A total of 60 age-matched inbred male Brown Norway (BN, RT1ⁿ) and 84 Lewis (LEW, RT¹) rats weighing 200 to 250 g were obtained from the Academy of Military Medical Science [Beijing, China; certificate no. SCXK (JUN) 2007-004]. These animals were maintained in a standard animal laboratory with free activity and free access to water and rodent chow. They were maintained in a temperature controlled environment at 22–24°C with a 12 h light/dark cycle. The rats were fasted for 12 h prior to surgery, and were provided with free access to 10% glucose water following surgery. All the surgical procedures were performed under sanitary conditions, and all the experiments were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals (16). The present study was approved (approval ID no. E2013008K) by the Ethics Committee of Tianjin First Central Hospital (Tianjin, China).

ADSCs were isolated from LEW rats (n=72) using a previously described method (17). Briefly, the rats were anesthetized with isoflurane inhalation (LUNAN Pharmaceutical Co., Ltd., Shandong, China) 14 days before mixed lymphocyte reaction (MLR) assay and autologous cell transplantation, and the subcutaneous adipose tissue was then obtained from the hemi-inguinal regions of these rats, which were carefully excised and minced into ~1 mm³ sections. The adipose tissue was digested in collagenase type I solution (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at 37°C with constant agitation (100 rpm). The stromal cells were separated from the floating adipocytes by centrifugation at 200 × g for 5 min at room temperature. The cells released were then resuspended in DMEM/F12 medium (Gibco, Waltham, MA, USA), then sieved through a 70 µm mesh (BD Falcon, Bedford, MA, USA). The resulting ADSCs were cultivated in DMEM/F12 medium containing 10% FBS (Gibco). When the cells reached confluence, the adherent cells were detached with trypsin/ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich) and reseeded for expansion. Following in vitro culture for 14 days at 37°C, 5% CO₂ and 95% humidity, we obtained a sufficient amount of ADSCs for autologous transplantation. The cultured ADSCs (3×10⁶) from each experimental rat were respectively labeled and cryopreserved in liquid nitrogen [Air Products and Chemicals (Tianjin) Co., Ltd., Tianjin, China] prior to injection.

The cultured ADSCs were characterized for the expression of hematopoietic markers, CD34 and CD45, and mesenchymal cell markers, CD90, CD73 and CD105 by fluorescence-activated cell sorting (FACS) analysis using a flow cytometer (FACSCalibur flow cytometer; Becton-Dickinson, Franklin Lakes, NJ, USA), and data were analyzed using the CellQuest software program.

DSCs were also confirmed by their capacity to differentiate into adipogenic, islet and osteogenic lineages as previously described (17). Briefly, the ADSCs were seeded in medium at 2×10⁴ cells/cm² in 6-well tissue culture plates. When the cells reached 100% confluency, DMEM/F12 was subsequently replaced with specific inducer medium. Adipogenic inducer medium is DMEM/F12 containing 1 µmol/l dexamethasone, 0.5 mmol/l 3-isobutyl-1-methylxanthine (Sigma-Aldrich), 5 mg/l insulin (Sigma-Aldrich), 100 µmol/l indomethacin (Sigma-Aldrich). Islet inducer medium is DMEM (high glucose; Gibco) containing 10 mmol/l nicotinamide (Sigma-Aldrich), 100 µg/l conophylline (BioBioPha, Yunnan, China), 10 µg/l betacellulin (PeproTech, Rocky Hill, NJ, USA), 10 µg/l hepatocyte growth factor (HGF) (PeproTech), 2% B27 supplement (Gibco), 1% N2 supplement (Gibco). Osteogenic inducer medium is DMEM/F12 containing 100 nmol/l dexamethasone (Sigma-Aldrich), 10 mmol/l β-sodium glycerophosphate (Sigma-Aldrich) and 50 µg/ml vitamin C (Sigma-Aldrich). Following a 21-day induction period, adipocytes, islet-like cells, and osteoblasts were identified by Oil Red O staining (Sigma-Aldrich), dithizone staining (Sigma-Aldrich), and Alizarin Red S staining (Genmed, Shanghai, China), respectively.

Briefly, the NCBI database was screened for coding sequences of rat OX40 extracellular domains. RNA was extracted from rat peripheral blood mononuclear cells (PBMCs) using TRIzol reagent (Life Technologies, Carlsbad, CA, USA). cDNA was synthesized using a reverse transcription system (Promega, Madison, WI, USA). The cycling conditions for polymerase chain reaction (PCR) were 94°C for 4 min; followed by 32 cycles of 94°C for 60 sec, 58°C for 60 sec, and 72°C for 60 sec; and then 72°C for 10 min. OX40 primers, designed with Primer Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA), were added into the cDNA mixture for PCR amplification (Promega). The primers used were designed as follows (forward and reverse, respectively): 5′-ACGGGATCCACCACCATGGTTACAGTGAAGCTCAAC-3′ and 5′-CGGAATTCAGGGCCCTCAGGAGCCACC-3′ (underlined letters denote restriction endo-nuclease recognition sequences). These PCR products were inserted into the pcDNA3.1(+)/linker (Life Technologies) plasmid using BamHI and EcoRI restriction sites to generate the recombinant plasmid, pcDNA3.1(+)/OX40-linker. The plasmid was double-checked by BamHI-EcoRI digestion with electrophoresis on agarose gel and sequencing (Sangon Biotech Co., Ltd., Shanghai, China).

A PCR-amplified cDNA fragment encoding the human IgG Fc fragment was inserted into the XhoI and XbaI sites of the pcDNA3.1(+)/OX40-linker vector to obtain the eukaryotic expression vector (plasmids) pcDNA3.1(+)/OX40-IgG Fc (pcDNA3.1/OX40Ig). The human IgG Fc primers used were designed as follows (forward and reverse, respectively): 5′-GTCAGCTCGAGGCAAGCTTCAAGGGCC-3′ and 5′-GCTCTAGACTATTTACCCGGAGACAGGGAGAG-3′ (the underlined letters denote restriction endonuclease recognition sequences). The constructed plasmids were verified by double restriction endonuclease digestion and DNA sequence analysis to confirm the sequence accuracy. The cytomegalovirus (CMV) promoter pcDNA3.1(+)/GFP (Invitrogen, Carlsbad, CA, USA) was used to gauge the transfection efficiency.

The nucleofection of the ADSCs was performed according to the optimized protocols provided by the manufacturer (Amaxa Biosystems, Cologne, Germany). Briefly, prior to nucleofection, a Petri dish culture containing 1 ml of DMEM was incubated in the CO₂ incubator at 37°C. The ADSCs were digested and centrifuged following the adjustment of the density of the suspended cells to 2×10⁶/ml. The cells (5×10⁵) and plasmid (2 µg) were suspended in 100 µl prewarmed nucleofector solution (Amaxa Biosystems). For nucleofection, the program U-23 was selected. Immediately, following nucleofection, the cells were transferred into prewarmed fresh medium in six-well plates, and incubated in a CO₂ incubator at 37°C and monitored daily. The cells were analyzed 24 h post-nucleofection for transfection efficiency by FACS (FACSCalibur flow cytometer; Becton-Dickinson) and a Nikon Ts100 fluorescence microscope (Nikon Corp., Tokyo, Japan) for GFP expression. The ADSCs transduced with pcDNA3.1/OX40Ig or pcDNA3.1/GFP are referred to as ADSCs^OX40Ig or ADSCs^GFP, respectively.

The transduced OX40Ig was confirmed by western blot analysis, as previously described (18). Briefly, the transduced ADSCs (ADSCs^OX40Ig or ADSCs^GFP) were fractionated on a 12% SDS-polyacrylamide gel, and the fractionated proteins were electrophoretically transferred onto nitrocellulose membranes (Millipore, Billerica, MA, USA). The protein samples were then incubated with primary anti-OX40 rabbit polyclonal antibodies (Cat no. ab203220) and thereafter with a horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Cat no. ab6721) (both from Abcam, Cambridge, MA, USA). Protein bands were visualized using an ECL western blotting kit (Amersham Pharmacia Biotech, Piscataway, NJ, USA).

To test the allostimulatory activity of the gene-transduced ADSCs, one-way MLR were performed. Splenocytes from the 24 BN and 24 LEW rats were filtered through nylon cell strainers (BD Falcon). The lymphocyte population was purified by density gradient centrifugation using a commercially lymphocyte separation medium (Sigma-Aldrich) and centrifuged at 400 × g for 25 min. The LEW lymphocytes (1×10⁵, responders) and BN lymphocytes (2×10⁵, stimulator) were co-cultured with untransduced ADSCs (ADSCs^native), ADSCs^GFP, or ADSCs^OX40Ig (1×104) in 0.2 ml of culture medium. The stimulator was mitotically inactivated with 50 µg/ml mitomycin C (Sigma-Aldrich) at 37°C for 30 min prior to MLR co-culture, while the responder was not treated. The mitomycin C pre-treated ADSCs^native, ADSCs^GFP, or ADSCs^OX40Ig were allowed to adhere to the plate for 2 h before the lymphocytes were added to allow attachment. The cultures were maintained at 37°C in a humidified atmosphere of 5% CO₂. Following routine culture for 4 days, MTT colorimetry was used to measure the absorbance values at 570 nm in each well. The inhibitory rate was calculated according to the following formula: inhibitory rate (%) = (1 − average absorbance for experimental group/average absorbance for control group) ×100.

The cells were analyzed for Treg markers using mouse anti-rat monoclonal antibody to CD4 (Cat no. 554843) and CD25 (Cat no. 554866) (both from BD Biosciences, San Diego, CA, USA). A FACSCalibur flow cytometer was used to determine the CD4⁺CD25⁺ cells, and data analysis was performed using CellQuest software (both from BD Biosciences, Franklin Lakes, NJ, USA).

For orthotopic renal transplantation, the 36 LEW rats received a kidney from the 36 BN rats using a previously described technique (19). In brief, following humane animal sacrifice by deep anesthesia with isoflurane inhalation (LUNAN Pharmaceutical Co., Ltd.), the kidneys from the BN rats were harvested, perfused with hypertonic citrate-adenine preservation (HC-A) solution (provide by the Long March Hospital of Shanghai, China) to remove blood from the vascular beds and maintained at 4°C until implantation. The kidney grafts were transplanted orthotopically into left nephrectomized LEW rat recipients, and the blood flow was restored using standard microsurgical techniques. The contralateral kidneys were removed immediately following the implantation of the left kidney graft. After the effect of graft reperfusion was observed, the abdomen was closed. No immunosuppressive agents were provided. All microsurgical procedures were performed by one surgeon. Five days post-transplantation, the rats were euthanized by deep anesthesia as described above, the grafts were removed and subjected to morphological, reverse transcription-quantitative PCR (RT-qPCR) and biochemical analysis.

In the BN-LEW allograft models, the LEW recipient rats were injected with autologous 2.0×10⁶ ADSCs diluted in 1 ml PBS or 1 ml PBS alone via the penile vein 4 days prior to transplantation, and intrarenal injection performed immediately following reperfusion, followed by an intravenous injection 6 h after the ischemia/reperfusion (I/R) procedure through the penile vein. The LEW-LEW syngeneic transplant models (n=12) were used as a control group.

The animals were randomly divided into 4 groups as follows: the isografts control group (n=12), the PBS control group (n=12), the ADSCs^native-treated group (n=12), and the ADSCs^OX40Ig-treated group (n=12).

The 2 ml of blood was collected from the inferior vena cava at days 5 post-transplantation. Serum creatinine (SCr) levels were determined as a measure of renal function via an enzymatic colorimetric method using an automatic biochemistry analyzer (Hitachi High-Technologies Corp., Tokyo, Japan).

The excised kidneys were fixed in formalin overnight and embedded in paraffin wax. Five-micrometer-thick kidney sections were deparaffinized and fixed. For histological analysis, the sections were stained with hematoxylin and eosin (H&E; Sigma-Aldrich), and observed using a Nikon Ni-U fluorescence microscope (Nikon Corp.).

Total RNA was extracted from the frozen kidney tissue samples using TRIzol reagent (Invitrogen) and then subjected to reverse transcription using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems Life Technologies, Foster City, CA, USA). qPCR was performed using an ABI 7500 Sequence Detection System (Applied Biosystems Life Technologies) with SYBR-Green (Takara, Tokyo, Japan). Rat primer sequences for intragraft interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, transforming growth factor-β (TGF-β), forkhead box protein 3 (Foxp3) and β-actin were as follows: IFN-γ sense, 5′-AGGCCATCAGCAACAACATAAGTG-3′ and antisense, 5′-GACAGCTTTGTGCTGGATCTGTG-3′; IL-4 sense, 5′-AGAAGCTGCACCGTGAATGA-3′ and antisense, 5′-TCGTAGGATGCTTTTTAGGCTTTC-3′; IL-10 sense, 5′-AAGGCCATGAATGAGTTTGACAT-3′ and antisense, 5′-CGGGTGGTTCAATTTTTCATTT-3′; TGF-β sense, 5′-CAAGGGCTACCATGCCAACT-3′ and antisense, 5′-CCGGGTTGTGTTGGTTGTAGA-3′; Foxp3 sense, 5′-ACCGTATCTCCTGAGTTCCAT-3′ and antisense, 5′-GTCCAGCTTGACCACAGTTTAT-3′; and β-actin sense, 5′-CGTTGACATCCGTAAAGACCTC-3′ and antisense, 5′-TAGGAGCCAGGGCAGTAATCT-3′. The level of expression was calculated using the 2^−ΔCt method, in which ΔCt was calculated as the Ct value of the target molecule - the Ct value of β-actin.

Data are expressed as the means ± standard deviation. Comparisons among treatment groups were analyzed by one-way analysis of variance (ANOVA). Animal survival analysis was performed using Kaplan-Meier survival estimates, and statistical significance was analyzed by the log-rank test. Statistical analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA). A value of P <0.05 was considered to indicate a statistically significant difference.

The recombinant vector, pcDNA3.1/OX40Ig, was identified by restrict enzyme digestion assay (Fig. 1). The DNA sequencing data of the pcDNA3.1/OX40Ig was consistent with DNA sequences of OX40 (extra) and IgG Fc listed in GenBank (data not shown).

The adherent ADSCs had a spindle-shaped fibroblastic morphology following expansion. The rat ADSCs expressed typical markers and differentiation profiles. They strongly expressed the stem cell markers, CD90, CD73 and CD105, but were negative for the hematopoietic markers, CD34 and CD45, as shown by flow cytometric analysis (Fig. 2A). In addition, the culture-expanded ADSCs were also functionally capable of differentiating into adipocytes, islet-like cells and osteoblasts under inductive culture conditions, and this was confirmed using Oil Red O, dithizone and Alizarin Red S staining (Fig. 2B).

To confirm the nucleofection efficiency, the vector pcDNA3.1/green fluorescent protein (GFP) encoding for GFP was used and observed with fluorescence microscopy and flow cytometry. Green fluorescent ADSCs^GFP could be observed (Fig. 3A and B). The expression of OX40Ig in the ADSCs was examined at the protein level. At 48 h post-transduction, OX40Ig protein was specifically detected in the ADSCs^OX40Ig, but not in the ADSCs^GFP and ADSCs^native (Fig. 3C).

The data indicated that the ADSCs^OX40Ig group markedly inhibited the allostimulatory T cell proliferation compared with the ADSCs^GFP and ADSCs^native groups (P<0.01) (Fig. 4). Flow cytometric analysis of CD4⁺CD25⁺ Tregs revealed that this population was significantly increased in the ADSCs^native, ADSCs^GFP and ADSCs^OX40Ig groups as compared with allogeneic T cells cultured alone (P<0.01). The percentage of CD4⁺CD25⁺ Tregs significantly increased in the ADSCs^OX40Ig group as compared with ADSCs^native and ADSCs^GFP groups (P<0.01) (Fig. 5). These results indicate that OX40Ig genetic modification may substantially enhance the immunosuppressive ability of ADSCs to allogeneic T cell proliferation and may increase the number of Tregs.

Renal transplantation induced a substantial increase in the SCr levels in the PBS control group than in the isografts control group (P<0.01). The administration of ADSCs^OX40Ig and ADSCs^native significantly attenuated the increase in the SCr levels compared with the PBS control group (P<0.01). However, the ADSCs^OX40Ig treated group had significantly lower SCr levels compared with the ADSCs^native treated group (P<0.05) (Fig. 6). Our results indicated that the isograft survival was >90 days. The survival of the allografts in the ADSCs^native treated group (10.2 ± 1.6 days) was slightly, but significantly prolonged compared to that of the PBS control group (6.2±0.8 days) (P<0.05). The administration of ADSCs^OX40Ig markeldy improved allograft survival, increasing the mean graft survival time to 14.2±2.2 days. The survival time of all recipients is shown in Fig. 7.

To determine the reason for graft failure, we then evaluated renal morphologies. The allogeneic PBS control group exhibited the histological characteristics of acute rejection, as shown by dense parenchymal mononuclear cell infiltration, extensive tubulitis and interstitial edema. By contrast, these changes were significantly attenuated (the signs of acute rejection) in the kidneys from the ADSCs^native- and ADSCs^OX40Ig-treated groups, although mild interstitial edema and a small amount of inflammatory cell infiltration was observed, and some tubular dilatation, or some epithelial swelling and degeneration were present. The kidneys from the isografts control group lacked the histological signs of rejection. Consistent with the functional analysis data, the histological examination also confirmed the beneficial effects of the administration of ADSCs^OX40Ig. Representative light microscopic findings are shown in Fig. 8.

Cytokines are key mediators in the induction and effector phases of the immune and inflammatory responses in kidney transplantation. In order to detect the effects of ADSCs on the level of rejection or tolerance-associated cytokines in allograft kidneys, we prepared total allograft mRNA for the measurement of cytokine transcript expression by RT-qPCR. Compared with the isografts control group, the allogeneic PBS control group exhibited a significantly increased mRNA expression of IFN-γ, IL-4, IL-10, TGF-β and Foxp3 in the allografts. Compared with the allogeneic PBS control group, the ADSCs^native- and ADSCs^OX40Ig-treated groups exhibited a significantly decreased expression of IFN-γ in the allografts, and there was a marked downregulation in the IFN-γ mRNA level in the ADSCs^OX40Ig-treated group. Furthermore, compared with the allogeneic PBS control group, the mRNA expression levels of IL-10, TGF-β and Foxp3 were significantly increased in the ADSCs^native- and ADSCs^OX40Ig-treated groups (Fig. 9). A significantly higher mRNA level of the Treg marker, Foxp3, was observed in the ADSCs^OX40Ig-treated group compared with the ADSCs^native-treated group (Fig. 9).