Measurement of Placental and Renal NADPH Oxidase

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and superoxide anion (O₂^●−) sources were measured in placenta and renal cortex homogenates by lucigenin-enhanced chemiluminescence [21 (link)]. Samples were preincubated for 5 min at 37 °C with the following inhibitors of potential O₂^●−sources (0.1 mmol/L; Sigma-Aldrich): DPI (inhibitor of flavoproteins); oxypurinol (inhibitor of xanthine oxidase); and rotenone (inhibitor of the mitochondrial electron transport chain). Different inhibitors of NADPH oxidase (Sigma-Aldrich) were also used as follows to explore the relative contributions of each NOX isoform in O₂^●− production: specific NOX1 inhibitor (0.5 µmol/L, ML171, cat. # 175226); NOX1/4 inhibitor (0.1 µmol/L, GKT136901, cat. # 492000); and a pan-NADPH oxidase inhibitor (10 µmol/L, VAS2870, cat. # 5340320001). All measurements were normalized to the protein content in the samples, and results were expressed in relation to the NP group.