The Molecular Mechanism Of Imbalance Of TH17 And TREG Cells In The Pathogenesis Of Primary Nephrotic Syndrome In Children

PART ONE: ROLE OF INTERLEUKIN-17 IN GLOMERULO- SCLEROSIS SECONDARY TO ADRIAMYCIN-INDUCED NEPHROPATHY IN RATSObjective To investigate the role of interleukin-17(IL-17) in glomerulosclerosis secondary to Adriamycin-induced nephropathy in rats.Methods Male Sprague-Dawley rats were randomly divided into normal control group, adriamycin-induced nephropathy group (model group). Eight rats were sacrificed at second ,4th,8th,12th week respectively. Histopathological changes of renal tissue were observed under light microscope and glomerulosclerosis index was evaluated.The expression of IL -17 was measured by RT-PCR, ELISA and immunohistochemistry. The expression of interleukin -1β(IL-1β) was detected by ELISA.Results Compared with control group, the expressions of IL-17mRNA , IL-17 and IL-1βprotein were higher in model group of 8th week (P<0.05),even they were further higher in model group of 12 th week than control group (all P<0.01). In model group of 12th week,the level of IL-17 protein had positive correlation with IL-1βand glomerulosclerosis index (P<0.05).Conclusion IL-17 might contribute to glomerulosclerosis in Adriamycin-induced nephropathy.PART TWO: ROLE OF TH17 CELL AND CD4+CD25+ FOXP3+ REGULATR T CELL IN CHILDREN WITH PRIMARY NEPHROTIC SYNDROMEObjective To investigate the levels and functions of Th17 cells and CD4+CD25+Foxp3+ regulate cells(Treg) in PBMC and renal tissues and explore their roles in pathogenesis of primary nephrotic syndrome(PNS) in children.Methods The circulating frequencies of Th17 cells and Treg were measured by FCM.Real-time PCR were used to analyze the mRNA expressions of RORC, IL-23p19 and Foxp3 in PBMC. The serum of IL-1β,IL-6,IL-10 and TGF-β1 were measured by ELISA.The expression of IL-17 and IL-1βin renal tissue of PNS were measured immunohistochemistry.Results Circulating frequencies of Th17 cells ,the mRNA levels of RORC,IL-23p19 and the serum of IL-1β,IL-6 were higher in SNS and NNS groups than control group(all P<0.05),and they were higher in NNS group than SNS group(all P<0.05). Compared with control group , the circulating frencency of Treg cells ,the Foxp3 mRNA level and the serum level of TGF-β1 were lower in NNS and SNS groups (all P<0.05), and the circulating frencency of Treg cells ,the Foxp3mRNA level were lower in NNS group than SNS group(all P<0.05),but NNS and SNS group had no statistical difference in the serum level of TGF-β1 .The expression of IL-17 and IL-1βin renal biopsy of PNS were increasing gradually in the renal tissue of FSGS,MsPGN and MCNS(FSGS>MsPGN>MCNS).Conclusion Imbalance of Th17 and Treg cells might contribute to the pathogenesis of PNS in children and had associated with clinical presentation, pathological type, glucocorticoid sensitivity and prognosis of the disease. PART THREE : THE EFFECT OF IL-17 ON PODOCYTE- ASSOCIATED FACTORS AND APOPTOSIS AND MOLECULAR MECHANISIMObjective To investigate the effect of IL-17 on podocyte-associated factors and apoptosis in podocytes and explore the molecular mechanism.Methods The apoptosis of podocytes induced by IL-17 in a dose and time way and the protein expression of Fas and FasL of podocytes induced by IL-17 were detected by Flow Cytometrey. The expression of mRNA of Nephrin, WT1, Synaptopodin , Podocylaxin, Fas and FasL were measured by Real-time PCR.The protein of WT1,Caspases8,Caspases3 in podocyte were detected by immunocytochemistry.Results IL-17 promoted the apopotosis of podocyte in a dose and time way(P<0.01),and decreased the expression of Podocylaxin (P<0.05). IL-17 increased the expression of Fas, Caspases8 and Caspases3 in podocyte(P<0.01),but had no effect on Nephrin,WT1,Synaptopodin, and FasL in podocyte(P>0.05).Conclusion IL-17 may cause to renal injure by inducing the apoptosis of podocyte and decreased the expression of Podocylaxin in podocyte. PART FOUR: THE EFFECT OF IL-17 ON HUMAN MESANGIAL CELL LINES AND MOLECULAR MECHANISMObjective To investigate the effect of IL-17 on preliferation and secretion of IL-1β, TNF-a and MCP-1 in the human mesengial cell line(HMCL) and explore the molecular mechanism.Methods The proliferation of HMCL induced by IL-17 were detected by MTT in a dose and time way.The secretion of IL-1β,TNF-a and MCP-1 in HMCL induced by IL-17 were measured by ELISA. The expression of NF-κB in HMCL was detected by immunocytochemistry.Results IL-17 did not increase HMCL proliferation(P>0.05). IL-17 can induce the secretion of IL-1βin HMCL in a dose and time way ,and IL-17 can promote the expression of NF-κB in HMCL(P<0.05).But IL-17 had no effect on the secretion of TNF-a and MCP-1 in HMCL(P>0.05)Conclision IL-17 may cause renal injure by inducing the secretion of IL-1βin a way of NF-κB in HMCL.