DnaJ Homolog Subfamily B Member 9 Is a Putative Autoantigen in Fibrillary GN

Abstract

Fibrillary GN is a rare form of GN of uncertain pathogenesis that is characterized by the glomerular accumulation of randomly arranged, nonbranching fibrils (12-24 nm) composed of Ig and complement proteins. In this study, we used mass spectrometry to comprehensively define the glomerular proteome in fibrillary GN compared with that in controls and nonfibrillary GN renal diseases. We isolated glomeruli from formalin-fixed and paraffin-embedded biopsy specimens using laser capture microdissection and analyzed them with liquid chromatography and data-dependent tandem mass spectrometry. These studies identified DnaJ homolog subfamily B member 9 (DNAJB9) as a highly sampled protein detected only in fibrillary GN cases. The glomerular proteome of fibrillary GN cases also contained IgG1 as the dominant Ig and proteins of the classic complement pathway. In fibrillary GN specimens only, immunofluorescence and immunohistochemistry with an anti-DNAJB9 antibody showed strong and specific staining of the glomerular tufts in a distribution that mimicked that of the immune deposits. Our results identify DNAJB9 as a putative autoantigen in fibrillary GN and suggest IgG1 and classic complement effector pathways as likely mediators of the destructive glomerular injury in this disease.

Keywords: Fibrillary glomerulonephritis; autoantigen; immunohistochemistry; mass spectrometry; renal biopsy.

Graphical abstract

Figure 1.

DNAJB9 is present in glomeruli of FGN cases but not in controls. Mass spectrometry–based quantification of a DNAJB9 peptide in FGN and control samples. The DNAJB9 protein normalized spectra abundance factor (NSAF) is significantly increased in FGN cases (seven of eight samples) compared with controls (114 samples). UPR, a conserved response related to endoplasmic reticulum stress, is highlighted by proteins that are induced (red) or repressed (green). Amyloid P serum component and apo E (APOE) were detected in some but not all FGN samples and are present in the majority of amyloid samples. COL4A3, COL4A4, and COL4A5 NSAFs were similarly distributed among all samples. The gray background indicates that the protein was not detected in the sample.

Figure 2.

Mass spectrometry–based quantification shows significantly increased DNAJB9 protein in FGN cases, which was not detected in controls. The DNAJB9 protein normalized spectra abundance factor (NSAF) is significantly increased in FGN cases (A) (eight samples) compared with controls (114 samples). COL4A3 (B), COL4A4 (C), and COL4A5 (D) NSAF values did not differ significantly between FGN and control cases (Mann–Whitney U test).

Figure 3.

Mass spectrometry detected DNAJB9 peptides between amino acids 68 and 195 in seven of eight FGN samples from five of five patients. The predicted signal peptide is amino acids 1–23, and the predicted mature protein is amino acids 24–223. The findings are consistent with the mature form of the protein being associated with FGN. Peptides detected in each of the FGN samples are underlined.

Figure 4.

Diagnostic characteristics of FGN. (A) Diffuse mesangial expansion predominantly due to deposition of eosinophilic, silver negative material with extension into the capillary walls. Original magnification, ×400. (B) Electron microscopy reveals deposition of abundant haphazardly arranged fibrils with an average diameter of 13 nm. Glomerulus with confluent, pseudolinear staining in the peripheral capillary loops and mesangium by IF (C) anti-DNAJB9 and (D) anti-IgG. Original magnification, ×400.

Figure 5.

Detection of DNAJB9 in glomeruli from FGN cases. Anti-DNAJB9 shows strong, discrete peripheral capillary wall and mesangial staining in (A and B) FGN but not in (C) MN, (D) immunotactoid glomerulopathy, (E) diabetic glomerulopathy, or (F) normal controls (IHC). Most cases show weak podocyte cytoplasmic staining by IHC and background staining in the tubulointerstitium. By IF, anti-DNAJB9 shows (G) bright peripheral capillary wall and mesangial staining in FGN but is (H) negative in normal controls. Original magnification, ×400.

Figure 6.

Dual staining reveals colocalization of IgG and DNAJB9 in immune deposits in FGN. IgG highlights (A) glomerular and (D) extraglomerular vascular deposits in FGN. DNAJB9 shows similar staining in (B) glomeruli and (E) rare vascular deposits. Merged images show substantial colocalization of these molecules in (C) glomerular and (F) extraglomerular deposits in FGN.