Cyclosporine A but not tacrolimus induces pro-apoptotic endoplasmic reticulum stress in renal tubular cells.

Calcineurin inhibitors (CNI), cyclosporine A (CsA) or tacrolimus (Tac), belong to the first-line immunosuppressive strategies in organ-transplanted patients. Both CNI may exert renal side effects, which are typically stronger in patients receiving CsA. Continued clinical demand for CsA requires improved understanding of its nephrotoxicity. Calcineurin inhibition by CsA occurs via complexes with cyclophilins, whereas Tac recruits FKBP12 instead. We hypothesized that impaired chaperone function of cyclophilins may aggravate CsA cytotoxicity due to induction of endoplasmic reticulum (ER) stress and pro-apoptotic unfolded protein response (UPR). To address this hypothesis, effects of CsA vs. Tac (10 µM for 6 h) on the UPR signaling were evaluated in cultured human embryonic kidney cells (HEK293), human renal proximal tubular (PT) epithelial cells (HRPTEpC), and isolated rat PTs using quantitative PCR, immunoblotting, and immunofluorescence staining. An established ER stress inducer, thapsigargin (Tg) served as a positive control. CsA and Tg, but not Tac, induced ER stress and pro-apoptotic UPR in HEK293 cells, which was reflected by increased levels of key UPR products (BiP, CHOP, spliced XBP1, and phosphorylated IRE1a) and cleaved caspase 3 (cCas3). Similar effects were observed in HRPTEpC cells and isolated rat PTs. Knockdown of cyclophilin A or B isoforms in HEK293 cells using siRNA augmented CHOP and cCas3 to an extent comparable with CsA treatment suggesting relevance of cyclophilin activity for intact proteostasis. Application of chemical chaperones, TUDCA or 4-PBA, alleviated the CsA-induced UPR suggesting that boosting the protein folding may have protective effects against CsA cytotoxicity. Along the same line, genetic suppression of UPR in HEK293 cells by CRISPR/Cas9-mediated deletion of the key ER stress sensors, PERK or ATF6, blunted the pro-apoptotic UPR in response to CsA. In summary, these results suggest that nephrotoxic effects of CsA are partially mediated by suppression of cyclophilins, ER stress, and pro-apoptotic UPR. Pharmacological modulation of UPR bears the potential to alleviate CsA nephrotoxicity.