Abstract: PO2385
Calcineurin Inhibitor Cyclosporine A but Not Tacrolimus Induces Proapoptotic Endoplasmic Reticulum Stress in Kidney Epithelial Cells
Session Information
- What's Hot, What's New in Transplantation: Basic and Translational
October 22, 2020 | Location: On-Demand
Abstract Time: 10:00 AM - 12:00 PM
Category: Transplantation
- 1901 Transplantation: Basic
Authors
- Yilmaz, Duygu Elif, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Kirschner, Karin Michaela, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Demirci, Hasan, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Bachmann, Sebastian, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
- Mutig, Kerim, Charite Universitatsmedizin Berlin, Berlin, Berlin, Germany
Background
Current immunosuppressive strategies in solid organ transplantation rely on the calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac). Their nephrotoxicity is a major limitation for long-term usage. We hypothesized that CsA compared to Tac exerts more pronounced toxic side effects at the cellular level, with endoplasmic reticulum (ER)-stress and maladaptive unfolded protein response (UPR) as the most prominent landmarks.
Methods
To test this hypothesis we treated human embryonic kidney (HEK293) cells with CsA (10 µM) or Tac (10 µM) for 6h. The established ER stress-inducing agent, thapsigargin, served as positive control. To study the molecular mechanisms of CNI-induced cell toxicity we studied genetically-modified HEK293 cells lacking the crucial UPR elements, PERK or ATF6. Abundances of the ER-stress sensor IRE1a, adaptive transcription factor XBP1, and proapoptotic transcription factor CHOP were evaluated as endpoints.
Results
Treatment of native HEK293 cells with CsA or Tac equally increased phosphorylation of the known calcineurin substrate, NFAT, verifying the treatment protocols. CsA increased levels of activating IRE1a phosphorylation (pIREa) and stimulated both CHOP and spliced XBP1 (sXBP1) products. In contrast, Tac enhanced pIRE1a abundance only. CsA but not Tac significantly increased the number of cleaved caspase 3-positive cells suggesting enhanced apoptosis rate. Treatment with the chemical chaperone, TUDCA, partially abolished the CsA-induced increases of CHOP but did not affect sXBP1, suggesting alleviation of ER-stress. Knockdown of CsA binding partners, cyclophilin A and B, by siRNA reduced their expression approximately by half and increased CHOP expression suggesting that suppression of cyclophilins may contribute to CsA-induced cellular toxicity. PERK- or ATF6-deficiency blunted the increases of CHOP and sXBP1 in response to CsA, suggesting an implication of these pathways in CsA-induced ER-stress and UPR.
Conclusion
In summary, CsA but not Tac induces pronounced ER-stress and proapoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CNI nephrotoxicity.
Funding
- Government Support - Non-U.S.