Exostosin 1/Exostosin 2–Associated Membranous Nephropathy

CLINICAL RESEARCH www.jasn.org

Sanjeev Sethi ,1 Benjamin J. Madden,2 Hanna Debiec,3 M. Cristine Charlesworth,2 LouAnn Gross,1 Aishwarya Ravindran,1 Amber M. Hummel,4 Ulrich Specks,4 Fernando C. Fervenza ,5 and Pierre Ronco3,6

1Department of Laboratory Medicine and Pathology, 2Medical Genome Facility, Proteomics Core, and Divisions of 4Pulmonary and Critical Care Medicine and 5Nephrology and Hypertension, Mayo Clinic, Rochester, Minnesota; 3Sorbonne Université, Université Pierre et Marie Curie Paris 06, and Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S1155, Paris, France; and 6Department of Nephrology and Dialysis, Assistance Publique-Hôpitaux de Paris, Tenon Hospital, Paris, France

ABSTRACT Background In membranous nephropathy (MN), which is characterized by deposition of immune complexes along the glomerular basement membrane (GBM), phospholipase A2 receptor (PLA2R) and thrombospondin type 1 domain–containing 7A are target antigens in approximately 70% and 1%–5% of cases of primary MN, respectively. In other cases of primary MN and in secondary MN, the target antigens are unknown. Methods We studied 224 cases of biopsy-proven PLA2R-negative MN and 102 controls (including 47 cases of PLA2R-associated MN) in pilot and discovery cohorts. We also evaluated 48 cases of PLA2R-negative presumed primary MN and lupus MN in a validation cohort. We used laser microdissection and mass spectrometry to identify new antigens, which were localized by immunohistochemistry. Results Mass spectrometry detected exostosin 1 (EXT1) and exostosin 2 (EXT2) in 21 cases of PLA2R- negative MN, but not in PLA2R-associated MN and control cases. Immunohistochemistry staining revealed bright granular GBM staining for EXT1 and EXT2. Clinical and biopsy findings showed features of autoimmune disease, including lupus, in 80.7% of the 26 EXT1/EXT2-associated MN cases we identified. In the validation cohort, we confirmed that EXT1/EXT2 staining was detected in pure class 5 lupus nephritis (eight of 18 patients) and in presumed primary MN associated with signs of autoimmunity (three of 16 patients); only one of the 14 cases of mixed class 5 and 3/4 lupus nephritis was positive for EXT1/EXT2. Tests in seven patients with EXT1/EXT2-associated MN found no circulating anti-exostosin antibodies. Conclusions A subset of MN is associated with accumulation of EXT1 and EXT2 in the GBM. Autoimmune disease is common in this group of patients.

JASN 30: 1123–1136, 2019. doi: https://doi.org/10.1681/ASN.2018080852

Membranous nephropathy (MN) is the commonest cause of nephrotic syndrome in white adults and Received August 21, 2018. Accepted March 24, 2019. results because of formation of antigen-antibody immune complexes in the subepithelial region of F.C.F. and P.R. contributed equally to this work. 1 the glomerular basement membrane (GBM). It is Published online ahead of print. Publication date available at characterized by thickening of the GBM on light www.jasn.org. microscopy, bright granular staining for IgG and Correspondence: Dr. Sanjeev Sethi, Department of Laboratory C3 along the glomerular capillary walls on immu- Medicine and Pathology, 200 1st Street SW, Mayo Clinic, Rochester, noﬂuorescence microscopy, and subepithelial GBM MN 55905. Email: [email protected] electron dense deposits on electron microscopy. Copyright © 2019 by the American Society of Nephrology

JASN 30: 1123–1136, 2019 ISSN : 1046-6673/3006-1123 1123 CLINICAL RESEARCH www.jasn.org

Depending on the cause, MN is classified as primary or sec- Significance Statement ondary MN, which account for 75%–80% and 20%–25% of MN, respectively.2 M-type phospholipase A2 receptor In approximately 70%–80% of cases of primary membranous nephrop- (PLA2R) and Thrombospondin Type-1 Domain–Containing athy(MN),phospholipaseA2receptor(PLA2R)/ThrombospondinType-1 – 7A (THSD7A) were identified as target antigens in primary Domain Containing 7A (THSD7A) and anti-PLA2R/THSD7A antibodies form immune complexes along the glomerular basement 3–5 – – MN, and account for approximately 70% 80% and 1% 5% membrane (GBM) that characterize the condition. In other cases of of primary MN, respectively.6,7 Secondary MN is associated with primary MN and all secondary MN, the target antigen is unknown. autoimmune diseases, malignancies, infections, and drugs.2 The Using proteomics and immunohistochemistry, the authors detected target antigen in PLA2R- and THSD7A-negative MN and in two proteins, exostosin 1 (EXT1) and exostosin 2 (EXT2), in the GBM secondary MN has remained elusive. The aim of this study of PLA2R-negative MN. EXT1 and EXT2 were absent in all cases of PLA2R-associated MN and controls. Clinical and biopsy findings was to determine the antigen in these cases of MN. Building showed features of autoimmune disease, including membranous on our expertise in mass spectrometry characterization and lupus nephritis, in 81% of the 26 EXT1/EXT2-associated MN cases discovery of new types of amyloid, complement, and other the authors identified. These findings suggest that EXT1/EXT2- glomerular deposits, we reasoned that we could identify new associated MN represents a distinct subtype of MN, most commonly MN antigens using the same approach. associated with autoimmune diseases (secondary MN).

membrane laser microdissection slide and, using a Zeiss Palm METHODS Microbeam microscope, the glomeruli were microdissected to reach approximately 250–500,000 mM2 per case. Resulting Patients FFPE fragments were digested with trypsin and collected for We initially selected a pilot cohort of seven cases of PLA2R- tandem mass spectrometry (MS/MS) analysis. The trypsin- associated MN and 15 cases of PLA2R-negative MN on kidney digested peptides were identified by nano-flow liquid chroma- biopsy for mass spectrometry studies. We detected unique tography electrospray MS/MS using a Thermo Scientific proteins exostosin 1 (EXT1) and exostosin 2 (EXT2) in five Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, cases of PLA2R-negative MN by mass spectrometry that we Bremen, Germany) coupled to a Thermo Ultimate 3000 confirmed by immunohistochemistry (IHC) (Figure 1). Sub- RSLCnano HPLC system. All MS/MS samples were analyzed sequently, we analyzed an additional 209 PLA2R-negative MN using Mascot and X! Tandem set up to search a Swissprot cases including eight cases of PLA2R-negative membranous human database. Scaffold (version 4.8.3; Proteome Software lupus nephritis by IHC for EXT1 and EXT2 staining. For con- Inc., Portland, OR) was used to validate MS/MS-based peptide trols, we used 95 cases that included: Ten cases of day zero and protein identifications. Peptide identifications were ac- protocol transplant kidney biopsy samples that were normal cepted at .95.0% probability by the Scaffold Local FDR al- on kidney biopsy sample examination, 40 cases of PLA2R- gorithm, with protein identifications requiring a two-peptide associated MN, eight cases of minimal change disease, 12 cases minimum and a 95% probability using Protein Prophet.8 De- of FSGS (six primary and six secondary), five cases of tails of laser capture and MS/MS are given in the Supplemental IgA nephropathy, seven cases of diabetic nephropathy, and Material. 13 cases of proliferative lupus nephritis without a membranous component which included two, two, and nine cases of Immunohistochemical Staining for EXT1, EXT2, and class 2, 3, and 4 lupus nephritis, respectively. Subsequently, we Exostosin like 2 confirmed the cases that were positive for EXT1/EXT2 on IHC FFPE tissues were sectioned at 5 mm and IHC staining was by mass spectrometry studies (Figure 1). performed online using EXT1, EXT2, and Exostosin like These biopsy specimens were received in the Renal Pathol- 2 (EXTL2) primary antibodies and a Polymer Refine De- ogy Laboratory, Department of Laboratory Medicine and Pa- tection System (Leica) that included hydrogen peroxidase thology, Mayo Clinic, for diagnosis and interpretation between block, post primary and polymer reagent, DAB, and hema- January of 2015 and May of 2018. Light microscopy, immu- toxylin staining steps. Details of IHC staining are given nofluorescence microscopy including PLA2R studies, and elec- in the Supplemental Material. In addition, a single case tron microscopy were performed in each case of MN. The (#16 from the validation cohort, see below) of EXT1/ clinical information was obtained from the accompanying EXT2-associated MN was also confirmed by immunofluo- charts. The study was approved by the Mayo Clinic Institu- rescence microscopy on paraffin sections and analyzed by tional Review Board. confocal microscopy.

Protein Identification by Laser Capture Western and Native Blotting Microdissection, Trypsin Digestion, and Nano LC- Patient serum antibodies for EXT1, the heterodimer EXT1/ Orbitrap Tandem Mass Spectrometry EXT2, and PLA2R were detected by western and native For each case, 10-mm-thick formalin-fixed paraffin-embedded blotting using standard procedures (see the Supplemental (FFPE) sections were obtained and mounted on a special PEN Material).

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PILOT COHORT (MS/MS) VALIDATION COHORT (IHC) n = 22 n = 48

15 PLA2R neg MN 7 PLA2R pos MN 16 18 14 PLA2R neg pMN Class V LN Mixed class LN MS/MS

5 Ext 1/2 pos All EXT 1/2 neg 3 8 1 (2)* IHC EXT 1/2 pos EXT 1/2 pos EXT 1/2 pos

EXT1/EXT2 pos EXT1/EXT2 neg

DISCOVERY COHORT (IHC) n = 304

209 PLA2R neg MN 95 controls

201 8 13 40 42 Non lupus Lupus MN Proliferative LN PLA2R Other pos MN 21 EXT 1/2 pos All negative for EXT 1/2 16/16 confirmed by MS/MS

Figure 1. Flowchart of the pilot, discovery, and validation cohorts. Initial pilot studies were done by mass spectrometry studies using 15 PLA2R- negative MN and seven PLA2R-positive MN cases. After detection of EXT1/EXT2 in ﬁve cases conﬁrmed by IHC, we studied a large number (n=209) of PLA2R-negative MN cases and controls (n=95) for expression of EXT1/EXT2 by IHC. Controls included 13 cases of proliferative lupus nephritis (LN) without a membranous component and 40 cases of PLA2R-positive MN. Finally, we studied 48 cases of MN in a validation cohort that included PLA2R-negative primary MN (pMN), membranous (class 5) LN, and mixed class LN with a membranous component. *One case (patient #16) started with pure MN with signs of autoimmunity and then shifted to mixed class. Neg, negative; pos, positive.

Validation Cohort the pilot cohort and in all 16 cases of the discovery cohort. The Forty-eight unstained kidney biopsy specimen slides of FFPE average total spectral count for EXT1 was 65.3 (SD634.6, tissue were provided by Inserm UMR-S1155 (H.D. and P.R., median 71, range 11–155) and the average total spectral count Tenon Hospital, Paris) and analyzed in the Renal Pathology for EXT2 was 83.4 (SD638.4, median 83, range 19–160). Laboratory, Mayo Clinic, where IHC for EXT1 and EXT2 was MS/MS did not detect EXT-like (EXTL) proteins in any of performed. The diagnosis of the biopsy samples was not known the EXT1/EXT2-associated MN. Also, MS/MS showed only at the time of receiving the slides. Subsequently, after the stain- baseline spectral counts of PLA2R in EXT1/EXT2-associated ing, the breakdown of the MN was as follows: 18 cases belonged MN. Both the average EXT1 and EXT2 total spectral numbers to class 5 membranous lupus nephritis, 14 cases were class 3/4 in EXT1/EXT2-associated MN were comparable to total spec- lupus nephritis with a component of lupus class 5 MN, and tral counts of PLA2R in PLA2R-associated cases. All control 16 were primary (nonlupus) cases of MN that were negative for and PLA2R-associated MN cases were negative for EXT1 or both PLA2R and THS7DA (Figure 1). EXT2 spectra (Supplemental Figure 2). MS/MS spectral matches to sequences from EXT1 and EXT2 are shown in Supplemental Figure 3. RESULTS All four classes of Ig were detected in EXT1/EXT2-associated MN: IgG1 was the most abundant Ig (average 97.5, Mass Spectrometry Results SD635.9,median106,range32–173), followed by IgG2 Detection of EXT1 and EXT2 in PLA2R-Negative (average 75, SD629.5, median 77, range 23–124), IgG3 Biopsy Specimens (average 74.4, SD630.3, median 69, range 33–146), and IgG4 We performed MS/MS studies in the five cases of the pilot (average 70.8, SD635.2, median 8, range 12–129). The average cohort and in 16 of 21 cases of the discovery cohort that stained spectral count of IgG1 was higher than IgG4 in EXT1/EXT2- positive for EXT1/EXT2 by IHC (Figure 2). We identified high associated MN (P,0.01), and also when compared with the total total spectral counts of both EXT1 and EXT2 in five cases of spectral counts of IgG1 in the PLA2R-associated MN (P=0.04).

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A A BC

B Probability Legend: over 95% 80% to 94% 50% to 79% 20% to 49% 0% to 19% Bio View: 2373 Proteins in 2143 Clusters Molecular weight Protein Grouping Ambiguity Combined control cases Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 With 2372 Filtered Out Accession Number Secretory phospholipase A2 receptor OS=Homo sapiens GN=PLA2R1 PE=1 SV=2 sp⏐Q13018⏐PLA2R_HUMAN 169 kDa 5 94 45 82 89 134 66 93 Ig gamma-1 chain C region OS=Homo sapiens GN=IGHG1 PE=1 SV=1 sp⏐P01857⏐IGHG1_HUMAN 36 kDa 56 47 79 80 60 79 64 66 Ig gamma-2 chain C region OS=Homo sapiens GN=IGHG2 PE=1 SV=2 sp⏐P01859⏐IGHG2_HUMAN 36 kDa 45 36 57 50 43 49 55 52 Ig gamma-3 chain C region OS=Homo sapiens GN=IGHG3 PE=1 SV=2 sp⏐P01860⏐IGHG3_HUMAN 41 kDa 45 51 52 69 45 63 96 75 Ig gamma-4 chain C region OS=Homo sapiens GN=IGHG4 PE=1 SV=1 sp⏐P01861⏐IGHG4_HUMAN 36 kDa 22 109 57 77 108 132 96 61 C

Figure 2. Proteomic identification of PLA2R in PLA2R-associated MN and EXT1 and EXT2 in EXT1/EXT2-associated MN. Glomeruli were microdissected and analyzed using mass spectrometry as described in the Methods section. (A) Laser microdissection of glomeruli. One case of MN showing (A) unmarked glomerulus, (B) glomerulus marked for dissection, and (C) vacant space on the slide after microdissection. (B) PLA2R- associated MN: Protein identification report from seven cases is shown. Numbers in green boxes represent spectral counts of MS/MS matches to a respective protein. All seven cases show large total spectral counts for PLA2R and Ig. For comparison, the average total spectral counts from six control cases (day 0 protocol transplant biopsy specimens) are also shown. (C) Representative sequence coverage map of PLA2R from one case. Amino acids highlighted in bold letters over yellow background are the amino acids detected. Note the extensive coverage. Green highlighted boxes indicate amino acids with artifactual chemical modification induced by mass spectrometry such as oxidation of methionine. (D) EXT1/ EXT2-associated MN: Protein identification from all 21 cases showing total spectral counts for both EXT1 and EXT2. For comparison, the average total spectral counts in six control cases (day 0 protocol transplant biopsy specimens) are also shown. IgG1 was the dominant IgG present. Higher spectral IgG counts in the control cases in the bottom panel compared with the top panel reflect normalization, with fewer EXT1/EXT2 cases in the lower panel compared with the top panel. (E and F) Sequence coverage maps: Representative EXT1 and EXT2 sequence coverage map from a case of EXT1/EXT2-associated MN showing the extensive amino coverage of both (E) EXT1 and (F) EXT2 by MS/MS. Amino acids highlighted in bold letters over yellow background are the amino acids detected. Note the extensive coverage. Green highlighted boxes indicate amino acids with artifactual chemical modification induced by mass spectrometry such as oxidation of methionine.

Detection of PLA2R in PLA2R-Associated MN 66, range 47–80), IgG3 (average 64.4, SD617.5, range 45–96), All seven cases showed large total spectral counts for PLA2R (Fig- and IgG2 (average 48.9, SD67.2, range 36–57). ure 2). The average PLA2R total spectra count was 86.1 (SD627.5, median 89, range 45–134). In comparison, the average PLA2R Immunohistochemical Staining for EXT1 and EXT2 spectral count in control cases and EXT1/EXT2-associated MN We performed IHC staining for EXT1 and EXT2 in 224 cases of was only 7.1 (SD65.2, median 8, range 0–19). IgG4 was the most PLA2R-negative MN (15 in the pilot and 209 in the discovery abundant Ig (average spectral count 91.4, SD627.6, median 96, cohorts). Twenty-six (11.6%) cases were positive for EXT1 and range 47–132), followed by IgG1 (average 67.9, SD612.3, median EXT2(ﬁveinthepilotand21inthediscoverycohorts),whereasthe

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D Probability Legend: over 95% 80% to 94% 50% to 79% 20% to 49% 0% to 19% Bio View: 2916 Proteins in 2640 Clusters Accession Number With 2914 Filtered Out Molecular weight Protein Grouping Ambiguity combined_controls Case 1 Case 2 Case 3 Case 4 Case 5 Case 6 Case 7 Case 8 Case 9 Case 10 Case 11 Case 12 Case 13 Case 14 Exostosin-2 OS=Homo sapiens GN=EXT2 PE=1 SV=1 sp⏐Q93063⏐EXT2_HUMAN 82 kDa 0 58 142 98 54 115 47 102 113 38 82 41 160 70 19 Exostosin-1 OS=Homo sapiens GN=EXT1 PE=1 SV=2 sp⏐Q16394⏐EXT1_HUMAN 86 kDa (0) 22 108 74 44 88 53 83 82 27 62 41 155 60 12 lg gamma-1 chain C region OS=Homo sapiens GN=IGHG1 PE=1 SV=1 sp⏐P01857⏐IGHG1_HUMAN 36 kDa 50 53 119 72 112 87 58 71 84 32 63 60 121 123 173 lg gamma-2 chain C region OS=Homo sapiens GN=IGHG2 PE=1 SV=2 sp⏐P01859⏐IGHG2_HUMAN 36 kDa 40 35 97 62 88 78 61 44 52 23 47 51 124 102 115 lg gamma-3 chain C region OS=Homo sapiens GN=IGHG3 PE=1 SV=2 sp⏐P01860⏐IGHG3_HUMAN 41 kDa 40 35 86 47 146 69 57 45 64 33 53 47 103 82 114 lg gamma-4 chain C region OS=Homo sapiens GN=IGHG4 PE=1 SV=1 sp⏐P01861⏐IGHG4_HUMAN 36 kDa 19 20 40 125 44 100 30 129 53 12 94 54 123 73 90 Secretory phospholipase A2 receptor OS=Homo sapiens GN=PLA2R1... sp⏐Q13018⏐PLA2R_HUMAN 169 kDa 58(0)10 (0) 10 5194 12 8 4 (0)15 (0)

Probability Legend: over 95% 80% to 94% 50% to 79% 20% to 49% 0% to 19% Bio View: 2732 Proteins in 2459 Clusters With 2730 Filtered Out Accession Number Molecular Weight Protein Grouping Ambiguity Combined_controls Case 17 Case 18 Case 19 Case 21 Case 22 Case 24 Case 25 Exostosin-2 OS=Homo sapiens GN=EXT2 PE=1 SV=1 sp⏐Q93063⏐EXT2_HUMAN 82 kDa 0 110 102 19 109 74 86 112 Exostosin-1 OS=Homo sapiens GN=EXT1 PE= 1 SV=2 sp⏐Q16394⏐EXT1_HUMAN 86 kDa (0) 94 75 11 71 40 78 92 lg gamma-1 chain C region OS=Homo sapiens GN=IGHG1 PE=1 SV=1 sp⏐P01857⏐IGHG1_HUMAN 36 kDa 62 129 135 71 108 106 129 141 lg gamma-2 chain C region OS=Homo sapiens GN=IGHG2 PE=1 SV=2 sp⏐P01859⏐IGHG2_HUMAN 36 kDa 49 70 100 44 94 77 88 123 lg gamma-3 chain C region OS=Homo sapiens GN=IGHG3 PE=1 SV=2 sp⏐P01860⏐IGHG3_HUMAN 41 kDa 50 72 111 46 79 66 96 112 Ig gamma-4 chain C region OS=Homo sapiens GN=IGHG4 PE=1 SV=1 sp⏐P01861⏐IGHG4_HUMAN 36 kDa 24 116 68 33 55 72 70 86 Secretory phospholipase A2 receptor OS=Homo sapiens GN=PLA2R1 PE=1 SV=2 sp⏐Q13018⏐PLA2R_HUMAN 169 kDa 6 10 8 10 11 2 8 5

Figure 2. Continued.

remaining 198 (88.4%) were negative. All 26 positive cases showed shown in Figure 3B. Figure 3C shows positive staining for EXT1 bright (2–3+/3) granular staining for EXT1 and EXT2 along the and EXT2 in a case of membranous lupus nephritis and negative GBM. Importantly, the staining was along the GBM with no sig- staining in two cases of proliferative lupus nephritis. Representa- nificant mesangial staining. There was no staining along the tive EXT1 and EXT2 staining in the remaining EXT1/EXT2- Bowman’s capsule, tubular basement membranes, or in vessel associated MN and control cases is shown in Supplemental Figure 1. walls. EXT1 and EXT2 staining in three cases is shown in Figure 3A. The positive granular staining mirrored the granular IgG Clinical and Kidney Biopsy Findings of EXT1/EXT2- along the GBM seen in each case. All 95 control cases were neg- Associated MN ative for both EXT1 and EXT2. Representative negative staining We identified 26 cases of EXT1/EXT2-associated MN on the for EXT1 and EXT2 in three cases of PLA2R-associated MN is basis of IHC. There were 21 (80.8%) female and five (19.2%)

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A EXT1 EXT1 EXT1

20μm 20μm 20μm EXT2 EXT2 EXT2

20μm 20μm 20μm

B EXT1 EXT1 EXT1

20μm 20μm 20μm EXT2 EXT2 EXT2

20μm 20μm 20μm

Figure 3. Immunohistochemical stain for EXT1 and EXT2 in EXT1/EXT2-associated MN, PLA2R-associated MN, and lupus nephritis. Each column is one case with top panel showing EXT1 and bottom panel showing EXT2 staining. (A) EXT1/EXT2- associated MN: Bright granular staining for EXT1 and EXT2 along the GBMs in three cases of EXT1/EXT2-associated MN. The first column is case 4, second column is case 7, and third column is case 25. (B) PLA2R-associated MN: Negative staining for EXT1 and EXT2 in three cases of PLA2R-associated MN. (C) Lupus nephritis: Bright positive staining for EXT1 and EXT2 in a case (case 26) of membranous lupus nephritis (column 1) and negative staining for EXT1 and EXT2 in a case of class 2 lupus nephritis (column 2) and class 4 lupus nephritis (column 3). (D) Validation cohort: Bright positive staining for EXT1 and EXT2 in two cases of membranous class 5 lupus nephritis and negative staining in one case of membranous class 5 lupus nephritis (note the focal proliferative features in this case). male patients with a ratio of 4.2:1. The mean age at presentation subepithelial deposits on electron microscopy. Overall, an av- was 35.7 (SD613.4). The mean serum creatinine and protein- erage of 22 (SD614.4) glomeruli were present, of which 2.3 uria at presentation were 1.0 mg/dl (SD60.9) and 5.9 g/24 h (SD63.9) were globally sclerosed. Immunofluorescence mi- (SD64.8), respectively. Seventeen (70.8%, n=24) had abnor- croscopy showed bright staining for IgG (2–3+/3) and C3 mal laboratory values for either anti-nuclear antibodies, anti– (2–3+/3) in all cases. Twenty-two (84.6%) of 26 cases also double-stranded DNA antibodies, anti-Smith antibodies, showed staining for IgA (1–3+/3)orIgM(1–3+/3) or both. anti–Sjögren syndrome–related antigen A or B (SSA or SSB), Nineteen (73.0%) of 26 cases showed staining for C1q that or anti-ribonucleoprotein antibodies. Nine (34.6%) patients ranged from 1+/3 to 3+/3. All cases showed staining for k had a clinical diagnosis of SLE. None of the patients had hep- (2–3+/3) and l (2–3+/3) light chains. Immunofluorescence atitis. One patient had a history of breast cancer and one had study for PLA2R was negative in all cases. Electron microscopy lung cancer. showed subepithelial deposits in all cases, subendothelial de- The kidney biopsy specimens of all cases of EXT1/EXT2- posits in nine (34.6%), and mesangial deposits in 25 (96.1%) associated MN showed the characteristic findings of thickened of 26 cases. Tubuloreticular inclusions were present in GBM on light microscopy, bright IgG and C3 staining along 21 (80.7%) of the 26 cases. The clinical and pathology findings the capillary wall on immunofluorescence microscopy, and are shown in Tables 1 and 2.

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C ABC

20μm 20μm 20μm DEF

20μm 20μm 20μm

D EXT1 EXT1 EXT1

20μm 20μm 20μm EXT2 EXT2 EXT2

20μm 20μm 20μm

Figure 3. Continued.

Validation Cohort EXT1/EXT2 staining remained positive throughout evolu- IHC tion and histologic classes. The clinical and laboratory Eight of 18 (44%) cases of lupus class 5 membranous nephritis findings of the EXT1/EXT2-associated MN of the valida- were positive for EXT1 and EXT2 staining along the GBM, tion cohort are given in Supplemental Tables 1 and 2. Rep- whereas only one of the 14 cases of mixed class 5 and class 3/4 resentative EXT1 and EXT2 staining of positive cases is lupus nephritis was positive for EXT1 and EXT2 along shown in Figure 3D. the GBM. Three (19%) of 16 cases of PLA2R- and THS7DA- negative and nonlupus MN cases were positive for EXT1 and Western and Native Blotting EXT2 staining along the GBM. These three cases had features of We then searched for circulating antibodies to EXT1 and the autoimmune disease on chart review. Patients #14 and #16 are heterodimer EXT1/EXT2 in seven EXT1/EXT2-positive pa- of particular interest because they both later developed a full- tients with available sera (one from the discovery and six blown lupus. In patient #14, the initial diagnosis was MN stage from the validation cohorts), and in one control patient 1–2withanimmunofluorescence pattern of primary MN; with glomerular PLA2R deposits. Western blotting was per- 6 years later, she developed full-blown clinical lupus but formed in SDS gels under nonreducing (Figure 5A) and re- the lesions and immunofluorescence pattern were unchanged. ducing (not shown) conditions. Despite strong reactivity of Patient #16 also had a presentation and an immunofluores- the recombinant protein with a control rabbit anti-human cence pattern of primary MN, but she developed a year later EXT1 and EXT2 serum, we failed to detect any reactivity very active clinical lupus disease with a mixed pattern of with the seven tested sera, as well as with one serum from a class 3 plus 5. She was referred 10 years later for nephrotic patient with PLA2R-related MN. In contrast, the serum from syndrome with asymptomatic lupus and class 5 MN on bi- this patient strongly reacted with a 185-kd band when it was opsy (Figure 4). In the two patients mentioned above, incubated with the PLA2R antigen.

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Table 1. Clinical and laboratory data of EXT1/EXT2-associated MN

Case Age/Sex/Ethnicity Rash/Arthritis/Other Serum Cr, mg/dl Proteinuria, g/24 h C3/C4 ANA/dsDNA Other Lupus Hepatitis/Malignancy www.jasn.org 1 41/M/white 2/2/2 1.1 12.0 N/N 2/2 No 2/2 2 32/F 2/2 0.7 5.0 N/N +/+ Yes 2/2 3 60/M/Hispanic 2/2/2 1.9 20.0 N/N 2/2 No 2/2 4 20/F/native American 2/2/2 0.5 8.0 N/N 2/2 No 2/2 5 59/F/white 2/+/sicca 0.6 5.7 N/L +/2 SSA+, SSB+ No 2/Breast cancer 6 29/F/white 2/2/2 0.8 6.0 N/N +/2 No 2/2 7 19/F/Indian-Hispanic 2/+/2 0.6 2.0 N/N +/2 No 2/2 8 30/M/black 2/2/2 0.7 13.0 N/N 2/2 No 2/2 9 55/F 2/+/2 0.7 6.0 N/N 2/2 No 2/SCC lung 10 39/F/Indian 2/2/2 0.5 3.0 ND +/2 SSA+, SSB+ No 2/2 11 30/F/white 2/2/2 0.5 8.4 N/N +/2 No 2/2 12 32/F/black 2/2/2 3.2 15.9 L/N +/2 No 2/2 13 51/F/white 2/+/2 0.7 3.0 N/N +/+ SSA+ Noa 2/2 14 21/F/Hispanic 2/2/lymphadenopathy 1.7 11 N/N 2/2 No 2/2 15 34/F/black 2/+/2 0.7 5.1 N/N +/2 SSA+, SSB+ Noa 2/2 16 31/F/black 2/+/allergies 0.7 2.2 N/N 2/2 No 2/2 17 17/M/white +/+/myositis 0.9 NR N/N +/+/+anti-Smith, + RNPb SLE/MCTD overlap 2/2 18 25/F +/+/ 0.8 NA N/L +/+/+SSA+ anti-Smith Yes 2/2 19 32/F/white 2/+/ﬁbromyalgia 0.6 11 ND +/+ Yes 2/2 20 67/F/black — 1.1 7.8 ND 2/2 No 2/2 21 32/F/Hispanic 2/2/pericardial effusion 0.4 3 L/L 2/2 No (treated as lupus) 2/2 22 38/M 2/+ 4.6 5 N/N +/+ Yes 2/2 23 20/F 2/+/2 0.7 3 ND +/+ Yes 2/2 24 36/F/Asian +/2/2 0.9 6 N/L +/+ Yes 2/2 25 43/F/black 2/+/2 0.7 2 L/L +/+ Yes 2/2 26 34/F +/+/pleurisy 0.9 2.2 N/N +/+/+ anti-Smith Yes 2/2 ANA, anti-nuclear antibody; dsDNA, anti–double-stranded DNA antibody; M, male; N, normal; F, female; L, low; SCC, squamous cell carcinoma; NR, nephrotic range, ﬁve males 20 females; NA, not available; ND, no data/data not available. aMixed connective tissue disorder. JASN bAnti-Smith/ribonucleoprotein antibody. 30: 1123 – 16 2019 1136, JASN 30: 1123 – 16 09ET/X2Ascae ebaosNephropathy Membranous EXT1/EXT2-Associated 2019 1136,

Table 2. Kidney biopsy findings of EXT1/EXT2-associated MN Mesangial or Interstitial Glomeruli/ Immunofluorescence Electron Microscopy Tubuloreticular Case Endocapillary Inflammation/ Arteries Sclerosed Microscopy Deposits SE/SU/ME Inclusion Hypercellularity IFTA 1 10/0 Not present 0/0 Moderate sclerosis IgG (3+) C1q (1+) C3 (3+) +/2/+ + 2 22/0 Not present, two small crescents 0/0 Normal IgG (3+) IgA (3+) IgM (1+) C1q (2+) C3 (3+) +/2/+ + 3 8/2 Not present 0/0 Normal IgG (2+) IgM (1+) C3 (1+) +/2/2 – 4 38/0 Not present 0/0 Normal IgG (3+) IgM (1+) C3 (2+) +/2/+ + 5 19/1 Not present 0/0 Normal IgG (3+) IgA (1+) IgM (1+) C1q (1+) C3 (3+) +/+/+ 2 6 7/0 Not present 0/0 Normal IgG (3+) IgA (2+), C1q (1+) C3 (2+) +/+/+ + 7 64/0 Not present 0/0 Normal IgG (3+) C3 (1+) +/+/+ + 8 23/1 Not present 0/10 Normal IgG (3+) IgA (2+) IgM (1+) C1q (2+) C3 (3+) +/+/+ + 9 26/6 Not present 0/0 Normal IgG (3+) IgM (1+) C3 (2+) +/2/+ 2 10 48/4 Not present 0/10 Normal IgG (3+) IgA (2+) IgM (1+) C1q (1+) C3 (2+) +/2/+ + 11 29/5 Not present 0/0 Normal IgG (2+) IgM (1+) C3 (3+) +/2/+ 2 12 54/18 Not present 10/30 Mild sclerosis IgG (3+) IgM (1+) C1q (1+) C3 (3+) +/2/a + 13 18/1 Not present 0/0 Normal IgG (3+) IgA (2+) IgM (1+) C1q (2+) C3 (3+) +/2/+ + 14 12/0 Not present, three small crescents 0/20 Normal IgG (3+) IgM (1+) C1q (2+) C3 (3+) +/2/+a + 15 12/2 Not present 25/25 Normal IgG (3+) IgM (1+) C1q (1+) C3 (3+) +/2/+a + 16 23/1 Not present 0/0 Normal IgG (3+) C3 (3+) +/2/+ + 17 17/0 Not present 0/0 Normal IgG (3+) IgA (2+) C1q (1+) C3 (3+) +/2/+ + 18 4/0 Not present 0/0 Normal IgG (3+) IgM (2+) C1q (1+) C3 (3+) +/+/+ + 19 14/0 Not present 0/0 Normal IgG (2+) C3 (2+) +/2/- 2 20 18/2 Not present 0/0 Normal IgG (3+) IgA (2+) IgM (1+) C1q (2+) C3 (3+) +/2/+ + 21 20/1 Not present 0/0 Normal IgG (3+) IgA (1+) IgM (1+) C1q (2+) C3 (3+) +/2/+ +

22 17/9 Not present 0/80 Severe sclerosis IgG (3+) IgM (2+) C1q (1+) C3 (3+) +/+/+ + www.jasn.org 23 14/0 Present 10/30 Normal IgG (3+) IgA (3+) IgM (3+) C1q (2+) C3 (3+) +/+/+ + 24 19/2 Not present 0/0 Normal IgG (3+) IgA (3+) IgM (3+) C1q (2+) C3 (3+) +/+/+a + 25 27/4 Present 0/10 Moderate IgG (3+) IgM (1+) C1q (2+) C3 (3+) +/+/+ + 26 16/1 Not present 0/25 Normal IgG (3+) IgM (1+) C1q (2+) C3 (2+) +/2/+ + ﬁ

IFTA, interstitial brosis and tubular atrophy; SE, subepithelial; SU, subendothelial; ME, mesangial. RESEARCH CLINICAL aTubular basement membrane deposits. 1131 CLINICAL RESEARCH www.jasn.org

2007 nephrotic syndrome MN stage I

EXT1 2007 EXT2 2007

50 µm 50 µm

2008 clinical lupus Class III S-A + V

EXT1 2008 EXT2 2008

2018 asymptomatic lupus Class V

EXT1 2018 EXT2 2018

Figure 4. EXT1 and EXT2 staining by immunoﬂuorescence microscopy of a case of MN that was initially weakly positive for EXT1/EXT2 and subsequently turned strongly positive at the time of development of clinical lupus. The inserts show the EXT1 and EXT2 deposits by confocal microscopy. The year of the biopsy and the clinical and histologic diagnoses are indicated above each picture.

To investigate the presence of epitopes sensitive to denatur- complexes. On the basis of identiﬁcation of the target antigen, ation by SDS, native blotting method was used. However, none MN is also classiﬁed as PLA2R-positive, THSD7A-positive, or of the seven tested sera showed reactivity with recombinant PLA2R/THSD7A double-negative, which represent approxi- EXT1 and EXT1/2 by this method (Figure 5B). We concluded mately 20% of MN. Although PLA2R- and THSD7A-associated that, under our experimental conditions, we were unable to MN usually present as primary disease, the double-negative MN detect circulating anti-EXT1/EXT2 antibodies. is often associated with autoimmune manifestations including SLE. The target antigen in the double-negative MN has remained elusive. In this study, we have used an original combination of DISCUSSION laser microdissection, MS/MS, and IHC techniques to identify twonovelproteins,EXT1andEXT2,that accumulatealongthe MN is the most common cause of nephrotic syndrome in white GBM in a subset of PLA2R-negative MN. The staining pattern adults. It results from accumulation of target antigens in mirrors the granular IgG staining along the GBM, indicating the GBM and subsequent formation of antigen-antibody that EXT1 and EXT2 are the likely antigens in the immune

1132 JASN JASN 30: 1123–1136, 2019 www.jasn.org CLINICAL RESEARCH

A Western blots (denatured non-reduced conditions)

Patient’s sera

Rabbit anti Human 123 4 5 6 Ext1 Ex1/2 Ext1 Ex1/2 Ext1 Ex1/2 Ext1 Ex1/2 Ext1 Ex1/2 Ext1 Ext1/2 Ext1 Ext1/2 250

130 100 70 55

Biopsies Ext1 and Ext1/2 positive, PLA2R negative

Patient’s sera

Ext1 Ext1/2 PLA2R

Ext1 Ex1/2 PLA2R Ext1 Ext1/2 PLA2R

Ext1 and 1/2 positive Ext1 and 1/2 negative Ext1 and 1/2 negative PLA2R negative PLA2R positive PLA2R negative

Biopsies

B Slot blots (non-denatured non-reduced conditions)

12 3 4 5 6 7

Ext1 Rabbit anti Human Ext1/2

Patient’s sera

Figure 5. Western and native blotting analyses of sera from patients with EXT1/EXT2 glomerular deposits and a patient with PLA2R- related MN. (A) Western blots (denatured, nonreduced conditions). The upper panel shows lack of reactivity with recombinant EXT1 and heterodimer EXT1/EXT2 of six sera from patients with EXT1- and EXT2-positive biopsies, contrasting with strong reactivity of the rabbit anti-human EXT1 and EXT2. Here are shown ﬁve of six available sera from the validation cohort; the last serum on the right is the only one available from the discovery cohort. The sixth available serum from the validation cohort is shown in the lower panel (left). The lower panel shows reactivity of sera from three patients with or without EXT1 and EXT1/2 deposits or PLA2R deposits in the kidney biopsy specimens. For this western blot, EXT1, EXT1/2, and PLA2R recombinant antigens were run in three different, consecutive lanes before transfer, and the blots were then incubated with each of the patient sera. Note the strong reactivity with PLA2R of the serum from PLA2R-related MN and lack of reactivity with EXT1 and EXT1/2 of the three sera. (B) Slot blots (nondenaturing, nonreduced conditions). Slot blotting analysis conﬁrms lack of reactivity in nondenaturing conditions with recombinant EXT1 and EXT1/2 protein of seven sera from patients with EXT1- and EXT2-positive biopsy specimens.

JASN 30: 1123–1136, 2019 EXT1/EXT2-Associated Membranous Nephropathy 1133 CLINICAL RESEARCH www.jasn.org complexes. Most importantly, the clinical features and kid- The clinical and kidney biopsy findings revealed an inter- ney biopsy findings show that EXT1/EXT2-associated MN esting pattern in the EXT1/EXT2-associated MN. A total of is mostly present in patients with autoimmune manifesta- 80.8% of the patients were women and the average age was tions or diseases including lupus. Taken together, our find- 35.7 years. Furthermore, 70.8% of the patients had abnormal ings show that EXT1 and EXT2 represent novel biomarker autoimmune laboratory findings, such as positive anti- proteins and possibly the target antigens in secondary (au- nuclear, anti–double-stranded DNA, anti-SSA/SSB, or anti- toimmune) MN. ribonucleoprotein antibodies. Nine (35%) of the patients Laser microdissection with mass spectrometry is an impor- had a clinical diagnosis of lupus and three (12%) had mixed tant new methodology that allows for screening of a large connective tissue disorder. Kidney biopsy findings revealed number of proteins.9 Mass spectrometry also allows for semi- features suggestive of a secondary MN related to autoimmune quantitative measurement of the proteins and compares the disease in 84.6% of the patients. These included staining for relative abundance of the proteins. In this study, we were able C1q and/or staining for IgA/IgM on immunofluorescence to identify 1500–2000 glomerular proteins per case; most were studies, subendothelial and mesangial deposits, and the find- house-keeping proteins and many were present in low total ing of tubuloreticular inclusions in endothelial cells on elec- spectral counts. We found that PLA2R was among the most tron microscopy. Furthermore, IgG1 was the dominant IgG abundant proteins in PLA2R-associated MN compared with with spectral counts significantly greater than for IgG4 within the house-keeping proteins. Next, we found that exostosin EXT1/EXT2-associated MN and also when compared with proteins EXT1 and EXT2 were among the most abundant IgG1 in PLA2R-associated MN. Taken together, these findings proteins in 21 cases of PLA2R-negative MN. MS/MS of 21 of suggestthatEXT1/EXT2representpotentialbiomarkersor 26 cases of EXT1/EXT2-associated MN showed high average target antigens in secondary autoimmune MN. spectral counts of both EXT1 (65.3) and EXT2 (83.4); in the We also studied other cases of primary glomerular disease remaining five cases there was inadequate tissue to perform with nephrotic syndrome, including minimal change disease, mass spectrometry studies. The total spectral counts for EXT1 primary FSGS and immune complex GN including lupus ne- and EXT2 were the highest among all glomerular proteins phritis (without a membranous component), and IgA ne- except for some complement and Ig proteins and basement phropathy by IHC and MS/MS. All cases were negative for membrane proteins. The high PLA2R (in PLA2R-associated EXT1 and EXT2 (Supplemental Figure 1F). Most importantly, MN) and EXT1/EXT2 (in EXT1/EXT2-associated MN) spec- cases of lupus nephritis that did not have a membranous com- tral counts were also validated by the extensive sequence cov- ponent were negative for EXT1 and EXT2. On the other hand, erage found for PLA2R and the EXT1 and EXT2 proteins. The nine of the 26 positive cases of EXT1/EXT2-associated MN EXT1 and EXT2 spectral counts in EXT1/EXT2-associated were consistent with membranous (class 5) lupus nephritis on MN were comparable to the PLA2R (86.1) spectral counts in the basis of clinical and kidney biopsy findings. Thus, cases PLA2R-associated MN cases. Most importantly, EXT1 and with pure proliferative lupus nephritis (class 2, 3, or 4) were EXT2 were absent in all PLA2R-associated MN and control negative for EXT1/EXT2 GBM staining by IHC. We found two cases. IHC confirmed bright granular staining for EXT1 and cases (cases #23 and #25) that had a proliferative component EXT2 along the GBM with no significant mesangial staining. (class 3/4) along the membranous (class 5) component. Larger It is important to note that the staining was evenly spread studies are needed to determine the percentage of positive throughout the thickened GBM and mirrored the Ig staining, EXT1/EXT2-associated MN in this group of patients. as would be expected in MN. The granular capillary wall stain- These findings were confirmed by EXT1/EXT2 IHC staining ing for EXT1 and EXT2 was similar to the staining pattern of of the validation cohort. For the validation studies, on the basis PLA2R in PLA2R-associated MN cases. The uniformity of our findings, we investigated cases of pure lupus class 5 of EXT1/EXT2 staining along the GBM and the subepithelial nephritis, proliferative class 3/4 lupus nephritis with a mem- deposits suggests that these proteins are shed from the branous class 5 component, and nonlupus MN being negative podocytes rather than representing circulating antigens or im- for both PLA2R and THS7DA. IHC staining showed that 44% mune complexes. It is unlikely that they are shed from me- of cases with class 5 lupus nephritis were positive for EXT1 and sangial cells or endothelial cells because there is no mesangial EXT2, whereas only two cases (#16 and #35) with a mixed class or subendothelial staining. It is also important to point out (3/4 plus 5, n=14) of lupus nephritis were positive for EXT1/ that we did not detect spectra for THSD7A in any of the EXT2. However, in the case of patient #16, EXT1/EXT2 stain- cases of EXT1/EXT2-associated MN or in any of the ing appeared at the firstbiopsywhenthediagnosisof PLA2R-associated MN cases. Although we found that some “primary” MN with signs of autoimmunity was made. Fur- basement membrane proteins were also present with high thermore, evaluation of 16 cases of double-negative (PLA2R- spectral counts, including laminin, basement membrane– and THS7DA-negative) nonlupus MN detected three positive specific heparan sulfate core proteins, nidogen, type IV collagen, cases of EXT1/EXT2-positive MN, all of them with biologic and structural proteins such as vinculin, these proteins had large features of an underlying autoimmune disease. It is noticeable spectral counts in both PLA2R-associated MN and in EXT1/ that two (#14 and #16) of those three patients developed full- EXT2-associated MN (data not shown). blown lupus. Further studies are needed to determine whether

1134 JASN JASN 30: 1123–1136, 2019 www.jasn.org CLINICAL RESEARCH

EXT1/EXT2 staining is a predictor of such evolution in pa- biomarker proteins for MN associated with autoimmune dis- tients with apparently “primary” MN with biologic signs of ease rather than target antigens. Further studies are required to autoimmunity and to identify the “missing” antigeninthe answer these questions. EXT1/EXT2-negative patients with lupus MN. In conclusion, we have identified a set of novel proteins, EXT1 The GBM is made up of mostly type IV collagen, laminin, and EXT2,associatedwithPLA2R andTHSD7Adouble-negative nidogen, and heparan sulfate proteoglycans.10–17 Exostosins MN in a subset of adult patients with clinical and biopsy findings are glycosyltransferases that are responsible for the synthesis of autoimmune disease including lupus. EXT1 and EXT2 may of the heparin sulfate backbone that add glycosaminoglycan represent the target antigens or biomarker proteins of secondary residues to the core protein resulting in the generation of (autoimmune) MN. This important finding has clinical impor- complex polysaccharides.18,19 There are five genes that encode tance for the molecular classification of MN and possibly for the the EXT proteins—EXT1, EXT2, EXTL1, EXTL2,andEXTL3. prediction of lupus occurrence in patients with so-called primary EXT1 and EXT2 show structural similarities, and EXT1 and MN but with features of autoimmunity. EXT2 can exist as heterodimers and act as copolymerases in the elongation of the heparin sulfate chain.20 The heterodimer 21 of EXT1/EXT2 also has increased stability and activity. This ACKNOWLEDGMENTS is the likely reason that EXT1/EXT2 (in the heterodimer form) are found together in our studies. The EXTL proteins show WewouldliketothanktheMayoClinicGenomeFacility amino acid sequence homology with EXT1 and EXT2, and are Proteomics Core (a shared resource of the Mayo Clinic Cancer Center also likely involved in heparan sulfate synthesis although their [National Cancer Institute P30 CA15083]), the Department of Lab- function is less well known. MS/MS did not detect EXTL pro- oratory Medicine and Pathology and the Pathology Research Core, and teins in any of the EXT1/EXT2-associated MN cases, although the Fulk Family Foundation. Dr. Ronco is a recipient of European there was mild staining in one case that showed very bright Research Council ERC-2012-ADG_20120314 grant 322947, the Sev- EXT1/EXT2 staining, raising the possibility of crossreactivity enth Framework Programme of the European Community contract of the EXTL2 antibody to EXT1/EXT2 (Supplemental Figure 2012-305608 (European Consortium for High-Throughput Research 1E). The EXT proteins are well conserved, especially in their in Rare Kidney Diseases), and the National Research Agency grant C-terminal regions. Except for EXTL1, the EXT proteins are Membranous Nephropathy aims (ANR-17- CE17-0012-01). We are ubiquitously expressed in various mammalian tissues. EXT greatly indebted to the clinicians who took care of the patients in the proteins are also expressed in podocytes, and a homozygous Department of Nephrology and Dialysis, and to the pathologists David knockout of EXT1 specifically in podocytes did not lead to Buob and Isabelle Brocheriou from the Department of Pathology, all at significant defects in glomerular filtration, although changes Tenon Hospital, Paris, France. in podocyte architecture and focal thickening of GBM were Dr. Sethi and Dr. Fervenza designed the study. Dr. Sethi wrote the noted.22,23 EXT proteins are transmembrane proteins in en- manuscript and interpreted the kidney biopsy, immunohistochem- doplasmic reticulum, and whether the EXT1 and EXT2 de- istry, and mass spectrometry data. Dr. Madden and Dr. Charlesworth tected in EXT1/EXT2-associated MN are full-length proteins performed thelasermicrodissection and mass spectrometry.Dr.Gross or represent shed partial or truncated proteins or are proteins performed the immunohistochemistry. Dr. Ravindran helped in with post-transitional modifications needs to be further stud- gathering the clinical information. Dr. Hummel and Dr. Specks ied.24 Finally, mutations in EXT1 and EXT2 are associated with performed the western blotting studies. Dr. Ronco and Dr. Debiec an autosomal dominant disorder, hereditary multiple exosto- provided tissue for the validation cohort and also performed ses, which is one of the most common inherited skeletal dis- the western blot analysis and the immunofluorescence studies. orders.19,25 To the best of our knowledge, we are not aware of Dr. Fervenza and Dr. Ronco also critically read the manuscript. All any disorder associated with accumulation of EXT1 and EXT2. authors read the manuscript. By western and native blotting analysis in nonreducing conditions, we could not detect circulating anti-EXT1/EXT2 antibodies. This does not exclude a role for anti-EXT1/EXT2 DISCLOSURES antibodies for the following reasons. First, we used recombi- Dr. Sethi, Dr. Fervenza, Dr. Madden, and Dr. Charlesworth have patent nant EXT1/EXT2 proteins which might not appropriately pre- pending for identifying, testing and treatment of Membranous nephropathy sent the epitopes reactive with the patients’ antibodies. Second, associated with EXT1/EXT2. Dr. Ronco reports research fees from Alexion, the antibodies may circulate at very low concentrations and/or outside of the submitted work. be cleared from the blood due to a sink effect of the deposited antigen. It is unlikely that EXT1 and EXT2 are shed from the podocyte in response to the formation of immune complexes SUPPLEMENTAL MATERIAL involving other antigens, because EXT1/EXT2 appear to be specifictoawelldefined subset of MN with autoimmune This article contains the following supplemental material online at disease. The absence of detectable antibodies to EXT1/EXT2 http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ASN.2018080852/-/ raises the possibility that EXT1/EXT2 proteins may represent DCSupplemental.

JASN 30: 1123–1136, 2019 EXT1/EXT2-Associated Membranous Nephropathy 1135 CLINICAL RESEARCH www.jasn.org

Supplemental Table. Details of laser microdissection of EXT1/ 10. Miner JH: The glomerular basement membrane. Exp Cell Res 318: EXT2-associated MN cases. 973–978, 2012 Supplemental Figure 1. Immunohistochemical staining for EXT1 11. Chen YM, Miner JH: Glomerular basement membrane and related glomerular disease. Transl Res 160: 291–297, 2012 and EXT2 in EXT1/EXT2-associated MN cases and control cases. 12. Miner JH: Glomerular basement membrane composition and the fil- EXTL2 staining is also shown in three EXT1/EXT2-associated MN tration barrier. Pediatr Nephrol 26: 1413–1417, 2011 cases. 13. Raats CJ, Van Den Born J, Berden JH: Glomerular heparan sulfate al- Supplemental Figure 2. Representative mass spectrometry findings terations: Mechanisms and relevance for proteinuria. Kidney Int 57: – of control cases. 385 400, 2000 14. Sugar T, Wassenhove-McCarthy DJ, Orr AW, Green J, van Kuppevelt Supplemental Figure 3. Example of MS/MS spectra match to se- TH, McCarthy KJ: N-sulfation of heparan sulfate is critical for syndecan- quence from EXT1 and EXT2. 4-mediated podocyte cell-matrix interactions. Am J Physiol Renal Details of methods. Physiol 310: F1123–F1135, 2016 Clinical, laboratory and kidney biopsy findings of validation 15. McCarthy KJ, Wassenhove-McCarthy DJ: The glomerular basement cohort. membrane as a model system to study the bioactivity of heparan sulfate glycosaminoglycans. Microsc Microanal 18: 3–21, 2012 16. Sugar T, Wassenhove-McCarthy DJ, Esko JD, van Kuppevelt TH, Holzman L, McCarthy KJ: Podocyte-specificdeletionofNDST1,akeyenzymeinthe REFERENCES sulfation of heparan sulfate glycosaminoglycans, leads to abnormalities in podocyte organization in vivo. Kidney Int 85: 307–318, 2014 1. De Vriese AS, Glassock RJ, Nath KA, Sethi S, Fervenza FC: A proposal 17. Chen S, Wassenhove-McCarthy D, Yamaguchi Y, Holzman L, van for a serology-based approach to membranous nephropathy. JAmSoc Kuppevelt TH, Orr AW, et al.: Podocytes require the engagement of Nephrol 28: 421–430, 2017 cell surface heparan sulfate proteoglycans for adhesion to extracellular 2. Couser WG: Primary membranous nephropathy. Clin J Am Soc Nephrol matrices. Kidney Int 78: 1088–1099, 2010 12: 983–997, 2017 18. Busse-Wicher M, Wicher KB, Kusche-Gullberg M: The exostosin family: 3. Beck LH Jr., Bonegio RG, Lambeau G, Beck DM, Powell DW, Cummins Proteins with many functions. Matrix Biol 35: 25–33, 2014 TD, et al.: M-type phospholipase A2 receptor as target antigen in idi- 19. Ahn J, Lüdecke HJ, Lindow S, Horton WA, Lee B, Wagner MJ, et al.: opathic membranous nephropathy. N Engl J Med 361: 11–21, 2009 Cloning of the putative tumour suppressor gene for hereditary multiple 4. Tomas NM, Beck LH Jr., Meyer-Schwesinger C, Seitz-Polski B, Ma H, exostoses (EXT1). Nat Genet 11: 137–143, 1995 Zahner G, et al.: Thrombospondin type-1 domain-containing 7A in id- 20. Busse M, Kusche-Gullberg M: In vitro polymerization of heparan sulfate iopathic membranous nephropathy. NEnglJMed371: 2277–2287, backbone by the EXT proteins. JBiolChem278: 41333–41337, 2003 2014 21. Busse M, Feta A, Presto J, Wilén M, Grønning M, Kjellén L, et al.: 5. Tomas NM, Hoxha E, Reinicke AT, Fester L, Helmchen U, Gerth J, et al.: Contribution of EXT1, EXT2, and EXTL3 to heparan sulfate chain Autoantibodies against thrombospondin type 1 domain-containing 7A elongation. J Biol Chem 282: 32802–32810, 2007 induce membranous nephropathy. J Clin Invest 126: 2519–2532, 2016 22. Miner JH: Glomerular filtration: The charge debate charges ahead. 6. Ronco P, Debiec H: Pathophysiological advances in membranous ne- Kidney Int 74: 259–261, 2008 phropathy: Time for a shift in patient’scare.Lancet 385: 1983–1992, 23. Chen S, Wassenhove-McCarthy DJ, Yamaguchi Y, Holzman LB, van 2015 Kuppevelt TH, Jenniskens GJ, et al.: Loss of heparan sulfate glycos- 7. Ronco P, Debiec H: Pathogenesis of membranous nephropathy: Recent aminoglycan assembly in podocytes does not lead to proteinuria. advances and future challenges. Nat Rev Nephrol 8: 203–213, 2012 Kidney Int 74: 289–299, 2008 8. Nesvizhskii AI, Keller A, Kolker E, Aebersold R: A statistical model for 24. Gajjala PR, Fliser D, Speer T, Jankowski V, Jankowski J: Emerging role identifying proteins by tandem mass spectrometry. Anal Chem 75: of post-translational modifications in chronic kidney disease and car- 4646–4658, 2003 diovascular disease. Nephrol Dial Transplant 30: 1814–1824, 2015 9. Sethi S, Vrana JA, Theis JD, Dogan A: Mass spectrometry based 25. Cook A, Raskind W, Blanton SH, Pauli RM, Gregg RG, Francomano CA, proteomics in the diagnosis of kidney disease. Curr Opin Nephrol et al.: Genetic heterogeneity in families with hereditary multiple ex- Hypertens 22: 273–280, 2013 ostoses. Am J Hum Genet 53: 71–79, 1993

1136 JASN JASN 30: 1123–1136, 2019 Supplement: Table of contents

Page 2.Supplement table showing details of laser microdissection of EXT1/EXT2-associated MN cases

Page 3-7. Supplement figure 1: Immunohistochemical staining for EXT1 and EXT2 in EXT1/EXT2-associated MN cases and control cases. EXTL2 staining is also shown in 3 EXT1/EXT2-associated MN cases.

Page 8.Supplement figure 2: Representative mass spectrometry findings of control cases.

Page 9-10. Supplement figure 3: Example of MS/MS spectra match to sequence from EXT1 and EXT2.

Page 11-14. Details of methods.

Page 15-20. Clinical, laboratory and kidney biopsy findings of validation cohort.

Supplement table 1. Laser microdissection: number of glomeruli dissected and cut area/case.

Case Number of Cut area number glomeruli square dissected microns Case 1 8 127231 Case 2 25 642922 Case 3 8 143755 Case 4 30 572387 Case 5 28 570610 Case 6 10 234746 Case 7 28 573762 Case 8 17 255959 Case 9 21 335912 Case 10 23 350584 Case 11 35 589132 Case 12 20 614729 Case 13 21 531275 Case 14 9 132207 Case 15 NA NA Case 16 NA NA Case 17 25 538594 Case 18 6 169703 Case 19 12 142972 Case 20 NA NA Case 21 28 534491 Case 22 13 195501 Case 23 NA NA Case 24 19 542453 Case 25 18 544770 Case 26 NA NA

Supplement figure 1A: EXT1/EXT2-associated MN: Granular staining for EXT1 and EXT2 along the GBM in 5 cases of EXT1/EXT2 associated MN. The first column is case 1, column 2 is case 2, column 3 is case 5, column 4 is case 6 and column 5 is case 8 (top row EXT1, bottom row EXT2).

Supplement figure 1B: EXT1/EXT2-associated MN: Bright granular staining for EXT1 and EXT2 along the GBM in 5 cases of EXT1/EXT2 associated MN. The first column is case 9, column 2 is case 10, column 3 is case 11, column 4 is case 12 and column 5 is case 13 (top row EXT1, bottom row EXT2).

Supplement figure 1C: EXT1/EXT2-associated MN: Bright granular staining for EXT1 and EXT2 along the GBM in 5 cases of EXT1/EXT2 associated MN. The first column is case 15, column 2 is case 16, column 3 is case 17, column 4 is case 18 and column 5 is case 19 (top row EXT1, bottom row EXT2).

Supplement figure 1D: EXT1/EXT2-associated MN: Bright granular staining for EXT1 and EXT2 along the GBM in 5 cases of EXT1/EXT2 associated MN. The first column is case 20, column 2 is case 21, column 3 is case 22, column 4 is case 23 and column 5 is case 26 (top row EXT1, bottom row EXT2).

Supplement figure 1E: EXTL2 staining in 3 cases of EXT1/EXT2-associated MN: 2 cases of EXT1/EXT2-associated MN are negative for EXTL2and one shows minimal (1+) granular staining for EXTL2 (case 5, that showed very bright EXT1/EXT2 staining).

Supplement figure 1F: EXT1/EXT2 staining in control cases compared to EXT1/EXT2-associated MN: Bright granular staining for EXT1/EXT2 along the GBM in a case of EXT1/EXT2-associated MN (column 1- case 24), and negative in control cases (column 2- IgA nephropathy, column 3- minimal change disease, column 4- FSGS and column 5-diabetic glomerulosclerosis) (top row EXT1, bottom row EXT2).

Supplement figure 2. Representative mass spectrometry findings of control cases: Low spectral counts of PLA2R and no detectable spectral counts of EXT1 or EXT2 are present in the control cases. The last 2 columns are from 2 cases of PLA2R-negative MN that were also negative for EXT1 and EXT2. Proteins inherent to the GBM such as laminin, nestin, nidogen are also shown (MCD-minimal change disease; PLA2R negative case-PLA2R-negative primary membranous nephropathy).

Supplement Figure 3.

A. The MS/MS spectra figure from Scaffold viewer of ion 867.96 [M+2H]2+ highlighting the detected b-ions (red) and y-ions (blue) matching the theoretical fragment masses listed in the table for the Exostosin-1 peptide IAESYQNILAAIEGSR.

B. The MS/MS spectra figure from Scaffold viewer of ion 1278.56 [M+2H]2+ highlighting the detected b-ions (red) and y-ions (blue) matching the theoretical fragment masses listed in the table for the Exostosin-2 peptide EYNELLMAISDSDYYTDDINR.

Supplement Methods

LASER MICRODISSECTION AND MASS SPECTROMETRY

Formalin fixed paraffin embedded (FFPE) renal biopsy materials were sent to the Mayo Clinic renal biopsy laboratory for diagnosis of membranous nephropathy. For each case, 10µM thick paraffin sections were obtained and mounted on PEN membrane laser microdissection slides. The sections were deparaffinized using xylene and alcohol. Using a Zeiss Palm Microbean microscope and Robopalm software, multiple glomeruli were microdissected to reach approximately 250-500,000µM2 per case, and catapulted into 35µl of digest buffer (100mM Tris, pH 8.5/0.002% Zwittergent Z3-16) in the cap of a 0.5 ml tube. The tube was removed from the collection plate and spun at 14000g x 2minutes. The samples were frozen until all samples were collected. Upon thawing, samples were heated to 98°C, then proteins were reduced and alkylated by sequential addition of TCEP (Tris(2-carboxyethyl) phosphine hydrochloride) and iodoacetamide to 10mM for 30 minutes each. Trypsin (0.05 µg) was added to each tube and proteins were digested overnight at 37°C for 16-18 hours. After digestion, the samples were acidified with trichloroacetic acid, dried down and resolubilized with A solvent for mass spectrometry.

The trypsin digested peptides were identified by nano-flow liquid chromatography electrospray tandem mass spectrometry (nanoLC-ESI- MS/MS) using a Thermo Scientific Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a Thermo Ultimate 3000 RSLCnano HPLC system. The peptide mixture was loaded onto a 250nl OPTI-PAK trap (Optimize Technologies, Oregon City, OR) custom packed with Michrom Magic C 8, 5µm solid phase (Michrom Bioresources, Auburn, CA). Chromatography was performed using 0.2 % formic acid in both the A solvent (98%water/2%acetonitrile) and B solvent (80% acetonitrile/10% isopropanol/10% water), and a 5%B to 40%B gradient over 90 minutes at 400 nl/min through a PicoFrit (New Objective, Woburn, MA) 100µm x 35cm column handpacked with Agilent Poroshell 120 EC C18 packing. The Q-Exactive mass spectrometer experiment was a data dependent set up with the MS1 survey scan from 340-1500 m/z at resolution 70,000 (at 200m/z), followed by HCD MS/MS scans on the top 15 ions having a charge state of +2, +3, or +4, at resolution 17,500. The ions selected for MS/MS were placed on an exclusion list for 30 seconds. The MS1 AGC target was set to 1e6 and the MS2 target is set to 1e5 with max ion inject times of 50ms for both.

DATABASE SEARCHING

Tandem mass spectra was extracted by msconvert version 3.0.9134. Charge state deconvolution and deisotoping was not performed. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.4.0) and X! Tandem (The GPM, thegpm.org; version X!Tandem Sledgehammer (2013.09.01.1)). Mascot and X! Tandem were set up to search a Swissprot human database with reverse decoy (40570 entries) assuming the digestion enzyme strict trypsin and with a fragment ion mass tolerance of 0.020 Da and a parent ion tolerance of 10.0 PPM. Glu- >pyro-Glu of the n-terminus, ammonia-loss of the n-terminus, gln->pyro-Glu of the n-terminus, oxidation of methionine is specified in X! Tandem as variable modifications and carbamidomethyl of cysteine was specified as a fixed modification. Oxidation of methionine and carbamidomethyl of cysteine were specified in Mascot as variable modifications and fixed modifications respectively.

CRITERIA FOR PROTEIN IDENTIFICATION Scaffold (version Scaffold_4.8.3, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least 2 identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm.7The protein decoy false discovery rate (FDR) was <1.5.In general, over 1500-2500protein were identified in each sample. Proteins that contain similar peptides and cannot be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. Proteins sharing significant peptide evidence were grouped into clusters. Protein comparisons were made with ratios of Scaffold normalized total spectral counts. The ‘Spectra’ value indicates the total number of mass spectrum collected on the mass spectrometer and matched to the protein using the proteomics software. A higher number of mass spectra is indicative of greater abundance and will typically yield greater amino acid sequence coverage. A higher mass spectra value also indicates a higher confidence in the protein identification.

IMMUNOHISTOCHEMICAL STAINING FOR EXOSTOSIN 1 (EXT1),EXOSTOSIN 1 (EXT2)AND EXOSTOSIN-LIKE 2 (EXTL2):

Tissue sectioning and immunohistochemical (IHC) staining was performed at the Pathology Research Core (Mayo Clinic, Rochester, MN) using the Leica Bond RX stainer (Leica). FFPE tissues were sectioned at 5 microns and IHC staining was performed on-line. Slides for the EXT1 stain were retrieved for 20 minutes using Epitope Retrieval 2 (EDTA; Leica) and incubated in Protein Block (Dako) for 5 minutes. The EXT1 primary antibody (rabbit polyclonal, Thermo Scientific #PA5-60699) and EXT2 antibody (rabbit polyclonal; Abcam) was diluted to 1:100 in Background Reducing Diluent (Dako) and incubated for 15 minutes. Slides for the EXTL2 stain were retrieved for 20 minutes using Epitope Retrieval 1 (Citrate; Leica) and incubated in Protein Block (Dako) for 5 minutes. The EXTL2 primary antibody (rabbit polyclonal, Origene #TA590761) was diluted to 1:1400 in Background Reducing Diluent (Dako) and incubated for 15 minutes. The detection system used was Polymer Refine Detection System (Leica). This system includes the hydrogen peroxidase block, post primary and polymer reagent, DAB, and Hematoxylin. Immunostaining visualization was achieved by incubating slides 10 minutes in DAB and DAB buffer (1:19 mixture) from the Bond Polymer Refine Detection System. To this point, slides were rinsed between steps with 1X Bond Wash Buffer (Leica). Slides were counterstained for five minutes using Schmidt hematoxylin and molecular biology grade water (1:1 mixture), followed by several rinses in 1X Bond wash buffer and distilled water, this is not the hematoxylin provided with the Refine kit. Once the immunochemistry process was completed, slides were removed from the stainer and rinsed in tap water for five minutes. Slides were dehydrated in increasing concentrations of ethyl alcohol and cleared in 3 changes of xylene prior to permanent cover slipping in xylene-based medium.

IMMUNOFLUORESCENCE STAINING FOR EXT1 AND EXT2

Immunofluorescence staining was performed on FFPE sections retrieved for 30 min using target retrieval solution high pH (Dako). The EXT1 primary antibody (rabbit polyclonal, Thermo Scientific #PA5-60699) and EXT2 primary antibody (rabbit polyclonal; Abcam) were diluted to 1:100 in blocking solution (2% fetal calf serum and 2% normal goat serum) and incubated overnight at 4°C with retrieved biopsy sections . Next, the slides were incubated with goat Alexa488-conjugated anti-rabbit Fab IgG antibodies as secondary antibody (Life technologies). Finally, slides were mounted in mounted medium (Thermo Scientific) and covered with LDS2460EP cover glasses. Stained sections were evaluated by using inverse microscope Olympus IX83.

WESTERN BLOTTING

The protein samples, recombinant human EXT1, recombinant human EXT1/EXT2 heterodimer (both R&D Systems) and recombinant human PLA2R1 (Origene Technologies), were diluted with non-reducing Laemmli sample buffer (Bio-Rad) and boiled for 5 min. Samples were loaded into Criterion 4-15% TGX gels (Bio-Rad) and electrophoresed in Tris-glycine-SDS running buffer. Proteins were transferred to poly (vinylidene difluoride) membranes according to standard protocols, then membranes were blocked with Pierce Protein-Free Blocking buffer (Thermo Scientific). Membranes were incubated overnight at 4°C with sera from patients, controls (dilution 1:50) and rabbit polyclonal antibodies against EXT1 and EXT2 (Thermo Scientific and Abcam, respectively). Subsequently, blots were washed and incubated for 2 h at room temperature with goat anti-human IgG, HRP conjugate (Millipore) or with goat anti-rabbit HRP conjugated secondary antibody. Immunoreactive proteins were visualized with SuperSignal West Pico Chemiluminescent substrate (Pierce) followed by luminescence detection with Ozyme Syngene LED imager.

Slot blot native blotting. Protein slot blotting was done using Bio-Dot SF assembly apparatus (Bio-Rad). EXT1 and EXT1/2 proteins were added to the wells. We used nitrocellulose membrane to slot recombinant EXT1 and EXT1/2 proteins under vacuum. All blocking and washing steps of the membrane were done according to manufacturer's instruction. Subsequently, membranes were removed from the apparatus and small pieces with antigens were prepared for overnight incubation with sera from patients (dilution 1:50) and rabbit polyclonal antibodies against EXT1 and EXT2. Subsequently slots were washed and incubated with goat-anti human or goat anti-rabbit IgG, AP conjugate (Sigma). Immunoreactive proteins were visualized with BCIP/NBT liquid substrate system (Sigma).

Table 1: Clinical and laboratory findings of Validation Cohort

C3 Age at kidney biopsy/ Serum Year of Proteinuria (0.74 -1.43)/ ANA/ dsDNA/ Clinical N° Code Exostosin Gender/ Rash/Arthritis/Other Creatinine biopsy (g/24 hours) C4 Other* presentation** Ethnicity (mg/dL) (0.19-0.43g/L)

1 "Primary" (double negative PLA2R and THSD7A) MN 1 T2N00129 2012 - 28/M/African N/N/N 0.7 5.3 2.18/0.62 80/neg/NS Nephrotic Sd 2 12N00288 2012 - 58/M/African N/N/N 1 2.9 1.19/0.49 ANA neg/neg/NS Nephrotic Sd N/N/ polyneuropathy, 3 14TN330 2014 - 61/M/Caucasian Sjögren 0.8 4.8 NA/NA ANA neg/neg/NS Nephrotic Sd 4 15TN00498 2015 - 27/F/African N/N/N 0.6 6.1 NA NA Nephrotic Sd 5 11N00262 2011 - 54/F/Caucasian N/N/N 0.6 12 1.7/0.41 1280/neg/Anti-centromere Nephrotic Sd 6 T08E00791 2008 - 40/F/Caucasian Y/N/N 0.9 17.3 1.12/0.26 80/neg/neg Nephrotic Sd N/N/Y myalgias, 7 05H07788 2005 - 58/F/Sri Lanka polyadenopathies 0.7 2.8 1.47/NA 200/neg/neg Nephrotic Sd 8 09H05897 2008 - 42/M/Caucasian Y/N/Y Raynaud 0.9 0.75 NA/NA 160/neg/neg Proteinuria 9 08H0466 2008 - 76/M/Maghrebian N/N/N 1 6.0 NA/NA ANA neg/neg/neg Nephrotic Sd N/N/Raynaud, sicca 10 07E 00106 2007 - 34/F/Caucasian syndrome 0.7 1.5 0.93/0.18 160/neg/neg Proteinuria 11 06H06087 2006 - 84/F/Caucasian N/N/N 0.7 0.7 0.97/0.13 NA Nephrotic Sd 12 18TN00253 2018 - 26/F/African N/N/N 0.6 3.4 1.23/0.20 1280/neg/SSA Proteinuria 13 12N00418 2012 - 41/M/African N/N/N 0.9 0.2 1.12/0.12 640/neg/NS Nephrotic Sd 14 06H11586 2006 + 24/F/Maghrebian N/N/N 0.6 1.2 0.96/0.17 ANA neg/neg/SSA Nephrotic Sd

Y/Y/thrombocytopenic 12N271 2012 + purpura 0.5 1.8 0.73/0.11 1280/neg/Sm.RNP.SSA.B Clinical lupus

15 18TNN00285 2018 + 70/M/African N/N/N 2.2 13.3 0.70/0.30 1280/neg/Sm.RNP.SSA Nephrotic Sd 16 T07H03197 2007 + 21/F/Caucasian N/N/N 0.7 NA 1.43/0.35 1000/neg/RNP Nephrotic Sd T08H07707 2008 + Y/N/Y pleuritis 0.7 5.7 0.86/0.37 320/neg/RNP Clinical lupus 18TN00273 2018 + N/N/N 0.5 4.8 0.71/0.12 1200/neg/RNP Asymptomatic lupus

2.Class V lupus MN

17 17TN00433 2017 + 20/F/Caucasian Hispanic N/Y/Y 0.5 6.3 1.04/0.30 No/8/SSA.Ro Clinical lupus N/N/thrombocytopenic 18 18TN00197 2018 + 49/F/African purpura 0.7 2.6 N/0.15 320/ neg/Sm. SSA. RNP Proteinuria 19 T08H10866 2008 + 27/F/African Y/Y/N 0.8 5.3 1.26/low C4 1280/NA/SSA Clinical lupus 17TN00426 2017 + Y/N/N 0.7 0.5 0.68/0.10 1280/26/Sm. SSA. RNP Clinical lupus

20 T05H08855 2005 + 26/F/Guyane Y/Y/Y lung emboli 0.8 10 0.52/0.04 1000/340/Sm. SSA. RNP Clinical lupus T06H02989 2006 + N/N/N 0.8 2 0.71/0.08 1000/68/SSA Asymptomatic lupus

21 T00H02015 2000 + 46/F/African Y/Y/Y sicca syndrome 0.5 3 0.74/0.2 1000/neg/RNP APLS & clinical lupus N/N/Y hemolysis 22 T01H01069 2001 + 38/F/African Coombs pos 0.7 7 N/0.16 1000/23/SSA Renal lupus N/Y/Y lung, lymph 23 T13N00303 2013 + 38/M/African nodes 1.0 1.4 N/N 1280/neg/Sm. RNP. SSA Clinical lupus 17TN00061 2017 + N/N/Y lymph nodes 0.7 2.8 0.19/0.07 1280/neg/Sm. RNP. SSA Clinical lupus

24 T13N00177 2013 + 45/F/African Y/Y/Y pericarditis 0.7 3.2 0.85/0.15 1280/neg/Sm.RNP Clinical lupus 25 14TN00361 2014 - 32/F/African&Caucasian Y/Y/N 0.7 0.7 0.62/0.15 1280/119/SSA Clinical lupus 26 T02H10849 2002 - 40/F/Maghrebian Y/Y/Y vasculitis 0.7 1.1 0.49/0.08 4000/27/neg Clinical lupus 27 T03H00890 2003 - 41/F/Caucasian Y/Y/N 0.9 <0.1 1.09/0.29 200/neg/NS Clinical lupus N/N/Y diarrhea, lymph 28 T04H09991 2004 - Pakistani node 1.0 6.1 N/ 0.04 1000/neg/Sm. RNP Clinical lupus 29 T11N00384 2011 - 35/F/Guyana N/Y/N 0.7 0.4 0.93/0.13 1280/150/SSA Clinical lupus Y/N/Y colitis, spinal 30 T05H01461 2005 - 54/M/Maghrebian amyotrophy 0.7 0.4 0.51/0.05 NA/NA Clinical lupus 31 18TN00204 2018 - 58/F/African N/Y/Y pericarditis 0.6 2 1.68/0.33 1280/neg/Sm. RNP Clinical lupus 32 T12N00232 2012 - 45/F/Caucasian Y/Y/Y neurolupus 0.5 3 0.74/0.22 320/24/NS Clinical lupus 33 17TNN00327 2017 - 35/M/African Y/Y/Y 0.6 0.8 0.58/0.12 1280/157/Sm. RNP.SSA Clinical lupus

34 12N00258 2012 - 55/F/Caucasian N/Y/Sjögren 0.7 8.9 1.28/0.24 160/neg/SSA Clinical lupus

3. Mixed class (V+III/IV) lupus nephritis

35 13N00201 2013 + 34/M/African Y/N/Y stroke 1.1 1.4 0.78/0.04 1280/231/Sm.RNP Clinical lupus 36 T03H10353 2003 - 48/F/African N/Y/Y seritis, Sjögren 1.1 1 0.66/0.18 1000/180/NS Clinical lupus N/N/ Y seritis, pseudo- 37 03H00679 2003 - 17/F/African lymphoma 1.5 6 0.51/0.03 NA Clinical lupus 38 07E 00028 2007 - 16/F/ Caucasian NA NA NA NA NA Proteinuria N/N/Y Raynaud 39 T1N00246 2011 - 42/F/Chinese syndrome 0.6 5.9 1.08/0.21 1280/neg/neg Clinical lupus Y/Y/Y hematological, 40 T13N00124 2013 - 38/F/African pancreatitis 0.7 5.0 0.81/0.18 NA/985/RNP.SSA Nephrotic Sd 41 T13N00089 2013 - 64/F/Maghrebian NA ESKD ESKD NA NA ESKD N/Y/ hematological, 42 14TN00098 2014 - 49/M/Asian (Vietnamese) pericarditis 1.9 0.8 0.14/0.02 2800/44/ECT Clinical lupus Y/Y/Y seritis, 43 14TN00109 2014 - 29/ F/Asian hematological 0.8 1.55 0.42/0.06 640/46/SSA Proteinuria/hematuria 14TN00312 sept-14 - 1 0.4 0.96/0.24 NA/NA Asymptomatic lupus Y/N/Y 44 14TN00316 2014 - 26/M/Caucasian hematological&SAPL 0.9 1.1 0.17/0.02 1280/50/RNP.SSA Clinical lupus 45 17N00320 2017 - 55/F/Maghrebian Y/Y/N 1.3 2 C3 normal/0.18 160/neg/NS Asymptomatic lupus 46 18TN00279 2018 - 30/F/Asian (Cambodian) Y/Y/Raynaud 0.8 3.0 0.61/0.07 1280/109/Sm. RNP. SSA Clinical lupus Y/Y/Y hematological, 47 14TN00235 2014 - 43/F/Caucasian neurolupus 0.8 3.3 0.6/0.08 320/50/NS Nephrotic Sd 48 17834 2000 - 34/M/Maghrebian Y/Y/Y 66 0.2 0.47/0.10 1000/16/RNP.SSA Asymptomatic lupus

*ANA=antinuclear antibody, dsDNA= anti-double stranded DNA antibody; RNP=ribonucleoprotein, SSA/B=Sjögren syndrome antibody A/B, NA=not available, NS=not specified

**Sd=syndrome

Table 2: Kidney biopsy findings of Validation Cohort

Glomeruli/ Mesangial or endocapillary Interstitial Inflammation/ Immunofluorescence N° Code Arteries Pathological diagnosis IgG Subclass sclerosed hypercellularity* IFTA microscopy

1. "Primary" (double negative PLA2R and THSD7A) MN

1 T2N00129 6/0 Not present 0/0 Normal G subepithelial MN stage I Ig4=3>1 2 12N00288 19/6 Not present Mild/mild Intimal fibrosis G subepithelial, mesangial MN stage II IgG1=3>2 3 14TN330 7/0 Not present 0/0 Intimal fibrosis G, A (weak), C3 subepithelial MN stage II IgG1 4 15TN00498 18/1 Not present 0/0 Intimal fibrosis G subepithelial MN stage I IgG4 5 11N00262 26/0 Not present 0/0 Normal G,C3 subepithelial MN stage I

6 T08E00791 19/0 Not present 0/0 Normal G,C3,C1q subepithelial MN stage I

7 05H07788 5/1 Not present 0/0 Normal G,C3,C1q subepithelial, MN stage I , segmental

8 09H05897 12/0 Not present 0/0 Normal G,M,C3 subepithelial MN stage II,III

9 08H0466 15/4 Not present Mild/mild Intimal fibrosis G subepithelial MN stage II

10 07E 00106 22/4 Not present 0/mild Normal G subepithelial MN stage III

11 06H06087 8/3 Not present Severe (CLL)/mild Normal G,C3 subepithelial kappa & lambda MN stage III

12 18TN00253 9/0 Not present 0/0 Normal G,M,C3 MN stage I IgG4>G1=G2 13 12N00418 28/1 Not present 0/mild Normal G subepithelial, C3 arterioles MN stage II IgG1>3>2 14 06H11586 31/0 Prolif. mes., small crescents 0/mild Normal G, C3 subepithelial MN stage I-II

12N271 22/1 Not present 0/0 Normal G,C3 subepithelial MN stage I-II

15 18TNN00285 10/1 Not present 0/0 Normal G,C3 subepithelial MN stage I IgG4>G1 16 T07H03197 19/0 Not present 0/0 Normal G, C3 subepithelial; M, mesangial MN stage I

T08H07707 26/0 Prolif.endo., thrombi Mild/0 Normal G,A,M,C3,C1q Class III S-A+ V

18TN00273 18/1 Not present 0/0 Normal G,C3,C1q Class V

2. Class V lupus MN

17 17TN00433 29/0 Not present 0/0 Normal G,A,M,C3 MN stage I

18 18TN00197 12/0 Not present 0/0 Normal G, C3, C1q MN stage I IgG1 >IgG2 19 T08H10866 15/0 Not present 0/0 Normal G,C3,C1q subepithelial, M mesangial Class V

17TN00426 8/0 Not present 0/0 Normal G,C3 MN stage I

20 T05H08855 10/0 Prolif. endo., crescents Mild/mild Normal G,A,C3,C1q Class IV-(G)A+ V

T06H02989 6/0 Prolif. decreased 0/0 Normal G,A,C3,C1q Class V G,A,C3,C1q subepithelial, G,M 21 T00H02015 10/1 Not present 0/0 Normal mesangial Class V G, (A), C3, C1q subepithelial, C1q 22 T01H01069 8/0 Not present 0/0 Normal endomembranous Class V

23 T13N00303 20/0 Not present 0/0 Normal G,C3,C1q MN stage 1 (Class V?) IgG1 17TN00061 34/2 Not present Severe/0 Normal G,C3, (A), (C1q) MN stage 2 IgG1,2,3 &4 G,C3 subepithelial, G M C3C1q 24 T13N00177 11/0 Not present 0/0 No artery mesangial Class V G,C3, C1q, subepithelial; GAMC3C1q 25 14TN00361 15/2 Mild mes. prolif. 0/0 Normal mesangial Class V G,A, C3,C1q subepithelial, M 26 T02H10849 9/2 Not present 0/mild Normal mesangial Class V G,A,M,C3,C1q subepithelial, 27 T03H00890 23/2 Mild mes. prolif. Mild/mild Normal mesangial(small) Class V G,A,C3, C1q subepith, G, A, M, C3 28 T04H09991 17/2 Prolif. mes. Focal /0 Mild endarteritis subendothelial Class V

29 T11N00384 08/1 Not present 0/0 Intimal fibrosis G subepithelial, M, C3 parietal Class V G,A,C3,C1q, subepithelial and 30 T05H01461 19/3 Not present mild/mild Fibrous endarteritis vascular Class V

31 18TN00204 27/2 Not present 0/mild Intimal fibrosis G,A,C3 subepithelial MN stage 1 (Class V?)

32 T12N00232 18/5 Not present 0/APS-related fibrosis Thrombosis (APS) G, C3, C1q subepithelial Class V + APS

33 17TNN00327 15/2 Not present 0/mild Normal G,C3 subepithelial Class V IgG3 34 12N00258 20/2 Not present 0/0 Intimal fibrosis G subepithelial, mesangial MN stage 1 IgG4=2>1=3

3. Mixed class (V+III/IV) lupus nephritis

35 13N00201 6/0 Prolif.mes. (1G)+ 1 crescent Severe (C/M)/tubulitis Intimal fibrosis G,C3 subepithelial, M,C3 mesangial Class III+V IgG2>1=3=4 36 T03H10353 23/2 Prolif.endo., necrosis, crescent Mild/mild Endarteritis (APLS) NA Class III+V

37 03H00679 26/0 Prolif.endo., mes., crescent 0/mild One thrombosis G,A,M,C3,C1qsubepitelial, mesangial Class III+V

38 07E 00028 14/5 Not present 0/mild Normal G,A,C3,C1q subepithelial Class IVc +V

39 T1N00246 24/0 Prolif. endo. 0/mild Intimal fibrosis G,M, C3, C1q subepithelial Class III + V stage II, III G,A,M,C3,C1q subepithelial and 40 T13N00124 18/3 Scars, synechias Mild/o N endomembranous Class IV-S (C:68%) + V Class IV-G (A, 27%; C, 41 T13N00089 18/9 Prolif. endo. &extracapillary Severe/severe Intimal fibrosis G,A,C3 subepithelial 78%) + V G=C1q> A=C3, subepithelial and 42 14TN00098 16/2 Prolif. endo. & thrombi 0/mild Intimal fibrosis endomembranous Class IV-S (A) + V Class III (A,14%; C, 27%) + 43 14TN00109 22/1 Prolif. endo. & extracapillary Mild/0 Intimal fibrosis G,A, C3, C1q subepithelial and mes. V G,A,C3 subepithelial, C1Q 14TN00312 21/5 Scars 0/0 Normal endomembranous segmental Class IIIC + V G,C1q,C3 subepithelial &endo, A,M 44 14TN00316 9/0 Prolif. endo. 0/0 Normal endomembranous Class IIIA + V

45 17N00320 11/5 Not present 0/mild Intimal fibrosis G,A subepithelial Class IVC + V G,A,M,C3,C1q subepithelial & 46 18TN00279 19/0 Prolif. endo. Mild/mild Normal endomembranous Class IIIA + V

47 14TN00235 17/4 Prolif. endo. 0/mild Normal G,A,M,C3,C1q, subepithelial & endo. Class IV-G (A/C) + V

48 INSERM 17834 21/0 Prolif. endo. &extracapillary Mild/mild Intimal fibrosis NA Class III+V

*Prolif.=proliferation, mes.=mesangial, endo.=endothelial, 1G=one glomerulus