Ethics statement
All experiments were performed using C57BL/6J mice and were conducted according to the Huazhong University of Science and Technology Guide for the Care and Use of Laboratory Animals. All experimental animal procedures were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology, Wuhan, China.
Animals
Experiments were performed with 7- to 8-week-old male C57BL/6 mice (weighing 20-25 g) purchased from Hua Fukang Company (Beijing, China). The animals were housed according to institutional animal research guidelines and were maintained under constant temperature, a 12-hour light/dark cycle, and 50±5% humidity with standard mouse chow and water ad libitum.
Cisplatin-induced acute kidney injury (AKI) model and treatment protocol
The experimental design is shown in Figure 1. AKI was induced in C57BL/6J mice via intraperitoneal injection of 20 mg/kg body weight cisplatin (Sigma Chemical Co.) which was dissolved in in saline. Mice were randomly allocated to the following groups: saline alone (0.9% NaCl) (control) (n=12), Cis alone (cisplatin, 20 mg/kg) (n=12), Cis+G-CSF (200 μg/kg/d intraperitoneally; Peprotech, Rocky Hill, NJ) (n=12), and Cis+G-CSF/AMD3100 (5mg/kg subcutaneously; Sigma-Aldrich, St. Louis, MO) (n=12). G-CSF treatment was administered over five consecutive days, and AMD3100 was administered 60 min before AKI induction. At 96 hours after the last cisplatin injection, the mice were euthanized. Blood and tissue samples were collected to assess renal function and tissue damage.
Ablation of BM stem cells in mice
To confirm BM (bone marrow) stem cell mobilization following G-CSF/AMD3100 administration, mice were irradiated with 2 separate doses of 4.5 Gy of whole-body γ-radiation at a 2-hour interval for bone marrow ablation (BMA). Mice were randomly assigned to the following groups: irradiation + saline group, irradiation + G-CSF/AMD3100 group, non-irradiation + saline group, and non-irradiation + G-CSF/AMD3100 group. Beginning 1 day after irradiation, G-CSF/AMD3100 was administered as described in the above-mentioned treatment protocol. Control mice that were not subjected to irradiation were injected with an equivalent dose of saline (n=6 per group). The mice were euthanized at 96 hours after treatment, and peripheral blood samples were collected for subsequent flow cytometry (FCM) analyses.
Peripheral blood flow cytometric analysis
Mice were anesthetized with chloraldurat, and peripheral blood samples were collected for FCM analyses. Red blood cells were lysed using Lysing Buffer (Sigma). The remaining cells were labeled with APC-labeled rat anti-mouse CD34, CD133 and CD44 antibodies and PE-labeled rat anti-mouse CXCL4 and c-kit antibodies (all from BD Pharmingen).
Measurement of blood urea nitrogen and serum creatinine levels
Serum samples were obtained from mice 4 d after cisplatin injection. Blood urea nitrogen (BUN) and creatinine levels were subsequently measured using an autoanalyzer (Hitachi 7150 Auto-analyzer; Hitachi, Tokyo, Japan).
Tissue processing and histopathological scoring
After the mice were euthanized, kidney specimens were fixed immediately in 10% buffered formalin for 24 hours at room temperature and then embedded in paraffin, and some kidney specimens were embedded in OCT compound for freezing, then were rapidly frozen in liquid nitrogen. Kidney tissues were cut into 3μm thick sections and were stained with PAS staining for immunohistochemical analysis. Tubulars injury was diagnosed based on the presence of tubular epithelial necrosis, cast formation, tubular dilatation and brush border loss. Renal injury severity was scored in a blinded fashion as described in a previous study17 based on the percentage of tubule lesions in ten randomly selected, non-overlapping fields (magnification, 200x) as follows: 0, 0%; 1, ≤10%; 2, 11-25%; 3, 26-45%; 4, 46-75%; and 5, 76-100%.
Immunofluorescence and immunohistochemical (IHC) studies
Mice were euthanized, and their kidneys were perfused with precooled PBS via the left ventricle. Specimens were embedded in paraffin and were cut into 3µm thick sections and processed for immunostaining. IHC labeling was performed to identify Ki-67-positive cells, which were counted by a blinded investigator in 20 randomly selected cortical and outer medullary (OSOM) fields at a magnification of 400×. In addition, kidney tissue sections were subjected to immunofluorescence staining for BrdU using mouse anti-BrdU monoclonal antibodies (Roche) and Dylight 594-conjugated secondary antibodies (Amyjet) to evaluate tubular epithelial cell proliferation. The number of BrdU-positive cells was determined by counting the numbers of positive nuclei in 20 randomly selected, non-overlapping cortical and OSOM fields at a magnification of 400×.
Apoptosis assay
Apoptosis was evaluated using a terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) assay kit (Roche, Indianapolis, IN, USA). Briefly, C57BL/6J mouse kidney sections were deparaffinized, rehydrated, digested with proteinase K and labeled with a TUNEL reaction mixture for 60 minutes at 37°C. The TUNEL-positive cells corresponding to apoptotic tubular epithelial cells were counted in 20 randomly selected cortical fields at high-power magnification (x400). All tissue sections were viewed and labeled by a blinded examiner
Western blot analysis
A 50µg quantity of total renal cell lysate was separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore; Billerica, MA, USA). Western blotting was performed as described in our previous study18. The following primary antibodies were used: PCNA (Ruiyng, China), Bcl-2 (Santa Cruz Biotechnology), and Bax (Santa Cruz Biotechnology). Anti-mouse or goat HRP-conjugated secondary antibodies (Amyjet) were used to detect protein using an ECL Assay Kit (Bipec Biopharma). β-Tubulin or β-actin was used as an internal control. Band intensity was quantified using ImageJ software (1.44 P).
Quantitative RT-PCR
Total RNA was extracted from tissues with TRIzol reagent (TaKaRa) according to the manufacturer’s instructions. RNA reverse transcription was conducted using a PrimeScript™ RT Reagent Kit (TaKaRa). PCR enzymes and master mixes (DBI Bioscience) were used for real-time PCR, along with primers specific for mouse GAPDH, TNF-α, IL-6, IL-10, Kim1, and Ngal. Relative expression levels were normalized to GAPDH and calculated using the 2−ΔΔCt method. The primer sequences were as follows:
IL-6 forward (F): 5′-TCCAGTTGCCTTCTTGGGAC-3′
reverse (R): 5′-TGCACAACTCTTTTCTCATTTCCAC-3′
IL-10 forward (F): 5′-ATCAGCAGGGGCCAGTAC-3′
reverse (R): 5′-AAGGCTTGGCAACCCAAGT-3′
Kim1 forward (F): 5′-TACCTGGAGTAATCACACTGAAGCA-3′
reverse (R): 5′-TTCAATCTTAGAGACACGGAAGGC-3′
Ngal forward (F): 5′-GGCAGCTTTACGATGTACAGCA-3′
reverse (R): 5′ -TCTGATCCAGTAGCGACAGCC -3′
TNF-α forward (F): 5′-TCACAAAACTTGAGAGTCGTGGTG-3′
reverse (R): 5′-AAAGTGGCTCTACGTTATATTCTGCC-3′
GAPDH forward (F): 5′-GCCAGCCTCGTCTCATAGACA-3′
reverse (R): 5′-AGAGAAGGCAGCCCTGGTAAC-3′
Statistical analysis
Results are presented as the mean ± SEM. For normally distributed data, differences within groups were assessed by ANOVA, and differences between groups were assessed using Tukey’s post hoc test. Student’s t-test was performed to analyze differences between two groups. For data that were not normally distributed, differences within groups were evaluated using the Kruskal–Wallis Htest, and differences between groups were evaluated using the Mann–Whitney U-test. All tests were two-tailed, and a P-value <0.05 was considered significant.