title: Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling.
abstract: 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 H2O2 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.
authors.affiliation: Nephrology and Hypertension, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Georg
authors.initials: G
authors.lastname: Kern
authors.affiliation: Clinical Immunology and Infectious Diseases, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Sabine M
authors.initials: SM
authors.lastname: Mair
authors.affiliation: Nephrology and Hypertension, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Susie-Jane
authors.initials: SJ
authors.lastname: Noppert
authors.affiliation: Physiology, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Paul
authors.initials: P
authors.lastname: Jennings
authors.affiliation: Nephrology and Hypertension, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Herbert
authors.initials: H
authors.lastname: Schramek
authors.affiliation: Nephrology and Hypertension, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Michael
authors.initials: M
authors.lastname: Rudnicki
authors.affiliation: Rheumatology and Nephrology, University of Goettingen, Goettingen, Germany.
authors.forename: Gerhard A
authors.initials: GA
authors.lastname: Mueller
authors.affiliation: Nephrology and Hypertension, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Gert
authors.initials: G
authors.lastname: Mayer
authors.affiliation: Nephrology and Hypertension, Innsbruck Medical University, Innsbruck, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Koppelstaetter
mesh_terms: D015153:Blotting, Western; D065095:Calcineurin Inhibitors / Q000494:pharmacology; D002460:Cell Line; D024062:Collagen Type V / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects* / Q000378:metabolism; D005355:Fibrosis / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects*; D020869:Gene Expression Profiling; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000235:genetics* / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000187:drug effects; D011347:Procollagen / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D051899:Smad2 Protein / Q000378:metabolism; D016559:Tacrolimus / Q000494:pharmacology*
chemical_list: D065095:Calcineurin Inhibitors; D024062:Collagen Type V; D011347:Procollagen; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; D051899:Smad2 Protein; D006861:Hydrogen Peroxide; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D016559:Tacrolimus