Figure 2: Crystal cytotoxicity involves the necroptosis pathway.

(a): Protein expression of TNFR1, RIPK1 and RIPK3 was determined by western blot from total proteins isolated 18 h after stimulation of mouse tubular epithelial cells with crystals of CaOx (1,000 μg ml⁻¹), MSU (500 μg ml⁻¹), CPPD (500 μg ml⁻¹) and cystine (500 μg ml⁻¹). β-actin was used as loading control. (b) Mouse tubular epithelial cells were exposed to different concentrations of CaOx, MSU, CPPD or cystine crystals as indicated in the presence or absence of necrostatin (Nec)-1 (100 μM) together with the pan-caspase inhibitor ZVAD–FMK–FMK (10 μM). Cell viability was assessed by MTT assay 24 h later. Data are expressed as mean cell viability±s.e.m. of three independent experiments. Baseline viability is set as 100%. (c) In similar experiments necrotic tubular epithelial cells were identified via propidium iodide positivity and the results were expressed as mean fluorescent intensity on digital analysis of pictures taken from culture dishes. Representative images are shown at an original magnification of × 200, scale bar, 40 μm. Data were analysed using Student’s t-test. *P<0.05, **P<0.01 and ***P<0.001 versus respective medium control. NS, not significant. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.