Abstract: PO1694
A Noncanonical Role for IRE1α in Podocyte Endoplasmic Reticulum (ER)-Phagy
Session Information
- Podocyte Pathobiology: Basic Science Studies and Animal Models
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Navarro-Betancourt, José R., McGill University, Montreal, Quebec, Canada
- Papillon, Joan, McGill University, Montreal, Quebec, Canada
- Guillemette, Julie, McGill University, Montreal, Quebec, Canada
- Iwawaki, Takao, Kanazawa Medical University, Kahoku-gun, Ishikawa, Japan
- Cybulsky, Andrey V., McGill University, Montreal, Quebec, Canada
Background
Glomerular diseases involving podocyte (glomerular epithelial cell; GEC) injury feature endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR). Inositol requiring enzyme-1α (IRE1α), a UPR transducer, mediates chaperone production and autophagy in podocytes during ER stress. Selective autophagy of the ER (ERphagy) is dependent on ER-resident adaptors (e.g. RTN3L) and is stimulated by ER stress. ER-derived coat protein complex II (COPII) vesicles may participate in the delivery of ER cargo to autophagosomes; however, regulation and importance of ERphagy in glomerular disease are not understood.
Methods
We employed mice with podocyte-specific deletion of IRE1α and littermate controls. IRE1α knockout (KO) and control GECs were produced from these mice. GECs were incubated with tunicamycin (TM) to induce ER stress.
Results
Mass spectrometry analysis of TM-stimulated control and IRE1α KO GECs showed that in addition to ER chaperones, proteins in the secretory pathway, including the COPII component Sec23B, were increased in an IRE1α-dependent manner. By immunoblotting, TM enhanced Sec23B and RTN3L expression in control, but not IRE1α KO GECs. By immunofluorescence microscopy, in control GECs, TM increased the biogenesis of LC3 and Sec23B particles, as well as colocalization of Sec23B with LC3 and RTN3L with LC3; increases were attenuated in IRE1α KO GECs. Thus, deletion of IRE1α impaired delivery of COPII vesicles and RTN3L-coated ER fragments to autophagosomes. Knockdown of Sec23B with siRNAs reduced autophagosome formation in TM-treated control GECs. After blocking protein synthesis with cycloheximide, TM stimulated degradation of RTN3L in control GECs, consistent with ERphagy flux, but RTN3L degradation was impaired in IRE1α KO cells. Similarly, TM induced degradation of α3,4,5 collagen IV in control, but not IRE1α KO GECs, suggesting that collagen IV is an IRE1α-dependent ERphagy substrate. In adriamycin nephrosis, where IRE1α activates an adaptive UPR and autophagy, expression of Sec23B and RTN3L was increased in glomeruli of control, but not IRE1α KO mice.
Conclusion
During ER stress, IRE1α redirects a subset of Sec23B-positive COPII vesicles to deliver RTN3L-coated ER fragments to autophagosomes. ERphagy is a novel outcome of the IRE1α pathway in podocytes and may play a cytoprotective role in glomerular diseases.
Funding
- Government Support – Non-U.S.