Bone remodeling serum markers in children with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune multisystem disease with high disability rate and mortality. Osteoporosis is one of the most common complications of SLE [1]. Bone remodeling is a dynamic equilibrium biological process of the interaction between osteoblast and osteoclast. When the balance of bone remodeling is disturbed, and bone resorption exceeds bone formation, it results in conditions like bone loss and osteoporosis [2].

Many childhood chronic diseases have impact on bone health, such as type 1 diabetes mellitus [3], chronic kidney disease [4], rheumatic arthritis [2], and SLE [1], involving bone remodeling degeneration. GC can contribute to bone deterioration through the suppression of bone formation and osteoclast activity. Paradoxically, GC can also contribute to reduce bone loss by the anti-inflammatory effect that may counteract the negative effect on bone [5]. Thus, more markers of bone remodeling are needed.

Receptor activator of nuclear factor-κB ligand (RANKL) and Osteoprotegerin (OPG) are a pair of important bioactive proteins in regulating the balance of bone metabolism. RANKL binds to RANK on cells of the myeloid lineage and induces diferentiation and activation of osteoclast precursors, which leads to increased bone resorption, while the apoptosis of osteoclasts was inhibited. OPG is a soluble decoy receptor for RANKL which prevents the RANKL-RANK binding and inhibits osteoclast maturation, suggesting that the ratio of RANKL/OPG plays a key role in the process of bone remodeling. A variety of cytokines, hormones, growth factors can regulate the expression of RANKL [6].

Glucocorticoid (GC) and inflammatory mediators could impact bone remodeling by OPG/RANKL/RANK signal system [2]. There have been few studies on the interactions of the OPG/RANK/RANKL system in children with lupus. In this study, we detected RANKL, OPG and 25(OH)VitD₃ in 40 children with SLE, while correlation between RANKL and other indexes were analysed, to explore the changes of bone remodeling in condition of lupus.

The vast majority of children with SLE need lifelong treatment, and the mortality is still high in young patients [7]. SLE is prone to recurrence and injury kidney, cardiovascular, nervous system and other organs, so it harms children's physical and mental health, and places a heavy burden on families and society. GC is widely used in patients with inflammatory, autoimmune and allergic diseases, which is one of the most common and irreplaceable drugs for SLE at present. More than 80% of patients with SLE need long-term maintenance treatment [8, 9]. In recent decades, the mortality of SLE patients has decreased significantly and the survival time has been greatly prolonged, mainly due to the use of GC and other immunosuppressants. The chronic complications like bone problem caused by medications and disease itself get more attention. Osteoporosis and fragility fractures occur frequently among SLE patients, including juvenile patients [1].

The ratio of RANKL/OPG plays a key role in the process of bone remodeling. Therefore, RANKL and OPG may become new sensitive biomarkers to evaluate bone metabolism in children. Many studies have shown that RANKL and OPG were changed in SLE and other immune-related diseases (Table 4). Ali et al. found that OPG, RANKL and RANKL/OPG were significantly increased in the SLE group, and OPG level was related to the activity of the disease [10]. But another study on children with SLE showed that RANKL level was not related to disease activity [11]. Therefore, our study selected the patients with disease activity controlled to reduce the interference of inflammatory activity with RANKL/OPG. The results showed that the significantly dfferences in serum RANKL and OPG concentrations between children with disease remission and normal (healthy) children. It suggested that dynamic balance regulation of bone metabolism still persisted even in the remission state, partially proved that RANKL is not related to disease activity. But the effect on bone in the active phase of the disease still needs to be further explored and clarified.

GC stimulates RANKL expression and inhibit OPG expression, which promotes osteoclast differentiation and osteolysis, while it induces osteocyte apoptosis and inhibits osteocyte generation [17]. Bone loss caused by the GC can be roughly divided into two stages, bone mineral density loss fast which is about 6 ~ 12% in the first year of treatment, and about 3% a year later [18]. At the later stages, GC increased apoptosis of osteoblasts and osteocytes, and then the expression of RANKL decreased [19], while the number of osteoclasts decreased by apoptosis and autophagy. Then bone loss was attenuate at the later stage as the disease got controlled and the GC dosage was reduced. Gupta et al. found that increased urine OPG level in active Lupus Nephritis (LN) and consequently decreased when the disease was controlled, suggesting that OPG may be increased by inflammatory stimulation, but decreased by GC [12]. Our study showed that RANKL expression was increased while OPG expression was decreased in the remission state in children with SLE, and respectively they were positively and negatively correlated with cumulative dose of GC. It proved that bone remodeling markers could be affected by GC dosage. Since the durations of the disease in our patients are all less than 2 years, longer follow-up and observation are needed. It is hard to distinguish whether the observed differences were present due to disease itself or glucocorticoids, despite exclusion of disease activity at the time of sampling. Thus, we should collect more samples of patients with SLE and other autoimmune diseases when they are not treated with glucocorticoids and dynamically detect the changes of the markers.

The correlation between BMD and RANKL/OPG levels is still inconclusive, but the correlation of increased RANKL/OPG ratio and BMD has been confirmed in many childhood diseases [13‐15, 20]. SLE may lead to decreased BMD, but the effect of RANKL and OPG on BMD in active stage of SLE is not clear. In our study, all the 40 children with SLE were in remission, so chronic inflammation in SLE might have little effect on RANKL and OPG. Meanwhile, the correlation between GC accumulation and RANKL/OPG suggested that GC played a very important role in regulating the balance of RANKL and OPG, while other blood biochemical indexes were not correlated with them. At present, the most commonly used method for BMD assessment is dual-energy (DXA). But local DXA is insufficient to reflect the condition, and the whole body DXA is difficult to perform in children because of the high dose of radiation and cooperation problem in young children. Considering that, BMD was not performed in these cases, which is a limitation and the correlation between RANKL/OPG and BMD cannot be determined. Thus, further animal and cell studies are warranted to find out the potential machanisms of the bone loss by GC in SLE model.

Vitamin D is a marker of bone metabolism, which regulates the balance of calcium and phosphorus. Vitamin D also has immunomodulatory functions, which plays an important role in both infectious and autoimmune diseases [21]. Serum 25(OH)VitD₃ level is considered to be the best reflection of vitamin D status. Low vitamin D level is a risk factor of osteoporosis. Our study also found that children in SLE with have low vitamin D levels, which were correlated with cumulative dose of GC, RANKL and OPG levels. It speculated that GC may participate in bone remodeling process in SLE mediated by the transformation of RANKL/OPG, which characterized by low levels of vitamin D status. But the specific regulatory mechanism still needs further research.

Low levels of vitamin D₃ and bone metabolic abnormalities still persist in children with SLE even if the disease is in remission, while serum RANKL level was elevated, OPG expression was reduced. These changes are associated with duration of SLE and cumulative dose of GC. It states that GC is involved in the process of the occurrence and development of bone metabolic abnormalities through RANKL/OPG in condition of disease remission.