CK-666 protects against ferroptosis and renal ischemia-reperfusion injury through a microfilament-independent mechanism

doi:10.21203/rs.3.rs-2749491/v1

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CK-666 protects against ferroptosis and renal ischemia-reperfusion injury through a microfilament-independent mechanism

https://doi.org/10.21203/rs.3.rs-2749491/v1

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Ferroptosis is a type of regulated cell death caused by iron-dependent accumulation of lipid peroxidation, exhibiting unique morphological changes. Actin microfilaments are crucial for various cellular processes, including morphogenesis, motility, endocytosis, and cell death. However, the role of actin microfilaments in ferroptosis is not well understood. Here, we found that actin microfilaments undergo remodeling and disassembly during ferroptosis. Interestingly, inhibitors targeting actin microfilament remodeling did not impact cell sensitivity to ferroptosis, except for CK-666. Notably, CK-666 attenuated ferroptosis independently of its canonical function of inhibiting the ARP2/3 complex. Further investigation revealed that CK-666 modulates the ferroptosis proteome and prevents lipid degradation during ferroptosis. Liquid chromatography-mass spectrometry analysis and functional assays demonstrated that CK-666 mitigates ferroptosis by directly eliminating lipid peroxidation. Importantly, CK-666 significantly ameliorated renal ischemia-reperfusion injury and ferroptosis in renal tissue. In summary, our findings provide new insights into the relationship between cytoskeleton and ferroptosis, and suggest potential applications of CK-666 in the treatment of ferroptosis-related diseases.

Biological sciences/Cell biology/Cell death

Health sciences/Diseases/Kidney diseases

Biological sciences/Chemical biology/Natural products

Ferroptosis is a type of regulated cell death characterized by iron dependence and the accumulation of lipid peroxidation^1–3. Ferroptosis plays an important role in various physiological and pathological contexts⁴. Therefore, small molecules that inhibit or induce ferroptosis with clinical potential are valuable for treating multiple ferroptosis-related diseases such as ischemia-reperfusion injury, acute liver injury, and cancer⁵. Ferroptotic cells exhibit unique morphological features, including abnormal mitochondrial structure, round up and lack chromatin condensation or fragmentation, while maintaining normal nuclear size⁶.

Microfilaments, consisting of actin filament and binding proteins, are the thinnest and smallest components of the cytoskeleton in cells. Microfilament remodeling plays a pivotal role in cell morphology, motility, polarization, and endocytosis, all of which rely on actin polymerization, depolymerization, and different architectures of actin networks⁷. Formin, an actin-binding protein, guides the nucleation and elongation of unbranched actin filaments⁸, while the Arp2/3 complex initiates branching at the end of the actin filament with the help of nucleation-promoting factors such as N-WASP⁹. The organization of actin filaments has been recognized to be crucial in different types of cell death such as apoptosis and necroptosis^10–12. However, the involvement of actin microfilaments in ferroptosis has yet to be fully elucidated.

Here, we discovered that ferropotic cells undergo actin microfilaments remodeling, but actin intervention drugs targeting actin polymerization or branching ferroptosis did not impact ferroptosis. However, CK-666, which is known for inhibiting the ARP2/3 complex, blocked ferroptosis independent of its canonical function in microfilaments. Mechanistically, CK-666 mitigated ferroptosis through directly inhibiting lipid peroxidation. Additionally, CK-666 significantly alleviated ferroptosis-related renal ischemia-reperfusion injury in vivo.

Differential effects of actin cytoskeleton inhibitors on ferroptosis

Actin filament remodeling has been shown to play significant roles in different types of cell death such as apoptosis and necroptosis. To investigate actin dynamics during ferroptosis, we expressed TdTomato-labeled actin chromobody in HT1080 cells. This actin chromobody specifically binds actin and enables live cell imaging of actin¹³. Ferroptosis was induced with RSL3, a commonly used inhibitor of the ferroptosis suppressor glutathione peroxidase 4 (GPX4). Live cell imaging showed that as the cells underwent ferroptosis, they gradually shrank, lost adherence, and finally underwent rupture. Simultaneously, the actin microfilaments underwent dynamic changes and disassembled. Finally, the chromobody leaked out after cell rupture (Figure 1a and Supplementary video). The basic dynamics of actin filaments are regulated by their polymerization and depolymerization.

To further test the role of microfilament in ferroptosis, we treated cells with different actin cytoskeleton inhibitors. Latrunculin A and Latrunculin B are marine toxins that bind to G-actin monomer, preventing actin polymerization¹⁴, while cytochalsin D binds to the barbed end of actin filament, blocking further polymerization of actin¹⁴. In contrast, jasplakinolide, a potent inducer of actin polymerization, stabilizes the pre-existing actin filaments and inhibits their disassembly¹⁵. Surprisingly, none of these inhibitors affected cell sensitivity to ferroptosis (Figure 1b). Formin is an actin binding protein that drives the elongation of unbranched actin filaments. However, cells treated with SMIFH2, a cell-permeable compound that specifically targets the FH2 domain of formin¹⁶, showed similar sensitivity to ferroptosis as control (Figure 1c). In addition to the elongation of unbranched actin filaments, actin filament branches to generate reticular structure mediated by the ARP2/3 complex and its nucleation-promoting factors such as N-WASP. However, cell ferroptosis was not affected by the N-WASP specific inhibitor Wiskostatin¹⁷ (Figure 1d).

Interestingly, the small molecular inhibitor CK-666, which binds to and stabilizes the inactive state of ARP2/3 complex¹⁸, significantly reduced ferroptosis induced by either RSL3 (Figure 1e and Supplementary Figure 1a) or erastin (Figure 1f and Supplementary Figure 1b). Overall, these findings suggest that changes in the actin cytoskeleton are associated with ferroptosis, but not the key driver of ferroptosis.

CK-666 attenuates ferroptosis independent of the ARP2/3 complex

Next, we investigated the effect of other ARP2/3 complex inhibitors on ferroptosis. CK-636, the structural and functional analogue of CK-666, exhibited a similar ability to attenuate RSL3-induced ferroptosis (Figure 2a). Conversely, the inactivation control compound, CK-689, did not mitigate RSL3-induced ferroptosis (Figure 2a). Interestingly, CK-869, a methoxy group containing compound that inhibits the ARP2/3 complex through a distinct structural mechanism compared to CK-666^18,19, slightly sensitized cells to RSL3 (Figure 2a). Similarly, CK666/636 rescued erastin-induced ferroptosis in HT1080 cells (Figure 2b), RSL3-induced ferroptosis in 293T cells (Figure 2c), and GPX4 deletion-induced ferroptosis in Pfa1 cells (Figure 2d). Next, we induced apoptosis (Supplementary Figure 2a), necroptosis (Supplementary Figure 2b), and pyroptosis (Supplementary Figure 2c) in different cell lines, but CK-666 did not affect these types of cell death, indicating the specificity of CK-666 to ferroptosis.

To further investigate whether CK-666/636 rescued ferroptosis through the ARP2/3 complex, we knocked down ARP2 and ARPC2, two main components of the ARP2/3 complex (Figure 2e). Surprisingly, neither ARP2 nor ARPC2 deficiency protected cells from ferroptosis induced by RSL3 (Figure 2f) or erastin (Figure 2g), unlike CK-666. Furthermore, CK-666 ameliorated ferroptosis even when ARP2 or ARPC2 was knocked down (Figure 2h). Thus, CK-666 inhibits ferroptosis independent of the ARP2/3 complex.

CK-666 rescues ferroptosis signatures in proteome and lipidome

To better understand the effects of CK-666 on ferroptosis, we conducted mass spectrometry to detect the proteome of cells. The levels of several ferroptosis-associated proteins, including TFRC, a biomarker of ferroptosis²⁰, were shown to be reversed by CK-666 treatment (Figure 3a). we identified 57 differentially expressed proteins between the RSL3 vs DMSO groupds and RSL3+CK-666 vs RSL3 groups (Figure 3b). KEGG analysis showed that HIF-1α signaling and ROS related proteins, including HIF-1α, HOMX1, TIMP1, and others were enriched (Figure 3c). Consistently, we found the transcriptional levels of HIF-1α downstream genes, including HILPDA and HMOX1 (Figure 3d), increased after RSL3 treatment and returned to control levels after CK-666 treatment. HIF-1α signaling has been reported to promote ferroptosis through HILPDA or HMOX1^21,22. Next, we examined whether the HIF-1α signal affects the function of CK-666 on ferroptosis with its activator DMOG and its inhibitor echinomycin. However, neither DMOG nor echinomycin blocked the effect of CK-666 on RSL3 induced ferroptosis (Figure 3e). Thus, CK-666 rescued ferroptosis signature in proteome but may not inhibit cell ferroptosis depending on HIF-1α signal.

Phospholipids containing polyunsaturated fatty acids are prone to be oxidized, resulting in ferroptosis, which leads to the degradation of these lipids. Consistently, we found that the majority of lipids decreased upon RSL3 treatment through lipidome analysis (Supplementary Figure 3a). However, most of the fatty acids, including saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA), remained unchanged by CK-666 treatment compared to control (Supplementary Figure 3b). In addition, all the phosphatidylethanolamines (PEs) that contain arachidonic acid (20:4) and adrenic acid (22:4), which are critical phospholipids in ferroptosis, showed no change upon CK-666 treatment compared to control (Supplementary Figure 3c). These results indicate that CK-666 reversed the ferroptosis signature in lipidome, but it may not affect cell ferroptosis through lipid remodeling.

CK-666 eliminates lipid peroxidation accumulation in cells

NRF2 is a crucial transcriptional factor that responds to ROS stress and regulates the expression of multiple antioxidant genes in cells²³. While both RSL3 and erastin increased NRF2 protein level (Supplementary Figure 4a) and NRF2 inhibitor ML385 sensitized cell to ferroptosis, CK-666 ameliorated ferroptosis regardless of ML385 (Supplementary Figure 4b). Whereas, CK-666 prevented glutathione (GSH) depletion induced by RSL3 (Figure 4a), indicating that CK-666 affected GSH independent of NRF2. Additionally, the antioxidant activity of CK-666 was tested using the cell-free DPPH assay. CK-666 and CK-636, but not CK-689, scavenged radicals similar to Fer-1 and Lip-1, demonstrating the antioxidant activity of CK-666 (Figure 4b).

To further investigate the effect of CK-666/636 on membrane oxidative rupture, we conducted the liposome leakage assay²⁴. Briefly, Tb³⁺ was enclosed into liposomes composed of soy phospholipids, which contain polyunsaturated fatty acids that are susceptible to peroxidation. The Liposome membrane isolates the enclosed Tb³⁺ from external solution, so liposome rupture can be detected by Tb³⁺ release. We triggered liposome rupture by Fenton reaction-mediated lipid peroxidation. Once ferrous iron and hydrogen peroxide were mixed with the liposome in solution, Tb³⁺ gradually released, and the fluorescence signal of Tb³⁺/DPA chelates was detected. Interestingly, CK-666/636 protected liposome from leakage as efficiently as Fer-1 and Lip-1 (Figure 4c).

To investigate the impact of CK-666 on lipid peroxidation in cells, we utilized the fluorescent probe C₁₁-BODIPY to determine the level of cell lipid peroxidation³. Interestingly, treatment with CK-666/636 rescued ferroptosis but resulted in a high level of oxidative C₁₁-BODIPY after 2 h (Supplementary Figure 5a) or 24 h (Supplementary Figure 5b) treatment. Similar results were obtained when lipid peroxidation was detected using Liperfluo, another widely used fluorescent probe of lipid peroxidation²⁵ (Supplementary Figure 5c). In addition, although CK666/636 rescued ferroptosis in Pfa1 cell (Figure 2d), the levels of oxidative C₁₁-BODIPY in Pfa1 cells treated with 4-OHT+CK-666/636 remained high, similar to Pfa1 cells treated with 4-OHT only (Supplementary Figure 5d). To examine whether the concentration of ferroptosis inhibitor affects the reliability of C₁₁-BODIPY, we titrated the concentrations of CK-666, Fer-1, and Lip-1 to inhibit RSL3-induced ferroptosis in HT1080 cells. We found 100 μM, a routinely used concentration⁹, CK-666 showed similar effectiveness with 31 nM Fer-1 or 12.5 nM Lip-1 in mitigating cell ferroptosis (Supplementary Figure 5e-g). Nevertheless, the percentages of oxidative C₁₁-BODIPY positive cells remained high in Fer-1 and Lip-1 treated cells, as well as in CK-666 treated cells (Supplementary Figure 5h-i).

To accurately measure lipid peroxidation in cells, we conducted LC-MS-based redox phospholipidomics to examine oxygenated phospholipids²⁶. Principal component analysis indicated that treatment with CK-666 resulted in oxidized phospholipids with a composition similar to that of Lip-1 (Fig. 4d). Furthermore, RSL3 treatment induced a significant increase in oxygenated phospholipids in the cells, which was effectively mitigated by both CK-666 and Lip-1 (Fig. 4e-f). Notably, CK-666 demonstrated a similar ability to Lip-1 in reversing RSL3-induced oxidation of PEs and phosphorylcholines (PCs), both of which contain polyunsaturated fatty acids like arachidonic acid (20:4) or adrenic acid (22:4) (Fig. 4g). Thus, CK-666 efficiently prevents the accumulation of lipid peroxidation in cells.

CK-666 alleviates renal ischemia-reperfusion injury

To further investigate the function of CK-666 in vivo, we induced ferroptosis in the kidney in mice through ischemia-reperfusion. Following renal ischemia-reperfusion, both serum creatinine and serum urea nitrogen levels increased in mice (Figure 5a-b). Intriguingly, CK-666 effectively alleviated the levels of serum creatinine and urea nitrogen, even better than Lip-1 (Figure 5a-b). As the final product of lipid peroxidation, MDA level reflects the extent of lipid peroxidation in tissues. We observed a significant increase in MDA levels in renal tissues following renal ischemia-reperfusion, while both Lip-1 and CK-666 reversed the elevation of MDA levels (Figure 5c). In addition, H＆E staining in renal tissue revealed obvious renal tubular injuries, which were rescued by Lip-1 or CK-666 (Figure 5d). Moreover, the results of TUNEL assay demonstrated that renal ischemia-reperfusion resulted in a significant increase in the proportion of dead cells in the renal tissues (Figure 5e). While Lip-1 treatment did not mitigate the cell death, CK-666 treatment significantly decreased the number of TUNEL-positive cells (Figure 5e). These results suggest that CK-666 efficiently alleviated renal ischemia-reperfusion injury and ferroptosis in renal tissue.

Previous reports have linked different types of cell death to cytoskeleton filaments. The early stages of apoptosis are associated with actin depolymerization, while microfilaments are redistributed and largely preserved during autophagy-induced cell death²⁷. Additionally, studies have shown that actin cytoskeleton is involved in the morphology change such as membrane blebbing and the formation of apoptotic bodies during apoptosis ²⁸, and manipulating the actin filament by jasplakinolide or cytochlasin D induces apoptosis^29–31. During necroptosis, actin participates in the traffic of MLKL to cell membranes¹². Here, we observed actin microfilaments remodeling and disassembly during ferroptosis. However, actin filaments inhibitors did not interfere with ferroptosis as they do with apoptosis or necroptosis. One possible explanation is that ferroptosis may be induced by lethal lipid peroxidation on the membrane, and as yet, no protein is recognized to execute the final step of ferroptosis, ruling out the possibility that actin impacts ferroptosis through regulating the action of an executor. Nevertheless, it has recently been reported the ESCRT III system³², which may cooperate with actin filaments, repairs small membrane damage caused by lipid peroxidation and blocks ferroptosis³³. Thus, we do not rule out the possibility that actin cytoskeleton may affect ferroptosis in other contexts. In addition, it is worth further investigating whether microtubules or intermediate filaments are involved in ferroptosis.

Both C₁₁-BODIPY and Liperfluo are hydrophobic fluorescent dyes that can be incorporated into lipid membranes and indirectly sense ROS, allowing the detection of lipid peroxidation. Oxidation of the polyunsaturated butadienyl portion of C₁₁-BODIPY results in a shift in fluorescence emission with a high quantum yield³⁴. The oxidized form of Liperfluo emits green fluorescence in lipophilic sites and is almost nonfluorescent in aqueous media, making it more specific to lipid peroxides with minimized photo-damage and auto-fluorescence³⁵. However, both C₁₁-BODIPY and Liperfluo are antioxidant reagents that reduce lipid hydroperoxides to lipid alcohols. It has been reported that C₁₁-BODIPY may overestimate the lipid peroxidation and underestimate the antioxidant effect of α-tocopherol due to its own antioxidant effect^36,37. Consistently, here we found that the oxidized form of C₁₁-BODIPY and Liperfluo remained high despite CK-666 blockinng cell ferroptosis. Similar results were found when the concentration of Fer-1 and Lip-1 were low enough to inhibit ferroptosis to the same level as CK-666. LC-MS, although limited by its low throughput and technical difficulties, directly analyzes specific oxidized lipids and is recognized as the most accurate method to measure lipid peroxidation. LC-MS analysis uncovered that lipid peroxidation is reduced in cells treated with CK-666. Therefore, our study suggests that when using fluorescent probes to assess lipid peroxidation during ferroptosis, the concentration of antioxidant reagent should be titrated carefully.

In summary, we have reported that in addition to its well-known role as an inhibitor of the ARP2/3 complex, CK-666 also acts as a radical-trapping antioxidant. Functionally, CK-666 inhibits ferroptosis and protects against I/R damage both in vitro and in vivo.

Mice

C57BL/6 WT mice were purchased from GemPharmatech Company (Jiangsu, China). 2-4-month age mice of both sexes were utilized, and all experimental protocols were approved by the Animal Care and Ethics Committee of Jinan University.

Cell culture

All cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37 °C with atmosphere of 5% CO₂. To promote differentiation into BMDM cells, 20 ng/ml M-CSF (PerproTech) were used to treat bone marrow cells from C57BL/6 WT mice for 7-9 days.

Chemicals

The compounds used to treat cells are as follow: RSL3, erastin, CK636, Ferrostatin-1, Liproxstatin-1, Necrostatin-1, and Z-VAD-FMK were purchased from Selleck; Wiskostatin, ML385, echinomycin, and DMOG were purchased from Targetmol; Deferoxamine (MCE), CK-666 (Abcam), CK-869 (Macklin), CK-689 (Merck), Jasplakinolide (Invitrogen), Cytochalsin D (Aladdin), Latrunculin A (APExBIO)，Latrunculin B (Cayman), SMIFH2 (Sigma), 4-Hydroxytamoxifen (Sigma).

Measurement lipid ROS with C₁₁-BODIPY and Liperfluo

Cells were seeded on 6-well plate. After treated with compounds for indicated times, cells were digested by trypsin and washed by PBS. Then, cells were resuspended in 200 μl PBS containing 5 μM C₁₁-BODIPY (Life Technology) or 1 μM Liperfluo (Dojindo). Incubate cells at 37°C for 30 min. After washing with PBS, cells were stained with DAPI and detected by flow cytometry with BD Fortessa. The levels of lipid ROS were measured in the FITC channel.

siRNA-mediated gene knockdown

siRNAs were transfected into HT1080 cells by Lipofectamine™ RNAiMAX Transfection Reagent (Life Technology). Cells were cultured into a 6-well plate in DMEM with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin and reach 60% confluent at transfection. siRNAs were mixed with Lipofectamine™ RNAiMAX Transfection Reagent in Opti-MEM™ medium (Life Technology) according to the manufacturer’s instruction. Then the mixture was gently added to the medium. The culture medium was refreshed by siRNA-free medium after 12h incubation. Cells were then transferred into a 96-well plate the next day for viability measurement.

Cell viability measurement

Cell viability was measured via Hoechst 33342 and PI staining. Briefly, cells were cultured into a 96-well plate. After treatment with different compounds, cells were stained by Hoechst 33342 and PI at 37°C for 15min. Then the cell images were captured, and the viability was measured with ImageXpress Micro Confocal (Molecular Devices).

DPPH assay

The radical trapping activity of chemicals was assessed using DPPH radical Scavenging Assay Kit (Solarbio, BC4755) following the manufacturer’s instruction. Briefly, compounds in extracting solution were added to DPPH and rotated for 30min at room temperature. Absorbance at 412nm was detected by a microplate reader.

Q-PCR

Total RNA was purified using RNAiso Plus (Takara) and then reverse transcribed with PrimeScript™ RT Master Mix (TaKaRa). Samples were analyzed through the QuantStudio 6 Flex system (Applied Biological Systems). Relative threshold cycle (Ct) to β-actin was used to detect gene expression levels.

Western blot

Cell pellets and tissues were lysed in RIPA buffer and the protein concentration was determined by BCA protein assay kit (Thermo). Then, the proteins were run on SDS-PAGE gels and transferred onto PVDF membranes (Bio-Rad). and antibodies against GPX4 (Abcam, ab125066), ARP2 (CST, 3128T), ARPC2 (Abcam, ab133315), NRF2 (Abcam, ab62352), HIF-1α (Novus Biologicals, NB100-105SS), β-actin (ABclonal, AC026), GAPDH (CST, 5174) were used. Signals were detected on an Amersham Imager 600 System (GE). The data were analyzed using ImageJ.

GSH level measurement

GSH level measurement was performed using Total Glutathione Assay Kit (S0053, Beyotime). Cells were seeded into 12-well plates (1.5 × 10⁵ per well). After drug treatment, cells were collected by scraping and prepared for measurement of glutathione according to the manufacturer’s instruction.

Liposome Leakage Assay

Liposome leakage assay was performed as previously described²⁴. To prepare liposomes, 2 mg of soy phospholipid (Avanti) dissolved in 500 μL chloroform was transferred into a round bottomed flask. A thin lipid film was formed after chloroform was evaporated by flask spinning (150 rpm) and nitrogen purging. 1mL buffer TL which contains 1 M HEPES (pH 7.4), 5M NaCl, 2M sodium citrate, and 1.5 M TbCl₃ was added into the flask. The lipid film was detached by vortex. Then the lipid film was extruded 30 times by an Avanti Mini-Extruder (Avanti) with 100 nm polycarbonate membranes to generate morphologically uniform liposomes. To remove unencapsulated Tb3⁺ ions, liposomes were washed with buffer L which contains 1M HEPES (pH 7.4) and 5M NaCl in a 100 KD Cut-off ultrafiltration tube (Millipore) and centrifugation at 4℃ for at least 8 times. The collected liposomes were used for liposome leakage assays. 10 μM hydrogen peroxide and 50 μM FeSO₄ were used to induce liposome leakage through Fenton reaction. Compounds to be tested were mixed with liposomes before the addition of hydrogen peroxide and FeSO₄. To assess liposome leakage, the release of Tb3⁺ was detected by the addition 50 μM of DPA and the fluorescent signals were measured through a microplate reader (λex=270 / λem=620 nm).

Living cell imaging

HT1080 cells were infected with Lentivirus HBLV-actin chromobody-TdTomato-puro generated by HANBIO (Shanghai, China). After puro selection, HT1080 cells expressed actin chromobody-TdTomato were obtained. Live cell imaging was acquired with Zeiss LSM 880 equipped with a definite focus system. Movies were analyzed with ImageJ.

Lipidomic analysis

Homogenize cell sample with 1mL mixture (including methanol，MTBE and internal standard mixture). Extract 500 uL supernatant and concentrate it. Dissolve powder with 100 uL mobile phase B. To perform UPLC, the sample extracts through ACQUITY UPLC HSS T3 C18 column (Waters). Next, LIT and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive and negative ion mode and controlled by Analyst 1.6.3 software (Sciex). Instrument tuning and mass calibration were performed with 10 and 100 μmol/L polypropylene glycol solutions in QQQ and LIT modes, respectively. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.

Proteomic analysis

Protein in cell samples were extracted through sonication in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail). The protein solution was then reduced with 5 mM dithiothreitol and alkylated with 11 mM iodoacetamide. Trypsin was added to digest proteins twice. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. Peptides were then fractionated by high pH reverse-phase HPLC using Thermo Betasil C18 column. Next, the peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4.

Analysis of oxidized phospholipids by LC-MS

Lipids were extracted using Folch method. A comprehensive analysis of oxidized phospholipidomics was conducted, followed previously established method³⁸. Samples were subjected to LC-MS analysis utilizing a Dionex Ultimate 3000 LC system coupled with a Q-Exactive mass spectrometer (Thermo Fisher). A Luna Silica (2) column (3 μm, 150 × 2.0 mm) (Phenomenex) was used to separate the samples at a flow rate of 0.2 mL/min and a column temperature of 35°C. The mobile phase consists of LC/MS-grade solvents, including 10 mM ammonium formate in isopropanol/hexane/water (285:215:5, V/V/V, solvent A) and isopropanol/hexane/water (285:215:40, V/V/V, solvent B). The gradient elution schedule was set according to the following parameters: 0 min, 10% B; 23 min, 32%; 32 min, 65%; 35 min, 100%; 70 min, 100%. The injection volume was 5 μL. Ionization was performed in negative ion mode with a capillary spray voltage of -2.8 kV, while the capillary temperature was set at 320 °C. The S-lens Rf level was set to 60. Data were acquired at a resolution of 70,000 for the full MS scan and 17,500 for the MS/MS scan in data-dependent mode, with the scan range set at m/z 400-1,800 and a maximum injection time of 200 ms using 1 microscan. For MS/MS analysis, a maximum injection time of 500 ms was utilized, with collision energy set to 24 eV and an isolation window of 1.0 Da.

Raw LC-MS data preprocess was conducted using MZmine 2.5.3³⁹, following a custom analysis workflow and database. Peaks with a signal-to-noise ratio above three were searched and identified against an in-house oxidized PL database. To identify lipid species, features were matched to m/z values with a tolerance of 5 ppm, further refined by retention time, and verified via MS/MS analysis using fragments for identification (https://www.lipidmaps.org/). Quantitative analysis relied on calibration curves created using known quantities of reference standards, including PA(18:1/18:1), PC(18:1/18:1), PE(18:1/18:1), PG(18:1/18:1), PI(18:1/18:1), PS(18:1/18:1), CL(16:0/18:2/18:2/20:4), and their corresponding internal standards PA(16:0-d31/18:1), PC(16:0-d31/18:1), PE(16:0-d31/18:1), PG(16:0-d31/18:1), PI(16:0-d31/18:1), PS(16:0-d31/18:1), CL(14:0/14:0/14:0/14:0).

Renal ischemia-reperfusion

Mice were intraperitoneally injected with Lip-1 (10 mg/kg) or CK-666 (30 mg/kg) before surgery. The mice were anesthetized and bilateral renal pedicles were clamped for 35min to induce renal ischemia-reperfusion injury, during which the body temperature of the mice was maintained on a heat plate at 38℃. The mice in the Sham group underwent the same procedures as those in the renal I/R group except for drug administration and clamping. The mice were sacrificed 24 h after surgery. Serum and tissue samples were collected for subsequent testing.

H&E staining

The kidney tissues were fixed with 4% paraformaldehyde and stained with H＆E. After scanning the images, three fields were randomly selected for each mouse's kidney section to calculate the proportion of injured renal tubules, and finally the statistical results were obtained.

TUNEL staining

The kidney tissues were fixed with 4% paraformaldehyde and stained with TUNELl. After scanning the images, three fields were randomly selected for the proportion of TUNEL positive cells in the kidney section of each mouse, and the statistical results were finally obtained.

Statistics

FlowJo version 10 was used to analyze the flow cytometric data. GraphPad Prism 8 was utilized for statistical analysis. Image J was used to analysis images and movies. The results are shown as mean ± SD. For data that were fitted with a Gaussian distribution, unpaired Student’s two-tailed t-test was used to determine the statistical significance. (ns: not significant, * P<0.05, **P<0.01, ***P<0.001.)

Acknowledgements

We thank Dr. Yilong Zou (Westlake University) for providing insightful suggestions.

Conflict of Interest

The authors declare no conflict of interest.

Author contribution

Z.J. oversaw the project and developed the concept of paper. Q.H., W.S., Z.O., W.D., Z.Q., Y.Z. performed the experiments. Q.H., W.S., Z.J., S.W. analyzed and interpreted the data. Q.H. wrote the manuscript with help from W.S., Y.G., D.T., X.C.. Y.G., D.T., T.C., R.H., X.C. provided insightful suggestions.

Ethics statement

No human participants, human data, or human tissue is involved in this study.

Funding

This work was supported by the National Key R&D Program of China [2021YFA0804903, 2021YFA1100103], the National Natural Science Foundation of China [92049304, 82030039, 82230047, 82271590, 82274403], the Guangdong Basic and Applied Basic Research Foundation [2020A1515110479, 2023B1515020020], the Program for Guangdong Introducing Innovative and Entrepreneurial Teams [2017ZT07S347], Innovative Team Program of Guangzhou Regenerative Medicine and Health Guangdong Laboratory [2018GZR110103002], the Open Project of the State Key Laboratory of Trauma, Burn and Combined Injury, Third Military Medical University [SKLKF202002].

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CK-666 protects against ferroptosis and renal ischemia-reperfusion injury through a microfilament-independent mechanism

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