Effects of calcium oxalate monohydrate crystals on expression and function of tight junction of renal tubular epithelial cells

Tight junction has a crucial role in regulating paracellular transports (as a barrier) and in separating apical from basolateral compartments to maintain cell polarity (as a fence). Tight junction can be disrupted by various stimuli, including oxidative stress, pathogens and proinflammatory cytokines. However, association of defective tight junction with kidney stone pathogenesis remains unknown. We therefore examined whether calcium oxalate monohydrate (COM) crystals, which are the major crystalline composition in kidney stones, have any effects on expression and function of tight junction of polarized renal tubular epithelial cells. Western blot analysis revealed marked decrease in levels of occludin and zonula occludens-1 (ZO-1) in COM-treated polarized Madin-Darby canine kidney (MDCK) cells. Immunofluorescence staining revealed not only the decline of these tight junction proteins but also their redistribution and dissociation in COM-treated cells. Additionally, transepithelial resistance was significantly decreased, indicating impaired tight junction barrier and increased paracellular permeability in COM-treated cells. Subcellular fractionation followed by western blot analysis of Na+/K+-ATPase-alpha 1 revealed that this basolateral membrane marker was also detectable in apical membrane fraction of COM-treated cells, but not in apical membrane fraction of control cells. Immunofluorescence study confirmed the translocation of Na+/K+-ATPase-alpha 1 (from basolateral to apical membranes) in COM-treated cells, indicating impaired fence function of the tight junction. Moreover, dihydrorhodamine assay using flow cytometry revealed the significantly higher level of hydrogen peroxide in the COM-treated cells. These data provide the first evidence to demonstrate decreased expression and defective barrier and fence functions of the tight junction of renal tubular epithelial cells exposed to COM crystals that may be fundamental for subsequent renal tubulointerstitial injury, which in turn enhances the stone pathogenesis. Laboratory Investigation (2011) 91, 97-105; doi:10.1038/labinvest.2010.167; published online 20 September 2010