The Berberine Derivative BBR684 Inhibits VDAC Oligomerization to Suppress Ferroptosis in Acute Kidney Injury
Zihao Jiang, Wenhao Zhang, Jiawei Zhu, Cong Wang, Guo Chen, Yongjian Guo
TL;DR
BBR684, a berberine derivative, prevents cell death in kidney injury by blocking a key protein involved in ferroptosis.
Contribution
BBR684 is shown to inhibit VDAC oligomerization, offering a new mechanism for suppressing ferroptosis in acute kidney injury.
Findings
BBR684 is more effective than berberine in reversing ferroptosis in HK-2 cells.
BBR684 reduces oxidative stress by restoring GSH and GPX4 levels and inhibiting VDAC activity.
In mice, BBR684 alleviates folic acid-induced AKI and reduces inflammation and lipid peroxidation.
Abstract
Ferroptosis is an iron-dependent form of programmed cell death driven by lipid peroxidation and is implicated in acute kidney injury (AKI). Here, we investigated the therapeutic potential of BBR684, a derivative of berberine, in suppressing ferroptosis and alleviating AKI. The anti-ferroptotic activity in HK-2 cells was assessed by Western blot, flow cytometry, and immunofluorescence. Renal fibrosis and the expression of related proteins were evaluated using Masson staining, PI staining, and immunohistochemistry. Our findings suggest that BBR684 exhibits superior efficacy compared to berberine in reversing erastin-induced ferroptosis in HK-2 cells. Mechanistically, BBR684 confers cytoprotection by restoring glutathione (GSH) levels and glutathione peroxidase 4 (GPX4) expression, thereby reducing oxidative stress. Moreover, BBR684 directly binds to the voltage-dependent anion channel…
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Taxonomy
TopicsFerroptosis and cancer prognosis · Autophagy in Disease and Therapy · Inflammasome and immune disorders
Introduction
Acute kidney injury (AKI) is a prevalent clinical syndrome characterized by tubular epithelial cell death and inflammation.1^,^2 With population aging and the rising prevalence of cardiovascular diseases, diabetes, and chronic kidney disease, along with elevated exposure to risk factors such as sepsis, contrast agents, and other nephrotoxins, the incidence of AKI continues to increase.3 Globally, AKI-related mortality now exceeds that of cancer, heart failure, and diabetes, with reported morbidity and mortality rates approaching 21%.4 AKI frequently arises as a complication of primary diseases and may progress to chronic kidney disease (CKD) or end-stage renal disease (ESRD).5^,^6 Currently, no clinically approved pharmacological therapies are available for the prevention or treatment of AKI, aside from supportive dialysis or kidney transplantation.7
Previous studies have identified that lipid peroxidation and dysregulation of glutathione metabolism–related proteins are closely associated with folic acid (FA)-induced AKI in mice, suggesting ferroptosis in AKI pathogenesis.8^,^9 Ferroptosis is a distinct form of programmed cell death driven by iron-dependent lipid peroxidation and reactive oxygen species (ROS), first described by Brent R. Stockwell in 2012.10 Although lipid peroxidation is a common feature of multiple cell death modalities, ferroptosis is specifically distinguished by its strict dependence on iron and the functional inactivation of glutathione peroxidase 4 (GPX4), an essential enzyme that protects cellular membranes from iron-driven peroxidative damage.11 Consequently, ferroptosis represents the convergence of iron, thiol, and lipid metabolism pathways.12^,^13 The core regulatory machinery governing ferroptosis is the System Xc–/GSH/GPX4 axis. The System Xc– antiporter, composed of the SLC7A11 and SLC3A2 subunits, mediates cystine uptake required for glutathione (GSH) synthesis. GSH serves as an essential cofactor for GPX4, which detoxifies phospholipid hydroperoxides and prevents lethal lipid peroxidation. Disruption of this axis—through inhibition of System Xc–, GSH depletion, or GPX4 inactivation—leads to uncontrolled peroxidation of membrane polyunsaturated fatty acids and ferroptotic execution. Consequently, SLC7A11, GSH, and GPX4 are recognized as hallmark molecular markers and therapeutic targets for ferroptosis modulation.14 The voltage-dependent anion channel (VDAC) is a pivotal regulator of mitochondrial Ca²⁺ homeostasis and metabolic cross-talk. Emerging studies underscore its critical involvement in ferroptosis. Activation of VDAC by ferroptosis inducers such as erastin potentiates Ca²⁺ influx, perturbs mitochondrial metabolism and amplifies ROS via the TCA cycle and electron transport chain. This cascade promotes lipid peroxidation and iron accumulation, which are defining hallmarks of ferroptotic cell death.15 The kidney is particularly vulnerable to redox imbalances, and ferroptotic signals can rapidly propagate between neighboring cells.16, 17, 18 In preclinical models of AKI induced by ischemia-reperfusion (IR) and folate (FA), tubular cell death occurs in a highly synchronous manner.19^,^20 Therefore, targeting ferroptosis pathway to modulate cell fate represents a promising therapeutic strategy to prevent AKI progression, attenuate renal inflammation, and accelerate the recovery of kidney disease by limiting parenchymal cell loss.21
Berberine (BBR) is an isoquinoline alkaloid isolated from Coptis chinensis and other Berberis species and is well recognized for its diverse pharmacological properties, including antioxidant and anti-inflammatory effects.22^,^23 Emerging evidence indicates that BBR exerts anti-ferroptotic activity and improves cognitive function in Alzheimer’s disease (AD) mouse models24, demonstrating its potential as a promising lead compound for the development of ferroptosis-targeted therapeutics. Moreover, the therapeutic efficacy of BBR in AKI remains insufficiently characterized. To develop a novel compound with independent intellectual property rights and enhanced cost-effectiveness, we modified the chemical structure of BBR. Screening of BBR-derived analogs led to the identification of BBR684, which exhibited markedly greater anti-ferroptosis activity. In this study, we systematically explored the anti-ferroptotic effects and underlying mechanisms of BBR684, highlighting its potential as a promising therapeutic candidate for AKI.
Materials and Methods
Cell Culture and Treatment
Human embryonic kidney HEK293T cells were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's Modified Eagle Medium (DMEM; Catalog #KGL1002-500, KeyGen Biotech, Nanjing, China) supplemented with 10% fetal bovine serum (FBS; Catalog #FCS500, Excell Bio, Shanghai, China). Human renal proximal tubular HK-2 cells were obtained from the China Cell Bank and maintained in DMEM/F-12 medium (Catalog #KGL1042-500, KeyGen Biotech) containing 10% FBS. All cells were incubated at 37°C in a humidified atmosphere with 5% CO₂.
Cell Viability Assay
HK-2 cells were seeded into 96-well plates and treated with indicated compounds for 24 hours. Subsequently, the medium was replaced with fresh medium, and 10 µl of CCK-8 solution (Catalog #KGA1606-1000, KeyGen Biotech) was added to each well. After incubation at 37°C for 1 hour, the absorbance at 450 nm was measured using a microplate reader. Cell viability was directly proportional to the absorbance value.
Cell Death Analysis by Propidium Iodide (PI) Staining
Following a 12-hour treatment, HK-2 cells were trypsinized, washed with phosphate-buffered saline (PBS), and transferred to flow cytometry tubes. Cells were then co-incubated with PI (50 µg/mL; Catalog #KGA1101-100, KeyGen Biotech) in PBS containing 2% FBS for 5 minutes in the dark. Cell death was quantified by flow cytometry using an Accuri C6 cytometer (BD Biosciences).
Lipid Peroxidation Assay
Lipid peroxidation was assessed using the fluorescent probe BODIPY 581/591 C11 (Catalog #D3861, Thermo Fisher Scientific, Waltham, MA, USA). Briefly, cells were seeded in 6-well plates at a density of 1 × 10^6^ cells per well and were treated with Erastin, with or without BBR684 for 12h. Cells were then incubated with 10 µM BODIPY 581/591 C11 in complete medium at 37°C for 20 minutes. After washing with PBS, cells were analyzed by flow cytometry (Accuri C6, BD Biosciences).
Malondialdehyde (MDA) Assay
The MDA level was determined using a commercial assay kit (Beyotime Biotechnology, Shanghai, China), according to the manufacturer's instructions. Cell lysate or mouse serum samples were reacted with thiobarbituric acid (TBA) to form the MDA-TBA adduct. The absorbance of the resulting pink-colored product was measured at 535 nm, which is proportional to the MDA concentration.
Reduced Glutathione (GSH) and Oxidized Glutathione Disulfide (GSSG) Assay
Total GSH and GSSG levels were measured using a GSH and GSSG Assay Kit (Catalog #S0053, Beyotime Biotechnology). In brief, cell lysates or serum were deproteinized. For total glutathione (GSH + GSSG) measurement, the sample was incubated with a working solution containing glutathione reductase and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB). The reduction of GSSG to GSH and the subsequent reaction of GSH with DTNB generates a yellow-colored 5-thio-2-nitrobenzoic acid (TNB), the absorbance of which was read at 412 nm. For GSSG measurement, GSH was first scavenged by a specific masking reagent prior to the same reaction. GSH concentration was calculated by subtracting GSSG from total glutathione.
Cellular Ferrous Iron Colorimetric Assay
Intracellular Fe²⁺ levels were quantified using a Ferrous Iron Colorimetric Assay Kit (Catalog #E-BC-K773-M, Elabscience, Wuhan, China) per the manufacturer's protocol. Cell lysates were mixed with 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ) to form a blue-colored Fe²⁺-TPTZ complex. Absorbance was measured at 593 nm and was proportional to the Fe²⁺ concentration.
Immunofluorescence
Cells (2 × 10⁵) seeded on coverslips were treated as indicated agents for 8 hours. After washing with PBS, cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature (RT), permeabilized with 0.5% Triton X-100 for 15 minutes, and blocked with 10% goat serum for 1 hour at 37°C. Cells were then incubated with primary antibodies overnight at 4°C, followed by incubation with appropriate fluorescent secondary antibodies for 1 hour at RT in the dark. Nuclei were counterstained with DAPI, and images were captured using a fluorescence microscope.
Western Blot Analysis
Treated cells were harvested, washed with PBS, and lysed in EBC lysis buffer (50 mM Tris-HCl, pH 7.6–8.0, 150 mM NaCl, 0.5% NP-40, 1 mM EDTA) supplemented with protease inhibitor cocktail (Topscience, Shanghai, China) by sonication. After centrifugation at 13,200 × g for 15 minutes at 4°C, protein concentrations in the supernatants were determined. Equal amounts of protein were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% non-fat milk and probed overnight at 4°C with the following primary antibodies: anti-VDAC (1:1000; 10866-1-AP, Proteintech), anti-GPX4 (1:1000; 67763-1-Ig, Proteintech), anti-NRF2 (1:2000; 16396-1-AP, Proteintech), and anti-HO-1 (1:1000; 10701-1-AP, Proteintech). After incubation with appropriate HRP-conjugated secondary antibodies, protein bands were visualized using an ECL chemiluminescence substrate (Catalog #P0018M, Beyotime Biotechnology).
Microscale Thermophoresis (MST) Binding Assay
HEK293T cells were transfected with either GFP-VDAC1 or an empty GFP vector plasmid. Cell lysates were collected 48 hours post-transfection. Lysates containing GFP-VDAC1 or GFP alone were mixed with a serial dilution of BBR684, loaded into the glass capillaries (MO-K022, NanoTemper Technologies, München, Germany), and subjected to MST analysis at 22.8 °C using medium MST power and 20% LED power.25 Dissociation constant (Kd) values were calculated using the mass action equation in NanoTemper Analysis software.
The Cellular Thermal Shift Assay (CETSA)
HEK293T cells were transfected with GFP-VDAC1 were lysed 48 hours post-transfection as described in Section 2.9. The cleared lysate was divided into 2 aliquots and incubated with either BBR684 or DMSO (vehicle control) for 2 hours at 4°C. Each aliquot was then subdivided, heated at different temperatures (49.5, 51.5, 52.5, 54.5, 55.5°C) for 10 minutes using a thermal cycler, and centrifuged at 13,200 × g for 5 minutes at 4°C. The soluble fractions were analyzed by Western blotting to detect remaining VDAC1/2 protein levels.
Reactive Oxygen Species (ROS) Detection
IntracellularROS levels were measured using a ROS Assay Kit (Catalog #S0033M, Beyotime Biotechnology) following the manufacturer's instructions. Briefly, after treatment, cells were collected, washed with PBS and incubated with 20 µM 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) at 37°C for 30 minutes in the dark. Cells were washed, resuspended in PBS with 2% FBS, and analyzed immediately by flow cytometry (Accuri C6, BD Biosciences).
Intracellular Calcium (Ca2+) Detection
Cytosolic Ca²⁺ levels were measured using a Fluo-4 AM Calcium Assay Kit (Catalog #S1061S, Beyotime Biotechnology). Post-treatment, cells were harvested, washed, and loaded with 2 µM Fluo-4 AM at 37°C for 30 minutes. After washing, cells were resuspended in PBS with 2% FBS, and fluorescence intensity was measured by flow cytometry (FITC channel, Accuri C6, BD Biosciences), which correlates with intracellular Ca²⁺ concentration.
Protein Crosslinking Assay
Cells were treated with hydrogen peroxide for 6 hours, harvested in PBS, and incubated with 250 µM ethylene glycol bis(succinimidyl succinate) (EGS; MCE, Monmouth Junction, NJ, USA) crosslinker for 15 minutes at RT. The reaction was quenched by adding Tris-HCl (pH 8.0) to a final concentration of 20 mM. Cells were pelleted, lysed, and subjected to Western blot analysis.
Cell Transfection
When cells reached 70-80% confluence, the medium was replaced with serum- and antibiotic-free DMEM. For transfection, 10 µg of GFP-VDAC1 plasmid and 20 µL of Exfect Transfection Reagent (Catalog #T101-01, Vazyme, Nanjing, China) were separately diluted in 500 µL of serum-free DMEM, incubated for 5 minutes, mixed, and incubated for an additional 15 minutes at RT. The transfection complex was added dropwise to the cell culture. The medium was replaced with complete growth medium 8 hours post-transfection.
Animal Model
All animal experiments were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (Approval No. 2024-07-020) and conducted in accordance with institutional guidelines for animal welfare. Six-week-old female C57BL/6 mice were purchased from GemPharmatech Co.,Ltd. (Nanjing, China) and housed under specific pathogen-free conditions with a 12-hour light/dark cycle. Mice were randomly divided into 3 groups (n = X per group): (1) Control group: received an intraperitoneal (i.p.) injection of PBS; (2) FA-AKI group: received a single i.p. injection of folic acid (FA, 200 mg/kg in PBS) to induce AKI; (3) FA-AKI + BBR684 group: FA injection followed by oral gavage of BBR684 (20 mg/kg) 6 hours later. Mice in the control and FA-AKI groups received vehicle (PBS) orally. All mice were euthanized 72 hours after FA injection for serum and kidney tissue collection.26
Blood Urea Nitrogen (BUN) Measurement
Serum BUN levels were measured using a commercial assay kit (Catalog #ml076479, mlbio, Shanghai, China). Serum samples were mixed with the provided reagents and heated in a boiling water bath for 10 minutes. The absorbance of the resulting colored product was measured at 540 nm, which is proportional to the BUN concentration.
Creatinine (CRE) Measurement
Serum creatinine was measured using a Creatinine Assay Kit (Catalog #ml059158, mlbio) according to the manufacturer's instructions. Serum was incubated with the working reagents at 37°C for 5 minutes. The absorbance of the reaction product was read at 546 nm and was proportional to the creatinine concentration.
Immunohistochemistry (IHC)
Kidney tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 µm thickness. After deparaffinization and rehydration, endogenous peroxidase activity was quenched with 3% H₂O₂. Antigen retrieval was performed using sodium citrate buffer (pH 6.0). Sections were blocked with 2.5% horse serum and incubated overnight at 4°C with primary antibodies. After washing, sections were incubated with appropriate secondary and tertiary antibodies. Color development was performed using a 3,3′-diaminobenzidine (DAB) substrate kit, followed by counterstaining with hematoxylin. Images were acquired using a light microscope.
Histopathological Evaluation of Renal Injury
Periodic acid–Schiff (PAS)-stained kidney specimens were used for histopathological assessment. Tubular injury, including tubular dilation, cast formation, brush border loss, and tubular necrosis, was evaluated in 5 randomly selected cortical fields (× 200 magnification) per animal. Each parameter was scored from 0 to 10 based on the percentage of affected tubules (0–1: none; 1–2: <11%; 2–4: 11–25%; 4–6: 26–45%; 6–8: 46–75%; 8–10: >75%). The scores for all parameters were summed to obtain the total acute tubular necrosis (ATN) score for each sample.
Reagents and Antibodies
Antibodies against β-Actin (Catalog #GB15003-100) were from Servicebio (Wuhan, China). Antibodies against GPX4 (Catalog #3F5G5), HO-1 (Catalog #10701-1-AP), NRF2 (Catalog #16396-1-AP), VDAC (Catalog #10866-1-AP), Cleaved Caspase-3 (Catalog #68773-1-Ig), and CD45 (Catalog #98035-1-RR) were purchased from Proteintech. The 4-Hydroxynonenal (4-HNE) antibody (Catalog #AB5605) was from Merck Millipore (Darmstadt, Germany). The PAS Staining Kit (Catalog #C0142S), Masson's Trichrome Staining Kit (Catalog #C0142S), and Mouse NGAL/Lipocalin-2 ELISA Kit (Catalog #PN757) were obtained from Beyotime Biotechnology.
Results
BBR684, A Novel Berberine Derivative, Exhibits Superior Anti-Ferroptotic Activity in HK-2 Cells
Berberine has been shown to exert anti-ferroptosis activity in multiple cell types. To develop more potent ferroptosis inhibitors based on berberine, we synthesized a series of berberine analogues, including BBR684. The introduction of a chlorine substituent increases lipophilicity and membrane permeability, thereby facilitating cell uptake and target engagement. In addition, the electron-withdrawing effect of chlorine alters molecular charge distribution and polarity (Figure 1A and B). We then compared the anti-ferroptotic effects of BBR684 and berberine at an identical concentration (2.5 µM) in erastin-induced ferroptosis of HK-2 cells. PI staining revealed that BBR684 almost completely inhibited erastin-induced cell death, whereas berberine conferred only partial protection (Figure 1C and D). Remarkably, in HK-2 cells undergoing Erastin-induced ferroptosis, treatment with increasing concentrations of BBR684 or BBR for 24 hours revealed that BBR684 was more potent in promoting cell survival. (Figure 1E). Furthermore, BBR684 at 0.5µM reversed more than 80% of cell death, while 2.5 µM nearly completely prevented erastin-induced cell death (Figure 1F). Taken together, BBR684 demonstrated superior efficacy over berberine in protecting HK-2 cells from Erastin-induced ferroptosis.Figure 1. The berberine derivative BBR684 is superior to berberine in (BBR) in protecting against Erastin-induced ferroptosis in HK-2 cells. (A-C)Representative phase-contrast microscopy images showing the morphological changes of HK-2 cells treated with the indicated compounds for 24 hours. (D) Quantification of cell death by flow cytometry using propidium iodide (PI) staining. HK-2 cells were pre-treated with BBR684 or BBR for 1 hour, followed by co-incubation with Erastin for 12 hours. (E) Cells viability assessed by the CCK-8 assay after treating HK-2 cells with increasing concentrations of BBR684 for 24 hours. (F) Flow cytometry analysis of cell death (PI-positive cells) in HK-2 cells treated with Erastin in the presence or absence of BBR684.Figure 1 dummy alt text
BBR684 Suppresses Erastin-Induced Lipid Peroxidation and Restores the Antioxidant System
Iron-dependent phospholipid peroxidation is a key driver of ferroptosis, making lipid metabolism a crucial regulatory determinant of this process.28 Ferroptosis was induced in HK-2 cells with erastin, followed by treatment with increasing concentrations of BBR684. Lipid peroxidation was assessed using BODIPY™ 581/591 C11 staining. Erastin significantly increased lipid peroxidation in HK-2 cells, whereas BBR684 suppressed this effect in a dose-dependent manner (Figure 2A). Additionally, intracellular ferrous ion (Fe^2+^) levels were elevated by erastin but progressively reduced by BBR684 (Figure 2B). Glutathione (GSH) is a critical intracellular antioxidant that scavenges ROS under GPX4 catalysis.29, 30, 31 Accumulation of glutathione disulfide (GSSG) is positively associated with ferroptosis. GSH and GSSG levels were measured following treatment with increasing concentrations of BBR684. In erastin-treated cells, both GSH and GSSG were almost depleted; however, BBR684 dose-dependently restored their levels, indicating effective inhibition of ferroptosis (Figure 2C). We further analyzed ferroptosis-related lipid peroxidation biomarkers, including malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).32 MDA levels peaked following erastin treatment and decreased progressively with increasing concentrations of BBR684. Similarly, immunofluorescence analysis revealed a marked reduction in intracellular 4-HNE levels upon BBR684 treatment (Figure 2D-F). Finally, we assessed the expression of key ferroptosis-related proteins, including GPX4, HO-1, and NRF2. Erastin significantly reduced GPX4 expression while upregulating HO-1 and NRF2.33^,^34 These alterations were reversed by BBR684, which restored GPX4 expression and suppressed HO-1 and NRF2 levels (Figure 2G). Collectively, these findings suggest that BBR684 effectively counteracts erastin-induced ferroptosis in HK-2 cells by inhibiting lipid peroxidation.Figure 2BBR684 attenuates Erastin-induced ferroptosis in HK-2 cells by suppressing lipid peroxidation and activating the antioxidant response. (A) Flow cytometric analysis of lipid peroxidation using the C11-BODIPY probe in HK-2 cells treated as indicated for 12 hours. Data are presented as mean fluorescence intensity (MFI). (B) Intracellular Fe²⁺ levels measured by a ferrous iron colorimetric assay. (C, D) Levels of malondialdehyde (MDA, C) and reduced glutathione (GSH, D) in cell lysates. (E, F) Representative immunofluorescence images (E) and quantification (F) of 4-hydroxynonenal (4-HNE) adducts (red) in HK-2 cells. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. (G) Western blot analysis of key ferroptosis-related proteins: glutathione peroxidase 4 (GPX4), heme oxygenase-1 (HO-1), and nuclear factor erythroid 2–related factor 2 (NRF2). β-actin served as the loading control. Data are from 3 independent biological replicates (n = 3) and presented as mean ± SEM. Significance was determined by one-way ANOVA followed by Tukey's HSD post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant.Figure 2 dummy alt text
BBR684 Directly Binds to VDAC1/2 and Inhibits Its Oligomerization
The voltage-dependent anion channel (VDAC) is a crucial mediator linking mitophagy and ferroptosis.15 Erastin binds to VDAC, promotes channel opening, induces excessive ROS production, and induces oxidative stress, thereby initiating ferroptosis.35^,^36 VDAC comprises 3 isoforms, including VDAC1, VDAC2 and VDAC3, which share high sequence and structural similarity. Using microcalorimetry (MST), we discovered that BBR684 directly binds to VDAC1/2 (Figure 3A). This interaction was further validated using a Cellular Thermal Shift Assay (CETSA). Across a temperature range of 49.5°C to 55.5°C, BBR684 significantly improved VDAC thermal stability compared with the DMSO control, with the strongest effect observed at 52.5°C. Consequently, 52.5°C was selected for subsequent concentration-gradient analyses, which revealed a concentration-dependent stabilizing effect of BBR684 on VDAC. These data indicate strong target engagement between BBR684 and VDAC (Figure 3B). Molecular docking analysis revealed that BBR684 interacts with the N-terminal α-helix of VDAC1/2, a region critical for VDAC oligomerization37 (Figure 3C).Figure 3BBR684 directly binds to VDAC and modulates its oligomerization and function.(A)Microscale thermophoresis (MST) binding curve. Recombinant GFP-tagged VDAC1 protein was titrated withincreasing concentrations of BBR684 (B) Cellular thermal shift assay (CETSA). Lysates from HEK293T cells overexpressing VDAC1 were incubated with DMSO (vehicle) or BBR684 and heated at the indicated temperatures. (C)BBR684 with the molecule docking of VDA Computational molecular docking model predicting the binding pose of BBR684 within the putative binding pocket of VDAC1. (D) Analysis of VDAC oligomerization by chemical crosslinking. HK-2 cells treated as indicated were incubated with the membrane-permeable crosslinker EGS. Cell lysates were then subjected to Western blot under non-reducing conditions to detect VDAC monomers and higher-order oligomers. (E, F) BBR684 attenuates mitochondrial dysfunction-associated stress. Flow cytometric analysis of intracellular reactive oxygen species (ROS, E) and calcium ion (Ca²⁺, F) levels in HK-2 cells treated with Erastin in the presence or absence of BBR684. Data are presented as mean fluorescence intensity (MFI).Figure 3 dummy alt text
Oxidative stress promotes the formation of VDAC oligomers. On the outer mitochondrial membrane, VDAC can assemble into dimers, trimers, tetramers, and higher-order oligomers, forming large pores that drive excessive ROS production.38, 39, 40 Additionally, VDAC oligomerization also regulates Ca^2+^ transport and cellular energy metabolism by modulating key enzymes of the TCA cycle.15^,^41 VDAC oligomerization was induced using peroxide peroxide, followed by treatment with increasing concentrations of BBR684.42 Following EGS crosslinking, immunoblot analysis showed that BBR684 dose-dependently reduced VDAC oligomer formation (Figure 3D). After erastin-induced ferroptosis, intracellular ROS and Ca^2+^ levels were measured following treatment with varying concentrations of BBR684. BBR684 significantly reduced intracellular ROS and Ca^2+^ accumulation in a concentration-dependent manner (Figure 3E and F). These results suggest that BBR684 inhibits VDAC oligomerization, thereby reducing ROS production and Ca^2+^ influx. BBR684 treatment led to a concentration-dependent reduction in intracellular ROS and Ca²⁺ levels.
VDAC Overexpression Abolishes the Anti-Ferroptotic Effect of BBR684
To further determine whether VDAC binding underlies the anti-ferroptotic effect of BBR684, VDAC was overexpressed in 293T cells, and its expression was confirmed by immunoblotting (Figure 4A). Subsequently, ferroptosis was then induced in both control and VDAC-overexpressing 293T cells using erastin, followed by treatment with 10 µM BBR684. After 12 hours of treatment, morphological examination revealed significant cell death in VDAC-overexpressing 293T cells treated with BBR684 (Figure 4B). Cells were subsequently collected for PI staining, and cell mortality was quantified using flow cytometry. The PI-positive population was significantly increased in VDAC-overexpressing 293T cells treated with BBR684 compared to normal 293T cells (Figure 4C).Figure 4. Overexpression of VDAC abolishes the protective effects of BBR684 against ferroptosis.(A) Western blot analysis validating the overexpression of VDAC1 in HEK293T cells. (B) Representative phase-contrast microscopy images showing the morphology of HEK293T cells with or without VDAC1 overexpression after the indicated treatments for 24 hours. (C) Quantification of cell death by flow cytometry (propidium iodide staining) in HEK293T cells treated with Erastin and BBR684. (D) Cells viability assessed by the CCK-8 assay in HEK293T cells treated with increasing concentrations of BBR684 for 24 hours. (n = 3, 3 biological replicates). (E, F) Levels of malondialdehyde (MDA, F) and reduced glutathione (GSH, E) in cell lysates. (n = 3, 3 biological replicates). (G) Intracellular Fe²⁺ levels measured by a colorimetric assay. (n = 3, 3 biological replicates).Figure 4 dummy alt text
Next, after erastin-induced ferroptosis, both cell groups were treated with increasing concentrations of BBR684. Cell viability was assessed after 24 hours using the CCK-8 assay. VDAC-overexpressing 293T cells exhibited significantly reduced viability compared to normal cells (Figure 4D). This indicates that VDAC overexpression can reverse the inhibitory effect of BBR684 on ferroptosis.
Next, we validated alterations in lipid peroxidation in VDAC-overexpressing cells. Following VDAC overexpression in 293T cells, ferroptosis was induced in both control and VDAC-overexpressing cells using erastin, followed by treatment with 10 µM BBR684. Measurement of GSH levels revealed that, compared to normal cells, VDAC-overexpressing cells treated with BBR684 had lower GSH levels (Figure 4E). Additionally, intracellular MDA and Fe^2+^ levels were elevated in VDAC-overexpressing cells, consistent with enhanced lipid peroxidation (Figure 4F and G).
These results indicate that VDAC overexpression abrogates the protective effect of BBR684 against ferroptosis, further supporting a VDAC-dependent mechanism underlying BBR684-mediated ferroptosis inhibition.
BBR684 Ameliorates Renal Injury and Ferroptosis in a Mouse Model of Folic Acid-Induced AKI
To verify whether BBR684 could alleviate AKI in vivo, kidney injury was induced in mice by intraperitoneal injection of FA at a dose of 200 mg/kg. In this model, ferroptosis is a key pathogenic mechanism underlying AKI.21^,^43 Following FA administration, mice were treated with BBR684. After treatment for 72 hours, mice were euthanized, and kidneys and serum were collected for subsequent analyses (Figure 5A). Compared to the control group, FA-treated mice exhibited a significant increase in kidney size and weight (Figure 5B). However, BBR684 treatment significantly reduced both kidney size and weight compared to the FA group. Additionally, creatinine (CRE) and blood urea nitrogen (BUN) levels were significantly elevated in FA-treated mice, while BBR684 effectively reversed these FA-induced increases (Figure 5C and D).Figure 5BBR684 ameliorates folic acid-induced acute kidney injury in mice. (A)Schematic timeline of the experimental design. Mice were orally administered BBR684 (20 mg/kg) or vehicle 6 hours after a single intraperitoneal injection of folic acid (FA, 200 mg/kg). Samples were collected 72 hours post-FA injection. (B) Mouse kidney weight in the treatment groups. Kidney weight ratio. (n = 5 mice per group). Serum levels of blood urea nitrogen (BUN, C) and creatinine (D), indicators of renal function. (n = 5, mice per group). (E, F) Levels of malondialdehyde (MDA, E) and reduced glutathione (GSH, F) in kidney tissue homogenates, reflecting lipid peroxidation and antioxidant capacity. (n = 5 mice per group). (G) Representative images of hematoxylin and eosin (H&E) staining and neutrophil gelatinase-associated lipocalin (NGAL) immunohistochemical staining in kidney sections (n = 5 mice per group). Significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, **P < 0.001; n.s., not significant.Figure 5 dummy alt text
Moreover, consistent with the in vitro findings, the levels of malondialdehyde (MDA) were significantly elevated in FA-treated mice, and this elevation was markedly suppressed by BBR684. Assessment of GSH content showed that FA treatment significantly reduced GSH levels, which were restored by BBR684 administration (Figure 5E and F). Histopathological alterations were further examined following FA-induced AKI and BBR684 treatment. HE staining revealed significant dilations of renal tubules and marked tubular injury in the FA group, which were substantially alleviated by BBR684 treatment. Neutrophil gelatinase-associated lipocalin (NGAL), a well-established biomarker of AKI, was significantly increased after FA treatment, while BBR684 reduced NGAL expression, consistent with the HE staining results (Figure 5G). These findings indicate that BBR684 can alleviate FA-induced AKI in mice.
BBR684 Attenuates Renal Inflammation and Lipid Peroxidation in AKI Kidneys
To further investigate histological changes in FA-induced AKI and the effects of BBR684 treatment, renal tubular dilation, brush border injury, and protein casts formation were evaluated using PAS staining.27^,^44 Compared to the FA group, BBR684-treated group exhibited significantly lower scores for renal tubular dilation and brush border injury, indicating effective attenuation of FA-induced tubular damage (Figure 6A and F). These findings were further confirmed by Masson trichrome staining44^,^45, which similarly demonstrated reduced tissue injury following BBR684 treatment (Figure 6B). Subsequently, we examined the changes in inflammatory marker CD45 and the lipid peroxidation product 4-HNE using immunohistochemical analysis. FA treatment markedly increased CD45 and 4-HNE levels, while BBR684 effectively reduced the expression of both markers (Figure 6C, D, G, and H). In AKI, ferroptosis can propagate among renal tubular cells, thereby promoting apoptosis and necrosis in adjacent tubules. Renal tubular cell apoptosis was assessed by caspase-3 staining, which revealed markedly increased caspase-3 expression after FA treatment. This increase was effectively reversed by BBR684 treatment (Figure 6E and I). Collectively, these findings indicate that BBR684 provides renal protection by inhibiting inflammatory responses and lipid peroxidation.Figure 6BBR684 mitigates renal pathological damage, inflammation, and ferroptosis markers in FA-induced AKI. (A)Representative periodic acid–Schiff (PAS)-stained kidney sections, highlighting tubular brush borders and protein casts. (B)Representative Masson’s trichrome-stained sections for collagen deposition (blue). (C-E) Representative immunohistochemical staining of kidney sections for (C) CD45 (leukocyte common antigen, indicating inflammation), (D) 4-hydroxynonenal (4-HNE, a lipid peroxidation adduct), and (E) cleaved caspase-3 (an apoptosis marker). (F) Quantification assessment of renal histopathological injury. Tubular dilation and brush border loss were semi-quantitatively scored (left y-axis), and the area of protein casts is presented as a percentage of the field (right y-axis). (G-I) Quantitative analysis of the positive staining area for (G) CD45, (H) 4-HNE, and (I) cleaved caspase-3. Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. *P < 0.05, **P < 0.01, **P < 0.001; n.s., not significant.Figure 6 dummy alt text
Discussion
Ferroptosis has emerged as a critical pathogenic mechanism in acute kidney injury (AKI),46 yet specific and effective therapeutic interventions remain elusive. In this study, we identified BBR684, a novel berberine derivative, as a potent ferroptosis inhibitor that protects against renal injury both in vitro and in vivo by targeting the voltage-dependent anion channel (VDAC). Our findings not only present a promising therapeutic candidate but also elucidate a mechanistic pathway centered on VDAC oligomerization, a relatively underexplored regulatory node in ferroptosis.
BBR684 confers superior protection against ferroptosis compared to its parent compound, berberine, primarily by suppressing the core biochemical events of this cell death process. It effectively inhibited Erastin-induced accumulation of lipid peroxidation products (MDA, 4-HNE) and lipid ROS,32 while restoring the depleted antioxidant system, as evidenced by increased glutathione (GSH) levels and GPX4 expression in HK-2 cells.29 The upregulation of GPX4 is likely an indirect consequence of the reduced oxidative burden, highlighting BBR684’s role in re-establishing cellular redox homeostasis.
A key mechanistic insight from this study is the identification of VDAC as a direct molecular target of BBR684. Through MST and CETSA assays, we demonstrated that BBR684 physically binds to VDAC, enhances its thermal stability, and critically inhibits its oligomerization. Since VDAC oligomerization on the outer mitochondrial membrane facilitates excessive ROS production and calcium influx,47 its inhibition by BBR684 directly explains the observed reduction in mitochondrial oxidative stress and dysregulation. This finding positions BBR684 as a modulator of a specific event in ferroptosis initiation. The functional necessity of VDAC binding for BBR684’s action was unequivocally established by gain-of-function experiments. Overexpression of VDAC in 293T cells completely abolished the protective effects of BBR684 against Erastin-induced cell death, lipid peroxidation, and antioxidant depletion. This evidence solidifies the claim that BBR684 exerts its anti-ferroptotic effects primarily through engaging VDAC, rather than through off-target mechanisms.
Translating these cellular findings to a disease-relevant model, BBR684 demonstrated significant efficacy in alleviating folic acid-induced AKI in mice. Treatment with BBR684 not only improved functional parameters (serum creatinine and BUN) and histological damage but also directly suppressed renal lipid peroxidation and inflammation, as shown by reduced 4-HNE and CD45 staining. The concordance between in vitro and in vivo outcomes strongly supports the pathophysiological relevance of the BBR684-VDAC-ferroptosis axis in AKI. Despite the compelling evidence, certain limitations of this study warrant consideration and guide future directions. First, while multiple lines of evidence support ferroptosis inhibition, the absence of a direct comparison with canonical inhibitors like Ferrostatin-1 limits the specificity claim. Second, the therapeutic efficacy was validated primarily in a single chemical-induced AKI model; its effectiveness in other prevalent models such as ischemia-reperfusion injury awaits investigation. Third, the detailed structural interaction between BBR684 and VDAC, along with the precise downstream consequences on mitochondrial metabolism, requires further elucidation through structural biology and metabolomic studies. Finally, comprehensive pharmacokinetic and long-term safety profiles are essential precursors to clinical translation.
In conclusion, this study unveils BBR684 as a novel and potent ferroptosis inhibitor that targets VDAC oligomerization. By integrating in vitro cell-based assays and in vivo FA-induced AKI model, we provide a coherent mechanistic narrative linking VDAC modulation to ferroptosis suppression and renal protection. These findings underscore the therapeutic potential of targeting mitochondrial VDAC in ferroptosis-driven diseases such as AKI and pave the way for the rational development of more specific VDAC-directed therapeutics.
Ethics Declarations
6-week female C57BL/6 mice were procured from GemPharmatech Co., Ltd. (Jiangsu, China). All experimental protocols were approved by the Institutional Animal Care and Use Committee of China Pharmaceutical University (Approval 2024-07-020) and conducted in compliance with the university's Guidelines for Animal Welfare.
Author Contributions
Zihao Jiang and Wenhao Zhang made the major contribution to this study in the conceptualization. Jiawei Zhu and Cong Wang contributed to methodology and data acquisition. Yongjian Guo and Guo Chen designed the experiments. Zihao Jiang, Wenhao Zhang and Jiawei Zhu performed the in vitro experiments. All authors have read and agreed to the published version of the manuscript.
Funding
This study was supported in part by Natural Science Foundation of China (NSFC) (NO. 82204442).
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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