Long-term follow-up including extensive complement analysis of a pediatric C3 glomerulopathy cohort

Over the last decade, the role of the complement system in many kidney disorders has been elucidated. C3 glomerulopathy (C3G) is one of these disorders that could be further characterized by increased knowledge of the underlying (complement) pathophysiology. C3G is a rare and severe kidney disease caused by dysregulation of the complement alternative pathway (AP) [1‐3]. Although several clinical symptoms, such as proteinuria, hematuria, and hypertension, possibly combined with low serum C3 levels, can be indicative of C3G, the diagnosis of C3G is still solely based on kidney biopsy. The defining characteristic in the kidney biopsy is the dominant immunofluorescence staining for C3 depositions in the glomerulus of at least two orders of magnitude greater than the intensity for other immune reactants [1]. Based on electron microscopy findings, C3G is further subdivided into its two major entities: dense deposit disease (DDD) and C3 glomerulonephritis (C3GN) [1, 3].

In addition to the prominent glomerular C3 staining in kidney biopsies, the presence of autoantibodies known as C3 nephritic factors (C3NeFs) is indicative of the AP dysregulation in C3G. In 50–80% of C3G patients, these C3NeFs can be found [4‐7]. C3NeFs prolong the activity of the AP convertases, leading to an overactive complement system via increased C3 conversion [8]. Low serum C3 levels indicate complement consumption in the majority of patients, but also other complement components and complement activation products may show an abnormal yet highly heterogenous pattern in patients with C3G [9]. In addition, in up to 20% of C3G cases, genetic aberrations are found in AP complement (regulatory) genes [4, 7, 10].

Since C3G is associated with a poor prognosis and high rate of recurrence after kidney transplantation, targeted therapy is needed. In recent years, a sharp increase in the development of complement-directed therapeutics can be noted, but many still have to pass the clinical trials [11]. Several phase II clinical trials with different complement inhibitors are currently including C3G patients [12]. Eculizumab, a C5 inhibitor and the first complement therapeutic drug that gained market approval, has proven its value in the treatment of another complement-mediated kidney disease, i.e., atypical hemolytic uremic syndrome. Although some case studies report a positive clinical response, the benefit of eculizumab treatment in patients with C3G remains unproven [13‐15].

In light of these (future) therapeutic opportunities, it has become very important to fully characterize the patient with C3G [3]. Recent advances in the development of assays measuring markers of complement activation and C3NeF types enable such improved patient characterization. Moreover, a recent study has shown that the incorporation of complement biomarker analyses, in addition to clinical and pathological characteristics, can be used to identify novel subgroups of C3G with distinct pathogenic patterns [16]. Furthermore, complement biomarkers such as C3 are increasingly used as inclusion criteria and/or as outcome measures of clinical trials with novel complement-targeted therapies.

Most of the cohort studies of patients with C3G comprise a mixed and thus heterogeneous group of both children (mostly adolescents) and adults [4, 7, 10, 17‐20]. Only a few studies on C3G in a pediatric cohort have been published [21‐26]. In this study, we describe the long-term follow-up of 29 Dutch children with C3G, including clinicopathological features, complement aberrations, and complement biomarker profiles.

In this study, we describe the long-term follow-up of a large pediatric C3G cohort, combined with an in-depth complement analysis over time. With our extensive retrospective analyses, we add important information regarding the relation between the presence of C3NeFs and clinical parameters such as C3 levels and disease recurrence. Moreover, C3NeF presence remained detectable over time despite immunosuppressive treatment. Profound analyses showed higher sC5b-9 and lower properdin levels over time in patients with C3GN than in DDD, suggestive of terminal complement pathway activation. Furthermore, in contrast to other cohort studies, a benign outcome was observed and only a few patients had an impaired kidney function.

Various complement aberrations have been described as cause of the complement dysregulation in patients with C3G, with one of the most important being C3NeFs. With our validated functional assay, we detected C3NeFs in 71% of the patients tested, which is consistent with reports in the literature [4‐7]. When focusing on the pediatric population only, our findings are in line with those of Marinozzi et al., who reported C3NeFs in 72% of the 57 pediatric patients they describe [37]. In most other cohorts describing the presence of C3NeFs in children, the number of patients tested is relatively low (≤ 11) and the percentage of C3NeF-positive patients ranges between 25 and 100% [13, 21‐23, 25, 38]. Some studies have reported C3NeFs more often in DDD than in C3GN [4, 7], but in our pediatric cohort no significant difference was found.

Rare variants in AP complement regulatory genes were found in only three patients, but no (likely) pathogenic genetic variants were identified in our cohort. In two patients with genetic variants, C3NeFs were also detected, leading to the question whether the genetic variation or rather the presence of C3NeF is driving the complement dysregulation in these patients. The role of rare genetic complement variants in C3G is further questioned by a recent study of Levine et al. that did not find enrichments of rare variants in complement (regulatory) genes in C3G patients. Instead, an HLA type was found to be associated with C3G, implicating that the disease should be considered an autoimmune process rather than a primary genetic disorder [39].

Importantly, a positive C3NeF finding in patients was linked to significantly lower C3 levels during follow-up and with disease relapses. Furthermore, C3NeFs remained detectable during follow-up despite immunosuppressive treatment, and their presence was consistently associated with lowered C3 levels in six out of the eight patients. Such longitudinal data on C3NeF presence in association with C3 levels are scarce in the literature [40]. Nonetheless, we as well as others have also shown that a normal C3 level does not exclude the presence of C3NeFs, and that C3NeFs can disappear (spontaneously) during the disease course [4, 26, 40]. Interestingly, we observed that patients who experienced a relapse were significantly more often C3NeF positive. This suggests a continuous pathogenic role of these autoantibodies during the course of disease and this could potentially influence treatment decisions. To further address the prognostic value of C3NeF, prospective data collection, of both clinical and chemical features, will be required. Moreover, one could question the (sub)classification of C3G solely based on pathology and maybe could rather identify novel subgroups of C3G based on the complement biomarker and autoantibody profile [16].

In C3GN patients, a significantly higher sC5b-9 level and lower properdin level was found using a linear mixed model to analyze the complement profiles with the longitudinal data of C3bBbP, C3bc, sC5b-9, C5, properdin, FH, and FI in DDD versus C3GN patients. Interestingly, these two markers are also the only two markers that were found significantly different between the C3G subgroups in a previous extensive biomarker study performed by Zhang et al. [9]. The combination of lowered properdin with elevated sC5b-9 may indicate that properdin is (partly) “consumed,” i.e., used by AP convertases to facilitate formation of C5 convertases and sC5b-9; a hypothesis that is supported by reports showing more pronounced activation of the terminal complement pathway in patients with C3GN [9, 37].

Despite the variety of treatment regimens, the outcome remains relatively benign in this pediatric cohort. Only one patient progressed to kidney failure requiring kidney replacement therapy. In total, six patients (n = 5 DDD, n = 1 C3GN) had an eGFR below 90 ml/min/1.73 m² at last follow-up. Of note, lower eGFR at presentation, as we observed mainly in DDD patients, has been described as a risk factor for kidney sequelae during follow-up [17]. These findings, statistically not significant due to the small group of patients, suggest a more severe presentation and outcome in patients with DDD compared to C3GN patients, and this is in line with results reported in other cohort studies [4, 17]. The overall benign outcome we described is in contrast to numerous reports in the literature pointing to a much worse outcome for C3G, with up to 50% of patients developing kidney failure within 10 years after diagnosis [4, 7, 10, 17, 37, 41]. Nevertheless, when looking at the pediatric population specifically, several reports also indicate that the outcome of children may be better than adults and that an older age of onset is associated with a higher chance of kidney failure [10, 17, 21, 25, 26, 37, 38]. An early detection and intervention in C3G and the lower presence of other, often age-related, comorbidities might contribute to this good outcome in children.

Even though the general outcome for these pediatric patients is relatively benign, proteinuria is still present in almost half of our patients despite immunosuppressive therapy (48%) and renin–angiotensin–aldosterone system inhibition (80%). This might contribute to chronic kidney disease later in life. The ongoing proteinuria as well as our findings of (persistent) complement abnormalities in time emphasize the need for targeted complement-directed therapy. The effect of the C5 inhibitor eculizumab in patients with C3G has been studied with variable results [13, 21, 42‐45]. Some studies report a potential therapeutic effect of eculizumab, especially in patients with elevated sC5b-9 levels, yet no clear benefit is observed [14]. In our study, five patients were treated with eculizumab during follow-up and these patients were described separately by Oosterveld et al. [27]. Yet, no significant difference could be observed in outcome (e.g., eGFR, C3 levels, proteinuria, hypertension) when compared to the other patients who did not receive eculizumab. In contrast to atypical hemolytic syndrome, in C3G patients, it might be necessary to intervene at an earlier level in the complement cascade, for example at the level of C3 conversion. Moreover, with the marked differences in complement biomarker profiles in patients with C3GN or DDD, different complement inhibitors should be considered for subtypes of C3G patients. At this moment, a wide variety of complement therapeutics are in the pipeline and subsequently studied in various phases of clinical trials [11, 12]. Furthermore, as C3NeFs are present in the vast majority of patients, one could argue to target these autoantibodies. However, reports in the literature targeting the production of C3NeFs with anti-CD19/CD20 monoclonal antibodies such as rituximab are scarce, and the described outcome varies highly [46‐48].

This study contains several limitations, mainly due to its retrospective character. The availability of patient material was limited, especially samples at presentation. Therefore, mixed model analysis was performed for the specialized complement biomarkers to include all available samples irrespective of disease state. The complement data were too limited to analyze relationships between complement marker profiles and clinical parameters. Furthermore, since there is no universal treatment scheme for patients with C3G, a highly diverse treatment regimen was represented in our study, with the choice of therapy depending on the preference of the treating physician at that time. Therefore, the effect of treatment on outcome was difficult to predict in this retrospective multicenter study.

In conclusion, this study is one of the largest C3G cohorts including solely pediatric patients and describes a follow-up period in which longitudinal complement analysis, including complement marker analysis and C3NeF detection, could be performed. Although the outcome of patients was relatively benign, persistent signs of complement (AP) dysregulation and persistent kidney injury underline the need for adequate targeted treatment to prevent decline in kidney function. Future prospective longitudinal studies are needed to further elucidate the different pathogenic mechanisms underlying this rare kidney disease, and to guide (and monitor) personalized treatment and predict outcome.