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Keywords:

  • horse;
  • laminitis;
  • laminar keratinocyte;
  • phenotypic marker;
  • disease progression;
  • ENaC;
  • GLUT1;
  • GLUT4;
  • immunohistochemistry

Summary

Reasons for performing study: Equine laminitis is a multifactorial connective tissue disorder with major implications for the welfare of horses. There are few published studies on phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques.

Objectives: To establish whether the epithelial sodium channel (ENaC) and the GLUT1 and GLUT4 facilitative glucose transporters may be used as phenotypic markers for identification of equine laminar keratinocytes using immunohistochemical techniques to monitor changes in the keratinocyte population in laminitis.

Methods: Histology and immunohistochemistry using polyclonal antibodies to the α subunit of ENaC (αENaC), GLUT1 and GLUT4 were used to compare the distribution of these proteins in normal and laminitic equine laminae.

Results: Immunohistochemistry with antibodies to αENaC, GLUT1 and GLUT4 confirmed the abundant expression of all 3 membrane proteins in healthy laminar keratinocytes. However, in laminitis, the Haematoxylin Van Gieson (HVG) technique revealed disordered laminar arrays and replacement with fibrous scar tissue. Immunostaining of laminitic samples confirmed the loss of αENaC, GLUT1 and GLUT4 positive keratinocytes. Other connective tissue cells did not stain positive for these proteins.

Conclusions: This is the first report of αENaC and GLUT1/GLUT4 protein expression in equine laminar keratinocytes, which also confirms that the loss of laminar structure and function in chronic laminitis is accompanied by the loss of laminar keratinocytes.

Potential relevance: αENaC, GLUT1 and GLUT4 may be used as phenotypic markers of metabolically active, differentiated equine laminar keratinocytes. Further in vitro studies are necessary to determine the effects of hypoxia, bacterial endotoxins, vasoactive amines, lactic acid and prostaglandins on the expression and activity of these plasma membrane keratinocyte markers.