Low serum vitamin D concentration is correlated with anemia, microinflammation, and oxidative stress in patients with peritoneal dialysis

Background Peritoneal dialysis (PD) is a form of dialysis to replace the function of kidney, that uses the peritoneum as a dialysis membrane to remove metabolites and water retained in the body. Vitamin D deficiency is prevalent in patients treated with PD. This research investigated the correlation between serum 25-hydroxyvitamin D [25(OH)D] concentration and anemia, microinflammation, and oxidative stress in PD patients. Methods 62 PD patients and 56 healthy volunteers were recruited in this research. Serum concentrations of 25(OH)D and basic parameters of anemia were detected. The correlation between serum 25(OH)D concentration with anemia, oxidative stress, and microinflammatory state were analyzed. Results In the PD group, the concentration of 25(OH)D was lower than the healthy control (HC) group (p < 0.001). Hemoglobin, red blood cell count (RBC), and total iron binding capacity (TIBC) in the PD group was significantly lower (all p < 0.001), while high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α) concentrations were significantly higher, than the HC group (all p < 0.001). In the PD group, malondialdehyde (MDA) concentration was higher than in the HC group (p < 0.001), while superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were lower (both p < 0.001). Serum 25(OH)D exhibited positive correlation with hemoglobin (r = 0.4509, p = 0.0002), RBC (r = 0.3712, p = 0.0030), TIBC (r = 0.4700, p = 0.0001), SOD (r = 0.4992, p < 0.0001) and GSH-Px (r = 0.4312, p = 0.0005), and negative correlation with hs-CRP (r = − 0.4040, p = 0.0011), TNF-α (r = − 0.4721, p = 0.0001), IL-6 (r = − 0.5378, p < 0.0001) and MDA (r = − 0.3056, p = 0.0157). Conclusion In conclusion, reduced serum 25(OH)D concentrations in PD patients contribute to anemia, oxidative stress and microinflammatory state. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-021-03077-w.


Background
Peritoneal dialysis (PD) is a form of dialysis to replace the function of kidney, that uses the peritoneum as a dialysis membrane to remove metabolites and water retained in the body [1]. Recent studies have found that as the duration of PD increases, the homeostasis of the body's internal environment is disrupted, and systemic inflammatory responses and oxidative stress gradually appear, forcing some patients to discontinue PD due to serious complications [2][3][4].
In patients, microinflammation is a state of sustained low-level inflammation, clinically manifested by the elevated levels of inflammatory factors [5]. Microinflammation is mostly mediated by intravascular inflammation caused by relative inflammatory substances, and its impact on the patient is manifested in several ways, closely related to anemia, malnutrition and low quality of life [6]. A high prevalence of various cardiovascular events is characteristic of end-stage renal diseases and is common in dialysis patients [7]. Existing studies have shown that the early intervention of microinflammatory status can inhibit cardiovascular complications and alleviate anemia and malnutrition status in hemodialysis patients [8]. However, studies on the effect of early intervention of microinflammation on PD patients are still limited.
Patients with lower serum 25-hydroxyvitamin D [25(OH)D] concentrations were treated with PD. Reasons for vitamin D deficiency in PD patients include chronic renal dysfunction, reduced sunlight exposure, restricted dietary and peritoneal effluent [9,10]. Vitamin D deficiency is reported to correlate with enhanced inflammation in stable hemodialysis patients [11].
This research aimed to explore the relationship between vitamin D deficiency and anemia, microinflammation and oxidative stress in PD patients.

Patients
In this research, 62 patients who received PD in the Affiliated Suqian Hospital of Xuzhou Medical University were selected as the PD group, and 56 healthy volunteers were recruited as the healthy control (HC) group. All patients provided written informed consent. Inclusion and exclusion criteria were shown in Additional file 1: Figure S1. This research was approved by the Ethics Committee of the Affiliated Suqian Hospital of Xuzhou Medical University.

Measurements
Blood samples were collected and stored at -80 °C. The concentration of 25(OH)D was analyzed using Roche Cobas E601 ECL analyzer (Roche, Geneva, Switzerland) and Roche Cobas Vitamin D total assay reagent (Roche Diagnostics GmbH, Mannheim, Germany). Calibration curves were constructed using calibrators provided in the kits.
Hemoglobin and red blood cell count (RBC) were measured through the UniCel DxH 600 Coulter Cellular Analysis System hematology analyzer (Beckman Coulter, Miami, FL). Total iron binding capacity (TIBC) was measured by AU5810 Chemistry Analyzers (Beckman Coulter).
Serum concentrations of high-sensitivity C-reactive protein (hs-CRP), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) were detected with high-sensitivity enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, USA). Malondialdehyde (MDA) concentration was determined through thiobarbituric acid method and superoxide dismutase (SOD) concentration was determined by pyrogallol autoxidation method, and the kits were purchased from Sichuan Vichy Biotechnology Co. Serum glutathione peroxidase (GSH-Px) concentration was determined by fluorometric assay, and the kit was purchased from Shanghai Yuanye Biotechnology Co. All the internal controls were provided by the kits.
Statistical analysis SPSS 22.0 was used for data analysis. Data were expressed as median with interquartile range or n (percentage, %). Differences for the two groups were compared using Mann-Whitney test. Proportions were compared using Chi-square (χ 2 ) test. Linear correlations were verified using the Spearman's correlation analysis. Receiver operating characteristic (ROC) analyses was employed to analyze the predictive value of serum 25(OH)D level on the state of peritoneal dialysis patients. The probability p < 0.05 was considered as the minimum condition of statistical significance.

Demographics and clinical characteristics
The demographics and clinical characteristics of the participants were shown in Table 1. Based on the results of statistical analyses, these two groups were homogenous for age, gender, body mass index, blood pressure, proportion of diabetes mellitus and cardiovascular disease, and the concentrations of serum albumin, triglycerides, and cholesterol (all p > 0 0.05). The median dialysis duration was 27 (19-39) months (Table 1). Chronic glomerulonephritis was the most frequent primary kidney disease (48.4%), followed by diabetic nephropathy (24.19%) and hypertensive nephropathy (9.7%) ( Table 1).

25(OH)D concentrations are downregulated in PD patients
In this research, we first analyzed the serum concentration of 25(OH)D in both groups. As shown in Fig. 1A, the median concentration of 25(OH)D was 15.8 (10.2-24.9) ng/mL in the PD group and 21.9 (13.8-32.2) ng/mL in the HC group. Thus, the PD group showed significantly lower 25(OH)D concentration than the HC group (p = 0.0009) (Fig. 1A). Figure 1B showed the ROC curve analysis for serum 25(OH)D. The area under the curve was 0.6683 (p = 0.0016) and cut-off concentration was 17.93 ng/mL, with 66.07% specificity and 58.06% sensitivity (Fig. 1B).

Low serum 25(OH)D concentration is correlated with anemia, microinflammation and oxidative stress in PD patients
Based on the cut-off value of serum 25(OH)D concentration, patients with PD were divided into high concentration group (> 17.93 ng/mL) and low concentration group  Table S1).

Discussion
Consistent with previous studies, we found that, compared with the healthy control group, PD patients showed lower 25(OH)D concentrations in the peripheral blood. Vitamin D deficiency can be observed in most dialysis patients and is usually associated with malnutrition [12,13]. Vitamin D has been shown to play a role in the regulation of the innate and adaptive immune systems [14,15]. Vitamin D deficiency has been shown to be associated with an increased risk of PD-associated peritonitis [12]. Intervention studies have also shown that oral vitamin D supplementation can reduce the rate of respiratory infections [16]. There is evidence supporting that 25(OH)D deficiency is also associated with an increased risk of anemia [17]. In adults with chronic kidney diseases, lower 25(OH)D concentrations are associated with lower hemoglobin concentrations and anemia, and vitamin D has also been shown to play a role in erythropoiesis [18]. It has been reported that PD patients with low serum 25(OH)D concentrations have a reduced quality of life [19]. Anemia of chronic kidney diseases is a state of anemia caused by various factors that interfere with the production and metabolism of RBC [20]. Patients with uremia have significant toxin accumulation, and most of the toxins can be effectively removed by PD [21]. However, some of the medium and large molecules may remain and inhibit the function of renal erythropoietin [22]. The inhibited synthesis of erythropoietin in the kidneys has a negative impact on the normal hematopoietic process [23], including reduced hemoglobin, RBC and TIBC [24].
The concentrations of hemoglobin, RBC and TIBC in the peripheral blood of the participants in both groups were compared. We found that, compared with the HC group, hemoglobin, RBC and TIBC concentrations in the peripheral blood were significantly lower in the PD group. Decreased anemia-related indexes in PD patients confirmed the existence of a certain degree of anemia. In patients with PD, those with high 25(OH)D concentration showed significantly higher concentrations of hemoglobin, RBC and TIBC in the peripheral blood than those with low 25(OH)D concentration. The Spearman correlation analysis further revealed that the concentrations of hemoglobin, RBC and TIBC in the peripheral blood of patients with PD were positively correlated with the concentrations of 25(OH)D, indicating that 25(OH)D concentration could reflect the severity of anemia.
The microinflammatory state refers to the stimulation of multiple inflammatory factors by toxin production and prolonged presence in the blood circulation, resulting in mild inflammation [25,26]. The microinflammatory state is commonly found in various chronic kidney diseases and may increase the risk of cardiovascular events in the long term [27,28]. Hs-CRP, IL-6 and TNF-α are molecules that are closely associated with the inflammatory response. CRP is synthesized by hepatocytes and is closely related to the process of inflammatory response, while IL-6 and TNF-α are involved in the regulation of inflammatory cell activation and infiltration during the inflammatory response [29].
We found that, compared with the HC group, serum concentrations of hs-CRP, IL-6 and TNF-α in the PD The decreased antioxidants and increased toxins can lead to enhanced oxidative stress [30]. The presence of large amounts of toxins in the body of PD patients can directly stimulate the generation of reactive oxygen species and enhance lipid peroxidation, while decreased antioxidants may also exacerbate oxidative stress [31]. The indicator of oxidative stress, MDA, and the antioxidant indicators SOD and GSH-Px, were therefore analyzed in this research.
In this study, we compared the differences in the serum concentrations of oxygenation indicators MDA and antioxidant indicators SOD and GSH-Px between the two groups. We found that, compared with the HC group, MDA concentration was higher and SOD and GSH-Px concentrations were lower in the PD group. These results indicated that there was still an oxidative/antioxidative imbalance in the PD group. In the PD group, those with high 25(OH)D concentration showed lower MDA concentration and higher SOD and GSH-Px concentrations than those with low 25(OH)D concentration. The Spearman correlation analysis further revealed that 25(OH)D concentration in PD patients was negatively correlated with MDA concentration, and positively correlated with SOD and GSH-Px concentrations. Thus, 25(OH)D concentration could indicate oxidative stress in patients.
There were some limitations in this research that should be mentioned. First, serum 25(OH)D concentrations were not monitored over an extended duration. Future research should be designed to evaluate serum 25(OH)D concentrations and the other parameters over a longer study period in PD patients. Second, the mechanism of vitamin D action in PD patients was not investigated in this research. Third, the effects of the supplementation of vitamin D on anemia, oxidative stress and microinflammatory state in PD patients should be explored in future work. Although vitamin D  19:411 has been used in the treatment of PD patients, vitamin D toxicity is a potential risk [32,33]. Several studies have investigated the influence of vitamin D3 supplementation on serum 25(OH)D concentration and indicated the safe dose of vitamin D3 ranging from 5000 to 50,000 IUs/day [34][35][36]. Plasma 25(OH)D concentrations can be increased to 30-40 ng/mL by vitamin D3 supplementation, and changes in the corresponding symptoms in patients before and after vitamin D3 supplementation should be observed and analyzed.

Conclusion
In conclusion, PD patients have shown low 25(OH) D concentration in the peripheral blood. The presence of reduced 25(OH)D concentrations in PD patients is related to anemia, oxidative stress and microinflammatory state. The supplementation of vitamin D may be a