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-beta 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-beta-like effects, manifested by increased expression of NAD(P) H-oxidase 4 (Nox4), transgelin, tropomyosin 1, and procollagen alpha 1(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-beta as confirmed by the use of neutralizing antibodies, and thus most likely caused by aberrant TGF-beta receptor signaling, where binding of tacrolimus to the regulatory FKBP12 protein results in a "leaky'' TGF-beta receptor. The myofibroblast marker alpha-smooth muscle actin was neither induced by tacrolimus nor by TGF-beta 1, indicating an incomplete activation of TK-173 fibroblasts under culture conditions. Tacrolimus-and TGF-beta 1-induced Nox4 protein upregulation was confirmed by Western blotting, and was accompanied by a rise in intracellular H2O2 concentration. Si-RNA mediated knockdown of Nox4 expression prevented up-regulation of procollagen alpha 1(V) mRNA in tacrolimus-treated cells, but induced procollagen alpha 1(V) expression in control cells. Nox4 knock-down had no significant effect on the other genes tested. TGF-beta 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.