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. 2026 Sep 16;11(38):57378–57393. doi: 10.1021/acsomega.6c05732

Quercetin Alleviates Ferroptosis-Induced Renal Fibrosis by Modulating the PRDM9/HMOX1/PTGS2 Axis

Qiongqiong Zhu †, Zhibin Chen †, Xianxian Yu †, Qiu Ye ‡, Xu Chen ‡,*
PMCID: PMC13629372  PMID: 42825118

Abstract

Ferroptosis-induced renal fibrosis is a key driver of chronic kidney disease (CKD) progression, yet effective therapies remain limited. This study investigates the therapeutic potential of quercetin (QCT), a bioflavonoid derived from Ginkgo biloba fruit peel, in mitigating ferroptosis-associated renal fibrosis. Network pharmacology identified HMOX1 and PTGS2 as core candidate genes associated with the therapeutic response to QCT in renal fibrosis. In a unilateral ureteral obstruction (UUO)-induced CKD mouse model, QCT treatment attenuated tubular injury, improved renal function, and reduced fibrosis markers such as fibronectin, Col1a1, and α-SMA. Mechanistically, QCT suppressed ferroptosis and epithelial–mesenchymal transition (EMT) by downregulating HMOX1. Further analysis revealed that PRDM9, a histone methyltransferase and transcription factor, drives HMOX1 expression through both transcriptional activation and H3K4me3-mediated epigenetic modification. Knockdown of PRDM9 reduced HMOX1 expression and alleviated fibrosis. These findings highlight a novel epigenetic mechanism underlying ferroptosis-induced renal fibrosis and position QCT as a promising therapeutic candidate for CKD.

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1. Introduction

Chronic kidney disease (CKD) is defined as a progressive condition marked by enduring structural and functional impairments of the kidneys. In the absence of timely intervention, CKD may advance to end-stage renal disease (ESRD). The major histopathological manifestations of CKD encompass glomerulosclerosis, tubular atrophy, interstitial fibrosis, and infiltration of inflammatory cells. , Despite extensive research, the mechanisms underlying kidney fibrosis remain inadequately understood, hindering the development of targeted therapies for CKD.

Ferroptosis, a form of iron-dependent, nonapoptotic cell death first described by Dixon in 2012, is characterized by reduced activity of glutathione peroxidase 4 (GPX4) and lipid peroxide accumulation. This process involves the light-chain subunit SLC7A11 of the amino acid antiporter system Xc–, which maintains glutathione (GSH) levels by exchanging extracellular cystine for intracellular glutamate. GPX4 then reduces phospholipid hydroperoxides to corresponding phospholipid alcohols. Both GPX4 and SLC7A11 act as negative regulators of ferroptosis and are crucial in ferroptosis-induced renal fibrosis. ,

Beyond the core regulatory axes of GPX4 and SLC7A11, emerging evidence has established a critical link between ferroptosis and the pathological progression of renal fibrosis. Ferroptosis has been recognized as a key driver of renal interstitial fibrosis originating from tubular epithelial cells (TECs) in the setting of UUO. Sustained ferroptotic activity within TECs has been shown to exacerbate fibrotic progression via the TGF-β/Smad signaling cascade. At the mechanistic level, ferroptosis in TECs orchestrates the release of pro-fibrotic cytokines, promotes macrophage infiltration, and facilitates TGF-β1-mediated extracellular matrix (ECM) accumulatio. Smad3, a key downstream effector of TGF-β signaling, has been shown to mediate renal fibrosis by directly binding to the GPX4 promoter to repress its transcription, thereby promoting ferroptosis. Conversely, inhibition of tubular ferroptosis effectively rescues the accumulation of profibrotic cytokines and alleviates renal fibrosis. Furthermore, ferroptosis has been closely implicated in the pathogenesis of renal fibrosis across various kidney diseases, including glomerulonephritis, renal ischemia-reperfusion injury, diabetic nephropathy, and renal calculus. Importantly, ferroptosis can trigger tubular atrophya hallmark pathological feature of fibrotic kidneysand is associated with the epithelial-to-mesenchymal transition (EMT) of TECs, a key cellular process driving fibrogenesis. , These findings collectively underscore that ferroptosis is not merely a consequence of kidney injury but an active driver of fibrotic remodeling. Given this pivotal role, elucidating the upstream epigenetic and transcriptional regulators of ferroptotic pathways, such as HMOX1 and its modifiers, is essential for understanding CKD pathogenesis.

Heme oxygenase-1 (HMOX1/HO-1), regulated by the transcription factor Nrf2, is a rate-limiting enzyme in heme degradation, producing carbon monoxide, biliverdin, and ferrous ions. , While HMOX1 is protective against oxidative stress, its excessive activation may promote ferroptosis. , Prostaglandin-endoperoxide synthase 2 (PTGS2) is a marker of ferroptosis, catalyzing polyunsaturated fatty acids to produce prostaglandins and lipid mediators, thus facilitating lipid peroxidation. Epigenetic modifications, such as DNA methylation, histone modification, noncoding RNAs, and microRNAs, influence gene expression without altering DNA sequence and are prevalent in kidney diseases. − Histone modifications, particularly acetylation and methylation of lysine residues, affect chromatin conformation and gene accessibility, Various histone methylation modifications, including, H3K9me3, and H3K27me3, are implicated in CKD pathogenesis. , Our preliminary research identified PRDM9 (PR domain containing 9), a transcription factor upstream of HMOX1, as a key player. PRDM9, a methyltransferase, catalyzes trimethylation of lysine 4 on histone H3 (H3K4) and contains C2H2 zinc finger motifs with transcription factor activity, The C-terminal region contains multiple tandem C2H2 zinc finger motifs, which also confer transcription factor activity. We hypothesize that PRDM9 may influence ferroptosis mediated by HMOX1 through both epigenetic and transcriptional regulation, thereby affecting renal fibrosis.

Quercetin is a bioflavonoid derived from various traditional Chinese medicines, including Ginkgo biloba, , and is known for its antioxidant, antitumor, and immunomodulatory properties. Previous research has demonstrated the therapeutic efficacy of quercetin across multiple disease models, including renal fibrosis. Mechanistically, quercetin influences renal tubular epithelial–mesenchymal transition and fibrosis through several signaling pathways, such as amphiregulin/EGFR, mTORC1/p70S6K, , and the SIRT1/PINK1/mitochondrial autophagy axis, as well as by modulating the TGF-β pathway. , However, the potential of quercetin to inhibit renal fibrosis through the ferroptosis pathway in chronic kidney disease remains unexplored. It has been shown in recent studies that quercetin attenuates ferroptosis in renal tubular epithelial cells through modulation of the Nrf2/HO-1 signaling axis. Given the established role of oxidative stress as a bridging factor between ferroptosis and fibrotic progression, we postulated that quercetin might represent a viable therapeutic candidate for counteracting ferroptosis-driven fibrosis. Accordingly, the present work was designed to explore the impact of quercetin on both ferroptosis and fibrotic remodeling in the context of CKD.

In summary, this study employed a unilateral ureteral obstruction (UUO) CKD model and a TGF-β-induced HK-2 cell model to demonstrate that quercetin treatment ameliorates ferroptosis-related renal injury and fibrosis. Mechanistically, quercetin inhibited the epigenetic modifications and transcriptional regulation of the HMOX1/PTGS2 signaling pathway mediated by PRDM9, thereby mitigating ferroptosis-induced renal fibrosis.

2. Material and Methods

2.1. Experimental Animal Model and Treatment

Quercetin (Merck, Q4951) was prepared as a suspension in 0.5% CMC-Na for injection. Male C57BL/6 mice (8 weeks old) were randomized into Sham, UUO, UUO + QCT, UUO + oeHMOX1, and UUO + siHMOX1 groups (n = 6 initial). After pentobarbital anesthesia (30 mg/kg i.p.), UUO or sham surgery was performed as described. 2.1.

For overexpression, AAV9-Ggt-HMOX1 (2.37 × 1013 v g/mL) was delivered renally 14 days presurgery, with controls receiving empty vector. For knockdown, HMOX1 siRNA (50 pmol·mL–1, i.v.) was given immediately postsurgery to the siHMOX1 group, while controls received nontargeting siRNA. QCT (18 mg/kg) was injected subcutaneously daily from Day 0 to Day 13; the Sham and UUO groups received vehicle alone. Mice were sacrificed on Day 14 for tissue/blood collection. Animals with severe distress, >20% weight loss, or early postoperative mortality were excluded; one UUO mouse died of infection on Day 3 (final n = 5/group). All procedures were approved by the Yueqing Hospital of Wenzhou Medical University Animal Ethics Committee.

2.2. Cell Culture and Treatment

HK-2 cells (ATCC, CRL-2190) were cultured in DMEM/F-12 (1:1) with 10% FBS and penicillin/streptomycin at 37 °C/5% CO2. Cells were allocated to seven conditions: control, erastin, erastin + QCT, erastin + QCT + oeHMOX1, TGF-β + QCT + siHMOX1, erastin + siPTGS2, and erastin + siPRDM9. Serum starvation (1% FBS, 24 h) was followed by QCT treatment (24 h). siRNA transfections (HMOX1/PTGS2/PRDM9) were conducted with Lipofectamine 2000, and 24 h later, cells were exposed to 20 μM erastin (MCE, HY-15763) for 24 h prior to harvest.

2.3. Detection of Lipid Peroxidation by Fluorescence Microscopy

Cellular lipid ROS were detected with 10 μM C11-BODIPY 581/591 (Invitrogen) in complete medium (1 h incubation), followed by PBS wash. Images of random fields were acquired promptly under a fluorescence microscope, and oxidized probe fluorescence (green channel) was quantified with ImageJ.

2.4. Network Pharmacology Analysis

2.4.1. Screening for Active Constituents and Target Genes Screening

Using two authoritative Traditional Chinese Medicine databases, HERB (http://herb.ac.cn/) and TCMIP (http://www.tcmip.cn/TCMIP/index.php/), herbal medicines with therapeutic effects on renal fibrosis were screened. Compounds were initially screened based on their Absorption, Distribution, Metabolism, and Excretion (ADME) properties. Thresholds of oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18, which are standard criteria in network pharmacology, were applied to ensure that the selected compounds possessed reasonable potential for in vivo activity. The common components identified by both databases were considered as candidate herbal medicines for the treatment of renal fibrosis. Subsequently, to identify therapeutic targets for kidney fibrosis, the top 500 genes were selected from the GeneCards (https://www.genecards.org/) of human disease gene database based on their relevance scores. Additionally, therapeutic targets of G. biloba were identified using the Traditional Chinese Medicine Integrative Pharmacology (TCMIP) database. The intersection of these two sets of targets was analyzed to identify potential targets for the treatment of kidney fibrosis with G. biloba. These common targets will be subjected to further analysis and experimental validation to explore their therapeutic potential. The active compounds of G. biloba used in this experiment were identified based on their therapeutic targets through screening with BATMAN-TCM (http://bionet.ncpsb.org.cn/batman-tcm).

2.4.2. Pathway and Functional Enrichment Analysis

Gene Ontology (GO) classification and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were carried out using the clusterProfiler R package. A significance threshold of P < 0.05 was employed to identify statistically over-represented terms.

2.4.3. PPI Network Construction

Protein–protein interaction (PPI) networks were predicted using the STRING database (version 12.0, available at https://string-db.org). with an interaction confidence cutoff set at 0.4. The resulting network was then visualized and further analyzed with Cytoscape software (version 1.0.4).

2.4.4. JASPAR Database Predicts PRDM9 Binding Sites on the HMOX1 Promoter

Query the conserved binding sites of the transcription factor PRDM9 in the JASPAR database. Then, based on Rauluseviciute et al.’s report, select a 2000 bp fragment upstream of the transcription start site of HMOX1 as the promoter region, and predict the potential binding sites of PRDM9 within the HMOX1 promoter.

2.5. Scr and BUN Assay

Renal injury markers, specifically serum creatinine (Scr) and blood urea nitrogen (BUN), were measured using commercial kits (Nanjing Jiancheng, cat no. C013-2 and cat no. C011-2-1, Nanjing, China) following the manufacturer’s instructions. Similarly, the determination of iron, MDA, and GSH content associated with ferroptosis assay kits (cat no. A039-2-1, cat no. A003-1, cat no. A005-1-2) were measured using the kits all obtained from Nanjing Jiancheng.

2.6. Histological Analyses of Mouse Kidney Tissue

2.6.1. Hematoxylin & Eosin Staining

To assess renal morphology, kidney tissues from six mice per cohort were fixed, paraffin-embedded, and sectioned (3–5 μm) after standard dehydration. H&E staining was performed with a Solarbio kit (G1120). Cortical sections (≥3 per mouse) were imaged at 200× using an Olympus VS120 virtual microscope. Tubular injury was scored semiquantitatively (0–5) by blinded observers using ImageJ V1.8.0, based on the percentage of affected tubules: 0 (normal), 1 (<10%), 2 (10–25%), 3 (26–50%), 4 (51–75%), and 5 (>75% with dilation, necrosis, or luminal destruction).

2.6.2. Masson’s Trichrome Staining

Renal fibrosis was evaluated by Masson’s trichrome staining (Solarbio, G1340). Images captured at 200× (VS120, Olympus) were processed with ImageJ V1.8.0 in a blinded manner to quantify the fibrotic area.

2.6.3. Immunohistochemistry of Mouse Kidney Tissue

Antigen retrieval was performed on rehydrated paraffin sections by microwave heating, and endogenous peroxidases were blocked with 10% H2O2 (30 min). Primary antibodies were applied overnight at 4 °C to detect KIM-1 (1:200, CST, 14971S), fibronectin (1:200, Abmart, TA5335M), α-SMA (1:200, Boster, BM0002), Col1a1 (1:200, Abcam, 138492), HMOX1 (1:500, Proteintech, 10701-1-AP), GPX4 (1:4000, Proteintech, 67763-1), SLC7A11 (1:200, Abmart, TD12509M), PTGS2 (1:500, Proteintech, 12375-1-AP), and H3K4me3 (1:4000, CST, 9751). Immunoreactivity was visualized with HRP-labeled secondary antibodies and DAB using a ZSGB-BIO detection kit.

2.6.4. Immunofluorescence (IF)

For IF analysis, HK-2 cells grown on slides for 24 h were fixed in ice-cold methanol/acetone (10 min), blocked with 5% nonfat milk, and incubated with anti-PRDM9 antibody (2 h, RT). After secondary antibody exposure (1 h) and DAPI counterstaining (10 min), the cells were imaged using an Olympus confocal system as per ref .

2.6.5. FISH/IF Double Staining

Fluorescence in situ hybridization combined with immunofluorescence (FISH-IF) was employed to examine the colocalization of nucleic acid molecules and proteins within cells, thereby elucidating the interaction between HMOX1 and PRDM9 during transcription. Fluorescent probes specific to HMOX1 were procured from Exiqon (Woburn, MA, USA) and hybridized to mouse renal tissue sections. In brief, paraffin-embedded sections were digested with proteinase K, hybridized with the HMOX1 probe at 37 °C, incubated with primary and secondary antibodies, and finally counterstained with DAPI. The fluorescent signals were then visualized using a confocal imaging system (Olympus, Tokyo, Japan).

2.7. Western Blotting

Western blotting analysis were performed as described previously. Primary monoclonal antibodies against PRDM9 (1:1000, A20340, cat no. A20340, China), KIM-1 (1:100, Abcam, cat no. ab78494, USA), Col1a1 (1:200, CST, cat no. 72026S, USA), α-SMA (1:200, Boster, cat no. BM0002, China), fibronectin (1:200, Abmart, cat no. TA5335M, China), HMOX1 (1:10000, cat no. 66743-1-Ig, Proteintech, China), SLC7A11 (1:200, Abmart, cat no. TD12509M, China), GPX4 (1:100, Abmart, cat no. T56959S, China), KLF15 (1:200, Abcam, cat no. ab167192, USA), E-cadherin (1:1000, Merck, cat no. ZRB1692, Germany), Vimentin (1:1000, Merck, cat no. SAB4503081, Germany), Snail-1 (1:5000, Merck, cat no. ABD38, Germany), Twist-1 (1:2000, Merck, cat no. SAB5701071, Germany), ZEB-1 (Abcam, cat no. ab81972, China), H3K4me3 (1:1000, Merck, no.07-473, Germany), were used for immunoblotting. The intensity of each band was determined using ImageJ software (version 1.53q; National Institutes of Health, Bethesda, MD).

2.8. Gene Expression, siRNA Transfection, and HMOX1 Overexpression

2.8.1. Gene Expression

Following the established protocol, we carried out qRT-PCR to quantify target transcripts. Total RNA was extracted from kidney cortex specimens with TRIzol (Invitrogen) and quantified spectrophotometrically (NanoDrop 8000, Thermo Scientific). Reverse transcription of 1 μg RNA aliquots was performed with the TAKARA PrimeScript kit (RR047A), and the resulting cDNA was amplified using SYBR Green (Vazyme, Q511-02) on a QuantStudio 3 cycler (Thermo Fisher). Primer details appear in Supporting Information, and relative mRNA levels were calculated by the 2–ΔΔCt method with β-actin as the reference.

2.8.2. siRNA Interference

For knockdown experiments, we obtained validated siRNAs targeting HMOX1, PTGS2, or PRDM9, along with nontargeting controls (Santa Cruz). Transfections were performed with Lipofectamine 3000 (Invitrogen, L3000001) according to the recommended protocol. Briefly, cells were seeded in 24-well plates at 5 × 104/well, cultured overnight, and then exposed to siRNA (50 pmol/mL) complexed with Lipofectamine for 6 h, after which the medium was replenished. In vivo, we administered the corresponding siRNA (or scrambled control) via tail-vein injection over 30 s just after UUO. Western blotting verified silencing efficiency.

2.8.3. HMOX1 Overexpression Cell and Mice

The pcDNA3.1-based HMOX1 overexpression construct was obtained from Sangon Biotechnology. For cell assays, we transfected 2000 ng of this plasmid or empty vector. For animal studies, we used AAV9 vectors (GeneChem) bearing either the Ggt-HMOX1 cassette (2.37 × 1013 v g/mL) or the control CV232 empty vector, injected into the renal parenchyma of 8 week-old mice. Following a 2 week transduction period, we performed UUO surgery as described earlier. Overexpression was confirmed by Western blot.

2.9. ChIP Assay

ChIP experiments were performed with a specific anti-H3K4me3 monoclonal antibody (Abcam, EPR20551-225) to evaluate the H3K4me3 level at the HMOX1 genomic locus. The antibody–chromatin mixtures were allowed to react overnight at 4 °C. The eluted DNA was subsequently purified with the QIAquick column (Qiagen, Valencia, CA) and subjected to PCR analysis targeting the HMOX1 proximal promoter.

2.10. Co-Immunoprecipitation (Co-IP)

For Co-IP experiments, 293FT cells were cultured for 48 h post-transfection with the relevant plasmids (PTGS2-HA, H3K4me3-HA, HMOX1-FLAG, or vector control) mediated by PEI (MW 40,000; Yeasen, 40816ES02). Cells were then lysed in NP-40 buffer (50 mM Tris, 150 mM NaCl, 0.1% NP-40, 0.5% glycerol, pH 7.5) for 30 min. After centrifugation, the cleared lysates were recovered and allowed to react with HA- or FLAG-conjugated magnetic beads (MCE) at RT for 2 h. The beads were subsequently washed five times with the lysis buffer, and the captured proteins were eluted by boiling in 1× SDS loading buffer for 10 min, prior to Western blot analysis as described in the preceding section.

2.11. Luciferase Reporter Assay

The proximal promoter region of HMOX1 was subcloned into the pGL3-basic luciferase reporter vector. A mutant construct harboring alterations in the PRDM9-binding site was generated via site-directed mutagenesis. For transient transfection, HEK293T cells were treated with the designated plasmids using Lipofectamine 3000 reagent, after which the luciferase activities were measured.

2.12. Statistical Analysis

Quantitative data are expressed as mean ± standard deviation (SD). All statistical evaluations were carried out using GraphPad Prism 9 (La Jolla, CA, USA). Intergroup differences between two groups were assessed via two-tailed unpaired Student’s t tests, while comparisons involving multiple groups were conducted using one-way analysis of variance (ANOVA), complemented by Tukey’s post hoc test at a 95% confidence interval. A probability value of p < 0.05 was considered to indicate statistical significance.

3. Results

3.1. Network Pharmacology Prediction of Potential Mechanisms of G. biloba in the Treatment of Renal Fibrosis

Network pharmacology analysis was employed to identify potential traditional Chinese medicines (TCMs) for treating renal fibrosis and to elucidate their mechanisms of action. Initially, five TCMs were selected from the HERB and TCMIP databases: Pinellia ternata (Thunb.) Breit., Croton tiglium L., Lycium barbarum L., Panax ginseng C. A. Mey., and G. biloba L. Among these, G. biloba emerged as a potential candidate (Figure A). The therapeutic targets of Ginkgo identified from the TCMIP database are significantly associated with redox processes (Supporting Information). These targets were cross-referenced with renal fibrosis-related targets in the GeneCards database, resulting in the identification of 11 potential drug targets (Figure B). A protein–protein interaction (PPI) network comprising 16 candidate drug targets was constructed using the STRING database version 12.0 and visualized with Cytoscape software (Figure C). The PPI network consisted of 33 interaction edges and exhibited an average node degree of 4.12. Topological analysis highlighted key nodes, including HMOX1 and PTGS2, which demonstrated high degrees of connectivity within the network. KEGG pathway analysis revealed that these proteins are enriched in pathways such as ferroptosis, nitrogen metabolism, VEGF signaling, and HIF-1 signaling (Figure D). Gene Ontology (GO) functional annotation indicated that the key nodes and modules are primarily involved in biological processes such as transcriptional regulation (Figure E). The results of the PPI network pharmacology analysis suggest that G. biloba may exert therapeutic effects on renal fibrosis by regulating key node proteins, including HMOX1 and PTGS2, and modulating signaling pathways, particularly those related to ferroptosis.

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Network pharmacology analysis of therapeutic targets for renal fibrosis. (A) Venn diagram illustrating renal fibrosis-related herbs identified from the HERB and TCMIP databases. (B) Venn diagram showing the intersection of target genes from G. biloba components and renal fibrosis-related genes. (C) Protein–protein interaction (PPI) network depicting common targets regulated by G. biloba components against renal fibrosis through ferroptosis. (D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways involved in the treatment of renal fibrosis by G. biloba components. (E) Gene Ontology (GO) biological processes associated with G. biloba components in the treatment of renal fibrosis.

3.2. QCT Alleviates Tubular Injury and Improves Renal Function in CKD Model

The chemical structure of quercetin is illustrated in Figure A. In the UUO-induced chronic kidney disease (CKD) mouse model, Scr and BUN levels were significantly elevated in the UUO group compared with the Sham group, indicating substantial kidney tissue damage. However, QCT treatment markedly reduced these levels (Figure B). Histological analysis using HE staining revealed that UUO-induced CKD mice exhibited pronounced renal tubular dilatation, tubular epithelial injury, and lumen destruction, which were accompanied by immune cell infiltration, compared to the control group. Notably, QCT administration significantly ameliorated these histological abnormalities (Figure C,D). Immunohistochemical analysis also demonstrated high KIM-1 expression in the kidney tissues of UUO-induced CKD mice, which was substantially diminished following QCT treatment (Figure E,F). Furthermore, we assessed kidney injury molecule-1 (KIM-1), a sensitive marker of proximal tubular injury, at both the mRNA and protein levels. As anticipated, QCT treatment led to a significant reduction in KIM-1 expression in the UUO group (Figure G,H). Collectively, these findings suggest that QCT alleviates tubular injury and improves renal function in a CKD model.

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QCT administration alleviated renal tubular injury and improved renal function in CKD models. (A): Chemical structure of quercetin (QCT). (B) Serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr). (C) Hematoxylin and eosin (HE) staining of renal tissues from mice in each group (scale bar = 100 μm). (D) Renal histopathological lesion score. (E) Immunohistochemical staining of KIM-1 in renal tissues from mice in each group (scale bar = 50 μm). (F) Quantitative analysis of KIM-1 positive area percentage. (G) mRNA expression level of KIM-1 in renal tissues from mice in each group. (H) Protein expression level of KIM-1 in renal tissues from mice in each group (representative Western blot bands and quantitative analysis). All experiments were independently repeated at least 3 times. Data are presented as mean ± SD N = 5 per group from an initial n = 6, with one exclusion per group due to surgical complications. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.3. QCT Treatment Alleviates Renal Fibrosis in CKD Model

Masson’s trichrome staining demonstrated that QCT treatment led to a significant reduction in extracellular matrix deposition compared to the UUO group (Figure A,B). Consistent with these findings, immunohistochemistry analyses also revealed that renal protein levels of fibronectin, Col1a1, and α-SMA were markedly decreased in QCT-treated UUO mice relative to the UUO group (Figure C). Furthermore, these proteins were downregulated at both the transcriptional and translational levels (Figure D,E). Collectively, these data indicate that QCT treatment attenuates UUO-induced renal fibrosis, as evidenced by the reduced expression of fibronectin, Col1a1, and α-SMA.

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Treatment with quercetin ameliorated renal fibrosis in UUO models. (A) Representative images of Masson’s trichrome staining of renal tissues from mice in each group (scale bar = 50 μm). (B) Quantitative analysis of fibrotic area in renal tissues. (C) Immunohistochemical staining of Fibronectin, Col1a1, and α-SMA in renal tissues (scale bar = 50 μm) and quantitative analysis of their positive area percentages. (D) Relative mRNA expression levels of Fibronectin, Col1a1, and α-SMA in renal tissues. (E) Representative Western blot bands and quantitative analysis of relative protein expression levels of Fibronectin, Col1a1, and α-SMA in renal tissues. All experiments were independently repeated at least 3 times. Data are presented as mean ± SD N = 5 per group from an initial n = 6, with one exclusion per group due to surgical complications. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. ***P < 0.001, ****P < 0.0001.

3.4. QCT Inhibits Ferroptosis in CKD Model by Reducing HMOX1 Expression

HMOX1 was identified as a key drug target through network pharmacology analysis to explore how QCT influences renal fibrosis from the perspective of ferroptosis. Initially, we measured iron content in kidney tissues, revealing that iron levels were significantly elevated in the UUO group compared to the control group. However, QCT treatment markedly reduced these elevated iron levels (Figure A). Additionally, we assessed oxidative stress by quantifying MDA and GSH levels. QCT treatment resulted in a decreased level of MDA and an increased level of GSH (Figure B,C). Furthermore, overexpression of HMOX1 effectively reversed the inhibitory effects of QCT on ferroptosis. We also thoroughly examined the transcriptional expression levels of two key proteins involved in ferroptosis inhibition, GPX4 and SLC7A11. Immunohistochemistry revealed significantly reduced expression of GPX4 and SLC7A11 in the erastin-induced ferroptosis cell model. Notably, QCT treatment significantly increased GPX4 and SLC7A11 expression, restoring it to normal levels; however, HMOX1 overexpression reversed this effect (Figure D). Similarly, RT-PCR and Western blot analyses demonstrated that the expression trends of HMOX1 at both the transcriptional and translational levels were consistent with these results (Figure E,F). To directly visualize lipid peroxidation, the hallmark event of ferroptosis, we performed C11-BODIPY lipid ROS fluorescence staining. UUO operation induced robust accumulation of lipid peroxides in renal tissues (Figure G). QCT intervention remarkably attenuated lipid ROS generation, whereas oeHMOX1 abolished the lipid peroxidation-suppressing capacity of QCT, with markedly elevated C11-BODIPY fluorescence intensity in the UUO + QCT + oeHMOX1 group. To further validate the causal link between ferroptosis and renal fibrotic phenotype in vivo, we treated UUO mice with the ferroptosis inducer Erastin to activate ferroptosis, while simultaneously administering the specific ferroptosis inhibitor Ferrostatin-1 (Fer-1) for intervention. Erastin treatment significantly exacerbated lipid peroxidation and upregulated the expression of the fibrosis marker α-SMA in kidney tissues, whereas cotreatment with Fer-1 effectively reversed these effects (Figure H), confirming that ferroptosis activation drives renal fibrotic transformation in vivo. Collectively, these findings suggest that QCT effectively inhibits ferroptosis in renal tissues of UUO-induced CKD model mice via downregulating HMOX1 expression, thereby mitigating lipid peroxidation and subsequent renal fibrosis.

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QCT inhibited ferroptosis in CKD models via HMOX1. (A) Iron content in renal tissues from mice in each group. (B) Malondialdehyde (MDA) content in renal tissues. (C) Glutathione (GSH) content in renal tissues. (D) Immunohistochemical staining of HMOX1, GPX4, and SLC7A11 in renal tissues (scale bar = 50 μm) and quantitative analysis of their positive area percentages. (E) Relative mRNA expression levels of HMOX1, GPX4, and SLC7A11 in renal tissues. (F) Representative Western blot bands and quantitative analysis of relative protein expression levels of HMOX1, GPX4, and SLC7A11 in renal tissues. (G) C11-BODIPY 581/591 fluorescence staining was used to detect lipid peroxidation in mouse kidney tissues (green fluorescence indicates lipid ROS levels). Scale bar = 100 μm. (H) To validate the causal relationship between ferroptosis and renal fibrosis in vivo, UUO mice were treated with the ferroptosis inducer Erastin and/or the specific inhibitor Ferrostatin-1 (Fer-1). Scale bar = 100 μm. All experiments were independently repeated at least 3 times. Data are presented as mean ± SD N = 5 per group from an initial n = 6, with one exclusion per group due to surgical complications. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.5. Knockdown of HMOX1 Improves Renal Fibrosis in CKD Mice and Partially Reverses Epithelial–Mesenchymal Transition

To further explore the role of HMOX1 in renal fibrosis within CKD mouse model, we employed RNA interference technology to examine the effects of HMOX1 knockdown on fibrosis. Masson’s trichrome staining revealed extensive fibrotic protein deposition in the renal tubular interstitium of UUO-induced mice, which was significantly ameliorated following HMOX1 knockdown (Figure A). Correspondingly, the levels of fibrotic proteins, including fibronectin, Col1a1, and α-SMA, were markedly reduced in the kidneys of UUO mice after HMOX1 knockdown (Figure B). Additionally, in the ferroptosis cell model, we observed significant changes in the expression of proteins associated with epithelial and mesenchymal cells, including decreased E-cadherin and KLF15 expression, and increased Vimentin and α-SMA expression. Notably, these alterations were reversed upon HMOX1 knockdown (Figure C). Furthermore, we assessed the transcription levels of Snail-1, Twist-1, and ZEB-1, key transcription factors in the epithelial–mesenchymal transition (EMT) process, and found that they were highly expressed in the ferroptosis cell model; however, their expression levels decreased following HMOX1 knockdown (Figure D). Finally, we measured the levels of iron, MDA, and GSH, which are indicators of ferroptosis, and the results were consistent with our hypothesis. HMOX1 knockdown reduced intracellular iron and MDA levels while increasing GSH levels (Figure E–G). In conclusion, HMOX1 may mitigate renal fibrosis by inhibiting the EMT process and ferroptosis.

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Knockdown of HMOX1 improves renal fibrosis and partially reverses epithelial-mesenchymal transition in CKD mice. (A) Representative images of Masson’s trichrome staining of renal tissues (scale bar = 50 μm) and quantitative analysis of fibrotic area. (B) Representative Western blot bands and quantitative analysis of relative protein expression levels of Fibronectin, Col1a1, and α-SMA in renal tissues. (C) Representative Western blot bands and quantitative analysis of relative protein expression levels of KLF15, E-cadherin, Vimentin, and α-SMA in vitro. (D) Relative mRNA expression levels of Snail-1, Twist-1, and ZEB-1 in vitro. (E) Iron content in renal tissues in vitro. (F) MDA content in renal tissues in vitro. (G) GSH content in renal tissues in vitro. All experiments were independently repeated at least 3 times. Data are presented as mean ± SD N = 5 per group from an initial n = 6, with one exclusion per group due to surgical complications. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.6. HMOX1 Induces Ferroptosis through PTGS2, Leading to Renal Fibrosis in CKD Mice

The network pharmacology analysis suggests that HMOX1 may interact with PTGS2 or participate in its signaling pathways (Figure C). Initially, an upregulation of PTGS2 expression was observed in the renal tissues of UUO model mice (Figure A). To validate the interaction between HMOX1 and PTGS2, coimmunoprecipitation (Co-IP) was performed, confirming that HMOX1 indeed interacts with PTGS2 in 293FT cell and HK-2 cell (Figure B). Consistent with previous findings, in HK-2 cells overexpression of HMOX1 in the ferroptosis cell model led to increased levels of iron and MDA, accompanied by a decrease in GSH levels. However, following PTGS2 knockdown, these changes were partially reversed, though not completely restored to control levels, indicating that HMOX1 may collaborate with PTGS2 in mediating ferroptosis (Figure C). Similarly, while HMOX1 overexpression elevated the expression of fibrosis-related proteins such as fibronectin, Col1a1, and α-SMA, PTGS2 knockdown significantly reduced these levels (Figure D,E). These findings imply that HMOX1 and PTGS2 may work together to induce ferroptosis, thereby contributing to renal fibrosis in CKD mice.

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HMOX1 and PTGS2 synergistically induce ferroptosis and cause renal fibrosis in CKD mice. (A) Immunofluorescence staining of PTGS2 in renal tissues (scale bar = 50 μm) and quantitative analysis of its positive area percentage. (B) Co-IP assay showing the interaction between HMOX1 and PTGS2. (C) Iron, MDA, and GSH contents in renal tissues. (D) Representative Western blot bands and quantitative analysis of relative protein expression levels of Fibronectin, Col1a1, and α-SMA in vitro. (E) Relative mRNA expression levels of Fibronectin, Col1a1, and α-SMA in vitro. All experiments were independently repeated at least 3 times. Data are presented as mean ± SD N = 5 per group from an initial n = 6, with one exclusion per group due to surgical complications. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

3.7. Inhibition of the H3K4 Histone Methyltransferase PRDM9 Alleviates Renal Fibrosis

Based on our findings, we further explored the biological role of histone modifications in renal fibrosis. Initially, we utilized the JASPAR database to analyze the regulatory effect of the methyltransferase PRDM9, acting as a transcription factor, on the expression of HMOX1. Predictive analysis revealed C- and G-rich sequences, suggesting multiple potential PRDM9 binding sites in the promoter region of HMOX1 (Figure A). Overexpression of PRDM9 significantly upregulated HMOX1 expression, while knockdown of PRDM9 exerted the opposite effect, indicating that PRDM9 positively regulates HMOX1 (Figure B). Immunofluorescence analysis demonstrated that PRDM9 is localized in the nucleus and is abnormally overexpressed in the renal tissues of UUO mice (Figure C). To ascertain whether PRDM9 regulates HMOX1 expression as a transcription factor, we cloned the HMOX1 promoter region into a pGL3 vector. Our results revealed that PRDM9 significantly activated the luciferase activity of the pGL3 vector carrying the wild-type HMOX1 promoter region, whereas this activation was markedly reduced following mutation of the binding sites (Figure D). To investigate whether HMOX1 is influenced by PRDM9-mediated H3K4me3 histone modification, we treated PRDM9-overexpressing HK-2 cells with the H3K4me3 inhibitor MRK-740. Western blot analysis showed that H3K4me3 expression was increased by PRDM9 overexpression and decreased by MRK-740 treatment (Figure E). Correspondingly, the upregulation of HMOX1 mRNA induced by PRDM9 overexpression was significantly reversed by MRK-740 (Figure F). Further immunohistochemical analysis revealed that H3K4me3 is highly expressed in the renal tissues of UUO mice, and its levels decreased after quercetin treatment, but were restored by overexpression of PRDM9 (Figure G). Immunofluorescence staining of RhoNox-1, a marker of lipid peroxidation, showed that UUO-induced ferroptosis was alleviated by quercetin, and this effect was partially reversed by PRDM9 overexpression (Figure H). Western blot analysis confirmed that quercetin treatment downregulated the expression of PRDM9, HMOX1, PTGS2, and fibrosis-related proteins (Fibronectin, Col1a1, α-SMA), while upregulating the ferroptosis marker GPX4 in UUO mice. Overexpression of PRDM9 reversed these changes induced by quercetin (Figure I). These findings suggest that HMOX1 is dually regulated by PRDM9, which has both transcription factor activity and histone methyltransferase function, in the promotion of renal fibrosis.

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Inhibition of the H3K4 histone methyltransferase PRDM9 alleviates renal fibrosis. (A) JASPAR analysis of the HMOX1 promoter region and the relative scores of top 6 transcription factor binding sites. (B) Relative mRNA expression of HMOX1 in renal cells after overexpression or knockdown of PRDM9. (C) IHC staining of HMOX1 in renal tissues from Sham and UUO mice (scale bar = 50 μm). (D) Relative luciferase activity of HMOX1-WT and HMOX1-Mut reporter constructs in vitro. (E) Western blot analysis of H3K4me3 expression in renal cells after PRDM9 overexpression and treatment with the H3K4me3 inhibitor MRK-740. (F) Relative mRNA expression of HMOX1 in renal cells after PRDM9 overexpression and MRK-740 treatment. (G) Immunohistochemical staining of H3K4me3 in renal tissues (scale bar = 50 μm). (H) Immunofluorescence staining of RhoNox-1 in renal tissues (scale bar = 50 μm). (I) Western blot analysis of PRDM9, HMOX1, GPX4, Fibronectin, Col1a1, and α-SMA expression in renal tissues from mice in different treatment groups. (J) Schematic diagram illustrating the mechanism by which quercetin alleviates UUO-induced renal fibrosis and ferroptosis via the PRDM9/HMOX1/PTGS2 axis. All experiments were independently repeated at least 3 times. Data are presented as mean ± SD N = 5 per group from an initial n = 6, with one exclusion per group due to surgical complications. One-way ANOVA followed by Tukey’s post hoc test was used for comparisons between groups. ****P < 0.0001.

4. Discussion

In this study, we observed that quercetin, a prominent pharmacologically active component of G. biloba, effectively ameliorated renal injury in UUO-induced chronic kidney disease mouse models. This improvement was evidenced by significant reductions in Scr and BUN levels. Moreover, QCT treatment notably suppressed abnormal ferroptosis and fibrosis in CKD UUO mice, suggesting a potential link between ferroptosis and fibrosis in renal parenchymal cells, such as HK2 cells. Mechanistically, QCT exerts its effects by inhibiting the positive transcriptional and epigenetic regulation of HMOX1 by the methyltransferase PRDM9 at both histone modification and transcriptional levels. This inhibition of HMOX1 subsequently suppresses the HMOX1/PTGS2 signaling pathway, ultimately preventing ferroptosis-induced renal fibrosis (Figure J).

Ferroptosis, an iron-dependent and novel mode of programmed cell death distinct from apoptosis and autophagy, is primarily characterized by lipid peroxidation. It has been implicated in renal fibrosis, a crucial pathological change in CKD. However, the precise mechanism by which ferroptosis contributes to renal fibrosis remains inadequately understood. Our study initially identified QCT’s therapeutic potential for renal fibrosis and highlighted key targets, including HMOX1, through network pharmacology screening. This approach enabled us to explore the therapeutic effects and mechanisms of QCT in renal fibrosis. Given that CKD pathology involves glomerulosclerosis, tubular atrophy, and interstitial fibrosis resulting from renal injury and abnormal tissue repair, , we utilized the UUO model to simulate renal fibrosis in mice. Kidney injury molecule-1 (KIM-1), a type I transmembrane glycoprotein, is a recognized biomarker for tubular injury in various renal diseases. C Consistent with our expectations, UUO-induced CKD mice displayed significant renal injury, as indicated by elevated serum creatinine levels. Histological examination also revealed notable changes such as tubular dilation, epithelial damage, and immune cell infiltration. Although previous studies have documented QCT’s efficacy in treating diabetic nephropathy and its potential in ameliorating renal fibrosis through plant extracts, the therapeutic efficacy and mechanisms of quercetin as a single agent in renal fibrosis treatment remain underexplored. Remarkably, our study demonstrated that QCT treatment significantly mitigated these pathological changes in renal tissue, indicating its potential as a therapeutic agent for renal fibrosis.

Renal tubulointerstitial fibrosis, a hallmark of chronic kidney disease, can be ameliorated by QCT. Additionally, QCT has been reported to alleviate liver fibrosis and pulmonary fibrosis. − Studies have highlighted the involvement of HMOX1 in ferroptosis, though its exact mechanism remains unclear. In our study, we explored the mechanism of QCT in renal fibrosis from the perspective of ferroptosis. Ferroptosis is characterized by specific biochemical changes, including increased accumulation of ferrous ions, MDA, and lipid peroxides such as 2-HNE, alongside reduced intracellular GSH levels. , In HK-2 cells treated with erastin, we observed increased intracellular Fe and MDA levels, as well as decreased GSH levels, indicative of oxidative stress and ferroptosis. Notably, QCT treatment significantly reversed these alterations, restoring cellular stability. When HMOX1 was overexpressed, intracellular Fe and MDA levels increased and GSH levels decreased, even with QCT treatment, resembling the state of cells treated with erastin. This further confirmed HMOX1’s involvement in ferroptosis. HMOX1, activated by nuclear factor erythroid 2-related factor 2 (Nrf2), has dual roles during oxidative stress: its catalytic product biliverdin provides antioxidant effects, while HMOX1 also promotes ferroptosis by generating excessive Fe2+ through the Fenton reaction. , Although some studies suggest HMOX1 mitigates ferroptosis, others indicate it accelerates it. In line with these findings, HMOX1 expression was upregulated in kidney tissues of UUO-induced CKD model mice, potentially contributing to increased intracellular Fe levels. Furthermore, the expression of SLC7A11 and GPX4, key proteins in the ferroptosis signaling pathway, was downregulated, aligning with Zhu et al.

QCT treatment in UUO mice activated System Xc-, leading to notable inhibition of ferroptosis and suppression of HMOX1-induced ferroptosis. Interestingly, despite QCT treatment, artificially elevated HMOX1 expression reduced System Xc- and antioxidant protein levels (Figure ). These observations highlight the pivotal role of HMOX1 in ferroptosis and suggest that QCT may alleviate renal fibrosis in CKD models by mitigating cellular ferroptosis through HMOX1 downregulation. Further studies validated our hypothesis, showing that HMOX1 knockdown led to significant reductions in renal fibrosis and ferroptosis markers, including Fe and MDA, while enhancing antioxidant capacity. Previous research has identified renal fibrosis as characterized by abnormal extracellular matrix protein deposition in glomeruli and interstitial regions, associated with epithelial–mesenchymal transition (EMT). − Our study assessed epithelial and mesenchymal cells and extracellular matrix biomarkers, revealing a loss of epithelial phenotype, decreased adhesion, increased mesenchymal cells, and extracellular matrix accumulation. These changes indicate a significant EMT process in both UUO mice renal tissues and QCT-induced ferroptosis cell models, closely linked to renal fibrosis progression.

Analysis of the PPI network revealed a potential interaction between HMOX1 and PTGS2. PTGS2 catalyzes the conversion of arachidonic acid to prostaglandin E2 (PGE2), a process leading to lipid peroxidation and cell death. Our study observed elevated PTGS2 expression in the UUO-induced CKD mouse model, suggesting its involvement in renal fibrosis related to ferroptosis. We confirmed the HMOX1-PTGS2 interaction and further explored their relationship by overexpressing HMOX1 and knocking down PTGS2. Our results indicated that these interventions synergistically promoted ferroptosis, akin to findings by Zhu et al. Notably, PTGS2 knockdown significantly reduced renal fibrosis, demonstrating that both HMOX1 and PTGS2 are integral to ferroptosis-induced renal fibrosis.

To elucidate HMOX1’s role in ferroptosis-induced renal fibrosis, we investigated the transcription factor PRDM9, previously predicted to be involved, and examined its correlation with HMOX1. PRDM9 functions as a transcription factor, regulating gene expression through its zinc finger domain, and exhibits histone methyltransferase activity involved in epigenetic modifications. We hypothesized that PRDM9 regulates HMOX1 through transcriptional and epigenetic mechanisms. Indeed, RT-PCR and immunofluorescence analyses revealed that PRDM9 knockdown reduced HMOX1 transcriptional levels. Additionally, PRDM9 was highly expressed in the UUO-induced CKD model. These findings suggest PRDM9’s involvement in ferroptosis-induced renal fibrosis. Luciferase reporter assays confirmed that PRDM9 binds to the HMOX1 promoter region, regulating its transcription. Our hypothesis that PRDM9 acts as a transcription factor positively regulating HMOX1, contributing to ferroptosis-induced renal fibrosis, is thus supported. PRDM9 also mediates histone H3K4me3 modifications. We observed increased H3K4me3 levels in UUO mice and ferroptosis cell models, while PRDM9 knockdown significantly reduced H3K4me3 levels. This suggests a close association between H3K4me3 levels and renal fibrosis. Co-IP experiments confirmed the PRDM9-H3K4me3 interaction. ChIP analysis further assessed HMOX1 expression in ferroptosis cell models. Additionally, PRDM9 mutation reduced fibrosis in erastin-induced ferroptosis cell models, likely due to PRDM9’s dual role as both a transcription factor and histone methyltransferase. Histone arginine methylation mediated by PRMT1 and PRMT3, , as well as histone methylation by G9a and other enzymes, also contribute to renal fibrosis, indicating a close association with various epigenetic modifications. A comprehensive understanding of these mechanisms will help elucidate the pathogenesis of renal fibrosis and identify potential drug targets.

In conclusion, our study provides novel insights into the therapeutic potential of QCT for chronic kidney disease (CKD). QCT modulates critical proteins involved in ferroptosis, specifically HMOX1 and PTGS2, while PRDM9 functions both as a transcription factor and as a mediator of histone methylation that regulates HMOX1-induced ferroptosis. These findings enhance our understanding of QCT’s mechanism of action and pave the way for developing innovative and effective treatments for CKD.

Supplementary Material

ao6c05732_si_001.pdf (458.9KB, pdf)

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05732.

  • Supplementary Figure 1: GO enrichment analysis of ginkgo therapeutic targets based on the TCMIP database: (A) Biological processes (Top 20). (B) Molecular functions (Top 20). (C) Cellular components (Top 20). Supplementary Table 1: Specific primer sequences for real-time PCR Genes: KIM-1, α-SMA, Col1a1, Fibronectin, HMOX1, GPX4, SLC7A11, Snail-1, Twist-1, ZEB-1 Forward and reverse primer sequences provided for each gene (PDF)

Qiongqiong Zhu: conception, investigation methodology, supervision, resources, project administration, writingoriginal draft. Zhibin Chen, Xianxian Yu and Qiu Ye: data curation, formal analysis, software, validation, visualization. Xu Chen: writingreview and editing, funding acquisition.

No.

Ethical and Legal Declarations: All experimental protocols were approved by the Institutional Animal Care and Use Committee of Yueqing Hospital of Wenzhou Medical University.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao6c05732_si_001.pdf (458.9KB, pdf)

Data Availability Statement

The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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