【作者】 张丹；

【导师】 蒋更如；

【作者基本信息】 上海交通大学 ， 内科学（肾脏病学）， 2018， 博士

【摘要】 目的:特发性膜性肾病(idiopathic membranous nephropathy,IMN)是成人肾病综合征(nephrotic syndrome,NS)的主要病因之一,且近年来我国IMN的发病率呈明显上升趋势。虽然部分患者可自发缓解,但仍有近1/3的患者将进展至终末期肾脏病(End-Stage Renal Disease,ESRD)。IMN是一种器官特异性自身免疫性疾病,2009年M型磷酯酶A2受体(phospholipase A2 receptor,PLA2R)被首次证实在肾小球足细胞表达,是IMN的重要靶抗原。近来又发现了IMN的另一靶抗原1型血小板反应蛋白7A域(thrombospondintype-1 domain containing 7A,THSD7A)。本研究旨在探讨肾小球PLA2R和THSD7A及其抗体用于诊断IMN的价值,IMN患者血清抗PLA2R抗体与肾小球PLA2R抗原之间的关系,以及分析PLA2R和THSD7A及其抗体与疾病临床预后的相关性。并进一步对MN合并肿瘤的患者进行临床病理和自身抗原抗体分析,进一步探索肿瘤与MN之间的关系。本研究还通过比较PLA2R相关性IMN和非PLA2R相关性IMN患者中补体系统C4d和甘露糖结合凝集素(mannose binding lectin,MBL)的表达以及分析PLA2R1,HLA-DQA1基因多态性,深入了解PLA2R参与IMN的发病机制。方法:选取临床和病理明确诊断为MN的患者以及其它肾小球疾病患者为研究对象,收集临床病理资料;用免疫组织化学方法检测肾组织PLA2R、THSD7A和IgG4;同时分别通过酶联免疫吸附检测和间接免疫荧光法检测血清抗PLA2R抗体和THSD7A抗体的水平;在肿瘤合并MN的患者中检测肾组织PLA2R、THSD7A和IGG4的阳性率以及血清PLA2R抗体和THSD7A抗体,分析其在鉴别肿瘤相关性MN中的价值;通过免疫组化法对肾组织进行C4d和MBL的检测,探索补体途径在PLA2R相关性IMN和PLA2R非相关性IMN中的差异;通过对6个PLA2R1基因中单核苷酸多态性(single nucleotide polymorphism,SNP)和1个HLA-DQA1 SNP检测,了解PLA2R相关性IMN的基因型特点。结果:肾小球PLA2R阳性率在IMN组、非IMN组和非典型MN组中分别为85.4%(164/192)、15%(9/60)和75%(15/20),IMN组和非IMN组间具显著差异(p<0.05);12例HBV-LN患者中PLA2R抗原染色阳性4例,14例肿瘤合并MN中PLA2R抗原阳性5例,V型LN和非MN患者PLA2R抗原均为阴性;在IMN中,PLA2R抗原阳性组较PLA2R抗原阴性组患者高血压发生率更高,血浆白蛋白水平更低(p<0.05);随访后PLA2R阳性和阴性组平均24hUPro、血浆Alb、SCr和eGFR均无统计学差异(p>0.05),PLA2R抗原阴性组缓解率较PLA2R抗原阳性组高,但无统计学差异(p>0.05);PLA2R抗原阴性的IMN患者中发现3例肾组织THSD7A阳性,占IMN患者的1.6%;儿童IMN中肾小球PLA2R阳性率占42.1%(16/38),其中少儿组占25.9%(7/27),青少年组占81.8%(9/11),组间有明显差异(p<0.05);少儿组与青少年组比较,血尿发生率更高,蛋白尿和血肌酐水平更低,eGFR水平更高(p<0.05)。血清抗PLA2R抗体阳性率在IMN组和非IMN组中分别为65.8%(50/76)和11.6%(5/43)(p<0.05),5例HBV-MN中血清PLA2R抗体阳性2例,7例肿瘤合并MN中3例PLA2R抗体阳性;76例IMN患者中肾小球PLA2R抗原阳性70例(92.1%),肾小球PLA2R抗原阳性患者中血清抗PLA2R抗体阴性21例(30%);抗PLA2R抗体阳性患者较抗体阴性患者24hUPro水平更高,Alb水平更低(p<0.05),且抗体滴度水平与24hUPro(p=0.047,r=0.372)和血Alb均有相关性(p=0.036,r=-0.258);PLA2R抗体高滴度组、低滴度组和PLA2R抗体(-)患者总缓解率分别为68.4%(13/19)、80.0%(12/15)和92.3%(12/13),其中CR者分别为31.6%,13.3%和46.2%,但无统计学差异(p>0.05);3例肾组织THSD7A抗原染色阳性的患者中,1例患者同时检测出血清THSD7A抗体阳性,随访后此患者蛋白尿部分缓解,同时测THSD7A抗体为阴性。与IMN患者相比,MN合并恶肿瘤的患者平均年龄明显升高,sCr水平更高,肾功能损伤程度更严重(p<0.05);13例恶性肿瘤合并MN患者中9例肾小球PLA2R抗原染色阴性,且不伴肾组织IgG4沉积;4例肾组织PLA2R阳性,同时IgG4染色阳性。其中2例同时伴血清抗PLA2R抗体阳性,且肿瘤切除后蛋白尿均未缓解,另2例为MN复发同时发现肿瘤;肾组织THSD7A染色及其抗体均为阴性;另发现一例多发性神经纤维瘤合并MN患者PLA2R和THSD7A抗原双阳性。IMN患者中肾组织C4d阳性率为82%(41/50),其中肾组织MBL阳性患者为29.3%(12/41)。C4d阴性患者中未发现MBL阳性;PLA2R相关性IMN和PLA2R非相关性IMN中,C4d阳性率分别为83.3%(25/30)和80%(16/20),其中MBL阳性率分别为26.7%(8/30)和20%(4/20)(p>0.05)。本队列IMN患者与健康人群相比,rs4664308、rs3828323、rs3749117和rs2715928在基因频率上均有显著性差异(p<0.05);PLA2R相关性和非相关性IMN组人群rs3828323均以CC型为主,前组CC型比例更高,组间具统计学差异(P<0.05);两组rs4664308、rs35771982、rs3749117和rs3749119多以AA型、GG型、TT型和CC型为主,HLA-DQA1基因rs2187668多为GG型,前组患者上述基因型比例更高,但组间均无统计学差异(P>0.05)。结论:IMN患者中肾小球PLA2R抗原染色阳性率明显高于非IMN患者,可作为鉴别IMN的有效手段;IMN中PLA2R阳性患者高血压比例更多,血浆Alb水平更低,提示PLA2R抗原与疾病严重程度有一定关系;虽然IMN患者肾组织THSD7A抗原阳性率较低,但与PLA2R抗原联合检测对IMN诊断仍有重要价值;在儿童IMN中肾小球PLA2R阳性率明显低于成人。少儿组(25.9%)与青少年组(81.8%)相比有明显差异,青少年IMN患者临床表现及PLA2R阳性率均与成人IMN更为接近,提示少儿IMN可能有与成人不同的发病机制,青少年组IMN发病机制可能与成人IMN相似。IMN患者中血清抗PLA2R抗体阳性率明显高于非IMN患者,提示抗PLA2R抗体在IMN中有较高特异性;血清抗PLA2R抗体与肾小球PLA2R抗原相比,抗体阳性者其抗原表达绝大多数为阴性,而抗原阳性者中部分抗体为阴性,提示PLA2R抗原敏感性相对更高;血清抗PLA2R抗体和THSD7A抗体可反映疾病严重程度,且可能与疾病预后相关。恶性肿瘤合并MN患者中4例PLA2R抗原阳性患者中2例PLA2R抗体阳性,肾组织同时存在IgG4沉积,2例为MN复发同时发现肿瘤,根据病程间距及预后判断可能为肿瘤伴随IMN同时存在。提示肾小球PLA2R及其抗体和IgG4检测一定程度上可反映肿瘤和MN间的关系。IMN患者中肾组织C4d阳性率82%,其中MBL阳性率为29.3%,提示IMN患者中除了旁路途径,经典途径和凝集素途径均可能参与补体激活;但补体激活在PLA2R相关性IMN与PLA2R非相关性IMN中未有明显差异。PLA2R相关性和非相关性IMN组rs3828323基因型具显著性差异,提示PLA2R相关性IMN和PLA2R非相关性IMN在基因易感性方面存在一定差异。更多还原

【Abstract】 Objective Idiopathic membranous nephropathy(IMN)is one of the main causes of nephrotic syndrome(NS)in adults.In the Chinese population,the prevalence of membranous nephropathy(MN)has increased rapidly in recent years.Although some of the patients with IMN may undergo a spontaneous remission of disease,one-third of cases progress to end-stage renal disease(ESRD).IMN is regarded as an organ-specific autoimmune disease.In 2009,M-type phospholipase A2 receptor(PLA2R)was considered as the major target antigen in IMN and Thrombospondin type-1 domain-containing 7A(THSD7A)is the most recently recognized new target antigen in patients with MN.The purpose of our study is to estimate the value of PLA2 R and THSD7 A in the diagnosis of IMN and to analyze the correlation between the expression of serum anti-PLA2 R antibody and renal PLA2 R.We also assess the relationship among glomerular PLA2 R expression with anti PLA2 R autoantibodies,the severity of the disease and the outcomes as well.Up to date,it lacks specific biomarkers for differentiating the malignancy associated MN from IMN coincident with cancer,we investigate clinicalpathological characteristics and the expression of antigens and autoantibodies of MN patients with cancer to evaluate the relationship between MN and malignancy.Since the different pathogenesis between PLA2 R related MN and PLA2 R unrelated MN remains unknown,we aim to understand the involvement of complements pathway and role of gene polymorphisms between them.Methods Adults and children patients with biopsy-proven MN and other glomerular diseases at Xin Hua Hospital and Shanghai Children’s Hospital were included.Clinicalpathological data were obtained from all of the patients at the time of renal biopsy.Serum specimens and urine samples were also collected at baseline and during the follow-up.IHC staining of PLA2 R,THSD7A,Ig G4,C4 d,MBL,S-100 and neurofibromin were performed and circulating anti-PLA2 R antibodies and THSD7 A antibodies were tested using enzyme linked immunosorbent assay(ELISA)and indirect immunofluorescence test(IFT)respectively.Genotype analysis was done by Taq Man assays for seven single-nucleotide polymorphisms in PLA2R1 and HLA-DQA1.Results A total of 85.4% of patients(164/192)with IMN had positive glomerular PLA2 R deposits,while 15% and 75% of cases showed positive of PLA2 R expression in non IMN and UAMN(undetermined atypical membranous nephropathy)group respectively.4 MN patients secondary to HBV infection and 5 MN with cancer showed positive of glomerular PLA2 R deposits.Among IMN patients,the proportion of hypertension were significantly higher whereas serum albumin was significantly lower in the PLA2R-associated patient group,compared with those in the non-PLA2R-associated group(p<0.05).There were no significant differences between two groups with respect to 24-h urinary protein level,serum albumin,serum creatinine levels,e GFR and remission rates during the follow-up.THSD7 A deposits were found in 3(1.6%)IMN patients and all of them presented PLA2 R deposits negative.In the child IMN patients only 16(42.1%)were positive for PLA2 R expression and a significantly higher rate of PLA2 R staining was also observed in the adolescent group when compared to the young child group(81.8% versus 25.9%;p<0.05).Hematuria,proteinuria,serum creatinine levels and e GFR were all significantly different between the two groups(p <0.05).65.8% of patients(50/76)with IMN had positive PLA2 R antibodies,two of five MN patients secondary to HBV infection and three of seven MN with cancer showed positive of anti-PLA2 R antibodies.Of 76 patients,positive rates of glomerular PLA2 R deposition were 92.1%(70/76).30%(21/70)of IMN patients with positive glomerular PLA2 R deposition were negative for anti-PLA2 R antibodies.Patients with PLA2 R antibodies had a higher level of proteinuria and low level of serum albumin than those who lacked PLA2 R antibodies(p <0.05).Patient antibody levels were weakly correlated with baseline proteinuria(p=0.047,r=0.372)and serum albumin(p=0.026,r=-0.358).There were no significant differences with respect to remission rates between patients with PLA2 R antibodies positive and negative during the follow-up.One of three patients with glomerular THSD7 A deposits positive was found to be positive for anti-THSD7 A antibodies.IFT was repeated at the point of partial remission after immunosuppressive therapy,and serum anti-THSD7 A antibody levels turned negative.MN Patients with cancer were significantly older than patients with IMN(p<0.05).MN patients with cancer had significantly higher serum creatinine and lower e GFR than IMN patients(p<0.05).Glomerular PLA2 R and Ig G4 deposits showed positive in 4 of 13 cases with cancer.Meanwhile,2 of 4 patients had serum PLA2 R antibodies positive.All of patients showed negative for glomerular THSD7A deposits and anti THSD7A antibodies.One case with neurofibromatosis 1 was identified with duel PLA2 R and THSD7 A antigen.In cases with PLA2 R related IMN and PLA2 R unrelated IMN,glomerular C4 d deposits were detected in 83.3%(25/30)of patients and 80%(16/20)of patients,respectively.In 41 patients with C4 d positive,activation of the lectin pathway was observed in 12(29.3%)patients.All of 9 C4 d negative patients showed negative for MBL.Allelic frequency distributions of 4 SNPs within PLA2R1 rs3828323,rs4664308,rs3749117 and rs2715928)were significantly different between IMN subjects and healthy control.There were significant differences in genotypic frequency distributions of rs3828323 between patients with PLA2 R related IMN and PLA2 R unrelated IMN.Conclusion The expression of glomerular PLA2 R is significantly higher in IMN patients than in non IMN patients,hence it could be considered as an available method for diagnosis of IMN.IMN patients with positive glomerular PLA2 R expression are associated with more severe proteinuria and higher proportion of hypertension,indicating that glomerular PLA2 R expression is correlated with the severity of disease.Although the incidence of THSD7 A expression is relatively low in IMN cases,it is still valuable for the diagnosis of IMN.The clinical features and prevalence of PLA2 R staining in adolescents with IMN were similar to what was seen in the adult patients,indicating that they probably have a closer etiology and pathogenesis of the disease.Most of the young children with IMN were negative for PLA2 R expression,with a more varied and underlying etiology than in the adults.Most of cases with serum PLA2 R autoantibody positive showed enhanced expression of glomerular PLA2 R,whereas serum PLA2 R antibody could be negative in patients with glomerular PLA2 R positive,suggesting that serum PLA2 R antibody has a good specificity but a little lower sensitivity for diagnosis of IMN.Serum PLA2 R antibody as glomerular PLA2 R expression may reflect the disease activity.One patient with anti-THSD7 A antibodies positive showed negative for anti-THSD7 A antibody at the point of partial remission after immunosuppressive therapy,indicating that anti-THSD7 A antibody may also reflect the severity of disease.Absence of glomerular PLA2 R deposition,together with Ig G4 could be useful clues to differentiate malignancy associated MN from IMN.The positivity of glomerular C4 d and MBL expression suggested that the lectin and/or classical pathway of complement were activated in IMN cases,which showed no difference between PLA2 R related IMN and PLA2 R unrelated IMN.Allelic frequency distributions of 4 SNPs within PLA2R1 rs3828323,rs4664308,rs3749117 and rs2715928)were significantly different between the IMN and healthy control.There were significant differences in genotypic frequency distributions of rs3828323 between patients with PLA2 R related IMN and PLA2 R unrelated IMN,suggesting that PLA2 R related IMN may have specific genetic factor to cause the disease.更多还原