Figure 5. Rk1 inhibits AGE-induced retinal endothelial permeability.

(A) HRECs were plated onto a Transwell filter . After reaching confluence, HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. HRECs were cultured with 50 µl (0.8 µCi/ml) of [³H] sucrose (1 µCi/µl) added to the upper compartment. The amount of radioactivity that diffused into the lower compartment was determined after 30 min by liquid scintillation counter. Data are the mean ± S.E. **, P < 0.01 vs. AGE alone. (B) HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. Total mRNAs were isolated and RT-PCR was performed with specific primers for human ZO-1, ZO-2, and occludin. GAPDH served as an internal control. (C) Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. Western blots were probed with anti-ZO-1, anti-ZO-2, anti-occludin and anti-Actin. (D) Densitometric analyses are presented as the relative ratio of tight junction proteins in membrane and cytosol and quantified with NIH ImageJ software. (E) Confluent HRECs were pretreated for 40 min with or without Rk1 (10 µg/ml) prior to stimulation with 5 µM AGE-BSA for 10 h. The cells were then fixed and stained with anti-ZO-1, anti-ZO-2, and anti-occludin antibodies. Arrowheads indicate disruption of tight junction proteins. (F) Tight junction protein intensity at cell borders was analyzed and quantified with NIH ImageJ software. Data are the mean ± S.E. **, p < 0.01 vs. Control. ^##, p < 0.01 vs. AGE alone.