NO₂-OA protects against hemin-induced renal injury in SCD. (A-B) Nrf2-dependent gene expression in HK2 cells treated with NO₂-CLA and NO₂-OA (n = 3). ∗P < .05, ∗∗P < .001, ∗∗∗P < .0001 by 1-way ANOVA and Dunnett posttest. (C) Protein expression 16 hours after NO₂-OA in HK2 cells. (D) DCF oxidation by hemin (8 μM) with or without NO₂-OA pretreatment (16 hours). ∗∗∗P < .0001 by 2-way ANOVA. (E) HK2 cell viability 48 hours after 40 μM hemin challenge with or without NO₂-OA pretreatment (16 hours) and 10 μM HO-1-IN-1 (n = 7). ∗∗∗P < .0001 by 2-way ANOVA and Dunnett posttest. (E, inset) No effect of HO1-IN-1 on HK2 cell viability. (F) Cytokine production by J774a1 macrophages after 40 μM hemin in the presence or absence of NO₂-OA (n = 4-6). ∗P < .05, ∗∗P < .001, ∗∗∗P < .0001 by 1-way ANOVA and Tukey posttest. (G-H) Representative micrographs and quantification of NQO1 and HO1 expression in the kidney of Townes’ SCD mice that received 2.5 mg/kg per day NO₂-OA or SO for 3 weeks by oral gavage. Each point is a single field of view from 3 mice per group. The bar represents 100 μm. ∗∗P < .001 by unpaired t test. (I-J) Representative micrographs and apoptotic cell quantification by TUNEL assay in SCD mouse kidney 48 hours after hemin challenge (20 μM/kg IV) in the presence or absence NO₂-OA treatment as before. The bar represents 20 μm. ∗∗P < .001 by t test. (K-M) Plasma creatinine, BUN, and urinary KIM-1 levels before and 48 hours after hemin challenge in SCD mice that received vehicle or NO₂-OA pretreatment (n = 7-8 per group). ∗P < .05, ∗∗∗P < .0001 by 2-way repeated measures ANOVA and Fisher posttest. BUN, blood urea nitrogen; DCF, 2,7-dichlorodihydrofluorescein diacetate; GCLM, regulatory subunit of glutamate-cysteine ligase; HO1-IN-1, heme oxygenase-1-IN-1; h, hour; KIM-1, kidney injury molecule-1; MCP-1, monocyte chemoattractant protein-1; MFI, mean fluorescence intensity; mRNA, messenger RNA; NQO1, NAD(P)H:quinone dehydrogenase-1; SO, sesame oil vehicle; TNF-α, tumor necrosis factor-α.