Figure 6. Loss of FoxO3 results in reduced autophagic adaptation.

Tubular FoxO3 was deleted from day 8 to day 21 following a 35-minute left kidney IRI and right nephrectomy. Kidneys were analyzed 4 weeks after IRI. (A) Representative EM images of autophagosomes and autolysosomes (white arrows) in tubules of FoxO3^ctl mice 4 weeks after IRI. (B) Lower levels of LC3II, LC3I, and Bnip3 proteins were detected, but no significant changes in ULK1 protein levels were seen in FoxO3^tub kidneys (n = 4). *P ˂ 0.05 comparing FoxO3^tub with FoxO3^ctl, by 2-tailed Student’s t test. (C) The autophagy reporter CREL mice were bred with FoxO3^tub and FoxO3^ctl mice and subjected to IRI, as above. Fewer RFP dots were visible in renal tubules labeled with E-cadherin (green) that appeared atrophic in FoxO3^tub mice. Tubules in FoxO3^ctl mice contained a greater abundance of RFP dots. (D) Quantification of RFP dots in proximal tubules (labeled in cyan with LTA) indicated that the majority of autophagic cells in FoxO3^tub kidneys contained 10 or fewer dots per cell, whereas the majority of autophagic cells in FoxO3^ctl kidneys contained 10 or more dots per cell. Nuclei were counterstained with DAPI in blue in C and D. Scale bars: 20 μm (C and D).