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. 2021 Sep 10:12:720183.
doi: 10.3389/fimmu.2021.720183. eCollection 2021.

FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy

Nóra Garam  1 , Marcell Cserhalmi  2 , Zoltán Prohászka  1   3 , Ágnes Szilágyi  1 , Nóra Veszeli  1   3 , Edina Szabó  1 , Barbara Uzonyi  4 , Attila Iliás  4 , Christof Aigner  5 , Alice Schmidt  5 , Martina Gaggl  5 , Gere Sunder-Plassmann  5 , Dóra Bajcsi  6 , Jürgen Brunner  7 , Alexandra Dumfarth  8 , Daniel Cejka  8 , Stefan Flaschberger  9 , Hana Flögelova  10 , Ágnes Haris  11 , Ágnes Hartmann  12 , Andreas Heilos  13 , Thomas Mueller  13 , Krisztina Rusai  13 , Klaus Arbeiter  13 , Johannes Hofer  7   14   15 , Dániel Jakab  16 , Mária Sinkó  16 , Erika Szigeti  16 , Csaba Bereczki  16 , Viktor Janko  17 , Kata Kelen  18 , György S Reusz  18 , Attila J Szabó  18 , Nóra Klenk  19 , Krisztina Kóbor  19 , Nika Kojc  20 , Maarten Knechtelsdorfer  21 , Mario Laganovic  22 , Adrian Catalin Lungu  23 , Anamarija Meglic  24 , Rina Rus  24 , Tanja Kersnik Levart  24 , Ernesta Macioniene  25 , Marius Miglinas  25 , Anna Pawłowska  26 , Tomasz Stompór  26 , Ludmila Podracka  27 , Michael Rudnicki  28 , Gert Mayer  28 , Romana Rysava  29 , Jana Reiterova  29 , Marijan Saraga  30   31 , Tomáš Seeman  32 , Jakub Zieg  32 , Eva Sládková  33 , Natasa Stajic  34 , Tamás Szabó  35 , Andrei Capitanescu  36 , Simona Stancu  36 , Miroslav Tisljar  37 , Kresimir Galesic  37 , András Tislér  38 , Inga Vainumäe  39 , Martin Windpessl  40 , Tomas Zaoral  41 , Galia Zlatanova  42 , Mihály Józsi  2   4 , Dorottya Csuka  1   3
Affiliations

FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy

Nóra Garam et al. Front Immunol. .

Abstract

Background: Factor H-related protein 5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in the pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement, and follow-up data.

Methods: A total of 120 patients with a histologically proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger sequencing, and selected variants were studied as recombinant proteins in ELISA and surface plasmon resonance (SPR).

Results: Eight exonic CFHR5 variations in 14 patients (12.6%) were observed. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period; furthermore, it showed clear association with signs of complement overactivation and clinically meaningful clusters.

Conclusions: Our observations raise the possibility that the FHR-5 protein plays a fine-tuning role in the pathogenesis of IC-MPGN/C3G.

Keywords: C3 glomerulonephritis (C3GN); C3 glomerulopathy; dense deposit disease (DDD); immune complex-mediated glomerulonephritis; membranoproliferative glomerulonephritis.

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Conflict of interest statement

Author VJ was employed by company Medimpax. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Factor H-related protein 5 (FHR-5) on Western blot. Two serum samples obtained from patients with wild-type FHR-5 protein were analyzed.
Figure 2
Figure 2
Factor H-related protein 5 (FHR-5) levels in patients with C3 glomerulopathy (C3G)/immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and in healthy individuals. 1p-value was determined with Kruskal–Wallis test. *p < 0.05 by Dunn’s posttest.
Figure 3
Figure 3
Localization of missense and frame-shift CFHR5 variations and the serum level of Factor H-related protein 5 (FHR-5).
Figure 4
Figure 4
(A) Comparison of serum Factor H-related protein 5 (FHR-5) levels and serum FHR-5 C3b-binding ability of patients with only FHR-5WT and those carrying FHR-5G278S or FHR-5R356H in ELISA. C3b was immobilized, then serum was added, and FHR-5 was detected by polyclonal anti-FHR-5. Dots represent individual patients. Data are mean of two measurements and are normalized to a calibrator sample containing only FHR-5WT. Subjects expressing FHR-5G278S show a tendency of lower FHR-5 levels and decreased C3b binding compared to the FHR-5WT-expressing group. Serum FHR-5 levels were measured in a sandwich ELISA. Circles represent individual patients. Data are mean of two measurements. (B) Dose-dependent binding of recombinant FHR-5WT, FHR-5G278S, and FHR-5R356H to purified C3b measured in ELISA. The FHR-5G278S variant binds significantly weaker to C3b (one-way ANOVA with Bonferroni’s posttest, p < 0.01). Data are mean of four measurements ± SEM. (C) Interaction of C3b–FHR-5 variants measured by surface plasmon resonance (SPR). C3b in serial dilutions was flown over immobilized FHR-5 variants. The KD of the C3b–FHR-5G278S is one order greater compared to that of C3b–FHR-5R356H and C3b–FHR-5WT. Data are representative of two experiments.
Figure 5
Figure 5
(A) Correlation heat map of different complement parameters. Number in boxes indicates Spearman correlation r. *p < 0.05. (B) Significant correlation of Factor H-related protein 5 (FHR-5) and other complement parameters.
Figure 6
Figure 6
Bubble plot of patients’ various complement parameters and Factor H-related protein 5 (FHR-5) levels.
Figure 8
Figure 8
Patients’ Factor H-related protein 5 (FHR-5) and C3 levels along with the presence of CFHR5 variations and end-stage renal disease (ESRD). Dotted lines indicate the threshold of high and low C3 (0.9 g/L) and FHR-5 (1.565 mg/L) levels. Rates of ESRD during follow-up - C3 <0.9 g/L, FHR-5 <1.56 mg/L: 0.032/event/patient/year, median (min–max) follow-up: 1.6 (0.11–6) - C3 <0.9 g/L, FHR-5 >1.56 mg/L: 0.11/event/patient/year, median (min–max) follow-up: 1.5(0.05–6) - C3 >0.9 g/L, FHR-5 <1.56 mg/L: 0/event/patient/year; median (min–max) follow-up: 1.57 (0.21–6) - C3 >0.9 g/L, FHR-5 >1.56 mg/L: 0.099 event/patient/year; median (min–max) follow-up: 1.5 (0.13–6).
Figure 7
Figure 7
Patients’ renal survival according to their Factor H-related protein 5 (FHR-5) serum levels. * p-value was determined by log-rank test comparing patients with high and low FHR-5 serum levels.
Figure 9
Figure 9
(A) Factor H-related protein 5 (FHR-5) protein levels in the previously described clusters of C3 glomerulopathy (C3G)/immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) patients (19, 37). The clinically meaningful clusters were generated based on clinical, histological, genetic, and complement data of patients as described by Iatropoulos et al. (19). Each dot represents one patient. 1p-value was determined with ANOVA for patients without CFHR5 variations comparing patients in clusters 1, 3, and 4. *p < 0.05 by Dunn’s posttest, for patients without CFHR5 variations. (B) C3 and FHR-5 levels according to cluster membership and the presence of CFHR5 variations.
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References

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