Abstract: TH-PO1100
Expression Profile of the cGAS-STING Pathway in Podocytes: Implications for Glomerular Diseases
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1204 Podocyte Biology
Authors
- Fontanella, Antonio Miguel, University of Miami, Enterprise, Alabama, United States
- Mallela, Shamroop Kumar, University of Miami, Enterprise, Alabama, United States
- Molina David, Judith T., University of Miami, Enterprise, Alabama, United States
- Burke, George William, University of Miami Miller School of Medicine, Miami, Florida, United States
- Merscher, Sandra M., University of Miami, Enterprise, Alabama, United States
- Fornoni, Alessia, University of Miami, Enterprise, Alabama, United States
- Mitrofanova, Alla, University of Miami, Enterprise, Alabama, United States
Background
Recent studies suggest that podocytes express elements of the innate immune system which may be involved in the local immune response and contribute to glomerular damage. As part of the innate immune system, the cGAS-STING pathway is activated in response to cytosolic DNA and is involved in disease pathogenesis for systemic lupus erythematosus. Whether or not podocytes express genes in the pathway remains unknown. This study aims to investigate if genes in the cGAS-STING pathway are expressed in murine and human podocytes and if activation of this pathway by cyclic dinucleotides (CDNs) contributes to podocyte injury in glomerular diseases.
Methods
Immortalized human and murine podocytes were cultured. For treatments, podocytes were differentiated for 14 days and serum starved for 24h starting day 12. STING knockdown human podocytes were established by lentiviral infection with siRNA. c-diAMP treatment was performed in RPMI medium at concentration 10 uM for 24h starting day 13. Plasma membranes were separated by ultracentrifugation (100,000xg, 1h). Several mouse models of glomerular disease of metabolic (db/db, ob/ob) and non-metabolic origin (Alport, NFAT) were used. mRNA was extracted using Qiagen RNAeasy kit according to the manufacturer’s protocol. Proteins were separated in 4-20% SDS-PAGE gels (BioRad) and transferred to nitrocellulose membranes for Western blot analysis. All monoclonal antibodies were obtained from Cell Signaling.
Results
Both murine and human podocytes showed expression of cGAS-STING pathway genes under physiological conditions. Treatment with c-diAMP increased expression of cGAS and phosphorylation of STING, IRF3 and TBK1. Notably, inactive STING was localized in the endoplasmic reticulum, while activate STING is translocated to the cytosol. STING knockdown podocytes did not respond to pathway activation after c-diAMP treatment. Among the mouse models of glomerular disease used in this study, only db/db and Alport mice showed increased cGAS-STING activity.
Conclusion
Genes of the cGAS-STING pathway are expressed in murine and human podocytes and the pathway can be activated by CDNs. Activation of the cGAS-STING pathway in mouse models of glomerular disease suggests a possible contribution of this pathway to podocyte injury.
Funding
- Other NIH Support