The epithelial cell tight junction structure is the site of the transepithelial movement of solutes and water between epithelial cells (paracellular permeability). Paracellular permeability can be divided into two distinct pathways, the Pore Pathway mediating the movement of small ions and solutes and the Leak Pathway mediating the movement of large solutes. Claudin proteins form the basic paracellular permeability barrier and mediate the movement of small ions and solutes via the Pore Pathway. The Leak Pathway remains less understood. Several proteins have been implicated in mediating the Leak Pathway, including occludin, ZO proteins, tricellulin, and actin filaments, but the proteins comprising the Leak Pathway remain unresolved. Many aspects of the Leak Pathway, such as its molecular mechanism, its properties, and its regulation, remain controversial. In this review, we provide a historical background to the evolution of the Leak Pathway concept from the initial examinations of paracellular permeability. We then discuss current information about the properties of the Leak Pathway and present current theories for the Leak Pathway. Finally, we discuss some recent research suggesting a possible molecular basis for the Leak Pathway.

Paracellular permeability is mediated by the epithelial cell tight junction. Studies in intestinal and other epithelia have suggested that the activity of src family kinases (SFKs) increases epithelial paracellular permeability through its action on the tight junction protein, occludin, but the involvement of SFKs and occludin in regulation of renal epithelial paracellular permeability is unclear. In this study, the role of SFKs in regulation of renal epithelial paracellular permeability and the involvement of occludin protein in this regulatory event was examined in two renal epithelial cell lines, LLC-PK(1) (proximal tubule-like) and MDCK (distal tubule-like). The effect of broad spectrum SFK inhibitors on paracellular permeability of calcein and fluorescein-dextran3000 were examined. SFK inhibitor treatment increased paracellular movement of both compounds in both renal epithelial cell lines. The SFK inhibitor effect was concentration-dependent and, at low concentrations, was not associated with cell damage/death. Response to SFK inhibitors was acquired progressively after cell populations attained confluence suggesting maturation of the regulatory mechanism. Increased paracellular permeability was not associated with dramatic changes in total cell content of occludin protein, its partitioning between detergent-soluble and -insoluble fractions, or its subcellular localization. Further, the SFK-induced increase in paracellular permeability was unaffected by either occludin protein overexpression or occludin protein knockdown. These results demonstrate that SFK activity decreases paracellular permeability of renal epithelial cells, as opposed to its effect in intestinal epithelial cells, and that this regulation is not mediated by occludin protein.

Hydrogen peroxide (HO) increases paracellular permeability of Madin-Darby canine kidney (MDCK) cells, but the mechanism mediating this effect remains unclear. Treatment of MDCK cells with HO activated ERK 1/2. Inhibition of ERK 1/2 activation blocked the ability of HO to increase paracellular permeability. Knockdown of zonula occludens-1 (ZO-1) protein but not occludin eliminated the ability of HO to increase paracellular permeability. HO treatment did not, however, affect the total cell content or contents of the Triton X-100-soluble and -insoluble fractions for occludin, ZO-1, or ZO-2. HO treatment decreased the number of F-actin stress fibers in the basal portion of the cells. Similar to wild-type MDCK cells, HO increased ERK 1/2 activation in ZO-1 knockdown and occludin knockdown cells. Inhibition of ERK 1/2 activation blocked the increase in paracellular permeability in occludin knockdown cells. ZO-1 knockdown cell paracellular permeability was regulated by PP1, an src inhibitor, indicating that the loss of response to HO was not a general loss of the ability to regulate the paracellular barrier. Inhibition of myosin ATPase activity with blebbistatin increased paracellular permeability in ZO-1 knockdown cells but not in wild-type MDCK cells. HO treatment sensitized wild-type MDCK cells to inhibition of myosin ATPase. Knockdown of TOCA-1 protein, which promotes formation of local branched actin networks, reproduced the effects of ZO-1 protein knockdown. These results demonstrate that HO increases MDCK cell paracellular permeability through activation of ERK 1/2. This HO action requires ZO-1 protein and TOCA-1 protein, suggesting involvement of the actin cytoskeleton.

The ability of hydrogen peroxide (H2O2) to increase paracellular permeability of renal epithelial cell monolayers was examined and the role of occludin in this regulation was investigated. H2O2 treatment increased the paracellular movement of calcein, a marker for the leak pathway permeability, across monolayers of two renal epithelial cell lines, MDCK and LLC-PK1, in a concentration-dependent manner. At the same concentrations, H2O2 did not alter transepithelial resistance (TER) nor increase cell death. The magnitude of the H2O2-induced increase in leak pathway permeability was inversely related to cellular occludin protein content. H2O2 treatment did not produce any major change in total cellular content or Triton X-100-soluble or –insoluble fraction content of occludin protein. Occludin protein staining at the tight junction region was diminished following H2O2 treatment. The most dramatic effect of H2O2 was on the dynamic mobility of GFP-occludin into the tight junction region. H2O2 treatment slowed lateral movement of GFP-occludin into the tight junction region but not on the apical membrane. Further, removal of the cytoplasmic C-terminal region of occludin protein eliminated the effect of H2O2 on GFP-occludin lateral movement into the tight junction region. An increase in the mobile fraction of GFP-occludin was associated with a loss of response to H2O2. These data indicate that the H2O2-induced increase in renal epithelial cell paracellular permeability is mediated, at least in part, through occludin protein, possibly through a slowing of the rate of occludin movement into the tight junction region.