**6.1. Phosphate**

Osteogenic differentiation of VSMCs is prevented under normal conditions by physiological inhibitors, such as MGP, OPN, and OPG [216, 217], and regulated by monocyte‐ and mac‐ rophage‐osteoclast differentiation within the vascular wall. The growth of crystals is also hindered thermodynamically and inhibited by PPi [183, 218]. Unlike OPN, OPG, and MGP, which function in the vessel wall, fetuin A is a circulating inhibitor of calcification that has a high affinity for hydroxyapatite crystals and is thought to function by binding small CaP par‐ ticles via a domain particularly rich in acidic residues, stabilizing and clearing them to phago‐ cytes for removal [218]. *In vitro*, fetuin A inhibits the *de novo* formation of hydroxyapatite crystals, but does not affect crystals that have already formed [219]. Fetuin A also has an anti‐ inflammatory function, dampening the effects of CaP particles in neutrophil stimulation, and is responsible also in macrophage cytokine release and induction of apoptosis. Additionally, fetuin‐A has been shown to accumulate in VSMC‐derived matrix vesicles, preventing them

26 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

Given the complexity of the systems that regulate vascular calcification, it is likely that many of these factors are at work simultaneously, but in some situations the physiological balance

An alternative mechanism for vascular calcification has recently been suggested. The "circu‐ lating cell theory" [220] postulates that circulating cells coming from sources such as bone

**Figure 7.** Physiopathological mechanisms promote cellular differentiation and mineral deposition during vascular calcification. Vascular smooth muscle cells undergo differentiation to osteoblast‐like cells when exposed to different

factors. These osteoblast‐like cells participate in vascular calcification.

from initiating and propagating calcification.

is disrupted and vascular calcification can progress (**Figure 7**).

The role of phosphate in the osteoblastic differentiation process is well established [176, 177, 181, 185, 187, 222]. *In vitro*, high extracellular phosphate concentrations induce a rise in intra‐ cellular phosphate concentrations actively mediated by three types of sodium‐dependent phosphate cotransporter, of which the type III transporters Pit‐1 and Pit‐2 are ubiquitously expressed and predominant in humans. Only Pit‐1 is required for the osteogenic differentia‐ tion of VSMCs [177, 223–225]. Increasing phosphate concentrations in the VSMCs induce their phenotypic switch to osteoblast‐like cells [177, 178, 184]. In the event of renal failure, phos‐ phate plays a key part in this mechanism [165, 168]. Vascular SMCs exposed to pro‐calcifying levels of phosphate (akin to what may happen in patients with chronic kidney disease (CKD)) lose their expression of the smooth muscle contractile proteins, SM22α and SMα‐actin, and express the bone markers Runx2, OPN, OCN, and ALP instead [178].

As well as phosphate, many other factors can influence the osteoblastic‐like phenotype. A long‐term exposure of VSMCs to a variety of chronic stresses and ionic disorders (especially hyperphosphatemia and hypercalcemia), for example, can override the action of some endog‐ enous inhibitors, such as MGP, OPN, OPG and PPi [217], inducing differentiation [226]. Oxidative stress, inflammation, hormonal perturbations, and metabolic disorders can lead to vascular calcification too.

#### **6.2. Oxidative stress**

Oxidative stress and endoplasmic reticulum stress have both been implicated in vascular cal‐ cification and shown to promote smooth muscle cell (SMC) differentiation. In particular, oxi‐ dative stress generated in VSMCs by hyperlipidemia and oxidized lipoproteins, or a uremic milieu [227] prompts the expression of BMP2, Runx2 [228], and osterix, and governs Wnt sig‐ naling [207]. Reactive oxygen species (ROS) signaling can also induce other markers of osteo‐ blastic differentiation. In the vascular wall, the induction of oxidative stress can recapitulate osteogenesis in the VSMC from their undifferentiated state [229]. The role of ROS formation and signaling in vascular calcification may also reveal a link between inflammation and vascu‐ lar calcification, since inflammatory cytokines induce calcification via the Msx2/Wnt/β‐catenin pathway [202]. It has also been found that calcium deposits colocalize with inflammatory cells both *in vitro* [230, 231] and *in vivo* [232]. Mineral crystals may therefore be pro‐inflammatory per se, prompting and exacerbating the inflammation and calcification [233, 234].

#### **6.3. Hormones**

Hormones have pleiotropic effects on calcific vasculopathy. For example, the adipose‐derived factor, leptin, promotes vascular cells *in vitro* [235] and *in vivo* [236], while adiponectin‐defi‐ cient mice have increased levels of vascular calcification [237]. The influence of parathyroid hormone (PTH), which is involved in the bone turnover process, is also well known. PTH has a crucial role in calcium homeostasis, and so does PTH‐related peptide (PTHrP), and the two may function as pathological calcification mediators. Both PTH and PTHrP prevent VSMC calcification in a dose‐dependent manner by inhibiting ALP activity [238]. In addition, PTHrP is secreted from VSMCs, an action that is impaired by calcitriol (1,25‐dihydroxyvitamin D, the active form of vitamin D) [239]. PTH not only promotes the release of calcium from bone but also mobilizes salts, including bicarbonate and phosphate and impairs renal phosphate excretion, leading, for example, to advanced nephron loss in CKD patients, and thus result‐ ing in severe hyperphosphatemia [240]. Accrued high levels of serum phosphate then further stimulate the secretion of PTH, forming a vicious cycle [241]. Hyperphosphatemia increases FGF23 (a protein released by bone), which—together with its co‐receptor Klotho (a trans‐ membrane protein expressed by the kidney and blood vessels)—may also be a pathogenic factor in vascular calcification [242, 243]. Klotho maintains the balance of circulating calcium and phosphate [244]. Activation of the vitamin D receptor increases the expression of Klotho and FGF23 to promote renal phosphate excretion by downregulating the sodium phosphate transporters Slc34A1/NaPi‐2a and Slc34A3/NaPi‐2c. Intriguingly, Klotho inhibits vascular calcification by preventing VSMC differentiation while disrupting Klotho‐FGF23 signaling results in hyperphosphatemia with ectopic calcification [244, 245].

Calcitriol may also exacerbate dystrophic calcification. Vitamin D toxicity is a common ani‐ mal model used to study vascular calcification [246]. Calcitriol dose‐dependently increases both calcification and ALP activity in VSMCs [239]. In response to interferon‐γ, macrophages express 25‐hydroxyvitamin D 1α‐hydroxylase, the enzyme needed to convert 25‐hydroxyvi‐ tamin D into calcitriol [239]. Once calcitriol binds to its receptor, signaling through this path‐ way has pleiotropic effects. The vitamin D receptor influences many genes in the vessel wall, including vascular endothelial growth factor (VEGF), matrix metalloproteinase 9, myosin, and structural proteins (including elastin and type I collagen [247–250], and this explains some of the effects of calcitriol on vascular calcification.

Glucocorticoids, a class of steroid hormones with anti‐inflammatory properties, have also been shown to mediate osteoblastic differentiation and thereby promote ectopic calcification. Long‐term glucocorticoid use has been associated with osteoporosis, however, and these compounds have been shown to initiate differentiation to an osteochondrogenic phenotype in vascular cells [251, 252]. Similarly, pericytes exposed to dexamethasone exhibit a weaker expression of MGP and OPN, and an increased ALP activity and calcium deposition.
