Abstract: SA-PO0713
N-Terminomic Analysis Elucidates High-Temperature Requirement Factor A1 (HTRA1)-Complement Interactions in HTRA1-Associated Membranous Nephropathy
Session Information
- Glomerular Diseases: Profiling Through Multiomics
November 08, 2025 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology
Authors
- Al-Rabadi, Laith, University of Utah Health, Salt Lake City, Utah, United States
- Demir, Fatih, Aarhus Universitet, Aarhus, Central Denmark Region , Denmark
- Takayama, Suguru, University of Utah Health, Salt Lake City, Utah, United States
- Beck, Laurence H., Boston University, Boston, Massachusetts, United States
- Rinschen, Markus M., Aarhus Universitet, Aarhus, Central Denmark Region , Denmark
Background
Previously identified as a novel antigen in membranous nephropathy (MN), HTRA1 is a serine protease that regulates extracellular matrix breakdown and tissue barrier integrity. Misregulated proteolytic processing is implicated in the podocyte damage that occurs in kidney disease as well as being a crucial factor for the complement pathway. Elucidating the pathological interaction between the HTRA1-complement systems in HTRA1-associated MN should accelerate developing therapeutic breakthroughs.
Methods
The following steps outline implementing N terminomic analysis. First, non-transplantable human kidneys were subjected to either active serine protease HTRA1 or mutant inactive HTRA1 (S328A) treatment, at both 2 hours and o/N for in vitro analysis. Second, forward N terminomic was used to compare cleavage patterns between WT mice and HTRA1 KO mice, by using the whole mouse kidney. Subsequently, Mouse glomeruli were isolated using intra-aorticmagnetic bead injection to assess the proteolysis of glomerular proteins and to validate results from whole cortex analysis. Finally, downstream in vitro proteomic degradation assays were performed.
Results
More than 20448 N-termini were identified in mouse whole kidney representing 4381 proteins that are involved in different molecular pathways. Most of these N-termini did not correspond to already known fragments and a considerable proportion displayed a substantial alteration in KO status (at least 50% alteration & p-value 0.05). We identified specific substrates of HTRA1 in the human complement system and linked this information to our identified N-termini from HTRA1-associated MN. Regarding complement inhibitors, such as clusterin and vitronectin, observed cleavage was significantly altered, which confirmed as substrates for HTRA1. They were also more frequently observed in the KO mice group. We also identified calreticulin, a known complement regulator, as a new substrate (L51) for HTRA1. Moreover, we established and confirmed a previously assumed link between Complement Factor H and HTRA1. While not a direct substrate, CFH fragments were more enriched in WT mice compared to KO mice.
Conclusion
The use of N Terminomic HUNTER guides insights into a more comprehensive understanding of the pathological interactions concerning the HTRA1-complement system in HTRA1-associated MN.
Funding
- NIDDK Support