Diabetes and Metabolism Research Unit, Institut de Recerca Hospital Vall d'Hebron, Universitat Autònoma de Barcelona, Pg. Vall d'Hebron 119-129, 08035 Barcelona, Spain.
Exp Eye Res. 2009 Dec;89(6):913-20. doi: 10.1016/j.exer.2009.07.017. Epub 2009 Aug 4.
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.
目前尚无高葡萄糖浓度对视网膜色素上皮（RPE）屏障功能的直接影响的信息。本研究旨在探讨高葡萄糖浓度对人 RPE 细胞系（ARPE-19）通透性和紧密连接蛋白（occludin、zonula occludens-1（ZO-1）和 claudin-1）表达的影响。为此，将 ARPE-19 细胞在含有 5.5 mM D-葡萄糖（模拟生理条件）和 25 mM D-葡萄糖（模拟糖尿病患者发生的高血糖）的培养基中培养 3 周。通过测量跨上皮电阻（TER）和葡聚糖的顶底侧向运动来评估通透性。通过实时 PCR（RT-PCR）和 Western blot 评估紧密连接蛋白的表达。与维持在 5.5 mM D-葡萄糖的细胞相比，在 25 mM D-葡萄糖中生长的细胞显示出显著更高的 TER 和显著更低的葡聚糖扩散。在维持在 5.5 mM 和 25 mM D-葡萄糖的培养物中，occludin 和 ZO-1 mRNA 水平和蛋白含量相似。相比之下，高葡萄糖浓度诱导 claudin-1 的显著过表达（mRNA：1.03 +/- 0.48 与 2.29 +/- 0.7 RQ；p = 0.039，在第 21 天。蛋白水平：0.92 +/- 0.12 与 1.14 +/- 0.28 任意单位；p = 0.03，在第 21 天）。然而，使用 siRNA 阻断 claudin-1 表达后，TER 和通透性没有变化。我们得出结论，高葡萄糖浓度导致 ARPE-19 细胞通透性降低。此外，我们的结果表明，高葡萄糖浓度诱导的 claudin-1 过表达不参与葡萄糖增加紧密连接密封功能的机制。需要进一步的研究来阐明 RPE 通透性调节的复杂性。
