Patients

Patients diagnosed with DN (n = 50) and healthy volunteers (n = 50) from the general population willing to undergo physical examinations were enrolled in this study at the Longhua Hospital during the period extending from December 2020 to March 2022. Patients diagnosed with DN were separated into two groups: those with microalbuminuria (UAER between 30 mg/24 h and 300 mg/24 h) and those with macroalbuminuria (UAER over 300 mg/24 h). Table 1 displays the clinical characteristics of the DN participants that were studied. Blood was collected from patients as well as normal healthy volunteers and centrifuged to obtain serum samples, which were then kept in a refrigerator at − 80 °C until further experimental procedures. Written informed consent was obtained from each of the participant. Our research was approved by the Ethics Committee of Longhua Hospital, Shanghai University of Traditional Chinese Medicine.

Table 1 Characteristics of the participants stratified Animal experiment

All procedures using animals in this study were performed according to the protocol approved by the Institutional Animal Care and Use Committee of Longhua Hospital, Shanghai University of Traditional Chinese Medicine, China. The animal ethics number is 20180702A. Eight-week-old male C57BLKS/J db/db and C57BLKS/J db/m mice obtained from the Aier Matt Experimental Animal Company (Suzhou, China). The mice were adapted for 2 weeks in specific situations. The db/m mice were randomly divided into two groups (n = 8 per group): normal group (db/m group) and normal + CRD group (400 mg/kg) (db/m + CRD group), while db/db mice were assigned into five groups (n = 8 per group): (1) DN group, (2) DN + glibenclamide group (Gli, 5 mg/kg, DN + Gli group), (3) DN + CRD group (400 mg/kg), (4) DN + CRD (400 mg/kg) + inhibitor NC group, and (5) DN + CRD (400 mg/kg) + miR-193b-5p inhibitor group. The mice in the CRD treatment groups were administrated by means of intragastric administration with indicated dose of CRD. The mice in db/m group were given the same dose of normal saline for gavage. The CRD treatment lasted for the next 12 weeks. MiR-193b-5p inhibitor or its negative control inhibitor NC was intravenously injected into the tail vein of db/db mice at 9th week intravenously via tail vein at doses of 5 µg/mice three times per week for 4 weeks. During the study, fasting blood glucose (FBG) was measured weekly, the 24 h urine from the mice was collected in metabolic cages to quantify urinary protein. All mice were fasted for 12 h to sacrifice under anesthesia, blood samples and renal tissues were collected to measure corresponding biochemical parameters and renal pathological changes, the kidney weight (KW) was also recorded. Serum was isolated from whole blood samples through centrifugation (3000 rpm for 10 min). Serum creatinine (Scr) or blood urea nitrogen (BUN) level was respectively detected using Creatinine Colorimetric Assay Kit (Cayman Chemical) or BUN detection kit (StressMarq Biosciences, Victoria, British Columbia) following the instructions of the manufacturer.

Immunofluorescence

The cells (3 × 104) were cultured on coverslips in 35 mm dishes. The preparations were washed with phosphate-buffered saline (PBS) and fixed with 3.7% formaldehyde for 30 min. The cells were washed with PBS again and permeabilized with 0.5% Triton X-100 for 10 min. After this time, the cells were washed with PBS, and the primary antibodies (MCL-1, Abcam, ab32087, 1:500) were added. The incubations with antibodies were performed in a humidified chamber overnight. The cells were washed with PBS again, and the secondary antibody was added and incubated for 3 h. The images were obtained using a confocal laser-scanning microscope (LSM 510-ZEISS).

Immunohistochemistry

Kidney tissues were initially fixed in 4% paraformaldehyde and further embedded in paraffin. Tissues were sectioned at 5 μm thickness, deparaffinized with xylene and rehydrated with alcohol in a gradient manner. Internal peroxidase activity was inactivated using 3% hydrogen peroxide in 100% methanol for 20 min at room temperature after antigen retrieval for 5 min at 121 °C using 10 mM citrate buffer (pH 6.0). Further, the sections were blocked using 10% goat serum for 30 min at RT. Further, incubation with primary antibody MCL-1 (Abcam, ab32087, 1:500) was achieved overnight at 4 °C. Further, the sections were washed using PBS. Sections were also incubated with biotinylated secondary antibody and horse radish peroxidase conjugated with streptavidin. Incubation with diaminobenzidine at RT for 2 min allowed formation of the brown color. The sections were further counter stained with hematoxylin. The slides were visualized using a light microscope.

Anthropometric and biochemical

Venous blood from upper limb of the enrolled subjects were collected after fasting. Age, gender, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), blood lipid indexes such as total cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C), albuminuria, eGFR, high-sensitivity C-reactive protein were all detected and recorded.

Cell culture and hyperglycemic induction

Human renal proximal tubular cells (HK-2) were obtained from Bena Culture Collection, Suzhou, China. Cells were further cultured in DMEM (Gibco, Thermo Fisher) medium containing 10% FBS (Gibco), 100 U/mL penicillin, and 100 U/mL streptomycin. The cells were further maintained at 37 °C and 5% CO2. The cells were treated with normal glucose (5.5 mM d-glucose, NG group), high-glucose (30 mM d-glucose, HG group), or osmotic control conditions (5.5 mM glucose + 24.5 mM d-mannitol, OC group) for 12, 24 or 36 h. Further the cells were treated with CRD at 0–100 µM concentration for 1 h prior to incubation with NG or HG.

Cell viability assay-MTT assay

HK-2 cells were initially seeded onto 96 well plates and post 24 h, cells were treated with varying concentrations of CRD. Further, cells were exposed to 10 µL of MTT. Finally, the purple formazan crystals formed were dissolved using DMSO and colorimetric assessment at 590 nm was performed.

5-Ethynyl-2′-deoxyuridine (EdU) staining assay

EdU staining of the cells were performed using BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 488 (Beyotime). The cells were briefly treated with 10 µM EdU reagent for 2 h and counterstained with DAPI. The EdU stained cells were visualized and imaged for further assessment.

Cell transfection

miR-193b-5p mimics and inhibitors and their respective controls were designed and synthesized by Gene Pharma (Shanghai, China). Cells were seeded onto a 24-well plate and were transfected with the mimics, inhibitors or the respective controls using lipofectamine 2000 reagent according to the manufacturer’s protocol.

siRNA interference assay

Three pairs of small interfering RNAs (siRNAs) were designed to knockdown the expression of MCL-1. All siRNAs were synthesized by Ribobio (Guangzhou, China). The targeting sequences are listed followed: MCL-1#1-F: 5′-GCUUGUAAAUGUAUUUGUAAA-3′, MCL-1#1-R: 5′-UACAAAUACAUUUACAAGCUG-3′; MCL-1#2-F: 5′-GGUUACUGAUGACUUACAAAU-3′, MCL-1#1-R: 5′-UUGUAAGUCAUCAGUAACCUU-3′; MCL-1#1-F: 5′-GGGUUAGGACCAACUACAAAU-3′, MCL-1#3-R: 5′-UUGUAGUUGGUCCUAACCCUU-3′. The si-NC sequences were 5′-UUCUCCGAACGUGUCACGU-3′ (F) and 5′-ACGUGACACGUUCGGAGAAdTdT-3′ (R). siRNAs were transfected into HK-2 cells for 48 h using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions.

Flow cytometric assessment

Assessment of apoptosis was performed using Annexin V-FITC Apoptosis Detection Kit (#556570, BD Pharmingen). Cells were exposed to HG, miR inhibitors, mimics or CRD for 24 h and the cells were detached, centrifuged, washed and stained with 5 µL of Annexin V and 5 µL of PI with incubated at RT for 15 min in dark. Fluorescence was measured using flow cytometry and apoptosis levels were assessed.

The annexin-V/PI apoptosis assay kit (#556570, BD Pharmingen) was used according to the manufacturer’s recommendation to examine the apoptotic fraction of HK-2 cells. HK-2 cells were washed twice with PBS and resuspended in 100 µL of 1× binding buffer mixed with 5 µL of annexin-V-FITC and 5 µL of a PI staining solution for 15 min in the dark at room temperature. After 15 min of incubation, another 400 µL of binding buffer was added, and the cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Ten thousand cells from the sample were scanned, and the data were analyzed using CellQuest software (BD Biosciences).

TUNEL staining

To further assess level of apoptosis, we performed TUNEL staining using Cell Death Fluorescein Detection Kit (11684795910, Roche Molecular Biochemicals, Mannheim, Germany). Using the Olympus BX-51 light microscope, images were captured. TUNEL-positive cells were assessed from five images per section from each group. For the purpose of identifying apoptosis in renal cells, we employed a TUNEL staining kit (C10619, Invitrogen, USA). After slicing, renal tissues were deparaffinized with xylene twice for 5 min. Following rinsing in distilled water, the sections were dehydrated in a gradient of ethanol (100, 95, 80, 75, and 50%). The tissue sections were then treated in a proteinase K/10 mM Tris solution at 37 °C for 30 min. After applying TUNEL detection solution, each segment was incubated for 60 min at 37 °C in the dark. Anti-fluorescence quenching mounting reagent was used to seal the slices after they were washed three times in PBS. Finally, a fluorescent microscope was used to analyse the sections.

Enzyme-linked immunosorbent assay

The enzyme-linked immunosorbent assay (ELISA) kits of inflammatory cytokine levels such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) were purchased from R&D Systems, Minneapolis, MN, USA.

Assessment of oxidative stress

The assay kits of oxidative stress markers superoxide dismutase (SOD), phospholipid hydroperoxide glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and catalase (CAT) in cell supernatant as well as kidney tissues were measured by commercially available kits, according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

PAS and Masson staining

Renal tissue sections at 4 μm were subjected to Periodic acid Schiff (PAS) and Masson’s trichrome staining to assess glycogen and collagen deposition, respectively. Assessment of glomerular mesangial dilation and sclerosis were performed using previously published protocol [23].

MitoSox staining

MitoSox red (Thermo Fisher Scientific, Waltham, MA, USA) was used to assess mitochondrial ROS levels in HK-2 cells, based on the manufacturer’s instructions. Initially, cells were cultured in 24 well plate for 48 h and subsequently incubated with MitoSox red at a final concentration of 5 µM at 37 °C for 30 min. Finally, cells were washed thrice with PBS before visualization under a light microscope.

DCFH-DA staining

ROS levels were also assessed by staining the cells with dichloro-dihydro-fluorescein diacetate (DCFH-DA, Beyotime Biotechnology) and measured by flow cytometry. HK-2 cells were briefly cultured for 48 h in a 6 well plate. Further, 10 µM of DCFH-DA dye was added to the cells and incubated for 20 min at 37 °C in dark. Post washing the cells with PBS, fluorescent images were obtained at 488/525 excitation/emission wavelengths.

JC-1 staining

JC-1 fluorescence staining was performed to assess the membrane potential of HK-2 cells. Initially, the cells were incubated with JC-1 (10 µg/mL) for 20 min in dark at 37 °C. Post wash, the cells were immediately and visualized using a light microscope.

Dual-luciferase assay

HK-2 cells were co-transfected with wt or mut MCL-1 3′UTR dual-luciferase reporter (100 ng) with miR-193b-5p mimic or NC (50 nM) by using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) in 96-well plates based on the manufacturer’s instructions. The activities of luciferase were measured using Dual-luciferase reporter assay system according to the manufacturer’s instructions (Promega) after 36 h transfection. Firefly luciferase activity was normalized to renilla luciferase activity to adjust for transfection efficiency. Data are expressed as relative luciferase activities.

Western blot

HK-2 cells were thoroughly lysed using RIPA lysis buffer. Total protein in the lysate was assessed using Bradford assay. Equal amounts of proteins were loaded onto sodium dodecyl sulphate polyacrylamide gel. The separated samples were then transferred onto a PVDF membrane. The membrane was further blocked using 5% skim milk and incubated with primary antibodies (MCL-1, Abcam, ab32087, 1:1000), PCNA (Abcam, ab29, 1:1500), Cyclin D1 (Abcam, ab16663, 1:1000), BAX (Abcam, ab32503, 1:1000), Bcl-2 (Abcam, ab182858, 1:1000), Cleaved-caspase-3 (Cell signaling, #9664, 1:1500), NOX1 (Abcam, ab121009, 1:1000), NOX2 (Abcam, ab310337, 1:1500), SOD1 (Abcam, ab51254, 1:1000), SOD2 (Abcam, ab68155, 1:1000), Caspase-3 (Abcam, ab184787, 1:1000), β-Actin (Abcam, ab8226, 1:3000), and GAPDH (Abcam, ab8245, 1:2000), overnight at 4 °C. Further, the blots were washed and incubated with secondary antibody for 1 h at RT. Finally, the bands were visualized using ECL western blotting substrate (Invitrogen, 32109, USA). Western blot images were quantified by using ImageJ (V1.8.0.112, National Institutes of Health, Bethesda, MD), with the density of each band normalized to that of β-Actin or GAPDH (Additional file 2).

Quantitative real-time PCR (qRT-PCR)

The total RNA was isolation using RNA isolation kit (Qiagen, Valencia, CA, USA). Reverse transcription of mRNA was carried out using cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). qRT-PCR were carried out using the QuantiTect Probe RT-PCR Kit (QIAGEN, Valencia, CA, USA), based on the manufacturer’s instructions. The relative expression levels were calculated using the 2−ΔΔCt method. GAPDH was used as the internal control. The primers used in the PCR reactions are listed in Table 2.

Table 2 Primer sequences used in this study Statistical analysis

Data are provided as mean ± standard deviation (SD) of at least three experimental repeats performed independently. All analysis were performed using Graphpad Prism 8.0 software (GraphPad software Inc). Differences among groups were carried out by the one-way ANOVA followed by the Bonferroni post hoc test. Pearson’s correlation coefficients were calculated for correlation analysis. P < 0.05 was considered statistically significant.