Osmotic expression of aldose reductase in retinal pigment epithelial cells: involvement of NFAT5.

Abstract

Diabetic retinopathy is associated with osmotic stress resulting from hyperglycemia and intracellular sorbitol accumulation. Systemic hypertension is a risk factor of diabetic retinopathy. High intake of dietary salt increases extracellular osmolarity resulting in systemic hypertension. We determined the effects of extracellular hyperosmolarity, chemical hypoxia, and oxidative stress on the gene expression of enzymes involved in sorbitol production and conversion in cultured human retinal pigment epithelial (RPE) cells. Alterations in the expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH) genes were examined with real-time RT-PCR. Protein levels were determined with Western blot analysis. Nuclear factor of activated T cell 5 (NFAT5) was knocked down with siRNA. AR gene expression in RPE cells was increased by high (25mM) extracellular glucose, CoCl2 (150μM)-induced chemical hypoxia, H2O2 (20μM)-induced oxidative stress, and extracellular hyperosmolarity induced by addition of NaCl or sucrose. Extracellular hyperosmolarity (but not hypoxia) also increased AR protein level. SDH gene expression was increased by hypoxia and oxidative stress, but not extracellular hyperosmolarity. Hyperosmolarity and hypoxia did not alter the SDH protein level. The hyperosmotic AR gene expression was dependent on activation of metalloproteinases, autocrine/paracrine TGF-β signaling, activation of p38 MAPK, ERK1/2, and PI3K signal transduction pathways, and the transcriptional activity of NFAT5. Knockdown of NAFT5 or inhibition of AR decreased the cell viability under hyperosmotic (but not hypoxic) conditions and aggravated the hyperosmotic inhibition of cell proliferation. The data suggest that sorbitol accumulation in RPE cells occurs under hyperosmotic, but not hypoxic and oxidative stress conditions. NFAT5- and AR-mediated sorbitol accumulation may protect RPE cells under conditions of osmotic stress.