Rimadyl Biopsy/Pathology Report
Cornell University Liver Histopathology relating to
Rimadyl Risks Liver Failure, the heartbreaking loss of Rimadyl-treated dog
May 15, 2017 | TheDogPress.com
Digital Copper Quantification: 2,756 ug/gm dry wt. (normal < 400 ug/gm)
Clinical History: acute onset anorexia with intermittent regurgitation. This dog was treated with Rimadyl for chronic LS pain. There is a history of chronic increased ALT activity with progressive development of hypoalbuminemia, low BUN, and increased bile acid values. Abdominal CT completed. Abdominal ultrasound: WNL. Laparoscopic liver biopsies 1/5/17 disclosed ascites and an irregular liver with diffusely disseminated nodules (red/tan).
Laboratory Synopsis - 1/5/2017:
CBC: PCV= 50%, MCV= 70 fL, WBC= 10.5 k/uL, platelets = 141 k/uL.
Chemistry: ALP= 268 u/L, GGT= 12 u/L, ALT= 117 u/L, AST= 61 u/L, TB= 0.4 mg/dL, BUN= 5 mg/dL, creatinine = 0.8 mg/dL, cholesterol = 90 mg/dL, triglycerides = 53 mg/dL, albumin = 2.3 g/dL, globulin = 2.9 g/dL.
Urinalysis: SG = 1.013, protein, glucose, ketones, bilirubin, blood = negative, inactive sediment, no ammonium biurates described,
Bile Acids: Pre= 84 μmol per liter, Post= 172 μmol per liter
Tick titers: Negative with the exception of a vaccinal Borrelia titer
T4 Post Pill: 8.5 ug/dL is high, presumably dose of thyroxin was reduced. High T4 can drive increased liver enzyme activity.
One vial with 4 liver laparoscopic biopsies is received.
Cornell Clinical Interpretation of Liver Histopathology: On routine H&E staining of all sections mounted on a single slide there are similar features in each biopsy. Hepatic parenchyma is severely disrupted by widespread dissecting sinusoidal fibrosis that septates parenchyma into individual packets/islands of hepatocytes. The pattern is one of limited nodular regeneration. Regenerative nodules are widely spaced and separated by chaotically remodeled parenchymal structure. Regenerative nodules are characterized by disorganized double-wide hepatic cords, a thin mantling fibrotic capsule, and compressive expansion against adjacent parenchyma. Coalescing regions of Parenchymal Extinction are scattered across biopsy sections. These areas reflect loss of functional hepatic mass where parenchyma is replaced by remnant deformed bile ducts reflecting impact of parenchymal remodeling causing obstruction/traction/compression of ductal elements, increased deposition of Extracellular Matrix (ECM), residual inflammatory cells and numerous pigmented macrophages (lipofuscin pigment & iron). Viable parenchyma displays a variable mild lymphoplasmacytic inflammatory infiltrate and scattered pigment laden macrophages. Hepatocytes and macrophages often display cytosolic refractile eosinophilic aggregates consistent with copper-protein particles. The nature of these aggregates are confirmed with special stains-see Rhodanine staining below. Co-localization of copper aggregates with Lipofuscin aggregates (brown/tan non-refractile granules) is widely evident. As Lipofuscin is the sentinel pigment of oxidative membrane injury- this implicates severe oxidative stress across all lobular zones. This reconciles with panlobular copper accumulation and history of chronic Rimadyl administration causing a Collision Toxicity Injury. Numerous hepatocytes display microvesicular lipid vacuolation consistent with mitochondrial dysfunction. This is a morphologic change frequently associated with hepatotoxicity and known to accompany severe pathologic copper accumulation. Necrotic hepatocytes are observed at a density of on 2-3/10 hpf (normal ¸1/10 hpf, hpf= single 400-X field of view). Mitotically active hepatocytes are equivalently observed but these have not organized into regenerative nodules for the most part.
Mesothelium of the liver capsule is hypertrophied reflecting the irritation induced by ascitic effusion. Severe remodeling across all hepatic zones is consistent with severe acquired sinusoidal hypertension and has most certainly led to development of severe splanchnic hypertension generating Acquired Portosystemic Shunts (APSS).
Special Stains allow better assessment of hepatic structural modification and metal accumulation.
Reticulin staining (substructural reticulin scaffolding) delineates few scattered regenerative nodules separated by coalescing parenchyma with wildly disturbed hepatic cord architecture- best described as Chaotic Disorganization. Hepatocytes are individualized by thick reticulin partitions forming small isolated islands of hepatic parenchyma. Some hepatic parenchyma is so severely remodeled it is classifiable as Parenchymal Extinction-that is loss of functional hepatic mass. Diffuse remodeling in this liver is consistent with severe sinusoidal hypertension and development of acquired portosystemic shunts (APSS). The remodeling is so severe that the changes are consistent with cirrhosis. Widely dilated lymphatics are observed scattered near large vascular structures- reflecting increased ultralymph formation contributing to ascites.
Masson’s trichrome, used to confirm deposition of fibrillar collagen, additionally also provides tinctorial contrast that enhances evaluation of architectural details. This stain demonstrates severe dissecting sinusoidal fibrosis individualizing hepatocytes in a pattern consistent with Lobular Dissecting Hepatitis- a descriptive term for the pattern of hepatocellular injury and fibrosis- NOT a unique diagnosis. Regenerative nodules are marginated by a thin fibrotic mantle. The tinctorial contrast afforded by this stain also details diffuse microvesicular lipid vacuolation likely caused by severe copper accumulation. It is well established that pathologic copper accumulation injures mitochondria responsible for microvesicular lipid vacuolation.
Prussian Blue confirms co-localization of Lipofuscin pigment with stainable iron in macrophages distributed across biopsy sections. Hepatocytes also display a positive “blush” for iron likely reflecting upper regulation of cytoplasmic ferritin as well as cytosolic lipofuscin-iron binding. Lipofuscin maintains a molecular configuration where iron may form covalent bonds-consequently, large amounts of iron & lipofuscin often co-localize. Regenerative nodules do not display any stainable iron in fixed sinusoidal macrophages (Kupffer cells) or hepatocytes. Otherwise, the plentiful iron sequestration in macrophages reflects severe architectural remodeling and release of iron from hepatocellular cytochromes. This iron is subsequently phagocytosed and sequestered under the influence of inflammatory cytokines. It is also possible that iron derived from systemic sources -depending upon other concurrent illnesses or anemia (copper can induce hemolysis). Substantial amounts of iron in this liver contribute to an oxidative redox imbalance. Like copper, iron catalyzes the Fenton and Haber Weiss reactions which degenerate injurious superoxide & hydroxyl radicals. Fortunately, d-penicillamine chelation also will remove a substantial amount of this iron during the process of copper chelation.
Rhodanine staining confirms widespread copper distribution in these biopsies. Hepatocytes marginating regenerative nodules display numerous fine cytosolic copper aggregates whereas more central regions of these nodules- containing newly regenerated cells- are negative. Otherwise, all areas including those classified as Extinct Parenchyma are strongly positively for pathologic copper accumulation. Based on the uniformity of copper distribution, the number of hepatocytes affected, and the density & size of copper aggregates a qualitative severity score of 4-5/5 is warranted
Histologic Features of Clinical Concern
1. Severe Panlobular Injury Reflecting Collision Impact of Chronic NSAID Administration & Widespread Pathologic Copper Accumulation & Foci of Parenchymal Extinction. The pattern of Lobular Dissecting Hepatitis describes a morphologic pattern of liver injury and is not a specific disease entity. Features are not consistent with an immune mediated hepatitis. The severity of copper accumulation and large amounts of lipofuscin pigment in residual hepatocytes and macrophages strongly implicate ongoing oxidative injury and Collision Toxicity imposed by copper accumulation & Rimadyl. Treatment must be focused on copper chelation, lifelong dietary restriction of copper intake, with mandatory avoidance of any future NSAID administration.
Remodeling in these biopsies corresponds to the clinician’s gross description and is consistent with designation as Cirrhosis- but don’t give up with that term applied. There is no evidence of an independent immune-mediated liver injury. The severity of remodeling is consistent with development of APSS due to sinusoidal hypertension causing severe splanchnic portal hypertension. As the portal circulatory system is a low pressure capacitance vascular bed without valves, portal circulation flows in the path of least resistance. In this case there is tremendous resistance created by hepatic parenchymal remodeling & fibrosis. The liver capsular changes confirm historic exposure to ascitic effusion consistent with the clinician’s observations. Absence of obvious immune mediated hepatocyte injury argues against initial institution of an immunomodulatory protocol. The severity of the sinusoidal hypertension causing APSS is reflected in the low cholesterol & BUN and high bile acids. The loss of perfusion to functional hepatic mass and provoked PU/PD likely contribute to the low BUN and creatinine.
Can you recover this dog ? Maybe- but the first 3-4 mths will be pivotal in balancing drug tolerance (d-Pen, antioxidants, Ursodiol, PhosChol™, pyridoxine, furosemide, spironolactone, water soluble vitamins without minerals, possibly vitamin K supplementation) and tailoring the appropriate diet and controlling severity of ascites. Can there be remodeling and recovery- Yes- to a degree. This liver will never be normal. The longer I have been a vet the more cases I have seen make remarkable recoveries- but that is highly dependent on the motivation of the owner and diligence of the clinician. No promises here for this dog.
Copper primarily manifests hepatotoxicity through oxidant injury and we have commonly recognized the impact of a collision toxicity between NSAIDs and pathologic copper accumulation. This is one of the most dramatic injuries that I have yet observed secondary to this deadly combination of hepatotoxic injuries. It is possible that by copper Chelation and future avoidance of Rimadyl there can be remodeling and healing. I doubt very much however, that this liver will entirely resolve observed changes. Copper chelation with the protocol I use & meticulous supportive care will be needed to bridge this dog to some degree of parenchymal remodeling /regeneration. Follow-up scrutiny of enzyme activity & synthetic markers-albumin, cholesterol, urea concentrations will be used as surrogate indicators of reparative processes, as repeat liver biopsy is unlikely. Based on the amount of copper observed in this dog I expect chelation to take ~9-12 mths with good treatment compliance.
Note: d-Pen can sometimes provoke a glycogen-type vacuolar hepatopathy and ALP activity- so this may be encountered. There are quite a few RARE side effects of D-Pen reported in humans- I have seen drug related proteinuria, granulocytopenia, Lupus-like effects, and hepatopathy in dogs. It is a good idea to evaluate for proteinuria before initiating this drug. I would start with 50% dose reduction of D-Pen in this dog and gradually work up to the recommended dose in the abbreviated protocol attached below.
Wedgewood Pharmacy or Canada Drug and now several other compounding companies can be sources of d-Penicillamine that are more affordable. I do not recommend primary treatment with zinc- as it will not mobilize the copper as chelation can. Ensure lifelong avoidance of NSAID administration in this dog -as toxic adduct formation has augmented the oxidative impact of pathologic copper. D- Pen chelation also will remove some of the sequestered iron that also contributes to risk for GSH redox imbalance.
Antioxidants to consider include: vitamin E (alpha-tocopherol mixed, 10 u/kg PO q24hr with food- no higher dose as this may result in accumulation of the injurious tocopheroxy radical), s'adenosylmethionine (20 mg/kg PO q24-48hrs on an empty stomach which improves bioavailability), +/- milk thistle (silibinin complexed with phosphatidylcholine [PPC], silibinin has no strong beneficial evidence in human medicine and none in veterinary medicine. However, it has many theoretical benefits, a wide safety margin, and is non-toxic.
I use phosphatidylcholine (PPC) as an antifibrotic (to reduce activation of sinusoidal myofibrocytes), as an antioxidant, and for its membranoprotective effects- as discussed above under VH management protocol. I use PhosChol™ - available via internet sites as this form of PhosChol provides ~52% dilinolylphosphatidylcholine (DLPC) the active antifibrotic component of phosphatidylcholine. This nutrient is likely more effective when co-administered with s'adenosylmethionine (SAMe) as noted above under VH discussion. Use a PhosChol dose of 25 mg/kg PO q24hr given with food and SAME at a dose of 15-20 mg/kg PO on an empty stomach (use enteric coated, bioavailable brand such as Denosyl or Denamarin). I have confirmed remarkable response to PPC as an antifibrotic in dogs with Copper Associated Hepatopathy based on follow-up liver biopsies in dogs with histologic injury as severe as observed in this dog.
There also are alternative approaches to control of fibrosis. It is salient to point out that the best antifibrotic is control of the instigating inflammatory process. I do not often use colchicine for control of fibrosis because it can inhibit neutrophil function, cell replication important for hepatocellular regeneration, and cause adverse drug interactions. Losartan and Telmisartan are alternative approaches that intervene in the RAS system as antifibrotics. Both drugs are undergoing clinical evaluation as antifibrotics in humans. At least in humans, there is more impressive data accumulating for Telmisartan, and there are now several canine publication regarding Telmisartan and we are commonly using this drug in dogs with pathologic proteinuria (see JVIM paper [S. Brown was one author] a few yrs ago - good response in a dog with pathologic proteinuria [no biopsy done to confirm type of disease]- but safe dose adjustment is cited-also see below). Telmisartan is an angiotensin receptor blocker-2 used widely in humans as an antihypertensive with partial PPAR-γ agonist activity; it also is nephro-protective in diabetes and renal injury and prevents effects of drug-induced hepatotoxicity and hepatic fibrosis. ARBs, selectively antagonizes the angiotensin receptor bypassing intermediary activation steps within the RAAS cascade. Telmisartan administration PO to healthy dogs at 1.0 mg/kg/day significantly increased urine volume and sodium excretion and was safely used in a single case report for amelioration of pathologic proteinuria in a dog- as noted above. Treatment was effective and non-toxic with escalating doses of 0.43 mg/kg PO q24hr to 0.79 mg/kg in AM and 0.38 mg/kg in PM; then 0.9 mg/kg/day. In this case, I would proceed with PPC even if you also use Telmisartan. However, this can become complicated if in dogs with ascites where spironolactone is used to manage the effusion. I have now witnessed 5 dogs treated with spironolactone and Telmisartan that have become hypotensive & lethargic, and several have collapsed with signs of ARF (resolved with drug discontinuation- caused by double effect on the RAS). So, if the ascites needs management and you plan to use spironolactone, I would not be willing to start Telmisartan.
I also would start this dog on Ursodiol for its numerous protective effects and attenuation of signaling that may reduce fibrosis. Use 10-15 mg/kg PO BID given with food for best bioavailability.
I usually use a combination of diuretics to mobilize and manage ascites: Spironolactone (1-4 mg/kg PO BID, initial doubled loading dose of spironolactone combined with Lasix (1-2 mg/kg PO BID)-use starting diuretic doses in the low dose range, recheck at 3-5 days to reassess: body wt., girth, PCV, TS, BUN, if no ill effects and ascites not mobilized, increase dose incrementally, again reassessing as before. I usually use diuretics intermittently, transitioning to every-other-day administration if all goes well with mobilization and dietary sodium restriction. I may discontinue diuretics if all ascites resolves for several weeks, and restart them if/when ascites is a recurrent problem- each treatment being individualized to the patient. The combined diuretic regimen helps avoid potassium wasting and metabolic alkalosis which can promote hepatic encephalopathy.
Diuretics must be coupled with sodium restriction (intake < 100 mg/100 kcal food, see below). AVOID: concurrent metoclopramide with spironolactone as it will negate the effect. The table below shows sodium content per dry matter (DM) dog foods recommended by AAFCO- note these are for a maintenance energy diet. You want the Low Na Diet < 0.2% DM basis:
Sodium Content per DM (dry matter) Dog Foods
Energy: 3.5 - 4.5 kcal / gm % DM
AAFCO Minimum Na Diet: 0.06
High-Na+ Diet: 1.13
Moderate-Na+ Diet 0.4-0.5
Low-Na+ Diet < 0.2
Very Low-Na+ Diet: < 0.1
Dietary copper restriction is important as a lifelong husbandry change to manage this case. As you restrict dietary copper- try to retain a positive nitrogen balance- this dog likely has a higher nitrogen / protein need than dogs studied to determine maintenance requirements in shunt management- the basis for the liver diet formulation. However, this dog is at risk for hepatic encephalopathy because it already has established APSS. Baseline prescription liver diets are restricted in copper and are the core of nutritional support- these diets were formulated to control hepatic encephalopathy in dogs with surgically created portosystemic shunts. Considering the patient’s nitrogen tolerance-I typically increase the baseline liver diet protein content from 2.2-2.5 gm protein/kg body weight when consumed for maintenance energy up to 3.5 gm protein/ kg body weight using low copper food and amounts determined by two different freeware web sites: USDA food tables are used to determine copper content of foods and the USDA food free webware- SuperTracker used to calculate protein content of the selected food. Determine how much of a selected low Cu food can be used to supplement the baseline low protein Cu restricted diet to incrementally increase it by 0.25 increments up to 1.5 gm protein /kg body weight (if the patient does not have hepatic insufficiency, portal hypertension, or evidence of hepatic encephalopathy [HE]). In the case of hepatic insufficiency- as in this case- I titrate protein to the dog’s tolerance – activity, apparent cognitive function/behavior, & appearance of ammonium biurate crystalluria- using 7-10 day intervals for adjustments (0.25 g protein/kg body wt added to baseline diet). Ammonium biurate crystalluria is unacceptable and indicates need for nitrogen restriction or need for lactulose/metronidazole (7.5 mg/kg PO BID avoiding neomycin as it may be absorbed leading to ototoxicity or renal injury [I have observed each of these complications in dogs]). In some dogs, unneeded protein restriction can provoke a Glycogen-type VH morphology. So you need to be attentive to deciding the level of protein allowance for this patient – based on meticulous owner observation of this dog’s activity and cognitive functions.
Considerations regarding Zinc Therapy: While there are numerous peer reviewed published manuscripts describing the response of humans with Wilson’s disease to monotherapy treatment with zinc, I am particularly impressed by the data presented in a 2011 paper published in Gastroenterology- n =288; median follow-up time, 17.1 years (Gastroenterology 2011;140:1189–1198) where poor response to zinc therapy vs chelation with D-Penicillamine or Trientine is demonstrated. Initial work in dogs was completed with 5 dogs where zinc dosing was assessed with radiolabeled copper. Unfortunately, zinc administration to dogs often leads to gastritis, vomiting, and inappetence. Zinc acetate is the least noxious form of zinc for oral medication of dogs. Theoretically, zinc increases enteric metallothionein which binds consumed dietary copper resulting in its fecal elimination with effete enterocytes. If you try zinc, use 5-7 mg elemental zinc/kg body weight divided BID- (dose thus depends on the molecular wt of the product and contribution of zinc to the total molecular weight). Measure plasma zinc at baseline and then at 2-6 wks of treatment to document a minor increase in plasma zinc concentration- if no increase is observed, then the zinc dose should be increased. Efficacy of long term zinc therapy for complete control of copper accumulation and copper associated hepatopathy remains questionable. Zinc has been shown to be less effective in humans with Wilson’s disease than chronic chelation therapy. In dogs, dose optimization is hard to prove- in man the dose is confirmed with radioisotope studies or urinary copper excretion. Zinc therapy does not quickly mobilize copper.
If you elect to chelate to remove Copper be sure not to use d-penicillamine and zinc simultaneously as neither therapy will provide benefit (d-pen chelates zinc). A complicated timing schedule for drug administration would be needed.
Summary of Therapeutic Strategies: Copper Associated Hepatopathy: Cornell University, S Center
1. Copper Chelation: remove Cu from tissues & blood
a. D-Penicillamine (10-15 mg/kg PO q12hr=gold standard, first line Rx) or Trientine (5-7 mg/kg PO q12hr).
Give chelator 30 min before meal time. Sometimes d-pen will require low dose prednisone to stimulate appetite. If inappetence, give dose with a small piece of meat. If vomiting, inappetence, drug intolerance, withdraw and reinitiate treatment with 50% dose reduction and gradually titrate upward.
b. Do NOT combine zinc treatment with D-Penicillamine chelation: zinc uptake by chelator
c. Supplement pyridoxine (25 mg/day B6) during D-penicillamine chelation
2. Reduce Systemic Copper Uptake
a. limit dietary Cu intake: use prescription diets with known low Cu content or home cook using Cu food content in USDA food tables as a guide- see below
Supplement low Cu containing hepatic diets (e.g., LD or Royal Canin Hepatic Support, Purina NF,
Hepatic diets restrict protein intake to 2.2 to 2.5 gm protein/kg body weight; I raise this to 3.5 gm protein/kg body weight by addition of low copper containing foods- This also keeps the liver diet interesting for the patient.
USDA Food Tables- Provides the National Nutrient Database for Standard Reference. Select Copper as the nutrient to search. This shows Cu content per unit/measure. Sort all foods, use nutrient content rather than name, set the measured food at 100g. Then use the SuperTracker USDA webware function to determine how much of a selected low Cu food can be used to supplement the base low protein Cu restricted diet, raising it by 0.5 to 1.5 gm protein /kg body weight if the patient does not have hepatic insufficiency, portal hypertension, or hepatic encephalopathy (HE). Use a more conservative supplement initially in dogs with compromised liver function (HE) or with acquired shunts. It is not clear if this applies to this case.
b. limit water Cu intake: < 0.1 ppm
c. zinc supplementation: chronic therapy AFTER chelation removal of Cu;
Efficacy of zinc remains questionable, recent large retrospective study in humans with Wilson’s disease has demonstrated serious treatment failure with zinc.
3. Glucocorticoids: ONLY dogs with crisis hepatocellular necrosis / hemolysis OR if dog has lymphoplasmacytic inflammatory foci thought to represent immune-mediated inflammation OR dog given d-Penicillamine that becomes hyporexic.
4. Avoid Vitamin C Supplementation: may augment transition metal induced tissue injury
a. vitamin E 10 IU /kg / day PO
b. SAMe (as DenosylTM) 20 mg/kg PO / day on an empty stomach
6. Ursodeoxycholic Acid: if cholestatic injury / high bile acids / severe tissue remodeling: 10-15 mg/kg/day, divided BID with food to improve bioavailability. Ursodiol has better bioavailability than Actigall.
Vitamin E: 10 IU/kg/24hr PO)
SAMe: 20 mg/kg/24hr PO)
Polyunsaturated phosphatidylcholine: metabolized to dilinolylphosphatidylcholine (DLPC) 20-50 mg/kg PO per day; preferably use PhosChol which provides 52% DLPC: 25 mg/kg PO per day. Combined in this treatment protocol this has resulted in remarkable remission in cases based on re-biopsy.
Losartan & Telmisartan- RAAS receptor-2 blockers that are undergoing clinical evaluation as antifibrotics. Currently, Telmisartan is the preferred drug for this application- which has now become commonly used in management of pathologic proteinuria in dogs without complication.
However, combination of Telmisartan with Spironolactone used to treat ascites has demonstrated an adverse drug interaction- with a dual hit on the RAS system leading to serious hypotension, lethargy, collapse, and ARF that responds to drug withdrawal and IV fluid therapy.
8. Milk Thistle- Silibinin or Silymarin: as an hepatoprotectant, role unresolved. If used, select silibinin combined with phosphatidylcholine to provide bioavailability.
Efficacy of chelation is monitored using ALT activity. Best assessment is by liver biopsy, not often pursued.
Most dogs have an ALT that normalizes within 8 weeks, but most take up to 6 mths to clear copper.
Dogs with hepatic copper concentrations > 3,000 ppm may take 9 to 12 mths to clear.
Stay on copper restricted diet
Chronic chelation with 50% dose reduction and administration every other day or 2x weekly has been used in some dogs.
Or change therapy to zinc acetate, use 5-7 mg elemental zinc/kg body weight. Measure plasma zinc at baseline and then at 2-6 wks of treatment to document increase in plasma zinc concentration. Efficacy of long term zinc therapy for complete control of copper accumulation and copper associated hepatopathy remains questionable. Zinc has been shown to be less effective in humans with Wilson’s disease than chronic chelation. In dogs, dose optimization is hard to prove. Zinc commonly leads to gastric irritation and inappetence.
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