HPMCs Induce Greater Intercellular Delocalization of Tight Junction-Associated Proteins Due to a Higher Susceptibility to H 2 O 2 Compared with HUVECs

Abstract

Background

Reactive oxygen species (ROS) have been speculated as possible inducers of structural or functional changes that lead to a hyperpermeable state in patients on long-term peritoneal dialysis. This study aimed to compare localization of tight junction-associated proteins (TJPs), which relate to solute permeability characteristics, between human peritoneal mesothelial cell (HPMC) monolayers and human umbilical vein endothelial cell (HUVEC) monolayers under oxidative stress.

Methods

HPMCs and HUVECs were cultured on a polymer mesh until transepithelial electrical resistance reached a plateau. Solute permeation tests were conducted using FITC-labeled dextrans. Localization of TJPs was observed under a confocal laser scanning microscope. These experiments were carried out with/without 0.1 mmol/L H₂O₂. In addition, ROS production as well as the amounts of intracellular reductive glutathione (GSH) and oxidative glutathione were measured.

Results

When the monolayers were exposed to 0.1 mmol/L H₂O₂/medium for 2 hours, the HPMC monolayer revealed a significant reduction in transepithelial electrical resistance (from 32.5 ± 3.4 to 17.4 ± 4.9 Ω cm) with delocalization of TJPs, particularly occludins. The HUVEC monolayer remained stable and exhibited an unremarkable change in TJP organization. Compared to the HUVEC monolayer, the HPMC monolayer exhibited two- to threefold higher 2′,7′–dichlorofluorescein intensities that increased in a dose-dependent manner. HUVECs contained approximately 2.5-times more GSH than HPMCs. This supported the lesser production of ROS when exposed to 0.1 mmol/L H₂O₂ for 24 hours. HUVECs used 8.03 nmol/mg GSH protein to maintain TJP localization, while only 3.75 nmol/mg GSH protein was available for the HPMCs.

Conclusion

The HUVEC monolayer, which was less permeable to middle-to-high molecular weight solutes, was more tolerant against ROS stress than the HPMC monolayer. Availability of intracellular GSH is an important issue in maintaining the integrity of the mesothelium.

Keywords

Human peritoneal mesothelial cells human umbilical vein endothelial cells solute permeability occludin zonula occludens-1 (ZO-1)transepithelial electrical resistance oxidative stress 2′,7′–dichlorofluorescein (DCF)glutathione

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