Mice and AKI Model

Male C57BL/6 mice, aged 8–10 weeks and weighing 20–25 g, were supplied by the Laboratory Animal Center of Xi’an Jiaotong University (Xi’an, China). The mice were accommodated in a specific-pathogen-free facility featuring a regulated environment. To induce AKI, a bilateral ischemia-reperfusion injury (IRI) model was used. Mice were anesthetized through isoflurane inhalation (2–3% for induction and 1–1.5% for maintenance) with oxygen assistance. A midline abdominal incision was performed to reveal the kidneys. Both renal pedicles were clamped using non-traumatic microvascular clamps for 45 minutes to induce ischemia. Following this period, the clamps were removed to allow reperfusion. The abdomen was closed with sutures, and the mice were allowed to recover under a warming lamp. At 24 hours post-reperfusion, mice were subjected to euthanasia through carbon dioxide asphyxiation, which was subsequently followed by cervical dislocation to confirm mortality. The kidneys were then carefully excised, rinsed in cold saline, and prepared for further examinations. All animal procedures were approved by the Institutional Animal Care and Use Committee of Xi’an Jiaotong University and carried out in accordance with the ARRIVE guidelines and the National Research Council’s Guide for the Care and Use of Laboratory Animals.

Cells and H/R Model

The human proximal tubular epithelial cell line, HK-2, was sourced from Procell (Wuhan, China). Cells were cultivated in DMEM/F-12 supplemented with 10% FBS and 1% penicillin-streptomycin and maintained at 37 °C in a humidified atmosphere containing 5% CO₂. To simulate H/R injury, HK-2 cells were exposed to a hypoxic environment for a duration of 24 hours, after which they were subjected to reoxygenation for a period of 6 hours. For the hypoxia phase, cells were placed in a modular hypoxic chamber maintained at 1% O2, 5% CO2, and 94% N2, with serum-free and glucose-free DMEM/F-12 medium. After 12 hours, the cells were returned to normoxic conditions (5% CO₂, 95% air) and cultured in complete medium for 6 hours to allow reoxygenation. This H/R protocol was designed to mimic the ischemia-reperfusion injury observed in vivo.

Western Blotting

Proteins were extracted from either mouse kidney tissue or cultured HK-2 cells. Kidney tissues were first homogenized in ice-cold RIPA buffer containing protease and phosphatase inhibitors. Similarly, HK-2 cells were lysed in RIPA buffer after being washed with cold PBS. The supernatant was collected as the protein extract for the subsequent analysis. The protein concentration of each sample was determined using the BCA assay kit (TianGen Biotech, Beijing, China). Equal quantities of protein (30 μg per sample) were subjected to separation using SDS-PAGE. After the completion of electrophoresis, proteins were subsequently transferred to PVDF membranes utilizing a wet transfer apparatus. Membranes were first treated with 5% non-fat milk at room temperature for 1 hour, followed by an overnight incubation at 4 °C with the designated primary antibodies: March6 (1:1000, Affinity Biosciences, Changzhou, China), GPX4 (1:1000, Proteintech, Wuhan, China), PTGS2 (1:1000, Proteintech), SLC7 A11 (1:1000, Abcam, Shanghai, China), ACSL4 (1:2000, Thermo Fisher Scientific, Waltham, MA, USA), p53 (1:500, Abcam), or GAPDH (1:5000, Proteintech). Subsequent to the washing process, the membranes were exposed to the relevant secondary antibodies (1:5000, Proteintech) for a duration of 1 hour at room temperature. The membranes underwent a washing process and were subsequently developed utilizing an ECL kit (TianGen Biotech). The signals were detected using a ChemiDoc Imaging System (Bio-Rad), with images acquired under uniform exposure parameters across all samples to guarantee uniformity.

Generation of March6-Overexpresing or Silencing HK-2 Cells

Recombinant lentiviruses expressing March6 cDNA for overexpression (LV-March6) and specific short hairpin RNA (shRNA) targeting March6 for gene knockdown (LV-shMarch6) were acquired from GenePharma (Shanghai, China). For the transduction process, HK-2 cells were seeded in 6-well plates at a density of 1 × 106 cells per well and incubated overnight to ensure optimal adherence. The following day, cells were incubated with the recombinant lentiviruses at a multiplicity of infection (MOI) of 50 to facilitate efficient transduction. To further enhance the transduction efficiency, Polybrene (8 μg/mL) was added to the viral solution prior to incubation. The cells were incubated with the viral mixture for 24 hours at 37 °C in a humidified atmosphere with 5% CO2. After the incubation period, the medium containing the lentiviruses was carefully replaced with fresh complete culture medium to remove excess virus and Polybrene. The cells were then incubated for an additional 48 hours to allow for optimal gene expression. To confirm the successful overexpression or knockdown of March6, western blotting was performed to assess March6 protein levels. The successfully transduced cells were utilized for subsequent experiments.

Cell Viability Assay

HK-2 cells infected with LV-March6 or LV-shMarch6 were seeded into a 96-well plate at a density of 5 × 103 cells per well and permitted to attach overnight. Cells were subjected to various experimental treatments to assess cell viability under different conditions. Cell viability was assessed utilizing the CCK-8 assay kit (Solarbio, Beijing, China). After desired treatments, a volume of 10 μl of the CCK-8 solution was introduced into each well, followed by incubation of the plates at 37 °C for a duration of 2 hours. The CCK-8 reagent interacts with dehydrogenase enzymes in live cells to produce a colorimetric change, allowing for the quantification of viable cells. The optical density (OD) was recorded at a wavelength of 450 nm utilizing a microplate reader.

Propidium Iodide (PI) Staining

To assess cell death, PI staining was performed. After desired treatments, cells were washed twice with PBS to eliminate any floating cells and residual debris. A total of 1× 105 cells was collected by centrifugation and resuspended in 500 μl of PI staining solution (Solarbio, Beijing, China). Cells were incubated for 15 minutes at 37 °C in the dark to ensure optimal staining. Following the incubation period, the cells were transferred to flow cytometry tubes. The samples were analyzed immediately using a flow cytometry instrument. Data acquisition was performed, and the results were analyzed using FlowJo software. The percentage of PI-positive (dead) cells was quantified relative to the total number of cells.

Detection of Lipid Peroxidation Using BODIPY 581/591 C11 Staining

To assess lipid peroxidation levels in HK-2 cells, the fluorescent dye BODIPY 581/591 C11 (Abmole Bioscience, Shanghai, China) was employed. After the appropriate experimental treatments, the cells were rinsed with pre-warmed PBS. Thereafter, the cells were treated with 10 μmol/l of BODIPY 581/591 C11 in serum-free medium for 30 minutes at 37 °C in the dark. Post-incubation, cells were washed with PBS to remove excess dye. Cells were collected, washed again with PBS, and resuspended in 500 μl of PBS. Fluorescence intensity was analyzed using a flow cytometer with appropriate settings to detect the fluorescence intensity.

Measurement of MDA and GSH Levels in HK-2 Cells and Kidney Tissue

MDA levels were measured using a commercial MDA assay kit (Beyotime, Shanghai, China). For HK-2 cells, cells were harvested and lysed in an appropriate lysis buffer. For kidney tissue, samples were homogenized in the same lysis buffer. After homogenization, the lysates from both the HK-2 cells and kidney tissues were centrifuged at 12,000×g for 10 minutes at 4 °C to remove cellular debris and insoluble material. The supernatant was collected for MDA quantification, following manufacturer’s protocols. The collected supernatants (100 μl) were mixed with 200 μl of MDA detection reagents. The samples were then heated in a boiling water bath for 15 minutes to facilitate the reaction. After heating, the samples were cooled to room temperature. Following cooling, the samples were centrifuged (1000×g, 10 minutes) to remove any precipitate formed during the reaction. Subsequently, 200 μl of supernatant was transferred to a 96-well plate, and the absorbance was measured at 532 nm using a microplate reader. GSH levels were assessed using a GSH assay kit (Jiancheng Bioengineering Institute, Nanjing, China). HK-2 cells were collected and lysed in the lysis buffer supplied with the kit. Kidney tissue samples were similarly homogenized in the same buffer. The lysates and homogenates were centrifuged to clear the samples of debris. The supernatant was used to determine GSH content in accordance with the protocols provided by the manufacturer. Brefly, 100 μl of supernatant was transferred to a 96-well plate, following by the addition of 125 μl of GSH detection reagent. The samples were then incubated at room temperature for 5 minutes. After the incubation period, the absorbance of the resulting solution was measured at a wavelength of 405 nm using a microplate reader. Both MDA and GSH measurements were normalized to protein concentration.

Detection of Ferrous Ion Levels HK-2 Cells and Kidney Tissue

Ferrous ion (Fe2+) concentrations in cultured cells and renal tissues were determined utilizing the Ferrous Iron Colorimetric Assay Kit (Elabscience, Wuhan, China). HK-2 cells were lysed utilizing the lysis buffer provided in the kit. Kidney samples were homogenized in the same lysis buffer using a homogenizer. Both cell lysates and tissue homogenates were centrifuged at 15,000×g for 10 minutes at 4 °C to remove any debris, and the supernatants were collected for further analysis. The supernatants were added to a 96-well plate at 80 μl per well, followed by the addition of 80 μl of detection reagents to each well. The samples were incubated for 30 minutes at 37 °C. After the incubation period, the absorbance was measured at a wavelength of 593 nm using a plate reader.

Detection of ROS Levels in Kidney Tissue

ROS levels in kidney tissue were quantified utilizing the DCF ROS Assay Kit (Abcam, Shanghai, China). Kidney tissue samples were prepared by homogenizing in lysis buffer. Subsequently, the homogenates underwent centrifugation at 10,000×g for a duration of 5 minutes to remove any particulate matter, after which the supernatants were carefully extracted. The supernatants were added to a 96-well plate at 50 μl per well, followed by the addition of 50 μl of Catalyst. After incubation for 5 minutes at room temperature, 100 μl of DCFH solution was added to each well, and the plates were then incubated for 30 minutes at room temperature in the dark. The fluorescence intensity, reflecting ROS levels, was measured using a fluorescence plate reader with an excitation wavelength of 480 nm and an emission wavelength of 530 nm.

Adeno-Associated Virus (AAV) Transduction In Vivo

For the in vivo overexpression study of March6, a recombinant AAV encoding the March6 gene under the control of a kidney-specific tubular epithelial cell promoter Ksp (GenePharma, Shanghai, China) was utilized. The AAV carrying the March6 gene was administered through renal vein injection. Mice were anesthetized through isoflurane inhalation. Following a midline abdominal incision, the target renal vein was carefully identified, and a syringe fitted with a fine needle (31G) was used to administer the AAV solution. Each mouse received a total of 1 × 1011 viral genomes (AAV-Ctrl or AAV-March6) in a volume of 50 μl. After injection, the needle was withdrawn, and the abdominal cavity was closed using absorbable sutures. Two weeks following the AAV injection, the transduction efficiency was assessed by examining March6 expression in mouse renal tissues using western blotting. Upon verification of overexpression, the IRI model was constructed to study the effects of March6 overexpression in the context of AKI. The study was conducted using four experimental groups: AAV-Ctrl + Sham, AAV-March6 + Sham, AAV-Ctrl + IRI, AAV-March6 + IRI, with each group consisting of six mice. Blood and kidney tissue samples were collected from the mice after euthanasia. These samples were preserved and processed for subsequent biochemical, histological, and molecular analyses to evaluate the impact of March6 overexpression on kidney function and pathology.

Assessment of Renal Function

Renal function assessment was conducted by quantifying serum creatinine and blood urea nitrogen concentrations through the utilization of commercially accessible assay kits (Jiancheng Bioengineering Institute). The serum creatinine and blood urea nitrogen concentrations were determined in accordance with the protocols provided by the kit manufacturer, and results were expressed in mg/dl. Blood samples were collected and allowed to clot at room temperature for 30 minutes. Following clotting, the samples were centrifuged at 3000×g for 10 minutes to separate the serum. The resulting serum supernatant was carefully transferred to a clean tube. For detection of serum creatinine, aliquots of serum were transferred to a 96-well plate at 12 μl per well, followed by the addition of 180 μl of Reagent I. After incubating for 5 minutes at 37 °C, the absorbance was measured at a wavelength of 515 nm using a plate reader. Subsequently, Reagent II was added to the plate at 60 μl per well. After an 8-minnute incubation at 37 °C, the absorbance was again measured at 515 nm using a plate reader. For the detection of blood urea nitrogen, aliquots of serum were transferred to a 96-well plate at 4 μl per well, followed by the addition of 50 μl of buffer solution. After incubating for 10 minutes at 37 °C, 125 μl of Chromogenic Agent and 125 μl of Alkaline NaClO were added to the sample, followed by incubation at 37 °C for 10 minutes. The absorbance was measured at 580 nm using a plate reader.

Histopathological Examination

To assess renal pathology, kidney tissues were preserved using 10% formalin, subsequently embedded in paraffin, and then sectioned to a thickness of 4 μm. Tissue sections were subjected to staining with hematoxylin and eosin (HE) in order to assess histopathological alterations. The tissue sections were placed on glass slides and allowed to dry for several hours at 37 °C to facilitate better adhesion. The slides were first immersed in xylene for 2 changes of 5 minutes each to completely dissolve the paraffin. This was followed by rehydration in a graded series of alcohol solutions, starting with 100% ethanol, then 95%, 80%, and finally 70% ethanol, with each step lasting 5 minutes. During the staining process, the sections were first incubated with hematoxylin for approximately 5 minutes. Following hematoxylin staining, the slides were rinsed in running water to remove excess dye and then immersed in eosin solution for about 3 minutes. After staining, the slides were washed again, dehydrated through a graded series of alcohol concentrations (70%, 80%, 95%, and 100%), and then cleared in xylene to remove the alcohol. Finally, a coverslip was applied using a mounting medium to preserve the samples for microscopic examination.

Detection of Inflammatory Cytokines

The quantification of pro-inflammatory cytokines, specifically IL-1β and IL-8, in kidney tissue homogenates was performed utilizing ELISA kits (Elabscience, Wuhan, China) specific for mouse IL-1β and IL-6. Briefly, kidney tissues were harvested from experimental animals and immediately placed in ice-cold PBS. The samples were then minced into small pieces (1 mm3) using surgical blades. The minced tissue was later homogenized in a lysis buffer containing protease inhibitors. After homogenization, the samples were centrifuged at 5000×g for 10 minutes at 4 °C. The supernatant was carefully collected and added to the ELISA plate at 100 μl per well. The plates were incubated at 37 °C for 90 minutes. Then, 100 μl of biotinylated detection Ab working solution was added to each well, and the plate was incubated at 37 °C for 1 hour. The solution was then decanted, and the plate was rinsed three times with wash buffer. Subsequently, 100 μl of HRP conjugate working solution was added to each well, and the plate was incubated at 37 °C for 30 minutes. After incubation, the plate was rinsed five times with wash buffer. Then, 90 μl of substrate reagent was added to each well. After incubation at 37 °C for 15 minutes, 50 μl of stop solution was added to each well. The optical density of each well at 450 nm was determined with a plate reader.

Real-Time Quantitative PCR for Tubular Injury Markers

Real-time quantitative PCR was utilized to assess the mRNA expression levels of tubular injury markers, specifically kidney injury molecule-1 (Kim-1) and neutrophil gelatinase-associated lipocalin (Ngal), in kidney tissue samples. Total RNA was extracted from the kidney tissues using a commercially available RNA extraction kit (TransGen, Beijing, China) according to the manufacturer’s instructions. The harvested kidney tissues were homogenized in the provided lysis buffer, followed by the addition of chloroform to separate the RNA from DNA and proteins. The RNA was subsequently precipitated with isopropanol, washed with 70% ethanol, and dissolved in RNase-free water. Reverse transcription was carried out to convert the extracted RNA into complementary DNA (cDNA) using a reverse transcription kit (TransGen). For the quantification of Kim-1 and Ngal mRNA levels, qPCR was performed using a real-time quantitative PCR kit (TransGen). The qPCR reaction was set up in a 20 μl volume, which included 10 μl of PCR Master Mix, 0.2 μmol/l of each primer (Kim-1, Ngal, and GAPDH), and 2 μl of cDNA template. The thermal cycling conditions were as follows: an initial denaturation step at 95 °C for 5 minutes, followed by 40 cycles of amplification consisting of 95 °C for 15 seconds (denaturation), and 60 °C for 30 seconds (annealing/extension). A melt curve analysis was performed at the end of the amplification to verify the specificity of the PCR products. The threshold cycle (Ct) values obtained from the qPCR were analyzed using the 2-ΔΔCt method to quantify the relative expression levels of Kim-1 and Ngal. GAPDH was selected as the housekeeping gene for normalization. The results were expressed as fold change relative to control samples.

Transmission Electron Microscopy for Mitochondrial Morphology

Kidney tissue specimens were initially preserved in a 2.5% glutaraldehyde solution prepared in a 0.1 mol/l phosphate buffer (pH 7.4) at 4 °C for an overnight duration. Subsequent to the primary fixation, the samples underwent washing in the same phosphate buffer and were then subjected to post-fixation in 1% osmium tetroxide for a period of 2 hours at room temperature. Following the fixation process, the tissues were dehydrated utilizing a sequential series of ethanol solutions (30%, 50%, 70%, 90%, and 100%). Following dehydration, the tissues were infiltrated with epoxy resin. The samples were first placed in a mixture of 1:1 epoxy resin and 100% ethanol for several hours, followed by pure epoxy resin overnight. Subsequently, the samples were embedded in fresh epoxy resin and cured at 60 °C for 24 hours to achieve complete polymerization, resulting in a solid block that encapsulated the tissues. Ultrathin sections, approximately 70 nm in thickness, were obtained using an ultramicrotome and subsequently placed on copper grids. The sections were first incubated with a 2% uranyl acetate solution for 10 minutes, followed by rinsing with distilled water. This was followed by a staining step with lead citrate, which involved a 5-minute incubation at room temperature. After staining, the grids were rinsed again to remove excess stain. The stained ultrathin sections were analyzed under a transmission electron microscope, where images were obtained for the evaluation of mitochondrial morphology.

Co-Immunoprecipitation (co-IP) Assay

To investigate the direct interaction between March6 and p53 or ACSL4 in HK-2 cells, we performed a Co-IP assay. Cells were transfected with a Flag-tagged March6 expression vector constructed using the pcDNA3.1–3 × Flag backbone, which facilitates the overexpression of March6. Transfection was performed using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s protocol. After 48 hours of transfection, cells were subjected to H/R treatment to induce cellular stress conditions in the presence of 10 μmol/l of MG132. Post-treatment, cells were washed with ice-cold PBS and lysed using a lysis buffer containing protease inhibitors. The lysates were collected by centrifugation and precleared with control agarose resin for 1 h at 4 °C with gentle rocking. The anti-Flag antibody and control IgG (Abcam) was immobilized with agarose resin. Approximately 500 μg of protein lysate was incubated with 2 μg of anti-Flag antibody (Abcam) overnight at 4 °C on a rotary shaker to facilitate the formation of immune complexes. The antibody-coupled resin was harvested by centrifugation and the immune complex was eluted using the elution buffer. For detection, eluted proteins were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was probed with antibodies against p53, ACSL4, and Flag to detect March6 binding partners. Protein bands were visualized using an ECL detection system.

Statistical Analysis

The data are expressed as mean values accompanied by standard deviations, derived from a minimum of three independent experiments. Statistical evaluations were performed utilizing GraphPad Prism. For comparisons between pairs of groups, Student’s t test was implemented. In the case of experiments with three or more groups, one-way analysis of variance (ANOVA) was applied, followed by Tukey’s post-hoc tests to ascertain statistical significance. A p value threshold of less than 0.05 was established to denote statistically significant differences.