Occludin: structure, function and regulation.

Epithelial and/or endothelial barriers play a critical role in animal, including human, life forms. The tight junction (TJ) is an essential component of these barriers. Occludin is a major component of the TJ. The structure of occludin, including its gene splice variants and protein essential components have been elucidated. Phosphorylation/dephosphorylation plays a major role in regulation of occludin and TJ. Disruption of occludin regulation is an important aspect of a number of diseases. Strategies to prevent and/or reverse occludin downregulation may be an important therapeutic target.

Transepithelial resistance and inulin permeability as endpoints in in vitro nephrotoxicity testing.

Transepithelial electrical resistance (RT) and the flux of fluorescein isothiocyanate (FITC) across Madin Darby canine kidney (MDCK) strain 1 cells and porcine epithelial kidney (LLC-PK1) monolayers were compared between three laboratories for a range of nephrotoxins. The precision of the REMS AutoSampler was similar to that of the Ussing chamber and the ENDOHM technique, but superior to using chopstick electrodes, for measurements of resistance. The nephrotoxins used were selective for the proximal tubule, and in all cases, LLC-PK1 cells were more sensitive than MDCK cells. In most cases, change in RT was a more sensitive indicator of damage than alterations in FITC flux. The REMS system provides high intra-plate precision for RT measurements and is a higher throughput system, which is applicable to screening for nephrotoxicity in vitro.

Role for TGF-beta in cyclosporine-induced modulation of renal epithelial barrier function.

It was previously shown that cyclosporine A (CsA) increases transepithelial resistance in MDCK cells. Activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) cascade seems to be pivotal to the CsA-induced increase in transepithelial electrical resistance (TER). This study examined the role played by TGF-beta in mediating the CsA-induced activation of ERK1/2 and the resulting increase in TER in MDCK cells. Paracellular permeability across MDCK monolayers after various treatments was assessed by measurement of TER. TGF-beta secretion was measured by Western blot and ELISA. Activation of the ERK1/2 pathway and tight junction protein expression were also assessed by Western blot analysis. CsA increased production and secretion of TGF-beta and expression of the TGF-beta receptor II. Exogenous addition of TGF-beta1 activated ERK1/2 and increased TER across MDCK monolayers, both of which were attenuated by the MEK inhibitor U0126. Neutralizing antibodies against TGF-beta1 and the TGF-beta receptor II significantly reduced the CsA-induced increase in TER. Both CsA and TGF-beta1 increased expression of tight junction proteins claudin-1 and zonula occludens 2. Inhibition of the p38 MAPK pathway also attenuated the TGF-beta1-induced increase in TER. The results presented here suggest that the CsA-induced modulation of paracellular permeability may be mediated, at least in part, by an increase in TGF-beta production.

Modulation of renal epithelial barrier function by mitogen-activated protein kinases (MAPKs): mechanism of cyclosporine A-induced increase in transepithelial resistance.

BACKGROUND: Cyclosporine A (CsA) has been shown to increase transepithelial resistance in Madin-Darby canine kidney (MDCK) cells, and the mechanism may involve altered phosphorylation of junctional proteins. In this study, we examine the effect of the extracellular signal-regulated protein kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (MAPK) pathways on the basal transepithelial resistance (TER) and on the CsA-induced increase in TER across MDCK monolayers. Here we present evidence that CsA may be mediating some of its effects through activation of the ERK 1/2 MAPK pathway. METHODS: MDCK cells were treated with CsA (4.2 micromol/L) and paracellular permeability was assessed by measuring TER. The role of the ERK 1/2 and the p38 MAPK pathways in modulating TER was investigated using the inhibitors PD98059 and U0126 for ERK 1/2 and SB203580 for p38. ERK 1/2 and p38 phosphorylation/activation was also examined by Western blot analysis. RESULTS: CsA (4.2 micromol/L) increased the TER of MDCK monolayers. The ERK 1/2 inhibitor PD98059 decreased basal TER and also ameliorated the CsA-induced increase in TER. Similar results were found with the U0126 inhibitor of ERK 1/2. The p38 inhibitor SB203580 had no effect on the basal TER of the monolayers, however, SB203580 significantly augmented the CsA-induced increase in TER. CsA was shown to significantly activate ERK 1/2 and this activation by CsA was prevented by PD98059. Inhibition of the p38 pathway by SB203580 also resulted in activation of ERK 1/2 and this activation of ERK 1/2 was further enhanced by CsA. No effect of CsA or the inhibitors PD98059 or SB203580 on p38 phosphorylation was detected. CONCLUSION: The results presented here suggest that activation of the ERK 1/2 MAPK cascade is important in the regulation of the paracellular permeability in MDCK cells. Activation of this pathway appears to be pivotal to the CsA-induced increase in TER.