Factor H-related protein 3 (FHR-3) deposition in kidney allografts – localization and correlation with complement activation

Introduction Factor H-related proteins (FHRs) have emerged as novel players in complement-mediated diseases, as they exhibit structural resemblances to factor H but lack the regulatory domains, enabling them to antagonize factor H and increase complement activation through several activities. Despite the widely importance of the complement system in kidney transplantation, FHRs have not been studied in this context. Utilizing a novel monoclonal antibody, we investigated the presence of FHR-3 in kidney allografts. Methods The RTEC-2 monoclonal antibody was validated using immunohistochemistry, Western Blot analysis, and immunoprecipitation combined with mass spectrometry. FHR-3 deposition, localization, and the relationships to complement activation were analyzed in human kidney biopsies obtained pre-transplantation from living and deceased donors, and post-transplantation in cases with acute tubular necrosis, acute cellular and vascular rejection, or chronic rejection. Results Glomerular FHR-3 deposition was detected in kidneys from deceased, but not living, donors before transplantation. Additionally, we observed FHR-3 deposition in post-transplant settings, both in cases of rejection and non-rejection. While tubular and vascular deposition of FHR-3 was observed in some cases, FHR-3 was predominantly seen in the glomeruli, where it was primarily localized to podocytes. Moreover, co-localization of FHR-3 and C3d was rarely detected, with most cases exhibiting separate and non-overlapping staining patterns for both antigens, However, there was a moderate correlation between the staining intensity of the FHR-3 and C3d in the kidney biopsies ( r =0.38, P=0.01). Conclusion We detected FHR-3 deposition in kidney allografts under inflammatory conditions, primarily colocalizing with podocytes in both the presence and absence of complement activation. ### Competing Interest Statement FP owns or owned stock in Apellis Pharmaceuticals, Chemocentryx, InflaRx, Iveric Bio, and Omeros and has served as a consultant for Invizius and Alnylam Pharmaceuticals. JMT and VMH are consultants for Q32 Bio, Inc., a company developing complement inhibitors. Both also hold stock and may receive royalty income from Q32 Bio, Inc. The remaining authors of this paper declare that they have no competing interests. ### Funding Statement Felix Poppelaars was supported by the European Federation of Immunological Societies-Immunology Letters (EFIS-IL Short-Term Fellowship), and the European Society for Organ Transplantation (ESOT Study Scholarship). This project has received funding from the European Union`s Horizon 2020 research and innovation programme under grant agreement No 899163 - Screening of inflammation to enable personalized Medicine (SciFiMed, ). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The use of human biopsies was approved by the Institutional Review Board (METc 2014/077)of the University Medical Center Groningen (UMCG), Groningen, The Netherlands. Patient data were processed and stored according to the Declaration of Helsinki. Clinical and research activities followed the principles of the Declaration of Istanbul on Organ Trafficking and Transplant Tourism. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes * ΔCFHR3,1 : Gene deletion of FHR-3 and FHR-1 ATN : Acute tubular necrosis BSA : Bovine serum albumin CCP : Complement control protein domains CFH : Complement Factor H gene CFHR : Complement Factor H-related protein gene CR : Chronic rejection CR1 : Complement receptor 1 DAPI : 4,6-diamidino-2-phenylindole DBD : Donation after brain death DCD : Donation after circulatory arrest FHR-1 : Factor H-related protein 1 FHR-3 : Factor H-related protein 3 FHRs : Factor H-related proteins HRP : Horseradish peroxidase PDGFR-β : Platelet-derived growth factor receptor-β pAb : Polyclonal antibody mAb : Monoclonal antibody TRITC : Tetramethylrhodamine