Introduction: Ischemia reperfusion injury (IRI) of donation After Cardiac death (DCD) transplanted kidney is the key factor of acute renal injury, acute and chronic rejection of renal transplantation and transplanted kidney disease.The latest researches show that mesenchymal stem cells (MSCs) have the function of protecting damaged tissues and organs and strong immune regulation. Induced pluripotent stem (iPS) cells have higher proliferative capacity and exosome secretion potential than MSCs. In addition to the functions of stem cells, exosomes have the unique ability to cross the blood-brain barrier and deliver remotely, which means that exosomes have better advantages in clinical disease treatment compared with the therapeutic potential of stem cells. Therefore, the main purpose of this study is to clarify the role of IPS derived exosomes in protecting the IRI of DCD transplanted kidney and regulating its local immune microenvironment.

Methods: The iPS cells were passaged to the third passage and massively expanded. After 48 hours of culture, 100 mL of cell culture supernatant was collected and exosomes were extracted from the supernatant by the kit method, and the morphology and surface membrane marker proteins of the exosomes were identified. The SD rats were established with DCD renal transplantation model and 10, 50, and 100 μg of exosomes were infused into that SD rats respectively, and a PBS control group was set at the same time. On the 3rd, 5th, 7th, 14th and 28th days after transplantation, the content of cytokines in the serum of the recipient rats, the proportion of lymphocyte subsets CD4+CD25+Foxp3+Tregs in the spleen and the relative expression of Foxp3 mRNA were detected. The proliferation of renal tubular cells was detected by PCNA immunofluorescence. The pathological changes of renal tissue were determined by HE staining, and the apoptosis of renal tubular epithelial cells was detected by Tunel staining. The infiltration of inflammatory cells was detected by immunohistochemistry. The expression changes of regulatory T cells in renal tissue and macrophage phenotypic characteristics were analyzed by flow cytometry.

Results: The experimental amount of exosomes was successfully obtained, and the morphology was observed by transmission electron microscope. The exosomes were round or oval membranous vesicles with uniform size, and the edges were clearly visible. The TSG101, CD63, and CD9 proteins were highly expressed on the exosome membrane surface by western-blot. A DCD rat kidney transplantation model was successfully established. The expression levels of IL-12, IL-2, IFN-γ, and TNF-α in the serum of each group were increased to varying degrees from the 3rd day after operation. On the 3rd and 7th days, the levels of IL-12, IL-2, IFN-γ, and TNF-α in the 100 μg exosome infusion group were significantly lower than those in the 10 μg, 50 μg and PBS groups (P<0.05), and the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than 10μg, 50μg and PBS groups (P<0.05). On the 28th day, the expression level of IL-10 in the 100 μg exosome infusion group was significantly higher than other groups (P<0.01). Compared with other groups, the CD4+CD25+Tregs and CD4+CD25+Foxp3+Tregs accounted for the highest proportion of CD4+T cells in the spleen of the 100μg infusion group (P<0.05). On the 28th day, the expression level of Foxp3 mRNA in the 100 μg infusion group was significantly higher than that in the 50 μg group (P<0.05). The stainings of PCNA, HE and Tunel showed that SD rats infused with 100μg exosomes could significantly reduce the pathological damage, inflammatory response and apoptosis of renal tubular epithelial cells in the transplanted kidney, promote the proliferation of renal tubular epithelial cells, and have a protective effect on IRI kidney. On the 28th day, the expression of CD4+CD25+Foxp3+Tregs cells in the transplanted kidney tissue was significantly increased, which further promoted the differentiation of macrophages into the immunomodulatory M2 subset.

Conclusions: The infusion of iPS-derived high-dose exosomes is involved in the protection of the DCD kidney transplant during IRI process, and alleviates the acute and chronic injury of the DCD kidney transplant, and promotes the formation of the local immunosuppressive microenvironment in the DCD kidney transplantation.