Abstract: TH-PO084
Potential Regulator of KIM-1 Mediated Autophagy
Session Information
- AKI: Mechanisms - I
November 03, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
Authors
- Li, Yilong, Western University, London, Ontario, Canada
- Tutunea-Fatan, Elena, Western University, London, Ontario, Canada
- Gunaratnam, Lakshman, Western University, London, Ontario, Canada
Background
Kidney injury molecule 1 (KIM-1) is a phosphatidylserine receptor expressed on injured proximal tubule epithelial cells (PTECs) during acute kidney injury (AKI). KIM-1 mediates the phagocytic clearance of apoptotic cells by PTECs and targets the engulfed apoptotic corpses to autophagosomes by triggering autophagy. However, the detailed signaling mechanisms are poorly understood. This study aims to uncover KIM-1-interacting proteins that regulate autophagy in PTECs.
Methods
KIM-1 immunoprecipitates from human PTECs (HK-2), which endogenously express KIM-1, stimulated with apoptotic cells were separated using SDS-PAGE. The bands of putative KIM-1 interacting proteins were visualized using Coomassie blue staining and analyzed using liquid chromatography with tandem mass spectrometry. Proteins were identified using PEAKS DB proteomics software, which uses de novo sequencing assisted database searching. Protein-protein interactions were confirmed using western blot analysis.
Results
The immunoprecipitation of KIM-1 was confirmed by both western blot and mass spectrometry analyses, in which KIM-1 was only present in the anti-KIM-1 group and absent in the IgG control. We identified the phosphoinositide-3-kinase regulatory subunit 4 (PIK3R4) with a PEAKS peptide score of 99.1 (minimum threshold at 26.7) and a sequence coverage of 6%. Western blot analysis showed that PIK3R4 co-immunoprecipitated with KIM-1 in both apoptotic cells-stimulated and unstimulated HK-2 cells.
Conclusion
PIK3R4 interacts with KIM-1 constitutively in PTECs. It has previously been implicated in the regulation of autophagy including autophagosome formation and endocytic trafficking but not in PTECs. Further investigation will elucidate whether PIK3R4 is required for KIM-1 mediated autophagy.
Figure 1. Co-immunoprecipitation of KIM-1 and PIK3R4 from HK-2 cells.