Salivary gland amyloidosis: Proteomic identification and clinicopathologic characterization of 57 cases.

Salivary gland amyloidosis is an uncommon diagnosis. Most studies have focused on minor salivary gland biopsies as a surrogate site for diagnosing systemic amyloidosis, while only few studies have investigated major salivary gland amyloidosis. We retrospectively identified 57 major and minor salivary gland amyloidosis cases typed using a proteomics-based method between 2010 and 2022. Frequency of amyloid types, clinicopathologic features, and distribution patterns of amyloid deposits were assessed. The indication for salivary gland biopsy/resection (known in 34 cases) included suspected amyloidosis (N = 14; 41.2%), lesion/mass (N = 12; 35.3%), swelling/enlargement (N = 5; 14.7%), and rule out Sjogren syndrome (N = 3; 8.8%). Concurrent pathology was reported in 16 cases, and included chronic sialadenitis (N = 11), extranodal marginal zone lymphoma (N = 3), plasma cell neoplasm (N = 1), and pleomorphic adenoma (N = 1). We identified 3 types of amyloidosis: immunoglobulin light chain/AL (N = 47; 82.5%); immunoglobulin heavy chain/AH (N = 1; 1.8%), and transthyretin/ATTR (N = 9; 15.8%). The patterns of amyloid deposits (assessed in 35 cases) included: 1) Perivascular and/or periductal distribution (N = 18; 51.4%); 2) Mass formation (N = 9; 25.7%); 3) Stromal micronodule formation (N = 7; 20.0%); and 4) Diffuse interstitial involvement (N = 1; 2.9%). We also identified one case of AL amyloidosis localized to the major salivary gland, where only 6 other cases with adequate staging workup to exclude systemic amyloidosis were previously reported. In conclusion, salivary gland amyloidosis is an uncommon diagnosis but may be underrecognized due to low index of suspicion. Most cases of salivary gland amyloidosis are AL type, but a minority are ATTR. Therefore, proteomics-based typing remains essential for treatment and prognosis.

Clinicopathologic Characteristics of Crystalglobulin-Induced Nephropathy: A Case Series.

RATIONALE & OBJECTIVE: Crystalglobulinemia is a rare syndrome characterized by intravascular crystallization of monoclonal immunoglobulins (MIg). Data on kidney involvement are limited to case reports. This series characterizes the clinicopathologic spectrum of crystalglobulin-induced nephropathy (CIN). STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Nineteen CIN cases identified from the nephropathology archives of Mayo Clinic and Columbia University. CIN was defined by intravascular (extracellular) MIg crystals visible by light microscopy (LM) and electron microscopy (EM). RESULTS: Among the cases, 68% were male, and 65% were Caucasian (median age, 56 years). Most patients presented with severe acute kidney injury (AKI) (median creatinine, 3.5mg/dL), hematuria, and mild proteinuria (median, 1.1g/day). Common extrarenal manifestations were constitutional (67%), cutaneous (56%), and rheumatologic (50%). Fifty percent of cases had hypocomplementemia. The hematologic disorders were monoclonal gammopathy of renal significance (MGRS) (72%), lymphoma (17%), or myeloma (11%), with 65% of these disorders discovered concomitantly with CIN. All patients had MIg identified on serum protein electrophoresis/immunofixation (IgGκ in 65%). The serum free light chain ratio was outside the renal range in 40%, and bone marrow biopsy detected the responsible clone in 67%. On LM, crystals involved glomeruli (100%) and vessels (47%), often with an inflammatory reaction (89%) and fibrin (58%). All cases exhibited crystal substructures (mostly paracrystalline) by EM. Immunofluorescence on paraffin-embedded tissue was more sensitive than frozen tissue (92% vs 47%) for demonstrating the crystal composition (IgGκ in 63%). Follow-up observation (median, 20 months) was available in 16 patients. Eighty-one percent received steroids, 44% plasmapheresis, 38% hemodialysis, and 69% chemotherapy. Ninety-percent of patients who received clone-directed therapy achieved kidney recovery versus 20% of those who did not (P=0.02). LIMITATIONS: Retrospective design, small sample size. CONCLUSIONS: CIN is a rare cause of nephropathy associated with lymphoplasmacytic disorders (mostly MGRS) and typically presents with severe AKI and extrarenal manifestations. Diagnosis often requires immunofluorescence performed on paraffin-embedded kidney tissue. Prompt initiation of clone-directed therapy, coupled with corticosteroids and plasmapheresis, may lead to recovery of kidney function.

Monoclonal immunoglobulin crystalline nephropathies.

Monoclonal Ig crystalline nephropathies are rare lesions resulting from precipitation of monoclonal Igs in the kidney as crystalline inclusions. They can be categorized into lesions with predominant intracellular crystals (light chain [LC] proximal tubulopathy, LC crystal-storing histiocytosis, and LC crystalline podocytopathy) and lesions with predominant extracellular crystals (crystalglobulin-induced nephropathy and crystalline variant of LC cast nephropathy). The majority of these lesions are associated with low tumor burden lymphoproliferative disorders, with the exception of crystalline variant of LC cast nephropathy. Extrarenal involvement (e.g., skin and cornea) is frequent. Kidney biopsy is the cornerstone for diagnosis, which often requires electron microscopy and antigen retrieval. A thorough hematologic workup and evaluation of extrarenal involvement is mandatory for management. Treatment of these lesions is with clone-directed therapy, with the goal of achieving hematologic very good partial response or complete response, which preserves or improves kidney function. In vitro and in vivo studies, animal models, and novel sequencing techniques have been invaluable tools to understand the pathogenesis of LC proximal tubulopathy and can be used to increase our limited knowledge of the pathogenesis of the other monoclonal Ig crystalline nephropathies. This review provides an update on the pathology, renal and hematologic characteristics, extrarenal manifestations, prognosis, treatment, and pathogenesis of monoclonal Ig crystalline nephropathies.

Apolipoprotein E is enriched in dense deposits and is a marker for dense deposit disease in C3 glomerulopathy.

C3 glomerulopathy (C3G) is a rare disease resulting from dysregulation of the alternative pathway of complement. C3G includes C3 glomerulonephritis (C3GN) and dense deposit disease (DDD), both of which are characterized by bright glomerular C3 staining on immunofluorescence studies. However, on electron microscopy (EM), DDD is characterized by dense osmiophilic mesangial and intramembranous deposits along the glomerular basement membranes (GBM), while the deposits of C3GN are not dense. Why the deposits appear dense in DDD and not in C3GN is not known. We performed laser microdissection (LCM) of glomeruli followed by mass spectrometry (MS) in 12 cases each of DDD, C3GN, and pretransplant kidney control biopsies. LCM/MS showed marked accumulation of complement proteins C3, C5, C6, C7, C8, C9 and complement regulating proteins CFHR5, CFHR1, and CFH in C3GN and DDD compared to controls. C3, CFH and CFHR proteins were comparable in C3GN and DDD. Yet, there were significant differences. First, there was a six-to-nine-fold increase of C5-9 in DDD compared to C3GN. Secondly, an unexpected finding was a nine-fold increase in apolipoprotein E (ApoE) in DDD compared to C3GN. Most importantly, immunohistochemical and confocal staining for ApoE mirrored the dense deposit staining in the GBM in DDD but not in C3GN or control cases. Validation studies using 31 C3G cases confirmed the diagnosis of C3GN and DDD in 80.6 % based on ApoE staining. Overall, there is a higher burden of terminal complement pathway proteins in DDD compared to C3GN. Thus, our study shows that dense deposits in DDD are enriched with ApoE compared to C3GN and control cases. Hence, ApoE staining may be used as an adjunct to EM for the diagnosis of DDD and might be valuable when EM is not available.

Autosomal dominant chronic tubulointerstitial nephropathy: do not forget amyloidosis.

Amyloidosis is a rare cause of inherited kidney disease, with most variants responsible for prominent glomerular involvement. In this issue, Kmochová et al. reported the first description of autosomal dominant medullary amyloidosis due to apolipoprotein A4 variants, resulting in slowly progressive chronic kidney disease with minimal proteinuria. Combining next-generation sequencing with histopathological studies incorporating Congo red staining and mass spectrometry should be considered in the diagnostic workup of hereditary tubulointerstitial disorders not identified after routine genetic testing.

2024 Update on Classification, Etiology, and Typing of Renal Amyloidosis: A Review.

Amyloidosis is a protein folding disease that causes organ injuries and even death. In humans, 42 proteins are now known to cause amyloidosis. Some proteins become amyloidogenic as a result of a pathogenic variant as seen in hereditary amyloidoses. In acquired forms of amyloidosis, the proteins form amyloid in their wild-type state. Four types (serum amyloid A, transthyretin, apolipoprotein A-IV, and ß2-macroglobulin) of amyloid can occur either as acquired or as a mutant. Iatrogenic amyloid from injected protein medications have also been reported and AIL1RAP (anakinra) has been recently found to involve the kidney. Finally, the mechanism of how leukocyte cell-derived chemotaxin 2 (ALECT2) forms amyloid remains unknown. This article reviews the amyloids that involve the kidney and how they are typed.

Clinicopathologic Characteristics, Etiologies, and Outcome of Secondary Oxalate Nephropathy.

OBJECTIVE: To report the clinicopathologic characteristics, prognostic indicators, prognosis, and transplant outcome of secondary oxalate nephropathy (ON). PATIENTS AND METHODS: We performed a retrospective analysis of 113 consecutive patients with secondary ON diagnosed at Mayo Clinic in Rochester, Minnesota, between January 1, 2001, and March 1, 2023. RESULTS: The incidence of secondary ON among all native biopsies from Mayo Clinic patients over the study period (n=11,617) was 0.97%. ON was attributed to enteric hyperoxaluria in 60% of the 113 patients (68; most commonly Roux-en-Y gastric bypass), excessive ingestion of foods high in oxalate or oxalate precursors in 23% (26) (most commonly vitamin C), and idiopathic in 17% (19). Most patients presented with acute kidney injury (AKI) (particularly in the ingestion group) or AKI on chronic kidney disease, and 53% (60 of 113) were diabetic. Calcium oxalate crystals were accompanied by acute tubular injury, inflammation, and interstitial fibrosis and tubular atrophy. Concurrent pathologic conditions were present in 53% of the patients (60 of 113), most commonly diabetic nephropathy. After a median follow-up of 36 months, 27% of the patients (30 of 112) had kidney recovery, 19% (21 of 112) had persistent kidney dysfunction, 54% (61 of 112) had development of kidney failure, and 29% (32 of 112) died. The mean kidney survival was worse for patients with a concurrent pathologic lesion (30 months vs 96 months for those without a concurrent pathologic lesion; P<.001). Independent predictors of kidney failure were the degree of interstitial fibrosis and tubular atrophy and nadir estimated glomerular filtration rate but not the degree of crystal deposition. After a median follow-up of 58 months in 23 patients who received kidney transplant, 4 had graft loss (due to ON in 3). The 2-, 5-, and 10-year graft survivals were 90% (18 of 20), 79% (11 of 14), and 50% (6 of 12). CONCLUSION: ON is a rare cause of AKI or AKI on chronic kidney disease. Most patients have comorbid pathologic conditions, particularly diabetic nephropathy, which worsen the prognosis. Recurrence in the renal allograft and graft loss may occur if hyperoxaluria is not controlled.

Monoclonal Immunoglobulin Crystalline Membranous Nephropathy.

Monoclonal immunoglobulin (MIg) crystalline nephropathies are rare lesions resulting from precipitation of MIgs in the kidney as intracellular or extracellular crystals. We describe a patient with multiple myeloma (IgGλ) and diabetes who presented with nephrotic range proteinuria. Kidney biopsy revealed membranous nephropathy superimposed on diabetic glomerulosclerosis. Glomeruli were negative for PLA2R, THSD7A, and NELL-1. Ultrastructurally, the subepithelial deposits were composed of crystals (ranging from rhomboid to rod to needle shaped), which failed to stain for immunoglobulins by routine immunofluorescence but stained for IgG+λ by paraffin immunofluorescence after pronase digestion. RNA-based immunoglobulin repertoire sequencing performed on bone marrow aspirate identified an IgGλ (γ1) clone, which was highly atypical, combining an extensively mutated (23.6%) Ig heavy chain derived from the IGHV1-24 with low pI and unusual mutations and a light chain derived from an extremely rare germline gene (IGLV10-54). This report expands the pathologic spectrum of MIg crystalline nephropathies by describing a unique case of crystalline nephropathy with IgGλ deposits manifesting as membranous nephropathy.

Atypical Anti-Glomerular Basement Membrane Nephritis: A Case Series From the French Nephropathology Group.

RATIONALE & OBJECTIVE: Atypical anti-glomerular basement membrane (GBM) nephritis is characterized by a bright linear immunoglobulin staining along the GBM by immunofluorescence without a diffuse crescentic glomerulonephritis nor serum anti-GBM antibodies by conventional enzyme-linked immunosorbent assay (ELISA). We characterized a series of patients with atypical anti-GBM disease. STUDY DESIGN: Case series. SETTING & PARTICIPANTS: Patients identified by the French Nephropathology Group as having atypical anti-GBM nephritis between 2003 and 2022. FINDINGS: Among 38 potential cases, 25 were included, of whom 14 (56%) were female and 23 (92%) had hematuria. The median serum creatinine at diagnosis was 150 (IQR, 102-203) µmol/L and median urine protein-creatinine ratio (UPCR) was 2.4 (IQR, 1.3-5.2) g/g. Nine patients (36%) had endocapillary proliferative glomerulonephritis (GN), 4 (16%) had mesangial proliferative GN, 4 (16%) had membranoproliferative GN, 2 (8%) had pure and focal crescentic GN, 1 (4%) had focal segmental glomerulosclerosis, and 5 had glomeruli that were unremarkable on histopathology. Nine patients (36%) had crescents, involving a median of 9% of glomeruli. Bright linear staining for IgG was seen in 22 cases (88%) and for IgA in 3 cases (12%). The 9 patients (38%) who had a monotypic staining pattern tended to be older with less proteinuria and rarely had crescents. Kidney survival rate at 1 year was 83% and did not appear to be associated with the light chain restriction. LIMITATIONS: Retrospective case series with a limited number of biopsies including electron microscopy. CONCLUSIONS: Compared with typical anti-GBM disease, atypical anti-GBM nephritis frequently presents with an endocapillary or mesangial proliferative glomerulonephritis pattern and appears to have a slower disease progression. Further studies are needed to fully characterize its pathophysiology and associated clinical outcomes. PLAIN-LANGUAGE SUMMARY: Atypical anti-glomerular basement membrane (GBM) nephritis is characterized histologically by bright linear immunoglobulin staining along the GBM without diffuse crescentic glomerulonephritis or circulating anti-GBM antibodies. We report a case series of 25 atypical cases of anti-GBM nephritis in collaboration with the French Nephropathology Group. Compared with typical anti-GBM disease, we observed a slower disease progression. Patients frequently presented with heavy proteinuria and commonly had evidence of endocapillary or mesangial proliferative glomerulonephritis. About half of the patients displayed a monotypic immune staining pattern; they tended to be older, with less proteinuria, and commonly without glomerular crescents in biopsy specimens. No concomitant circulating monoclonal gammopathy was detected. Further studies are needed to fully characterize its pathophysiology and associated clinical outcomes.

Recurrent atypical antiglomerular basement membrane nephritis in the kidney transplant.

Atypical antiglomerular basement membrane (anti-GBM) nephritis can be defined as linear GBM staining for monotypic or polytypic immunoglobulin (Ig) by immunofluorescence (IF) without a diffuse crescentic pattern. We describe the clinicopathologic features of 6 patients (18 biopsies) in this first series of recurrent atypical anti-GBM nephritis after kidney transplantation. Recurrent glomerulonephritis occurred at a mean of 3.8 months posttransplant (range 1-7 months). Three index biopsies were for clinical indication, and 3 were protocol biopsies. Glomerular histologic changes were mild, with 2 showing segmental endocapillary hypercellularity, 1 focal glomerular microangiopathy, and the others no significant glomerular histologic changes. All 6 allografts showed monotypic linear glomerular Ig staining by IF: IgG kappa (n = 2), IgG lambda, IgA kappa, IgA lambda, and IgM lambda. Follow-up biopsies were available for 5 patients and showed similar histologic and IF findings without evidence of significant progression. No patients had detectable serum anti-GBM antibody or monoclonal proteins. The mean serum creatinine level on follow-up (24-62 months posttransplant) was 1.8 (range 0.93-2.77) mg/dL; no grafts were lost to recurrent disease. This series demonstrates that monotypic atypical anti-GBM recurs in the allograft and supports the idea that this disease is due to a circulating monoclonal protein.

Pathologic-genomic correlation identified a novel variant in FN1 and established the diagnosis of recurrent fibronectin glomerulopathy in the kidney allograft.

Fibronectin glomerulopathy is a rare inherited kidney disease, characterized by abnormal accumulation of fibronectin in the glomeruli. We report an exceptional case of recurrent fibronectin glomerulopathy first diagnosed in the kidney allograft. The presence of IgA staining in the native kidney biopsy and the reported family history of IgA nephropathy had led to initial pretransplant diagnosis of IgA nephropathy. At 4.5 years posttransplant, the patient presented with kidney insufficiency and minimal proteinuria. The allograft biopsy revealed glomerular deposits with very weak staining for immunoglobulins and vague filamentous material. Immunostaining for fibronectin was positive, and genetic studies showed a variant of unknown significance in the fibronectin 1 gene. Proteomic analyses of the glomeruli in the native kidney biopsy demonstrated large amount of fibronectin with abundant accumulation of the peptide synthesized by the detected variant. These findings established the diagnosis of recurrent fibronectin glomerulopathy secondary to a novel variant in the fibronectin 1 gene. This report sheds light on recurrent fibronectin glomerulopathy in the allograft, highlights the diagnostic pitfalls of the disease, and underscores the importance of pathologic-genomic correlation to establish the correct diagnosis.

Anakinra-Associated Systemic Amyloidosis.

OBJECTIVE: To describe a 41-year-old woman with a history of neonatal onset multisystem inflammatory disease, on treatment with daily subcutaneous injections of 600 mg of recombinant interleukin-1 receptor antagonist (IL-1Ra) protein, anakinra, since the age of 28, who presented with golf-ball size nodules at the anakinra injection sites, early satiety, new onset nephrotic syndrome in the context of normal markers of systemic inflammation. METHODS: Clinical history and histologic evaluation of biopsies of skin, gastric mucosa, and kidney with Congo-red staining and proteomic evaluation of microdissected Congo red-positive amyloid deposits by liquid chromatography-tandem mass spectrometry. RESULTS: The skin, stomach, and kidney biopsies all showed the presence of Congo red-positive amyloid deposits. Mass spectrometry-based proteomics demonstrated that the amyloid deposits in all sites were of AIL1RAP (IL-1Ra protein)-type. These were characterized by high spectral counts of the amyloid signature proteins (apolipoprotein AIV, apolipoprotein E, and serum amyloid P-component) and the amyloidogenic IL-1Ra protein, which were present in Congo red-positive areas and absent in Congo red-negative areas. The amino acid sequence identified by mass spectrometry confirmed that the amyloid precursor protein was recombinant IL-1Ra (anakinra) and not endogenous wild-type IL-1Ra. CONCLUSION: This is the first report of iatrogenic systemic amyloidosis due to an injectable protein drug, which was caused by recombinant IL1Ra (anakinra).

Mayo Clinic consensus report on membranous nephropathy: proposal for a novel classification.

Membranous nephropathy (MN) is a pattern of injury caused by autoantibodies binding to specific target antigens, with accumulation of immune complexes along the subepithelial region of glomerular basement membranes. The past 20 years have brought revolutionary advances in the understanding of MN, particularly via the discovery of novel target antigens and their respective autoantibodies. These discoveries have challenged the traditional classification of MN into primary and secondary forms. At least 14 target antigens have been identified, accounting for 80%-90% of cases of MN. Many of the forms of MN associated with these novel MN target antigens have distinctive clinical and pathologic phenotypes. The Mayo Clinic consensus report on MN proposes a 2-step classification of MN. The first step, when possible, is identification of the target antigen, based on a multistep algorithm and using a combination of serology, staining of the kidney biopsy tissue by immunofluorescence or immunohistochemistry, and/or mass spectrometry methodology. The second step is the search for a potential underlying disease or associated condition, which is particularly relevant when knowledge of the target antigen is available to direct it. The meeting acknowledges that the resources and equipment required to perform the proposed testing may not be generally available. However, the meeting consensus was that the time has come to adopt an antigen-based classification of MN because this approach will allow for accurate and specific MN diagnosis, with significant implications for patient management and targeted treatment.

Mayo Clinic Consensus Report on Membranous Nephropathy: Proposal for a Novel Classification.

Membranous nephropathy (MN) is a pattern of injury caused by autoantibodies binding to specific target antigens, with accumulation of immune complexes along the subepithelial region of glomerular basement membranes. The past 20 years have brought revolutionary advances in the understanding of MN, particularly via the discovery of novel target antigens and their respective autoantibodies. These discoveries have challenged the traditional classification of MN into primary and secondary forms. At least 14 target antigens have been identified, accounting for 80%-90% of cases of MN. Many of the forms of MN associated with these novel MN target antigens have distinctive clinical and pathologic phenotypes. The Mayo Clinic consensus report on MN proposes a 2-step classification of MN. The first step, when possible, is identification of the target antigen, based on a multistep algorithm and using a combination of serology, staining of the kidney biopsy tissue by immunofluorescence or immunohistochemistry, and/or mass spectrometry methodology. The second step is the search for a potential underlying disease or associated condition, which is particularly relevant when knowledge of the target antigen is available to direct it. The meeting acknowledges that the resources and equipment required to perform the proposed testing may not be generally available. However, the meeting consensus was that the time has come to adopt an antigen-based classification of MN because this approach will allow for accurate and specific MN diagnosis, with significant implications for patient management and targeted treatment.

Clinicopathologic Spectrum of Lysozyme-Associated Nephropathy.

Introduction: Lysozyme-associated nephropathy (LyN), a rare cause of kidney injury in patients with chronic myelomonocytic leukemia (CMML), has not been well described to date. We report the clinicopathologic spectrum of LyN from a multi-institutional series. Method: We identified 37 native kidney biopsies with LyN and retrospectively obtained clinicopathologic data. Results: Thirty-seven patients had a median age of 74 years and included 78% males. Their most common presentation was acute kidney injury (AKI) or AKI on chronic kidney disease (CKD) (66%) with median estimated glomerular filtration rate (eGFR) of 21.7 ml/min per 1.73 m2, and proteinuria of 1.7 g. A minority (15%) had partial Fanconi syndrome. Serum lysozyme levels were elevated in all tested. Hematologic disorder (n = 28, 76%) was the most common etiology, including CMML (n = 15), acute myeloid leukemia (n = 5), and myelodysplastic syndrome (MDS) (n = 5). Nonhematologic causes (n = 5, 14%), included metastatic neuroendocrine carcinoma (n = 3), sarcoidosis, and leprosy. Etiology was unknown in 4 (11%). Pathology showed proximal tubulopathy with abundant hypereosinophilic intracytoplasmic inclusions, with characteristic staining pattern by lysozyme immunostain. Mortality was high (8/30). However, among the 22 alive, including 85% treated, 7 had improved kidney function, including 1 who discontinued dialysis and 6 with increase in eGFR >15 ml/min per 1.73 m2 compared with eGFR at the time of biopsy. Conclusion: Increased awareness of the full clinicopathologic spectrum of LyN may lead to prompt diagnosis, earlier treatment, and potentially improved outcome of this rare entity.

Heterogeneity of Target Antigens in Sarcoidosis-Associated Membranous Nephropathy.

Introduction: Membranous nephropathy (MN) is the most common glomerular disease associated with sarcoidosis. The target antigen M-type phospholipase A2 receptor 1 (PLA2R) has been identified in a subset of sarcoidosis-associated MN. The target antigen is not known in the remaining sarcoidosis-associated MN. Methods: Data of patients with history of sarcoidosis and biopsy-proven MN were retrieved and analyzed. Mass spectrometry (MS/MS) was performed on all kidney biopsies of sarcoidosis-associated MN to detect the target antigens. Immunohistochemistry (IHC) studies were performed to confirm and localize the target antigens along the glomerular basement membrane (GBM). Results: Eighteen patients with history of sarcoidosis and biopsy-proven MN were identified, of whom 3 were known to be PLA2R-negative, and in the remaining patients the target antigen was unknown. Thirteen (72%) patients were males; the median age at MN diagnosis was 54.5 years. The median proteinuria at presentation was proteinuria 9.8 g/24 h. Eight patients (44.4%) had concurrent sarcoidosis. Using MS/MS, we detected PLA2R and neural epidermal growth factor-like-1 protein (NELL1) in 7 (46.6%) and 4 (22.2%) patients, respectively. In addition, 1 case each (5.5%) was positive for thrombospondin type 1 domain-containing 7A (THSD7A), protocadherin-7 (PCDH7), and putative antigen Serpin B12. No known target antigen was detected in the remaining 4 patients (22.2%). Conclusion: Patients with sarcoidosis and MN exhibit heterogeneous target antigens. We identified, along with PLA2R, the presence of previously unreported antigens, including NELL1, PCDH7, and THSD7A. The incidence of the target antigens in sarcoidosis appears to mirror the overall incidence of target antigens in MN. MN in sarcoidosis may be the result of a heightened immune response and is not associated with a single target antigen.

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