Fig. 5: Podocyte-specific expression of REDD1 was required for increased renal macrophage infiltration in the kidney of diabetic mice.
A, B Differentiated 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 as an osmotic control (OC) for 48 h. Transwell migration assay was used to evaluate chemotaxis in a co-culture model with CIHP-1 and THP-1 macrophages (A). Macrophages were stained with crystal violet and cells that migrated across the Transwell were counted (B). C Cre-lox recombination was used to achieve conditional podocyte-specific REDD1 knockout (REDD1 PodKO). D–H Diabetes was induced in REDD1fl/fl and REDD1 PodKO mice by streptozotocin (STZ) administration. Non-diabetic groups were administered a vehicle (Veh) control. All assessments were performed after 16 weeks of diabetes. Urine albumin to creatinine ratio (ACR) was determined (D). Kidney sections from diabetic and non-diabetic mice were immunolabeled for REDD1 (red) and the podocyte marker Nephrin (green) (E). Protein abundance of CCL2 was determined in renal homogenates by western blotting (F). Representative blots are shown with protein molecular mass in kDa indicated at right of each blot. Kidney sections were immunolabelled for F4/80 (red) and nuclei were counterstained with Hoechst 33342 (blue) (G). Representative micrographs (scale bar 50 µm) are shown. Immune cell populations of CD11b + F4/80+ macrophages (H) and CD86 + M1 macrophages (I) were determined by flow cytometry. 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.
