Expression of phospholipase A2 receptor and IgG4 in patients with membranous nephropathy
Authors Liu L, Chang B, Wu X, Guo Y, Pan Y, Yang L
Received 26 December 2017
Accepted for publication 9 March 2018
Published 29 May 2018 Volume 2018:14 Pages 103—108
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 4
Editor who approved publication: Prof. Dr. Amudha Kadirvelu
Lei Liu,1 Baochao Chang,1 Xueping Wu,1 Yaling Guo,1 Yan Pan,1 Lijuan Yang2
1Department of Nephrology, The First Affiliated Hospital of Bengbu Medical College, Anhui, People’s Republic of China; 2Department of Physiology, Bengbu Medical College, Anhui, People’s Republic of China
Objectives: The aims of this study were to detect the expression of M phospholipase A2 receptor (PLA2R) in the kidney tissue of patients with idiopathic membranous nephropathy (IMN), secondary membranous nephropathy (SMN), and the nonmembranous nephropathy (non-MN), to evaluate the value of PLA2R in the kidney tissue and serum anti-PLA2R antibody in the diagnosis of membranous nephropathy (MN), and to explore the relationship between PLA2R of the kidney tissue or serum anti-PLA2R antibody and clinical features of MN.
Methods: The kidney tissue was collected by kidney biopsy. Immunofluorescence assay was used to detect the level of PLA2R and IgG4 antigen in kidney tissue. Furthermore, the level of the PLA2R was detected using the enzyme-linked immunosorbent assay (ELISA). The positive and negative rates of PLA2R and IgG4 in different diseases and the sensitivity and specificity, were calculated using the statistical method. The specificity and coincidence rate of PLA2R or anti-PLA2R used in the differential diagnosis of IMN and SMN were evaluated.
Results: The expression intensities of anti-PLA2R antibody and IgG4 were significantly higher in patients with IMN than in patients with SMN but are not non-MN. There was no significant difference in anti-PLA2R antibody and IgG4 in patients with SMN and non-MN. The coincidence rate of serum anti-PLA2R antibody and PLA2R in kidney tissue was 100%.
Conclusion: The expression of PLA2R and IgG4 antibody had great significance in the pathological diagnosis of MN. The detection of the serum anti-PLA2R antibody had great diagnostic value in diagnosing MN.
Keywords: membranous nephropathy, PLA2R, IgG4
Membranous nephropathy (MN) is one of the most common pathological types of the nephrotic syndrome in China. With the gradual improvement of the utilization rate of pathological diagnosis technology in the diagnosis of kidney diseases, the increasing incidence of MN has been reported in many different medical centers1 and has become the most common cause of nephrotic syndrome.
Usually, the MN refers to the idiopathic MN (IMN), which is also named the primary MN. Approximately 80% of patients were diagnosed with nephrotic syndrome characterized by large number of proteinuria and hypoalbuminemia, complicated with infection, thrombosis, and embolism events. Without a timely and effective diagnosis and treatment, patients may progress to end-stage renal failure after 5–10 years. Therefore, it is of high urgency to improve the diagnosis and treatment effect.
In the past many authors have studied how to improve the accurate diagnosis rate of MN. In 2009, Beck et al2 first discovered that the extracellular domain of phospholipase A2 receptor (PLA2R) was used as the mutant antigen to activate autoimmunity response. Combined with the PLA2R antibodies produced in the body, PLA2R forms the insitus immune complex, resulting in the injury of basement membrane, which was the major pathogenic factor of the majority of IMN patients. Many literature reported that the detection of peripheral blood in patients with serum, results positive rate of anti PLA2R antibodies in patients with IMN was 50–80%,3–5 and was about 20%6 in patients with secondary MN (SMN), in the patients with non-MN and normal population was almost to zero.7 Recent studies have pointed out that the titers of anti-PLA2R antibodies in peripheral blood were associated with the disease activity.8–10 Thus, the detection of serum anti-PLA2R antibodies had great significance for diagnosis, treatment, and prognosis of the disease.
In recent years, more and more studies have confirmed that the immunoglobulin IgG subtypes play an vital role in the identification of MN caused by different antibodies, such as IgG4 mainly in IMN kidney tissue deposits11 and IgGl and IgG3 mainly in SMN.12,13 Beck et al2 first identified IgG4 as the major component of anti-PLA2R antibody in IMN patients, which is consistent with kidney pathology. Thus, the detection of glomerular IgG4 expression could serve as the reference index in the diagnosis and differential diagnosis of kidney diseases. In this study, we analyzed the data of patients with MN diagnosed in our hospital and the expression of PLA2R and IgG subtype in blood and kidney tissue, respectively. The presence of IgG subtype and PLA2R in the identification of IMN and non-MN was assessed, and the values of both expressions were also evaluated.
A total of 39 (including 27 males and 12 females) patients who underwent renal biopsy were selected from the Department of Nephrology, The First Affiliated Hospital of Bengbu Medical College, Bengbu, China, from August 2016 to December 2016. Signed informed consent was obtained from all the patients. This study was approved by the ethics committee of The First Affiliated Hospital of Bengbu Medical College (2016094).
Inclusion and exclusion criteria for patients
Patients aged between 12 and <65 years and those who satisfied the standard diagnostic criteria of chronic kidney disease (CKD) were included in the study. The diagnosis of CKD was made when one of the following two criteria was met: 1) structural or functional abnormalities of the kidney lasting for at least 3 months, and 2) glomerular filtration rate (GFR) <60 mL/min/1.73 m2 for at least 3 months.14 Patients were excluded based on the following two criteria: 1) patients who were not willing to perform kidney pathology, and 2) patients who had contraindications to kidney pathology. The risk of the kidney pathology examination is higher in patients older than 65 years, hence, the examination was not done in these patients.
Inclusion and exclusion criteria for IMN and SMN
The inclusion criteria for IMN were as follows: the pathological diagnosis of the kidney tissue was MN, except for clinical factors such as systemic lupus erythematosus (SLE), hepatitis B virus (HBV), tumor, and drug-induced secondary membranous nephropathy. The inclusion criteria for SMN were as follows: 1) membranous lupus nephritis (MLN): ≥ SLE classification revised by Systemic Lupus International Collaborating Clinics (SLICC) in 2012 and the pathological diagnosis of the kidney tissue was MN; 2) HBV-related glomerulonephritis (HBV-GN): serum HBsAg was positive, except for clinical factors such as SLE, tumor, and drugs, the pathological diagnosis of the kidney tissue was MN, and hepatitis B surface antigen (HBsAg) and/or hepatitis B core antigen (HBcAg) in the kidney tissue were positive.15
Age, gender, serum creatinine, serum albumin, and 24 h protein excretion were used for this study.
Kidney histopathology examination
The kidney tissues were collected from the patients, and the pathological specimens examined under light immunofluorescence microscopy were sent to KingMed Diagnostics.
Kidney pathology and immunofluorescent examination
Immunofluorescent staining was performed for PLA2R and IgG4 on 3 μm sections of formalin-fixed tissue using a kit, according to manufacturer’s instructions. Briefly, the rabbit antihuman PLA2R polyclonal antibody (1:100 dilution; Sigma-Aldrich Co., St Louis, MO, USA) was added to the sections. After 1 h of incubation, the goat antirabbit IgG (Abcam, Cambridge, UK), which had been diluted to 1:50, was added to the sections. After 1 h of incubation, the sections were washed with water, dried, glycerol mounted, and then observed under fluorescence microscopy. The method and steps of IgG4 detection were as follows: the first antibody mouse antihuman CD138-FITC (Maixin, FuZhou, China) and the second antibody (1:200 dilution) IgG4 monoclonal antibody (Abcam) were added to the sections. After 1 h of incubation, the sections were washed with water, dried, glycerol mounted, and then observed under fluorescence microscopy. Fluorescence intensity determination: observed by fluorescence microscopy, -, negative, ±/+~ 3 +, positive.16
Serum anti-PLA2R antibody detection
According to the kit instructions (Euroimmun, Lübeck, Germany), the blood was extracted from the peripheral vein and the serum was detected by ELISA after centrifugation. The ELISA detector measured the absorbance value at the 450 nm wavelength. The serum antibody level was calculated according to the curve equation, with 0–14 RU/mL as the standard reference value of the serum antibody.
Normally distributed data are presented as the mean ± standard deviation. The data were statistically analyzed using the SPSS 17.0 software package (SPSS Inc., Chicago, IL, USA). Parametric data were analyzed using one-way analysis of variance and Student’s t-test. The sensitivity and specificity, negative predictive value, and positive predictive value were calculated by Fisher’s exact test. P<0.05 was considered as a statistically significant difference.
Clinical data and biochemical parameters
There were 39 patients enrolled in this study from August 2016 to December 2016. All patients underwent kidney biopsy, including 27 males and 12 females, with mean age 45.8±13.6 years, and were divided into the following three groups according to the pathological results: primary MN group (IMN; 27 patients), SMN group (five patients), and non-MN group (seven patients). No statistically significant differences were observed for serum albumin and 24 h urine protein in the three groups. There was one case of acute renal injury and one case of severe lupus in the non-MN group; the serum creatinine concentration of two patients was significantly higher than normal, so there was a significant difference compared with IMN and SMN groups (P<0.01) (Table 1).
PLA2R and IgG4 expressions in the kidneys of the three groups
The positive expressions of PLA2R (88.89%) and IgG4 (81.48%) were higher in the IMN group than in the SMN group and non-MN group, and the difference was statistically significant (P<0.01). There was no significant difference in PLA2R-positive expression and IgG4-positive expression between the SMN group and non-MN group (P>0.05) (Table 2).
Comparison of PLA2R between kidney tissue and serum in IMN patients
The results showed that 24 patients whose kidney tissue PLA2R were positive and their anti-PLA2R antibodies in serum were also detected positive. Three cases of IMN kidney tissue PLA2R-negative patients with anti-PLA2R antibodies in serum were also negative, suggesting that serum anti-PLA2R antibody and kidney tissue PLA2R detection consistent rate was 100% (Table 3).
Evaluation of indicator of PLA2R and IgG4 expressions in the kidney tissue
According to the results, the detection of the PLA2R and IgG4 expression in the kidney tissue had higher sensitivity compared with SMN group and non-MN groups. The detection of the PLA2R or IgG4 had higher specificity compared with SMN group and non-MN groups, which may be due to the specificity of expression of PLA2R in the non-IMN group. Compared with the sensitivity, specificity, negative predictive value, positive predictive value, and coincidence rate in the three groups, no statistically significant difference was observed (P=0.459, 0.222, 0.387, 0.718, and 0.843). This was a significant limitation in our study; the number of patients included was small due to the shorter research period. We will include more cases in the future to improve research persuasiveness (Table 4).
Immunofluorescence changes in the three groups
Immunofluorescence showed that there were significant differences in the fluorescence values among the three groups (Figure 1). Immunofluorescence-labeled immunocomplex was more abundant in the glomerular capillary loops in the IMN group but not in the SMN and non-MN groups.
MN is a common pathological type of nephrotic syndrome, and the incidence increases year by year. Some causes such as the hepatitis, drug, high incidence of cancer, environmental pollution and so on could cause MN.17–20 In the present study, we collected all patients who underwent kidney pathology within 5 months in our hospital were collected; the statistical results showed that the incidence of MN was up to 69.2%, and the incidence of MN was higher than IgA nephropathy, which had been the first cause in chronic glomerular disease.
Phospholipase A2 (PLA2) is a group of enzymes distributed in multiple organs of the human body. It has many subtypes and can bind to PLA2R to form an immune complex, which plays a key role in regulating cell proliferation, adhesion, and activity factor secretion in the inflammatory reaction process. PLA2R was overexpressed in renal tissue of IMN patients, and anti-PLA2R antibody produced by its expression was also observed. A series of studies confirmed that anti-PLA2R antibody levels in IMN patients were significantly elevated compared with the normal and non-IMN patients.21,22 Anti-PLA2R antibodies bind to PLA2R on kidney podocytes to cause complement activation, podocyte damage, and basement membrane damage.23,24 In this study, we found that the PLA2R from kidney tissue was positive in 88.89% of IMN patients, while PLA2R from kidney tissue was positive in 40 and 0% of SMN and non-MN patients, respectively. Beck et al reported that IgG4 was mainly anti-PLA2R antibodies in IMN patients. Many studies showed that the pathogenesis of IMN is because the recognition of IgG4 recognition and the glomerular podocyte PLA2R, the formation of in situ immune complexes, and the activation of the complement system, which led to immune injury, podocyte morphological change, proteinuria, hypoproteinemia, and other clinical symptoms.25 Our study showed that the positive rate of IgG4 expression in kidney tissue of IMN patients was 81.48%, while the positive rate of IgG4 expression in kidney tissue of SMN and non-MN patients was 20 and 14.9%, respectively. However, there were two cases of SMN positive for PLA2R and one case of SMN and non-MN positive for IgG4. This may result from: 1) there was a possibility of IMN combined with another disease;26 2) the combined detection of PLA2R and IgG4 could not completely exclud the secondary causes; and 3) the mechanism of MN was not completely clear and the explanation of the role of PLA2R and IgG4 in patients with MN was difficult.27 Still, we have reasons to believe that the positive rate of IgG4 in kidney tissue consistent with PLA2R, and the detection of IgG4 combined with PLA2R improved the diagnostic accuracy of MN. Therefore, IgG4-related disease (IgG4-RD) is recently gaining a lot of attention. It is worth mentioning that IgG4 found in IMN is not IgG4-related disease. In 2012, IgG4-related MN was proposed by Alexander et al.28 In patients with MN secondary to IgG4-RD, an immunofluorescence assay showed granular deposits of C3 and IgG, of which IgG4 was the dominant subclass, along the glomerular basement membrane. In these patients, the kidney tissue is typically negative when staining with anti-PLA2R antibodies, similar to the serum anti-PLA2R staining. Currently, there is still no uniform diagnostic standard for MN secondary to IgG4-RD. The diagnosis of MN secondary to IgG4-RD should be made in the context of IgG4-RD in other organs or IgG4-related tubulointerstitial nephritis (IgG4-TIN).29
IgG4 and PLA2R detection in kidney tissue was used as an important diagnostic tool. For those patients who were not or refused to perform kidney pathology, anti-PLA2R antibody detection in serum was used as a convenient and rapid detection method to provide guidance for clinical diagnosis. In this study, the number of patients included was less because of the shorter period of study. We will continue this study and include more patients to improve the scientific nature of this study.
This work was supported by the grants from the Program of Natural Science Foundation of Higher Education of Anhui Province (KJ2015B021by and KJ2015B036by) and the key Program of Natural Science Foundation of Anhui Province (KJ2016A481). The funding organizations played no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the article.
The authors report no conflicts of interest in this work.
Xie J, Chen N. Primary glomerulonephritis in mainland China: an overview. Contrib Nephrol. 2013;181:1–11.
Beck LH Jr, Bonegio RG, Lambeau G, et al. M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy. N Engl J Med. 2009;361(1):11–21.
Wang LJ, Shang MH, Zhuge YF, et al. Expression of renal PLA2R in patients with idiopathic membranous nephropathy and its relationship with the curative effect of immunotherapy. Zhonghua Yi Xue Za Zhi. 2016;96(1):4–8.
Lin W, Li H, Li X, et al. The relationship between anti-phospholipase A2 receptor antibody and idiopathic membranous nephropathy. Zhonghua Nei Ke Za Zhi. 2015;54(9):783–788.
Radice A, Trezzi B, Maggiore U, et al. Clinical usefulness of autoantibodies to M-type phospholipase A2 receptor (PLA2R) for monitoring disease activity in idiopathic membranous nephropathy (IMN). Autoimmun Rev. 2016;15(2):146–154.
Hofstra JM, Wetzels JF. Phospholipase A2 receptor antibodies in membranous nephropathy: unresolved issues. J Am Soc Nephrol. 2014;25(6):1137–1139.
Dahnrich C, Komorowski L, Probst C, et al. Development of a standardized ELISA for the determination of autoantibodies against human M-type phospholipase A2 receptor in primary membranous nephropathy. Clin Chim Acta. 2013;421:213–218.
Ruggenenti P, Debiec H, Ruggiero B, et al. Anti-phospholipase A2 receptor antibody titer predicts post-rituximab outcome of membranous nephropathy. J Am Soc Nephrol. 2015;26(10):2545–2558.
Quintana LF, Blasco M, Seras M, et al. Antiphospholipase A2 receptor antibody levels predict the risk of posttransplantation recurrence of membranous nephropathy. Transplantation. 2015;99(8):1709–1714.
Hoxha E, Harendza S, Pinnschmidt H, Panzer U, Stahl RA. PLA2R antibody levels and clinical outcome in patients with membranous nephropathy and non-nephrotic range proteinuria under treatment with inhibitors of the renin-angiotensin system. PLoS One. 2014;9(10):e110681.
Schilt U. Differentiation of herpes simplex virus type 1 and type 2 by immunofluorescence: discriminative staining by labelled IgG preparations. Z Immunitatsforsch Immunobiol. 1979;155(5):411–419.
Lönnbro-Widgren J, Ebefors K, Mölne J, Nyström J, Haraldsson B. Glomerular IgG subclasses in idiopathic and malignancy-associated membranous nephropathy. Clin Kidney J. 2015;8(4):433–439.
Vivarelli M, Emma F, Pelle T, et al. Genetic homogeneity but IgG subclass-dependent clinical variability of alloimmune membranous nephropathy with anti-neutral endopeptidase antibodies. Kidney Int. 2015;87(3):602–609.
Kidney Disease Outcomes Quality Initiative(K/DOQI) Group. K/DOQI clinical practice guidelines for managing dyslipidemias in chronic kidney disease. Am J Kidney Dis. 2003;41:S1–S91.
Jiang W, Liu T, Dong H, et al. Relationship between serum DNA replication, clinicopathological characteristics and prognosis of hepatitis B virus-associated glomerulonephritis with severe proteinuria by lamivudine plus adefovir dipivoxil combination therapy. Biomed Environ Sci. 2015;28(3):206–213.
Zambol L, Hermanova M, Adamkova Krakorova D, et al. Nestin expresion in high-grade osteosarcomas and its clinical significance. Oncol Rep. 2012;27(5):1592–1598.
Xu X, Zhu X, Yuan S, et al. Role of M-type phospholipase A2 receptor and its antibody in hepatitis B virus-associated membranous nephropathy. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2016;41(10):1064–1068.
Yokoyama H, Narita I, Sugiyama H, et al. Drug-induced kidney disease: a study of the Japan Renal Biopsy Registry from 2007 to 2015. Clin Exp Nephrol. 2016;20(5):720–730.
Hoxha E, Wiech T, Stahl PR, et al. A mechanism for cancer-associated membranous nephropathy. N Engl J Med. 2016;374(20):1995–1996.
Xu X, Wang G, Chen N, et al. Long-term exposure to air pollution and increased risk of membranous nephropathy in China. J Am Soc Nephrol. 2016;27(12):3739–3746.
Zhu SS, Zhou SL, Zhou CM, et al. Detection of PLA2R1 in renal biopsy specimens of patients with idiopathic membranous nephropathy. Nan Fang Yi Ke Da Xue Xue Bao. 2015;35(4):526–529.
Gopalakrishnan N, Abeesh P, Dineshkumar T, et al. Prevalence of serum anti M-type phospholipase A2 receptor antibody in primary membranous nephropathy: a single center experience. Indian J Nephrol. 2016;26(4):257–261.
Sugahara G, Kamiie J, Kobayashi R, Mineshige T, Shirota K. Expression of phospholipase A2 receptor in primary cultured podocytes derived from dog kidneys. J Vet Med Sci. 2016;78(5):895–899.
Kao L, Lam V, Waldman M, Glassock RJ, Zhu Q. Identification of the immunodominant epitope region in phospholipase A2 receptor-mediating autoantibody binding in idiopathic membranous nephropathy. J Am Soc Nephrol. 2015;26(2):291–301.
Debiec H, Ronco P. Immunopathogenesis of membranous nephropathy: an update. Semin Immunopathol. 2014;36(4):381–397.
Larsen CP, Messias NC, Silva FG, Messias E, Walker PD. Determination of primary versus secondary membranous glomerulopathy utilizing phospholipase A2 receptor staining in renal biopsies. Mod Pathol. 2013;26(5):709–715.
Qin W, Beck LH, Zeng C, et al. Anti-phospholipase A2 receptor antibody in membranous nephropathy. J Am Soc Nephrol. 2011;22:1137–1143.
Alexander MP, Larsen CP, Gibson IW, et al. Membranous glomerulonephritis is a manifestation of IgG4-related disease. Kidney Int. 2013;83(3):455–462.
Wada Y, Saeki T, Yoshita K, et al. Development of IgG4-related disease in a patient diagnosed with idiopathic membranous nephropathy. Clin Kidney J. 2013;6(5):486–490.
This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.Download Article [PDF]