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Epigenetic regulators, including SETD7, as new targets for the treatment of chronic kidney disease
Poster Abstracts
Epigenetic regulators, including SETD7, as new targets for the treatment of chronic kidney disease Ian Logan, Victoria Shuttleworth, Luke Gaughan, Neil Sheerin
Abstract Published Online February 25, 2016 Poster 27 Newcastle University, Newcastle upon Tyne, UK (I Logan PhD, V Shuttleworth BSc, L Gaughan PhD, Prof N Sheerin PhD) Correspondence to: Dr Ian Logan, Institute of Cellular Medicine, Newcastle University, Medical School, Newcastle upon Tyne NE2 4HH, UK [email protected]
Background Fibrosis is common to all forms of chronic kidney disease and leads to kidney failure. 1·3% of the National Health Service budget and 7% of the US Medicare budget is spent on managing complications of the disease, yet there is no available treatment for the underlying fibrosis. We studied specific epigenetic regulators, including SETD7, to provide a paradigm for these enzymes as future targets in the treatment of chronic kidney disease. Methods Human renal fibroblasts (obtained after ethics approval), mesangial cells, and mouse embryonic fibroblasts were used to study myofibroblast transformation, expression of extracellular matrix proteins by immunofluorescence, wound healing, and global gene expression with whole-genome microarrays (Illumina, San Diego, CA, USA). Tubular epithelial cells were used to study the SETD7 lysine methyltransferase interaction with SMAD3 by immunoprecipitation, SMAD3 stability, and SMAD3 nuclear import. Findings SETD7-deficient cells failed to undergo myofibroblast transformation in response to profibrotic transforming growth factor β1 (TGF β1). They also expressed lower concentrations of fibrotic collagen and fibronectin-1 than SETD7-proficient cells and showed impaired wound healing. In response to TGFβ1, SMAD3 interacted with SETD7 resulting in enhanced SMAD3 stability in cells. Additionally, SMAD3 nuclear import was dependent on SETD7, as determined by immunofluorescence and subcellular fractionation. SETD7 overexpression enhanced SMAD3 transcriptional activity on natural (plasminogen activator inhibitor-1) and synthetic (CAGA) cis-regulatory elements, whereas SETD7 depletion reduced SMAD3 transcriptional activity, as demonstrated in reporter gene assays. Gene Ontology analysis indicated that SETD7 regulated expression of profibrotic genes. Interpretation We conclude that SETD7 is crucial in the development of the fibrotic phenotype that characterises chronic kidney disease and other fibrotic disorders. Pharmacological inhibition of this epigenetic regulator might therefore be of clinical use in this context. Our development of in-vitro models to study fibrosis, including human primary kidney fibroblasts, should overcome translational difficulties with animal models, thereby allowing further assessment of epigenetic enzymes as targets in the treatment of chronic kidney disease. Funding Northern Counties Kidney Research Fund, Academy of Medical Sciences. Contributors IL and VS designed and conducted the experiments. LG generated reagents and designed experiments. NS intepreted the data and contributed to the discussion. Declaration of interests We declare no competing interests.
66
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Epigenetic regulators, including SETD7, as new targets for the treatment of chronic kidney disease Ian Logan, Victoria Shuttleworth, Luke Gaughan, Neil Sheerin
Abstract Published Online February 25, 2016 Poster 27 Newcastle University, Newcastle upon Tyne, UK (I Logan PhD, V Shuttleworth BSc, L Gaughan PhD, Prof N Sheerin PhD) Correspondence to: Dr Ian Logan, Institute of Cellular Medicine, Newcastle University, Medical School, Newcastle upon Tyne NE2 4HH, UK [email protected]
Background Fibrosis is common to all forms of chronic kidney disease and leads to kidney failure. 1·3% of the National Health Service budget and 7% of the US Medicare budget is spent on managing complications of the disease, yet there is no available treatment for the underlying fibrosis. We studied specific epigenetic regulators, including SETD7, to provide a paradigm for these enzymes as future targets in the treatment of chronic kidney disease. Methods Human renal fibroblasts (obtained after ethics approval), mesangial cells, and mouse embryonic fibroblasts were used to study myofibroblast transformation, expression of extracellular matrix proteins by immunofluorescence, wound healing, and global gene expression with whole-genome microarrays (Illumina, San Diego, CA, USA). Tubular epithelial cells were used to study the SETD7 lysine methyltransferase interaction with SMAD3 by immunoprecipitation, SMAD3 stability, and SMAD3 nuclear import. Findings SETD7-deficient cells failed to undergo myofibroblast transformation in response to profibrotic transforming growth factor β1 (TGF β1). They also expressed lower concentrations of fibrotic collagen and fibronectin-1 than SETD7-proficient cells and showed impaired wound healing. In response to TGFβ1, SMAD3 interacted with SETD7 resulting in enhanced SMAD3 stability in cells. Additionally, SMAD3 nuclear import was dependent on SETD7, as determined by immunofluorescence and subcellular fractionation. SETD7 overexpression enhanced SMAD3 transcriptional activity on natural (plasminogen activator inhibitor-1) and synthetic (CAGA) cis-regulatory elements, whereas SETD7 depletion reduced SMAD3 transcriptional activity, as demonstrated in reporter gene assays. Gene Ontology analysis indicated that SETD7 regulated expression of profibrotic genes. Interpretation We conclude that SETD7 is crucial in the development of the fibrotic phenotype that characterises chronic kidney disease and other fibrotic disorders. Pharmacological inhibition of this epigenetic regulator might therefore be of clinical use in this context. Our development of in-vitro models to study fibrosis, including human primary kidney fibroblasts, should overcome translational difficulties with animal models, thereby allowing further assessment of epigenetic enzymes as targets in the treatment of chronic kidney disease. Funding Northern Counties Kidney Research Fund, Academy of Medical Sciences. Contributors IL and VS designed and conducted the experiments. LG generated reagents and designed experiments. NS intepreted the data and contributed to the discussion. Declaration of interests We declare no competing interests.
66
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