Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy
Authors: Al-Rabadi Laith Farah , Caza Tiffany , Trivin-Avillach Claire , Rodan Aylin R. , Andeen Nicole , Hayashi Norifumi , Williams Brandi , Revelo Monica P. , Clayton Fred , Abraham Jo , Lin Edwin , Liou Willisa , Zou Chang-Jiang , Ramkumar Nirupama , Cummins Tim , Wilkey Daniel W. , Kawalit Issa , Herzog Christian , Storey Aaron , Edmondson Rick , Sjoberg Ronald , Yang Tianxin , Chien Jeremy , Merchant Michael , Arthur John , Klein Jon , Larsen Chris , Beck Laurence H. 
Published: May 2021

Significance Statement
Membranous nephropathy (MN) is a glomerular disease that often leads to nephrotic syndrome and is caused by autoantibodies that target podocyte proteins. Recent work has identified new targets in this disease, although many cases remain untyped, making clinical monitoring difficult. A combination of conventional and more novel techniques reveals a new target podocyte antigen in MN, serine protease HTRA1. Fourteen patients were identified in a demographic group with mean age of 67.3 years. The titer of circulating anti-HTRA1 antibodies appears to correlate with the disease course, suggesting serial monitoring could facilitate diagnostic and therapeutic decisions. Study of the function of HTRA1 may provide important clues to podocyte biology and the underlying pathogenesis of this type of MN.

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.