ClC-2 is required for rapid restoration of epithelial tight junctions in ischemic-injured murine jejunum.
OBJECTIVE
Involvement of the epithelial chloride channel ClC-2 has been implicated in barrier recovery following ischemic injury, possibly via a mechanism involving ClC-2 localization to the tight junction. The present study investigated mechanisms of intestinal barrier repair following ischemic injury in ClC-2(-/-) mice.


METHODS
Wild type, ClC-2 heterozygous and ClC-2(-/-) murine jejunal mucosa was subjected to complete ischemia, after which recovery of barrier function was monitored by measuring in vivo blood-to-lumen clearance of (3)H-mannitol. Tissues were examined by light and electron microscopy. The role of ClC-2 in re-assembly of the tight junction during barrier recovery was studied by immunoblotting, immunolocalization and immunoprecipitation.


RESULTS
Following ischemic injury, ClC-2(-/-) mice had impaired barrier recovery compared to wild type mice, defined by increases in epithelial paracellular permeability independent of epithelial restitution. The recovering ClC-2(-/-) mucosa also had evidence of ultrastructural paracellular defects. The tight junction proteins occludin and claudin-1 shifted significantly to the detergent soluble membrane fraction during post-ischemic recovery in ClC-2(-/-) mice whereas wild type mice had a greater proportion of junctional proteins in the detergent insoluble fraction. Occludin was co-immunoprecipitated with ClC-2 in uninjured wild type mucosa, and the association between occludin and ClC-2 was re-established during ischemic recovery. Based on immunofluorescence studies, re-localization of occludin from diffuse sub-apical areas to apical tight junctions was impaired in ClC-2(-/-) mice.


CONCLUSIONS
These data demonstrate a pivotal role of ClC-2 in recovery of the intestinal epithelium barrier by anchoring assembly of tight junctions following ischemic injury.
Nighot
Prashant K
PK
Department of Clinical Science, College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street Raleigh, NC 27606, USA.
Moeser
Adam J
AJ
Ryan
Kathleen A
KA
Ghashghaei
Troy
T
Blikslager
Anthony T
AT
D000818:Animals; D000090007:CLC-2 Chloride Channels; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels / Q000172:deficiency / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D047468:Immunoprecipitation; D007511:Ischemia / Q000378:metabolism* / Q000503:physiopathology; D007583:Jejunum / Q000098:blood supply* / Q000473:pathology* / Q000648:ultrastructure; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D062793:Occludin; D021381:Protein Transport; D019108:Tight Junctions / Q000378:metabolism* / Q000648:ultrastructure; D014945:Wound Healing
D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D008565:Membrane Proteins; D062793:Occludin; C568986:Ocln protein, mouse; D008353:Mannitol