Abstract: TH-PO296

ZO-1 Protein Is Required for H2O2-Induced, ERK 1/2-Dependent Increase in MDCK Cell Paracellular Permeability

Session Information

Category: Acute Kidney Injury

  • 001 AKI: Basic

Authors

  • Bilal, Sahar, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States
  • Jaggi, Shirin, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States
  • Voronina, Angelina, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States
  • Watari, Jessica, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States
  • Axis, Josephine, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States
  • Janosevic, Danielle, Indiana University School of Medicine, Indianapolis, Indiana, United States
  • Amsler, Kurt, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States
Background

Tight Junctions (TJ) are complexes of multiple proteins on the apicolateral membranes of adjacent epithelial cells that interact to form a selectively permeable paracellular barrier. Hydrogen peroxide (H2O2) treatment increases renal epithelial paracellular permeability but the mechanism(s) mediating this effect are unclear. Previous studies suggest kinase-mediated regulation may influence paracellular permeability. In this study, we examined the roles of ERK 1/2 activation and of specific TJ proteins (occludin, ZO-1, ZO-2) in H2O2-induced paracellular permeability in renal epithelial cell monolayers.

Methods

Paracellular permeability via the leak pathway (large solutes) was measured as transepithelial movement of calcein, a fluorescent dye, across monolayers of wild type and knockdown MDCK cells grown on permeable membrane filters. H2O2 and inhibitors were added prior to measurement of calcein flux. ERK 1/2 activation and TJ protein content were monitored by immunoblot.

Results

Treatment of MDCK cells with H2O2 at non-toxic concentrations increased both ERK 1/2 activation and paracellular calcein flux rate in a concentration-dependent manner. ERK 1/2 activation occurred within 30’. Inhibition of ERK 1/2 activation by U0126 blocked the ability of H2O2 to increase paracellular calcein movement. Knockdown of either occludin or ZO-2 protein did not block the ability of H2O2 to increase paracellular calcein movement nor its inhibition by U0126. In contrast, knockdown of ZO-1 protein, which links the TJ to the actin cytoskeleton, blocked the ability of H2O2 to increase paracellular calcein flux. H2O2 treatment also altered F-actin organization of confluent MDCK cells, including disruption of actin stress fibers.

Conclusion

We demonstrate that H2O2 treatment of renal epithelial cells activates ERK 1/2 which is required for H2O2 to increase MDCK cell paracellular permeability. ZO-1 protein, but not ZO-2 or occludin protein, is required for H2O2 to increase MDCK cell paracellular permeability. Since ZO-1 protein links the TJ to the actin cytoskeleton, these results may implicate ERK 1/2 modulation of the actin cytoskeleton in mediating the ability of H2O2 to increase MDCK cell paracellular permeability.

Funding

  • NIDDK Support