Epithelial to mesenchymal transformation in tubular epithelial cells undergoing anoxia.

ABSTRACT

AIM: Epithelial-mesenchymal transformation (EMT) has been proved to be a critical event in fibrogenesis of renal allografts. This study sought to determine whether anoxia could induce EMT from tubular epithelial cells (TEC). METHODS: Rat TEC-line (NRK-52E) was cultured in Dulbelco's modified Eagle's medium (DMEM) without glucose under 100% N2 for 4 hours. After 6, 12, 24, 48, and 72 hours, the expressions of connective tissue growth factor (CTGF) mRNA and protein were measured by real-time RT-PCR and Western blot, respectively. Morphologic changes and cytoskeleton remodeling were observed in NRK-52E cells under laser confocal microscopy. Immunohistochemistry and flow cytometry were used to detect expression changes of E-cadherin, alpha-smooth muscle actin (SMA), types I and IV collagen, all of which are involved in TEC, EMT. RESULTS: After stimulation by anoxia, NRK-52E cells became round and enlarged with a remodeled cytoskeleton. The expressions of CTGF mRNA and protein were upregulated after 6 hours, reaching their peak at 48 hours. The expressions of types I and IV collagen, and alpha-SMA were all upregulated except for E-cadherin. CONCLUSIONS: Anoxia upregulated the expression of CTGF and other EMT-associated genes in NRK-52E cells.