Tacrolimus induces ligand independent TGFβ receptor signaling to promote renal fibrosis

Background Although calcineurin inhibitors (CNIs) such as tacrolimus and cyclosporin have dramatically improved the quality of patient care, long‐term therapy causes irreversible damage to the kidneys in the form of renal fibrosis. These morphologic changes ultimately lead to a decline in renal function and can progress to end‐stage renal failure. These detrimental outcomes present a critical need to identify the driving mechanisms by which CNIs cause renal damage. Objective It is well established that TGFb is a major contributor to CNI‐induced renal fibrosis. However, the underlying mechanisms remain unknown. The objectives of this study are to 1) investigate whether TGFb secretion is required to stimulate TGFb receptor signaling in a model of CNI‐induced renal fibrosis and 2) investigate whether calcineurin plays a critical role in regulating TGFb receptor activity. Experimental Design To examine the role of calcineurin inhibition in altered TGFb receptor signaling, wild type mice were treated with either vehicle (0.01% ethanol) or 10 mg/kg tacrolimus for 7 days. To confirm in vivo findings, wild‐type mouse renal cortical fibroblasts were treated with either vehicle (0.01% ethanol) or 1nM tacrolimus for 24 hours in the presence and absence of anti‐TGFb neutralizing antibodies. TGFb receptor expression and activation, TGFb receptor downstream signaling mediators, profibrotic markers and calcineurin activity were analyzed. Results Findings demonstrate that tacrolimus‐induced loss of calcineurin activity is accompanied with enhanced TGFb receptor activation and signaling. Notably, increasing concentrations of anti‐TGFb neutralizing antibodies failed to abolish aberrant TGFb signaling and increased expression of profibrotic markers. Conclusions Together, these results demonstrate that 1) CNIs promote ligand‐independent TGFb signaling and 2) calcineurin plays a functional role in regulating TGFb receptor activity. Significance This study elucidates a direct pharmacological mechanism by which CNIs promote renal fibrosis and provides a physiological role of calcineurin in regulating TGFb receptor activity. Further insight into the role of calcineurin in regulating TGFb receptor activity could improve the long‐term outcomes of patients on chronic CNI therapy.