Effects of high glucose concentration on the barrier function and the expression of tight junction proteins in human retinal pigment epithelial cells

Marta Villarroel, Marta García-Ramírez, Lidia Corraliza, Cristina Hernández, Rafael Simó

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Abstract

There is no information on the direct effect of high glucose concentrations on the barrier function of retinal pigment epithelium (RPE). The aim of this study was to explore the effect of high glucose concentrations on the permeability and the expression of tight junction proteins (occludin, zonula occludens-1 (ZO-1) and claudin-1) in a human RPE line (ARPE-19). For this purpose, ARPE-19 cells were cultured for 3 weeks in a medium containing 5.5 mM d-glucose (mimicking physiological conditions) and 25 mM d-glucose (mimicking hyperglycemia that occurs in diabetic patients). The permeability was evaluated by measuring transepithelial electrical resistance (TER) and apical-basolateral movements of dextran. The expression of tight junction proteins was evaluated by real-time PCR (RT-PCR) and Western blot. Cells grown at 25 mM of d-glucose showed a significant higher TER and a significant lower dextran diffusion than the ones maintained at 5.5 mM of d-glucose. Occludin and ZO-1 mRNA levels and protein content were similar in cultures maintained in 5.5 mM and 25 mM d-glucose. By contrast, high glucose concentrations induced a significant overexpression of claudin-1 (mRNA: 1.03 ± 0.48 vs 2.29 ± 0.7 RQ; p = 0.039, at 21 days. Protein levels: 0.92 ± 0.12 vs 1.14 ± 0.28 arbitrary units; p = 0.03, at 21 days). However, after blocking claudin-1 expression using siRNA no changes in TER and permeability were observed. We conclude that high glucose concentration results in a reduction of permeability in ARPE-19 cells. In addition, our results suggest that the overexpression of claudin-1 induced by high glucose concentrations is not involved in the mechanisms by which glucose increases the tight junction sealing function. Further studies addressed to unravel the complexity of permeability regulation in RPE are needed. © 2009 Elsevier Ltd. All rights reserved.
Original languageEnglish
Pages (from-to)913-920
JournalExperimental Eye Research
Volume89
Issue number6
DOIs
Publication statusPublished - 1 Dec 2009

Keywords

  • blood-retinal barrier
  • cell culture
  • retinal pigment epithelium
  • tight junction

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