Proprotein convertase subtilisin/kexin type 6 (PCSK6) is a likely antigenic target in membranous nephropathy and nonsteroidal anti-inflammatory drug use

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, and 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 cases of PLA2R-negative MN by All cases were negative for spectral counts for EXT1/EXT2, NELL1, 1 and and spectral counts for PLA2R were present in 1 of the cases. For cases, we performed MS/MS on cases that included cases of kidney cases of cases of 7 cases of cases of and cases of MN. of the cases showed spectral counts for PCSK6. The 250 PLA2R-negative MN cohort) and cases have been characterized and for the discovery of EXT1/EXT2, NELL1, and 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 were obtained and on a membrane laser microdissection and using a the glomeruli were to case. were with and for MS/MS The were by tandem MS/MS using a to an All MS/MS were using and to a was used to and protein were at by the with protein a and a using for A. A. E. et for by tandem mass PubMed Scopus Google Scholar The glomerular for PCSK6-associated MN were as case case case case and case staining was performed at the Pathology (Mayo Clinic, using the were at and staining was performed for PCSK6 were for using 1 and in for The PCSK6 was to in and for The detection used was This the and and was by in and from the this were between with were for using and followed by in and is not the with the the was were from the and in for were in of and in of to in IgG was from frozen kidney biopsy of antibodies from biopsy PubMed Scopus Google Scholar The IgG was obtained from the 2 patients with PCSK6-associated MN that were The IgG was obtained from 2 patients with MN. of were side by side on a The were with for at and for with The were with of a and in a at The was with a and with to a of A protein to antigenic in PCSK6 was used The target and is at The protein was with and for The were and in were to membrane to were at with PCSK6 and eluates from PCSK6-associated MN and MN. were and 1 at with a secondary IgG and IgG was detected at the and in the The was for in The validation cohort cases were obtained from the clinical case at a approved by the Institutional Review Board. Kidney were with the diagnosis of membranous membranous lupus nephritis of Pathology lupus of glomeruli on tissue and glomeruli frozen and interstitial and We included cases that were PLA2R-positive and as for known antigen type for Biopsies negative for antigens were used for discovery with protein G was performed to complexes from frozen kidney biopsy tissue as 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 biopsy were in and were a protein in a protein G were to the was at for 1 with were in followed by with for were of the complexes to protein G was used for analysis as was used to on a with a The were with a of A from a to a 1 was used to the on an mass of a were on the mass with target in a to a were the of the For from were target followed by at target using a with was used to were the spectral from with a and to an with a and protein of were the For confocal microscopy, tissue were with PCSK6 and IgG by as tissue were followed by antigen with K. were with PCSK6 at for at were with and IgG was as a secondary at a for were by IgG at for were in and a confocal laser PLA2R-positive MN cases were in to cases of as negative We detected a by MS/MS in the glomeruli of cases of MN from the 250 PLA2R-negative cases of the discovery cohort cases The total spectral counts of PCSK6 from to in the cases, with an total spectral of ± All cases including and MN cases were negative for PCSK6. The total spectral counts of cases of MN, along with a of are in and The MS/MS from 1 case is in to point is that of the cases show spectral counts for EXT1/EXT2, NELL1, PLA2R counts were detected in 1 of the identification of convertase subtilisin/kexin type 6 in phospholipase A2 receptor membranous nephropathy cohort were and using mass spectrometry as described in of PCSK6 in cases of PLA2R-negative MN in total spectral counts of tandem mass spectrometry (MS/MS) to a All show high total spectral counts for PCSK6 and and and spectral counts of PLA2R were detected in 1 of the cases. For the total spectral counts from 6 cases are PCSK6 is not present in the cases. of PCSK6 from 1 case. in are the The MS/MS from the of the detected and the in the for the PCSK6 cohort. PCSK6 protein G with the to cases of MN of known neural tissue encoding protein with epidermal growth factor repeats exostosin 2 and MN cases. staining was performed on biopsy in cases of the discovery cohort and showed granular staining along the glomerular basement membrane in cases staining was present in case A MN is that is negative for PCSK6 staining along the Western blot analyses were performed using PCSK6 to determine the of anti-PCSK6 antibodies in the obtained from 2 kidney of PCSK6-associated MN cases PCSK6 was detected by PCSK6 shows a at to PCSK6 by PCSK6 The was detected of PCSK6 to IgG obtained from the of PCSK6-associated MN antibodies were not detected in IgG obtained from patients with the cohort at was performed on cases of negative lupus nephritis protein G to antibodies from frozen biopsy was detected as a protein in 6 of the cases 1 and The of this protein target not be this as was for patients with a history of PCSK6 was not in PLA2R cases. PCSK6 was confirmed as a protein target in 2 of the 6 cases by on tissue laser The cases were confirmed by on to the cases, PCSK6 was as a target antigen in 2 additional patients with MN in the of NSAID use by and confocal microscopy were performed in the validation cohort to show staining for PCSK6 and of IgG and PCSK6 along the microscopy showed granular staining of PCSK6 along the in cases and confocal microscopy showed of IgG and PCSK6 along the MN was negative for PCSK6. The mean of PCSK6-associated MN was ± patients were and 6 were patients were 1 1 1 and in the was not known. The mean serum creatinine and proteinuria at kidney biopsy were 0.93 ± 0.47 and 6.5 ± 3.3 respectively. All cases of the discovery cohort had history of prolonged NSAID use. The NSAIDs included and the validation cases had history of NSAID 2 had no history of NSAID and 1 not have an available case of patients had history of NSAID use. of the cases of the validation 1 case had 2 had 1 had and had no associated the discovery 1 had and the patients had no associated Kidney biopsy revealed an MN in cases with and no was no and interstitial in of 13 patients revealed granular IgG and along the in cases. basement membrane staining for IgG in of the cases. microscopy revealed electron in in most cases, between was present MN of and were were present in a of patients. PLA2R staining was negative in cases. IgG subclass staining of cases from the validation cohort showed IgG1 and was available for patients patients 2 patients to have and 1 had a of the 2 patients that to have proteinuria was a case with a of 2 NSAID use was in patients in the discovery cohort and patients in the validation cohort had history of NSAID use. patients were treated received 1 received 1 received not and 1 received followed by the had a 1 and 2 received as of a clinical et in the of membranous nephropathy.N Engl J Med. 2019; PubMed Scopus Google Scholar for the discovery cohort was from to and of the validation cohort was from 2 to and were as proteinuria proteinuria and a from no from negative followed by interstitial and nonsteroidal anti-inflammatory lupus are from the discovery and patients are from the validation were as proteinuria proteinuria and a from no from in a interstitial and nonsteroidal anti-inflammatory lupus are from the discovery and patients are from the validation cohort. MN has in target antigen phospholipase A2 receptor and were in and et phospholipase A2 receptor as target antigen in membranous nephropathy.N Engl J Med. PubMed Scopus Google et in membranous nephropathy.N Engl J Med. PubMed Scopus Google Scholar Since target antigens NELL1, and have been in the Using laser microdissection and mass confocal microscopy, and Western blot et have novel antigens in 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 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 have been in novel target 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 et as a novel target antigen in membranous nephropathy.J Am Soc Nephrol. 2021; 32: PubMed Scopus Google M. M. et is a novel target antigen in membranous nephropathy associated with chronic Int. 2021; 100: Full Text Full Text PDF PubMed Scopus Google Scholar The target antigens the MN Although target such as and not have associated with target antigens are associated with in the of and in 1 in FAT1 in stem cell and in However, the target antigen in subgroup that is associated with prolonged NSAID this we show that a novel protein PCSK6 is the likely target MN antigen in patients with prolonged NSAID use. A protein PCSK6 with high total spectral counts was on MS/MS in cases in the discovery cohort of 250 PLA2R-negative MN cases. The cases were negative for known antigens. of the clinical the MS/MS results revealed that cases had a prolonged history of NSAID use. confocal microscopy, and Western blot analysis confirmed that PCSK6 is the likely target antigen in these cases. to is that discontinuation of the NSAID in cases, and in 2 patients and in in of nephrotic syndrome in and in 1 of the patients received were in a clinical and at the of the study were PLA2R-negative MN et in the of membranous nephropathy.N Engl J Med. 2019; PubMed Scopus Google Scholar of NSAID-associated MN is as patients use NSAID for a of study this in that NSAID use was in on of the clinical MS/MS revealed high spectral counts of in the PLA2R-negative MN cases the most cases of MN with history of NSAID use are likely to be negative for and NSAIDs likely are not associated with MN in most cases of MN with history of NSAID use. detection of PCSK6 on MS/MS studies in MN to clinical for NSAID including of NSAID use with of MN. For the validation MN cases were obtained from The had been in PCSK6 as a protein in study of MN. of cases revealed PCSK6 in PLA2R-negative MN. important point to is that the used for the identification of PCSK6 in the validation cohort is from that used in the discovery cohort. the validation protein IgG and to IgG were by protein G This was followed by mass spectrometry of the complexes to identify the binding to Thus, 2 were used in the discovery and validation and PCSK6 as a protein in a of PLA2R-negative MN cases. G the of anti-PCSK6 IgG along the as complexes are from the frozen kidney biopsy The 2 have The discovery cohort is not for type of PLA2R-negative MN and PLA2R-negative MN cases received at the Mayo the the PLA2R-negative MN cases from the validation cohort are for MN. This may for in as of cases to the discovery cohort in of patients patients and 1 NSAID use was in of the validation cohort cases, and not have history of NSAID that PCSK6 may be the target antigen in a of MN cases. Further studies are to this The detection of of in of 13 cases is to the of in patients with The of in PLA2R-positive membranous 2022; PubMed Scopus Google Scholar the discovery NSAIDs were used for in 2 chronic in 2 and in 1 the the disease to NSAID use in the validation as this group was for The study is in that we had tissue from frozen biopsy tissue to and we were to Western blot analysis PCSK6 is a that is the and including growth such as growth and adhesion et a with J. PubMed Scopus Google Scholar PCSK6 to the A. K. E. et and are are in the of in the of in the PubMed Scopus Google Scholar PCSK6 a in the growth that is for and that and a 14: PubMed Google Scholar PCSK6 a in S. 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R. et and the 2022; Scopus (5) Google R. et convertase is the of the in the 2021; PubMed Scopus Google Scholar This was in by to from the Department of Laboratory Medicine and Pathology at the Mayo Clinic for was obtained from of and and Kidney of for and was obtained from The the Mayo Clinic of the Mayo Clinic the Department of Laboratory Medicine and Pathology and the Pathology Mayo in the Renal Pathology Laboratory for with Laboratory, for laser in and in the and the the the kidney and Western blot in of cases and in of the kidney performed the confocal and performed the and Western blot performed Western blot and the clinical and biopsy and of the validation cohort. The was and by the with as from antigen to membranous nephropathy is an kidney disease characterized by the of target antigens have been including associated with and secondary anti-inflammatory drugs an important secondary cause of membranous this study by et identify convertase subtilisin/kexin type 6 as a target antigen in nonsteroidal anti-inflammatory membranous PDF