Anti-Phospholipase A2 Receptor Antibody in Membranous Nephropathy

CLINICAL RESEARCH www.jasn.org

Weisong Qin,* Laurence H. Beck, Jr.,† Caihong Zeng,* Zhaohong Chen,* Shijun Li,* Ke Zuo,* David J. Salant,† and Zhihong Liu*

*Research Institute of Nephrology, Jinling Hospital, Nanjing University School of Medicine, Nanjing, China; and †Boston University Medical Center, Boston, Massachusetts

ABSTRACT 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.

J Am Soc Nephrol 22: 1137–1143, 2011. doi: 10.1681/ASN.2010090967

Membranous nephropathy (MN) is a common nephropathy. This discovery proved that an antigen cause of idiopathic nephrotic syndrome in adults.1,2 on human podocytes could serve as the target for The formation of subepithelial immune deposits podocytopathic antibodies.14,15 In 2009, the M-type and complement activation result in alterations of phospholipase A2 receptor (PLA2R) was identified the basement membrane structure and damage to as the major target podocyte antigen involved in the filtration barrier, causing proteinuria.3,4 adult autoimmune idiopathic membranous ne- It has become clear that binding of circulating phropathy.16,17 The initial study by Beck and col- autoantibodies to target antigens on the podocyte leagues showed a 70% positivity rate in a cohort of initiates the disease process. In the past half century, American idiopathic MN patients, consisting pre- a significant amount of effort has been put into dominantly of Caucasians and African Americans. identifying the target antigens of idiopathic MN.5–7 In this study, we explored the prevalence of au- Studies of MN in a rat model (Heymann’s nephritis) established that the subepithelial immune deposits consisting of the target antigen, megalin, Received September 19, 2010. Accepted January 16, 2011. with circulating antimegalin antibodies are Published online ahead of print. Publication date available at formed in situ.8–12 However, megalin is not ex- www.jasn.org. pressed on human podocytes and is not the target Correspondence: Dr. Zhihong Liu, Research Institute of Ne- 13 phrology, Jinling Hospital, Nanjing University School of Med- antigen in human disease. icine, 305 East Zhongshan Road, Nanjing 210002, China. Tele- In 2002, neutral endopeptidase (NEP) was iden- phone: 86-25-80860218; Fax: 86-25-84801992; E-mail: tified as the responsible antigen in a rare subset of [email protected] infants with alloimmune antenatal membranous Copyright © 2011 by the American Society of Nephrology

J Am Soc Nephrol 22: 1137–1143, 2011 ISSN : 1046-6673/2206-1137 1137 CLINICAL RESEARCH www.jasn.org

toantibodies against PLA2R in Chinese patients with idiopathic MN. Typical secondary MN cases including lupus MN, HBV-associated MN, and tumor-associated MN were chosen to confirm the specificity of anti-PLA2R for idiopathic MN.

RESULTS

Detection of Anti-PLA2R in Idiopathic MN Anti-PLA2R autoantibodies in serum samples from 60 Chi- nese patients with idiopathic MN and Ͼ3.5 g/24 h proteinuria were examined by Western blotting using human glomerular extract (HGE; a source of native glomerular PLA2R) as the substrate. Serum samples from 49 patients (81.7%) were reactive with PLA2R, which was visualized as a 185-kD protein band. All these serum samples also recognized cell-expressed recombinant human PLA2R, which appears smaller, likely be- Figure 2. Idiopathic MN sera react with glycosylated and cause of incomplete glycosylation in vitro (Figure 1). No other deglycosylated PLA2R. Native PLA2R in HGE is heavily glyco- reactive bands were observed. As previously reported,16 IgG4 sylated and treatment with peptide N-glycosidase F (PNGaseF) was the predominant anti-PLA2R reactive subclass; however, caused a downward shift to approximately 145 kD. The anti- there was weaker reactivity with the other IgG subclasses in PLA2R antibodies from patients IMN1, IMN3, and IMN4 rec- most cases. ognized the deglycosylated as well as the native PLA2R. M. Protein standard (kD). PLA2R is heavily glycosylated and treatment with peptide N-glycosidase F causes a shift in its mobility to approximately 145 kD.16,18 As previously shown in the American cohort,16 the sample was decreased to 1:10, the HRP-labeled secondary an- anti-PLA2R antibody in Chinese idiopathic MN patients could tibodies were increased to 1:3000, and the exposure time was also recognize the deglycosylated PLA2R (Figure 2). increased to 10 minutes. Low titers of anti-PLA2R could be To determine if the remaining 11 patients had any evidence detected in 10 of these 11 patients. The strength of the band was of anti-PLA2R autoantibodies below the level of detection of much weaker on Western blot than that of a representative our standard Western blot protocol, the dilution of serum positive serum (defined as such using the standard assay) diluted to 1:1000 (Figure 3). No low titer of anti-PLA2R was detected in healthy controls (n ϭ 20). We compared the intensity of glomerular immunostaining for IgG4 in five patients with high and five patients with low titers of IgG4 anti-PLA2R. Although IgG4 was present in the glomeruli of all anti-PLA2R–positive cases, the intensity did not correlate with the titer of circulating antibodies (Supple- mental Table 1). During the follow-up of 60 patients with idiopathic MN (Supplemental Table 2), one patient without anti-PLA2R went into complete remission after treatment for 3 months. In 10 patients with low titers of anti-PLA2R, 5 patients (50%) went into remission within 1 year of treatment and developed no relapse. In 49 patients with high titers of anti-PLA2R antibodies, 15 patients (30.6%) went into remission without relapse. The average time to remission (14.47 Ϯ 7.62 months) was lon- Figure 1. Western blot analysis of representative serum samples from ger than that of patients with low titers of anti-PLA2R (6.60 Ϯ patients with idiopathic MN demonstrates reactivity with native and 3.58 months). These results suggested that patients who had recombinant PLA2R. Extracts of HGE and recombinant human PLA2R low levels of anti-PLA2R antibodies were prone to developing (rPLA2R) were electrophoresed under nonreducing conditions and im- remission within a shorter period of time (P Ͻ 0.01). munoblotted with patient serum (IMN1 through IMN4) at 1:25 and detected with anti-human IgG4. IMN1, IMN3, and IMN4 recognize PLA2R in HGE and the smaller cell-expressed rPLA2R. The recognition Anti-PLA2R in Idiopathic MN after Remission was confirmed using commercial polyclonal anti-PLA2R antibody. IMN2 Anti-PLA2R reactivity was examined in serum specimens col- in this figure is negative. No other reactive bands were observed. M. lected from 21 additional idiopathic MN patients who had Protein standard (kD). entered a clinical remission after treatment. Anti-PLA2R was

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Table 1. Anti-PLA2R in Chinese patients with membranous nephropathy n Anti-PLA2R % Idiopathic MN 60 49 81.7% Lupus MN 20 1 5.0% HBV-associated MN 16 1 6.3% Tumor-associated MN 10 3 30%

If we assume a similar proportion (Ͼ80%) of these patients were anti-PLA2R–positive at the time they were initially nephrotic, these results suggest that the presence of anti-PLA2R in idiopathic MN patients is associated with the disease activity and, conversely, that the autoantibodies decrease or disappear in remission (Figure 4). Two of the patients with high titers of anti-PLA2R antibodies relapsed 6 months after remission.

Anti-PLA2R in Secondary MN Figure 3. Increasing the sensitivity of the western blot assay reveals Although anti-PLA2R could be detected in most of the patients low titers of anti-PLA2R in initially negative idiopathic MN sera. Repre- with idiopathic MN, anti-PLA2R was largely negative in pa- sentative examples of immunoblots of HGE with two idiopathic MN tients with secondary causes of MN including lupus, HBV, and sera that were negative under standard conditions (IMN2 and IMN8) tumor-associated MN (Table 1, Figure 5). but became positive when the dilution of serum sample was reduced to 1:10 and the exposure time was prolonged to 10 minutes. PC is a MN In the serum samples from 20 patients with lupus MN, only serum that was positive under standard conditions and is diluted to one sample contained anti-PLA2R antibodies. No low titers of 1:1000 for this experiment. The downward shift in the size of the anti-PLA2R could subsequently be detected in the negative reactive band could be observed after PNGaseF treatment. HC, healthy patients using our more sensitive Western blot protocol. Sim- control, serum from healthy adult (1:10). M. Protein standard (kD). ilarly, only 1 of 16 HBV-associated MN samples was found to be positive. Low titers of anti-PLA2R were detected in 2 of the negative in 17 (81.0%) patients using the standard method. 15 HBV-associated MN samples initially considered negative Low titers of anti-PLA2R were detected in 7 patients using the by the standard method. IgG4 was the predominant PLA2R- enhanced sensitivity assay (Supplemental Table 3). However, it reactive subclass in these cases, whereas testing for reactivity is not known how many of these 21 were positive to start with. with IgG1, IgG2, and IgG3 did not disclose any additional cases of secondary MN with anti-PLA2R. Nor did we observe any other bands. Interestingly, obvious IgG4 immunostaining in glomeruli was observed in the lupus MN and HBV-associated MN patients that had detectable circulating anti-PLA2R anti-

Figure 4. The prevalence of anti-PLA2R in idiopathic MN (IMN) in remission is low by comparison to IMN patients with nephrotic syn- Figure 5. Anti-PLA2R is detected infrequently in secondary MN. drome. The prevalence of anti-PLA2R seropositivity was compared in The dark bars represent the prevalence of anti-PLA2R detected in the 60 patients with nephrotic syndrome to 21 additional patients in patients with idiopathic (IMN), lupus-associated (LMN), hepatitis remission (see Supplemental Tables 2 and 3 for details), using the B—associated (HBV-MN), and cancer-associated (Ca-MN) mem- standard assay. branous nephropathy, using the standard Western blot assay.

J Am Soc Nephrol 22: 1137–1143, 2011 Anti-PLA2R in Chinese MN 1139 CLINICAL RESEARCH www.jasn.org bodies, including the two patients with low titers of anti- cer. Moderate IgG4 deposition in glomeruli was observed in 2 PLA2R. However, there was no IgG4 immunostaining in those of 3 patients with circulating anti-PLA2R antibodies (with patients without anti-PLA2R antibodies (Figure 6). During the trace deposition in the third), but the staining for IgG4 in follow-up, none of the HBV-MN patients with anti-PLA2R glomeruli was negative in the 7 patients without circulating (including the two with low titers) entered into remission after anti-PLA2R antibodies (Figure 6). antiviral therapy, whereas most of the other patients without The clinical data for the 10 tumor-associated MN patients anti-PLA2R developed complete remission after such treat- are listed in Supplemental Table 6. Proteinuria manifested be- ment (Supplemental Table 5). fore or at the time of diagnosis of malignancy in 9 patients. The A higher proportion of the 10 patients with MN associated tumor was asymptomatic and recognized by systematic diag- with solid tumors were found to have anti-PLA2R antibodies. nostic procedures triggered by the diagnosis of MN. In one Anti-PLA2R was negative in 7 patients but positive in the other patient, MN was diagnosed 6 months after the diagnosis of 3 patients. No low titers of anti-PLA2R were further detected in lung cancer. During follow-up, all three patients with anti- the negative patients. Interestingly, the strength of the IgG4 PLA2R autoantibodies had persistent or relapse of proteinuria signal by immunofluorescence in the immune deposits was despite resection of the tumor. In those patients without anti- quite different between these two groups of patients with can- PLA2R autoantibodies, complete remission of the proteinuria was seen in two patients after tumor resection; three patients died of cancer; the remaining two patients had persistent proteinuria without tumor resection.

DISCUSSION

Membranous nephropathy is one of the most common glomerulopathies worldwide. We set out to determine the sensitivity and specificity of circulating anti-PLA2R autoantibodies in a Chinese cohort with MN. In this study, we determined the sensitivity of anti-PLA2R positivity in Chinese idiopathic MN. Typical secondary MN cases were chosen to address the specificity of anti-PLA2R. Our results show that using the standard Western blot protocol as described by Beck and colleagues, 49 of 60 (81.7%) biopsy- proven Chinese idiopathic MN patients had anti-PLA2R antibodies of the IgG4 subclass, the predominant Ig subclass in glomerular deposits.19–21 For the remaining idiopathic MN serum samples from the 11 patients in whom anti-PLA2R was undetectable by the standard method, the sensitivity of the immunoblot assay was enhanced by increasing the concentration of patient serum and HRP-labeled secondary antibody and prolonging the exposure time on films. Very low levels of anti-PLA2R antibodies were detected in 10 samples. These results indicated that anti- PLA2R autoantibody existed in nearly all of the 60 idiopathic MN patients. Then why was the low level of anti-PLA2R in these idiopathic MN patients not associated with a similarly low level of proteinuria? It is likely that the presence of antibodies does not perfectly parallel the disease activity during the clinical course, such that the presence of proteinuria in MN does not necessar- Figure 6. Staining for IgG4 deposition in glomeruli of secondary MN ily imply immunological activity. There was no significant dif- patients distinguishes those with circulating anti-PLA2R from anti- ference on staining of IgG4 in biopsies between patients with PLA2R-negative cases. (A,C,E) Negative immunostaining for IgG4 in glomeruli of patients with lupus-MN (A), HBV-MN (C), and tumor-asso- high titers of anti-PLA2R antibodies and low titers of anti- ciated MN (E) but without circulating anti-PLA2R antibodies. (B,D,F) PLA2R antibodies. There might have been an absence or sig- Bright IgG4 immunostaining in glomeruli of patients with lupus-MN (B), nificant attenuation of immunological activity at the time of HBV-MN (D), and tumor-associated MN (F) whose circulating anti- serum collection despite the requirement for Ͼ3.5 g/24 h pro- PLA2R antibodies were positive. teinuria in our cohort.16,22 Residual proteinuria in these pa-

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tients is possibly due to altered architecture of the filtration glomeruli was negative in the patients without circulating anti- barrier that results from remodeling of the glomerular base- PLA2R antibodies. The clinical follow-up of these two groups ment membrane in the progress of disease. It was demon- of patients also showed important differences. Those patients strated in the rat model of membranous nephropathy that pro- with anti-PLA2R autoantibodies had persistent proteinuria or teinuria did not necessarily imply the continued active relapse of proteinuria after resection of the tumor, suggesting formation and in situ deposition of immune complexes be- that the two processes were not causally related. In contrast, in cause irreversible structural changes in the glomerular capil- several patients without anti-PLA2R autoantibodies, complete lary wall and immune deposits can persist even in the absence remission of the proteinuria could indeed be observed after of circulating autoantibody to megalin.23,24 tumor resection and, in others, the proteinuria persisted in the On the other hand, anti-PLA2R antibodies were mostly absence of tumor resection. Additionally, Ͼ50% of the idio- negative or at low levels in idiopathic MN with remission after pathic MN patients were aged 40 to 60 years, which is also an treatment. Our results showed that 10 of 21 such patients were age group with a high frequency of tumors.29–31 Therefore, it is negative whereas 7 others had only low titers of anti-PLA2R. highly possible that coincidence of idiopathic MN and cancer These results suggest that the presence of high-titer anti- occurred in those tumor-associated MN patients with anti- PLA2R in idiopathic MN patients is associated with the disease PLA2R antibodies. More studies in this area are needed to reactivity and that a significant portion of individuals have lost solve this question. anti-PLA2R reactivity when they are in clinical remission. Additionally, the autoimmune process may also target However, the patients in remission were prone to relapse if more than one antigen in the initial or later phases of the dis- their anti-PLA2R antibodies were in high titers. ease.32 Recently, there have been other target antigens reported The formation of subepithelial immune complexes is in idiopathic MN.33,34 However, in our research no other con- thought to occur by the following potential mechanisms. Sub- sistent bands were observed during the detection of the anti- epithelial immune complexes may be formed in situ through PLA2R antibody, perhaps because of technical limitations of the reaction of circulating autoantibody to a native podocyte our assay. There also may be cryptic epitopes that were not antigen.25,26 Antibodies in the circulation may secondarily in- revealed under our extraction and electrophoretic conditions. teract with non-native antigens artificially planted in the sub- It is also possible that the antibodies to the intracellular en- epithelial space because of a biophysical or immunologic affin- zymes reported to date are the consequence of podocyte dam- ity for GBM structural elements.17,27,28 The high prevalence of age by anti-PLA2R and may contribute to ongoing injury.33,34 anti-PLA2R antibody that has been demonstrated in idio- In conclusion, our research suggests that anti-PLA2R is a pathic MN and the fact that PLA2R is expressed by the podo- specific marker of idiopathic MN in Chinese patients. It also cyte suggest a process in which the in situ subepithelial im- indicates that there is a common pathophysiology among pa- mune complex deposits mechanism is involved.16 Typical tients with idiopathic MN from the three population groups secondary MN cases, which may exhibit different pathophysi- studied to date: those of Caucasian, African, and Han Chinese ologic mechanisms of immune complex deposition, were used ancestry. Establishment of anti-PLA2R as a sensitive bio- to explore the specificity of anti-PLA2R antibody for idio- marker may provide new tools for diagnosis, disease activity pathic MN. Anti-PLA2R antibody was negative in most cases monitoring, and design of new antigen-driven therapeutic of secondary MN except in one patient with lupus and one with strategies in MN. HBV. Interestingly, IgG4 immunostaining in glomeruli could be observed in lupus MN and HBV-associated MN patients with high- or low-titer anti-PLA2R antibodies whereas there CONCISE METHODS was no IgG4 immunostaining in those patients without anti- PLA2R antibodies. Furthermore, HBV-associated MN pa- Patient Selection tients with either high- or low-titer anti-PLA2R antibodies did Membranous nephropathy in all cases was diagnosed by renal biopsy not enter remission after antiviral treatment whereas most of at the Research Institute of Nephrology, Nanjing University School of the other patients without anti-PLA2R developed complete re- Medicine (Nanjing, China). All patients including primary and sec- mission after antiviral therapy. Given the high prevalence of ondary MN had Ͼ3.5 g/24 h proteinuria. HBV disease in China, it is highly possible that these few pa- The diagnosis of idiopathic MN was established by the following tients represent a coincidental occurrence of the two disease criteria. Urinary protein was Ͼ3.5 g/24 h (Supplemental Table 2). processes. Immune and electron-dense deposits were present exclusively in a Anti-PLA2R antibody was also negative in tumor-associ- subepithelial location, with an absence of deposition in subendothe- ated MN except in three MN patients with cancer of lung, lial or mesangial regions. No glomerular infiltrating cells or prolifer- larynx, and stomach, respectively. It is very interesting that the ation of mesangial and endothelial cells were present. Immunofluo- degree of IgG4 deposition in glomeruli of these two groups is rescence demonstrated that IgG was deposited in a granular pattern quite different. In the few anti-PLA2R–positive patients with along the glomerular capillary walls in association with C3. No pa- tumor-associated MN, moderate immunostaining of IgG4 in tients had evidence of secondary features, such as antinuclear anti- glomeruli could be observed. However, the IgG4 deposition in bodies, anti–double-stranded DNA, antinuclear ribonucleoprotein,

J Am Soc Nephrol 22: 1137–1143, 2011 Anti-PLA2R in Chinese MN 1141 CLINICAL RESEARCH www.jasn.org anti-Smith, anti-Ro (SSA), anti-La (SSB), antitopoisomerase I (anti– tions and transferred to nitrocellulose membranes. Membranes were Scl-70), anti–Jo-1 antibodies, anticardiolipid antibodies, antithyro- blocked with 5% milk and then incubated with human serum at a globulin antibodies, serum HBV antigens and antibodies, HCV anti- dilution of 1:25. Sheep antibodies against human IgG4 (1:3000, Bind- bodies, HCV RNA, tumor markers, or exposure to toxic agents. ing Site) were used as a secondary antibody. Donkey anti-sheep per- Lupus MN was diagnosed on the basis of clinical diagnosis of SLE oxidase-conjugated antibody was used as the detecting antibody at with at least four American Rheumatic Association criteria (Supple- 1:5000 (Jackson ImmunoResearch). A commercial antibody to hu- mental Table 4). Renal pathology showed a membranous pattern of man PLA2R (Sigma-Aldrich) was used at 1:400 to confirm the posi- injury without mesangial hypercellularity or endocellularity. Immu- tion of the PLA2R bands. nofluorescence showed primarily subepithelial granular deposits. Blots were incubated in chemiluminescent substrate (100 mM Urinary protein was Ͼ3.5 g/24 h and urinary red blood cells were less Tris, pH 8.5, 250 mM Luminol, 90 mM p-coumaric acid, hydrogen than 1 ϫ 106 per ml. peroxide) for 3 minutes and exposed to HyBlot CL autoradiography HBV-associated MN was diagnosed by the following criteria. Hep- film (Denville Scienctific, Inc.). The exposure times were typically 10 atitis B serology tests and HBV DNA results were positive (Supple- to 30 seconds for positive bands and up to 10 minutes for weak or mental Table 5). HBeAg staining was positive on frozen sections. Uri- negative bands. The film was developed using a Kodak X-OMAT nary protein was initially Ͼ3.5 g/24 h. 2000A processor. Tumor-associated MN was diagnosed in 10 patients at the time of kidney biopsy. Most of these patients presented with proteinuria Deposits of IgG4 in Glomeruli by Immunofluorescence (Ͼ3.5 g/24 h) (Supplemental Table 6). Various types of solid tumors Cryosections of kidney biopsy specimens were prepared. The slides were present, including cancer of lung (five cases), stomach (two were blocked with 10% fetal calf serum. Mouse anti-human IgG4 cases), colon (one case), larynx (one case), and tongue (one case). monoclonal antibody (Sigma-Aldrich), diluted at 1:400, was used as the primary antibody and incubated for 2 hours. After the slides were Serum Samples rinsed with PBS for 5 minutes, the FITC-labeled rabbit anti-mouse Serum samples were collected from patients with idiopathic MN (n ϭ IgG secondary antibody (1:50; DAKO) was added and incubated for 60), lupus MN (n ϭ 20), HBV-MN (n ϭ 16), and tumor associated 30 minutes. The slides were dried and sealed with glycerin for obser- MN (n ϭ 10), which were approved by the institutional review board vation by a fluorescence microscope. of Jinling Hospital. Serum samples from 21 idiopathic MN patients in Ͻ remission (urinary protein 1.0 g/24 h) after treatment were also Statistical Analysis collected (Supplemental Table 3). Serum samples from 20 healthy Data are presented as mean Ϯ SEM. The t test was used for compar- volunteers were used as controls (Supplemental Table 7). isons between two groups.

Human Glomerular Extracts (HGEs) Kidneys from deceased donors, which were unsuitable for transplan- ACKNOWLEDGMENTS tation, were obtained from the New England Organ Bank. HGEs were prepared as previously reported.16 Briefly, glomeruli were collected This work was carried out in the laboratory of Drs. David Salant and using a series of graded sieves and resuspended in an equal volume of Laurence Beck, Jr., at Boston University Medical Center and was sup- 100 mM Tris (pH 8, 1 mM MgCl ) and freeze-thawed at Ϫ80°C. An 2 ported by NIH grant DK 30932 (to D.J.S.) and an award from the equal volume of RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM ASN/Halpin Foundation (to L.H.B.). The authors would especially NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS; Boston Bio- like to thank Dr. Ge´rard Lambeau (Institut de Pharmacologie Products) was added with Protease Inhibitor Cocktail Set I (Calbio- Mole´culaire et Cellulaire, Valbonne, France) for providing the full- chem/EMD Chemicals, Inc.) and homogenized on ice. RIPA-insolu- length human PLA2R cDNA used in these studies. ble debris was removed by centrifugation. Contaminating human IgG was removed with Immobilized Protein G Plus (Fisher Scientific). N-linked carbohydrates were removed with peptide N-glycosidase DISCLOSURES F (PNGase F; New England Biolabs) in the absence of reducing agent. L.H.B. and D.J.S. report having a patent pending for a diagnostic immuno- assay to detect anti-PLA2R autoantibodies in membranous nephropathy. Extracts of HEK Cells Expressing Recombinant PLA2R L.H.B. and D.J.S. have received consulting fees and research support from A plasmid vector containing full-length human PLA2R under the Questcor Pharmaceuticals, Inc. D.J.S. has received consulting fees from Tali- control of the cytomegalovirus promoter was transiently transfected gen Therapeutics, Inc. into HEK293T cells. Cells were collected after culture and lysed with Tris buffer plus protease inhibitors. Insoluble cell debris was dis- carded after centrifugation. REFERENCES

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J Am Soc Nephrol 22: 1137–1143, 2011 Anti-PLA2R in Chinese MN 1143 Supplementary Table 1 Comparison of glomerular IgG4 staining in patients with high and low titers of anti-PLA2R antibodies

Months Glomerular IgG4 Cr Alb Urine Sample Age Sex before Diagnosis staining Anti-PLA2R mg/dl g/L protein biopsy ( Luminosity units)

IMN4 38 M 0.9 29 4.93 1 Idiopathic MN 52.37±11.11 High titer IMN53 69 M 0.98 21.9 6.93 3 Idiopathic MN 47.58±10.87 High titer

IMN55 53 M 0.65 20.6 5.57 6 Idiopathic MN 64.02±11.78 High titer

IMN58 34 M 0.75 22 8.75 4 Idiopathic MN 56.22±9.55 High titer

IMN60 60 M 0.8 23.7 7.56 26 Idiopathic MN 43.82±12.17 High titer

IMN17 27 M 0.73 15.2 13.4 4 Idiopathic MN 50.20±9.53 Low titer

IMN20 31 F 0.67 23.2 3.51 3 Idiopathic MN 56.33±11.60 Low titer

IMN29 64 F 0.7 28.1 4.16 36 Idiopathic MN 46.92±10.10 Low titer

IMN54 29 F 0.68 30.9 4.37 2 Idiopathic MN 61.57±8.61 Low titer

IMN59 44 M 0.92 22.8 3.78 6 Idiopathic MN 39.55±7.42 Low titer

IMN, idiopathic membranous nephropathy. Numbers refer to the patient number with idiopathic MN. Cr, Serum creatinine. Alb, Serum albumin. Urine protein is presented as urine protein-to-creatinine ratio collected during 24 hours at the time point when biopsy was performed and serum sample was collected. Months before biopsy reflect the time in months between the initial proteinuria and renal biopsy.

Glomerular IgG4 staining: The sections were examined by epifluorescent microscopy that used a Nikon Pan Fluor lens. The images were captured with a Spot

CCD camera and exported into Adobe Photoshop. All exposure settings were kept constant for each group of kidneys. Fluorescence intensity was measured by outlining the perimeter of six glomeruli in each section and reading the luminosity from the Histogram command in Adobe Photoshop. Calibration of the CCD exposure time assured that the settings chosen were in the linear range and well below saturation. There was no difference of IgG4 immunostaining in glomeruli of patients with high titers and low titers of anti-PLA2R antibodies (P>0.05).

Supplementary Table 2 Clinical information for patients with idiopathic MN

Time Cr Alb Urine Treatment after Remission Sample Age Sex before Diagnosis stages Relapse Anti-PLA2R mg/dl g/L protein biopsy (mo) biopsy IMN1 64 M 2.5 24.8 4.18 5 Idiopathic MN NA CSA,TW CR(48) Yes POS

IMN3 40 F 0.45 25.1 5.22 18 Idiopathic MN NA CSA CR(26) No POS

IMN4 38 M 0.9 29 4.93 1 Idiopathic MN NA FK CR(7) Yes POS

IMN5 56 F 0.41 26.9 9.92 12 Idiopathic MN NA CSA CR(4) No POS

IMN6 53 M 0.8 24.1 4.48 3 Idiopathic MN NA CSA CR(10) No POS

IMN7 47 M 0.72 24.6 5.08 2 Idiopathic MN NA CTX CR(4) No POS

IMN9 25 M 0.91 23.5 6.38 6 Idiopathic MN NA CTX,TW CR(9) No POS

IMN10 40 M 0.75 19.8 7.43 1 Idiopathic MN Ⅱ CSA NR POS

IMN11 37 F 0.76 31.3 7.4 1 Idiopathic MN NA TW CR(10) No POS

IMN13 57 M 0.84 26.4 4.1 12 Idiopathic MN NA CTX CR(12) Yes POS

IMN16 50 M 0.85 14.2 4.48 2 Idiopathic MN Ⅰ-Ⅱ TW CR(4) No POS

IMN19 40 M 0.61 17.2 9.24 1 Idiopathic MN NA TW CR(14) NA POS

IMN21 24 M 1.06 20.8 6.77 17 Idiopathic MN Ⅱ CTX,TW PR(20) No POS IMN22 59 M 0.59 20.8 4.73 2 Idiopathic MN NA CTX NR POS

IMN23 61 F 0.78 21.8 6.89 9 Idiopathic MN NA TW CR(14) No POS

IMN24 72 M 1.4 21.9 8.24 3 Idiopathic MN NA TW NR POS

IMN25 59 M 1.5 31.2 3.51 10 Idiopathic MN NA TW CR(24) No POS

IMN26 18 F 0.4 17.4 6.5 4 Idiopathic MN Ⅱ CSA,TW PR(24) Yes POS

IMN27 49 M 3.92 37.2 6.5 12 Idiopathic MN Ⅲ TW CR(20) No POS

IMN28 47 M 0.92 20.8 11.27 1 Idiopathic MN Ⅱ TW NR POS

IMN30 59 M 0.84 27.9 9.89 2 Idiopathic MN NA TW NA POS

IMN31 77 M 0.89 24.9 8.49 NA Idiopathic MN NA TW NR POS

IMN32 71 M 0.66 31.3 5.11 1 Idiopathic MN NA TW CR(20) No POS

IMN33 66 M 0.65 21.2 6.01 1 Idiopathic MN NA TW NR POS

IMN34 22 M 0.6 23.8 10.96 5 Idiopathic MN NA TW CR(24) No POS

IMN35 54 M 0.57 29.6 4.23 5 Idiopathic MN NA TW NA POS

IMN36 42 M 0.79 26.7 3.72 6 Idiopathic MN Ⅰ-Ⅱ TW NR POS

IMN37 64 F 0.98 20 7.63 2 Idiopathic MN Ⅱ CTX,CSA CR(24) Yes POS

IMN38 62 M 0.8 26.9 9.52 3 Idiopathic MN Ⅱ CTX,MMF,TW NR POS IMN39 39 M 0.75 26.5 4.38 36 Idiopathic MN NA TW NR POS

IMN40 46 M 0.81 33 6.75 3 Idiopathic MN Ⅱ TW NR POS

IMN41 34 M 0.75 21.8 6.87 2 Idiopathic MN Ⅲ-4 CTX,TW NR POS

IMN42 42 M 0.98 26.4 4.41 1 Idiopathic MN Ⅱ CSA NA POS

IMN43 68 M 0.94 29.7 3.63 84 Idiopathic MN NA TW NA POS

IMN44 27 M 0.64 22.4 4.84 2 Idiopathic MN Ⅰ-Ⅱ TW CR(36) NA POS

IMN45 58 M 0.64 22.5 6.37 18 Idiopathic MN NA TW CR(12) NA POS

IMN46 39 M 0.83 30 7.47 3 Idiopathic MN Ⅲ-Ⅳ TW CR(12) NO POS

IMN47 39 M 0.90 23.9 3.98 1 Idiopathic MN NA TW NR POS

IMN48 39 F 0.58 33.5 3.99 5 Idiopathic MN Ⅱ TW CR(16) NO POS

IMN49 61 M 0.91 19.4 4.57 16 Idiopathic MN NA TW NR POS

IMN50 18 M 0.91 28.7 9.42 1 Idiopathic MN NA TW PR(12) Yes POS

IMN51 46 F 0.41 27.4 5.06 10 Idiopathic MN Ⅰ TW NR POS

IMN52 64 M 0.98 22.9 4.33 1 Idiopathic MN Ⅲ-Ⅳ TW NA POS

IMN53 69 M 0.98 21.9 6.93 3 Idiopathic MN NA TW NR POS

IMN55 53 M 0.65 20.6 5.57 6 Idiopathic MN NA TW NR POS IMN56 43 M 0.64 18.9 13.5 1 Idiopathic MN NA TW CR(18) NA POS

IMN57 67 F 0.72 23.5 3.61 5 Idiopathic MN NA TW NR POS

IMN58 34 M 0.75 22 8.75 4 Idiopathic MN Ⅱ TW NA POS

IMN60 60 M 0.8 23.7 7.56 26 Idiopathic MN NA CTX,TW NR POS

IMN18 18 M 0.88 19.7 5.07 2 Idiopathic MN Ⅱ TW CR(3) NA NEG

IMN2 29 M 0.74 37.3 4.8 6 Idiopathic MN Ⅱ MMF CR(4) No Low titer

IMN8 66 F 0.62 28.4 4.44 12 Idiopathic MN NA TW,CSA NR Low titer

IMN12 37 F 0.46 24.8 5.27 1 Idiopathic MN NA TW CR(3) No Low titer

IMN14 42 F 0.73 26.6 9.85 4 Idiopathic MN Ⅱ TW CR(24) Yes Low titer

IMN15 44 F 0.8 17.8 8.2 1 Idiopathic MN Ⅱ TW NA NA Low titer

IMN17 27 M 0.73 15.2 13.4 4 Idiopathic MN NA TW CR(4) Yes Low titer

IMN20 31 F 0.67 23.2 3.51 3 Idiopathic MN Ⅲ-Ⅳ FK CR(6) No Low titer

IMN29 64 F 0.7 28.1 4.16 36 Idiopathic MN NA TW NR Low titer

IMN54 29 F 0.68 30.9 4.37 2 Idiopathic MN Ⅱ TW CR(8) No Low titer

IMN59 44 M 0.92 22.8 3.78 6 Idiopathic MN Ⅱ TW CR(12) No Low titer

Months before biopsy reflect the time in months between the initial proteinuria and renal biopsy. Stages were determined by electron microscope observations.

NA, not available. Treatment indicates immunosuppressive therapy after the diagnosis of idiopathic MN based on biopsy. TW, Tripterygium wilfordii hook F.

CTX, cyclophosphamide. MMF, mycophenylate mofetil. FK, FK506(tacrolimus). CSA, cyclosporine. None, no immunosuppressive therapy. Most proteinuric patients were also being treated with angiotensin-converting enzyme inhibitors and/or angiotensin II receptor blockers, and diuretics. Remission time reflects the time in months after the diagnosis of idiopathic MN, either complete remission (CR) or partial remission (PR). Complete remission was defined by proteinuria <0.3 g/24hr and normalized serum albumin concentration at least for 1 month. Partial remission was defined as proteinuria less than 3.5 g/24 hr and 50% lower than baseline proteinuria. NR, no remission.

NEG: negative. POS, positive.

Supplementary Table 3 Clinical information for patients with idiopathic MN in remission Months Cr Alb Urine Remission Sample Age Sex before Stages Diagnosis Treatment Relapse Anti-PLA2R mg/dl g/L protein time(mo) biopsy MNR1 49 F 0.77 34 0.75 6 NA Idiopathic MN None 6 No NEG

MNR3 52 F 0.54 35.4 0.6 8 Ⅱ Idiopathic MN MMF,TW 10 No NEG

MNR4 34 F 0.46 40.9 0.44 4 Ⅱ-Ⅲ Idiopathic MN TW 4 No NEG

MNR5 28 M 0.74 38.8 0.6 NA NA Idiopathic MN TW 12 NA NEG

MNR7 49 F 0.62 31.6 0.38 4 Ⅰ Idiopathic MN TW 10 No NEG

MNR8 42 F 0.67 36.5 0.72 4 Ⅱ Idiopathic MN TW 12 NA NEG

MNR10 54 F 0.34 43.6 0.59 6 NA Idiopathic MN TW 5 No NEG

MNR12 41 M 0.75 47.9 0.53 2 NA Idiopathic MN NA 4 No NEG

MNR13 37 F 0.65 33.2 0.86 10 NA Idiopathic MN TW 6 No NEG

MNR17 38 F 0.49 31.1 0.64 5 Ⅰ-Ⅱ Idiopathic MN TW 5 No NEG

MNR2 67 M 0.96 38.1 0.56 2 NA Idiopathic MN TW 6 No Low titer

MNR9 40 M 0.78 32.1 0.63 NA NA Idiopathic MN TW 5 No Low titer

MNR14 23 F 0.46 30.1 0.84 5 NA Idiopathic MN NA 5 No Low titer MNR15 45 F 0.71 43.3 0.1 36 NA Idiopathic MN TW 6 No Low titer

MNR19 65 M 0.86 33.2 0.53 2 NA Idiopathic MN TW 12 NA Low titer

MNR21 53 F 1.07 32.6 0.77 24 Ⅱ Idiopathic MN TW 12 No Low titer

MNR22 32 F 0.55 35.9 0.86 5 NA Idiopathic MN NA 6 No Low titer

MNR6 37 F 0.58 31.4 0.23 1 Ⅱ Idiopathic MN TW 6 No POS

MNR11 43 M 0.8 34.6 0.77 6 Ⅱ Idiopathic MN FK,TW 6 Yes POS

MNR16 36 F 0.45 35.3 0.47 NA Ⅱ-Ⅲ Idiopathic MN TW 6 Yes POS

MNR23 47 M 0.78 39.5 0.41 12 NA Idiopathic MN TW 10 No POS

MNR, idiopathic membranous nephropathy in remission. Numbers refer to the patient number. Cr, Serum creatinine. Alb, Serum albumin.

Urine protein is presented as urine protein-to-creatinine ratio collected during 24 hours.

Months before biopsy reflect the time in months between the initial proteinuria and the date of first biopsy. Stages were determined by electron

microscope observations. NA, not available. Treatment indicates immunosuppressive therapy after the diagnosis of idiopathic MN based on biopsy. TW,

Tripterygium wilfordii hook F. MMF, mycophenylate mofetil. FK,FK506 (tacrolimus). None, no immunosuppressive therapy. Most proteinuric

patients were also being treated with angiotensin-converting enzyme inhibitors and/or angiotensin II receptor blockers, and diuretics. Remission time

reflect the time in months after the diagnosis of idiopathic MN.

NEG: negative. POS, positive.

Supplementary Table 4 Clinical information for patients with Lupus-MN Months Cr Alb Urine C3 C4 Sample Age Sex ANA Ads-DNA ACL before Treatment Anti-PLA2R mg/dl g/L protein g/L g/L biopsy LMN1 38 F 0.63 17.5 9.44 1:1024 NEG NEG 0.34 0.13 2 MMF NEG

LMN2 51 F 0.50 20.9 4.79 1:256 NEG NEG 0.45 0.25 3 TW NEG

LMN3 26 F 0.72 28.2 6.60 1:256 NEG NEG 1.04 0.23 96 NA NEG

LMN4 22 F 0.65 28 5.76 1:512 NEG + 0.56 0.22 2 NA NEG

LMN5 31 F 0.64 15.5 14.43 1:1024 + (1:10) + 0.38 0.11 15 TW NEG

LMN6 42 F 0.61 22.9 3.56 1:64 NEG NEG 0.92 0.11 84 NA NEG

LMN7 21 F 0.60 18.7 6.52 1:256 NEG NEG 0.80 0.10 5 FK NEG

LMN8 58 F 1.17 17.8 4.22 1:256 NEG + 0.77 0.09 8 NA NEG

LMN9 15 M 0.83 19.0 7.56 1:256 NEG NEG 0.84 0.25 6 FK,TW NEG

LMN10 16 F 0.46 21.2 4.79 1:256 NEG + 0.67 0.17 1 FK NEG

LMN11 37 F 0.57 27.9 5.62 1:512 NEG NEG 0.78 0.12 12 CTX NEG

LMN12 22 F 0.72 23.4 3.98 1:16 NEG NEG 0.43 0.22 48 NA NEG

LMN13 38 F 1.05 33.8 4.97 1:256 + (1:10) NEG 0.98 0.22 12 TW NEG LMN14 30 M 0.52 27.7 4.51 1:1024 + (1:10) NEG 0.27 0.05 7 TW NEG

LMN15 21 M 0.69 29.2 4.96 1:1024 + (1:10) NEG 0.45 0.08 1 CTX NEG

LMN16 65 F 1.02 22.6 4.39 1:128 NEG NEG 0.80 0.20 36 TW NEG

LMN17 29 F 1.46 15.7 4.73 1:512 NEG NEG 0.56 0.23 36 TW NEG

LMN18 13 M 0.98 20.8 5.73 1:512 NEG NEG 0.61 0.19 NA NA POS

LMN19 43 F 0.65 24.2 3.51 1:128 NEG + 0.75 0.21 8 TW NEG

LMN20 32 F 0.70 30.3 4.26 1:256 + (1:10) NEG 0.47 0.12 60 TW NEG

LMN, lupus membranous nephropathy. Numbers refer to the patient number. Cr, Serum creatinine. Alb, Serum albumin. Urine protein is presented as urine protein-to-creatinine ratio collected during 24 hours at the time point when biopsy was performed and serum sample was collected. ANA, antinuclear antibody. Ads-DNA, anti-double-stranded DNA. ACL, anticardiolipid antibody.

Months before biopsy reflect the time in months between the initial proteinuria and renal biopsy. Treatment indicates immunosuppressive therapy after the diagnosis of lupus membranous nephropathy. TW, Tripterygium wilfordii hook F. CTX, cyclophosphamide. MMF, mycophenylate mofetil. FK,FK506

(tacrolimus). CSA, cyclosporine. NA, not available. Most proteinuric patients were also being treated with angiotensin-converting enzyme inhibitors and/or angiotensin II receptor blockers, and diuretics.

NEG: negative. POS, positive. Supplementary Table 5 Relevant clinical information about patients with hepatitis B-associated MN

Hepatitis B serology tests Months Cr Alb Urine ALT Anti- Sample Age Sex before Treatment Remission mg/dl g/L protein HBSAg HBeAb HBeAg HBeAb HBcAb (IU/L) PLA2R biopsy HBMN1 37 M 0.62 23.4 4.18 + - - + + 81 NA Lamivudine CR NEG

HBMN2 43 F 0.79 25.6 8.06 - - + - + 90 NA Lamivudine NA NEG

Lamivudine, HBMN3 42 M 0.22 29.8 5.91 + - - + + 100 60 CR NEG TW HBMN4 41 M 0.85 20.6 6.30 + - - + + 86 3 Lamivudine CR NEG

Lamivudine, HBMN5 30 M 0.68 21.0 10.39 + - - + + 28 2 CR NEG TW HBMN6 23 M 0.79 15.2 6.20 + - + - + 23 6 Lamivudine CR NEG

HBMN7 8 M 0.31 21.7 4.13 + - + - + 22 1 IFN-alpha CR NEG

HBMN8 50 M 0.67 16.3 10.68 + - - + + 30 3 Lamivudine NR Low titer

HBMN9 29 M 0.52 17.6 4.70 + - + - + 152 8 Lamivudine CR NEG

Lamivudine, HBMN10 22 M 0.78 18.8 9.62 + - - + + 22 4 NR Low titer TW HBMN11 16 M 0.46 30.8 5.51 + - + - + 67 5 Lamivudine CR NEG

HBMN12 27 M 1.45 29.1 10.01 + - + - + 22 36 Lamivudine NR POS HBMN13 51 F 0.44 24.7 6.06 + - + - + 57 2 Lamivudine CR NEG

HBMN14 14 F 0.45 30.8 3.58 + - + - + 44 2 Telbivudine CR NEG

HBMN15 45 M 0.85 24.9 3.83 + - + - + 91 2 Entecavir CR NEG

HBMN16 37 M 0.80 26.6 4.67 + - + - + 65 1 Lamivudine NA NEG

HBMN, hepatitis B-associated MN. Numbers refer to the patient number. Cr, Serum creatinine. Alb, Serum albumin. Urine protein is presented as urine protein-to-creatinine ratio collected during 24 hours at the time point when biopsy was performed and serum sample was collected. ALT, Alanine aminotransferase.

Months before biopsy reflect the time in months between the initial proteinuria and renal biopsy.

Treatment indicates anti-viral therapy after the diagnosis of hepatitis B-associated MN based on biopsy. TW, Tripterygium wilfordii hook F. NA, not available. CR, complete remission, which was defined by proteinuria <0.3 g/24hr and normalized serum albumin concentration at least for 1 month. NR, no remission.

NEG: negative. POS, positive.

Supplementary Table 6 Relevant clinical information about patients with tumor-associated MN

Cr Alb Urine Time of Associated Type of Histology of Anti- Sample Age Sex Resection Remission Outcome C3 IgG1 IgG4 mg/dl g/L protein tumor therapies cancer cancer PLA2R

TUMN1 55 M 0.53 25.6 6.31 0 no none NR death lung Adenocarcinoma 2+ 2+ - NEG

TUMN3 47 F 0.55 23.7 7.06 0 surgery Chemotherapy NR death colon Adenocarcinoma 2+ 2+ - NEG clear-cell TUMN5 59 M 0.80 16.4 6.97 0 surgery TW CR alive tongue 2+ 2+ - NEG carcinoma Squamous cell TUMN7 40 M 1.13 42.4 1.72 6 surgery Chemotherapy CR alive lung 1+ 2+ - NEG carcinoma

TUMN8 54 M 0.58 28.5 3.14 0 surgery Chemotherapy NR death stomach Adenocarcinoma 1+ 2+ - NEG Squamous cell TUMN9 52 M 0.90 26.6 5.18 0 no Chemotherapy NR alive lung 2+ 2+ - NEG carcinoma Squamous cell TUMN10 53 M 0.62 22.3 3.94 0 no Chemotherapy NR alive lung 2+ 2+ - NEG carcinoma Chemotherapy, TUMN2 65 M 0.64 31.1 2.11 0 surgery NR alive stomach Adenocarcinoma 2+ 2+ 2+ POS TW Chemotherapy, Squamous cell TUMN4 56 M 0.90 27.9 3.86 0 surgery Relapse alive lung 2+ 2+ +/- POS TW carcinoma Chemotherapy, Squamous cell TUMN6 55 M 1.05 23.6 6.37 0 surgery Relapse alive larynx + 2+ 2+ POS TW carcinoma

TUMN, tumor-associated MN. Numbers refer to the patient number. Cr, Serum creatinine. Alb, Serum albumin. Urine protein is presented as urine protein-to-creatinine ratio collected during 24 hours at the time point when biopsy was performed and serum sample was collected.

Time of tumor reflects the time between the diagnosis of tumor and that of MN. In most of the patients, tumor was asymptomatic and recognized by systematic diagnostic procedures triggered by the diagnosis of MN. In one patient (TUMN7), MN was diagnosed 6 months after the diagnosis of lung cancer. Associated therapies, most patients using general chemotherapy protocol after surgery. TW, Tripterygium wilfordii hook F.

Time to remission reflect the time in months after the diagnosis of MN. CR,complete remission, which was defined by proteinuria <0.3 g/24hr and normalized serum albumin concentration at least for 1 month. NR, no remission. In 7 patients without anti-PLA2R autoantibodies, 2 patients went into complete remission of proteinuria after tumor remission, three patients died as a direct or indirect result of tumors; the other 2 patients remained alive but had persistent proteinuria without tumor resection. Relapse, two patients had complete remission of proteinuria after resection of their tumors, but relapse of proteinuria. C3,IgG1,IgG4 reflect the immunofluorescence staining in renal biopsies.

NEG, negative. POS,positive.

Supplementary Table 7 Information about healthy controls

Cr Alb Sample Age Sex Urine protein mg/dl g/L

HC1 22 M 0.67 43.9 NEG

HC2 57 M 0.74 40.3 NEG

HC3 59 F 0.79 41.2 NEG

HC4 70 M 0.76 39.2 NEG

HC5 57 F 0.68 39.6 NEG

HC6 18 F 0.69 47.7 NEG

HC7 46 M 0.77 42.3 NEG

HC8 45 M 0.79 46.9 NEG HC9 60 F 0.96 36.5 NEG HC10 55 M 0.74 39.2 NEG HC11 70 M 0.87 39.8 NEG HC12 59 F 0.64 46.4 NEG HC13 62 M 0.63 39.7 NEG HC14 25 F 0.69 47.4 NEG HC15 55 M 0.77 42.5 NEG HC16 40 M 0.72 44.8 NEG HC17 62 F 0.96 43.9 NEG HC18 61 M 0.71 39.5 NEG HC19 40 M 0.79 49.4 NEG HC20 45 F 0.86 45.7 NEG

HC, healthy control. Numbers refer to the control number. Cr, Serum creatinine. Alb, Serum albumin. Urine protein is presented as spot urine determined by urine dipstick. NEG: negative.