Investigating the contribution of cytokinesis to population-based measurements of epithelial Leak Pathway permeability

Abstract

The mechanism mediating the paracellular movement of macromolecules across epithelial cell layers, the Leak Pathway, remains poorly understood. Two recent studies have demonstrated that epithelial cell cytokinesis allows the transient paracellular passage of macromolecules (Stephenson et al. 2019. Dev Cell 48:445-459, Richter et al. 2022. Ann NY Acad Sci 1516:151- 161). It is unclear, however, if this cytokinesis-mediated transepithelial passage of macromolecules can contribute significantly to the Leak Pathway permeability measured in a post-confluent epithelial cell population. The renal epithelial cell line, Madin Darby Canine Kidney (MDCK II), develops tight junctions and forms a typical paracellular permeability barrier upon reaching a confluent density. MDCK II cells have been reported to maintain a basal rate of cell proliferation, even in post-confluent cell populations. To investigate the role of cytokinesis in contributing to population-based measurements of Leak Pathway permeability, we examined the effect of arresting and releasing cytokinesis in post-confluent populations of MDCK II cells on Leak Pathway permeability. We confirmed that post-confluent populations of MDCK II cells exhibit a low level of cell proliferative activity. Leak Pathway permeability was determined by measuring the flux of 20 kDa fluorescein-dextran across post-confluent populations of MDCK II cells grown on permeable membrane filters. Inhibiting cytokinesis of MDCK II cells by overnight treatment with RO-3306, a CDK-1 inhibitor, decreased modestly the paracellular flux of 20 kDa fluorescein-dextran compared to untreated cell populations. Washing out RO-3306 1 hour prior to the flux assay produced an increase in paracellular flux of 20 kDa fluorescein-dextran to a level higher than that observed in untreated cell populations. Our results support the working hypothesis that cytokinesis can contribute to Leak Pathway permeability measured in epithelial cell populations. They suggest that more proliferative epithelial cell populations will exhibit higher rates of Leak Pathway permeability compared to epithelial cell populations with very low levels of proliferative activity. The significant Leak Pathway permeability measured in MDCK II cell populations maintained in the presence of RO-3306 suggests that cytokinesis is not the sole mechanism mediating Leak Pathway permeability across epithelial cell populations. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.