抗肾小球基底膜GBM病

1、抗肾小球基底膜(GBM)病1抗抗GBM病的背景病的背景 抗抗GBM病：病：循环中出现抗循环中出现抗GBM抗体、抗体、脏器中沉积为特征的自身免疫病脏器中沉积为特征的自身免疫病 1919: Goodpasture首先报道首先报道 1例例18岁男性病人，咯血、急性肾衰竭岁男性病人，咯血、急性肾衰竭 主要累及肺和肾脏：主要累及肺和肾脏：Goodpasture病病 内科危重症：危及生命内科危重症：危及生命 80%就诊时已进入尿毒症（就诊时已进入尿毒症（ESRD）2Goodpasure EM. Am J Med Sci 1919;158: 863-870Cui Z, Zhao MH. Nat Rev Ne

2、phrol. 2011 Dec 7:697-706 少见病：少见病：1-2/百万人口百万人口 本研究所：累计诊断本研究所：累计诊断500余例余例 国际上最大的临床资源库国际上最大的临床资源库 治疗依赖血浆置换：昂贵，但多为时已晚治疗依赖血浆置换：昂贵，但多为时已晚抗抗GBM病仍然是我病仍然是我国国内科医生的重大挑内科医生的重大挑战战3抗GBM病的发生情况 Cui Z, Zhao MH. Nat Rev Nephrol. 2011 Dec 7:697-706抗抗GBM病研究现状病研究现状4Hudson GB. Vanderbilt UniversityGBM molecular architec

3、ture of conformational epitopesPusey CD. HammersmithImperial College LondonPE in anti-GBM diseaseWKY rat modelGenetics of EAG modelsSegelmark M & Wieslander JLund UniversityRecombinant antigensDetection of anti-GBM diseaseZhao MH & Cui ZPeking UniversityHuman anti-GBM diseaseLinear epitopesMolecular

4、 mimicryKitching AR. Monash University.MHC and T cell activationAnimal modelsLou YH. University of TexasT cell epitopeAnimal model抗抗GBM病是典型的自身免疫病病是典型的自身免疫病 靶抗原靶抗原 3(IV)NC1 （肺、肾）（肺、肾） Epitope Ea和和Eb-构象性构象性Saus J, et al. J Biol Chem 1988;15;263:13374-80Salant DJ. N Engl J Med 2010;363;4:381-39156抗抗GBM

5、病的科学问题病的科学问题 病因病因表型差异表型差异病因遗传易感背景自身免疫T细胞B细胞3(IV)NC1表型？6免疫耐受？诱发因素？7抗抗GBM病的科学问题病的科学问题 病因病因 表型差异表型差异肾受累轻重肾受累轻重1/3合并合并ANCA少数合并少数合并MN7病因遗传易感背景自身免疫T细胞B细胞3(IV)NC1表型？免疫耐受？诱发因素？823/M间断咯血 4 个月，加重1个月HGB: 71g/L; PO2 58mmHg; Scr 94.0 mol/l尿常规: protein (+), RBC 5-8/HPF血清抗GBM抗体 (+), ANCA (-)肾活检: IgG沿GBM线样沉积,肾小球轻微病

6、变治疗：Pred 1 mg/kg/d x 8w, 无PE和CTX随访7年肾功能正常Cui Z, et al. Kidney Int 2007;72:1403-8 8肾受累轻患者介于正常人与重症患者之间？转换机制？v既往：健康人血清无抗GBM抗体v发现天然抗GBM抗体：n中国和瑞典：各10名献血员nIgG成分-亲和层析n“阴性选择”？n如何发展成致病性抗体？Cui Z, et al. Kidney Int 2006:69:894-9Cui Z, et al. Kidney Int 2010;78:590-7Natural anti-GBM ab9抗抗GBM抗体如何转变成致病性？抗体如何转变成致病

7、性？天然抗GBM抗体Anti-GBM (+)严重肾受累Anti-GBM (+)正常肾功能正常人病人A病人 CIntra-moleculeEpitope spreading3, 4 1、2、3、4和5Subclass switchingIgG2、IgG4IgG1、IgG2、IgG3和IgG4治疗个体化T细胞调控3 Ea、 Eb3内其他位点10Anti-GBM (+)轻度肾受累病人BCui Z, et al. Kidney Int 2006;69:894-9.Yang R, et al. J Am Soc Nephrol 2007;18(4):1338-43.Cui Z, et al. Kidne

8、y Int 2007;72(11):1403-8.Zhao J & Cui Z, et al. Kidney Int. 2009;76:1108-15.Cui Z, et al. Kidney Int 2010;78(6):590-7.Chen JL & Hu SY, et al. Clin J Am Soc Nephrol. 2013;8(1):51-8.Inter-moleculeEpitope spreading NATURE REVIEWS | NEPHROLOGY Cui Z, Zhao MH. Nat Rev Nephrol. 2011 Dec;7:697-706. 抗抗GBM病的

9、科学问题病的科学问题 病因病因 表型差异表型差异肾受累轻重肾受累轻重1/3合并合并ANCA少数合并少数合并MN12病因遗传易感背景自身免疫T细胞B细胞3(IV)NC1表型？免疫耐受？诱发因素？13抗抗GBM病合并病合并MN 个例报道个例报道 MN GBM damage：释放：释放3 抗抗GBM 病病 抗抗GBM病病足细胞损伤：表达足细胞损伤：表达M-PLA2R MN13 8 patients with MN and anti-GBM disease Sequential or simultaneous Better prognosis Anti-3 (+): narrow antigen sp

10、ectrum Anti-PLA2R (-)Jia XY, et al. Kidney Int2014 Apr;85(4):945-52抗抗GBM病的科学问题病的科学问题 病因病因易感性：易感性：HLA？诱发因素诱发因素病因病因表型差异表型差异15病因遗传易感背景自身免疫T细胞B细胞3(IV)NC1表型？免疫耐受？诱发因素？16抗抗GBM病的免疫学发病机制病的免疫学发病机制?Linear toConformational 涉及感染、抗原递呈、抗原决定簇扩展、分子模拟涉及感染、抗原递呈、抗原决定簇扩展、分子模拟17Background ( HLA )HLA geneuLocation: CHR 6

11、p21.3uClassical HLA gene MHC class II molecular: uDistribution : DCs、B cells、MuStructure: hetero-dimerurecognized by CD4+ T cell uAg processed 、presentation MHC & disease: u MS、RA、IDDM、 SLE et al. 18(Rees, Kid Int, 1999)Dominantly protective alleles DR1 and DR7No gene dosage effectMHC II dominant pr

12、otectionHLA-DRB1*01:01 generates 3136-146 specific regulatory T cells.HLA-DRB1*15:01 generates 3136-146 specific effector T cell precursors.In HLA-DRB1*15:01x01:01 mice, 3136-146 specific effector T cell precursors are dominantly suppressed by 3136-146 specific regulatory T cellsRees et al, Kidney I

13、nt 1999 Ooi et al, J Am Soc Nephrol 2013 DRB1*1501 allele：p=1.597107 DRB1*0404 allele：p=0.037 Patients with DRB1*1501 or *0404 had more crescent formation. (p=0.021).Yang R. et al. Clin Immunol 2009;133:245-25019Association of HLA alleles (4 digits, P3.55E-4)AlleleCase_freControl_freORP_valDRB1*1501

14、0.43840.14694.5495.658E-28DQB1*06020.38400.15443.3362.032E-17DQA1*05020.0255470.000834730.696.987E-7DQB1*03030.04710.14440.28858.575E-6DRB1*09010.065220.14940.39111.611E-420Determine the significant variation marker of genotypeCase: 138 vs. Control: 599rs41541412: the only significant SNP, belongs t

15、o DQA1*0502 nonsense mutation, change the 82th AA of DQ polypeptide. CHRSNPCaseControlCHISQP-valOR632609249.C0.02550.00083524.626.99E-730.69632609249.G0.02550.00083524.626.99E-730.69Association of a novel HLA SNP(P3:Confirmed linkage. LOD-2: No linkage. LOD=0: the possibility is equal Linkage analys

16、is among the significant allelesUnpublished data抗抗GBM病的科学问题病的科学问题 病因病因易感性易感性诱发因素：环境？诱发因素：环境？病因病因表型差异表型差异23病因遗传易感背景自身免疫T细胞B细胞3(IV)NC1表型？免疫耐受？诱发因素？24抗抗GBM病的科学问题病的科学问题 病因病因易感性易感性诱发因素诱发因素病因：感染？病因：感染？表型差异表型差异24病因遗传易感背景自身免疫T细胞B细胞3(IV)NC1表型？免疫耐受？诱发因素？25假说：微生物可能是抗假说：微生物可能是抗GBM病的病因之一病的病因之一1919: Goodpasture首先

17、报道 1例18岁男性病人，咯血、急性肾衰竭 流感？60%的患者发病前有前驱感染症状 病原微生物-分子模拟?Goodpasure EM. Am J Med Sci 1919;158: 863-87025分子模拟B细胞表位T细胞表位26B B细胞的线性抗原决定簇细胞的线性抗原决定簇合成24条重叠肽段： 覆盖3(IV)NC1的234aa 起始的线性抗原决定簇：P14（aa129-150） 2422212019181716151413121110987654321Linear peptides along 3(IV)NC1100%80%60%40%20%0%Frequency of recogniti

18、onInitiation epitope?Risk epitope?Jia XY, et al. Clin J Am Soc Nephrol 2012 Jun;7(6):926-33P14(22mer)诱发诱发WKY大鼠抗大鼠抗GBM肾炎肾炎P14（aa129-150） 分子内抗原决定簇扩展分子内抗原决定簇扩展 诱发自身免疫性诱发自身免疫性T细胞增殖细胞增殖 T/B细胞共同抗原决定簇细胞共同抗原决定簇27Unpublished dataB B细胞的关键抗原决定簇与核心氨基酸基序细胞的关键抗原决定簇与核心氨基酸基序 P14氨基酸序列氨基酸序列: P14: TDIPPCPHGWISLWKGFSFI

19、MF P14a: TDIPPCPHGWISL P14b: CPHGWISLWKGFS P14c: ISLWKGFSFIMFT28P14c逐个氨基酸突变B细胞识别的关键氨基酸基序 GFxF p14cISLWKGFSFIMFT-0.20.00.81.01.21.4Residues of substitutionNet OD value at 405nmUnpublished data29Critical motif on P14 for pathogenicityP14-1 ADIPPCPHGWISLWKGFSFIMFP14-2 TAIPPCPHGWISLWKGFSFIMFP14

20、-3 TDAPPCPHGWISLWKGFSFIMFP14-4 TDIAPCPHGWISLWKGFSFIMFP14-5 TDIPACPHGWISLWKGFSFIMFP14-6 TDIPPAPHGWISLWKGFSFIMFP14-7 TDIPPCAHGWISLWKGFSFIMFP14-8 TDIPPCPAGWISLWKGFSFIMFP14-9 TDIPPCPHAWISLWKGFSFIMFP14-10 TDIPPCPHGAISLWKGFSFIMFP14-11 TDIPPCPHGWASLWKGFSFIMFP14-12 TDIPPCPHGWIALWKGFSFIMFP14-13 TDIPPCPHGWISA

21、WKGFSFIMFP14-14 TDIPPCPHGWISLAKGFSFIMFP14-15 TDIPPCPHGWISLWAGFSFIMFP14-16 TDIPPCPHGWISLWKAFSFIMFP14-17 TDIPPCPHGWISLWKGASFIMFP14-18 TDIPPCPHGWISLWKGFAFIMFP14-19 TDIPPCPHGWISLWKGFSAIMFP14-20 TDIPPCPHGWISLWKGFSFAMFP14-21 TDIPPCPHGWISLWKGFSFIAFP14-22 TDIPPCPHGWISLWKGFSFIMAUnpublished dataTryptophan138,

22、 Isoleucine139, Leucine141, and Tryptophan142P14129-150: TDIPPCPHGWISLWKGFSFIMF抗GBM病-病因研究 针对致病微生物的研究 细菌、病毒等培养（尚无来源） 合成抗原分子 利用生物信息学预测可能的T/B细胞抗原决定簇 确定抗GBM病患者是否感染 血清抗致病微生物蛋白抗体 动物实验验证其致病性30 8 patients with MN and anti-GBM disease Sequential or simultaneous Better prognosis Anti-3 (+): narrow antigen spe

23、ctrum Anti-PLA2R (-) 原因？原因？Jia XY, et al. Kidney Int2014 Apr;85(4):945-52人3(IV)NC1诱发了小鼠膜性肾病 DBA/1 mice (vs. WKY rat) rh-a3(IV)NC1 Nephrotic Syndrome EM：MN32Zhang JJ, et al. J Immunol. 2012 Apr 1;188(7):3268-77Membranous nephropathy induced by P13, P14, P15 on DBA/1 mice33Unpublished dataMicrobial?HL

24、A-DRB1*1501Like H-2?MHC?Anti-GBM diseaseMNPodocyte protein?Podocyte 3？ 抗原递呈-MHC-II: DBA1 mice vs. WKY ratHuman？靶抗原小鼠3？足细胞抗原？病因-关键氨基酸与分子模拟？Human 3Intra-molecule epitope spreadingInter-molecule epitope spreadingRat 335小结 抗GBM病并非罕见, 仍为肾科医生的挑战 自身抗体免疫学特性的变化与疾病进展密切相关 抗GBM病的病因有待明确和证实353623/M间断咯血 4 个月，加重1个月

25、HGB: 71g/L; PO2 58mmHg; Scr 94.0 mol/l尿常规: protein (+), RBC 5-8/HPF血清抗GBM抗体 (+), ANCA (-)肾活检: IgG沿GBM线样沉积,肾小球轻微病变治疗：Pred 1 mg/kg/d x 8w, 无PE和CTX随访7年肾功能正常Cui Z, et al. Kidney Int 2007;72:1403-8 36肾受累轻患者介于正常人与重症患者之间？转换机制？ 8 patients with MN and anti-GBM disease Sequential or simultaneous Better progno

26、sis Anti-3 (+): narrow antigen spectrum Anti-PLA2R (-)Jia XY, et al. Kidney Int2014 Apr;85(4):945-5238Background ( HLA )HLA geneuLocation: CHR 6p21.3uClassical HLA gene MHC class II molecular: uDistribution : DCs、B cells、MuStructure: hetero-dimerurecognized by CD4+ T cell uAg processed 、presentation MHC & disease: u MS、RA、IDDM、 SLE et al. Association of HLA alleles (4 digits, P3.55E-4)AlleleCase_freControl_freORP_valDRB1*15010.43840.14694.5495.658E-28DQB1*06020.38400.15443.3362.032E-17DQA1*05020.0255470.000834730.696.987E-7DQB1*03030.04710.14440.28858.575E-6DRB1*09010.