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.
Highlights
Different renal tubular segments have specific epithelial characteristics and functions
To investigate the contributory role of tight junctions (TJ) proteins in the leaky proximal tubule, we xamined the effect of inhibiting claudin-2, occludin, and zonula occludens (ZO)-1 expression on transepithelial electrical resistance (TER) and paracellular permeability using the immortalized human proximal tubule epithelial cell line HK-2
Control HK-2 monolayers achieved a steady-state TER of 6–8 ΩÁcm2 when grown in 12-well Transwell filters, which are compatible with leaky epithelia
Summary
Different renal tubular segments have specific epithelial characteristics and functions. The proximal tubule has leaky epithelia, whereas collecting ducts have more effective tight junctions (TJs). Paracellular permeability decreases from the proximal tubule to the collecting ducts because of a unique array of claudins expressed in each segment, with higher levels of occludin and ZO-1 in distal tubules [1]. Claudin-2, occludin, and zonula occludens (ZO)-1 are major components of TJs in proximal tubules [2], their roles in maintaining “loose” epithelia have yet to be defined. Claudin-2, ZO-1, and occludin are major components of tight junctions (TJs) in the proximal tubule. Their roles in maintaining paracellular permeability as leaky epithelia have yet to be defined.
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