Regulation of Fibre Cell Volume: Implication for Lens Transparency

Abstract

Maintenance of a constant volume is vital for lens transparency and members of the Cation Chloride Cotransporter (CCC) family have been implicated in regulating cell volume in the lens. In this thesis, I have confirmed that the K+ Cl‐ Cotransporter (KCC) facilitates ion efflux in the lens periphery at the anterior and equatorial cortex, while the Na+ K+ Cl‐ Cotransporter (NKCC1) facilitates ion influx in the peripheral and deeper equatorial and posterior cortex. The third member of the CCC family, the Na+ Cl‐ Cotransporter (NCC) appears to play a role in the deeper lens, but only under hypertonic conditions. The activity of the CCC family is regulated by their phosphorylation status in other tissues, and in this thesis I show that the key phospho‐regulators of CCC activity are also expressed in the rat lens. The key kinases, With No Lysine (K) kinase (WNK) 1, 3, and 4, STE‐20 like Proline Alanine‐rich Kinase (SPAK) and Oxidative Stress Response kinase 1 (OSR1) were identified by RT‐PCR at the transcript level. Furthermore, WNK1, 3, 4, SPAK and OSR1 were also identified at the protein level, while the key protein phosphatases (PP) 1 and 2A were also confirmed in the rat lens. To test the functionality of this pathway, the effects of its inhibition on lens transparency, mass and cellular morphology were investigated. Incubating lenses in N‐ethylmaleimide (NEM), an activator of KCC activity through the inhibition of an upstream kinase, revealed deeper lens cortical cell swelling and peripheral cell shrinkage. In addition, the inhibition of PP1 and PP2A by calyculin A produced vesiculation of cortical fibre cells. This result indicates that in addition to their previously documented role in apoptosis in the lens, the phosphatase may be modulating lens volume through regulation of the phosphorylation status of KCC and NKCC. The data in this thesis has added to the current understanding of lens cell volume regulation, and mechanisms for facilitating circulating ion fluxes and cell elongation are proposed. The damage phenotypes observed by manipulation of the phospho‐regulatory pathway mimic those observed in a streptozotocin‐induced rat model of diabetic cortical cataract, suggesting a role for the dysfunction of CCC regulation in the initiation of cell volume disruption that manifests as diabetic cortical cataract.