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EA Walker, S Rauz, PI Murray, PM Stewart; Expression and Distribution of the Serum and Glucocorticoid Regulated Kinase, and the Epithelial Sodium Channel Subunits in the Human Cornea . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3196.
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Purpose: The sodium transporting capacity of the corneal endothelium is vital for preserving corneal transparency. The clarity of the cornea has traditionally been attributed to the Na+K+ATPase located in the corneal endothelium, maintaining the cornea in a dehydrated state. Recent studies have shown that the enzyme serum and glucocorticoid regulated kinase isoform 1 (SGK1) plays a role in sodium transporting epithelial tissues, through activation of the epithelial sodium channels (ENaC). This study was designed to identify whether these elements were present within the human cornea. Methods: In-situ hybridization (ISH) studies were performed using non-radioactively labelled digoxigenin (DIG) antisense cRNA probes for SGK1 and the three ENaC subunits (α, ß, γ) generated from cDNA constructs sub-cloned into pGEMT-Easy Vector (Promega). Labelling efficiency was determined by chemiluminescence and ISH was conducted on paraffin embedded 5µm thick sections of six human eyes. Control experiments were carried out in parallel using three methods: no probe; competition of the antisense DIG-labelled cRNA probe in the presence of a 60-fold excess unlabelled anti-sense cRNA; and sense DIG-labelled cRNA probe. The hybridized probe was visualised by 4-nitroblue-terazolium chloride (NBT)/ 5-bromo-4-chloro-3-indolylyphosphate (BCIP) chromagen precipitation. Results: There was marked expression of the SGK1 and all three ENaC subunits in the corneal endothelial cytoplasm. There were low levels of expression for the SGK in the central basal cells of the corneal epithelium in the perinuclear region, and more intense cytoplasmic expression in the limbal epithelial cells. There was no chromagen precipitation in the Bowman's membrane, corneal stroma, or Descemet's membrane. Control analyses revealed no hybridization signal throughout the corneal layers. Conclusion: These data suggest that the sodium transport across the corneal endothelium may in part be dependent on the induction of the SGK1 and the activation of the epithelium sodium channels.
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