Read-Only Case Details Reviewed: Feb 2009

JPC SYSTEMIC PATHOLOGY

URINARY SYSTEM

January 2024

U-T10

SLIDE A: SIGNALMENT (JPC# 1965664): A sheep.

HISTORY: This tissue is from a sheep that died during a hemolytic crisis. Levels of copper in the liver were 1,183 ppm wet weight (normal=70-188 ppm wet weight).

MICROSCOPIC DESCRIPTION: Kidney: Multifocally, numerous cortical and medullary tubular lumina, and less commonly Bowman’s space, are ectatic and contain a bright red, hyaline to granular material (hemoglobin casts) or less often, a pale eosinophilic homogenous to proteinaceous fluid that is often admixed with erythrocytes. Multifocally, tubular epithelial cells demonstrate one or more of the following changes: are attenuated; contain pale vacuolated cytoplasm and swollen nuclei (degeneration); contain hypereosinophilic cytoplasm and pyknotic nuclei (necrosis); or contain numerous eosinophilic intracytoplasmic granules (hyaline droplets). Multifocally, medullary tubular lumina are expanded by variably sized aggregates of deeply basophilic granular material (mineral). There are occasional interstitial aggregates of few lymphocytes and plasma cells. Multifocally the capsular surface is mildly undulant.

MORPHOLOGIC DIAGNOSIS: Kidney: Tubular degeneration and necrosis, acute, diffuse, severe, with hemoglobin and protein casts, hyaline droplet formation, and medullary tubular mineralization, breed not specified, ovine.

ETIOLOGY: Chronic copper toxicity

ETIOLOGIC DIAGNOSIS: Hemoglobinuria-associated acute tubular injury (previously hemoglobinuric nephrosis) secondary to chronic copper toxicity

SLIDE B: SIGNALMENT (JPC # 2292838): A dog.

HISTORY: Tissue from a dog that had several coins composed of zinc (pennies) in the stomach, identified at necropsy.

MORPHOLOGIC DESCRIPTION: Kidney: Multifocally, numerous cortical and medullary tubules are ectatic and lined by attenuated epithelium, with lumina containing bright red, hyaline to granular material (hemoglobin casts) or less often a pale eosinophilic homogenous material (protein). Multifocally, tubular epithelial cells demonstrate one or more of the following changes: contain pale vacuolated cytoplasm and swollen nuclei (degeneration); contain hypereosinophilic cytoplasm and pyknotic nuclei (necrosis); or contain variably sized light brown intracytoplasmic globules (hemosiderin or lipofuscin). Within more severely affected tubules, there is variable disruption of basement membranes (tubulorrhexis). The interstitium is expanded and partially effaced by fibrin, hemorrhage, edema, and cellular debris with scattered aggregates of few lymphocytes and plasma cells. Multifocally within Bowman’s space, there is attachment of visceral to parietal epithelium (synechiae).

MORPHOLOGIC DIAGNOSIS: Kidney: Tubular degeneration and necrosis, acute, diffuse, severe, with hemoglobin casts, tubulorrhexis, and glomerular synechia, breed not specified, canine.

ETIOLOGY: Zinc toxicity

ETIOLOGIC DIAGNOSIS: Hemoglobinuria-associated acute tubular injury (previously hemoglobinuric nephrosis) secondary to zinc toxicity

GENERAL DISCUSSION:

Acute tubular injury (ATI) is an important cause of acute renal failure, most often caused by ischemia (hypotension and/or marked renal ischemia) or nephrotoxins
1. Endogenous nephrotoxins include bile, hemoglobin, and myoglobin
  1. Exogenous nephrotoxins include, but are not limited to, antimicrobials (e.g. aminoglycosides, Amphotericin B, cephalosporins); antineoplastic agents (e.g. cisplatin); cantharidin (blister beetle); ethylene glycol, metals (e.g. arsenic, bismuth, lead, mercury, thallium); monensin, mycotoxins (e.g. ochratoxin A); paraquat; and plants (e.g. Lantana camara, many oxalate-containing plants, Amaranthus retroflexus/pigweed)
Specifically, massive hemolysis can cause ATI and produces a specific pattern of injury known as hemoglobinuria-associated ATI (i.e. tubular degeneration and necrosis in association with pigmentary tubular casts).

Copper/toxicosis

Copper is a heavy-metal and an essential trace element involved in numerous biologic processes (e.g. mitochondrial respiration, antioxidant defense, melanin synthesis, iron metabolism, neurotransmitter biosynthesis)
The liver is the primary regulator of copper levels
Excess copper concentrations (secondary to dietary intake) are toxic, with significant species variation in susceptibility to copper toxicosis
1. Sheep: Prone to copper toxicosis due to reduced biliary excretion of copper; may develop hemolytic crisis (with ATI) secondary to chronic copper toxicosis
2. Excess hepatitis copper accumulation is associated with chronic hepatitis in dogs (D-M17)

Zinc/toxicosis

Zinc is an essential element for multiple biochemical processes in both plants and animals, with a narrow margin between safe levels and toxicity
1. Zinc is a component of the antioxidant enzyme copper-zinc superoxide dismutase
2. Zinc is required for synthesis, storage, and secretion of insulin and glucagon by β and α cells in the pancreas
Metallic zinc is present in alloys (post-1982 pennies) and galvanized products, zinc compounds are used widely in pesticides and herbicides, and zinc salts have been associated with treatment of dermatologic disease in cattle and sheep (also human; zinc oxide in diaper rash products)
The pancreas is the major organ involved in zinc excretion; in animals, zinc toxicity most often affects the pancreas, liver, kidney, and erythrocytes
Susceptibility to zinc toxicity is also species dependent
1. Sheep, other ruminants: Pancreatic ductular elements most susceptible
2. Pigs: Pancreatic acinar tissue most susceptible

PATHOGENESIS:

Copper

Sheep: Excess dietary copper concentrations are toxic due to the two (2) redox states that mediate free-radical production
1. Three (3) environmental factors are involved in ovine copper toxicosis:
  - Excessive copper intake due to contaminated feed, water, or pasture
  - Low dietary levels of molybdenum contributes to increased availability of dietary copper; low dietary sulfur and other minerals (calcium, zinc, iron) can also play a role in limiting copper excretion in bile and urine.
  - Presence of other hepatoxins, including pyrrolizidine alkaloids (Heliotropium, Crotalaria, Senecio sp.; Eastern Australia), phomopsin from lupines (western Austrailia, possibly South Africa)
Copper metabolism: Copper absorbed from the small intestine by divalent metal transporter (DMT1) and copper transporter (Ctr1) > bound to ceruloplasmin, transcuprein, and albumin > transported to liver > sequestered in metallothionein or glutathione > excretion in bile or blood (via proteins such as ATP7A), with excess stored in lysozomes
Copper toxicity:
1. Elevated dietary intake (in conjunction with low dietary molybdenum and presence of other hepatotoxin in sheep) > copper sequestration in hepatocellular lysosomes; if excess copper stored > production of reactive oxygen species > destructive lipid peroxidation > loss of lyosomal membrane integrity and damage to mitochondria > hepatocellular apoptosis > elevated liver-specific enzymes in plasma; once hepatocellular loss exceeds ability of liver to sequester and phagocytose cell debris > elevated plasma copper levels with free copper ions > oxidation of hemoglobin in erythrocytes > intravascular hemolysis > anemia
2. Combined intravascular hemolysis and anemia > accelerated rate of hepatocellular necrosis > increased rate of copper release into blood > acute toxic crisis

Zinc toxicosis

Pathogenesis unclear; may be partially related to inhibition of glutathione reductase and enzymes of the hexose-monophosphate-shunt pathway as absence of these enzymes' function would make RBCs more susceptible to oxidative damage
1. May not be the only factor as not every animal develops Heinz body anemia
Zinc phosphide toxicity: Ingestion (usually powder or pellet used for rodent control) > hydrolyzed to phosphine gas in an acidic environment > directly toxic
The acidic environment of the stomach allows for formation of soluble salts of zinc and systemic absorption after ingestion

TYPICAL CLINICAL FINDINGS:

Sheep with liver copper concentrations >1,000 ug/g (ppm) may be clinically and hematologically normal; paroxysmal intravascular hemolysis and liver failure may result in death within ~6 hours
Icterus
Clinical pathology:
1. Hemoglobinemia (principal feature of intravascular hemolysis)
2. Heinz body anemia; Heinz bodies are formed when oxidized hemoglobin undergoes conformation change and forms membrane-bound precipitates
3. Elevated liver enzymes/bilirubin
4. Hemoglobinuria

Zinc toxicity:

Dogs: Anemia and hyperbilirubinemia are most common clinicopathologic findings
1. Increase in BUN can reflect gastric hemorrhage, pre-renal azotemia (hypovolemia), and acute tubular (pigmentary) injury secondary to hemoglobinuria

TYPICAL GROSS FINDINGS:

Copper toxicity

Liver: Soft, swollen & deep orange; fibrotic & nodular with chronicity
Kidney: Very dark, almost black “gunmetal blue” discoloration due to entrapped hemoglobin and deep red urine
Spleen: Enlarged, soft, dark in color
Icteric carcass with superimposed red or brown coloration due to free hemoglobin/methemoglobin

Zinc toxicity

Kidney: Reddish-brown kidneys (dogs)
Pancreas: Enlarged and pale with lobulation in acute toxicity; shrinkage and nodularity with chronicity
Liver: Reduced size, with prominent fatty degeneration and sunken region of necrosis/collapse with acute lesions and scarring in chronic lesions

TYPICAL LIGHT MICROSCOPIC FINDINGS:

Intravascular hemolysis from either copper or zinc results in ischemic acute tubular injury characterized by:
1. Severe acute focal necrosis of tubules (especially proximal tubule) with disruption of the basement membrane (tubulorrhexis)
2. Fine red granules (hemoglobin) may also present in tubular epithelial cells
3. Occlusion of tubular lumina by eosinophilic hyaline and hemoglobin casts (orange-red granular material)
4. Tubular regeneration
5. Often, normal glomeruli

Copper toxicity

Liver: Multifocal centrilobular and midzonal (to possibly massive) necrosis with foci of macrophages, plasma cells, neutrophils, and intracytoplasmic copper, predominately within centrilobular region
North Ronaldsay sheep: Hepatic periportal and portal tract fibrosis, with cholangiolar proliferation, and mixed inflammatory infiltrates

Zinc toxicity

Pancreas
1. Acute: Degeneration and necrosis of exocrine pancreas; endocrine pancreas unaffected; interstitial edema
2. Chronic: Exocrine acinar cells become atrophic and form dilated ductule-like structures without discernible zymogen granules (tubular complexes) often mistaken for hyperplastic ductules; interstitial fibrosis
Liver: Post-necrotic scarring with chronicity

Kidneys: Acute tubular injury similar to copper, but generally targets the loops of Henle

ADDITIONAL DIAGNOSTIC TESTS:

Copper: Positive rhodanine or rubeanic acid stains; blood and kidney copper levels
Zinc: Abdominal radiographs for ingested zinc products
Heavy metal concentrations in tissue
JMS silver stain: To identify kidney tubulorrhexis

DIFFERENTIAL DIAGNOSIS:

Histologic differentials, red pigment in kidneys

Hemoglobinuric nephrosis (intravascular hemolysis)

Cattle: Leptospirosis or babesiosis
Horses: Red maple (Acer rubrum) toxicity, neonatal isoerythrolysis
Dogs: Autoimmune hemolytic anemia

Myoglobinuric nephrosis (indistinguishable from hemoglobin casts)

Horses: Exertional rhabdomyolysis, polysaccharide storage myopathy, monensin toxicity, plant toxicity (e.g. white snakeroot, coffee senna)
Wildlife and zoo animals: Capture myopathy
Severe trauma in any species

Heinz body hemolytic anemia

Onion ingestion (dog, cattle, horse)
Acetaminophen (cat)
Rape or kale (cattle, sheep)
Phenothiazine (horse)
Methylene blue, a urine acidifier (cat)
Red maple (horse)
Inherited glucose-6-phosphate deficiency (rarely, sheep and dog)
Postparturient hemoglobinuria (cattle)
Selenium deficiency
Normal finding (cat 5%)

COMPARATIVE PATHOLOGY:

Copper toxicity

Dogs: Hepatic copper accumulation is most important mechanism (D-M17)
1. Primary accumulation disease due to a metabolic defect that impairs biliary copper excretion; accumulation is centrilobular and concentrations greater than 2,000 ppm
2. Bedlington terriers – mutation of the COMMD1 gene (autosomal recessive)
3. Breed associated copper accumulation hepatitis (mutations unknown) – Skye terrier, Dalmation, West Highland white terrier, Doberman pinscher, Cocker spaniel, and Labrador retriever
4. Accumulation secondary to hepatic inflammation that alters biliary excretion of copper; accumulation is primarily periportal, and less than 2,000 ppm
5. Dietary excess
Other ruminants, and pigs: Acute poisoning most often seen after accidental administration of large doses of copper; chronic poisoning most often due to feed or diet supplementation errors
1. Copper toxicity reported in a 8-month-old crossbred calf with ataxia and altered mentation; gross lesions included diffusely yellow-orange liver, diffuse, marked brown-black discoloration of renal cortex and medulla, urinary bladder filled with dark red urine; microscopic lesions included centrilobular hepatocellular degeneration lacking necrosis, intraluminal bright eosinophilic fluid and red-orange granular casts that stained positive for hemoglobin (Dunn-Thompson method) (Wong, J Vet Diagn Invest. 2020)
Goats: Considered resistant to chronic copper toxicosis (adults especially) and do not develop a hemolytic crisis; meat breeds generally more resistant; exception is Boer goats
Rabbits: Considered sensitive to copper toxicosis; hemolytic anemia with intravascular hemolysis, erythrophagocytosis in spleen, hematuria with tubular casts, hepatic centrilobular necrosis with Kupffer cells containing blue-green granules (granules stain positive for rhodanine)

Wildlife and zoo animals:
1. Invertebrates: Copper toxicity reported in aquatic invertebrates, with variation in susceptibility based upon species; larval oysters, adult and larval mussels, and some echinoderms are considered some of the most copper sensitive taxa
  - Gross lesions: “Distress syndrome” in freshwater pulmonate snails (immobility, muscular spasms, extension of the cephalopedal mass, inability to attach the foot to substrate, swelling of tentacles, epidermal sloughing
  - Microscopic lesions: Zooxanthella expulsion in cnidarians, branchial epithelial necrosis, amoebocytic inflammation in connective tissue, necrosis and sloughing of mucosa in gastropods; mucosal necrosis in stomach, digestive gland atrophy in oysters; inflammation, necrosis, vacuolation in digestive diverticula, kidney, and intestinal tract of clams; branchial necrosis in crabs; epidermal ulceration and irregular epidermal contour in numerous aquatic invertebrate species

Zinc toxicity

Pigs: Pancreatic signs are the most consistently reported, but many other are possible with chronic exposure to include bone/joint changes
1. Chronic pancreatitis associated with excess dietary zinc oxide administered on a pig farm in Japan; gross lesions included a shrunken pancreas, slightly swollen livers, and the cranial portion of each lung lobe was dark and purple; microscopic lesions included replacement of pancreatic parenchyma by fat cells, decreased number of exocrine acinar cells with increased irregular cuboidal epithelial duct-like cells separated by collagen, fibroblasts, and few lymphocytes; pancreatic islet cells were also decreased and shrunken and IHC with glucagon and somatostatin antibodies revealed shrunken α and β cells (Komatsu T, et al., J Vet Diagn Invest. 2020)

Ferrets:
1. Clinical: Pale mucous membranes secondary to anemia, posterior weakness, and lethargy
2. Gross: Kidneys are large, pale, and soft with capsular depressions; liver is orange; gastric erosions are present
3. Histology: Kidneys contain collapsed glomeruli and dilated tubules; liver contains periacinar fatty infiltration; gastric erosions and hemorrhage; bone marrow contains reduced erythroid precursors

Birds: Pancreas is the target organ of zinc toxicity, +/- ventricular ulceration; renal and hepatic lesions are similar to mammals

Wildlife and zoo animals:
1. Ostrich: Pancreatic exocrine atrophy and fibrosis with regenerative ducts
2. Anseriformes, Ciconiiformes, Charadriiformes, Gruiformes: Variable pancreatic granule depletion and acinar cell atrophy and/or necrosis with disruption of normal acinar architecture in acute toxicity; progressive interstitial and parenchymal fibrosis, and exocrine pancreatic regeneration and/or ductular hyperplasia with chronic lesions; other lesion include degeneration and necrosis of the koilin, glandular epithelium and smooth muscle in the ventriculus; and hepatic hemosiderosis
3. Captive striped hyena (case report): Zine toxicosis secondary to ingestion of pennies; severe nonregnerative anemia, jaundice, and gallbladder edema; multifocal thrombosis; centrilobular hemosiderosis, cholestasis, Kupffer cell hyperplasia, and single-cell necrosis in the liver; intraluminal casts and lymphoplasmacytic, tubulointerstitial nephritis in the kidney

REFERENCES:

1. Barthold SW, Griffey SM, Percy DH. Pathology of Laboratory Rodents and Rabbits. 4th ed. Ames, IA: Wiley Blackwell; 2016:315.

2. Brown DL, Van Wettere AJ. Hepatobiliary system and exocrine pancreas. In: Zachary JF, ed. Pathologic Basis of Veterinary Disease. 7th ed. St. Louis, MO: Elsevier; 2022:512, 529, 533.

3. Burrough ER, De Mille C, Gabler NK. Zinc overload in weaned pigs: tissue accumulation, pathology, and growth impacts. J Vet Diagn Invest. 2019;31(4):537-545

4. Cianciolo RE, Mohr FC: Urinary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2, 6th ed. St. Louis, MO: Elsevier; 2016:422-424,429-430.

5. Church ME, Terio KA, Keel MK. Procyonidae, viverridae, hyenidae, herpestidae, eypleridae, and prionodontidae. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 307.

6. Cullen JM, Stalker MJ. Liver and biliary system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2, 6th ed. St. Louis, MO: Elsevier; 2016:342-343.

7. Fox JG. Other systemic diseases. In: Fox JG, Marini RP, eds. Biology and Diseases of the Ferret. 3rd ed. Ames, IA: John Wiley & Sons, Inc; 2014:435-436.

8. Jubb KVF, Stent AW. Pancreas. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 2, 6th ed. St. Louis, MO: Elsevier; 2016:356-357.

9. Komatsu T, et al. Chronic pancreatitis in farmed pigs fed excessive zinc oxide. J Vet Diagn Invest. 2020; 32(5): 689-694.

10. Newton AL, Smolowitz R. Invertebrates. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 1013, 1015-1016.

11. Schmidt RE, Reavill DR, Phalen DN. Pathology of Pet and Aviary Birds. 2nd ed. Ames, IA: Wiley Blackwell; 2015:73,88,140.

12. Smith DA. Palaeognathae: Apterygiformes, casuariiformes, rheiformes, struthioniformes, tinamiformes. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018:637

13. Valli VEO, Kiupel M, Bienzle D: Hematopoietic system. In: Maxie MG, ed. Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals. Vol 3. 6th ed. St. Louis, MO: Elsevier; 2016:125-126.

14. Viner TC, Kagan RA. Forensic wildlife pathology. In: Terio KA, McAloose D, St. Leger J, eds. Pathology of Wildlife and Zoo Animals. San Diego, CA: Elsevier; 2018: 33.

15. Watson JL, George LW. Copper toxicosis. In: Smith BP, ed. Large Animal Internal Medicine. 6th ed. St. Louis, MO, Elsevier , 2020:1174-1176.

16. Wong A, Wilson-Frank CR, Hooser SB, Burcham GN. Chronic copper toxicosis in a crossbred heifer calf. J Vet Diagn Invest. 2020; 32(3):458-462.

17. Zabolotzky SM, Walker DB. Peripheral Blood Smears. In: Valenciano, ed. Diagnostic Cytology and Hematology of the Dog and Cat. 5^th ed. St. Louis, MO: Elsevier; 2020:445-456.