Effects of high glucose concentration on the barrier function and the expression of tight junction proteins in human retinal pigment epithelial cells.
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.
Villarroel
Marta
M
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.
García-Ramírez
Marta
M
Corraliza
Lidia
L
Hernández
Cristina
C
Simó
Rafael
R
D001813:Blood-Retinal Barrier / Q000187:drug effects / Q000502:physiology; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects; D002478:Cells, Cultured; D062445:Claudin-1; D017097:Electric Impedance; D005136:Eye Proteins / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D062793:Occludin; D010539:Permeability / Q000187:drug effects; D010750:Phosphoproteins / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D055213:Retinal Pigment Epithelium / Q000187:drug effects* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019108:Tight Junctions / Q000187:drug effects* / Q000378:metabolism; D062826:Zonula Occludens-1 Protein
C568108:CLDN1 protein, human; D062445:Claudin-1; D005136:Eye Proteins; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; D010750:Phosphoproteins; D034741:RNA, Small Interfering; C569042:TJP1 protein, human; D062826:Zonula Occludens-1 Protein; D005947:Glucose