Selective Targeting of Calcineurin Isoforms by Calcineurin Inhibitors

Abstract

Background Calcineurin 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, their side effects are also noteworthy: long-term CNI therapy is nephrotoxic and is a growing concern for clinicians and patients. 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. 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. Objective It is well established that long-term CNI treatment causes an increase in profibrotic markers leading to renal damage. 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. Experimental Design To determine dose-dependent isoform selectivity of CNIs, wild-type mouse renal cortical fibroblasts (WT FB) were treated with varying doses of either vehicle (100% ethanol) or CNI (tacrolimus, cyclosporin) 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. Results : CNI treatment decreases activation of calcineurin isoforms in a dose-dependent manner. Notably, there were striking differences in isoform activity with tacrolimus treatment versus cyclosporin in a dose-dependent manner. Additionally, CNIs altered expression of calcineurin isoforms and profibrotic markers. Conclusions Together, these results demonstrate that 1) isoform selectivity differs amongst each CNI and 2) inhibiting calcineurin isoforms affects total protein expression and 3) CNIs have distinct profibrotic profiles. Significance This 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. We expect that development of future CNIs targeting specific calcineurin isoforms will prevent kidney damage and maintain renal function with long-term CNI use.