Acute kidney injury (AKI) due to ischemia-reperfusion injury (IRI) remains a major clinical challenge without effective targeted therapies. Gut microbiome profiling of mice with IRI-AKI revealed distinct microbial community structures with prominent enrichment of tryptophan-associated metabolic pathways, implicating tryptophan as a candidate renoprotective mediator. Tryptophan is an essential amino acid extensively metabolized by gut microbiota through the indole, kynurenine, and serotonin pathways, with emerging roles in organ protection. We therefore systematically investigated whether tryptophan supplementation attenuates IRI-AKI and explored its underlying mitochondrial effects.

Gut microbiome was analyzed by 16S rRNA sequencing with principal component analysis (PCA) and pathway enrichment. IRI-AKI was established in 129S1/SvImJ mice by unilateral nephrectomy followed by contralateral renal pedicle clamping, with or without tryptophan supplementation. In renal tubular epithelial cells under hypoxic conditions, mitochondrial integrity was assessed by MitoTracker staining and mitochondrial respiratory function by Seahorse extracellular flux analysis.

Through metabolic pathway analysis, the gut microbiome PCA identified a distinct microbial cluster in healthy mice within the IRI-AKI model that was significantly enriched with tryptophan, thereby providing a rationale for targeted tryptophan supplementation. Tryptophan supplementation significantly attenuated IRI-AKI, with improved serum creatinine, BUN, histological injury scores, and tubular injury markers on immunohistochemistry. In vitro, tryptophan preserved mitochondrial membrane potential and morphology under hypoxia. Seahorse analysis demonstrated significantly improved oxygen consumption rate in tryptophan-treated hypoxic cells, indicating restoration of mitochondrial respiratory function. Canonical mitophagy markers (LC3, PINK1, p62) were not significantly altered by tryptophan, suggesting a mitophagy-independent mechanism of mitochondrial protection.

Tryptophan ameliorates IRI-AKI and preserves mitochondrial function through a mitophagy-independent pathway. These findings identify a gut microbiota–tryptophan–mitochondrial axis as a novel renoprotective mechanism and support further investigation of tryptophan as a potential therapeutic strategy in acute renal injury.