| Summary: | Retinoids, including the prototypic vitamin A and its main bioactive form, alltrans retinoic acid (tRA), have both anti- and pro-fibrotic effects in renal disease models. To understand and prevent the pro-fibrotic effects of retinoids it is important to establish in vitro models. This work aimed to explore the mechanisms behind the fibrogenic effects of tRA in renal fibroblasts. A picro-Sirius red-based in vitro assay was used to determine the effects of retinoids on total collagen accumulation with and without transforming growth factor (TGF)-β1 in NRK-49F normal rat kidney fibroblasts. Individual fibrotic markers and nuclear receptors were investigated using molecular biology approaches, activity assays and chemical agonists and inhibitors. tRA dose-dependently increased total collagen deposition with and without TGF-β1 in NRK-49F cells. At the level of gene expression tRA showed dual potential, down-regulating mRNAs encoding a range of extracellular matrix proteins and matrix metalloproteinases (MMPs), while up-regulating others including plasminogen activator inhibitor (PAI)-1 and transglutaminase 2 (TG2). tRA alone and additively, with TGF-β1, reduced MMP activity and increased PAI-1 protein; the PAI-1 inhibitor tiplaxtinin reduced the increase in total collagen caused by tRA and TGF-β1 treatment. TG2 protein was not modulated by tRA and a TG2 inhibitor did not reduce tRA’s pro-fibrotic effect. NRK-49F cells expressed retinoid nuclear receptors and PPARβ/δ and nuclear receptor mRNAs were differentially regulated by tRA and TGF-β1. RAR and RXR antagonists reduced tRA’s pro-fibrotic effect while a pan-RXR agonist more than a pan-RAR or PPARβ/δ agonist increased total collagen deposition. RAR isotype-selective agonists had less, if any, effect on fibrosis. In summary, an in vitro model for the pro-fibrotic effects of retinoids has been established, which is associated with modulation of MMPs, PAI-1 and RAR/ RXR. Further studies of RAR isotype-selective agonists might be of merit as they had reduced pro-fibrotic activities compared to less selective retinoids.
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