An in vitro model for the pro-fibrotic effects of retinoids: mechanisms of action.

background and purposeRetinoids, including all-trans retinoic acid (tRA), have dose-dependent pro-fibrotic effects in experimental kidney diseases. To understand and eventually prevent such adverse effects, it is important to establish relevant in vitro models and unravel their mechanisms. EXPERIMENTAL APPROACH: Fibrogenic effects of retinoids were assessed in NRK-49F renal fibroblasts using picro-Sirius red staining for collagens and quantified by spectrophotometric analysis of the eluted stain. Other methods included RT-qPCR, immunoassays and matrix metalloproteinase (MMP) activity assays. KEY

resultsWith or without TGF-β1, tRA was dose-dependently pro-fibrotic, notably increasing collagen accumulation. tRA and TGF-β1 additively suppressed expression of mRNA for MMP2, 3 and 13 and suppressed MMP activity. tRA, in the presence of TGF-β1, induced plasminogen activator inhibitor-1 (PAI-1) mRNA and they additively induced PAI-1 protein expression. A PAI-1 inhibitor, a pan-retinoic acid receptor (RAR) antagonist and a pan-retinoid X receptor (RXR) antagonist each partially prevented the pro-fibrotic effect of tRA. The dose-dependent pro-fibrotic effects of a pan-RXR agonist were similar to those of tRA. A pan-RAR agonist showed weaker, less dose-dependent pro-fibrotic effects and the pro-fibrotic effects of RARα and RARβ-selective agonists were even smaller. An RARγ-selective agonist did not affect fibrogenesis. CONCLUSIONS AND IMPLICATIONS: An in vitro model for the pro-fibrotic effects of retinoids was established in NRK-49F cells. It was associated with reduced MMP activity and increased PAI-1 expression, and was probably mediated by RXR and RAR. To avoid or antagonize the pro-fibrotic activity of tRA, further studies on RAR isotype-selective agonists and PAI-1 inhibitors might be of value.