Roles for Cation-Chloride Cotransporters in Maintenance of Lens Volume & Transparency

Abstract

Visual acuity is reliant on lens transparency. In diabetic cataract it is well known that the ability of the lens to effectively regulate its volume is disrupted. This results in a localised zone of cellular damage, which distorts the precise tissue architecture of the lens producing light scattering, loss of transparency, and eventually blindness. The volume regulatory mechanisms disrupted in cataract formation are unknown. Therefore the major goal of my thesis is to provide a better understanding of transport systems responsible for controlling the volume of the normal lens. This information is urgently required if we are to understand the processes that initiate diabetic lens cataract. My thesis classifies for the first time the presence and involvement of members of the cation-chloride cotransporter superfamily (CCC) in the maintenance of lens volume and transparency. The potassium-dependent chloride cotransporters (KCCl, 3, 4), the sodium-dependent potassium chloride cotransporters (NKCCl), and the sodium-dependent chloride cotransporters (NCC) were identified in the lens at the transcript and protein levels using RT-PCR and Western analysis respectively. Immunocytochemistry was performed to map the spatial distributions of the different transporters. This analysis showed that each isoform had distinctive spatial distributions. KCC1,3 and 4 were differentially expressed in the cortex but only KCC4 was found in the core. NKCC1 and NCC were also present in the cortex but only NCC was located in the core. To assess the functional activity of these different transporters organ cultured lenses were exposed to specific inhibitors. DIOA mediated inhibition of KCC's resulted in peripheral cell swelling suggesting a role in ion efflux. In contrast, bumetanide inhibition produced a deeper zone of extracellular space dilations suggesting that NKCC mediates ion influx in differentiating fibre cells. The inhibition of NCC with thiazide produced inconclusive results. Taken together this molecular and functional data indicates that the above members of the CCC family mediate spatially distinct chloride influx and efflux pathways in the lens. This information was used to develop an integrative model of the transport processes in the lens. The complex and dynamic nature of these transport systems are illustrated and highlight, the integrated functions of these spatially distinct transporters to maintain the volume set point of the fibre cells. These transport processes are involved not only in steady state volume maintenance, but have roles in cell growth and elongation, and activation under hypotonic stress conditions are elucidated. As well as contributing to a more comprehensive understanding of normal lens volume maintenance, my results have provided a novel theory into the combination of both osmotic and oxidative stresses imposed on the lens under hyperglycaemic trauma. Along with the cotransporters isolated in this thesis, the mechanisms for their control can also be marked as potential targets for innovative anti-cataract therapies.