C-Reactive Protein Levels Are Associated with Complement C4 Deposits and Interstitial Arteritis in ANCA-Associated Renal Vasculitis

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a potentially life-threatening systemic small-vessel vasculitis that is characterized by pauci-immune glomerulonephritis in case of kidney involvement, representing a major denominator of AAV mortality. Innate immunity with complement system activation is increasingly recognized in the pathogenesis of AAV and as an attractive therapeutic target. Although C-reactive protein (CRP) was thought to be a passive, nonspecific marker of inflammation, recent studies indicate that CRP plays a key role in the innate immune system by recognizing pathogens and altered self-determinants. Elevated baseline CRP at disease onset of AAV has already been described as a determinant of poor long-term outcomes. However, its clinical implications at disease onset of AAV, with respect to vasculitis manifestations and complement system activation that might also affect long-term outcomes, remain elusive. CRP levels were retrospectively analyzed in 53 kidney-biopsy-confirmed cases of ANCA-associated renal vasculitis; a total of 138 disease controls were also evaluated. Univariate and multivariate regression analysis was performed on clinicopathological parameters associated with CRP levels in ANCA-associated renal vasculitis. Results: Compared to disease controls, CRP elevation was common in ANCA-associated renal vasculitis and associated with de novo disease (p = 0.0169), critical illness (p = 0.0346), and severe deterioration of kidney function (p = 0.0167), independent of extrarenal disease manifestations. As confirmed by multiple regression analysis, CRP levels were correlated with active lesions predominated by interstitial arteritis in renal vasculitis, specifically with MPO-ANCA seropositivity (p = 0.0017). Based on analysis of systemic complement system activation and intrarenal complement deposits, CRP elevation was correlated specifically with complement C4 deposits in interstitial arteries in the subgroup with myeloperoxidase (MPO)-ANCA seropositivity (p = 0.039). Finally, this association was independent of systemic complement system activation, as reflected by the consumption of respective complement components. Here, we expand our current understanding of CRP in ANCA-associated renal vasculitis not only as an inflammatory marker, but potentially also as being involved in the pathogenesis of kidney injury by interaction with the complement system.


CRP Elevation Was Correlated with Complement C4 Deposits in Interstitial Arteries in MPO-ANCA-Associated Renal Vasculitis
Based on our observation that elevated CRP levels were correlated with active glomerular and tubulointerstitial renal vasculitis, we finally analyzed their association with systemic complement system activation and intrarenal complement deposits ( Figure 3A). We did not observe any association between CRP levels and systemic complement system activation reflected by serum C3c and C4 measurements ( Figure 3B). Among in- CRP elevation is correlated with complement C4 deposits in interstitial arteries in MPO-ANCA-associated renal vasculitis. Correlations between CRP levels, serum C3 and C4 measurements, and intrarenal complement C3 and C4 deposits localized to different vascular compartments in ANCA-associated renal vasculitis are shown by heatmaps reflecting the mean values of Spearman's ρ; circle size represents the significance level.

CRP Elevation Was Correlated with Complement C4 Deposits in Interstitial Arteries in MPO-ANCA-Associated Renal Vasculitis
Based on our observation that elevated CRP levels were correlated with active glomerular and tubulointerstitial renal vasculitis, we finally analyzed their association with systemic complement system activation and intrarenal complement deposits ( Figure 3A). We did not observe any association between CRP levels and systemic complement system activation reflected by serum C3c and C4 measurements ( Figure 3B). Among intrarenal complement deposits, the only significant correlation was observed between CRP elevation and complement C4 deposits in interstitial arteries, specifically in the subgroup with MPO-ANCA seropositivity (ρ = +0.45, p = 0.039, Figure 3B,C). This observation was not attributed to significant differences in complement C4 deposits between MPO-ANCA and PR3-ANCA seropositivity per se (Table 3). In summary, CRP elevation was correlated with interstitial arteritis along with complement C4 deposits in interstitial arteries, specifically in the subgroup with MPO-ANCA seropositivity. Furthermore, this association was independent of systemic complement system activation, as reflected by the consumption of respective complement components.
subgroup with MPO-ANCA seropositivity (ρ = +0.45, p = 0.039, Figure 3B,C). This observation was not attributed to significant differences in complement C4 deposits between MPO-ANCA and PR3-ANCA seropositivity per se (Table 3). In summary, CRP elevation was correlated with interstitial arteritis along with complement C4 deposits in interstitial arteries, specifically in the subgroup with MPO-ANCA seropositivity. Furthermore, this association was independent of systemic complement system activation, as reflected by the consumption of respective complement components.

Discussion
CRP is part of the innate immunity involved in nonspecific opsonization and neutralization of antigens, in contrast to the antigen-specific adaptive/acquired immunity maintained by antibodies [25]. While elevated baseline CRP at disease onset of AAV has already been described as a determinant of poor long-term outcomes, its clinical implications at disease onset of AAV with respect to vasculitis manifestations and complement system activation remain elusive [21][22][23][24]. Therefore, in this study, we aimed to systematically describe clinicopathological findings in association with baseline CRP levels at disease onset of AAV in kidney-biopsy-confirmed severe ANCA-associated renal vasculitis. Baseline CRP elevation was common in ANCA-associated renal vasculitis and associated with de novo disease, critical illness, and severe deterioration of kidney function. This is especially relevant because a higher initial mortality rate of critically ill patients within the first hospital stay was observed, while the long-term mortality of hospital survivors did not differ between the ICU and non-ICU groups [26]. Regarding the kidneys, severe renal dysfunction has already been associated with poor long-term outcomes in ANCA-associated renal vasculitis [2]. Our observation that we did not find any association between CRP levels at AAV onset and extrarenal disease manifestations further supports the predominant role of CRP levels in severe kidney involvement. Furthermore, we found that CRP levels were correlated with active lesions predominated by interstitial arteritis in renal vasculitis, specifically with MPO-ANCA seropositivity. We and others have recently described how interstitial arteritis represents a subtype of ANCA-associated renal vasculitis affecting a considerable subset of patients [27,28]. In addition, the presence of interstitial arteritis was associated with poor long-term renal outcomes and significantly increased the risk prediction by established scoring systems [28,29]. Interestingly, our observations confirm those of previous reports that the presence of interstitial arteritis was correlated with higher levels of CRP [28]. Our observation that CRP elevation was also correlated with complement C4 deposits in interstitial arteries could therefore further improve our current understanding of the underlying mechanisms contributing to interstitial arteritis in ANCA-associated renal vasculitis. Regarding the complement system, CRP can bind to the C1q globular recognition domain to activate the classical complement pathway, as well as to ficolins, mannosebinding lectins, and factor H to regulate the alternative and lectin-dependent complement pathways [25]. In kidney allografts, previous studies suggested that CRP can mediate complement activation in vivo [30]. In terms of distinct complement components, C3 is critical for activation of the complement system as a whole, while C4 is the major protein of the classical cascade [31]. Anaphylatoxin C3a stimulates inflammation by inducing an oxidative burst in macrophages, eosinophils, and neutrophils [32][33][34]. Furthermore, C3a and C5a directly activate basophils and mast cells, resulting in histamine production [35,36]. Although the pro-inflammatory effects of C3a are not in question, studies have also highlighted the anti-inflammatory role of C3a in different contexts [37]. Migration of neutrophils and degranulation are prevented in the presence of anaphylatoxin C3a, regardless of whether other granulocytes are activated by C3a [38,39]. The C4 activation product C4a has also been shown to exert a functional activity on macrophages and monocytes [40,41]. Because no C4a receptor has yet been reported, the physiological role of anaphylatoxin C4a and its contribution to autoimmune diseases such as AAV remain elusive [42]. On a mechanistic level, the ability of CRP to activate the complement system is by binding to an appropriate ligand, such as complement C4 [12,19]. We found that this association between CRP elevation, interstitial arteritis, and corresponding complement C4 deposits was specifically present in the subgroup with MPO-ANCA seropositivity. In this context, specific binding of CRP to MPO (and not PR3) has already been described and experimentally linked to complement system activation [43]. In addition, CRP is capable of stimulating the release of MPO in vitro and in vivo [44]. These findings might indicate a pathomechanistic link between MPO seropositivity and CRP in ANCA-associated renal vasculitis. The alternative pathway plays a predominant role in AAV pathogenesis, and it has already been reported that alternative pathway activation of the complement system requires the presence of C4 [45][46][47][48]. We have previously shown that intrarenal complement deposits were not associated with corresponding consumption of serum levels, supporting the concept of intrarenal synthesis of distinct complement system components [49,50]. Therefore, this study expands our current understanding of CRP in ANCA-associated renal vasculitis not only as an inflammatory marker, but also as potentially being involved in the pathogenesis of kidney injury by interaction with the complement system, and possibly local complement synthesis in the kidney. This is especially relevant because targeted therapies to block complement system activation in AAV are currently emerging.
Our study has several limitations, such as the small number of patients and the retrospective study design. Furthermore, our observations are associative and do not prove causality, which would require mechanistic studies. Nevertheless, this study provides a clinically approached link between CRP elevation, complement C4 deposits, and interstitial arteritis limited to MPO-ANCA-associated renal vasculitis, thereby broadening our current pathophysiological understanding.

Study Population and Subgroup Formation
A cohort of 53 kidney-biopsy-confirmed cases of ANCA-associated renal vasculitis was retrospectively enrolled between 2015 and 2020 in a single-center observational study at the University Medical Center Göttingen, Göttingen, Germany [51][52][53][54][55]. In addition, a total of 138 disease controls-including AL amyloidosis, tubulointerstitial nephritis, diabetic kidney disease, focal segmental glomerulosclerosis (FSGS), immunoglobulin A (IgA) nephropathy, IgA vasculitis, hypertensive nephropathy, lupus nephritis, membranous nephropathy, minimal change disease, monoclonal dense deposit disease (DDD), post-infectious glomerulonephritis (GN), and thrombotic microangiopathy-were also evaluated. While no formal approval was required for the use of routine clinical data, a favorable ethical opinion was granted by the local ethics committee (no. 22 February 2014 and 28 September 2017). All participants provided their written informed consent for the utilization of routinely collected data for research purposes as part of their regular medical care. Medical records were used to collect data on age, sex, medication, comorbidities, and laboratory findings, including baseline CRP at the time of kidney biopsy.
Moreover, all injured glomeruli (crescentic and/or necrotic) were screened for the presence of a Bowman's capsule rupture, the extent of which was further quantified as previously described in [59][60][61].

Statistical Analysis
Normal distribution was evaluated using the Shapiro-Wilk test; statistical comparisons were not formally powered or pre-specified. Probability values (p-values) below 0.05 were considered to be statistically significant. Parameters are shown as the median and interquartile range (IQR); median comparisons were performed with the Mann-Whitney U-test. Heatmaps reflect the mean values of Spearman's ρ in the univariate linear regression analysis; circle size represents the significance level. For stepwise multiple linear regression, covariates were retained to significant differences in the linear regression model to avoid model overfitting. Data analyses were performed using GraphPad Prism (version 9.4.1 for MacOS, GraphPad Software, San Diego, CA, USA) and IBM SPSS Statistics (version 28.0.1.1 for MacOS, IBM Corporation, Armonk, NY, USA).

Conclusions
This study expands our current understanding of CRP in ANCA-associated renal vasculitis not only as an inflammatory marker, but also as potentially being involved in the pathogenesis of kidney injury by interaction with the complement system. Funding: This study was supported by the Else-Kröner research program entitled "molecular therapy and prediction of gastrointestinal malignancies", grant number 7-67-1840876. We also acknowledge support by the Open Access Publication Funds of Göttingen University. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Informed Consent Statement: Patient consent for participation was not required for this study, in accordance with the national legislation and the institutional requirements.

Data Availability Statement:
The original contributions presented in this study are included in the article; further data and materials are available from the corresponding author upon reasonable request.