Significance Statement: Vascular calcification contributes to high cardiovascular mortality in patients with CKD. Although research findings have suggested an association between the uremic toxins indoxyl sulfate and p-cresyl sulfate and cardiovascular disease, direct evidence has been lacking. In this study, the authors demonstrate in a rat model of CKD that continuous exposure to indoxyl sulfate or p-cresyl sulfate promotes moderate to severe calcification in the aorta and peripheral vessels. Activation of inflammation and coagulation pathways in the arterial wall plays a pivotal role in toxin-induced calcification and strongly associates with hyperglycemia and insulin resistance. These findings provide etiologic evidence for indoxyl sulfate and p-cresyl sulfate as major contributors to vascular calcification and suggest new avenues for identifying novel therapeutic targets to prevent or treat calcification in the vessel wall of patients with CKD. Background: Protein-bound uremic toxins indoxyl sulfate (IS) and p-cresyl sulfate (PCS) have been associated with cardiovascular morbidity and mortality in patients with CKD. However, direct evidence for a role of these toxins in CKD-related vascular calcification has not been reported. Methods: To study early and late vascular alterations by toxin exposure, we exposed CKD rats to vehicle, IS (150 mg/kg per day), or PCS (150 mg/kg per day) for either 4 days (short-term exposure) or 7 weeks (long-term exposure). We also performed unbiased proteomic analyses of arterial samples coupled to functional bioinformatic annotation analyses to investigate molecular signaling events associated with toxin-mediated arterial calcification. Results: Long-term exposure to either toxin at serum levels similar to those experienced by patients with CKD significantly increased calcification in the aorta and peripheral arteries. Our analyses revealed an association between calcification events, acute-phase response signaling, and coagulation and glucometabolic signaling pathways, whereas escape from toxin-induced calcification was linked with liver X receptors and farnesoid X/liver X receptor signaling pathways. Additional metabolic linkage to these pathways revealed that IS and PCS exposure engendered a prodiabetic state evidenced by elevated resting glucose and reduced GLUT1 expression. Short-term exposure to IS and PCS (before calcification had been established) showed activation of inflammation and coagulation signaling pathways in the aorta, demonstrating that these signaling pathways are causally implicated in toxin-induced arterial calcification. Conclusions: In CKD, both IS and PCS directly promote vascular calcification via activation of inflammation and coagulation pathways and were strongly associated with impaired glucose homeostasis.

Sclerostin is a negative regulator of the Wnt/β-catenin signaling and is, therefore, an important inhibitor of bone formation and turnover. Since ectopic vascular calcification develops in a similar way to bone formation, one might reasonably attribute a role to sclerostin in this pathological process. Ectopic calcification, especially vascular calcification, importantly contributes to mortality in elderly and patients with diabetes, osteoporosis, chronic kidney disease (CKD) and hypertension. The central players in this ectopic calcification process are the vascular smooth muscle cells that undergo dedifferentiation and thereby acquire characteristics of bone-like cells. Therefore, we hypothesize that depletion/de-activation of the Wnt/β-catenin signaling inhibitor sclerostin, may promote the development of ectopic calcifications through stimulation of bone-anabolic effects at the level of the arteries. We investigated the role of sclerostin (encoded by the Sost-gene) during vascular calcification by using either Sost^-/- mice or anti-sclerostin antibody. Sost^-/- and wild-type mice (C57BL/6J background) were administered an adenine-containing diet to promote the development of CKD-induced vascular calcification. Calcifications developed more extensively in the cardiac vessels of adenine-exposed Sost^-/- mice, compared to adenine-exposed WT mice. This could be concluded from the cardiac calcium content as well as from cardiac tissue sections on which calcifications were visualized histochemically. In a second experiment, DBA/2J mice were administered a warfarin-containing diet to induce vascular calcifications in the absence of CKD. Here, warfarin-exposure led to significantly increased aortic and renal tissue calcium content. Calcifications, which were present in the aortic medial layer and renal vessels, were significantly more pronounced when warfarin-treatment was combined with anti-sclerostin antibody treatment. This study demonstrates a protective effect of sclerostin during vascular calcification.

The protein-bound uremic toxins, indoxyl sulfate (IS) and p-cresyl sulfate (PCS), are considered to be harmful vascular toxins. Arterial media calcification, or the deposition of calcium phosphate crystals in the arteries, contributes significantly to cardiovascular complications, including left ventricular hypertrophy, hypertension, and impaired coronary perfusion in the elderly and patients with chronic kidney disease (CKD) and diabetes. Recently, we reported that both IS and PCS trigger moderate to severe calcification in the aorta and peripheral vessels of CKD rats. This review describes the molecular and cellular mechanisms by which these uremic toxins induce arterial media calcification. A complex interplay between inflammation, coagulation, and lipid metabolism pathways, influenced by epigenetic factors, is crucial in IS/PCS-induced arterial media calcification. High levels of glucose are linked to these events, suggesting that a good balance between glucose and lipid levels might be important. On the cellular level, effects on endothelial cells, which act as the primary sensors of circulating pathological triggers, might be as important as those on vascular smooth muscle cells. Endothelial dysfunction, provoked by IS and PCS triggered oxidative stress, may be considered a key event in the onset and development of arterial media calcification. In this review a number of important outstanding questions such as the role of miRNA’s, phenotypic switching of both endothelial and vascular smooth muscle cells and new types of programmed cell death in arterial media calcification related to protein-bound uremic toxins are put forward and discussed.

Sclerostin is a well-known inhibitor of bone formation that acts on Wnt/β-catenin signaling. This manuscript considers the possible role of sclerostin in vascular calcification, a process that shares many similarities with physiological bone formation. Rats were exposed to a warfarin-containing diet to induce vascular calcification. Vascular smooth muscle cell transdifferentiation, vascular calcification grade, and bone histomorphometry were examined. The presence and/or production of sclerostin was investigated in serum, aorta, and bone. Calcified human aortas were investigated to substantiate clinical relevance. Warfarin-exposed rats developed vascular calcifications in a time-dependent manner which went along with a progressive increase in serum sclerostin levels. Both osteogenic and adipogenic pathways were upregulated in calcifying vascular smooth muscle cells, as well as sclerostin mRNA and protein levels. Evidence for the local vascular action of sclerostin was found both in human and rat calcified aortas. Warfarin exposure led to a mildly decreased bone and mineralized areas. Osseous sclerostin production and bone turnover did not change significantly. This study showed local production of sclerostin in calcified vessels, which may indicate a negative feedback mechanism to prevent further calcification. Furthermore, increased levels of serum sclerostin, probably originating from excessive local production in calcified vessels, may contribute to the linkage between vascular pathology and impaired bone mineralization.

Arterial calcification, the deposition of calcium-phosphate crystals in the extracellular matrix, resembles physiological bone mineralization. It is well-known that extracellular nucleotides regulate bone homeostasis raising an emerging interest in the role of these molecules on arterial calcification. The purinergic independent pathway involves the enzymes ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs), ecto-nucleoside triphosphate diphosphohydrolases (NTPDases), 5′-nucleotidase and alkaline phosphatase. These regulate the production and breakdown of the calcification inhibitor—pyrophosphate and the calcification stimulator—inorganic phosphate, from extracellular nucleotides. Maintaining ecto-nucleotidase activities in a well-defined range is indispensable as enzymatic hyper- and hypo-expression has been linked to arterial calcification. The purinergic signaling dependent pathway focusses on the activation of purinergic receptors (P1, P2X and P2Y) by extracellular nucleotides. These receptors influence arterial calcification by interfering with the key molecular mechanisms underlying this pathology, including the osteogenic switch and apoptosis of vascular cells and possibly, by favoring the phenotypic switch of vascular cells towards an adipogenic phenotype, a recent, novel hypothesis explaining the systemic prevention of arterial calcification. Selective compounds influencing the activity of ecto-nucleotidases and purinergic receptors, have recently been developed to treat arterial calcification. However, adverse side-effects on bone mineralization are possible as these compounds reasonably could interfere with physiological bone mineralization.