γδT Cell Exacerbates Podocyte Injury Via CD28/B7-1-phosphor-SRC Pathway In PNS

PART ONE γδT CELL EXACERBATES KIDNEY INJURY IN LPS NEPHROPATHY MICEObjective: γδT cell plays an important role in several immunological diseases.Previous studies have confirmed immunologic derangement participants in PNS mechanism.Our previous study found γδT cells increase in peripheral blood in PNS children.We designed this part of experiments to determine whether γδT cell takes part in LPS nephropathy mechanism.Methods: WT mouse nephropathy in C57 background was induced by LPS injection.Urine protein and kidney pathology were detected.The infiltration of γδT cells in mouse kidney was detected by flow cytometry and immunofluorescence.Furthermore,TCRδ KO mouse were enrolled and nephropathy was induced by LPS injection as well.To confirm the function of γδT cell in LPS nephropathy,we compare the urine protein and kidney injury among control group,WT nephropathy group and TCRδ KO nephropathy group.Results: Compared with control group,the urine protein increased at 24 hours after LPS injection and severe foot process fusion was observed by electron microscope in WT nephropathy group,which verified LPS nephropathy could mimic human MCD.Compared with control group,the γδT cells ratio in kidney increased significantly in WT nephropathy group(P<0.01).The immunofluorescence result revealed γδT cells were located to glomeruli.Although urine protein increased in TCRδ KO nephropathy group when compared with control group.The urine protein decreased significantly in TCRδ KO nephropathy group when compared with WT nephropathy group(P<0.01).And foot process effacement alleviated in TCRδ KO nephropathy group when compared with WT nephropathy group.Conclusion: γδT cell promotes kidney injury in LPS nephropathy,PART TWO γδT CELL EXACERBATES KIDNEY INJURYVIA CD28/B7-1-PHOSPHOR-SRC PATHWAYObjective: Previous studies have found B7-1 expression on podocyte.We designed this part of experiment to determine whether B7-1 participant in LPS nephropathy and whether γδT cell can have interaction with podocyte and activate phosphor-SRC through CD28/B7-1 combination.Methods: Mouse kidney and podocyte B7-1 m RNA expression were detected by PCR.And podocyte B7-1 protein expression was detected by Western Blotting.CD28 loss of γδT cells in WT nephropathy group was detected by flow cytometry.Co-location of γδT cells and B7-1 was detected by immunofluorescence.Furthermore,the phosphor-SRC protein level of control group,WT nephropathy group and TCRδ-/-nephropathy group was detected by western blotting to elucidate the relation between γδT cells and phosphor-SRC activation.Results: Compared to control group,both B7-1 m RNA expression(P< 0.01)and B7-1 protein level(P < 0.05)of podocytes increased significantly in LPS treatment group.B7-1 m RNA expression was significantly higher in WT nephropathy group than control group(P<0.01).Co-location detection of B7-1 and synaptopodin in kidney of WT nephropathy group revealed B7-1 was restricted to podocytes.Co-location detection of B7-1 and γδT cells in kidney of WT nephropathy group revealed the location of B7-1 and γδT cells was near and even overlapped.The loss of CD28 on γδT cells was detected in WT nephropathy group rather than control group by flow cytometry.Finally,western blotting result determined significantly higher phosphor-SRC protein level in WT nephropathy group than control group and TCRδ-/-nephropathy group(P<0.05).Conclusion: The pathway which induced γδT cells to promote kidney injury may be CD28/B7-1-phosphor-SRC pathway.To determine the pathway,experiment in vitro was required.PART THREE γδT CELL EXACERBATES PODOCYTEINJURY VIA CD28/B7-1-PHOSPHOR-SRC PATHWAYObjective: To determine whether γδT cell exacerbates podocyte injury via CD28/B7-1-phosphor-SRC pathway,experiments in vitro were designed.Methods: Mouse spleen γδT cells were isolated.Mouse podocytes were divided into five groups,including control group(A group),LPS treatment group(B group),podocytes cultured with γδT cell using transwell(C group),podocytes co-cultured with γδT cell group(D group),and podocytes co-cultured with γδT cell and treated with CTLA4-Ig group(E group).Among them,group B,C,D,E groups were treated with LPS(100ug/ml).Forty-eight hours after treatment,podocyte apoptosis rate was detected by flow cytometry.Phosphor-SRC expression of podocytes were detected by immunofluorescence.And podocyte F-actin was detected by immunofluorescence.Results: Compared with control group,the early and late apoptosis of podocytes from group D were significantly higher(P<0.01).However,compared with group D,the early and late apoptosis of podocytes from group E were much lower(P<0.01).Only group D had higher phosphorSRC expression when compared with control group(P<0.01).There was no significant difference between control group and group E.Furthermore,podocyte F-actin loss was observed in group D,and F-actin injury recovered in Group E.Conclusion: Results of experiments in vitro confirmed only when the direct interaction between γδT cell and podocyte existed,phosphor-SRC expression increased and severe podocyte injury was observed.And CD28/B7-1 blocker CTLA4-Ig could alleviate phosphor-SRC expression and podocyte injury.The results revealed γδT cell exacerbates podocyte injury via CD28/B7-1-phosphor-SRC pathway.