**7. Evidence of cell‐driven renal calcification**

It is worth considering the possibility of ectopic renal calcification being an osteogenic‐like process. Evidence to support the notion that resident renal cells could be prompted to trans‐ differentiate, or differentiate along an osteogenic lineage, comes from the following observa‐ tions. Madin‐Darby canine kidney (MDCK) cells grown in monolayers directly on a plastic dish, or a dish coated with collagen gel, developed small blisters/domes/nodules after 21 days that became more prominent after 30 days [269, 270]. Microscopic examination showed that the nodules were CaP crystals. MDCK cells grown in agar produced spherical colonies in which layers of epithelial cells, with their apical surface on the outside, enclosed CaP crystal deposits on the basal side of the epithelium [90, 93, 270–272].

Kumar et al. [71] found that rat inner medullary collecting duct cells grown in a calcifying medium formed calcifying nodules that were positive for typical bone proteins. Miyazawa et al. [273, 274] reported finding that CaOx crystals upregulated vimentin (VIM) in normal rat kidney proximal cells and that other genes, such as OPN, fibronectin (FN), cathepsins B and L, and mitogen‐activated protein kinase, related to the pathogenesis of stone formation. Using MDCK cells grown for 28 days in the presence of 10 mM β‐glycerophosphate, Azari et al. identified a mineralization process with an increased ALP activity and the presence of small aggregates of hydroxyapatite crystals within membrane‐bounded vesicles [275]. Other related osteogenic genes (RUNX1 and 2, osterix, BMP2 and 7, bone morphogenetic protein receptor 2, collagen, OCN, osteonectin (ON), OPN, MGP, OPG, cadherins, FN, and VIM) were found upregulated in the kidney of hyperoxaluric rats [276, 277]. Khan et al., again, showed a pronounced expression of MGP, together with that of collagen, OPN and FN, in renal medul‐ lary peritubular vessels of hyperoxaluric rats [111], confirming that the tubular epithelial cells of hyperoxaluric kidneys acquired a number of osteoblastic features, and suggesting a dedif‐ ferentiation of epithelial cells to the osteogenic phenotype [278].

Mezzabotta et al. were the first to provide evidence of human renal cells transdifferentiating into an osteogenic‐like phenotype, producing CaP deposits [64]. They found spontaneous instances of calcification phenomena in primary papillary renal cells derived from a patient with medullary sponge kidney (MSK) and medullary nephrocalcinosis, who carried a muta‐ tion in the GDNF gene. To investigate whether this spontaneous mineralization was merely a physicochemical phenomenon or a well‐organized biomineralization process, they searched for any sign of the bone mineralization machinery being expressed in the cells. They found the cells positive for osteogenic markers such as ON, ALP, collagen I, laminin and Runx2, and weakly positive for OCN, but negative for OPN (a known inhibitor of crystal forma‐ tion). The upregulation of ON and downregulation of OPN were also demonstrated at mRNA level. Investigating which cells were the main actors behind the phenomenon observed, the authors found that the cells were mesenchymal stroma cells (MSCs), which are very similar to pericytes. The microvasculature of the renal papilla is particularly rich in pericytes, which regulate microvascular integrity in the peritubular capillary network and give the descending vasa recta its contractile function [279]. Thus, like VSMCs, papillary MSCs associated with the perivascular niche may be capable of driving an osteogenic process under certain conditions.

In the same paper, the Authors demonstrated that human renal tubular HK‐2 cells exposed to an osteogenic medium displayed the ability to produce Ca2 PO4 by regulating the ON/OPN ratio in favor of ON.

Overall, all these very interesting data underscore that renal cells may acquire an osteoblast‐ like phenotype, and that a process very similar to vascular calcification may have a role in the development of human nephrocalcinosis.
