Flow cytometry identification of hucMSCs

Characterization of the hucMSCs phenotype was performed by flow cytometry. Briefly, cells were collected in phosphate buffer saline (PBS) (Zhejiang Senrui Biotechnology, Huzhou, China) at a density of 1 × 106 cells/ml and incubated with the following antibodies: P-phycoerythrin (PE)-conjugated anti-CD73, PE-conjugated anti-CD90, PE-conjugated anti-CD105, PE-conjugated anti-CD34, PE-conjugated anti-CD45, PE-conjugated anti-CD11b, PE-conjugated anti-CD19, and PE-conjugated anti-HLA-DR at 4 °C in dark for 30 min. The isotope controls used were PE-Mouse IgG1 and PE-Mouse IgG2a. The labeled cells with the above antibodies and their corresponding isotope control were separately analyzed through a multicolor flow cytometer (BD Accuri C6, NY, USA), and then the result pictures were fitted and compared.

Animals

Specific pathogen-free and healthy male Sprague–Dawley rats (200 ± 10 g, 6 weeks, Shanghai SLAC Laboratory Animal Co. Ltd., China) were used in this study. During the experiment, animals' rooms were regularly cleaned up, and sufficient feed and water were provided twice a day. Rats were monitored for signs of disease (illness, injury, or abnormal behavior) at least once a day by animal care and veterinary staff, and the frequency of observation is increased when animals showed signs of abnormality. Rats were transferred to individual cages when their condition deteriorated to such an extent that access to water/food was compromised or that they might be harmed by other animals. Dying or dead animals were quickly removed from their cages. This study was approved by the Committee of Animal Care and Use of Zhejiang Chinese Medical University (Animal Ethics No: IACUC-20211101–13). Rats were maintained under standard feeding conditions (temperature 20 ± 2 °C; humidity 45 ~ 55%; 12 h light and dark cycle) with free access to food and water and acclimatized for two weeks.

Animal experiments

The sample size (32 SD rats) in this study was determined based on the preset minimum number of rats in each group (n = 8), and no a priori sample size calculation was performed before the study.

To evaluate the in vivo efficacy of hucMSCs, 32 rats were randomly (random number table method) divided into four groups (n = 8): (i) normal group (Normal), (ii) diabetic model group (Model), (iii) low dose hucMSCs-treated group (MSC-L), and (iv) high dose hucMSCs-treated group (MSC-H). With the animal cage as experimental unit, four units were set and rats in same group were raised in the same cage. Diabetic model was induced by a single intraperitoneal injection of 50 mg/kg streptozocin (STZ) (dissolved in 0.1 mM citrate buffer, pH 4.5) in rats, as described previously [18]. The normal group received equal volume of citrate buffer. The rats with tail blood glucose concentration ≥ 16.1 mM for 3 consecutive days after 1 week were included as successful modeling, otherwise, the rats that did not meet this inclusion criteria were excluded. During the therapy, blood glucose levels of all rats in four experimental unit were monitored once a week, and rats were eliminated if their blood glucose level did not fulfill the aforementioned screening criteria over two weeks. Then, rats in the MSC-L (5 × 106 cells per rat) and MSC-H (1 × 107 cells per rat) groups were injected with hucMSCs intravenously and rats in the normal and model group were injected with equal volume of PBS once a week for 4 weeks in SPF animal room. Body weight (BW) and fasting blood glucose (FBG) were measured weekly. After 4 weeks, urine samples were collected using metabolic cages from the rats for the detection of 24 h urine protein, urine creatinine and urinary albumin/creatinine ratio. After the physical and biochemical measurements, all rats were anesthetized with CO2 at a flow rate of 2 ~ 5 l/min and euthanized for the following experiments (Fig. 1).

Fig. 1

Timetable and flowchart of rat modeling and cell therapy

The rat DN model construction and its following treatments were performed by four experienced operators in a blinded manner. The first operator designed the experimental protocol and grouping. The second operator was responsible for the treatment of all groups of rats. The third operator was responsible for collecting and recording blood glucose, body weight and metabolism of rats in all groups. The fourth investigator anesthetized all animals and collected samples. To reduce possible confounders, treatments were done at the same time and in a random sequence, and all rats in each cage were housed in the same animal room and given ad libitum access to the same food and water.

Histopathological analysis and immunohistochemical staining

Immediately after euthanasia, kidneys of the rats were removed for weighing. Both sides of kidneys were placed in liquid nitrogen and then preserved in -80℃ for subsequent experiments. And the bilateral kidney were fixed with 4% formaldehyde for 24 h, dehydrated, and embedded in paraffin. Then, 4 μm thick sections were cut in succession and stained using hematoxylin and eosin (HE) and periodic acid Schiff (PAS) for morphology evaluations. For immunohistochemical staining, the paraffin-embedded slices were first dewaxed and antigen retrieval with 0.01 mol/l citrate buffer (pH 6.0, Solarbio, Beijing, China) was performed. After blocking with 8% goat serum for 30 min at room temperature, slices were incubated overnight at 4 °C with p-AMPK and p-mTOR primary antibodies (1:50, Cell Signaling Technology, USA). Subsequently, the sections were treated with horseradish peroxidase-conjugated secondary antibody (PV-9001) for 30 min. After 3,3′-diaminobenzidine (DAB) staining, sections were stained with hematoxylin. For all sections, a microscope was used to obtain the images (Invitrogen EVOS M7000, NY, USA). Image J software (Version 1.49, Bethesda, USA) was used to process the images and quantify the average optical density/area of p-AMPK and p-mTOR. Semiquantitative scoring was performed to assess the degree of renal tissues injury. Vacuolation of tubular epithelial cells and extracellular matrix precipitation were assessed according to the degree of renal tissues injury (score from 0 to 4: 0, normal; 1, minor; 2, mild; 3, moderate and 4, severe), and the renal injury score was calculated by scoring each glomerulus and calculating a weighted average of these scores [22].

Imaging of fluorescently labeled hucMSCs and tracking

To evaluate tissue distribution and in vivo kinetic of hucMSCs, hucMSCs were stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate (DiD) cell labeling solution (Invitrogen, NY, USA) according to the reagent's instructions and washed twice with PBS. The fluorescently labeled hucMSCs were injected intravenously into rats (5 × 106 cells per rat). At 12 h, 24 h, 48 h, 120 h, and 168 h after injection, rat tissues including kidneys, lungs, liver, spleen, brain, and heart were collected for ex vivo imaging. The intensity of fluorescence was quantified using the IVIS® Spectrum system and Living Image Software (PerkinElmer, Massachusetts, USA).

Cell culture and conditioned medium preparation

Rat podocytes were purchased from the Chinese Academy of Sciences (Beijing, China) and cultured in Dulbecco’s modified eagle’s medium (DMEM) (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, NY, USA) and 5% penicillin–streptomycin solution in a 37 °C, 5% CO2 incubator. HucMSCs were obtained from the Cell Resource Bank and Integrated Cell Preparation Center of Xiaoshan District (Hangzhou, China) and were grown in minimum essential medium-alpha modification (α-MEM) with Glutamax™-1 (Gibco, NY, USA). Conditioned medium (CM) of rat podocytes and of hucMSCs were prepared for in vitro experiments. Briefly, rat podocytes and hucMSCs were seeded at a density of 2 × 106 cells/15 cm dish with complete medium. The medium was refreshed at cell confluency of 80% and the cells were allowed to further grow for 48 h. Then 12 ml of the cell culture medium was collected, centrifuged for 10 min at 1200 rpm. The RP-CM and MSC-CM were filtered through a 0.22 µm cell strainer, and stored at -80℃ for further use.

In vitro experiment

To investigate the effects and action mode of hucMSCs on rat podocytes, both MSC-CM and co-cultured hucMSCs were applied for the in vitro treatment. Rat podocytes were divided into four groups as follows: (i) normal group; (ii) model group; (iii) MSC-CM group, and (iv) MSC group. To establish an in vitro cellular model of high glucose-induced damage, rat podocytes in model and MSC-CM groups were pre-treated with high glucose DMEM (33 mM glucose) for 72 h, while the normal group with normal DMEM (17.5 mM glucose) for 72 h. Subsequently, rat podocytes in the MSC-CM group were treated with MSC-CM for 48 h, while rat podocytes in normal and model groups were treated with RP-CM for 48 h. For co-cultured hucMSCs treatment, rat podocytes were inoculated into 6-well plates and cultured in high glucose DMEM for 72 h. After discarding the supernatant, the upper chamber was placed in the co-culture group and hucMSCs (3 × 105 cells/well) were added to the upper chamber and high glucose DMEM medium was added to the lower chamber, followed by co-culture for another 48 h. Moreover, an autophagy inhibitor chloroquine (CQ) (Selleck, Houston, USA) was applied in the MSC-CM + CQ group to verify the autophagy-related actions of MSC-CM. Rat podocytes in the MSC-CM + CQ group were pre-treated with high glucose DMEM for 72 h, followed by the treatment of MSC-CM containing 70 μM CQ for 48 h. All rat podocytes were washed twice with PBS after intervention for follow-up experiments.

Cell viability assay

The cell viability of rat podocytes was determined by using Cell Counting Kit-8 (CCK-8) assay. Briefly, cells were seeded into 96-well plates at a density of 2 × 103 cells/well in 200 µl medium, followed by high glucose or MSC-CM intervention as described above. Aliquots of each 20 µl CCK-8 solution (Beyotime, Nanjing, China) were added to each well and incubated at 37 °C for 2 h, until the color turned to orange. The optical density (OD) was measured at 450 nm by a microplate reader (SpectraMax i3x, Shanghai, China). Cell viability was calculated according to the following formula: [(OD value of each group/ average OD value of the normal group) × 100%]. Each experiment was conducted in triplicate.

Wounding healing assay

To perform wounding healing assay, rat podocytes in the logarithmic growth phase were inoculated into six-well plates at a density of 4 × 104 cell/well. The cell monolayer was then scraped with sterile 100 µl pipette tips to form a cell-free rectangular zone, followed by high glucose or MSC-CM treatment as described above and the mannitol group was set up as an osmotic pressure control (17.5 mM glucose plus 15.5 mM mannitol). Then, cells were washed twice with PBS and replaced with fresh serum-free medium. The cells were observed and imaged at two different time points (0 h and 24 h) by using an inverted microscope (Olympus IX73, Japan). The scratch area was measured by using ImageJ software (Version 1.49, Bethesda, USA). Each experiment was conducted in triplicate.

Cell senescence assay

Cell senescence of rat podocytes was observed by using senescence-associated β-galactosidase (SA-β-gal) staining. Briefly, cells were seeded in six-well plates (4 × 104 cell/well), followed by high glucose or MSC-CM treatment as described above. Cells were then fixed with 2% formaldehyde and stained using SA-β-gal staining kit (Beyotime, Nanjing, China), according to the manufacturer’s instructions. The percentage of SA-β-gal-positive cells was calculated as (the number of SA-β-gal-positive cells / the total number of cells) × 100%. Each experiment was conducted in triplicate.

Real-time PCR (qPCR) analysis

Total RNA from rat podocytes was extracted by using RNAiso Plus reagent (Takara Bio, Beijing, China), quantified by a NanoDrop™ 2000 spectrometer (Thermo Fisher Scientific, MA,USA), and reverse transcribed to cDNA by using a PrimeScript™ RT reagent kit (Takara Bio, Beijing, China). The cDNA was amplified by using the TB Green PCR kit (Takara Bio, Beijing, China). qPCR was performed on LightCycler® 480 system (Roche, Shanghai, China) in a 20 µl qPCR reaction system with the following procedure: pre-incubation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 30 s. GAPDH was used as a reference gene and the primer sequences for all genes are shown in Table 1. The relative mRNA expression was measured by the 2−∆∆Cq method. Each experiment was conducted in triplicate.

Table 1 Primer sequences used for qPCR analysisWestern blot (WB) analysis

The protein expressions of rat podocytes and kidney tissues were determined by using western blot (WB) analysis. Total protein of rat podocytes from each group was extracted with RIPA lysis buffer (Beyotime, Nanjing, China). To ensure the reproducibility of the in vivo WB assays, kidney samples were obtained from three separate rats in the normal, model, MSC-L, and MSC-H groups, respectively. For the protein isolation of the kidney, part of the rat kidney tissue was cut with sterilized scissors and placed in lysate with multi-tissue homogenizer (Tissuelyser-24, Shanghai, China) at 65 Hz for 90 s, followed by lysis at 4℃ for 30 min. The lysate was centrifuged at 4℃ for 15 min, and the supernatant was transferred to the precooled centrifuge tube. The protein concentration was determined by using Bicinchoninic Acid (BCA) Kit (Beyotime, Nanjing, China). 30 μg total protein lysate was loaded onto 10% or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels for separation and transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, MA, USA). The membranes were blocked with 5% skimmed milk for 2 h, followed by incubation overnight at 4℃ with primary antibodies. The primary antibodies used in this experiment are shown in Table 2. The membranes were then probed with the corresponding horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000, cat. no. 14708; Cell Signaling Technology) secondary antibody for 90 min at room temperature. Bands were visualized with ECL reagent (Biological Industries) and blots were displayed on X-ray film (Beyotime, Nanjing, China) and detected by Gel Doc XR + Imaging Systems (Bio-Rad, California, USA). The density of each strip was analyzed by using ImageJ software (version 1.49, Bethesda, USA). Each experiment was conducted in triplicate.

Table 2 Antibodies used for WB analysisStatistical analysis

All data were analyzed by using SPSS 22.0 software (SPSS, Chicago, USA). Firstly, the analysis data were tested for normality. If the data conformed to normality, the Student′s t test was performed when analyzed the statistical significance of two groups and the one-way analysis of variance (ANOVA) based on the least significant difference (LSD) method was used for the multiple comparisons analysis. If the data did not conform to normality, nonparametric statistics were applied. Data were expressed as mean ± standard deviation (SD). Differences were considered statistically significant when P < 0.05 and < 0.01.