| BackgroundKidney transplantation have been the primary therapeutic option for the end stage of renal disease (ESRD) patients.The acute rejection haved been reduced and short-term graft survival rates been improved greately over the last two decades with the use of new immunosuppressants.However, the long-term graft survival rate have not changed significantly, and chronic allograft rejection now remains as the major cause of kidney functional decline after transplantation. The drugs currently used for chronic renal allograft rejecton are nonspecific immunosuppressants with severe side effects and have no satisfying effect.There is still no single agent known to be capable of preventing and reversing chronic allograft rejection. It is very necessary to explore new approaches for treating chronic allograft rejection without the need for nonspecific immunosuppressants.Bone marrow mesenehymal stem cells reside in bone marrow as a nonhematopoietic cell population. They can differentiate into osteoblasts, chondrocytes, tendons, myocytes and adipocytes under some conditions. MSCs have characteristics of hypoimmunogenicity, immunosuppressive effects and may play a role in tissue repair.It is suggested that MSCs can be used to induce the allograft tolerance and prolong graft survival.Although many studys in vitro showed that MSCs may have effect of suppressing immune rejection, there were few studies in vivo and the conclusion is very different. In some studies, MSCs could promote acceptance of allograft. Some researchs shew that it had not effect and even worse.It have been proven that MSCs can prolong the survival of skin graft in baboon and can be used for treating graft versus host disease(GVHD) and the later have been initiated for phaseâ… andâ…¡trial and shown as a good effect. In heart transplantation, the results are very different. One research found that MSCs could reduce rejection, another found that MSCs even accelerated the rejection. The timing of MSC application as well as the cumulative number of cells administered might be the cause of the different effects of MSCs. The different immune microenvironment under different experimental condiction may be the other important cause of this difference. Studies in animal model have also shown that MSCs can reduce the rejection of transplanted islet cells. Most studies found that MSCs can induce allograft acceptance under certain condictions, especially combined with short course of immunosuppressant treatment.The few studies about the effect of MSCs on kidney transplantation shew that MSCs could alleviate actute rejection of renal allograft, reduce the production of inflammation mediators such as IL-1, TGF-β1 and protect early transplanted kidney.A nother research about human renal transplantation found that donor-derived adipose MSCs can significantly suppress the mixed lymphocyte reaction between donors and recipients,and the proliferation of T lymphocyte in vitro.But the effcts of MSCs on transplanted kidney during chronic rejection and its immunoregulation mechanism in vivo is still unkonw by now. The well-established rat model of chronic renal allograft rejection is established under the condition of a short course of immunosuppressant treatment and can manifest typical histological changes of chronic renal allograft rejection within 12 weeks after transplantation.The model can satisfy the condition of being combined with immunosuppressant treatment, whicn may be able to help MSCs protect transplanted kidney better. Hence, this kind of model was adopted in this study. A cell dosage which have been proven be able to suppress immune response in vito and in vitro and a timing of MSCs which was made mainly according to its passaging pattern were chosen for administration. The effect of donor-derived bone marrow MSCs on histological damages and TGF-β1 expression of transplanted kidney and its adverse effect was observed to evaluate the therapeutic effect. The serum and transplanted tissue concentration of Thl/Th2 associated cytokine was detected to investigate the immunomodulation mechanism of MSCs in vivo.The study may be able to provide some useful data for the clinical application of MSCs to prevent and treat chronic renal allografts.METHOD1 Establishment and modification of a rat model of chronic renal allgraft rejection in SD-Wistar strainsOrthotopic left kidney transplantations were performed in rat strain combinations of SD->Wistar using microsurgery technique. After the animals were anesthetized, the left renal artery, vein, aorta and left ureter of donor rats were separated from each other. The left kidney of the donor rat was perfused via the abdominal aorta with 4℃heparinized Ringer's lactate solution and harvested en bloc, after transecting the ureter, the renal artery close to the aorta, and the renal vein near the vena cava. The donor kidney was preserved in 4℃Ringer's lactate solution. The left kidney of the recipient was removed after transecting the left ureter, the left renal artery and the renal vein close to kidney. End-to-end anastomoses of the donor and recipient left renal artery, vein and ureter were performed. The right native kidney of the recipient was left intact and used as an internal control for each individual rat. All alive recipients received a short course treatment of cyclosporine A (CsA microemulsion) (2 mg/kg/day for 10 days i.p) to suppress acute rejection and one time intramuscular injection of ampicillin sodium(50mg/kg) to prevent infection after transplantation.2 recipient rats were killed and kidney were harvested at week 4,8,12 after transplantation. The specimens were fixed in 10% buffered formalin and embedded in paraffin, then stained with hematoxylin-eosin, periodic Acid-Schiff and PASM-Masson's staining. The graft histology was evaluated to determine whether there were histological changes of chronic rejection according to the Banff 2007 criteria.8 recipient rats were chosed randomly for detecting the lowest CsA concentration on day 4 post-operation.2 Isolation, culture, purification and identification of rat MSCs from the bone marrowThe MSCs were isolated by adhesion separation method according to their ability to adhere to cell culture plastic. Four weeks old male SD rats was killed and degermed to get long bones, and bone marrow cells were collected by flushing the long bones. Cell suspensions were centrifugated at 1000r/min for 3 minutes and cultured in culture flask at 37℃,50mL/L CO2 with L-DMEM medium(supplemented with 20% fetal bovine serum).The adherent cells were chosen for serial subcultivation. The third generation cells in good growth condition was harvested and observed the cell morphology using inverted microscope. The cell cycle of the third generation cells in good growth condition was measured by flow cytometry. The positive rate of CD29, CD34, D44, CD45 was detected to identify the cell phenotype of MSCs by flow cytometry. The mouse IgGl-FITC and IgGl-PE was chosed as isotype control antibody. Adipocyte differentiation was performed to help identify MSCs. The third generation cells in good growth condition was cultured with adipocyte differentiation medium for two weeks. Adipocyte differentiation was confirmed by oil red O staining.3 Effect of donor-derived bone marrow MSCs on histological changes of chronic renal allograft rejectiong in ratsOrthotopic left kidney transplantations were performed in rat strain combinations of SD->Wistar.Male SD rats was as dornor rats and female Wistar rats as recipients. The right native kidney of the recipient was left intact and used as an internal control for each individual rat. All recipients received one time intramuscular injection of ampicillin sodium(50mg/kg) to prevent infection after transplantation.The 23 well-transplanted recipient rats were divided into three groups:MSCs and CsA therapy group(therapy group,n=8),CsA control group(control group, n=8), no therapy group (n=7). All recipients of therapy group and control group received a short course treatment of cyclosporine A (CsA microemulsion) (2 mg/kg/day for 10 days i.p) to suppress acute rejection.The recipients of therapy group received a injection of SD donor-derived bone marrow MSCs suspension at a dosage of 1×10个/kg via left iliac vein before close abdomen during transplantation, and another two dosages of MSCs was injected via tail vein at day 3 and day 7 post-operation.The recipients of control group was injected saline at the same volume and in the same time by the same methods as therapy group.The adverse effect in therapy group was observed after the injection of MSCs.The recipients of no therapy group didn't recived CsA or MSCs treatment.The kidneys of no therapy group were harvested at week 4 post-operation. The recipients of therapy group and control group were killed and the kidneys were harvested at week 12 post-operation. The specimens were stained with hematoxylin-eosin, PAS and PASM-Masson's staining.Histological changes of each kidney were evaluated in a blinded manner by two investigator. Renal histologic changes were semiquantitatively scored on a scale (0-3) for mononuclear cell interstitial inflammation (i), interstitial fibrosis(ci), tubular atrophy (ct), allograft glomerulopathy(cg) and vascular fibrous intimal thickening(cv) based on criteria according to the Banff 2007 and Banff 1997 criteria. The sum score was 0-15. Glomerulosclerosis rates of each specimen were calculated.Anatomy of the recipients of therapy group and control group was performed to determine whether there were tumors caused by MSCs.4 Immunohistology for transforming growth factor-betal (TGF-β1) expressionImmunohistology of each kidney of therapy group and control group at week 12 post-operation were performed to detect TGF-β1 expression.5 Detection of serum and transplanted kidney cytokine concentration by Enzyme linked immunosorbent assay(ELISA)The serum and kidney tissues of therapy group,control group and 8 healthy Wistar rats were collected at week 12 post-operation for detection of IFN-γTNF-α,IL-4,IL-10 concentration by ELISA.Result1 65 rat renal transplantations were performed in the whole study.37 transplantations were performed,20 recipients and only 12 transplanted kidney survived in the pilot study.The rate of success was only 32%.28 transplantations were performed,24 recipients and 23 transplanted kidney survived in the formal study. The rate of success was 82%. In the formal study, the warm ischemia time was about 10S～15S and the cold ischemia time was 40min-60 min, the operative time of the whole operation was 2h-2.5h, the recipients operative time was 1.5h～2h.Transplanted kidneys survived and the size was smaller than normal kidney and the whole graft was paler at week 12 after transplantation. The typical histological changes of chronic renal allograft rejection were observed under microscope, characterized by vascular fibrous intimal thickening. No histolological changes were observed in all the native right kidneys of the recipients.The lowest CsA concentration was 153.2±17.1 ng/ml on day 4 post-operation.2 When primary cells passaged for 3 generations, adherent MSCs had a spindle-shaped or star-shaped fibroblastic morphology. The detection of cell cycle of passage 3 of MSCs by FCM shew that the G0/G1 cells was over 81.5%. Phenotypic analysis of passage 3 of MSCs by FCM shew that the MSCs were positive for CD29 and the positive rate was 98.3%, and positive for CD44, the rate was 99.2%; But lacked expression of CD34 and CD45, the negative rate was 98.9% and 96.6% respectively. After the adipocyte differentiation induction for 2 weeks, the accumulation of lipid-rich vacuoles within cells was observed and the cells turned into round shape or polygon. The inductions were confirmed by oil red O staining.3 All the transplanted kidneys of no therapy group were rejected and got swollen,augmented and dark pale, and become necrotic ultimately within 4 weeks after transplantation (data no shown). No adverse reaction including chill, dyspnea and infection was observed after MSCs injection in therapy group.No tumors were observed in the recipients of therapy group and control group at week 12 after transplantaion. All the transplanted kidneys of therapy group and control group survived and the size were smaller than normal kidney and the whole graft was paler. The various histological changes of chronic renal allograft rejection were observed under microscope.The histological changes of therapy group(5.13±0.99)were lighter compared with control group(10.13±0.99) (P=0.000).The glomerulosclerosis rate of therapy group (%,12.44±1.01) was lower compared with control group(%,27.21±3.23)(P=0.000).4 TGF-β1 expression was observed in tubular epidermal cell, interstitial cell and glomerulus of therapy group and control group.The TGF-β1 expression of therapy group was lower compared with control group (P=0.043).5 The serum IL-4 concentration of therapy group(pg/mL,48.18±5.43)was higher compared with control group (38.53±7.36) (P=0.008).The serum IL-10 and IFN-γconcentration of this two groups was no significantly different(P=0.105 P=0.455).The serum TNF-a concentration of therapy group(pg/mL,15.53±9.63)was lower compared with control group (48.81±19.17)(P=0.000).The serum Th1/Th2 (IFN-y/IL-4) (2.113±1.134) of therapy group was lower compared with control group (3.587±1.292) (P=0.040).The concentrations of IL-4,IL-10,IFN-y and TNF-a in the transplanted kidney of therapy group and control group were higer compared with normal group(all P=0.000).The concentrations of IL-4 in the transplanted kidney of therapy group(311.54±53.45) were higer compared with control group(210.82±56.01)(P=0.000). The concentrations of IL-10, IFN-y and TNF-a in the transplanted kidney of therapy group were no significantly different compared with control group(P=0.254, P=0.349, P=0.480).The Thl/Th2 (IFN-γ/IL-4) (1.41±0.42) in the transplanted kidneys of therapy group was lower compared with control group Thl/Th2 (IFN-γ/IL-4) (2.40±0.83) (P=0.009).Conclusion1 An experimental rat model of renal chronic allgraft rejection model in SD-Wistar strains can be established by a short course treatment of cyclosporine A (CsA microemulsion) (2 mg/kg/day for 10 days i.p) for recipeients.The native rignt kidneys of recipients was kept intact as an internal control for each individual rat. The transplanted kidney of this model can survive for a long time and present typical histological changes of chronic renal allograft rejection within 12 weeks after transplantation.2 Highly pure MSCs can be collected by adhesion separation in vitro.The cultured cells in this study were idendified as marrow mesenchymal stem cells by their morphology, cell phynotype and capacity to differentiate into adipocytes.3 Donor-derived bone marrow MSCs can effectly reduce the histological damages of chronic renal allograft rejection and have a distant effect on protecting transplanted kidney.The dosage and timing of MSCs in this stdudy was not enough to induce graft immune tolerance.No adverse reaction including tumor and infection was observed after MSCs injection in this study.The dosage of MSCs in this stdudy is safe for injection.4 Donor-derived bone marrow MSCs can reduce the expression of TGF-β1 in renal allograft with chronic rejection in vivo.The downregulation of TGF-β1 expression may be the consequence of other mechanism that protect the transplanted kidney with chronic rejection by MSCs and not the cause.5 Thl and Th2 cytokines of rat renal allograft with chronic rejection can be both highly expressed. Donor-derived bone marrow MSCs can promoting a Thl to Th2 shift in a condition of both high level of Thl and Th2 cytokine.Maybe it is the one important mechanism for MSCs to reduce the histological damages of chronic renal allograft rejection and prolong the survival of graft.
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