Abstract: PO0198
Targeting Gadd34 Upstream Open Reading Frame to Treat Sepsis-Induced Kidney Injury
Session Information
- AKI Mechanisms - 2
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Kidwell, Ashley N., Indiana University School of Medicine, Indianapolis, Indiana, United States
- Maier, Bernhard, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Dagher, Pierre C., Indiana University School of Medicine, Indianapolis, Indiana, United States
- Hato, Takashi, Indiana University School of Medicine, Indianapolis, Indiana, United States
Background
Sepsis-induced acute kidney injury remains a major clinical problem with no effective therapy to date. We have previously shown that bacterial sepsis causes global translation shutdown via phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Under physiological conditions, this eIF2α phosphorylation is counter-regulated by two eIF2α holophosphatases. Of the two holophosphatases, Growth Arrest DNA-inducible Gene 34 (Gadd34) is the only stress-inducible regulatory subunit. Using ribo-seq, we found that Gadd34 is translationally repressed during late phase sepsis even though eIF2α is heavily phosphorylated. We postulate that this blunted Gadd34 induction could explain the sustained phosphorylation of eIF2α and translation shutdown that contributes to delayed renal recovery in sepsis.
Methods
The 5’-untranslated region (UTR) of Gadd34 has an upstream open reading frame (uORF) that is conserved across mammalian species. Our ribo-seq analysis of the kidney from septic mice revealed high ribosomal occupancy of the uORF, but not the main protein coding sequence (CDS), consistent with a model in which the uORF serves as a translational inhibitor of the downstream CDS. To investigate the inhibitory role of the Gadd34 uORF, we designed plasmid constructs consisting of a full length Gadd34 5’UTR where a single nucleotide mutation was introduced to abolish the uORF start codon. We also designed antisense oligonucleotides (ASO) that are complementary to a specific portion of the uORF to modulate ribosomal scanning on the native Gadd34 mRNA.
Results
The uORF point mutation led to a two-fold increase in the readout luciferase signal compared with the wild-type control, confirming the inhibitory property of the Gadd34 uORF. We also found that masking of uORF by ASO resulted in sequence-specific increases in translation of the downstream CDS, possibly due to enhanced leaky ribosomal scanning. Finally, we tested the applicability of antisense approach in vivo using a mouse model of endotoxin-induced kidney injury. Despite late intervention (8 hrs post endotoxin challenge), the administration of uORF-targeted ASOs rescued translation and reduced kidney injury.
Conclusion
These findings indicate that translational suppression of Gadd34 in late phase sepsis is a maladaptive response that could be therapeutically modulated by targeting its uORF.
Funding
- NIDDK Support