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 CnAα and CnAβ. Although the renal phenotype of CnAα-/- mice substantially mirrors CNI-induced nephrotoxicity, the mechanisms downstream of CnAα are poorly understood. Since NADPH oxidase-2 (Nox2)-derived oxidative damage has been implicated in CNI-induced nephrotoxicity, we hypothesized that CnAα inhibition drives Nox2 upregulation and promotes oxidative stress. To test the hypothesis, Nox2 regulation was investigated in kidneys from CnAα-/-, CnAβ-/-, and wild-type (WT) littermate mice. To identify the downstream mediator of CnAα, nuclear factor of activated T cells (NFAT) and NF-κ B regulation was examined. To test if Nox2 is transcriptionally regulated via a NF-κ B pathway, CnAa-/- and WT renal fibroblasts were treated with the NF-κ B 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 CnAα-/- mice. In these mice, NF-κ B but not NFAT activity was increased. In CnAα-/- renal fibroblasts, NF-κ B inhibition prevented Nox2 upregulation and reactive oxygen species (ROS) generation. In conclusion, these findings indicate that 1) CnAα loss stimulates Nox2 upregulation, 2) NF-κ B is a novel CnAα-regulated transcription factor, and 3) NF-κ B mediates CnAα-induced Nox2 and ROS regulation. Our results demonstrate that CnAα 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 CnAα-sparing CNIs, ultimately circumventing the CNI nephrotoxicity. [ABSTRACT FROM AUTHOR]