Fig. 7: Urinary detection of bleomycin-induced lung fibrosis.
a, Schematic representation of bleomycin-induced pulmonary fibrosis model. Briefly, C57BL/6 mice were either intratracheally administered with bleomycin for 7 days (incipient fibrosis) or 14 days (established fibrosis) or left untreated. At the endpoint, mice were administered with nanoprobes i.v. Urine was collected 2 h p.i. of nanoprobes, and the renally cleared AuNC was detected using alloy formation assay. Created in BioRender. Fruk, L. (2026) https://BioRender.com/6j83ai8. b, Representative CT scans of lungs from untreated- versus bleomycin-treated mice. c,d, Representative histological images (c) and staining quantification (d) of lungs at the treatment endpoint, stained for fibrosis markers (Masson’s trichrome and α-SMA), senescence markers (p21 and p16) and MMP-7, from untreated mice and mice after undergoing 14-day bleomycin treatment (n = 8 independent mice per group, mean ± s.e.m, unpaired two-tailed t-test). Scale bar = 25 μm. e, Photograph of the alloy formation assay on urine samples from untreated or bleomycin-treated mice injected with nanoprobes. f, Absorbance values (A414 nm) from alloy formation assay of urine samples collected from untreated or bleomycin-treated mice 2 h p.i. with nanoprobes (n = 8 mice per group, mean ± s.e.m, unpaired two-tailed t-test). g, ROC analysis showing the diagnostic specificity and sensitivity of nanoprobes and alloy formation assay in detecting established fibrosis between bleomycin-treated (14 days) and untreated mice group. The solid line represents the ROC curve for distinguishing untreated controls (n = 8 independent mice) from bleomycin-treated patients (n = 8 independent mice). The dashed diagonal line represents the performance of a random classifier (AUC = 0.5). The area under the curve (AUC) was 0.86 (standard error = 0.1019; 95% confidence interval 0.6597–1.000; P = 0.0157). h,i, Correlation analysis between the percentage of intensity of signals from urine samples detected using the alloy formation assay with (h) fibrosis markers, Masson’s+ area (r = 0.7485; P = 0.0327) and α-SMA+ cells (r = 0.701; P = 0.0321), and (i) senescence markers, p16 (r = 0.3910; P = 0.3382) and p21 (r = 0.7904; P = 0.0196), in the lungs of 14-day bleomycin-treated mice. For (h) and (i), solid lines indicate linear regression fits (least-squares fit; estimated mean relationship between variables), and dashed lines denote the 95% confidence intervals of the regression line; n = 8 independent mice per group, two-tailed Pearson correlation; Pearson correlation coefficients (r) and corresponding one-tailed P values are listed. j,k, Representative histological images (j) and staining quantification (k) of lungs at the treatment endpoint, stained for fibrosis markers (Masson’s trichrome and α-SMA), senescence markers (p21 and p16) and MMP-7, from untreated mice and mice after undergoing 7-day bleomycin treatment (n = 4 individual mice (untreated) and n = 5 individual mice (7-day bleomycin) (biological replicates); mean ± s.e.m, unpaired two-tailed t-test). Scale bar = 25 μm. l, Absorbance values (A414 nm) from alloy formation assay of urine samples collected from untreated or bleomycin-treated (7 days) mice 2 h p.i. with nanoprobes (mean ± s.d., n = 4 individual mice (untreated) and n = 5 individual mice (7-day bleomycin) (biological replicates), mean ± s.e.m, unpaired two-tailed t-test). m, ROC analysis showing the diagnostic specificity and sensitivity of nanoprobes and alloy formation assay in detecting incipient fibrosis between bleomycin-treated (7 days) and untreated mice groups. The solid line represents the ROC curve for distinguishing untreated controls (n = 4 independent mice) from bleomycin-treated mice (n = 5 independent mice). The dashed diagonal line represents the performance of a random classifier (AUC = 0.5). The area under the curve (AUC) was 0.90 (standard error = 0.118; 95% confidence interval 0.6809 – 1; P = 0.0432). n,o, Correlation analysis between the percentage of intensity of signals from urine samples detected using the alloy formation assay with (n) fibrosis markers, Masson’s+ area (r = −0.3905; P = 0.5158) and α-SMA+ cells (r = 0.9268; P = 0.0235), and (o) senescence markers, p16 (r = 0.5264; P = 0.3622) and p21 (r = 0.8847; P = 0.0462), in the lungs of 7-day bleomycin-treated mice. For (n) and (o), solid lines indicate linear regression fits (least-squares fit; estimated mean relationship between variables), and dashed lines denote the 95% confidence intervals of the regression line; n = 5 individual mice (7-day bleomycin); two-tailed Pearson correlation.
