Animals models of AKI and treatment

Male C57BL/6 J mice were bought from Three Gorges University's Experimental Animal Center. The bilateral renal pedicle was clamped for 28 min with a microaneurysm clamp, as indicated previously [28]. In the sham surgery, the renal pedicle was exposed without being clamped. Reperfusion was then conducted at various time intervals of 0.5 h, 2 h, 6 h, and 24 h. P110 peptides and fragment of the HIV transactivator protein (TAT) (dissolved in saline) were synthesized by Selleck and Genscript company. P110 and TAT was intraperitoneally injected into mice at various time points and dosages based on our prior research, whereas control animals received the same quantity of saline daily to mimic a similar therapy.

Eight Bamach miniature pigs weighing 12.65 ± 1.12 kg each were isolated (provided by Wuhan Wuguan Medical Technology Co., Ltd.) and allowed to acclimate for 2 weeks. The pigs were randomly divided into two groups, TAT group and P110 group. At 24 h and 1 h before surgery, 0.4 mg/kg of P110 and TAT were intravenously injected into the respective groups. The animals were fasted for 12 h before the surgery. Prior to the start of the surgery, anesthesia was induced using Zoletil 50 (Virbac, France) through intramuscular injection. Each pig underwent endotracheal intubation and was maintained under anesthesia with 2% isoflurane. They were connected to a ventilator and a sophisticated electrocardiogram monitoring device. Additionally, a venous catheter was placed in the ear. A left-sided dorsal incision was made, and the left renal hilum was exposed through the retroperitoneum. The renal artery of the left kidney was clamped for 120 min. Subsequently, a right-sided dorsal incision was made, and the right renal hilum was exposed through the retroperitoneum for right nephrectomy, obtaining the right kidney as the sham group. At 24 and 48 h post-surgery, venous blood samples were collected to assess renal function. After 72 h of reperfusion, the left kidney and serum were collected under anesthesia. Kidney tissues at the cortex-medulla junction were rapidly frozen or preserved in 4% formaldehyde. Euthanasia was performed by intravenous injection of potassium chloride.

Folic acid-induced nephropathy was induced with a single intraperitoneal injection of folic acid (250 mg/kg body weight in 0.3 M sodium bicarbonate solution) in C57/Bl6 male mice. Kidney tissue and serum samples were extracted 48 h after folic acid treatment for kidney function, protein expression and histological analyses.

Renal histology

For paraffin embedding, fresh kidneys were taken and treated with 4% paraformaldehyde overnight at 4 °C. Hematoxylin and eosin (H&E) and Periodic Acid Schiff (PAS)  staining was applied to 4-mm slices of the paraffin-embedded tissues. Histopathologists scored the degree of kidney injury in a blind manner. According to renal tubular injury estimation, the following histological damage was graded: Damage levels are 0, no damage; 1, < 25; 2, 25–50%; 3, 50–75%; and 4, > 75%.

Renal function measurement

The central laboratory of the Center People's Hospital of Yichang (Roche Diagnostics GmbH, Penzberg and Mannheim, Germany) analyzed the levels of plasma creatinine and BUN.

Immunofluorescence and immunohistochemistry analysis

The paraffin sections were deparaffinized and subjected to antigen retrieval by steaming with sodium citrate buffer. mPTCs grown on collagen-coated coverslips were fixed with 4% paraformaldehyde. Primary antibodies for immunofluorescence included Ki67 (Cell Signaling Technology, 9129T), Bax (Abcam, ab182733), DLP1(BD Bioscience, 611112), Fis1 (Proteintech, 10956-1-AP) and dsDNA (Abcam,ab27156). Anti-rabbit or anti-goat or Alexa Fluor 647 conjugated secondary antibodies were used, and the fluorescent signals were visualized under a fluorescence microscope. Primary antibodies for immunohistochemical staining included KIM-1(R&D System, AF1750), PGC1 α (Novusbio, NBPI1-04676SS), COX I (Bioss, bs-3953R), STING(Cell Signaling Technology, 13647S),F4/80(Starter, S0B0227) and NGAL(R&D System, AF1757). The nucleus was counterstained with hematoxylin, dried, and mounted after being counterstained with DAB for color development. Using Image-Pro plus 6.0 software, positive staining regions were quantified.

Examination of mitochondrial fragmentation in kidney tissues

The kidney tissue was perfused with 1 ml of 10 U/ml heparin, followed by 2 ml fixative containing 100 mM sodium cacodylate, 2 mM calcium chloride, 4 mM magnesium sulfate, 4% paraformaldehyde, and 2.5% glutaraldehyde through the abdominal aorta. From each kidney, a tissue block measuring roughly 1 mm3 was taken, along with some renal cortex and outer medulla tissue for standard processing for electron microscopy. To find representative proximal tubules, the tissue block was initially examined at a modest magnification (× 3000). Then, high-magnification (× 15,000) examination of the cells in these tubules was performed to get electron micrographs.

Analysis of mitochondrial fragmentation

Plasmid of pDsRed2-mito was transfected into mPTC using Lipofecmin 2000 to visualize mitochondria. Using confocal microscopy, mitochondrial morphology was analyzed after cells were treated with 4% paraformaldehyde. For each slide, 150–200 cells from ten randomly chosen places were evaluated to determine the percentage of cells with mitochondrial fragmentation in accordance with the previous research [11, 29, 30].

Cell culture and treatments

The mouse proximal tubular cells (mPTCs) line was provided by Sciencell Research Laboratories, and all cells were grown with 10% fetal bovine serum + 1% dual antibody + DMEM in an incubator with 5% CO2 at 37 °C. A 35 mm culture dish of 1.0 × 106 mPTCs was used, and intervention was carried out when the cell growth density reached 80%–90%. The cells were treated in antimycin 2 μM and oligomycin 2 μM in Krebs–Ringer bicarbonate solution (in mM: 115 NaCl, 1 KH2PO4, 4 KCl, 1 MgSO4, 1.25 CaCl2, 25 NaHCO3, pH 7.4) with for 3 h to cause ATP depletion, followed by 2 h of recovery in normal culture media, to create the ATP-depletion Recovery (ATP-DR) model [31]. P110 or TAT was added at a 2 μM concentration three hours before to the intervention.

Isolation of cytosolic and mitochondrial fractions

Cells were divided into cytosolic and mitochondrial fractions using a mitochondria isolation buffer that contained, with some minor modifications from our earlier work, 225 mM mannitol, 75 mM sucrose, 1 mM ethylene glycol tetraacetic acid, 10 mM Tris-hydrochloride, and protein inhibitor cocktail (pH 7.4). In brief, cells were rinsed with ice-cold PBS and suspended in a cold mitochondria isolation buffer.Then the cells were homogenized by passing through a syringe with a 27-gauge needle five times. The homogenates were centrifuged at 800 × g for 10 min at 4 °C to remove cell debris and nuclei followed by centrifugation at 15,000 × g for 10 min to supernatant the supernatant as cytosolic fraction and the pellet as mitochondrial fraction Fresh kidney tissues were homogenized in a mitochondria isolation buffer containing 0.1% BSA. The homogenates underwent several rounds of centrifugation at 1000 × g for 10 min to eliminate cell debris and nuclei, followed by centrifugation at 15,000 × g for 15 min to collect the supernatant as the cytosolic fraction and the pellet as the mitochondrial fraction.

Measurement of mitochondrial membrane potential and ROS

The mitochondrial membrane potential in living cells was measured with the mitochondrial membrane potential sensitive dye JC-1,which was purchased from MCE (CAS No. 3520–43-2). The cells were treated, and 20 uM of JC-1 (dissolved in DMSO) was added. The cells were then cultured in a 5% CO2 incubator at 37 °C for 15–20 min. An inverted fluorescence microscope was used to measure the fluorescence of the samples.

MitoSOX (Thermo Fisher) was employed for the visualization of mitochondrial reactive oxygen species (ROS). Following the instructions, 5 μM MitoSOX dye (dissolved in DMSO) was applied to the treated cells. The cells were then incubated at 37 °C for 10 min in a 5% CO2 incubator and subsequently washed three times with gentle PBS. Finally, fluorescence images were acquired and examined using an inverted fluorescence microscope.

Co-immunoprecipitation

Thermo Scientific's Pierce's Classic IP Kit, item number 88828, was employed. The pre-treated cells were first lysed with lysate, the antibodies were then incubated with magnetic beads for 30 to 60 min, the lysate was then incubated with the coupled magnetic beads for 2 h or overnight at 4 °C, and a single antibody was added. Finally, the magnetic beads were eluted with eluent to obtain the target protein. Electrophoresis using SDS-PAGE was used to evaluate the related proteins.

RNA extraction and quantitative real-time PCR

The kidney tissue or cell RNA was extracted with RNeasy Plus Extraction Kit (TranGen Biotech) and mRNA was obtained by reverse transcription using PrimeScript RT Master Mix (Vazyme). The StepOnePlus Real-Time PCR System (Applied Biosystems) was used to carry out the quantitative real-time PCR using Fast SYBR Green Master Mix (Vazyme) and the cDNA as the template. The internal check for the processes employed glyceraldehyde 3-phosphate dehydrogenase. The comparative cycle threshold (2-ΔΔCt) approach was used to calculate the relative levels of gene expression. Additional file 7: Table S1 contains a list of primer sequences.

Immunoblotting analysis

The protein content of kidney lysates or cultured cells was assessed using the Pierce bicinchoninic acid reagent from Thermo Fisher Scientific. A similar number of protein samples were loaded for electrophoresis on an SDS–polyacrylamide gel and electroblotting onto a PVDF membrane. The blots were incubated with the main antibody at 4 °C overnight after blocking in 5% non-fat milk or bovine serum albumin, and then with the HRP-conjugated secondary antibody. Following an incubation period with Super Signal West Pico Chemiluminescent Substrate from Thermo Fisher Scientific, the blots were scanned using either a KwikQuant Imager from Kindle Bioscience or a MyECL Imager from Thermo Fisher Scientific to capture the signals. The primary antibodies used for immunoblotting were as follows: p-Drp1(Ser616) (Cell Signaling Technology, 3455S), Cleaved-Caspase-3(Cell Signaling Technology, 9661T), Sirt3(Cell Signaling Technology, 2627 s), cGAS (Cell Signaling Technology, 31659), STING (Cell Signaling Technology, 13647) NF-kappaBp65(Cell Signaling Technology, 8242 T), p-NF-kappaBp65(s536) (Cell Signaling Technology, 3033T), p-IRF3 (Cell Signaling Technology, 4947), TBK1/NAK(Cell Signaling Technology, 3504), Phospho-TBK1/NAK(Ser172)(Cell Signaling Technology, 5483), STING(Abcam, ab288157), DLP1(BD Bioscience, 611116), COX IV(Abcam, ab16056), Bax(Abcam, ab182733), PGC1α(ABclonal, A20995), TFAM(ABclonal, A3173), IRF3((ABclonal, A19717), phospho-IRF3-S386(ABclonal, AP0995),MCP-1(Santa Cruz, sc-52701),IL-1β/IL-1F2(RnDSystems,AF-401-SP), F4/80(Invitrogen,14–4801-82),MFF(Proteintech,17090-1-AP) and MID51(Proteintech, 20164-1-AP).

Cellular respiration assessment using the Seahorse XF24 Analyzer

Mitochondrial respiration was assessed using a Seahorse XF24 extracellular flux analyzer (Seahorse Bioscience, Copenhagen, Denmark). Cells were seeded in Seahorse XF24 cell culture microplates and treated 24 h before analysis. The analysis was conducted in growth media supplemented with 1 mM sodium pyruvate, 1 mM glutamine, and 10 mM glucose. After a 1-h acclimatization period, basal oxygen consumption was recorded for 20 min. Subsequently, OCR measurements were taken following the sequential addition of mitochondrial inhibitors: (1) oligomycin (1.5 mM) to evaluate ATP synthesis coupling efficiency; (2) the uncoupler carbonyl cyanide 4-trifluoromethoxy-phenylhydrazone (0.5 mM) to assess spare respiratory capacity; and (3) a combination of rotenone (0.5 mM) and antimycin A (0.5 mM) to measure nonmitochondrial consumption of O2.

Statistical analyses

ANOVA was utilized for multigroup comparison, and the t test was employed to demonstrate a difference between two groups that was statistically significant. For one-way ANOV A and two-way ANOVA, respectively, the Fisher least significant difference test and the Dunn multiple comparisons test were applied. P values less than 0.05 were deemed significant. The data was presented as mean ± SD. All computations were performed using GraphPad Prism 9.