Purification And Identification Of Anti-nonmuscular Myosin Heavy Chain ⅡA Antibody In Idiopathic Membranous Nephropathy&Rat Passive Membranous Nephropathy Model Induced By Anti-nonmuscular Myosin Heavy Chain ⅡA Antibody From Imn Patients

Part 1:Purification and Identification of Anti-Nonmuscular Myosin Heavy Chain ⅡA Antibody in Idiopathic Membranous NephropathyObjective:To purify Anti-Nonmuscular Myosin Heavy Chain ⅡA Antibody in vivo of idiopathic membranes nephropathy,and identify it.Methods:10 patients with the clinical manifestations of massive proteinuria(≥ 3g/24 hours)and with the pathology results confirming the diagnosis of idiopathic membranous nephropathy(IMN)were chosen.Before using hormones and other immunosuppressive therapy,we extracted 10ml EDTA anticoagulant blood,and keep its serum after centrifugation.We selected 10 health people as controls at the same time.Those serum was mix in ratios respectively.First,we isolated serum IgG with serum IgG isolation kit,and confirmed by SDS-PAGE electrophoresis,then detected its concentration.By further doing co-IP on kit,we reversely extracted and purified anti-human nonmuscular myosin heavy chain Ⅱ A antibodies,which were confirmed the main composition in the extract by Western blot,using podocyte protein and commercialization Myosin9 protein fragments(molecular weight 110KD)as antigen.Results:Anti-human nonmuscular myosin heavy chain ⅡA antibodies in vivo of idiopathic membranes nephropathy can be purified to some extent by this way,and can reach a certain centrifugation,which was significantly higher than the normal control level,showing clinical significance.Conclusions:With efforts of graduate students and instructors in our department,anti-human nonmuscular myosin heavy chain ⅡA antibodies has been proven to be one of the most likely newly-found target antigen for IMN.In this study,with the principle of co-immunoprecipitation,we purified a certain concentration of non-muscle myosin heavy chain ⅡA antibodies in patients mainly through two steps,having significant differences with the normal control group extract concentration.According to the purification results,it can be found that there are differences between the antibodies and normal controls,which confirmed the important role of non-muscle myosin heavy chain Ⅱ antibodies in patients with idiopathic membranous nephropathy.With former studies,it seems that non-muscle myosin heavy chain ⅡA was one of the target antigens for idiopathic membranous nephropathy.Therefore,this antibody extracted and purified from the IMN patients can be used to study the immune mechanisms and disease transfer of idiopathic membranous nephropathy.Part 2:Rat Passive Membranous Nephropathy Model Induced by Anti-Nonmuscular Myosin Heavy Chain Ⅱ A Antibody from IMN PatientsObjective:To observe the clinical and renal pathological features in the rat model induced by anti-nonmuscular myosin heavy chain ⅡA antibody,which was purified from IMN patients.Methodology:Male Sprague-Dawley rats were randomly divided into 9 groups:normal control group;the time points of 3days group,5 days group,7 days group,14 days group,21 days group,28 days group,35 days group and 42 days group after being injected with anti-nonmuscular myosin heavy chain Ⅱ A antibodies,5 rats per group.The levels of 24 hour proteinuria were measured.The pathological;changes of rats kidneys were observed under the light microscope,immunofluorescence microscope,and transmission electron microscope.Results:Compared with the normal control group(0 day),the level of 24-hour urine protein of the rats injected the anti-nonmuscular myosin heavy chain ⅡA antibody from IMN patients in day 7 group increased.The urine protein levels showed gradually increasing.The urine protein levels is 13.34±1.22mg/d in the 14 days group after injiection,which was significantly higher than in the control group(P<0.05).In the 21 days group,the total urinary protein was significantly increased[(17.20±2.58)mg/d,P<0.01].The highest level was in the 42 days group[(35.15±2.58)mg/d,P<0.01],By light microscope there was no obvious changes of rats kidneys in 7 days group,14 days group,21 days group and 28 days group after injected anti-nonmuscular myosin heavy chain II A antibodies which were dyed with HE,PAS,Masson,PASM.There were no obvious differences from normal control group in addition to mild mesangium hyperplasia.The immunofluorescence results showed that:Only 1 and 3 days after rats were injected antibody when can we detect granular deposition of anti-NMMHC ⅡA antibody from IMN patients along the glomerular basement membrane in renal tissues.The own IgG gradually increased after 7 day,reached the most on the 21st day,which mainly showed granular deposition along the basement membrane,without fluorescence intensity attenuation.By electron microscope,after injected the anti-NMMHC ⅡA antibody,some rats podocytes gradually turned flat or disappeared,and basement membrane began to appear focal thickening on the 28thday,while foot processes fusion increased.Lesions increased on 42nd day,the subepithelial dense appearing and gradually increasing,focal thickening sites on basement membrane increasing,which is obviously different from the normal control group.Conclusion:Rat Passive Membranous Nephropathy Model Induced by Human Anti-Nonmuscular Myosin Heavy Chain ⅡA Antibody has successfully been built.