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• W4367316319 abstract "Drugs are an important secondary cause of membranous nephropathy (MN) with the most common drugs associated with MN being nonsteroidal anti-inflammatory drugs (NSAIDs). Since the target antigen in NSAID-associated MN is not known, we performed laser microdissection of glomeruli followed by mass spectrometry (MS/MS) in 250 cases of PLA2R-negative MN to identify novel antigenic targets. This was followed by immunohistochemistry to localize the target antigen along the glomerular basement membrane and western blot analyses of eluates of frozen biopsy tissue to detect binding of IgG to the novel antigenic target. MS/MS studies revealed high total spectral counts of a novel protein Proprotein Convertase Subtilisin/Kexin Type 6 (PCSK 6) in five of the 250 cases in the discovery cohort. A validation cohort using protein G immunoprecipitation, MS/MS, and immunofluorescence detected PCSK6 in eight additional cases. All cases were negative for known antigens. Ten of 13 cases had a history of heavy NSAID use with no history available in one case. The mean serum creatinine and proteinuria at kidney biopsy were 0.93 ± 0.47 mg/dL and 6.5 ± 3.3 gms/day, respectively. Immunohistochemistry/immunofluorescence showed granular staining for PCSK6 along the glomerular basement membrane, and confocal microscopy showed co-localization of IgG and PCSK6. IgG subclass analysis in three cases revealed codominance of IgG1 and IgG4. Western blot analysis using eluates from frozen tissue showed IgG binding to PCSK6 in PCSK6-associated but not in PLA2R-positive MN. Thus, PCSK6 may be a likely novel antigenic target in MN in patients with prolonged NSAID use. Drugs are an important secondary cause of membranous nephropathy (MN) with the most common drugs associated with MN being nonsteroidal anti-inflammatory drugs (NSAIDs). Since the target antigen in NSAID-associated MN is not known, we performed laser microdissection of glomeruli followed by mass spectrometry (MS/MS) in 250 cases of PLA2R-negative MN to identify novel antigenic targets. This was followed by immunohistochemistry to localize the target antigen along the glomerular basement membrane and western blot analyses of eluates of frozen biopsy tissue to detect binding of IgG to the novel antigenic target. MS/MS studies revealed high total spectral counts of a novel protein Proprotein Convertase Subtilisin/Kexin Type 6 (PCSK 6) in five of the 250 cases in the discovery cohort. A validation cohort using protein G immunoprecipitation, MS/MS, and immunofluorescence detected PCSK6 in eight additional cases. All cases were negative for known antigens. Ten of 13 cases had a history of heavy NSAID use with no history available in one case. The mean serum creatinine and proteinuria at kidney biopsy were 0.93 ± 0.47 mg/dL and 6.5 ± 3.3 gms/day, respectively. Immunohistochemistry/immunofluorescence showed granular staining for PCSK6 along the glomerular basement membrane, and confocal microscopy showed co-localization of IgG and PCSK6. IgG subclass analysis in three cases revealed codominance of IgG1 and IgG4. Western blot analysis using eluates from frozen tissue showed IgG binding to PCSK6 in PCSK6-associated but not in PLA2R-positive MN. Thus, PCSK6 may be a likely novel antigenic target in MN in patients with prolonged NSAID use. Lay SummaryMembranous nephropathy (MN) results from accumulation of antigen–antibody complexes along the glomerular basement membranes. Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been associated with MN. However, the target antigen in these patients is not known. Using mass spectrometry studies, this study shows that a novel protein—proprotein convertase subtilisin/kexin type 6 (PCSK6)—is the likely target antigen in MN in patients with prolonged use of NSAIDs. Thus, the finding of PCSK6 on biopsy in MN can point to the underlying trigger—that is, NSAID use in this subgroup of MN patients. Further studies are needed to determine whether circulating antibodies to PCSK6 can be detected in the serum and whether patients can be treated and followed based on determination of anti-PCSK6 titers in the serum. The discovery of PCSK6 in MN in patients with prolonged NSAID use as the likely target antigen provides an etiology and framework for future studies in this group of patients. Membranous nephropathy (MN) results from accumulation of antigen–antibody complexes along the glomerular basement membranes. Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been associated with MN. However, the target antigen in these patients is not known. Using mass spectrometry studies, this study shows that a novel protein—proprotein convertase subtilisin/kexin type 6 (PCSK6)—is the likely target antigen in MN in patients with prolonged use of NSAIDs. Thus, the finding of PCSK6 on biopsy in MN can point to the underlying trigger—that is, NSAID use in this subgroup of MN patients. Further studies are needed to determine whether circulating antibodies to PCSK6 can be detected in the serum and whether patients can be treated and followed based on determination of anti-PCSK6 titers in the serum. The discovery of PCSK6 in MN in patients with prolonged NSAID use as the likely target antigen provides an etiology and framework for future studies in this group of patients. Chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with the entity described as analgesic nephropathy and is characterized by a chronic interstitial nephritis.1De Broe M.E. Elseviers M.M. Analgesic nephropathy.N Engl J Med. 1998; 338: 446-452Crossref PubMed Scopus (130) Google Scholar A less common side effect of chronic NSAID use is its association with development of nephrotic syndrome and membranous nephropathy (MN).2Clive D.M. Stoff J.S. Renal syndromes associated with nonsteroidal antiinflammatory drugs.N Engl J Med. 1984; 310: 563-572Crossref PubMed Scopus (992) Google Scholar, 3Radford Jr., M.G. Holley K.E. Grande J.P. et al.Reversible membranous nephropathy associated with the use of nonsteroidal anti-inflammatory drugs.JAMA. 1996; 276: 466-469Crossref PubMed Google Scholar, 4Mérida E. Praga M. NSAIDs and nephrotic syndrome.Clin J Am Soc Nephrol. 2019; 14: 1280-1282Crossref PubMed Scopus (20) Google Scholar, 5Nawaz F.A. Larsen C.P. Troxell M.L. Membranous nephropathy and nonsteroidal anti-inflammatory agents.Am J Kidney Dis. 2013; 62: 1012-1017Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 6Bakhriansyah M. Souverein P.C. van den Hoogen M.W.F. et al.Risk of nephrotic syndrome for non-steroidal anti-inflammatory drug users.Clin J Am Soc Nephrol. 2019; 14: 1355Crossref PubMed Scopus (22) Google Scholar, 7Tattersall J. Greenwood R. Farrington K. Membranous nephropathy associated with diclofenac.Postgrad Med J. 1992; 68: 392Crossref PubMed Scopus (11) Google Scholar MN associated with NSAID use is thought to be reversible with discontinuation of NSAID use.3Radford Jr., M.G. Holley K.E. Grande J.P. et al.Reversible membranous nephropathy associated with the use of nonsteroidal anti-inflammatory drugs.JAMA. 1996; 276: 466-469Crossref PubMed Google Scholar,7Tattersall J. Greenwood R. Farrington K. Membranous nephropathy associated with diclofenac.Postgrad Med J. 1992; 68: 392Crossref PubMed Scopus (11) Google Scholar,8Campistol J.M. Galofre J. Botey A. et al.Reversible membranous nephritis associated with diclofenac.Nephrol Dial Transplant. 1989; 4: 393-395Crossref PubMed Scopus (20) Google Scholar Although rare case reports have been made of phospholipase A2 receptor (PLA2R) positivity in NSAID-associated MN,5Nawaz F.A. Larsen C.P. Troxell M.L. Membranous nephropathy and nonsteroidal anti-inflammatory agents.Am J Kidney Dis. 2013; 62: 1012-1017Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar in most cases, the target antigen in NSAID-associated MN is not known. We have recently used laser microdissection and tandem mass spectrometry (MS/MS) to detect novel target antigens in MN, such as neural tissue encoding protein with epidermal growth factor (EGF)–like repeats (NELL1), exostosin 1/exostosin 2 (EXT1/EXT2), semaphorin 3B (SEMA3B), protocadherin 7 (PCDH7), neural cell adhesion molecule (NCAM1), protocadherin FAT1 (FAT1), and neuron-derived neurotrophic factor (NDNF).9Sethi S. New ‘antigens’ in membranous nephropathy.J Am Soc Nephrol. 2021; 32: 268Crossref PubMed Scopus (89) Google Scholar, 10Sethi S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2–associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (141) Google Scholar, 11Sethi S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Sethi S. Debiec H. Madden B. et al.Semaphorin 3B–associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients.Kidney Int. 2020; 98: 1253-1265Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Sethi S. Madden B. Debiec H. et al.Protocadherin 7-associated membranous nephropathy.J Am Soc Nephrol. 2021; 32: 1249-1261Crossref PubMed Scopus (47) Google Scholar, 14Sethi S. Madden B. Casal Moura M. et al.Hematopoietic stem cell transplant-membranous nephropathy is associated with protocadherin FAT1.J Am Soc Nephrol. 2022; 33: 1033-1044Crossref PubMed Scopus (16) Google Scholar, 15Caza T.N. Hassen S.I. Kuperman M. et al.Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis.Kidney Int. 2021; 100: 171-181Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Sethi S. Madden B. Casal Moura M. et al.Membranous nephropathy in syphilis is associated with neuron-derived neurotrophic factor.J Am Soc Nephrol. 2023; 34: 374-384Crossref PubMed Scopus (5) Google Scholar Using similar methodology, we now show that a novel protein—proprotein convertase subtilisin/kexin type 6 (PCSK6)—is the likely target antigen in MN in patients with prolonged use of NSAIDs. Biopsies received in the Renal Pathology Laboratory, Department of Laboratory Medicine and Pathology, Mayo Clinic, for diagnosis and interpretation between 2017 and 2021 were evaluated. The diagnosis of MN was confirmed by light microscopy, immunofluorescence (IF) microscopy including PLA2R studies, and electron microscopy (EM). The clinical information was obtained from the accompanying charts. The study was approved by the Mayo Clinic Institutional Review Board. For detection of novel proteins, we performed MS/MS in 250 cases of PLA2R-negative cases (Mayo Clinic discovery cohort) that included cases used for identification of EXT1/EXT2, NELL1, SEMA3B, PCDH7, FAT1, and NDNF.10Sethi S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2–associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (141) Google Scholar, 11Sethi S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Sethi S. Debiec H. Madden B. et al.Semaphorin 3B–associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients.Kidney Int. 2020; 98: 1253-1265Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Sethi S. Madden B. Debiec H. et al.Protocadherin 7-associated membranous nephropathy.J Am Soc Nephrol. 2021; 32: 1249-1261Crossref PubMed Scopus (47) Google Scholar, 14Sethi S. Madden B. Casal Moura M. et al.Hematopoietic stem cell transplant-membranous nephropathy is associated with protocadherin FAT1.J Am Soc Nephrol. 2022; 33: 1033-1044Crossref PubMed Scopus (16) Google Scholar We detected a novel protein PCSK6 in 5 cases of PLA2R-negative MN (PCSK6-associated MN) by MS/MS. All 5 cases were negative for spectral counts for thrombospondin type-1 domain-containing 7A (THSD7A), EXT1/EXT2, NELL1, SEMA3B, HTRA1, contactin 1 (CNTN1), NCAM1, PCDH7, and FAT1, and baseline spectral counts for PLA2R were present in 1 of the 5 cases. For control cases, we performed MS/MS on 116 cases that included 15 cases of time-zero kidney transplant biopsies, 17 cases of minimal change disease, 44 cases of focal segmental glomerulosclerosis, 7 cases of diabetic glomerulosclerosis, 5 cases of IgA nephropathy, and 28 cases of PLA2R-associated MN. None of the control cases showed any spectral counts for PCSK6. The initial 250 PLA2R-negative MN (discovery cohort) and control cases have been characterized and previously utilized for the discovery of EXT1/EXT2, NELL1, SEMA3B, PCDH7, FAT1, and NDNF.10Sethi S. Madden B.J. Debiec H. et al.Exostosin 1/exostosin 2–associated membranous nephropathy.J Am Soc Nephrol. 2019; 30: 1123-1136Crossref PubMed Scopus (141) Google Scholar, 11Sethi S. Debiec H. Madden B. et al.Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy.Kidney Int. 2020; 97: 163-174Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 12Sethi S. Debiec H. Madden B. et al.Semaphorin 3B–associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients.Kidney Int. 2020; 98: 1253-1265Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 13Sethi S. Madden B. Debiec H. et al.Protocadherin 7-associated membranous nephropathy.J Am Soc Nephrol. 2021; 32: 1249-1261Crossref PubMed Scopus (47) Google Scholar, 14Sethi S. Madden B. Casal Moura M. et al.Hematopoietic stem cell transplant-membranous nephropathy is associated with protocadherin FAT1.J Am Soc Nephrol. 2022; 33: 1033-1044Crossref PubMed Scopus (16) Google Scholar For each case, 10-micron–thick formalin-fixed paraffin-embedded (FFPE) sections were obtained and mounted on a special PEN membrane laser microdissection slide (Zeiss), and using a ZEISS PALM MicroBeam microscope (Zeiss), the glomeruli were microdissected to reach approximately 250–550,000 μM2 per case. Resulting FFPE fragments were digested with trypsin and collected for MS/MS analysis. The trypsin-digested peptides were identified by nano-flow liquid chromatography electrospray tandem MS/MS using a Thermo Fisher Scientific Q-Exactive or Exploris480 Mass Spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000 RSLCnano HPLC system. All MS/MS samples were analyzed using Mascot and X! Tandem set up to search a Swissprot human database. Scaffold (version 4.8.3, Proteome Software) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted at greater than 95.0% probability by the Scaffold Local FDR algorithm (Proteome Software) with protein identifications requiring a 2-peptide minimum and a 95% probability using ProteinProphet (Institute for Systems Biology).17Nesvizhskii A. Keller A. Kolker E. et al.A statistical model for identifying proteins by tandem mass spectrometry.Anal Chem. 2003; 75: 4646-4658Crossref PubMed Scopus (3686) Google Scholar The glomerular areas dissected for each PCSK6-associated MN were as follows: case 1— 85952 μM2; case 2—534119 μM2; case 3—537081 μM2; case 4—693343 μM2; and case 5—659849 μM2. IHC staining was performed at the Pathology Research Core (Mayo Clinic, Rochester, MN) using the BOND RX stainer (Leica Biosystems). FFPE tissues were sectioned at 5 microns, and IHC staining was performed online. Slides for PCSK6 stain were pretreated for 5 minutes using Enzyme 1 (AR9551; Leica Biosystems) and incubated in Protein Block (Dako) for 5 minutes. The PCSK6 primary antibody (Rabbit Polyclonal; Novus Biologicals, catalog #NBP1-87354) was diluted to 1:50 in Background Reducing Diluent (Dako) and incubated for 30 minutes. The detection system used was Polymer Refine Detection System (Leica Biosystems). This system includes the hydrogen peroxidase block, post primary and polymer reagent, 3,3′-diaminobenzidine, and hematoxylin. Immunostaining visualization was achieved by incubating slides 10 minutes in 3,3′-diaminobenzidine and 3,3′-diaminobenzidine 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 Biosystems). Slides were counterstained for 5 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 Leica Biosystems Refine kit). Once the immunochemistry process was completed, slides were removed from the stainer and rinsed in tap water for 5 minutes. Slides were dehydrated in increasing concentrations of ethyl alcohol and cleared in 3 changes of xylene prior to permanent coverslipping in xylene-based medium. IgG was acid eluted from frozen (tissue remaining after IF microscopy) kidney biopsy specimens.18Feltkamp T.E. Boode J.H. Elution of antibodies from biopsy tissue.J Clin Pathol. 1970; 23: 629-631Crossref PubMed Scopus (39) Google Scholar The eluate containing anti-PCSK6F IgG was obtained from the 2 patients with PCSK6-associated MN that were pooled together. The eluate containing control IgG was obtained from 2 patients with PLA2R-associated MN. Serial cryostat sections of 4-μm thickness were mounted side by side on a glass slide. The slides were thawed, fixed with pre-chilled 100% acetone for 10 minutes at room temperature, and then washed for 5 minutes with phosphate-buffered saline (0.01 M; phosphate, pH 7.2). The slide sections were covered with 0.2 ml of a 0.02 M citrate buffer (pH 3.2) and incubated overnight in a humid chamber at 4 °C. The eluate was extracted with a calibrated syringe and neutralized with 0.4-M NaOH to a pH of 7.2. A recombinant protein corresponding to antigenic determinants in human PCSK6 (Abnova) was used under reducing conditions. The target molecular weight and dominant band is expected at 37 kDa. The protein (1600 ng) was diluted with reduced (with β-mercaptoethanol) Laemmli sample buffer (BioRad) and boiled for 5 minutes. The samples were loaded into Criterion 12% TGX gels (Bio-Rad Laboratories) and electrophoresed in Tris-glycine-sodium dodecylsulfate running buffer. Proteins were transferred to nitrocellulose membrane (0.20-μm pore size), according to standard protocols. Membranes were incubated overnight at 4 °C with mouse anti-human PCSK6 (1:5000, recommended dilution, Antibodies-Online) and eluates from PCSK6-associated MN and PLA2R-associated MN. Subsequently, blots were washed and incubated 1 hour at room temperature with a secondary anti-mouse IgG Fc (1:15000) and anti-human IgG Fc (1:5000). Near-infrared fluorescence was detected at the 700- and 800-nm channels in the Odyssey Infrared Imaging System (LI-COR Biosciences). The reading was done for 10 minutes in both channels (total: 30 minutes). The validation cohort cases were obtained from the clinical case archives at Arkana Laboratories under a protocol approved by the Solutions Institutional Review Board. Kidney biopsies were chosen with the following inclusion criteria: (i) diagnosis of membranous glomerulopathy or membranous lupus nephritis (International Society of Nephrology/Renal Pathology Society [ISN/RPS] class V, without concurrent proliferative lupus nephritis); (ii) adequacy of ≥ 10 glomeruli on FFPE tissue and ≥ 5 glomeruli within frozen tissue; and (iii) ≤ 50% interstitial fibrosis and tubular atrophy. We included cases that were PLA2R-positive (n = 96), THSD7A-positive (n = 60), EXT1/2-positive (n = 52), and NELL1-positive (n = 57) as controls for known antigen type for specificity. Biopsies negative for all 4 antigens were used for discovery (n = 118). Immunoprecipitation with protein G was performed to recover immune complexes from residual frozen kidney biopsy tissue as previously described.15Caza T.N. Hassen S.I. Kuperman M. et al.Neural cell adhesion molecule 1 is a novel autoantigen in membranous lupus nephritis.Kidney Int. 2021; 100: 171-181Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar Briefly, biopsy cores were washed 3 times in phosphate-buffered saline and were then dissociated into a protein lysate through bead-beating in a 250-μl Pierce lysis buffer (Thermo Fisher Scientific, catalog #87787) containing 1% Halt protease inhibitor (Thermo Fisher Scientific, catalog #78440). Then, 50-μl protein G magnetic beads (Thermo Fisher Scientific, catalog #1003D) were added to the supernatant, which was incubated at room temperature for 1 hour with shaking. Beads were washed in phosphate buffered saline, followed by digestion with trypsin for MS preparation. Peptides were produced through trypsin digestion of the immune complexes adhered to protein G beads post-immunoprecipitation. Data-independent acquisition (DIA) MS was used for analysis as follows. An UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) was used to separate tryptic peptides on a 150 x 0.075 mm column packed with a reverse phase XSelect CSH C18 2.5-μm resin. The peptides were eluted with a gradient of buffer A (0.1% formic acid + 0.5% acetonitrile)/buffer B (0.1% formic acid + 99.9% acetonitrile), from a 97:3 ratio to a 60:40 ratio over 1 hour. Electrospray (2.25 kV) was used to ionize the eluted peptides on an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific). Six gas-phase fractions of a pooled sample were acquired on the Orbitrap Exploris mass spectrometer with 4 m/z DIA spectra (30,000 resolution, normalized automated gain control [AGC] target 100%; maximum inject time, 66 ms) in a staggered window pattern to assemble a chromatogram library. Precursor spectra were acquired after each DIA duty cycle, spanning the m/z range of the gas-phase fraction (i.e., 496–602 m/z; 596–702 m/z). For wide-window acquisitions, precursor spectra from each DIA cycle were acquired (385–1015 m/z; 60,000 resolution; normalized automated gain control target 100%; maximum injection time, 50 ms), followed by 50 x 12 m/z DIA spectra (12 m/z precursor isolation windows at 15,000 resolution; normalized automated gain control target 100%; maximum injection time, 33 ms) using a staggered window pattern with optimized window placements. ScaffoldDIA (Proteome Software) was used to configure DIA library searches. Narrow-window DIA files were searched against the human predicted spectral library from Prosit (2019/04 Uniprot) with a 10 parts per million precursor and fragment ion tolerances to generate an empirically corrected chromatogram library with a peptide and protein false-discovery rate of 1%. Wide-window DIA files subsequently were searched against the generated empirically corrected chromatogram library. For confocal microscopy, tissue sections were costained with PCSK6 and IgG by paraffin IF as follows: 4-μm FFPE tissue sections were deparaffinized, followed by antigen retrieval with proteinase K. Sections were incubated with PCSK6 antibody (Novus Biologicals, catalog #NBP1-87354) at 1:50 dilution for 30 minutes at room temperature. Tissue sections were washed with phosphate buffered saline and rhodamine Red X Affinipure goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, catalog #111-295-144) was applied as a secondary antibody at a 1:100 dilution for 30 minutes. Tissue sections were then costained by adding fluorescein isothiocyanate–conjugated polyclonal anti-human IgG (Agilent Technologies) at 1:40 dilution for 30 minutes. Slides were coverslipped in anti-fade mounting medium and imaged under a Leica SBA DMI8 confocal laser scanning microscope (Leica Biosystems). PLA2R-positive MN cases were costained in parallel to cases of interest as negative controls. We detected a unique protein, PCSK6, by MS/MS in the glomeruli of 5 cases (2%) of MN from the 250 PLA2R-negative cases of the discovery cohort cases (Figure 1). The total spectral counts of PCSK6 ranged from 21 to 122 in the 5 cases, with an average total spectral count of 55.4 (SD ± 45.8). All control cases including 15 time-zero transplant biopsies, 73 other glomerulopathies, and 28 PLA2R-associated MN cases were negative for PCSK6. The total spectral counts of 5 cases of PCSK6-positive MN, along with a representative sequence coverage map of PCSK6, are shown in Figure 2a and b . The MS/MS spectra match from 1 case is shown in Figure 2c. Important to point out is that none of the PCSK6-positive cases show any spectral counts for EXT1/EXT2, THSD7A, NELL1, NCAM1, contactin 1, HTRA1, SEMA3B, PCDH7, FAT1, or NDNF, whereas baseline PLA2R counts were detected in 1 of the 5 cases.Figure 2Proteomic identification of proprotein convertase subtilisin/kexin type 6 (PCSK6) in phospholipase A2 receptor (PLA2R)–negative membranous nephropathy (MN). Discovery cohort (a–c): Glomeruli were microdissected and analyzed using mass spectrometry as described in Methods. (a) Detection of PCSK6 in 5 cases of PLA2R-negative MN (top row). Numbers in green boxes represent total spectral counts of tandem mass spectrometry (MS/MS) matches to a respective protein. All 5 biopsies show high total spectral counts for PCSK6 and IgG1, IgG2, and IgG3 subtypes, and baseline spectral counts of PLA2R were also detected in 1 of the 5 cases. For comparison, the pooled total spectral counts from 6 control cases (time 0 protocol transplant biopsies) are also shown. PCSK6 is not present in the control cases. (b) Representative sequence coverage map of PCSK6 from 1 case. Amino acids highlighted in bold letters over yellow background are the amino acids detected. (c) The MS/MS spectra figure from the Scaffold viewer of ion 721.90 [M+2H]2+ highlighting the detected b-ions (red) and y-ions (blue) matching the theoretical fragment masses listed in the table for the PCSK6 peptide NVVVTILDDGIER. (d) Validation cohort. Volcano plot showing PCSK6 enriched within protein G immunoprecipitates with the highest fold-change (FC; red) compared to other cases of MN of known types (PLA2R, thrombospondin type-1 domain-containing 7A [THSD7A], neural tissue encoding protein with epidermal growth factor [EGF]–like repeats [NELL1], exostosin 2 [EXT2]) and other “quadruple negative” MN cases. Ctrl, control.View Large Image Figure ViewerDownload Hi-res image Download (PPT) IHC staining was performed on biopsy samples in all 5 cases of the discovery cohort and showed granular staining (2–3+) along the glomerular basement membrane (GBM) in all cases (Figure 3a). Segmental staining was present in case 1. A control PLA2R-associated MN is shown that is negative for PCSK6 staining along the GBM. Western blot analyses were performed using recombinant human PCSK6 to determine the presence of anti-PCSK6 antibodies in the eluate obtained from 2 pooled kidney biopsies of PCSK6-associated MN cases (Figure 4). PCSK6 was detected by mouse anti-human PCSK6 under reducing conditions. Figure 4 shows a dominant band at approximately 37 kDa to reduced PCSK6 by mouse anti-human PCSK6 (lane 1, positive control). The same band was detected after exposure of reduced human PCSK6 to IgG obtained from the eluate of PCSK6-associated MN (lane 2). Anti-PCSK6 antibodies were not detected in IgG eluate obtained from patients with PLA2R-associated-MN (lane 3). In the cohort at Arkana Laboratories, MS was performed on 118 cases of PLA2R-/THSD7A-/NELL1-/EXT2-quadruple negative MN/membranous lupus nephritis biopsies following protein G immunoprecipitation to elute antibodies from frozen biopsy tissue. PSCK6 was independently detected as a uniquely enriched protein in 6 of the cases (Figure 1 and Figure 2d). The frequency of this protein target could not be determined within this MS cohort, as it was enriched for patients with a history of autoimmune disease. PCSK6 was not identified in PLA2R (n = 96), THSD7A (n = 60), NELL1 (n = 57), or EXT2-positive (n = 52) control cases. PCSK6 was confirmed as a protein target in 2 of the 6 cases by performing MS on paired FFPE tissue following laser capture microdissection. The remaining 4 cases were confirmed by immunostaining on paraffin IF. In addition to the MS cases, PCSK6 was identified as a target antigen in 2 additional patients with MN in the setting of high-dose NSAID use by IF microscopy. Both paraffin IF and confocal microscopy were performed in the validation cohort to show staining for PCSK6 and colocalization of IgG and PCSK6 along the GBM. IF microscopy showed granular staining of PCSK6 along the GBM in all cases (Figure 3b), and confocal microscopy showed colocalization of IgG and PCSK6 along the GBM (Figure 3c). Control PLA2R-associated MN was negative for PCSK6. The mean age of PCSK6-associated MN was 46.9 ± 11.8 years (range: 32–66; median, 50). Seven patients were male, and 6 were female. Eight patients were white, 1 black, 1 Alaskan native, 1 American Indian, and in 2, the race was not known. The mean serum creatinine and proteinuria levels at kidney biopsy were 0.93 ± 0.47 mg/dl and 6.5 ± 3.3 g/d, respectively. All 5 cases of the discovery cohort had history of prolonged (>2 years) NSAID use. The NSAIDs included naproxen, ibuprofen, and meloxicam. In the validation cohort, 5 cases had history of NSAID use, 2 had no history of NSAID use, and 1 did not have an available case history. Taken together, 10 of 12 patients (83.3%) had history of NSAID use. In addition, of the 8 cases of the validation cohort, 1 case had Hashimoto’s thyroiditis, 2 had Sjogren’s syndrome, 1 had lupus, and 4 had no associated autoimmune disease. In the discovery cohort, 1 patient had rheumatoid arthritis, and the remaining patients had no associated disease. Kidney biopsy revealed an MN in all cases with thickened GBMs and no proliferative findings. There was no or minimal (≤10%) tubular atrophy and int" @default.
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• W4367316319 title "Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a likely antigenic target in membranous nephropathy and nonsteroidal anti-inflammatory drug use" @default.
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• W4367316319 doi "https://doi.org/10.1016/j.kint.2023.04.006" @default.
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