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.

sBioSITe enables sensitive identification of the cell surface proteome through direct enrichment of biotinylated peptides.

BACKGROUND: Cell surface proteins perform critical functions related to immune response, signal transduction, cell-cell interactions, and cell migration. Expression of specific cell surface proteins can determine cell-type identity, and can be altered in diseases including infections, cancer and genetic disorders. Identification of the cell surface proteome remains a challenge despite several enrichment methods exploiting their biochemical and biophysical properties. METHODS: Here, we report a novel method for enrichment of proteins localized to cell surface. We developed this new approach designated surface Biotinylation Site Identification Technology (sBioSITe) by adapting our previously published method for direct identification of biotinylated peptides. In this strategy, the primary amine groups of lysines on proteins on the surface of live cells are first labeled with biotin, and subsequently, biotinylated peptides are enriched by anti-biotin antibodies and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: By direct detection of biotinylated lysines from PC-3, a prostate cancer cell line, using sBioSITe, we identified 5851 peptides biotinylated on the cell surface that were derived from 1409 proteins. Of these proteins, 533 were previously shown or predicted to be localized to the cell surface or secreted extracellularly. Several of the identified cell surface markers have known associations with prostate cancer and metastasis including CD59, 4F2 cell-surface antigen heavy chain (SLC3A2) and adhesion G protein-coupled receptor E5 (CD97). Importantly, we identified several biotinylated peptides derived from plectin and nucleolin, both of which are not annotated in surface proteome databases but have been shown to have aberrant surface localization in certain cancers highlighting the utility of this method. CONCLUSIONS: Detection of biotinylation sites on cell surface proteins using sBioSITe provides a reliable method for identifying cell surface proteins. This strategy complements existing methods for detection of cell surface expressed proteins especially in discovery-based proteomics approaches.

Writers and readers of H3K9me2 form distinct protein networks during the cell cycle that include candidates for H3K9 mimicry.

Histone H3 lysine 9 methylation (H3K9me), which is written by the Euchromatic Histone Lysine Methyltransferases EHMT1 and EHMT2 and read by the heterochromatin protein 1 (HP1) chromobox (CBX) protein family, is dysregulated in many types of cancers. Approaches to inhibit regulators of this pathway are currently being evaluated for therapeutic purposes. Thus, knowledge of the complexes supporting the function of these writers and readers during the process of cell proliferation is critical for our understanding of their role in carcinogenesis. Here, we immunopurified each of these proteins and used mass spectrometry to define their associated non-histone proteins, individually and at two different phases of the cell cycle, namely G1/S and G2/M. Our findings identify novel binding proteins for these writers and readers, as well as corroborate known interactors, to show the formation of distinct protein complex networks in a cell cycle phase-specific manner. Furthermore, there is an organizational switch between cell cycle phases for interactions among specific writer-reader pairs. Through a multi-tiered bioinformatics-based approach, we reveal that many interacting proteins exhibit histone mimicry, based on an H3K9-like linear motif. Gene ontology analyses, pathway enrichment, and network reconstruction inferred that these comprehensive EHMT and CBX-associated interacting protein networks participate in various functions, including transcription, DNA repair, splicing, and membrane disassembly. Combined, our data reveals novel complexes that provide insight into key functions of cell cycle-associated epigenomic processes that are highly relevant for better understanding these chromatin-modifying proteins during cell cycle and carcinogenesis.

Differential proteomic expression profiles in vulvar lichen planus as compared to normal vulvar tissue, vulvar lichen sclerosus, or oral lichen planus: An exploratory study.

Vulvar lichen planus (VLP) is a chronic inflammatory disease which adversely affects patients' quality of life. The pathogenesis of VLP is unknown although Th1 immune response has been implicated. We aimed to discover specific tissue-based protein biomarkers in VLP compared to normal vulvar tissue (NVT), vulvar lichen sclerosus (VLS) and oral lichen planus (OLP). We used laser capture microdissection-liquid chromatography- tandem mass spectrometry to assess protein expression in fixed lesional mucosal specimens from patients with VLP (n = 5). We then compared proteomic profiles against those of NVT (n = 4), VLS (n = 5), OLP (n = 6) and normal oral mucosa (n = 5), previously published by our group. IL16, PTPRC, PTPRCAP, TAP1 and ITGB2 and were significantly overexpressed in VLP compared to NVT. Ingenuity pathway analysis identified antigen presentation and integrin signalling pathways. Proteins overexpressed in both VLP versus NVT and OLP versus NOM included IL16, PTPRC, PTPRCAP, TAP1, HLA-DPB1, HLA-B and HLA-DRA. This proteomic analysis revealed several overexpressed proteins in VLP that relate to Th1 autoimmunity, including IL16. Overlapping pathways, including those involving IFNγ and Th1 signalling, were observed between VLP, VLS, and OLP.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

An atlas of the bone marrow bone proteome in patients with dysproteinemias.

Multiple myeloma (MM) bone disease is a significant cause of morbidity but there is a paucity of data on the impact of malignant plasma cells on adjacent trabecular bone within the BM. Here, we characterize the proteome of trabecular bone tissue from BM biopsies of 56 patients with monoclonal gammopathy of undetermined significance (MGUS), smoldering (SMM), newly diagnosed (NDMM), relapsed MM (RMM), and normal controls. Proteins involved in extracellular matrix (ECM) formation and immunity pathways were decreased in SMM and active MM. Among the proteins most decreased were immunoglobulins, type IV collagen, and TIMP3, suggesting increased immunoparesis and decreased ECM remodelling within trabecular bone. Proteins most increased in SMM/MM were APP (enhances osteoclast activity), ENPP1 (enhances bone mineralization), and MZB1 (required for normal plasmablast differentiation). Pathway analyses showed that proteins involved in gamma -carboxylation, a pathway implicated in osteocalcin function, osteoblast differentiation, and normal hematopoiesis, were also overexpressed in SMM/MM. This study is the first comprehensive proteomic atlas of the BM bone proteome in dysproteinemias. We identify new key proteins and pathways for MM bone disease and potentially impaired hematopoiesis, and show for the first time that gamma -carboxylation pathways are increased in the bone tissue of SMM/MM.

Differential proteomic expression in indolent versus transforming oral lichen planus.

Oral lichen planus (OLP) confers an approximately 1% risk of transformation to oral squamous cell carcinoma (OSCC). Early identification of high-risk OLP would be very helpful for optimal patient management. We aimed to discover specific tissue-based protein biomarkers in patients with OLP who developed OSCC compared to those who did not. We used laser capture microdissection- and nanoLC-tandem mass spectrometry to assess protein expression in fixed lesional mucosal specimens in patients with indolent OLP (no OSCC after at least 5-year follow-up, n = 6), transforming OLP (non-dysplastic epithelium with lichenoid inflammation marginal to OSCC, n = 6) or normal oral mucosa (NOM, n = 5). Transforming OLP protein profile was enriched for actin cytoskeleton, mitochondrial dysfunction and oxidative phosphorylation pathways. CA1, TNNT3, SYNM and MB were overexpressed, and FBLN1 was underexpressed in transforming OLP compared with indolent OLP. Integrin signalling and antigen presentation pathways were enriched in both indolent and transforming OLP compared with NOM. This proteomic study provides potential biomarkers, such as CA1 overexpression, for higher-risk OLP. While further validation studies are needed, we propose that epithelial-mesenchymal transition may be involved in OLP carcinogenesis.

Molecular signatures of inherited and acquired sporadic late onset nemaline myopathies.

Acquired sporadic late onset nemaline myopathy (SLONM) and inherited nemaline myopathy (iNM) both feature accumulation of nemaline rods in muscle fibers. Unlike iNM, SLONM is amenable to therapy. The distinction between these disorders is therefore crucial when the diagnosis remains ambiguous after initial investigations. We sought to identify biomarkers facilitating this distinction and to investigate the pathophysiology of nemaline rod formation in these different disorders. Twenty-two muscle samples from patients affected by SLONM or iNM underwent quantitative histological analysis, laser capture microdissection for proteomic analysis of nemaline rod areas and rod-free areas, and transcriptomic analysis. In all iNM samples, nemaline rods were found in subsarcolemmal or central aggregates, whereas they were diffusely distributed within muscle fibers in most SLONM samples. In SLONM, muscle fibers harboring nemaline rods were smaller than those without rods. Necrotic fibers, increased endomysial connective tissue, and atrophic fibers filled with nemaline rods were more common in SLONM. Proteomic analysis detected differentially expressed proteins between nemaline rod areas and rod-free areas, as well as between SLONM and iNM. These differentially expressed proteins implicated immune, structural, metabolic, and cellular processes in disease pathophysiology. Notably, immunoglobulin overexpression with accumulation in nemaline rod areas was detected in SLONM. Transcriptomic analysis corroborated proteomic findings and further revealed substantial gene expression differences between SLONM and iNM. Overall, we identified unique pathological and molecular signatures associated with SLONM and iNM, suggesting distinct underlying pathophysiological mechanisms. These findings represent a step towards enhanced diagnostic tools and towards development of treatments for SLONM.

Histologic and proteomic remodeling of the pulmonary veins and arteries in a porcine model of chronic pulmonary venous hypertension.

AIMS: In heart failure (HF), pulmonary venous hypertension (PVH) produces pulmonary hypertension (PH) with remodeling of pulmonary veins (PV) and arteries (PA). In a porcine PVH model, we performed proteomic-based bioinformatics to investigate unique pathophysiologic mechanisms mediating PA and PV remodeling. METHODS AND RESULTS: Large PV were banded (PVH, n = 10) or not (Sham, n = 9) in piglets. At sacrifice, PV and PA were perfusion labelled for vessel-specific histology and proteomics. The PA and PV were separately sampled with laser-capture micro-dissection for mass spectrometry. Pulmonary vascular resistance [Wood Units; 8.6 (95% confidence interval: 6.3, 12.3) vs. 2.0 (1.7, 2.3)] and PA [19.9 (standard error of mean, 1.1) vs. 10.3 (1.1)] and PV [14.2 (1.2) vs. 7.6 (1.1)] wall thickness/external diameter (%) were increased in PVH (P < 0.05 for all). Similar numbers of proteins were identified in PA (2093) and PV (2085) with 94% overlap, but biological processes differed. There were more differentially expressed proteins (287 vs. 161), altered canonical pathways (17 vs. 3), and predicted upstream regulators (PUSR; 22 vs. 6) in PV than PA. In PA and PV, bioinformatics indicated activation of the integrated stress response and mammalian target of rapamycin signalling with dysregulated growth. In PV, there was also activation of Rho/Rho-kinase signalling with decreased actin cytoskeletal signalling and altered tight and adherens junctions, ephrin B, and caveolae-mediated endocytosis signalling; all indicating disrupted endothelial barrier function. Indeed, protein biomarkers and the top PUSR in PV (transforming growth factor-beta) suggested endothelial to mesenchymal transition in PV. Findings were similar in human autopsy specimens. CONCLUSION: These findings provide new therapeutic targets to oppose pulmonary vascular remodeling in HF-related PH.

Differential proteomic expression in indolent vulvar lichen sclerosus, transforming vulvar lichen sclerosus and normal vulvar tissue.

Vulvar lichen sclerosus (VLS) confers approximately 3% risk of malignant transformation to vulvar squamous cell carcinoma (VSCC). We used unbiased proteomic methods to identify differentially expressed proteins in tissue of patients with VLS who developed VSCC compared to those who did not. We used laser capture microdissection- and nanoLC-tandem mass spectrometry to assess protein expression in individuals in normal vulvar tissue (NVT, n = 4), indolent VLS (no VSCC after at least 5 years follow-up, n = 5) or transforming VSCC (preceding VSCC, n = 5). Interferon-γ and antigen-presenting pathways are overexpressed in indolent and transforming VLS compared to NVT. There was differential expression of malignancy-related proteins in transforming VLS compared to indolent VLS (CAV1 overexpression, AKAP12 underexpression), particularly in the EIF2 translation pathway, which has been previously implicated in carcinogenesis. Results of this study provide additional molecular evidence supporting the concept that VLS is a risk factor for VSCC and highlights possible future biomarkers and/or therapeutic targets.

Proteome-Wide Response of Dormant Caryopses of the Weed, Avena fatua, After Colonization by a Seed-Decay Isolate of Fusarium avenaceum.

Promoting seed decay is an ecological approach to reducing weed persistence in the soil seedbank. Previous work demonstrated that Fusarium avenaceum F.a.1 decays dormant Avena fatua (wild oat) caryopses and induces several defense enzyme activities in vitro. The objectives of this study were to obtain a global perspective of proteins expressed after F.a.1-caryopsis colonization by conducting proteomic evaluations on (i) leachates, soluble extrinsic (seed-surface) proteins released upon washing caryopses in buffer and (ii) proteins extracted from whole caryopses; interactions with aluminum (Al) were also evaluated in the latter study because soil acidification and associated metal toxicity are growing problems. Of the 119 leachate proteins classified as defense/stress, 80 were induced or repressed. Defense/stress proteins were far more abundant in A. fatua (35%) than in F.a.1 (12%). Avena defense/stress proteins were also the most highly regulated category, with 30% induced and 35% repressed by F.a.1. Antifungal proteins represented 36% of Avena defense proteins and were the most highly regulated, with 36% induced and 37% repressed by F.a.1. These results implicate selective regulation of Avena defense proteins by F.a.1. Fusarium proteins were also highly abundant in the leachates, with 10% related to pathogenicity, 45% of which were associated with host cell wall degradation. In whole caryopsis extracts, fungal colonization generally resulted in induction of a similar set of Avena proteins in the presence and absence of Al. Results advance the hypothesis that seed decay pathogens elicit intricate and dynamic biochemical responses in dormant seeds.

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 proteome of distal nerves: implication in delayed repair and poor functional recovery.

Chronic denervation is one of the key factors that affect nerve regeneration. Chronic axotomy deteriorates the distal nerve stump, causes protein changes, and renders the microenvironment less permissive for regeneration. Some of these factors/proteins have been individually studied. To better delineate the comprehensive protein expression profiles and identify proteins that contribute to or are associated with this detrimental effect, we carried out a proteomic analysis of the distal nerve using an established delayed rat sciatic nerve repair model. Four rats that received immediate repair after sciatic nerve transection served as control, whereas four rats in the experimental group (chronic denervation) had their sciatic nerve repaired after a 12-week delay. All the rats were sacrificed after 16 weeks to harvest the distal nerves for extracting proteins. Twenty-five micrograms of protein from each sample were fractionated in SDS-PAGE gels. NanoLC-MS/MS analysis was applied to the gels. Protein expression levels of nerves on the surgery side were compared to those on the contralateral side. Any protein with a P value of less than 0.05 and a fold change of 4 or higher was deemed differentially expressed. All the differentially expressed proteins in both groups were further stratified according to the biological processes. A PubMed search was also conducted to identify the differentially expressed proteins that have been reported to be either beneficial or detrimental to nerve regeneration. Ingenuity Pathway Analysis (IPA) software was used for pathway analysis. The results showed that 709 differentially expressed proteins were identified in the delayed repair group, with a bigger proportion of immune and inflammatory process-related proteins and a smaller proportion of proteins related to axon regeneration and lipid metabolism in comparison to the control group where 478 differentially expressed proteins were identified. The experimental group also had more beneficial proteins that were downregulated and more detrimental proteins that were upregulated. IPA revealed that protective pathways such as LXR/RXR, acute phase response, RAC, ERK/MAPK, CNTF, IL-6, and FGF signaling were inhibited in the delayed repair group, whereas three detrimental pathways, including the complement system, PTEN, and apoptosis signaling, were activated. An available database of the adult rodent sciatic nerve was used to assign protein changes to specific cell types. The poor regeneration seen in the delayed repair group could be associated with the down-regulation of beneficial proteins and up-regulation of detrimental proteins. The proteins and pathways identified in this study may offer clues for future studies to identify therapeutic targets.

Proteomic profiling of sporadic late-onset nemaline myopathy.

OBJECTIVE: To define the proteomic profile of sporadic late-onset nemaline myopathy (SLONM) and explore its pathogenesis. METHODS: We performed mass spectrometry on laser-dissected frozen muscle samples from five patients with SLONM, three of whom with an associated monoclonal protein (MP), and four controls, to determine the proteomic profile of SLONM. Furthermore, we assessed the role of the MP by evaluating the expression of the immunoglobulin light chain variable regions (IGVL). RESULTS: There were 294 differentially expressed proteins: 272 upregulated and 22 downregulated. Among the top 100 upregulated proteins, the most common categories were: nuclear or nucleic acid metabolism (24%), extracellular matrix and basal lamina (17%), immune response (13%), and actin dynamics (8%). Downregulated proteins consisted mostly of contractile proteins. Among upregulated proteins, there were 65 with a role related to the immune system, including eight proteins involved in major histocompatibility complex 1 (MHC1) and antigen processing, 15 in MHCII complex and phagocytosis, and 23 in B and/or T-cell function. Among nine upregulated immunoglobulin proteins, there were two IGVL genes. However, these were also detected in SLONM cases without an MP, with no evidence of clonally dominant immunoglobulin deposition. In muscle sections from SLONM patients, nemaline rods tended to accumulate in atrophic fibers with marked rarefaction of the myofibrils. Increased MHC1 reactivity was present in fibers containing nemaline rods as well as adjacent nonatrophic fibers. CONCLUSION: Our findings suggest that aberrant immune activation is present in SLONM, but do not support a direct causal relationship between the MP and SLONM.

Membranous Nephropathy With Extensive Tubular Basement Membrane Deposits Following Allogeneic Hematopoietic Cell Transplant: A Report of 5 Cases.

Tubular basement membrane (TBM) deposits are very uncommon in non-lupus membranous nephropathy. We report 5 patients with membranous nephropathy and extensive TBM deposits following allogeneic hematopoietic cell transplant. Patients presented with nephrotic syndrome (3 also had acute kidney injury) late post-transplant in association with chronic graft-versus-host disease (cGVHD). Kidney biopsies revealed global subepithelial and extensive TBM immune complex deposits, accompanied by acute tubular injury (n = 4) and tubulointerstitial inflammation (n = 4). Proteomic analysis of glomeruli in 4 cases identified PLA2R in 1, with no significant protein spectra for PLA2R, THSD7A, EX1/2, NELL-1, PCDH7, NCAM1, or SEMA3B detected in the remaining 3. On follow-up (for a mean 42 months), 4 patients had complete and 1 partial remission following prednisone and/or rituximab therapy. We propose that membranous nephropathy with extensive TBM deposits is a distinctive clinicopathologic lesion associated with allogeneic hematopoietic cell transplant. Pathogenesis likely involves cGVHD-driven antibodies against glomerular and TBM components, the identity of which remains to be elucidated.

1H Nuclear Magnetic Resonance Spectroscopy-Based Methods for the Quantification of Proteins in Urine.

We described several postprocessing methods to measure protein concentrations in human urine from existing 1H nuclear magnetic resonance (NMR) metabolomic spectra: (1) direct spectral integration, (2) integration of NCD spectra (NCD = 1D NOESY-CPMG), (3) integration of SMolESY-filtered 1D NOESY spectra (SMolESY = Small Molecule Enhancement SpectroscopY), (4) matching protein patterns, and (5) TSP line integral and TSP linewidth. Postprocessing consists of (a) removal of the metabolite signals (demetabolization) and (b) extraction of the protein integral from the demetabolized spectra. For demetabolization, we tested subtraction of the spin-echo 1D spectrum (CPMG) from the regular 1D spectrum and low-pass filtering of 1D NOESY by its derivatives (c-SMolESY). Because of imperfections in the demetabolization, in addition to direct integration, we extracted protein integrals by the piecewise comparison of demetabolized spectra with the reference spectrum of albumin. We analyzed 42 urine samples with protein content known from the bicinchoninic acid (BCA) assay. We found excellent correlation between the BCA assay and the demetabolized NMR integrals. We have provided conversion factors for calculating protein concentrations in mg/mL from spectral integrals in mM. Additionally, we found the trimethylsilyl propionate (TSP, NMR standard) spectral linewidth and the TSP integral to be good indicators of protein concentration. The described methods increase the information content of urine NMR metabolomics spectra by informing clinical studies of protein concentration.

Protocadherin 7-Associated Membranous Nephropathy.

BACKGROUND: Membranous nephropathy (MN) results from deposition of antigen-antibody complexes along the glomerular basement membrane (GBM). PLA2R, THSD7A, NELL1, and SEMA3B account for 80%-90% of target antigens in MN. METHODS: We performed laser microdissection and mass spectrometry (MS/MS) in kidney biopsies from 135 individuals with PLA2R-negative MN, and used immunohistochemistry/immunofluorescence and confocal microscopy to confirm the MS/MS finding, detect additional cases, and localize the novel protein. We also performed MS/MS and immunohistochemistry on 116 controls and used immunofluorescence microscopy to screen biopsy samples from two validation cohorts. Western blot and elution studies were performed to detect antibodies in serum and biopsy tissue. RESULTS: MS/MS studies detected a unique protein, protocadherin 7 (PCDH7), in glomeruli of ten (5.7%) PLA2R-negative MN cases, which also were negative for PLA2R, THSD7A, EXT1/EXT2, NELL1, and SEMA3B. Spectral counts ranged from six to 24 (average 13.2 [SD 6.6]). MS/MS did not detect PCDH7 in controls (which included 28 PLA2R-positive cases). In all ten PCDH7-positive cases, immunohistochemistry showed bright granular staining along the GBM, which was absent in the remaining cases of PLA2R-negative MN and control cases. Four of 69 (5.8%) cases in the validation cohorts (all of which were negative for PLA2R, THSD7A, EXT1, NELL1, and SEMA3B) were PCDH7-positive MN. Kidney biopsy showed minimal complement deposition in 12 of the 14 PCDH7-associated cases. Confocal microscopy showed colocalization of PCDH7 and IgG along the GBM. Western blot analysis using sera from six patients showed antibodies to nonreduced PCDH7. Elution of IgG from frozen tissue of PCDH7-associated MN showed reactivity against PCDH7. CONCLUSIONS: MN associated with the protocadherin PCDH7 appears to be a distinct, previously unidentified type of MN.

In Patients with Membranous Lupus Nephritis, Exostosin-Positivity and Exostosin-Negativity Represent Two Different Phenotypes.

BACKGROUND: In patients with secondary (autoimmune) membranous nephropathy, two novel proteins, Exostosin 1 and Exostosin 2 (EXT1/EXT2), are potential disease antigens, biomarkers, or both. In this study, we validate the EXT1/EXT2 findings in a large cohort of membranous lupus nephritis. METHODS: We conducted a retrospective cohort study of patients with membranous lupus nephritis, and performed immunohistochemistry studies on the kidney biopsy specimens against EXT1 and EXT2. Clinicopathologic features and outcomes of EXT1/EXT2-positive versus EXT1/EXT2-negative patients were compared. RESULTS: Our study cohort included 374 biopsy-proven membranous lupus nephritis cases, of which 122 (32.6%) were EXT1/EXT2-positive and 252 (67.4%) were EXT1/EXT2-negative. EXT1/EXT2-positive patients were significantly younger (P=0.01), had significantly lower serum creatinine levels (P=0.02), were significantly more likely to present with proteinuria ≥3.5 g/24 h (P=0.009), and had significantly less chronicity features (glomerulosclerosis, P=0.001 or interstitial fibrosis and tubular atrophy, P<0.001) on kidney biopsy. Clinical follow-up data were available for 160 patients, of which 64 (40%) biopsy results were EXT1/EXT2-positive and 96 (60%) were EXT1/EXT2-negative. The proportion of patients with class 3/4 lupus nephritis coexisting with membranous lupus nephritis was not different between the EXT1/EXT2-positive and EXT1/EXT2-negative groups (25.0% versus 32.3%; P=0.32). The patients who were EXT1/EXT2-negative evolved to ESKD faster and more frequently compared with EXT1/EXT2-positive patients (18.8% versus 3.1%; P=0.003). CONCLUSIONS: The prevalence of EXT1/EXT2 positivity was 32.6% in our cohort of membranous lupus nephritis. Compared with EXT1/EXT2-negative membranous lupus nephritis, EXT1/EXT2-positive disease appears to represent a subgroup with favorable kidney biopsy findings with respect to chronicity indices. Cases of membranous lupus nephritis that are EXT1/EXT2-negative are more likely to progress to ESKD compared with those that are EXT1/EXT2-positive.

Semaphorin 3B-associated membranous nephropathy is a distinct type of disease predominantly present in pediatric patients.

Membranous nephropathy results from subepithelial antigen-antibody complex deposition along the glomerular basement membrane. Although PLA2R, THSD7A, and NELL-1 account for a majority (about 80%) of the target antigens, the target antigen in the remaining cases is not known. Using laser microdissection of PLA2R-negative glomeruli of patients with membranous nephropathy followed by mass spectrometry we identified a unique protein, Semaphorin 3B, in three cases. Mass spectrometry failed to detect Semaphorin-3B in 23 PLA2R-associated cases of membranous nephropathy and 88 controls. Semaphorin 3B in all three cases was localized to granular deposits along the glomerular basement membrane by immunohistochemistry. Next, an additional eight cases of Semaphorin 3B-associated membranous nephropathy were identified in three validation cohorts by immunofluorescence microscopy. In four of 11 cases, kidney biopsy also showed tubular basement membrane deposits of IgG on frozen sections. Confocal microscopy showed that both IgG and Semaphorin 3B co-localized to the glomerular basement membrane. Western blot analysis of five available sera showed reactivity to reduced Semaphorin 3B in four of four patients with active disease and no reactivity in one patient in clinical remission; there was also no reactivity in control sera. Eight of the 11 cases of Semaphorin 3B-associated membranous nephropathy were pediatric cases. Furthermore, in five cases, the disease started at or below the age of two. Thus, Semaphorin 3B-associated membranous nephropathy appears to be a distinct type of disease; more likely to be present in pediatric patients.

Proteomic Analysis of Complement Proteins in Membranous Nephropathy.

INTRODUCTION: Membranous nephropathy (MN) is the most common cause of nephrotic syndrome in Caucasian adults. Phospholipase A2 receptor (PLA2R)- and exostosin 1 (EXT1)/exostosin 2 (EXT2)-associated MN represent the most common primary and secondary forms of MN. The complement profile using a proteomics approach has not been studied in these 2 common forms of MN. METHODS: We used laser microdissection and mass spectrometry (MS/MS) to dissect glomeruli and identify glomerular complement proteins in PLA2R-associated (n = 7), EXT1/EXT2-associated MN (n = 21), and 11 control cases (time 0 transplant biopsies). RESULTS: MS/MS identified high total spectral counts for PLA2R and EXT1/EXT2 in corresponding cases of PLA2R- and EXT1/EXT2-positive MN. Both PLA2R- and EXT1/EXT2-associated MN had high spectral counts of complement proteins C3, C4, C5, C6, C7, C8, and C9. Complement protein C1 was present in low spectral counts in EXT1/EXT2-associated MN. Regulators of complement activation that were detected in MN included higher spectral counts of FH, FHR-1, FHR-5, clusterin, vitronectin and lower spectral counts of FHR-3, FHR-4, and CD59. Low spectral counts of FB and properdin, key components of the alternative pathway, also were detected. IgG4 and IgG1 were the most abundant IgG subclasses in PLA2R- and EXT1/EXT2-associated MN. Lower spectral counts for C3, C4, and C5 were detected in control cases when compared with MN. CONCLUSION: Significant complement activation is present in MN as evidenced by large spectral counts of complement proteins from C3- and C4-based pathways, including regulatory proteins of complement pathways. These data suggest that anticomplement drugs may be effective in treatment for MN.