Acquired dysfunction of CFTR underlies cystic fibrosis-like disease of the canine gallbladder.

Mucocele formation in dogs is a unique and enigmatic muco-obstructive disease of the gallbladder caused by amassment of abnormal mucus that bears striking pathological similarity to cystic fibrosis. We investigated the role of CFTR in the pathogenesis of this disease. The location and frequency of disease-associated variants in the coding region of CFTR was compared using whole genome sequence data from 2,642 dogs representing breeds at low-risk, high-risk, or with confirmed disease. Expression, localization, and ion transport activity of CFTR was quantified in control and mucocele gallbladders by NanoString, Western blotting, immunofluorescence imaging, and studies in Ussing chambers. Our results establish significant loss of CFTR-dependent anion secretion by mucocele gallbladder mucosa. A significantly lower quantity of CFTR protein was demonstrated relative to E-cadherin in mucocele compared to control gallbladder mucosa. Immunofluorescence identified CFTR along the apical membrane of epithelial cells in control gallbladders but not in mucocele gallbladder epithelium. Decreases in mRNA copy number for CFTR was accompanied by decreases in mRNA for the Cl-/HCO3- exchanger SLC26A3, K+ channels (KCNQ1, KCNN4), and vasoactive intestinal polypeptide receptor (VIPR1) which suggest a driving force for change in secretory function of gallbladder epithelial cells in the pathogenesis of mucocele formation. There were no significant differences in CFTR gene variant frequency, type, or predicted impact comparing low risk, high risk, and definitively diagnosed groups of dogs. This study describes a unique, naturally occurring muco-obstructive disease of the canine gallbladder, with uncanny similarity to cystic fibrosis, and driven by underlying failure of CFTR function.

Identification of a novel missense mutation in the fibroblast growth factor 5 gene associated with longhair in the Maine Coon Cat.

Hair length can be a highly variable trait within the Felis catus species, varying between and within different cat breeds. Previous research has demonstrated this variability is due to recessive mutations within the fibroblast growth factor 5 (FGF5) gene. Following a genetic screen, four longhaired Maine Coons were identified that had only one copy of a known FGF5 mutation. We performed DNA sequencing on samples from two of these Maine Coons and identified a missense mutation in FGF5 c.577G > A p.Ala193Thr. Genetic screening via restriction digest was then performed on samples from the other two Maine Coons and an additional 273 cats of various breeds. This screening found that only the two additional Maine Coons were heterozygous for the novel variant. Furthermore, the novel variant was not identified after in silico analysis of 68 whole genome cat sequences from various breeds, demonstrating that this novel mutation is most likely a breed-specific variant for the Maine Coon, contributing to the longhair phenotype in about 3% of these cats.

Use of whole genome analysis to identify shared genomic variants across breeds in canine mitral valve disease.

Familial mitral valve prolapse in human beings has been associated with several genetic variants; however, in most cases, a known variant has not been identified. Dogs also have a naturally occurring form of familial mitral valve disease (MMVD) with similarities to the human disease. A shared genetic background and clinical phenotype of this disease in some dog breeds has indicated that the disease may share a common genetic cause. We evaluated DNA from 50 affected dogs from five different dog breeds in a whole genome sequencing approach to identify shared variants across and within breeds that could be associated with MMVD. No single causative genetic mutation was found from the 50 dogs with MMVD. Ten variants were identified in 37/50 dogs around and within the MED13L gene. These variants were no longer associated with MMVD when evaluated with a larger cohort including both affected and unaffected dogs. No high/moderate impact variants were identified in 10/10 miniature poodles, one was identified in 10/10 Yorkshire Terriers and 10/10 dachshunds, respectively, 14 were identified in 10/10 Miniature schnauzers, and 19 in 10/10 CKCS. Only one of these could be associated with the cardiac valve (Chr12:36801705, COL12A1; CKCS) but when evaluated in an additional 100 affected CKCS the variant was only identified in 84/100 affected dogs, perhaps indicating genetic heterogeneity in this disease. Our findings indicate that development of MMVD in the dog may be related to a combination of genetic and environmental factors that impact specific molecular pathways rather than a single shared genetic variant across or within breeds.

A defect in the NOG gene increases susceptibility to spontaneous superficial chronic corneal epithelial defects (SCCED) in boxer dogs.

BACKGROUND: Superficial chronic corneal epithelial defects (SCCEDs) are spontaneous corneal defects in dogs that share many clinical and pathologic characteristics to recurrent corneal erosions (RCE) in humans. Boxer dogs are predisposed to SCCEDs, therefore a search for a genetic defect was performed to explain this susceptibility. DNA was extracted from blood collected from Boxer dogs with and without SCCEDs followed by whole genome sequencing (WGS). RNA sequencing of corneal tissue and immunostaining of corneal sections from affected SCCED Boxer dogs with a deletion in the NOG gene and affected non-Boxer dogs without the deletion were performed. RESULTS: A 30 base pair deletion at a splice site in Noggin (NOG) (Chr 9:31453999) was identified by WGS and was significantly associated (P < 0.0001) with Boxer SCCEDs compared to unaffected non-Boxer dogs. NOG, BMP4, MMP13, and NCAM1 all had significant fold reductions in expression and SHH was significantly increased in Boxers with the NOG deletion as identified by RNA-Seq. Corneal IHC from NOG deletion dogs with SCCEDs had lower NOG and significantly higher scores of BMP2. CONCLUSIONS: Many Boxer dogs with SCCED have a genetic defect in NOG. NOG is a constitutive protein in the cornea which is a potent inhibitor of BMP, which likely regulate limbal epithelial progenitor cells (LEPC). Dysregulation of LEPC may play a role in the pathogenesis of RCE.

Canine junctional epidermolysis bullosa due to a novel mutation in LAMA3 with severe upper respiratory involvement.

BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone. OBJECTIVES: To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant. ANIMALS: Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing. METHODS AND MATERIALS: Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant. RESULTS: Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL RELEVANCE: A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.

A missense variant in the titin gene in Doberman pinscher dogs with familial dilated cardiomyopathy and sudden cardiac death.

The dog provides a large animal model of familial dilated cardiomyopathy for the study of important aspects of this common familial cardiovascular disease. We have previously demonstrated a form of canine dilated cardiomyopathy in the Doberman pinscher breed that is inherited as an autosomal dominant trait and is associated with a splice site variant in the pyruvate dehydrogenase kinase 4 (PDK4) gene, however, genetic heterogeneity exists in this species as well and not all affected dogs have the PDK4 variant. Whole genome sequencing of a family of Doberman pinchers with dilated cardiomyopathy and sudden cardiac death without the PDK4 variant was performed. A pathologic missense variant in the titin gene located in an immunoglobulin-like domain in the I-band spanning region of the molecule was identified and was highly associated with the disease (p < 0.0001). We demonstrate here the identification of a variant in the titin gene highly associated with the disease in this spontaneous canine model of dilated cardiomyopathy. This large animal model of familial dilated cardiomyopathy shares many similarities with the human disease including mode of inheritance, clinical presentation, genetic heterogeneity and a pathologic variant in the titin gene. The dog is an excellent model to improve our understanding of the genotypic phenotypic relationships, penetrance, expression and the pathophysiology of variants in the titin gene.

Angiotensin-converting enzyme activity in Cavalier King Charles Spaniels with an ACE gene polymorphism and myxomatous mitral valve disease.

OBJECTIVES: Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is particularly common in the Cavalier King Charles Spaniel (CKCS) breed and affected dogs are frequently managed with angiotensin-converting enzyme inhibitors (ACE-I). We have previously identified a canine ACE gene polymorphism associated with a decrease in angiotensin-converting enzyme (ACE) activity. The aim of this study was to evaluate for the prevalence of the ACE polymorphism in CKCS with mitral valve disease and to determine whether the presence of the polymorphism is associated with alterations in ACE activity at different stages of cardiac disease. METHODS: Seventy-three dogs with a diagnosis of mitral valve disease were evaluated and a blood sample was drawn for ACE polymorphism genotyping and ACE activity measurement. RESULTS: Forty-three dogs were homozygous for the ACE polymorphism; five were heterozygous and 25 were homozygous wild type. The mean age and the median severity of disease were not different for dogs with the polymorphism and dogs with the wild-type sequence. The median baseline ACE activity was significantly lower for the ACE polymorphism (27.0 U/l) than the wild-type sequence dogs (31.0 U/l) (P=0.02). Dogs with more severe disease and the ACE polymorphism had significantly lower levels of ACE activity than dogs with the wild-type sequence (P=0.03). CONCLUSION: The CKCS appears to have a high prevalence of the ACE variant. Dogs with the ACE variant had lower levels of ACE activity even in more advanced mitral valve disease than dogs without the variant. The clinical significance of this finding and its impact on the need for ACE-I in dogs with the polymorphism and heart disease deserves further study.

Intracoronary allogeneic cardiosphere-derived stem cells are safe for use in dogs with dilated cardiomyopathy.

Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells.

Evaluation of the genetic basis of primary hypoadrenocorticism in Standard Poodles using SNP array genotyping and whole-genome sequencing.

Primary hypoadrenocorticism, also known as Addison's disease, is an autoimmune disorder leading to the destruction of the adrenal cortex and subsequent loss of glucocorticoid and mineralocorticoid hormones. The disease is prevalent in Standard Poodles and is believed to be highly heritable in the breed. Using genotypes derived from the Illumina Canine HD SNP array, we performed a genome-wide association study of 133 carefully phenotyped Standard Poodles (61 affected, 72 unaffected) and found no markers significantly associated with the disease. We also sequenced the entire genomes of 20 Standard Poodles (13 affected, 7 unaffected) and analyzed the data to identify common variants (including SNPs, indels, structural variants, and copy number variants) across affected dogs and variants segregating within a single pedigree of highly affected dogs. We identified several candidate genes that may be fixed in both Standard Poodles and a small population of dogs of related breeds. Further studies are required to confirm these findings more broadly, as well as additional gene-mapping efforts aimed at fully understanding the genetic basis of what is likely a complex inherited disorder.

Evaluation of a DLA-79 allele associated with multiple immune-mediated diseases in dogs.

Immune-mediated diseases are common and life-threatening disorders in dogs. Many canine immune-mediated diseases have strong breed predispositions and are believed to be inherited. However, the genetic mutations that cause these diseases are mostly unknown. As many immune-mediated diseases in humans share polymorphisms among a common set of genes, we conducted a candidate gene study of 15 of these genes across four immune-mediated diseases (immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, immune-mediated polyarthritis (IMPA), and atopic dermatitis) in 195 affected and 206 unaffected dogs to assess whether causative or predictive polymorphisms might exist in similar genes in dogs. We demonstrate a strong association (Fisher's exact p = 0.0004 for allelic association, p = 0.0035 for genotypic association) between two polymorphic positions (10 bp apart) in exon 2 of one allele in DLA-79, DLA-79*001:02, and multiple immune-mediated diseases. The frequency of this allele was significantly higher in dogs with immune-mediated disease than in control dogs (0.21 vs. 0.12) and ranged from 0.28 in dogs with IMPA to 0.15 in dogs with atopic dermatitis. This allele has two non-synonymous substitutions (compared with the reference allele, DLA-79*001:01), resulting in F33L and N37D amino acid changes. These mutations occur in the peptide-binding pocket of the protein, and based upon our computational modeling studies, are likely to affect critical interactions with the peptide N-terminus. Further studies are warranted to confirm these findings more broadly and to determine the specific mechanism by which the identified variants alter canine immune system function.

Evaluation of artificial selection in Standard Poodles using whole-genome sequencing.

Identifying regions of artificial selection within dog breeds may provide insights into genetic variation that underlies breed-specific traits or diseases-particularly if these traits or disease predispositions are fixed within a breed. In this study, we searched for runs of homozygosity (ROH) and calculated the d i statistic (which is based upon F ST) to identify regions of artificial selection in Standard Poodles using high-coverage, whole-genome sequencing data of 15 Standard Poodles and 49 dogs across seven other breeds. We identified consensus ROH regions ≥1 Mb in length and common to at least ten Standard Poodles covering 0.6 % of the genome, and d i regions that most distinguish Standard Poodles from other breeds covering 3.7 % of the genome. Within these regions, we identified enriched gene pathways related to olfaction, digestion, and taste, as well as pathways related to adrenal hormone biosynthesis, T cell function, and protein ubiquitination that could contribute to the pathogenesis of some Poodle-prevalent autoimmune diseases. We also validated variants related to hair coat and skull morphology that have previously been identified as being under selective pressure in Poodles, and flagged additional polymorphisms in genes such as ITGA2B, CBX4, and TNXB that may represent strong candidates for other common Poodle disorders.

Cardiac regenerative potential of cardiosphere-derived cells from adult dog hearts.

The regenerative potential of cardiosphere-derived cells (CDCs) for ischaemic heart disease has been demonstrated in mice, rats, pigs and a recently completed clinical trial. The regenerative potential of CDCs from dog hearts has yet to be tested. Here, we show that canine CDCs can be produced from adult dog hearts. These cells display similar phenotypes in comparison to previously studied CDCs derived from rodents and human beings. Canine CDCs can differentiate into cardiomyocytes, smooth muscle cells and endothelial cells in vitro. In addition, conditioned media from canine CDCs promote angiogenesis but inhibit cardiomyocyte death. In a doxorubicin-induced mouse model of dilated cardiomyopathy (DCM), intravenous infusion of canine CDCs improves cardiac function and decreases cardiac fibrosis. Histology revealed that injected canine CDCs engraft in the mouse heart and increase capillary density. Out study demonstrates the regenerative potential of canine CDCs in a mouse model of DCM.

Impact of the canine double-deletion ß1 adrenoreceptor polymorphisms on protein structure and heart rate response to atenolol, a ß1-selective ß-blocker.

OBJECTIVE: ß-Adrenergic receptor antagonists are widely utilized for the management of cardiac diseases in dogs. We have recently identified two deletion polymorphisms in the canine adrenoreceptor 1 (ADRB1) gene.We hypothesized that canine ADRB1 deletions would alter the structure of the protein, as well as the heart rate response to the ß-adrenergic receptor antagonist, atenolol. The objectives of this study were to predict the impact of these deletions on the predicted structure of the protein and on the heart rate response to atenolol in a population of healthy adult dogs. METHODS: Eighteen apparently healthy, mature dogs with (11) and without (seven) ADRB1 deletions were evaluated. The heart rate of the dogs was evaluated with a baseline ambulatory ECG before and 14-21 days after atenolol therapy (1 mg/kg orally q12 h). Minimum, average, and maximum heart rates were compared between groups of dogs (deletions, controls) using an unpaired t-test and within each group of dogs using a paired t-test. The protein structure of ADRB1 was predicted by computer modeling. RESULTS: Deletions were predicted to alter the structure of the ADRB1 protein. The heart rates of the dogs with deletions were lower than those of the control dogs (the average heart rates were significantly lower). CONCLUSION: ADRB1 deletions appear to have structural and functional consequences. Individual genome-based treatment recommendations could impact the management of dogs with heart disease.

A single codon insertion in PICALM is associated with development of familial subvalvular aortic stenosis in Newfoundland dogs.

Familial subvalvular aortic stenosis (SAS) is one of the most common congenital heart defects in dogs and is an inherited defect of Newfoundlands, golden retrievers and human children. Although SAS is known to be inherited, specific genes involved in Newfoundlands with SAS have not been defined. We hypothesized that SAS in Newfoundlands is inherited in an autosomal dominant pattern and caused by a single genetic variant. We studied 93 prospectively recruited Newfoundland dogs, and 180 control dogs of 30 breeds. By providing cardiac screening evaluations for Newfoundlands we conducted a pedigree evaluation, genome-wide association study and RNA sequence analysis to identify a proposed pattern of inheritance and genetic loci associated with the development of SAS. We identified a three-nucleotide exonic insertion in phosphatidylinositol-binding clathrin assembly protein (PICALM) that is associated with the development of SAS in Newfoundlands. Pedigree evaluation best supported an autosomal dominant pattern of inheritance and provided evidence that equivocally affected individuals may pass on SAS in their progeny. Immunohistochemistry demonstrated the presence of PICALM in the canine myocardium and area of the subvalvular ridge. Additionally, small molecule inhibition of clathrin-mediated endocytosis resulted in developmental abnormalities within the outflow tract (OFT) of Xenopus laevis embryos. The ability to test for presence of this PICALM insertion may impact dog-breeding decisions and facilitate reduction of SAS disease prevalence in Newfoundland dogs. Understanding the role of PICALM in OFT development may aid in future molecular and genetic investigations into other congenital heart defects of various species.

An imminent need for veterinary medical educators: are we facing a crisis?

A potential emerging shortage of veterinary medical educators requires the profession to acknowledge and understand the factors leading to this outcome. Expanding class sizes within existing schools and colleges of veterinary medicine and the expected expansion of new programs seeking AVMA-Council of Education accreditation have heightened the need to address an impending shortage of veterinary medical educators. A solution-oriented approach that accurately projects educator workforce needs and identifies factors contributing to the shortage requires effective collaboration across various partnering organizations to develop innovations in pedagogy and educational delivery methods. The veterinary profession must also identify and reduce disincentives that deter students and post-DVM trainees from pursuing careers in education. Finally, efforts at the state and federal level are critical to advocate for financial support and incentives for expansion of the veterinary medical educator workforce. Through these collective approaches and partnerships, the veterinary medical educator workforce can be strengthened to overcome obstacles for educating the next generation of veterinarians to meet societal needs.

Extent of linkage disequilibrium in large-breed dogs: chromosomal and breed variation.

The aim of this study was to better define the extent of linkage disequilibrium (LD) in populations of large-breed dogs and its variation by breed and chromosomal region. Understanding the extent of LD is a crucial component for successful utilization of genome-wide association studies and allows researchers to better define regions of interest and target candidate genes. Twenty-four Golden Retriever dogs, 28 Rottweiler dogs, and 24 Newfoundland dogs were genotyped for single-nucleotide polymorphism (SNP) data using a high-density SNP array. LD was calculated for all autosomes using Haploview. Decay of the squared correlation coefficient (r (2)) was plotted on a per-breed and per-chromosome basis as well as in a genome-wide fashion. The point of 50 % decay of r (2) was used to estimate the difference in extent of LD between breeds. Extent of LD was significantly shorter for Newfoundland dogs based upon 50 % decay of r (2) data at a mean of 344 kb compared to Golden Retriever and Rottweiler dogs at 715 and 834 kb, respectively (P < 0.0001). Notable differences in LD by chromosome were present within each breed and not strictly related to the length of the corresponding chromosome. Extent of LD is breed and chromosome dependent. To our knowledge, this is the first report of SNP-based LD for Newfoundland dogs, the first report based on genome-wide SNPs for Rottweilers, and an almost tenfold improvement in marker density over previous genome-wide studies of LD in Golden Retrievers.

The effect of taurine supplementation on the renin-angiotensin-aldosterone system of dogs with congestive heart failure.

The role of taurine in the treatment of congestive heart failure (CHF) in dogs without systemic deficiency is unexplored. Taurine might have beneficial cardiac effects aside from deficit replacement. We hypothesized that oral taurine supplementation administered to dogs with naturally-occurring CHF would suppress the renin-angiotensin aldosterone system (RAAS). Oral taurine was administered to 14 dogs with stable CHF. Serum biochemical variables, blood taurine concentrations, and comprehensive analysis of RAAS variables were compared before and 2 weeks after taurine supplementation added to background furosemide and pimobendan therapy for CHF. Whole blood taurine concentrations increased after supplementation (median 408 nMol/mL, range 248-608 before and median 493 nMol/mL, range 396-690 after; P = .006). Aldosterone to angiotensin II ratio (AA2) was significantly decreased after taurine supplementation (median 1.00, range 0.03-7.05 before and median 0.65, range 0.01-3.63 after; P = .009), but no other RAAS components significantly differed between timepoints. A subset of dogs showed marked decreases in RAAS metabolites after supplementation and these dogs were more likely to have been recently hospitalized for CHF treatment than dogs that did not show marked decreases in classical RAAS metabolites. Overall, taurine only lowered AA2 in this group of dogs, however, response heterogeneity was noted, with some dogs showing RAAS suppression.

Investigation of the cardiac effects of exercise testing on apparently healthy Boxer dogs.

BACKGROUND: Holter electrocardiographic monitoring is a cornerstone of diagnostic testing for arrhythmogenic cardiomyopathy (ACM) in Boxer dogs, but physical activity during monitoring is not controlled. In humans, exercise testing (ExT) can identify latent tachyarrhythmias associated with cardiomyopathy, and exercise increases serum cardiac troponin-I concentrations ([hs-cTnI]). These effects have not yet been investigated in Boxer dogs. HYPOTHESIS/OBJECTIVES: Subjecting Boxer dogs to brief, moderate-intensity ExT can identify changes in Holter recordings and [hs-cTnI] compared to baseline results. ANIMALS: Thirty overtly healthy, client-owned Boxer dogs. METHODS: Prospective interventional study. Dogs underwent baseline diagnostic testing including 24-hour Holter monitoring and [hs-cTnI], followed by brief ExT (accompanied, brisk stair-climbing and -descending for <5 minutes). RESULTS: Eleven dogs (37%) had >100 premature ventricular complexes (PVCs)/24 hours at baseline (3), ExT (3), or both (5). After ExT, these dogs had more PVCs/24 hours and greater increases in [hs-cTnI] compared to those with ≤100 PVCs/24 hours. Dogs with the striatin mutation had more PVCs/24 hours and a greater increase in [hs-cTnI] after ExT than did dogs without the striatin mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: Exercise testing may improve the binary classification of Boxer dogs with or without ACM by increasing the number of PVCs and [hs-cTnI] in affected dogs to a greater degree than in unaffected dogs. This effect also is associated with presence or absence of the striatin mutation. Exercise should be a controlled variable when screening Boxer dogs for ACM.

Pharmacokinetics of pimobendan after oral administration to dogs with myxomatous mitral valve disease.

BACKGROUND: Pimobendan is an important therapy for dogs with myxomatous mitral valve disease (MMVD). The pharmacokinetics are reported in healthy dogs but not in dogs with heart disease. HYPOTHESIS/OBJECTIVES: To determine if dog characteristics such as age, breed, body condition score, ACVIM stage of heart disease or biochemical laboratory value alter the pharmacokinetics of orally administered pimobendan and its metabolite in a cohort of dogs with naturally occurring MMVD. ANIMALS: Fifty-seven client-owned dogs with MMVD ACVIM Stage B2, C, or D and administered pimobendan to steady state blood concentrations. METHODS: Prospective, observational study. Samples were collected using a sparse-sampling protocol at specific intervals after administration of pimobendan. Plasma pimobendan and the active metabolite (O-desmethyl-pimobendan, ODMP) concentrations were determined via high-pressure liquid chromatography and fluorescence detection. Data was analyzed via a population pharmacokinetic approach and nonlinear mixed effects modeling (NLME). Numerous covariates were examined in the NLME model. RESULTS: The absorption and elimination half-lives (t1/2 ) were approximately 1.4 and 1 hour for pimobendan and 1.4 and 1.3 hours for ODMP, respectively. Pharmacokinetic parameters were highly variable, especially the values for pimobendan absorption and elimination rate, and absorption rate of ODMP with coefficients of variation of 147.84%, 64.51% and 64.49%, respectively. No covariate evaluated was a significant source of variability. CONCLUSIONS AND CLINICAL IMPORTANCE: The pharmacokinetic parameters were highly variable among this group of dogs with MMVD. The variability was not associated with the dog's age, body weight or condition score, stage of heart disease, dose, serum creatinine, or alkaline phosphatase.

Evaluation of Renin-Angiotensin-Aldosterone System Components and Enzymes in Systemically Hypertensive Cats Receiving Amlodipine.

BACKGROUND: Chronic renin-angiotensin-aldosterone system (RAAS) activation is harmful. Amlodipine activates RAAS in humans and dogs, but contradictory data exist for systemically hypertensive (SHT) cats. HYPOTHESIS: Cats with SHT and chronic kidney disease treated with amlodipine (SHT/CKD-A) are RAAS activated. ANIMALS: Client-owned cats: unmedicated normotensive (NT) cats (n = 9); SHT/CKD-A cats (n = 5) with median systolic blood pressure of 170 mmHg (vs. 195 mmHg, pre-treatment), chronic kidney disease, and receiving no RAAS-suppressive therapy. METHODS: Serum was frozen (-80 °C) until RAAS analysis via equilibrium analysis. The RAAS variables (reported as median (minimum-maximum)) were compared between groups, using Mann-Whitney U test. RESULTS: Angiotensin 1, angiotensin 1,7, angiotensin III, and angiotensin 1,5, and angiotensin-converting enzyme (ACE)-2 activity were higher in SHT/CKD-A cats compared to NT cats, while ACE activity was lower in SHT/CKD-A cats compared to NT cats (p < 0.05 all). A marker for alternative RAAS influence (ALT-S) was significantly higher (69; 58-73 pmol/pmol) in SHT/CKD-A cats compared to NT cats (35; 14-63 pmol/pmol; p = 0.001). Aldosterone concentrations were significantly higher (393; 137-564 pmol/L) in SHT/CKD-A cats compared to NT cats (129; 28-206 pmol/L; p = 0.007). CONCLUSION AND CLINICAL IMPORTANCE: Circulating RAAS is activated in systemically hypertensive cats receiving amlodipine. Although this study did not parse out the individual contributions of SHT, chronic kidney disease, and amlodipine, the findings suggest that the use of concurrent RAAS-suppressant therapy, specifically aldosterone antagonism, in amlodipine-treated SHT cats with chronic kidney disease might be indicated.