Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a multisystemic disease that causes inflammation of small blood vessels, often affecting the kidneys. The disease is triggered by ANCA binding to proteins like proteinase 3 (PR3) and myeloperoxidase (MPO) on neutrophils, which leads to neutrophil degranulation and the release of inflammatory molecules.1 Treatment involves intensive immunosuppressive therapy during the induction phase, followed by a maintenance phase. Anti-CD20 targeted therapy with rituximab has demonstrated its efficacy both in the induction to remission phase as in the maintenance phase.2 Rituximab not only abrogates ANCA production but also inhibits neutrophil responses, although the exact mechanism remains unclear. Some researchers have proposed that this may be associated with changes in stromal-derived factor-1 (SDF-1) levels during B-cell recovery, potentially affecting neutrophil egress from the bone marrow. Others have suggested alternative mechanisms, including neutropenia caused by autoantibody production following rituximab treatment and the expansion of large granular lymphocytes that may induce neutrophil apoptosis.3 While guidelines suggest a maintenance period of at least 24 months, the optimal duration remains unclear. In this setting, biomarkers to assess disease activity, predict relapses, and tailor treatment plans accordingly are a topic of interest.

Calprotectin is a protein released by activated monocytes and neutrophils that promotes the inflammatory response in AAV leading to increased transcription of proinflammatory cytokines and facilitating leukocyte recruitment to inflammation sites.4 Due to its low molecular weight (36.5kDa), calprotectin diffuses into the bloodstream, where it can be measured, though it has a short half-life of only 5h.5 Some authors have observed higher serum calprotectin concentration in AAV patients in the acute phase of the disease compared to patients in remission.6–8 Moreover, Pepper et al. found that, in PR3-ANCA patients in the acute phase treated with rituximab, and increase in serum calprotectin by month 2 or 6 compared to the moment of the diagnostic was associated to a higher risk of relapse at 18 months.9 On the contrary, the dynamics of calprotectin levels during remission have been less extensively studied. Our group examined the evolution of serum calprotectin levels in a cohort of patients in stable remission over a 2-year period.6 Additionally, we observed higher calprotectin levels in patients who showed a greater decline in glomerular filtration rate (GFR) and worsening proteinuria during the 2-year follow-up in remission, but we were unable to correlate calprotectin levels with an increased risk of relapse. Romand et al., in a post-hoc analysis of the MAINTRITSAN study, demonstrated that patients in remission who showed increasing calprotectin levels during the first 6 months after initiating maintenance treatment had worse renal outcomes during follow-up and a tendency toward a higher risk of AAV relapse.10 In both studies, the maintenance treatment was not homogenous and included rituximab in different schedules of administration or the administration of antiproliferative drugs such as azathioprine or mycophenolate, together with low-dose steroids.

CD163 is a membrane protein found on the surface of monocytes and macrophages. The soluble form of CD163 (sCD163) is generated through enzymatic cleavage during inflammation and is associated with excessive M2 macrophage activation.11 O’Reilly et al. reported that urinary sCD163, shed by macrophages, correlates with active glomerular inflammation in AAV.12 B-cell depletion by rituximab is mediated by various immune mechanisms, including the phagocytosis of rituximab-opsonized B cells by M2 macrophages.13 In a cohort of 24 incident AAV patients, Villacorta et al. observed a decline in usCD163 levels during the first year after diagnosis in all patients who responded to treatment and reached remission.11 In contrast, 5 patients who relapsed during follow-up showed a significant increase in usCD163 compared to previous levels. Although some authors have reported significantly lower usCD163 levels in remission compared to active AAV, the dynamics of CD163 during stable remission have not been studied.

This study aims to comprehensively describe the dynamics of two recognized biomarkers of AAV activity – calprotectin and usCD163 – during periods of stable remission. Additionally, it seeks to explore the correlation between these biomarkers and with other markers of inflammation and disease activity, their relationship with relapses, and with rituximab administration.

AAV is a multisystemic disease characterized by inflammation of small vessels, which often affects the kidneys. Treatment consists of immunosuppressive therapy, which is more intense during the induction phase and is subsequently reduced in the maintenance phase of disease remission. The optimal duration of maintenance treatment has not been established by randomized controlled trials, although clinical guidelines recommend at least 24 months. Given the chronic, relapsing nature of AAV, several researchers have sought to identify biomarkers that can detect subclinical disease activity and predict future relapses during the remission phase, with the goal of individualizing the treatment schedule. The kinetics of several biomarkers have been extensively studied in the acute phase and early remission phase, but little is known about long-term remission. This latter phase is of the utmost importance, as it is when the clinician must decide whether to maintain or withdraw immunosuppressive treatment. Our findings provide new insights into the behavior of these biomarkers over time, their association with systemic inflammation and kidney function, and their potential to anticipate relapse. We also investigated how immunosuppressive maintenance therapy, particularly rituximab, may influence their dynamics.

Our first objective was to assess the correlation between serum calprotectin and usCD163 during remission. We found a statistically significant positive correlation between both biomarkers, suggesting that while they may reflect overlapping inflammatory pathways, they likely represent distinct immune processes. This is consistent with previous literature reporting that calprotectin is a marker of neutrophil and monocyte activation,15 whereas usCD163 reflects macrophage activity in renal tissue and urinary compartments.16 The concept of combining biomarkers that reflect different inflammatory pathways is not new and has been explored in previous studies aiming to improve diagnostic or prognostic accuracy. In the context of AAV, where disease activity can be heterogeneous and driven by multiple immune mechanisms, using a multimarker approach may enhance the performance of individual biomarkers. Several publications, from our group and other authors, have already proposed combining biomarkers such as calprotectin, CD163, and others to capture distinct aspects of disease pathophysiology, with the goal of achieving better sensitivity and specificity in detecting subclinical activity or predicting relapse.7,17,18 Thus, the fact that both biomarkers show a positive correlation supports their combined use – not only during active disease but also during remission – where they may capture complementary aspects of subclinical inflammation.

Secondly, these biomarkers showed consistency in correlating with other markers of activity, such as CRP,19 lymphocyte count20 and ESR. Importantly, we observed that only calprotectin correlated with kidney function parameters, highlighting its possible dual role in reflecting systemic inflammation and renal involvement.

A key goal of our study was to investigate whether elevated levels of these biomarkers during remission were associated with subsequent relapse. In our cohort, patients who relapsed had higher levels of both biomarkers during remission, although only usCD163 reached statistical significance. Most existing studies on usCD163 have focused on its potential as a biomarker for early detection of disease relapse, investigating its rise shortly before renal flares. In one prospective study, only a single patient exhibited an elevation in usCD163 levels prior to the clinical onset of relapse,11 suggesting limited predictive value based on current evidence. In contrast, our study specifically examines the dynamics of usCD163 during periods of stable remission, an aspect that has been less thoroughly explored. We observed that patients who eventually experienced a relapse had slightly higher usCD163 levels already during remission, compared to those who remained in sustained remission. However, these levels still fell within the low range typically associated with remission. This finding suggests that subtle variations in usCD163 expression during remission may be associated with an increased risk of future relapse.

Our group previously investigated serum calprotectin levels in AAV patients in remission and found that higher levels were associated with worse renal outcomes during follow-up.6 These observations are consistent with findings by Romand et al.,10 which suggest that persistent elevation of calprotectin during remission may be linked to adverse renal outcomes. In the present study, we confirmed these findings by observing a positive correlation between serum calprotectin and markers of kidney dysfunction – specifically, serum creatinine and proteinuria – during remission. Although calprotectin levels did not significantly predict relapse, their association with declining renal function suggests a possible role as an early indicator of chronic disease progression or subclinical inflammation.

Most existing studies have focused on demonstrating elevated levels of serum calprotectin and suCD163 at diagnosis, where their elevation has been linked to active glomerular inflammation and relapse, and where levels tend to decrease as patients enter the remission phase of the disease.4,12,21,22 Our observations revealed a negative correlation between biomarker levels and the number of days since disease activity. This finding reflects that, despite remission is achieved and no clinical or analytical activity is detectable, the levels of these biomarkers continue to decrease over time. Our data suggest that the clinical definition of remission, based on the absence of clinical activity and a BVAS score of zero, may not fully capture the complete resolution of disease. Thus, the current definition of disease remission may be insufficient and warrants reconsideration. Incorporating information provided by new biomarkers could enhance the precision of the remission definition.

Importantly, we observed that the administration of rituximab influenced serum calprotectin levels, which were higher when the time elapsed since administration was shorter. In contrast, the administration of rituximab did not affect CD163 levels. During NETosis, neutrophil extracellular trap (NET) formation and release occur, involving the liberation of web-like structures composed of decondensed chromatin, with lytic enzymes and proinflammatory molecules – originally contained in the neutrophil cytoplasm – embedded within them. Calprotectin is one of the neutrophil cytoplasmic molecules released during NETosis.23 In an in vitro study, Hoffman et al. demonstrated that rituximab therapy induces NETosis and triggers NET formation and release24 which may help explain the findings in our study. There are no data regarding the direct effect of rituximab on macrophages, which aligns with the absence of influence of rituximab over suCD163 levels. This finding further supports the notion that calprotectin and suCD163 reflect distinct immune processes. Moreover, our results highlight a potential confounding effect when interpreting biomarker values solely in relation to disease activity, emphasizing the need to consider treatment timing when using biomarkers to monitor disease status. Therefore, we suggest that calprotectin should be used primarily in patients receiving treatments other than rituximab, where its interpretation is not compromised. In contrast, due to the stability of suCD163 levels, it may serve as a more reliable biomarker for monitoring inflammation and the immune microenvironment in patients treated with rituximab. However, it is important to note that suCD163 levels increase only in the presence of renal activity, and thus its clinical utility is limited to detecting renal relapses. Taken together, these findings underscore the importance of a multibiomarker strategy, rather than relying on a single biomarker, to achieve a more comprehensive and accurate assessment of disease activity across different organ systems and treatment contexts.

This exploratory and proof-of-concept study is based on the analysis of serial samples obtained during 100 outpatient follow-up visits from patients with AAV in remission: 17 of them treated with rituximab, and 6 treated with mycophenolate and low-dose corticosteroids as maintenance therapy. Although the number of patients recruited is relatively small and the number of relapses recorded is low – and this constitutes one of the limitations –, the strength of the study lies in the high degree of cohort homogeneity, the consistency of treatment protocols, and the structured serial sampling across an extended follow-up period. Additionally, the recruitment of control patients treated with mycophenolate allowed us to contextualize the biomarker trends observed in AAV patients receiving rituximab. This design enabled a temporal analysis of biomarker dynamics during remission – an aspect that remains novel in AAV biomarker research. The number of control cases treated with mycophenolate is limited; however, their inclusion still provides valuable comparative context, particularly given the current challenges in recruiting patients receiving immunosuppressants other than rituximab. Notably, even with a limited number of relapse events, we were able to detect meaningful differences in biomarkers between patients who later relapsed and those who remained in sustained remission, as well as significant differences in biomarker behavior depending on treatment. These findings suggest that, despite the limited sample size, the study was sufficiently powered to uncover relevant biological signals, supporting the potential utility of these biomarkers in risk stratification during remission. However, at the present moment these markers serve primarily as a complementary tool, with their broader incorporation into routine clinical practice expected to evolve as validation emerges from larger cohorts with longer follow-up periods.

The continued decline in these biomarkers over time, even after remission, the correlation between calprotectin levels and renal function, and the higher levels of biomarkers in patients who later experienced a relapse during follow-up (although only differences in suCD163 reached statistical significance), provide new insights into the chronic nature of the persistent immune activity associated with this disease. Taken together, our results reinforce the potential utility of calprotectin and usCD163 as complementary biomarkers for understanding disease dynamics in AAV. While neither marker alone clearly predicts relapse, elevated values during remission may indicate patients at higher risk, particularly when interpreted in the context of other clinical parameters. Further prospective studies in larger cohorts are warranted to validate these findings and to determine whether these biomarkers can be integrated into individualized treatment strategies to improve outcomes in AAV.

This study is supported by Sociedad Española de Nefrologia (Ayudas Sociedad Española de Nefrologia a la Investigación en Nefrologia 2022), Ministerio de Ciencia e Innovación ISCIII (PI24/01357, co-funded by FEDER funds/European Regional Development Fund – a way to build Europe) and has been funded by Instituto de Salud Carlos III through the grant CM21/00170, RD24/0004/0027, and JR21/00059 [co-funded by European Social Fund (ESF investing in your future)].

None declared.