Hk 2 human proximal tubular epithelial cells

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.

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 17. VHL‐defective clear cell renal carcinoma cells (VHL−/− ccRCC) and VHL+/+ ccRCC cells have been previously described 18. For experiments, cells were rested in serum‐free media 24 hr prior to the addition of stimuli and throughout the experiment. Five hundred thousand cells were seeded in 60 mm diameter wells for RNA extraction, Western blot or flow cytometry experiments. Cells were treated with 100 ng/ml TWEAK (Millipore, Watford, UK), 1 ng/ml TGFβ1 (Peprotech, London, UK), 100 nmol/l paricalcitol (Abbot, Chicago, Illinois), 100 μmol/l TGFβ1 receptor 1 (ALK5) inhibitor (SB431542; Sigma‐Aldrich, Sigma, St. Louis, MO) or 1 ng/ml neutralizing anti‐TGFβ1 antibody (ab100NA; R&D Systems, Minneapolis, MN), based on prior dose–responses studies from our lab.