Animals and Experimental Protocol

Cyagen Biotechnology Co., Ltd. (Suzhou, China) provided 40 C7ORF41 knockout mice (KOCMP-10672-Mturn), and Beijing HFK Bioscience Co., Ltd. (Beijing, China) supplied 40 C57BL mice. Male mice that were 8 weeks old and weighed between 23 and 25 g were utilized. The Ethics Committee of Renmin Hospital of Wuhan University reviewed and granted approval for the animal study. In order to create a mouse model of nonfatal kidney injury caused by endotoxemia, wild-type (WT) and C7ORF41 knockout (KO) mice were given a single injection of 5 mg/kg Escherichia coli LPS (serotype 0111 B4, Sigma) intraperitoneally. The control group was administered an equivalent amount of sterile saline. C7ORF41 knockout mice or wild-type mice were randomly assigned to two groups: control group and LPS group, each consisting of a minimum of 20 mice. After a 24 h period, the mice were euthanized and their samples were collected for future utilization. Anesthesia is provided as follows: Mice were anesthetized with sodium pentobarbital (30 mg/kg). Carbon dioxide induction is the method employed for euthanizing the mice. At the beginning, the flow of air remain consistent with a carbon dioxide level ranging from 30 to 70% V/min for approximately one minute. Furthermore, it is crucial to uphold the circulation of air for a minimum duration of 1 min subsequent to clinical death in order to prevent reversal. A minimum of three replications were conducted for all animal experiments.

Ethics Declarations

The Ethics Committee of Renmin Hospital of Wuhan University reviewed and granted approval for the animal study. All experiments were performed in accordance with relevant named guidelines and regulations. The authors complied with the ARRIVE guidelines.

Cell Culture

In these studies, HK-2 cells, acquired from the Cell Bank of the Chinese Academy of Sciences in Shanghai, China, were employed. These cells were cultivated in an incubator designated for cell culture, maintained at a temperature of 37 °C and an atmosphere containing 5% CO2. The growth medium used was Dulbecco’s Modified Eagle Medium (DMEM)/F-12, enriched with 10% fetal bovine serum (FBS) and 1% penicillin, to nourish the cells. To assess the extent of cellular impairment in EA-AKI, the HK-2 cells underwent treatment with 10 µg/mL LPS obtained from Sigma (Product code L3129).

Cell Lines

HK-2 cells underwent transfection with lentiviral particles carrying C7ORF41 short hairpin RNA (shC7ORF41) or with control viral particles (C7ORF41-vector) obtained from Shanghai Shenggong Bioengineering Co., Ltd., China. Adenovirus vector carrying human C7ORF41 (C7ORF41-OE) to over express C7ORF41 were constructed and amplified by the iBioscience Company (Jinan, China).

The infection of HK-2 cells was executed at a multiplicity of infection (MOI) of 50, using 5 µg/mL of polybrene over a duration of 18 h. Following this, the HK-2 cells were seeded into 6-well plates and subjected to 5 µg/mL of puromycin treatment for 72 h to ensure cell selection. The efficiency of the transfection was evaluated by quantitative Polymerase Chain Reaction (qPCR) and Western blot analyses.

Total RNA Extraction and qRT-PCR

To extract total RNA from HK-2 cells, the RNAiso Plus reagent from TaKaRa (Product code 9108Q) was used. This RNA was then reverse-transcribed into complementary DNA (cDNA) using reverse transcription kits provided by Invitrogen (Kit K1621). The resultant cDNA was subsequently stored at a temperature of −20 °C. Real-time PCR assays were conducted utilizing a qPCR kit supplied by Novoprotein (Catalogue #E096-01 A). To normalize the expression levels of the target genes, the housekeeping gene GAPDH was used as an internal control.

The PCR protocol entailed an initial pre-denaturation step at 95 °C for 30 s, followed by 40 cycles of PCR reaction, which consisted of denaturation at 95 °C for 5 s and annealing/extension at 60 °C for 30 s, and concluded with a melt curve analysis stage. Gene expression levels were quantified as fold changes relative to the control by applying the 2-AACT method. The specific forward primer used in the qPCR are detailed subsequently:

C7ORF41: 5`-GCAGGATGGATTTCTACGCCGA-3`

NRF2: 5`-GGAUGGAUUUCUACGCCGACC-3`

GAPDH: 5`-GTCTCCTCTGACTTCAACAGCG-3`

Then cDNA was used as the template for SYBR qRT-PCR.

Western Blot Analysis

To extract total proteins from HK-2 cells and mouse kidney tissues, the process adhered to the protocol provided by the manufacturer. Each sample contributed approximately 60 micrograms of proteins, which were then separated via SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and subsequently transferred onto PVDF (Polyvinylidene Fluoride) membranes. The membranes, once prepared, were blocked with 5% non-fat milk for one hour to prevent non-specific binding. Following the blocking step, they were incubated overnight with the primary antibody tailored to the protein of interest.

On the subsequent day, the membranes underwent incubation with the secondary antibody, specific to the primary antibody, for one hour. This was done to enable the detection of the protein of interest by enhancing the signal. The detection of protein bands was facilitated by a protein development instrument, indicating the successful binding of the antibodies to their corresponding proteins.

Cell Viability Assay

The Cell Counting Kit-8 (CCK-8) is a widely used colorimetric assay for evaluating cell viability and proliferation. Around 3,000 cells were placed into each well of a 96-well plate and were incubated for 24 h in the presence of various concentrations of LPS (Lipopolysaccharide). After each treatment, 10 µl of CCK-8 solution (Product Number CK04; obtained from Dojindo Molecular Technologies, Inc.) was added to each well. The cells were then incubated for an additional hour with the CCK-8 solution, which facilitates the assessment of cell viability based on metabolic activity.

Following the incubation with the CCK-8 solution, the absorbance was measured at a wavelength of 450 nm to determine cell viability. To ensure the reliability and reproducibility of the results, the entire procedure was repeated three times.

Transmission Electron Microscopy

After the necessary steps, the Kidney tissues were trimmed to a dimension of 1 mm3 and then fixed in a 2% glutaraldehyde solution for 1 h at room temperature. Subsequently, the samples were exposed to a dimly illuminated setting and treated with 2% uranium acetate for a period of 10 min at room temperature. Afterwards, they were washed for a duration of 20 s. Subsequently, the samples underwent a 10-minute treatment with lead citrate at room temperature, followed by a 20-second rinse. In the end, the samples were recognized at the electron microscopy center located in Renmin Hospital, which is associated with Wuhan University. Acquisition of images was done using the HITACHI Transmission Electron Microscope (model H7650B). Representative images are displayed for each structure of interest, with a minimum of 10 images acquired for each.

ROS Analysis

The assessment of ROS levels in HK-2 cells was conducted using a ROS detection kit (from Beyotime Institute of Biotechnology, catalog# S0033M), adhering strictly to the guidelines provided by the manufacturer. The HK-2 cells were initially seeded into six-well plates with a cell density of 600,000 cells per well. They were then exposed to LPS at a concentration of 10 µg/mL for a 24-hour period for the purpose of ROS induction. To further investigate the role of ferroptosis in ROS levels, HK-2 cells were also treated with RSL3 (a ferroptosis inducer) and ferrostatin-1 (a ferroptosis inhibitor) at 37 °C for 2 h. Subsequently, post this pre-treatment, cells were again subjected to LPS (10 µg/mL) for another 24 h at the same temperature of 37 °C. For the detection of ROS, after the second LPS treatment, cells were incubated with 10 µM of the fluorescent probe 2,7-dichlorofluorescein diacetate (H2DCF-DA), at 37 °C for one hour. This dye is utilized for the intracellular detection of ROS due to its oxidation by ROS into a highly fluorescent compound. Post-incubation, the cells were meticulously washed twice with PBS to remove any residual dye and then resuspended in 200 µL of PBS. The quantification of ROS was done through Flow Cytometry, using equipment from BD Biosciences, and data analysis was conducted with FlowJo 7.6 software. For each treatment condition, a minimum of 10,000 cells were analyzed to ensure statistical relevance and accuracy.

The Levels of MDA and GSH in Samples of Cells and Kidney Tissues

The concentrations of MDA and GSH within both cell and renal tissues were quantified utilizing respective assay kits for MDA and GSH, adhering to the guidelines set by the providers. These substances’ levels were detected at wavelengths of 450 nm and 405 nm, correspondingly, via a microplate fluorometer. Moreover, to determine the total protein content in the samples, the Bradford assay was employed, a method provided by the Beyotime Institute of Biotechnology in Haimen, China.

Measurement of BUN, sCr

Kidney tissue and blood were collected for biochemical analysis. Serum levels of BUN and sCr were assessed by employing the Mouse BUN Elisa kit provided by Shanghai Huabang Biotechnology Co. (HB-P9S949X), following the guidelines provided by the manufacturer.

Kidney Tissue Histopathological, Immunohistochemistry (IHC)

Harvested recently obtained kidney tissues were promptly preserved in formalin and subsequently encased in paraffin. The tissues were then sliced into sections with a thickness of 5 μm, which were utilized for hematoxylin-eosin (H&E) staining, IHC. To evaluate the extent of renal interstitial injury in mice, various factors are considered, including the shape of tubular endothelial cells, the integrity of the brush border, the presence of renal epithelial casts, and the number of necrotic cells in the lumen. The kidney injury was evaluated by using the following criteria (0–4): 0-no cellular or tubular damage visible; 1-damage visible, but less than 25% of the tubular damage; affected; 2–25–50% tubular damage; 3–50–75% tubular damage; and 4-more than 75% damage. Image J software detected the photographs.

Iron Measurements

The cells were homogenized using an iron assay kit (Abcam, China) in iron assay buffer, following the provided instructions.Briefly, all cell lysates and standards were added to 96-well plates, and assay buffer was also added.Incubation of all samples took place at a temperature of 37 °C for a duration of 30 min.Afterwards, the samples were supplemented with iron probes and incubated at a temperature of 37 °C for a duration of 1 h. Finally, the OD value was measured with a colorimetric microplate reader at 593 nm.

Statistical Analysis

To ensure the reliability and precision of the results, each experiment was repeated three times. For the purpose of statistical evaluation, SPSS software (version 22.0) was utilized. Normal distribution of all data was confrmed, and results were presented as mean ± SD. Specifically, the Analysis of Variance (ANOVA) statistical test was conducted. A P-value threshold of 0.05 was used to determine statistical significance.