Increasing the Magnesium Concentration in Various Dialysate Solutions Differentially Modulates Oxidative Stress in a Human Monocyte Cell Line

Abstract

es (M.A.) † These authors share senior authorship.Abstract: Oxidative stress is exacerbated in hemodialysis patients by several factors, including the uremic environment and the use of dialysis fluids (DFs). Since magnesium (Mg) plays a key role in modulating immune function and in reducing oxidative stress, we aimed to evaluate whether increasing the Mg concentration in different DFs could protect against oxidative stress in immunocompetent cells in vitro. Effect of ADF (acetate 3 mM), CDF (citrate 1 mM), and ACDF (citrate 0.8 mM + acetate 0.3 mM) dialysates with Mg at standard (0.5 mM) or higher (1, 1.25, and 2 mM) concentrations were assessed in THP-1 monocyte cultures. Reactive oxygen species (ROS) and malondialdehyde (MDA) levels were quantified under basal and uremic conditions (indoxyl sulfate (IS) treatment). Under uremic conditions, the three DFs with 0.5 mM Mg promoted higher ROS production and lipid damage than the control solution. However, CDF and ACDF induced lower levels of ROS and MDA, compared to that induced by ADF. High Mg concentration (1.25 and/or 2 mM) in CDF and ACDF protected against oxidative stress, indicated by reduced ROS and MDA levels compared to respective DFs with standard concentration of Mg. Increasing Mg concentrations in ADF promoted high ROS production and MDA content. Thus, an increase in Mg content in DFs has differential effects on the oxidative stress in IS-treated THP-1 cells depending on the dialysate used. renal function and the prevalence of cardiovascular events through altered leukocyte activation and intensified oxidative stress [3][4][5][6][7].Classically, UTs are divided into three categories: low-molecular-weight water-soluble solutes (<500 Da), middle-molecular-weight solutes (>500 Da), and protein-bound solutes [8]. These uremic toxins, represented by IS, is extremely toxic and difficult to eliminate by conventional dialysis; consequently, high IS levels may trigger oxidative-inflammatory stress, playing a key pathophysiologic role in CKD. In fact, several studies have confirmed that the occurrence of CVD induced by CKD is closely related to the IS accumulation [3].Uremia promotes a state of enhanced oxidative stress, which is the result of pro-oxidant molecules, such as reactive oxygen species (ROS), overwhelming the antioxidant defense mechanisms [9]. An increase in oxidative stress occurs during the dialysis session owing to the loss of antioxidants (e.g., uric acid, etc.) and the activation of leukocytes, which trigger both inflammatory compounds and ROS production, contributing to oxidative damage of biomolecules (e.g., lipid peroxidation, DNA damage, etc.) [3,[9][10][11][12].Clinical and experimental evidence demonstrates that several factors inherent to the dialysis procedure itself, such as dialysis modality, iron administration, biocompatibility, and types of dialyzer membrane and dialysates, can play a key role promoting chronic inflammation and oxidative stress in HD patients [9][10][11][12]. Bio-incompatibility during HD induces leukocyte activation...

A high magnesium concentration in citrate dialysate prevents oxidative stress and damage in human monocytesin vitro

“…Intracellular ROS production was measured in THP-1 monocytes using a method [ 30 ] based on the fluorescence emitted by 2′,7′-dichlorodihydrofluorescein (DCF-DA, Sigma-Aldrich) which is oxidized in the cytoplasm by ROS to 2′,7′-dichlorofluorescein, with several modifications [ 25 ]. Briefly, THP-1 cell suspension adjusted to 0.2 × 10 6 cells/well in the different DFs or control solution were loaded into 96-well plates (Nunclon Delta, Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 30 min (37°C) with or without LPS (1 μg/mL, Escherichia coli serotype 0127: B8 endotoxin, Sigma-Aldrich).…”

“…Both reduced (GSH) and oxidized (GSSG) glutathione levels were quantified using a fluorimetric method [ 33 , 34 ], with several modifications [ 25 ]. This assay was carried out on frozen THP-1 monocytes (3 × 10 6 cells) aliquots, which were previously cultivated in CDF or control solution with different Mg concentrations (0.5, 1, 1.25 and 2 mM) and incubated (4 h; 37°C; 5% CO 2 ) with or without LPS (1 μg/mL).…”

“…In all experiments, the viability of THP-1 monocytes was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay kit (Sigma-Aldrich) [ 25 ]. THP-1 cells (0.2 × 10 6 cells/well) in 96-well, flat-bottomed plates were incubated in ADF, CDF or control solution with different Mg concentrations (0.5, 1, 1.25 and 2 mM) and then stimulated or not with LPS (1 μg/mL) for 4 h. Later, the solutions were replaced with MTT solution (0.5 mg/mL) for 4 h (37°C; 5% CO 2 ).…”

“…A recent multicentre, randomized, prospective study has described Mg reduction in relation to the use of CDF [ 24 ]. Several investigators suggested a CDF formulation with an Mg concentration higher than usual (0.5 mM), because higher Mg levels might be beneficial or indeed needed when using 1 mM citrate concentrates [ 18 , 25 ]. Furthermore, the increase of Mg concentration in DFs such as in ADF is reportedly associated with a decrease of intra- and post-dialysis arrhythmias [ 26 ], the serum’s propensity to inhibit calcification [ 27 ] and cardiovascular mortality in HD patients [ 28 , 29 ].…”

“…Furthermore, the increase of Mg concentration in DFs such as in ADF is reportedly associated with a decrease of intra- and post-dialysis arrhythmias [ 26 ], the serum’s propensity to inhibit calcification [ 27 ] and cardiovascular mortality in HD patients [ 28 , 29 ]. Therefore, higher Mg concentrations in DFs could be a potential mechanism to modify oxidative–inflammatory stress in HD patients [ 25 ]. However, more studies are necessary to reinforce clinical translation in this field.…”