Fig. 4: REDD1 deletion prevented diabetes-induced NF-κB activation in the kidney. | Cell Death & Disease

Fig. 4: REDD1 deletion prevented diabetes-induced NF-κB activation in the kidney.

From: REDD1 expression in podocytes facilitates renal inflammation and pyroptosis in streptozotocin-induced diabetic nephropathy

Fig. 4

A, B Diabetes was induced in REDD1+/+ and REDD1−/− mice by administration of streptozotocin (STZ). Non-diabetic control mice received vehicle (Veh). A Nuclear isolates were prepared from kidney homogenates. NF-κB and Lamin B were examined in nuclear isolates by western blotting and NF-κB activity was quantified by DNA-binding ELISA. Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. B REDD1 (red) and Nephrin (green) were visualized in kidneys by immunofluorescence microscopy. White box indicates area shown at increased magnification. Representative micrographs are shown (scale bar 50 μm). C–I Wild-type (WT) and REDD1 knockout (KO) CIHP-1 were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol (OC) for 48 h. NF-κB phosphorylation at S536 and REDD1 protein abundance was determined in cell lysates by western blotting (C). Nuclear localization of NF-κB p65 (white arrowheads) was evaluated by immunofluorescence (D). Nuclei were visualized with DAPI (scale bar 25 μm). NF-κB activity was measured in lysates from cells expressing NF-κB firefly luciferase/Renilla luciferase reporter plasmids by dual luciferase assay (E). Relative expression of IL1B and CCL2 mRNA were determined by qPCR (F). IL-1β secreted into culture media was determined by ELISA (G). Chromatin immunoprecipitation (ChIP)-PCR analysis was carried out in WT and REDD1 KO podocytes to determine binding of p65 NF-κB to the promoter region of the CCL2 gene (H). CCL2 protein levels were determined in cell lysates by western blotting (I). J NF-κB p65 phosphorylation and NF-κB luciferase reporter activity was evaluated in REDD1 KO cells expressing either an empty vector control (EV) or hemagglutinin (HA)-tagged REDD1. Individual data points are presented as means ± SD (n = 4–6). Differences between groups were identified by two-way ANOVA. *p < 0.05 versus Veh or NG; #p < 0.05 versus REDD1+/+, WT, or EV.

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