Fig. 6: REDD1 expression in podocytes was required for NLRP3 inflammasome activation in diabetic mice.
A–E Differentiated wild-type (WT) and REDD1 knockout (KO) CIHP-1 cells were exposed to culture media containing either 30 mM glucose (HG) or 5 mM glucose plus 25 mM mannitol as an osmotic control (OC) for 48 h. Relative expression of NLRP3 mRNA was determined in cell lysates by qPCR (A). NLRP3 protein content relative to GAPDH was estimated by western blotting (B). Representative blots are shown with molecular mass in kDa indicated to the right of the blot. Active caspase-1 (green) was visualized by immunofluorescence microscopy using FAM-YVAD-FMK FLICA (fluorescently labeled inhibitor of caspase) probe (C; scale bar 25 µm). Gasdermin D (GSDMD) N-terminal cleavage (GSDMD-N) was determined in cell lysates by western blotting (D). LDH released into cell culture supernatant was quantified (E). F-I, Diabetes was induced in REDD1fl/fl and REDD1 PodKO mice by streptozotocin (STZ) administration. Non-diabetic mice were administered a vehicle (Veh) control. Nlrp3 mRNA expression in glomerular isolates was quantified by qPCR (F). NLRP3 and GSDMD protein in glomerular isolates were determined by western blotting (G). Immunofluorescence microscopy was used to determine colocalization of NLRP3, GSDMD, and WT-1 with the podocyte marker nephrin (H). IL-1β protein content in renal homogenates was quantified by ELISA (I). Representative micrographs (scale bar 50 µm) are shown. Individual data points are plotted. Significance was analyzed by two-way ANOVA and pairwise comparisons were made using the Tukey’s test for multiple comparisons. *p < 0.05 versus OC or Veh; #p < 0.05 versus WT or REDD1fl/fl.
