The Function Of Endometrial Regenerative Cells In Treatment Of Renal Ischemia Reperfusion Injury

Background: Renal ischemia-reperfusion injury(IRI) is a major cause of clinical acute kidney injury(AKI), which is a frequent and serious problem in transplantation, renal surgery, chronic renal diseases and trauma. It damaged renal structure, metabolism, function and injured long-term renal function seriously. Vigorous research on IRI has been conducted, but the mortality rate of AKI remains high. Mesenchymal stem cells(MSCs) are a group of self-renewing and pluripotent stromal cells which derived from mesoderm. The researchers found them in a variety of tissues and they can be expanded in vitro and can be induced under certain conditions to differentiate into neural cells, osteoblasts, cartilage cells, cardiac muscle cells, fat cells, etc. They have a broad clinical application prospects. They participate in the repair of kidney injury, reduce renal damage and protect renal function. Endometrial regenerative cells(ERCs) are a novel type of adult MSCs. More studies have reported that ERCs effectively prevent critical limb ischemia and attenuate ulcerative colitis. However, whether ERCs could be feasible to simultaneously suppress inflammatory and immune responses, and repair the damaged tissue following IRI remains unclear. Thus, the aim of this study was to explore the potential role of ERC therapy in prevention of renal IRI.Objective: To investigate the therapeutic effect and mechanism of endometrial regenerative cells to attenuate renal ischemia reperfusion injury in a mouse renal ischemia reperfusion injury model.Methods:(1)We isolate and culture the human endometrial regenerative cells by the density gradient centrifugation and observe the cells morphology.(2)Eight-week-old male C57BL/6 mice weighing 20 to 25 g were housed under conventional experimental environment. Renal IRI in C57BL/6 mice was induced by clipping bilateral renal pedicles for 30 minutes, followed by reperfusion for 48 hours. ERCs were injected(1 million/mouse, i.v.) into mice 2 hours prior to IRI induction. C57BL/6 mice were randomly assigned to three groups(n=6 per group): sham control group, mice were operated by opening and closing the abdomen, without clipping the renal pedicles; untreated IRI group, through a middle abdomen incision, bilateral renal pedicles were clipped for 30 minutes, then released;ERC-treated IRI group, 1 ×106 ERCs were intravenously injected 2 hours before clipping bilateral renal pedicles.(3)After 48 h, the mice were sacrificed and the whole blood, kidney and spleen were collected. Serum levels of both blood urea nitrogen(BUN) and creatinine(Cr) were measured when the mice were sacrificed 48 hours after renal reperfusion using multichannel analyzer. Paraffin sections of kidneys stained with Hematoxylin and eosin(H&E) were used for morphology analysis under light microscopy. We performed immunohistochemical staining by using anti-CD3 and anti-Ly6 G antibodies for detecting intra-renal cellular infiltration of T cells and neutrophils, respectively. The levels of TNF-?, IFN-?, IL-6 or IL-4 in the serum samples taken from mice 48 hours after IRI were measured using an ELISA Kit. Phenotypic analysis of various immune cells was performed using a flow cytometry with staining with antibodies against CD3, CD4, CD8, CD25, F4/80, CD206, CD11 b,Ly6C, and Ly6 G. Results:(1)ERCs are spindle, spindle and fibroblast like cells in the microscope and they grow quickly.(2)Compared with Sham group, untreated IRI mice displayed wide spread tubular necrosis, loss of brush border, cast formation, tubular dilatation, expansion of Bowman's capsule and interstitial edema. In comparison with untreated IRI group, infused ERCs significantly reduced serum levels of BUN and Cr; Infused ERCs significantly attenuates pathological damage and the mices show normal histology.(3)Compared with Sham group, neutrophil and CD3+ T cell infiltrations were markedly increased in untreated IRI group. In comparison with untreated IRI group, administration of ERCs suppressed neutrophil and CD3+ T cell infiltration.(4)Compared with Sham group, the population of CD3+ T cell and Ki67 were significantly increased in the untreated IRI group. In comparison with the untreated IRI group, the population of CD3+ T cell and Ki67 were significantly decreased in ERC-treated group.(5)Compared with Sham group, the percentages of CD4+ and CD8+ T cells were increased in the untreated IRI group in both spleen and kidney.The percentages of CD4+ and CD8+ T cells were significantly decreased in ERC-treated group compared with those of untreated IRI group in both spleen and kidney.(6)Compared with untreated IRI group, CD4+CD25+ Treg population was significantly increased by ERC treatment in mice with renal IRI. compared splenic Treg population in different groups. There was no significant difference in the number of Tregs between untreated IRI group and sham control group.(7)Compared with Sham group, CD11b+Ly6C+ and CD11b+Ly6G+ cell was significantly increased in ERC-treated group; Compared with Sham group, the percentages of CD11b+Ly6C+ and CD11b+Ly6G+ cell was slightly increased in untreated IRI group.(8)ERCs effectively suppressed the percentage of total macrophages(F4/80+ cell) compared with those of untreated IRI group(p<0.05), even though slightly higher than those of mice in sham control group(p<0.05). M2 macrophage(CD206+ cell) population was significantly increased in ERC-treated mice compared to that of untreated IRI mice(p<0.05). Compared with Sham group, the percentage of total macrophages(F4/80+ cell) was significantly increased in untreated IRI group.(8)Compared with group, the serum levels of pro-inflammatory cytokines(TNF-?, IFN-? and IL-6) were markedly increased in untreated IRI group. But treatment with ERCs increased the level of the anti-inflammatory cytokine IL-4 in the sera(p<0.05) In comparison with untreated IRI group, ERC treatment significantly reduced the pro-inflammatory cytokine levels in the sera(p<0.05).Conclusions:(1)We have used the model of renal ischemia reperfusion injury in mice successful and have confirmed that ERC can effectively improve renal function and reduce renal pathological injury.(2)ERCs may reduce the proportion of CD3+, CD4+ and CD8+T cells in the kidney and CD4+ and CD8+T cells in the spleen and attenuate renal ischemia reperfusion injury in mice.(3) ERCs attenuate injury and promote the repair of renal injury,which is related to increasing the percentages of spleen Tregs cells.(4)ERCs reduce the percentages of renal F4/80+ macrophages, but increase the percentages of CD206+ cells(M2 macrophages) to protect renal tissue.(5)ERCs increase the percentages of CD11b+Ly6C+ and CD11b+Ly6G+ cells to attenuate the renal pathological damage and improve the renal function.(6)ERCs restore renal injury by affecting the level of inflammatory cytokines.