Regenerative and fibrotic pathways in canine hepatic portosystemic shunt and portal vein hypoplasia, new models for clinical hepatocyte growth factor treatment

BACKGROUND: We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls. RESULTS: We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor β1 (TGF-β1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-β1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-β1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-β1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis. CONCLUSION: The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic inflammatory diseases and cirrhosis

Detection of Helicobacter pylori in bile of cats

Lymphocytic cholangitis (LC) in cats is a biliary disease of unknown etiology. Helicobacter spp. were recently implicated in human primary sclerosing cholangitis (PSC) and primary biliary cirrhosis (PBC). Because of the similarities between PSC/PBC with LC, we hypothesized that Helicobacter spp. are involved in feline LC. A PCR with Helicobacter genus-specific 16S rRNA primers was performed on DNA isolated from feline bile samples. Four of the 15 (26%) LC samples were positive, whereas only 8/51 (16%) of non-LC samples were PCR positive (p=0.44). Sequence analysis of the amplicons revealed a 100% identity with the Helicobacter pylori specific DNA fragments. Our data suggest an etiological role of H. pylori in feline LC and that cats are a potential zoonotic reservoir

Козацькі могили у творчості Тараса Шевченка

The detoxification of ammonia occurs mainly through conversion of ammonia to urea in the liver via the urea cycle and glutamine synthesis. Congenital portosystemic shunts (CPSS) in dogs cause hyperammonemia eventually leading to hepatic encephalopathy. In this study, the gene expression of urea cycle enzymes (carbamoylphosphate synthetase (CPS1), ornithine carbamoyltransferase (OTC), argininosuccinate synthetase (ASS1), argininosuccinate lyase (ASL), and arginase (ARG1)), N-acetylglutamate synthase (NAGS), Glutamate dehydrogenase (GLUD1), and glutamate-ammonia ligase (GLUL) was evaluated in dogs with CPSS before and after surgical closure of the shunt. Additionally, immunohistochemistry was performed on urea cycle enzymes and GLUL on liver samples of healthy dogs and dogs with CPSS to investigate a possible zonal distribution of these enzymes within the liver lobule and to investigate possible differences in distribution in dogs with CPSS compared to healthy dogs. Furthermore, the effect of increasing ammonia concentrations on the expression of the urea cycle enzymes was investigated in primary hepatocytes in vitro. Gene-expression of CPS1, OTC, ASL, GLUD1 and NAGS was down regulated in dogs with CPSS and did not normalize after surgical closure of the shunt. In all dogs GLUL distribution was localized pericentrally. CPS1, OTC and ASS1 were localized periportally in healthy dogs, whereas in CPSS dogs, these enzymes lacked a clear zonal distribution. In primary hepatocytes higher ammonia concentrations induced mRNA levels of CPS1. We hypothesize that the reduction in expression of urea cycle enzymes, NAGS and GLUD1 as well as the alterations in zonal distribution in dogs with CPSS may be caused by a developmental arrest of these enzymes during the embryonic or early postnatal phase

Long-Term Adult Feline Liver Organoid Cultures for Disease Modeling of Hepatic Steatosis.

Hepatic steatosis is a highly prevalent liver disease, yet research is hampered by the lack of tractable cellular and animal models. Steatosis also occurs in cats, where it can cause severe hepatic failure. Previous studies demonstrate the potential of liver organoids for modeling genetic diseases. To examine the possibility of using organoids to model steatosis, we established a long-term feline liver organoid culture with adult liver stem cell characteristics and differentiation potential toward hepatocyte-like cells. Next, organoids from mouse, human, dog, and cat liver were provided with fatty acids. Lipid accumulation was observed in all organoids and interestingly, feline liver organoids accumulated more lipid droplets than human organoids. Finally, we demonstrate effects of interference with β-oxidation on lipid accumulation in feline liver organoids. In conclusion, feline liver organoids can be successfully cultured and display a predisposition for lipid accumulation, making them an interesting model in hepatic steatosis research

Inherited liver shunts in dogs elucidate pathways regulating embryonic development and clinical disorders of the portal vein

Congenital disorders of the hepatic portal vasculature are rare in man but occur frequently in certain dog breeds. In dogs, there are two main subtypes: intrahepatic portosystemic shunts, which are considered to stem from defective closure of the embryonic ductus venosus, and extrahepatic shunts, which connect the splanchnic vascular system with the vena cava or vena azygos. Both subtypes result in nearly complete bypass of the liver by the portal blood flow. In both subtypes the development of the smaller branches of the portal vein tree in the liver is impaired and terminal branches delivering portal blood to the liver lobules are often lacking. The clinical signs are due to poor liver growth, development, and function. Patency of the ductus venosus seems to be a digenic trait in Irish wolfhounds, whereas Cairn terriers with extrahepatic portosystemic shunts display a more complex inheritance. The genes involved in these disorders cannot be identified with the sporadic human cases, but in dogs, the genome-wide study of the extrahepatic form is at an advanced stage. The canine disease may lead to the identification of novel genes and pathways cooperating in growth and development of the hepatic portal vein tree. The same pathways likely regulate the development of the vascular system of regenerating livers during liver diseases such as hepatitis and cirrhosis. Therefore, the identification of these molecular pathways may provide a basis for future proregenerative intervention

Canine models of copper toxicosis for understanding mammalian copper metabolism

Hereditary forms of copper toxicosis exist in man and dogs. In man, Wilson’s disease is the best studied disorder of copper overload, resulting from mutations in the gene coding for the copper transporter ATP7B. Forms of copper toxicosis for which no causal gene is known yet are recognized as well, often in young children. Although advances have been made in unraveling the genetic background of disorders of copper metabolism in man, many questions regarding disease mechanisms and copper homeostasis remain unanswered. Genetic studies in the Bedlington terrier, a dog breed affected with copper toxicosis, identified COMMD1, a gene that was previously unknown to be involved in copper metabolism. Besides the Bedlington terrier, a number of other dog breeds suffer from hereditary copper toxicosis and show similar phenotypes to humans with copper storage disorders. Unlike the heterogeneity of most human populations, the genetic structure within a purebred dog population is homogeneous, which is advantageous for unraveling the molecular genetics of complex diseases. This article reviews the work that has been done on the Bedlington terrier, summarizes what was learned from studies into COMMD1 function, describes hereditary copper toxicosis phenotypes in other dog breeds, and discusses the opportunities for genome-wide association studies on copper toxicosis in the dog to contribute to the understanding of mammalian copper metabolism and copper metabolism disorders in man

Efficacy of orally administered sodium benzoate and sodium phenylbutyrate in dogs with congenital portosystemic shunts

Background: Hyperammonemia can result in hepatic encephalopathy, which in severe cases eventually can lead to coma and death. In dogs, congenital portosystemic shunts (CPSS) are the most common cause for hyperammonemia. Conservative treatment consists of a protein modified diet, nonabsorbable disaccharides, antibiotics, or some combinations of these. Sodium benzoate (SB) and sodium phenylbutyrate (SPB) both are used in the acute and long-term treatment of humans with hyperammonemia caused by urea cycle enzyme deficiencies. Both treatments are believed to lower blood ammonia concentrations by promoting excretion of excess nitrogen via alternative pathways. Objectives: To evaluate the efficacy and safety of PO treatment with SB and SPB on hyperammonemia and clinical signs in CPSS dogs. Methods: Randomized, double-blind, placebo-controlled crossover trial. Concentrations of blood ammonia and bile acids were measured in CPSS dogs before and after a 5-day treatment with SB, SPB, and placebo. A wash-out period of 3 days was used between treatments. A standard questionnaire was developed and distributed to owners to evaluate clinical signs before and after each treatment. Results: Blood ammonia concentrations were not influenced by any of the treatments and were comparable to those observed during placebo treatment. In addition, SB and SPB treatment did not result in improvement of clinical signs. Adverse effects during treatment included anorexia, vomiting, and lethargy. Conclusions and Clinical Importance: Based on our results, we conclude that SB or SPB are not useful in the conservative treatment of hyperammonemia in dogs with CPSS