Recombinant human soluble tweak

HK-2 human proximal tubular epithelial cells (ATCC, Rockville, MD, USA) were grown on RPMI 1640 (Life Technologies, Grand Island, NY, USA) with 10% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/ml sodium selenite and 5 ng/ml hydrocortisone in 5% carbon dioxide at 37 °C. For experiments, cells were cultured in serum-free media 24 h prior to addition of stimuli and throughout the experiment. Exposure of cultured tubular cells to bovine serum albumin (Sigma, St. Louis, MO, USA) was used as a surrogate for the in vivo exposure of tubular cells to albumin in proteinuric nephropathies. Recombinant human soluble TWEAK (Millipore, Billerica, MA, USA) was used at 100 ng/ml, murine TNF-α (PrePotech, London, UK) at 30 ng/ml and INF-γ (PrePotech, London, UK) at 30 U/ml.

MCT cells are a cultured line of proximal tubular epithelial cells harvested originally from the renal cortex of SJL mice and have been extensively characterized [27 (link)]. They were cultured in RPMI 1640 (GIBCO, Grand Island, NY, USA), 10% decomplemented fetal bovine serum (FBS), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin, in 5% CO2 at 37 °C [27 (link)]. Recombinant human soluble TWEAK (Millipore, Billerica, MA) was used at 10 to 100 ng/ml, based on prior dose-response experiments [5 (link)]. DDT (Prof. Bucala, Yale University School of Medicine) was used at 10 ng/mL based on prior experience activating the CD74 receptor on renal cells with MIF and on circulating levels of DDT [21 (link)]. The NFκB inhibitor parthenolide (Sigma, St. Louis, MO) was used at 10 μM based on previous dose-responses studies [28 (link)].