All methods were performed in accordance with the relevant guidelines and regulations. Human AMSC samples were collected with the written consent of all subjects, and the protocol was approved by the Institutional Review Board of the Affiliated Hospital of Xuzhou Medical University. All animal experiments were performed by procedures approved by the Ethics Committee for Animal Research at the Xuzhou Medical University.
The cells were obtained from human abdominal subcutaneous adipose tissues and cultured in a modified minimum essential medium (Hyclone, Logan, NY, USA) supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% Mycillin (Beyotime, Shanghai, China), plated in 25 cm2 T-flasks and maintained at 37°C in a 5% CO2 incubator. The culture medium was changed every 2 or 3 days.
Lentivirus vector production
A green fluorescent protein (GFP) label for a lentivirus vector plasmid system carrying the GDNF gene was constructed by the Ji Kai Gene Company (Shanghai, China). AMSCs at the third passage were transfected with adenovirus vectors at the most appropriate multiplicity of infection (MOI)=20 following standard procedures. GFP expression was observed and detected at 1d, 2d, 3d after lentiviral vector transfection.
For MSCs' characterization, cells were analyzed by flow cytometry (BD, Franklin Lakes, N.J., USA). Cells were detached by treatment with 0.25% trypsin/EDTA, washed with (PBS), and incubated in the dark for (30 mins) at room temperature with the hematopoietic and mesenchymal antibodies, PE-CD34, FITC-CD45, FITC-HLA-DR, APC-CD105, PE-Cy7-CD90, PE-CD73, (BD, USA). Approximately 1×104 cells were then washed with wash flow buffer and re-suspended in (500μl of 1%) formaldehyde solution, examined using flow cytometer and analyzed by Flowjo software (Flowjo, Ashland, Oregon, USA).
Tube Formation Assay
Tube formation was performed as previously described. In brief, endothelial cells (1 × 104) were seeded in a 48-well plate coated with 100μl of growth factor-reduced Matrigel TM (BD, USA) and incubated with basic culture medium, AMSCs culture medium, GDNF-AMSCs culture medium for tube stabilization for 24 h at 37 °C to allow for gelling. Tube formation was observed and quantified by measuring the total tube loops in five random microscopic fields with a computer-assisted microscope (OLYMPUS, JAPAN).
Vascular Ring Assay
The thoracic aorta was removed after euthanasia. The vessels were dissected free of fat and connective tissue and cut into ring segments approximately 2-3 mm in width. The dissection was performed on the tension transducer in the groove full of Krebs solution (K3753, Sigma) and was maintained at 37°C. The aortic segments were equilibrated for 90 minutes and following which they were exposed to standard concentrations of KCl (60Mm) for two times. KCl is washed away immediately after the vasoconstriction is stable. The aortic segments were equilibrated for 20 minutes, afterwards the vasoconstriction was stimulated sequentially by phenylephrine (PE) at a cumulative concentration of 10-9 mol/L-10-4mol/L. After the vasoconstriction was stabilized, the vasodilatation was administered sequentially with acetylcholine (Ach) at a cumulative concentration of 10-9 mol/L-10-4 mol/L and was recorded.
Vasodilatation rate= (The maximum tension before Ach administration－The tension recorded after Ach administration)/ (The maximum tension before Ach administration-The basic tension)* 100%
Endothelial junctions test
AMSCs culture medium, GDNF-AMSCs culture medium was generated as follows. AMSCs and GDNF-AMSCs were cultured in culture flasks. Afterward, culture media from Passage 3–5 were harvested and then stored at −80 °C until use. HUVECs were cultured in 4 conditions, normoxia condition, hypoxia condition for 12h, preconditioned with the AMSCs culture media and GDNF-AMSCs culture media under hypoxic condition. Afterwards, immunofluorescence staining was conducted. The procedure is the same as that described in FMA.
In vivo tracking of AMSCs
Green Fluorescence Protein (GFP)-labelled AMSCs (5*105) were intravenously injected after the UUO model established. The kidney samples were harvested at days 3 and 7 and embedded by optimum cutting temperature (OCT) after fixation and dehydration. OCT-embedded kidneys were sectioned into 10 μm sections and mounted on Superfrost slides to follow the localization of MSCs in kidneys. Slides were stained with DAPI (4’6-diamidino- 2-phenylindole) and were observed under a confocal laser microscope (FV1000; Olympus, Tokyo, Japan)
Nu/nu mice (body weight: 18–22 g, age: 6–8 weeks) were purchased from the Laboratory Animal Centre of Xuzhou Medical University (Jiangsu, China). The animals were maintained at a temperature (22±1°C) and lighting (12 h light-dark cycle) controlled room with free access to food and water. After one week of acclimatization, the mice were randomly divided into four following groups (n=10 in each group): sham-operated mice (sham group), UUO mice treated with intravenous injection of saline solution (UUO group), UUO mice treated with intravenous injection of AMSCs immediately after model establishment (AMSC group), and UUO mice treated with intravenous injection of GDNF-modified AMSCs immediately after the model was established (GDNF-AMSC group). UUO was performed using an established procedure as described. The mice in the AMSC group were intravenously injected through the tail vein with 5×105 GFP labeled AMSCs in 150 μl of saline solution, and the GDNF-AMSC group was injected with 5×105 GFP labeled GDNF-AMSCs in 150 μl of a saline solution via the same route. As a control, the mice in the UUO group were injected with a saline solution.
At days 3 and 7 post-surgery, mice from four groups were sacrificed. Mice blood was collected from the retro-orbital plexus to measure the renal function, including serum creatinine (Scr) and blood urea nitrogen (BUN), then the left kidneys were respectively extracted and decapsulated in saline solution. One part of the kidneys was fixed in 10% formaldehyde for the pathological test, while others were stored at -80°C for later PCR and Western blot analysis.
To evaluate renal morphology, paraffin-embedded mouse kidney sections (4-μm-thick sections) were prepared by a routine procedure. The sections were stained with hematoxylin-eosin, Masson’s trichrome and picrosirius red reagents by standard protocol. The areas of interstitial fibrosis were detected using Masson’s trichrome staining, which was stained dark blue and picrosirius red staining. Ten microscopic visual fields of renal tissues were randomly selected in the sections under high-power magniﬁcation (×40).
FMA was performed as previously described. Briefly, mice were anesthetized with chloral hydrate (10.0%, 0.003 ml/g intraperitoneal) in a supine position. The thoracic cavity was exposed after cutting along bilateral ribs. All solutions were prewarmed to 41°C according to Rafael Karmann et al. One milliliter of heparinized saline followed by 1 ml of 3 M KCl was injected in the beating left ventricle using a vein needle. The right atrium was then cut and the mouse was perfused with 41°C prewarmed PBS (10 ml), immediately followed by 5 ml of the agarose-microbead mixture (500 ml 0.02 mm FluoSpheres plus 4.5 ml 1% agarose/mouse). Kidneys were removed after perfusion for about 1min and carefully placed in a small container surrounded by ice for 10 min. Thereafter, the kidneys were fixed in 4% paraformaldehyde on ice for 2 hours, then incubated in 30% sucrose in PBS at 4°C overnight and embedded by OCT. OCT-embedded kidneys were sectioned into 10 μm sections and mounted on Superfrost slides. For immunofluorescence staining, selected sections were blocked with 10% donkey serum containing 0.4% Triton-X-100 for 1h, and then the slides were respectively immunostained with primary antibodies, including anti-CD31 rabbit antibody (1:20, ab28364, Abcam), anti-alpha smooth muscle actin rabbit antibody (1:100, ab32575, Abcam), anti-alpha smooth muscle actin mouse antibody (1:200, ab7817, Abcam), Anti-VE-Cadherin antibody (1:200, ab33168, Abcam). To visualize the primary antibodies, slides were stained with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (1:2000, #A-21202, ThermoFisher) and Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 594 (1:200, #A-11037, ThermoFisher). Thereafter, all slides were viewed with a confocal laser microscope (FV1000; Olympus, Tokyo, Japan).
Kidney tissue samples were lysed in RIPA and PMSF on ice for 30 min and then centrifuged at 12,000 rpm for 15 min at 4°C. After the protein samples were heated in boiling water for 5 min, approximately 50 μg of total protein was loaded on 8%, 10% or 12% sodium dodecyl sulfate-polyacrylamide (SDS) gels and transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting. Non-specific binding was blocked by incubating the membrane in 5% non-fat dry milk for 1h at room temperature. The membrane was then incubated with primary antibodies against HIF-1α(1:500, 20960-1-AP, Proteintech), gp91-phox (1:800, ab180642, Abcam), p67-phox (1:2000, sc-374510, santa cruze), VEGF1(1:200, ab39256, Abcam), TGF-β1(1:500, ab92486, Abcam), α-SMA (1:1000, #19245, Cell Signaling Technology), p-enos (1:1000, #9570, Cell Signaling Technology), enos (1:1000, #32027, Cell Signaling Technology), PI3K (1:1000, #4249,Cell Signaling Technology), p-Akt (1:2000, #4060, Cell Signaling Technology), Akt (1:1000, #9272, Cell Signaling Technology) at 4°C overnight, followed by an incubation with ECL secondary antibodies. The signal was detected with ImageQuant LAS 4000 mini and was quantified by beta-actin.
RNA from kidney samples was isolated using the RNeasy kit (Roche, USA). Quantitative RT-PCR was carried out using an ABI PRISM 7300 Sequence Detection System. The final reaction contained template complementary DNA, iTaq SYBR Green Supermix with ROX (Bio-Rad, Hercules, CA, USA) and gene-specific primers. The sequences of the mouse primer pairs used in qRT-PCR are as follows. TGF-β1 (mouse) forward primer, 5’- TCGCTTTGTACAACAGCACC-3’, reverse primer, 5’-ACTGCTTCCCGAATGTCTGA -3’; HIF-1 a (mouse), forward primer, 5’-CTCACCAGACAGAGCAGGAA-3’, reverse primer, 5’-AAGGGAGCCATCATGTTCCA -3’; gp91-phox (mouse), forward primer, 5’- GAGGTTGGTTCGGTTTTGGC-3’, reverse primer, 5’-TGCACAGCAAAGTGATTGGC-3’; p67-phox (mouse), forward primer, 5’- AACATAGGCTGCGTGAACACTATCC-3’, reverse primer,5’- GCAAGGTCGTACTTCTCCATTCTGTAG -3’. The conditions, 50◦C for 2 min and 95◦C for 10 min, followed by 40 cycles for 30 sec at 95◦C, 45 sec at 60◦C and 30sec at 72◦C were applied. GADPH was used as an internal control. The cycle threshold values of GADPH and other specific genes were acquired after PCR. The normalized fold expression was obtained using the 2-ΔΔCT method. The results were expressed as the normalized fold expression for each gene.
Data are presented as the mean±SD, and statistical analysis was performed with Graph- Pad Prism software, version 6.0c. Comparisons between groups were analyzed by one-way analysis of variance (ANOVA) with Turkey’s or Dunnett’s post hoc test or post hoc Bonferroni correction. A P-value less than 0.05 was considered statistically significant.