Epithelial barrier characteristics and expression of cell adhesion molecules in proximal tubule-derived cell lines commonly used for in vitro toxicity studies

Recent studies indicate that the actions of several nephrotoxic substances involve alterations in the function of cell adhesion molecules and changes in the paracellular permeability of the proximal tubule. In light of these findings, there is a need for appropriate in vitro model systems to study these phenomenae in greater detail. In the present study, the transepithelial resistance (TER), paracellular permeability of 14C-mannitol and immunofluorescent labeling of cell adhesion molecules (E-cadherin, N-cadherin, ZO-1, occludin, and claudins-2 and -7) were evaluated in several proximal tubule-derived cell lines that have been commonly used as model systems for in vitro toxicity studies. The cell lines studied included: LLC-PK 1, OK, NRK-52E and HK-2, along with commercially available primary cultures of human renal proximal tubule epithelial cells (HRPTE). LLC-PK 1 cells developed the highest TER followed by the OK cells and NRK-52E cells. The other cell lines failed to develop a TER even after 2 weeks in culture. There was a direct correlation between TER and ability to restrict paracellular movement of 14C-mannitol. Labeling studies showed that the LLC-PK 1 and NRK-52E cells expressed high levels of E-cadherin while the HRPTE cells expressed lower levels. OK cells expressed moderate levels of N-cadherin while LLC-PK 1 and NRK-52E cells expressed lower levels in isolated patches of cells. All cell lines expressed moderate-high levels of ZO-1. LLC-PK 1 also expressed the tight-junction proteins occludin and claudin-7; OK cells also expressed moderate levels of occludin. All other cell lines had weak claudin-7 and occludin labeling. None of the cell lines expressed claudin-2. These results show that the LLC-PK 1, OK and NRK-52E cell lines exhibit characteristics that most closely resembled those of the proximal tubule in vivo, and they indicate that these cell lines would be appropriate models for studying the effects of toxicants on cell–cell junctions and cell adhesion molecules.