Isolation of podocytes from CD36 knockout mice (on C57BL6/J background, kindly provided by Dr. Deneys van der Westhuyzen at University Kentucky), TSP1 knockout mice (Jackson laboratory), or control wild type mice was performed as described previously [20 (link), 21 (link)]. Briefly, glomeruli were isolated from mice [21 (link)] and plated on collagen type I-coated dishes at 37°C in RPMI-1640 medium with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, 100 mM HEPES, 1mM sodium bicarbonate, and 1mM sodium pyruvate to allow glomerular podocytes to grow out. Subculture of primary podocytes was performed by detaching the glomerular cells with 0.25% trypsin-EDTA, followed by sieving thought 40 µm cell strainer, and cultured on type 1 collagen coated dishes. Podocytes of passages 1 or 2 were used for the experiments.
CD36−/− podocytes or wild type podocytes were treated with 125 µM of palmitate (Sigma) or control BSA for 24 h. After treatment, cells were harvested. TSP1 expression in both mRNA levels and protein levels were determined by PCR and western blotting, respectively. TSP1−/− podocytes or wild type podocytes were treated with 125 µM of palmitate (Sigma) or control BSA for 24 hours. After treatment, caspase 3 activity was analyzed by using caspase-3 colorimetric assay kit (R&D). BSA filter assay was also analyzed in these cells.
CD36−/− podocytes or wild type podocytes were treated with 125 µM of palmitate (Sigma) or control BSA for 24 h. After treatment, cells were harvested. TSP1 expression in both mRNA levels and protein levels were determined by PCR and western blotting, respectively. TSP1−/− podocytes or wild type podocytes were treated with 125 µM of palmitate (Sigma) or control BSA for 24 hours. After treatment, caspase 3 activity was analyzed by using caspase-3 colorimetric assay kit (R&D). BSA filter assay was also analyzed in these cells.