HNF4α promotes urate excretion by activating ABCG2 and mediates the anti-hyperuricemic effect of saffron floral bio-residue.

BACKGROUND: Current pharmacotherapies for hyperuricemia (HUA) are limited by variable efficacy and adverse effects, highlighting the urgent need for novel therapeutic targets and agents. The natural product saffron floral bio-residue (SFB) has demonstrated anti-hyperuricemic potential, but the underlying molecular mechanism remains unclear. METHODS: To elucidate the molecular targets and mechanisms of SFB, quantitative proteomic profiling was performed in a HUA rat model, identifying hepatocyte nuclear factor 4 alpha (HNF4α) and ATP-binding cassette subfamily G member 2 (ABCG2) as key regulatory targets. The role of HNF4α was validated using kidney-specific knockdown and overexpression rat models, as well as in vitro cellular models. The regulatory relationship between HNF4α and ABCG2 was mechanistically dissected using pharmacological activation of ABCG2 and dual-luciferase reporter assays. The dependence of SFB and its major bioactive constituent, kaempferol-3-O-sophoroside (KS), on the HNF4α/ABCG2 axis was systematically evaluated both in vivo and in vitro. RESULTS: Integrated proteomic and functional analyses identified HNF4α and the urate transporter ABCG2 as key regulatory targets mediating the anti-hyperuricemic effect of SFB. Kidney-specific knockdown of HNF4α markedly suppressed ABCG2 expression, leading to elevated serum uric acid (UA) level, increased oxidative stress, heightened inflammation, and aggravated renal injury. Conversely, HNF4α overexpression in HUA rats restored ABCG2 level as well as ameliorated HUA-associated pathological changes. Further, the ABCG2 agonist rifampicin demonstrated that ABCG2 functions downstream of HNF4α in HUA and renal protection. Dual-luciferase reporter assays showed that HNF4α probably binds to the ABCG2 promoter and transcriptionally activates its expression. Notably, the anti-hyperuricemic effect of SFB was abolished upon HNF4α knockdown in vivo, indicating that HNF4α was essential for SFB's therapeutic action. Additionally, SFB also exerted anti-inflammatory and antioxidant effects that might not be entirely dependent on HNF4α signaling. Subsequent investigation revealed that KS, the primary active flavonoid in SFB, acted as a potential activator of HNF4α and protected HK-2 cells against UA-induced injury. CONCLUSION: This study identifies the HNF4α/ABCG2 axis as a potential pathway governing renal urate excretion and positions HNF4α as a promising therapeutic target for HUA. SFB and its active constituent KS function as natural activators of HNF4α, providing a novel mechanistic basis for the development of natural product-based therapies against HUA.