Development and handling of the animal model

All procedures involving animals were conducted following the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were subjected to approval from the animal experimentation ethics of Youjiang Medical University for Nationalities (ethical batch number: 2021103001). The C57BL/6 mice (NO. SYXK 2015-0008) were provided by Shanghai Genechem Animal Co., Ltd. These mice were housed at the SPF facility of Youjiang Medical University for Nationalities (NO. SYXK 2017‐0004) under controlled conditions, including a 12-h light/dark cycle, 50% humidity, and temperatures between 20 and 24 °C. The mice had unrestricted access to sterilized water and food. A CX3CL1-Knockout (CX3CL1-KO) mouse model was established, as indicated in a previous study (Gong et al. 2021). Each mouse was randomly assigned to one of three groups (n = 5): control group, cisplatin group, and cisplatin + CX3CL1-KO group. The animals received a single intraperitoneal dose of cisplatin (20 mg/kg) (Zhou et al. 2022). 48 h after receiving cisplatin, mice were humanely killed by cervical dislocation, and samples of blood and renal tissues were obtained.

Cell culture and treatment

The AB8/13 human immortalized podocyte cell line was generously provided by Dr. Moin A. Saleem from Bristol, U.K. Podocytes were cultured following previously established protocols (Gong et al. 2022). Lentiviral vector Ubi-MCS-CBh-gcGFP-IRES-Puro-CX3CL1 was employed to induce CX3CL1 knockdown (CX3CL1-KD) in podocytes, following the manufacturer’s instructions from Shanghai Genechem Co., Ltd. Three distinct podocyte groups were established: the control group, the cisplatin-treated group (20 μM/ml, 24 h), and the CX3CL1-KD + cisplatin group.

Bioinformatic analysis

Published RNA-seq data in Total Productive Maintenance (TPM) format were downloaded from the Gene Expression Omnibus (GEO) database (accession number: GSE106993). TPMs were converted to log2 (TPM + 1) for further analysis. Differentially expressed genes (DEGs) between cisplatin-treated and wild-type (WT)-group mice were examined with the limma package. Genes with LogFC > 1.5 and adjusted p-value < 0.05 were statistically significant. Functional enrichment analysis was conducted with Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) for all differential genes. The functional differences between the two treatment groups were further investigated by conducting additional Gene Set Enrichment Analysis (GSEA). The MSigDBR package was used to acquire the hallmark gene set (c2.all.v7.5.1.symbols.gmt).

Renal function assessment

The serum levels of blood urea nitrogen (BUN) and serum creatinine (Scr) in mice were measured by urease and sarcosine oxidase assays, respectively, according to the manufacturer (Jiancheng Bioengineering Institute, Nanjing, China; kit numbers C013-2-1 and C011-2-1, respectively).

MDA, SOD, and iron content and GSH/GSSG ratio analysis

The contents of malondialdehyde (MDA), superoxide dismutase (SOD), and iron and the glutathione/oxidized glutathione (GSH/GSSG) ratio in kidney tissues and podocytes were detected using MDA (thiobarbituric acid method) and SOD assay kits (hydroxylamine method; Jiancheng Bioengineering Institute, Nanjing, China), an Iron content assay Kit (ferrozine microplate method; Leagene Biotechnology, Beijing, China), and a GSH/GSSG assay kit (fluorometric—green; ab205811, Abcam), respectively, following the corresponding kit instructions.

Enzyme-linked immunosorbent assay (ELISA)

The serum levels of IL-6 and TNF-α were quantified using ELISA kits (Cusabio Biotech Co., Ltd) according to the manufacturer’s instructions.

Hematoxylin and eosin (HE) and periodic acid-Schiff (PAS) staining

The kidney tissues were fixed in 4% formaldehyde and embedded in paraffin. The tissues were then sliced into 4 μm thickness sections for HE and PAS staining. Light microscopy (BX53, Olympus, Tokyo, Japan) was used to study renal tissue pathology. The pathological changes in the kidney tissues were evaluated by a board-certified pathologist. To ensure impartiality, the pathologist was blinded to the treatment conditions during the evaluation, eliminating potential biases. A scoring system (ranging from 0 to 4 points), based on the extent of injury (< 25% injury, 25–50% injury, 50–75% injury, and > 75% injury), was employed to gauge the severity of glomerular sclerosis through PAS staining (Liu et al. 2022).

Immunohistochemistry (IHC)

Renal tissue section (4 μm) were fixed in 4% formaldehyde. After deparaffinization and rehydration, IHC was performed on the sections using anti-TNF-α, anti-F4-80 (mouse EGF-like module-containing mucin-like hormone receptor-like 1), anti-4-HNE (4-Hydroxynonenal), anti-3-NT (3-nitrotyrosine), or anti-GPX4 primary antibody (all from Affinity Biosciences, OH, USA; 1:200). The primary antibodies were incubated at 4 °C overnight, followed by subsequent incubation with horseradish peroxidase-conjugated secondary antibody (Dako, Glostrup, Denmark) at a 1:200 dilution at room temperature (RT) for 1 h. The sections were washed with PBS following each process. In the absence of the primary antibody or upon the addition of excessive antigen, the staining condition was assessed to ensure specificity. The fluorescence image was captured by a fluorescence microscope (BX53, Olympus, × 400). Staining intensities were quantified with ImageJ software.

Reactive oxygen species (ROS) analysis

Renal tissue section (4 μm) were first incubated with 20 μM dihydroethidium (DHE-DA; Meilunbio, Dalian, China) for half an hour at RT. The ROS level of podocytes was determined using a 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe (Beyotime, Shanghai, China). To study its effects, the podocytes were first treated with cisplatin; then, they were subjected to incubation at RT for half an hour with 2 μL/well of DCFH-DA. The ROS levels in renal tissues and podocytes were observed using fluorescence microscopy (Olympus, Tokyo, Japan).

Transmission electron microscopy (TEM)

Fresh renal tissues (1 mm3) were placed in a TEM fixative (P1126, Servicebio, Beijing, China) for 2 h at 4 °C. After tissues were sliced into the ultrathin section (60–80 nm), the specimens were stained with uranyl acetate and lead citrate before being examined by TEM (HT7700, HITACHI, Tokyo, Japan).

Immunofluorescence (IF) staining

Renal cryosections (3 μm) and podocyte sections were fixed using 4% paraformaldehyde (PFA), followed by blocking using 10% fetal bovine serum (FBS). The sections were then immunostained with anti-CX3CL1, anti-nephrin, anti-TNF-α, anti-eIF2α, anti-p-eIF2α, anti-CHOP, anti-HO-1, anti-HIF1A, anti-GPX4 or anti-XCT primary antibody overnight at 4 °C. Next, the sections were subjected to incubation with goat anti-rabbit secondary antibody for an hour. The stained slice images were then captured under a fluorescence microscope (Olympus, Tokyo, Japan). Image pro-plus 6.0 software was used to analyze the integrated optical density (IOD) of each fluorescent image, and finally, the average IOD was calculated.

Western blotting

Renal tissues and podocytes were first lysed utilizing RIPA solution on ice. The total proteins isolated were then examined using the specific anti-CX3CL1 (1:2000, ab25088, Abcam), anti-nephrin (1:1000, AF7951, Affinity Biosciences), anti-podocin (1:1000, DF8593, Affinity Biosciences), anti-WT1 (Wilms tumor protein; 1:800, DF6331, Affinity Biosciences), anti-TNF-α (1:1000, AF7014, Affinity Biosciences), anti-IL-6 (1:1000, DF6087, Affinity Biosciences), anti-F4/80 (1:750, DF2789, Affinity Biosciences), anti-UCP2 (uncoupling protein 2; 1:750, DF8626, Affinity Biosciences), anti-Mfn2 (Mitofusin 2; 1:800, DF8106, Affinity Biosciences), anti-PGC1α (peroxisome proliferators-activated receptor γ coactivator lalpha; 1:1000, Af5395, Affinity Biosciences), anti-GPX4 (1:1000, DF6701, Affinity Biosciences), anti-XCT (1:1000, DF12509, Affinity Biosciences), anti-GRP78 (1:1000, BF8024, Affinity Biosciences), anti-eIF2α (1:1000, AF6216, Affinity Biosciences), anti-p-eIF2α (1:1000, AF3216, Affinity Biosciences), anti-CHOP (1:1000, AF6277, Affinity Biosciences), anti-HO-1 (1:750, AF5393, Affinity Biosciences), anti-HIF1A (1:1500, ab179483, Abcam) and anti-β-actin (1:5000, AF7018, Affinity Biosciences) primary antibody followed by IRDye 800RD goat anti-rabbit (SA535571, Invitrogen, USA) secondary antibody. Stained proteins were then detected using a Licor Odyssey scanner (Licor Bioscience, Lincoln, USA).

Mitochondrial membrane potential (MMP) measurement

The JC-1 fluorescence staining assay was conducted to determine the MMP. Briefly, podocytes were harvested and incubated with JC-1 (Sigma-Aldrich, MO, USA) for 20 min at RT. Podocyte fluorescence intensity was then measured using fluorescence microscopy (Olympus, Tokyo, Japan). The presence of red fluorescence within podocytes indicated the aggregation dependent on mitochondrial potential, reflecting the mitochondrial membrane potential (ΔΨm). A transition from red to green fluorescence indicated depolarization in the mitochondria.

Statistical analysis

Data were presented as mean ± standard deviation (SD). GraphPad Prism 8 was utilized for statistical analyses. The statistical significance of the difference (variation) between groups was assessed using the Student’s two-tailed unpaired t-test and one-way analysis of variance (ANOVA). Tukey’s test was performed for multiple comparisons. p values < 0.05 indicated statistically significant differences between groups.