EMT and NF-κB Pathway in Renal Cells

NRK-52E cells, a rat renal tubular cell line, were obtained from ATCC and cultured in the presence of DMEM/Ham’s F12 (DMEM/F12) medium supplemented with 10% fetal calf serum (FCS), glutamine (2 mM), penicillin (100 IU/ml), and streptomycin (100 μg/ml). Cells were cultured at 37 °C in a humidified atmosphere of 5% CO2 in air. For EMT experiments, cells were treated with 20μg/ml rat albumin (Sigma) for 48 h; for NF-κB translocation experiments, cells were treated with 20μg/ml rat albumin for 3 h.
Mesenchymal stem cells (MSCs) were generous gifts from Texas A&M Health Science Center. Passenger 7 MSCs were cultured according to the instruction in Eagle’s alpha minimum essential medium (α-MEM; Sigma), supplemented with 20% FBS (FBS, Invitrogen), 4mM L-glutamine (Invitrogen-Gibco), 100U/ml penicillin and 100ug/ml streptomycin (Invitrogen-Gibco). After 72 h, the medium was collected and used as stem cell conditioned media (SCM). The control conditioned media (CCM) were obtained from culturing rat renal medullary interstitial cells for 72 h using the same medium. Rat renal medullary interstitial cells were prepared as we described previously [22 (link), 23 (link)]. In preliminary experiments, cells treated with CCM did not show significant difference in the EMT markers described below compared with naive cells.