Animals and establishment of CLP model

All animal experiments in this study were conducted in accordance with the guidelines for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Yancheng No.1 People’s Hospital. Wistar rats (160–200 g; 8–10 weeks old) were purchased from the SLAC Laboratory Animal Co., Ltd, China, and were used to establish an in vivo model through ceacal ligation and puncture (CLP). The CLP model was constructed according to a previous report [20]. Briefly, all rats were anesthetized with isoflurane (2.5%, Sigma-Aldrich, USA) inhalation, and a 2-cm midline laparotomy was made to expose the caecum under aseptic conditions. The caecum was then ligated with a 4 − 0 silk suture and pierced twice with a 20-gauge needle to mimic the sepsis-associated AKI clinical symptoms. The rats in the Sham group had their caecum exposed without perforation. After surgery, rats were placed in individual cages.

To explore the effects of circ_001653 knockdown on SA-AKI progression in vivo, adenovirus carrying shRNA against circ_001653 or comparable control (sh-NC) was constructed and obtained from Ribobio (Guangzhou, China). The rats were injected with adenovirus carrying sh-circ_001653 (1 × 109 TU) or sh-NC via the tail vein [20]. Two days after the surgery, rats were euthanized and the serum, urine, and kidney tissue samples were collected for subsequent experiments.

Hematoxylin and eosin (H&E) staining

Kidney tissues were fixed in 4% paraformaldehyde, embedded with paraffin, and then cut into 5 μm thick sections. The sections were then stained with Hematoxylin and Eosin Staining Kit (Beyotime, Shanghai, China) following the manufacturer’s protocol, respectively. Briefly, sections were dewaxed with xylene for 10 min, and immersed in 100%, 90%, 80%, and 70% gradient alcohol for 5 min each. Then the sections were stained with hematoxylin for 10 min and eosin for 2 min at room temperature. Next, sections were dehydrated with gradient alcohol, permeabilized with xylene, and mounted using neutral balsam. The degree of kidney injury was then evaluated by morphological changes detected under a light microscope. The scoring criteria from 0 to 5 were as follows [21]: 0 = normal morphology; 1 = Only degeneration without necrosis; 2 (25%) = necrosis; 3 (50%) = vacuolar degeneration; 4 (75%) = tubular dilatation; 5 (75%) = hemorrhage.

Cell culture and treatment

Human kidney-2 (HK-2) cells were purchased from ATCC (CRL-2190, USA) and were cultured in DMEM/F12 (Dulbecco’s modified Eagle’s medium; Gibco, USA) supplemented with 10% fetal bovine serum (FBS, DMEM, Gibco, Grand Island, USA) and 1% penicillin-streptomycin and incubated at 37 °C with 5% CO2 atmosphere.

To construct an in vitro cell model of SA-AKI, HK-2 cells (5 × 105 cells/well) were seeded into a 96-well plate and cultured for 24 h until 80% confluence. Subsequently, the cells were stimulated with lipopolysaccharide (LPS, SMB00610, Sigma-Aldrich) for 24 h at different concentrations (0, 2.5, 5, and 10 µg/mL) [22,23,24]. HK-2 cells without LPS treatment were used as the control group.

Cell viability

Cell viability of HK-2 cells was assessed using a CCK-8 assay kit (Beyotime, Shanghai, China). Briefly, cells were grown in a 96-well plate and incubated for 24 h. Then each well was added with 10 µL of CCK8 reagent and cultured for another 2 h at 37 °C. The absorbance was determined at 450 nm using a microplate reader (Promega, USA). Our initial result showed that 10 µg/mL of LPS had more prominent suppression on HK-2 cell viability, thus we used this dose in subsequent experiments.

Cell transfection

To evaluate the role of circ_001653, BUD13, and KEAP, sh-circ_001653 (-1, -2 and − 3) (OBiO Technology, China), sh-BUD13 (OBiO Technology, China), or oe-KEAP1 (GenePharma, China) vectors or the corresponding negative control vectors (sh-NC or oe-NC) were designed and synthesized by GenePharma (Shanghai, China), respectively. HK-2 cells were seeded into a 6-well plate at a density of 1 × 105 cells/well and then transfected with these vectors using Lipofectamine 2000 (11668027, ThermoFisher Scientific (CA, USA) following the manufacturer’s protocol. The cells were then cultured for 48-hours in the incubator at 37 °C with 5% CO2. After 48 h of transfection, cells were then left untreated or treated with 10 µg/mL of LPS for another 24 h. Finally, the cells were harvested for subsequent assays.

Quantitative real-time polymerase chain reaction (qRT-PCR)

The total RNA was extracted from HK-2 cells or kidney tissues using TRIzol® Reagent (15596026, ThermoFisher, CA, USA). iScript™ cDNA Synthesis Kit (1708890, Bio-Rad, USA) was used for reverse transcription into cDNA. The PCR reaction was performed in triplicate using a 7500 Real-Time PCR System with SYBR™ Green PCR Master Mix (4309155, ThermoFisher, USA). The target gene expression level was normalized to β-actin or U6 and calculated using the 2−ΔΔCT method. Primer sequences used in this study are shown in Table 1.

Table 1 Primer sequences used in the studyRibonuclease (RNase) R treatment

RNase R assay was used to assess the stability of circ_001653 and DUSP22 mRNA in HK-2 cells. Briefly, 4 µg of RNAs extracted from HK-2 cells were incubated with RNase R (3 U/µg, RNR07250, Biosearch) for 1 h at 37 °C. After treatment, RNA was extracted with TRIzol, and qRT-PCR was conducted to determine the expression of circ_001653 and circ_001653 linear mRNA (DUSP22) levels in HK-2 cells.

Actinomycin D treatment

To block transcription, 2 mg/ml actinomycin D (SBR00013, Sigma-Aldrich) or negative control DMSO was added into the cell culture medium and cultured for 0, 6, 12, 18, and 24 h. After that, qRT-PCR was carried out to determine the expression level of circ_001653 and KEAP1.

Subcellular localization

Cytoplasmic and nuclear extracts of HK-2 cells were separated using a Cytoplasmic and Nuclear RNA Purification Kit (Norgen Biotek, USA) according to the manufacturer’s protocol. U6 was utilized as the nuclear endogenous control and β-actin as the cytoplasmic endogenous control. The distribution of RNA (U6, β-actin, and circSEMA5A) in the nucleus and cytoplasm of HK-2 cells was measured using qRT-PCR.

Fluorescence in situ hybridization (FISH) assay

FAM-labeled circ_001653 probes were designed and synthesized by Foco (Guangzhou, China). The subcellular localization of circ_001653 in HK-2 cells was determined using a FISH Tag™ RNA Red Kit (F32954, ThermoFisher, USA) following the manufacturer’s protocols. Briefly, cells were seeded on slides at the bottom of a 24-well plate and cultured to reach 70% confluence. Then cells were fixed with 4% paraformaldehyde at room temperature for 10 min, and incubated with pre-hybridization buffer at 37 °C for 30 min, followed by incubation with probes in hybridization buffer at 37 °C overnight in the dark. After that, cells were washed, and dehydrated, and DAPI was used to stain the nuclei. Images were taken with a laser scanning confocal microscope (Eclipse E6000; Nikon, Corporation, Tokyo, Japan).

TUNEL assay

TUNEL assay was used to determine the cell apoptotic rate. In brief, HK-2 cells (2 × 105 per group) were seeded on coverslips in a 24-well plate. The cells were then fixed with 4% paraformaldehyde before being treated with methanol and 30% H2O2 at a 50:1 ratio at room temperature for 30 min. After that, a One-step TUNEL In Situ Apoptosis Kit (E-CK-A320, Elabscience, USA) was used to detect TUNEL-positive cells in each group, according to the manufacturer’s procedure. Slides were incubated with 100 µL TdT Equilibration Buffer at 37 °C for 20 min. After removing the TdT Equilibration Buffer, cells were then stained with 50 µL working solution containing 35 µL TdT Equilibration Buffer, 10 µL Labeling Solution, and 5 µL TdT Enzyme at 37 °C for 60 min in the dark. Thereafter, the cell nuclei were stained with DAPI at room temperature in the dark and rinsed with PBS thrice. Coverslips were mounted with mounting medium and images were taken using a Zeiss Axioplan 2 microscope.

EdU assay

The proliferation rate of HK-2 cells was determined using an EdU Staining Proliferation Kit (iFluor 647) (ab222421, Abcam, USA). Briefly, HK-2 cells were grown in 96-well plates and cultured for one day. Then cells were stained with 100 µL of EdU solution (50 µM) for 2 h under optimal growth conditions. Following a washing step, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 15 min and then mixed with 0.5% Triton-X-100 (Sigma-Aldrich) for 20 min. After that, the nucleus was stained with DAPI (Sigma-Aldrich). Finally, the EdU-positive cells were observed under a fluorescence-inverted microscope (Olympus, Tokyo, Japan) and the percentage of EdU-positive cells was calculated.

Immunofluorescence measurement of ROS levels

A DCFDA/H2DCFDA-Cellular ROS Assay Kit (ab113851, Abcam) was used to determine the oxidative stress level in HK-2 cells. Briefly, HK-2 cells were seeded at a density of 3 × 105 cells/well into a 6-well plate. Following the indicated treatment, HK-2 cells were washed with PBS before being stained for 30 min at 37 °C with a 5 µmol/L solution of 2′,7′-dichlorofluorescin diacetate (DCFDA) in the dark. Finally, an Array Scan VII microscope (BX53, Japan) was used to scan the signal with the excitation wavelength at 488 nm and the emission wavelength at 525 nm. Ten randomly selected fields were chosen, and the target area of each section was quantified independently using the Indica Labs - Area Quantification FL v2.1.2 module in Halo v3.0.311.314 analysis software, and the mean intensity was calculated.

Determination of oxidative stress parameters

Lithium-heparin tubes were used to collect blood samples, which were then centrifuged for 10 min at 1,500 × g to separate the serum. The supernatant of cells after indicated transfection or treatment was collected from the medium. The contents of malondialdehyde (MDA, #A003-1-2), glutathione (GSH, #A006-2-1), superoxide dismutase (SOD, #A001-3-2), and catalase (CAT, #A007-1-1) in rat serum or cell supernatants were determined with relevant commercial kits according to the manufacturer’s protocol (Nanjing Jiancheng Bioengineering Institute). The optical density (OD) was measured using a spectrophotometer (SpectraMax® M5).

Evaluation of renal functional markers

Rat blood samples were obtained and subjected to centrifugation at 5000 g to separate the serum. To assess kidney function, levels of blood urea nitrogen (BUN) and serum creatinine (sCr) were measured using a colorimetric technique (#C013-1-1, Nanjing Jiancheng) and a creatinine test kit (#C011-2-1, Nanjing Jiancheng, China), following established protocols.

The urine samples were obtained and subjected to centrifugation at 600 g for 5 min. The levels of urinary neutrophil gelatinase-associated lipocalin (uNGAL, #E-EL-R3055, Elabscience) and urinary kidney injury molecule‐1 (uKIM‐1, #CSB-E08808r, Cusabio Biotech, Zhengzhou, China) were then measured using the commercial ELISA kits according to the provided instructions.

Enzyme-linked immunosorbent assay (ELISA) to measure inflammation indexes

The levels of IL-1β, IL-6, and TNF-α proinflammatory cytokines in the serum and supernatant of cultured HK-2 cells were measured using IL-1β ELISA kit (#CSB-E08055r, #CSB-E08053h, Cusabio Biotech, Wuhan, China), IL-6 ELISA kit (#CSB-E04640r, #CSB-E04638h, Cusabio Biotech), and TNF-α ELISA kit (#CSB-E11987r, CSB-E04740h, Cusabio Biotech), following the manufacturer’s instruction, respectively.

RNA immunoprecipitation (RIP) assay

Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (17–701, Sigma-Aldrich, USA) was used for RIP assay as the manual instructed. In brief, following HK-2 cell lysis with lysis buffer (Thermo Fisher) for 5 min, and centrifuged for 10 min at 14,000 rpm at 4 °C to remove the supernatant. Then the lysate was cocultured with magnetic beads coupled to anti-BUD13 (A303-320 A, ThermoFisher, USA), anti-EIF4A3 (PA5-117930, ThermoFisher, USA), anti-RANGAP1 (MA5-34762, ThermoFisher, USA), anti-SLTM (A302-834 A, ThermoFisher, USA), and negative control anti-IgG (Millipore, Massachusetts, USA) overnight at 4 °C. After digestion using Proteinase K buffer for 30 min at 55 °C, immunoprecipitated RNA was extracted and subject to qRT-PCR analysis.

Western blot (WB)

Protein in HK-2 cells or kidney tissue samples was extracted with RIPA lysis buffer (Sigma-Aldrich). The concentration of protein was measured using a Pierce™ BCA Protein Assay Kit (23225, Thermo Fisher, USA). A total of 20 µg of protein was loaded and separated on sodium dodecyl sulfate-polyacrylamide gel and then wet transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were incubated with primary antibodies at 4 °C overnight. One day later after washing, the membranes were incubated with goat anti-rabbit IgG secondary antibody (ab7090, Abcam, Cambridge, UK). Finally, the proteins were visualized using a BM Chemiluminescence Western Blotting Kit (11520709001, Sigma-Aldrich, USA). The primary antibodies were all purchased from Abcam (Cambridge, UK), including KEAP1 (ab227828, 1/2000), Nrf2 (ab62352, 1/1000), HO-1 (ab52947, 1/2000) and β-actin (ab6276, 1/5000).

Statistical analysis

SPSS 22.0 software (SPSS Inc., Chicago, US) was used for statistical analysis. The data were presented as the mean ± SD and all assays were carried out in triplicate. For the comparison of two groups, a student’s two-tailed t-test was used while one-way or two-way ANOVA followed by a post-hoc test was used for the comparison of more than two groups. P value < 0.05 was regarded as statistically significant.