High glucose concentration leads to differential expression of tight junction proteins in human retinal pigment epithelial cells

espanolUna de las primeras consecuencias de la retinopatia diabetica es la rotura de la barrera hematorretiniana (BHR) causada por la interrupcion de las tight junctions. Mientras que la alteracion de las proteinas implicadas en la interrupcion de las tight junctions de la BHR interna ha sido estudiada extensamente, la informacion sobre este proceso en la barrera hematorretiniana externa (constituida por el epitelio pigmentario de la retina) es escasa. El objetivo de este trabajo es estudiar el cambio de expresion (ARNm y proteina) de ocludina, zonula occludens-1 y claudina-1 en celulas de epitelio pigmentario de retina (EPR) humanas con dos concentraciones diferentes de glucosa. Materiales y metodos: Se utilizo una linea de celulas de EPR humano (ARPE-19), cultivada durante 3 semanas en un medio suplementado con un 10% de suero bovino fetal y con una concentracion de 5,5 mmol de D-glucosa (simulando condiciones fisiologicas) o 25 mmol de D-glucosa (simulando la hiperglucemia que ocurre en pacientes diabeticos). Las proteinas de las tight junctions ocludina, zonula occludens-1 y claudina-1 se estudiaron por Western blot y PCR real time. Todas las determinaciones se hicieron a los 14 y 21 dias. Resultados: La expresion de ARNm y proteina de ocludina y ZO-1 fue similar en cultivos mantenidos a 5,5 y 25 mmol de Dglucosa. Por el contrario, en condiciones de hiperglucemia (25 mmol) se produjo un claro aumento en la expresion de ARNm de la claudina-1 y en el contenido de esta proteina a los 21 dias (concentraciones de ARNm, 1,03 frente a 2,29; proteina, 0,92 frente a 1,14). Conclusiones: La elevada concentracion de glucosa produce una expresion diferencial de las proteinas de las tight junctions en celulas del epitelio pigmentario de la retina humana. Ademas, nuestros resultados sugieren que la hiperexpresion de la claudina-1 mediada por glucosa participaria en la funcion de sellado de las tight junctions. Las consecuencias funcionales y la traduccion clinica de estos hallazgos seran motivo de futuras investigaciones. EnglishOne of the early features of diabetic retinopathy is the breakdown of the blood-retinal barrier (BRB) due to disruption of the tight junctions. Whereas impairment of the proteins involved in the disruption of the tight junctions of the internal BRB has been extensively studied, there is no information on the direct effect of high glucose concentration on the barrier function of the outer blood-retinal barrier (formed by the retinal pigment epithelium [RPE]). The aim of this study was to explore the effect of high glucose concentration on the expression of tight junction proteins (occludin, zonula occludens-1 [ZO-1] and claudin-1) in a human RPE line under two distinct glucose concentrations. Materials and methods: An RPE cell line (ARPE-19) were cultured for 3 weeks in a medium supplemented with 10% fetal calf serum containing 5.5 mmol D-glucose (mimicking physiological conditions) or 25 mmol D-glucose (mimicking the hyperglycemia that occurs in diabetic patients). Occludin, ZO-1 and claudin-1 were studied by real-time polymerase chain reaction and Western blot at 14 and 21 days. Results: Occludin and ZO-1 mRNA levels and protein content were similar in cultures maintained at 5.5 mmol and 25 mmol of D-glucose. In contrast, high glucose concentration (25 mmol) induced a clear upregulation in claudin-1 mRNA expression and protein content at 21 days (mRNA level: 1.03 vs 2.29; protein content: 0.92 vs 1.14). Conclusions: High glucose concentration leads to differential expression of tight junction proteins in ARPE-19 cells. In addition, our results suggest that the upregulation of claudin-1by glucose is involved in the increase of tight junction sealing function. The functional consequences and clinical applicability of these findings require further investigation.