Abstract
Autophagy has been shown to be important for normal homeostasis and adaptation to stress in the kidney. Yet, the molecular mechanisms regulating renal epithelial autophagy are not fully understood. Here, we explored the role of the stress-responsive transcription factor forkhead box O3 (FoxO3) in mediating injury-induced proximal tubular autophagy in mice with unilateral ureteral obstruction (UUO). We show that following UUO, FoxO3 is activated and displays nuclear expression in the hypoxic proximal tubules exhibiting high levels of autophagy. Activation of FoxO3 by mutating phosphorylation sites to enhance its nuclear expression induces profound autophagy in cultured renal epithelial cells. Conversely, deleting FoxO3 in mice results in fewer numbers of autophagic cells in the proximal tubules and reduced ratio of the autophagy-related protein LC3-II/I in the kidney post-UUO. Interestingly, autophagic cells deficient in FoxO3 contain lower numbers of autophagic vesicles per cell. Analyses of individual cells treated with autophagic inhibitors to sequentially block the autophagic flux suggest that FoxO3 stimulates the formation of autophagosomes to increase autophagic capacity but has no significant effect on autophagosome-lysosome fusion or autolysosomal clearance. Furthermore, in kidneys with persistent UUO for 7 days, FoxO3 activation increases the abundance of mRNA and protein levels of the core autophagy-related (Atg) proteins including Ulk1, Beclin-1, Atg9A, Atg4B, and Bnip3, suggesting that FoxO3 may function to maintain components of the autophagic machinery that would otherwise be consumed during prolonged autophagy. Taken together, our findings indicate that FoxO3 activation can both induce and maintain autophagic activities in renal epithelial cells in response to injury from urinary tract obstruction.
Highlights
Autophagy has been shown to be important for normal homeostasis and adaptation to stress in the kidney
Using the autophagy reporter mice CAG-RFP-EGFP-LC3, we examined epithelial autophagy by the presence of RFP puncta that were known to be present in all stages of autophagic vesicles
We show that persistent obstructive injury and hypoxia to renal tubules activates the forkhead box O3 (FoxO3) transcription factor to regulate epithelial autophagy
Summary
Autophagy has been shown to be important for normal homeostasis and adaptation to stress in the kidney. We explored the role of the stress-responsive transcription factor forkhead box O3 (FoxO3) in mediating injury-induced proximal tubular autophagy in mice with unilateral ureteral obstruction (UUO). We focused on FoxO3, which is a stress-induced transcription factor known to play an important role in energy metabolism, stress adaptation, longevity, cell proliferation, and death [8]. Exposing renal tubular cells to serum obtained from caloric restricted aging mice activates FoxO3 and induces the expression of a core autophagy protein Bnip, which plays a role in mitophagy. This FoxO3-mediated autophagic effect provides a mechanism for cytoprotection against hypoxia during aging [16]. Given the importance of FoxO3 in stress response across many cell types
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