Sodium glucose transporter 2 inhibitors: Will these drugs benefit non-diabetic veterinary patients with cardiac and kidney diseases?

Sodium glucose transporter type 2 (SGLT2) inhibitors have been introduced into human medicine where their beneficial effects go beyond the expected improvement in blood glucose control. These drugs appear to prevent progression of both cardiovascular and kidney diseases, not only in diabetic but also in non-diabetic human patients. As these drugs have received conditional approval for use in diabetic cats and are being used in other veterinary species, the intriguing question as to whether they will have similar cardioprotective and nephroprotective effects in dogs and cats is being asked. The primary mechanism(s) by which SGLT2 inhibitors are cardio- and nephroprotective remain to be fully characterized. This paper reviews these suggested mechanisms in the context of the pathophysiology of progressive cardiovascular and kidney diseases in dogs and cats with the goal of predicting which categories of non-diabetic veterinary patients these drugs might be of most benefit.

Effect of metastatic calcification on complication rate and survival in 74 renal transplant cats (1998-2020).

OBJECTIVE: To report the incidence of metastatic calcification in cats with renal failure presenting for renal transplantation, and to determine if metastatic calcification detected prior to renal transplantation is associated with complication rates and patient survival. STUDY DESIGN: Retrospective case series. SAMPLE POPULATION: Seventy-four cats. METHODS: In imaging studies, 178 feline renal transplant recipients from 1998 to 2020 were evaluated for metastatic calcification. Demographic, clinicopathological data, intraoperative complications, postoperative complications, need for hemodialysis, and survival times were recorded. Exclusion criteria were cats lacking imaging reports and cats with gastric, renal, or tracheal/bronchial calcification alone. Univariable and multivariable analyses were performed to determine variables that were independently associated with survival. Kaplan-Meier analysis was used to generate survival plots and estimate median survival times with a 95% confidence interval (CI). RESULTS: Seventy four of 178 cats met the inclusion criteria. Fifteen of 74 (20.3%) cats had metastatic calcification prior to renal transplantation. Twelve of 74 (16.2%) cats developed calcification following transplantation, and 47 of 74 (63.5%) cats had no calcification during the study period. Median follow-up time was 472 days, with a range of 0-1825 days. Cats with pretransplant calcification had shorter median survival times (147 days) than cats without calcification (646 days) (p = .0013). Metastatic calcification pretransplant was associated with an increased risk of death by 240% (95% CI, 1.22-4.71). CONCLUSION: Metastatic calcification in renal transplant cats is a negative prognostic indicator and is associated with decreased survival times. CLINICAL SIGNIFICANCE: These findings may help guide therapeutic recommendations and owner expectations in cats undergoing renal transplantation.

Serotonin receptor 2B signaling with interstitial cell activation and leaflet remodeling in degenerative mitral regurgitation.

AIMS: Mitral valve interstitial cells (MVIC) play an important role in the pathogenesis of degenerative mitral regurgitation (MR) due to mitral valve prolapse (MVP). Numerous clinical studies have observed serotonin (5HT) dysregulation in cardiac valvulopathies; however, the impact of 5HT-mediated signaling on MVIC activation and leaflet remodeling in MVP have been investigated to a limited extent. Here we test the hypothesis that 5HT receptors (5HTRs) signaling contributes to MVP pathophysiology. METHODS AND RESULTS: Diseased human MV leaflets were obtained during cardiac surgery for MVP; normal MV leaflets were obtained from heart transplants. MV RNA was used for microarray analysis of MVP patients versus control, highlighting genes that indicate the involvement of 5HTR pathways and extracellular matrix remodeling in MVP. Human MV leaflets were also studied in vitro and ex vivo with biomechanical testing to assess remodeling in the presence of a 5HTR2B antagonist (LY272015). MVP leaflets from Cavalier King Charles Spaniels were used as a naturally acquired in vivo model of MVP. These canine MVP leaflets (N=5/group) showed 5HTR2B upregulation. This study also utilized CB57.1ML/6 mice in order to determine the effect of Angiotensin II infusion on MV remodeling. Histological analysis showed that MV thickening due to chronic Angiotensin II remodeling is mitigated by a 5HTR2B antagonist (LY272015) but not by 5HTR2A inhibitors. CONCLUSION: In humans, MVP is associated with an upregulation in 5HTR2B expression and increased 5HT receptor signaling in the leaflets. Antagonism of 5HTR2B mitigates MVIC activation in vitro and MV remodeling in vivo. These observations support the view that 5HTR signaling is involved not only in previously reported 5HT-related valvulopathies, but it is also involved in the pathological remodeling of MVP.

Effect of sodium-glucose cotransporter 2 inhibitor canagliflozin on interstitial glucose concentration in insulin-treated diabetic dogs.

BACKGROUND: The utility of sodium-glucose cotransporter 2 inhibitors (SGLT2i) has not been reported in insulin-treated diabetic dogs. HYPOTHESIS: Canagliflozin, a PO-administered SGLT2i, decreases interstitial glucose concentration (IG) in insulin-treated diabetic dogs. ANIMALS: Five insulin-treated diabetic dogs. METHODS: Uncontrolled open label longitudinal study. Canagliflozin (2-4 mg/kg/day PO) was added to an unchanged insulin dose for 7 days. Fractional excretion of glucose was calculated by dividing the product of urine glucose and serum creatinine concentrations by the product of serum glucose and urine creatinine concentrations. Hypoglycemia was defined as IG <60 mg/dL. RESULTS: Median IG in 2869 measurements obtained while dogs were treated with insulin and canagliflozin was 87 mg/dL (range, 40-500 mg/dL) and was significantly lower than median IG in 1426 measurements obtained while dogs were treated with insulin alone (212 mg/dL; range, 41-500 mg/dL; P < .001). Median fractional excretion of glucose when dogs were treated with insulin and canagliflozin was 1.1% (range, 0.9%-2.0%), significantly higher than when dogs were treated with insulin alone (0.3%; range, 0.01%-1.0%; P = .04). The frequency of hypoglycemia was higher in dogs treated with insulin and canagliflozin (544 of 2869 IG measurements, 19%) compared with the frequency of hypoglycemia in dogs treated with insulin alone (52 of 1426 IG measurements, 4%; P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Canagliflozin may have a role in improving glycemic control in insulin-treated diabetic dogs, but the dose of insulin should be decreased when adding canagliflozin to insulin treatment.

Longitudinal analysis of echocardiographic and cardiac biomarker variables in dogs with atrial fibrillation: The optimal rate control in dogs with atrial fibrillation II study.

BACKGROUND: Rate control (RC; meanHRHolter ≤ 125 bpm) increases survival in dogs with atrial fibrillation (AF). The mechanisms remain unclear. HYPOTHESIS/OBJECTIVES: Investigate echocardiographic and biomarker differences between RC and non-RC (NRC) dogs. Determine if changes post-anti-arrhythmic drugs (AAD) predict successful RC in subsequent Holter monitoring. Evaluate if early vs late RC affects survival. ANIMALS: Fifty-two dogs with AF. METHODS: Holter-derived mean heart rate, echocardiographic and biomarker variables from dogs receiving AAD were analyzed prospectively at each re-evaluation and grouped into RC or NRC. The primary endpoint was successful RC. Between group comparisons of absolute values, magnitude of change from admission to re-evaluations and end of study were performed using Mann-Whitney tests or unpaired t-tests. Logistic regression explored variables associated with inability to achieve RC at subsequent visits. Kaplan-Meier survival analysis was used to compare survival time of early vs late RC. RESULTS: At visit 2, 11/52 dogs were RC; at visit 3, 14/52 were RC; and at visit 4, 4/52 were RC. At the end of study, 25/52 remained NRC. At visit 2, both groups had increased cardiac dimensions, but NRC dogs had larger dimensions; biomarkers did not differ. At the end of study, RC showed decreased cardiac dimensions and end-terminal pro-brain natriuretic peptide (NT-proBNP) compared with NRC. No variables were useful at predicting RC success in subsequent visits. Survival analysis found no differences between early vs late RC. CONCLUSIONS AND CLINICAL IMPORTANCE: The RC dogs had decreased cardiac dimensions and NT-proBNP, suggesting HR-mediated reverse-remodeling might benefit survival, even with delayed RC achievement. Pursuit of RC is crucial despite initial failures.

Observed and expected reliability of echocardiographic volumetric methods and critical change values for quantification of mitral regurgitant fraction in dogs.

BACKGROUND: Reliability of echocardiographic calculations for stroke volume and mitral regurgitant fraction (RFMR) are affected by observer variability and lack of a gold standard. Variability is used to calculate critical change values (CCVs) that are thresholds representing real change in a measure not associated with observer variability. HYPOTHESIS: Observed intra- and interobserver accuracy and variability in healthy dogs help model CCV for RFMR. ANIMALS: Reliability cohort of 34 healthy dogs; allometric scaling cohort of 99 dogs with heart disease and 25 healthy dogs. METHODS: Accuracy, variability, and CCV of 2 observers using geometric and flow-based echocardiography were prospectively compared against a standard of RFMR = 0% and extrapolated across a range of expected RFMR values in the reliability cohort partly derived from cardiac dimensions predicted by the allometric cohort. RESULTS: Accuracy of methods to determine RFMR in descending order was 4-chamber bullet (Bullet4CH), mitral inflow, cube formula, and Simpson's method of disks. Intraobserver variability was relatively high. The CCV for RFMR ranged from 28% to 88% and was inversely related to RFMR when extrapolated for use in affected dogs. For both observers, the Bullet4CH method had the lowest intraobserver CCV (Operator 1:28%, Operator 2:41%). Interobserver strength of agreement was low with intraclass correlation coefficients ranging from 0.210 to 0.413. CONCLUSIONS AND CLINICAL IMPORTANCE: Echocardiographic volumetric methods used to calculate stroke volume and RFMR have low accuracy and high variability in healthy dogs. Extrapolation of observed CCV to a range of expected RFMR suggests observers and methods are not interchangeable and variability might hinder routine clinical usage. Individual observers should be aware of their own variability and CCV.

Urine sodium concentration after intravenous furosemide in dogs with acute congestive heart failure and correlation with treatment efficacy.

BACKGROUND: Poor natriuresis is a potential marker of diuretic resistance in dogs with acute congestive heart failure (CHF) but little is known about the relationship between urine sodium concentration (uNa) and frequency of successful decongestion. Supplemental O2 is a common treatment in dogs with severe CHF. The time from start to discontinuation of supplemental O2 therapy (DCSO2 ) typically reflects the time course and ease of decongestion. HYPOTHESIS/OBJECTIVES: Urine Na concentration after IV administration of furosemide will be correlated with duration of treatment with supplemental O2 (timeO2 ) and the cumulative frequency of successful DCSO2 during hospitalization. ANIMALS: Fifty-one dogs with acute CHF. METHODS: Retrospective observational single center study. RESULTS: Dogs with low uNa had significantly longer mean timeO2 than dogs with high uNa (uNa <87 mmol/L, 24.2 ± 2.6 hours vs uNa ≥87 mmol/L, 16.6 ± 1.7 hours; P = .02). Low uNa was correlated with lower cumulative frequency of DCSO2 (12 hour, 28%; 24 hour, 42%; 36 hour, 73%) compared to high uNa (12 hour, 28%; 24 hour, 88%; 36 hour, 96%; P = .005). History of PO loop diuretics, low serum chloride concentration (sCl), and high PCV were associated with low uNa. Urine Na concentration outperformed other metrics of diuretic responsiveness including weight loss. CONCLUSIONS AND CLINICAL IMPORTANCE: Urine Na concentration after IV furosemide predicted timeO2 and cumulative frequency of DCSO2 in dogs with acute CHF, which likely reflects important aspects of diuretic responsiveness. Urine Na can assess diuretic responsiveness and treatment efficacy in dogs with CHF.

Utility of focused cardiac ultrasonography training in veterinary students to differentiate stages of subclinical myxomatous mitral valve disease in dogs.

BACKGROUND: Differentiation of the subclinical phases of myxomatous mitral valve disease (MMVD) in dogs relies heavily on echocardiography. Focused cardiac ultrasonography (FCU) is a point-of-care technique that can assess heart size. HYPOTHESIS/OBJECTIVES: Veterinary students trained in FCU can differentiate dogs with subclinical MMVD based on left ventricular (LV) and left atrial (LA) dimensions. ANIMALS: Forty-eight dogs with subclinical MMVD. METHODS: Veterinary students were trained to measure LV dimension and LA-to-aortic root dimension ratio (LA : Ao) using FCU. Dogs were categorized into 2 cohorts based on whether or not the LV normalized internal diastolic dimension was ≥1.7 and LA : Ao was ≥1.6. Agreement between FCU and echocardiographic studies performed by cardiologists was evaluated. RESULTS: One-hundred and forty-six FCU examinations were performed by 58 veterinary students on 48 dogs. Overall agreement between students and cardiologists was moderate (Fleiss' kappa, 0.54; 95% confidence interval [CI], 0.39-0.69; P < .001). Percentage accuracy in observations with heart dimensions less than the cutoffs (86/89, 97%) was significantly higher than in observations in with larger hearts (31/57, 54%; P < .001). Agreement increased from moderate to good as heart sizes became more extreme. Degree of confidence by students in performing FCU was significantly higher at the end vs start of the study. CONCLUSIONS AND CLINICAL IMPORTANCE: Categorization of dogs with subclinical MMVD by veterinary students using FCU was associated with moderate to good agreement with echocardiography. Focused cardiac ultrasonography is a point-of-care method that can help assess clinical stage in dogs with subclinical MMVD.

Toward quantification of loop diuretic responsiveness for congestive heart failure.

Diuretics, such as furosemide, are routinely administered to dogs with congestive heart failure (CHF). Traditionally, dose and determination of efficacy primarily are based on clinical signs rather than quantitative measures of drug action. Treatment of human CHF patients increasingly is guided by quantification of urine sodium concentration (uNa) and urine volume after diuretic administration. Use of these and other measures of diuretic responsiveness is associated with decreased duration of hospitalization, complication rates, future rehospitalization, and mortality. At their core, loop diuretics act through natriuresis, and attention to body sodium (Na) stores and handling offers insight into the pathophysiology of CHF and pharmacology of diuretics beyond what is achievable from clinical signs alone. Human patients with low diuretic responsiveness or diuretic resistance are at risk for difficult or incomplete decongestion that requires diuretic intensification or other remedial strategies. Identification of the specific etiology of resistance in a patient can help tailor personalized interventions. In this review, we advance the concept of loop diuretic responsiveness by highlighting Na and natriuresis. Specifically, we review body water homeostasis and congestion in light of the increasingly recognized role of interstitial Na, propose definitions for diuretic responsiveness and resistance in veterinary subjects, review relevant findings of recent studies, explain how the particular cause of resistance can guide treatment, and identify current knowledge gaps. We believe that a quantitative approach to loop diuretic usage primarily involving natriuresis will advance our understanding and care of dogs with CHF.

Animal Trauma Triage Score, Modified Glasgow Coma Scale, age, and weight were associated with outcome in feline bite wounds (1,065 cases): a VetCOT registry study.

OBJECTIVE: To identify associations between admission variables, Animal Trauma Triage (ATT) score, and Modified Glasgow Coma Scale (MGCS) score with need for transfusion or surgical interventions and survival to discharge in cats with bite wounds. ANIMALS: 1,065 cats with bite wounds. PROCEDURES: Records of cats with bite wounds were obtained from the VetCOT registry from April 2017 to June 2021. Variables included point of care laboratory values, signalment, weight, illness severity scores, and surgical intervention. Associations between admission parameters, terciles of MGCS, quantiles of ATT scores, and death or euthanasia were assessed using univariable and multivariable logistic regression analysis. RESULTS: 872 cats (82%) survived to discharge, while 170 (88%) were euthanized and 23(12%) died. In the multivariable model, age, weight, surgical treatment, ATT and MGCS scores were associated with nonsurvival. For every 1 year of age, odds of nonsurvival increased by 7% (P = .003) and for every 1 kg of body weight, odds of nonsurvival decreased by 14% (P = .005). Odds of dying increased with lower MGCS and higher ATT scores (MGCS: 104% [95% CI, 116% to 267%; P < .001]; ATT: 351% [95% CI, 321% to 632%; P < .001). Odds of dying decreased by 84% (P < .001) in cats that underwent surgery versus those that did not. CLINICAL RELEVANCE: This multicenter study indicated association of higher ATT and lower MGCS with worse outcome. Older age increased the odds of nonsurvival, while each kilogram increase in bodyweight decreased odds of nonsurvival. To our knowledge, this study is the first to describe associations of age and weight with outcome in feline trauma patients.

Optimal rate control in dogs with atrial fibrillation-ORCA study-Multicenter prospective observational study: Prognostic impact and predictors of rate control.

BACKGROUND: The optimal heart rate (HR) in dogs with atrial fibrillation (AF) is unknown. Impact of HR on survival needs elucidation. HYPOTHESIS/OBJECTIVES: Dogs with a 24 hours Holter-derived meanHR ≤125 beats per minute (bpm; rate controlled) survive longer than dogs with higher meanHR. We further aimed to determine which variables predict ability to achieving rate control. ANIMALS: Sixty dogs with AF. METHODS: Holter-derived meanHR, clinical, echocardiographic, and biomarker variables were analyzed prospectively. Survival was recorded from time of rate control, with all-cause mortality as primary endpoint. Cox proportional hazards analysis identified variables independently associated with survival; Kaplan-Meier survival analysis estimated the median survival time of dogs with meanHR ≤125 bpm vs >125 bpm. Logistic regression explored baseline variables associated with inability to achieve rate control. RESULTS: Structural heart disease was present in 56/60 dogs, 50/60 had congestive heart failure, and 45/60 died. Median time to all-cause death was 160 days (range, 88-303 days), dogs with meanHR >125 bpm (n = 27) lived 33 days (95% confidence interval [CI], 15-141 days), dogs with meanHR ≤125 bpm (n = 33) lived 608 days (95% CI, 155-880 days; P < .0001). Congenital heart disease and N-terminal pro-B-type natriuretic peptide were independently associated with higher risk of death (P < .01 and <.0001, respectively) whereas meanHR ≤125 bpm decreased the risk of death (P < .001). Increased left atrial size, increased C-reactive protein concentration and lower blood pressure at admission were associated with failure to achieve rate control. CONCLUSIONS AND CLINICAL IMPORTANCE: Rate control affects survival; an optimal target meanHR <125 bpm should be sought in dogs with AF. Baseline patient variables can help predict if rate control is achievable.

Effect of a specially formulated diet on progression of heart enlargement in dogs with subclinical degenerative mitral valve disease.

BACKGROUND: Previous studies in dogs with degenerative mitral valve disease (DMVD) have identified altered myocardial energy metabolism and oxidation, which might contribute to cardiac hypertrophy. Diets rich in medium chain fatty acids and antioxidants are a potential means of treatment. A previous clinical study found significantly smaller left atrial diameter (LAD) and left atrium-to-aorta diameter ratio (LA : Ao) in dogs with subclinical DMVD fed a specially formulated diet vs control diet for 6 months. HYPOTHESIS/OBJECTIVES: A specially formulated diet will slow or arrest left heart enlargement in dogs with subclinical DMVD over 365 days. ANIMALS: One hundred twenty-seven dogs with unmedicated subclinical DMVD; 101 dogs in the per protocol cohort. METHODS: Randomized double-blinded controlled multicenter clinical trial. RESULTS: The study's primary composite outcome measure was the sum of percentage change in LAD and left ventricular internal dimension at end-diastole (LVIDd) at day 365. In the per protocol cohort, the outcome measure increased by 8.0% (95% confidence interval [CI], 2.9%-13.1%) in dogs receiving the test diet vs 8.8% (95% CI, 5.1%-12.5%) in dogs receiving control diet (P = .79). Neither component of the primary outcome measure was significantly different between groups (LAD, P = .65; LVIDd, P = .92). No difference was found in mitral valve E wave velocity (P = .36) or the proportion of dogs withdrawn from the study because of worsening DMVD and heart enlargement (P = .41). CONCLUSIONS AND CLINICAL IMPORTANCE: Feeding a specially formulated diet for 365 days was not associated with a significantly different rate of change of left heart size in dogs with subclinical DMVD as compared to control.

Decreased serotonin transporter activity in the mitral valve contributes to progression of degenerative mitral regurgitation.

Degenerative mitral valve (MV) regurgitation (MR) is a highly prevalent heart disease that requires surgery in severe cases. Here, we show that a decrease in the activity of the serotonin transporter (SERT) accelerates MV remodeling and progression to MR. Through studies of a population of patients with MR, we show that selective serotonin reuptake inhibitor (SSRI) use and SERT promoter polymorphism 5-HTTLPR LL genotype were associated with MV surgery at younger age. Functional characterization of 122 human MV samples, in conjunction with in vivo studies in SERT-/- mice and wild-type mice treated with the SSRI fluoxetine, showed that diminished SERT activity in MV interstitial cells (MVICs) contributed to the pathophysiology of MR through enhanced serotonin receptor (HTR) signaling. SERT activity was decreased in LL MVICs partially because of diminished membrane localization of SERT. In mice, fluoxetine treatment or SERT knockdown resulted in thickened MV leaflets. Similarly, silencing of SERT in normal human MVICs led to up-regulation of transforming growth factor ß1 (TGFß1) and collagen (COL1A1) in the presence of serotonin. In addition, treatment of MVICs with fluoxetine not only directly inhibited SERT activity but also decreased SERT expression and increased HTR2B expression. Fluoxetine treatment and LL genotype were also associated with increased COL1A1 expression in the presence of serotonin in MVICs, and these effects were attenuated by HTR2B inhibition. These results suggest that assessment of both 5-HTTLPR genotype and SERT-inhibiting treatments may be useful tools to risk-stratify patients with MV disease to estimate the likelihood of rapid disease progression.

A splice site mutation in a gene encoding for PDK4, a mitochondrial protein, is associated with the development of dilated cardiomyopathy in the Doberman pinscher.

Familial dilated cardiomyopathy is a primary myocardial disease that can result in the development of congestive heart failure and sudden cardiac death. Spontaneous animal models of familial dilated cardiomyopathy exist and the Doberman pinscher dog is one of the most commonly reported canine breeds. The objective of this study was to evaluate familial dilated cardiomyopathy in the Doberman pinscher dog using a genome-wide association study for a genetic alteration(s) associated with the development of this disease in this canine model. Genome-wide association analysis identified an area of statistical significance on canine chromosome 14 (p(raw) = 9.999e-05 corrected for genome-wide significance), fine-mapping of additional SNPs flanking this region localized a signal to 23,774,190-23,781,919 (p = 0.001) and DNA sequencing identified a 16-base pair deletion in the 5' donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 gene in affected dogs (p < 0.0001). Electron microscopy of myocardium from affected dogs demonstrated disorganization of the Z line, mild to moderate T tubule and sarcoplasmic reticulum dilation, marked pleomorphic mitochondrial alterations with megamitochondria, scattered mitochondria with whorling and vacuolization and mild aggregates of lipofuscin granules. In conclusion, we report the identification of a splice site deletion in the PDK4 gene that is associated with the development of familial dilated cardiomyopathy in the Doberman pinscher dog.

Effect of angiotensin receptor blockers and angiotensin converting enzyme 2 on plasma equilibrium angiotensin peptide concentrations in dogs with heart disease.

BACKGROUND: The pathophysiology of heart failure involves maladaptive angiotensin peptides (APs) and enzymes, including angiotensin 2 (AT2) and angiotensin converting enzyme (ACE), as well as recently described alternative components, such as angiotensin 1-7 (Ang1-7) and angiotensin converting enzyme 2 (ACE2). The relative effects of different neurohormonal-targeting drugs on balance of APs in dogs with heart disease are unknown. HYPOTHESIS/OBJECTIVES: Plasma AP concentrations differ in dogs receiving angiotensin converting enzyme inhibitors (ACEIs) vs angiotensin receptor blockers (ARBs) and recombinant human ACE2 (rhACE2) will further increase these differences. ANIMALS: Eight dogs with degenerative mitral valve disease (DMVD). METHODS: Prospective open-label trial. Equilibrium concentrations of APs from plasma during PO ACEI treatment and then after 14 days of PO ARB treatment using telmisartan were measured using liquid chromatography-tandem mass spectroscopy before and after in vitro incubation with rhACE2. RESULTS: Concentration of Ang1-7 was increased during ARB treatment (Ang1-7: 443 pg/mL; 95% confidence interval [CI] = 247-794 pg/mL) vs ACEI (Ang1-7: 182 pg/mL; 95% CI = 66.2-503 pg/mL; P = .01). Incubation with rhACE2 decreased traditional APs while increasing beneficial alternative APs, and Ang1-7 was significantly higher in the ARB + rhACE2 (880 pg/mL; 95% CI = 560-1383 pg/mL) vs ACEI + rhACE2 (455 pg/mL; 95% CI = 188-1104 pg/mL; P = .03) group. The most favorable theoretical AP profile was achieved in the ARB + rhACE2 group. CONCLUSIONS AND CLINICAL IMPORTANCE: The AP profile during telmisartan treatment is associated with higher plasma Ang1-7 as compared with during ACEI. This favorable shift is potentiated in vitro by combination of ARB + rhACE2. These data support potential AP-targeting strategies and drugs in dogs with DMVD.

Effect of angiotensin receptor blockers and angiotensin-converting enzyme 2 on plasma equilibrium angiotensin peptide concentrations in cats with heart disease.

BACKGROUND: Little is known about the effect of renin angiotensin aldosterone system-inhibiting (RAASi) drugs on alternative angiotensin peptides (APs) such as angiotensin 1-7 (Ang1-7), which are mediated by angiotensin-converting enzyme 2 (ACE2). HYPOTHESIS/OBJECTIVES: Angiotensin receptor blockers (ARBs) would alter balance of APs and differences would be magnified in vitro by incubation of plasma samples with recombinant human ACE2 (rhACE2). ANIMALS: Six cats with cardiomyopathy (CM), 8 healthy cats. METHODS: Prospective open label trial. Plasma equilibrium concentrations of APs were measured in healthy cats as well as in CM cats that first received no RAASi drugs (CMnoRAASi ) and then after 14 days of PO telmisartan (CMARB ). Plasma APs also were measured after in vitro incubation with rhACE2. RESULTS: No significant differences were found between healthy and CMnoRAASi groups. Concentrations of several APs, including angiotensin I (AT1) and angiotensin II (AT2) were significantly different between CMnoRAASi and CMARB groups. Incubation with rhACE2 decreased AT1 and AT2 in both groups. The geometric mean concentration of Ang1-7 was significantly higher in CMARB (4.9 pg/mL; 95% confidence interval [CI], 3.7-6.4 pg/mL) vs CMnoRAASi (3.2 pg/mL; 95% CI, 2.2-4.7 pg/mL; P = .01) and in CMARB + ACE2 (5.0 pg/mL; 95% CI, 3.9-6.4 pg/mL) vs CMnoRAASi + ACE2 (3.0 pg/mL; 95% CI, 1.7-5.5 pg/mL; P = .01). The most favorable theoretical AP profile that maximized Ang1-7 and other alternative APs was CMARB + ACE2. CONCLUSIONS AND CLINICAL IMPORTANCE: Balance between traditional and alternative APs can be favorably shifted using ARBs and in vitro incubation with rhACE2. These data shed light on new AP-targeting strategies in cats with CM.

Gut Dysbiosis and Its Associations with Gut Microbiota-Derived Metabolites in Dogs with Myxomatous Mitral Valve Disease.

Gut dysbiosis and gut microbiota-derived metabolites, including bile acid (BA), short-chain fatty acid, and trimethylamine N-oxide (TMAO), are associated with cardiovascular disease. Canine myxomatous mitral valve disease (MMVD) is a model for human MMVD. The aim of the study is to evaluate gut microbial dysbiosis and its relationship with gut-produced metabolites in dogs with MMVD. Fecal samples from 92 privately owned dogs, including 17 healthy, 23 and 27 asymptomatic MMVD dogs without (stage B1) and with (stage B2) secondary cardiac enlargement, respectively, and 25 MMVD dogs with history of congestive heart failure (stage C or D), were analyzed by 16S rRNA sequencing. Alpha and beta diversities were different between healthy and MMVD dogs (adjusted P < 0.05). The average dysbiosis indexes were -1.48, -0.6, 0.01, and 1.47 for healthy, B1, B2, and C/D dogs, respectively (P = 0.07). Dysbiosis index was negatively correlated with Clostridium hiranonis (P < 0.0001, r = -0.79). Escherichia coli, capable of trimethylamine production in the gut, had an increased abundance (adjusted P < 0.05) and may be responsible for the increased circulating TMAO levels in stage B2 and C/D MMVD dogs. Primary and secondary BAs showed opposite associations with C. hiranonis, a key BA converter (P < 0.0001 for both, r = -0.94 and 0.95, respectively). Secondary BAs appeared to promote the growth of Fusobacterium and Faecalibacterium but inhibit that of E. coli Multivariate analysis revealed significant but weak associations between gut microbiota and several circulating metabolites, including short-chain acylcarnitines and TMAO.IMPORTANCE Our study expands the current "gut hypothesis" to include gut dysbiosis at the preclinical stage, prior to the onset of heart failure. Gut dysbiosis index increases in proportion to the severity of myxomatous mitral valve disease (MMVD) and is inversely associated with Clostridium hiranonis, a key bile acid (BA) converter in the gut. Secondary BAs appear to promote the growth of beneficial bacteria but inhibit that of harmful ones. An intricate interplay between gut microbiota, gut microbiota-produced metabolites, and MMVD pathophysiological progression is implicated.

Metabolomics Analysis Reveals Deranged Energy Metabolism and Amino Acid Metabolic Reprogramming in Dogs With Myxomatous Mitral Valve Disease.

Background Myxomatous mitral valve disease (MMVD), a naturally occurring heart disease, affects 10% to 15% of the canine population. Canine MMVD shares many similarities with human MMVD. Untargeted metabolomics was performed to identify changes in metabolic pathways and biomarkers with potential clinical utilities. Methods and Results Serum samples from 27 healthy, 22 stage B1, 18 stage B2 preclinical MMVD dogs, and 17 MMVD dogs with a history of congestive heart failure (CHF) were analyzed. Linear regression analysis identified 173 known metabolites whose concentrations were different among the 4 groups (adjusted P<0.05), of which 40% belonged to amino acid super pathways, while 30% were lipids. More than 50% of significant metabolites were correlated with left atrial diameter but not left ventricular dimension. Acylcarnitines, tricarboxylic acid cycle intermediates, and creatine accumulated in proportion to MMVD severity. α-Ketobutyrate and ketone bodies were increased as MMVD advanced. Nicotinamide, a key substrate of the main nicotinamide adenine dinucleotide (NAD+) salvage pathway, was decreased, while quinolinate of the de novo NAD+ biosynthesis was increased in CHF dogs versus healthy dogs. 3-Methylhistidine, marker for myofibrillar protein degradation, was higher in CHF dogs than non-CHF dogs. Trimethylamine N-oxide (TMAO) and TMAO-producing precursors, including carnitine, phosphatidylcholine, betaine, and trimethyllysine, were increased in CHF dogs versus non-CHF dogs. Elevated levels of uremic toxins, including guanidino compounds, TMAO, and urea, were observed in CHF dogs. Pathway analysis highlighted the importance of bioenergetics and amino acid metabolism in canine MMVD. Conclusions Our study revealed altered energy metabolism, amino acid metabolic programming, and reduced renal function in the development of MMVD and CHF. Complex interplays along the heart-kidney-gut axis were implicated.

Classification and Grading of Melanocytic Lesions in a Mouse Model of NRAS-driven Melanomagenesis.

The mouse line carrying the Tg(Tyr-NRAS*Q61K)1Bee transgene is widely used to model in vivo NRAS-driven melanomagenesis. Although the pathological features of this model are well described, classification and interpretation of the resulting proliferative lesions-including their origin, evolution, grading, and pathobiological significance-are still unclear and not supported by molecular and biological evidence. Focusing on their classification and grading, this work combines histopathology and expression analysis (using both immunohistochemistry [IHC] and quantitative PCR) of selected biomarkers to study the full spectrum of cutaneous and lymph nodal melanocytic proliferations in the Tg(Tyr-NRAS*Q61K)1Bee mouse. The analysis of cutaneous and lymph nodal melanocytic proliferations has demonstrated that a linear correlation exists between tumor grade and Ki-67, microphthalmia-associated transcription factor (MITF), gp100, and nestin IHC, with a significantly increased expression in high-grade lesions compared with low-grade lesions. The accuracy of the assessment of MITF IHC in melanomas was also confirmed by quantitative PCR analysis. In conclusion, we believe the incorporation of MITF, Ki-67, gp100, and nestin analysis into the histopathological classification/grading scheme of melanocytic proliferations described for this model will help to assess with accuracy the nature and evolution of the phenotype, monitor disease progression, and predict response to experimental treatment or other preclinical manipulations.

Accuracy of history, physical examination, cardiac biomarkers, and biochemical variables in identifying dogs with stage B2 degenerative mitral valve disease.

BACKGROUND: Treatment is indicated in dogs with preclinical degenerative mitral valve disease (DMVD) and cardiomegaly (stage B2). This is best diagnosed using echocardiography; however, relying upon this limits access to accurate diagnosis. OBJECTIVES: To evaluate whether cardiac biomarker concentrations can be used alongside other clinical data to identify stage B2 dogs. ANIMALS: Client-owned dogs (n = 1887) with preclinical DMVD prospectively sampled in Germany, the United Kingdom, and the United States. METHODS: Dogs that met inclusion criteria and were not receiving pimobendan (n = 1245) were used for model development. Explanatory (multivariable logistic regression) and predictive models were developed using clinical observations, biochemistry, and cardiac biomarker concentrations, with echocardiographically confirmed stage B2 disease as the outcome. Receiver operating characteristic curves assessed the ability to identify stage B2 dogs. RESULTS: Age, appetite, serum alanine aminotransferase activity, body condition, serum creatinine concentration, murmur intensity, and plasma N-terminal propeptide of B-type natriuretic peptide (NT-proBNP) concentration were independently associated with the likelihood of being stage B2. The discriminatory ability of this explanatory model (area under curve [AUC], 0.84; 95% confidence interval [CI], 0.82-0.87) was superior to NT-proBNP (AUC, 0.77; 95% CI, 0.74-0.80) or the vertebral heart score alone (AUC, 0.76; 95% CI, 0.69-0.83). A predictive logistic regression model could identify the probability of being stage B2 (AUC test set, 0.86; 95% CI, 0.81-0.91). CONCLUSION AND CLINICAL IMPORTANCE: Our findings indicate accessible measurements could be used to screen dogs with preclinical DMVD. Encouraging at-risk dogs to seek further evaluation could result in a greater proportion of cases being appropriately managed.