Copper-associated hepatitis (CAH) is a well-recognized disease in dogs. Since the original description of copper toxicosis in Bedlington Terriers in 1975, other affected breeds, including West Highland White Terriers, Doberman Pinschers, and Dalmatians, have been identified.[1-4] More recently, several reports have described CAH in Labrador Retrievers.[5-7] Although a specific genetic mutation has only been described in Bedlington Terriers, CAH is presumed to be hereditary in other affected breeds.[8, 9]
The pathophysiologic consequences of excess hepatic copper, as well as therapies to address this problem, are well described. Yet, little remains known about mechanisms of copper accumulation in non-Bedlington Terrier breeds.[2, 5, 8] Given that sex predisposition, signalment, and clinical course vary among breeds, it is probable that different genetic mutations are involved.[1-3, 8] Furthermore, it remains unknown whether copper can accumulate in other organs and result in dysfunction in those organs. Specific organ or tissue accumulation could vary depending on the mutation. There are a variety of copper storage disorders in humans. Wilson's disease is the best-described disorder in humans and is characterized by reduction or absence of ATP-7B gene expression resulting in copper accumulation in the brain, eyes, and kidneys in addition to the liver. In fact, approximately 40–50% of patients present for neurologic disease or signs unrelated to hepatic copper accumulation.[11, 12]
Recently, 2 case reports (not in Labrador Retrievers) have identified concurrent CAH and transient Fanconi syndrome that resolved with d-penicillamine chelation therapy.[13, 14] Renal copper was only quantitated in 1 dog and found to be abnormally high at 212 ppm (reference range, 17.5–60 ppm), and the semiquantitative copper determination (rhodanine-stained kidney sections) was negative in this dog. Another report documented Fanconi syndrome in a Labrador Retriever with transiently increased liver enzyme activity, but hepatic biopsy was not performed. In another series of 24 Labrador Retrievers with chronic hepatitis, 3 dogs were noted to have transient renal glucosuria. However, it remains unclear if these dogs had CAH. Furthermore, renal histopathology and follow-up information were not reported. Studies characterizing concurrent proximal renal tubular dysfunction and CAH in Labrador Retrievers have not been reported. The objective of this study was to describe clinical features, diagnostic findings, renal and hepatic histopathology, quantitative tissue copper concentrations, and outcomes in 9 Labrador Retrievers with concurrent CAH and proximal renal tubular dysfunction.
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- Materials and Methods
Copper-associated hepatitis is a common liver disease in Labrador Retrievers.[5-8] Diagnosis requires hepatic histopathology with either semiquantitative or quantitative copper determinations. Both the amount and location of copper accumulation are important in reaching a diagnosis. Eight dogs in our series had classic CAH characterized by centrilobular hepatitis and copper accumulation. One dog had moderate increases (1200 ppm) in quantitative copper. However, the presence of fibrosis decreases the relative copper concentration, and regenerative nodules often do not accumulate copper. This dog had severe fibrosis and nodular regeneration. It is possible that the tissue analyzed quantitatively contained fibrosis or regenerative nodules, which may explain the discrepancy between rhodanine staining and quantitative copper concentrations. This case still was considered to be CAH given the breed, rhodanine grading, and association of centrilobular inflammatory changes with hepatocellular copper.
Acquired diseases of the proximal renal tubule are rare in veterinary medicine. Primary renal glucosuria, Fanconi syndrome, and type II renal tubular acidosis have all been described.[15, 20, 21] Fanconi syndrome is characterized by failure of normal proximal tubular reabsorptive function resulting in excessive loss of glucose, amino acids, water, bicarbonate, phosphate, and other electrolytes. Renal glucosuria often is the initial finding before the development of overt Fanconi syndrome.[19, 22] Osmotic diuresis results in PU/PD in many of these dogs. With disease progression, hyperchloremic metabolic acidosis and renal azotemia often develop. Although substantial information is available on Fanconi syndrome in Basenjis, there is little published information characterizing acquired renal tubulopathies. Several reports have described acquired, sometimes transient tubulopathies in dogs in association with a variety of diseases, including acute kidney injury, renal toxins, primary hypoparathyroidism, and CAH.[13, 14, 24, 25]
Eight dogs in our series had PU/PD. A previous report of Labrador Retrievers with hepatitis only documented PU/PD in 2 of 24 dogs. On the basis of our inclusion criteria (renal tubular dysfunction), the presence of PU/PD is not unexpected. Glucosuria resulting in an osmotic diuresis is 1 explanation. However, PU/PD is known to occur in various liver diseases in which there is no evidence of renal tubular dysfunction. Although exact mechanisms are unknown, renal medullary washout secondary to decreased blood urea nitrogen concentrations, stimulation of thirst receptors secondary to hepatic encephalopathy, and increased endogenous cortisol production have all been proposed. Three dogs in our series had decreased blood urea nitrogen concentrations, 2 dogs had increased fasting ammonia concentrations, and 2 dogs were azotemic. Furthermore, the trace glucosuria observed in 2 dogs was unlikely to induce substantial osmotic diuresis. As such, the PU/PD in our series of dogs is likely multifactorial. Nevertheless, PU/PD is an important historical finding that could be indicative of underlying renal tubular dysfunction.
The presence of renal glucosuria localized disease to the proximal renal tubules in all dogs in our report. The findings of low to low normal serum phosphorous concentration (≤3.0 mg/dL; 6/7), mild proteinuria (8/9), and low to low normal serum TCO2 (≤20 mEq/L; 7/7) support more global dysfunction of the proximal renal tubules and are suggestive of Fanconi syndrome. The presence of hyperchloremic metabolic acidemia documented in 4 dogs in our series also is a classic finding associated with both proximal renal tubular acidosis (type II) and progressive Fanconi syndrome. However, confirmatory testing for Fanconi syndrome only was performed in 2 dogs. In previously reported cases of concurrent Fanconi syndrome and CAH in dogs, proteinuria and glucosuria also were detected. However, acid base status and urine pH were only reported in 1 dog (hyperchloremic metabolic acidemia with alkalinuria).[13, 14] In our series, we documented metabolic acidemia in all dogs in which blood pH determinations were made. Interestingly, the majority of dogs (78%) in this study paradoxically had neutral to alkaline urine despite evidence of systemic acidosis (low normal or decreased serum TCO2). In an animal with only proximal tubular dysfunction, distal urinary acidification mechanisms are still intact, and an acidic urine pH can be achieved, especially in the face of acidemia. This is consistent with distal renal tubular acidosis (type I). As such, it is possible that some dogs had defects in both proximal and distal tubular function. The documented copper accumulation in the renal medulla supports this possibility. The normal anion gap hyperchloremic metabolic acidosis present in the majority of dogs in our study is consistent with renal tubular acidosis. However, it is probable that some dogs had mixed acid base disturbances as evidenced by 1 dog (patient 3) that had hyperchloremic metabolic acidosis and metabolic alkalosis. Furthermore, the presence of renal tubular acidosis with concomitant vomiting, diarrhea, dehydration, and azotemia could contribute to mixed acid base disturbances in other dogs. Despite this possibility, the predominant clinical and laboratory picture was that of a hyperchloremic (normal anion gap) metabolic acidosis consistent with renal tubular acidosis.
Renal histopathologic abnormalities in our series of dogs were similar to those in 2 previous reports, but it remains unclear from these reports if increased renal copper was associated with the renal tubulopathy.[13, 14] Of the 4 previously reported cases, increased renal copper was documented in 2 dogs. Staining for renal copper was negative in 1 dog; 1 dog did not undergo renal biopsy.[13, 14] In our series of dogs, increased copper deposition was observed in renal tissues of all 4 dogs in which kidney specimens were available. Our observations suggest that renal tubular abnormalities are due, at least in part, to renal copper accumulation. The decision to perform rhodanine staining or ICP-AES was based on the amount of tissue available. Quantitative copper analysis was deemed most important if only small, but sufficient, amounts of tissue were available. Given that the normal amount of renal copper is low (17.5–60 ppm, dry matter basis), histochemical staining methods (rubeanic acid or rhodanine) may not consistently detect increased renal copper. In liver tissue, histochemical staining usually is not positive until copper concentration exceeds 300–400 ppm.[3, 8]
Abnormalities of renal tubular function have been identified in humans with Wilson's disease and Menkes disease, an X-linked recessive condition caused by mutations in the ATP-7A gene.[27-30] Although renal tubular dysfunction in Labradors with CAH and humans with Wilson's and Menkes disease is associated with renal copper accumulation,[28, 30] the exact pathogenic mechanism of copper-induced injury is unclear. The proximal tubule is the site of nephrotoxicity caused by many metals. Copper is known to increase the rate of the normally slow-occurring Haber–Weiss reaction, leading to enhanced free radical formation and subsequent oxidative damage, cellular necrosis, and inflammation. Copper also may alter Na-K-ATPase activity in tubular epithelial cells. It is likely that these prooxidant and enzyme altering effects of copper are involved in the pathogenesis of copper-induced renal tubular disease. In rodent models of Wilson's and Menkes disease, proximal tubular epithelial copper accumulation was observed under both acute and chronic copper loading conditions, whereas other components of the nephron were relatively spared.[32-34] In 1 study, proximal tubular cell disarray and irreversible nuclear damage were correlated with increasing intranuclear copper accumulation. In the same study, decreases in copper and tubular recovery were associated with lysosomal sequestration and excretion of copper into the tubular lumen. However, in a different model of Wilson's disease, it was suggested that renal dysfunction was independent of increased renal copper. Their findings suggested that copper-metallothionein complexes released from the damaged liver were freely filtered at the glomerulus and then induced dysfunction of brush border membranes of the proximal tubular cells after the complexes split into free copper and amino acids. Similarly, copper-metallothionein complexes also were demonstrated in excess in the proximal tubular cells in a model of Menkes disease. However, the finding of metallothionein mRNA in the kidney suggested that formation of these complexes occurred in situ. Although proposed mechanisms of copper-induced renal injury vary with different models, most reports are in agreement that copper plays a causative role, and resolution of disease depends on reduction of tissue copper.[27-29] The source of renal copper in our dogs remains unknown. However, hepatic release of excess copper is an appealing hypothesis in these Labradors given the degree of hepatic necrosis noted in 6 dogs. Nevertheless, we cannot exclude the possibility that chronic renal copper accumulation observed in our dogs resulted from processes independent of hepatic copper or that multiple mechanisms may be involved. Additional studies are needed to determine the source of renal copper in dogs with CAH.
On the basis of the previous descriptions of concurrent Fanconi syndrome and CAH, it would seem that copper chelation is essential for resolution of renal tubular disease.[13, 14] Studies of Wilson's disease also support this assumption. However, in our series of dogs, resolution of urinalysis abnormalities (proteinuria and glucosuria) occurred in 2 dogs without copper chelation. In another surviving dog, proteinuria and glucosuria had nearly resolved by the time copper chelation had commenced. These findings suggest that renal tubular dysfunction may resolve with or without copper chelation. There are several plausible explanations including dietary intervention and effects of other pharmacologic agents. All dogs were fed a low copper diet which has been shown to decrease hepatic copper concentrations in Labradors with CAH. However, gradual reduction by dietary restriction is unlikely to be the sole explanation. All dogs were treated with antioxidants, which potentially could ameliorate the oxidative damage induced by excess tissue copper. The use of steroids in 2 of the dogs may have decreased hepatic inflammation and decreased the amounts of copper released from the damaged liver. Alternatively, steroids may have directly decreased renal inflammation, but there was only mild inflammation initially. Finally, administration of ursodeoxycholic acid to 3 dogs may have either altered tissue copper concentrations or blunted the deleterious effects of excess tissue copper by increasing bile flow, inhibiting apoptosis, modulating immune responses, or increasing glutathione and metallothionein production. Although treatment was varied and sometimes nonspecific, some type of treatment is likely necessary for the resolution of copper-induced tubular disease. This hypothesis is supported by the observation that 3 of these dogs had been glucosuric for weeks, and improvement was not seen until medical therapy was initiated. However, the most important aspect of treatment remains unknown as all dogs received multiple therapeutic interventions. None of the surviving dogs required alkali administration or other specific treatments for their tubulopathy.
The prevalence of renal tubular disease and its relation to the prognosis of CAH in dogs is unknown. On the basis of increases in bilirubin concentrations in all 9 dogs, it is possible that development of renal tubular dysfunction is associated with more severe or advanced liver disease. This is further supported by the fact that all dogs had overt clinical signs relating to liver disease as opposed to serendipitous discovery of increased liver enzyme activity. An excellent long-term prognosis has been previously reported for dogs with concurrent CAH and Fanconi syndrome.[13, 14] This somewhat contrasts with our series in which 4 of 9 dogs died near the time of diagnosis. However, some dogs did have long-term survival with resolution of clinical renal and hepatic disease. Additional studies are needed to more accurately define the prognostic importance of renal tubular dysfunction in dogs with CAH. Even if renal tubular abnormalities correlate with more advanced disease, a favorable prognosis still is possible in affected dogs.
In summary, concurrent renal tubular dysfunction and CAH is likely more common than previously recognized (33% of the MSU-VTH Labradors reviewed for inclusion in this manuscript), although the overall incidence remains unknown. Detection of renal tubular abnormalities in a Labrador Retriever with biochemical evidence of liver disease is strongly suggestive of CAH and indicates the need for hepatic histopathology. Although renal tubular lesions are present in affected dogs and appear to be associated with copper accumulation, the necessity of renal histopathology is less clear as it is unlikely to alter patient management. The exact mechanism of renal copper accumulation and injury remains unknown and requires additional study. Despite the association of renal tubular dysfunction with more advanced disease, successful long-term outcome is possible in these dogs with a variety of therapies with or without d-penicillamine.