Molecular cloning and expression of canine hepcidin.

Background: Hepcidin is a recently identified acute phase protein with antimicrobial and iron regulatory functions. It has been suggested that hepcidin may be the key mediator of anemia of chronic disease. Our research group is interested in developing a diagnostic assay to measure hepcidin in dogs.

Objectives: The objectives of this study were to clone and sequence the canine hepcidin gene and to gather preliminary data about tissue expression of hepcidin in dogs.

Methods: RNA was extracted from fresh canine liver tissue and cDNA was generated and amplified. Standard reverse transcription polymerase chain reaction techniques were used with degenerate primers based on sequence homology between several other species. The amino acid (AA) sequence was compared with known sequences in other species. Tissue expression of canine hepcidin was determined by Western blot.

Results: The canine hepcidin cDNA sequence encoded a highly conserved protein of 85 AAs with 8 cysteine residues at the C-terminal end. This protein was likely the precursor form (pro-hepcidin) of a smaller secreted peptide. Phylogenetic analysis showed that human hepcidin was more homologous with canine than with rodent hepcidin. In dogs, as in people, hepcidin was expressed most strongly in the liver. Western blotting showed a clear band of approximately 9 kDa, consistent with pro-hepcidin. Weak expression was also detected in canine kidney and lung tissues.

Conclusion: The results of this study establish the basis for future investigation involving canine hepcidin and suggest that the dog may be a suitable model for studying the role of hepcidin in human health and disease.