2.1 Cell Culture and In Vitro Experiments
NRK-52E cells were cultured in Dulbeccos’s Modified Eagle’s Medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) with antibiotics (100 U/mL penicillin G and 100 g/mL streptomycin) at 37°C in a humidified atmosphere of 5% CO2 and 95% air. When the cell density reached about 90%, the medium was replaced with Hank's balanced salt solution (HBSS). Hypoxia-reoxygenation(H/R) AKI cell model, in the H/R group, a mixture of 1.5 µmol/L calcium and 10 µmol/L antimycin A was added for chemical hypoxia treatment. The aforementioned mixture was not added to the control group. After the two groups of cells were cultured for 2 h, HBSS was replaced as the complete medium for reoxygenation treatment. The cells were collected after 2 h of hypoxia and 2 h of reoxygenation. To evaluate death of NRK-52E cells induced by copper ion (Cu2+) carrier, namely elesclomol (ES), we established a control group, Cu2+ group, ES group, and Cu2+ + ES group. When the cells exhibited 80–90% confluence, Cu2+, ES, and ES + Cu2+ were added to respective groups (1:1 ratio, 10 nM), followed by incubation for 16 hours. The cells in the control group were not subjected to any treatment. Subsequently, cell viability was evaluated following cell growth in fresh medium.
2.2 Animals and Experimental Design
In this study, male C57 BL/6J mice of SPF grade (8–12 weeks old, body weight 22–25 g) were purchased from the Experimental Animal Center of the Army Characteristic Medical Center. The temperature of the breeding environment was 23 ± 3°C, the relative humidity was 40%-70%, and the light was alternating for 12 hours. AKI models of ischemia-reperfusion (I/R) were established as follows, Male mice were randomly divided into two groups, namely the sham operation group and I/R operation group. The mice were intraperitoneally injected with 5% chloral hydrate at a dose of 0.1 mL/10 g. After successful anaesthesia, openings of approximately 0.5 cm were made in mice in the I/R group below the ribs bilaterally, the skin was cut in turn until the retroperitoneum, the kidney pedicles were freed bilaterally, and vascular clips were used to clamp them and removed after 28 minutes. During this period, the anal temperature was maintained at 36.5°C-37.0°C, and after the kidney turned bright red, the incision was closed with continuous sutures. In the sham operation group, only the skin was cut to the back of the peritoneum, the kidney pedicles were dissociated bilaterally but were not clipped with vascular clips, and the abdominal cavity was closed with continuous sutures. Blood and renal tissue samples were obtained from mice in the I/R group 24 hours after the experiment. To collect specimen, we anesthetized mice by intraperitoneally injecting them with sodium pentobarbital, and then 800 ~ 1000 µL of blood was collected from the eye orbit and the thoracic cavity was opened for cardiac perfusion. Renal-tissue specimens were obtained from both kidneys, the kidney capsule was removed, and part of the kidney was fixed in 4% paraformaldehyde for renal pathological tissue staining. The remaining part of the kidney was stored at − 80℃ for western blot and RT-qPCR analyses.
2.3 Assessment of Renal Function
Collected blood was stored in a refrigerator at 4℃ for 1 hour, and then centrifuged at 4℃ in a low-temperature centrifuge (3,000×g for 10 min). Subsequently, supernatants were collected and used to determine serum creatinine and urea nitrogen levels using the Beckman 5800 Automatic Analyzer (Beckman, Krefeld, Germany). Kidney damage molecule-1 (KIM-1) and neutrophil gelatinase-associated adiponectin (NGAL) levels were determined by qPCR.
KIM-1(F) 5'-AAGGTTGGTGGAGAGGCAGGTC-3'
KIM-1(R) 5'-TCCGATAGGTGACGTGGGTTCC-3'
NGAL(F) 5'-GCAGCAGAACTTCCAGGACAACC-3'
NGAL(R) 5'-CAGGACGGAGGTGACATTGTAGC-3'
2.4 Study Population and Pathological Data
We reviewed the renal pathology data of patients with AKI from 1 January 2021 to 30 December 2021 in the hospital records Renal Pathology Archives. The diagnosis of AKI was based on the necrosis and abscission of the RTECs under a light microscope, which was further confirmed by electron microscopy. Normal renal tissue samples from patients with renal cell carcinoma with minimal pathological changes served as controls. All renal biopsy specimens were independently re-evaluated by two experienced pathologists. The Ethics and Program Review Committee of Chongqing Medical University approved this study.
2.5 Western Blot Analysis
After protein extraction from mouse kidney tissues and cells, the protein concentration was measured with the BCA kit. The SDS buffer solution was added, mixed well, and heated in a 100°C metal bath for 10 min to denature the protein. Following this, 12% SDS polyacrylamide gel was used to separate 30–80 mg of protein, following this, 30–80 mg of protein was separated on 12% SDS polyacrylamide gel and then transferred onto a membrane by applying 260 mA, 80 V, and constant pressure. Target strip was sealed with 5% bovine serum albumin solution. The membrane was washed thrice for 5 min each. Primary antibodies, namely CTR1 (1:1000, catalog no. 13086S, Cell Signaling Technology, Danvers, MA, USA), FDX1 (1:1000, catalog no. NBP1-89227, Novus, Colorado Springs, CO, USA), and β-actin (1:2000, catalog no. T40104S, Abbart, Shanghai, China) were added, and incubation was conducted overnight in a shaker at 4°C. Next, horseradish peroxidase-conjugated anti-mouse IgG (1:3000, catalog no. 7076S, Cell Signaling Technology) and horseradish peroxidase-linked goat anti-rabbit (1:30000, Cat# 7074, RRID: AB_2099233, Cell Signaling Technology) were added. Protein concentration in the bands was quantified using ImageJ software and normalized against that of the loading control, i.e, β-actin.
2.6 RT-qPCR
TRIzol was used to extract total RNA from the renal tissue, and quantitative RNA (optical density, OD 260 nm) was used. The Evo M-MLV reverse transcriptase kit (Accurate Biology, Hunan, China) was used for reverse transcription into cDNA. The NovoStart SYBR qPCR SuperMix Plus (Novoprotein, China) was used to amplify the cDNA RT-qPCR primers designed and synthesised by Bioengineering Co., Ltd (Shanghai). The primer sequence was as follows:
Primer name | Base sequence |
CDKN2A(F) | 5'-GGCACCAGAGGCAGTAACCATG-3' |
CDKN2A(R) | 5'-AGTTGTGGCCCTGTAGGACCTTC-3' |
GLS(F) | 5'-GGCTGCGACACTGGCTAATGG-3' |
GLS(R) | 5'-TGGAAAGCAAACTGCCCTGAGAAG-3' |
MTF1(F) | 5'-GATGACAACGGACTGGTGCCTTC-3' |
MTF1(R) | 5'-GTTCTCCACACTGTCCATCGTCTTC-3' |
PDHB(F) | 5'-CGAGGGCTGTGGAAGAAATATGGAG-3' |
PDHB(R) | 5'-GGCTTGCATGGAGAAATTGAAGGTC-3' |
PDHA1(F) | 5'-CACAGACCATCTCATCACAGCCTAC-3' |
PDHA1(R) | 5'-CCTCCTTTCCCTTTAGCACAACCTC-3' |
DLAT(F) | 5'-TTGATGTCAGTGTTGCGGTCAGTAC-3' |
DLAT(R) | 5'-GTGGCTGTAGTTTACCCTCTCTTGC-3' |
DLD(F) | 5'-GTCGTGTGTACTGCTCCTTGGC-3' |
DLD(R) | 5'-AATCGGCTGATCTGCGTAAGTTCTC-3' |
LIPT1(F) | 5'-TCTGAACTGAATCTCGCTCTGTTGC-3' |
LIPT1(R) | 5'-GAGGCAGGAGAATCGCTTGAACC-3' |
LIAS(F) | 5'-AATAACAGAGGTGGTGCCAGAATGC-3' |
LIAS(R) | 5'-GTGCTGGGATTACAGGCGTGAG-3' |
FDX1(F) | 5'-AACCCTGGCTTGTTCAACCTGTC-3' |
FDX1(R) | 5'-CCAACCGTGATCTGTCTGTTAGTCC-3' |
2.7 LDH Release Rate
An appropriate amount of cells was inoculated into 96-well plates, ensuring that the cell density did not exceed 80–90% when tested. The pores were divided into the following groups: blank control, sample control, maximum release pore and treated cell pore, and marked. Based on the needs of the experiment, appropriate drug treatment (the same as the in vitro experiment) was initiated, and the routine culture was continued. One hour before the detection time point, LDH release reagent was added into the maximum release hole, blown repeatedly and mixed well, incubated in the cell incubator until the predetermined time, and the cell culture plate was centrifuged with a 400 g porous plate for 5 min. The supernatant was obtained from each hole (120 µL) to determine the sample. The LDH release rate was analysed at 490 nm using multifunctional enzyme labelling, and the calculation was as follows: LDH release rate=(measured pore OD value, spontaneous pore OD value)/(maximum pore OD value, spontaneous pore OD value, 100%).
2.8 Detection of Copper Concentration in the Cells
We added 0.5 mL of HNO3 and 0.5 mL of hydrogen peroxide to the cells. The cells were then heated and digested at 90°C for 1 h, and the solution was cooled to room temperature, diluted with ultrapure water to 5 mL, and mixed well. Copper quantification was performed by Beijing Zhongke Baise Testing Technology Co., Ltd. using the NexION 300D ICP-MS (PerkinElmer, Waltham, MA, USA).
2.9 Periodic Acid-Schiff (PAS) Staining
After fixing, the renal tissues were washed, dehydrated, embedded and sliced to a thickness of 2 µm and then soaked in absolute ethanol, 95% ethanol and 70% ethanol for 5 minutes in sequence. The slices were immersed in periodate oxidation solution for 10–20 minutes, washed, dyed with Schiff solution at 37°C for 20 minutes, and washed again. The nucleus was stained with haematoxylin for 3 minutes, differentiated in hydrochloric, and washed with tap water until the nucleus turned blue. Following this, the tissue sections were dehydrated and blocked. Two nephrologists observed each sample in a double-blind manner, and each sample was scored using a light microscope at 200× magnification. Each sample was observed under at least 10 non-repetitive visual fields. The degree of renal tubular injury was evaluated by estimating the percentage of tubular epithelial necrosis, as follows: 0 points for no injury; 1 point (0–10%) and 2 points (11–20%) for minor injury; 3 points (21–40%) and 4 points (41–60%) for moderate injury; and 5 points (61–75%) and 6 points (> 75%) for serious injury.
2.10 Immunohistochemistry and Immunofluorescence Staining
The renal tissues were dehydrated, embedded and sectioned 24 hours after being fixed in a 10% neutral buffered formaldehyde solution (4 µm). Baked slices were dewaxed and dehydrated. After adding EDTA, the slices were heat repaired under high temperature and high pressure for 120 s. Goat serum was added for 30 min, and FDX1 (1:100) was added for more than 12 hours. A drop of HRP-labelled goat anti-rabbit/mouse secondary antibody (catalog no. PV-8000, Gorigene, Beijing, China) was added, followed by incubation for 40 min. The immunofluorescence secondary-antibodies was dripped with FITC-labelled goat anti-rabbit (1:50, catalog no. SA00003-2, Proteintech Group, Wuhan, China). After cleaning and sealing the films, we observed the sections by electron microscopy and fluorescence microscopy. Finally, the sections were observed using the DS-i2 type light microscope (Nikon, Tokyo, Japan). Two nephrologists observed each sample in a double-blind manner, and each sample was scored using a 200 light microscope. Each sample was observed under at least 10 non-repetitive visual fields. The proportion of FDX1-positive renal tubules in all renal tubules was determined.
2.11 Transfection
FDX1 siRNA, SF1 siRNA, and the corresponding negative control were provided by Sangon Biotech (Shanghai, China), and cell transfection was performed according to the manufacturer's instructions. The culture medium was replaced with Opti-MEM when the cells exhibited 70% confluence. The transfection reagents were added to continue the culture of the cells for 6 hours based on the number of NRK-52E cells, following which the Opti-MEM was changed to a P/S free culture medium containing 10% FBS. The H/R model was constructed after 48 hours.
2.12 Statistical Analysis
All quantitative data are expressed as the means ± SD using GraphPad Prism 8.0 software (GraphPad Software, Inc., San Diego, CA, USA). One-factor analysis of variance (ANOVA) followed by Tukey's post hoc test was used to analyse the variance for multiple group comparisons. A single asterisk indicates statistical significance at P < 0.05.