Gain-of-function factor H–related 5 protein impairs glomerular complement regulation resulting in kidney damage

Figures

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  Fig. 1.. delFH-FHR mice develop spontaneous C3 glomerulopathy, whereas delFHR mice do not. (A) Schematic of the mouse FH-FHR locus and proteins depicting the genomic deletions and strains. The delFH-FHR strain is deficient in both FH and the FHR proteins. The delFHR strain is deficient in the FHR proteins only. Fiber-FISH using four probes (circles) across the locus confirmed the deletions. gRNAs (red arrows) and bridging repair oligonucleotides (delFHR: yellow/blue bars; delFH-FHR: yellow/green bars) are indicated, and genome coordinates are derived from Ensembl Mus Musculus version 100.38, Genome Reference Consortium Mouse Build 38 patch release 6. (B) Western blot of plasma FH and FHR-C (arrows) in delFH-FHR mice. Control lanes: plasma from wild-type (WT), FH-deficient, and Murphy Roths Large (MRL) (FHR-C–deficient) mice. In the upper blot image, plasma FH is detected with rat anti-mouse FH. In the lower blot image, FHR-C is detected with a sheep anti-mouse FH antibody. FH and FHR-C are reduced and absent in heterozygous and homozygous delFH-FHR mice, respectively. (C) Plasma C3 levels in delFH-FHR mice. Bars denote the mean, and whiskers denote the SD. P values are derived from ANOVA…

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  Fig. 2.. delFH-FHR mice coexpressing hFH and FHR5mut recapitulate CFHR5 nephropathy. (A) Schematic showing the generation of the hFH-FHR5 and hFH-FHR5mut strains. The transgenic cassette contained each cDNA in tandem, separated by an IRES, under the control of a loxP-dependent cytomegalovirus early enhancer/chicken β-actin promoter (pCAG). (B) Plasma hFH and (C) FHR5 levels. Bars denote the mean, and whiskers denote the SD. P values are derived from ANOVA with Bonferroni multiple comparisons test. (D) Western blot of FHR5 demonstrating the expression of WT and mutant FHR5 protein. Normal mouse sera (NMS), normal human sera (NHS), and FHR5 nephropathy serum (R5M) are shown. (E) Plasma C3 levels by ELISA. Bars denote the mean, and whiskers denote the SD. P values are derived from ANOVA with Bonferroni multiple comparisons test. (F) Representative glomerular immunostaining images of C3b/iC3b/C3c, C3d, FHR5/FHR5mut, FH, IgG, and properdin in 3-mo-old mice. (G) Representative glomerular images from Periodic acid–Schiff–stained renal sections from 12-mo-old mice. hFH-FHR5mut male mice show conspicuous capillary wall thickening (arrows). Scoring of capillary wall double contours in 12-mo-old…

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  Fig. 3.. FHR5mut triggers glomerular C3 deposition in hFH-FHR5 mice. (A) Representative glomerular images and (B) staining scores for C3b/iC3b/C3c, C3d, FHR5/FHR5mut, FH, IgG, and properdin in male hFH-FHR5 mice treated with either AAV-GFP (n = 5, black dots) or AAV-FHR5mut (n = 6, red dots). Bars denote the median, and whiskers denote the interquartile range. *P = 0.0022; values are derived from Mann–Whitney U test. Plasma FHR5/FHR5mut (C), C3 (D), and FH (E) levels after administration of either AAV-FHR5mut or AAV-GFP to hFH-FHR5 male mice. Bars denote the mean, and whiskers denote the SD. P values are derived from two-way ANOVA with Bonferroni multiple comparisons test. (F) Representative glomerular images of C3b/iC3b/C3c, C3d, and FHR5mut in male hFH-FHR5mut mice expressing one copy of the transgene on the background of either heterozygous (delFH-FHRhet.hFH-FHR5mut) or homozygous (hFH-FHR5mut) deficiency of the mouse FH-FHR locus. Controls are male delFH-FHRhet.hFH-FHR5 and hFH-FHR5 animals.

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  Fig. 4.. HDM-FH ameliorates glomerular C3 in hFH-FHR5mut mice. (A) Representative glomerular images and (B) staining scores for C3b/iC3b/C3c, C3d, FHR5mut, FH, IgG, and properdin in male hFH-FHR5mut mice treated with either AAV-GFP (n = 5, black dots) or AAV-HDM-FH (n = 7). Bars denote the median, and whiskers denote the interquartile range. *P = 0.0013, derived from Mann–Whitney U test. Plasma hFH/HDM-FH (C), C3 (D), and FHR5mut (E) levels after administration of either AAV-GFP or AAV-HDM-FH to hFH-FHR5mut male mice. Bars denote the mean, and whiskers denote the SD. P values are derived from two-way ANOVA with Bonferroni multiple comparisons test.