In vitro assessment of pirfenidone anti-fibrotic properties in a humanized 3D bioprinted kidney tubulointerstitium model

Kidney fibrosis is a hallmark of chronic kidney disease (CKD), characterized by excessive extracellular matrix (ECM) deposition that progressively impairs kidney function. Fibrosis affects multiple segments of the nephron, particularly the tubulointerstitium, the space between tubule, capillaries and the intestinal tissue, where homeostasis is disrupted. Currently, no clinically approved therapies exist for kidney fibrosis. Conventional 2D cell cultures and animal models fail to replicate human disease progression, limiting therapeutic development. Three-dimensional (3D) models offer a promising alternative to better mimic pathophysiological conditions. Pirfenidone (PFD) is a clinically approved drug for the treatment of pulmonary fibrosis, and since its approval, multiple pre-clinical and clinical studies have been conducted to investigate its anti-fibrotic properties in other tissues' fibrosis. In this work, we developed a 3D bioprinted perfusable humanized model to mimic the tubulointerstitium, using human primary kidney proximal tubule epithelial and fibroblast cells. A fibrotic-like environment was induced with transforming growth factor-β1 (TGF-β1), and the anti-fibrotic effect of PFD was evaluated. Gene expression was performed, denoting a fold change decrease for LOX, SMAD1 and VIM2, together with a reduction of proteins and ECM deposition for conditions with PFD. However, no clear effect on the epithelial-mesenchymal transition (EMT) and cellular differentiation into myofibroblasts was shown with immunostaining for alpha-smooth muscle actin (α-SMA). In conclusion, we demonstrated that the developed bioprinted model could be used to test drugs for kidney fibrosis, offering an alternative to current models with more pathophysiological relevant conditions.