Autoantibodies Specific for the Phospholipase A2 Receptor in Recurrent and De Novo Membranous Nephropathy

Recent findings in idiopathic membranous nephropathy (MN) suggest that in most patients, the disease is because of anti‐phospholipase A₂ receptor (PLA₂R1) autoantibodies. Our aim was to analyze the prevalence and significance of anti‐PLA₂R1 antibodies in recurrent and de novo MN after transplantation. We assessed circulating PLA₂R1 autoantibodies by a direct immunofluorescence assay based on human embryonic kidney cells transfected with a PLA₂R1 cDNA, and the presence of PLA₂R1 antigen in immune deposits. We showed that PLA₂R1 was involved in 5 of 10 patients with recurrent MN, but in none of the 9 patients with de novo MN. We also showed a marked heterogeneity in the kinetics and titers of anti‐PLA₂R1, which may relate to different pathogenic potential. We provide evidence that some patients with PLA₂R1‐related idiopathic MN and anti‐PLA₂R1 antibodies at the time of transplantation will not develop recurrence. Because PLA₂R1 autoantibody was not always associated with recurrence, its predictive value should be carefully analyzed in prospective studies.     

Identification of a Major Epitope Recognized by PLA2R Autoantibodies in Primary Membranous Nephropathy

Phospholipase A₂ receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.     

Temporal IgG Subtype Changes in Recurrent Idiopathic Membranous Nephropathy

Determination of the IgG subtypes within the immune deposits in membranous nephropathy (MN) may be helpful in the differential diagnosis. IgG4 is the predominant subtype in idiopathic MN and recurrent MN, while IgG1, IgG2, and IgG3 subtypes are more common in secondary MN and de novo disease in the allograft. The temporal change of IgG subclasses in individual patients and its correlation with clinical variables have not been studied. We reviewed all posttransplantation protocol and indication biopsies (49) in 18 patients with recurrent MN who underwent transplantation at our center between 1998 and 2013 and performed IgG subtyping (IgG1–4). We tested serum for M‐type phospholipase A₂ receptor (PLA₂R) autoantibodies or performed PLA₂R antigen staining on the kidney biopsy. IgG4 was the (co)dominant IgG subtype in 10 of 14 biopsies at the diagnosis of recurrence regardless of PLA₂R association. In 8 of 12 transplantations with serial biopsies, the (co)dominant subtype did not change over time. There was a trend toward IgG1 and IgG3 (co)dominance in biopsies >1 year from recurrence and more IgG1 (co)dominant subtyping in the setting of more‐advanced EM deposits. Treatment with rituximab did not affect the IgG subtype. In conclusion, the dominant IgG subtype did not change over time in recurrent MN.     

Anti‐Phospholipase A2 Receptor Antibodies in Recurrent Membranous Nephropathy

About 70% of patients with primary membranous nephropathy (MN) have circulating anti‐phospholipase A₂ receptor (PLA₂R) antibodies that correlate with disease activity, but their predictive value in post‐transplant (Tx) recurrent MN is uncertain. We evaluated 26 patients, 18 with recurrent MN and 8 without recurrence, with serial post‐Tx serum samples and renal biopsies to determine if patients with pre‐Tx anti‐PLA₂R are at increased risk of recurrence as compared to seronegative patients and to determine if post‐Tx changes in anti‐PLA₂R correspond to the clinical course. In the recurrent group, 10/17 patients had anti‐PLA₂R at the time of Tx versus 2/7 patients in the nonrecurrent group. The positive predictive value of pre‐Tx anti‐PLA₂R for recurrence was 83%, while the negative predictive value was 42%. Persistence or reappearance of post‐Tx anti‐PLA₂R was associated with increasing proteinuria and resistant disease in 6/18 cases; little or no proteinuria occurred in cases with pre‐Tx anti‐PLA₂R and biopsy evidence of recurrence in which the antibodies resolved with standard immunosuppression. Some cases with positive pre‐Tx anti‐PLA₂R were seronegative at the time of recurrence. In conclusion, patients with positive pre‐Tx anti‐PLA₂R should be monitored closely for recurrent MN. Persistence or reappearance of antibody post‐Tx may indicate a more resistant disease.     

Phospholipase A2 Receptor Autoantibodies and Clinical Outcome in Patients with Primary Membranous Nephropathy

Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in adults, with an uncertain clinical outcome. The characterization of the phospholipase A₂ receptor (PLA₂R) as the major target antigen in primary MN and the detection of circulating autoantibodies in these patients is a major advance in understanding this disease. To test whether PLA₂R antibody levels reflect disease activity or clinical outcome, we performed a prospective multicenter study of 133 adult patients with primary MN and detectable serum PLA₂R antibodies who had not received immunosuppressive therapy. Patients were followed ≤24 months. PLA₂R antibody levels associated with clinical disease activity (proteinuria) in patients with immunosuppressive therapy (n=101) or supportive care (n=32). Within 3 months, immunosuppressive therapy led to a sustained 81% reduction in PLA₂R antibody levels paralleled by a 39% reduction in proteinuria. Patients who experienced remission of proteinuria after 12 months had significantly lower PLA₂R antibody levels at the time of study inclusion compared with patients with no remission. Patients with high PLA₂R antibody levels achieved remission of proteinuria significantly later than patients with low PLA₂R antibody levels. PLA₂R antibody levels fell over time in patients with spontaneous remission but remained elevated in patients who did not show a reduction in proteinuria. Multivariable Cox regression analysis confirmed PLA₂R antibody level as an independent risk factor for not achieving remission of proteinuria. We conclude that a decrease in PLA₂R antibody level is associated with a decrease of proteinuria in patients with primary MN.Since the landmark discovery that circulating autoantibodies against the phospholipase A₂ receptor (PLA₂R) are specific for patients with primary membranous nephropathy (MN) completely new paradigms for the diagnosis and clinical care of these patients are possible.¹ These are urgently needed because the clinical outcome of patients with primary MN varies and ranges from spontaneous clinical remissions to end stage renal failure.^2,3 Because of the absence of reliable predictors of clinical outcome, the best documented methods to predict outcome and hence make a decision to treat patients with an immunosuppressive agent or maintain them on supportive medications currently require prolonged follow-up measurements of proteinuria.^4,5 Furthermore, in patients who receive immunosuppressive therapy, the intensity and duration of the treatment currently depend on changes in proteinuria, which do not necessarily reflect the severity or activity of the immunologic disease. On the other hand, patients who clinically do not respond to immunosuppressive agents may have insufficient therapy and still have active immunologic disease. A marker that reflects immunologic disease activity in real time and indicates clinical outcome could substantially improve the care of these patients. The availability of recently developed and easily applicable assays to measure PLA₂R antibody levels in the serum^6–8 makes it possible to study patients prospectively and to analyze whether PLA₂R antibody levels are related to disease activity. To address this question, we conducted a multicenter open prospective study in patients with biopsy-proven MN.     

Circulating antibodies to α-enolase and phospholipase A2 receptor and composition of glomerular deposits in Japanese patients with primary or secondary membranous nephropathy

Background

Phospholipase A₂ receptor (PLA₂R) is recognized as a target antigen in primary membranous nephropathy (MN); Anti-α-enolase antibody in primary and secondary MN has been proposed, however, little is known about the potential contribution of α-enolase to the pathogenesis of MN.

Methods

We evaluated circulating antibodies to α-enolase by a dot blotting system and PLA₂R by indirect immunofluorescence, and glomerular deposition of these proteins in 25 patients with primary MN, 20 patients with secondary MN, 44 patients with collagen disease or severe infection, 60 patients with nephritis (each ten patients of IgA nephropathy, focal segmental gloemrulosclerosis, minimal change nephrotic syndrome, membranoproliferative glomeurlonephritis, diabetic glomerulosclerosis, and tubulointerstitial nephritis) as disease control, and 20 healthy subjects.

Results

In primary MN, 18 of 25 sera (72 %) showed anti-α-enolase antibody (IgG1 and IgG4, 11 pts; IgG4 alone, six pts; IgG1 alone, one pt). In secondary MN, 15 of 20 sera (75 %) contained anti-α-enolase antibody (IgG1 and IgG3, 13 pts; IgG3 alone, two pts). No circulating anti-α-enolase antibody was found in 44 collagen diseases or septic patients, 60 nephritis without MN, and 20 healthy subjects. Twelve of 25 sera (48 %) from patients with primary MN were positive for anti-PLA₂R antibody, whereas all patients with secondary MN were negative. Eight of the 12 PLA₂R-positive patients (67 %) with primary MN also had anti α-enolase antibody. Although PLA₂R antigen was present in a subepithelial pattern in 10 of 19 (52 %) patients with primary MN, α-enolase was never detected in glomerular deposits in 19 and ten patients with primary and secondary MN, respectively.

Conclusions

Circulating anti-α-enolase antibodies are highly present in both primary and secondary MN (about 70 %, respectively), while anti-PLA₂R antibodies are specific for primary MN (48 %) with a prevalence apparently lower in the Japanese population than in Chinese and Caucasian populations. The absence of α-enolase from subepithelial immune deposits suggests that anti-α-enolase antibodies do not contribute directly to immune-deposit formation, although they may have other pathogenic effects.

     

特发性膜性肾病的分子发病机制

特发性膜性肾病( IMN)是国内外常见的引起成人肾病综合征的病理类型之一。在中国人群中,近年来特发性膜性肾病在原发性肾小球疾病中所占比例明显升高。由于病因及发病机制仍不清楚,以及缺乏预测疾病活动性的生物学标志物,因此目前治疗所带来的经济负担以及药物毒性仍具有争议和挑战。IMN是一种器官特异性的自身免疫性疾病,非炎症性自身抗体与足细胞上的靶抗原结合,在基底膜外侧上皮下形成原位免疫复合物,激活补体,从而引起足细胞损伤和蛋白尿。Heymann肾炎模型是经典的膜性肾病动物模型,然而其靶抗原Megalin并不是人类膜性肾病发生的原因。之后,中性肽链内切酶(NEP)被证实为母胎同种异体产前膜性肾病的靶抗原。直到2009年,磷脂酶A2受体(PLA2R)被证实为IMN的靶抗原。在IMN患者血浆中检测到抗PLA2R-IgG4,其特异性高达100%,敏感性约为70%～80%。抗PLA2R-IgG4滴度可以预测疾病的活动性,其与PLA2R抗原结合形成原位免疫复合物,激活补体凝集素途径,形成C5b-9膜攻击复合物沉积在足细胞上,引起足细胞损伤,导致蛋白尿形成。HLA-DQA1与PLA2R基因多态性均与IMN的发病相关,二者的风险基因具有叠加效应。综上所述,IMN的发生是多种因素共同作用的结果,包括易感基因、靶抗原、自身抗体、补体等,这些研究进展对于IMN的诊断和治疗具有重要意义。     

成人膜性肾病患者血清抗PLA2R抗体与病情的相关性

目的 分析成人膜性肾病患者血清抗M型磷脂酶A2受体(PLA2R)抗体与特发性膜性肾病( IMN)实验室指标的相关性,探讨抗PLA2R抗体在IMN发病中的作用.方法 选取经肾活检证实的46例肾小球疾病患者,包括20例IMN、7例IgA肾病、6例乙型肝炎病毒相关性膜性肾病( HBV-MN)、6例微小病变性肾病、4例局灶性节段性肾小球硬化、3例Ⅴ型狼疮肾炎.应用Western印迹法检测血清抗PLA2R抗体,并对其与IMN患者血清白蛋白、24 h尿蛋白量、血清总胆固醇和Scr作相关性分析.结果 20例IMN患者中15例血清抗PLA2R抗体阳性,阳性比例为75％;7例IgA肾病患者中1例抗PLA2R抗体阳性,阳性比例为14.29％;6例HBV-MN患者中1例阳性,阳性比例为16.67％;其余患者均为阴性.IMN患者血清抗PLA2R抗体阳性比例显著高于继发性膜性肾病和其他肾小球肾炎(均P＜0.01),且抗PLA2R抗体水平与IMN患者尿蛋白量呈正相关(r=0.803,P＜0.01);与血清白蛋白呈负相关(r=-0.816,P＜0.01).结论 IMN患者血清抗PLA2R抗体阳性比例高,提示抗PLA2R抗体可能是IMN的特异性抗体.该抗体与尿蛋白量呈正相关,提示抗PLA2R抗体可能是IMN的致病性抗体.     

Characteristics of patients with coexisting IgA nephropathy and membranous nephropathy

Background: Coexistence of IgA nephropathy (IgAN) and membranous nephropathy (MN) in the same patient is rare. Few studies have reported the clinical and pathological features of patients with combined IgAN and MN (IgAN–MN).

Methods: The clinico-pathological features, levels of galactose-deficient IgA1 (Gd-IgA1) and autoantibodies against M-type transmembrane phospholipase A₂ receptor (anti-PLA₂R) in sera were compared among IgAN–MN, IgAN, and MN patients.

Results: Twenty-six patients with biopsy-proven IgAN–MN were enrolled. The mean age at biopsy was 43.6?±?15.9?years, and 65.4% were male. Proteinuria and estimated glomerular filtration rate (eGFR) levels in patients with IgAN–MN were similar to that of MN patients. Compared with the IgAN patients, IgAN–MN patients showed a higher median proteinuria level (4.3 vs. 1.2?g/day, p?2, p?p?=?.801). Percentage of IgAN–MN patients with detectable serum levels of anti-PLA₂R was lower than that of MN patients (38.5% vs. 68.6%, p?=?.011).

Conclusions: IgAN–MN patients display similar clinical features to MN patients and milder pathological lesions than IgAN patients. IgAN–MN patients have similar levels of Gd-IgA1 to those of IgAN patients, and a lower proportion of anti-PLA₂R than MN patients.     

Anti -PLA2R autoantibodies in serum and IgG subclass deposits on glomeruli in undetermined atypical membranous nephropathy

ObjectiveTo investigate the characteristic of autoantibodies of M – type phospholipase A2 receptor (PLA2R) in serum and the glomerular IgG subclass deposits in undetermined atypical membranous nephropathy (MN) patients. MethodsFrom Feb 2004 to Nov 2011, 53 cases diagnosed as MN by kidney puncture biopsy in our hospital were included into the study. There were 20 undetermined atypical membranous nephropathy (UAMN), 20 idiopathic membranous nephropathy (IMN) and 13 secondary membranous nephropathy (SMN) which were composed of lupus membranous nephropathy (LMN) and HBV related membranous nephropathy (HBV-MN). Clinlical and pathological characteristics were analyzed. The autoantibodies of PLA2R in serum were detected and the glomerular IgG subclass deposits were observed. Results(1) The average age underwent renal biopsy was (37.9±3.8) years of UAMN, (50.1±3.0) years of IMN and (49.5±4.5) years of SMN. The difference in onset average age at disease was significant between UAMN and IMN (P＝0.0178). The female/male ratio (F/M) in UAMN, IMN and SMN was 0.8∶1, 0.7∶1 and 0.6∶1(P＞0.05). (2) Compared with SMN, the level of 24-hours urinary protein excretion (3.47 g vs 7.89 g, P＝0.023), the ratio of amount urinary protein patients (50.0% vs 84.6%, P＝0.043), the level of serum IgG [(8.40±3.58) g/L vs (10.09±4.69) g/L, P＝0.025] and the positive rate of ANA in serum (10.0% vs 53.8%, P＝0.006) in UAMN were all much lower. There were no significant statistical differences in serum albumin, serum creatinine, eGFR, positive rate of HBsAg, HBeAg or HCV, as well as the ratio of hypo - albuminemia and nephritic syndrome among the three groups. (3) IF positive rate of IgA, IgM and C1q in UAMN were all significantly higher than that in IMN (P＜0.01). There were no significant differences in IF positive rate of IgA, IgM, C1q, IgG and C3 between UAMN and SMN. The IF strength of IgA, IgG, IgM, C3 and C1q in UAMN showed no significant differences between UAMN and SMN. (4) The serum autoantibodies of PLA2R were only detected in 10 cases of IMN group (50%) with all the other cases negative. This detection rate of serum autoantibodies of PLA2R showed significant statistical differences among the three groups (P＜0.01), but no differences between UAMN and SMN (the detection rate in both groups were 0%). (5) IgG1 deposits was the dominant IgG on the glomeruli in UAMN group (40%), as well as in SMN group (76.9%). IgG4 deposits was the dominant IgG on the glomeruli in IMN group (60%). The positive rate of IgG1 and IgG3 in UAMN showed no significant statistical differences when compared with IMN or SMN. The positive rate of IgG2 in UAMN was significantly lower than in SMN（30.0% vs 69.2%, P＜0.05). The positive rate of IgG4 in UAMN was significantly lower than in IMN (20% vs 60%, P＜0.05). The positive rate of IgG1, IgG2 and IgG3 in SMN were all significantly higher than in IMN. ConclusionsNone of the UAMN group had autoantibodies of PLA2R in serum, and IgG1 deposits was the dominant IgG subclass on the glomeruli which indicated the similarity with the SMN group. At the same time, UAMN was significantly different from SMN in clinical manifestations.     

Interaction between PLA2R1 and HLA-DQA1 Variants Associates with Anti-PLA2R Antibodies and Membranous Nephropathy

Risk alleles at genome loci containing phospholipase A2 receptor 1 (PLA2R1) and HLA-DQA1 closely associate with idiopathic membranous nephropathy (IMN) in the European population, but it is unknown whether a similar association exists in the Chinese population and whether high-risk alleles promote the development of anti-PLA2R antibodies. Here, we genotyped 2132 Chinese individuals, including 1112 patients with IMN and 1020 healthy controls, for three single nucleotide polymorphisms (SNPs) within PLA2R1 and three SNPs within HLA genes. We also selected 71 patients, with varying genotypes, to assess for circulating anti-PLA2R antibody and for PLA2R expression in glomeruli. Three SNPs within PLA2R1 and one SNP within HLA-DQA1 strongly associated with IMN, and we noted gene–gene interactions involving these SNPs. Furthermore, these risk alleles strongly associated with the presence of anti-PLA2R antibodies and glomerular PLA2R expression. Among individuals who carried risk alleles for both genes, 73% had anti-PLA2R antibodies and 75% expressed PLA2R in glomeruli. In contrast, among individuals who carried protective genotypes of both genes, none had anti-PLA2R antibodies and glomerular expression of PLA2R was weak or absent. In conclusion, the interaction between PLA2R1 and HLA-DQA1 risk alleles associates with the development of IMN in the Chinese population. Individuals carrying risk alleles are predisposed to the generation of circulating anti-PLA2R autoantibodies, which may contribute to the development of IMN.Idiopathic membranous nephropathy (IMN), characterized by subepithelial glomerular immune deposits and glomerular membrane thickening, is one of the most common reasons for adult nephrotic syndrome.^1–3 It is now recognized that IMN is an organ-specific autoimmune disease. To date, two major antigens have been identified in human membranous nephropathy. The first is neutral endopeptidase, the alloantigen involved in neonatal cases of membranous nephropathy that occur in newborn infants from neutral endopeptidase-deficient mothers.⁴ The second is the M-type phospholipase A2 receptor (PLA2R), the first autoantigen identified in adult IMN patients.⁵ PLA2R is a type I transmembrane protein expressed on glomerular podocytes, forming subepithelial deposits in situ through binding of circulating anti-PLA2R autoantibodies. Another key finding in membranous nephropathy comes from a genome-wide association study (GWAS) using European white ancestry. Stanescu et al. identified risk alleles at two genome loci containing PLA2R1 and HLA-DQA1, which both contribute to the risk of membranous nephropathy.⁶ These results strongly support an interaction between HLA-DQ and PLA2R in the pathogenesis of membranous nephropathy. It has been postulated that certain genetic variants of PLA2R1 yield peptides with strong affinity for specific HLA-DQA1 variants that subsequently confer a predisposition to anti-PLA2R autoantibody generation. Although there are studies reporting an association between PLA2R1 gene polymorphisms and IMN in Asian populations from Korea and Taiwan,^7,8 no study thus far has evaluated whether the gene interaction between PLA2R1 and HLA-DQA1 contributes to production of anti-PLA2R autoantibodies and development of IMN in an independent cohort.In China, IMN accounts for >25% of nephrotic syndrome and 6.7% of all biopsy glomerular disease,⁹ which is not as prevalent as that reported in Western countries.^9–14 In this study, we aim to evaluate the association between these risk alleles and the development of IMN in a large Chinese cohort with >2000 participants and to further explore their roles in the generation of anti-PLA2R antibodies and expression of PLA2R in glomeruli.     

Autoantibodies Against Glomerular Mesangial Cells and Their Target Antigens in Lupus Nephritis

Mesangial proliferation and deposition of immunoglobulins and complement components within glomerular mesangium was one of the important pathological features of lupus nephritis. Autoantibodies against human mesangial cells could be detected in the sera of patients with IgA nephropathy (IgAN) and Henoch-Schöenlein nephritis. We speculated that autoantibodies against human glomerular mesangial cells might play a role in the development of lupus nephritis. Objective. To screen autoantibodies against human glomerular mesangial cells in sera from patients with lupus nephritis and to identify their target antigens. Methods. Sera were collected from 96 patients with lupus nephritis as well as 25 patients with IgAN and 20 patients with idiopathic membranous nephropathy (IMN). Cell lysates of in vitro cultured human glomerular mesangial cells were used as antigens in Western-blot analysis to detect autoantibodies against human mesangial cells in sera from patients with lupus nephritis as well as IgAN and IMN. The clinical and pathological significance of the autoantibodies were further investigated. Results. Autoantibodies against human mesangial cells could be detected in 94/96 (97.9%) of the sera from patients with lupus nephritis in Western-blot analysis. Twelve protein bands could be blotted by the sera from patients with lupus nephritis. The prevalence of autoantibodies against human mesangial cells in IgAN was 14/25 (56.0%) and only seven protein bands could be blotted. Five autoantibodies (anti-18, 24, 36, 46, and 91 kD) could be detected only in sera from patients with lupus nephritis. In patients with lupus nephritis, some autoantibodies might have some relationship with gender, hematuria, ANA, anti-dsDNA or anti-ENA antibodies. Conclusions. There are autoantibodies directly against heterogeneous antigens of human glomerular mesangial cells in sera from patients with lupus nephritis, and some of them might be associated with different clinical manifestations.     

Intramolecular epitope spreading in Heymann nephritis

Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.     

血、尿中PLA2R抗体以及THSD7A抗体在特发性膜性肾病中的临床意义

目的通过检测膜性肾病(MN)患者中血清、尿中M型磷脂酶A2受体(PLA2R)抗体以及1型血小板反应蛋白7A域(THSD7A)抗体浓度,探讨其在特发性膜性肾病(IMN)中的临床意义。 方法选取2015年7月至2017年12月在山西医科大学第二医院肾内科经过肾活检确诊为MN的患者,通过酶联免疫吸附方法(ELISA)测定血清、尿中PLA2R抗体以及THSD7A抗体浓度水平。采用SPSS 21.0软件对数据进行统计分析。 结果经临床、病理确诊为MN的患者189例,其中IMN组165例(87.3%)。(1)IMN患者血清、尿PLA2R抗体的阳性率分别64.8%、60.0%,血清、尿THSD7A抗体的阳性率分别为8.4%、5.4%;(2)IMN患者血清、尿PLA2R抗体浓度与24 h尿蛋白定量呈正相关(P<0.05),与血清白蛋白呈负相关(P<0.05);(3)IMN患者中血清、尿THSD7A抗体浓度与24 h尿蛋白定量呈正相关(P<0.05),与血清白蛋白无相关性(P>0.05);(4)IMN患者中血清PLA2R、抗体浓度与尿PLA2R抗体浓度呈正相关(P<0.05),血清THSD7A抗体浓度与尿THSD7A抗体浓度呈正相(P<0.05)。 结论血清、尿中PLA2R抗体以及THSD7A抗体均可作为IMN诊断的特异性指标,相对于THSD7A抗体而言,PLA2R抗体检出率更高,且这两种抗体在血清、尿中浓度水平与临床指标存在一定的相关性。     

GAD65-specific autoantibodies enhance the presentation of an immunodominant T-cell epitope from GAD65

GAD65 autoantibodies (GAD65Ab) are highly prevalent in type 1 diabetes, but their functional role in the pathogenesis of the disease and their relationship to T-cell reactivity to GAD65 is still unclear. We tested the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. T-cell hybridoma T33.1, which recognizes the GAD65 274-286 epitope in the context of HLA-DRB 1*0401, was incubated with antigen-presenting cells exposed to recombinant human GAD65 alone or complexed with GAD65Ab' or GAD65Ab- sera. Stimulation of the T33.1 hybridoma was greatly enhanced by multiple GAD65Ab+ sera. The enhancement effect was most prominent with sera from patients with high GAD65 autoantibody levels. Sera from GAD65Ab- subjects had no effect. The correlation between T-cell stimulation and GAD65Ab levels was not absolute, suggesting that other variables such as autoantibody recognition of different regions of GAD65 and variable effects on processing of the 274-286 epitope may contribute. Uptake of antibody-complexed GAD65 was Fc receptor (FcR)-mediated because the enhancement of presentation was inhibited by monoclonal antibodies against FcR. Our results support the hypothesis that GAD65Ab modulate presentation of GAD65 to T-cells. Increased antigen uptake and heterogeneity in the autoantibody specificity may provide a mechanism for antibody-facilitated T-cell response influencing the progression of type 1 diabetes.     

Anti-Phospholipase A2 Receptor Antibody Titer Predicts Post-Rituximab Outcome of Membranous Nephropathy

Rituximab induces nephrotic syndrome (NS) remission in two-thirds of patients with primary membranous nephropathy (MN), even after other treatments have failed. To assess the relationships among treatment effect, circulating nephritogenic anti-phospholipase A₂ receptor (anti-PLA₂R) autoantibodies and genetic polymorphisms predisposing to antibody production we serially monitored 24-hour proteinuria and antibody titer in patients with primary MN and long-lasting NS consenting to rituximab (375 mg/m²) therapy and genetic analyses. Over a median (range) follow-up of 30.8 (6.0–145.4) months, 84 of 132 rituximab-treated patients achieved complete or partial NS remission (primary end point), and 25 relapsed after remission. Outcomes of patients with or without detectable anti-PLA₂R antibodies at baseline were similar. Among the 81 patients with antibodies, lower anti-PLA₂R antibody titer at baseline (P=0.001) and full antibody depletion 6 months post-rituximab (hazard ratio [HR], 7.90; 95% confidence interval [95% CI], 2.54 to 24.60; P<0.001) strongly predicted remission. All 25 complete remissions were preceded by complete anti-PLA₂R antibody depletion. On average, 50% anti-PLA₂R titer reduction preceded equivalent proteinuria reduction by 10 months. Re-emergence of circulating antibodies predicted disease relapse (HR, 6.54; 95% CI, 1.57 to 27.40; P=0.01), whereas initial complete remission protected from the event (HR, 6.63; 95% CI, 2.37 to 18.53; P<0.001). Eighteen patients achieved persistent antibody depletion and complete remission and never relapsed. Outcome was independent of PLA2R1 and HLA-DQA1 polymorphisms and of previous immunosuppressive treatment. Therefore, assessing circulating anti-PLA₂R autoantibodies and proteinuria may help in monitoring disease activity and guiding personalized rituximab therapy in nephrotic patients with primary MN.     

Protocadherin 7–Associated Membranous Nephropathy

BackgroundMembranous nephropathy (MN) results from deposition of antigen-antibody complexes along the glomerular basement membrane (GBM). PLA2R, THSD7A, NELL1, and SEMA3B account for 80%–90% of target antigens in MN.MethodsWe performed laser microdissection and mass spectrometry (MS/MS) in kidney biopsies from 135 individuals with PLA2R-negative MN, and used immunohistochemistry/immunofluorescence and confocal microscopy to confirm the MS/MS finding, detect additional cases, and localize the novel protein. We also performed MS/MS and immunohistochemistry on 116 controls and used immunofluorescence microscopy to screen biopsy samples from two validation cohorts. Western blot and elution studies were performed to detect antibodies in serum and biopsy tissue.ResultsMS/MS studies detected a unique protein, protocadherin 7 (PCDH7), in glomeruli of ten (5.7%) PLA2R-negative MN cases, which also were negative for PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B. Spectral counts ranged from six to 24 (average 13.2 [SD 6.6]). MS/MS did not detect PCDH7 in controls (which included 28 PLA2R-positive cases). In all ten PCDH7-positive cases, immunohistochemistry showed bright granular staining along the GBM, which was absent in the remaining cases of PLA2R-negative MN and control cases. Four of 69 (5.8%) cases in the validation cohorts (all of which were negative for PLA2R, THSD7A, EXT1, NELL1, and SEMA3B) were PCDH7-positive MN. Kidney biopsy showed minimal complement deposition in 12 of the 14 PCDH7-associated cases. Confocal microscopy showed colocalization of PCDH7 and IgG along the GBM. Western blot analysis using sera from six patients showed antibodies to nonreduced PCDH7. Elution of IgG from frozen tissue of PCDH7-associated MN showed reactivity against PCDH7.ConclusionsMN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN.     

Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti–aldose reductase (AR) and anti–manganese superoxide dismutase (SOD2) IgG₄ in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG₄ from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG₄ and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.Primary membranous nephropathy (MN) is a common glomerular disease in humans with no universally effective clinical therapy. Treatments are entirely empirical, and the disease evolves toward renal failure in a significant number of patients.^1,2 The presence of glomerular subepithelial immune deposits is the distinctive pathologic feature of MN, thus supporting the concept of an immunologic origin. It is also known that inflammatory compounds such as complement, oxygen radicals,^3,4 or intracellular protein kinase Cβ⁵ may participate, having a key role in disease progression.In the past few decades, studies of experimental models, with a particular emphasis on the Heymann nephritis (HN) model,^6–8 have led to the identification of antigens of the autoantibody response in rats (megalin),⁷ mice (aminopeptidase A),⁹ and rabbits (neutral endopeptidase [NEP]),^10–12 but limited data are available for humans. Moreover, megalin, which is the target antigen in HN, is absent in human glomeruli, and the LDL receptor, its human homolog, is only partially co-localized with MN IgG deposits.^5,13–15 Seminal studies by Debiec et al.^11,12 support the formation of immune deposits against NEP, in particular, cases with metallomembrane endopeptidase mutations that lead to NEP deficiency and alloimmunization during pregnancy. More recently, Beck et al.¹⁶ reported the presence of specific IgG₄ against the M-type phospholipase A2 receptor (PLA2R) in glomerular eluates and in plasma of a significant percentage of patients with MN, suggesting PLA2R is a major antigen in this disease.Unequivocal identification of coexisting antigens in the podocyte membrane and in subepithelial deposits is essential for any progression in the understanding of the mechanisms of MN in humans. The aim of this study was to identify podocyte proteins recognized by circulating autoantibodies in patients with MN, to define their expression in glomeruli, and to quantify the levels of specific antibodies in sera and in renal biopsies. Results provide first evidence for de novo expression of specific autoantibodies against aldose reductase (AR) and superoxide dismutase 2 (SOD2) in sera and glomeruli of patients with MN.     

Anti-phospholipase A2 receptor antibody in membranous nephropathy

The M-type phospholipase A2 receptor (PLA2R) is a target autoantigen in adult idiopathic membranous nephropathy (MN), but the prevalence of autoantibodies against PLA2R is unknown among Chinese patients with MN. Here, we measured anti-PLA2R antibody in the serum of 60 patients with idiopathic MN, 20 with lupus-associated MN, 16 with hepatitis B (HBV)-associated MN, and 10 with tumor-associated MN. Among patients with idiopathic MN, 49 (82%) had detectable anti-PLA2R autoantibodies using a Western blot assay; an assay with greater sensitivity detected very low titers of anti-PLA2R in 10 of the remaining 11 patients. Using the standard assay, we detected anti-PLA2R antibody in only 1 patient with lupus, 1 with HBV, and 3 with cancer, producing an overall specificity of 89% in this cohort limited to patients with secondary MN. The enhanced assay detected low titers of anti-PLA2R in only 2 additional samples of HBV-associated MN. In summary, these results suggest that PLA2R is a major target antigen in Chinese idiopathic MN and that detection of anti-PLA2R is a sensitive test for idiopathic MN.     

Autoantibodies against phospholipase A2 receptor in Brazilian patients with glomerular diseases

Background

Recently, great progress has been made in understanding the pathogenesis of membranous nephropathy (MN) with the discovery of autoantibodies (Abs) to M-type phospholipase A2 receptor (PLA2R) in serum and in immunocomplexes deposited in glomerulus in most adult patients with primary MN.

Objective

To evaluate the diagnostic performance of anti-PLA2R in Brazilian patients with MN, as well as to verify the possible association of anti-PLA2R serum levels with disease activity.

Methods

117 patients with glomerular diseases confirmed by renal biopsy underwent routinely clinical and laboratory evaluation (serum creatinine and albumin, 24-h proteinuria, urinalysis, tests for etiological investigation) and determination of serum anti-PLA2R by ELISA.

Results

67.5% of the patients had MN, 9.4% focal segmental glomerulosclerosis, 7.7% lupus nephritis class V and 15.4%, other proteinuric glomerular diseases. The mean level of glomerular filtration rate (estimated by the CKD-EPI formula) was 79.43 mL/min (12.00–151.20 mL/min), 24 h proteinuria of 2.89 g (0–14.90 g), serum albumin of 3.79 g/dL (1.20–4.80 g/dL). Anti-PLA2R was detected in 27 patients, all with active MN, being 26 primary and 1 secondary MN. Sensitivity and specificity rates for the test were 60.5–94.7%, and positive (PPV) and negative (NPV) predictive values were 92.9 and 67.9%, respectively.

Conclusions

Anti-PLA2R showed high specificity and PPV for the diagnosis of primary MN in Brazilian patients. There was a strong correlation between disease activity and positive anti-PLA2R. This biomarker represents an important diagnostic tool for primary MN and may contribute to the monitoring of disease activity in such patients.