Kidney Week

Abstract: TH-PO296

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

Session Information

• AKI Basic: Oxidative Injury and Nephrotoxins
  November 02, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Acute Kidney Injury

• 001 AKI: Basic

Authors

• Bilal, Sahar, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States

Sahar Bilal,

• Jaggi, Shirin, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States

Shirin Jaggi,

• Voronina, Angelina, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States

Angelina Voronina,

• Watari, Jessica, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States

Jessica Watari,

• Axis, Josephine, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States

Josephine Axis,

• Janosevic, Danielle, Indiana University School of Medicine, Indianapolis, Indiana, United States

Danielle Janosevic,

• Amsler, Kurt, NYIT College of Osteopathic Medicine, Old Westbury, New York, United States

Kurt Amsler,

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