Abstract

BACKGROUND
In mammals, cellular protection from hypertonic damage is achieved by increasing the expression of genes, such as aldose reductase(AR) and Na+/myo-inositol transporter(SMIT). Recent studies have revealed that tonicityresponsive enhancer binding protein(TonEBP) regulates the increased transcription of these genes. Increased AR gene expression leads to hyperglycemiainduced cellular impairment, and the degree of basal aldose reductase gene expression influences the development of diabetic complication. The alteration of cellular protection genes, such as AR, SMIT, HSP(Heat Shock Protein) 70 and TonEBP, from hypertonic stress in the presence of sustained hyperglycemia was examined in human retinal pigment epithelial(hRPE) and placental cells. METHODS: After the cultures became confluent, the hRPE cells were exposed to 25mM glucose, 100mM NaCl or both for 1, 2 and 3 days. The expressions of AR, SMIT and HSP70 were determined by northern blot analysis. Decreased inductions of the hypertonicity in AR and SMIT mRNA were first evident after exposure to 25mM glucose after 1 day, achieving near maximal level after 3 days; however, no significant alteration in the HSP70 mRNA was noted at any time. For these reasons, the alteration of cellular protection genes, such as AR, SMIT and TonEBP, in hRPE and placental cells were examined after 3-days exposure to the various experimental conditions. The cells were incubated in serum-free media, containing 5.5 or 25mM glucose, 100mM NaCl(in hRPE cells) or 75mM NaCl(in placental cells) and 25mM glucose+100mM NaCl(in hRPE cells) or 75mM NaCl(in placental cells), with or without 20muM tolrestat for 72hrs, at which time the expressions of AR, SMIT and TonEBP were determined. To examine the role of cellular ionic strength in hyperglycemic hRPE cells, excess(5mM) betaine was added to the medium to accelerate the accumulation of the compatible osmolyte, which should lead to a strength reduction. RESULTS: In the hRPE and placental cells, incubated in media with 25mM glucose+100mM(in hRPE cells) or 75mM(in placental cells) NaCl, the AR and SMIT mRNA levels were decreased compared to those cells incubated in media with 100mM(in hRPE cells) or 75mM(in placental cells) NaCl alone. Significant prevention of these decreased AR and SMIT mRNA levels were also observed in those cells incubated with tolrestat. The addition of betaine reduced the abundance of AR and SMIT mRNA in hypertonic stress, similar to in hypertonic and hyperglycemic cells. The inhibition of aldose reductase due to tolrestat in hyperglycemic and hypertonic media was complete in the media not containing betaine. CONCLUSION: Under conditions where sorbitol rapidly increases in hypertonic and hyperglycemic medium should be an important new system for exploring the mechanism of cellular impairment to the effects of hyperglycemia. The expressions of AR and SMIT genes that may influence the development of diabetic complication were down-regulated by the intracellular accumulation of sorbitol in sustained hyperglycemia, and the reliable effect of AR inhibitors on the biological improvements were verified in hyperglycemic cells.

• Keywords: Hyperglycemia;Hypertonicity;AR;AR inhibitor;Tolrestat;SMIT;