Tacrolimus Increases Nox4 Expression in Human Renal Fibroblasts and Induces Fibrosis-Related Genes by Aberrant TGF-Beta Receptor Signalling

Chronic nephrotoxicity of immunosuppressives is one of the main limiting factors in the long-term outcome of kidney transplants, leading to tissue fibrosis and ultimate organ failure. The cytokine TGF-β is considered a key factor in this process. In the human renal fibroblast cell line TK-173, the macrolide calcineurin inhibitor tacrolimus (FK-506) induced TGF-β-like effects, manifested by increased expression of NAD(P)H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen α1(V) mRNA after three days. The macrolide mTOR inhibitor rapamycin had similar effects, while cyclosporine A did not induce fibrose-related genes. Concentration dependence curves were sigmoid, where mRNA expression was induced already at low nanomolar levels of tacrolimus, and reached saturation at 100–300 nM. The effects were independent of extracellular TGF-β as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-β receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a “leaky” TGF-β receptor. The myofibroblast marker α-smooth muscle actin was neither induced by tacrolimus nor by TGF-β1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus- and TGF-β1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H(2)O(2) concentration. Si-RNA mediated knock-down of Nox4 expression prevented up-regulation of procollagen α1(V) mRNA in tacrolimus-treated cells, but induced procollagen α1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-β is a key molecule in fibrosis, and the constant activation of aberrant receptor signaling by tacrolimus might contribute to the long-term development of interstitial kidney fibrosis in immunosuppressed patients. Nox4 levels possibly play a regulatory role in these processes.