Female specific pathogen-free level Sprague Dawley (SD) rats (weight, ~180 g) were obtained from the Experimental Animal Centre of Wuhan University (Wuhan, China). All animal experimental protocols were approved by the Animal Care and Use Committee of Wuhan University. Fusin (V340) antibody, specific to the C-X-C family, was a product of Bioworld Technology, Inc. (St. Louis Park, MN, USA). Monoclonal TGF-β1 antibody was purchased from R&D Systems (Minneapolis, MN, USA). Antibodies for MSC characterization were from Biolegend, Inc. (San Diego, CA, USA).

Bilateral thigh bones were taken under aseptic conditions from 2–3-week-old normal female SD rats. Bone marrow was flushed out by serum-free low glucose Dulbecco’s modified Eagle’s medium (DMEM) after two washes with phosphate-buffered saline (PBS). Next, Percoll lymphocyte separating medium was added (at a 1:1 ratio) and cells were isolated according to the combined method of adherent cell isolation and density gradient centrifugation. Mononuclear cells were absorbed from the cell interface for inoculation. Cells were cultured at 37°C in a humidified incubator with 5% CO₂. After 48–72 h, the nonadherent cells were removed. Following this, culture medium was renewed every other day and cells with a confluency of ~80% were subcultured.

The second passage MSCs were used for flow cytometry (FCM) analysis. The MSC surface markers, CD29, CD105, CD34 and CD45, were analyzed. Antibodies for CD34 and CD45 were labeled with fluorescein isothiocyanate, while antibodies for CD29 and CD105 were labeled with phycoerythrin (PE). Cell surface CXCR4 was detected by PE-labeled antibody through FCM.

Kidney ischemia reperfusion was performed under standard conditions. The two renal pedicles were clamped for 40 min and reperfusion was subsequently performed for 60 min. Following this, kidney homogenate was prepared in PBS buffer at a final concentration of 20 g/l. The homogenate was centrifuged at a speed of 500 × g for 15 min. Supernatant was collected and filtered using a 0.22-μm filter. Aliquots were stored at −70°C for further use. SD rats of ~8 weeks old were used for experiments carried out under aseptic conditions.

MSC total RNA was extracted by TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA). cDNA was synthesized using the RevertAid H Minus First Strand cDNA Synthesis kit (Fermentas, Waltham, MA, USA) and 2 μg RNA templates were applied. Using SYBR Green PCR master mix (Invitrogen Life Technologies), qPCR was performed and products were detected on a Step One Plus Real-Time PCR system (Invitrogen Life Technologies). A 100 nM primer concentration was selected for the PCR system and β-actin was used as the internal reference. Primers used were as follows: CXCR4 forward, 5′-GCTAACACTTACGCAAAGACAT-3′ and reverse, 5′-CGTGAAACAGACAAACAACAG-3′; β-actin forward, 5′-CGTTGACATCCGTAAAGACCTC-3′ and reverse, 5′-TAGGAGCCAGGGCAGTAATCT-3′. PCR was performed for 40 cycles of 15 sec at 95°C, 15 sec at 59°C and 45 sec at 72°C. CXCR4 protein levels were determined by WB following normal protocols. Cells were lysed with buffer containing 50 mM Tris HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 0.5% Triton X-100, 10 mM sodium fluoride, 20 mM 2-ME, 250 μM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride and complete protease inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA), and were incubated at 4°C for 1 h. The lysates were ultrasonicated and centrifuged at 12,000 ×g for 10 min. Protein concentrations were determined using the BCA method. Proteins (50 μg) were separated on eight 10% polyacrylamide sodium dodecyl sulfate gels and electroblotted onto nitrocellulose membranes (Hybond ECL; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Subsequent to blocking with Tris-buffered saline and 5% nonfat dried milk for 2 h, the membrane was incubated overnight at 4°C with antibodies specific to the CXC family (Bioworld Technology, Inc., St. Louis Park, MN, USA) followed by incubation with a horseradish peroxidase-conjugated secondary antibody (goat anti mouse; 1:2,000; Pierce, Rockford, IL, USA) for 45 min at room temperature, and the signals were visualized by enhanced chemiluminescence detection. As a loading control, the blots were reprobed with a specific antibody against human β-actin (mouse anti human; 1:5,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).

MSC migration was evaluated using a 24-well chamber system. In total, 20 μg Matrigel was added to the top chamber prior to injection of MSCs (3×10⁴/ml). Next, 600 μl DMEM containing 10% fetal calf serum with SDF-1α (100 ng/ml) was applied in the bottom chamber. Various contents were added to the top chamber and cells were incubated at 37°C for 12 h. Next, the Matrigel and cells above the microporous film were removed. Cells which had migrated to the lower surface were fixed by pure ethanol for 20 min, stained by DAPI and photographed and counted under fluorescence microscopy.

Statistical significance was assessed by a two-tailed Student’s t-test or analysis of variance test. P<0.05 was considered to indicate a statistically significant difference and P<0.01 was considered to indicate a marked statistically significant difference.

Freshly inoculated MSCs presented a round shape of non-uniform size in the suspension. After 72 h, the number of adherent monolayer fibroblasts with a fusiform shape increased. On day 14, cells reached 90% confluence. Following this, cells were passaged in a 1:3 ratio. After 24 h, passage cells had adhered and exhibited a rapid rate of proliferation. The majority of cells presented a tenuous spindle shape, and protuberant pancake and fibroblast-like shapes were also observed. The third passage of cells with a tenuous spindle shape demonstrated good refractivity. The cell boundary was clear and nuclei were plump in the center. Cells were arranged with a relative directivity which exhibited a radial or vortex pattern. Cells in subsequent passages were similar to those of passage 3, with a uniform size and a tenuous spindle shape (Fig. 1).

Cells of passage 2 were used for analysis and MSC surface markers, CD29, CD105, CD34 and CD45, were applied for MSC characterization. Labeled antibodies were incubated with cells and continued FCM analysis was performed. The results demonstrated a 97.18% positive rate for CD29 and CD105, while the positive rate for CD34 and CD45 was 1.37 and 0.95%, respectively. These results are characteristic of MSC surface features (Fig. 2).

MSCs were subjected to various treatments and FCM analysis was carried out (Fig. 3). The results showed that normal MSCs presented CXCR4 on the membrane (MSC group relative fluorescence intensity, 5.89). The negative control group demonstrated low fluorescence (the primary antibody was replaced by PBS). Homogenate from the ischemia reperfusion renal tissue markedly increased the CXCR4 surface presentation (homogenate group relative fluorescence intensity, 6.22). This increased presentation was inhibited by TGF-β1 antibody (TGF-β1 antibody group relative fluorescence intensity, 4.89), which indicates that TGF-β1 in the homogenate can promote CXCR4 surface presentation. In addition, the positive expression rate may be greatly reduced by CXCR4 antibody (CXCR4 group relative fluorescence intensity, 0.88).

qPCR results revealed that homogenate markedly upregulated CXCR4 mRNA levels (P<0.01) compared with the MSC group. Cells treated with homogenate and CXCR4 antibody demonstrated a small difference from the MSC group (no statistical significance). TGF-β1 antibody partially neutralizes TGF-β1 in the homogenate, which can decrease CXCR4 mRNA levels. The degree of inhibition depends on the antibody concentration (Fig. 4). WB of CXCR4 protein levels in various groups was performed following treatment. Homogenate markedly increased the CXCR4 protein levels, while TGF-β1 neutralization downregulated protein levels, as with mRNA. Inhibition was antibody concentration- (Fig. 5A) and time-dependent (Fig. 5B). CXCR4 antibody can decrease CXCR4 protein levels, which corresponds in part to mRNA levels following antibody treatment.

Normal MSCs also exhibit migration ability to SDF-1 (18.47±5.02 cells/cm²) (Fig. 6). Homogenate from ischemia reperfusion renal tissue markedly increased MSC migration to SDF-1 (38.92±6.79 cells/cm²). TGF-β1 antibody decreased the effects caused by homogenate, in a concentration-dependent manner (Table I). CXCR4 antibody markedly inhibited MSC migration (7.03±2.14 cells/cm²), which suggests that the interaction of CXCR4 and SDF-1 plays an important role in renal ischemia reperfusion-induced MSC chemotaxis.