Systemic lupus erythematosus (SLE) is a prototypic systemic autoimmune disease characterized by a wide array of autoantibodies, mainly against nuclear components. Autoantibody production is associated with various clinical manifestations and among these manifestations, renal involvement, that is, lupus nephritis (LN), is the most serious clinical problem predicting morbidity and mortality [1, 2]. The mechanisms underlying the pathogenesis of LN are not fully elucidated. However, LN has often been considered an inflammatory disease resulting from deposition of preformed immune complexes or binding of autoantibodies to antigens localized to glomeruli, so-called in situ complex formation [3–5]. Among the many antibodies potentially participating in the formation of immune complexes, antibodies against DNA are the hallmark of SLE. Recently, it has been shown that these DNA-containing immune complexes constitute among others, high mobility group box 1 (HMGB1), which has been suggested to be involved in binding of these immune complexes to renal tissue and initiate renal injury [6].

HMGB1 is a nuclear DNA-binding protein that resides inside the nucleus and can be released to the extracellular space under specific conditions [7, 8]. Whereas HMGB1 is actively released from lipopolysaccharide (LPS), TNF-, and IL-1 activated monocytes and macrophages, its release also occurs passively during the late phase of apoptosis as well as during necrosis [7, 9, 10]. Extracellularly, HMGB1 acts as an alarmin involved in inflammatory reactions through binding to its functional receptors, that is the receptor of advanced glycation end products (RAGE) and toll-like receptors (TLR)-2, -4, and -9 [11–14].

There is accumulating evidence that HMGB1 contributes to the pathogenesis of inflammatory and autoimmune diseases, especially SLE [15–17]. This is related to the fact that apoptotic cells accumulate in SLE and are the main source of autoantigens, including HMGB1 [7, 18]. We and others showed that serum levels of HMGB1 are elevated in SLE patients and correlate with SLE disease activity score and, inversely, with levels of the complement components C3 and C4. Moreover, we could demonstrate that serum levels of HMGB1, in particular, were increased in SLE patients with active renal disease and correlated with proteinuria [15]. The origin of the increased serum levels of HMGB1 is not known, and HMGB1 could possibly result from release from damaged and/or inflamed renal tissue. As such, HMGB1 could appear in the urine during (active) LN. In this study, we hypothesize that urinary excretion of HMGB1 reflects renal inflammatory injury in SLE. We investigated this by measuring HMGB1 levels in the urine of SLE patients and correlating this to clinical and biochemical measures of renal and systemic disease activity.

In the present study we demonstrated for the first time that HMGB1 levels are significantly increased in the urine of SLE patients especially in those with active proliferative LN. Moreover, urinary levels of HMGB1 were correlated with disease activity scores, and reversely with levels of complement C3.

LN is a serious manifestation in patients with SLE that can progress to end-stage renal failure. Despite extensive studies, the pathogenesis of LN has not been fully clarified. Recently, the role of HMGB1 has been demonstrated in autoimmunity and chronic inflammatory diseases, especially those with renal manifestations such as antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, chronic kidney disease (CKD) and SLE [15, 17, 20–25]. However, until now no study has been conducted to evaluate urinary levels of HMGB1 as a reflection of renal inflammation. Thus, in the current study we investigated urine levels of HMGB1, in comparison to serum levels, in SLE patients with and without renal involvement. In accordance with our previous study, serum levels of HMGB1 were significantly increased in active LN patients compared to patients without active renal disease and controls [15]. Similarly, urinary levels of HMGB1 were increased in patients with active LN. Of note, urinary HMGB1 levels were also detectable, but at a lower level, in patients without active LN, but not in HC. Detectable amounts of HMGB1 in the urine of patients without active LN might be explained in two ways. A possibly ongoing low-grade renal inflammatory activity could contribute to the release of HMGB1. Indeed, Zickert et al. showed that extracellular expression of HMGB1 is detectable in renal biopsies taken from quiescent SLE patients at follow up [26]. Furthermore, some patients with SLE may have negative urine findings despite histological evidence of lupus nephritis [27]. Second, increased levels of serum HMGB1 might lead to urinary excretion of HMGB1, particularly in patients with a history of LN and slight persisting proteinuria. HMGB1 has been recognized as a new autoantigen and as an important inflammatory mediator in SLE as exemplified by increased serum levels and presence of antibodies against this protein [15–17]. Both HMGB1 and anti-HMGB1 antibodies have been shown to be associated with SLE disease activity, decreased complement levels, and proteinuria [15]. In our present study however, we did not find a positive correlation of HMGB1 serum levels with proteinuria. Now a large group of patients with newly diagnosed active LN without induction therapy was included in contrast to our previous study. This may explain the discrepancy in findings between both studies. Nevertheless, the absence of a correlation in the present study is not fully explained.

Interestingly, in the current study urinary HMGB1 levels were correlated with SLEDAI, particularly in patients with active LN. Furthermore, urinary levels of HMGB1 were inversely correlated with complement levels. We were not able to determine prospectively if urine levels of HMGB1 can be used for monitoring renal disease activity in patients with active LN. Nevertheless, increased urine levels of HMGB1 might indicate that HMGB1 is an important inflammatory mediator and that urinary HMGB1 might be an additional biomarker for assessment of renal disease activity in SLE. However, more studies are needed to ascertain that urinary levels of HMGB1 are a clinically useful marker.

The exact events leading to glomerular inflammation and damage are still not known. Anti-dsDNA or anti-nucleosome antibodies, present in immune complexes, have been suggested as important players. It has been shown that HMGB1 is an important component of these circulating DNA-containing immune complexes [6]. HMGB1 is responsible for immune-inflammatory activity and for binding of DNA-containing immune complexes, containing anti-dsDNA and/or anti-HMGB1 antibodies, to renal tissue, causing injury. However, the origin of HMGB1, whether it is produced outside the kidney or locally within the inflamed kidney, is still unresolved. In the current study, the percentage of HMGB1-negative nuclei, probably resulting from HMGB1 release, was higher in renal tissue of patients with active LN compared to control renal tissue, suggesting active release of HMGB1 in pro-inflammatory processes within the kidney. Our results are in line with the findings of Qing et al. [6]. In their study, specific data on the characteristics of their LN patients were not mentioned, and levels of HMGB1 in serum were not detectable. Also, urinary levels of HMGB1 were not measured. We were not able to definitely identify the cells releasing HMGB1. Release of HMGB1 could result from infiltrating cells as indicated by immunohistochemical staining, but could also result from either activation or cell death of constitutive renal tissue. Also, we cannot exclude the possibility that at least some of urinary HMGB1 might emerge from systemic inflammation. This might explain the lack of correlation between urinary HMGB1 and HMGB1 released from nuclei in the kidney. Further support for the role of HMGB1 in renal inflammation comes from the increased expression of the HMGB1 receptors TLR2, TLR4 and RAGE in the renal tissue of LN patients. To the best of our knowledge, this is the first study describing expression of the functional receptors (TLR2, TLR4 and RAGE) of HMGB1 in lupus nephritis. The low intrinsic expression of these receptors in control tissue suggests that TLR2, TLR4, and RAGE are all part of the innate immune system in the kidney. The intense and altered expression of TLR2, TLR4, and RAGE in patients with active LN, however, could reflect a role of these receptors in the development and severity of lupus nephritis. In combination, all these data indicate that HMGB1 might be an important player in the pathogenesis of LN and, possibly, a therapeutic target. Indeed, Mao et al. demonstrated that targeting HMGB1 by monoclonal antibodies in a murine adenovirus-accelerated SLE model inhibited the development of proteinuria and proved protective [28]. Clinical studies targeting HMGB1, however, have not been performed.

The present study provides evidence that HMGB1 expression is increased not only in the sera but also in the urine of active LN patients as well as at the site of local renal inflammation. Urine levels of HMGB1 could reflect both local and systemic inflammation. Urinary biomarkers might be more relevant than serum biomarkers because they may directly reflect renal pathology. However, further studies are needed to evaluate the clinical significance of measuring urinary HMGB1 and its value as a biomarker in lupus patients with renal involvement.

The present study demonstrated increase in urine HMGB1 levels in SLE patients, in particular in those with active LN. Increase in urine HMGB1 levels correlated to SLE disease activity index (SLEDAI). Accordingly, renal tissue of LN patients showed increase in nuclear HMGB1 release compared to control tissue. Furthermore, expression of TLR2, TLR4 and RAGE was increased in a renal biopsy from active LN patients. Together, we suggest that HMGB1 may play an important role in renal pathology in SLE patients.