2.1 Preparation of MSCs and MSC-ACE2
The processes of isolation, culture and characterization of MSCs were as previously described [17]. MSCs were transfected with a lentiviral plasmid overexpressing ACE2 (LV-ACE2), which was purchased from Shanghai Genechem Company, at a multiplicity of infection (MOI) of 80 as previously described [20], and the collected cells were referred to MSCs-ACE2.
The MSC line overexpressing ACE2 was established and confirmed by detecting the expression of ACE2 mRNA and protein in MSCs-ACE2 and the expression of ACE2 protein in culture medium as previously described [20].
2.2 Animal model, experimental design and sample collection
Sixty female Wistar rats (weight 200–250 g) were obtained from the Animal Center of Shandong University. The animal care and experimental protocol complied with the guidelines of the Ethics Committee of the Second Hospital of Shandong University, which are consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
The diabetes model was established as previously described [17]. Eight weeks after successful establishment of the diabetes model, MSCs or MSCs-ACE2 were administered in 0.5 mL serum-free medium at a cell concentration of 2×106 once a week for 2 weeks via tail vein injection. The rats with diabetes were further randomly divided into three groups (n = 12 in each group): the no treatment group that served as the positive controls (DN); the MSC group that received intravenous injection of MSCs; and the MSC-ACE2 group that received intravenous injection of MSCs-ACE2. Twelve rats without diabetes were used as negative controls. Similarly, 0.5 mL of normal saline was injected into the DN and normal control groups instead of stem cells.
Twenty-four-hour urine samples were collected, and body weights were measured at 16 weeks after STZ injection. Blood samples and kidneys were collected and processed as previously described [17].
2.3 Cell studies
The rat glomerular mesangial cell (GMC) line HBZY-1 was purchased from the Center of Type Culture Collection and cultured in low glucose DMEM (0.3 g/L L-glutamine, 10000 u/ml penicillin and streptomycin) containing 10% FBS in an atmosphere of 5% CO2 at 37°C. One portion of GMCs was transfected with LV-ACE2 at an MOI of 20, and these cells were named GMCs-ACE2. GMCs were cocultured indirectly with stem cells (MSCs or MSC-ACE2) in a collagen-coated Transwell system with a 0.4-µm pore size (Corning, Inc., NY) as previously described [20]. Afterward, the cocultures were stimulated with 100 nmol/L Ang II for 24 hours. The dose of Ang II was determined according to preliminary studies [21]. GMCs cultured in normal media were used as negative controls. GMCs treated with Ang II but without coculture with stem cells were used as positive controls (Ang II-GMC). The experimental groups included GMCs-ACE2 stimulated by Ang II (Ang II-GMC-ACE2), coculture of GMCs and MSCs stimulated by Ang II (Ang II-GMC-MSC), and coculture of GMCs and MSC-ACE2 stimulated by Ang II (Ang II-GMC-MSC-ACE2).
2.4 Parameter detection
2.4.1 Biochemical measurement
Glucose level, serum creatinine level, urinary creatinine level and 24-hour urinary albumin excretion were measured. Renal function was assessed by measurement of creatinine clearance (Ccr). The renal mass index, which is the ratio of kidney weight to body weight, was also calculated.
2.4.2 Light microscopy study
Some renal tissues were embedded in paraffin and cut into 4-µm sections, and TGF-β (Bioworld, USA), FN (Abcam, USA), collagen I (Abcam, USA) and MCP-1 expression (Abcam, USA) in the kidney was detected by immunohistochemistry staining as previously described [17]. Periodic acid Schiff (PAS) staining was used to assess glomerular sclerotic injury. Glomerulosclerosis (GS) was defined and evaluated as described previously [20].
2.4.3 Gene expression
IL-6, IL-1β, TNFα, MCP-1, Ang II and ACE2 mRNA expression was detected by real-time reverse transcription-polymerase chain reaction (RT–PCR) using a sequence detection system (Eppendorf, Hamburg, Germany). The primers used for RT–PCR were designed and generated by TaKaRa Biotechnology. The sequences were as follows: IL-6 forward was 5'-ATTGTATGAACAGCGATGATGCAC-3', and the reverse 5'-CCAGGTAGAAACGGAACTCCAGA-3'; IL-1β forward was 5'-CTACCTATGTCTTGCCCGTGGAG-3', and the reverse 5'-GGGAACATCACACACTAGCAGGTC-3'; TNFα forward was 5'-ATACACTGGCCCGAGGCAAC-3', and the reverse 5'-CCACATCTCGGATCATGCTTTC-3'; MCP-1 forward was 5'-TGTTGATGTGAAACATTATGCC-3', and the reverse 5'-AATGATTCTTGCAAAGACCCTC-3'; Ang II forward was 5'-TGTGCTGAGTCTGGTTGCGA-3', and the reverse 5'-GTTCTGGGGTGAGGGGAGAT-3'; ACE2 forward was 5'-AATCGTAGGCTCTGGGCTTGG-3', and the reverse 5'-TTCGATCAACTGGTTTCGGTTGTA-3'. GAPDH was used as an internal control with the forward and reverse primers being 5’-ACAAGATGGTGAAGGTCGGTG-3’ and 5’-AGAAGGCAGCCCTGGTAACC-3’, respectively.
2.4.4 Protein expression
Total protein was extracted from renal tissues and GMCs and quantified using the Bradford method (Biyotime, Shanghai, China). IL-6, IL-1β, TNFα, Ang II, ACE2 and Ang1-7 protein expression in renal tissues; ACE2 and Ang1-7 protein expression in the supernatant of cell culture media; and IL-6, IL-1β, TNFα, MCP-1, ACE2 and Ang1-7 protein expression in GMCs were detected using a commercial peptide ELISA kit (Bio-Swamp Life Science, China) following the manufacturer’s instructions.
Protein expression in cells was detected by Western blotting (WB) as previously described [17]. The following primary antibodies were employed: p38 (1:1000, Cell Signaling Technology, USA), p-p38 (1:1000, Cell Signaling Technology, USA), p-IκB-α protein (1:50, Santa Cruz Biotechnology, Inc., USA), and β-actin (1:100, Abcam, USA).
2.4.5 Oxidative stress assays
The malondialdehyde (MDA) content and the total superoxide dismutase (SOD) activity in the supernatant of cell culture media and renal tissues were measured using the thiobarbituric acid method and xanthine oxidase method, respectively, with commercially available kits following the manufacturer’s instructions (Jiancheng Institute of Biotechnology, Nanjing, China). GSH and GSSG levels were quantified using a GSH/GSSG Assay Kit (Jiancheng Institute of Biotechnology, Nanjing, China). Freshly dissected kidney tissues were cut into 5-µm sections, and ROS levels were detected via the chemifluorescein method using commercial kits (Genmed, Shanghai, China). ROS in GMCs were determined using the fluorescent probe dihydroethidium (DHE, 1 µmol/L, Biyotime, Shanghai, China).
2.5 Statistical analysis
Statistical analysis was performed using the statistical software package SPSS (version 21.0; Chicago, IL, USA). Differences between groups were evaluated using one-way analysis of variance (ANOVA). For each test, all the data are expressed as the mean ± SD, and a P value < 0.05 was considered a significant difference.