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

Abstract

Fibrillary GN (FGN) is a rare primary glomerular disease. Histologic and histochemical features of FGN overlap with those of other glomerular diseases, and no unique histologic biomarkers for diagnosing FGN have been identified. We analyzed the proteomic content of glomeruli in patient biopsy specimens and detected DnaJ heat shock protein family (Hsp40) member B9 (DNAJB9) as the fourth most abundant protein in FGN glomeruli. Compared with amyloidosis glomeruli, FGN glomeruli exhibited a >6-fold overexpression of DNAJB9 protein. Sanger sequencing and protein sequence coverage maps showed that the DNAJB9 protein deposited in FGN glomeruli did not have any major sequence or structural alterations. Notably, we detected DNAJB9 in all patients with FGN but not in healthy glomeruli or in 19 types of non-FGN glomerular diseases. We also observed the codeposition of DNAJB9 and Ig-γ Overall, these findings indicate that DNAJB9 is an FGN marker with 100% sensitivity and 100% specificity. The magnitude and specificity of DNAJB9 overabundance in FGN also suggests that this protein has a role in FGN pathogenesis. With this evidence, we propose that DNAJB9 is a strong biomarker for rapid diagnosis of FGN in renal biopsy specimens.

Keywords: DNAJB9; biomarker; fibrillary GN; immunofluorescence; proteomics; renal.

Graphical abstract

Figure 1.

Proteomics identifies DNAJB9 as an FGN-specific tissue marker. (A) Proteins codeposited with amyloids of all types are highlighted with blue stars. Proteins highlighted 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 in a sample. (B) Portions of the DNAJB9 amino acid sequence detected in patient #3 were highlighted with bold black letters on yellow background. The first 23 amino acids represent the signal peptide and are absent from the mature protein. Amino acids highlighted in green are post-translational modifications due to sample handling (oxidation of methionine and formation of pyroglutamate from N-terminal glutamine). (C) “Amyloid” represents renal amyloidosis types listed in the text. “NFGNGD” represents non-FGN glomerular diseases listed in the text. “Norm-Gloms” represents healthy glomerulus samples. ****Mann–Whitney U rank sum test P ≤0.001. AL, light chain amyloidosis; MS/MS, tandem mass spectrum; Nom., normal.

Figure 2.

Glomerular deposition of DNAJB9 and immune complexes in a patient with FGN. This figure is from the biopsy sample of patient #7 in Supplemental Table 1. (A) PAS stain shows glomerular mesangial hypercellularity and expansion. IF shows global smudgy mesangial and glomerular capillary wall staining for IgG (B), κ light chain (C), and λ light chain (D). (E) Electron micrograph shows randomly oriented fibrils expanding the glomerular basement membrane. (F) Proteomic analysis of the laser microdissected glomeruli shows expression of DNAJB9 protein and lack of universal amyloid tissue markers (APOE, APOA4, and SAP). Replicate dissections were performed for the patient. PreBlank refers to a blank LC-MS/MS run that ensures data integrity. (G) IHC shows bright smudgy global mesangial and glomerular capillary wall staining for DNAJB9. Original magnification is ×400 for (A) and (G), ×200 for (B) and (C), and ×21,000 for (E). Supplemental Figure 2 shows the staining specificity of the DNAJB9 antibody used for IHC.

Figure 3.

DNAJB9 and IgG dual IF showing colocalization. A single FFPE section from a patient with FGN in the cohort was used to stain for IgG (green) and DNAJB9 (red) in order to show their colocalization. IF utilized the same DNAJB9 primary antibody that was validated 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. Original magnification for all panels was ×20.