2.1 The data of participants
40 diabetic patients hospitalized in the Second Hospital of Hebei Medical University from February 2022 to October 2022 were selected, including 20 patients without DKD (DM group) and 20 patients with DKD confirmed by renal biopsy (DKD group). 15 healthy subjects from medical examination department of the same hospital during the same period were selected as the normal control (NC group). All specimens were obtained with the consent of the patients and the protocols were approved by the Clinical Research Ethics Committee of the Second Hospital of Hebei Medical University, Ethical review number: 2022-R059. The clinical data were collected, including age, gender, body mass index (BMI). Fasting for 8 h, venous bloods of all participants were collected for biochemical indicators, including glycosylated hemoglobin (HbA1c), serum creatinine (SCr), total cholesterol (TC), triglyceride (TG). The morning urine of each participant were collected for the measure of ACR. 100 mL of first-morning urine were collected in sterile centrifuge tubes and centrifuged at 3,000 g for 30 min at 4℃ to separate cells from debris, and 0.22µm filter for filtering to remove bacteria, residual cells and debris. The pellet was stored at − 80℃ for subsequent applications. All the samples were processed within 1 h after collection. In order to gain an expression profile of urinary exosomal miRNAs, 4 of DM group and 5 of DKD group respectively were randomly selected for miRNA sequencing.
2.2 Exosome extraction and identification
The urine samples were centrifuged at 15,000g for 20 min at 4°C. The 17,000 g supernatant was ultracentrifuged at 200,000g for1 h at 25°C. The ultracentrifugation steps were repeated 3 times, adding new 17,000g supernatant volume each time to each of the 12 tubes. The suspensions were pooled together. The abundant urinary protein uromodulin or Tamm-Horsfall protein forms very high molecular weight complexes through disulfide linkages. These complexes sediment in the 200,000g spin unless denatured. The resuspended pellet was added to an ultracentrifuge tube, and isolation solution was added to increase the volume to 8 ml. The sample was centrifuged at 200,000g for 1 h at 25°C. The pellet was suspended in 50ul of isolation solution and frozen at -80°C. The exosome samples were diluted 5 times with PBS and then applied to 200-mesh nickel grids. Samples were stained with 2% phosphotungstic acid for 5 min at room temperature, and air-dried. The exosomes were identified by transmission electronic microscope analysis at Shanghai Umibio biotechnology Co Ltd. The exosomal surface markers CD63 and CD9 were detected via western blot. The exosomes from the NC, DM and DKD groups were respectively defined as the NC-Exo, DM-Exo and DKD-Exo.
2.3 Cell culture
Both human tubular epithelial cells (HK-2) and podocytes are purchased from ATCC (American Type Culture Collection). Podocytes were cultured in 5.5 mM D-glucose RPMI-1640 (Gibco, USA) medium containing with 10% fetal bovine serum (Gibco, USA) and 0.5% penicillin/streptomycin 100X (Solarbio, China), then were incubated in the humidified incubator at 37℃ with 5% CO2. HK-2 cells were cultured in average DMEM and DMEM-F12 medium (DMEM; DMEM-F12, Gibco, USA), containing 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.), 10% L-glutamine, 0.5% penicillin/ streptomycin, with 5% CO2 at 37°C. When cell fusion reached about 60%, the cells were respectively treated with NC-Exo, DM-Exo and DKD-Exo, namely, the NC-Exo, DM-Exo and DKD-Exo groups.
2.4 Exosome uptake assay
Exosomes were labeled with diluent C solution (50µl, Life Technologies) and PKH67 (4µl, Sigma-Aldrich Corp., USA) for 4 min at room temperature, then were added to exosomes suspension (DKD-Exo: 50ul) respectively, incubated for 5 min and resuspended in 1% BSA. HK-2 cells and podocytes were respectively treated with PHK67-labeled exosomes for 3 h, 7 h and 24 h, the uptake of exosomes was measured by using an immunofluorescence microscope (Nikon Instruments, Tokyo, Japan), and the pictures were taken using a Nikon inverted fluorescence microscope (Eclipse TE200 microscope).
2.5 Cell viability and apoptosis of podocytes
Cell viability was analyzed by cell counting kit (CCK-8, Dojindo, Japan) according to manufacturer's instructions. HPCs in the logarithmic growth were collected and inoculated into a 96 -well plate and incubated. After culturing for 24 h, experimental samples (NC-Exo; DM-Exo; DKD-Exo) were allocated to each group, with each group comprising six replicate wells. Urinary exosome concentrations were established at 20ug/ul, 40ug/ul, 60ug/ul, and 80ug/ul, respectively. Following a 48-hour incubation period, 10 µL of CCK-8 reagent was introduced to each well of the 96-well plate under sterile conditions, followed by a further 4-hour cell incubation. Optical density (OD) values for each group were determined at a wavelength of 450 nm using an automated microplate reader (Molecular Devices Corporation). Measurements were conducted at least thrice to obtain an average OD value for analytical calculations.
Apoptosis was analyzed using PE Annexin V/7-ADD detection kits. The study categorized samples into three groups: NC-Exo; DM-Exo;DKD-Exo. Following the experimental protocol, HPCs in each group underwent stimulation for 48 hours, were thrice washed with PBS, incubated with trypsin devoid of EDTA, and examined microscopically for round cell morphology prior to terminating digestion with fetal bovine serum-containing cell culture medium. HPC cells were harvested into EP tubes at a density of 1×10^5 cells per tube and subjected to two rounds of centrifugation with precooled PBS. After the addition of 1×binding buffer to each tube, 5ul of PE Annexin V and 7-ADD were gently pipetted into the tubes for mixing. The samples were incubated in the dark at room temperature for 15 minutes, followed by the addition of 400ul 1×binding buffer, thorough mixing, and transfer to flow cytometry tubes equipped with a filter. Apoptosis rates for each group were determined within one hour via flow cytometry(BD, Biosciences, USA), with subsequent data analysis conducted using FlowJo software.
2.6 Western blotting analysis
Total proteins were separated from podocytes and HK-2 cells or exosomes via using radio immunoprecipitation assay (RIPA) lysis buffer (Solarbio, China), the concentrations were estimated by BCA protein analysis kit. 30 ug denatured proteins from each sample were separated by 10% SDS-polyacrylamide gel electrophoresis (PAGE) (Epizyme, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes were blocked by 5% skim milk (BioFroxx, Germany) for 2 h at 24℃, then incubated with primary antibodies: CD63 (1:1000, Abcam), CD9 (1:1000, Abcam), Bax (1:1000, Protein Tech), Bcl-2 (1:1000, Protein Tech ), cleaved caspase-3 (1:500, Cell Signaling), α-SMA(1:1000, Protein Tech);Collagen I (1:500, Immunoway), Fibronectin (1:2000, Immunoway ),β-actin (1:1000, Protein Tech ), GAPDH (1:5000, Abcam) at 4℃ overnight. Next day, the membranes were incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit or anti-mouse secondary antibody (1:5000, Affinity) for 1h at 24℃. The protein bands were detected by using the chemiluminescence (ECL) reagents (General Electric, USA), and quantified by ImageJ software (Bio-Rad, USA).
2.7 MiRNA sequencing
The experimental procedures were conducted by Kangchen Biotech Co., Ltd. (Shanghai, China) as follows: Total RNA was extracted from tissues using TRIzol® Reagent, according to Invitrogen's instructions. Degradation and contamination were assessed on 1% agarose gels, and RNA concentration was measured using the ND-2000 spectrophotometer (NanoDrop Technologies). RNA integrity was subsequently assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). A total of 3 µg of RNA per sample was used for the small RNA library preparation. Sequencing libraries were generated using the NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (NEB, USA), following the manufacturer’s recommendations, and index codes were added to each sample. PCR amplification was carried out using LongAmp Taq 2X Master Mix, SR Primer for Illumina, and index (X) primer. Library quality was evaluated using the Agilent Bioanalyzer 2100 system with DNA High Sensitivity Chips. Following cluster generation, the libraries were sequenced on an Illumina platform, generating 50bp single-end reads. The hairpin structure characteristics of miRNA precursors can aid in predicting novel miRNAs. The expression levels of miRNAs were quantified using the transcripts per million reads (TPM) method. Significantly differentially expressed (DE) miRNAs were identified using DEseq2, with criteria of |log2FC| >1 and FDR < 0.05.
2.8 Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted using RNA-easyTM Isolation Reagent (Vazyme, Nanjing, China). cDNA was synthesized using Bulge-LoopTM miRNA RT-qPCR Starter kit (Ribobio, guangzhou, China). RT-qPCR was performed on a CFX96 PCR system (Bio-Rad, USA) using GoTaq® qPCR Master Mix (Promega, USA). The relative expression of miRNAs were normalized by internal references U6, and then quantified using by 2−△△Ct method. The primers were as follows:
miR-371a-3p: Forward: 5’-ACUCAAACUGUGGGGGCACU-3, Reverse: 5’-AAGUGCCGCCAUCUUUUGAGUGU-3’;
miR-483-5p: Forward: 5’-GCGAAGACGGGAGGAAAGA-3’, Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’;
miR-124-3p: Forward: 5’-CGUGUUCACAGCGGACCUUGAU-3’, Reverse: 5’-UAAGGCACGCGGUGAAUGCCAA-3’;
miR-371a-5p: Forward: 5’-ACUCAAACUGUGGGGGCACU-3’, Reverse: 5’-AAGUGCCGCCAUCUUUUGAGUGU-3’;
U6: Forward: 5’-CTCGCTTCGGCAGCACAU-3’ Reverse: 5’-AACGCTTCACGAATTTGCGT-3’.
2.9 Target gene analyses of differential miRNAs
Target gene predictions for miRNAs were conducted using the miRanda algorithm (http://www.miranda.org/). Predicted target genes were aligned using BLAST (http://blast.ncbi.nlm.nih.gov/)and annotated using theGO (http://www.geneontology.org/)and KEGG(http://www.genome.jp/kegg/)databases. Functional-enrichment analyses, including those involving GO and KEGG, were conducted to identify targets significantly enriched in GO terms and metabolic pathways, with a Bonferroni-corrected P-value ≤ 0.05, compared to the whole-reference gene background. GO functional enrichment and KEGG pathway analyses were conducted using Goatools(https://github.com/tanghaibao/Goatools) and KOBAS (http://kobas.cbi.pku.edu.cn/home.do).
3.0 Statistical analysis
Statistical analysis was performed using Prism (GraphPad 8.0 Software). All data were tested for normality and presented as mean ± standard error of the mean (SEM). The data between the two groups were analyzed by independent-samples t-test. The data in multiple groups were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s test. A level of p<0.05 was identified statistically significant.