Saline is as effective as nitrogen scavengers for treatment of hyperammonemia.

Urea cycle enzyme deficiency (UCED) patients with hyperammonemia are treated with sodium benzoate (SB) and sodium phenylacetate (SPA) to induce alternative pathways of nitrogen excretion. The suggested guidelines supporting their use in the management of hyperammonemia are primarily based on non-analytic studies such as case reports and case series. Canine congenital portosystemic shunting (CPSS) is a naturally occurring model for hyperammonemia. Here, we performed cross-over, randomized, placebo-controlled studies in healthy dogs to assess safety and pharmacokinetics of SB and SPA (phase I). As follow-up safety and efficacy of SB was evaluated in CPSS-dogs with hyperammonemia (phase II). Pharmacokinetics of SB and SPA were comparable to those reported in humans. Treatment with SB and SPA was safe and both nitrogen scavengers were converted into their respective metabolites hippuric acid and phenylacetylglutamine or phenylacetylglycine, with a preference for phenylacetylglycine. In CPSS-dogs, treatment with SB resulted in the same effect on plasma ammonia as the control treatment (i.e. saline infusion) suggesting that the decrease is a result of volume expansion and/or forced diuresis rather than increased production of nitrogenous waste. Consequentially, treatment of hyperammonemia justifies additional/placebo-controlled trials in human medicine.

Sensitivity and Specificity of Plasma ALT, ALP, and Bile Acids for Hepatitis in Labrador Retrievers.

BACKGROUND: Biochemical indicators for diagnosing liver disease are plasma alanine aminotransferase activity (ALT), alkaline phosphatase activity (ALP), and bile acid concentration (BA). OBJECTIVES: To determine the sensitivity and specificity of ALT, ALP, and BA for detecting primary hepatitis (PH) in clinically healthy Labrador retrievers and investigate whether ALT and ALP can discriminate between dogs with PH and nonspecific reactive hepatitis (RH). ANIMALS: 191 clinically healthy and 51 clinically ill Labrador retrievers with hepatic histopathology. METHODS: Retrospective study. Medical records were reviewed for ALT, ALP, preprandial BA, liver histopathology, and hepatic copper concentrations. RESULTS: In 64% (122/191) of the clinically healthy Labrador retrievers, hepatic histology revealed inflammatory infiltrates. This frequency might be biased because part of them was included as first-line relatives of dogs with copper-associated hepatitis. Sensitivity of ALT, ALP, and BA in this population for detecting acute hepatitis was 45, 15, and 15%, respectively. For chronic hepatitis, sensitivity was 71, 35, and 13%, respectively. Specificity of ALT, ALP, and BA was >90% for AH, CH, and RH. When increased liver enzymes were present, median ALT was significantly higher in PH cases (312 U/L, range 38-1,369) compared to RH cases (91 U/L, range 39-139) (P < .001). There was no difference in ALP between dogs with a PH and a RH (P = .361). CONCLUSIONS AND CLINICAL IMPORTANCE: Histopathologic abnormalities in the liver were present in the majority of apparent clinically healthy Labrador retrievers. The sensitivity of ALT, ALP, and BA for detecting acute and chronic hepatitis in this population was low. More sensitive biomarkers are needed for early detection of liver disease in apparent clinically healthy dogs.

Erythrocyte copper chaperone for superoxide dismutase and superoxide dismutase as biomarkers for hepatic copper concentrations in Labrador retrievers.

Hereditary hepatic copper accumulation in Labrador retrievers leads to hepatitis with fibrosis and eventually cirrhosis. The development of a non-invasive blood-based biomarker for copper status in dogs could be helpful in identifying dogs at risk and to monitor copper concentrations during treatment. In this study, two cellular copper metabolism proteins, Cu/Zn superoxide dismutase (SOD1) and its chaperone (copper chaperone for SOD1, CCS) were measured in erythrocytes and tested for association with hepatic copper concentrations in 15 Labrador retrievers with normal or increased hepatic copper concentrations. Antibodies against CCS and SOD1 were applicable for use in canine specimens. This was demonstrated by the loss of immune-reactive bands for CCS and SOD1 in siRNA treated canine bile duct epithelial cells. Erythrocyte CCS and CCS/SOD1 ratios were decreased 2.37 (P <0.001) and 3.29 (P <0.001) fold in the high copper group compared to the normal copper group. Erythrocyte CCS and CCS/SOD1 ratio are potential new biomarkers for hepatic copper concentrations in Labrador retrievers and could facilitate early diagnosis and treatment monitoring for copper-associated hepatitis in dogs.

Use of Serum MicroRNAs as Biomarker for Hepatobiliary Diseases in Dogs.

BACKGROUND: Current biochemical indicators cannot discriminate between parenchymal, biliary, vascular, and neoplastic hepatobiliary diseases. MicroRNAs are promising new biomarkers for hepatobiliary disease in humans and dogs. OBJECTIVE: To measure serum concentrations of an established group of microRNAs in dogs and to investigate their concentrations in various types of hepatobiliary diseases. ANIMALS: Forty-six client-owned dogs with an established diagnosis of hepatobiliary disease and stored serum samples and eleven client-owned healthy control Labrador Retrievers. METHODS: Retrospective study. Medical records of dogs with parenchymal, biliary, vascular, or neoplastic hepatobiliary diseases and control dogs were reviewed. Concentrations of miR-21, miR-122, miR-126, miR-148a, miR-200c, and miR-222 were quantified in serum by real-time polymerase chain reaction. RESULTS: No different microRNA concentrations were found in the adenoma and congenital portosystemic shunt groups. In all other diseases, miR-122 concentrations were elevated with the highest concentration in the mucocele group (267-fold, CI: 40-1,768, P < .001). In dogs with biliary diseases, miR-21 and miR-222 were only increased in dogs with mucoceles (26-fold, CI: 5-141, P = .005 and 13-fold, CI: 2-70, P = .025, respectively). Uniquely increased microRNAs were found in the hepatocellular carcinoma group (miR-200c, 35-fold increase, CI: 3-382, P = .035) and the chronic hepatitis group (miR-126, 22-fold increase, CI: 5-91, P = .002). CONCLUSIONS AND CLINICAL IMPORTANCE: A microRNA panel consisting of miR-21, miR-122, miR-126, miR-200c, and miR-222 can distinguish between parenchymal, biliary, and neoplastic hepatobiliary diseases. Serum microRNA profiling is a promising new tool that might be a valuable addition to conventional diagnostics to help diagnose various hepatobiliary diseases in dogs.

Hepatocyte-derived microRNAs as sensitive serum biomarkers of hepatocellular injury in Labrador retrievers.

Common parenchymal liver diseases in dogs include reactive hepatopathies and primary hepatitis (acute or chronic). In chronic hepatitis, there is usually a long subclinical phase. Specific clinical signs become overt only when liver damage is severe and in this phase, treatment is usually less effective. Limited data are available regarding the sensitivity of liver enzyme activity or biomarkers for early detection of subclinical hepatitis. Hepatocyte-derived microRNAs (HDmiRs) were recently identified as promising biomarkers for hepatocellular injury in multiple species. Here, the potential of the HDmiRs miR-122 and miR-148a as sensitive diagnostic biomarkers for hepatocellular injury in Labrador retrievers was investigated. Samples from 66 Labrador retrievers with histologically normal livers, high hepatic copper, and with various forms of liver injury were evaluated for serum alanine aminotransferase (ALT) activity and microRNA values. Median values of HDmiR-122 were 34.6 times higher in dogs with liver injury and high ALT than in normal dogs (95% confidence intervals [CI], 13-95; P <0.001). HDmiR-122 values were significantly increased in dogs with liver injury and normal ALT (4.2 times; 95% CI, 2-12; P <0.01) and in dogs with high hepatic copper concentrations and unremarkable histopathology (2.9 times; 95% CI, 1.1-8.0; P <0.05). Logistic regression analyses demonstrated that miR-122 and miR-148a were both predictors of hepatocellular injury. The sensitivity of miR-122 was 84% (95% CI, 73-93%), making it superior to ALT (55%; 95% CI, 41-68%) for the detection of hepatocellular injury in Labrador retrievers (P <0.001). This study demonstrated that serum HDmiR, particularly miR-122, is a highly sensitive marker for the detection of hepatocellular injury in Labrador retrievers and is a promising new biomarker that may be used for early detection of subclinical hepatitis in dogs.

Diagnostic value of the rectal ammonia tolerance test, fasting plasma ammonia and fasting plasma bile acids for canine portosystemic shunting.

Portosystemic shunting (PSS) often results in hyperammonaemia and, consequently, hepatic encephalopathy. This retrospective study evaluated the sensitivity, specificity, positive and negative predictive values (PPV and NPV, respectively) and other test performance metrics for the ammonia tolerance test (ATT), serum fasting bile acids (FBA), serum fasting ammonia concentration (FA), and combinations of these tests for their association with PSS in dogs. Medical records of 271 dogs suspect for PSS (symptomatic group) and 53 dogs returning for evaluation after surgical closure of a congenital PSS (CPSS post-surgical control group) were analysed. In the symptomatic group, ATT at 40 min (T40), and the FBA had the highest sensitivity (100% and 98%, respectively) and NPV (100% and 96%, respectively) for PSS. The combination of increased FBA and FA had the highest specificity (97%), with a PPV of 97%, and a positive likelihood ratio of 29. In the CPSS post-surgical control group, the specificity and PPV of FA and the combination of increased FBA/FA were both 100%. In purebred populations, the NPV of all tests was 100%. Consequently, PSS would be ruled out in a symptomatic dog with normal FBA or ATT (T40) and would be highly probable when both FBA and FA are increased. Increased FA was conclusive for PSS in dogs evaluated for post-surgical closure of a CPSS. FBA was the most suitable test for screening purposes.

Canine hepacivirus and idiopathic hepatitis in dogs from a Dutch cohort.

Liver diseases are highly prevalent in the general dog population, though the etiology is often unknown. Recently a homolog of human hepatitis C virus was discovered in dogs with respiratory infections. Although this canine hepacivirus (CHV) was detectable in some liver samples, a clear link with liver disease has not been established. A recent study by Bexfield et al. showed that in a large cohort of dogs from the UK with idiopathic hepatitis, no evidence can be found for exposure to, or carrier state of CHV both in liver and in serum. The authors however state that 'the absence of CHV infection on dogs from the UK might not represent the global ecology of the virus'. We investigated CHV-infection in 267 liver biopsies from 120 dogs idiopathic hepatitis and 135 control animals, in a population from the Netherlands. Using a highly sensitive PCR assay for CHV-NS3, no CHV was detected in all 267 liver samples. Our data show that the lack of association between canine hepacivirus and chronic liver disease in dogs is not limited to the UK, but is also found in an independent cohort from the European continent.

The canine hepatic progenitor cell niche: molecular characterisation in health and disease.

Hepatic progenitor cells (HPCs) are an adult stem cell compartment in the liver that contributes to liver regeneration when replication of mature hepatocytes is insufficient. In this study, laser microdissection was used to isolate HPC niches from the livers of healthy dogs and dogs with lobular dissecting hepatitis (LDH), in which HPCs are massively activated. Gene expression of HPC, hepatocyte and biliary markers was determined by quantitative reverse transcriptase PCR. Expression and localisation of selected markers were further studied at the protein level by immunohistochemistry and immunofluorescent double staining in samples of normal liver and liver from dogs with LDH, acute and chronic hepatitis, and extrahepatic cholestasis. Activated HPC niches had higher gene expression of the hepatic progenitor markers OPN, FN14, CD29, CD44, CD133, LIF, LIFR and BMI1 compared to HPCs from normal liver. There was lower expression of albumin, but activated HPC niches were positive for the biliary markers SOX9, HNF1ß and keratin 19 by immunohistochemistry and immunofluorescence. Laminin, activated stellate cells and macrophages are abundant extracellular matrix and cellular components of the canine HPC niche. This study demonstrates that the molecular and cellular characteristics of canine HPCs are similar to rodent and human HPCs, and that canine HPCs are distinctively activated in different types of liver disease.

American Cocker Spaniel chronic hepatitis in Japan.

BACKGROUND: American Cocker Spaniels are predisposed to chronic hepatitis. OBJECTIVE: To describe the clinical and histological features of chronic hepatitis in Japanese American Cocker Spaniels. ANIMALS: Thirteen cases examined from 2003 to 2009. METHODS: Retrospective study. Medical records were searched for American Cocker Spaniels with chronic liver diseases. History, physical examination, clinicopathologic features, hepatic ultrasonographic findings, hepatic histopathology, and immunohistochemistry were evaluated. RESULTS: The median age was 4.6 (1.9-10.7) years. Clinical signs included inappetence (11/13), ascites (11/13), lethargy (9/13), diarrhea (7/13), and melena (2/13). Only 1/13 dogs was jaundiced. Clinicopathological abnormalities were increased liver enzymes (gamma-glutamyl transpeptidase: 9/12, aspartate aminotransferase: 7/10, alanine aminotransferase: 6/13, alkaline phosphatase: 6/13), increased total serum bile acid concentrations (10/12), and hypoalbuminemia (10/13). The liver had an irregular surface in all dogs and acquired portosystemic collaterals were verified in 11/13 dogs by abdominal ultrasound (2), laparoscopy (4), or both (5). Liver histology revealed severe fibrosis and cirrhosis in all cases, subdivided in lobular dissecting hepatitis (7), periportal fibrosis (1), micronodular cirrhosis (3), and macronocular cirrhosis (2). Inflammatory activity was low to mild. Immunohistochemical stains showed ductular proliferation. The median survival time was 913 (range: 63-1981) days. CONCLUSION AND CLINICAL IMPORTANCE: Hepatitis in Japanese American Cocker Spaniels is clinically silent until an advanced stage and is associated with severe hepatic fibrosis leading to cirrhosis, extensive ductular/putative hepatic progenitor cell proliferation, portal hypertension, and acquired portosystemic collateral shunting, but relatively long survival times. Lobular dissecting hepatitis seems more prevalent than in previously reported cases from other countries.

Copper-induced hepatitis: the COMMD1 deficient dog as a translational animal model for human chronic hepatitis.

Chronic inflammatory liver disease regardless of aetiology leads to failing regeneration and fibrosis, ending in cirrhosis. Both in man and in animals this worldwide health problem has no definitive cure. Chronic liver injury causes hepatic stellate cells to proliferate and differentiate into matrix-producing cells. New therapeutic options will be developed upon detailed understanding of the molecular mechanisms driving liver fibrosis. This may lead to new anti-fibrotic therapies which need to be tested in suitable models before application in the veterinary and human clinic. On the other side, to restore the failing regenerative capacity of the diseased liver cells, adult progenitor cells are of interest, as an alternative to whole organ transplantation. In order to find the most suitable large animal model it is important to recognise that the typical histopathological reaction pattern of the liver can differ between mammalian species. It is therefore imperative that specialists in veterinary internal medicine and pathology, being familiar with the diseases and pathologies of the liver in different animal species, are teaming-up in finding the best models for veterinary and human liver diseases. Several large animal models have been mentioned, like pigs, sheep, and dogs. Based on the observations that man and dog share the same hepatopathies and have identical clinical, pathological and pathogenetic reaction patterns during the development of liver disease, the dog seems to be a properly suited species to test new therapeutic strategies for pets and their best friends.

Expression of hepatocyte growth factor and the proto-oncogenic receptor c-Met in canine osteosarcoma.

Hepatocyte growth factor (HGF) and the proto-oncogenic receptor c-Met are implicated in growth, invasion, and metastasis in human cancer. Little information is available on the expression and role of both gene products in canine osteosarcoma. We hypothesized that the expression of c-Met is associated with malignant histologic characteristics, a short survival time, and a reduced disease-free interval in canine osteosarcoma. Quantitative real-time polymerase chain reaction was used to analyze the messenger RNA (mRNA) expression of both HGF and c-Met in 59 canine osteosarcoma samples. The relationship between HGF and c-Met expression, patient outcome, and histologic characteristics of the tumor were studied. Western blot analysis was performed to investigate the presence of active HGF protein. The expression pattern of c-Met in 16 slides of canine osteosarcoma was identified by immunohistochemistry. Coexpression of HGF and c-Met mRNA in all canine osteosarcoma samples suggested autocrine or paracrine receptor activation. A significant, moderately positive correlation was found between c-Met and HGF mRNA expression. c-Met mRNA expression was not associated with survival time or disease-free interval. Expression of c-Met was significantly associated with metastasis via the lymphogenic route. Immunolabeling with c-Met revealed a cytoplasmic staining pattern in all osteosarcoma cell types. In this study, c-Met mRNA expression in canine osteosarcoma was found to be of no influence on survival time and disease-free interval. Further studies are necessary to confirm the involvement of the c-Met pathway in the lymphogenic route of metastasis.

Minimal external masculinization in a SRY-negative XX male Podenco dog.

Normal mammalian sex differentiation takes place in three genetically controlled steps: chromosomal sex determination (XX or XY), gonadal differentiation and development of the phenotypic sex. Animals are considered to be sex reversed if chromosomal sex determination and gonadal development are not in agreement. In this report, sex reversal is described in a 1.5-year-old Podenco dog that was referred because of suspected recurrent growth of a previously removed os clitoridis in the vulva. With that exception the dog was phenotypically female, but had never been in oestrus and exhibited male behaviour. Abdominal ultrasonography showed a small tubular structure dorsal to the bladder, consistent with a uterus. An ovoid structure resembling a gonad was visible between the right kidney and inguinal canal. Plasma testosterone concentrations before and after GnRH administration indicated the presence of functional testicular tissue. Two testes, each with its epididymis and ductus deferens, and a complete bicornuate uterus were removed surgically. Cytogenetic analysis of peripheral blood lymphocytes showed a normal female karyotype (78, XX). These findings are consistent with the diagnosis of an XX male. PCR analysis of genomic DNA revealed that the SRY gene was absent. In summary, this report describes the first SRY-negative XX male Podenco dog with an almost complete female phenotype despite high basal and stimulated plasma testosterone concentrations. It is hypothesized that the clinical observations in this dog may have been caused by reduced and delayed Müllerian-inhibiting substance secretion and the absence of conversion of testosterone to dihydrotestosterone due to 5alpha-reductase deficiency.

Functional consequences of RNA interference targeting COMMD1 in a canine hepatic cell line in relation to copper toxicosis.

A deletion in the copper metabolism (Murr1) domain containing 1 (COMMD1) gene is associated with hepatic copper toxicosis in dogs, yet evidence of copper retention in COMMD1-depleted hepatic cells has not been shown. In a dog hepatic cell line, we analysed the copper metabolic functions after an 80% (mRNA and protein) COMMD1 reduction with COMMD1-targeting siRNAs. Exposure to 64Cu resulted in a significant increase in copper retention in COMMD1-depleted cells. COMMD1-depleted cells were almost three times more sensitive to high extracellular copper concentrations. Copper-mediated regulation of metallothionein gene expression was enhanced in COMMD1-depleted cells. Based on the increased copper accumulation and enhanced cellular copper responses upon COMMD1 reduction, we conclude that COMMD1 has a major regulatory function for intracellular copper levels in hepatic cells.

Chronic hepatitis in Doberman pinschers. A review.

Chronic hepatitis in Doberman pinschers is predominantly seen in female dogs, usually between 4 and 7 years of age and was first recognized in the early eighties. The histopathological characteristics of Doberman hepatitis are those of micronodular cirrhosis with histological features of fibrosis, piece meal necrosis and progressive lymphocyte and plasma cell infiltration of the portal triads. Currently there are two hypotheses on the pathogenesis although neither of them has been elucidated. The first hypothesis is that of a copper toxicosis. The second is that of autoimmunity. Similarities and differences with other breeds and studies on both hypotheses are reviewed, as well as results of recent research of our group. Based on recent findings chronic hepatitis in Doberman pinschers is most likely to be a form of copper toxicosis. Although there are several indications that suggest autoimmunity as well, this still remains unclear.

Characterisation of 11 beta-hydroxysteroid dehydrogenases in feline kidney and liver.

11 Beta-hydroxysteroid dehydrogenases type 1 and 2 (11 beta-HSD1 and 11 beta-HSD2) are microsomal enzymes responsible for the interconversion of cortisol into the inactive form cortisone and vice versa. 11 beta-HSD1 is mainly present in the liver, and has predominantly reductase activity although its function has not yet been elucidated. 11 beta-HSD2, present in mineralocorticoid target tissues such as the kidney, converts cortisol into cortisone. Reduced activity due to inhibition or mutations of 11 beta-HSD2 leads to hypertension and hypokalemia resulting in the Apparent Mineralocorticoid Excess Syndrome (AMES). Like humans, cats are highly susceptible for hypertension. As large species differences exist with respect to the kinetic parameters (K(m) and V(max)) and amino acid sequences of both enzymes, we determined these characteristics in the cat. Both enzyme types were found in the kidneys. 11 beta-HSD1 in the feline kidney showed bidirectional activity with predominantly dehydrogenase activity (dehydrogenase: K(m) 1959+/-797 nM, V(max) 766+/-88 pmol/mg*min; reductase: K(m) 778+/-136 nM, V(max) 112+/-4 pmol/mg*min). 11 beta-HSD2 represents a unidirectional dehydrogenase with a higher substrate affinity (K(m) 184+/-24 nM, V(max) 74+/-3 pmol/mg*min). In the liver, only 11 beta-HSD1 is detected exerting reductase activity (K(m) 10462 nM, V(max) 840 pmol/mg*min). Sequence analysis of conserved parts of 11 beta-HSD1 and 11 beta-HSD2 revealed the highest homology of the feline enzymes with the correspondent enzymes found in man. This suggests that the cat may serve as a suitable model species for studies directed to the pathogenesis and treatment of human diseases like AMES and hypertension.

Comparison of the response of renal sodium excretion to almitrine and hypoxia in conscious normotensive and spontaneously hypertensive rats.

The effect of hypobaric hypoxia and almitrine on renal sodium excretion and systolic blood pressure was investigated in conscious adult normotensive control rats (NCR) and spontaneously hypertensive rats (SHR). Moderate hypoxia caused a significant natriuresis within the first two days in both NCR and SHR. The systolic blood pressure was significantly diminished in SHR only. Oral application of almitrine (1.0 mg/kg b.w.) induced natriuresis in SHR and NCR. However, there are considerable differences in the pattern of natriuresis between the animals of both strains and when compared to the effect of hypoxia. The urinary sodium excretion by almitrine was not associated with a decrease in systolic blood pressure neither in SHR nor in NCR. The results indicate that both hypoxia and almitrine induce a strong natriuresis in conscious rats apparently by different mechanisms. However, there is no real evidence at present as to whether the peripheral arterial chemoreceptors are indeed involved in these mechanisms, or not, and if so, to what degree.