Tacrolimus induces fibroblast-to-myofibroblast transition via a TGF-β-dependent mechanism to contribute to renal fibrosis.

Use of immunosuppressant calcineurin inhibitors (CNIs) is limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesized that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the transforming growth factor-β (TGF-β)/Smad signaling axis. To test this, kidney damage-resistant mice (C57BL/6) received tacrolimus (10 mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1 nM) or vehicle for 24 h. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-smooth muscle actin expression was significantly increased, suggesting the presence of fibroblast activation. TGF-β receptor activation and downstream Smad2/3 signaling were also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-smooth muscle actin, actin stress fibers, cell motility, and collagen type IV expression were significantly increased. These findings were accompanied by concomitant induction of TGF-β signaling. Pharmacological inhibition of the downstream TGF-β effector Smad3 attenuated tacrolimus-induced phenotypic changes. Collectively, these findings suggest that 1) tacrolimus inhibits the calcineurin/nuclear factor of activated T cells axis while inducing TGF-β1 ligand secretion and receptor activation in renal fibroblasts; 2) aberrant TGF-β receptor activation stimulates Smad-mediated production of myofibroblast markers, notable features of FMT; and 3) FMT contributes to extracellular matrix expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts into the growing list of CNI-targeted cell types and identify renal FMT as a process mediated via a TGF-β-dependent mechanism.NEW & NOTEWORTHY Renal fibrosis, a detrimental feature of irreversible kidney damage, remains a sinister consequence of long-term calcineurin inhibitor (CNI) immunosuppressive therapy. Our study not only incorporates renal fibroblasts into the growing list of cell types negatively impacted by CNIs but also identifies renal fibroblast-to-myofibroblast transition as a process mediated via a TGF-β-dependent mechanism. This insight will direct future studies investigating the feasibility of inhibiting TGF-β signaling to maintain CNI-mediated immunosuppression while ultimately preserving kidney health.