Enhanced expression of the M-type phospholipase A2 receptor in glomeruli correlates with serum receptor antibodies in primary membranous nephropathy
2012; Elsevier BV; Volume: 82; Issue: 7 Linguagem: Inglês
10.1038/ki.2012.209
ISSN1523-1755
AutoresElion Hoxha, U. Kneissler, Gesa Stege, Gunther Zahner, I. Thiele, Ulf Panzer, Sigrid Harendza, Udo Helmchen, Rolf A.K. Stahl,
Tópico(s)Amyloidosis: Diagnosis, Treatment, Outcomes
ResumoThe M-type phospholipase A2 receptor (PLA2R) is the major target antigen in idiopathic membranous nephropathy with detectable autoantibodies in the serum of up to 70% of patients. In retrospective studies, the PLA2R-autoantibody titer in the serum was sometimes negative indicating their measurement alone may be inconclusive. In order to better differentiate between primary and secondary membranous nephropathy, we conducted a prospective study that included 88 patients with a histologic diagnosis of membranous nephropathy. Immunohistochemical analysis for PLA2R was faintly positive in kidneys from normal individuals and patients with various other glomerular injuries. In 61 of the 88 patients, PLA2R expression was strongly positive in glomeruli, and in 60 of these patients PLA2R autoantibodies were also detected in the serum. The 27 patients negative for serum PLA2R autoantibodies were faintly positive for PLA2R staining in glomeruli and in 15 of these patients a secondary cause was found. The remaining 12 patients have a yet undetected secondary cause of membranous nephropathy or have different glomerular antigens other than PLA2R. Thus, increased staining for PLA2R in glomeruli of renal biopsies tightly correlates with the presence of PLA2R autoantibodies in the serum and this may help discriminate between primary and secondary membranous nephropathy. The M-type phospholipase A2 receptor (PLA2R) is the major target antigen in idiopathic membranous nephropathy with detectable autoantibodies in the serum of up to 70% of patients. In retrospective studies, the PLA2R-autoantibody titer in the serum was sometimes negative indicating their measurement alone may be inconclusive. In order to better differentiate between primary and secondary membranous nephropathy, we conducted a prospective study that included 88 patients with a histologic diagnosis of membranous nephropathy. Immunohistochemical analysis for PLA2R was faintly positive in kidneys from normal individuals and patients with various other glomerular injuries. In 61 of the 88 patients, PLA2R expression was strongly positive in glomeruli, and in 60 of these patients PLA2R autoantibodies were also detected in the serum. The 27 patients negative for serum PLA2R autoantibodies were faintly positive for PLA2R staining in glomeruli and in 15 of these patients a secondary cause was found. The remaining 12 patients have a yet undetected secondary cause of membranous nephropathy or have different glomerular antigens other than PLA2R. Thus, increased staining for PLA2R in glomeruli of renal biopsies tightly correlates with the presence of PLA2R autoantibodies in the serum and this may help discriminate between primary and secondary membranous nephropathy. Membranous nephropathy (MN) is the leading cause of a nephrotic syndrome in Caucasians. The disease is characterized by the presence of immune complexes in the subepithelial space of the glomerular filtration barrier. An in situ immune complex formation is considered to be the pathogenetic mechanism, which means that circulating antibodies bind to an antigen expressed on glomerular podocytes.1.Couser W.G. Steinmuller D.R. Stilmant M.M. et al.Experimental glomerulonephritis in the isolated perfused rat kidney.J Clin Invest. 1978; 62: 1275-1287Crossref PubMed Scopus (265) Google Scholar,2.Kerjaschki D. Miettinen A. Farquhar M.G. Initial events in the formation of immune deposits in passive Heymann nephritis. gp330-anti-gp330 immune complexes form in epithelial coated pits and rapidly become attached to the glomerular basement membrane.J Exp Med. 1987; 166: 109-128Crossref PubMed Scopus (112) Google Scholar In ∼25% of patients, MN is associated with an underlying disease, such as systemic lupus erythematodes, malignant tumors, or disease caused by exposure to certain drugs or due to a variety of viral or bacterial infections.3.Ronco P. Debiec H. Antigen identification in membranous nephropathy moves toward targeted monitoring and new therapy.J Am Soc Nephrol. 2010; 21: 564-569Crossref PubMed Scopus (79) Google Scholar When no secondary cause is apparent, the disease is classified as primary. However, in some patients, MN can appear months or even years before a secondary cause is detected. In these patients, particularly those older than 65 years, great uncertainty exists about the clinical management, as an undetected malignant tumor could be the cause.4.Burstein D.M. Korbet S.M. Schwartz M.M. Membranous glomerulonephritis and malignancy.Am J Kidney Dis. 1993; 22: 5-10Abstract Full Text PDF PubMed Scopus (150) Google Scholar Furthermore, in some patients, MN and a potential secondary cause of MN (e.g., malignoma) may develop independently of each other and present chronologically within a short period of time.4.Burstein D.M. Korbet S.M. Schwartz M.M. Membranous glomerulonephritis and malignancy.Am J Kidney Dis. 1993; 22: 5-10Abstract Full Text PDF PubMed Scopus (150) Google Scholar,5.Beck L.H. Membranous nephropathy and malignancy.Semin Nephrol. 2010; 30: 635-644Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar Hence, the differential diagnosis between primary and secondary MN is of great clinical importance. In patients with primary MN, a conservative or an immunosuppressive treatment is chosen depending on the clinical status, whereas in patients with secondary MN treatment is directed toward the causal disease.6.Cattran D. Management of membranous nephropathy: when and what for treatment.J Am Soc Nephrol. 2005; 16: 1188-1194Crossref PubMed Scopus (186) Google Scholar A reliable differentiation between primary and secondary MN would substantially improve further management of the patients. This could include less diagnostic procedures in the case of a primary MN or a more aggressive diagnostic approach to search for underlying causes in secondary forms of MN. The discovery of the M-type phospholipase A2 receptor (PLA2R) as a target antigen in primary MN7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar is an important finding, which could help improve the differential diagnosis of MN. In a series of retrospective analyses in patients with biopsy-proven MN, the levels of PLA2R-antibodies (PLA2R-AB) in the serum, however, ranged from 52 to 78%7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar, 8.Hofstra J.M. Beck L.H. Beck D.M. et al.Anti-phospholipase A2 receptor antibodies correlate with clinical status in idiopathic membranous nephropathy.Clin J Am Soc Nephrol. 2011; 6: 1286-1291Crossref PubMed Scopus (292) Google Scholar, 9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar, 10.Beck L.H. Fervenza F.C. Beck D.M. et al.Rituximab-induced depletion of anti-PLA2R autoantibodies predicts response in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1543-1550Crossref PubMed Scopus (358) Google Scholar, 11.Debiec H. Ronco P. PLA2R autoantibodies and PLA2R glomerular deposits in membranous nephropathy.N Engl J Med. 2011; 364: 689-690Crossref PubMed Scopus (247) Google Scholar and demonstrated that there may be uncertainty in the differential diagnosis. The reported discrepancies in these mostly retrospective studies might have several reasons: (i) immunosuppression could have removed the antibodies;7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar, 8.Hofstra J.M. Beck L.H. Beck D.M. et al.Anti-phospholipase A2 receptor antibodies correlate with clinical status in idiopathic membranous nephropathy.Clin J Am Soc Nephrol. 2011; 6: 1286-1291Crossref PubMed Scopus (292) Google Scholar, 9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar, 10.Beck L.H. Fervenza F.C. Beck D.M. et al.Rituximab-induced depletion of anti-PLA2R autoantibodies predicts response in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1543-1550Crossref PubMed Scopus (358) Google Scholar (ii) as in a large number of patients the measurement of PLA2R-AB was performed after a long time interval following renal biopsy, spontaneous remission with disappearance of the antibody might have occurred; and (iii) antigens other than the PLA2R may be relevant for MN. Thus, only a prospective analysis in which the PLA2R-AB levels are measured in close proximity to the time of renal biopsy will help to obtain a near estimation of the real number of PLA2R-AB-positive patients. Second, we hypothesized that the intensity of the glomerular expression of the PLA2R could be another parameter to increase the specificity of the differential diagnosis between primary and secondary MN, as antigen–antibody complexes should be present in active disease.7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar We therefore analyzed in this prospective study 88 patients with biopsy-proven MN for their PLA2R protein expression in glomeruli and analyzed their PLA2R-AB serum levels in close proximity to the time of biopsy. PLA2R staining is enhanced in glomeruli of those patients in whom antibodies are detectable in the serum and correlates with the detection of PLA2R-AB in the serum. PLA2R-AB and PLA2R-antigen were not found in patients with secondary MN. We conclude that the increased glomerular PLA2R staining and the detection of PLA2R-AB improve the differential diagnosis between primary and secondary MN. Between November 2009 and April 2011, 88 consecutive patients (64 men and 24 women) with the histologic diagnosis of MN made in our institution were included in this study (Supplemental Table S1 online). None of the patients had received immunosuppressive therapy before the renal biopsy or the measurement of PLA2R-AB levels. In 66 (75%) patients, serum PLA2R-AB was measured within 4 weeks after renal biopsy, in 16 (18%) patients between 4 and 24 weeks, and in the remaining 6 (7%) patients between 24 and 44 weeks following renal biopsy. At the time of PLA2R-AB measurement, mean proteinuria was 8.0±5.7g/24h. All, except three, patients were on angiotensin-converting enzyme inhibitors or AT2-R blockers at the time of PLA2R-AB measurement. In all, 66 (75%) patients received lipid-lowering medications, and 69 (78%) patients were on diuretics. Anticoagulation was initiated in 33 (37%) patients. Immunohistochemical staining for PLA2R revealed a faint positivity in normal kidneys (see Figure 1a). PLA2R was selectively expressed on podocytes in a fine granular pattern. In 61 (69%) out of the 88 patients with the histologic diagnosis of a MN, the immunohistochemistry revealed a markedly enhanced glomerular staining for the PLA2R when compared with the normal kidneys. Immunohistochemistry showed a more granular pattern (Figure 1d). The immune deposits are localized in the subepithelial space and appear in the same location as the staining for immunoglobulins (Figure 1e and h). The enhanced expression of the PLA2R was very similar and homogeneous between the individual patients and did not show any significant correlation with the serum antibody levels or the degree of proteinuria. It rather resembles an ‘on’–‘off’ phenomenon. When analyzed for the presence of PLA2R-AB in the serum, antibodies were detectable in 60 (98.4%) of the 61 patients with enhanced glomerular PLA2R (Figure 2). An enhanced glomerular PLA2R staining was found in Patient 18, but no PLA2R-AB was detected in his serum. PLA2R-AB measurement in this case was performed 9 months after the renal biopsy. Of the other 27 patients with the histologic diagnosis of MN who did not reveal an enhanced PLA2R expression in glomeruli (Figure 1g), and who had no detectable PLA2R-AB in the serum (Figure 2), an underlying disease was detected in 15 patients. Five patients had systemic lupus erythematodes, one patient had hepatitis B, seven patients had malignant tumors, and two patients had developed MN while taking nonsteroidal anti-inflammatory drugs. There was no significant difference in all important clinical parameters between patients with an enhanced PLA2R expression and those without an enhanced PLA2R expression in glomeruli (Table 1). Proteinuria and serum creatinine levels were slightly higher in patients with enhanced PLA2R expression in glomeruli, but these differences were not statistically significant.Table 1Clinical characteristics of the patients included in this study. Patients with enhanced PLA2R staining did not significantly differ in any of the clinical features from patients with normal PLA2R stainingEnhanced glomerular PLA2R stainingNormal (non-enhanced) glomerular PLA2R stainingP-valueNumber of patients6127–Age (mean±s.d.), years56.5±15.857.7±15.00.74Gender (m:f)49:1215:12–Serum creatinine, mg/dl (mean±s.d.)1.4±0.81.2±0.50.19Proteinuria, g/24h (mean±s.d.)8.6±6.16.7±4.60.15Time from renal biopsy to PLA2R-AB measurement in serum, months (mean±s.d.)1.6±2.81.7±2.50.94Abbreviations: AB, antibody; f, female; m, male; PLA2R, phospholipase A2 receptor. Open table in a new tab Abbreviations: AB, antibody; f, female; m, male; PLA2R, phospholipase A2 receptor. To study whether the PLA2R expression was an exclusive finding in PLA2R-AB-positive patients with MN, renal biopsies of 78 patients with various diseases (14 with minimal change disease, 11 with immunoglobulin IgA nephropathy, 9 with primary FSGS, 8 with sarcoidosis, 8 with diabetic nephropathy, 7 with lupus nephritis type IV, 6 with lupus nephritis type V, 6 with MPGN type I, 4 with fibrillary GN, and 5 normal kidneys) were tested for the expression of the PLA2R and the presence of PLA2R-AB in the serum. PLA2R expression in all renal biopsies from patients with diseases other than MN was similar to the expression in glomeruli of normal kidneys or in kidneys from patients with secondary MN (data not shown). None of the patients with renal diseases other than MN had detectable PLA2R-AB in the serum. In all PLA2R-AB-positive patients, IgG4-PLA2R-AB levels were detectable. In addition, in 80% (48 of 60 patients) of these patients other PLA2R-AB IgG subtypes were detectable (Table 2). In 20% (12 of 60 patients), the IgG4 subclass PLA2R-AB was the only detectable antibody, whereas in 40% (24 of 60) all IgG subclasses of PLA2R-AB were present.Table 2IgG subclasses of PLA2R-AB in patients with detectable PLA2R-AB in serum. The data show that all patients, who were positive for PLA2R-AB, had IgG4 subclass antibodies. In a large percentage of patients, however, other IgG subclass antibodies were presentPLA2R-AB subtypes in serumNumber of patients (% of PLA2R-AB positive)PLA2R-AB positive60 (100)IgG135 (58.3)IgG229 (48.3)IgG342 (70.0)IgG460 (100)Only one PLA2R-AB subtype (IgG4)12 (20.0)Two PLA2R-AB-IgG subtypes14 (23.3)Three PLA2R-AB-IgG subtypes10 (16.7)All four PLA2R-AB-IgG subtypes24 (40.0)Abbreviations: Ig, immunoglobulin; PLA2R-AB, phospholipase A2 receptor antibody. Open table in a new tab Abbreviations: Ig, immunoglobulin; PLA2R-AB, phospholipase A2 receptor antibody. When an analysis was performed to check whether proteinuria and PLA2R-AB levels show a potential interrelation and whether PLA2R-AB levels eventually indicate the severity of the clinical disease at the time of the measurement of proteinuria, no correlation could be detected by the Spearman's rank coefficient of correlation (r=-0.06; P=0.65; Figure 3). As one would assume, in the in vivo situation, when PLA2R-AB levels are detectable in the serum of the patients, all epitopes of the receptor would be occupied by the circulating antibodies. It was therefore surprising that in the in vitro situation of the immunohistochemical analysis of the renal biopsies from patients with PLA2R-AB in their serum an antibody could still detect the increased PLA2R in the glomeruli. We therefore tested in an in vitro approach, using PLA2R-overexpressing HEK 293 cells, whether human PLA2R-AB from serum was able to impair, or even abolish, the binding of the diagnostic antibody against PLA2R used for immunohistochemistry. Antiserum from patients with a high titer of the PLA2R bound to a receptor on the cells but did not interfere with the binding of the diagnostic antibody applied for immunohistochemistry (Figure 4). This may suggest that potentially pathogenetic human PLA2R-AB bind to (an) epitope(s) other than the antibody used in the immunohistochemistry. To address the question whether the enhanced expression of the PLA2R might be the result of an increased PLA2R formation, we measured the mRNA expression of PLA2R in kidneys from patients with MN and enhanced PLA2R staining (n=6) and kidneys from patients with MN and no enhanced PLA2R staining (n=6). As summarized in Figure 5, there was no difference between the mRNA levels of the PLA2R in the different groups (P=0.53). When stained for IgG4, 95% (58 of 61) of renal biopsies from patients with an enhanced PLA2R staining were positive (Table 3). In contrast, only 20% (3 of 15) of patients with a known secondary cause of MN had a positive IgG4 staining. Furthermore, 45% (5 of 11) of patients who did not have an enhanced PLA2R staining and in whom no secondary cause of MN was detectable had a positive IgG4 staining.Table 3Ninety-five percent of patients with enhanced PLA2R staining had a positive IgG4 staining in renal biopsies. Only 31% of the patients with non-enhanced PLA2R staining had a positive IgG4 stainingEnhanced PLA2R stainingNon-enhanced PLA2R stainingNumber of patients6127Number of patients with positive IgG4 staining (%)58 (95.0)8 (30.8)aIn one case with non-enhanced PLA2R staining and no PLA2R-AB in serum, no IgG4 staining could be performed because of lack of further renal tissue.Abbreviations: Ig, immunoglobulin; PLA2R, phospholipase A2 receptor.a In one case with non-enhanced PLA2R staining and no PLA2R-AB in serum, no IgG4 staining could be performed because of lack of further renal tissue. Open table in a new tab Abbreviations: Ig, immunoglobulin; PLA2R, phospholipase A2 receptor. This prospective study in patients with MN shows that patients with MN who had an enhanced glomerular expression of the PLA2R by immunohistochemistry also had detectable PLA2R-AB in their serum. In contrast, patients who did not have enhanced glomerular PLA2R expression had no detectable PLA2R-AB in their serum. Neither PLA2R-antigen in biopsy nor PLA2R-AB in serum was found in patients with a known cause of secondary MN. This observation allows separating what is currently called primary MN from secondary MN. It is of great clinical importance to discriminate between patients with primary and secondary MN. Diagnostic procedures and therapeutic strategies are completely different for these patients. Although therapy of the secondary form is directed to an underlying disease, in patients with primary MN an immunosuppressive strategy may be an option.6.Cattran D. Management of membranous nephropathy: when and what for treatment.J Am Soc Nephrol. 2005; 16: 1188-1194Crossref PubMed Scopus (186) Google Scholar The correct diagnosis is essential to avoid unnecessary drug exposure or extensive diagnostic procedures. To date, the diagnosis of primary MN is still made by exclusion of secondary causes, using medical history, physical examination, appropriate laboratory tests, and often invasive diagnostic procedures. A standardized diagnostic approach, which could distinguish between primary and secondary forms of MN, would therefore lead to a more focused investigation of secondary causes only when necessary. This would be particularly important in patients with MN over 65 years of age, as MN may be either associated or may be detected years before a diagnosis of a malignant tumor.4.Burstein D.M. Korbet S.M. Schwartz M.M. Membranous glomerulonephritis and malignancy.Am J Kidney Dis. 1993; 22: 5-10Abstract Full Text PDF PubMed Scopus (150) Google Scholar,5.Beck L.H. Membranous nephropathy and malignancy.Semin Nephrol. 2010; 30: 635-644Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar This study consists of the staining of renal biopsies from patients with the diagnosis of MN for the PLA2R and the concurrent measurement of PLA2R-AB in the serum. PLA2R-AB levels are detected only in sera from patients with a ‘primary’ form of MN.7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar,9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar Yet, not all patients with primary MN show PLA2R-AB.7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar,9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar This discrepancy could be due to spontaneous remission of the disease, immunosuppressive therapy, or the existence of other antigens. To avoid the possibility of spontaneous remission of proteinuria or immunosuppressive therapy, we chose a prospective approach and tried to measure antibody levels as close as possible to the time of renal biopsy. The mean time from renal biopsy to antibody measurements was 1.6 months, which is a very short time compared with all the published retrospective studies. With the use of a polyclonal PLA2R-AB, we were able to show an enhanced expression in 61 out of 88 patients with biopsy-proven MN, compared with only a faint expression in normal kidneys. Immunohistochemistry showed a granular pattern. The immune deposits are localized in the subepithelial space, whereas in normal kidneys the antigen is detectable only on the cell surface of the podocytes. The intensity of the expression was similar in all biopsies that were considered enhanced and has the characteristic of an ‘on’ or ‘off’ situation. The staining intensity was independent of the PLA2R-AB levels, which were very different in these patients and did not show any correlation with the level of proteinuria as well. A very similar observation of a histologic pattern of ‘negative’ or ‘positive’ for PLA2R1 staining was recently reported by others11.Debiec H. Ronco P. PLA2R autoantibodies and PLA2R glomerular deposits in membranous nephropathy.N Engl J Med. 2011; 364: 689-690Crossref PubMed Scopus (247) Google Scholar using immunofluorescence histology. These investigators also did not report a difference in the intensity of PLA2R staining in the presence of different serum antibody levels. Thus, both studies might suggest that there is a limit in receptor availability. To address this issue in some more detail, the mRNA expression of the PLA2R was studied. Renal biopsies from patients with MN and enhanced PLA2R staining did not show increased transcription of the receptor when compared with renal biopsies from patients with MN but non-enhanced PLA2R staining. This of course does not exclude the possibility of increased posttranscriptional synthesis or any other mechanism that may increase the availability of the antigen to the surface of the podocytes. The results, however, do not support the findings in experimental MN, in which it was shown that the receptor synthesis is increased.12.Makker S.P. Widstrom R. Huang J. Transcription and translation of gp600 and receptor-associated protein (RAP) in active Heymann nephritis.Am J Pathol. 1995; 146: 1481-1487PubMed Google Scholar To address these questions in more detail, in vivo and in vitro studies are necessary. To better understand, however, the finding that the glomerular PLA2R expression by immunohistochemical techniques is detectable in a situation in which excessive circulating antibodies in these patients probably bind all epitopes of the receptor, we performed additional in vitro studies in HEK 293 cells overexpressing the PLA2R. These in vitro experiments show that the naturally occurring antibodies in the patients bind to (a) epitope(s) on the PLA2R different from those epitopes which are bound by the antibody that was used for the detection of the receptor in the renal tissues. In a next step, we wanted to be certain that PLA2R expression is exclusive in patients with MN, as in other studies PLA2R–AB were detected in patients with sarcoidosis, systemic lupus erythematodes, or tumors.13.Knehtl M. Debiec H. Kamgang P. et al.A case of phospholipase A2 receptor-positive membranous nephropathy preceding sarcoid-associated granulomatous tubulointerstitial nephritis.Am J Kidney Dis. 2011; 57: 140-143Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar,14.Qin W. Beck Jr., L.H. Zeng C. et al.Anti-phospholipase A2 receptor antibody in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1137-1143Crossref PubMed Scopus (346) Google Scholar For this purpose, 78 biopsies from patients with various glomerular injuries other than MN were studied. In none of these patients, including 8 patients with sarcoidosis and 13 patients with systemic lupus erythematodes PLA2R-antigen, staining was increased in glomeruli. The immunohistochemistry in all of these biopsies shows the same pattern as in control kidneys (data not shown). As it has been demonstrated that the PLA2R-AB belongs primarily to the IgG4 subclass,7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar the renal tissues were stained for the presence of IgG4 antibodies. Although the vast majority of patients with enhanced PLA2R were positive for IgG4 staining (95%), this was not the case for patients who did not have an enhanced glomerular PLA2R staining. In patients with a secondary MN and a known cause of the disease, only 20% were positive for IgG4. In patients with non-enhanced glomerular PLA2R staining and without a known secondary cause of MN, in 45% of patients IgG4 was detectable in glomeruli. When the 61 patients with MN who had an enhanced glomerular PLA2R expression were compared with those patients who had detectable serum levels of the PLA2R-AB, 60 of the 61 patients did show both. It is currently unclear why patient 18 did not have detectable PLA2R-AB, but this patient might have been in immunologic remission with still detectable enhanced PLA2R expression in his glomeruli. If one accepts the hypothesis that primary MN is characterized by the presence of PLA2R-AB7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar and adds the information that these antibodies can become negative for several reasons,7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar, 8.Hofstra J.M. Beck L.H. Beck D.M. et al.Anti-phospholipase A2 receptor antibodies correlate with clinical status in idiopathic membranous nephropathy.Clin J Am Soc Nephrol. 2011; 6: 1286-1291Crossref PubMed Scopus (292) Google Scholar, 9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar, 10.Beck L.H. Fervenza F.C. Beck D.M. et al.Rituximab-induced depletion of anti-PLA2R autoantibodies predicts response in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1543-1550Crossref PubMed Scopus (358) Google Scholar then primary MN can be diagnosed by the histologic pattern that we show in our study—that is, an increased expression of the PLA2R in glomeruli in combination with the standard morphologic diagnostics of MN. This might be of clinical importance when the time of biopsy is far (several months to years) from antibody measurement. Our data, however, also show that under the conditions described in this manuscript a positive PLA2R-AB serum level allows the diagnosis of primary MN and supports the initial hypothesis of Beck et al.7.Beck L.H. Bonegio R.G. Lambeau G. et al.M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.N Engl J Med. 2009; 361: 11-21Crossref PubMed Scopus (1505) Google Scholar In all, 82% (60/73) of patients, in whom no secondary cause of MN could be detected, were positive for PLA2R-AB. This is the highest rate of PLA2R-AB positivity shown in a Caucasian population. By using a highly sensitive western blot approach in a Chinese cohort, an even higher percentage of seropositivity for PLA2R-AB was found.14.Qin W. Beck Jr., L.H. Zeng C. et al.Anti-phospholipase A2 receptor antibody in membranous nephropathy.J Am Soc Nephrol. 2011; 22: 1137-1143Crossref PubMed Scopus (346) Google Scholar The remaining 12 patients in our study in whom no clinical cause was found and who were negative for PLA2R and PLA2R-AB can currently only be classified as having secondary MN. IgG4 positivity has been suggested as a potential marker to differentiate primary from secondary MN.15.Doi T. Mayumi M. Kanatsu K. et al.Distribution of IgG subclasses in membranous nephropathy.Clin Exp Immunol. 1984; 58: 57-62PubMed Google Scholar, 16.Noel L.-H. Aucouturier P. Monterio R.C. et al.Glomerular and serum immunoglobin G subclasses in membranous nephropathy and anti-glomerular basement membrane nephritis.Clin Immunol Immunopathol. 1988; 46: 186-194Crossref PubMed Scopus (75) Google Scholar, 17.Imai H. Hamai K. Komatsuda A. et al.IgG subclasses in patients with membranoproliferative glomerulonephritis, membranous nephropathy, and lupus nephritis.Kidney Int. 1997; 51: 270-276Abstract Full Text PDF PubMed Scopus (176) Google Scholar, 18.Ohtani H. Wakui H. Okuyama S. et al.Distribution of glomerular IgG subclass deposits in malignancy-associated membranous nephropathy.Nephrol Dial Transplant. 2004; 19: 574-579Crossref PubMed Scopus (155) Google Scholar In these 12 patients, however, only 45% showed positivity for the IgG4 subclass in glomeruli. If other antigen–antibody pairs are identified, which have a role in the development of MN, the definition of primary and secondary MN might be modified to add the information of the antigen–antibody pair. Until this is clear, we suggest that patients with either enhanced PLA2R in glomeruli or elevated PLA2R-AB in the serum would have a primary MN. In this prospective study, 88 consecutive patients with the histologic diagnosis MN were studied. Patients were eligible to participate if the diagnosis of MN was confirmed by a renal biopsy and if they had not received any immunosuppressive treatment before renal biopsy and PLA2R-AB measurement. The study was conducted in accordance with the ethical principles stated by the Declaration of Helsinki Principles. An informed consent was obtained from all participating patients, and the study was approved by our local ethics committee. MN was diagnosed by renal biopsy at the Nierenregister Hamburg, Universitätsklinikum Hamburg-Eppendorf (Hamburg, Germany). Diagnosis was made on the basis of light microscopy, immunohistochemistry, and electron microscopy findings (see Figure 1). All patients underwent a screening for secondary causes of MN, including a detailed medical history consisting of medication, physical examination, serological analysis (i.e., for systemic lupus erythematodes, hepatitis B, hepatitis C and HIV), and the exclusion or presence of malignant diseases as intensive as possible. Measurement of PLA2R-AB and detection of subtype-specific IgG antibodies in the serum were performed with an indirect immunofluorescence test as described.9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar For detection of subtype-specific IgG antibodies, fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-human IgG1 (Sigma-Aldrich Chemie GmbH, München, Germany, product no. F0767), IgG2 (Sigma-Aldrich, product no. F4516), IgG3 (Sigma-Aldrich, product no. F4641), and IgG 4 (Sigma-Aldrich, product no. F9890) antibodies from mouse were used. In all patients, in addition to standard histology, kidney biopsies were immunohistochemically stained with a rabbit polyclonal anti-PLA2R–AB (Atlas Antibodies AB, Stockholm, Sweden). Staining for PLA2R, IgG1, and IgG4 was performed on formalin-fixed paraffin-embedded renal biopsies. For PLA2R staining, 2-μm-thick sections were deparaffinized, hydrated, and subjected to 15min of autoclave heating at 120°C. The POLAP-AP system (Zytomed Systems GmbH, Berlin, Germany) was used for detection of the primary antibody (Atlas Antibodies). For IgG1 and IgG4 staining, the primary antibodies (Dianova and Binding Site, respectively) were detected by the APAAP system (Progen, Biotechnik GmbH, Heidelberg, Germany) after pretreatment with protease (Sigma P 8038, type XXIV, Sigma-Aldrich) in 2-μm-thick sections. For descriptive statistics, the data are presented as the means (±s.d.). The correlation between proteinuria and PLA2R-AB levels was analyzed by Spearman's rank coefficient of correlation. The differences were considered significant with a P-value <0.05. Total RNA was isolated from five 8-μm-thick sections of formalin-fixed paraffin-embedded kidney biopsies of patients with MN and enhanced PLA2R staining in kidney biopsies and patients with MN and non-enhanced PLA2R staining in kidney biopsies using the High Pure FFPE RNA Micro Kit (Roche, Deutschland Holding GmbH, Grenzach-Wyhlen, Germany). RNA (0.1μg) served as template for complementary DNA synthesis using 100ng/μl Random Hexamer Primer (Invitrogen, Darmstadt, Germany), 10mmol/l deoxyribonucleotide triphosphates (Invitrogen), and 200U RevertAid Reverse Transcriptase and an appropriate buffer (both Fermentas GmbH, St Leon-Rot, Germany). For complementary DNA synthesis, RNA and Primer were incubated for 5min at 65°C. Thereafter, 15μl enzyme–buffer–dNTP mix was added up to a total volume of 20μl, and samples were incubated for 10min at 25°C, 60min at 42°C, and finally for 10min at 70°C. The relative expression of PLA2R was related to the expression of podocin as the reference gene. For analysis, the TaqMan gene expression assays Hs00234853_m1 (PLA2R) and Hs00922492_m1 (podocin; Applied Biosystems, Darmstadt, Germany) were used. As both assays contain intron-spanning primers, a detection of genomic DNA could be avoided. All samples were measured in triplicates. Real-time PCR was performed according to the protocol provided by the manufacturer. A biochip coated with HEK 293 cells expressing the PLA2R protein was used as described before.9.Hoxha E. Harendza S. Zahner G. et al.An immunofluorescence test for phospholipase-A2-receptor antibodies and its clinical usefulness in patients with membranous glomerulonephritis.Nephrol Dial Transplant. 2011; 26: 2526-2532Crossref PubMed Scopus (216) Google Scholar,19.Stahl R.A. Hoxha E. Fechner K. PLA2R autoantibodies and recurrent membranous nephropathy after transplantation.N Engl J Med. 2010; 363: 496-498Crossref PubMed Scopus (117) Google Scholar In the first step, the biochip was incubated with a 1:10 dilution of a human serum with very high levels of PLA2R-AB (titer 1:10.000). In the second step, the biochip was incubated with the rabbit polyclonal anti-PLA2R-AB, which was used for the immunohistochemistry (Figure 4a). A biochip incubated only with the rabbit polyclonal antibody against PLA2R, but without human serum containing PLA2R-AB, was used as a control (Figure 4b). Both biochips were developed with donkey FITC-conjugated anti-rabbit IgG antibodies. These FITC-conjugated anti-rabbit-IgG antibodies from donkey do not bind to human PLA2R-AB as shown in Figure 4c. To verify the binding of the human PLA2R-AB from the serum to PLA2R, these ABs were detected with an FITC-conjugated anti-human IgG antibody from goat (Figure 4d). We thank Eugen Kinzler, Daniela Bergleiter, and Catharina Fürstenau for their technical and administrative assistance. The following colleagues were involved in the recruitment of the patients in this study (in alphabetical order): Aedtner, Frank (Halberstadt); Altrogge, Hans (Hamburg); Amir-Kabirian, Djahangir (Hamburg); Arndt, Liane (Buchholz); Arnold, Paul (Siegen); Assenmacher, Achim (Kamp-Lintfort); Beckermann, Johannes (Vechta); Beckmann, Marie-Luise (Bocholt); Biernat, Sabine (Varel); Bohling, Margot (Wilhelmshaven); Bokemeyer, Dirk (Bochum); Bozkurt, Fuan (Daun); Bramstedt, Jörn (Bremerhaven); Brunkhorst, Reinhard (Hannover); Claus, Manfred (Essen); Dannemann, Ernst-Gerhard (Gelsenkirchen); Daul, Anton, (Mülheim an der Ruhr); David-Walek, Tilman (Kiel); Dellanna, Frank (Düsseldorf); Dose, Ulrich (Siegburg); Duvigneau, Daniel (Hamburg); Feyerabend, Gotthard (Reinbek); Fricke, Lutz (Bochum); Fricke, Lutz (Lübeck); Fritz, Heribert (Würselen); Gerhardt, Thomas (Bonn); Gladziwa, Ulrich, (Würselen); Gööck, Thomas (Arnstadt); Grosser, Sebastian (Hamburg); Hamadeh, Andy (Höxter); Heering, Peter (Solingen); Heidenreich, Stefan (Aachen); Hintzen-Kruse, Christiane (Chemnitz); Hochtritt, Marc Sören (Celle); Hollenbeck, Markus (Bottrop); Jabs, Wolfram (Berlin); Jacobson, Jewgeni (Dortmund); Kahlke, Dominik (Hamburg); Keller, Frieder (Ulm); Kleinecke, Rudolf (Bamberg); Koch, Michael (Velbert); Kohnle, Matthias (Mettmann); Kühns, Achim (Hamburg); Kunigk, Falk (Pinneberg); Lange, Doris (Heilbad Heiligenstadt); Meier-Sundhaußen, Gregor (Berlin-Marzahn); Meyer, Wiebke (Buxtehude); Meyer, Tobias (Hamburg); Müller, Ralf (Westerstede); Nöthen, Wolfgang (Siegburg); Özcan, Fedai (Dortmund); Pfalzer, Benjamin (Hamburg); Plöger, Angela (Bielefeld); Potratz, Jürgen (Rotenburg, Wümme); Rensinghoff, Eberhard (Bochum); Roch, Peter (Regensburg); Schaumann, Dirk (Hameln); Schilken, Peter (Paderborn); Schlee, Hendrik (Weißenfels); Schnegelsberg, Olaf (Buchholz); Schneidenbach, Rolf (Hamburg); Schneider, André (Neuwied); Schnitzler, Andreas (Lüneburg); Seyfried, Joachim (Pforzheim); Stahn, Andreas (Hamburg); Steinhauer, Hjalmar Bernulf (Cottbus); Toussaint, Kai (Hamburg); Treiber, Wolfgang (Neuwied); Tripps, Christine (Halberstadt); Tröster, Sibille (Westerstede); Vischedyk, Martin (Paderborn); Voßkühler, Andre (Bottrop); Weber, Michael (Bovenden); Weiß, Martin (Hamburg); Wolf, Gunter (Jena); Wollweber, Thomas (Wadersloh). This work is supported by the ‘Deutsche Forschungsgemeinschaft’ KFO 228 (Z project (EH) and STA193/9-1) and the Else-Kroener-Fresenius Foundation. Table S1. The clinical characteristics of the patients are included in this table. Supplementary material is linked to the online version of the paper at http://www.nature.com/ki Download .doc (.17 MB) Help with doc files Supplementary Table
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