Fig. 7: PDGFR inhibitors reduce fibrosis in ARPKD organoids.

Control or ARPKD organoid cultures were treated with vehicle or PDGFR inhibitors, and the extent of fibrosis developing in the cultures was measured by three different methods. a Whole-mount immunostaining of control HOs and of drug (10 μM) or vehicle-treated ARPKD organoids. Representative images of organoids stained with COL1A and CK8 antibodies, and counterstained with Hoechst 33342. Scale bar, 50 μm. b The fibrosis score was determined by measuring the ratio of the volume of COL1A and CK8 in 100 stacked images obtained from 4 whole-mount organoids per group. The PDGFR inhibitors (Crenolanib, Imatinib) decreased the fibrosis score in ARPKD organoids to levels that were similar to control organoids (n = 4 per group, ARPKD vs Imatinib p = 0.00089, ARPKD vs Crenolanib p = 0.0042, ARPKD vs DAPT p = 0.0014). c Representative images of COL1A and CK8 immunostained ARPKD organoids before and after treatment with 10 μM of the indicated inhibitor. For comparison, an image of an immunostained control organoid is also shown. Scale bar is 50 μm. d The fraction of the total area occupied by bile ducts (CK8⁺ area) within isogenic control (normal) and ARPKD organoids after treatment with 10 μM of the indicated drug (n = 5 per group, DMSO vs Crenolanib p = 0.37, DMSO vs Sunitinib p = 0.58, DMSO vs Imatinib p = 0.015, n.s. no significant difference). e COL1A1 mRNA levels in ARPKD organoids are significantly higher than in control organoids, and were decreased 17-fold after treatment with the PDGFR inhibitors. RT-PCR measurements made on 3 organoids per treatment group; and the values were normalized relative to simultaneously measured GAPDH mRNA levels (DMSO vs Crenolanib p = 0.00048, DMSO vs Sunitinib p = 0.0021, DMSO vs Imatinib p = 0.00071, ARPKD vs DAPT p = 0.00068). Box plots in b, d, and e (center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range). Statistical differences between the groups were determined by unpaired two-tailed t-test. *p < 0.05; **p < 0.01; and ***p < 0.001. f, g 4-OH Pro levels in ARPKD organoids are decreased by treatment with 10, 2, or 0.5 μM concentrations of PDGFRB inhibitors (Control vs DMSO p = 0.00097, DMSO vs Crenolanib p = 0.0046, DMSO vs Sunitinib p = 0.00082, DMSO vs Imatinib p = 0.0013; DMSO vs 2 μM Crenolanib p = 0.00047, DMSO vs 0.5 μM Crenolanib p = 0.0019, DMSO vs 2 μM Sunitinib p = 0.37, DMSO vs 0.5 μM Sunitinib p = 0.0014, DMSO vs 2 μM Imatinib p = 0.0064, DMSO vs 0.5 μM Imatinib p = 0.004). Box plots in b, d and e (center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range). Statistical differences between the groups were determined by unpaired two-tailed t-test. *p < 0.05; **p < 0.01; and ***p < 0.001.