Figure 4.

Calpastatin overexpression restores glomerular autophagic flux in the type I diabetes model. (A) Representative immunofluorescence of SQSTM1 (green) and PODXL (red) in the glomerulus from GFP-LC3 UniNx and GFP-LC3 CST^Tg UniNx mice 6 weeks after being injected or not with STZ. Nuclei are stained in blue with Hoechst. Scale bar: 50 μm (B) Quantification of the SQSTM1 area in the PODXL area from GFP-LC3 UniNx and GFP-LC3 CST^Tg UniNx mice 6 weeks after being injected or not with STZ. For ethical reasons, we did not duplicate GFP-LC3 UniNx and GFP-LC3 CST^Tg uniNx groups, and quantifications of these groups are already presented in Figure 1H and Supplemental Figure 1B. n=7 GFP-LC3 UniNx, n=10 GFP-LC3 UniNx+STZ, n=7 GFP-LC3 CST^Tg uniNx, n=8 GFP-LC3 CST^Tg UniNx+STZ mice. Two-way analysis of variance: genotype, P = 0.13; treatment, *P = 0.0223. Tukey multiple comparison test: **P = 0.002 for GFP-LC3 UniNx versus GFP-LC3 UniNx+STZ and **P = 0.007 for GFP-LC3 UniNx versus GFP-LC3 CST^Tg UniNx+STZ. SQSTM1 accumulates in WT diabetic mice but not in mice with calpastatin overexpression. (C) Representative immunofluorescence of GFP-LC3 (green) and PODXL (red) in glomeruli from GFP-LC3 UniNx and GFP-LC3 CST^Tg UniNx mice 6 weeks after being injected or not with STZ. Nuclei are stained in blue with Hoechst. (D) Quantification of the GFP-LC3 punctum area in the PODXL area from GFP-LC3 UniNx and GFP-LC3 CST^Tg UniNx mice 6 weeks after being injected or not with STZ. n=6 GFP-LC3 UniNx, n=10 GFP-LC3 UniNx+STZ, n=7 GFP-LC3 CST^Tg uniNx, n=8 GFP-LC3 CST^Tg UniNx+STZ mice. Two-way analysis of variance: genotype, P = 0.09; treatment,**P = 0.008. Tukey multiple comparison test: **P = 0.02 GFP-LC3 UniNx+STZ versus GFP-LC3 CST^Tg UniNx+STZ and **P = 0.02 for GFP-LC3 CST^Tg UniNx versus GFP-LC3 CST^Tg UniNx+STZ. We observe an increased number of LC3+ dots in podocytes from diabetic mice with calpastatin overexpression when compared with WT diabetic mice.