We evaluated the prognostic and diagnostic value of circulating anti-dsDNA, anti-C1q, and anti-C3b antibodies alone or in combination with other autoantibodies in SLE patients with different kidney involvements. The results indicated that antibodies against dsDNA and C1q were more frequent in active and inactive LN groups. In this regard, the prevalence of them was higher in active LN patients. Moreover, disease activity (SLEDAI scores) was higher for the patients having these autoantibodies. Conversely, C3 and C4 levels were lower for patients having anti-dsDNA and anti-C3b antibodies, while no significant differences were observed in patients with positive antibodies against C1q. Importantly, the frequency of anti-dsDNA and anti-C1q was superior to the other antibodies among histological classes of kidney biopsies. Of note, more than 75% of SLE patients with severe LN (Class IV) have anti-dsDNA or anti-C1q antibody.
These data indicated that the evaluation of the simultaneous existence of antibodies against dsDNA and C1q could be useful as diagnostic and predictive biomarkers for SLE patients with kidney involvement. Several studies are consistent with our findings [5, 12, 18, 20]. Recently, it has been demonstrated that any deficiency in complement activation through classic and alternative pathways strongly correlated with the progression and severity of LN. Among these complement components, C1q and C3b are the most widely explored factors and have a crucial role in facilitating the clearance of immune complexes and cell residue [9, 28]. In this regard, the production of antibodies against C1q may result in the formation of circulating or local complexes of C1q and anti-C1q antibodies. These complexes may deposit in the kidneys and lead to tissue damage. Moreover, anti-C1q may induce a pathogenic condition via disrupting the clearance activity of apoptotic cells, triggering autoimmune inflammatory responses [17, 22].
Although a clinical significance has been observed between the existence of anti-C1q antibodies and LN, the diagnostic value and pathogenic role of antibodies to C1q in LN remains controversial [21, 29]. Anti-C1q autoantibodies may be found in the sera of healthy individuals and patients with other autoimmune diseases such as rheumatoid vasculitis, mixed connective tissue disease (MCTD), Felty syndrome (FS), and autoimmune thyroid disease [22, 30, 31]. Particularly, these antibodies are detected at a high titer almost in all patients with hypocomplementemic urticarial vasculitis (HUV) syndrome [32, 33]. Available data suggest that anti-C1q antibodies on pathophysiological levels are essential, but not enough for beginning the immune inflammation in the renal glomeruli since some healthy persons and patients with UHV syndrome with high levels of anti-C1q antibodies do not experience kidney involvement throughout their lives [21, 30, 33].
Furthermore, Flierman et al. indicated that animal model experiments with the injection of anti-C1q antibodies or anti-glomerular basement membrane (GMB) alone did not lead to obvious kidney disease; however, the simultaneous injection of them is necessary for inducing kidney inflammation [34]. Nevertheless, several studies have shown the deposition of anti-C1q antibodies and C1q molecules along with tubular and glomerular basement membranes [35].
In the current study, we found that antibodies to dsDNA were the most prevalent autoantibody in patients with SLE. Furthermore, this frequency was higher when patients with SLE were classified based on kidney involvement with active (71.4%) and inactive (62.5%) LN. In this context, Fabrizio and colleagues categorized patients with SLE, according to the presence of anti-dsDNA antibodies, including persistent negativity (24.4%) and positivity (62.3%) for anti-dsDNA as well as patients with initial positivity and further negativity during the disease course (13.3%). In agreement with our data, they identified that kidney involvement was significantly more frequent in patients with SLE having anti-dsDNA antibodies [6]. Moreover, we showed the C4 and C3 levels were lower in patients with the positive anti-dsDNA antibody. The formation of immune complexes as a result of autoantibodies can fix complement proteins and induce systemic inflammation responses. The kidneys are one of the major sites of these inflammatory processes because deposited anti-C1q and anti-dsDNA antibodies are enriched in kidney elutes and its levels correlate with the severity of nephritis. In summary, the unique ability of these autoantibodies to induce kidney injury alone comprises the formation and deposition of circulating immune complexes in the kidney glomeruli, binding directly to kidney antigens, and induction of inflammatory cytokine afterward [5, 36, 37]. Consequently, evaluation of the simultaneous presence of antibodies against C1q and dsDNA can enhance the predictive power of detection of patients with active LN.
Recently, a few studies have revealed that anti-Sm antibodies are associated with the early poor outcome of LN [38, 39]. Accordingly, Ishizaki et al. [39] showed high levels of autoantibodies against Sm may predict silent LN without abnormal proteinuria. Notably, in our study, we did not recognize any significant association among the frequency of antibodies against SSA, Sm, and SSA with the severity of the disease (SLEDAI), serum complement levels, and kidney involvement. A possible description for this might be that we have fewer numbers of patients with silent LN (n = 5 of class II) contrary to 26 patients with class I and II of LN in Ishizaki study.
Antibodies against C3 fragments such as C3b have been identified as immunoconglutinins (IK) and reported in patients with SLE at different levels. Rare studies have reported an association between anti-C3b antibodies and LN. In our study, although anti-C3b compared with anti-C1q was less sensitive (34% vs. 43%) for SLE, anti-C1q was more specific (77.5% vs. 75%) for active LN. Indeed, anti-C3b IgG levels inversely correlated with C3 and C4 levels. However, there was no significant difference between the patients with and without anti-C3b antibodies for SLEDAI scores.
In a few numbers of cohort studies, the prevalence of anti-C3b antibodies was reported 30 to 35 percent in SLE patients with kidney involvement [26]. However, our results showed more frequent anti-C3b antibodies in the active LN (45.7%) compared with inactive LN (35%) condition.
These antibodies targeted ‘neoepitopes’ in C3b molecules. Neoepitopes are not available in the native protein and emerge by structural alterations. Some alterations that may lead to the production of neoepitopes occur in processes such as protein activation, proteolytic cleavage, or protein binding [17]. Vasilev et al. demonstrated that these neoepitopes share among C3, C3b, iC3b, and C3c molecules. Remarkably, they showed autoantibodies also reacted with C4 molecules, indicating that common neoepitopes may exist in all of them. These neoepitopes are formed on the immobilized C3 fragments at the surface of glomeruli and result in kidney damage [26].
This study is accompanied by some limitations. First, these autoantibodies were only detected in sera, while they were not directly evaluated in the pathogenesis of LN. However, it is accepted when they bind deposited complement components or immune complexes, they may develop the inflammation in the site of kidneys. Second, we evaluated limited ethnic group and sample size, thus a multi-racial investigation with a larger-scale population study is necessary to support our data. Third, we did not discriminate different isotypes (or subclasses) among autoantibodies.Finally, our study did not include any follow-up data (serial sampling), especially the assessment of the relationship between LN treatment and autoantibody levels.