| BackgroundMembranous nephropathy(MN)is the most common nephrotic syndrome in adults.Its typical histological manifestation consists of immunocomplex deposition in the glomerular subepithelium with basement membrane thickening.The 75%of cases have no known causes,and this subtype is known as idiopathic membranous nephropathy(IMN)or primary membranous nephropathy(PMN).The target antigens discovered so far include phospholipase A2 receptor,Thrombospondin type-1 domain-containing 7A,and nerual epidermal growth factor-like 1 protein.In view of the large heterogeneity of clinical manifestations,treatment response and prognosis of MN,the study of its pathogenesis is particularly important.Recent studies have shown that about 70%of patients with primary membranous nephropathy detected specific anti-podocyte target antigen PLA2R related antibodies,and the level of peripheral blood anti-PLA2R antibodies was closely related to the remission of proteinuria in patients with primary membranous nephropathy related.Studies at home and abroad have found that anti-PLA2R antibody was detected in the eluate of kidney tissue,and the antibody co-localized with PLA2R on podocytes.There is also evidence that the increase in the level of anti-PLA2R antibodies in the peripheral blood preceded the recurrence of proteinuria,and its decline preceded the remission of proteinuria.At the same time,studies have found that patients with positive anti-PLA2R antibodies in the serum of patients receiving kidney transplantation were more likely to relapse into membranous nephropathy.The research background suggested that anti-PLA2R antibody was involved in the occurrence and development of primary membranous nephropathy,but its pathogenic effect on podocytes was not clear.Research have found that more than 70%of patients with membranous nephropathy had local complement C3 deposition and co-localization with anti-PLA2R antibodies,suggesting that the activation of the complement system was involved in the renal damage of membranous nephropathy.It was the first observation in a rat model of Passive Heymann Nephritis that immune complexes was deposited under the kidney epithelium,and podocyte was damaged by activation of the local complement system,including podocyte cytoskeleton reorganization,slit membrane protein Loss and the appearance of a large amount of proteinuria,although the podocyte in situ antigen in this model was confirmed to be meglin protein,the role of the complement system in its podocyte injury is still worthy of attention.Complement system pathways include classic pathway,alternative pathway and mannose binding lectin pathway.It should be pointed out that more than 90%of patients with membranous nephropathy have C4d deposition under the glomerular epithelium.C4d is the product of cleavage of C4 when the classical pathway of complement or MBL pathway is activated,suggesting that the classical pathway or MBL pathway in membranous nephropathy was involved in local renal tissue damage.In addition,most studies have found that the renal tissue of patients with membranous nephropathy has significant C1q deposition,which further suggests that the activation of the complement MBL pathway played a key role in the renal tissue damage of patients with membranous nephropathy.In recent years,studies have found that there was an abnormal ratio of N-acetylglucosamine(GlcNAc)to galactose at the N-glycosylated end of the IgG4 type anti-PLA2R antibody in patients with membranous nephropathy,suggesting that the antibody molecule has N-glycosyl Modification of the possibility of abnormalities.Interestingly,it has been found in current studies of IgA nephropathy that IgA1 molecules lacking galactose in serum damaged mesangial cell by activating the MBL pathway.Moreover,research from patients with rheumatoid arthritis found that the deletion of IgG1 type rheumatoid factor N-glycosylation terminal galactose modification leaded to GlcNAc exposure,which could pass through mannose Combining MBL to activate complement aggravates disease damage.The above studies suggested that anti-PLA2R antibodies may interact with the MBL pathway.Therefore,it is particularly necessary to study the interaction between the two.It should be pointed out that since PLA2R is specifically expressed in mammals,there is no PLA2R-related IMN specific animal model.Podocytes are an important part of the glomerular filtration barrier.Podocyte injury is an important cause of glomerular filtration barrier barrier and proteinuria production,mainly manifested as podocyte foot process fusion,cytoskeleton reorganization and cell apoptosis.Therefore,it is particularly important to use the in vitro podocyte culture system to explore the pathogenicity of anti-PLA2R antibodies and their interaction with the MBL pathway.This study aims to explore the role of anti-PLA2R antibodies and MBL pathway in the pathogenesis of IMN,with a view to discovering potential clinical therapeutic targets.Purpose1.To explore the expression of MBL in the glomeruli of IMN patients and its relationship with the severity of the disease,treatment response,and clinical prognosis.2.To explore the damage effect of anti-PLA2R antibody from patients with IMN on human podocytes.3.To explore the synergistic effect of anti-PLA2R antibody from patients with IMN and MBL on human podocyte damage.Method1.The baseline kidney tissue of the IMN follow-up cohort was used to detect the deposition of MBL and anti-PLA2R antibodies in the kidney tissue using immunofluorescence.2.The baseline serum of the IMN follow-up cohort was used to detect Anti-PLA2R,anti-THSD7A,and serum MBL in serum with an ELISA kit.3.Purified IMN-derived anti-PLA2R antibodies were used to stimulate podocytes,and the expression of podocytes inherent proteins,the levels of inflammatory factors in the cell supernatant,cytoskeletal disorders,and apoptosis were observed.4.Purified IMN-derived anti-PLA2R antibodies and MBL components were used to co-stimμlate podocytes to further observe the activation of podocyte membrane complement,the expression of intrinsic proteins,cytoskeletal disorders,and apoptosis.Results1.79.1%(53/67)of IMN patients had positive MBL immunofluorescence staining in kidney tissue,and MBL was co-localized with anti-PLA2R antibody.There was no statistical difference in the positive rate of tissue anti-PLA2R antibody staining and the positive rate of serum anti-PLA2R antibody in the tissue MBL positive group and the MBL negative group.The median serum MBL concentrations after serum creatinine correction were 4.92(IQR,0.86,8.90)and 2.28(IQR,0.4,5.62),and there was no statistical difference between the two groups.Similarly,the serum albumin levels(26.5±6.6 and 28.6±6.1g/L),eGFR levels(104.8 ± 17.4 and 114.6±16.1ml/min.1.73m2),and urine protein quantitative levels(5.35 and 4.25g)and other clinical indicators between the two groups have not found significant statistical differences.According to the reference,we define ICR1 as urine protein quantitative between 0.3-1.0g/d,ICR2 as urine protein quantitative between 1.0-3.5g/d,or urine protein quantitative drop below half of the baseline.Complete remission(CR):urine protein quantitative<0.3g/d.Abnormal renal function is defined as a 30%decrease in eGFR from baseline.In univariate COX regression analysis,we found that MBL tissue deposition was a protective factor for ICR1((HR,2.78;95%CI,1.07-7.19;P=0.035),after correction for gender,age,serum anti-PLA2R antibody concentration,baseline eGFR,treatment methods,and systolic blood pressure,the multivariate COX regression results still suggested that MBL deposition had a protective effect on ICR1(HR,6.31;95%CI,1.10-36.14;P=0.039).However,MBL tissue deposition had no significance for CR(HR:8.00,95%CI,0.65-98.23;P=0.10)and the prognosis of renal function(HR,0.48;95%CI,0.16-1.40;P=0.18).It indicated that patients with MBL deposits in renal tissues at baseline IMN were likely to achieve partial remission of proteinuria.2.A protein affinity chromatography column was used to purify the anti-PLA2R antibody to construct a podocyte culture system.Establish IMN patient IgG stimulation group,healthy human IgG(Normal IgG)stimulation group and control group to stimulate podocytes respectively.We found that compared with the Normal IgG group,the expression of podocin protein in the podocytes of the IMN group decreased(P=0.016),the expression of podocin mRNA in podocytes decreased(P=0.005),the fluorescence expression of podocin protein on the surface of podocytes was weakened,the cytoskeleton was disordered and reorganized,and the ratio of podocytes apoptosis increased(P=0.008).It suggested that the anti-PLA2R antibody could damage podocytes.3.In order to further clarify whether there was an interaction between anti-PLA2R antibody and MBL aggravating podocyte damage,we constructed a co-stimulation group containing anti-PLA2R IgG antibody and MBL,a co-stimulation group containing healthy human IgG and MBL,containing anti-PLA2R IgG antibody single stimulation group,and MBL single stimulation group to explore the interaction between the two.The study had found that compared with containing anti-PLA2R antibody single stimulation group and the healthy human IgG and MBL costimulation group,anti-PLA2R antibody and MBL co-stimulation group had more C5b-9 expression on podocytes,podocin protein(P=0.002,P=0.002)and mRNA(P=0.019,P=0.032)lower expression,more disordered podocyte cell structure,and more podocyte apoptosis,suggesting that anti-PLA2R antibodies may exert podocyte pathogenic effects through MBL pathway.Conclusions1.MBL deposition in kidney tissue of IMN is more common and co-localized with anti-PLA2R antibody.Its deposition is related to the relief of IMN proteinuria.2.Anti-PLA2R IgG antibody has a damaging effect on IMN podocytes,and it interacts with complement MBL to participate in podocyte damage.Innovations1.This study confirmed that kidney tissue MBL is involved in disease progression in Chinese IMN patients..2.In this study,the pathogenicity of podocytes was explored by using anti-PLA2R IgG antibodies derived from IMN patients.3.This study confirms from the cellular level that anti-PLA2R IgG and MBL from IMN patients can cause podocyte damage.Insufficiency1.The clinical research part is a single-center retrospective analysis,with a small number of IMN follow-up samples and a relatively short follow-up time.2.As there is no IMN disease model,animal experiments have not been carried out for in vivo verification. |
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