Fig 2: The clinicopathological significance of WNT10A expression in patients with IPF. a An immunohistochemical analysis of the WNT10A protein expression in the healthy control and patients with IPF. The left panels show normal human lung tissue treated for the localization of WNT10A using immunohistochemistry reactivity in the smooth muscle of vessels (single arrows) and airways (double arrows). The right upper panels show a WNT10A-positive case, which demonstrated a specific expression of WNT10A in one part of the spindled cells (fibroblasts, indicated by arrows in a high-power view of the right panel) at a site of pulmonary fibroblastic foci (boxes, low-power view of left panel). The right bottom panels indicate a WNT10A-negative case in patients with IPF. b The results of the Kaplan-Meier survival analysis of the overall survival in IPF patients who were WNT10A-positive or negative

Fig 3: WNT10A protected fibroblasts against high glucose stress.(A) Proliferation assay. Control and WNT10A-overexpressing cells (COS1-10A-cl.1) (2×104 cells/well) were seeded into a 12-well plate, and incubated in normal DMEM. After 24 hours, cells (0hr) were harvested, and other cells were cultured in each medium (5.5 mM, 11 mM, and 22 mM of glucose concentration) for 72 hours. Proliferative rates for 72 hours were compared. Black bar (5.5 mM), white bar (11 mM), gray bar (22 mM). All values are the means of at least three independent experiments. Bars indicate standard deviation. *p<0.001 vs 5.5 mM, **p<0.001 vs 5.5 mM and 11 mM, #p<0.05 vs 5.5 mM (B) Caspase and PARP expression. Whole lysates obtained from control and WNT10A-overexpressing cells treated with each medium (5.5 mM, 11 mM, and 22 mM of glucose concentration) were analyzed by western blotting.

Fig 4: WNT10A protected fibroblasts against oxidative stress.(A) Western blot analysis of peroxiredoxin (PRDX)1, 2, 3, 4, and 5. Western blot analysis was performed using whole lysates obtained from COS1-vector (control) and COS1-10A cells. Only PRDX5 was increased with expression of WNT10A. (B) Relative expression of PRDX1, 2, 3, 4, and 5 normalized to β-actin expression. The black bar shows control cells, white and gray bars show COS1-10A-cl.1 and COS1-10A-cl.2 respectively. (C) The sensitivity of control and WNT10A-overexpressing cells (COS1-10A-cl.1) to H2O2. All values are the means of at least three independent experiments. Bars indicate standard deviation. *p<0.01 vs control. #p<0.05 vs control. (D) Downregulation of PRDX5 in WNT10A-overexpressing cells (COS1-10A-cl.1) by two kinds of siRNA. (E) The sensitivity of siRNA transfected cells to H2O2. All values are the means of at least three independent experiments. Bars indicate standard deviation. *p<0.05 vs PRDX5#1 and #3 siRNA. (F) Western blot analysis of caspase-3 and PARP expression. Western blot analysis was performed using whole lysates obtained from COS1-vector (control) and COS1-10A. Each cells were cultured in normal medium or medium containing hydrogen peroxide (30 µM) for 4 hours.

Fig 5: The relationship between WNT family gene expression and kidney function.(A) Patient selection method to evaluate eGFR among AIN patients. eGFR (mL/min per 1.73 m2) of AIN patients with (B) WNT1, (C) WNT3, (D) WNT4, and (E) WNT10A. (B) WNT1 negative-expression; n = 9, positive-expression; n = 11 (C) WNT3 negative-expression; n = 12, positive-expression; n = 8 (D) WNT4 negative-expression; n = 15, positive-expression; n = 5 (E) WNT10A negative expression; n = 10, positive-expression; n = 10. Light gray box indicates eGFR of patients without WNT protein expression, and dark gray box indicates eGFR of patients with WNT protein expression. The left panels show kidney function on kidney biopsy, and right panels show kidney function on discharge. Data of each category is shown in Table 2.