Roles of claudin-2, ZO-1 and occludin in leaky HK-2 cells

Abstract

BackgroundClaudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. However, their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined.MethodsTo investigate the contributory role of TJ proteins in the leaky proximal tubule, we xamined the effect of inhibiting claudin-2, occludin, and ZO-1 expression on transepithelial electrical resistance (TER) and paracellular permeability using the immortalized human proximal tubule epithelial cell line HK-2. For this, small-interfering RNAs (siRNAs) against claudin-2, occludin and ZO-1 were transfected into HK-2 cells. TER and transepithelial flux rates of dextrans (4 and 70 kDa) were determined after 24 h.ResultsTransfection of siRNAs (25 nM) knocked down TJ protein expression. Control HK-2 monolayers achieved a steady-state TER of 6–8 Ω·cm2 when grown in 12-well Transwell filters, which are compatible with leaky epithelia. Knockdown of claudin-2 decreased in TER and increased occludin expression. Transfection with siRNA against either occludin or ZO-1 increased TER and decreased claudin-2 expression. TER was decreased by co-inhibition of claudin-2 and ZO-1 but increased by co-inhibition of claudin-2 and occludin. TER was suppressed when claudin-2, occludin, and ZO-1 were all inhibited. Dextran flux rate was increased by claudin-2, occludin, or ZO-1 siRNA transfection. Increased dextran flux was enhanced by co-transfection of claudin-2, ZO-1, and occludin siRNA.ConclusionsThe depletion of claudin-2, occludin and ZO-1 in HK-2 cells had differential effects on TER and macromolecule flux. We demonstrated that integration of claudin-2, occludin and ZO-1 is necessary for maintaining the function of the proximal tubular epithelium.

Blocking Connexin-43 mediated hemichannel activity protects against early tubular injury in experimental chronic kidney disease

“…To delineate the functional consequences of these altered levels of expression, atomic force microscopy force spectroscopy and trans-epithelial electrical resistance assessed changes in cell-cell tethering and paracellular permeability respectively. Corroborating recent findings that depletion of Claudin-2 and ZO-1 is detrimental to proximal tubule epithelial cell function through a "leaky" epithelia, [49] both TGF-β1 and ATPγS independently reduce PTEC resistance, and ultimately impair barrier integrity. Furthermore, force spectroscopy confirmed ATPγS reduced the unbinding force required to uncouple two attached cells.…”

Worm-Based Alternate Assessment of Probiotic Intervention against Gut Barrier Infection

“…Therefore, differential clustering of transcription factors would lead to specific regulation of ZO-1 without affecting expressions of other TJ molecules. Moreover, genetic ablation of ZO-1 level had marginal impacts on expressions of other TJ molecules including claudins and occludin in the epithelial cell layer [36], indicating independent pathways of ZO-1 expression. In the present study, EcN improved expression of ZO-1, which is a crucial scaffold protein for direct linker molecules such as claudins and occludin in the intercellular gap.…”

Therapeutic Target and Cell-signal Communication of Chlorpromazine and Promethazine in Attenuating Blood–Brain Barrier Disruption after Ischemic Stroke

“…Shi et al thought that blood occludin could be a clinically viable biomarker for evaluating BBB damage during ischemia/reperfusion in patients, which put forward the clinical significance of occludin on the BBB 25 . The disruption of occludin led to functional changes of the TJs in rats 26,27 and was associated with hypoxia-induced vascular leakage 28 . The protective effects of occludin on BBB integrity, however, are not sufficiently understood.…”