Flow Cytometry of CD14, VDR, Cyp27 and Cyp24 and TLR4 in U937 Cells

Background

Chronic Kidney Disease (CKD) is characterized by the failure filtration rate resulting in serum accumulation of toxic substances, called uremic toxins (Vanholder et al., 2003). It has been shown that uremic toxins induce an inflammatory response in patients with CKD (Rossi et al., 2014; Borges et al., 20 16; Kaminski et al., 2017). Several studies have been reported a relationship between uremic toxins as indoxyl sulfate, p-cresyl sulfate, and indole-3-acetic acid with inflammation (Rossi et al., 2014; Lau et al., 2018; Onal et al., 2019). These toxins signalize NF-ĸB, oxidative stress, upregulated cytokines, intercellular adhesion molecule-1 (ICAM-1) (Bolati et al., 2013; Shimizu et al., 2013; Stockler-Pinto et al., 2018), and induce endothelial injury, contributing to endothelial dysfunction (Carmona et al., 2017). In addition, with the loss of renal function, there is no synthesis of the 1α-hidroxilase enzyme that converts calcidiol (25(OH)D/inactive form of vitamin D) to calcitriol (1,25(OH)₂D–active form of vitamin D). The uremic toxins and deficiency of calcitriol have been described to contribute to inflammation in this population (Franca Gois et al., 2018). The calcitriol has been described as anti-inflammatory (Tsoukas et al., 1984; Mathieu and Adorini, 2002). However, in the last 20 years, it has been reported that there is an extra renal production of this vitamin, mainly in monocytes (Stumpf et al., 1979; Schwartz et al., 1998; Cross et al., 2001; Holick, 2007, Gombart et al., 2007). The monocytes have the enzyme 1α-hydroxylase (CYP27) that convert calcidiol (25(OH)D–inactive form of vitamin D) to calcitriol (1,25(OH)₂D) and 24 hidroxilase (CYP24) that regulate 1,25(OH)₂D over production. The extrarenal synthesis of calcitriol might be linked to autocrine and paracrine biologic regulatory mechanisms, mainly in cells of the immunologic system (Aranow, 2011; Holick, 2012). Besides, the monocytes have TLR-4 and TLR-2 receptors that recognize antigens and activation of these receptors increased expression of VDR (vitamin D receptor) and CYP27, both regulate the conversion of inactive to active form of vitamin D (Kreutz et al., 1993).

As uremic toxins and hypovitaminosis D are important in inflammatory state in CKD environment, the aim of our study was to evaluate in vitro the effect of 25-vitamin D on TLR4, VDR, CYP27 and CYP24 in human monocyte lineage (U937). For this purpose, we evaluated TLR4 and the vitamin D intracellular mechanisms by flow cytometry.

In fact, in our previous study, we have already used this protocol and observed that 25(OH)D improved CYP27 and decreased IL-6, IFN-γ, TLR7 and TLR9 intracellular expression in lymphocytes from dialysis patients (Carvalho et al., 2017).

The VDR, CYP27 and CYP24 can be detected by other techniques such as RT-qPCR, Western-blot and immunohistochemistry as reported by Zou et al. (2019). In this study, authors investigated the effect of Shenyuan granules on the Klotho/FGFR23/Egr1 signaling pathway and calcium-phosphorus metabolism in diabetic nephropathy. Similarly, Chang et al. (Chang and Kim, 2019; Chang, 2019) investigated the effect of vitamin D on muscle cell (C2C12) proliferation and differentiation via VDR, CYP24 and CYP27 by RT-qPCR.

Although RT-qPCR, Western Blot and Immunofluorescence are techniques with high sensitivity and specificity to assess the expression of the TLR4, VDR, CYP27 and CYP24 gene, these are laborious techniques, requiring several stages for their performance (which can contribute to the failure of the techniques), require a large number of cells, high cost compared to the protocol described in the present study using flow cytometry, which is less laborious and easy to perform. Besides CKD patients, this protocol can also be applied in other researches and clinical areas such as oncology, auto immune disease, immune response, since vitamin D may modulate several cell types as lymphocytes that expression VDR, CYP27 and CYP24.