Paul Donaldson, Irene Vorontsova, Julie Lim

Purpose:
To identify the cellular pathways that regu-late lens fibre cell volume and whose dysfunction givesrise to the discrete zone of tissue damage in the outercortex of the lens which is a defining characteristic ofdiabetic cataract1.

Methods:
Ion channels and transporters plus the sig-nalling pathways that regulate their activity were iden-tified and localised in rat lenses using a combination ofRT-PCR, Western Blotting and immunohistochemistry.The functional activity of specific transport proteinsand signalling components were assessed either bymorphological analysis of whole rat lenses organ cul-tured in pharmacological modulators of their activity,or by the use of patch clamping to assess their activityin isolated fibre cells.

Results:
Our cumulative data shows that spatiallydistinct ion influx and efflux pathways exist in thelens and that they interact to control steady state lensvolume2. Furthermore, we can mimic the character-istic localised damage phenotype of diabetic cataractby stimulating ion and water uptake in the lens viathiol-inactivation of regulatory kinases (WNK/SPAK/OSR1) that control the phosphorylation status, andhence activity of the Cation Chloride Cotransporters(CCC) family that we have previously shown tocontrol lens volume3, 4. In addition, swelling thelens causes a release of ATP, which activates P2Xchannels localised to the influx zone5. These non-selective cation (NSC) channels produce membranedepolarisation and increased intracellular Ca2+,which in turn activates a normally quiescent hemi-channel like conductance6, which is permeable tointracellular metabolites, and ultimately causes thelocalised tissue liquefaction.

Conclusions:
Based on our studies of cell volumeregulation, we propose that in diabetics,hyperglycaemia-induced oxidative damage to theWNK/SPAK/OSR1 pathway that regulates CCC activityproduces the localised cell swelling that initiates dia-betic cataract formation. This initial insult then leads toa cascade of events involving ATP release, P2X mediateddepolarisation, hemi-channel activation, Ca2+ influxand activation of Ca2+-dependent proteases that resultsin the tissue liquefaction observed in the influx zone.This cascade of events if verified in humans will lead tothe identification of relevant drug targets to protecthumans against diabetic cataract.

This work was supported by the Health ResearchCouncil of New Zealand. Dr Vorontsova was a recipi-ent of stipend support from the Auckland MedicalResearch Foundation and the Kate Edger EducationalCharitable Trust.