**1. Introduction**

During chronic kidney disease (CKD), uremic retention solutes accumulate in the bloodstream due to progressive kidney function loss. Three classes of uremic retention solutes exist: (i) low-molecular-weight water-soluble solutes (<500 Da), (ii) middle-molecular-weight solutes (>500 Da), and (iii) protein-bound solutes. This latter class is characterized by a limited dialytic removal due to the high molecular weight of the protein complexes that complicates their movement across the dialysis membrane [1]. Both indoxyl sulfate (IS) and p-cresyl sulfate (PCS) belong to the protein-bound uremic toxins and originate from protein fermentation in the intestine. The intestinal microbiota facilitates the breakdown of tyrosine/phenylalanine and tryptophan into, respectively, p-cresol and indole, which are absorbed and detoxified by oxidation and conjugation with sulfate [2]. In the bloodstream, IS and PCS bind to albumin, which implies that glomerular filtration does not take

place and thus requires tubular transporter systems in the kidney to excrete these two protein-bound uremic toxins. Basolateral organic anion transporter 1 (OAT1) and 3 (OAT3), breast cancer resistance protein (BCRP) and multidrug resistance protein 4 (MRP4) belong to the IS and PCS tubular transport system [3]. Due to progressive kidney function loss, the concentration of IS and PCS increases with CKD stage in humans, as shown in Table 1 (adapted from Lin et al., *J. Food Drug Anal.*, 2019 [4]), ending up with levels of around 20-fold CKD stage 1. Moreover, free IS and PCS levels are 100-fold higher in pretreatment hemodialysis patients as compared to normal subjects [5]. There is, however, a high inter-individual variability, reflected by high standard deviations, which is also observed in other studies [6–8]. An explanation might be (i) alterations in colon microbiome attributed in part to dietary restrictions in CKD patients [9], (ii) modulation of IS and PCS transporters [10] and (iii) residual kidney function [11]. This inter-patient variability might influence the interpretation on the association between of IS and PCS concentrations and clinical outcomes. However, unbound PCS serum levels are suggested to hold a substantial predictive value for the survival in CKD patients [8]. Moreover, IS and PCS serum levels are also associated with cardiovascular disease and mortality [12,13]. Furthermore, Shafi et al. reported no association between total IS and PCS serum concentrations with cardiac death, sudden cardiac death, and first cardiovascular event [14]. These conflicting clinical associations demand for experimental study designs to unravel the role of IS and PCS in cardiovascular disease, which is crucial, as cardiovascular defects account for 50% of all deaths in CKD patients [15], having high serum IS and PCS levels.

**Table 1.** Serum levels of albumin bound (total) and unbound (free) IS and PCS.


Data represent the mean ± standard deviation.

### **2. Molecular Mechanisms by Which IS and PCS Induce Vascular Calcification**

Arterial media calcification is a life-threatening disease that manifests in elderly and patients with chronic kidney disease (CKD) and diabetes mellitus. The disease phenotype is characterized by a passive and active deposition of calcium phosphate crystals in the media layer of the arterial wall that leads to arterial sti ffness, which in turn induces hypertension, left ventricular hypertrophy, and impaired coronary perfusion. Arterial media calcification occurs already in the early stages of CKD, and more than half of the CKD patients on dialysis su ffer from it [16,17]. Moreover, calcifications in the arterial wall are also present in children with CKD [18]. Moreover, CKD patients who also su ffer from diabetes mellitus have a higher incidence of arterial calcification compared to nondiabetic hemodialysis patients [19]. Poor control of glucose levels is a predictor of arterial calcification in humans [19,20]. Our laboratory recently reported that both IS and PCS are important harmful vascular toxins, as they trigger moderate to severe arterial media calcification in CKD rats, which goes along with the activation of inflammation (i.e., acute phase response signaling pathway) and coagulation (i.e., intrinsic/extrinsic prothrombin activation pathway) pathways linked with increased circulating glucose levels and insulin resistance. These changes were even observed after four days of IS or PCS exposure, i.e., before arterial media calcifications had developed, indicating that the IS/PCS mediated upregulation of inflammation and coagulation precedes the vascular calcification process [21]. Additionally, in this study, escape from uremic-toxin-induced calcification was linked with liver X receptor and farnesoid X/liver X receptor signaling pathways, discussed more in detail below. This review focuses on these signaling pathways, as well as on the connection with endothelial dysfunction and the e ffects on microRNAs.
