Pathology in Practice

Simone de Brot School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, England.

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Jessica Adamany Pride Veterinary Centre, Riverside Road, Derby, Derbyshire DE24 8HX, England.

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Kerstin Baiker School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, England.

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Marc Dhumeaux Pride Veterinary Centre, Riverside Road, Derby, Derbyshire DE24 8HX, England.

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Cinzia Allegrucci School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, England.

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Laura Polledo School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, England.

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Llorenç Grau-Roma School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, England.

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History

A 13-month-old 8.8-kg (19.4-lb) neutered male orange roan English Cocker Spaniel (Canis familiaris) underwent clinical examination because of weight loss and lethargy. The owner reported an approximate weight loss of 1.5 kg (3.3 lb) within 1 month and a 4-day history of anorexia.

Clinical and Gross Findings

Serum biochemical and urinary analyses revealed severe azotemia (serum creatinine concentration, 740 mg/dL [reference range, 0.3 to 1.7 mg/dL]) and marked proteinuria (urine protein-to-creatinine ratio, 22 [reference range, 0 to 0.5]). Because of the poor prognosis for recovery, the dog was euthanized by means of IV injection of pentobarbital sodium and necropsied within 24 hours. The dog was in rather poor body condition. There was moderate subcutaneous edema, which mainly affected the ventral abdominal and inguinal areas. Approximately 150 mL of yellow-red transparent liquid was present within the abdomen (ascites), and 100 mL of similar fluid was present in the thorax (hydrothorax). The lungs were moderately edematous. Both kidneys were diffusely pale tan. The renal cortical surfaces appeared diffusely rough and granular with several multifocal to coalescing, irregular and small (up to 2 cm in length and 1 cm in width) pitted areas (Figure 1). On incision, the cortex had a generalized punctiform or granular appearance.

Figure 1—
Figure 1—

Photograph of the kidneys from a 13-month-old English Cocker Spaniel that underwent clinical examination because of weight loss of approximately 1.5 kg (3.3 lb) within 1 month. The dog had severe azotemia and marked proteinuria; because of a poor prognosis for recovery, the dog was euthanized. Notice that both kidneys are very pale and have a diffusely rough surface. The cortex has a diffuse granular appearance and contains moderate numbers of multifocal to coalescing irregular pitted areas. Scale length = 3 cm.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.661

Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page→

Histopathologic Findings

Samples of various tissues were routinely processed for histologic examination and stained with H&E stain. Additional kidney sections were stained with periodic acid–Schiff, Masson trichrome, or Congo red stains.

In sections of both kidneys, all renal glomeruli had marked segmental to global damage accompanied by mild to moderate multifocal interstitial inflammation and fibrosis. The Bowman spaces of most glomeruli were expanded by abundant brightly eosinophilic, homogeneous material (protein-rich glomerular filtrate [Figure 2]). This proteinaceous material stained bright red with Masson trichrome stain (Figure 3) and purple with periodic acid–Schiff stain (Figure 4); there was no uptake of Congo red stain. Often, glomeruli had multifocal to diffuse thickening of the Bowman capsule, segmental to global thickening of capillary basement membranes, and mild to moderate increase in cellularity. Adhesions between the glomerular tuft and Bowman capsule (synechiae) and periglomerular fibrosis were common. Some glomerular tufts were shrunken, eosinophilic, and hypocellular (global glomerulosclerosis [obsolescence]). There was mild to moderate interstitial (often periglomerular) lymphoplasmacytic inflammation; mild to moderate interstitial fibrosis often extended from the cortex to the medulla, forming radial streaks. Tubules were frequently distended with intraluminal eosinophilic material (proteinuria).

Figure 2—
Figure 2—

Photomicrograph of a section of a kidney from the dog in Figure 1. Renal glomeruli have abundant eosinophilic material (protein-rich glomerular filtrate) in the Bowman space (asterisks), marked mesangial cellularity, synechiae, and periglomerular fibrosis (arrow). Renal tubules often contain pale eosinophilic intraluminal material (dagger). H&E stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.661

Figure 3—
Figure 3—

Photomicrograph of a section of a kidney from the dog in Figure 1. There is moderate interstitial, periglomerular, and glomerular fibrosis (stained blue [asterisk]). The protein-rich glomerular filtrate within the Bowman space is stained bright red (dagger). Masson trichrome stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.661

Figure 4—
Figure 4—

Photomicrograph of a section of a kidney from the dog in Figure 1. There is moderate thickening of the Bowman capsule (arrow) and basement membranes of glomerular capillaries and renal tubules (stained purple-magenta [arrowhead]). The protein-rich glomerular filtrate within the Bowman space is stained purple (asterisk). Periodic acid–Schiff stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 251, 6; 10.2460/javma.251.6.661

Light microscopic examination of sections of other organs and tissues revealed moderate pulmonary and interstitial pancreatic edema. No other relevant histologic abnormalities were observed.

Additional Laboratory Findings

Deoxyribonucleic acid was extracted from some of the dog's hair follicles collected at necropsy. Extracted DNA was submitted to a commercial laboratorya for PCR assay detection of an A → T single nucleotide substitution at base 115 of the COL4A4 gene, as previously described.1 Results indicated that the dog was homozygous for this mutation.

Morphologic Diagnosis and Case Summary

Morphologic diagnosis: severe, chronic, generalized, segmental to global, bilateral membranoproliferative glomerulonephritis with glomerulosclerosis and proteinuria.

Case summary: hereditary nephropathy (familial nephropathy) in an English Cocker Spaniel.

Comments

The clinical signs and clinicopathologic abnormalities in the dog of the present report were suggestive of chronic renal failure. The severe azotemia and proteinuria were directly related to decreased renal function. In general, chronic renal failure in young animals can be classified as congenital, inherited, familial (develops in animals in a closely related group), or acquired.

Hereditary nephropathy (also called familial nephropathy or hereditary nephritis) is the most commonly used name for kidney diseases that develop in dogs as a result of type IV collagen abnormalities.1–3 This group of diseases is considered analogous to Alport nephropathy in humans, although the latter is usually associated with ocular and hearing abnormalities, which have not been reported for dogs with hereditary nephropathy.4–8 To date, 4 type IV collagen gene mutations have been identified in dogs with hereditary nephropathy. Breeds affected include English Cocker Spaniels,1 Samoyeds,9 mixed-breed dogs,10 and English Springer Spaniels.11

Hereditary nephropathy in English Cocker Spaniels is an invariably progressive and ultimately fatal renal disease, which typically causes renal failure in dogs that are 6 months to 2 years of age. The disease is inherited as an autosomal recessive trait and is associated with a single base substitution in the COL4A4 gene that causes premature truncation of the protein.1 Collagen type IV defects cause severe structural and functional alterations in glomerular basement membranes (GBMs).2,5,6,8 Unlike most collagens, type IV collagen is present only in the basement membranes and comprises up to 6 genetically distinct α-chains; these α-chains are designated as α1(IV) through α6(IV) and are encoded by 6 genes (COL4A1 through COL4A6).8 Three defined trimer combinations (α1α1α2, α3α4α5, and α5α5α6) are formed from the 6 α(IV) chains.2,6,8 The α1 and α2 chains are present in the basement membranes of all tissues, whereas the other 4 chains have restricted tissue distribution during development.6,8 The expression of collagen type IV chains is also subject to temporal regulation.8 In GBMs, genes encoding the α1 and α2 chains are expressed during embryonic development, but their expression levels gradually decrease as the expression of genes encoding the α3, α4, and α5 chains starts. This switch in gene expression is the reason that affected animals are born apparently healthy and have an early onset of disease. In affected English Cocker Spaniels, the mutation in COL4A4 introduces a premature stop codon, generating truncated α4 chain proteins. Truncated α4 chains are unable to combine with normal partners (ie, α3 and α5) to produce α3α4α5 collagen type IV heterotrimers in mature glomeruli.1

In affected English Cocker Spaniel puppies, the first clinical sign of disease (proteinuria) becomes evident at 2 to 8 months of age.1 Renal damage progresses and end-stage renal disease is often detected at 12 months of age (age range, 6 to 18 months),2 as illustrated by the case described in the present report.

The light microscopic lesions in the kidneys of dogs with hereditary nephropathy are reported to be segmental thickening of GBMs and mesangial expansion, progressing through glomerular and periglomerular fibrosis and glomerulosclerosis. Typically, these lesions are accompanied by tubulointerstitial nephritis, interstitial fibrosis, and tubular atrophy.2,3 All these light microscopic features were observed in the dog of the present report.

Immunohistochemical analysis and transmission electron microscopy are useful techniques to further characterize the lesions in dogs with hereditary nephropathy but were not performed in the case described in the present report. Normal mature GBMs are known to strongly express α3, α4, and α5 chains of type IV collagen. However, in English Cocker Spaniels with hereditary nephropathy, the expression of those chains is either absent (α4) or greatly reduced (α5). In addition, there is increased expression of 3 other chains (α1, α2, and α6) that would normally only be minor components of the mature GBMs and therefore weakly expressed.2 Ultrastructural changes in GBMs are common to all the genetically characterized forms of hereditary nephropathy. The principal change is an irregular thinning or thickening of GBMs with splitting and fragmentation of the lamina densa. There is also often fusion of visceral epithelial cell foot processes and wrinkling of glomerular capillary walls.2,12

For the dog of the present report, a definitive diagnosis was based on results of a DNA test offered by a commercial laboratory.a Genetic tests are recommended in cases of suspected hereditary nephropathy in English Cocker Spaniels and are widely available.13

Acknowledgments

Dr. Grau-Roma's research is funded by the People Programme (Marie Curie Actions) of the European Union's Seventh Framework Programme (FP7/2007–2013) under REA grant agreement No. PCOFUND-GA-2012-600181.

The case presented in this manuscript was submitted to the Joint Pathology Center Veterinary Pathology Service Wednesday Slide Conference, Silver Spring, Md, November 2015.

The authors thank Alan Lasslett, Joanne Sanders, and Natalie Watson for technical assistance.

Footnotes

a.

Antagene, La Tour-de-Salvagny, France.

References

  • 1. Davidson AG, Bell RJ, Lees GE, et al. Genetic cause of autosomal recessive hereditary nephropathy in the English Cocker Spaniel? J Vet Intern Med 2007; 21:394401.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Lees GE. Kidney diseases caused by glomerular basement membrane type IV collagen defects in dogs J Vet Emerg Crit Care (San Antonio) 2013; 23:184193.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Maxie MG, Newman SJ. Urinary system In: Jubb, Kennedy, and Palmer's pathology of domestic animals. 5th ed. Vol 2. St Louis: Saunders Elsevier, 2007;460.

    • Search Google Scholar
    • Export Citation
  • 4. Longo I, Porcedda P, Mari F, et al. COL4A3/COL4A4 mutations: from familial hematuria to autosomal-dominant or recessive Alport syndrome? Kidney Int 2002; 61:19471956.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Gubler MC. Inherited diseases of the glomerular basement membrane Nat Clin Pract Nephrol 2008; 4:2437.

  • 6. Heidet L, Cai Y, Guicharnaud L, et al. Glomerular expression of type IV collagen chains in normal and X–linked Alport syndrome kidneys? Am J Pathol 2000; 156:19011910.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Deltas C, Pierides A, Voskarides K. Molecular genetics of familial hematuric diseases Nephrol Dial Transplant 2013; 28:29462960.

  • 8. Khoshnoodi J, Pedchenko V, Hudson BG. Mammalian collagen IV Microsc Res Tech 2008; 71:357370.

  • 9. Zheng K, Thorner PS, Marrano P, et al. Canine × chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha 5 chain of collagen type IV? Proc Natl Acad Sci USA 1994; 91:39893993.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Cox ML, Lees GE, Clifford EK, et al. Genetic cause of X-linked Alport syndrome in a family of domestic dogs? Mamm Genome 2003; 14:396403.

  • 11. Nowend KL, Starr-Moss AN, Lees GE, et al. Characterization of the genetic basis for autosomal recessive hereditary nephropathy in the English Springer Spaniel? J Vet Intern Med 2012; 26:294301.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Lees GE, Wilson PD, Helman RG, et al. Glomerular ultrastructural findings similar to hereditary nephritis in 4 English Cocker Spaniels? J Vet Intern Med 1997; 11:8085.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. British Kennel Club website. Dog health. Breed specific DNA tests (up-dated April 2015). Available at: www.thekennelclub.org.uk/media/14688/dnatestsworldwide.pdf#sthash.eQZ2iMgB.dpuf. Accessed Jun 29, 2015.

    • Search Google Scholar
    • Export Citation
  • Figure 1—

    Photograph of the kidneys from a 13-month-old English Cocker Spaniel that underwent clinical examination because of weight loss of approximately 1.5 kg (3.3 lb) within 1 month. The dog had severe azotemia and marked proteinuria; because of a poor prognosis for recovery, the dog was euthanized. Notice that both kidneys are very pale and have a diffusely rough surface. The cortex has a diffuse granular appearance and contains moderate numbers of multifocal to coalescing irregular pitted areas. Scale length = 3 cm.

  • Figure 2—

    Photomicrograph of a section of a kidney from the dog in Figure 1. Renal glomeruli have abundant eosinophilic material (protein-rich glomerular filtrate) in the Bowman space (asterisks), marked mesangial cellularity, synechiae, and periglomerular fibrosis (arrow). Renal tubules often contain pale eosinophilic intraluminal material (dagger). H&E stain; bar = 50 μm.

  • Figure 3—

    Photomicrograph of a section of a kidney from the dog in Figure 1. There is moderate interstitial, periglomerular, and glomerular fibrosis (stained blue [asterisk]). The protein-rich glomerular filtrate within the Bowman space is stained bright red (dagger). Masson trichrome stain; bar = 50 μm.

  • Figure 4—

    Photomicrograph of a section of a kidney from the dog in Figure 1. There is moderate thickening of the Bowman capsule (arrow) and basement membranes of glomerular capillaries and renal tubules (stained purple-magenta [arrowhead]). The protein-rich glomerular filtrate within the Bowman space is stained purple (asterisk). Periodic acid–Schiff stain; bar = 50 μm.

  • 1. Davidson AG, Bell RJ, Lees GE, et al. Genetic cause of autosomal recessive hereditary nephropathy in the English Cocker Spaniel? J Vet Intern Med 2007; 21:394401.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 2. Lees GE. Kidney diseases caused by glomerular basement membrane type IV collagen defects in dogs J Vet Emerg Crit Care (San Antonio) 2013; 23:184193.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 3. Maxie MG, Newman SJ. Urinary system In: Jubb, Kennedy, and Palmer's pathology of domestic animals. 5th ed. Vol 2. St Louis: Saunders Elsevier, 2007;460.

    • Search Google Scholar
    • Export Citation
  • 4. Longo I, Porcedda P, Mari F, et al. COL4A3/COL4A4 mutations: from familial hematuria to autosomal-dominant or recessive Alport syndrome? Kidney Int 2002; 61:19471956.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 5. Gubler MC. Inherited diseases of the glomerular basement membrane Nat Clin Pract Nephrol 2008; 4:2437.

  • 6. Heidet L, Cai Y, Guicharnaud L, et al. Glomerular expression of type IV collagen chains in normal and X–linked Alport syndrome kidneys? Am J Pathol 2000; 156:19011910.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 7. Deltas C, Pierides A, Voskarides K. Molecular genetics of familial hematuric diseases Nephrol Dial Transplant 2013; 28:29462960.

  • 8. Khoshnoodi J, Pedchenko V, Hudson BG. Mammalian collagen IV Microsc Res Tech 2008; 71:357370.

  • 9. Zheng K, Thorner PS, Marrano P, et al. Canine × chromosome-linked hereditary nephritis: a genetic model for human X-linked hereditary nephritis resulting from a single base mutation in the gene encoding the alpha 5 chain of collagen type IV? Proc Natl Acad Sci USA 1994; 91:39893993.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 10. Cox ML, Lees GE, Clifford EK, et al. Genetic cause of X-linked Alport syndrome in a family of domestic dogs? Mamm Genome 2003; 14:396403.

  • 11. Nowend KL, Starr-Moss AN, Lees GE, et al. Characterization of the genetic basis for autosomal recessive hereditary nephropathy in the English Springer Spaniel? J Vet Intern Med 2012; 26:294301.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 12. Lees GE, Wilson PD, Helman RG, et al. Glomerular ultrastructural findings similar to hereditary nephritis in 4 English Cocker Spaniels? J Vet Intern Med 1997; 11:8085.

    • Crossref
    • Search Google Scholar
    • Export Citation
  • 13. British Kennel Club website. Dog health. Breed specific DNA tests (up-dated April 2015). Available at: www.thekennelclub.org.uk/media/14688/dnatestsworldwide.pdf#sthash.eQZ2iMgB.dpuf. Accessed Jun 29, 2015.

    • Search Google Scholar
    • Export Citation

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