Figure 9: Necroptosis is involved in human acute oxalate nephropathy.

(a–c) Primary renal human progenitor cells were pretreated with either ZVAD–FMK (10 μM) and Nec-1 (100 μM) or NSA (1 μM) before being exposed to CaOx (1000 μg ml⁻¹), MSU (500 μg ml⁻¹), CPPD (500 μg ml⁻¹) and cystine (500 μg ml⁻¹). Cell viability was assessed by MTT assay (a and b) and cell death was assessed quantifying PI positivity (c) 24 h later. Data are expressed as mean±s.e.m. of three independent experiments. Baseline viability is set as 100%. Data were analysed using Student’s t-test. *P<0.05, **P<0.01, ***P<0.001 either versus vehicle control. (d) Primary renal human progenitor cells were stimulated with different crystals as indicated above for 18 h. The expression of Phospho-mlkl and total mlkl was detected by western blot with β-actin as a loading control. (e) Selective cases of human oxalate crystal-related acute kidney injury were stained with PAS and for TNF-α, TNFR1 and phosphorylated MLKL. Representative images are shown at an original magnification of 1:400. Scale bar, 40 μm. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PAS, periodic acid-Schiff; PI, prodidium iodide.