Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy

Abstract

Background: Identification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%-20% of patients.

Methods: A multipronged approach, using traditional and modern technologies, converged on a novel target antigen, and capitalized on the temporal variation in autoantibody titer for biomarker discovery. Immunoblotting of human glomerular proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection followed by mass spectrometry, elution of immune complexes from renal biopsy specimen tissue, and autoimmune profiling on a protein fragment microarray.

Results: These approaches identified serine protease HTRA1 as a novel podocyte antigen in a subset of patients with primary MN. Sera from two patients reacted by immunoblotting with a 51-kD protein within glomerular extract and with recombinant human HTRA1, under reducing and nonreducing conditions. Longitudinal serum samples from these patients seemed to correlate with clinical disease activity. As in PLA2R- and THSD7A- associated MN, anti-HTRA1 antibodies were predominantly IgG4, suggesting a primary etiology. Analysis of sera collected during active disease versus remission on protein fragment microarrays detected significantly higher titers of anti-HTRA1 antibody in active disease. HTRA1 was specifically detected within immune deposits of HTRA1-associated MN in 14 patients identified among three cohorts. Screening of 118 "quadruple-negative" (PLA2R-, THSD7A-, NELL1-, EXT2-negative) patients in a large repository of MN biopsy specimens revealed a prevalence of 4.2%.

Conclusions: Conventional and more modern techniques converged to identify serine protease HTRA1 as a target antigen in MN.

Keywords: membranous nephropathy; nephrotic syndrome; podocyte.

Graphical abstract

Figure 1.

Identification of a 50-kDa target antigen present within human glomerular protein extract. Identical nitrocellulose membrane strips of transferred human glomerular protein extract were cut from the original membrane, separately Western blotted with patient serum samples (top labels) and reassembled prior to imaging. (A) Longitudinal serum samples from our index patient with MN (MN01) used for WB of human glomerular protein extract. The 50-kD band was detected with serum sampled at time of a relapse of nephrotic syndrome (lane 1), but it declined and disappeared (lanes 2–3) as the patient achieved clinical remission. This band, representing a putative target glomerular antigen, was not detected by the serum of a patient with anti-PLA2R antibodies (lane 4; the large 180-kD band is PLA2R) or by healthy controls (not shown). (B) WB of human glomerular protein extract using rabbit anti-human HTRA1 (leftmost lane) or with individual MN serum samples and detected with appropriate secondary antibodies. Rabbit anti-HTRA1 strongly recognizes a band migrating to just below 50 kD (arrow), which is identical in size to the band recognized by the index patient (MN01) and another patient (MN03). Additional sera from patients with uncharacterized MN (MN-A, -B, -C, and -D) or known PLA2R antibody–associated MN (PLA2R-Ab+) fail to recognize the approximately 50 kD band. Anti-Hu, anti-human; anti-Rb, anti-rabbit.

Figure 2.

Western blot detection and immunoprecipitation of glomerular proteins with sera from patients with HTRA1-associated MN and controls. (A) Paired lanes of recombinant HTRA1 and human glomerular extract (HGE) individually underwent WB with patient serum (represented by black bars and labels at bottom) and were detected for IgG4. Serial samples (A–C) from index patient MN01 showed increased reactivity for the recombinant and native protein over time. In addition, serum from MN03 is clearly shown to react with recombinant HTRA1. In contrast, there was no reaction in the approximately 50 kD region when recombinant HTRA1 or HGE was blotted with normal control human serum (NHS) or from patients with known THSD7A- or PLA2R-associated MN. The 250- and 180-kD bands demarcate the position of THSD7A and PLA2R, respectively. (B) Serum from the index patient (MN01) or a healthy control (CTL) was used to immunoprecipitate the antigen from HGE. The HGE input, the immunoprecipitates (IPs), and the residual HGE supernatant post-IP were gel electrophoresed and immunoblotted with a rabbit (Rb) antibody to human HTRA1. HTRA1 was identified in the IP from MN01 (corresponding to the size in the input lane) and was largely depleted from the supernatant. In contrast, CTL serum did not IP HTRA1 and the protein was retained in the supernatant (arrow; band is displaced downward due to large albumin band above). M, marker.

Figure 3.

Characterization of antibody reactivity with reduced and non-reduced forms of native and recombinant HTRA1. WB of recombinant HTRA1 or native human glomerular extract (HGE) within HGE electrophoresed under reducing and nonreducing conditions. (A) WB with PLA2R antibody (PLA2R-Ab)–containing serum shows loss of reactivity to PLA2R (180-kD band) under reducing (Red) conditions. (B) The polyclonal anti-HTRA1 antibody (HTRA1-Ab) detects recombinant and native HTRA1 under both reducing and nonreducing (NR) conditions. (C) Serum from the index patient MN01 reacts with recombinant and native HTRA1 under both reducing and nonreducing conditions. (D) The weaker-titer MN03 serum also recognizes recombinant HTRA1 under both conditions. The arrow demarcates the position of the HTRA1 monomer. Higher and lower bands in (B) are likely oligomers and proteolytic fragments, respectively. Hu, human; Rb, rabbit.

Figure 4.

Anti-HTRA1 antibody response in relation to clinical disease parameters in two patients with HTRA1-associated MN. Longitudinal serum samples from (A) the index patient MN01 and (B) MN03 were used to immunoblot equal amounts of recombinant HTRA1 and demonstrate the changes in anti-HTRA1 titer over time. Identical amounts of recombinant HTRA1 were gel electrophoresed and transferred to a nitrocellulose membrane, and after visualization of the protein bands with Ponceau S, the individual lanes were manually cut into strips with scissors. Each strip was incubated with a standard dilution of patient serum from different time points (letters at bottom of images and graphs) then detected for human IgG4. These images represent digital merges of the chemiluminescence image captures (dark bands at 50 kDa. Please see Supplemental Figure 3 and the detailed methods in the Supplemental Material. Urine protein-Cr ratio (blue) and serum albumin (red) are shown over the same time periods.

Figure 5.

Ig capture from frozen kidney biopsy specimens followed by MS identifies serine protease HTRA1 as a target antigen. (A) Sequence coverage maps representing peptides detected from MN01 showing the extensive coverage by MS/MS. Peptide sequences detected are highlighted in bold letters over yellow background. (B) Heat map of 13 peptides quantified from the same patients. These peptides were specific to HTRA1-associated MN and were not present in four patients with PLA2R-associated MN and three patients with THSD7A-associated MN included as controls. (C) The volcano plot showing fold-change (FC) and P value of proteins quantified in HTRA1 versus non-HTRA1 cases. The serine protease HTRA1 (red dot) is a potential protein of interest, with the highest fold-change and lowest P value relative to other quantified proteins. Adj. P. Val, adjusted P value; neg, negative; OS, organism name; OX, organism identifier; GN, gene name; PE, protein existence; SV, sequence version.

Figure 6.

Immunofluorescence and immunohistochemical staining in HTRA1-associated MN patients and controls. Immunofluorescence microscopy of MN biopsy tissue (MN03) demonstrates a strong, granular, peripheral capillary loop staining pattern in (A) HTRA1-associated MN but not in (B) PLA2R-associated MN. (C) Immunohistochemical staining similarly showed strong capillary wall staining for HTRA1 in a peripheral capillary loop pattern, (D) but much weaker staining, consistent with low-level baseline podocyte expression, was seen in PLA2R-associated MN. (E–I) HTRA1 staining is uniquely present in (E) HTRA1-associated MN, but not within other forms of MN of known type, including (F) PLA2R-associated MN, (G) THSD7A-associated MN, (H) NELL1-associated MN (H), and (I) EXT1/EXT2-associated MN. A representative glomerulus from three biopsy specimens is shown within each row.

Figure 7.

Dual immunofluorescence staining for IgG (green) and HTRA1 (red) shows colocalization (right panels, yellow) in HTRA1-associated MN (MN02) but not in PLA2R-associated MN.

Figure 8.

HTRA1 is a podocyte-expressed protein. (A) Protein extracts of differentiated, immortalized human podocytes were immunoblotted with rabbit anti-HTRA1 and they exhibited reactivity with HTRA1 in both the nonreduced (NR) and reduced (R) state. (B) Immunofluorescence imaging of primary podocytes using rabbit anti-HTRA1 revealed cellular expression of the HTRA1 (green) in a reticular and perinuclear pattern (nuclei in blue via 4′,6-diamidino-2-phenylindole staining). Original magnification, ×400. (C) Immunohistochemical staining of normal kidney tissue with rabbit anti-HTRA1 shows positive HTRA1 staining of podocyte cell bodies (arrows).