The Study Of The Mechanism On P2X7 Receptor And Its Antagonist In Childredn With Primary Nephrotic Syndrome

Background and aimPrimary nephrotic syndrome(PNS)is one of the common glomerular diseases in children.And most children with MCD accepted therapy are sensitive to steroid,but some of them are dependent or resistant to steroid,which leads to recurrence and eventually develops into glomerulosclerosis and end-stage kidney disease(ESRD).Hyperlipidemia is an important pathophysiological change in primary nephrotic syndrome,characterized by elevated plasma total cholesterol and low density lipoprotein.Hyperlipidemia leads to the deposition of ox-LDL in the kidney,which leads to the infiltration of inflammatory cells,the proliferation and injury of renal innate cells,the accumulation of extracellular matrix and the formation of foam cells,directly or indirectly facilitating to glomerulosclerosis.At present,the research was found that the basic principle of the model of nephrotic syndrome induced by Adriamycin is similar to the pathological type of human nephrotic syndrome.Nephrotic syndrome induced by Adriamycin that manifests minimal change disease in early stage and FSGS in late stage is a classic animal model of nephrotic syndrome.Podocytes are differentiated cells which are located in the lateral of basement membrane in the glomerulus and play an important role in maintaining the structure and function of the glomerular filtration barrier.Therefore,the intervention for podocytes have become an important target in chronic kidney diseases.It has been confirmed that the damage of podocytes is associated with minimal change nephrosis and focal segmental glomerulosclerosis(FSGS).Podocyte dysfunction may promote renal proteinuria and even glomerulosclerosis.The deposition of lipid in glomerular podocytes is an important risk factor for glomerulosclerosis.Particularly,ox-LDL plays an important role in the development of atherosclerosis and various glomerular diseases.In our previous study,the level of serum ox-LDL was increased in children with primary nephrotic syndrome and positively correlated with the level of serum CXCL16.Subsequently,it was shown that the expressions of ox-LDL and CXCL16 was increased in the glomerulus of mice with nephrotic syndrome induced by adriamycin in animal model experiments.And in vitro cell experiments it was confirmed that CXCL16 can act as scavenger receptor to uptake ox-LDL,promoting the accumulation of lipid in mouse podocytes and leading to podocyte injury.However,the mechanism of injury in podocyte by ox-LDL is not fully unclear.P2X7R,a member of the P2X receptor family,is a trimer ATP-gated ion channel Compared with other family members,its structure is specific,so it has been attracted much.The research has been found that P2X7R is involved in the onset and development of a variety of kidney diseases,such as diabetic nephropathy,polycystic kidney disease,hypertensive nephropathy and acute kidney injury.And it has been proved that the preventive use of P2X7R antagonists has a renal protective effect in many models of kidney diseases.Therefore,P2X7R has become an attractive therapeutic target for kidney diseases.Although the level of P2X7R in normal kidneys is very low,there is increased expression of P2X7R in models with cell damage and inflammation,such as hypertension,type 1 diabetes,and acute glomerulonephritis.It was found that the increased expression of P2X7R in the glomerulus may reflect the early inflammatory injury of glomerulus.P2X7R may play an important role in early glomerular injury and proteinuria.Furthermore,podocyte apoptosis is also a phenotype of early glomerular injury and plays an important role in the formation of proteinuria.P2X7R activated by ATP induces apoptosis of various cells,including macrophages,lymphocytes,dendritic cells and mesangial cell.Sha w.et al.found that P2X7R may participate in podocyte apoptosis through vitro experiments.Another study was found that P2X7R plays a key role in regulating lipid storage and metabolism in the body.Ox-LDL was the most likely mediator for up-regulating the expression of P2X7R in atherosclerosis.Using two independent studies(GSE108113 and GSE108109)in Gene Expression Omnibus(GEO),we found that the gene expression of P2X7R was positively correlated with CXCL16 in FSGS.However,whether P2X7R is involved in the occurrence of primary nephrotic syndrome in children and its specific mechanism have been rarely reported at home and abroad.In this study,the expression of P2X7R、ox-LDL and CXCL16 in renal tissue of children with primary nephrotic syndrome was observed.We established a nephrotic model induced by adriamycin(ADR)in young mice to observe the changes of P2X7R,ox-LDL,CXCL16,Bax,caspase-3;NLRP3,IL-1β,IL-18 and apoptosis in renal tissue,and the changes of body weight,biochemical indexes and urinary protein in mice.And we established the human podocyte injury model induced by ox-LDL to investigate the expressions of P2X7R,Bax,caspase-3 and CXCL16 in podocytes,and in the same time to observe the apoptosis and ROS production of podocytes and the uptake of ox-LDL by podocytes.Meanwhile we used P2X7R antagonist in vivo and vitro to observe the changes of the above indexes in nephrotic models of mice and podocyte injury model by P2X7R inhibition.Through the above experiments,we discussed the mechanism of P2X7R in primary nephrotic syndrome and its antagonist protecting against primary nephrotic syndrome,so as to provide a new theoretical basis for whether P2X7R can be used as a diagnosis and treatment target for primary nephrotic syndrome at the early stage of onset.Objects and methods:1.Fifteen children with PNS who have been undergone renal biopsy were selected as the objects of this study,male:female=2:1,aged 3～12 years old.Among them,Nine cases were minimal change disease(MCD),and six cases were focal segmental glomerulosclerosis(FSGS).While the control group was four children with nephroblastoma,male:female=1:1,aged 3～10 years old.2.Establishment of ADR nephropathy models:Thirty healthy male Balb/c mice aged 5 weeks,with an average body weight of(17.96±0.67)g,were randomly divided into 6 groups,namely the normal control group(NC group,n=5),ADR nephropathy group(ADR group,n=5),ADR+A43 8079(100μmol/kg)group(ADR+A100 group,n=5),ADR+A43 8079(200μmol/kg)group(ADR+A200 group,n=5),ADR+A438079(300μmol/kg)group(ADR+A300 group,n=5)and A438079(300 μmol/kg)group(A300 group,n=5).ADR(10.5mg/kg)was injected into the tail vein by one time for ADR nephropathy model.After 1 week,the mice with large amount of proteinuria(24h urinary protein ration≥50mg/kg)were considered ADR nephropathy.Treatment of A438079:A438079(100 mol/kg,200 mol/kg,300 mol/kg)was injection into intraperitoneal 6 hours before establishing models,and then A438079(100 mol/kg,200 mol/kg,300 mol/kg)was given every day for 1 week.3,Conditionally immortalized human podocytes(HPC)were incubated in 1640 complete medium without IFN-y at 37℃ for 2 weeks in vitro until their differentiation and maturation.They were incubated with ox-LDL(80μg/ml),P2X7R antagonist A438079(10μM),or the compound of A438079 and ox-LDL,respectively.Among them,the podocytes in the A438079 group and the ox-LDL+A438079 group were pretreated with A438079 for 15 min.Each group was incubated in 37℃incubator with 5%CO2 for 48h before treatment.4.Detection of renal tissue indexes in children:①The Hematoxin Eosin(HE)was performed to observe the pathological changes of glomeruli under light microscope;②The ultrastructural changes of podocyte,basement membrane and mesangial area were observed by transmission electron microscope;③The expressions of P2X7R,ox-LDL and CXCL16 in renal tissue was detected by immunohistochemistry with SABC method.5.Examed indexes of mice with ADR nephropathy:①Body weight of each group;②Serum albumin(ALB),total cholesterol(TC)and urine protein quantification for 24h were determined by automatic biochemical analyzer;③IL-1β and IL-18 in renal tissue were detected by ELISA;④P2X7R,ox-LDL,CXCL16,Bax,caspase-3 and NLRP3 in renal tissue were detected by immunohistochemistry with SABC method;⑤The apoptosis in renal tissue was observed by TUNEL method.6.Examed indexes of cell experiment in vitro:①The uptake of ox-LDL by podocytes was observed in ImageXpress Micro Confocal;②Cellular apoptosis and ROS were evaluated using flow cytometer;③P2X7R、Bax、caspase-3 and CXCL16 protein expression was detected by western blot and immunofluorescence analysis.Results:1.The results of renal tissue in clinical(1)The morphological changes of renal tissue in each group:the glomeruli with MCD were basically normal under light microscope,while the glomeruli with FSGS were shown focal and segmental sclerosis,increased mesangial matrix with capillary occlusion,sclerosis and hyalinosis,visible foam cells,segmental scar formation and adhesion to renal capsule;The podocytes with MCD were shown foot process fusion and disappear under electron microscope,with no obvious mesangial cell proliferation,increased matrix width and immunoglobulin deposition,while the podocytes with FSGS were shown foot process fusion generally,with increased mesangial matrix,the deposition of fine particle electron dense in mesangial area and beside the mesangial area,GBM distortion and thickened in segmental sclerosis and collapse of the capillary loops.(2)The expression changes of P2X7R,ox-LDL and CXCL16 in each group:Compared with normal renal tissue,the expressions of P2X7R,ox-LDL and CXCL16 in renal tissue of children with NS were significantly increased(P<0.01).And contrast to children with MCD,the expressions of P2X7R,ox-LDL and CXCL16 in renal tissue of children with FSGS were more obvious.2.The experimental results of mice with ADR nephropathy(1)The changes of weight in each group:Compared with the NC group,the weight of mice in the ADR group was gradually decreased(Day 3:P<0.05,Day 5 and 7:P<0.01).Compared with the ADR group,the loss of weight was significantly reduced in both the ADR+A200 group and the ADR+A300 group(P<0.05 and P<0.01),while there was no difference in the ADR+A100 group(P>0.05).There was no difference between the A300 group and the NC group(P>0.05).(2)The changes of the biochemical indexes of serum and urine in each group:Compared with the NC group,one week after ADR injection the mice in the ADR group had a large amount of proteinuria(P<0.01),decreased serum ALB(P<0.01),and increased serum TC(P<0.01).Compared with the ADR group,the changes of 24h proteinuria ration and TC in the ADR+A438079(200 μmol/kg,300 μmol/kg)groups were significantly alleviated(P<0.01),while there was no difference in the ADR+A100 group(P>0.05).And the changes of serum ALB were only reduced in the ADR+A300 group(P<0.05).There was no difference between the A300 group and the NC group(P>0.05).(3)The changes of IL-1β and IL-18 of renal tissue in each group:Compared with the NC group,the levels of IL-1β and IL-18 in renal tissue of the ADR group were significantly increased(P<0.01).While compared with the ADR group,the changes of renal IL-1β in the ADR+A438079(100μmol/kg,200 μmol/kg,300 μmol/kg)groups were alleviated(P<0.05 or P<0.01).However the change of renal IL-18 were only significantly reduced in the ADR+A300 group(P<0.01).There was no difference between the A300 group and the NC group(P>0.05).(4)The expression changes of P2X7R,ox-LDL,CXCL16,Bax,caspase-3 and NLRP3 of renal tissue in each group:Compared with the NC group,the expressions of P2X7R,ox-LDL,CXCL16,Bax,caspase3 and NLRP3 in renal tissue of the ADR group was significantly increased(P<0.01).While compared with the ADR group,the expressions of them in the ADR+A438079(200 μmol/kg,300μmol/kg)groups was reduced(P<0.05 or P<0.01),and there was no difference in the ADR+A100 group(P>0.05).Meanwhile,there was no difference between the A300 group and the NC group(P>0.05).(5)The changes of apoptosis of renal tissue in each group:Compared with the NC group,the apoptosis of glomeruli in the ADR group was increased(P<0.01).While compared with the ADR group,the apoptosis of glomeruli in the ADR+A438079(100 mol/kg,200 mol/kg,300 mol/kg)groups was decreased(P<0.01).There was no difference between the A300 group and the NC group(P>0.05).3.The results of cellular experiments in vitro(1)The changes of ox-LDL uptake by podocytes in each group:Compared with the NC group,the uptake of Dil-oxLDL by podocytes in the ox-LDL group was increased(P<0.05).While compared with the ox-LDL group,the uptake of Dil-oxLDL by podocytes in ox-LDL+A438079 group was decreased(P<0.05).There was no difference between the A438079 group and the NC group(P>0.05).(2)The changes of apoptosis and ROS production of podocytes in each group:Compared with the NC group,podocyte apoptosis and ROS in the ox-LDL group were increased(P<0.05).However,compared with the ox-LDL group,podocyte apoptosis and ROS in the ox-LDL+A438079 group were decreased(P<0.05).There was no difference between the A438079 group and the NC group(P>0.05).(3)The expression changes of P2X7R,Bax,caspase-3 and CXCL16 of podocytes in each group:Compared with the NC group,the expressions of P2X7R,Bax,caspase-3 and CXCL16 was increased in the ox-LDL group(P<0.05),while the expressions of them in the ox-LDL+A43 8079 group was decreased(P<0.05).There was no difference between the A438079 group and the NC group(P>0.05).Conclusion:1.The expressions of P2X7R,ox-LDL and CXCL16 in renal tissue of children with primary nephrotic syndrome and mice with ADR nephropathy are increased,and P2X7R antagonist decreased the expression of CXCL16 and ox-LDL in mice with ADR nephropathy.It is suggested that P2X7R,ox-LDL and CXCL16 may be associated with primary nephrotic syndrome in children and ADR nephropathy in mice;inhibited P2X7R may reduce the expression of ox-LDL through down-regulating CXCL16 pathway to alleviate kidney injury of mice with ADR nephropathy.2.Activated P2X7R may promote the release of inflammatory cytokines IL-1 0 and IL-18 through the downstream P2X7R/NLRP3 pathway and up-regulate the expression of Bax and caspase-3 to promote apoptosis,which participate in the process of ADR nephropathy;inhibited P2X7R may reduce the release of IL-1 0 and IL-18 through down-regulating the P2X7R/NLRP3 pathway and down-regulate the expression of Bax and caspase-3 and reduce the apoptosis,thereby alleviating kidney injury of mice with ADR nephropathy.3.Activated P2X7R by ox-LDL may increase the expression of CXCL16 and promote the uptake of ox-LDL by podocytes leading to the injury of podocytes;inhibited P2X7R may down-regulate the expression of CXCL16 and reduce the uptake of ox-LDL by podocytes to ameliorate the injury of podocytes.4.Activated P2X7R by ox-LDL may promote the expression of Bax and caspase-3 and the production of ROS leading to podocyte apoptosis;inhibited P2X7R may down-regulate the expression of Bax and caspase-3 and reduce the production of ROS to ameliorate podocyte apoptosis induced by ox-LDL.Innovations and meanings:1.This study firstly investigated the expression of P2X7R in renal tissue of children with primary nephrotic syndrome.At the same time,the expression and mechanism of P2X7R in renal tissue of mice with ADR nephropathy were observed through establishment of models in mice with ADR nephropathy.This will offer a new theoretical basis for early intervention treatment of primary nephrotic syndrome.2.This study firstly investigated the expression and mechanism of P2X7R in human podocytes induced by ox-LDL,confirming that podocyte injury induced by ox-LDL may be related to the up-regulated expression of CXCL16,the increased up-taken of ox-LDL and the increased expression of apoptotic protein and ROS.This will further confirm the mechanism of P2X7R on podocyte injury.3.This study firstly investigated inhibition P2X7R for the influence of kidney injury in mice with ADR nephropathy and human podocytes injury induced by ox-LDL through applying P2X7R antagonist in vivo and vitro.It is confirmed that inhibition P2X7R may protect the kidney injury of mice with ADR nephropathy and the injury of podocytes induced by ox-LDL through down-regulating P2X7R/NLRP3 pathway and CXCL16 pathway,decreasing the expression of apoptotic protein and the production of ROS.This will provide theoretical basis of the treatment of P2X7R antagonist for primary nephrotic syndrome.