Abstract:

Objective：Establishing mice’s model with ichemia-reperfusion (I/R) kidney injury, drawing bilateral renal cortex to make kidney homogenate, culturing mouse mesenchymal stem cells (mMSCs) with kidney homogenate to simulate acute kidney injury (AKI) microenvironment, and to investigate mMSCs’ differentiation and replication under this microenvironment. Methodology: To make AKI mice models by clamping bilateral renal pedicles 30 minutes and reopening 30 minutes. Then immediately drew bilateral renal cortex to make I/R kidney homogenate supernatant. C57BL/6 mice’s mMSCs had been successfully isolated by percoll density gradient centrifugation and adherence cultivation, their surface markers were identified by flow cytometry. Then P3-mMSCs were treated with different group: Control group (low glucose DMEM medium with 10% fetal bovine serum)，and intervention group (low glucose DMEM medium with 10% fetal bovine serum plus I/R kidney homogenate supernatant ). Each group was incubated for 1d, 3d, 5d, and 7d，morphological changes of these cells were observed by inverted microscope and ultrastructure changes were examined by transmission electron microscope. Cytokeratin-18，proliferation of mMSCs，and apoptosis were detected by flow cytometry，CCK-8 and TUNEL respectively. Results: The cells （P3-mMSC） were highly expressed CD29 and CD44, lowly expressed CD34 and CD45. Compared with the control group, the cells of intervention group presented oval shape, and short fusiform on the third day, round shape, oval shape, and short-pang fusiform on the 7th day. At the same time, there were much rough endoplasmic, lysosome, and mitochondria appearing in cytoplasm. There was only trace expression of CK18 in control group, while the expression rate was increased significantly after intervention of the I/R kidney homogenate supernatant. The I/R kidney homogenate supernatant could alleviate the proliferation ability of mMSCs while the apoptosis percentage increased significantly (P<0.01). Conclusion: AKI microenvironment can not only induce mMSCs partially to transfer to renal tubular epithelial-shaping cells，but also induce mMSCs’ apoptosis, weaken their proliferation ability, and as a result, decrease the number of mMSCs that can make trans-differentiation. It may be a reason that renal repair ability of MSCs’transplantation is limited.