The Protective Effects And Mechanism Of Bone Marrow Derived Mesenchymal Stem Cells Against Rhabdomyolysis-induced Acute Kidney Injury In Rats

Objective: BMSCs of SD rats were isolated, cultivated, purified and biologically characterized in vitro to provide the basis for their application. Lentiviral vectors were constructed and traced by GFP labeling. Feasibility of BMSCs transplantation for rhabdomyolysis-induced AKI(RIAKI) in rats was observed, and its effects on renal morphology and function of rats with RIAKI were assessed. Therapeutic efficacy of BMSCs on RIAKI was observed at the cellular level. Furthermore, effects of BMSCs on Th17/Treg cell balance and inflammatory mediators were observed,and therapeutic mechanisms of BMSCs for RIAKI were investigated.Methods:1、BMSCs were collected from bilateral hind limbs of healthy male SD rats in sterile conditions, isolated in vitro by density gradient centrifugation, cultured and passaged. P2-P6 generations of BMSCs were preserved, and P3 generation of BMSCs were used for treatment. Cell surface marker molecules and biological properties were detected by flow cytometry, including analyses of growth curve and cell cycle.Multilineage differentiation potential of BMSCs was identified by immunocytochemistry. BMSCs were labeled and amplified with lentiviral vector-mediated GFP, and infected at multiplicity of infection(5, 10, 25, 50) to achieve optimal infection efficiency and cell activity. 2、Therapeutic efficacy of BMSCs on rat models with RIAKI:(1) After deprivation of water(no solid fasting)for 24 h, healthy adult male SD rats were intramuscularly injected with 10 ml/Kg50% glycerin(diluted with normal saline) at the bilateral hind limbs to establish RIAKI rat model.(2) SD rats were randomized into four groups: normal control group(Normal group); sham operation group(NS group); model group(RIAKI group); and treatment group(BMSCs group). The rats in each group were observed for general conditions; serum biochemical indices: blood urea nitrogen(BUN), serum creatinine(Scr), creatine kinase(CK); morphological and pathological changes:compressed muscles(HE staining); kidney(HE, PAS staining) and renal injury pathological scoring; and renal cell apoptosis(TUNEL apoptosis analysis, Western Blotting of caspase3, caspase 9, Bcl-2 and cytochrome C expressions in renal tissues).3、Therapeutic mechanisms of BMSC transplantation in RIAKI rats:(1) Cellular level: proportion of Treg cells(CD25+Foxp3+CD4+T) to total CD4+T cells in spleen tissues of rats in each group was detected by flow cytometry, as well as the proportion of Th17 cells(CD4+, IL-17+representative Th17).(2) Genetic level: levels of Th17cell-specific transcription factor RORγtm RNA and Treg cell-specific transcription factor Foxp3 m RNA in spleen and kidney tissues of rats in each group were determined by RT-PCR.(3) cytokine level: specific cytokines released by Treg cells in sera and renal tissues of rats in each group were detected by ELISA, including:TGF-β, IL-10(anti-inflammatory); Th17 cell-released IL-17(pro-inflammatory);inflammation-associated cytokines TNF-α, IL-6.Results:1、BMSCs cultured by density gradient centrifugation combined with adherent culture were in anelongated spindle shape, with circular or oval nuclei located in the center. Flow cytometry revealed that the BMSCs expressed CD44 and CD90, with positive rates of 89.9% and 91.3% respectively; while CD45 was negative, with a positive rate of 2.8%. Growth curve of passage cells showed that 1-2d were the latent phase, 3-7 d were the logarithmic phase, and after 7 d, plateau phase was entered. Green fluorescence(high GFP expression) was visible just at MOI = 25.According to observations, MSC-GFP> 90%, and cells grew fast, so optimal MOI was determined to be 25 in this study.2、Biochemical index changes: blood BUN, SCr and CK in RIAKI group SD rats progressively elevated after injury, exhibiting significant differences from the pre-intervention period and the Normal group(P<0.01) at 6 h, 24 h, 48 h, 72 h, 96 h and 1 W later. BUN and Scr peaked at 72 h,whereas CK reached its peak at 24 h. The elevated levels and variation trends of the above biochemical indices were in line with the laboratory diagnostic criteria for rhabdomyolysis and acute kidney injury. After treatment with BMSCs, Blood BUN and SCr levels in the BMSCs group decreased significantly compared to the RIAKI group at 24 h, 48 h, 72 h, 96 h and 1 W(P <0.01). Blood CK levels, a muscle tissue damage index, at? ? 24 h, 48 h, 72 h and 96 h were significantly lower for the BMSCs group than the RIAKI group(P <0.01). No significant difference was found in any index between the NS and Normal groups(P> 0.05). Under light microscope,myolysis of local muscle tissues was visible in bilaterial hind limbs of rats in the RIAKI and BMSCs groups. At 24 h, 48 h and 72 h, severity of rhabdomyolysis in the BMSCs group markedly reduced compared to the RIAKI group. Besides, severities of AKI at 24 h, 48 h and 72 h in the BMSCs group mitigated compared to the RIAKI group, which were manifested as reduced renal tubular casts and alleviated epithelial cell necrosis. Apoptosis was detected by TUNEL assay. Under fluorescence microscope, decreased apoptotic renal cells and reduced apoptosis index(AI) were observed in the BMSCs group at 24 h, 48 h, 72 h and 1 w compared to the RIAKI group(P <0.01). WB results revealed extremely low levels of caspase 3, caspase 9and cytochrome C expressions and high Bcl-2 expression levels in the Normal and NS groups. At 24 h, 48 h, 72 h and 1 w, caspase 3, caspase 9 and cytochrome C levels were all lower in the BMSCs group than the RIAKI group(P <0.05). Bcl-2expression levels in the BMSCs group were higher than the RIAKI group at various time points(P <0.05).3 、 Flow cytometry detection of changes in the Treg cell proportion in spleens of rats revealed increased Treg cell proportions and decreased Th17 cell proportions in the BMSCs group at 24 h, 48 h and 72 h compared to the RIAKI group, showing significant differences(P <0.05). RT-q PCR detection of RORγtm RNA and Foxp3 m RNA expressions found down-regulated RORγt m RNA levels and up-regulated Foxp3 m RNA levels in spleen and kidney tissues in the BMSCs group compared to the RIAKI group, presenting significant differences(P<0.05). ELISA detection of serum and renal tissue IL-17, IL-10, TGF-β, IL-6 and TNF-α levels found decreased IL-17 levels(P <0.05), increased IL-10 levels(P<0.05), increased TGF-β levels(P <0.05) and decreased IL-6, TNF-α levels in the BMSCs group compared to the RIAKI group, showing significant differences(P<0.05).Conclusion : 1 、 BMSCs of SD rats were extracted, isolated, purified and identified by density gradient centrifugation combined with adherence screening to obtain sufficient quantities of purified BMSCs with high activity. GFP lentiviral vectors successfully transfected BMSCs, and efficiently expressed GFP, which did not affect the biological characteristics and functions of BMSCs such as growth and differentiation, and thus can be used for labeling and tracing of transplanted BMSCs.2、Rat model of RIAKI was successfully induced by intramuscular injection of 50%glycerol solution at the bilateral hind limbs. Treatment with BMSCs reduced blood BUN, SCr and CK levels in RIAKI rats, and somewhat improved the pathological injury resulted from acute tubular necrosis, thus confirming BMSCs’ therapeutic effect on RIAKI. Further observation of the RIAKI-improving effect of BMSCs at the cellular level found that treatment with BMSCs can reduce the number of apoptotic cells in kidneys of RIAKI rats, lower the AI, and improve the structure and function of kidneys. BMSC transplantation up-regulated the expression of anti-apoptotic protein Bcl-2, inhibited the release of pro-apoptotic factor cyt C, and down-regulated the expressions of apoptotic effectors caspase-9 and caspase-3 2 in renal tissues of RIAKI rats. BMSCs may inhibit renal cell apoptosis by influencing the mitochondrial pathway of apoptosis, and thereby play a renal protective role. No BMSC colonization was found in the kidneys, nor was it confirmed that the BMSCs promoted the repair of AKI by direct "differentiation mechanism" after colonization in the damaged kidneys. Presumably, BMSCs relieved RIAKI by "non-differentiation repair" such as paracrine secretion and immune regulation.3 、 BMSCs improved RIAKI by regulating Th17/Treg cell balance. On the basis of the experiment in part two, the possible "non-differentiation repair" mechanisms, i.e. paracrine secretion and immune regulation, of BMSCs for mitigating RIAKI were further studied. The results demonstrated the presence of Th17/Treg imbalance, changes in specific and inflammatory cytokine levels and presence of pro-/anti-inflammatory response imbalance in RIAK rats. Treatment with BMSCs up-regulated the proportion of Treg cells, down-regulated the expression of Th17 cell-specific RORγt m RNA at the transcriptional level, promoted the up-regulation of Treg cell-specific transcription factor Foxp3 m RNA, and facilitated the restoration of Th17/Treg imbalance.Treatment with BMSCs suppressed the expression of Th17-specific pro-inflammatory cytokine IL-17, and promoted the expressions of Treg anti-inflammatory cytokines IL-10 and TGF-β while suppressing the expressions of inflammatory effectors IL-6and TNF-α, thus further confirming the inflammatory response inhibitory effect of BMSCs at the cytokine level. Hence, the Th17/Treg balance regulating and inflammatory response inhibiting effects of BMSCs were verified at all the cellular,transcriptional and cytokine levels, which may be the important mechanisms for BMSCs to mitigate the severity of RIAKI and to play a renal protective role.