Aim: Vascular calcification (VC) is a risk factor of cardiovascular and all-cause mortality in patients with chronic kidney disease (CKD). CKD–mineral and bone metabolism disorder is an important problem in patients with renal failure. Abnormal levels of serum phosphate and calcium affect CKD–mineral and bone metabolism disorder and contribute to bone disease, VC, and cardiovascular disease. Hypercalcemia is a contributing factor in progression of VC in patients with CKD. However, the mechanisms of how calcium promotes intracellular calcification are still unclear. This study aimed to examine the mechanisms underlying calcium-induced calcification in a rat aortic tissue culture model.Methods: Aortic segments from 7-week-old male Sprague–Dawley rats were cultured in serum-supplemented medium for 10 days. We added high calcium (HiCa; calcium 3.0 mM) to high phosphate (HPi; phosphate 3.8 mM) medium to accelerate phosphate and calcium-induced VC. We used phosphonoformic acid and the calcimimetic R-568 to determine whether the mechanism of calcification involves Pit-1 or the calcium-sensing receptor.Results: Medial VC was significantly augmented by HPi+HiCa medium compared with HPi alone (300%, p < 0.05), and was associated with upregulation of Pit-1 protein. Pit-1 protein concentrations in HPi+HiCa medium were greater than those in HPi medium. Phosphonoformic acid completely negated the augmentation of medial VC induced by HPi+HiCa. R-568 had no additive direct effect on medial VC.Conclusion: These results indicated that exposure to HPi+HiCa accelerates medial VC, and this is mediated through Pit-1, not the calcium-sensing receptor.

Previous clinical and experimental studies have indicated that magnesium may prevent vascular calcification (VC), but mechanistic characterization has not been reported. This study investigated the influence of increasing magnesium concentrations on VC in a rat aortic tissue culture model. Aortic segments from male Sprague-Dawley rats were incubated in serum-supplemented high-phosphate medium for 10 days. The magnesium concentration in this medium was increased to demonstrate its role in preventing VC, which was assessed by imaging and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) mapping. Magnesium supplementation of high-phosphate medium dose-dependently suppressed VC (quantified as aortic calcium content), and almost ablated it at 2.4 mm magnesium. The FTIR images and SEM-EDX maps indicated that the distribution of phosphate (as hydroxyapatite), phosphorus and Mg corresponded with calcium content in the aortic ring and VC. The inhibitory effect of magnesium supplementation on VC was partially reduced by 2-aminoethoxy-diphenylborate, an inhibitor of TRPM7. Furthermore, phosphate transporter-1 (Pit-1) protein expression was increased in tissues cultured in HP medium and was gradually-and dose dependently-decreased by magnesium. We conclude that a mechanism involving TRPM7 and Pit-1 underpins the magnesium-mediated reversal of high-phosphate-associated VC.

The FGF23-Klotho signaling axis is known to exert anti-aging effects via calcium-phosphorus metabolism. In mice deficient in FGF23-Klotho signaling, however, the number of splenocytes is reduced. FGF23 is expressed in both bone and spleen, with regulation of its production differing in these organs. As FGF23-Klotho signaling may play an immunological role in the spleen, splenocytes in male C57BL/6J mice were assayed for expression of Klotho or FGF23 by flow cytometry and immunohistochemistry. Cells that expressed Klotho included CD45R/B220 CD21/CD35 CD1d CD43 marginal zone B cells. These cells also expressed FGF receptor 1, indicating that Klotho-positive B cells could respond to FGF23. Plasmacytoid dendritic cells (pDCs) with CD11c CD45R/B220 CD11b CD8α were found to produce FGF23. Klotho-positive cells and FGF23-producing cells were present in close proximity to each other, suggesting that FGF23 produced by pDCs may act within a limited area. These findings indicate that FGF23-Klotho signaling could play a biological or immunological role in the spleen.

Aim: High phosphorus conditions promote vascular calcification (VC) in both chronic kidney disease (CKD) patients and experimental models. However, the composition of medial calcification has not been accurately determined, so the objective of this study was to evaluate the mineral composition of calcification in a tissue culture model, not a cell culture system. Methods: Aortic rings obtained from male Sprague-Dawley rats were incubated in serum-supplemented medium for 10 days. The inorganic phosphate (Pi) concentration of the medium was increased to induce VC, which was assessed by histology, imaging, and spectroscopy. The mineral composition of the calcification was analyzed using Fourier transform infrared (FTIR) spectroscopic imaging, scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDX) mapping. Results: The calcium content significantly increased only in aortic rings cultured for 10 days in the high-Pi medium (HiP: 3.8 mmol/L). The concentration of the phosphate transporter Pit-1 in the aortic tissue exposed to HiP was higher than that in the control incubated sections. The FTIR images and spectra indicated that PO4 3 was mostly distributed as hydroxyapatite in the medial calcification of aortic rings cultured in HiP. A small quantity of carbonate was identified. The SEM -EDX overlay map demonstrated that phosphorus and calcium simultaneously accumulated and localized in the area of medial calcification induced by exposure to HiP. Conclusion: This is the first report of accurate determination of the chemical composition of aortic medial calcification. Exposure to high Pi concentration augments aortic calcification via an increase in Pit-1, which mainly contains calcium phosphate.

There are 2 types of vascular calcification in CKD patients. One is endothelial vascular calcification, a common type of vascular calcification. Another is medial vascular calcification, a specific type that is common in CKD patients. The former is mainly associated with atherosclerosis due to hyperlipidemia, especially hypercholesterolemia. The latter CKD-specific type is called Moenckeberg's arteriosclerosis. A known risk factor for this type of vascular calcification is hyperphosphatemia. In this review article, we mainly discuss a preventive strategy for Moenckeberg type vascular calcification in CKD, primarily involving the treatment of hyperphosphatemia. Several possible modalities are considered. However, at present, dietary restriction of phosphate is not recommended so as to avoid malnutrition in CKD patients. The first consideration is the enhancement of phosphate removal by renal replacement therapy in dialysis patients. Various phosphate binder therapies can be beneficial and effective. Surgical and pharmacological parathyroidectomies are also useful for treating secondary hyperparathyroidism. Good quality bone provides a good pool of calcium and phosphate. Thus, bone protection is another option for preventing vascular calcification. Several therapeutic agents have been developed to manage osteoporosis. These trial agents may be reasonably effective in impeding the progression of vascular calcification in CKD patients. Key Messages: We should make full use of several modalities so as to completely prevent vascular calcification.