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

• Published in: PloS one Vol. 9; no. 5; p. e96377
• Main Authors: Kern, Georg, Mair, Sabine M, Noppert, Susie-Jane, Jennings, Paul, Schramek, Herbert, Rudnicki, Michael, Mueller, Gerhard A, Mayer, Gert, Koppelstaetter, Christian
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 09-05-2014; Public Library of Science (PLoS)
• Subjects: Aberration; Actin; Activation; Antibodies; Blotting, Western; Bone morphogenetic proteins; Calcineurin; Calcineurin Inhibitors - pharmacology; Cell culture; Cell Line; Collagen; Collagen Type V - genetics; Collagen Type V - metabolism; Cyclosporins; Experiments; Extracellular matrix; Fibroblasts; Fibroblasts - drug effects; Fibroblasts - metabolism; Fibrosis; Fibrosis - genetics; FKBP12 protein; Gene expression; Gene Expression - drug effects; Gene Expression Profiling; Genes; Growth factors; Humans; Hydrogen peroxide; Hydrogen Peroxide - metabolism; Hypertension; Inhibitors; Kidney - metabolism; Kidney transplantation; Kidneys; Kinases; Laboratories; Limiting factors; Medicine and Health Sciences; Muscle proteins; Muscles; NAD; NADPH Oxidase 4; NADPH Oxidases - genetics; NADPH Oxidases - metabolism; Nephrology; NOX4 protein; Oligonucleotide Array Sequence Analysis; Oxidases; Phosphorylation - drug effects; Procollagen; Procollagen - genetics; Procollagen - metabolism; Proteomics; Rapamycin; Receptors, Transforming Growth Factor beta - genetics; Receptors, Transforming Growth Factor beta - metabolism; Reverse Transcriptase Polymerase Chain Reaction; Ribonucleic acid; RNA; RNA Interference; Signal Transduction - drug effects; Signal Transduction - genetics; Signaling; Smad2 Protein - metabolism; Smooth muscle; Tacrolimus; Tacrolimus - pharmacology; TOR protein; Transforming growth factor-b; Transforming growth factor-b1; Transforming growth factors; Transplantation; Transplants; Transplants & implants; Tropomyosin; Western blotting; Austria
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0096377

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.