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Kidney Week

Abstract: FR-PO1110

Calcineurin Inhibitors Induce Endoplasmic Reticulum Stress in Kidney Epithelial Cells via PERK- and ATF6-Dependent Mechanisms

Session Information

  • Transplantation: Basic
    November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
    Abstract Time: 10:00 AM - 12:00 PM

Category: Transplantation

  • 1901 Transplantation: Basic

Authors

  • Yilmaz, Duygu Elif, Charité - Universitätsmedizin Berlin, Berlin, Germany
  • Kirschner, Karin Michaela, Charité - Universitätsmedizin Berlin, Berlin, Germany
  • Thomson, Martin N., Charité - Universitätsmedizin Berlin, Berlin, Germany
  • Bachmann, Sebastian, Charité - Universitätsmedizin Berlin, Berlin, Germany
  • Mutig, Kerim, Charité - Universitätsmedizin Berlin, Berlin, Germany
Background

Calcineurin inhibitors such as cyclosporine A (CsA) or tacrolimus (Tac) form the core of immunosuppressive regimens in organ transplant recipients but may cause substantial nephrotoxicity with induction of endoplasmic reticulum (ER) stress and maladaptive unfolded protein response (UPR) in kidney epithelia. Calcineurin stimulates the activity of PKR-like ER kinase (PERK), which is an ER stress sensor suppressing protein translation. Calcineurin may also promote ATF6-induced autophagy to alleviate ER stress. We hypothesized that calcineurin inhibition interferes with functions of PERK and ATF6, thereby inducing ER stress and UPR.

Methods

To test this hypothesis, we generated PERK-deficient and ATF6-deficient human embryonic kidney (HEK293) cell lines using CRISPR/Cas9-mediated gene editing. Effects of CsA on expression and cellular distribution of two critical UPR transcription factors, CHOP and spliced XBP1, were monitored using quantitative PCR, immunoblotting and immunofluorescence.

Results

Treatment of control HEK293 cells with CsA (10 µM for 6h) induced a two-fold increase of CHOP abundance and a 70-fold increase of spliced XBP1 abundance in cell lysates. Parallel immunofluorescence analysis showed CsA-induced nuclear translocation of CHOP. In contrast, PERK-deficient and ATF6-deficient cells showed no significant CsA-induced increases of CHOP expression. Stimulation of spliced XBP1 was weaker in PERK-deficient (five-fold increase) and ATF6-deficient HEK293 cells (30-fold increase) compared to control cells. To extend these results, we evaluated effects of Tac in cultured murine distal convoluted tubule (DCT) cells. In this model, treatment with Tac (10 µM for 6h) increased CHOP expression by two-fold but did not affected spliced XBP1.

Conclusion

In sum, these results suggest that calcineurin inhibitors cause ER stress and UPR in kidney epithelial cells, which may in part depend on suppression of PERK or ATF6 functions.

Funding

  • Government Support - Non-U.S.