Tacrolimus increases Nox4 expression in human renal fibroblasts and induces fibrosis-related genes by aberrant TGF-beta receptor signalling

Georg Kern, Sabine M Mair, Susie-Jane Noppert, Paul Jennings, Herbert Schramek, Michael Rudnicki, Gerhard A Mueller, Gert Mayer, Christian Koppelstaetter

Research output: Contribution to JournalArticleAcademicpeer-review


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.

Original languageEnglish
Pages (from-to)e96377
JournalPLoS ONE
Issue number5
Publication statusPublished - 2014
Externally publishedYes


  • Blotting, Western
  • Calcineurin Inhibitors
  • Cell Line
  • Collagen Type V
  • Fibroblasts
  • Fibrosis
  • Gene Expression
  • Gene Expression Profiling
  • Humans
  • Hydrogen Peroxide
  • Kidney
  • NADPH Oxidase
  • Oligonucleotide Array Sequence Analysis
  • Phosphorylation
  • Procollagen
  • RNA Interference
  • Receptors, Transforming Growth Factor beta
  • Reverse Transcriptase Polymerase Chain Reaction
  • Signal Transduction
  • Smad2 Protein
  • Tacrolimus
  • Journal Article
  • Research Support, Non-U.S. Gov't


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