Selective Targeting of Calcineurin Isoforms by Calcineurin Inhibitors

Abstract

BackgroundCalcineurin Inhibitors (CNIs) have increased one‐year and five‐year post‐transplantation survival rates to ~90% and ~75%, respectively. While CNIs have dramatically improved the quality of patient care, its side effects are also noteworthy: Long‐term CNI therapy is nephrotoxic and is a growing concern for clinicians and patients. To address this concern, voclosporin, a newer generation CNI, was developed and has been reported to ameliorate the nephrotoxic side effects noted in its predecessors. Studies are necessary to investigate this phenomenon further. Research into calcineurin (CnA) activity has identified distinct actions of two main isoforms: CnAα and CnAβ. CnAα is required for kidney function, whereas CnAβ predominantly modulates the immune response. It is widely accepted that cyclosporin A and tacrolimus have similar inhibitory activity against both isoforms, while there is no published data on voclosporin selectivity for calcineurin isoforms. Moreover, the degree of isoform selectivity and mechanisms of CNI‐induced renal impairment still remains unknown. Investigating CNI dose‐dependent selective binding between catalytic isoforms will provide clues for further studies to narrow in on mechanisms by which the inhibition of calcineurin causes renal fibrosis.HypothesisIt is well established that long‐term CNI treatment causes an increase in profibrotic markers leading to renal fibrosis. It has also been established that the most predominant catalytic isoform expressed in the kidney is CnAa. The objectives of this study are to investigate whether CNI treatment is more selective for CnAa than CnAb in the kidney, and that CnAa inhibition promotes fibrosis via a TGFβ‐mediated cell signaling pathway.Experimental DesignTo determine dose‐dependent isoform selectivity of CNIs, wild‐type mice renal cortical fibroblasts (WT FB) were treated with varying doses of either vehicle (100% ethanol) or CNI (tacrolimus, cyclosporin or voclosporin) for 24 hours. Cell lysates were collected and calmodulin pull‐down assays were carried out for western blot analysis to analyze expression of activated and total calcineurin isoforms.ResultsCNI treatment decreases activation of calcineurin isoforms in a dose‐dependent manner. Notably, there were striking differences in isoform activity with tacrolimus treatment versus cyclosporin and voclosporin. Additionally, CNIs altered expression of calcineurin isoforms and profibrotic markers.ConclusionsTogether, these results demonstrate that 1) isoform selectivity differs amongst each CNI, 2) inhibiting calcineurin isoforms affects total protein expression and 3) CNIs have distinct profibrotic profiles.SignificanceThis study begins to characterize the behavior of two main active calcineurin isoforms with varying doses of CNI treatment. CNI selectivity for calcineurin isoforms directs future studies investigating molecular culprits and signaling pathways involved in CNI‐induced renal damage. Development of future CNIs targeting specific calcineurin isoforms will bypass activation of profibrotic pathways that diminish renal function with long‐term CNI use.Support or Funding InformationAHA‐16SDG27080009 and NIH‐R21DK119879