A novel zebrafish model of hyperuricemia established by combined genetic and dietary induction and its application in evaluating the XOD-targeting peptide KNRAK derived from tuna protein hydrolysate

With rising global hyperuricemia due to lifestyle changes, conventional assays and mouse models faced limitations in technical complexity and cost. In this study, we established a zebrafish model of hyperuricemia through a combined genetic and dietary induction approach. Wild-type zebrafish were treated with the uricase inhibitor potassium oxonate (PO) and the uric acid precursor xanthine, resulting in uric acid levels increasing to approximately 2-fold that of wild-type. To reduce reliance on PO and create a more stable model, we knocked out the uox gene encoding uricase using CRISPR/Cas9. The resulting mutant uox -/- increased the uric acid level to 2.83-fold compared to wild-type zebrafish. Treatment with xanthine further increased the uric acid to 3.79-fold compared to wild-type zebrafish, thereby generating a zebrafish hyperuricemia model with highly elevated uric acid. Subsequently, high-throughput screening of tuna protein hydrolysate identified a dominant peptide, Lys-Asn-Arg-Ala-Lys (KNRAK), predicted to target xanthine oxidase via molecular docking. KNRAK effectively reduced uric acid levels by 74.4% and 29.8% in hyperuricemia models derived from wild-type and uox -/- zebrafish, respectively. Transcriptomic analysis revealed that KNRAK reduced uric acid accumulation in the wild-type derived model by modulating purine metabolism, oxidative stress responses, and urate transport–related pathways, whereas in the uox -/- derived model, KNRAK attenuated NF-κB/MAPK-mediated inflammatory signaling. In summary, this study developed a zebrafish hyperuricemia model with high uric acid levels through combined genetic and dietary induction, and identifies KNRAK as a promising therapeutic peptide with dual mechanisms of action.