Abstract

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, https://scifimed.eu/).

Author Declarations

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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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.

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AbbreviationsΔCFHR3,1Gene deletion of FHR-3 and FHR-1ATNAcute tubular necrosisBSABovine serum albuminCCPComplement control protein domainsCFHComplement Factor H geneCFHRComplement Factor H-related protein geneCRChronic rejectionCR1Complement receptor 1DAPI4,6-diamidino-2-phenylindoleDBDDonation after brain deathDCDDonation after circulatory arrestFHR-1Factor H-related protein 1FHR-3Factor H-related protein 3FHRsFactor H-related proteinsHRPHorseradish peroxidasePDGFR-βPlatelet-derived growth factor receptor-βpAbPolyclonal antibodymAbMonoclonal antibodyTRITCTetramethylrhodamine