Manipulation of Actin Cytoskeleton and Tight Junction Protein Knockdown Differentially Interact to Modulate Basal and H2O2‐Induced Paracellular Permeability of Renal Epithelial Cells
Authors: Bilal Sahar , Sharma Rhea , Voronina Angelina , Axis Josephine , Amsler Kurt 
Published: June 2021

Renal epithelial tight junction permeability to large molecules (leak pathway) is increased by hydrogen peroxide (H2O2). Previous studies have suggested a role for the actin cytoskeleton in regulation of epithelial paracellular permeability but its possible involvement in H2O2‐induced regulation of renal epithelial paracellular permeability is unknown. To assess the role of the actin cytoskeleton, we examined the effect of two agents that manipulate differentially the actin cytoskeleton, blebbistatin (inhibits myosin II ATPase) and jasplakinolide (enhances F‐actin nucleation), on basal and H2O2‐induced renal epithelial leak pathway permeability, measured as calcein flux rate. The effects of these compounds was examined in wild type MDCK cells and in MDCK cells in which either occludin protein or ZO‐1 protein has been knocked down to determine if either of these tight junction proteins might be important in mediating actin network actions. Under basal conditions, treatment with 40 mM blebbistatin produced little or no change in calcein flux rate in wild type MDCK cells. H2O2 increased calcein flux rate, as expected. The combination of H2O2 and blebbistatin produced a synergistic increase in calcein flux rate. Jasplakinolide (200 nM) produced a marked increase in calcein flux rate. H2O2 and jasplakinolide did not increase calcein flux rate beyond the rate produced by jasplakinolide alone. Knockdown of occludin protein in MDCK cells did not affect basal calcein flux rate but enhanced the H2O2‐induced increase. In the absence of H‐2O2, blebbistatin produced little or no change in calcein flux rate. H2O2 and blebbistatin did not increase calcein flux rate further than that produced by H2O2 alone. Jasplakinolide increased the calcein flux rate but attenuated the increase produced by H2O2 when cells were incubated with both compounds. Knockdown of ZO‐1 protein increased basal calcein flux rate approximately two‐fold. Treatment with H2O2 did not further increase flux rate. Treatment of ZO‐1 knockdown cells with blebbistatin increased the calcein flux rate, in contrast to its lack of effect on wild type MDCK cells. Treatment with the combination of H2O2 and blebbistatin did not increase flux rate beyond that produced by blebbistatin alone. Jasplakinolide alone increased calcein flux rate in ZO‐1‐knockdown cells. The combination of H2O2 and jasplakinolide did not increase flux rate beyond that produced by jasplakinolide alone. These results suggest a complex role of the actin cytoskeleton in regulation of basal and H2O2‐stimulated leak pathway permeability in renal epithelial cells. First, they suggest that an intact F‐actin network is necessary to maintain low basal leak pathway permeability. Second, they suggest the tight junction is under inward actomyosin‐mediated tension, via the ZO‐1 protein, which contributes to maintenance of low leak pathway permeability, particularly in stress conditions. Third, they suggest that H2O2 treatment produces a state in MDCK cells that is functionally similar to knockdown of ZO‐1 protein. Fourth, they suggest that the effect of blebbistatin is mediated via occludin protein.

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NIDDK and institutional funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.