Abstract: TH-PO790
Characterization of a Novel Human Podocyte Cell Line as a Model for CKD Drug Development
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 02, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1403 Podocyte Biology
Authors
- Zheng, Gang, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Ren, Shuyu, Pfizer Inc, New York, New York, United States
- Myshkin, Eugene, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Kalyana-Sundaram, Shanker, Janssen Research and Development LLC, Raritan, New Jersey, United States
- Bukanov, Nikolay O., Janssen Research and Development LLC, Raritan, New Jersey, United States
- Magnone, Maria Chiara, Janssen Research and Development LLC, Raritan, New Jersey, United States
Background
Podocytes are terminally differentiated epithelial cells which act as a crucial component to form the glomerular filtration barrier. There are a number of challenges in working with human and mouse podocytes in culture for drug discovery programs as most cell lines do not present a typical podocyte morphology that is comparable with the in vivo situation, and some of the podocyte markers are not expressed in these cell lines. In this study, we characterized a novel human podocyte cell line PODO/TERT 256 from Evercyte that has several advantages over long-standing SV40 T antigen immortalized podocytes. We completed further evaluation of this cell line as a potential tool for target validation and drug discovery.
Methods
The mRNA expression of podocyte markers was detected using qPCR and the protein expression was confirmed by western blotting and immunofluorescence staining of cultured podocytes. Comparative RNA profiling with other cell lines was assessed by RNA-seq. The effect of podocyte injury was assessed using puromycin aminonucleoside. The cell viability study and adhesion assay were quantified using CellTiter-Glo. The adhesion of podocytes was evaluated using Laminin, a ligand for Integrin α3β1.
Results
We showed, that in PODO/TERT 256 cell line, the key podocyte markers NPHS1, SYNPO, PODXL, WT-1, KIRREL, KIRREL3 were detectable by qPCR. Expression of the nephrin, podocin, synaptopodin and podocalyxin proteins were confirmed by western blotting. The localization to the cellular membrane for synaptopodin and nephrin was confirmed by immunofluorescent staining. RNA profiling study indicated that this cell line possesses podocyte characteristics. Nephrin expression was stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) and suppressed by glucose. The cells demonstrated susceptibility to puromycin aminonucleoside (PAN) induced injury. Cell adhesion assays confirmed the suitability of this novel podocyte cell line for functional studies.
Conclusion
Characterization of novel podocyte cell line PODO/TERT 256 (Evercyte) confirmed expression of multiple podocyte-specific markers on RNA and protein levels. This new podocyte cell line will become a powerful tool for identification of the novel therapeutic targets and discovery of the new drugs for CKD.
Funding
- Commercial Support – Janssen Research & Development