Association of High Calcitriol Serum Levels and Its Hydroxylation Efficiency Ratio with Disease Risk in SLE Patients with Vitamin D Deficiency

Vitamin D (calcidiol) deficiency in systemic lupus erythematosus (SLE) is more frequent than in healthy subjects (HS); it is associated with clinical activity and damage in SLE. Although calcidiol is considered the best indicator of the vitamin D serum status, its deficiency could not reflect its hydroxylation efficiency ratio and calcitriol serum status. This study was aimed at assessing the association of calcidiol and calcitriol serum levels and its hydroxylation efficiency ratio with the risk to clinical and renal disease activities in SLE patients. A cross-sectional study was conducted in 308 SLE and HS women; calcidiol and calcitriol serum levels were evaluated by immunoassays. SLE patients showed lower calcidiol serum levels vs. HS (21.2 vs. 24.2 ng/mL; p < 0.001). Active SLE patients presented higher calcidiol/calcitriol ratio scores vs. inactive SLE patients (2.78 vs. 1.92 pg/ng; p = 0.02), and SLE patients with renal disease activity showed a pattern of calcidiol-deficient levels (19.5 vs. 25.3 ng/mL; p < 0.04) with higher calcitriol levels (47 pg/mL vs. 41.5 pg/mL; p = 0.02) and calcidiol/calcitriol ratio scores (2.13 vs. 1.54 pg/ng; p < 0.02) compared to SLE patients without renal disease activity. Calcidiol levels were negatively correlated with calcitriol levels (r = −0.26; p = 0.001) and urine proteins (mg/dL) (r = −0.39; p < 0.01). Regarding calcitriol levels, it was positively correlated with the blood lymphocyte count (r = 0.30; p < 0.001) and negatively correlated with the glomerular filtration rate (r = −0.28; p = 0.001). Moreover, the calcitriol/calcidiol ratio was positively correlated with urine proteins (r = 0.38; p < 0.01). The calcidiol deficiency (OR = 2.27; 95% CI = 1.15-4.49; p < 0.01), high calcitriol levels (T3rd, OR = 4.19, 95% CI = 2.23-7.90; p < 0.001), and a high calcitriol/calcidiol ratio score (T3rd, OR = 5.93, 95% CI: 3.08-11.5; p < 0.001) were associated with the risk for SLE. In conclusion, a pattern of calcidiol deficiency with high calcitriol serum levels and a high vitamin D hydroxylation efficiency ratio was associated with disease risk in SLE patients.


Introduction
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by tolerance loss to nuclear self-antigens, autoantibody production, damage organs, and vitamin D deficiency [1][2][3]. The substantial reduction in clearance activity and formation of immune complexes may lead to local inflammation and damage with an aberrant expression of autoreactive T helper (Th) cells and proinflammatory cytokines, which are critical factors associated with SLE pathogenicity and lupus nephritis (LN) development [4,5].
Several studies suggest that SLE is a Th type 2 (Th2) profile-driven disease. However, in SLE, different lymphocyte subsets drives its pathophysiology. The proinflammatory Th1, Th2, and Th17 profiles positively correlated with high clinical disease activity and were described as elevated in humans and murine models [4,6]. The contributions of Th2 response to renal disease activity and LN development include the IL-6 and IL-4 production by activated basophils, which leads to autoantibody deposition in the kidney, increasing in a positive feedback loop the Th2 response, and B cell activation [4].
Currently, it has been described that the nutrients could play a crucial role in the survival, proliferation, and activation of immune cells. Vitamin D is a widely studied candidate nutrient in pathologies with an immune component [7,8]. It can be obtained from the endogenous synthesis in the epidermis by exposure to UVB light and from foods and supplements in the form of ergocalciferol (vitamin D 2 ) and cholecalciferol (vitamin D 3 ) [9].
The main metabolite used to measure the vitamin D serum status is calcidiol [25 (OH)D]. However, its active metabolite calcitriol [1α,25(OH) 2 D] exerts its effects through genomic mechanisms modulated by the nuclear vitamin D receptor (VDR)/retinoid X receptor (RXR) complex to bind to vitamin D response elements (VDRE) in target genes of several immune cells [9]. Hence, calcitriol is considered an immunomodulator that participates in the control of selftolerance and influences immune cell differentiation and production of several cytokines [1,8,10].
Calcitriol suppresses the differentiation of Th1 cells while promoting the formation of tolerogenic dendritic cells, T regulatory cells (Tregs), Th2 differentiation, IL-4 production, the anti-inflammatory status, and reduction of autoantibodies [7,11,12]. Nevertheless, the effects of calcitriol on B cells that are pivotal in SLE are still unclear because it depends on its available quantity, cytokine microenvironment, activation, and differentiation status of B cells [11].
In a biphasic dose-dependent manner, high calcitriol concentrations stimulate the plasma cell development; when B cells are terminally differentiating, induce a high IL-10 production, B cell hyperactivity, and autoantibody production associated with high clinical disease activity in SLE [5,11,13], highlighting that vitamin D could be an immunomodulatory nutrient that plays a bimodal role during physiological and pathological events.
Most studies in SLE patients have focused on evaluating the calcidiol serum levels with controversial findings of its relationship with the clinical and renal disease activities or comorbidities, and the association of calcidiol with the active metabolite calcitriol has not been previously reported in other studies conducted in SLE patients so far.
Therefore, the physiological complexity of the synthetic pathway of vitamin D suggests that the efficient regulation of vitamin D hydroxylation might be more crucial than the concentration of any D metabolite alone, which could be altered in SLE. Although calcidiol is considered the best indicator of the serum vitamin D status, its deficiency could not reflect calcitriol's serum status. According to this, the calcitriol/calcidiol ratio assessment, which theoretically represents how many picograms of calcitriol are produced per nanogram of circulating calcidiol, could be considered representative of the vitamin D hydroxylation efficiency [20]. Based on these previous findings, the aim of our study was at assessing the association of calcidiol, calcitriol, and its hydroxylation efficiency ratio with the risk to clinical and renal disease activities in SLE patients.

Participants.
A cross-sectional study was conducted in 308 women from an unrelated Mexican-mestizo population, made up of two groups: the first group was made up of 157 SLE women patients classified according to the 1997 SLE American College of Rheumatology (ACR) [21], recruited between 2017 and 2020 from the Rheumatology Department of the Hospital Civil Fray Antonio Alcalde, Guadalajara, Jalisco, Mexico. SLE patients had no recent infections, trauma, surgery, pregnancy, or systemic autoimmune conditions unrelated to SLE.
The second group was composed of 151 healthy female subjects (HS) as a reference group, recruited from the same geographical area. The HS included did not refer to recent infections, trauma, surgery, pregnancy, or autoimmune conditions; they were also asked about the presence of autoimmune diseases in their families. They did not mention that their close relatives like siblings, parents, and grandparents presented autoimmune diseases. Both study groups presented an ancestry of at least back three generations in the same geographical region.  [24,25]. For calcitriol, the reference cutoff values were 15-60 pg/ mL [26,27]. However, for the evaluated sample, we stratified the calcitriol levels classified into tertiles: (a) low calcitriol serum levels = T1 st (0.33 to <33.6 pg/mL), (b) average calcitriol serum levels = T2 nd (≥33.6 to <48.7 pg/mL), and (c) high calcitriol serum levels = T3 rd (≥48.7 to 157.3 pg/mL).

Ethical
To estimate the vitamin D hydroxylation efficiency, we calculated the calcitriol/calcidiol ratio based on values of calcitriol (pg/mL) and calcidiol (ng/mL), which resulted in arbitrary units (pg/ng) that should indicate how many pg of calcitriol is produced per ng of circulating calcidiol [20]. The calcitriol/calcidiol ratio score was also stratified into tertiles: (a) low conversion rate of calcidiol to calcitriol = T1 st (0.01 to <1.36 pg/ng), (b) average conversion rate of calcidiol to calcitriol = T2 nd (≥1.36 to ≤2.23 pg/ng), and (c) high conversion rate of calcidiol to calcitriol = T3 rd (≥2.23 to 23.6 pg/ng).
Also, the glomerular filtration rate (GFR) was estimated with the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation based on serum creatinine (mg/ dL) and using the parameters sex, race, and age, expressed in mL/min/1.73 m 2 of the body surface area [28]. The GFR (mL/min/1. 2.6. Statistical Analysis. The statistical analyses were performed with the software STATA v 15 (College Station, TX, USA) and GraphPad Prism v 8.0 (San Diego, CA, USA). The statistical power was evaluated according to the calculation of the sample size, performed with an estimated error margin of 2% with a confidence degree of 95% and an expected prevalence of serum vitamin D insufficiency and deficiency excess in SLE patients of 83-96% and 3.1-4.6%, respectively, reported in a previous study in Mexican SLE patients [30].
The Shapiro-Wilk test was used to determine the nonparametric and parametric distribution of the continuous variables. For the descriptive analysis, the nominal discontinuous variables were expressed as frequencies; the continuous variables with parametric distribution were expressed as means ± standard deviation (SD) and the nonparametric variables as medians and percentiles 5 th -95 th . For the inferential analysis, the χ 2 test was used to compare proportions. For parametric quantitative determinations of two groups, Student's t-test was used and the Mann-Whitney U test was used for nonparametric quantitative determinations. We used linear and logistic regression models to evaluate the association and contribution of the calcidiol and calcitriol serum levels as well as the calcitriol/calcidiol ratio to the clinical and renal disease activities. The differences were considered significant with a p value < 0.05.
Regarding calcitriol serum levels, SLE patients presented higher values with 46.8 pg/mL vs. 34.4 pg/mL in HS (p < 0:001) (Figure 1(b)). The calcitriol serum levels were categorized in tertiles, and we observed in the SLE patients a higher frequency in the third tertile classified as high calcitriol serum levels (T3 rd = 43%), followed by the second tertile with the average calcitriol levels (T2 nd = 37%), and the first tertile with the low calcitriol levels (T1 st = 20%), compared with HS who presented a higher frequency in the first tertile (T1 st = 47%), followed in decreasing order by the T2 nd (30%), and the T3 rd (24%) (p < 0:001) (data not shown).

Journal of Immunology Research
The vitamin D hydroxylation efficiency was estimated by the calcitriol/calcidiol ratio, and the SLE patients showed a higher score of 2.16 pg/ng, interpreted as a high-calcidiolto-calcitriol conversion rate compared to HS (1.42 pg/ng) (p < 0:001) (Figure 1(c)).

Vitamin D Serum Levels Stratified by Clinical Disease
Activity. To evaluate the association of calcidiol serum levels with the clinical disease activity, the SLE patients were stratified according to the Mex-SLEDAI score in inactive SLE patients (Mex-SLEDAI < 2) and active SLE patients (Mex-SLEDAI ≥ 2). Active SLE patients showed a higher frequency of renal disease activity (45%; p = 0:01) than inactive SLE patients (Table 1). A trend of lower calcidiol serum levels (21.2 ng/mL; p = 0:14) and a higher frequency of calcidiol deficiency (48%; p = 0:34) was observed in active SLE patients in comparison to inactive SLE patients (Table 1).
No differences were observed on calcitriol serum levels between active and inactive SLE patients. However, active SLE patients showed a trend of higher frequency of calcitriol serum levels in the third tertile (T3 rd = 50%) compared to inactive SLE patients (p = 0:26) ( Table 1).

Vitamin D Serum Levels in SLE
Regarding the evaluation of vitamin D metabolites, SLE patients with renal disease activity showed lower calcidiol serum levels (19.5 ng/mL) classified as calcidiol deficiency in comparison to SLE patients without renal disease activity (25.3 ng/mL) classified as insufficiency (p < 0:04) (Figure 2(a)). Moreover, higher calcidiol deficiency was observed in SLE patients with renal disease activity (52%) compared to SLE patients without renal disease activity (25%) (p = 0:03) ( Table 2). By contrast, SLE patients with renal disease activity showed higher calcitriol levels (47 pg/ mL) (p = 0:02) (Figure 2(b)) and a higher frequency of high calcitriol levels in the third tertile (T3 rd : 41%) (p = 0:03) compared to SLE patients without renal activity ( Table 2).

Journal of Immunology Research
About the calcitriol/calcidiol ratio, SLE patients with renal disease activity showed a higher conversion rate of calcidiol to calcitriol (2.13 pg/ng), compared to SLE patients without renal disease activity (1.54 pg/ng; p < 0:02) (Figure 2(c)).

Calcitriol Serum Levels and Vitamin D Hydroxylation Efficiency Ratio Stratified by the Calcidiol Reference Values.
Based on the previous result, we evaluated the calcitriol serum levels and the calcitriol/calcidiol ratio pattern according to the calcidiol reference values stratified in deficiency, insufficiency, and sufficiency of vitamin D.
We observed significant differences with higher calcitriol serum levels in SLE patients vs. . This pattern highlights that in a serum calcidiol deficiency, the conversion rate of calcidiol to calcitriol increases and the conversion decreases as sufficiency is achieved (Figure 3(b)).
Additionally, when we compare the SLE patients according to the presence of clinical disease activity and stratified by the calcidiol reference values, we observed higher  (Figure 3(d)).
Because renal disease activity is one of the main variables contributing to the clinical disease activity, we made the same comparative analysis about the calcitriol serum levels and the vitamin D hydroxylation efficiency scores stratified by calcidiol serum reference values. We observed that SLE patients with renal disease activity and calcidiol deficiency showed higher calcitriol serum levels (renal disease activity = 48:7 pg/mL     (Figure 3(f)). This similar pattern observed in the comparison of SLE patients with renal activity shows that the calcidiol deficiency and insufficiency are related to higher calcitriol serum levels and higher conversion rate of calcidiol to calcitriol compared to renal inactive SLE patients.
Based on the findings observed, we estimated the association of vitamin D metabolites and the calcitriol/calcidiol ratio with the risk to SLE and clinical and renal disease activities, using multiple logistic and linear regression models.

Journal of Immunology Research
Calcidiol deficiency in SLE patients has been associated with clinical disease activity and damage, with controversial findings [31][32][33]. In our study, no association between calcidiol levels with the clinical disease activity was observed; however, there was a trend of lower calcidiol serum values in active SLE patients and calcidiol serum levels were negatively correlated with calcitriol; this correlation pattern was not observed in HS. Notably, calcitriol serum levels were higher in the SLE patients compared to HS and high calcitriol serum levels in SLE patients were associated with the risk to the disease. Recently, very few studies in the literature have evaluated the calcitriol serum levels in SLE, despite being the biologically active metabolite of vitamin D [34]. However, it has been described that in a serum calcidioldeficient status, calcitriol levels are often elevated due to local calcitriol synthesis in several organs and tissues to regulate the genes related to cellular proliferation and other homeostatic healthy important functions [24]. High calcitriol levels in pathological conditions could be indicative of active inflammatory disease; in a study of patients with granulomatous diseases, high calcitriol production by inflammatory cells has been associated with the inflammatory process, and in another study in patients with early diagnoses of tuberculous pleuritis, elevated calcitriol levels have been associated with active pulmonary tuberculosis [35].
Although similar data have not been reported in SLE or patients with other autoimmune diseases, high calcitriol serum levels have been described as a risk factor in other diseases such as cancer and allergies [35][36][37][38]. The antitumor properties of calcitriol have been reported in several studies; however, the calcitriol excess may not always be beneficial, particularly in cancer [36]. Murine studies about cancer showed that excess of calcitriol favors the stimulation of the metastasis of 4T1 mammary gland carcinoma in BALB/c mice [37]; besides, another study about the effects of calcitriol on immunity in 4T1 tumor-bearing mice showed an enhancement of Th2 response and stimulation of Th17 cell differentiation, with an increased local calcitriol synthesis in M2 tumor-associated macrophages. Thus, in cancer, the calcitriol may intensify the immunosuppression of the tumor niche, contributing to the stimulation of cancer progression [36].
In patients with asthma and chronic obstructive pulmonary disease (COPD), it has been reported that inflammatory processes dysregulate the vitamin D metabolism; these patients before and after a cholecalciferol supplementation showed lower calcidiol serum levels and higher calcitriol/calcidiol ratio; moreover, the patients with asthma presented higher calcitriol levels in comparison to controls. In these diseases, the expression of TNF-α, IL-1β, and TGF-β was increased and these proinflammatory cytokines have been reported to induce expression of the enzymes CYP24A1 and CYP27B1, which are related to calcidiol and calcitriol enzymatic hydroxylation, respectively [38].
Concerning the vitamin D hydroxylation efficiency evaluated in our study by the calcitriol/calcidiol ratio, the SLE patients showed a higher ratio than the HS, representing that SLE patients are converting more calcidiol to calcitriol, like the pattern described in patients with asthma and COPD [38]. In our study, this pattern of high vitamin D hydroxylation efficiency ratio and high calcitriol serum levels was more evidently observed in calcidiol-deficient SLE patients with clinical and renal disease activities. According to our findings, Pasquali et al. reported in subjects with risk to renal disease and calcidiol deficiency a similar pattern of high calcitriol/calcidiol ratio and high calcitriol serum levels; besides conforming that the calcidiol sufficiency was reached, the calcitriol/calcidiol ratio was lower compared to those of calcidiol-deficient subjects [20], highlighting a possible compensatory regulation mechanism that promotes a higher calcitriol synthesis when the calcidiol deficiency is presented.
Regarding this, it has been described that the inflammatory state modulated by TNF-α and IFN induces via Tolllike receptors (TLRs) the upregulation of the VDR expression on the macrophage surface, inducing the activity of the CYP27B1 enzyme related to the conversion of calcidiol to calcitriol in situ, which has a costimulatory effect on lymphocyte polarization [5,24,39,40]. This event might support that the immune cells follow a biphasic dose response of several cytokines and nutrients, as suggested for calcitriol concentrations [41]. In a murine model of experimental autoimmune encephalomyelitis (EAE), a moderate diet in vitamin D (1500 IU/kg food) decreased the severity of EAE, while high doses of cholecalciferol supplementation (75000 IU/kg food) enhance activation, proinflammatory differentiation of T cells, and phagocytic activity of peripheral myeloid antigen-presenting cells, characterized by an enhanced surface expression of MHC class II and costimulatory molecules CD40, CD80, and CD86 [42]. Additionally, mice with high vitamin D levels showed in the central nervous system higher IFN-γ and IL-17 levels with lower frequency of Treg cells [42,43]. Hence, the beneficial effect of vitamin D may be achieved at moderate doses.
Some studies have reported that calcitriol administration may prevent strong Th1 responses, whereas its effects on Th2 cells are more complex and not fully elucidated [11,44,45]. Also, as previously mentioned, a possible biphasic effect of vitamin D on allergies has been described; vitamin D deficiency may be related to allergic reactions; however, it has been hypothesized that the vitamin D excess leads to an increased allergy risk with a Th2 response predominance and high production of specific IgE against the allergen by the B cells, resulting in an acute inflammatory response [44,45], maybe modulated by the pattern of high calcitriol when the calcidiol deficiency is presented. Notably, in SLE, a Th2 response and activation of B cells have classically been associated with the pathophysiology of the disease.
The high calcitriol serum levels observed in our study could be promoting an increase in the Th2 cell response in the SLE patients. A subtle balance between Th1 and Th2 cells is provided under normal conditions. Nevertheless, in autoimmunity, a strong Th2 predominance leads to pathologic conditions [45]. Several studies suggest that SLE is a Th2 profile-driven disease [4,6] characterized by IL-4, IL-5, IL-6, IL-10, and IL-13 cytokines. In murine models, IL-4 promoted the survival of autoreactive B cells and the high expression of IL-5 promotes the proliferation and differential signaling of self-antigen-activated B cell [5]. Regarding IL-6 and IL-10, these cytokines have been associated with high clinical disease activity in SLE, while IL-13 induces the expression of IgE, IL-6, and surface antigen such as CD23, CD71, and MHC II, which together could contribute to the exacerbation of the disease [5].
This pattern of low calcidiol and high calcitriol serum levels observed in our study in SLE patients could be influenced by a high polyclonal B cell activation in active SLE patients. The activation of human B cells induces the CYP27B1 enzyme expression; these cells secrete large amounts of calcitriol, enhancing IL-10 expression from activated B cells more than threefold [11,46]. According to the biphasic functions of calcitriol, IL-10 is another molecule that has a biphasic function, which could be either protective or pathologic following a concentration gradient; in particular, high IL-10 serum levels in SLE are considered pathogenic. Increased IL-10 production induced by calcitriol may promote the B cell differentiation into plasmablasts, promoting B cells' hyperactivity, autoantibody production, and the antibody class switch to IgA, IgG, and IgM [5,13,46,47].
Notably, in our study, the SLE patients with renal disease activity also showed the same pattern of lower calcidiol serum levels with higher vitamin D hydroxylation ratio and higher calcitriol serum levels in comparison to SLE patients without renal activity. According to this, in SLE patients from Poland, 37% of patients with renal disorder showed lower calcidiol serum levels than patients without renal disorder (p = 0:006) [48]. In another study conducted in African-American and Caucasian SLE patients, 18% of patients showed low calcidiol serum levels (<10 ng/mL) and the presence of renal disease was a predictor to lower calcidiol serum levels (OR = 13:3, 95% CI 2.3-76.7, p < 0:001) [49], while in pediatric SLE patients from the U.S., the presence of proteinuria (urine protein/creatinine ratio ≥ 0:5) was associated with a decrease of 13 ng/mL of calcidiol and 19.7-fold increased risk of deficient calcidiol serum levels ≤ 10 ng/mL (95% CI 1.8-944.5) [50]. Nevertheless, in these previous studies, calcitriol serum levels were not quantified to be able to contrast the pattern observed in our results.
In our study, high calcitriol/calcidiol ratio scores and high calcitriol serum levels were negatively correlated with  In SLE, lower calcidiol serum levels (vitamin D deficiency) increase the vitamin D hydroxylation efficiency ratio and the extrarenal calcitriol synthesis; this event may occur due to a compensatory mechanism where the immune cell system in a proinflammatory autoimmune context increases the extrarenal hyperactivity of the CYP27B1 enzyme, contributing to an increase in the calcitriol serum concentrations by calcidiol hydroxylation and promotes the exacerbation of the pathological autoimmune response, characterized by a positive feedback loop of the aberrant self-antigenic presentation, the plasma cell differentiation, autoantibody production, autoreactive Th2 polarization, tolerance loss, and damage to tissues.
the GFR in SLE patients. These observed findings highlight that the kidney is not the only source of calcitriol in patients with renal compromise, is possible that the calcitriol/calcidiol ratio could represent the extrarenal synthesis of calcitriol by immune cells [20]; this event could explain why the SLE patients with renal disease activity had higher calcitriol serum levels in comparison to inactive renal patients. Regarding this, immune cells such as monocytes, DCs, and B cells can produce calcitriol by the local CYP27B1 expression [45]. Calcitriol enhances the polarization to the Th2 phenotype [11], and the contributions of Th2 cytokines to the renal disease include the production of IL-6 and IL-4 by activated basophils that leads to autoantibody deposition in the kidney via enhanced Th2 response and B cell activation, promoting the release of Th2 cytokines such as IL-10, IL-13, and IL-6; the influx of inflammatory cells; and renal manifestation in active SLE or SLE patients with LN, compared to patients without renal disorders [4,5].
Therefore, although calcidiol is considered the best indicator of the serum vitamin D status, its deficiency could not reflect calcitriol's serum status [20]. Despite that the half-life of calcitriol in the circulation is very short (12-36 h), its concentration appears to be relatively steady because, after a negative or positive stimulus, several weeks are necessary to return to the prestimulus calcitriol serum range. This indicates that one blood measurement at a specific time point is sufficient to estimate the range in which circulating calcitriol concentrations will be seen over several weeks [44]. Hence, the implementation of the quantification of both vitamin D metabolites, calcidiol and calcitriol, together with the evaluation of the calcitriol/calcidiol ratio, could be important biomarkers in SLE to elucidate in a better way the complex biological regulation of the vitamin D serum status in the clinical disease and renal activities in SLE.
Based on our results and previous findings reported about calcitriol, we hypothesized that the pattern of high calcitriol serum levels and increased synthesis of calcitriol in calcidiol-deficient SLE patients could be due to a compensatory mechanism to regulate the low amount of available calcidiol serum, and in an autoimmune context, the increase of calcitriol serum concentration in calcidiol-deficient SLE patients that present clinical and renal activity could contribute to the exacerbation of the autoimmune response. Notably, in physiological conditions, calcitriol's beneficial effects could be achieved at moderate calcitriol concentrations presented in the calcidiol sufficiency status ( Figure 6).
According to our results, the strength of the present study was that the sample of SLE patients and HS evaluated was homogeneous in the following characteristics: all participants were female, from the same geographic area (western Mexico), classified as the Mexican-mestizo population with three ancestors in the same geographic region, and of similar age, which reduces the bias regarding environmental and genetic factors of ancestry that could influence the results. Nevertheless, our methodological constraints were that our cross-sectional study design limited us by simply showing an association between high calcitriol serum levels and the calcitriol/calcidiol ratio with SLE and renal disease activities. Therefore, we do not suggest causality because our study only provides information on a specific time point. Moreover, the other limitations of the present study were that we did not quantify the serum levels of proinflammatory cytokine associated with pathogenic Th2 response in SLE and the molecules related to vitamin D metabolism, such as cholecalciferol, PTH, and phosphorus, that could also influence the vitamin D serum levels. Furthermore, to test our hypothesis, we were not able to evaluate the expression at the mRNA, protein level, and enzymatic activity of the vitamin D hydroxylase enzymes involved in the catabolism of calcidiol such as CYP24A1 and the calcitriol renal and extrarenal synthesis such as CYP27B1 in the kidney and leucocytes from SLE patients and HS with vitamin D deficiency.
Therefore, further studies in SLE focused on these points will be necessary to perform, to assess the causality of the relationship of low calcidiol, high-calcidiol-to-calcitriol conversion rate, and high calcitriol serum levels in SLE patients with clinical and renal disease activities described in the present cross-sectional study. These future studies will help to support the clinical and translational interventions with vitamin D supplementation in subsequent studies conducted in patients with autoimmune diseases.

Conclusions
In conclusion, a pattern of calcidiol deficiency with high calcitriol serum levels and high vitamin D hydroxylation efficiency ratio was associated with disease risk in SLE patients. Tertile.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon reasonable request.

Disclosure
The funding sources were not involved in any step of the study.