Clinicopathological Study And T Cell Receptor Repertoire Analysis Of Exostosin 1/Exostosin 2 Associated Membranous Nephropathy

Objective:Membranous nephropathy(MN)is a common cause of adult nephrotic syndrome.The pathogenesis is related to the deposition of immune complexes outside the glomerular basement membrane(GBM).Recently,several novel proteins have been detected as antigens/markers of the MN[1-7].Exostosin l/exostosin 2(EXT1/EXT2)have been reported as the main antigens/markers of autoimmune disease-associated MN by Sethi et al[8].EXTs are glycosyltransferases expressed on podocytes,which are responsible for the synthesis of the heparan sulfate[10].Immunohistochemistry(IHC)shows granular staining for EXT1 and EXT2 along the GBM.The majority(>80%)of EXT1/EXT2 positive MN patients has autoimmune finding.Some patients have autoimmune diseases(ADs),and some patients have positive autoantibodies(ANA,Anti-dsDNA,Anti-RNP,Anti-SSA or Anti-SSB).Till now,there are only four researches about EXT1/EXT2 associated MN[8,11-13].This is the first study with a large sample size targeting the Chinese population.EXT1/EXT2 can be detected in membranous lupus nephritis(MLN).MN patients with EXT1/EXT2 positive results could develop to MLN[8].Lupus nephritis(LN)are divided into proliferative lupus nephritis(PLN)and non-proliferative lupus nephritis(MLN).The treatment of lupus nephritis is based on its pathological type.Rituximab does not show additive efficacy in the treatment of PLN[15],but some studies have shown that rituximab is effective in the treatment of MLN[16-18].The rituximab is also effective in idiopathic membranous nephropathy(IMN)[24].T cell receptor(TCR)are receptors distributed on the surface of T cells.They can specifically recognize antigen-presenting cells(APC),participating in antigen recognition and immune response[19].Different diseases have different TCR clonal diversity,clonal expansion,TRBV,TRBJ gene usage,V-J pairing and shared clone.It has been used in the identification of tumors,inflammations,and different immune diseases[21-23].This study has enrolled MN patients with autoimmune background,including patients diagnosed with ADs and patients with only abnormal autoantibodies.IHC staining of EXT1/EXT2 has been conducted in all patients.This study has analyzed the clinical and pathological characteristics of EXT1/EXT2 associated MN,treatment and disease outcome has also been recorded.This study has sequenced the TCRβCDR3 of EXT1/EXT2 associated MN(including MLN),PLA2R associated IMN,and PLN patients,in order to compare the difference of TCR between groups.Methods:1.Research objects:A number of 189 MN patients were enrolled,including patients had at least one abnormal autoantibody(including:ANA,Anti-dsDNA,AntiU1RNP,Anti-SM,Anti-SSA,Anti-SSB,Anti-SLC-70,Anti-RIB,Anti-RO-52,Anti-JO-1)and patents diagnosed with MLN in West China Hospital of Sichuan University from January 2014 to September 2020.A number of 32 patients diagnosed with PLA2R associated IMN were enrolled as control from January 2016 to September 2020.2.IHC and IF staining:IHC staining of EXT1/EXT2 on paraffin sections were conducted in the 189 patients.Positive result showed bright granular staining for EXT1 or EXT2 along the GBM.The IF staining was conducted if the IHC staining was blurred.Calculated the positive rate of EXT1/EXT2.3.Localization of EXT1 in the glomerulus:IF staining of EXT1 with Nephrin or Agrin were conducted.Observed the localization of EXT1 in the glomerulus under a confocal microscope.4.Pathological indicators:Light microscope indicators included:mesangial hyperplasia,basement membrane thickening,basement membrane spike formation,renal tubule degeneration,renal interstitial fibrosis and tubule atrophy(IFTA),renal interstitial inflammatory cell infiltration,and renal blood vessels lesion.Immunofluorescence indicators included:IgG,IgM,IgA,C3,C4,Clq.Electron microscope indicators included:Ehrenreich-Churg classification.5.Clinical indicators and disease outcome:gender,age,rheumatic system symptoms,hemoglobin,platelets count,white blood cell count,lymphocyte count,serum creatinine,serum albumin,and estimate glomeruli filtration rate,complement C3,C4,24-hour urine protein quantification,urine proteincreatinine ratio,red blood cell count,serum immune findings,SLEDAI score,therapeutic response and disease outcomes.6.TCRβ CDR3 sequencing and data analysis:Randomly selected patients with EXT1/EXT2 associated MN,PLA2R associated IMN or PLN(LN Ⅲ-Ⅳ)for TCRβ CDR3 sequencing,each group had five patients.Five healthy control were enrolled as control.Compared the TCR clonal diversity,clonal expansion,TRB V,TRBJ gene usage,V-J pairing and shared clone between the four groups.Results:1.This was the first study about EXT1/EXT2 associated MN in China.A number of thirty-three EXT1/EXT2 positive MN patients were selected out from one hundred and eighty-nine MN patients with autoimmune background.2.The total rate of EXT1/EXT2 in the MN patients with autoimmune background was 17.46%.The rate in the MLN group was 24.49%(1997 ACR diagnostic criteria).The rate in group of patients with other ADs was 16.67%and the rate in group of patients with only abnormal autoantibody was 14.93%.The granular deposition of EXT1 was expressed on the podocyte.3.Pathological manifestations3.1 All MN patients with autoimmune background:the proportion and degree of IFTA in EXT1/EXT2 positive and negative patients were lower than IMN patients(P=0.011,0.013).The positive rate of IgA and IgM in EXT1/EXT2 positive and negative patients were higher than IMN patients(P=0.010,0.011),there were no significant differences between EXT 1/EXT2 positive and negative patients in the four indicators.The proportion of C3 and C1q≥2+in EXT1/EXT2 positive MN patients was significantly higher than EXT1/EXT2 negative and IMN patients(P=0.001,0.001).3.2 MLN group:The proportion of mesangial hyperplasia in EXT1/EXT2 positive patients was lower(P=0.025).The proportion of C3 and C1q≥2+in EXT1/EXT2 positive patients were higher(83.33%vs 56.76%,83.33%vs 62.16%),but the difference were not significant(P=0.189,0.315).3.3 Group of MN with other types of ADs(except SLE):There was only one EXT1/EXT2 positive patient with Primary Sjogren’s syndrome(PSS).She had obvious renal interstitial damage and immune complex deposition.3.4 4.4 Group of MN with only abnormal autoantibody:The proportion of C3,C1q≥2+in EXT1/EXT2 positive patients were significantly higher(P=0.026).4.Clinical manifestations4.1 All MN patients with autoimmune background:The positive rates of AntiU1RNP and Anti-SM in EXT1/EXT2 positive patients were significantly higher(P=0.001,0.019).The percentage of female patients in EXT1/EXT2 positive and negative patients were higher than that of IMN patients(P=0.001),but there was no difference between EXT1/EXT2 positive and negative patients.Complement C3 and C4 were lower in the EXT1/EXT2 positive and negative patients compared to the IMN group(P=0.019,0.004),but there was no significant difference between EXT1/EXT2 positive and negative patients.The serum albumin level of EXT1/EXT2 positive patients was significantly lower than that of EXT1/EXT2 negative patients(P=0.026),but there was no significant difference from IMN patients(P=1.000).4.2 MLN group:The positive rate of Anti-U1RNP in EXT1/EXT2 positive patients was significantly higher(P=0.002).The serum albumin of EXT1/EXT2 positive patients was lower(P=0.002),and the rate of proteinuria≥3.5 g/24 h was higher in EXT1/EXT2 positive patients,despite that the difference was not significant(66.67%vs 37.84%,P=0.081).4.3 Group of MN with other types of ADs(except SLE):The EXT1/EXT2 positive patient was positive for ANA,Anti-U1RNP,and Anti-SSA.The creatinine was 53umol/L,and the 24-hour urine protein was 1.18g/24h.4.4 Group of MN with only abnormal autoantibody:The positive rate of AntiU1RNP in EXT1/EXT2 positive patients was higher(P=0.005).5.Treatment responseA total of 110 patients were followed up for at least12 months,twenty in EXT1/EXT2 positive group and ninety in EXT1/EXT2 negative group.Eighteen IMN patients were followed up.Overall,MN patients with autoimmune background(EXT1/EXT2 positive and negative patients)had a higher overall response rate than IMN patients(100%vs 93.02%vs 61.54%,P=0.001),within the immunosuppressive therapy containing glucocorticoid(GC).GC might be effective for the treatment of MN patients with autoimmune background.The EXT1/EXT2 positive and negative patients with severe proteinuria responded similarly to immunosuppressive therapy containing GC.In addition,RTX might be effective for EXT1/EXT2 positive MN patients.6.Clinical outcome6.1 EXT1/EXT2 were not factors influenced the first time CR at 3 months,6 months,and 12 months.Multivariate logistic analysis showed that higher serum albumin was a predictor of the first time CR at 3 months(OR 1.106,95%CI 1.021,1.199,P=0.013)and 12 months(OR 1.067,95%CI 1.003,1.135,P=0.040).Anti-U1RNP was related to the first time CR at 6 months(OR2.603,95%CI 1.004,6.752,P=0.049).6.2 EXT1/EXT2 were not factors influenced the cumulative first time CR rate(Log-rank,P=0.710).IFTA and Anti-U1RNP were factors influenced the CR rate(Log-rank,P=0.003,0.045).COX regression showed that IFTA was a negative prognostic factor(HR0.477,95%CI 0.269,0.847,P=0.012).6.3 EXT1/EXT2 were not factors influenced the cumulative relapse rate(Log-rank,P=0.331).6.4 Only one patient developed to end-stage renal disease(ESRD)under irregular treatment.One patient who had malignant tumor died during the follow-up.7.TCRβ CDR3 sequencing results showed the cumulative frequency of Top 10 TCR clones in HC group was significantly lower than other groups(P=0.001).The usage frequency of TRBV11-3 and TRBV29-1 in EXT-MN group and IMN group were similar.There were 17 specific V-J gene pairs expressed similarly in the EXT-MN group and IMN group.There were 4.41%shared sequences(3468 species)in the EXT-MN group and IMN group,of which 2218 were only expresses in the two groups.In addition,the EXT-MN group and IMN group shared 12 common sequences.Conclusion:1.EXT1/EXT2 association MN had features of autoimmune diseases.Although the EXT1/EXT2 positive rate was higher in MLN patients,EXT1/EXT2 could also be detected in MN patients with other ADs and patients with only abnormal autoantibodies.2.EXT1/EXT2 associated MN had stronger complement C3 and Clq deposition,indicating that the pathogenesis might be related to stronger activation of the complement classical pathway.3.Anti-U1RNP was a characteristic antibody of EXT1/EXT2 associated MN.And Anti-U1RNP might be related to cumulative first time CR rate.More researches were needed to illuminate the pathogenesis.4.EXT1/EXT2 associated MLN patients had higher rate of proteinuria>3.5g/d and lower serum albumin.EXT1/EXT2 were not factors influenced the cumulative first time CR rate in patients with autoimmune background.5.Though EXT1/EXT2 associated MN(including MLN)and PLN had the same ADs,the similarity of TCR repertoire between EXT1/EXT2 associated MN and IMN indicated that the EXT1/EXT2 associated MN(including MLN)and IMN might be a type of disease with similar antibody recognition patterns.