inactive koc)

inactive koc)

Cell shrinkage nem/jgsh Cell swelling

Fig. 5. Mimicking diabetic cataract by activation of the potassium chloride cotransporter (KCC). (A) Equatorial section from a lens organ cultured in the KCC activator N-ethylmaleimide (NEM) for 18 hours results in shrinkage of peripheral fibre cells and the swelling of deeper lying fibre cells producing a band of localized tissue liquefaction that mimics that seen in diabetic cataract. (B) A close-up view of a region from (A) showing the two distinct phenotypes induced by activation of KCC. (C) Schematic diagrams outlining how activation of KCC via either NEM or depletion of glutathione (GSH) affects the phosphorylation status and activity of KCC in the lens periphery (left panel) and in deeper zones of tissue liquefaction (right panel), NEM or GSH depletion inhibits the activity of two kinase that either directly (Kinase 1), or indirectly (Kinase 2) via inhibition of a phosphatase (PP1A), phosphorylates KCC and renders it inactive. The resultant dephosphorylation and activation of KCC promotes cell shrinkage in the periphery and cell swelling in deeper fibre cells.

organ-cultured lenses to NEM, induces the overactivation of KCC and resulted in the shrinkage of peripheral fibre cells (Fig. 5B) in the efflux zone and the swelling of deeper fibre cells (Fig. 5A) in the influx zone that ultimately caused the formation of a localized band of tissue liquefaction (11). This indicates that the normally minimal KCC activity is massively upregulated in the influx zone by NEM activation. The activity of the KCC transporter is normally modulated by its phosphorylation status (51). Two separate kinases are involved in the phosphorylation-dependent inactivation of KCC, while dephosphorylation via a PP1A-type phosphatase causes an increase in KCC activity. NEM stimulates KCC transporter activity via thiol inactivation of the two kinases that control transporter activity (60) (Fig. 5C).

The similarity between the liquefaction zone seen in NEM-treated (Fig. 5A) and diabetic lenses (Fig. 3A) is so striking that it prompts speculation that overactivation of KCC transport is occurring in the diabetic cataract. In red blood cells it has been shown that depletion of glutathione (GSH) levels by oxidative stress, causes activation of KCC presumably via oxidation of the critical thiol groups in the two regulatory kinases (61). In the diabetic lens, hyperglycaemia is known to deplete GSH levels (46), thus it is possible that excess glucose compromises the ability of fibre cells to regulate their volume via two pathways: the accumulation of the impermeable osmolyte, sorbitol; and the stimulation of ion influx mediated by the activation of KCC. These osmotic stresses are exacerbated by the activation of volume-sensitive chloride and cation channels, which because of the direction of the ion gradients in this region of the lens, import more osmo-lytes and water, causing uncontrolled cell swelling (53). Finally, membrane leakage of calcium causes the activation of calcium-dependent proteases, leading to fibre cell vesiculation (62-64) and localized tissue liquefaction that is characteristic of diabetic cataract(43). The potential involvement of KCC in the initiation of diabetic cataract opens up new avenues for the development of novel anti-cataract therapies.

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