Study On The Mechanism Of CXCL16 Induced Podocyte Injury By Regulating ERK1/2 In Children With Primary Nephrotic Syndrome

Background and purpose of the study:Primary nephrotic syndrome(Primary nephrotic syndrome,PNS)is a common glomerular disease in children.Most children are sensitive to hormone therapy,the pathological type is Minimal Change Disease(MCD),and the prognosis is good,but some children are dependent or resistant to hormone therapy.If other drug interventions are not given in time,they may progress to kidney Glomerular sclerosis,the pathological type is focal segmental glomerular sclerosis(Focal segmental glomurular sclerosis,FSGS),which eventually leads to end-stage renal disease(ESDR).End-stage renal disease seriously affects the physical and mental health and quality of life of children,and brings a heavy economic burden to the family and society.Therefore,it is of great clinical significance to study the mechanism of primary nephrotic syndrome and its progression to glomerulosclerosis,and to intervene as early as possible.Studies have shown that podocyte injury plays an important role in the occurrence of PNS,and podocyte injury is involved in the formation of proteinuria and glomerulosclerosis.Podocytes are terminally differentiated visceral epithelial cells located outside the glomerular basement membrane(GBM),which are of great significance for maintaining the structure and function of the glomerular filtration barrier.Previous studies have found that many factors such as anti-capsular antibodies,metabolic factors,hemodynamics and infection can cause podocyte damage.Podocyte injury results in apoptosis,shedding,and proliferation inhibition,leading to glomerular sclerosis and renal function damage.Under certain conditions,podocyte damage can lead to phenotypic transition,in which the podocytes lost epithelial cell markers,such as P-cadherin and ZO-1,while acquiring mesenchymal marker proteins,such as vimentin(Vimentin)and N-cadherin This process is called epithelial mesenchymal transition(EMT)of podocytes.Recent studies have shown that EMT lead to podocytes move and eventually destruction of the fine structure of these cells,thereby weakening their filtering barrier function and leading to glomerular sclerosis.Cell migration is generally considered a hallmark of EMT.Studies have confirmed that epithelial-mesenchymal transition can be observed in patients with nephrotic syndrome.Harris JJ’s studies have proved that the plasma of patients with active FSGS can significantly increase the exercise capacity of podocytes in vitro.Zhai S et al.found that IL-17 participates in podocyte damage in children with primary nephrotic syndrome by inducing podocyte apoptosis.However,the specific mechanism of podocyte injury in children with PNS has not been deeply studied.Therefore,by studying podocyte apoptosis,proliferation,EMT and migration,in-depth exploration of the mechanism of podocyte injury in PNS will help to find therapeutic targets for delaying the occurrence of primary nephrotic syndrome and its progression to glomerular sclerosis,and providing a basis for precision medicine.CXC chemokine ligand 16(CXC chemokine 16,CXCL16)is a CXC chemokine,exists in a transmembrane or a soluble form,and only binds to its unique receptor CXCR6.More and more data indicate that CXCL16 is involved in kidney disease.During the previous research,we have proved that the serum CXCL16 level in children with primary nephrotic syndrome was higher than that in the remission period and normal control group.Serum CXCL16 can be used as a useful indicator or biomarker of disease activity in children with nephrotic syndrome.However,there are few reports on the expression of CXCL16 in PNS kidney tissue and its clinical significance.CXCL16 is highly expressed in podocytes and acted as a scavenger receptor of ox-LDL to assist podocytes to take up ox-LDL to mediate podocyte lipid damage.In addition,our research group established a CXCL16 gene silencing podocyte model and found that after CXCL16 gene silencing,the podocyte damage caused by lipid accumulation was reduced,ROS production decreased,and β1 integrin expression increased,which played a cytoprotective function.Previous studies on the mechanism of CXCL16’s damage to podocytes mostly mediate the lipid damage of podocytes.Our previous studies have found that after ox-LDL stimulation,lipid accumulation occurs in podocytes and the expression level of CXCL16 increases.It promotes podocyte migration by regulating the expression of CXCL16 and regulating the actin cytoskeleton.CXCL16 is a key factor for podocyte migration and damage.Therefore,we speculate that there are other mechanisms of CXCL16 on podocyte damage in children’s PNS,and further research is needed.Extracellular signal-regulated protein kinase(ERK)is a member of the mitogen-activated protein kinase(MAPK)family.It is widely involved in regulating cell growth,cell cycle,apoptosis and differentiation,and is an important signal molecule for intracellular signal transduction.The two main subtypes of ERK,ERK1 and ERK2,are proteins encoded by two splice variants of the same gene,which are 84%identical at the amino acid level.ERK1 and ERK2 signaling pathways are involved in many different cell functions,including proliferation,growth,differentiation,cell migration,cell survival,metabolism,and transcription.The research on the role of ERK in kidney disease mostly focuses on ERK’s involvement in the migration,invasion and epithelial mesenchyme of renal cancer cells.Various factors can induce ERK pathway to participate in renal tubular epithelial cell epithelial interstitialization,and ERK pathway participates in diabetic nephropathy.Studies by Bryan M.Grabias and others have confirmed that in the case of ERK2 protein expression,its downstream target protein Snail continues to be expressed,which in turn promotes the continuous expression of the marker proteins N-cadherin and vimentin of mesenchymal cells,suggesting that ERK2 can regulate EMT process and participate in renal fibrosis.Studies have found that the protein expression level of ERK1/2 in diabetic nephropathy mice were increased,and angiotensin II receptor antagonists can reduce its expression level.The activation of ERK1/2 signaling pathway induced by angiotensin Ⅱ(Ang Ⅱ)plays an important role in the apoptosis of podocytes in diabetic nephropathy.There are few studies on the role of ERK1/2 in podocyte injury in primary nephrotic syndrome.Zhang W et al.found that CXCL16 can induce renal tubular epithelial cell apoptosis,inhibit proliferation,and participate in acute kidney injury(AKI)by regulating ERK1/2.However,whether CXCL16 can induce podocyte apoptosis,migration,EMT and inhibit proliferation through ERK1/2,and then participate in the occurrence of podocyte injury in primary nephrotic syndrome,has not been reported previouslyTo Explore the mechanism of CXCL16 on PNS,we observed the protein and mRNA expression levels of CXCL16 and ERK1/2 in renal biopsy tissues of children with PNS,and compared the mRNA expression of CXCL16 and ERK1/2 in renal tissues of children with PNS diagnosed as MCD and FSGS.At the same time,the correlation between the expression of CXCL16 mRNA and ERK1/2 mRNA in children’s PNS kidney tissue with 24h urine protein quantification,serum albumin and serum cholesterol were analyzed,and we have explored whether CXCL16 and ERK1/2 are involved in the occurrence and development of children’s PNS.Stablely transduced human podocyte(HPC)strain were constructed,including CXCL16 overexpressed,CXCL16 knockdown,ERK1/2 knockdown,CXCL16 overexpressed and ERK1/2 knockdown and in these cells,proliferation,apoptosis,cell migration,and The expression of P-cadherin,Vimentin and N-cadherin,ZO-1,which is closely related to EMT were deteced to explore whether CXCL16 can induce PNS podocyte damage in children through ERK1/2 and its mechanism of action,and provide a theoretical basis for the prevention and treatment of PNS progression to end-stage renal disease.This research is divided into three parts:Part I Expression and significance of CXCL16 and ERK1/2 in renal tissue of children with primary nephrotic syndromeObjective:1.To detect the expression of CXCL16 and ERK1/2 in the renal tissue of children with primary nephrotic syndrome.2.Analyze the correlation between the expression of CXCL16 and ERK1/2 with serum albumin,serum cholesterol,24h urine protein,and explore whether they are involved in the pathogenesis of PNS.Objects and Methods:1.Research objects and specimen collection:15 cases of renal tissue biopsy specimens from children with primary nephrotic syndrome(PNS)were collected as the PNS group,and 15 cases of normal kidney tissue 3 cm outside the lesion of children with Wilms tumor were collected as the normal control group.Collect 3ml of fasting venous blood from children in PNS group and normal control group.Collect 24h urine of children in PNS group,and accurately record 24h total urine volume.2.Detection of serum albumin,serum total cholesterol and 24-hour urine protein quantification:The bromocresol green method is used to determine serum albumin(Albumin,Alb)on the AU5800 automatic biochemical analyzer,and the enzymatic method is used to determine serum total cholesterol(Concentration).of blood total cholesterol,CHOL),pyrophenol red method to determine 24-hour urine protein(24hUP).3.Detect the expression of CXCL16 and ERK1/2 in the renal tissues of children with primary nephrotic syndrome:immunohistochemistry(Immunohistochemistry,IHC),western blot(Western blot,WB)and real-time fluorescent quantitative PCR(Quantitative Real-time PCR,qRT-PCR)were used for detection.4.Analyze the correlation between the expression of CXCL16 and ERK1/2 and serum albumin,serum total cholesterol,and 24h urine protein quantification:the mRNA expression of CXCL16 and ERK1/2 were quantified in the renal tissue of the PNS group with serum albumin,serum total cholesterol,and 24h urine protein.Perform correlation analysis.Results:1.The results of immunohistochemical quantitative analysis showed that,compared with the normal control group,the protein expression of CXCL16 and the protein expression of ERK1/2 in children’s PNS kidney tissue were significantly increased,P<0.001.2.Western blot quantitative analysis results showed that compared with the normal control group,the protein expression of CXCL16 and ERK1/2 in children’s PNS kidney tissue were significantly increased,P<0.001.3.The results of qRT-PCR quantitative analysis showed that compared with the normal control group,the mRNA expression of CXCL16 and the mRNA expression of ERK1/2 in children’s PNS kidney tissue were significantly increased,P<0.001.4.Compared with the MCD group,the expression of CXCL16 mRNA and the mRNA expression of ERK1/2 in PNS kidney tissue of children diagnosed as FSGS by pathology were both increased,P<0.05.5.The results of correlation analysis showed that the mRNA expression of CXCL16 and the mRNA expression of ERK1/2 in children’s PNS kidney tissue were positively correlated with the quantification of small urine protein at 24 hours(P<0.001,P<0.01),and were negative with serum albumin.correlation(P<0.01,P<0.05);CXCL16 mRNA expression in children’s PNS kidney tissue was positively correlated with ERK1/2 mRNA expression and serum total cholesterol(P<0.05,P<0.05).Conclusion:1.CXCL16 and ERK1/2 were up-regulated in children with primary nephrotic syndrome(PNS).2.CXCL16 and ERK1/2 were positively correlated with 24-hour urinary protein and negatively correlated with serum albumin.3.The expression of CXCL16 and ERK1/2 in PNS was higher than that in MCD.4.CXCL16 and ERK1/2 may participate in the occurrence and development of PNS.Part Ⅱ Study on the role of CXCL16 in podocyte injuryObjective:1.Construct a CXCL16 stable overexpressed and a CXCL16 stable knockdown human podocyte cell line.2.Explore the role of CXCL16 in podocyte injury.Methods:1.Use the three-plasmid packaging system to package the lentivirus overexpressing CXCL16,and infect HPC cells with the successfully packaged lentivirus to construct a HPC cell line that stably overexpresses CXCL16.2.Construct a lentiviral plasmid that silences CXCL 16.The four-plasmid packaging system is used to package the lentivirus that silences CXCL16,and HPC cells were infected using the lentivirus with CXCL 16 knockdown to construct a HPC cell line model that stably silences CXCL 16.The expression of CXCL 16 in each cell line model was verified by qRT-PCR and WB.3.Through(Cell counting Kit 8,CCK8)experiments,the cell proliferation ability of HPC cell lines that stably overexpress CXCL 16 or silence CXCL 16 were tested.4.The apoptosis of HPC cell lines that stably overexpress CXCL 16 or knockdown CXCL 16 were detected by flow cytometry.5.Through the Transwell chamber,the cell migration ability of HPC cell lines that stably overexpress CXCL 16 or knockdown CXCL 16 are tested.Results:1.By double enzyme digestion and comparison of sequencing results,CXCL16 shRNA1 and CXCL 16 shRNA 2 plasmids were successfully constructed and varified.2.Western blot results showed that,compared with the control group,the protein expression of CXCL 16 in HPC cell lines overexpressing CXCL 16 was significantly increased,P<0.01;compared with the control group pLKO.1-HPC,The CXCL16 expression was significantly reduce in the cell line pLKO.1-shCXCL16 HPC,P<0.01.3.The qRT-PCR results showed that compared with the control group,the mRNA expression of CXCL16 was significantly increased in HPC cell lines overexpressing CXCL16,P<0.001;while compared with the control group pLKO.1-HPC,CXCL16 expression in the CXCL16 knockdown HPC cell line was significantly reduced,P<0.05,P<0.01.4.The results of the CCK-8 experiment showed that compared with the control group,the cell proliferation ability of the podocyte strain overexpressing CXCL16 was significantly reduced at the 3rd and 5th day after inoculation,P<0.01,P<0.001;compared with the control group,the cell proliferation ability of the CXCL 16-knockdown podocyte line was significantly increased at the 3rd and 5th day after inoculation,P<0.001.5.The results of flow cytometry showed that compared with the control group,the apoptosis rate of the podocyte line overexpressing CXCL 16 is increased significantly,P<0.05;compared with the control group,the apoptosis rate of the CXCL16 knockdown podocyte line is decreased,P<0.05.6.The results of the transwell chamber test showed that compared with the control group,the cell migration ability of the podocyte line overexpressing CXCL 16 was significantly increased,P<0.001;compared with the control group,the cell migration ability of the CXCL 16 knockdown podocyte line was significantly reduced.P<0.01.Conclusion:1.Human podocyte models with stable overexpression and knockdown of CXCL16 were successfully constructed.2.CXCL 16 overexpression of podocytes decreased cell proliferation and increased podocyte apoptosis and migration.3.CXCL 16 silenced podocytes showed increased cell proliferation,decreased podocyte apoptosis and migration.4.CXCL16 participates in podocyte injury in PNS by inhibiting podocyte proliferation,promoting podocyte apoptosis and migration.Part Ⅲ Study on the mechanism of CXCL16 induced podocyte injury by regulating ERK1/2Objective:1.To explore whether the expression of ERK1/2 in podocytes is regulated by CXCL16.2.Construct a HPC cell line model that overexpresses CXCL16 and silences ERK1/2.3.Explore the role of ERK1/2 in podocyte injury.4.Explore whether CXCL16 can induce podocyte damage and its mechanism by regulating ERK1/2.Methods:1.Use Western blot to verify the protein expression level of ERK1/2 in podocytes that overexpress or silence CXCL16.2.Construct a lentiviral plasmid that silences ERK1/2.After the constructed plasmid is successfully digested with double enzyme and sequenced analysis,the four-plasmid packaging system is used to package the lentivirus that silences ERK1/2,and the lentivirus silence ERK1/2 were seperately used for infect HPC cell lines and HPC cell lines overexpressing CXCL16 to construct HPC cell line models that silence ERK1/2,overexpress CXCL16 and silence ERK1/2.The expression of CXCL16 and ERK1/2 in each cell line model was verified by qRT-PCR and Western blot experiments.3.Through(Cell counting Kit 8,CCK8)experiments,the cell proliferation ability of HPC cell lines that silence ERK1/2,overexpress CXCL16 and silence ERK1/2 were tested.4.Use flow cytometry to detect the apoptosis of HPC cell lines that silence ERK1/2,overexpress CXCL16 and silence ERK1/2.5.Through the transwell chamber,the cell migration ability of HPC cell lines that silence ERK1/2,overexpress CXCL16 and silence ERK1/2 are tested.6.Use Western blot to detect the expression of ZO-1,P-cadherin,Vimentin and N-cadherin proteins in HPC cell lines that silence ERK1/2,overexpress CXCL16 and silence ERK1/2,which are closely related to the EMT process.Results:1.Compared with the control group,the expression of CXCL16 in HPC cell lines overexpressing CXCL16 was significantly increased,P<0.05,while the expression of ERK1/2 was significantly increased at the same time,P<0.01;compared with the control group,CXCL16 was downregulated in CXCL16 knockdown HPC cell lines,P<0.05,while the expression of ERK1/2 was significantly reduced,P<0.01.2.By double enzyme digestion and comparison of sequencing results,the ERK1/2 shRNA plasmid was successfully constructed and varified.3.Western blot results showed that compared with the control group,the protein expression of ERK1/2 in the HPC cell line that silenced ERK1/2 was reduced,P<0.05;the protein expression of CXCL16 in the HPC cell line that overexpressed CXCL16 and silenced ERK1/2 was significantly increased,P<0.001,while the protein expression of ERK1/2 was significantly reduced,P<0.001.4.qRT-PCR results showed that compared with the control group,the mRNA expression of ERK1/2 in the HPC cell line that silenced ERK1/2 was reduced,P<0.05;compared with the control group,the mRNA expression of ERK1/2 in the HPC cell line that overexpressed CXCL16 and silenced ERK1/2 was significantly reduced,P<0.01;compared with the control group,the mRNA expression level of ERK1/2 in the HPC cell line overexpressing CXCL16 was significantly increased,P<0.01.5.Compared with the control group,silencing ERK1/2 can significantly increase the proliferation ability of podocytes,P<0.01;overexpression of CXCL16 can significantly reduce the proliferation ability of podocytes,P<0.001;and in the CXCL16 overexpressing HPC cell line,knockdown ERK1/2;increase the proliferation significantly,compared with the HPC group overexpressing CXCL16 alone,P<0.001.6.Compared with the control group,silencing ERK1/2 reduce the apoptosis rate of podocytes,P<0.05;overexpression of CXCL16 significantly increase the apoptosis rate of podocytes,P<0.01;and in the HPC cell line overexpressing CXCL16,with further knock down the expression of ERK1/2,compared with the HPC group overexpressing CXCL16 alone,the apoptosis rate was significantly reduced,P<0.001.7.Compared with the control group,silencing ERK1/2 significantly reduce the migration ability of podocytes,P<0.01;overexpression of CXCL16 significantly increase the migration ability of podocytes,P<0.001;and further knock down ERK1/2 in the HPC cell line overexpressing CXCL16,results in the significantly reduced cell migration ability,compared with the HPC group overexpressing CXCL16 alone,P<0.001.8.Compared with the control group,the expression of ZO-1 and P-cadherin in the ERK1/2 knockdown HPC cell line were increased,and the expression of Vimentin and N-cadherin were decreased,P<0.05;In the HPC cell line overexpressing CXCL16,expression of P-cadherin,ZO-1 were decreased,Vimentin,N-cadherin were increased,P<0.05.In the HPC cell line overexpressing CXCL16,the expression of ERK1/2 was further knocked down.ZO-1 and P-cadherin protein were increased,Vimentin and N-cadherin were decreased,compared with the HPC group overexpressing CXCL16 alone,P<0.05.Conclusion:1.The protein expression and mRNA expression of ERK1/2 in podocytes may be regulated by CXCL16.2.Successfully constructed HPC cell lines knockdown ERK1/2 and HPC cell lines that overexpress CXCL16 and knockdown ERK1/2.3.knockdown ERK1/2 increased podocyte proliferation,decreased podocyte apoptosis,migration and epithelial mesenchymal transition.4.Further knockdown of ERK1/2 expression in podocytes overexpressing CXCL16 could prevent the inhibition of podocyte proliferation,the increase of podocyte apoptosis rate and migration ability,and the increase of epithelial mesenchymal transition induced by CXCL16.5.CXCL16 can inhibit podocyte proliferation,promote podocyte apoptosis,migration and EMT by regulating ERK1/2,and then participate in the occurrence of podocyte injury in PNS.Innovations and meaning:1.It is the first time to explore the expression and clinical effects of CXCL16 and ERK1/2 in the renal tissue of children with primary nephrotic syndrome.The significance of the bed provides an important theoretical basis for studying the pathogenesis of PNS in children.2.It was confirmed for the first time that CXCL16 not only mediates podocyte lipid damage,but also inhibits podocyte proliferation,Promote podocyte apoptosis,promote podocyte migration and induce epithelial-mesenchymal transition to participate in podocyte injury,providing important clues for the prevention and treatment of children PNS podocyte injury in early clinical.3.It is the first to prove that CXCL16 can inhibit podocyte proliferation by regulating ERK1/2 and promote apoptosis,migration and EMT,and then participate in the occurrence of PNS podocyte injury in children,help to find therapeutic targets that delay the occurrence and progression of primary nephrotic syndrome in children to glomerulosclerosis,and provide a basis for precision medicine.