldosterone system Rapid pressor response test Systolic blood pressure Systolic blood pressure at peak pressor response Time at peak pressor response

perimental disease, with some but not all affected cats having evidence of RAAS overactivity.10–13 Thus, the RAAS would seem to be a logical target for pharmacologic modulation of hypertension in at least some cats with hypertension associated with CKD. AJVR, Vol 74, No. 11, November 2013

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Numerous antihypertensive agents, with their effects mediated by RAAS inhibition, have been developed as a means of decreasing BP in hypertensive human patients; these include ACE-i, ARBs, and renin inhibitors. In addition to their antihypertensive properties, RAAS-modifying agents have renoprotective effects, largely attributed to their ability to reduce proteinuria and glomerular hypertension.14,15 Assessment of the efficacy of antihypertensive treatments requires a reproducible method of objective and accurate endpoint evaluation. In veterinary medicine, clinical trials designed to evaluate these drugs can be expensive and time-consuming and are confounded by issues such as unpredictable owner compliance, inaccuracy and imprecision of indirect BP measurement devices, difficulty in the detection of subtle yet important differences in BP, and patient comorbidities. To overcome these limitations, large case numbers are needed, which further extends study duration, especially if multiple doses and drugs are investigated. In human patients, exogenous angiotensin challenge during continuous BP measurement (ie, the RPRT) is a valid means for evaluating blockade of the RAAS by agents designed to modify this system.16 Pressor response, in addition to measurement of circulating hormone concentrations, is the preferred method of evaluation in phase I clinical trials examining these drugs.16 By use of this technique, there is good correlation between doses eliciting a 75% attenuation of the effect of angiotensin I or II in healthy volunteers and the doses with therapeutic efficacy in clinical patients.16 The goal of the study reported here was to evaluate BP responses to exogenously administered angiotensin I and II in healthy cats. The hypothesis was that a measurable, transient, dose-dependent increase in SBP and DBP would be observed in response to bolus administration of angiotensin I and II. Materials and Methods Cats—Seven adult sexually intact male purposebred domestic shorthair cats were used. Approximately 10 months prior to entrance in the study, these cats were inoculated with Brugia malayi as part of a project to induce microfilariasis.17 All cats included in the present study failed to become microfilaremic. Prior to entrance into the study, the cats’ general health was confirmed with a physical examination, CBC, serum biochemical profile, and urinalysis. All cats were vaccinated against common viral diseases and tested negative for FeLV antigens and antibodies against FIV. Cats were housed individually in cages, had access to water at all times, and were fed a commercially available adult feline rationa ad libitum. Ambient temperature was maintained from 20° to 22°C, and the cats were exposed to a 12-hour light to dark cycle. The Institutional Animal Care Committee of the University of Georgia approved all research activities. One cat was removed from the present study after a cardiac arrhythmia was identified following administration of 500 and 1,000 ng of angiotensin II/kg. This arrhythmia was transient, resolved as angiotensin II response waned, and was not observed during subsequent ECG studies. Echocardiographic evaluation AJVR, Vol 74, No. 11, November 2013

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revealed moderate concentric hypertrophy of the left ventricle. Given the cat’s age (3 years) and the absence of hypertension, a diagnosis of hypertrophic cardiomyopathy was made, prompting removal of the cat from the study. Surgical placement of radiotelemetry implants— The technique used for device implantation has been described in detail.18,19 Food was withdrawn 12 hours prior to surgery, and cats were allowed free access to water. Cats were premedicated with a combination of acepromazineb (0.01 mg/kg, IM), buprenorphinec (0.04 mg/kg, IM), and ketamined (7 mg/kg, IM). Anesthesia was induced with isofluranee in 100% oxygen supplied by a facemask, cats were endotracheally intubated, and anesthesia was maintained with isoflurane in 100% oxygen. A pressure-sensing telemetry devicef was surgically inserted into the right femoral artery of each cat. The body of the implant was sutured to the ventral aspect of the abdominal wall. In one of the cats, implant failure required repositioning to the left femoral artery during a separate anesthetic event. This was performed prior to data collection and analysis. Angiotensin I and II RPRTs—Each cat was premedicated with butorphanolg (0.2 mg/kg, IM). An IV catheterh was placed in either the right or left cephalic vein, through which propofoli (7 mg/kg, IV) was administered for induction of anesthesia. The trachea was intubated, and 100% oxygen was provided for inhalation during the anesthetic period. Following intubation, a stable plane of anesthesia was maintained by a constant rate infusion of propofol (0.3 mg/kg/min, IV). Forced-air warming, by use of a commercial convective warming unit,j was used to decrease body heat dissipation and prevent hypothermia. Anesthetic depth was determined by periodic evaluation of eye position, jaw tone, and palpebral reflex. Pulse and respiratory rates were obtained manually at 5-minute intervals. Rectal temperature was measured with a commercial digital thermometer at 15-minute intervals. A second IV catheter was placed in the other cephalic vein and used for angiotensin I or II administration. Cats were positioned in right lateral recumbency, with a radiotelemetry receiverk positioned beneath them. In anticipation that the RPRT would be used for assessment of the efficacy of orally administered antihypertensive agents, each cat received a gelatin capsule containing placebo,l orally, approximately 90 minutes prior to anesthetic induction. Angiotensin I and II were diluted as follows. Stock solutions (500 µg/mL) were prepared by adding 10 mg of anhydrous angiotensin Im or angiotensin IIn to 20 mL of PBS solution.o These solutions were filtered through a 0.2-µm filter,p aliquoted into sterile cryovials (250 µg of angiotensin/vial), and stored at –80°C. On the day that the RPRT was to be performed, 1 aliquot was thawed on ice and serially diluted in sterile PBS solution to produce solutions with final concentrations of 5 µg/mL, 0.05 µg/mL, and 0.0005 µg/mL. Time from angiotensin reconstitution to its use in the RPRT was 1 and 18 days for angiotensin II and angiotensin I, respectively. Each IV injection of angiotensin was administered as a bolus during a period of approximately 2 seconds 1393

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and was followed immediately by administration of 1 mL of saline (0.9% NaCl) solution. The concentration was increased with each dose administered (0.02, 0.2, 20.0, 100, 500, and 1,000 ng/kg of body weight). The subsequent angiotensin bolus was not administered until baseline BP was stable and a minimum 10-minute washout period was allowed. For 20 to 60 seconds prior to and up to 300 seconds following each bolus injection, digital BP data detected by the receiver as well as ambient barometric pressure data were routed to a dedicated computer-based acquisition systemq that recorded data in 2 formats. In the first format, continuous, real-time BP was displayed as a graphed tracing (pressor response curve), with time (seconds) and pressure (mm Hg) on the x- and y-axes, respectively (Figure 1). In the second format, these variables, as well as continuously derived HR and pulse pressure, were recorded in a spreadsheet as single values obtained by determining the mean of the data in intervals of 5 seconds. All BP measurements were expressed in millimeters of mercury and HR measurements in beats per minute. In any cat for which SBPmax exceeded 250 mm Hg in response to a given dose of angiotensin, further dose increases were not performed. Following data collection, propofol infusion was discontinued and cats were allowed to breathe 100% oxygen for 3 to 5 minutes before removal from the breathing circuit. Forced-air warming was continued until rectal tem-

perature was within reference range. Extubation was performed when vigorous reflex swallowing was elicited. Rectal temperature, respiratory rate, and HR were monitored manually at 5-minute intervals during the recovery period, until cats were able to maintain sternal recumbency. During a separate anesthetic event for each cat, this procedure was repeated in an identical manner for the same doses of angiotensin I. To evaluate repeatability, the RPRT was replicated on a later date by use of angiotensin I at doses from 20 to 1,000 ng/kg and angiotensin II at doses from 20 to 500 ng/kg. Telemetric implants were left in place at the conclusion of the study. Analysis of pressor response—Four observers (AEC, TLJ, SAB, and CWS) without knowledge of cat, date, time, angiotensin type, and angiotensin dose independently evaluated each pressor response curve (Figure 1) for the following variables: SBP at baseline, SBPmax, DBP at baseline, DBP at peak pressor response, and Tmax. From these values, ∆SBP, ∆DBP, ∆SBPrate, ∆PP, and time between angiotensin injection and Tmax were calculated for each response curve. In addition to these graph-derived variables, HR and pulse pressure at baseline and at Tmax were identified for each cat at representative low (0.02 ng/ kg), intermediate (20.0 ng/kg), and high (500.0 ng/ kg) angiotensin doses from continuous data (mean

Figure 1—Representative BP pressor-response curve generated following IV bolus administration (open arrow) of 500 ng of angiotensin II/kg to a healthy anesthetized cat. Notice the biphasic response to the high dose of angiotensin. DBPBL = DBP at baseline. DBPmax = DBP at peak pressor response. SBPBL = SBP at baseline. SBPmax = SBP at peak pressor response. Tinj = Time at injection. Tmax = Time at maximum systolic pressor response. 1394

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Table 1—Mean ± SEM values for variables in 6 anesthetized healthy cats administered various doses of angiotensin I or angiotensin II in a study of an RPRT. Angiotensin I Angiotensin dose (ng/kg) 0.02 0.2 2 20 100 500 1,000

∆SBP (mm Hg)

∆DBP (mm Hg)

0.3 ± 0.2a 0.7 ± 0.3a 1.5 ± 0.5a 8.8 ± 1.3a 25.7 ± 5.2b 69.4 ± 12.2 c 96.2 ± 13.2d

0.1 ± 0.1a 0.6 ± 0.2a 1.1 ± 0.4a 7.7 ± 1.1a 23.4 ± 4.7b 58.1 ± 9.4c 77.7 ± 9.7d

∆SBPrate (mm Hg/s) 0.003 ± 0.002a 0.017 ± 0.007a 0.037 ± 0.015a 0.249 ± 0.026a,b 0.441 ± 0.051b 0.708 ± 0.066c 0.907 ± 0.091c

Angiotensin II ∆PP (mm Hg)

∆SBP (mm Hg)

∆DBP (mm Hg)

0.1 ± 0.1a 0.2 ± 0.1a 0.3 ± 0.3a 1.1 ± 0.5a 2.3 ± 0.7a 11.6 ± 3.2b 18.5 ± 4.0c

1.6 ± 0.9a 2.5 ± 2.3a 4.5 ± 1.82a 18.2 ± 4.2b 45.0 ± 9.1c 99.5 ± 15.6d —

1.4 ± 0.7a 1.9 ± 1.7a 3.2 ± 1.2a 15.4 ± 3.9b 36.4 ± 7.8c 80.2 ± 12.6d —

∆SBPrate (mm Hg/s)

∆PP (mm Hg)

0.034 ± 0.013a 0.090 ± 0.073a 0.158 ± 0.062a 0.733 ± 0.102b 0.971 ± 0.107b 1.253 ± 0.114c —

0.3 ± 0.2a 0.6 ± 0.6a 1.3 ± 0.7a 2.8 ± 0.8a 8.6 ± 1.8b 19.3 ± 5.9c —

a–d Within each column, values with different superscript letters are significantly (P < 0.05) different. — = Not applicable.

values calculated during 5-second intervals) determined with the analysis software. Statistical analysis—Analyses were performed with commercial software.r A repeated-measures ANOVA that recognized multiple observations as belonging to the same cat was used to test for differences in ∆SBP, ∆DBP, ∆SBPrate, and ∆PP among hormones (angiotensin I or II), replicate day (1 or 2), hormone doses, and observers. The full model included fixed factors for hormone, hormone dose, observer, replicate and all 2-, 3-, and 4-way interaction terms, and a random factor of cat. Multiple comparisons were adjusted by means of the Tukey test. No significant (P < 0.05) observer effects or significant observer interactions were found; thus, observer was dropped from the model. An unstructured covariance was used in all repeated-measures models. All hypothesis tests were 2-sided, and the significance level was α = 0.05. The repeated-measures analysis was performed with a commercial software program.s Results are reported as mean ± SEM. A paired t test was used to compare HR and pulse pressure variables at Tmax versus a theoretical mean of zero. For these variables, the Kolmogorov-Smirnov test was used to test for normality. Results One cat was removed from the study after developing cardiac arrhythmia following the administration of high doses of angiotensin II. Of the 6 remaining cats, all were from 1 to 3 years of age. Mean ± SEM weight was 4.2 ± 0.3 kg. Mean ± SEM total anesthetic time per cat during data collection periods was 92 ± 4 minutes. No complications were detected during collection periods, and all cats recovered without complications from anesthesia. Pressor response curves and continuous HR and pulse pressure data were generated for each of the 6 cats that completed the study at doses from 0.02 to 1,000 ng of angiotensin I/kg and 0.02 to 500 ng of angiotensin II/kg (Table 1). An SBPmax > 250 mm Hg was observed with the administration of 500 ng of angiotensin II/kg in 2 cats, terminating any further dose increase in these cats; therefore, data obtained for the 1,000 ng/kg dose of angiotensin II were not included in analysis because of low numbers of observations. Identification or derivation of each variable of interest was possible for all pressor response curves, and no effect of observer was detected for any graph-derived variable. AJVR, Vol 74, No. 11, November 2013

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Table 2—Mean ± SEM values (mm Hg) of ∆SBP in 6 anesthetized healthy cats administered various doses of angiotensin I or angiotensin II on replicate days in a study of an RPRT. Dose Vasopressor Angiotensin I Replicate 1 Replicate 2 Angiotensin II Replicate 1 Replicate 2

20 ng/kg

100 ng/kg

8.8 ± 1.3 4.8 ± 0.8

25.7 ± 5.2 69.4 ± 12.2 96.2 ± 13.2 16.3 ± 4.23 61.55 ± 12.1 93.5 ± 15.4

18.2 ± 4.2 24.6 ± 4.8

500 ng/kg

45.0 ± 9.1 99.5 ± 15.6 36.1 ± 7.61 95.5 ± 11.86

1,000 ng/kg

— —

See Table 1 for key.

Transient, dose-dependent increases in ∆SBP, ∆DBP, ∆SBPrate, and ∆PP were detected following both angiotensin I and angiotensin II administration (Table 1; Figure 1). There was a significant (P < 0.001) effect of angiotensin type and dose on ∆SBP, ∆DBP, ∆SBPrate, and ∆PP. Increases were greatest at angiotensin doses > 20 ng/kg for ∆SBP, DBP, and ∆SBPrate and those > 100 ng/kg for ∆PP, with significant differences found among doses at these values. No significant difference in group means between replicate days were detected for any of the pressor response variables tested for angiotensin I or angiotensin II (Table 2). Angiotensin I and II doses ≥ 500 ng/kg in all cats and ≥ 100 ng/kg in 3 cats elicited a biphasic increase in BP (Figure 1). Response curves at these doses were characterized by an initial rapid increase, with a peak in approximately the first third (initial 15 to 40 seconds) of the total upstroke phase. This initial peak was terminated by a comparatively small decrease in BP of approximately 5 to 20 mm Hg and immediately followed by a second peak of greater magnitude, the latter characterized by a slower rate of increase. In these instances, peak values used for analysis were taken from the latter peak. At lower doses, a single peak in BP response was detected. Immediately following angiotensin injection and contemporaneous with increases of SBP, DBP, and pulse pressure, a dose-dependent decrease in HR was observed. This response was examined at representative low (0.02 ng/kg), medium (20 ng/kg), and high (500 ng/kg) angiotensin doses by comparing the mean baseline HR and pulse pressure with those at SBPmax. For angiotensin II, the HR decrease and pulse pressure in1395

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crease were significant at the 20 and 500 ng/kg doses, but not at 0.02 ng/kg. For angiotensin I, the HR decrease was significant at all doses examined, whereas the pulse pressure increase remained significant only at the 20 and 500 ng/kg doses. Changes in HR and pulse pressure were transient, with values returning to baseline coincident with the decrease in BP, generally within 2 to 5 minutes. Discussion Results of the present study confirmed the safety, feasibility, and repeatability of the RPRT in healthy anesthetized cats. Transient, measurable increases in systolic and diastolic BP occurred in a dose-dependent manner, and no serious adverse effects were detected. Pharmacodynamic profiling of drugs interfering with RAAS may be achieved through the use of biochemical methods that evaluate the anticipated hormonal consequences of blockade by measurement of BP or other clinical indicators of cardiac or renal function or by blockade of the cardiovascular response to challenge with exogenous angiotensin I or angiotensin II.20 The latter—the RPRT—is the preferred approach for phase I studies performed in normotensive human volunteers.20 By use of this technique, quantification of antagonist potency (specifically, degree and duration of RAAS blockade) can be determined by evaluating attenuation of invasively measured BP changes in response to repeated challenge with exogenous angiotensin I or angiotensin II, before and after administration of the drug of interest.16 Importantly, the RPRT provides relatively rapid determination of the minimal dose required to obtain adequate RAAS blockade. In healthy human volunteers, there appears to be good agreement between those doses that induce 75% attenuation of the effect of angiotensin and the doses that have efficacy in subsequent clinical trials.16 Blockade of the pressor response to angiotensin I is closely related to plasma concentrations of the ACE-i under investigation and ACE activity in vivo.21 A distinct advantage of the RPRT is that it permits quantitative assessment of a surrogate measure of clinical efficacy, which may substantially hasten clinical development of drugs that modify the RAAS.16 The RPRT will likely facilitate investigation of the efficacy of antihypertensive agents with a similar mechanism (eg, comparison of 2 ACE-i) as well as evaluation of different approaches to interference with RAAS activity (eg, ACE-i vs ARB vs both). In addition, determination of the onset and duration of action of these agents is possible when the test is performed at various time intervals after administration. Other methods for assessment of RAAS blockade have the advantage of being less invasive; however, there are several disadvantages to those methods. Accurate quantification of plasma ACE activity, typically accomplished through in vitro methods that make use of the ability of circulating ACE to cleave a natural or synthetic substrate, is generally difficult, with results varying considerably depending on the type of substrate and the assay condition used.22 A recent veterinary study23 corroborated these observations after evaluating 3 methods for measurement of ACE activity in equine plasma and finding poor agreement. This fact presents 1396

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a unique challenge when comparing the results of studies that used differing methodologies to quantify ACE activity. Perhaps more important are concerns about whether plasma ACE, which has a well-recogized, relatively minor role in the conversion of angiotensin I to angiotensin II, represents a proper surrogate for the more physiologically important endothelial and tissue ACEs.24 Studies25,26 in humans suggest that extravascular RAAS (ie, nonplasma tissue ACE) may be inhibited to varying degrees by ACE-i; therefore, in vitro ACE activity may not accurately reflect angiotensin II attenuation or the hemodynamic effects of these drugs. Concerns about possible in vitro dissociation of the ACE-i from ACE following blood sampling, as well as large interindividual variation in plasma ACE activity caused by genetic polymorphism of the enzyme, may further limit the value of this assay.27 Because ACE-i induce a predictable decrease in plasma angiotensin II concentration and increase in plasma angiotensin I concentration, the ratio of these can be used to estimate in vivo ACE activity. This ratio represents ACE inhibition more accurately than in vitro assays and avoids many of the methodological problems associated with the latter.22 However, quantification of plasma angiotensin II concentrations is problematic, requiring special precautions to halt conversion in vitro and a radioimmunoassay technique that is hampered by difficulties such as cross-reaction of the angiotensin II antibody with high angiotensin I concentrations and with angiotensin II metabolites.28 In the present study, pressor responses to both angiotensin I and angiotensin II were detected. In vivo, the biologically inactive angiotensin I undergoes rapid conversion to angiotensin II by way of ACE and other enzymatic pathways.29 The acute pressor response following administration of either of these peptides is mediated by angiotensin II–dependent activation of the angiotensin II subtype 1 receptor.29,30 In addition to direct vasoconstriction, activation of this receptor causes indirect vasoconstrictive effects, which are equal in importance and mediated by interactions with the autonomic nervous system.29,31 The doses of angiotensin I and angiotensin II used in the present study were selected on the basis of our pilot data from clinically normal cats, which revealed measurable and safe increases in SBP in response to bolus doses (IV) of angiotensin I ranging from 0.001 to 0.1 µg (approx 0.2 to 20 ng/ kg of body weight). To improve safety and attempt to identify the lowest angiotensin dose capable of inducing a measurable BP response, we increased doses after administering the first dose, which was 10% of the lowest used in these pilot studies. Further studies, in which attenuation of the pressor response is evaluated following pharmacologic intervention with RAAS modifiers, will be necessary to establish the true usefulness of the RPRT in cats. When comparing relative antagonist potencies of RAASmodifying agents by use of the RPRT, the choice of angiotensin peptide will clearly depend on the drug class under evaluation. Because ACE-i interfere with the RAAS by blocking conversion of angiotensin I to angiotensin II, comparison among specific agents of this class, or between ACE-i and those interfering with the AJVR, Vol 74, No. 11, November 2013

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RAAS further downstream (eg, ARB), will necessitate evaluation of the degree of response attenuation following angiotensin I administration. Conversely, comparison of efficacy among drugs of the ARB class may be more specifically evaluated by observation of the degree of attenuation achieved by use of angiotensin II. On the basis of responses detected in the present study, the authors propose the use of angiotensin doses from 20 to 1,000 ng of angiotensin I/kg and 20 to 500 ng of angiotensin II/kg to generate dose-response curves with varying doses of RAAS-modifying agents and establish quantitative estimation of inhibition. In investigations of healthy human volunteers, dose-response curves are generated by administering increasing doses of angiotensin to induce a maximum target BP response of 30 to 35 mm Hg.32–34 Doses up to 60 to 100 ng of angiotensin/kg are required in most cases, comparable to those reportedly required in dogs35 and those required in the present study to induce similar BP responses. The data reported here revealed variability in pressor response among individual cats, particularly in response to higher doses of angiotensin. Variation is expected in this system and has also been reported in human subjects.33 In the present study, maximum interindividual variation in systolic pressor response for a given dose of angiotensin I ranged from 0.75 mm Hg (0.02 ng of angiotensin I/kg) to 78 mm Hg (1,000 ng of angiotensin I/kg), whereas that for angiotensin II ranged from 5.5 mm Hg (0.02 ng of angiotensin II/kg) to 107 mm Hg (500 ng of angiotensin II/kg). This variability persisted when the test was repeated on different days. However, rank order of responses of individual cats was also preserved (data not shown), suggesting consistency of response within each subject. Thus, researchers and clinicians should use multiple repetitions when conducting the RPRT and take this variability into account when designing protocols and interpreting results. Conversion of angiotensin I to angiotensin II is a rapid process, with most conversion occurring in the seconds that it takes for blood to traverse the pulmonary vasculature.29 Further, in a murine study,36 the half-life of circulating angiotensin II was approximately 14.8 and 23.68 seconds for conscious and barbiturateanesthetized mice, respectively. Because at least 10 minutes and the return of BP to baseline were allowed between doses in the present study, hormone accumulation was not expected with the serial administration protocol that was used. In dogs, angiotensin administration intervals > 5 minutes are associated with good reproducibility of responses,31 and repeated administration of angiotensin I in human patients at intervals > 7 minutes does not induce tachyphylaxis.33,37 The observed dose-dependent decrease in HR coincident with SBPmax was expected to occur as a result of high-pressure baroreceptor-mediated reflex reduction of sympathetic input and augmentation of vagal stimulation of the sinoatrial node following the angiotensininduced spike in peripheral vascular resistance, with these changes acting to restore BP toward baseline values.38 A previous experimental study39 in animals has revealed opposing effects on HR of exogenous angiotensin II, the first mediated by baroreceptor stimulation and the second through a central action that inhibits AJVR, Vol 74, No. 11, November 2013

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vagal discharge. Typically, however, the central effect is concealed by the greater magnitude of the baroreceptor stimulation. Baroreceptor-mediated responses may also explain the pulse pressure widening detected in the present study because the reflex decreases in HR would be expected to decrease DBP to a greater degree than SBP.40 Interestingly, some decrease in peripheral vascular resistance (and therefore, increase in pulse pressure) may also be mediated, at least in part, by a vasodilatory effect attributable to the interaction of angiotensin II with peripheral angiotensin II receptors.41 Angiotensin I and II doses ≥ 500 ng/kg in all cats and ≥ 100 ng/kg in 3 cats elicited a biphasic pressor response in the present study. In isolated canine splenic artery specimens, exogenous angiotensin II administration leads to marked potentiation of a biphasic vasoconstrictor response to periarterial electrical nerve stimulation, suggesting an important role for angiotensin II in the regulation of autonomic neurotransmission.42–44 In those experiments, the first peak resulted from a transient purinergic phase, followed by a second, predominant, and longer-lived peak associated with an α1-adrenergic phase. The time course of these responses was similar to that noted in the present study, and the response was potentiated at higher doses of angiotensin II (ie, those capable of increasing basal perfusion pressures by > 100%).42 This mechanism may explain the double-peaked response observed in the present study at higher doses of angiotensin. Indeed, in conscious dogs in which baroreflex buffering is prevented by baroreceptor denervation, nearly half of the vasoconstrictive effects of angiotensin are mediated by the autonomic nervous system.31 Accurate evaluation of BP in awake, nonsedated cats presents a unique challenge. The confounding effects of physical restraint and environmental stressors on BP (the so-called white-coat effect) are well known and make reliable assessment of antihypertensive treatment efficacy in a clinical or laboratory environment problematic.18,45 To reliably evaluate experimentally induced changes in BP and pharmacological modification of these responses, it may be advantageous to perform such tests on anesthetized subjects. In this way, transient spikes in BP following angiotensin administration may be interpreted as a drug response, as opposed to simply reflecting coincident alterations attributable to physical activity or unpredictable alterations in neurohormonal effects on BP. Further, subtle responses may be identified, which might otherwise be lost in background BP fluctuations. Nevertheless, the potentially suppressive effects of anesthesia on these responses should be considered. Specifically, BP responses detected in the anesthetized cats of the present study may not accurately represent those of conscious cats, although in a murine study,36 barbiturate anesthesia resulted in no alterations in response to injected angiotensin II despite decreasing baseline arterial pressure. The development of cardiac arrhythmia in 1 cat of the present study highlights an important consideration when selecting feline subjects for the RPRT and monitoring them during administration of the test. Although ECG was not available to characterize the arrhythmia, it is suspected that the acute increase in cardiac after1397

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load imposed by angiotensin II–mediated vasoconstriction led to an increase in end-systolic myocardial wall tension (and therefore myocardial oxygen demand) in the setting of underlying cardiomyopathy, favoring the development of ventricular tachyarrhythmia.38 The arrhythmia was self-limiting and did not persist beyond the termination of the pressor response. Up to 15.5% of apparently healthy cats may be affected by occult cardiomyopathy,46 underscoring the need for careful subject screening prior to and monitoring during RPRT. In the present study, variables associated with peak pressor response were reported. The investigators would like to have measured BP until its return to baseline to calculate the area under the curve and duration of response. Unfortunately, 2 problems precluded accurate determination of these values. First, the BP measuring system that was used allows a maximum of 300 seconds of continuous, real-time, beat-to-beat BP recording; beyond this, the graphed curve is reset. At higher doses of angiotensin, the time for return of BP to baseline exceeded the allowable recording period, precluding measurement of area under the curve for all recorded responses. In addition, return of BP to baseline occurred gradually, making accurate identification of a specific time point at return to baseline problematic and introducing unacceptable interobserver variability for this variable. There were limitations to this study. Because neither arterial blood gas measurements nor capnography was monitored during the anesthetic period, it is unclear whether hypercarbia or hypoxia may have affected the BP and HR responses. Propofol administration, at doses used for both anesthetic induction and maintenance, is associated with respiratory depression and hypercapnea in cats.47,48 However, this depression was considered mild in a recent investigation48 of constant rate infusions in clinically normal cats, which studied maintenance propofol doses similar to those used in the present study. In addition, following an initial decrease in arterial BP, infusion of this drug is known to induce a stable BP decrease in cats.48 Observations in the present study confirmed these findings because a decrease in BP was detected after induction, with a plateau occurring within 10 to 15 minutes. To account for this effect, we ensured that baseline BP was stable for at least 5 minutes prior to commencing angiotensin administration. The lack of a control group to which propofol alone was administered was considered a limitation; however, this was believed to be minor because the primary variables of interest were changes in pressure relative to baseline values recorded immediately preceding angiotensin administration. The RPRT described in this study reliably induced dose-dependent increases in BP in clinically normal cats. The importance of screening individuals for underlying cardiac disease prior to an RPRT and monitoring them during its administration should be emphasized to ensure the safety of this test. The RPRT may prove useful for evaluating the efficacy of drugs that modify the RAAS, and the authors advocate its use in preclinical studies to establish the efficacy and appropriate dose range of RAAS blocking agents. Further studies that assess the degree of attenuation of the 1398

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pressor response following pharmacologic intervention with RAAS-modifiers will be necessary to establish the true usefulness of this test. a. b. c. d. e. f. g. h. i. j. k. l. m. n. o. p. q. r. s.

LabDiet, PMI Nutrition International LLC, Brentwood, Mo. PromAce injectable, 10 mg/mL, Fort Dodge Animal Health Inc, Fort Dodge, Iowa. Buprenex injectable, 0.3 mg/mL, Reckitt Benkiser Healthcare Ltd, Hull, Yorkshire, England. Ketaset injectable, 100 mg/mL, Fort Dodge Animal Health Inc, Fort Dodge, Iowa. IsoFlo, Abbott Laboratories Inc, Abbott Park, Ill. Model TA11 PA-C40, Data Sciences International, Saint Paul, Minn. Torbugesic injectable, 10 mg/mL, Fort Dodge Animal Health Inc, Fort Dodge, Iowa. Surflo Teflon IV Catheters, 22 and 24 gauge, Terumo Medical Corpo, Elkton, Md. Propoflo injectable, 10 mg/mL, Abbott Laboratories Inc, Abbott Park, Ill. Thermacare Convective Warming System, Model TC3000, Gaymar Industries Inc, Orchard Park, NY. Model RLA-2000, Data Sciences International Inc, Saint Paul, Minn. Lactose monohydrate, 250 mg, PCCA USA, Houston, Tex. Angiotensin I human acetate salt hydrate, Sigma-Aldrich, Co, St Louis, Mo. Angiotensin II human acetate salt hydrate, Sigma-Aldrich Co, St Louis, Mo. Cellgro phosphate-buffered saline without calcium and magnesium, Mediatech Inc, Manassas, Va. Steriflip Vacuum Filtration System with Millipore Express PLUS Membrane (0.22 µm), Millipore Corp, Billerica, Mass. Dataquest ART data acquisition system, version 2.0, Data Sciences Internation Inc, Saint Paul, Minn. SAS, version 9.2, SAS Institute Inc, Cary, NC. PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC.

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