Sex-Specific Pathophysiological Signatures in Allometric Dosing-Controlled Bleomycin Acute Lung Injury Model

In acute lung injury (ALI), clinical data show that while mortality rates are similar between sexes, women require shorter ventilation times and intensive care unit stays than men, yet preclinical studies show conflicting sex-specific vulnerabilities. We reasoned that a hidden dosing bias may explain the inconsistency, as intratracheal bleomycin is scaled to body weight, even though lung mass grows more slowly than total body mass, so age-matched males, whose body mass outpaces lung growth, inevitably receive more drug per gram of lung than females.

We compared age-matched (12-week) and body-weight-matched (~300g) Sprague-Dawley rats receiving intratracheal bleomycin (2.5mg/kg) or saline. Both cohorts underwent functional assessments (plethysmography, lung mechanics, arterial gases, histology) at day 7; weight-matched animals exclusively underwent mechanistic profiling (BALF analysis, cytokine multiplex, paired mRNA/miRNA-sequencing, immunoblotting).

Males developed worse hypoxemia (PaO₂: age-matched p = 0.045; weight-matched p = 0.027) with higher respiratory rates (both cohorts p < 0.05). Weight-matched males showed greater compliance loss (p = 0.029), increased BALF protein (p = 0.008), and elevated IL-1β (p =0.005) and TNF-α (p = 0.017). RNA-sequencing identified 2,393 male versus 1,533 female differentially-expressed genes, with males activating complement-coagulation cascades while females enriched ECM-remodeling/BMP-signaling pathways. Males exhibited significant miR-672-3p suppression (p < 0.0001), inversely correlating with inflammatory targets. SERPINA3 and its upstream regulator STAT3 showed significantly higher induction in males (both p < 0.0001), whereas females exhibited higher BMPR2 protein levels (p = 0.009) and preserved IL-10 (p = 0.023).

Body-weight matching corrects unrecognized allometric bias affecting preclinical ALI sex-difference studies. Both cohorts demonstrated male vulnerability with worse hypoxemia and increased respiratory rates. Weight-matched molecular analyses revealed distinct responses: males showed significant miR-672-3p suppression with concurrent inflammatory mediator upregulation, including higher SERPINA3, IL-1β, and TNF-α. In contrast, females maintained higher miR-672-3p levels alongside elevated BMPR2/IL-10, suggesting that divergent post-transcriptional regulation contributes to functional differences and may inform sex-specific therapeutic strategies.