Tier 3 — preclinical

Fisetin ameliorates fibrotic kidney disease in mice via inhibiting ACSL4-mediated tubular ferroptosis

Wang B, Yang L-N, Yang L-T, Liang Y, Guo F, Fu P, Ma L
Acta Pharmacologica Sinica 2024 Volume 45, issue 1, pages 150-165

Bibliography

PubMed
PMID 37696989
PubMed Central
PMC10770410
DOI
10.1038/s41401-023-01156-w
Funding
Science/Technology Project of Sichuan Province (grant nos. 2021YFQ0027 and 2022YFS0589) and the Health Commission of Sichuan Province (grant no. 20PJ048).
Competing interests
The authors declare no competing interests.

Study snapshot

DesignControlled animal studies (adenine diet-induced and UUO-induced CKD mouse models) with mechanistic in vitro validation in TCMK-1 renal tubular epithelial cells, plus immunohistochemical analysis of archived human CKD kidney biopsies.
ModelMale C57BL/6J mice (adenine diet-induced and unilateral ureteral obstruction CKD models); mouse renal tubular epithelial cell line TCMK-1 (adenine- or TGF-β1-stimulated); human kidney biopsy sections (IgA nephropathy, membranous nephropathy, focal segmental glomerulosclerosis, lupus nephritis, diabetic nephropathy vs para-cancerous control tissue).
SampleMouse experiments n=4-6 per group; in vitro assays performed in triplicate (n=3).
InterventionFisetin 50 or 100 mg/kg/day by oral gavage (adenine-diet model, 3 weeks) or 100 mg/kg/day (UUO model, 7 days); positive control pirfenidone 250 mg/kg in the adenine model; in vitro fisetin 20 µM with or without the ferroptosis inhibitor ferrostatin-1 (Fer-1, 1 µM) in adenine- or TGF-β1-stimulated TCMK-1 cells; ACSL4 siRNA knockdown and plasmid overexpression experiments.
Duration3 weeks (0.2% adenine diet model) or 7 days (UUO model); in vitro stimulation 48 hours.
EndpointsSerum creatinine, BUN and urinary albumin-to-creatinine ratio; Tubular injury markers NGAL and KIM-1; Ferroptosis markers ACSL4, COX2/PTGS2, GPX4 and HMGB1 (mRNA and protein); Renal iron, GSH/GSSG ratio and lipid peroxide (MDA) levels; Mitochondrial ultrastructure by transmission electron microscopy; Pro-inflammatory (IL-1β, IL-6, IL-8, TNF-α, MCP-1, CXCL-10) and pro-fibrotic (α-SMA, fibronectin, collagen I/IV/VI) markers; ACSL4 immunohistochemistry in human CKD biopsies vs control; Lipid ROS by BODIPY 581/591 C11 staining in vitro

What the study showed, in plain terms

This Sichuan University kidney-research group asked whether fisetin can slow the scarring (fibrosis) that chronic kidney disease causes, and if so, how. They used two classic mouse models of chronic kidney disease — a high-adenine diet and surgical ureter obstruction — plus kidney cells in a dish.

In both mouse models, oral fisetin (50-100 mg/kg/day) reduced kidney injury markers, inflammation and scarring compared with untreated diseased mice. The mechanism the authors zeroed in on is 'ferroptosis' — a form of iron-driven cell death distinct from classic apoptosis — which is increasingly recognised as a driver of tubular injury in chronic kidney disease.

Fisetin lowered a ferroptosis-driving enzyme called ACSL4 in both mouse models and in stressed kidney cells, restored the protective enzyme GPX4, and normalised iron, glutathione and lipid-peroxidation levels in the kidney. When the researchers forced ACSL4 back up with a gene-overexpression trick, fisetin's protective effect disappeared — strong evidence that blocking ACSL4-driven ferroptosis is how fisetin protects the kidney here.

They also stained human kidney biopsies from several chronic kidney diseases (IgA nephropathy, membranous nephropathy, FSGS, lupus nephritis, diabetic nephropathy) and found the same ACSL4 elevation seen in the mouse models, which strengthens the case that this pathway is relevant to human kidney disease — though no fisetin was actually given to the human tissue donors; this was purely correlative staining.

Key findings

  • Fisetin (50-100 mg/kg/day, oral) significantly improved kidney function markers (creatinine, BUN, UACR) and reduced tubular injury (NGAL, KIM-1) in both adenine diet-induced and UUO-induced chronic kidney disease mouse models.
  • Fisetin reduced pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNF-α, MCP-1, CXCL-10) and pro-fibrotic markers (α-SMA, fibronectin, collagen I/IV/VI) in both models.
  • Ferroptosis markers were reversed by fisetin: ACSL4, COX2 and HMGB1 were downregulated while the protective enzyme GPX4 was upregulated, at both mRNA and protein level.
  • Fisetin restored kidney iron, glutathione (GSH and GSH/GSSG ratio) and reduced lipid peroxide (MDA) to near-normal levels, and normalised mitochondrial ultrastructure on electron microscopy.
  • In adenine- or TGF-β1-stimulated TCMK-1 tubular cells, fisetin reduced inflammatory and fibrotic markers and lipid ROS, mirroring the ferroptosis inhibitor ferrostatin-1.
  • ACSL4 siRNA knockdown reproduced fisetin's protective effects, while ACSL4 overexpression blocked fisetin's anti-ferroptotic, anti-inflammatory and anti-fibrotic actions, confirming ACSL4 as the key mechanistic target.
  • ACSL4 was markedly elevated in human kidney biopsies across five different chronic kidney diseases (IgAN, MN, FSGS, LN, DN) compared with control tissue, supporting the clinical relevance of the ACSL4-ferroptosis axis.

What this study can and cannot tell us

This is a preclinical mouse and cell-culture study; no fisetin was administered to humans. The human biopsy data are purely correlative (elevated ACSL4 in diseased tissue) and do not show whether fisetin would have any effect in human chronic kidney disease.

Oral gavage doses of 50-100 mg/kg/day in mice are substantially higher, on a body-weight basis, than typical human oral fisetin supplement doses, and interspecies pharmacokinetic differences mean these results cannot be directly extrapolated to human dosing.

The two mouse models (adenine diet, UUO) represent tubulointerstitial injury but do not capture the full range of human chronic kidney disease aetiologies (e.g., diabetic nephropathy, glomerulonephritis) in an interventional sense.

Editorial review

Reviewed by the Biohack Blueprint research team

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Dr. Khaled Mahmoud, MBBChCritical Care Medicine, Longevity Science & Editorial Direction Dr. Amr Yasser, PhDCardiometabolic, Musculoskeletal, Gut & Bioavailability Dr. Abdulla Mostafa, PhDCellular Senescence, Neurological, Mitochondrial & Brain-Ageing Biology