From Patterns to Proteins: Mass Spectrometry Comes of Age in Glomerular Disease.

Laser capture microdissection and mass spectrometry (LCM/MS) is a technique that involves dissection of glomeruli from paraffin-embedded biopsy tissue, followed by digestion of the dissected glomerular proteins by trypsin, and subsequently mass spectrometry to identify and semiquantitate the glomerular proteins. LCM/MS has played a crucial role in the identification of novel types of amyloidosis, biomarker discovery in fibrillary GN, and more recently discovery of novel target antigens in membranous nephropathy (MN). In addition, LCM/MS has also confirmed the role for complement proteins in glomerular diseases, including C3 glomerulopathy. LCM/MS is now widely used as a clinical test and considered the gold standard for diagnosis and typing amyloidosis. For the remaining glomerular diseases, LCM/MS has remained a research tool. In this review, we discuss the usefulness of LCM/MS in other glomerular diseases, particularly MN, deposition diseases, and diseases of complement pathways, and advocate more routine use of LCM/MS at the present time in at least certain diseases, such as MN, for target antigen detection. We also discuss the limitations of LCM/MS, particularly the difficulties faced from moving from a research-based technique to a clinical test. Nonetheless, the role of LCM/MS in glomerular diseases is expanding. Currently, LCM/MS may be used to identify the etiology in certain glomerular diseases, but in the future, LCM/MS can play a valuable role in determining pathways of complement activation, inflammation, and fibrosis.

A reliable clinical test for detection of membranous nephropathy antigens using laser microdissection and mass spectrometry.

Membranous nephropathy (MN) results from accumulation of antigen-antibody immune complexes along the subepithelial region of the glomerular basement membranes. Over the last years, 13 target antigens have been discovered and include PLA2R, THSD7A, EXT1 and EXT2, NELL1, SEMA3B, NCAM1, CNTN1, HTRA1, FAT1, PCDH7, NTNG1, PCSK6 and NDNF, accounting for 80-90% of MN antigens. MN associated with many of these antigens have distinctive clinicopathologic findings. It is important to accurately identify the antigen in MN. Immunohistochemical (IHC) and/or immunofluorescence (IF) methods are currently used to detect PLA2R, THSD7A, NELL1, SEMA3B and EXT1/EXT2. However, for the remaining antigens, IHC/IF methods do not exist and are not practical for detection. Here, we developed laser microdissection-based mass spectrometry methodology (LMD/MS) as a one-stop clinical test for the detection of MN antigens using paraffin-embedded kidney biopsy tissue. The LMD/MS test was validated in two steps. LMD/MS was used to detect the antigen in 75 cases of MN with known antigens and correctly identified the antigen in all these cases. Next, LMD/MS was used to identify the antigen in 61 MN cases where the antigen was unknown and identified one of the known antigens in 40 of 61 cases including many of the less common antigens. This lower-than-expected detection rate is explained by intentional enrichment of the cohort with PLA2R-negative MN. Overall, PLA2R was identified in 16.4%, one of the other antigens detected in 49.1%, and in the remaining 34.5% of cases, none of the above antigens was detected. Thus, LMD/MS is an extremely useful and reliable method for the detection of known MN antigens and possibly indicating an unknown MN antigen for eventual discovery.

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.

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.

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.

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.

Granulomatosis With Polyangiitis With And Without Antineutrophil Cytoplasmic Antibodies: A Case-Control Study.

BACKGROUND: ANCA-negative GPA remains a diagnosis of exclusion. Clinical differences between patients with ANCA-negative vs ANCA-positive GPA have not been analyzed in sizable case-control studies, and the effects of ANCA-seroconversion from negative to positive are not well documented. METHODS: A single-center, sex, and age matched case-control study evaluated ANCA-negative vs ANCA-positive GPA from January 1, 1996, to December 31, 2015. Patients who experienced seroconversion were the subject of a case crossover study. Clinical data and outcomes were retrieved from electronic medical records. RESULTS: ANCA-negative GPA was identified in 110 patients; 65% were female; median age was 55 (IQR 39-65) years at time of diagnosis. Disease severity was milder in ANCA-negative GPA (BVAS/WG = 2 vs 6, p< 0.001). Mucous membranous/eye manifestations were more frequent in ANCA-negative GPA. General symptoms, pulmonary, and renal involvement were more frequent in ANCA-positive GPA. Patients with ANCA-positive GPA relapsed more over 60 months (21.8% vs.9.1%, p= 0.009) compared with ANCA-negative GPA and had shorter time to event (p= 0.043). Patients with general manifestations, BMI > 30kg/m2 and necrotizing granulomatous inflammation were more likely to relapse. The 16 patients who seroconverted into ANCA-positive during follow-up had higher mean BVAS/WG at time of diagnosis (p< 0.001) and increased incidence of relapses (p= 0.004) after seroconversion. Necrotizing granulomatous inflammation on biopsy in ANCA-negative GPA patients was identified as a risk factor for subsequent seroconversion to ANCA-positivity. CONCLUSION: Patients with ANCA-negative GPA have milder disease and a lower frequency of relapse than those with ANCA-positive GPA. ANCA appearance portended higher disease severity and an increased frequency of relapses.

Membranous nephropathy-diagnosis and identification of target antigens.

Membranous nephropathy (MN) is a common cause of nephrotic syndrome in adults. MN is characterized by subepithelial accumulation of immune complexes along the glomerular basement membrane. The immune complexes are composed of immunoglobulin G and a target antigen. PLA2R is the target antigen in approximately 60% of MN cases, and MN is traditionally classified as PLA2R-positive or PLA2R-negative MN. Over the last 7 years, additional target antigens have been identified, which have specific disease associations, distinctive clinical and pathologic findings, and therapeutic implications. The newly discovered target antigens include NELL1, EXT1/EXT2, NCAM1, SEMA3B, PCDH7, FAT1, CNTN1, NTNG1, PCSK6 and NDNF. To group all these antigens into a generic 'PLA2R-negative' MN group is imprecise and un-informative. We propose a logical approach for detection of the target antigen which includes (i) currently available serology-based testing to detect anti-PLA2R and anti-THSD7A antibodies; and (ii) kidney biopsy testing to detect the target antigens. Determination of the antigen on kidney biopsy can be done by immunohistochemistry or immunofluorescence studies. Alternatively, laser capture microdissection (LCM) of glomeruli followed by mass spectrometry (MS) can be used to identify a target antigen. LCM/MS has the advantage of being a one-stop test and is particularly useful for detection of rare target antigens. At the current time, while it is possible to detect the newer antigens by immunohistochemistry/immunofluorescence/LCM/MS, serology-based tests to detect serum antibodies to the new antigens are not yet available. It is critical that serology-based tests should be developed not just for accurate diagnosis, but as a guide for treatment. We review the current methodology and propose an algorithm for diagnosis and detection of target antigens in MN that may shape the current practice in the future. Membranous nephropathy (MN) results from accumulation of subepithelial immune complexes along the glomerular basement membrane.PLA2R is the most common target antigen, but newly discovered target antigens have filled the void of PLA2R-negative MN.MN associated with the newly discovered target antigens have distinctive clinical and pathologic findings, treatment and prognostic implications. These include NELL1, EXT1/EXT2, NCAM1, PCDH7, SEMA3B, CNTN1, FAT1, NDNF and PCSK6.Immunohistochemistry/immunofluorescence methodology is currently in use for detecting target antigens in kidney biopsy tissue, although we anticipate laser capture microdissection of glomeruli followed by mass spectrometry will become available soon.Serologic testing is currently available for only detecting antibodies to PLA2R and THSD7A. It is critical that serologic tests become available for detecting antibodies to the newly discovered antigens.

Membranous nephropathy treatment standard.

Membranous nephropathy (MN) is characterized by deposition of immune complexes leading to thickening of glomerular basement membranes. Over time, the understanding of MN has evolved, with the identification of specific autoantibodies against novel podocyte antigens and the unraveling of intricate pathogenic pathways. Although the anti-CD20 monoclonal antibody rituximab is favored as part of the initial therapy in MN, a subgroup of MN patients may be resistant to rituximab necessitating the use of alternative agents such as cytotoxic therapies. In addition, newer agents such as novel anti-CD20 monoclonal antibodies, therapies targeting the CD38-positive plasma cells and anti-complement therapy are being studied in patients who are resistant to traditional treatment strategies. This manuscript furnishes a review of the novel developments in the pathophysiology of MN including the identification of target antigens and current treatment standards for MN, concentrating on evidenced-based interventions designed to attain remission and to prevent disease progression.

Specific antigen-based stratification of membranous nephropathy in patients after haematopoietic stem cell allotransplantation - a case series and literature review.

BACKGROUND: Nephrotic syndrome (NS) is a rare complication that can occur after haematopoietic stem cell transplantation (HSCT). In patients with membranous nephropathy (MN) who have undergone allogeneic HSCT, a new antigen called protocadherin FAT1 has been identified. Our objective is to present a case series of MN patients after HSCT with a novel antigen-based stratification. CASE PRESENTATIONS: Patients who developed full-blown NS due to MN after an HSCT were enrolled in the University Hospital Centre Zagreb study. The first two patients were treated with an HSCT for acute myeloid leukaemia, and both developed NS after cessation of graft versus host disease (GVHD) prophylaxis. The first patient had reduced kidney function, while the second had completely preserved function. Kidney biopsy showed MN with only subepithelial deposits. A thorough examination revealed that there was no secondary cause of the disease. The patients achieved complete remission after undergoing immunosuppression treatment. The third patient underwent HSCT for acute lymphoblastic leukaemia. He developed both acute and chronic GVHD and also experienced avascular hip necrosis. After sixteen years, the patient developed NS with preserved kidney function. The kidney specimen showed membranous nephropathy (MN) with mesangial and subepithelial deposits. Extensive research was conducted, but no secondary cause for the MN was detected. All three cases tested negative for anti-PLA2R antibodies. Biopsy tissue samples were analysed using laser microdissection and tandem mass spectrometry of glomeruli for the detection of different specific antigens. Patients one and two tested positive for FAT1, whereas patient three tested positive for PCSK6. CONCLUSIONS: MN can develop at various time intervals after HSCT. Specific antigen testing can help establish the relationship between MN and HSCT. In the future, serum testing for anti-FAT1 antibodies in HSCT patients could be significant in diagnosing FAT1-associated MN, similar to how anti-PLA2R antibodies are significant in diagnosing PLA2R-associated MN.

Membranous Nephropathy in Syphilis is Associated with Neuron-Derived Neurotrophic Factor.

SIGNIFICANCE STATEMENT: Syphilis is a common worldwide sexually transmitted infection. Proteinuria may occur in patients with syphilis. Membranous nephropathy (MN) is the most common cause of proteinuria in syphilis. The target antigen of MN in syphilis is unknown. This study shows that MN in syphilis is associated with a novel target antigen called neuron-derived neurotrophic factor (NDNF). NDNF-associated MN has distinctive clinical and pathologic manifestations and NDNF appears to be the target antigen in syphilis-associated MN. BACKGROUND: Syphilis is a common sexually transmitted infection. Membranous nephropathy (MN) is a common cause of proteinuria in syphilis. The target antigen is not known in most cases of syphilis-associated MN. METHODS: We performed laser microdissection of glomeruli and mass spectrometry (MS/MS) in 250 cases (discovery cohort) of phospholipase A2 receptor-negative MN to identify novel target antigens. This was followed by immunohistochemistry/confocal microscopy to localize the target antigen along the glomerular basement membrane (GBM). Western blot analyses using IgG eluted from frozen biopsy tissue were performed to detect binding to target antigen. RESULTS: MS/MS studies of the discovery cohort revealed high total spectral counts of a novel protein, neuron-derived neurotrophic factor (NDNF), in three patients: one each with syphilis and hepatitis B, HIV (syphilis status not known), and lung tumor. Next, MS/MS studies of five cases of syphilis-MN (validation cohort) confirmed high total spectral counts of NDNF (average 45±20.4) in all (100%) cases. MS/MS of 14 cases of hepatitis B were negative for NDNF. All eight cases of NDNF-associated MN were negative for known MN antigens. Electron microscopy showed stage I MN in all cases, with superficial and hump-like deposits without GBM reaction. IgG1 was the dominant IgG subtype on MS/MS and immunofluorescence microscopy. Immunohistochemistry/confocal microscopy showed granular staining and colocalization of NDNF and IgG along GBM. Western blot analyses using eluate IgG of NDNF-MN showed binding to both nonreduced and reduced NDNF, while IgG eluate from phospholipase A2 receptor-MN showed no binding. CONCLUSION: NDNF is a novel antigenic target in syphilis-associated MN.

Mapping antigens of membranous nephropathy: almost there.

Membranous nephropathy (MN) is characterized by subepithelial accumulation of immune complexes along the glomerular basement membranes. The immune complexes compromise IgG and the corresponding target antigen. Recent advances have led to the discovery of novel target MN antigens. In this study, by Caza et al., 7 novel "putative" antigens are proposed. Target antigens can now be identified in approximately 90% of cases of MN. In addition to describing another 10 novel putative antigens, we propose a working algorithm for evaluating the target antigens in MN.

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.

Pathological characteristics of light chain crystalline podocytopathy.

Monoclonal immunoglobulin light chain (LC) crystalline inclusions within podocytes are rare, poorly characterized entities. To provide more insight, we now present the first clinicopathologic series of LC crystalline podocytopathy (LCCP) encompassing 25 patients (68% male, median age 56 years). Most (80%) patients presented with proteinuria and chronic kidney disease, with nephrotic syndrome in 28%. Crystalline keratopathy and Fanconi syndrome were present in 22% and 10%, respectively. The hematologic condition was monoclonal gammopathy of renal significance (MGRS) in 55% and multiple myeloma in 45%. The serum monoclonal immunoglobulin was IgG κappa in 86%. Histologically, 60% exhibited focal segmental glomerulosclerosis (FSGS), often collapsing. Ultrastructurally, podocyte LC crystals were numerous with variable effacement of foot processes. Crystals were also present in proximal tubular cells as light chain proximal tubulopathy (LCPT) in 80% and in interstitial histiocytes in 36%. Significantly, frozen-section immunofluorescence failed to reveal the LC composition of crystals in 88%, requiring paraffin-immunofluorescence or immunohistochemistry, with identification of kappa LC in 87%. The LC variable region gene segment, determined by mass spectrometry of glomeruli or bone marrow plasma cell sequencing, was IGKV1-33 in four and IGKV3-20 in one. Among 21 patients who received anti-plasma cell-directed chemotherapy, 50% achieved a kidney response, which depended on a deep hematologic response. After a median follow-up of 36 months, 26% progressed to kidney failure and 17% died. The mean kidney failure-free survival was 57.6 months and was worse in those with FSGS. In sum, LCCP is rare, mostly associates with IgG κappa MGRS, and frequently has concurrent LCPT, although Fanconi syndrome is uncommon. Paraffin-immunofluorescence and electron microscopy are essential to prevent misdiagnosis as primary FSGS since kidney survival depends on early diagnosis and subsequent clone-directed therapy.

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.

Acute glomerulonephritis.

Glomerulonephritis is a heterogeneous group of disorders that present with a combination of haematuria, proteinuria, hypertension, and reduction in kidney function to a variable degree. Acute presentation with full blown nephritic syndrome or rapidly progressive glomerulonephritis is uncommon and is mainly restricted to patients with post-infectious glomerulonephritis, anti-neutrophil cytoplasmic antibodies-associated vasculitis, and anti-glomerular basement membrane disease. Most frequently, patients present with asymptomatic haematuria and proteinuria with or without reduced kidney function. All glomerulonephritis disorders can show periods of exacerbation, but disease flairs characteristically occur in patients with IgA nephropathy or C3 glomerulopathy. The gold standard for the diagnosis of a glomerulonephritis is a kidney biopsy, with a hallmark glomerular inflammation that translates into various histopathological patterns depending on the location and severity of the glomerular injury. Traditionally, glomerulonephritis was classified on the basis of the different histopathological patterns of injury. In the last few years, substantial progress has been made in unravelling the underlying causes and pathogenetic mechanisms of glomerulonephritis and a causal approach to the classification of glomerulonephritis is now favoured over a pattern-based approach. As such, glomerulonephritis can be broadly classified as immune-complex glomerulonephritis (including infection-related glomerulonephritis, IgA nephropathy, lupus nephritis, and cryoglobulinaemic glomerulonephritis), anti-neutrophil cytoplasmic antibodies-associated (pauci-immune) glomerulonephritis, anti-glomerular basement membrane glomerulonephritis, C3 glomerulopathy, and monoclonal immunoglobulin-associated glomerulonephritis. We provide an overview of the clinical presentation, pathology, and the current therapeutic approach of the main representative disorders in the spectrum of glomerulonephritis.

Netrin G1 Is a Novel Target Antigen in Primary Membranous Nephropathy.

BACKGROUND: Primary membranous nephropathy (MN) is caused by circulating autoantibodies binding to antigens on the podocyte surface. PLA2R1 is the main target antigen in 70%-80% of cases, but the pathogenesis is unresolved in 10%-15% of patients. METHODS: We used native western blotting to identify IgG4 autoantibodies, which bind an antigen endogenously expressed on podocyte membranes, in the serum of the index patient with MN. These IgG4 autoantibodies were used to immunoprecipitate the target antigen, and mass spectrometry was used to identify Netrin G1 (NTNG1). Using native western blot and ELISA, NTNG1 autoantibodies were analyzed in cohorts of 888 patients with MN or other glomerular diseases. RESULTS: NTNG1 was identified as a novel target antigen in MN. It is a membrane protein expressed in healthy podocytes. Immunohistochemistry confirmed granular NTNG1 positivity in subepithelial glomerular immune deposits. In prospective and retrospective MN cohorts, we identified three patients with NTNG1-associated MN who showed IgG4-dominant circulating NTNG1 autoantibodies, enhanced NTNG1 expression in the kidney, and glomerular IgG4 deposits. No NTNG1 autoantibodies were identified in 561 PLA2R1 autoantibodies-positive patients, 27 THSD7A autoantibodies-positive patients, and 77 patients with other glomerular diseases. In two patients with available follow-up of 2 and 4 years, both NTNG1 autoantibodies and proteinuria persisted. CONCLUSIONS: NTNG1 expands the repertoire of target antigens in patients with MN. The clinical role of NTNG1 autoantibodies remains to be defined.

Prognostic Implications of a Morphometric Evaluation for Chronic Changes on All Diagnostic Native Kidney Biopsies.

BACKGROUND: Semiquantitative visual inspection for glomerulosclerosis, interstitial fibrosis, and arteriosclerosis is often used to assess chronic changes in native kidney biopsies. Morphometric evaluation of these and other chronic changes may improve the prognostic assessment. METHODS: We studied a historical cohort of patients who underwent a native kidney biopsy between 1993 and 2015 and were followed through 2021 for ESKD and for progressive CKD (defined as experiencing 50% eGFR decline, temporary dialysis, or ESKD). Pathologist scores for the percentages of globally sclerosed glomeruli (GSG), interstitial fibrosis and tubular atrophy (IFTA), and arteriosclerosis (luminal stenosis) were available. We scanned biopsy sections into high-resolution images to trace microstructures. Morphometry measures were percentage of GSG; percentage of glomerulosclerosis (percentage of GSG, ischemic-appearing glomeruli, or segmentally sclerosed glomeruli); percentage of IFTA; IFTA foci density; percentage of artery luminal stenosis; arteriolar hyalinosis counts; and measures of nephron size. Models assessed risk of ESKD or progressive CKD with biopsy measures adjusted for age, hypertension, diabetes, body mass index, eGFR, and proteinuria. RESULTS: Of 353 patients (followed for a median 7.5 years), 75 developed ESKD and 139 experienced progressive CKD events. Visually estimated scores by pathologists versus morphometry measures for percentages of GSG, IFTA, and luminal stenosis did not substantively differ in predicting outcomes. However, adding percentage of glomerulosclerosis, IFTA foci density, and arteriolar hyalinosis improved outcome prediction. A 10-point score using percentage of glomerulosclerosis, percentage of IFTA, IFTA foci density, and any arteriolar hyalinosis outperformed a 10-point score based on percentages of GSG, IFTA, and luminal stenosis >50% in discriminating risk of ESKD or progressive CKD. CONCLUSION: Morphometric characterization of glomerulosclerosis, IFTA, and arteriolar hyalinosis on kidney biopsy improves prediction of long-term kidney outcomes.

Hematopoietic Stem Cell Transplant-Membranous Nephropathy Is Associated with Protocadherin FAT1.

BACKGROUND: Membranous nephropathy (MN) is a common cause of proteinuria in patients receiving a hematopoietic stem cell transplant (HSCT). The target antigen in HSCT-associated MN is unknown. METHODS: We performed laser microdissection and tandem mass spectrometry (MS/MS) of glomeruli from 250 patients with PLA2R-negative MN to detect novel antigens in MN. This was followed by immunohistochemical (IHC)/immunofluorescence (IF) microscopy studies to localize the novel antigen. Western blot analyses using serum and IgG eluted from frozen biopsy specimen to detect binding of IgG to new 'antigen'. RESULTS: MS/MS detected a novel protein, protocadherin FAT1 (FAT1), in nine patients with PLA2R-negative MN. In all nine patients, MN developed after allogeneic HSCT (Mayo Clinic discovery cohort). Next, we performed MS/MS in five patients known to have allogeneic HSCT-associated MN (Cedar Sinai validation cohort). FAT1 was detected in all five patients by MS/MS. The total spectral counts for FAT1 ranged from 8 to 39 (mean±SD, 20.9±10.1). All 14 patients were negative for known antigens of MN, including PLA2R, THSD7A, NELL1, PCDH7, NCAM1, SEMA3B, and HTRA1. Kidney biopsy specimens showed IgG (2 to 3+) with mild C3 (0 to 1+) along the GBM; IgG4 was the dominant IgG subclass. IHC after protease digestion and confocal IF confirmed granular FAT1 deposits along the GBM. Lastly, Western blot analyses detected anti-FAT1 IgG and IgG4 in the eluate obtained from pooled frozen kidney biopsy tissue and in the serum of those with FAT1-asssociated MN, but not from those with PLA2R-associated MN. CONCLUSIONS: FAT1-associated MN appears to be a unique type of MN associated with HSCT. FAT1-associated MN represents a majority of MN associated with HSCT.

The characteristics of seronegative and seropositive non-hepatitis-associated cryoglobulinemic glomerulonephritis.

The clinicopathologic characteristics and long-term outcome of non-hepatitis-associated cryoglobulinemic glomerulonephritis (CryoGN) are not well-defined and cases with undetectable serum cryoglobulin (seronegative CryoGN) have not been investigated. To resolve this, we retrospectively identified 81 patients with biopsy-proven non-hepatitis CryoGN, including 22 with seronegative CryoGN. The median age was 61 years and 76% presented with nephritic syndrome. A hematologic condition was found in 89% of patients, including monoclonal gammopathy of renal significance (65%) and symptomatic lymphoproliferative disorder (35%). In the seropositive group, 56% had type II, 29% type I, and 8% type III cryoglobulin. Extrarenal manifestations, mostly of skin, were present in 64% and were significantly less common in seronegative CryoGN. Glomerular deposits by immunofluorescence were IgM dominant (84%) and polytypic (70%) in the seropositive group, whereas 52% of seronegative cases had monotypic deposits (i.e., type I cryoglobulin). Ultrastructurally, the deposits were organized in 77% of cases. Substructure appearance significantly differed according to the type of CryoGN, forming most commonly short cylindrical structures in type II and other organized substructures in type I CryoGN. Most patients were treated with clone-directed therapy. On follow up (median 33 months), 77% had partial or complete remission, 10% reached kidney failure and 14% died. Predictors of kidney failure on univariate analysis were AKIN stage 3, positive rheumatoid factor and biclonal gammopathy at diagnosis. We conclude that most CryoGN cases (types I and II) are due to a hematologic condition and are associated with favorable outcome after clone-directed therapy. Seronegative CryoGN accounts for about a quarter of cases and is mostly a kidney-limited disease. Thus, further investigations are needed to unravel the pathophysiology of seronegative CryoGN.