Advances in Small Animal Hepatology - The WSAVA International Award for Scientific Achievement
David C. Twedt, DVM, DACVIM
Prior to 1970, there was very little known about canine and feline liver disease, and what was known consisted of descriptions of histopathology such as hepatitis or cirrhosis. It was in the mid-1970s that a Bedlington terrier breeder in the US observed that many of her dogs were dying from liver disease. She was convinced that Bedlington terriers had some type of inherited liver disease. She contacted many veterinarians and veterinary universities begging someone to investigate her theory. At that time, The University of Minnesota happened to have a Bedlington terrier that had just died of cirrhosis. They further investigated the liver sample and noted there were strange granules in the hepatocytes. Applying different histochemical stains to determine what was in these granules, pathologists found that they contained copper.
It was shortly thereafter, while I was a resident at the Animal Medical Center in New York City, that I was treating a Bedlington terrier that had liver disease and we too identified copper granules in the dog's hepatocytes. I contacted the world's expert on Wilson's disease (a human copper-storage disease), Dr. Sternleib, at Albert Einstein's Medical School and Liver Research Center. Soon, a collaborative relationship was formed and I was later hired as a research associate in the Liver Research Center, where we investigated copper-associated liver disease in Bedlington terriers with the hope that this could become a research model for Wilson's disease.
We subsequently described the clinical, morphologic and chemical findings of copper toxicosis in 90 Bedlington terriers. Specifically, we showed copper accumulated with age, was sequestered in lysosomes and that lysosomal damage resulted in free copper leaking into the cytoplasm, causing mitochondrial oxidative injury and subsequent hepatocellular death. We were also the first to measure canine ceruloplasmin, the copper carrying protein, and found it to be normal in affected Bedlingtons, a characteristic different from Wilson's disease associated with low ceruloplasmin levels. Radioisotope CU64 excretion studies documented that affected dogs fail to excrete copper from the liver. Breeding studies demonstrated this copper excretion defect to be autosomal recessive with approximately 75% of the Bedlingtons being either carriers or affected with the disease.
Our long-term treatment studies found the copper chelator penicillamine to be effective in removing copper from the liver and excreting it through the kidneys. We also investigated a second copper chelator, trientine (2,2,2 tetramine), and found it to be equally effective in removing hepatic copper. Then, we developed a potent modification of trientine, a 2,3,2 tetramine, and demonstrated it to be the most effective agent in removing copper and successfully treated many cases. We presented our results at the American Association for the Study of Liver Disease and it generated considerable interest, but unfortunately the medication was never produced commercially. Other investigators have now identified the defective gene involved with copper excretion and, with genetic testing and counseling, this hepatic disease has almost been eradicated from the breed or affected dogs successfully managed. We also now know that the gene involved with Bedlington terrier copper-storage disease is different than the one associated with Wilson's disease.
We also identified and reported hepatitis with associated abnormal copper concentrations in both the Dalmatian and Doberman pinscher. Affected Dalmatians accumulate very high copper concentrations approaching the levels found in the Bedlington terrier and at much higher levels that are found in other breeds associated with abnormal copper. Others have shown that the affected Dobermans have copper accumulation, as well as an immune component to the disease. Many breeds have also now been found to be associated with hepatitis and abnormal copper accumulation. Most recently, the Labrador retriever was identified with abnormal copper and concurrent hepatitis. Researchers demonstrated that the copper accumulation in these dogs is controlled using short-term penicillamine therapy, followed by feeding a low-copper diet. They further found dietary copper in commercially available dog food can influence hepatic copper concentrations and can be a risk factor for the development of copper-associated hepatitis in Labrador retrievers with a genetic susceptibility to copper.
The role of dietary copper and hepatic copper has been an area of interest and speculation of mine over the years. My mentor, Dr. Sternleib, years ago told me that he believed that all dog food is supplemented with too much copper. He also pointed out that in the 1930s canine hepatic copper was approximately 50 µg/g dry weight liver, similar to that found in humans; today, normal hepatic copper concentrations in the dog are 4 to 8 times higher. We recently investigated hepatic copper concentrations in stray dogs from two underdeveloped countries, unlikely to have consumed commercial dog food. Compared to age- and sex-matched US dogs receiving a variety of commercial dog foods, the hepatic copper concentrations were significantly lower in stray dogs suggesting diet may contribute to copper levels and subsequent liver disease. Using quantitative PCR techniques, we are investigating if copper upregulates the hepatocyte metallothionein copper-binding protein that could be a potential marker for susceptibility of disease. To date, it is still unknown what ideal dietary copper concentrations should be.
My ongoing investigation also includes chronic hepatitis in standard poodles. Breeders have recognized that liver disease occurs in the breed and many suspect it to be inherited. Through funding from the Standard Poodle Club of America we have been able to obtain clinical information, as well as histological samples and DNA for clinical investigation. Our current understanding at this point is that the chronic hepatitis is likely inherited, occurs most commonly in middle-aged female dogs and that copper is not a component of the disease. We suspect there may be immune-mediated mechanisms associated with the condition, also supported by the fact that immune disease is so common in poodles. Preliminary studies suggest that hepatocytes may have aberrant MHC class II expression that may contribute to autoimmune hepatitis through the activation of CD4 T cells.
We are also studying the potential of cyclosporine in the treatment of suspected immune hepatitis. We are investigating the pharmacokinetics of cyclosporine and the in vitro effects of various compounds inhibiting intestinal cytochrome P-450 and P-glycoprotein to increase cyclosporine bioavailability. We also recently reported on 13 cases of chronic hepatitis treated with cyclosporine. In our small preliminary pilot study, 12/13 dogs had a greater than 70% reduction in ALT concentrations with 50% returning to normal. In 8/8 dogs with hepatitis, the hyperbilirubinemia or ascites resolved during therapy. Obviously, this is a small number of select cases, but our ongoing investigation supports cyclosporine being preferred to corticosteroids by negating steroid side effects and resulting secondary steroid hepatopathy.
Our knowledge of acute liver toxicity secondary to various toxins and drugs has also expanded over the years. Drug-associated liver damage is either idiosyncratic, being unpredictable in occurrence, or directly hepatotoxic, causing a predictable dose-dependent toxicity. We, first, reported two idiosyncratic drug reactions occurring in dogs being administered trimethoprim-sulfa and mitotane. Both cause acute liver damage and if identified early with subsequent drug withdrawal, the damage is often reversible. Generally, the treatment of many types of liver disease is only supportive. Oxidative damage within the hepatocyte appears to be a common pathway of most all types of liver insult. Hepatic oxidative damage has been an area of our investigation, coupled with the role of antioxidants in the therapy of liver disease. We developed a research model of acute acetaminophen toxicity in the cat. Giving doses far below the lethal doses, cats developed both hepatic and hematological changes, including Heinz bodies and methemoglobinemia. We were able to show that cats given S-adenosylmethionine (SAMe) were protected against both the hepatic and hematological changes. SAMe-treated cats, compared to placebo cats, had both higher hepatic and RBC glutathione concentrations. SAMe is a precursor for glutathione production, the important soluble antioxidant and also improves membrane fluidity and function and enhances cell renewal through other pathways. SAMe is now a common adjunct therapy for many types of liver disease. We also investigated milk thistle, a herbal therapy used for treatment of liver disease. We performed pharmacokinetic studies on the active isomer of milk thistle, silybin that was bound to phosphatidylcholine for improved GI absorption, and demonstrated the compound is readily absorbed and likely undergoes enterohepatic recycling. There was no evidence of toxicity and there was improved oxidant status in cat's RBCs and WBCs by measuring cellular glutathione concentrations using flow cytometry. We have also performed in vitro oxidative studies using isolated hepatocytes and demonstrated that vitamin E protected against hepatic damage when exposed to copper, bile acids and iron. We also found affected Bedlington terriers to have significantly decreased mitochondrial vitamin E concentrations with increased markers of oxidative damage. These findings spurred a clinical investigation on the effects of vitamin E (d-alpha tocopherol) supplementation in dogs with confirmed chronic hepatitis. This placebo-controlled study demonstrated less oxidative damage by measuring the ratio of oxidized to reduced glutathione when dogs were supplemented with vitamin E during a three-month-treatment period.
I was also a member of the WSAVA-sponsored Liver Standardization Group that published the textbook WSAVA Standards for Histological and Clinical Diagnosis of Canine and Feline Liver Diseases. Our aim was to obtain worldwide standardization for histological evaluation of liver diseases with unified nomenclature, well-defined histological diagnostic criteria, and precise definition of chronicity stages and grades of diseases. An area of great interest to me in this process was our classification and speculation into etiologies of feline biliary tract disease. We have subsequently reported in a retrospective review of the causes of icterus in the cat and reported cholangitis to be one of most common cholestatic categories in our series of 180 cases. Using the WSAVA classifications of feline cholangitis, we sought to investigate the suspected potential role of bacteria as an etiology of cholangitis. Using eubacterial fluorescence in situ hybridization (FISH), we demonstrated that enteric bacteria (most often E. coli) were significantly associated with both acute and chronic neutrophilic cholangitis (100% acute, 69% chronic). There is also a relationship of cholangitis with pancreatitis and inflammatory bowel disease. Years ago, I first coined the term "feline triaditis syndrome," referring to the relationship of these three diseases and now it has become common terminology. In a companion FISH study of pancreatitis, we found approximately 30% of the cases to have enteric bacteria within the pancreas. Our working theory is that bacteria may enter the liver and pancreas through intestinal translocation or by ascending biliary and pancreatic duct. This finding has changed my approach in management of feline triaditis and I'm more inclined to treat with antibiotics first before reaching for corticosteroids. We also reported on ultrasound findings in cats having cholangitis and published the first paper on magnetic resonance cholangiopancreatography (MRCP) in cats with pancreatitis and cholangitis finding duct changes characteristic in both.
The most recent and exciting area of my investigation is stem cell therapy for liver disease. As part of the Center of Regenerative Research at Colorado State University, we are investigating canine adipose-derived mesenchymal stem cells (MSC). Using optimized culture conditions, we hope to drive these cells to behave more like hepatocytes and then to investigate their characteristics in vitro. We have also begun work on creation of canine-induced pluripotent stem cells (iPSC). Using genetic reprogramming with protein transcription factors, pluripotent stem cells are created equivalent to embryonic stem cells that can differentiate into any cell type. Our pilot studies using labeled MSC injected into the spleen showed they reached the liver in normal dogs. We are about to begin clinical trials of stem cell therapy in dogs suffering from either acute or chronic liver disease by giving stem cells through splenic injection. Our goal is to determine if MSC or iPSC function in immunomodulation, hepatocyte replacement, improved function or hepatic regeneration.
As is evidenced by this brief glimpse of progress in the study and understanding of liver disease, we have certainly come a long way in the last 40 years.