Figure 1.

Mutation of ATG5 in kidney epithelium prevents autophagy and causes FSGS. (A) Ethidium-stained gel showing amplified PCR products of genomic DNA at the Atg5 locus to determine the genotype and deletion of floxed gene of the Atg5 locus. The upper gel represents an image with shorter exposure time, while the lower gel shows the one with longer exposure time. They indicate that in Six2-Cre; Atg5^flox/flox whole kidney most genomic DNA has undergone recombination and only a small portion of DNA contains the floxed exon. (B) Western blots of whole kidney at 2 months showing deletion of ATG5 protein, increase of P62 protein, and loss of LC3II (lower band), indicative of loss autophagy in the kidney. (C) Electron microscopic images showing an autophagosome in a Six2-Cre; Atg5^fl/fl kidney proximal tubule cell in (bar=100 nm). (D) Graph showing urine albumin-to-creatinine ratio in mice with time after birth. (E–H) Typical glomerular morphology at 4 months after birth, stained by periodic acid-Schiff or silver methenamine. Note the presence of segmental sclerotic lesions (fibrosis and capillary loop destruction) and focal glomerular involvement. (I–L) Examples of additional glomerular changes, including pseudocrescent formation, collapsing lesions, and hyalinosis. (M and N) Quantification of glomerulosclerosis by index or by percentage of glomeruli with sclerosis. (O) Quantification of glomerulosclerosis by severity score. (P) Quantification of WT1+ cells per glomerular cross-section. (Q) Representative electron microscopic images showing capillary loop coated with podocytes in the urinary space. Note in mutant mice that the podocytes show vacuolation (arrows) and there is wrinkling and collapse of the capillary loop structure and extensive foot process effacement (arrowheads). Data are mean±SEM. n=4–7/group. ***P<0.001; **P<0.01; *P<0.05. Bar=100 µm (E, G), 50 µm (F, H, I–L), 2.5 µm (Q).