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

SUBMITTER: Kern G

PROVIDER: S-EPMC4015940 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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

Kern Georg G Mair Sabine M SM Noppert Susie-Jane SJ Jennings Paul P Schramek Herbert H Rudnicki Michael M Mueller Gerhard A GA Mayer Gert G Koppelstaetter Christian C

PloS one 20140509 5

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 aft ...[more]

PMID: 24816588

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Project description:Fibrotic interstitial lung disease (ILD) are lung disorders characterized by the accumulation of extracellular matrix, ultimately resulting in the destruction of the pulmonary scaffold. Continuous pro-fibrotic signaling perpetuates the remodeling process, specifically targeting the epithelial cell compartment, thereby destroying the gas exchange area. Studies that address this detrimental crosstalk between lung epithelial cells and fibroblasts are key to understanding ILD. With the aim of identifying functionally relevant targets that drive mesenchymal-epithelial crosstalk and their potential as new avenues to therapeutic strategies, we developed an organoid co-culture system based on human induced pluripotent stem cell-derived alveolar epithelial type 2 cells and lung fibroblasts from ILD patients as well as IMR-90 controls. While organoid formation capacity and organoid size was comparable in the presence of ILD or control lung fibroblasts, metabolic activity was significantly increased in ILD co-cultures. Alveolar organoids cultured with ILD fibroblasts further demonstrated reduced stem cell function supported by reduced Surfactant Protein C gene expression together with an aberrant basaloid-prone differentiation program indicated by elevated Cadherin 2, Bone Morphogenic Protein 4 and Vimentin transcription. In order to identify key mediators of the misguided mesenchymal-to-epithelial crosstalk with a focus on disease-relevant inflammatory processes, we used secretome mass spectrometry to identify key signals secreted by end stage ILD lung fibroblasts. Over 2000 proteins were detected in a single-shot experiment with 47 differentially upregulated proteins when comparing ILD and non-chronic lung disease control fibroblasts. The secretome profile was dominated by chemokines of the C-X-C motif family, including CXCL1, -3, and -8, all interfering with (epithelial) growth factor signaling orchestrated by Interleukin 11 (IL11), steering fibrogenic cell-cell communication, and proteins regulating extracellular matrix remodeling including epithelial-to-mesenchymal transition. When in turn treating 3D monocultures of iAT2s with IL11 we recapitulated the co-culture results obtained with primary ILD fibroblasts including changes in metabolic activity as well as organoid formation capacity and size. In summary, our analysis identified mesenchyme-derived mediators likely contributing to the disease-perpetuating mesenchymal-to-epithelial crosstalk in ILD by using sophisticated alveolar organoid co-cultures indicating the importance of cytokine-driven aberrant epithelial differentiation and confirmed IL11 as a key player in ILD using an unbiased approach.

Dataset Information

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

Publications

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

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