Renin‐angiotensin aldosterone profile before and after angiotensin‐converting enzyme‐inhibitor administration in dogs with angiotensin‐converting enzyme gene polymorphism

Abstract Background An angiotensin‐converting enzyme (ACE) gene polymorphism occurs in dogs; however, functional importance is not well studied. Hypothesis We hypothesized that dogs with the polymorphism would show alternative renin‐angiotensin aldosterone system (RAAS) pathway activation and classical RAAS pathway suppression before and after ACE‐inhibitor administration, as compared to dogs without the polymorphism that would show this pattern only after ACE‐inhibitor administration. Animals Twenty‐one dogs with mitral valve disease that were genotyped for the ACE gene polymorphism. Methods This retrospective study utilized stored samples from 8 ACE gene polymorphism‐negative (PN) dogs and 13 ACE gene polymorphism‐positive (PP) dogs before and after enalapril administration. Equilibrium analysis was performed to evaluate serum RAAS metabolites and enzyme activities. Results were compared before and after enalapril, and between groups. Results The classical RAAS pathway was suppressed and the alternative RAAS pathway was enhanced for both genotypes after administration of enalapril, with no differences before enalapril administration. Aldosterone breakthrough occurred in both PN (38%) and PP (54%) dogs despite angiotensin II suppression. Aldosterone was significantly higher (P = .02) in ACE gene PP dogs (median, 92.17 pM; IQR, 21.85‐184.70) compared to ACE gene PN dogs (median, 15.91 pM; IQR, <15.00‐33.92) after enalapril. Conclusions and Clinical Importance The ACE gene polymorphism did not alter baseline RAAS activity. Aldosterone breatkthrough in some dogs suggests nonangiotensin mediated aldosterone production that might be negatively influenced by genotype. These results support the use of aldosterone receptor antagonists with ACE‐inhibitors when RAAS inhibition is indicated for dogs, especially those positive for the ACE gene polymorphism.


| INTRODUCTION
The classical renin-angiotensin aldosterone system (RAAS) is the neurohormonal cascade initiated by the release of renin that cleaves angiotensin I from angiotensinogen, followed by the conversion of angiotensin I to angiotensin II by the angiotensin-converting enzyme (ACE) and production of angiotensin II metabolites (angiotensin III and angiotensin IV) by aminopeptidates, and ending in the stimulation of aldosterone release from the adrenal glands. 1 Angiotensin II is the major stimulator of aldosterone synthesis and release from the adrenal cortex, although hyperkalemia and adrenocorticotrophic stimulating hormone can also induce aldosterone release. 2,3 The RAAS is activated in dogs with advanced heart disease, and contributes to disease progression and clinical signs. 1,[4][5][6][7] The major end-metabolites of the classical RAAS are angiotensin II and aldosterone, both of which directly or indirectly mediate sodium and water retention, vasoconstriction, and pathological remodeling of cardiac, vascular, and renal tissues. 1,4,6,7 Recent reports describe an alternative RAAS pathway mediated by ACE2, prolyl-carboxy-peptidase 16, prolyl-endopeptidase, and neprilysin, with production of the metabolites angiotensin 1-9, angiotensin 1-7 and angiotensin 1-5 in people and dogs. [8][9][10][11] Additional metabolites within this cascade have also been discovered including angiotensin 2-10, angiotensin 2-7, and angiotensin 3-7. 8,10,11 Whereas the functional importance of some metabolites is not yet known, angiotensin 1-7 has counterbalancing vasodilatory and natriuretic properties. 10 Angiotensin-converting enzyme-inhibitors prevent the conversion of angiotensin I to angiotensin II, and thereby mitigate the maladaptive effects of these neurohormones through reduced formation of angiotensin II and angiotensin-II-driven aldosterone production. 1 Polymorphisms of the ACE gene are recognized in human beings and dogs, which could influence the therapeutic strategies and outcome of diseases in which the RAAS plays a pathogenic role. [12][13][14] Directly measured ACE activity is lower in dogs that are homozygous for a single base pair ACE gene polymorphism compared to dogs without the polymorphism; however, ACE activity after administration of ACE-inhibitors is similarly suppressed regardless of genotype. 13,14 The functional effects and clinical importance of these findings are not well understood.
Although the RAAS has historically been challenging to investigate, renin-angiotensin system equilibrium analysis has emerged as a novel technique that allows for comprehensive evaluation of this complex neurohormonal system. Catalyzing enzymes other than ACE (such as ACE2, neprilysin, chymase, and aminopeptidases) mediate production of RAAS metabolites in this pathway, and recent publications have begun to shed light on blood and tissue activities in dogs. [8][9][10][11] This methodology has shown that ACE-inhibitors not only suppress angiotensin II formation, but also increase levels of the beneficial peptide hormone angiotensin 1-7, which is known to be metabolized to angiotensin 1-5 by the N-domain of ACE. 15

| Equilibrium analysis of RAAS components
The equilibrium concentrations of 6 different RAAS angiotensin peptide metabolites and aldosterone in canine serum samples were quantified by liquid chromatography-mass spectrometry/massspectroscopy performed at a service provider laboratory (Attoquant Diagnostics, Vienna, Austria), using previously validated and described methods. 8,10,17 Briefly, serum conditioning for equilibrium analysis was performed at 37 C followed by stabilization through the addition of an enzyme inhibitor cocktail (Waters, Milford, Massachusetts), as described previously. 8,10,17 Previous results have shown similar qualitative outcomes when comparing the quantification of circulating (stabilized immediately at blood drawing) and equilibrium angiotensin peptide levels. 8 The ratio of angiotensin II to angiotensin I was calculated as a marker for ACE activity (ACE-S). Angiotensin I and angiotensin II were summed as a marker for plasma renin concentration (PRA-S). 17 The ratio of angiotensin 1-5 to angiotensin 1-7 (Ang 1-5/Ang 1-7) was calculated as an indicator of ACE activity driven by its N-domain. 15 The ratio of aldosterone to angiotensin II (AA2) was calculated as an indicator of adrenal responsiveness to angiotensin II stimulation of aldosterone release. 9 Aldosterone breakthrough (ABT) was defined as any increase in serum aldosterone after enalapril compared to baseline (pre-enalapril) aldosterone. 18 T A B L E 1 Renin-angiotensin aldosterone system (RAAS) metabolites and ratios, pre-and post-enalapril, for ACE polymorphism negative dogs and ACE polymorphism positive dogs Note: Values are shown as median and interquartile range. Statistically significant P values are bolded. Values below the lower limit of quantification are shown as < the lowest reported value for each assay.

| Statistical analysis
Statistical analysis was performed using commercially available software (GraphPad Prism 8, San Diego, California). Data values that were below the lower limit of assay quantification were reported as half the lower limit for statistical analysis only. 19

| Genotype comparisons
No significant differences in the RAAS profile and enzyme activities were present between PN and PP dogs before enalapril treatment (  into angiotensin 1-5; Figure 1). Promotion of angiotensin 1-7 (predominantly through neprilysin conversion of angiotensin I to angiotensin 1-7) is believed to mediate ACE-inhibitor benefits of vasodilation and natriuresis. 8 Alternate RAAS metabolite production did not differ based on genotype.
The RAAS profile of some dogs in this study after treatment with enalapril is consistent with ABT; however, this importantly does not appear to be mediated by angiotensin II that was appropriately suppressed with enalapril treatment. Aldosterone breakthrough is the term used to describe inadequate or temporary suppression of aldosterone, despite the administration of appropriate doses of ACE-inhibitors. 21 The phenomenon of ABT has been shown to occur in both people and dogs. 18 and inhibition of angiotensin II formation or binding (ACE-inhibitors or angiotensin receptor blockers, respectively) is often necessary to provide comprehensive RAAS inhibition. 18,21,24 Additional study is required to elucidate other pathways that may be impacted by RAAS inhibitors.
Additionally, we found that the magnitude of ABT was greater in PP dogs compared to PN dogs despite suppression of angiotensin II formation with enalapril in both groups. This was also reflected in significantly greater AA2 ratio in PP dogs after enalapril treatment, indicating that the high aldosterone was independent of angiotensin II. This finding suggests an influence of the ACE gene polymorphism on an, as of yet unidentified angiotensin II-independent pathway of aldosterone stimulation. In addition to the many possible substrates for ACE, there is recent evidence that numerous non-angiotensin-mediated, nonelectrolyte factors can control aldosterone secretion from the adrenal cortex. 22 These paracrine regulatory factors include bioactive signals released from mast cells (eg, serotonin), nerve fibers (eg, catecholamines), chromaffin cells, adipocytes (eg, leptin), vascular endothelial cells (eg, endothelin 1), and steroidogenic cells (eg, prostaglandin E2). 22 The influence of genotype in this setting requires further study to explore the impact of this polymorphism on dogs receiving ACE inhibition.
The results of this study provide mechanistic insight into the phenomenon of ABT and hold clinical implications for dogs with advanced heart disease. Although ABT occurs in a substantial minority (30%-40%) of people and dogs receiving ACE-inhibitors, predicting which patients are or will be affected is elusive. 18,21 Aldosterone breakthrough occurring with short-term ACE-inhibitor monotherapy in this study was independent of angiotensin II, and the ACE gene polymorphism appeared to negatively influence aldosterone concentrations in this group of dogs with preclinical mitral valve disease. Dogs positive for the ACE gene polymorphism might be at greater risk for adverse effects associated with unopposed circulating aldosterone than dogs negative for the polymorphism, despite the effectiveness of enalapril in suppressing angiotensin II production in both groups. The genetic heterogeneity of the ACE gene and the nonuniversal occurrence of ABT in dogs could be an explanation for discordant outcome findings of previous studies reporting the long-term effects of ACE-inhibitors in dogs with heart disease. 25,26 This study is limited by the small study population, which could have affected the ability to find differences between groups for some parameters. We did not separately analyze dogs that were homozygous from those that were heterozygous for the ACE gene polymorphism because of small subject numbers. Future studies with larger numbers will be necessary to determine if there are RAAS profile differences in dogs with 1 or both abnormal alleles. Genotyping might also prove useful for determining subpopulations of dogs that would benefit the most from aldosterone antagonizing medications.
In conclusion, we demonstrated that ABT occurs independent of successful angiotensin II suppression with ACE-inhibitor treatment in some dogs, and that the ACE gene polymorphism seems to negatively influence this suppression. Additionally, the presence of the polymorphism appears to not reduce overall RAAS activation in the untreated dog, possibly because of upregulation of compensatory enzymes that might serve to stabilize angiotensin II levels. These findings not only improve our understanding of the RAAS in dogs, but also show functional importance of the ACE gene polymorphism in canine patients. The results of this study support the use of aldosterone receptor antagonists in conjunction with ACE-inhibitors in dogs requiring RAAS suppression.