Glomerular Complement Factor H–Related Protein 5 (FHR5) Is Highly Prevalent in C3 Glomerulopathy and Associated With Renal Impairment

Introduction Therapeutic agents that target complement are increasingly available for glomerular diseases. However, the mechanisms linking glomerular complement deposition with inflammation and damage are incompletely understood. Complement factor H–related protein 5 (FHR5) interacts with complement C3 and is considered to promote activation. Circulating and glomerular FHR5 associates with IgA nephropathy and abnormal FHR5 associates with familial C3 glomerulopathy (C3G). We characterized glomerular FHR5 staining in C3G and assessed its relationships with histological features of glomerular injury and clinical outcome. Methods We developed FHR5 staining protocols for formalin-fixed paraffin-embedded (FFPE) renal tissue and applied them to surplus biopsy sections from a C3G cohort. Results Glomerular FHR5 was highly prevalent in native and transplant C3G and correlated with glomerular C3 and C5b-9 staining. Glomerular FHR5 staining correlated negatively with estimated glomerular filtration rate (eGFR) (P = 0.04, difference of medians 19.7 ml/min per 1.73 m2; 95% confidence interval [CI] 1.1–43.0) and positively with a membranoproliferative glomerulonephritis pattern at diagnostic biopsy (odds ratio 18; 95% CI 1.6–201; P = 0.049). Glomerular FHR5 staining intensity positively correlated with glomerular complement C3b/iC3b/C3c (Pearson’s correlation coefficient [R] = 0.59; P = 0.0008), C3dg (R = 0.47; P = 0.02) and C5b9 (R = 0.44, P = 0.02). Conclusions Glomerular FHR5 is highly prevalent in C3G, interacts with glomerular C3, and is associated with markers of disease severity. Glomerular FHR5 likely exacerbates complement-mediated glomerular damage in C3G and its interaction with glomerular complement might be exploited to target complement therapeutic agents.

C omplement dysregulation is associated with renal disease, which includes atypical hemolytic uremic syndrome and C3G. 1,2 Drugs that target specific complement proteins and pathways have emerged as therapeutic options for complement-associated renal diseases. 3 Complement C5 inhibition is an effective therapy for atypical hemolytic uremic syndrome, a condition characterized by complement-mediated glomerular thrombosis. 2 However, the mechanisms through which complement dysregulation contribute to glomerular inflammation are incompletely understood. This limits our ability to select patients for specific complement inhibitors and monitor disease activity. For example, the response of C3G to complement C5 inhibition is heterogeneous. 2 C3G is associated with significant morbidity. Up to 50% of C3G patients progress to end-stage renal disease within 5 years of diagnosis, 4,5 and recurrent C3G frequently causes transplant dysfunction. 6,7 Abnormal regulation of complement alternative pathway activation is seen in C3G. [8][9][10] This can be the result of genetic and acquired factors. However, in many cases the cause is unknown. The key negative regulator of C3 activation through the alternative pathway is complement factor H (fH). The fH protein family includes the 5 fH-related proteins (FHR1-5), named because of their structural similarity to fH. 11 Because they lack the complementregulatory domains of fH but retain C3-binding domains, 12 FHR proteins are currently thought to act as positive regulators that promote complement activity. A heterozygous mutation in FHR5 is associated with a familial form of C3G, known as CFHR5 nephropathy. 13 A number of other FHR protein variants have been identified in association with cases of C3G. [14][15][16][17][18] C3G includes dense deposit disease (DDD) and C3 glomerulonephritis. The range of histopathology features encompassed by a diagnosis of C3G has uncertain relevance to outcome. In some studies, DDD has been associated with a higher incidence of progression to end-stage renal disease, whereas in a recent cohort no difference between these subsets was evident. 5,6 It is also unclear whether or not glomerular deposition of specific complement proteins influences C3G outcome. Complement activation results in the cleavage of C3 to C3b, which can covalently attach to glomerular tissue. The C3b can contribute to further C3b generation through a positive feed-back loop or it can be cleaved sequentially to iC3b and then to C3dg, releasing C3c. Glomerular C3c is cleared within 24 hours after stopping complement activation in experimental glomerulonephritis models, 19 whereas C3dg persists. Glomerular FHR5 has been documented in a similar staining distribution to glomerular C3 in a range of glomerulopathies, including C3G and IgA nephropathy. 20 FHR5 binds C3b, iC3b, and C3dg and demonstrates stronger binding than fH for iC3b and C3dg. 21 In addition to the associations between FHR5 variants and familial C3G, 13,15,22 evidence that glomerular FHR5 might increase glomerular injury includes in vitro studies demonstrating FHR5 can (i) bind extracellular matrix components and support assembly of the alternative pathway C3 convertase formation, thereby promoting further C3b generation, and (ii) antagonize the ability of fH to limit amplification of C3b (a process termed fH deregulation). 21,23,24 Furthermore, both circulating FHR5 levels 25,26 and the intensity of glomerular FHR5 deposition 27 associate with IgA nephropathy severity.
In this study, we characterized the spectrum of glomerular FHR5 deposition in C3G in native and transplant kidneys, its relationships with other complement components, and, where possible, its change over time in the same patient. We show that glomerular FHR5 is highly prevalent in both native and transplant kidney C3G, colocalizes with C3b/iC3b/C3c and C3dg in vivo, and associates with disease severity.

Patient Cohort
We reviewed the diagnosis of all kidney biopsies performed at Imperial College Healthcare NHS Trust over a 20-year period. This totaled 6592 native and 2577 transplant biopsies. We included all patients with a biopsy diagnosis of C3G. After review of each case, we excluded patients with thrombotic microangiography, immune-complex dependent disease, or membranoproliferative glomerulonephritis (MPGN) type 1. We derived clinical and pathology data from medical records. Ethical approval was through the Natural History Study of C3 Glomerulopathy: Discovery of Histological Predictors of Outcome study (UK National Research Ethics Service reference 16/WM/0497) and the Imperial College Healthcare Tissue Bank. The Imperial College Healthcare Tissue Bank is supported by the National Institute for Health Research Biomedical Research Centre based at Imperial College Healthcare NHS Trust and Imperial College London. The Imperial College Healthcare Tissue Bank is approved by the National Research Ethics Service to release human material for research (12/WA/0196). Outcome parameters were 50% of eGFR from presentation and endstage renal disease (defined as commencing dialysis or receiving a renal transplant). Patients with less than 12 months of follow-up were excluded from outcome analyses. eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration equation 28 for adults and the Schwartz formula 29 for children and corrected to a maximum of 120 ml/min per 1.73 m 2 . Nephrotic syndrome was defined as a combination of serum albumin of less than 32 g/l, edema, and urine protein:creatinine ratio of at least 250 mg/mmol or 24hour urine proteinuria of at least 3 g. We defined crescentic C3G as the presence of cellular or fibrocellular crescents in at least 50% of glomeruli.
Two-micron thickness sections from FFPE renal tissue were fixed on Polysine slides, deparaffinized, and hydrated (xylene for 10 minutes followed by an ethanol gradient), washed in distilled water, and subjected to antigen retrieval. For C3b/iC3b/C3c, C5b9, and FHR5 (Supplementary Figure 1): bacterial proteinase XXIV (#P8038; Sigma-Aldrich) at 37 C for 30 minutes. For C3dg, C4d, fH, and C1q: sodium citrate tribasic buffer pH 6 at 95 C for 30 minutes. For properdin, FHR1, and CD68: Tris-EDTA buffer at pH 9 and heating in a pressure cooker for 30 minutes. Sections were blocked for 60 minutes at room temperature with hydrogen peroxidase (EnVision, #K4007 or #K4011; Dako) followed by 30% normal goat serum. Primary antibody was applied overnight at 4 C followed by application of secondary antibodies for 60 minutes at room temperature: horseradish peroxidase-conjugated goat anti-mouse (EnVisionþSystem-HRP, #K4007; Dako) or anti-rabbit (EnVisionþSystem-HRP, #K4011; Dako) IgG. 3ʹ-Diaminobenzidine substrate (EnVi-sionþSystem-HRP, #K4007 or #K4011; Dako) was applied for up to 15 minutes and sections counterstained with filtered hematoxylin, washed and  dehydrated and cleared in xylene for 10 minutes before mounting using xylene-based media (Pertex, #SEA-0104-00A; CellPath, Newtown, UK). Antigen staining intensities were graded as previously described. 27 Because nonspecific staining was seen in sclerosed glomeruli in previous 20 and our studies, only nonsclerosed glomeruli were included in statistical analyses. We tested for correlations between staining intensities and clinicopathologic markers of disease severity: MPGN light microscopy pattern; urine protein:creatinine ratio at biopsy; eGFR at biopsy; loss of 50% eGFR during follow-up; change in eGFR during follow-up; end-stage renal disease during follow-up.
Staining was performed using 2-to 4mm-thick FFPE sections fixed on Polysine slides, deparaffinized (xylene for 10 minutes followed by an ethanol gradient), washed in distilled water, and subjected to antigen retrieval: immersion in sodium citrate tribasic buffer pH 6 at 95 C for 30 minutes and incubation with HistoReveal (#103720; Abcam) for 5 minutes at room temperature. Slides were blocked with 2% bovine serum albumin in phosphate-buffered saline for 60 minutes at room temperature and the first antibody applied overnight at 4 C. Sections were blocked again with 2% bovine serum albumin at 4 C for 8 hours followed by incubation of the second antibody overnight at 4 C. Sections were sequentially incubated with the appropriate secondary Alexa Fluor antibodies, nuclear stain (4ʹ,6-Diamidino-2-Phenylindole, Dilactate, #D3571; ThermoScientific, Waltham, MA), 0.1% Sudan Black B (#199664; Sigma, St. Louis, MO) in 70% ethanol to reduce autofluorescence and mounted using Mowial 4-88 (#475904; Calbiochem, San Diego, CA).
We used ImageJ software (National Institutes of Health, Bethesda, MD) and the COLOC2 plug-in 30 to calculate antigen location correlations. This analyzed the antigen colocalization of each pixel within the image region of interest. We ensured IF images were captured with identical microscope settings, images were of identical size, and the brightness threshold at which monochromatic (red or green) fluorescence was counted as positive deposition was standardized. The    glomerulus from each section was selected as the region of interest for analysis. Pearson's correlation coefficients were recorded for each antigen combination in all glomeruli. For each antibody combination, this provided a number of antibody location correlation coefficients, which quantified how closely 2 antibodies colocalized on the same tissue section. We wanted to compare how closely each pair of antibodies colocalized. For example, we questioned whether the C3b/ iC3b/C3c and C3d antibodies colocalized more or less closely than FHR5 and C3d. To do this, we calculated the median of the correlation coefficients for each antibody pair, and then compared these median values.

Statistical Analysis
We used Pearson's correlation coefficient to calculate IF antigen location correlations with ImageJ software. 30 We used GraphPad (La Jolla, CA) Prism Version 6.00 for Windows for all other statistical analyses. Normally distributed continuous variables were compared using either unpaired t-test or (for multiple groups) 1-way analysis of variance. Continuous variables with skewed distribution were compared using either Mann-Whitney U test or (for multiple groups) Kruskal-Wallis test. We calculated CIs using the Hodges-Lehmann method and adjusted for multiple analyses with the 2-stage linear step-up procedure of Benjamini et al. 31 Boxplots were used to display correlation data. We included repeat biopsies from the same patients because we were interested in the correlation of glomerular FHR5 with other complement proteins and eGFR at each biopsy point. The dependence of these outcomes on repeated measures is not known.

C3G Cohort
We identified 27 individuals with biopsy-proven C3G (Tables 1 32,33 and 2 (Figure 1a-c). We did not detect significant differences in complement protein immunostaining when comparing cases of DDD with C3GN.
We have previously reported the prevalence of glomerular FHR5 deposition in IgA nephropathy. 27 We did not detect glomerular FHR5 in 3 thin basement membrane cases (Supplementary Figure S2). We detected glomerular FHR5, C3, and C5b-9 in 2 cases of CFHR5 nephropathy, 1 with and 1 without glomerular inflammation and clinical signs of glomerulonephritis (Figure 1b). Although C5b-9 and FHR5 staining intensities were greater in the biopsy with glomerular inflammation, the detection of C5b-9 and FHR5 in a potential kidney donor with normal renal function but subclinical CFHR5 nephropathy shows that glomerular complement deposition can be detected in the absence of glomerular inflammation in CFHR5 nephropathy.
Two of the renal transplant biopsies were part of rejection surveillance programs ("protocol biopsy"), thereby providing an opportunity to characterize subclinical C3G (Figure 1c). None of the C3G patients with renal transplant had a diagnosis of antibodymediated rejection. In 1 protocol biopsy performed 6 months posttransplant, the glomeruli were normal by light microscopy and glomerular C3, C5b9, and CD68 staining were negative. However, glomerular FHR5 staining was detectable. Nine months later, the patient developed biopsy-proven C3G recurrence (surplus biopsy tissue unavailable to study). The other protocol biopsy was from an individual who had a 3 further transplant biopsies and treatment with eculizumab. The initial protocol biopsy revealed subclinical C3G recurrence with glomerular endocapillary hypercellularity and glomerular staining for C3b/iC3b/C3c, C3dg, C5b-9, FHR5, and CD68 ( Figure 2). A biopsy performed due to proteinuria and fall in eGFR 6 months later showed crescentic C3G and increased glomerular C3b/iC3b/C3c, C3dg, C5b-9, FHR5, and CD68 staining (Figure 2, transplant biopsy 2). A biopsy 4 months after starting eculizumab treatment showed reduced endocapillary hypercellularity and reduced glomerular CD68 infiltration (Figure 2, transplant biopsy 3). Proteinuria and eGFR had improved but glomerular FHR5, C3b/iC3b/C3c, C3dg, and C5b-9 staining were unchanged. Three months after stopping eculizumab, graft function deteriorated again, and a fourth transplant biopsy showed recurrent crescentic C3GN. Rapid clinical improvement was seen after reintroducing eculizumab treatment, but subsequently proteinuria increased despite ongoing eculizumab therapy.

Glomerular FHR5 and C5b9 Staining Intensity Associates With Loss of eGFR in C3G
We next examined whether intense glomerular staining (grade 3þ) associated with renal impairment (eGFR) at the time of biopsy (Figure 4). Both glomerular FHR5 and C5b9 negatively correlated with eGFR, whereas glomerular FHR1, C3b/iC3b/C3c, C3dg, and C4d did not. Our correlation excluded sclerosed glomeruli, so any relationships, such as that between glomerular FHR5 and eGFR at time of biopsy, were unlikely to be influenced by the prevalence of sclerosed glomeruli. Furthermore, we did not identify correlations between glomerular FHR5 intensity and the percentage of sclerosed glomeruli per biopsy (Pearson's correlation coefficient r ¼ 0.1993, P ¼ 0.27). Glomerular FHR5 also correlated with an MPGN pattern at diagnostic biopsy (90% patients with vs. 33.3% without MPGN pattern, P ¼ 0.049; odds ratio 18; 95% CI 1.6-201). Glomerular FHR5 deposition intensity did not correlate with age (P ¼ 0.5), gender (P > 0.99), urine protein:creatinine ratio (P ¼ 0.27), the presence of cellular crescents (P > 0.99), or endocapillary hypercellularity (P ¼ 0.27) at the time of biopsy. = Figure 2. (continued) C5b9, C3b/iC3b/C3c, and C3dg were detected. The estimated glomerular filtration rate (eGFR) was stable and there was no significant proteinuria. Approximately 6 months posttransplant, the patient developed proteinuria and a fall in eGFR. Biopsy showed crescentic C3GN and increased glomerular CD68-positive cells (Transplant biopsy 2). Glomerular FHR5, C5b9, C3b/iC3b/C3c, and C3dg were detected but at increased staining intensity compared with the first transplant biopsy. The eGFR and proteinuria improved with eculizumab and prednisolone treatment. After 4 months of eculizumab treatment, renal biopsy (Transplant biopsy 3) showed resolution of glomerular CD68 staining, but glomerular staining for FHR5, C5b9, C3b/iC3b/C3c, and C3dg remained unchanged. After a further 3 months, proteinuria increased and biopsy showed crescentic C3GN and the recurrence of glomerular CD68-positive cells (Transplant biopsy 4). Proteinuria improved with re-introduction of eculizumab. UPCR, urine protein:creatinine ratio. Original magnification Â200 or Â400. Bars ¼ 100 mm.  Tubulo-interstitial FHR1 But Not FHR5 Was Detectable in C3G We did not detect significant FHR5 staining in the tubulointerstitium of either native or transplant C3G ( Figure 5a); however, there was tubular cell staining for both FHR1 and properdin (Figure 5a). This staining did not correlate with proteinuria, eGFR, tubular atrophy, loss of 50% eGFR (data not shown), or tubular deposition of other complement proteins (Figure 5a). Tubular FHR1 staining was detected in non-C3G cases and was absent in a section from a patient with IgA nephropathy and FHR1 deficiency (Figure 5b). Notably, when we used pressure cooker heating for antigen retrieval, we were able to detect tubulointerstitial FHR5 staining in different glomerulopathies, including C3G (Supplementary Figure 1). However, unlike the glomerular FHR5 staining evident using enzyme antigen retrieval, tubulo-interstitial staining could not be inhibited by pre-incubation of the primary antibody with purified FHR5, suggesting it was nonspecific.

DISCUSSION
Glomerular FHR5 was the most prevalent protein in native and transplant renal biopsies in our C3G cohort. FHR5 staining intensity associated with eGFR at the time of biopsy and correlated with the intensities of glomerular C3b/iC3b/C3c, C3dg, and C5b9 deposition. These findings support a role for FHR5 in complementdependent glomerular injury, and FHR5 staining may improve C3G diagnosis. Murphy et al. 20 demonstrated the presence of FHR5 in glomerular diseases, and we have demonstrated an association between IgA nephropathy severity and glomerular FHR5 deposition. 27 It will be interesting to determine the prevalence of glomerular FHR5 deposition in a large population of patients with complement-dependent and complementindependent renal diseases. We identified an association between MPGN light microscopy pattern at diagnostic biopsy and a 50% loss of eGFR. Intense glomerular FHR5 deposition also associated with an MPGN pattern in our cohort. The interaction between glomerular FHR5 deposition and MPGN morphology requires further investigation. FHR5 and complement glomerular deposition would ideally be assessed in serial sections of the same glomeruli; however, the limited amount of surplus biopsy tissue precluded assessment of serial sections in most cases. Glomerular FHR5 staining intensity correlated positively with the staining intensity of both C3b/iC3b/C3c and C3dg, which identify ongoing and previous complement activity, respectively. Our double-staining immunofluorescence protocols identified areas of FHR5 codeposition with C3b/iC3b/C3c and C3d. Therefore, FHR5 may bind both areas of ongoing (C3b/ iC3b/C3c) and previous (C3dg) complement activity.
FHR5 and C5b9 were the only glomerular complement antigens to associate with renal impairment. The interplay of C3G activity, impaired glomerular filtration, and glomerular FHR5 and C5b9 deposition requires further investigation. Notably, glomerular C5b9 has been shown to persist in lupus nephritis following complement activity cessation, 34 so may not be a reliable marker of ongoing glomerular C5 activation in C3G as well.
We made a number of interesting observations from transplant cases. Glomerular FHR5 deposition was identified in the absence of and predated detectable C3b/iC3b/C3c deposition and glomerular inflammation in one transplant patient. Whether this implicates FHR5 in early C3G pathogenicity or represents the sensitivity and affinity of FHR5 for otherwise undetectable C3 fragments requires further investigation. Specifically, a comparison of FHR5 deposition in protocol biopsies from patients with and without clinical signs of C3G recurrence is needed. Serial transplant biopsies during recurrent crescentic C3G showed FHR5 deposition correlated with initial disease activity, but = Figure 3. (continued) comparisons. (b) Representative images of combined immunofluorescence staining in three C3G cases for glomerular FHR5 with either C3b/iC3b/C3c or C3dg, and C3b/iC3b/C3c with C3dg. Renal biopsies in all 3 cases showed C3-dominant membranoproliferative glomerulonephritis, and the biopsy indications together with the urine protein:creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), and serum C3 levels at the time of biopsy are listed. The staining patterns for C3b/iC3b/C3c and C3dg (right-hand column of images) showed areas of colocalization (arrows), areas of C3b/iC3b/C3c alone (stars), and areas of C3dg alone (triangles). The staining pattern for C3b/ iC3b/C3c and FHR5 (left-hand column of images) showed areas of colocalization (arrows), areas of C3b/iC3b/C3c alone (stars), and areas of FHR5 alone (triangles). The staining pattern for C3dg and FHR5 (middle column of images) showed areas of colocalization, particularly along capillary walls in cases 2 and 3 (arrows) and areas of FHR5 alone in cases 1 and 2 (triangles). Notably, we did not detect areas of C3dg without was unaltered by C5 inhibition. This is consistent with a role for FHR5 in alternative but not terminal complement pathway activity. As evidenced by improved CD68-positive cell infiltration, clinical improvement following eculizumab treatment is likely secondary to reduced C5a production and inflammatory cell recruitment. Whether continued terminal pathway blockade would cause long-term disease improvement is unclear. Evidence from C3G animal models suggests alternative pathway activity in the absence of C5 reduces glomerular inflammation, but C3 glomerulopathy persists. 35 The role of C5 inhibition in treating acute exacerbations of disease and its ability to modify longterm outcomes requires further study. Glomerular C4d was prevalent in both native and transplant C3G biopsies, suggesting the absence of C4d as a tool for diagnosing C3G is unreliable in our cohort, and the role of C4d in C3G requires further investigation.
In our protocols, glomerular FHR5 staining could identify complement activation in the absence of anti-C3b/iC3b/C3c staining. We speculate that, by comparing FHR5 and C3dg immunostaining patterns, it might be possible to identify areas within glomeruli where there is either ongoing (glomerular FHR5 without C3dg) or previous (glomerular FHR5 with C3dg) complement activation. Moreover, the combination of glomerular C5b9 and CD68 staining patterns could differentiate ongoing from previous glomerular C5 activation. This approach would rely on CD68 staining as a surrogate marker of complement C5a production. Consequently, ongoing C5 activation would be inferred if both C5b-9 and glomerular CD68 were positive; whereas previous C5 activation would be inferred if glomerular C5b-9 was positive in the absence of glomerular CD68.
Because FHR5 binds all immobilized C3 fragments in vitro (Supplementary Figure 2), the identification of glomerular C3b/iC3b/C3c deposits without FHR5 (Supplementary Figure 3) was unexpected. The structural conformation of C3c in vivo may hide the epitope that FHR5 binds in vitro. Alternatively, we speculate glomerular C3b/iC3b/C3c staining without FHR5 is C3c released from extrarenal complement activation that deposits in glomeruli but does not participate in complement activation 32 and therefore does not interact with FHR5. In this scenario, comparing C3b/iC3b/C3c and FHR5 deposition could differentiate local from extra-glomerular complement activation.
proteinuria. The significance of FHR1 in this context is unclear. Tubulo-interstitial staining for FHR5 was absent when we used enzymatic antigen retrieval but became detectable when we used pressure cooker antigen retrieval. We considered this to be nonspecific staining, as we were unable to inhibit the staining with pre-incubation of the anti-FHR5 antibody with purified FHR5. Nevertheless, the role of the FHR proteins in the tubulointerstitium requires further investigation.
Mass spectrometry analysis of laser-captured glomeruli has identified alternative and terminal complement pathway components and FHR proteins in C3G. 38 Using mass spectrometry spectra as semiquantitative evidence of glomerular protein abundance, Sethi et al. 38 found C9, C3, and, specifically, C3dg were abundant in C3G, and DDD and C3GN cases had similar complement proteomic profiles. These observations seem consistent with our findings. By mass  Figure 5. Tubulo-interstitial staining for FHR5 in C3 glomerulopathy. (a) Representative images of staining for factor H-related protein 5 (FHR5), factor H-related protein 1 (FHR1), factor H (fH), properdin, and C3b/iC3b/C3c. No tubulo-interstitial staining for FHR5 or fH was evident but there was strong tubulo-interstitial staining for both properdin and FHR1. (b) Tubulo-interstitial staining for properdin was also seen in other renal diseases (thin basement membrane disease [TBM], lupus nephritis [LN], and membranous nephropathy). Tubulo-interstitial staining for FHR1 was demonstrable in biopsies from patients with TBM and IgA nephropathy. Tubulo-interstitial FHR1 staining in TBM was still detectable but less intense when the proteinase XXIV enzyme was used instead of pressure cooker antigen retrieval. Glomerular and tubulo-interstitial FHR1 was absent in renal tissue from a patient with IgA nephropathy and FHR1 deficiency. C3G, C3 glomerulopathy; LN IV(A/C), lupus nephritis class 4, active and chronic. Original magnification Â200. Bars ¼ 100 mm.

CLINICAL RESEARCH
NR Medjeral-Thomas et al.: FHR5 in C3 Glomerulopathy spectrometry, FHR1 was more prevalent and detected with higher mass spectrometry spectral counts than FHR5. 38 In contrast, using immunohistochemistry, we found glomerular FHR5 to be more prevalent than FHR1; FHR5 was detected (at least 1þ intensity) in 96% (23 of 24) native and 100% (8 of 8) transplant biopsies; FHR1 was detected in 77% (17 of 22) native and 78% (7 of 9) transplant biopsies. Glomeruli FHR5 also appeared to be more abundant than FHR1; FHR5 was detected at 3þ staining in 71% (19 of 24) native and 63% (5 of 8) transplant biopsies, compared with FHR1 that we detected at 2þ intensity in 32% (7 of 22) native and 56% (5 of 9) transplant biopsies. Given the different research techniques and the experimental nature of protein quantification by spectra abundance, it is difficult to conclude why we did not replicate this finding in our cohort. Our observational data cannot unravel the mechanisms that result in glomerular FHR5 deposition or its relationship with glomerular damage; however, we speculate that glomerular FHR5 interacting with C3 impairs the ability of fH to downregulate glomerular complement C3 activation. In addition, C3dg-bound FHR5 could facilitate C3 convertase formation and local C3 activation. 24 FHR5 could have similar effects after binding directly to glomerular components, such as laminin-521 or -211. 39 In addition, C3-bound FHR5 could impair complement receptor-dependent phagocytosis of deposited C3-coated immune complexes by mesangial cells and macrophages. In these scenarios, inhibition of glomerular FHR5 would be predicted to improve glomerular complement regulation and ameliorate complement-mediated glomerular inflammation and injury.
In summary, glomerular FHR5 deposition associates with C3G severity and colocalizes with C3 fragments in vivo. The prevalence of glomerular FHR5 binding to C3 fragments could be exploited to target therapeutic complement inhibitors to areas of complement activation in C3G.

DISCLOSURE
All the authors declared no competing interests.