**5. Mechanisms of vascular calcification**

**Figure 6.** Mechanism of stone growing on Randall's plaque. The plaque appears in the interstitial tissue within the renal papilla, with no crystals present in any tubular lumens. The plaque is composed of calcium phosphate (CaP) in the mineral form of apatite. Papillary epithelium is lost, and the plaque can be exposed to urinary fluid in the renal calyx. The resulting calcium oxalate stone may grow and the plaque keeps the stone from flowing out with the urine, and the insolubility of the calcium oxalate makes the stone quite. Stones that are formed on Randall's plaques are released from

In the last decade, some researchers have attempted to clarify the effects of high oxalate and crystal concentrations on the biology of renal tubular cells because the exact role of the tubu‐ lar cells in response to the influx of these potentially precipitating ions is still uncertain.

A role in the pathogenesis of Randall's plaques has also been suggested for interstitial cells capable of transdifferentiating along the bone lineage, leading to the hypothesis that neph‐ rocalcinosis could be an osteogenic cell‐driven process, similar to that of vascular calcifica‐ tion [64, 165–168]. Tubular epithelial cells have a well‐known ability to differentiate into cells with the mesenchymal phenotype (for instance, renal interstitial myofibroblasts may origi‐ nate from renal tubular cells undergoing epithelial‐mesenchymal transformation) [169]. This capacity for differentiation is not exclusive to renal cells, or epithelial cells. It is shared with

**4. Cell‐driven calcification: the example of vascular calcification**

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

the papilla in the renal calyx.

Calcification may involve both osteogenic and chondrogenic differentiation. In humans, it is primarily osteogenic (with bone tissue formation), whereas in mice it is primarily chon‐ drogenic (with cartilage formation). Although osteoblasts and chondroblasts are distinct cell types, they have substantial similarities in mineralization mechanisms and gene expression, leading to the formation of a complex and highly structured extracellular matrix, which can also be found in the calcified vasculature.

There is evidence to indicate that the proteins controlling bone mineralization are involved in regulating vascular calcification as well. Many key bone formation regulators and bone structural proteins, including pro‐osteogenic factors like the bone morphogenetic proteins (BMP) [171–186], and inflammatory mediators such as tumor necrosis factor‐α (TNF‐α), are expressed in atherosclerotic plaques as well as during the osteogenic differentiation of VSMCs. These factors can induce calcification via Msx2 and Wnt signaling, which plays a crucial part in the commitment of pluripotent mesenchymal cells, activated during vascular calcification [198–202], and they have been implicated in the regulation of osteoblastic VSMC transdifferen‐ tiation [203, 204]. Wnt signaling induces an upregulated expression of the transcription factors Cfb1/Runx (core‐binding factor subunit1α/runt‐related transcription factor 2) and osterix [177, 178, 198–200, 205–207]. In turn, Runx2 increases the expression of the bone‐related proteins osteocalcin (OCN), sclerostin, and receptor activator of nuclear factor‐kappa β ligand (RANKL) [208]. Downstream from Runx2, osterix increases the expression of other bone‐related pro‐ teins, including bone sialoprotein, alkaline phosphatase (ALP) [206, 209, 210], OPN [211–213], matrix γ‐carboxyglutamic acid protein (MGP) [214], and osteoprotegerin (OPG) [215].

The cellular and systemic conditions that permit VSMC differentiation to osteoblast‐like cells are multifactorial. At cellular level, procalcifying conditions may occur because of the factors that increase cellular stress responses. Similarly, systemic factors, such as a loss of circulating inhibitors of calcification, or changes in levels of hormonal regulators of calcium and phos‐ phate homeostasis can also facilitate VSMC differentiation and vascular calcification.

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 from initiating and propagating calcification.

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

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.

marrow may have an active role in vascular calcification. A circulating immature bone‐mar‐ row‐derived cell population has been identified, and a small subset of this bone marrow population reportedly possesses bone‐forming properties *in vitro*. Under the influence of che‐ moattractants (released by damaged endothelium, for instance), these cells may home in on diseased arteries. Under pathological conditions such as an imbalance between promoters and inhibitors of vascular calcification, this population may undergo osteogenic differentiation in the lesions, promoting vessel mineralization [220]. In another study, it has also been claimed that multipotent vascular stem cells (MVSC) in the blood vessel wall might differentiate into osteoblast‐like cells [221]—though this theory remains highly controversial for the time being.
