Kidney Week

Abstract: TH-PO238

Hypoxia Induces Tubular Autophagy by Preventing Oxygen-Dependent Prolyl Hydroxylation and Degradation of Stress-Responsive Transcriptional Factor FoxO3

Session Information

• AKI Basic: Cell Death and Biomarkers
  November 02, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Acute Kidney Injury

• 001 AKI: Basic

Authors

• Li, Ling, Collumbia University College of Physicians and Surgeons, New York, New York, United States

Ling Li,

• Ha, Catherine, Collumbia University College of Physicians and Surgeons, New York, New York, United States

Catherine Ha,

• Al-Awqati, Qais, Collumbia University College of Physicians and Surgeons, New York, New York, United States

Qais Al-Awqati,

• Lin, Fangming, Collumbia University College of Physicians and Surgeons, New York, New York, United States

Fangming Lin,

Background

Renal hypoxia results in metabolic perturbations and cell stress which likely leads to CKD following AKI. Autophagy is an evolutionally conserved cellular response to stress. Recent finding in our lab has shown that the stress-responsive transcription factor, FoxO3, can activate renal epithelial autophagy.

Methods

We used the autophagy reporter line (CREL mouse), which expresses a tandem RFP and EGFP fused with LC3 protein, to study molecular regulation of autophagy during the AKI to CKD transition.

Results

At 2-4 weeks following left renal ischemic injury for 35 min and right nephrectomy, we found progressive increases in proximal tubular autophagy in areas of low capillary density and thus hypoxia. Concomitantly, FoxO3 was activated with a 4-fold increase in nuclear expression over controls in the hypoxic tubules. To test whether hypoxia is an upstream activator for FoxO3 leading to autophagy, we exposed primary cultures of proximal tubular cells to 1%O₂ and found an increase in FoxO3 protein levels (50% increase at 30 min and 115% increase at 60 min), as well as its prominent nuclear localization. In renal tubular cells isolated from CREL mice, hypoxia or nutrient deprivation (a potent stimulator for autophagy) led to a time-dependent appearance of autophagic dots and increased conversion of LC3I to LC3II proteins. How could hypoxia activate FoxO3? We found that FoxO3, like Hif proteins, is prolyl hydroxylated, which leads to its degradation. Immunoprecipitation with FoxO3 antibody followed by immunoblot analyses with pan-hydroxyl proline antibody showed that prolyl hydroxylated FoxO3 diminished in hypoxic or starved conditions. Treating tubular epithelial cells grown in normal conditions with a prolyl hydroxylase (Phd) inhibitor, dimethyloxalylglycine (DMOG), caused a 60% reduction in OH-FoxO3 and a 39% increase in FoxO3 protein abundance. Conversely, treating starved cells with DMKG, a cell permeable analogue of α-ketoglutarate, the Phd substrate, resulted in a 57% decrease in FoxO3 protein abundance.

Conclusion

Taken together, our results indicate that FoxO3 can be regulated by the O₂- and α-ketoglutarate-dependent Phd enzymes in the kidney. Hypoxia and metabolic perturbations inactivate Phd enzymes to inhibit FoxO3 degradation, thus allowing it to induce tubular autophagy.

Funding

• NIDDK Support