Abstract Rationale Arginine is a conditionally essential amino acid with roles in protein production, nitric oxide synthesis, and biosynthesis of proline and polyamines. Arginine is also an important signaling molecule, promoting activation of the mechanistic target of rapamycin complex 1 (mTORC1). We have recently shown that arginine promotes profibrotic activation of lung fibroblasts through mTORC1 activation, independent of its catabolism. How arginine deprivation affects fibrotic events in vivo is unknown. Here, we sought to determine how dietary restriction of arginine affects induction of lung fibrosis downstream of bleomycin instillation. Methods Fibrosis was induced by intratracheal bleomycin instillation into C57BL/6 mice using two protocols- single dose (0.7U/kg) and multidose (0.5U/kg x 3 doses with 14 days between doses). 8 days after final bleomycin instillation, mice were switched to either arginine-containing or arginine-deficient defined diets. At day 21 (single dose) or day 70 (multidose), we measured lung mechanics (Flexivent) and assessed the severity of lung fibrosis by histology and biochemical analysis. Lung collagen content was measured by hydroxyproline assay. Lung metabolite levels were measured by LC/MS (liquid chromatography/mass spectroscopy). Lungs from single dose experiments were also processed for single-cell RNA sequencing (scRNAseq). Results Mice fed an arginine-deficient diet had reduced plasma arginine levels compared with control diet-fed mice; however, lung arginine levels were not significantly changed. Despite this, mice on arginine-deficient diet had reduced fibrosis development in both single dose and multi dose models of bleomycin-induced fibrosis. Arginine-deficient mice displayed higher lung compliance and reduced hydroxyproline content, consistent with reduced fibrosis. scRNAseq analysis showed that arginine-deficient diet affected gene expression in multiple cellular populations. Conclusions Arginine deficient diets reduced bleomycin-induced fibrosis in both single dose (resolving) and multidose (progressive) models. Our findings highlight the effects of arginine on development of fibrosis in vivo. This abstract is funded by: NIH, DOD
Hamanaka et al. (Fri,) studied this question.