Dnaj Heat Shock Protein Family B Member 9 Is a Novel Biomarker for Fibrillary GN

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DnaJ Heat Shock Protein Family B Member 9 Is a Novel Biomarker for Fibrillary GN

Surendra Dasari,1 Mariam P. Alexander,2 Julie A. Vrana,2 Jason D. Theis,2 John R. Mills,2 Vivian Negron,2 Sanjeev Sethi,2 Angela Dispenzieri,2,3 W. Edward Highsmith Jr.,2 Samih H. Nasr,2 and Paul J. Kurtin2

Departments of 1Health Sciences Research, 2Laboratory Medicine and Pathology, and 3Internal Medicine, Mayo Clinic, Rochester, Minnesota BRIEF COMMUNICATION

ABSTRACT Fibrillary GN (FGN) is a rare primary glomerular disease. Histologic and histochem- Significance Statement ical features of FGN overlap with those of other glomerular diseases, and no unique histologic biomarkers for diagnosing FGN have been identified.Weanalyzedthe Fibrillary glomerulonephritis (FGN) is a proteomic content of glomeruli in patient biopsy specimens and detected DnaJ heat primary glomerular disease with a poor shock protein family (Hsp40) member B9 (DNAJB9) as the fourth most abundant prognosis. Currently FGN poses substan- tial diagnostic challenges, in part because protein in FGN glomeruli. Compared with amyloidosis glomeruli, FGN glomeruli there are no specific histological biomarkers. . exhibited a 6-fold overexpression of DNAJB9 protein. Sanger sequencing and This manuscript describes the discovery, protein sequence coverage maps showed that the DNAJB9 protein deposited in using proteomics, of a new potential bio- FGN glomeruli did not have any major sequence or structural alterations. Notably, marker, DNAJB9 (DnaJ Heat Shock Protein we detected DNAJB9 in all patients with FGN but not in healthy glomeruli or in 19 Family [Hsp40] Member B9). We demonstrate that DNAJB9 is present in over- types of non-FGN glomerular diseases. We also observed the codeposition of abundance in FGN glomeruli, but not in fi DNAJB9 and Ig-g. Overall, these ndings indicate that DNAJB9 is an FGN marker glomeruli from patients with other glo- with 100% sensitivity and 100% specificity. The magnitude and specificity of merular diseases or from healthy subjects. DNAJB9 overabundance in FGN also suggests that this protein has a role in FGN DNAJB9 is potentially a useful diagnostic pathogenesis. With this evidence, we propose that DNAJB9 is a strong biomarker marker for FGN. Study of its function may provide important clues to the underlying for rapid diagnosis of FGN in renal biopsy specimens. pathogenesis of this disease. J Am Soc Nephrol 29: 51–56, 2018. doi: https://doi.org/10.1681/ASN.2017030306

Fibrillary GN (FGN) is a rare glomerular detection of haphazardly arranged, stance, glomerular fibrillar deposits ob- disease seen in 0.5%–1% of native kidney straight fibrils measuring 10–30 nm in servedwithEMcanbeseeninotherlesions biopsy specimens.1,2 Patients with FGN thickness in the mesangium and/or like amyloidosis and diabetic fibrillosis. present with proteinuria, hematuria, re- along the glomerular basement mem- Also, theIFfindingscanoverlapwithother nal impairment, and hypertension. Most branes by electron microscopy (EM). cases are idiopathic,1–4 although a third The deposits are Congo red (CR) nega- of the cases occur in association with tive, distinguishing FGN from amyloid- Received March 20, 2017. Accepted July 22, 2017. hepatitis C infection, dysproteinemia, osis. IF in a typical patient with FGN S.D. and M.P.A. contributed equally to this work. 4,5 or autoimmune diseases. FGN has shows smudgy glomerular staining for S.H.N. and P.J.K. are co-senior authors. a poor prognosis, with nearly 50% of IgG (g), both k and l light chains, and Published online ahead of print. Publication date patients progressing to ESRD within C3. LM demonstrates mesangial expan- available at www.jasn.org. 4 years despite therapeutic interven- sion/hypercellularity with or without tions.4 The pathogenesis of FGN is un- duplication of the glomerular basement Correspondence: Dr. Samih H. Nasr, Mayo Clinic, Division of Anatomic Pathology, Hilton 10–20, 200 known. Traditionally, an FGN diagnosis membranes. To date, there is no unique First Street, SW, Rochester, MN 55905. Email: Nasr. relies on integrating ultrastructural, biomarker for diagnosing FGN. [email protected] fl fi immuno uorescence (IF), and light mi- The main pathologic ndings in FGN Copyright © 2018 by the American Society of croscopic (LM) findings. These include can overlap with other diseases. For in- Nephrology

J Am Soc Nephrol 29: 51–56, 2018 ISSN : 1046-6673/2901-51 51 BRIEF COMMUNICATION www.jasn.org diseases like immunotactoid glomerulop- transplant glomerulopathy, n=5 im- amyloidosis samples and to perform athy and lupus nephritis. The pathologic munotactoid GN, n=5 fibronectin differential expression analysis. We de- changes seen on LM are nonspecific, and glomerulopathy, n=4 lupus nephritis tected DNAJB9 (DnaJ Heat Shock Pro- glomeruli can be unremarkable on LM in class IV and V, n=4 cryoglobulinemic tein Family [Hsp40] Member B9) as the early stage FGN. Because of these limita- GN, n=2 lupus nephritis class IV, n=2 fourth most abundant protein (by tions, we assembled a large renal biopsy diabetic glomerulosclerosis, n=2 glo- normalized spectral counts) in FGN glo- cohort containing 253 patients to study merular thrombotic microangiopathy, meruli (Supplemental Table 2). Table 1 the proteomic characteristics of FGN in n=2 membranous nephropathy, n=2 presents a list of proteins that were over- search of an FGN-specificdiagnostic proliferative GN with monoclonal abundant in the FGN cohort when com- marker. The Mayo Clinic Institutional IgG deposits, n=2 Ig g heavy chain pared with the amyloidosis cohort. Review Board approved this study and deposition disease, and n=2 IgA ne- DNAJB9 was the top most overabun- it was conducted in accordance to the phropathy). All of the diagnoses in dant protein in FGN glomeruli when Declaration of Helsinki. The patients this group were established following compared with amyloidosis glomeruli in the study were distributed among standard clinical and pathologic criteria. (FDR corrected P value=3.79E2286 four different subcohorts: 4. Healthy subcohort (n=12). and log2[fold change]=6.68). We also detected an overabundance of complement 1. FGN subcohort (n=24). Supplemental The FGN proteome has never been factor proteins in FGN glomeruli when Table 1 shows the salient clinical char- fi compared with renal amyloidosis (Table fi de nitively determined. Hence, we wan- acteristics and pathologic ndings of ted to assess whether FGN glomeruli 1), consistent with the IF findings in these patients. contain unique protein expression FGN. A pathway analysis with pro- patterns that could provide specificdi- teins that were overabundant in FGN 2. Renal amyloidosis subcohort (n=145 agnostic biomarkers and/or clues to the glomeruli showed that these proteins distributed among n=32 AL-k, n=57 pathogenesis of FGN. To accomplish this, are involved in activation and regulation AL-l, n=31 ALect2, n=22 AA, n=2 we used laser microdissection (LMD) to of the C3 and C5 complement cascade AFib, and n=1 ALys). In each of these extract glomeruli from formalin-fixed (Supplemental Table 3). We did not de- patients, the kidney specimens con- paraffin-embedded (FFPE) biopsy sam- tect differential abundance of Ig light tained CR-positive amyloid deposits ples obtained from patients with FGN, chains in FGN glomeruli when com- that were typed using a previously renal amyloidosis, and NFGNGD and pared with amyloidosis because approx- published mass spectrometry–based healthy subjects. Proteins present in the imately 60% of the renal amyloidosis proteomics assay.6 dissections were analyzed using liquid samples were of AL type. However, a sep- 3. Non-FGN glomerular disease subcohort chromatography–assisted tandem mass arate differential expression analysis (NFGNGD; n=72 including: n=13 spectrometry (LC-MS/MS) and identi- comparing FGN and healthy glomeruli dense deposit disease, n=7 C3 GN, fied using a previously published bioin- samples showed overabundance of Ig-g, n=5 anti-GBM nephritis, n=5 bac- formatics pipeline.7 We used QuasiTel8 Ig-k, and Ig-l in FGN glomeruli (data terial infection–associated GN, n=5 software to normalize the protein not shown), which is another known fea- pauci-immune crescentic GN, n=5 spectral counts found in the FGN and ture of FGN. This proteomic confirmation

Table 1. Proteins overabundant in FGN glomeruli compared with renal amyloidosis a b Protein Accession Log2(Fold Change) P Value FDR Description gi|9558755|ref|NP_036460.1| 6.68056009 3.79E2288 3.09E2286 DNAJ homolog subfamily B member 9 gi|13540563|ref|NP_110414.1| 3.26054699 7.12E236 7.26E235 Complement factor H–related protein 5 precursor gi|5031695|ref|NP_005657.1| 2.9231304 1.26E219 8.21E219 Complement factor H–related protein 2 precursor gi|178557739|ref|NP_001002029.3| 2.65550872 1.85E2145 6.04E2144 Complement C4-B–like preproprotein gi|67190748|ref|NP_009224.2| 2.60177949 1.32E2139 3.59E2138 Complement C4-A preproprotein gi|4826657|ref|NP_004333.1| 2.48703128 3.29E225 2.82E224 Caldesmon isoform 2 gi|115298678|ref|NP_000055.2| 1.76396088 5.86E2130 1.36E2128 Complement C3 precursor gi|118442839|ref|NP_002104.2| 1.66684761 2.20E225 1.99E224 Complement factor H–related protein 1 precursor gi|7669550|ref|NP_054706.1| 1.62918471 2.21E222 1.71E221 Vinculin isoform VCL gi|62739186|ref|NP_000177.2| 1.3270844 6.43E213 3.61E212 Complement factor H isoform a precursor gi|145309326|ref|NP_002284.3| 1.06193732 1.85E212 1.00E211 Laminin subunit g-1 precursor Glomeruli were subjected to LMD–LC-MS/MS as described in the Concise Methods sections. Proteins present in the samples were identiﬁed using the bioinformatics pipeline described in the Concise Methods. QuasiTel software utilized the protein spectral counts to detect differentially expressed proteins between FGN and renal amyloidosis cohorts. FDR, false discovery rate. aProtein accessions are from the RefSeq protein database. b FDR obtained from the differential expression P value. The table was sorted by decreasing order of log2(fold change). Proteins with a log2(fold change) of $1.0 (where 0.0 signiﬁes no change) are shown here.

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expansion (Figure 2A). IF showed global smudgy mesangial and glomerular capillary wall staining for Ig-g (Figure 2B), Ig-k (Figure 2C), and Ig-l (Figure 2D). EM showed glomerular randomly oriented fibrils (Figure 2E). The overabundance of DNAJB9 in glomeruli was detected by both proteomics (Figure 2F) and IHC (Figure 2G). An amyloidosis diagnosis was excluded because the deposits were CR negative (not shown) and we did not detect all three of the typical biomarkers commonly codeposited with amyloids of all types11 (Figure 2F). We also performed an IF dual stain for Ig-g and DNAJB9 in an FFPE FGN case, which showed colocalization of DNAJB9 in a pattern that is consistent with IgG deposition in FGN (Figure 3). These data sug- gest that overabundant DNAJB9 in FGN glomeruli is deposited extracellularly and the immune complexes that are observed in patients with FGN are colocalized with DNJAB9. There are few case reports of familial fi fi Figure 1. Proteomics identi es DNAJB9 as an FGN-speci c tissue marker. (A) Proteins FGN in humans.12,13 We wanted to codeposited with amyloids of all types are highlighted with blue stars. Proteins highlighted check for the possibility that DNAJB9 with yellow stars are AL type specific markers. DNAJB9 protein was highlighted with a double star. Numbers in the boxes show the total number of MS/MS matched to the protein genetic alterations could potentially in a sample. (B) Portions of the DNAJB9 amino acid sequence detected in patient #3 were lead to the pathogenesis of FGN. For highlighted with bold black letters on yellow background. The first 23 amino acids represent this, DNAwas isolated from two subjects the signal peptide and are absentfrom the mature protein. Amino acids highlighted in green in the FGN subcohort and the DNAJB9 are post-translational modifications due to sample handling (oxidation of methionine and gene was sequenced using Sanger formation of pyroglutamate from N-terminal glutamine). (C) “Amyloid” represents renal sequencing. We did not detect any path- amyloidosis types listed in the text. “NFGNGD” represents non-FGN glomerular diseases listed ogenic mutations in either of the pa- in the text. “Norm-Gloms” represents healthy glomerulus samples. ****Mann–Whitney U rank tients. One of the patients had a single # sum test P 0.001. AL, light chain amyloidosis; MS/MS, tandem mass spectrum; Nom., normal. nucleotide polymorphism resulting in a synonymous amino acid change (Sup- of known FGN molecular features lends was exclusively present in the FGN sam- plemental Figure 1). These results lend technical credibility for the mass spec- ples and was not detectable in normal, support to the hypothesis that mutant trometry method to detect meaningful amyloidosis, and NFGNGD samples (Fig- DNAJB9 is unlikely to be the cause of protein changes that are present in the ure 1C). This supports the hypothesis that FGN. However, extensive epidemiologic FGN glomeruli. DNAJB9 can distinguish between FGN studies would be required to definitively Figure 1A shows a composite pro- and its mimics like amyloidosis, immuno- address this question; that task is out of tein identification report in representa- tactoid glomerulopathy,1,2,9,10 and lupus scope of this study. tive samples from all four subcohorts nephritis.9 Taken together, these data in- The histologic and histochemical fea- (FGN,healthy,renalamyloidosis,and dicate that FGN glomeruli contain over- tures of FGN overlap with those of other NFGNGD). DNAJB9 was only detected abundant DNAJB9 and its presence can glomerular diseases. Further, there are in the patients with FGN. The peptides serve as a specificdiagnosticbiomarker no descriptions of a single, specificbio- detected for DNAJB9 were distributed for FGN in renal biopsy samples. marker that can distinguish FGN from across the entire length of the mature Figure 2 demonstrates glomerular de- other entities in the differential diagnosis. protein (Figure 1B, showing sequence position of DNAJB9 using immunohisto- Hence, in current clinical practice, an coverage of the protein in patient #3). chemistry (IHC), and Ig-g heavy chain, integrated diagnostic approach combin- This result indicates that full-length and Ig-k and Ig-l light chains, using IF ing histology, IF, and ultrastructural protein was detected in the FGN glomeruli in a patient with FGN. LM showed glo- features is used to diagnose FGN. Our (i.e., there were no truncations). DNAJB9 merular mesangial hypercellularity and discovery of DNAJB9 overabundance in

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this, we believe that the discovery of DNAJB9 as a unique molecular feature of FGN is a solid ﬁrst step toward improving the clinical care of these patients.

CONCISE METHODS

Renal Biopsy Sample Workup All diagnoses were made using standard pathologic review of renal cortex and medulla of the FFPE renal biopsy tissues. Hematoxylin and eosin–stained FFPE sections were examined using LM. CR stain was performed on the FFPE sections following a standard pro- tocol and apple-green birefringence was eval- uated with polarized LM. IF for Ig-g,Ig-a, Ig-m,Ig-k,Ig-l, C3, and C1q was performed on frozen sections and in selected cases on pronase-treated paraffin sections using standard techniques. Standard processing was also used for EM. All FGN cases included in this study fulfilled the following previ- Figure 2. Glomerular deposition of DNAJB9 and immune complexes in a patient with FGN. ously established diagnostic criteria4:glo- This figure isfrom the biopsy sample of patient #7 in Supplemental Table 1. (A) PAS stain shows merular deposition of fibrils that were (1) glomerular mesangial hypercellularity and expansion. IF shows global smudgy mesangial randomly-oriented; (2) lacked hollow cen- and glomerular capillary wall staining for IgG (B), k light chain (C), and l light chain (D). ters at magnification of ,30,000; (3)were fi (E) Electron micrograph shows randomly oriented brils expanding the glomerular Congo-red negative; and (4) stained with basement membrane. (F) Proteomic analysis of the laser microdissected glomeruli antisera to Igs by IF. shows expression of DNAJB9 protein and lack of universal amyloid tissue markers (APOE, APOA4, and SAP). Replicate dissections were performed for the patient. Pre- Blank refers to a blank LC-MS/MS run that ensures data integrity. (G) IHC shows bright LMD-Assisted Shotgun Proteomics smudgy global mesangial and glomerular capillary wall staining for DNAJB9. Original We used a previously established proteomics magnification is 3400 for (A) and (G), 3200 for (B) and (C), and 321,000 for (E). Sup- method for characterizing the glomerular plemental Figure 2 shows the staining specificity of the DNAJB9 antibody used for IHC. proteomes.6 In brief, for amyloid tissues, CR-stained FFPE tissue sections for each patient were mounted on a Director slide FGN glomeruli, but not in other non- FGN. This is primarily because the precise (Nantomics, Rockville, MD) and examined FGN glomerular diseases or in healthy molecular cause of FGN is unknown. The for presence of amyloid in glomeruli. Stained glomeruli, is likely to prove extremely overabundance and extracellular codepo- slides were loaded on an LMD apparatus valuable in simplifying the diagnostic sition of DNAJB9 with Ig light and heavy (Lieca, Wetzlar, Germany) and glomeruli algorithms for FGN. For example, for chains in FGN glomeruli indicates that were visualized under fluorescent light for routine practices where mass spectrom- DNAJB9 is likely to be a key feature in dissection. For nonamyloid tissues, hematox- etry is unavailable, IHC for DNAJB9 the pathogenesis of FGN. Therapies that ylin and eosin–stained slides were loaded on could be developed and validated to target this particular protein may result in an LMD apparatus and glomeruli were visu- provide a practical ancillary test for the better outcomes for patients with FGN. alized under bright field. Multiple indepen- diagnosis of FGN. For instance, Lee et al.17 recently showed dent dissections (replicates), each configured FGN is treated with immunosuppres- that DNAJB9 is a negative regulator of p53 to capture material from an area of 60,000 mM,2 sive therapy with limited success. Hogan and is induced by the Ras/Raf/ERK path- were performed for each patient. Material from et al.14 observed that rituximab therapy way in response to p53 induction. Hence, each replicate dissection was captured in a was associated with nonprogression of Ras/Raf/ERK inhibitors might be effective tube containing 35 ml of Tris/EDTA/0.002% FGN in only a third of the 12 patients stud- in reducing the overabundance of Zwittergent buffer. Proteins were extracted ied. Other researchers have observed sim- DNAJB9 observed in FGN glomeruli. from the captured FFPE fragments using heat ilar limited gains with immunosuppressive However, the exact mechanism by which and sonication. Extracted proteins were diges- therapy in patients with FGN.15,16 To date, DNAJB9 is induced in FGN is still un- ted with trypsin and resulting peptides were there is no curative, targeted therapy for known and needs further study. Despite analyzed on an QExactive mass spectrometer

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using hybrid primers containing 20–22 bases of gene specific sequence and a universal sequencing primer (UPS) sequence (19 or 23 bases for the forward and reverse primers) at the 59 end. Amplified products, which included 610 bp of the intron-exon bound- aries of coding exons, were sequenced using universal sequencing primers, the ABI Big Dye terminators (Applied Biosystems, Foster City, CA), and capillary electrophoresis on an ABI 3730 sequencer. Sequencing traces were manually reviewed and visualized using Mutation Surveyor (SoftGenetics, College Station, PA) configured to use corresponding reference sequence obtained from GenBank Figure 3. DNAJB9 and IgG dual IF showing colocalization. A single FFPE section from a (with accession NM_012328.2). Variant re- patient with FGN in the cohort was used to stain for IgG (green) and DNAJB9 (red) in order to view was performed according to ACMG show their colocalization. IF utilized the same DNAJB9 primary antibody that was validated 2015 guidelines.20 for IHC (Supplemental Figure 2). (A) Green channel showing IgG, (B) red channel showing DNAJB9, and (C) blue channel showing Hoechst 33342 staining of the nuclei. (D) Merged DNAJB9 and IgG shows colocalization of DNAJB9 in a pattern similar to that of IgG. IHC of DNAJB9 Original magnification for all panels was 320. Slides were stained with an anti-DNAJB9 rabbit polyclonal antibody (cat# HPA040967; (Thermo Fisher Scientific, Waltham, MA), an counts) that was consistently detected in all 1/75 titer; Sigma-Aldrich, St. Louis, MO) on a LTQ-Velos mass spectrometer (Thermo Fisher replicate dissections.6,7,11 Theproteomepro- Ventana BenchMark XT system using Ventana Scientific), or an LTQ-Orbitrap mass spec- files of the rest of the cohorts were also re- OptiView Universal DAB Detection and trometer (Thermo Fisher Scientific) using viewed by a pathologist. OptiView Amplification Kits. LC-MS/MS. Differential Expression Analysis of Dual IF Staining for DNAJB9 Bioinformatics of Protein FGN and Amyloid Cohorts and IgG Identification LC-MS/MS data from FGN and amyloid co- All antigen retrieval and staining steps were We used a previously published bioinformatics hort samples were subjected to differential performed on a LeicaBondRXstainer pipeline for processing all of the LC-MS/MS expression analysis in order to find proteomic (Leica). Four-micrometer-thick sections data.7 In brief, LC-MS/MS data from each pa- differences. For this, Scaffold software expor- were obtained from the FFPE tissue blocks tient sample were analyzed using multiple ted the number of MS/MS spectra matched and mounted on standard IF slides. Slides database search engines configured to match to a protein as a semiquantitative measure of were retrieved for 5 minutes using Enzyme MS/MS against a composite protein sequence its abundance.19 QuasiTel software was mod- 2 pretreatment kit (Leica). Slides were first database containing SwissProt human pro- ified to read the Scaffold protein exports and incubated in 10% normal goat serum (Life teome (downloaded 08/2012) and common perform differential expression of proteins by Technologies, Carlsbad, CA) for 30 minutes, contaminants (e.g., wool, etc.). Reversed using a quasi-likelihood–based generalized followed by 60 minutes with an anti-DNAJB9 protein sequences were used to estimate false linear mixture model.8 The software auto- rabbit polyclonal antibody (cat# HPA040967; discovery rates. The search engines were con- matically normalizes the protein spectral Sigma-Aldrich) diluted to 1:50 in Back- figured to derive tryptic peptides from the counts present in each sample to account ground Reducing Diluent (Dako; Agilent sequence database and look for the follow- for protein loading differences between LC- Technologies, Santa Clara, CA). Next, slides ing variable modifications: oxidation of MS/MS experiments. Proteins with a correc- were incubated for 60 minutes in a goat anti- methionine (+15.994 D) and formation of ted differential expression P value of #0.05 rabbit IgG secondary antibody (Alexa Flour N-terminal pyroglutamic acid (217.023 D). and an absolute fold change $1.0 (where 0.0 568; Thermo Fischer Scientific) diluted to Scaffold software processed the results and as- signifies no change) were considered as sig- 1:200 in background reducing diluent. Slides sembled protein identifications with at least nificantly differentially expressed between were then incubated for another 30 minutes two confident (identification P.0.9) and the FGN discovery and amyloid cohorts. with anti–IgG-FITC rabbit polyclonal anti- unique peptide matches. For amyloid body (Dako; Agilent Technologies) diluted samples, a pathologist scrutinized the profile DNAJB9 Sequencing to 1:10 in background reducing diluent. Coun- for an amyloid confirming universal molecular Genetic evaluation for DNAJB9 was per- terstain was performed using Hoechst 33342 signature (APOE, SAP, and APOA4)11 and as- formed using white blood cells isolated (Life Technologies) for 10 minutes and slides signed the type on the basis of the most abun- from peripheral blood samples. The two cod- were coverslipped using ProLong Gold antifade dant amyloidogenic protein18 (by spectral ing exons of DNAJB9 gene were amplified mounting media (Life Technologies).

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ACKNOWLEDGMENTS in clinical biopsy specimens. Blood 114: P, Touchard G, Bridoux F: Long-term kidney 4957–4959, 2009 disease outcomes in fibrillary glomerulone- 7. Theis JD, Dasari S, Vrana JA, Kurtin PJ, phritis: A case series of 27 patients. Am J Kid- Financial support was provided by the De- Dogan A: Shotgun-proteomics-based clinical ney Dis 62: 679–690, 2013 partment of Laboratory Medicine and Pa- testing for diagnosis and classification of amy- 16. Kalbermatter SA, Marone C, Casartelli D, thology, Mayo Clinic (Rochester, MN). loidosis. J Mass Spectrom 48: 1067–1077, 2013 Hausberg M, Banfi G, Mihatsch M, Dickenmann 8. Li M, Gray W, Zhang H, Chung CH, Billheimer M: Outcome of fibrillary glomerulonephritis. D, Yarbrough WG, Liebler DC, Shyr Y, Slebos Swiss Med Wkly 142: w13578, 2012 DISCLOSURES RJ: Comparative shotgun proteomics using 17. Lee HJ, Kim JM, Kim KH, Heo JI, Kwak SJ, spectral count data and quasi-likelihood mod- Han JA: Genotoxic stress/p53-induced None. eling. J Proteome Res 9: 4295–4305, 2010 DNAJB9 inhibits the pro-apoptotic function 9. Alpers CE, Kowalewska J: Fibrillary glomer- of p53. Cell Death Differ 22: 86–95, 2015 ulonephritis and immunotactoid glomerul- 18. Sipe JD, Benson MD, Buxbaum JN, Ikeda S, REFERENCES opathy. JAmSocNephrol19: 34–37, 2008 Merlini G, Saraiva MJ, Westermark P: No- 10. Bridoux F, Hugue V, Coldefy O, Goujon JM, menclature 2014: Amyloid fibril proteins and ’ fi 1. Fogo A, Qureshi N, Horn RG: Morphologic Bauwens M, Sechet A, Preud Homme JL, clinical classi cation of the amyloidosis. – and clinical features of fibrillary glomerulo- Touchard G: Fibrillary glomerulonephritis Amyloid 21: 221 224, 2014 nephritis versus immunotactoid glomerul- and immunotactoid (microtubular) glomer- 19. Liu H, Sadygov RG, Yates JR 3rd: A model for opathy. Am J Kidney Dis 22: 367–377, 1993 ulopathy are associated with distinct immu- random sampling and estimation of relative – 2. Rosenstock JL, Markowitz GS, Valeri AM, nologic features. Kidney Int 62: 1764 1775, protein abundance in shotgun proteomics. – Sacchi G, Appel GB, D’Agati VD: Fibrillary 2002 Anal Chem 76: 4193 4201, 2004 and immunotactoid glomerulonephritis: Dis- 11. Vrana JA, Theis JD, Dasari S, Mereuta OM, 20.RichardsS,AzizN,BaleS,BickD,DasS, tinct entities with different clinical and patho- Dispenzieri A, Zeldenrust SR, Gertz MA, Gastier-Foster J, Grody WW, Hegde M, Lyon logic features. Kidney Int 63: 1450–1461, 2003 Kurtin PJ, Grogg KL, Dogan A: Clinical di- E, Spector E, Voelkerding K, Rehm HL, ; 3. Iskandar SS, Falk RJ, Jennette JC: Clinical agnosis and typing of systemic amyloid- ACMG Laboratory Quality Assurance Com- and pathologic features of fibrillary glomer- osis in subcutaneous fat aspirates by mass mittee: Standards and guidelines for the in- ulonephritis. Kidney Int 42: 1401–1407, 1992 spectrometry-based proteomics. Haemato- terpretation of sequence variants: A joint 4. Nasr SH, Valeri AM, Cornell LD, Fidler ME, logica 99: 1239–1247, 2014 consensus recommendation of the American Sethi S, Leung N, Fervenza FC: Fibrillary 12. Chan TM, Chan KW: Fibrillary glomerulone- college of medical genetics and genomics and glomerulonephritis: A report of 66 cases from a phritis in siblings. Am J Kidney Dis 31: E4, 1998 the association for molecular pathology. Genet – single institution. Clin J Am Soc Nephrol 6: 13. Ying T, Hill P, Desmond M, Agar J, Mallett A: Med 17: 405 424, 2015 775–784, 2011 Fibrillary glomerulonephritis: An apparent 5. Markowitz GS, Cheng JT, Colvin RB, Trebbin familial form? Nephrology (Carlton) 20: 506– WM, D’Agati VD: Hepatitis C viral infection is 509, 2015 associated with fibrillary glomerulonephritis 14. Hogan J, Restivo M, Canetta PA, Herlitz LC, See related editorial, “Glomerular Disease Pathol- and immunotactoid glomerulopathy. JAm Radhakrishnan J, Appel GB, Bomback AS: ogy in the Era of Proteomics: From Pattern to Soc Nephrol 9: 2244–2252, 1998 Rituximab treatment for fibrillary glomerulo- Pathogenesis,” on pages 2–4. 6. Vrana JA, Gamez JD, Madden BJ, Theis JD, nephritis. Nephrol Dial Transplant 29: 1925– fi Bergen HR 3rd, Dogan A: Classi cation of 1931, 2014 This article contains supplemental material online at amyloidosis by laser microdissection and 15. Javaugue V, Karras A, Glowacki F, McGregor B, http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ mass spectrometry-based proteomic analysis LacombeC,GoujonJM,RagotS,Aucouturier ASN.2017030306/-/DCSupplemental.

56 Journal of the American Society of Nephrology J Am Soc Nephrol 29: 51–56, 2018 Supplemental Figure 1. DNAJB9 Sequencing Detected Synonymous SNP. White blood cells were isolated from two FGN patients and the two exons of DNAJB9 gene were sequenced using Sanger sequencing. A single nucleotide change in codon 61 resulting in a synonymous amino acid change was detected in one of the subjects (location highlighted with blue arrow in the below figure). No mutations were detected in the second subject.

Patient

Control Supplemental Figure 2. DNAJB9 Primary Antibody Specificity. We tested the specificity of DNAJB9 primary antibody using immunohistochemistry (IHC). A total of 6 FGN cases, 9 amyloid cases (3 AL-κ, 3 AL-λ, and 3 ALect2), and 9 healthy controls were chosen from the LC-MS/MS cohort for antibody testing. (A-B) DNAJB9 staining from two FGN cases showing smudgy glomerular staining (sparing the nuclei). All FGN cases showed similar staining pattern. (C-D) Serial sections from the case shown in (B) were stained with control diluent and polyclonal rabbit IgG as primary stain, instead of anti-DNAJB9 antibody, respectively. This showed that the DNAJB9 staining observed in FGN cases was not due to non-specific secondary antibody staining. (E-F) DNAJB9 staining in example cases of AL-λ and healthy control, respectively. As expected, we observed tubular staining, but glomeruli were negative for DNAJB9 in all amyloid and healthy controls.

(A) (B)

(E) (F) Supplemental Table 1. Clinical and Pathologic Characteristics of 24 cases of Fibrillary Glomerulonephritis Case # DNAJB9 Age/ S. Cr. Proteinuria Full Hematuria Concurrent Glomerular # of glomeruli Degree of Positive immune reactants in Extraglom Fibril Mean fibril method of gender (mg/dl) (g/day) NS conditions morphology on sampled for LM/ % TA&IF glomeruli by IF erular distribution on thickness testing LM global staining EM glomerulosclerosis for IgG

1 MS + IHC 44/M 12.8 NA (>300 no yes HTN mes. proliferative 12/83% marked IgG (3+), C3 (2+), kappa (3+), lambda no mes, GBM 18 mg/dl on UA GN (3+) 2 MS + IHC 60/F NA NA no yes none mes. proliferative 101/20% mild IgG (2+), C3 (2+), kappa (+/-), lambda no mes, GBM 20 (subnephrotic) GN (1+) 3 MS + IHC 71/M 2.6 2.5 no no hepatocellular Ca, mes. proliferative 22/23% mild IgG (3+), C3 (3+), kappa (2+), lambda no mes, GBM 19 HTN GN (2-3+), IgA (1-2+), C1q (2+) 4 MS + IHC 59/F 2.3 NA (2+ on UA) no yes HTN, hemoptysis crescentic GN 8/25% none IgG (3+), C3 (3+), kappa (1-2+), lambda no mes, GBM 18 (1-2+), IgM (+/-), C1q (+/-) 5 MS + IHC 64/M 1.2 1.7 no yes prostate CA mes. proliferative 24/0% mild IgG (1-2+), C3 (1+), IgA (1-2+), IgM (1-yes (focal mes, GBM 17 GN 2+), kappa (+/-), lambda (1-2+) TBM

6 MS + IHC 53/F 2.8 20 no no DM, HTN, COPD mes. proliferative 13/15% moderate IgG (3+), C3 (2-3+), kappa (3+), lambda no mes, GBM 19 GN (3+) 7 MS + IHC 58/F 1.6 9 no yes none membranoproliferat 25/72% moderate IgG (2-3+), IgM (1+), IgA (+/-), C3 no mes, GBM 20 ive GN (1+), kappa (2+), lambda (2-3+) 8 MS 55/M 1.9 6.7 no yes HCV, HIV, HTN mes. proliferative 12/42% moderate IgG (3+), kappa (3+), lambda (3+)(by no mes, GBM not measured GN pronase IF), C3 (2+) 9 MS 75/M 1.4 6.6 no yes HTN mes. proliferative 18/39 mild IgG (2+), C3 (1+), IgM (1+), kappa no mes, GBM 16 GN (1+), lambda (2+) 10 MS 73/M 5.5 8.8 yes yes DM endocapillary 15/ 20% mild IgG (3+), C3 (2+), C1q (1+), IgA (+/-); no mes, GBM 12 proliferative GN staining for kappa and lambda not done

11 MS 55/M 0.7 3.4 no yes cutaneous lupus mes. proliferative 12/0% mild IgG (3+), C3 (1+), IgM (1+), IgA (1+), no mes, GBM 15 GN C1q (2+); staining for kappa and lambda not done 12 MS 53/M 2.4 17 NA yes HCV, HTN, mes. proliferative 20/25% IgG (1+), C3 (1+), kappa (+/-), lambda no mes, GBM not measured morbid obesity GN (1+) 13 MS 44/F 0.8 5 no yes undifferentiated mes. proliferative 28/11% none IgG (2-3+), C3 (3+), IgM (1-2+), IgA no mes, GBM 10 MCTD GN (1+), C1q (2+); kappa (2+), lambda (3+)

14 MS 37/M 0.9 2.4 no no none mes. proliferative 16/0% none IgG (3+), C3 (1+), kappa (2+), lambda no mes, GBM 20 GN (3+) 15 MS 59/F 4.1 NA (>300 NA yes HTN, CAD mes. proliferative 53/75% marked IgG (2-3+), C3 (1+), kappa (1+), lambda no mes, GBM 17 mg/dl on UA GN (+/-) 16 MS 71/F 1.7 1.7 no yes HTN, gout mes. proliferative 17/88% moderate IgG (2+), C3 (1-2+), IgM (1-2+), kappa no mes, GBM 17 GN (1-2+), lambda (1-2+) 17 MS 67/F 6.8 NA NA NA none membranoproliferat 30/53% marked IgG (3+), C3 (3+), kappa (1+), lambda no 19 ive GN (3+) 18 MS 76/F 1.6 NA NA NA NA mes. proliferative 11/45% mild IgG (3+), C3 (2+), kappa (3+), lambda no mes, GBM 17 GN (3+) 19 MS 63/F 1.4 0 no yes COPD mes. proliferative 6/50% mild IgG (2+), C3 (1+), kappa (1-2+), no mes, GBM 16 GN lambda (2+) 20 MS 66/M 1 3.7 NA yes DM, HTN mes. proliferative 19/5% mild IgG (2+), C3 (1+), kappa (+/-), lambda no mes, GBM 18 GN (+/-) 21 MS 62/M 1.3 6 no yes HTN mes. proliferative 25/20% mild IgG (2-3+), C3 (2+), kappa (3+), lambda no mes, GBM 14 GN (1-2+), IgA (2-3+) 22 MS 50/F 0.9 0.6 no yes HTN mes. proliferative 34/26% mild IgG (3+), C3 (3+), kappa (1-2+), lambda yes (TBM) mes, GBM, 13 GN (1+), C1q (1+) TBM 23 MS 65/M 2.4 5.3 NA NA DM endocapillary 32/34% marked IgG (3+), IgM (2+), C3 (2+), kappa no mes, GBM 14 proliferative GN (3+), lambda (2+) 24 MS 40/F 2.3 2.7 no yes NSIP mes. proliferative 31/0% mild IgG (3+), C3 (3+), kappa (2+), lambda no mes, GBM 13 GN (1-2+) CA, carcinoma; CAD, coronary artery disease; COPD, chronic obstructive pulmonary disease; DM, diabetes; GBM, glomerular basement membrane; HTN, hypertension; IHC, immunohistochemistry; MCTD, mixed connective tissue disorder; mes, mesangial; NSIP, nonspecific interstitial pneumonia; mes, mesangial; NS, nephrotic syndrome; MS, mass spectrometry; NA, not available; TBM, tubular basement membranes; Median SpCa Proteins FGN Amyloid Description gi|4501885|ref|NP_001092.1| 132 93 actin, cytoplasmic 2 [Homo sapiens] gi|4501889|ref|NP_001606.1| 93 69 actin, aortic smooth muscle [Homo sapiens] gi|62414289|ref|NP_003371.2| 77 91 vimentin [Homo sapiens] gi|9558755|ref|NP_036460.1| 54 0 dnaJ homolog subfamily B member 9 [Homo sapiens] gi|115298678|ref|NP_000055.2| 54 18 complement C3 precursor [Homo sapiens] gi|119703755|ref|NP_002283.3| 49 15 laminin subunit beta-2 precursor [Homo sapiens] gi|4557325|ref|NP_000032.1| 47 118 apolipoprotein E precursor [Homo sapiens] gi|4504349|ref|NP_000509.1| 45 44 hemoglobin subunit beta [Homo sapiens] gi|12667788|ref|NP_002464.1| 41 21 myosin-9 [Homo sapiens] gi|240255535|ref|NP_476507.3| 39 10 collagen alpha-3(VI) chain isoform 4 precursor [Homo sapiens]

Supplemental Table 2: This table displays top 10 proteins detected in the glomeruli of fibrillary glomerulonephritis (FGN). (a) Total number of spectra matched to a protein is considered as a semi-quantitative measure of its abundance in a sample. Protein spectral counts were normalized to account for protein loading differences between LC-MS/MS experiments. Median protein spectral counts (SpC) for each the protein detected in FGN and amyloid cohorts was computed. Rows were ordered by the decreasing abundance of the protein in FGN cohort. ID Name #Gene FDR R-HSA-977606 Regulation of Complement cascade 5 4.39E-08 R-HSA-166658 Complement cascade 5 2.71E-07 R-HSA-174577 Activation of C3 and C5 3 2.05E-05 R-HSA-166663 Initial triggering of complement 3 4.83E-04 R-HSA-168249 Innate Immune System 6 1.39E-02

Supplemental Table 3: This table displays the pathway enrichment results for proteins that are overabundant in FGN glomeruli. For this, proteins that had a corrected differential expression p-value of <=0.05 and a log2(fold change)>=0.3 were uploaded to WebGestalt for pathway enrichment analysis (http://www.webgestalt.org/option.php). Pathways that had an corrected enrichment FDR <=0.05 were considered as significant for reporting. SIGNIFICANCE STATEMENT

Fibrillary glomerulonephritis (FGN) is a primary glomerulardisease with apoor prognosis. Currently FGNposessubstantialdiagnosticchallenges,inpart because there are no speciﬁc histological biomarkers. This manuscript describes the discovery, using proteomics, of a new potential biomarker, DNAJB9 (DnaJ Heat Shock Protein Family [Hsp40] Member B9). We demonstrate that DNAJB9 is present in overabundance in FGN glomeruli, but not in glomeruli from patients with other glomerular diseases or from healthy subjects. DNAJB9 is potentially a useful diagnostic marker for FGN. Study of its function may provide important clues to the underlying pathogenesis of this disease.