Interruption of RAAS activation is an important goal in the pharmacological treatment of congestive heart failure. Hypotension, decreased cardiac output, sympathetic stimulation, and low blood sodium concentrations all stimulate the RAAS. Stimulation of the RAAS in the short term may allow for restoration of blood volume and perfusion of vital organs and is therefore a critical response to acute hypovolemia or hypotension. Although RAAS activation may allow for beneficial compensation in early congestive heart failure, chronic activation of the RAAS becomes maladaptive, promotes and perpetuates heart failure, and is associated with a poor outcome.1–4 The role of chronic RAAS activation as a cause of fluid retention, endothelial and baroreceptor dysfunction, and vascular and myocardial remodeling in patients with chronic heart failure is clearly established.5–12 Also, diuretics and vasodilators used in the pharmacological treatment of heart failure potentiate RAAS activation.2,13–15
The benefits of interrupting RAAS activation through the use of ACEIs have been demonstrated in multiple controlled clinical trials in dogs with heart failure attributable to both degenerative mitral valve disease and dilated cardiomyopathy.16–19 A consensus panel consisting of 10 board-certified veterinary cardiologists outlined guidelines for the treatment of chronic valvular disease in dogs.20 In that report,20 ACEIs were recommended for the treatment of dogs with chronic or refractory congestive heart failure (stages C-chronic and D). Aldosterone receptor blockers can be effective in managing heart failure in humans21–23 and dogs24 by further suppressing the RAAS and have been recommended by most of the consensus panelists for treatment of dogs in stage C-chronic and stage D-chronic.20
The authors’ research group currently uses a pharmacological (furosemide) method to experimentally approximate RAAS activation in heart failure. Although pharmacological RAAS activation in healthy dogs does not likely exactly mimic the time course and magnitude of RAAS activation in dogs with naturally occurring heart disease, this method allows for the study of pharmacological activation and interruption of the RAAS. Importantly, this method uses drugs to stimulate the RAAS that are also used to treat heart failure, thereby likely playing a role in RAAS activation in patients. Efficacy of standard dosages of 2 ACEIs (benazepril and enalapril) for suppression of the RAAS has been evaluated in other studies15,25,26,a conducted by our research group. Activation of the RAAS can be evaluated via measurement of the UAldo:C by use of radioimmunoassays; the UAldo:C correlates closely with 24-hour urinary aldosterone excretion.27 The efficacy of ACEIs in suppression of ACE activity is evaluated by measuring the percentage suppression of ACE activity in each dog. Theoretically, successful RAAS blockade with ACEIs should reduce the secretion of aldosterone because angiotensin II is a potent secretogogue for aldosterone. Other studies15,25,26,a conducted by our research group that involved assessment of the UAldo:C have revealed failure of standard-dose ACEIs to consistently inhibit the pharmacologically activated RAAS in healthy dogs. In 2 of those studies,26,a ACE activity was significantly suppressed; however, despite effective ACE suppression, the mean UAldo:C increased after furosemide stimulation. Thus, results of those 2 studies26,a indicated the phenomenon of aldosterone breakthrough (defined as the incomplete pharmacological blockade of the RAAS system by ACEIs or angiotensin II receptor blockers [or both]) for this pharmacological method of RAAS activation in dogs. Although this phenomenon is widely accepted for humans with heart failure, chronic renal disease, and hypertension, the mechanisms are poorly understood.28–32 In domestic animals, there is neither a widely accepted definition for aldosterone breakthrough nor an understanding of the mechanisms and prevalence. Therefore, the purpose of the study reported here was to investigate whether high dosages of ACEIs might be more effective than standard dosages in suppression of the furosemide-activated RAAS, thereby preventing or lessening the degree of aldosterone breakthrough.
Materials and Methods
Animals
Six mature (> 1 year old) Beagles (2 sexually intact females and 4 sexually intact males) were enrolled in the study. Mean ± SD body weight was 9.37 ± 1.89 kg (range, 6.7 to 11.8 kg). Mean age of the dogs was 38.91 ± 997 months (range, 26.1 to 53.1 months). Each dog was considered healthy on the basis of evaluation of the medical history, results of a complete physical examination (including measurement of systemic BP), and results of clinicopathologic testing, which consisted of a CBC, serum biochemical analysis, and urinalysis. These assessments were performed on day -2 of each of 2 experiments (day 0 was the first day of drug administration for each experiment).
The dogs were housed in a facility approved by the Association for Assessment and Accreditation of Laboratory Animal Care International. The light-dark cycle was controlled, and dogs received a standard commercial dietb (0.42% sodium and 0.84% chloride on a dry-matter basis). The study was approved by the North Carolina State University College of Veterinary Medicine Institutional Animal Care and Use Committee.
Study design
During the first experiment, all 6 dogs received furosemidec,d (2 mg/kg, PO, q 12 h) and enalaprile,f (1 mg/kg, PO, q 12 h) for 8 days (F + E experiment). After a 2-week washout period, the second experiment was conducted, and all dogs received furosemidec,d (2 mg/kg, PO, q 12 h) and benazeprilg (1 mg/kg, PO, q 12 h) for 8 days (F + B experiment). Drug administration was on days 0 through 7 for both experiments. Body weight and heart rate were measured on days -2, -1, 1, 3, and 7 of each experiment. Renal variables (concentrations of SUN, serum creatinine, and serum electrolytes) were measured on days 1, 3, and 7 of each experiment. Baseline values for serum biochemical variables were determined from results for the day -2 sample. For variables for which data were collected on days -2 and -1, baseline values represented a mean of the values for both days.
Systemic BP
Systemic (diastolic, systolic, and mean) BP was measured on days -2, -1, 1, 3, and 7 of each experiment. Dogs were allowed an acclimatization period of 10 to 15 minutes. Dogs were then restrained in a standing position, and BP was measured oscillometricallyh by use of an appropriately sized cuff placed over the coccygeal artery. The value recorded was the mean calculated for 3 consecutive measurements obtained within 5 minutes and that had results within 10% of one another. Baseline values represented a mean of the values for days -2 and -1.
Serum ACE activity and serum aldosterone concentration
To assess the time course and magnitude of the response of the RAAS to stimulation, serum ACE activity was measured on days -1, 3, and 7 and serum aldosterone concentration was measured on days -2, -1, 1, 3, and 7 in both experiments. Blood samples (2 to 3 mL) were centrifuged within 30 minutes after collection, and serum was separated and stored at −80°C until batch analysis of ACE activity and aldosterone concentration. Baseline values were results for the day -1 sample for serum ACE activity and a mean of the results for days -2 and -1 for the serum aldosterone concentration.
Serum samples for assessment of peak inhibition of ACE activity were obtained approximately 6 hours after ACEI administration. A commercially available kiti was used in accordance with the manufacturer's instructions. The principle of this assay was that ACE mediated cleavage of a synthetic substrate (3H-hippuryl-glycylglycine) into 3H-hippuric acid and the glycylglycine dipeptide. The sample was then acidified by the addition of HCl, and titrated hippuric acid was separated from unreacted substrate by extraction with scintillation cocktail and measured in a β counter.j
Briefly, scintillation vials were labeled in duplicate for the standards and serum samples. The frozen serum samples were thawed on ice and then mixed by use of a vortex device. Then, 10 μL of each standard and sample was pipetted into the corresponding vial. An aliquot (100 μL) of the titrated substrate was added to each vial, and vial contents were mixed well by use of a vortex device. Vials then were incubated in a water bath at 37°C for 1 hour. After incubation was complete, 50 μL of 1N HCl was added to each vial and each vial again was mixed by use of a vortex device. Next, 1.5 mL of this scintillation cocktail was added to each vial; vials were thoroughly mixed by use of a vortex device and allowed to sit undisturbed at room temperature (20°C) for 1 hour. Results were determined by counting in a β counter for 5 minutes. The %ΔACE (ie, percentage change from the baseline value) was calculated. The %ΔACE results for the F + E and F + B experiments were compared with results for 2 previous studies26,a of similar design that had been conducted by our research group. Calibration curves were generated,k which allowed the calculation of unknowns.
Serum aldosterone concentrations were measured with a commercially available ELISA kit.l This assay used a competitive binding method, in which an enzyme-labeled antigen competed with unlabeled antigen (aldosterone) for a limited number of antibody binding sites on each microwell plate. An enzyme substrate was added, and when the reaction was terminated, absorbance was quantitated. Color intensity was inversely proportional to the concentration of aldosterone in the sample. No pretreatment of serum samples was required for this assay, as per the manufacturer's instructions. Briefly, 50 μL of sample and 100 μL of conjugate working solution were pipetted into wells; plates were incubated on a plate shaker at 200 revolutions/min for 1 hour at room temperature. Enzyme substrate (150 μL) was pipetted into each well at scheduled intervals, after which plates were incubated for 10 to 15 minutes at room temperature. Stop solution (50 μL) was pipetted into each well, and the plates were assessed on a microwell plate reader at 450 nm within 20 minutes after the addition of stop solution. Calibration curves were generated,k which allowed calculation of the serum aldosterone concentration.
UAldo:C
Five milliliters of urine was obtained from each dog in the morning and evening of days −2, −1, 1, 3, and 7 of each experiment for determination of the UAldo:C. Baseline values were the mean calculated for days −2 and −1. Each sample was refrigerated within 10 minutes after collection and frozen at −70°C within 3 hours after collection. Four months after study completion, urine samples were thawed and equal aliquots of the morning and evening urine samples of each dog were mixed, refrozen, and submitted for determination of the UAldo:C.
Urine aldosterone concentrations were measured with a commercially available kitm in accordance with the manufacturer's instructions by personnel at a veterinary diagnostic laboratory.n Urine samples (250 μL) were incubated in the dark with 3.2 N HCl for 24 hours to cause acid hydrolysis of the glucuronide of aldosterone-18-glucuronide, which allowed detection of this metabolite. Extraction was then performed by the addition of 2.5 mL of ethyl acetate. Tubes were capped, placed in a mechanical rotator for 60 minutes, and then centrifuged at 600 × g for 5 minutes. The volume of an aliquot of solvent phase was measured and then evaporated to dryness. Residues were resuspended by the addition of 500 μL of the zero standard, and the solution was mixed in a vortex device. Results were corrected for the dilution factor associated with hydrolysis and reconstitution after extraction. A standard curve was generated, with the percentage bound plotted against the logarithm of the concentration. Unknown values were calculated by interpolation. Urine creatinine concentration was measured by use of a standard colorimetric assay at a veterinary diagnostic laboratory.n Urine aldosterone concentrations (in μg) were indexed to the urine creatinine concentration (in g) and expressed as the UAldo:C.
Statistical analysis
Systemic BP, body weight, heart rate, serum aldosterone concentration, urine creatinine concentration, urine aldosterone concentration, and the UAldo:C were assessed at 5 time points. Serum concentrations of electrolytes (phosphorus, sodium, potassium, calcium, and chloride), albumin, creatinine, total protein, and bicarbonate and the SUN concentration were assessed at 4 time points, and serum ACE activity was assessed at 3 time points. These outcomes were analyzed by use of a mixedmodel ANOVA to assess the effect of time within a treatment. The linear model included treatment, time, and the treatment-by-time interaction as fixed effects, whereas dog was a random effect. Variance component was used for dog, and spatial power was used for the repeated-measures effect of time (ie, day of the experiment), allowing the association between days to decrease as the lag between days increased, which accommodated unequal spacing among the levels of the effect for time. The log-likelihood test was used to compare independence versus spatial power, and spatial power was almost always found to be significant (P < 0.05). Tukey-adjusted P values were used for comparisons. The %ΔACE of the F + E and F + B experiments was compared by use of a nonparametric Wilcoxon 2-sample test with the %ΔACE reported for 2 other studies26,a with similar protocols.
All analyses were performed by use of a commercially available statistical program.o Values were considered significant at P < 0.05.
Results
Drug dosages
The mean dosage of furosemide for experiments 1 and 2 was 2.10 mg/kg, PO, every 12 hours and 2.11 mg/kg, PO, every 12 hours, respectively. The mean dosage of enalapril for experiment 1 was 1.09 mg/kg, PO, every 12 hours. The mean dosage of benazepril for experiment 2 was 1.10 mg/kg, PO, every 12 hours.
Systemic BP, heart rate, and body weight
Mean BP decreased over time during both experiments, with the BP on day 7 lower than the baseline value. None of the dogs developed clinically relevant hypotension. For the F + E experiment, systolic BP was significantly lower than the baseline value on days 3 (P = 0.02) and 7 (P < 0.001). For the F + B experiment, systolic BP did not differ from the baseline value on any day of the experiment. Diastolic BP measured on days 1, 3, and 7 did not differ significantly from the baseline value for either experiment. Systolic and diastolic BPs for any given day did not differ significantly between the 2 experiments.
For both experiments, heart rate increased on days 1 and 3 and then decreased on day 7. Mean body weight decreased slightly, but not significantly, during both experiments.
Serum biochemical analysis
Significant differences were detected for several variables (Table 1). Mild elevation of the SUN concentration outside the reference interval was detected on days 1 and 3 of both experiments. The serum creatinine concentration remained within the laboratory's reference interval for all dogs throughout both experiments. Clinically relevant changes, such as persistent increases in SUN and serum creatinine concentrations outside the reference interval or hyperkalemia, were not detected.
Mean ± SD values of variables for 6 clinically normal dogs receiving furosemide (2 mg/kg, PO, q 12 h) and enalapril (1 mg/kg, PO, q 12 h [F + E]) and then furosemide (2 mg/kg, PO, q 12 h) and benazepril (1 mg/kg, PO, q 12 h [F + B]) for 7 days.
| Experiment | Variable | Reference interval | Baseline | Day 1 | Day 3 | Day 7 |
|---|---|---|---|---|---|---|
| F + E | Systolic BP (mm Hg) | 110–160 | 138.4 ± 11.6 | 132.5 ± 10.7 | 125.1 ± 10.5* | 120.2 ± 11.7† |
| Heart rate (beats/min) | 70–160 | 112.5 ± 15.8 | 118.7 ± 26.7 | 118.2 ± 22.7 | 104.3 ± 15.6 | |
| Body weight (kg) | NA | 9.5 ± 1.8 | 9.3 ± 1.8 | 9.4 ± 1.9 | 9.3 ± 1.8 | |
| SUN (mg/dL) | 6–26 | 17.3 ± 2.7 | 22.5 ± 5.4* | 24.0 ± 4.0† | 19.8 ± 3.5 | |
| Creatinine (mg/dL) | 0.7–1.5 | 0.8 ± 0.1 | 0.9 ± 0.2 | 0.9 ± 0.1 | 0.9 ± 0.1 | |
| Sodium (mmol/L) | 140–156 | 145.7 ± 1.5 | 141.7 ± 2.7† | 140.5 ± 3.6† | 144.3 ± 2.4 | |
| Potassium (mmol/L) | 4.0–5.3 | 4.6 ± 0.3 | 4.8 ± 0.3 | 4.6 ± 0.3 | 4.5 ± 0.5 | |
| Chloride (mmol/L) | 108–122 | 112.0 ± 2.7 | 106.7 ± 2.3† | 105.7 ± 3.1† | 105.5 ± 0.5† | |
| Bicarbonate (mmol/L) | 18–25.8 | 21.7 ± 2.4 | 22.5 ± 1.5 | 23.3 ± 2.0 | 24.5 ± 1.2* | |
| F + B | Systolic BP (mm Hg) | 110–160 | 130.3 ± 8.9 | 123.2 ± 7.5 | 120.8 ± 11.6 | 123.5 ± 8.6 |
| Heart rate (beats/min) | 70–160 | 105.9 ± 18.5 | 117.3 ± 14.7 | 118.8 ± 23.0 | 107.7 ± 18.5 | |
| Body weight (kg) | NA | 9.7 ± 1.7‡ | 9.6 ± 1.6 | 9.6 ± 1.9 | 9.6 ± 1.8 | |
| SUN (mg/dL) | 6–26 | 21.5 ± 2.4 | 22.8 ± 7.5 | 20.0 ± 5.0 | 21.5 ± 3.1 | |
| Creatinine (mg/dL) | 0.7–1.5 | 0.8 ± 0.1 | 0.9 ± 0.1 | 0.9 ± 0.1 | 0.9 ± 0.1 | |
| Sodium (mmol/L) | 140–156 | 140.8 ± 3.7‡ | 144.3 ± 1.6† | 145.5 ± 1.0†‡ | 139.3 ± 2.3‡ | |
| Potassium (mmol/L) | 4.0–5.3 | 4.8 ± 0.2 | 4.8 ± 0.3 | 4.6 ± 0.5 | 4.5 ± 0.4 | |
| Chloride (mmol/L) | 108–122 | 109.2 ± 4.6 | 108.5 ± 1.0 | 107.2 ± 17 | 105.8 ± 1.2 | |
| Bicarbonate (mmol/L) | 18–25.8 | 20.7 ± 1.0 | 25.2 ± 1.5†‡ | 25.8 ± 1.5†‡ | 24.8 ± 1.6† |
Day 0 = First day of drug administration for each experiment; there was a 2-week washout period between experiments. Baseline values for systolic BP, body weight, and heart rate represent the mean value for results obtained on days -2 and −1; baseline values for serum biochemical variables represent results for a sample obtained on day -2. NA = Not applicable.
Within a row, value differs significantly (P < 0.05) from the baseline value.
Within a row, value differs significantly (P = 0.01) from the baseline value.
Within a time point, value differs significantly (P < 0.05) from the corresponding value for the F + E experiment.
Serum ACE activity
For both experiments, serum ACE activity was significantly (P < 0.001) lower on days 3 and 7, compared with the baseline value (Figure 1). For both experiments, ACE activity was lowest on day 3. Mean ACE activity increased between days 3 and 7 for both experiments; however, the mean ACE activity on day 7 was still significantly lower than the baseline value. Percentage reduction of ACE activity from the baseline value was significantly (P = 0.04) greater on days 3 and 7 for the F + B experiment than for the F + E experiment. The %ΔACE for the F + E and F + B experiments did not differ significantly from the %ΔACE reported for 2 studies26,a of similar design but that involved use of a standard dose of ACEI and pharmacological RAAS activation.
Mean ± SD serum ACE activity measured 6 hours after drug administration to 6 clinically normal dogs that received furosemide (2 mg/kg, PO, q 12 h) and enalapril (1 mg/kg, PO, q 12 h; A) and then furosemide (2 mg/kg, PO, q 12 h) and benazepril (1 mg/kg, PO q, 12 hours; B) for 8 days (day 0 = first day of drug administration for each experiment). There was a 2-week washout period between experiments. Baseline values represent results for a sample obtained on day −1. *Within an experiment, value differs significantly (P < 0.001) from the baseline value.
Citation: American Journal of Veterinary Research 76, 12; 10.2460/ajvr.76.12.1041
UAldo:C
Change in the UAldo:C for both experiments was characterized by an increase from the baseline value on days 1 and 3 and then a decrease on day 7 (Figure 2). The UAldo:C increased significantly from the baseline value on all days for the F + B experiment (P ≤ 0.01) and on days 3 and 7 for the F + E experiment (P < 0.05). The UAldo:C for any given day did not differ significantly between the 2 experiments.
Mean ± SD UAldo:C determined with urine collected in the morning and evening from 6 clinically normal dogs receiving furosemide and enalapril (A) and then furosemide and benazepril (B) for 8 days. Urine aldosterone concentrations (in μg) were indexed to the urine creatinine concentration (in g) and expressed as the UAldo:C. Baseline values represent the mean of results for samples obtained on days −2 and −1. *†Within an experiment, value differs significantly (*P = 0.01; †P < 0.05) from the baseline value. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 76, 12; 10.2460/ajvr.76.12.1041
Serum aldosterone concentration
For both experiments, the serum aldosterone concentration increased from the baseline value on days 1 and 3 and then decreased on day 7 (Figure 3); this mirrored the results for the UAldo:C. The mean serum aldosterone concentration on day 7 remained higher than the baseline value for both experiments. For the F + B experiment, the serum aldosterone concentration was significantly increased on days 1 (P < 0.001) and 3 (P = 0.02). For the F + E experiment, the serum aldosterone concentration was not significantly increased on any day, compared with the baseline value. The serum aldosterone concentration for the F + B experiment was significantly (P = 0.002) greater than that for the F + E experiment on day 1, but there were no significant differences between the experiments on days 3 and 7.
Mean ± SD serum aldosterone concentration measured 6 hours after drug administration on days 3 and 7 to 6 clinically normal dogs receiving furosemide and enalapril (A) and then furosemide and benazepril (B) for 8 days. Baseline values represent the mean of results for samples obtained on days −2 and −1. See Figures 1 and 2 for remainder of key.
Citation: American Journal of Veterinary Research 76, 12; 10.2460/ajvr.76.12.1041
Discussion
For the experimental method of pharmacological RAAS activation reported here, high doses of the ACEIs enalapril and benazepril effectively suppressed circulating ACE activity but were no more effective than standard doses for suppression of ACE activity.26,a Also, aldosterone breakthrough, as determined on the basis of lack of suppression of the UAldo:C and serum aldosterone concentration, was not prevented by high doses of ACEIs.
The high doses of enalapril and benazepril used in the present study led to significant suppression of circulating ACE activity on all study days (Figure 1). The suppression of ACE activity (percentage change from the baseline value) for experiments F + E and F + B was compared with results for 2 other studies26,a conducted by the authors’ research group; those previous studies had the same study design but involved the use of lower doses of enalapril (0.5 mg/kg, PO, q 12 h) and benazepril (1 mg/kg, PO, q 24 h). In those previous studies,26,a dosages of enalapril and benazepril more closely approximated dosages used in other studies16–19 of ACEIs in patients with naturally occurring heart disease. When suppression of ACE activity was compared between the present F + E experiment and a standard-dose F + E treatmenta and the present F + B experiment and a standard-dose F + B treatment,26 the only difference was evident on day 7, for which there was significantly greater suppression of ACE activity in the standard-dose studies. This may have been attributable to the fact that the baseline ACE activity was higher in dogs in those previous studies, compared with the baseline values for the present study. This is a weakness of evaluating percentage change from the baseline value because investigators cannot account for imbalances between groups at baseline. Overall, high doses of enalapril and benazepril efficiently suppress RAAS activation, and despite the shortcomings of comparing the percentage change, it appears that they do not confer greater suppression of the circulating RAAS when compared with results for standard doses.
The increase in the UAldo:C over the experimental period was consistent with results of other studies25,26,33,34,a conducted by the authors’ research group in which there was furosemide activation of the RAAS in clinically normal dogs. Spot urine samples and radioimmunoassays have been used to determine the correlation between the UAldo:C and 24-hour urine aldosterone excretion.27 Validation of the UAldo:C as a measure of RAAS activation is in the expected response of the UAldo:C to perturbations, such as reductions in blood volume or BP, changes in sodium intake, and stress. In the experiments reported here, the serum aldosterone concentration and UAldo:C were typically higher than baseline values at the end of each experiment, and this increase was evident despite RAAS blockade with ACEIs and suppression of circulating ACE activity. This incomplete or temporary pharmacological blockade of the RAAS system by ACEIs and aldosterone-receptor blockers has been referred to as both aldosterone escape and aldosterone breakthrough.
In studies35,36 that involved the use of ACE inhibition as an endpoint for evaluating the pharmacokinetic and pharmacodynamic relationship of benazepril in dogs, it was found that there was nearly complete inhibition of circulating ACE activity after 2 relatively low doses of benazepril (0.125 mg/kg, q 24 h). This likely reflects the high affinity of ACEIs for ACE (both circulating ACE and ACE that has been cleaved from the vascular endothelium). Therefore, higher doses of ACEIs are unlikely to provide additional benefit in inhibition of circulating ACE activity. However, it is estimated that approximately 90% of the ACE in a body is tissue bound (noncirculating).35,37 Tissue RAAS and local generation of angiotensin II and aldosterone play an important role in physiologic and pathophysiological processes in the kidneys, vasculature, myocardium, and brain.37–39 Measurement of tissue ACE and other components of the tissue RAAS requires a tissue homogenate,40,41 which is difficult to obtain from clinical patients and thus makes it difficult to evaluate the efficacy of ACEIs on the tissue RAAS.
The ACEIs differ in their affinities for binding of tissue ACE. Differences in this affinity are related to dosage, bioavailability, half-life, tissue penetration, and tissue retention.37 Tissue affinity (from greatest to least) of the commonly used ACEIs in veterinary medicine are as follows: benazepril > ramipril > lisinopril > enalapril > captopril.37,42 It is not known whether increasing the dosage of ACEIs will increase efficacy for blunting the circulating and tissue RAAS. The dosages used in the study reported here were twice the upper limit of the recommended dosage for enalapril and benazepril in dogs. One hypothesized mechanism for aldosterone breakthrough is a local effect (ie, tissue generation of angiotensin II); thus, it is possible that aldosterone breakthrough may be explained by inadequate suppression of tissue ACE activity, even at the higher dosages used in the present study. Comparison of the efficacy of various ACEIs, such as detecting changes in pharmacological properties (eg, tissue-binding affinity in patients), can be accomplished by measuring clinical endpoints such as heart failure mortality rate, degree of cardiac remodeling, or control of systemic hypertension. Two studies42,43 conducted to compare various ACEIs in humans suggest that a higher tissue-binding capacity does not decrease the risk for several important cardiovascular outcomes.42,43 However, in rats with experimentally induced volume overload, quinapril was more effective than enalapril for blunting the development of left ventricular hypertrophy.44 In that study,44 both ACEIs prevented the increase in circulating angiotensin II concentrations in untreated control rats, but only quinapril prevented the increase in left ventricular (tissue) angiotensin II concentrations. Further studies will be required to determine whether ACEIs with greater tissue-binding capacity confer clinical benefits to humans and domestic animals with heart disease.
Studies45,46 conducted to compare the effect of high versus low dosages of ACEIs on clinical outcome and neurohormones in humans with chronic heart failure and reduced ejection fraction have yielded conflicting results. Those studies found a dose-dependent suppression of circulating ACE activity in which a high dosage of ACEIs was associated with greater suppression of ACE activity than was achieved with a lower dosage of ACEIs. In one of those studies,46 a higher dosage of lisinopril for 2 weeks led to a greater reduction in plasma aldosterone concentrations, compared with results for people receiving a low dosage of ramipril. In the other study,45 which was conducted to evaluate a high versus low dosage of enalapril for humans over a 6-month period, no significant benefit was found for the high dosage of the ACE when a composite clinical endpoint was evaluated. Also, there was no significant difference in plasma angiotensin II and serum aldosterone concentrations between the 2 groups, and these hormones remained persistently elevated.45 The authors of that report45 considered this to be aldosterone breakthrough (defined as a serum aldosterone concentration greater than the upper limit of the reference interval), despite treatment with an ACEI or angiotensin-receptor blocker. When the incidence of aldosterone breakthrough was compared between the high- and low-dosage enalapril groups, the prevalence of aldosterone breakthrough was higher (39% vs 30%), but not significantly, for the low-dosage group.45 The investigators also found a significant increase in the plasma renin activity over the study period for the high-dosage group. Comparison of tissue ACE activity on the final day of the study revealed higher values for the high-dose group, which supported the hypothesis of excessive renin production in response to an ACEI as a mechanism for aldosterone breakthrough.
There are several other hypothesized mechanisms for aldosterone breakthrough. One hypothesis is tissue production of angiotensin II, a major secretogogue of aldosterone, via non–ACE-mediated pathways that involve chymase or kallikrein. This ACE-independent tissue production of angiotensin II is thought to cause continued aldosterone production despite ACEI administration. It is possible that high dosages of ACEIs may increase renin release, as has been reported in 1 study45; however, renin activity was not measured in that study. The increase in circulating renin concentrations could influence tissue components of the RAAS and cause or contribute to aldosterone breakthrough. However, excessive tissue production of angiotensin II is not likely the sole mechanism for aldosterone breakthrough because the use of angiotensin-receptor blockers does not appear to decrease the prevalence of aldosterone breakthrough.32,47 Similarly, a recent study32 conducted to evaluate aldosterone breakthrough in patients with proteinuric renal diseases found no difference in the prevalence of aldosterone breakthrough for patients receiving the direct renin inhibitor aliskiren and those receiving a placebo.
Potassium, another potent secretogogue of aldosterone, has been implicated in the mechanism for aldosterone breakthrough. The hypothesized mechanism is that increasing serum potassium concentrations attributable to treatment with ACEIs or angiotensin-receptor blockers or declining renal function serve as potent stimulation for aldosterone release from the zona glomerulosa of the adrenal cortex. This hypothesis has not been supported in clinical studies32,47 in humans and is not supported by results for the dogs of the present study because serum potassium concentrations did not change significantly over the study period. Obesity may also contribute to excess aldosterone production because adipocytes are capable of synthesizing aldosterone, and increased adiposity has been associated with aldosterone breakthrough.32,48 Finally, because ACTH exerts a permissive effect on aldosterone secretion, endocrine diseases such as hyperadrenocorticism may contribute to aldosterone breakthrough. None of these hypotheses apply to aldosterone breakthrough for the experimental methods used by the authors.
The efficacy of ACEI suppression of aldosterone secretion may differ among dogs. Mechanisms underlying this variation among dogs are not known. Polymorphisms in the ACE and other RAAS genes exist in dogs49 and may lead to differences in circulating ACE activity and in the affinity of ACEI for both circulating and tissue ACE among dogs. There also may be individual variation in efficacy of pharmacological stimulation (diuretic or vasodilatory) and suppression (ACEI or angiotensin-receptor blocker) of the RAAS. Also, serum ACE activity only reflects the circulating pool, and it is difficult to evaluate the efficacy for ACEI suppression of tissue RAAS.
The UAldo:C may be a useful clinical tool in determining those patients that would benefit from additional RAAS blockade with combinations of ACEIs, angiotensin-receptor blockers, or mineralocorticoid-receptor blockers (spironolactone or eplerenone). In a field trial,24 investigators found a significant reduction in the risk of cardiac morbidity and death attributable to cardiac causes in dogs with chronic degenerative mitral valve disease that were treated with spironolactone in addition to an ACEI, furosemide, and sometimes digoxin, compared with outcomes for dogs treated with furosemide, an ACEI, and sometimes digoxin but without spironolactone. The effect of spironolactone in combatting aldosterone breakthrough is 1 explanation for the benefits detected in that study.24 The UAldo:C is a good indicator of aldosterone breakthrough because it combines results for 2 (morning and evening) urine samples and provides a mean value, with each urine sample reflecting several hours of aldosterone secretion. Conversely, spot test blood measurements of RAAS components have substantial minute-to-minute variation attributable to changes in sympathetic stimulation and posture changes.50–54 For this reason, the reference interval for blood aldosterone concentration is relatively wide. Changes in serum aldosterone concentrations in the present study mirrored those of the UAldo:C (Figure 3). The mean baseline serum aldosterone concentrations in this group of research dogs was within the reference interval of the commercial laboratoryn but exceeded this reference interval after initiation of furosemide and ACEI administration. The relatively high baseline serum aldosterone concentrations in this particular group of research dogs may have reflected the fact that these dogs were not conditioned to the experimental procedures and may have been overly excited or stressed.
Systolic BP was significantly decreased from the baseline value on several days during the experiments (Table 1). These changes in mean BP can be compared with results for previous studies26,a conducted by our research group to evaluate the recommended ACEI dosage for this method of furosemide-induced RAAS activation. In those standard-dosage studies,26,a there were no significant changes in BP. Thus, there appears to be a greater reduction in BP with higher doses of ACEI for this experimental method.
Mean body weight decreased slightly, but not significantly, during both experiments. Body weight was maintained in previous studies26,33,a conducted by our research group on furosemide-induced RAAS in healthy dogs. Although water intake and urine output were not quantified in the study reported here, dogs were likely drinking more water than usual. Shortterm mechanisms of diuretic resistance, such as the braking phenomenon and increased sodium and fluid reabsorption in the proximal tubules, may mitigate substantial weight loss.55
Mean serum sodium and chloride concentrations decreased in the F + E experiment, and the bicarbonate concentration increased over the experimental period, which is consistent with administration of a loop diuretic.56 The serum chloride and bicarbonate concentrations responded similarly in the F + B experiment. However, the serum sodium concentration increased on days 1 and 3 before decreasing on day 7. The serum albumin concentration followed the same pattern as for the serum sodium concentration; thus, it is possible that hemoconcentration led to these discordant findings in the serum sodium concentration.
The aforementioned findings indicated that high doses of enalapril and benazepril were tolerated well by healthy dogs receiving furosemide. If such doses were to be used in dogs with heart failure and known or suspected kidney disease, gradual titration of the dosage with concurrent monitoring of renal function and BP would be recommended.
One limitation of the study reported here was that the experiments were of short duration, which does not mimic the time course for pharmacological treatment of chronic heart failure. In another study25 conducted by the authors’ research group, it was reported that the UAldo:C (RAAS activation) plateaus after diuretic treatment for approximately 5 days. However, it is possible that dynamics of the RAAS for this experimental method could change over time, which could lead to a decrease or an increase in the prevalence of aldosterone breakthrough. It is worth mentioning that the prevalence of aldosterone breakthrough in humans increases with an increasing duration of disease and treatment.31 Another limitation of the present study was the use of healthy research dogs. In comparison, RAAS activation in dogs with naturally occurring heart failure is usually a chronically evolving response accompanying deterioration of cardiac performance. Although the dogs of the present study did not have underlying cardiac dysfunction, it could be argued that the presence of such dysfunction would likely further stimulate the RAAS and contribute to aldosterone breakthrough. Although high doses of 2 ACEIs were tolerated well by these healthy dogs, the doses were not associated with detectable clinically relevant benefits. The dosages should be evaluated for safety in clinical patients with heart failure, particularly those receiving other vasodilators or high doses of furosemide.
Another study limitation was the lack of a control group (administration of only an ACEI). The effect of high doses of an ACEI on the circulating RAAS would best be evaluated via measurement of renin activity, serum aldosterone and angiotensin II concentrations, and the UAldo:C. It is possible that high doses of ACEIs may paradoxically potentiate RAAS activation via preferential dilation of efferent arterioles and increased renin release. However, in the present study, changes in indices (concentrations of SUN, serum creatinine, and serum potassium) that would indicate a decrease in glomerular filtration rate did not occur between baseline and day 7, thereby failing to support this hypothesis. The method of furosemide-induced RAAS activation has been evaluated by the authors’ research group, and the effects of furosemide on the serum ACE activity and UAldo:C have been described.25–27,33,a For this reason, a furosemide-only group was not included in the study. Finally, the present study was not designed to evaluate differences in efficacy between classes of ACEIs for suppression of aldosterone secretion because the chosen doses may not have been equipotent.
For the study reported here, it can be concluded that furosemide-induced RAAS activation was achieved, and aldosterone suppression with twice the recommended dosages of the 2 most commonly used ACEIs in veterinary medicine was inadequate. This was true despite significant suppression of ACE activity by 6 hours after treatment. Thus, it appears that aldosterone breakthrough is an ACEI class effect and that higher ACEI dosages will be unlikely to eliminate aldosterone breakthrough.
Acknowledgments
Supported by a grant from the Jane Lewis Seaks Endowment.
The authors thank Allison Klein, Anne Crews, and Susan Beyerlein for technical assistance.
ABBREVIATIONS
| %ΔACE | Percentage change in angiotensin-converting enzyme activity |
| ACE | Angiotensin-converting enzyme |
| ACEI | Angiotensin-converting enzyme inhibitor |
| BP | Blood pressure |
| RAAS | Renin-angiotensin-aldosterone system |
| UAldo:C | Urinary aldosterone-to-creatinine ratio |
Footnotes
Lantis AC, Atkins CE, Ames M. The effect of enalapril on furosemide-activated renin-angiotensin-aldosterone system (RAAS) in normal dogs (abstr). J Vet Intern Med 2012;26:715.
ProActive Health Adult MiniChunks, Iams Co, Dayton, Ohio.
Furosemide, 12.5 mg, Vedco Inc, St Joseph, Mo.
Furosemide, 20 mg, Mylan Pharmaceuticals Inc, Morgantown, Wva.
Vasotec, 2.5 mg, Valeant Pharmaceuticals LLC, Bridgewater, NJ.
Vasotec, 10 mg, Valeant Pharmaceuticals LLC, Bridgewater, NJ.
Lotensin, 10 mg, Validus Pharmaceuticals LLC, Parsippany, NJ.
Cardell model 9405, Paragon Medical Inc, Coral Springs, Fla.
Serum ACE activity REA, ALPCO Diagnostic, Salem, NH.
Tri-Carb 2900 TR beta counter, PerkinElmer Inc, Waltham, Mass.
SoftMax Pro, version 5.0, Molecular Devices Corp, Sunnyvale, Calif.
Serum aldosterone ELISA, ALPCO Diagnostics, Salem, NH.
Coat-a-Count aldosterone RIA, Siemens Medical Diagnostic Solutions, Los Angeles, Calif.
Diagnostic Center for Population and Animal Health, Michigan State University, Lansing, Mich.
SAS/STAT, version 9.4, SAS Institute Inc, Cary, NC.
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