| Objective To elucidate the interaction between T cells and maturation differentiation of DCs in ischemia/reperfusion AKI,and the mechanism of their protective effect in DIPCs.Also,we investigated whether DIPCs remained protective against acute kidney injury from ischemia/reperfusion in CD11c~+conditional deletion transgenic mice,as well as its related changes in immune cells,to provide evidence for DC s to become a potential target for the treatment of ischemia/reperfusion AKI.Methods1.Construction of a wild-type mouse IRI/DIPC model,and these mice were randomly divided into 3 groups:(1)sham operation group(SHAM+SHAM group);(2)ischemia/reperfusion injury group(SHAM+IR group);(3)delayed ischemic preconditioning group(DIPC+IR group).Verification of the protective effect of DIPC by serum creatinine levels and renal pathological changes;2.Study of the relationship between the protective effect of DIPC and the maturation of DCs and the proportion of T cells differentiated in wild-type mice.The phenotypic changes and quantity ratio of DCs and T cells in each group were analyzed by flow cytometry;Western Blotting was wont to find the expression of CCR7 within the urinary organ of mice in every group;the expression of CD11c~+dendritic cells within the urinary organ was detected by immunohistochemistry;SOD and MDA detection kits were used to detect the changes of oxidative stress levels in serum and renal tissues of mice in each group;various cytokines(such as IL-6,IL-10,IFN-γ,TNF,etc.)in the serum of mice were detected by multiple liquid protein quantification techniques(CBA);the levels of IL-10 and TNF-αin the kidney of mice were detected by ELISA;3.DTR mice with conditional deletion of CD11c~+DCs were screened and identified by PCR and flow cytometry;and to verify the appropriate concentration,duration of action,and drug toxicity of diphtheria toxin(DT),the experiment was divided into 3 groups:(1)WT mice with intraperitoneal injection of DT(WT+DT group);(2)DTR mice with intraperitoneal inje ction of PBS(DTR+PBS group);(3)DTR mice with intraperitoneal injection of DT(DTR+DT group);4.Research the link between DCs and T cells when DIPC in DTR mice.To construct an IRI/DIPC model of DTR mice,male DTR mice were divided into 4groups,all of which were injected intraperitoneally with DT or control PBS 16h before surgery for kidney injury:(1)sham-operated group with injection of DT(DT+SHAM+SHAM group);(2)ischemia/reperfusion injury group with injection of DT(DT+SHAM+IR group);(3)delayed ischemic preconditioning group with injection of DT(DT+DIPC+IR group);(4)delayed ischemic preconditioning group with injection of identical volume of PBS(PBS+DIPC+IR group).When CD11c~+DCs were eliminated,the role played by DIPC was verified by serum creatinine levels and renal pathological changes;flow cytometry was applied to analyze the phenotypic changes and numerical ratios of DCs and T cells in each group of mice,and further observe the relationship between DIPC and the degree of kidney injury and Tregs after DCs deletion by WB,oxidative stress,and inflammatory factor assays;5.Study of the effect of mature/immature DCs on the action of HK-2 cells under different culture conditions:HK-2 cells were cultured alone or co-cultured with mature/immature DCs under normoxic and hypoxia/reoxygenation conditions respectively,and the levels of apoptosis and necrosis of HK-2 cells were detected by flow cytometry and immunofluorescence.Results1.The DIPC model was successfully constructed,and DIPC significantly reduces creatinine levels and the degree of pathological damage after IRI in the kidney;2.DIPC inhibits inflammatory cell aggregation in the kidney,inhibits the maturation and differentiation of DCs as well as their chemotactic capacity,and initiates immune action in peripheral immune organs,increasing the proportion of Tregs in renal tissues;it also reduces the level of oxidative stress in the kidney after IRI and increases the expression level of the anti-inflammatory factor IL-4;3.The DTR transgenic mouse model was successfully constructed.After exploration of DT gradient concentration and gradient duration of action,it was found that 16h after intraperitoneal injection of 16ng/g(mouse weight)DT significantly reduced the proportion of CD11c~+cells in the spleen and kidney of mice,and did not cause drug-related kidney damage in mice;4.After the DCs of DTR mice injected with DT were eliminated,DIPC lost its target of action and its functional and histological protection against post-IRI renal IR was diminished,the inhibitory effect on inflammatory cell aggregation was weakened,the inhibition of the maturation and chemotactic ability of DCs and the recruitment of Tregs were significantly weakened,the effect of reducing the level of oxidative stress after renal IRI was also weakened,and the expression level of inflammatory factors was decreased.5.The HK-2 cell hypoxia-reoxygenation model was successfully constructed,and im DCs and m DCs were successfully isolated and cultured.When H/R HK-2was co-cultured with im DCs/m DCs,the apoptosis and necrosis rates were significantly lower compared to H/R HK-2 cells,with the decrease in apoptosis and necrosis rates being more pronounced when co-cultured with im DCs.Conclusion1.DIPC stimulates the differentiation of T cells towards Tregs by inhibiting the maturation of DCs,thereby producing a protective effect against renal IRI;2.The protective effect of DIPC on ischemia/reperfusion AKI was significantly diminished in terms of creatinine levels,changes in oxidative stress levels,and pathomorphological changes when DCs was removed;3.DCs play a crucial role in renal IRI as well as in DIPC,but vary with the degree and state of disease damage,and different local microenvironments. |
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