Calcineurin A-a suppression drives nuclear factor-jB-mediated NADPH oxidase-2 upregulation

Abstract

Calcineurin inhibitors (CNIs) are vital immunosuppressive therapies in the management of inflammatory conditions. A long-term consequence is nephrotoxicity. In the kidneys, the primary, catalytic calcineurin (CnA) isoforms are CnAa and CnAb. Although the renal phenotype of CnAa-/- mice substantially mirrors CNI-induced nephrotoxicity, the mechanisms downstream of CnAa are poorly understood. Since NADPH oxidase-2 (Nox2)-derived oxidative damage has been implicated in CNI-induced nephrotoxicity, we hypothesized that CnAa inhibition drives Nox2 upregulation and promotes oxidative stress. To test the hypothesis, Nox2 regulation was investigated in kidneys from CnAa-/-, CnAb-/-, and wild-type (WT) littermate mice. To identify the downstream mediator of CnAa, nuclear factor of activated T cells (NFAT) and NF-κB regulation was examined. To test if Nox2 is transcriptionally regulated via a NF-κB pathway, CnAa-/- and WT renal fibroblasts were treated with the NF-κB inhibitor caffeic acid phenethyl ester. Our findings showed that cyclosporine A treatment induced Nox2 upregulation and oxidative stress. Furthermore, Nox2 upregulation and elevated ROS generation occurred only in CnAa-/- mice. In these mice, NF-κB but not NFAT activity was increased. In CnAa-/- renal fibroblasts, NF-κB inhibition prevented Nox2 upregulation and reactive oxygen species (ROS) generation. In conclusion, these findings indicate that 1) CnAa loss stimulates Nox2 upregulation, 2) NF-κB is a novel CnAa-regulated transcription factor, and 3) NF-κB mediates CnAa-induced Nox2 and ROS regulation. Our results demonstrate that CnAa plays a key role in Nox2 and ROS generation. Furthermore, these novel findings provide evidence of divergent CnA isoform signaling pathways. Finally, this study advocates for CnAa-sparing CNIs, ultimately circumventing the CNI nephrotoxicity.