Human Umbilical Cord Mesenchymal Stem Cells Repair Renal Ischemia Reperfusion Injury Via Macrophage Participation

Objective:Mesenchymal stem cell (MSC) administration has a protective and repair effect on renal injury after ischemia-reperfusion and is considered a beneficial strategy for cell therapy, but the underlying mechanism is still unknown. The aim of this study is to investigate the immunomodulation effect of MSCs on macrophages and the involvement of macrophages in the mechanism of renoprotective role mediated by MSCs. This will suggest a novel mechanism for the therapy role of MSC administration in renal injury, which will provide a necessary theory for the clinical application of MSCs.Methods:Human umbilical cord mesenchymal stem cells (hucMSCs) were administrated to renal injury induced by ischemia-reperfusion (I/R) in C57BL/6mice via intravenous infusion by the caudal vein. The renoprotective effect of hucMSCs was evaluated. The samples of blood and tissues were collected from mice before surgery and at different time-points during1and10days after reperfusion. The concentrations of serum Cr and BUN were detected by a biochemistry analysis instrument, which was considered as an index to evaluate the amelioration of renal function by hucMSC administration. Histology of kidney sections stained by H&E and tubular injury score were employed to reveal the protective role of hucMSCs in the integrity of renal tissues. The effect of hucMSCs on the proliferation and apoptosis of tubular cells were investigated by PCNA immunostaining and TUNEL, respectively. The macrophages infiltrated in kidneys of mice with different treatments were detected by immunohistochemistry for F4/80antigen in order to elucidate the effect of hucMSC treatment on the quantity of infiltrated macrophages after I/R. Meanwhile, we further investigated the underling mechanism through detecting the expression of monocyte chemoattractant protine-1(MCP-1) in the kidneys by immunostaining. We utilized flow cytometry for analyzing the percentage of CD206-positive cells among CD11b-positive macrophages infiltrated in renal tissues. Intravenously injection of liposome-dichloromethylene bisphosphonate (lipo-Cl2MBP) was performed to deplete monocytes and macrophages in the kidneys during injury and repair phase post-reperfusion and the protective role of hucMSCs was evaluated by the histology of kidney sections. Real-time RT-PCR was used to determine the mRNA copies of the cytokines IL-1β, IL-6and IL-10in renal tissues of mice treated with hucMSCs or with macrophage depletion, which was explored to indicate the alternation of inflammatory state at the injury local. In vitro experiment, RAW264.7cells were analyzed for phenotype and cytokine profile after cocultured with hucMSCs. The expression of CD206, a marker for M2macrophage, was detected on the surface of RAW264.7cells in the coculture experiment by flow cytometry. The effect of hucMSCs on both mRNA and protein levels of the cytokines IL-1β, IL-6, arginase-1and IL-10in RAW264.7cells were analyzed by RT-PCR and ELISA, respectively.Results:HucMSC treatment led to a decrease in the level of serum Cr or BUN, and the tubular injury score of renal tissues at different time-points after reperfusion. Moreover, hucMSC infusion could enhance the proliferation, whereas reduce the apoptosis of tubular epithelial cells. Compared with PBS-treated mice, macrophages infiltrated in the kidneys of mice given hucMSC administration highly decreased, which was most significant at5days after reperfusion. On the other hand, the results by immunohistochemistry demonstrated that MCP-1, which has recruitment activity for macrophages, highly expressed in tubular epithelial cells subjected to IRI. HucMSC treatment led to a reduction of MCP-1expression in renal tissues. The determination of macrophage phenotypes in renal tissues during the late repair phase by flow cytometry showed that the proportion of M2macrophage with repair property significantly increased after hucMSC infusion compared with PBS-group. Depletion of macrophages at the early stage of IRI in vivo resulted in a promotion of repair progression medicated by hucMSCs. However, depletion of macrophages at the late stage of IRI almost eliminated the renoprotective effect of hucMSCs. Results of RT-PCR demonstrated that renal tissues of hucMSCs-treated mice expressed high levels of the pro-inflammatory cytokines and low levels of the anti-inflammatory cytokines, which conduces to the resolution of inflammation at injury local. In vitro, RAW264.7cells showed a highly increase in the expression of CD206when cocultured with hucMSCs in a Transwell system or in a directly contact manner. In addition, RAW264.7cells cocultured with hucMSCs provided a decrease in the expression of IL-1β and IL-6, whereas an increase in the expression of arginase-1and IL-10.Conclusions:HucMSC administration has both protective and repair effects on renal ischemia-reperfusion injury in C57BL/6mice. The therapy activity of hucMSCs is concerned with their roles in reducing the infiltrated macrophages, enhancing the resolution of inflammation at injury local and mediating the phenotype switch of macrophages in the kidneys. HucMSCs may exert the repair effect on renal injury through their regulation in the phenotype and cytokine profile of macrophages both in renal tissue and a coculture system, which led to the M2polarization assigned with repair function. Compared with the mechanism related to differentiation and paracrine of hucMSCs, our findings indicate here that macrophages with distinct phenotypes are involved in the repair progress of acute kidney injury (AKI) by hucMSCs, which can be considered as a novel mechanism for the promotion effect of MSCs on tissue repair.