| Background and aims:Diabetic kidney disease(DKD)is a metabolic disease mainly characterized by glomerulosclerosis and proteinuria.It is one of the most common microvascular complications of diabetes and has become one of the main causes for end-stage nephropathy.The current treatments including strict control of blood glucose and blood pressure,restriction of protein intake,reduction of urinary protein excretion,use of RAS system blockers and SGLT-2 inhibitors,etc.were still limited and are difficult to prevent the occurrence and progress of DKD.Immune inflammation is the driving factor of DKD progression,and this study is aim to explore a new strategy for DKD based on the perspective of immune inflammation.Recently,regenerative medicine such as stem cell therapy has a promising application prospect in the prevention and treatment for DKD.In particular,mesenchymal stem cells(MSCs)have been paid great attention because of their low immunogenicity,strong immunomodulatory properties,rich sources and non-ethics.Evidence has demonstrated that MSCs can promote DKD damage repair by alleviating oxidative stress,paracrine trophic factors,and regulating immune microenvironment of the kidney.However,most of the studies focus on the interaction between MSCs and innate immune cells.Whether they can play a therapeutic role on DKD by regulating the balance of adaptive immune cells such as Th17/Treg cells are not reported.Based on the above research background,this project aims to study the repair effect of P-MSCs on DKD through immune inflammation injury and explore the possible molecular mechanism.Methods:This study screened the DKD renal tissue expression datasets from the GEO database,and used the CIBERSORT and single sample gene set enrichment score(ss GSEA)algorithm to analyze the immune cell infiltration.Principal component analysis(PCA)was used to discriminate immune infiltration characteristics between DKD and normal control groups,and analyzed the relationship between various immune cells in DKD microenvironment using R“psych”packages.Then,the DKD and control kidney wax samples were selected for pathological staining and multiple immunofluorescence analysis,and analyzed programmed death receptor-1(PD1)expression level in renal tissues.Next,we isolated and identified P-MSCs,and established the model of streptozotocin(STZ)-induced type 1 diabetes mellitus(T1DM)rats and were randomly divided into two groups(T1DM renal injury group,P-MSCs treatment group),and normal rats were used as control group.P-MSCs cell suspension(1×10~6/cells,once a week,3 times)or the same amount of saline were injected via tail vein at8th weeks after the STZ injection.Urine,serum and kidney tissue samples were collected for subsequent analysis at 8th weeks after P-MSCs infusion.Serum creatinine,urea nitrogen,blood glucose level were detected by automatic biochemical analyzer,serum cystatin C level and urine microalbumin level were detected by ELISA method,and urine creatinine level was detected by picric acid method.The renal pathological changes were evaluated by HE staining and PAS staining.The P-MSCs was detected by immunofluorescence,the proportion of peripheral blood Th17/Treg cells was detected by flow cytometry,and the levels of serum and renal tissue cytokines(IL-17A、IL-6、IL-10)were detected by liquid chip technique.Moreover,the significantly enriched pathways were analyzed by transcriptome sequencing of renal tissue in model group and P-MSCs treatment group.Finally,the spleen lymphocytes of rats were isolated and co-cultured with P-MSCs,which were divided into 4 groups including control group,IL-2(10ng/m L)+PHA(10ug/m L)stimu Lated lymphocyte group,direct contact between lymphocytes and P-MSCs group(4:1 ratio),indirect contact between lymphocytes and P-MSCs(upper chamber is lymphocyte,lower chamber is P-MSCs)group.The proportion of Th17/Treg cells and the expression level of PD1 in lymphocytes were detected by flow cytometry,and thus to analyze the possible pathways of regulating Th17/Treg balance.In addition,the PD-L1 si RNA sequence was used to down-regulated PD-L1expression on P-MSCs surface,and then detect the proportion of Th17/Treg cells by flow cytometry,so as to verify whether the P-MSCs regulates the cell balance through the PD1/PD-L1 pathway.Results:This study included 25 controls and 19 DKD samples from the GEO database.The results showed that the infiltration levels of cytotoxic T cells,Th2、Tfh、mucosa-associated T cells,monocytes and macrophages significantly increased;Treg cells,NK cells and neutrophils significantly decreased;Th1 and Th17 cells elevated without statistical difference in DKD tissues when compared to control group.Moreover,the results of PCA and correlation analysis suggest that the pattern of immune cell infiltration can distinguish the DKD from the control samples,and various immune cells have a network regulatory relationship in the renal microenvironment.The results of 6 DKD and 6 control samples collected in our hospital showed that infiltration level of macrophages and Treg cells was consistent with the predicted results,Th2 cells were scarce in the kidney tissue,and the expression of PD-1 was remarkably elevated in the renal microenvironment.The isolated P-MSC is spindle-like in cell morphology,with high expression of CD73、CD90、CD105 markers and low expression of CD19、CD31、CD34、CD45、CD11b、HLA-DR markers on the cell surface.P-MSC can differentiate into fat,cartilage and bone tissue,thus it meets the MSCs identification criteria.Immunofluorescence results showed that P-MSCs were mainly homing in immune organs such as thymus and spleen,but the number of colonization in pancreas and kidney tissues were scarce.P-MSCs treatment can effectively improve blood glucose,creatinine,urea nitrogen,urinary albumin/creatinine ratio,renal hypertrophy index and renal pathological injury in T1DM rats.Also,P-MSCs treatment can significantly decrease the proportion of Th17 cells and the level of IL-17 and IL-6 cytokines,increase the proportion of Treg cells and the level of IL-10 factor.Moreover,the resu Lts of renal transcriptome analysis exhibited that the adaptive immune pathway,cytokine pathway(IL-17 signaling pathway and TNF-αsignaling pathway)were remarkably enriched in the P-MSCs treatment group,thus supporting the mechanism of MSCs improving DKD by regulating immune inflammation.The results of MSCs and T lymphocyte co-culture showed that the proportion of Th17 cells was significantly higher and Treg cells was lower in phytohemagglutinin(PHA)stimulation group than that in control group.The proportion of Th17 cells was significantly lower and Treg cells was higher in P-MSCs direct co-culture group than that in PHA stimulation group,and the expression of PD1 in T lymphocytes was up-regulated,while the proportion of Th17 and Treg cells was not significantly changed in the indirect co-culture group.The above results suggest that P-MSCs can improve Th17/Treg cell balance through direct cell-cell contact.Moreover,compared with the si RNA NC group,the proportion of Th17 cells was significantly increased and Treg cells was decreased in the si RNA PD-L1 transfection P-MSCs group,suggesting that P-MSCs potentially regulates Th17/Treg cell balance through the PD1/PD-L1 pathway.Conclusions:This study showed that P-MSCs promote DKD damage repair by regulating immune inflammation,and that PD-1/PD-L1 pathway may be a molecular mechanism mediating P-MSCs regulation of Th17/Treg cell balance.This project will provide new experimental data concerning MSCs treatment for DKD. |
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