Evaluation of the novel ALK5 inhibitor EW-7197 on therapeutic efficacy in renal fibrosis using a three-dimensional chip model.

ABSTRACT

Background: EW-7197, a potent oral ALK5 inhibitor, was assessed for its impact on transforming growth factor beta 1 (TGF-ß1)-induced fibrosis in a three-dimensional (3D) renal fibrosis-on-a-chip and a mouse model. The evaluation included tubular epithelial-mesenchymal transition, angiogenesis, and inflammatory cytokine expression. Methods: In a 3D renal fibrosis-on-a-chip model, three cell types (kidney fibroblasts, human proximal tubular cell line, and human umbilical vein endothelial cells) were cultured and treated with TGF-ß1 and EW-7197. Expression of alpha smooth muscle actin (α-SMA) and keratin 8 (KRT-8) was assessed, angiogenesis was observed via confocal microscopy, and cytokine levels were measured using real-time polymerase chain reaction, immunoassay, and enzyme-linked immunosorbent assay. In a cisplatin-induced renal fibrosis mouse model, blood urea nitrogen levels, TGF-ß, and Smad 2/3 were determined, and renal fibrosis was assessed with Masson's trichrome stain. Results: The α-SMA expression was significantly lower in the EW-7197 group than in the TGF-ß fibrosis group. TGF-ß decreased the expression of the epithelial marker KRT-8, an effect that was reversed by EW-7197 and SB431542. In the TGF-ß-induced fibrosis model, the length of the thick vessels was reduced, and the diameter of both thick and thin vessels was decreased, but EW-7197 reversed these effects. EW-7197 significantly reduced the messenger RNA expression of TGF-ß and increased the levels of vascular endothelial growth factor receptor 2, interleukin (IL)-10, and IL-6. EW-7197 reduced the levels of secretory cytokines TGF-ß1, TGF-ß3, IL-1ß. In the cisplatin-induced renal fibrosis mouse model, EW-7197 reduced renal fibrosis by down-regulating TGF-ß signaling. Conclusion: EW-7197 attenuated the TGF-ß1-induced fibrotic cellular response in the 3D chip model and animal model. These findings indicate the potential effect of EW-7197 in attenuating renal fibrosis.