Formononetin ameliorates ferroptosis-associated fibrosis in renal tubular epithelial cells and in mice with chronic kidney disease by suppressing the Smad3/ATF3/SLC7A11 signaling

Aims: Chronic kidney disease (CKD) is characterized by interstitial fibrosis, while limited treatment drugs are available. Ferroptosis is a newly identified process that can trigger tubular atrophy and fibrosis. The aim of this study is to investigate the effects of formononetin (FN), a bioflavonoid, on Ferroptosis and renal fibrosis.

Main methods: In in vivo experiments, unilateral ureteral obstruction (UUO)- and folic acid (FA, 250 mg/kg)-induced CKD models were constructed in C57BL/6 mice of 6-8 weeks old, followed by the administration with 40 mg/kg/day FN by gavage. For in vitro experiments, Ferroptosis was induced with RSL3 or erastin in primary mouse renal tubular epithelial cells (TECs), followed by the addition of indicated concentrations of FN. Then, the levels of Ferroptosis and fibrosis were analyzed. The translocation of SMAD3, ATF3, and Nrf2 from the cytoplasm to the nucleus was checked by western blotting. The interaction of SMAD3 and ATF3 was detected by Co-immunoprecipitation.

Key findings: FN dramatically ameliorated tubular injury along with reduced expression of the profibrotic genes including α-SMA, Col1a1, and fibronectin in both two CKD mouse models and RSL3/erastin-treated TECs. Furthermore, FN administration also significantly suppressed Ferroptosis, as evidenced by increased expression of SLC7A11 and GPX4, and decreased levels of 4-HNE. In mechanism, FN disrupted the interaction between SMAD3 and ATF3, resulting in the blocking of their translocation from the cytoplasm to the nucleus. In addition, FN also promoted the separation of the Nrf2/Keap1 complex and enhanced Nrf2 nuclear accumulation.

Significance: FN alleviates CKD by impeding ferroptosis-associated fibrosis by suppressing the SMAD3/ATF3/SLC7A11 signaling and could serve as a candidate therapeutic drug for CKD.

ATF3; CKD; Ferroptosis; Formononetin; Nrf2; Smad3.