Renal ischemia-reperfusion injury results in oxidative stress-induced alterations in barrier function. Activation of the mitogen-activated protein (MAP) kinase pathway during recovery from oxidative stress may be an effector of oxidant-induced tight junction reorganization. We hypothesized that tight junction composition and barrier function would be perturbed during recovery from oxidative stress. We developed a model of short-term H 2 O 2 exposure followed by recovery using Madin Darby Canine Kidney cells (MDCK II). H 2 O 2 perturbs barrier function without a significant cytotoxic effect except in significant doses. ERK-1/2 and p38, both enzymes of the MAP kinase pathway, were activated within minutes of exposure to H 2 O 2 . Transient exposure to H 2 O 2 produced a biphasic response in transepithelial electrical resistance (TER). An initial drop in TER at 6 hours was followed by a significant increase at 24 hours. Inhibition of ERK-1/2 activation attenuated the increase in TER observed at 24 hours. Expression of occludin initially decreased followed by partial recovery at 24 hours. In contrast, claudin-1 levels decreased and failed to recover at 24 hours. Claudin-2 levels markedly decreased at 24 hours; however, inhibition of ERK-1/2 activation was protective. Occludin and claudin-1 localization at the apical membrane on immunofluorescent images was fragmented at 6 hours after H 2 O 2 exposure with subsequent recovery of appropriate localization by 24 hours. MDCK II cell recovery after H 2 O 2 exposure is associated with functional and structural modification of the tight junction that are mediated in part by activation of the MAP kinase enzymes, ERK-1/2 and p38.

Loss of barrier function in kidney epithelial cells due to alterations in the tight junction (TJ) may be a contributory mechanism of ischemia‐reperfusion injury. The objective of this study was to demonstrate the changes in TJ function and protein composition during recovery after exposure to oxidative stress. Oxidative stress was induced in MDCK Type II cells by exposure to H2O2, and cells were subsequently recovered for 24 hours. Transepithelial electrical resistance (TER) and solute flux were used as measures of barrier function. Protein expression and localization was determined for TJ proteins during the recovery period. Moderate doses of H2O2 perturbed TJ function without exhibiting a significant cytotoxic effect. ERK‐1/2, an enzyme of the mitogen activated protein kinase (MAPK) pathway, was activated within minutes of exposure to H2O2. Exposure to H2O2 with subsequent recovery resulted in initially decreased TER followed by recovery of function and was accompanied by changes in expression and localization of the TJ proteins occludin and claudin‐1. Inhibition of ERK activation significantly attenuated recovery changes in TER and TJ protein composition induced by H2O2. Our findings suggest that the TJ in kidney epithelial cells is altered by H2O2 exposure and recovery. These changes appear to be mediated by activation of the MAPK pathway. This work was supported by NIH Grant DK065652 and the HHMI Foundation.

Oxidative stress has been linked to many disease processes, including ischemia‐reperfusion injury and toxicant damage. Loss of barrier function in renal epithelial cells due to alterations in the tight junction (TJ) may complicate the effects of oxidative stress. The mitogen‐activated protein kinase (MAPK) pathway has been shown to be an early cellular signal during hydrogen peroxide exposure oxidant injury and may be linked to TJ protein alterations. In this study, we are interested in effects on cell barrier function due to a brief exposure to hydrogen peroxide and the ensuing recovery period. Transepithelial electrical resistance and paracellular flux were used as a measure of barrier integrity, while the lactate dehydrogenase activity assay was used to assess cytotoxicity. Protein expression was determined via Western blot analysis, and immunofluorescent staining was used for localization of TJ proteins and examination of the actin cytoskeleton. A positive relationship between dose of hydrogen peroxide and cytotoxicity was observed. Low doses of hydrogen peroxide perturb TJ function without exhibiting a cytotoxic effect. ERK‐1/2, an enzyme of the MAPK pathway, was activated within minutes of exposure to hydrogen peroxide as determined by phospho‐ERK content. Exposure to hydrogen peroxide with subsequent recovery was also associated with changes in expression and localization of the TJ proteins occludin and claudin‐1. These findings demonstrate that the TJ in MDCK cells is altered by exposure to hydrogen peroxide and these changes are preceded by activation of the MAPK pathway.