Subjects and samples

This retrospective study was performed using existing clinical information and samples from eligible subjects in Yantai Affiliated Hospital of Binzhou Medical University from December 2021 to December 2022. This study selected trauma-induced kidney injury patients from the cohort who had at least one outpatient serum creatinine measurement between 7 days prior to admission and at least one inpatient creatinine data, with hospitalization greater than 24 h, as the AKI cohort. AKI was defined as an increase in serum creatinine by more than 0.3 mg/dl (26.5 µmol/l) within 48 h or an increase in serum creatinine to over 1.5 times that at baseline within the prior 7 days. The exclusion criteria of participants were: (1) patients who had end-stage renal disease; (2) patients who required renal replacement therapy (dialysis or renal transplantation); (3) change of serum creatinine didn’t attribute to AKI; (3) patients with hospital stay < 48 h or > 30 days; (4) patients with incomplete medical records or follow-up information; (5) Patients dead during 7 days; (6) patients with other comorbidities. Eventually, seventy-two individuals were selected for the final AKI cohort. At the same time, we collected information and samples of health volunteers during the same time period. The medical research ethics committee at the Yantai Affiliated Hospital of Binzhou Medical University approved this retrospective study. The information of participants was deidentified (Table 1). All participants or their legally authorized representatives have provided written informed consent. Recovery was defined as the serum creatinine concentrations falling below 90% of the baseline. Non-recovery was defined as plasma creatinine remaining above 90% of the serum creatinine at presentation or whenever the patient became dialysis dependent.

Table 1 Baseline data for the subjects

The supernatants of peripheral venous blood samples were drawn at the time of AKI diagnosis. Briefly, fresh whole blood was collected in an anticoagulant tube, gently inverted several times to thoroughly mix with the anticoagulant, and placed in an upright position at 4℃ for 15 min. Then, after centrifugation at 3000 rpm for 10 min, the supernatant was taken and placed in a centrifuge tube. The obtained serum was quickly frozen in liquid nitrogen and transported to a -80℃. To perform RNA detection, samples were mobilized from the frozen condition.

Cell culture

HK-2 proximal tubule epithelial cells were cultured at 37ºC, 5% CO2 in keratinocyte serum-free medium (Invitrogen, USA) supplemented with 10% FBS, 100 U/mL penicillin/streptomycin, 5 ng/mL epidermal growth factor and 50 µg/mL BPE. Cell experiments were performed at passages 5–12 when 70–80% confluent. HK-2 cells were seeded in 6-well plates in K-SFM without any supplements, and treated for 24 h with LPS (10 µg/mL). At the end of treatment, media was collected and filtered through 0.2 μm filter.

Cell transfection

Knockdown of hsa_circ_0005519 in HK-2 was performed by transfecting the specific siRNAs (anti-CIRC) (Ribo Bio, China) at the concentration of 50 nM using X-treme siRNA transfection reagent (Roche Applied Science, Germany). Inhibition of miR-98-5p was performed by transfecting the cells with 50 nM miR-98-5p inhibitor (GenePharma, China), using the negative inhibitor served as controls (in-NC). The knockdown efficiency was assesse by reverse transcription-qPCR.

RNA extraction and reverse transcription-qPCR

The extraction and quantification of total RNA were processed using TRIzol reagent (Thermo Fisher Scientific, USA) and a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA), respectively. Then, one µg of total RNA was converted into cDNA using the HiScript® II Q Select RT SuperMix for qPCR (Vazyme; China). Next, the qPCR reaction was performed using 1 µL cDNA in 5 µL SYBR®Green Master Mix (Vazyme; China), using GAPDH or U6 as an endogenous control. Primers used were listed in supplementary Table 1. The expression levels of RNA were calculated by the 2−∆∆Ct method.

In vitro inflammation-related factor assay

TNF-α, IL-6, and IL-10 were quantified in the filtrate of media from HK-2 cells 24 h after stimulation with LPS, by kit-based ELISA (R&D Systems, USA) according to standard protocols provided by the manufacturer.

Assays for myeloperoxidase (MPO) activity and malondialdehyde (MDA) content

MPO activity and MDA content were analyzed to assess the oxidative stress level, using respective assay kits (Nanjing Jiancheng, China) following the manufacturer’s instructions.

Cell viability assay

Cell viability was assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. In brief, HK-2 cells were seeded into 96-well plates for 24-hour culture. MTT solution was then supplemented to the medium, followed by a culture of 2 h. Then, the medium was removed. Cells were resuspended and measured at 570 nm using a plate-reader (Bio-Rad Laboratories). The data was calculated as relative value of control group.

Bioinformatics analysis

ENCORI was used for the prediction of downstream miRNAs for hsa_circ_0005519. The predicted miRNAs were then intersected with those differentially expressed miRNAs from GSE125305 dataset from GEO database (logFC>1 or logFC<-1, P.Value <0.05). ENCORI (clipExpNum >1), TargetScan (Total context + + score <-0.01), and miRDB (Target Score >60) were used for the prediction of miR-98-5p target genes. Then, the target genes were intersected with the AKI-related genes from GeneCards database (Relevance score more than the mean value). The common genes were subjected to OmicShare Tools for KEGG and GO enrichment analyses. The protein-protein interaction (PPI) network was constructed using STRING and Cytoscape. The hub genes were analyzed by Cytoscape.

Dual-luciferase reporter assay

Dual-luciferase reporter plasmids, carrying wild-type or mutant hsa_circ_0005519 or IGF1R, were purchased from Hanbio (China). HK-2 cells were co-transfected with luciferase reporter plasmid and miR-93 mimics or mimic control using Lipofectamine 3000 (Invitrogen, USA). 48 h later, luciferase activity of cells was measured with a Dual Luciferase Reporter Gene Assay Kit (Beyotime, China) after 48 h. The ratio of FLUC activity to RLUC activity was calculated and then normalized to the ratio of the control group.

Western blot analysis of IGF1R protein expression

For analysis of IGF1R protein expression, Western blotting technique was used. HK-2 cells were grown to 70% confluency. Next, the cells were washed with ice-cold PBS and lysed using radioimmunoprecipitation assay buffer (Absin, China) for 15 min at 4℃. The lysis solution was centrifuged at 12,000 rpm for 10 min. The supernatant was discarded, and precipitated protein was measured using the Pierce Coomassie (Bradford) Protein Assay Kit (Thermo Fisher Scientific, USA). Next, 20 µg of protein were loaded in each lane on a tris-glycine gel and transferred to nitrocellulose membranes. The menbrance were then blocked and incubated overnight with the indicated primary antibodies diluted 1:300 and 1:200 of the rabbit-derived polyclonal anti-IGF1R antibody (Santa Cruz, USA), and mouse β-actin (LI-COR Biosciences, USA). After secondary antibody incubation, membranes were scanned on an Odyssey infrared imaging system (LI-COR Biosciences, USA) at 800 nm wavelength. The scanned images were subjected to ImageJ for measurement of integrated density.

Data analysis

The categorical parameters were compared between the control and AKI groups, using Pearson’s chi-squared test. The continuous variables were compared using the Mann-Whitney U test, after verification of non-normality by the Shapiro-Wilk test. The diagnostic capacity of hsa_circ_0005519 to differentiate patients with AKI from health, or recovered patients from those non-recovered ones, was evaluated using an receiver operating characteristic (ROC) curve-based analysis. The criterion for per-comparison significance was set at two-sided p < 0.05.