Abstract: FR-PO589
Chemical Modulation of the Ire1α-Xbp1 Pathway Reduces Cyst Size in ADPKD Mouse and Human Three-Dimensional Spheroids
Session Information
- Cystic Kidney Diseases: Mechanisms and Models
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1201 Genetic Diseases of the Kidneys: Cystic
Authors
- Hasan, Fatema, Yale University School of Medicine, New Haven, Connecticut, United States
- Bhardwaj, Rishi, Yale University School of Medicine, New Haven, Connecticut, United States
- Rehman, Michael, Yale University School of Medicine, New Haven, Connecticut, United States
- Cai, Yiqiang, Yale University School of Medicine, New Haven, Connecticut, United States
- Dong, Ke, Yale University School of Medicine, New Haven, Connecticut, United States
- Cordido, Adrian, Yale University School of Medicine, New Haven, Connecticut, United States
- Pioppini, Carlotta, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Yilmaz, Duygu Elif, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Tian, Xin, Yale University School of Medicine, New Haven, Connecticut, United States
- Somlo, Stefan, Yale University School of Medicine, New Haven, Connecticut, United States
- Krappitz, Matteus, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Fedeles, Sorin V., Yale University School of Medicine, New Haven, Connecticut, United States
Background
The Ire1α -XBP1 pathway is the most conserved UPR branch from yeast to mammals. Genetic inactivation of XBP1 leads to a significant improvement in disease severity in neonatal and adult Pkd1-dependent ADPKD mouse models through a pro-apoptotic mechanism driven by Ire1α kinase domain. The endoribonuclease domain of Ire1α splices an intron from the XBP1 mRNA leading to the formation of active XBP1 which acts as a transcription factor to mitigate ER stress. Here we set forth to test whether chemical inhibition of the Ire1α endoribonuclease domain through toyocamycin (previously tested in a Phase 1 study with a favorable safety profile) can alleviate cyst growth in 3D spheroid cyst models derived from ADPKD human and mouse kidney tubule cells.
Methods
Human (PKD1: 10594C>T (Gln3 532*)) and mouse wild-type or Pkd1 deficient cells were grown in matrigel. At Day 1, cells were administered either ddAVP (human cells) or forskolin (mouse cells) to promote cyst growth. Following that, cells were treated with various concentrations of toyocamycin over several days followed by fixation and image processing. We calculated lumen and spheroid area (um^2) in addition to measuring cell viability via CellTiterGlo.
Results
Mouse cells (in the presence or absence of the adenylate cyclase activator, forskolin) displayed a clear genotype/phenotype correlation with the Pkd1-KO cells forming abundant spheroids with large lumen sizes as compared with their WT counterparts which did not exhibit almost any lumen formation. In the presence of toyocamycin (up to 500nM), we found an almost complete normalization of the lumen/spheroid area ratio compared with the untreated cells. When the human spheroids were treated with toyocamycin (from 30nM to 1uM), the spheroid area was reduced to baseline levels at all concentrations tested. This was accompanied by an increase in cell death as well as decrease in ATP levels.
Conclusion
Our results describe a relevant 3D spheroid model system in mouse and human ADPKD tubule cells that was employed to test the effect of the Ire1α-XBP1 endoribonuclease inhibitor, toyocamycin, on spheroid formation/growth. We found a very potent effect of toyocamycin on spheroid and lumen size which warrants further in vivo investigations.
Funding
- Other U.S. Government Support