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Abstract: FR-OR35

High Temperature Recombinant Protein A1 (HTRA1): A Novel Antigen in Membranous Nephropathy

Session Information

Category: Glomerular Diseases

  • 1202 Glomerular Diseases: Immunology and Inflammation

Authors

  • Al-Rabadi, Laith, University of Utah Health Hospitals and Clinics, Salt Lake City, Utah, United States
  • Caza, Tiffany, Arkana Laboratories, Little Rock, Arkansas, United States
  • Avillach, Claire, Boston University, Boston, Massachusetts, United States
  • Rodan, Aylin R., University of Utah Health Hospitals and Clinics, Salt Lake City, Utah, United States
  • Williams, B., University of Utah Health Hospitals and Clinics, Salt Lake City, Utah, United States
  • Abraham, Josephine, University of Utah Health Hospitals and Clinics, Salt Lake City, Utah, United States
  • Revelo Penafiel, Monica Patricia, University of Utah Health Hospitals and Clinics, Salt Lake City, Utah, United States
  • Andeen, Nicole K., Oregon Health and Science University School of Medicine, Portland, Oregon, United States
  • Kawalit, I., Jordan University of Science and Technology, Irbid, Jordan
  • Clayton, Frederic, University of Utah Health Hospitals and Clinics, Salt Lake City, Utah, United States
  • Cummins, Timothy, University of Louisville, Louisville, Kentucky, United States
  • Merchant, Michael, University of Louisville, Louisville, Kentucky, United States
  • Klein, Jon B., University of Louisville, Louisville, Kentucky, United States
  • Larsen, Christopher Patrick, Arkana Laboratories, Little Rock, Arkansas, United States
  • Beck, Laurence H., Boston University, Boston, Massachusetts, United States
Background

Previous discoveries in MN included identification of PLA2R, THSD7A and NELL-1 as target antigens in 80-85% of primary MN cases. However, target antigens in the remaining 15-20% of cases remain unidentified.

Methods

We present a multi-pronged approach using traditional and modern technologies to converge on a novel target antigen. Instead of a case-vs-control design, we capitalized on the temporal variation in autoantibody titer for our biomarker discovery. Western blotting (WB) of human glomerular extract (HGE) proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection / mass spectrometry as well as autoimmune profiling on a protein expressed sequence tag microarray. Commercial antibodies to the candidate antigen were used for immunostaining MN biopsies, and reactivity of patient sera with a recombinant HTRA1 (rHTRA1) by WB and ELISA was assessed.

Results

Using these combined approaches, we identified HTRA1 as a novel antigen in a subset of patients with primary MN. Serum from those patients reacted by WB with a 51 KDa protein in non-reduced HGE as well as rHTRA1, which correlated with clinical disease activity.(Fig A and B) Consistent with PLA2R and THSD7A, anti-HTRA1 antibodies were predominantly IgG4. HTRA1 specifically was detected in a capillary loop fine granular pattern. (Fig C) and colocalized with IgG4. Whole-proteome peptide microarrays detected significantly higher titer (6.9 SD) of anti-HTRA1 antibody in the active stage as opposed to the remission stage which was informative of its candidacy as a podocyte targeted protein. We have 3 confirmed cases of HTRA1-associated MN and are currently screening several large biopsy cohorts of MN that are negative for PLA2R, THSD7A, and NELL-1 to better assess the prevalence of this novel form of MN.

Conclusion

This report demonstrates the convergence of conventional with more modern techniques to identify HTRA1 as a target podocyte antigen in MN.

Funding

  • Private Foundation Support