Roles for Cation Chloride Cotransporters in Diabetic Cataract

Abstract

Cataract is the leading cause of blindness, and diabetes mellitus (DM) is one of the main risk factors associated with cataract development. DM/hyperglycaemia induced cortical cataract has been associated with dysregulation of cation chloride cotransporters (CCCs) that regulate lens fibre cell volume. In other cell types, the activity of the CCCs such as the Na+K+Cl- cotransporter 1 (NKCC1) and KCl cotransporter (KCC) are reciprocally regulated by modulation of their phosphorylation status. I have characterised a panel of antibodies and utilised Western blotting to detect the phosphorylation status of NKCC1 and KCC3, and found that the phosphorylation status of NKCC1 is increased and decreased in response to hypertonic and hypotonic stress, respectively. Having optimised the Western blotting protocols, lenses were subsequently cultured in high glucose-artificial aqueous humour (AAH) to mimic the hyperglycaemia observed in diabetes. In these lenses, fibre cells in the lens periphery demonstrate minor swelling at 2 hours, and with increased periods (at 72 hours) of exposure to high glucose, cells lose their ability to regulate their volume resulting in cell swelling and cell damage reminiscent of diabetic cataract. At 2 hours, NKCC1 phosphorylation appeared to decrease, but this was not statistically significant. However, incubation of lenses in high mannitol-AAH, a membrane impermeable non-hydrolysable sugar, for 2 hours caused a significant increase in NKCC1 phosphorylation. My results show NKCC1 phosphorylation can be manipulated in the lens and suggests a role for the dysfunction of CCC regulation in the initiation of cell volume disruption that manifests as diabetic cortical cataract.