There is increasing interest in the effects of chronic kidney disease (CKD)-mineral and bone disorder (CKD-MBD) (1). CKD is known to be associated with a bone and mineral disorder manifested by: (i) laboratory abnormalities of calcium (Ca), phosphorus (P), parathyroid hormone (PTH), and vitamin D; (ii) evidence of bone disease; and (iii) vascular calcification (Fig. 1). Vascular calcification is associated with many adverse clinical outcomes, including ischemic cardiac events and subsequent vascular mortality (2). As shown in Figure 2, the pathogenesis of vascular calcification in CKD is complex, and instead of occurring by a simple process of Ca and P precipitation, is produced by an active process in which vascular smooth muscle cells (VSMCs) undergo apoptosis and vesicle formation and are transformed into osteoblast-like cells that induce matrix formation and attract local factors that are involved in the mineralization process (3). Vascular calcification is usually seen in the elderly, after vascular injury, and in a variety of clinical conditions, including diabetes, atherosclerosis, and Monckeberg's medial sclerosis. The etiology of the complex multifactorial interactions between ageing and amplification of calcification remains uncertain. However, there is no doubt that CKD patients are at high risk of, and have a high prevalence of, vascular calcification because of multiple risk factors that induce the phenotypic transformation of VSMCs into osteoblast-like cells capable of performing the tissue mineralization process (4). Vascular calcification has been associated with numerous traditional cardiovascular (CV) risk factors such as older age, hypertension, diabetes and dyslipidemia, as well as with non-traditional CV risk factors, including hyperphosphatemia, hyperparathyroidism, hypervitaminosis D, and excessive administration of Ca salts (5). The hemodynamic consequences of vascular calcification include loss of arterial elasticity, an increase in pulse wave velocity (6), the development of left ventricular hypertrophy (7), a decrease in coronary artery perfusion, and myocardial ischemia (Fig. 3). Current strategies that are used to delay vascular calcification are focused on the correction of mineral metabolism markers of bone disease, such as P, Ca, parathyroid hormone (PTH), and vitamin D. The therapeutic drugs such as bisphosphonates (8) and cinacalcet have shown great promise, but there is an urgent need for additional clinical data. The ADVANCE study has recently demonstrated that cinacalcet plus low-dose vitamin D attenuate vascular and cardiac valve calcification in hemodialysis patients with moderate to severe secondary hyperparathyroidism (9). Cutting-edge scientific research on the mechanisms underlying vascular calcification is increasingly being undertaken, and further insight into the mechanisms may lead to the development of several types of therapeutic agents that will improve the CV outcome in CKD patients. This review article summarizes current knowledge regarding the pathogenic determinants and methods of assessing and managing vascular calcification in CKD patients.
Vascular calcification is very prevalent in patients with chronic kidney disease (CKD). In addition to having more traditional cardiovascular (CV) risk factors, CKD patients also have a number of non-traditional CV risk factors that may play a prominent role in the pathogenesis of vascular calcification. The transformation of vascular smooth muscle cells into osteoblast-like cells seems to be a key element in the pathogenesis of vascular calcification in the presence of calcium (Ca) and phosphorus (P) deposition due to abnormal bone metabolism and impaired renal excretion. Vascular calcification causes increased arterial stiffness, left ventricular hypertrophy, decreased coronary artery perfusion, myocardial ischemia, and increased cardiovascular morbidity and mortality. Although current treatment strategies focus on correcting abnormal Ca, P, parathyroid hormone, or vitamin D levels in CKD, a better understanding of the mechanisms of abnormal tissue calcification may lead to the development of new therapeutic agents that are capable of reducing vascular calcification and improving the CV outcome of CKD patients. This review article summarizes the following: (i) the pathophysiological mechanism responsible for vascular calcification; (ii) the methods of detecting vascular calcification in CKD patients; and (iii) the treatment of vascular calcification in CKD patients.
PATHOPHYSIOLOGY AND BONE-ASSOCIATED PROTEINS IN VASCULAR CALCIFICATION
The pathophysiology of vascular disease in CKD is increasingly recognized as distinct from the pathophysiology of atherosclerosis in the general population (10). Vascular calcification increases throughout ageing, and accumulation of Ca in the elastin-rich layer of the media is ≥30-times more in the thoracic aorta at 90 years of age than that at 20 years of age (11). Age-related medial elastocalcinosis in arteries is also associated with hypertension, diabetes and dyslipidemia (12). Age-related vascular calcification is specific for arteries and does not involve other soft tissues in the general population. Vascular calcification was initially viewed as a passive phenomenon, but it has subsequently been recognized as an active cell-mediated process (13–15). There are two patterns of vascular calcification. One pattern occurs in the intimal layer and the other pattern occurs in the medial layer of the vessel wall as in Monckeberg's sclerosis, which is very common in CKD patients (16), and both are associated with increased mortality in CKD patients (17). Intimal calcification is associated with inflammation and the development of plaques and occlusive lesions, while adjacent regions of the vessel wall may remain remarkably normal. This intimal form of calcification is an indicator of the advanced stage of atherosclerosis and is seen in the aorta, coronary arteries, and other large arteries. Medial calcification is characterized by diffuse mineral deposition throughout the vascular tree and can occur completely independently of atherosclerosis or together with it, and it is commonly observed in muscle-type conduit arteries, such as the femoral, tibial, and uterine arteries (13,16).
The mechanisms underlying the accelerated vascular calcification in CKD are not completely understood. Changes in the arterial wall, fibro-elastic intimal thickening, calcification of elastic lamellae, an increased extracellular matrix, and deposition of collagen and a relatively small elastic fiber content, are all well known to cause arterial remodeling in CKD patients (17). Many bone-associated proteins, including osteocalcin, osteopontin and osteoprotegerin, and many bone morphogenetic proteins, are involved in the remodeling process, and they are expressed in calcified arterial lesions and are associated with vascular calcification (18). VSMCs are the major component of the media of arteries and they can differentiate into osteoblast-like cells as a result of up-regulation of transcription factors, such as Runt-related transcription factor 2 (Runx2) and Msh homeobox 2 (Msx2), which are critical factors for normal bone development (19). This phenotypic transformation may lead to the calcification of VSMC, in a process similar to bone formation, indicating that this pattern of vascular calcification is actually ectopic ossification. Moreover, uremia induces differentiation of VSMCs into an osteoblast-like phenotype and inhibits the differentiation of monocyte macrophages into osteoclasts (20). The arteries of dialysis patients show increased alkaline phosphatase activity and increased expression of Runx2 and Osterix (Osx), which are indicative of osteogenic transformation of VSMCs (21).
METABOLISM OF Ca AND P AND VASCULAR CALCIFICATION
Numerous risk factors for vascular calcification have been reported (22) (Table 1). Several risk factors are traditional, such as older age, hypertension, diabetes, and dyslipidemia and a number of non-traditional factors, including mineral metabolism abnormalities, extremely high PTH serum levels, excess administration of calcium salts, inflammation, malnutrition, and oxidative stress have been described in CKD patients (3). Patients with advanced CKD develop hyperphosphatemia secondary to impaired renal phosphate excretion. There is strong evidence that vascular calcification is closely associated with high serum Ca and P levels and a high serum Ca × P product (16,21,23). High serum P levels can be considered a vascular toxin (24), and clinical studies have shown that patients with the poorest P control experience the most rapid progression of vascular calcification (25). Two different mechanisms of vascular calcification have been proposed to explain the relationship between Ca and P disorders and vascular calcification. Experimental studies have demonstrated that Ca plays a role in the development of vascular calcification by stimulating mineralization of VSMCs under normal P conditions (26), and when the P levels are elevated, this Ca-driven mineralization has been found to be accelerated synergistically (27). Hyperphosphatemia may directly induce vascular injury, and it indirectly stimulates osteoblastic differentiation through a type III sodium-dependent phosphate co-transporter (PiT-1). Jono et al. (28) suggested that an elevated intracellular P concentration may directly stimulate VSMCs to transform into calcifying cells by activating genes associated with osteoblastic functions. Additionally, an important recent paper (29) has reported a good animal model of CKD-related vascular calcification in which extensive arterial calcification develops only after the animals are placed on a high-P diet, suggesting that hyperphosphatemia is a powerful accelerator of this process. These findings provide strong evidence that an excess P and Ca load is probably the most important pathogenetic factor in vascular calcification.
Secondary hyperparathyroidism (SHPT) is common in CKD patients and occurs even in the early stage of the disease. Progression of CKD is associated with disorders of mineral metabolism (hyperphosphatemia and hypocalcemia), leading to the development of SHPT, which is characterized by increased serum PTH levels and parathyroid gland hyperplasia (30). High PTH levels are responsible for the increased number and activity of osteoclasts, and also responsible for the increased bone resorption in CKD. A significant decrease in serum 1,25-dihydroxyvitamin D level is observed in the early stage of CKD (31), and the decrease is attributable to renal and non-renal factors, including reduced sun exposure, impaired production of the 25-hydroxy vitamin D precursor molecule, and reduced dietary intake (32). As vitamin D deficiency progresses in CKD, the parathyroid glands become maximally stimulated, which causes SHPT (30). Low 25-hydroxy vitamin D levels affected mortality independently of vascular calcification and stiffness, suggesting that 25-hydroxy vitamin D may influence survival in CKD patients via additional pathways that need to be further explored (33). Vitamin D stimulates gastrointestinal absorption of Ca and P and induces the proliferation and osteoblastic differentiation of VSMCs. Moreover, 1,25-dihydroxy vitamin D has been shown to act as a negative hormonal regulator of the renin-angiotensin system, which plays an important role in the cardiovascular system by modulating volume and electrolyte homoeostasis (34). Although administration of supra-physiological doses of 1,25-dihydroxy vitamin D has been reported to induce vascular calcification (35), physiological doses have been found to protect against aortic calcification in animal models of CKD (36).
FIBROBLAST GROWTH FACTOR-23 (FGF-23) AND VASCULAR CALCIFICATION
FGF-23 is a novel factor produced by osteoblasts that is involved in the regulation of P and vitamin D metabolism (37). FGF-23 appears to impair the synthesis and accelerate the degradation of 1,25(OH)2D, because expression of renal 25-hydroxyvitamin D-1α-hydroxylase mRNA changes within 1 h after injecting mice with recombinant FGF-23 (38). The increased degradation of 1,25(OH)2D by 24-hydroxylase may be associated. Recombinant FGF-23 also has a phosphaturic effect, which is attributable to reduced renal P reabsorption. FGF-23 down-regulates the expression of both type IIa and type IIc sodium-P cotransporters on the apical surface of renal proximal tubular epithelial cells in vivo (38,39).
The Klotho gene is a 130-kDa transmembrane β-glucuronidase that catalyzes the hydrolysis of steroid β-glucuronides and was discovered by Kuro-o et al. in 1997 (40). The Klotho gene is expressed in a limited number of tissues, mainly the kidneys, and mutations cause multiple aging-related disorders in nearly all organs and tissues (41). Because FGF-23-KO mice exhibit phenotypes similar to those of Klotho-KO mice (42,43), a common signaling pathway has been postulated. Indeed, FGF-23 exerts its biological effects through activation of FGF receptors (FGF-Rs) in a Klotho-dependent manner, because a Klotho/FGF-R complex binds to FGF-23 with higher affinity than FGF-R or Klotho alone (44). FGF-23 has rather low affinity for its widely represented receptors and the presence of circulating Klotho is essential to facilitate the binding of FGF-23 to its receptors (45). Thus, activation of FGF-23 receptors requires not only presence of the circulating FGF-23 as their ligand, but the presence of Klotho as a specific promoter whose affinity dictates the selectivity on its targets.
Klotho is mainly expressed in the kidneys, whereas FGF-23 comes from bone cells, and this functional bone-kidney axis is of physiological and pathological relevance. Based on available knowledge, this axis seems to exert a prevailing regulation of Ca balance with Klotho and to exert a more specific and direct effect on P homeostasis through FGF-23. Both Klotho and FGF-23, linked by the receptor mechanism described above, affect vitamin D synthesis and parathyroid hormone (PTH) secretion, and both are expressed in the parathyroid glands, suggesting that FGF-23 might regulate PTH secretion. In support of this possibility, data obtained in vitro suggest that FGF-23 decreases PTH mRNA transcription and protein secretion in a dose-dependent manner (46). Conversely, PTH may stimulate FGF-23 secretion by osteoblasts, because the FGF-23 levels of rodents with primary hyperparathyroidism (HPT) are increased, which may be reduced by parathyroidectomy (47).
Both human and animal studies have shown a close association between reduced FGF-23 or Klotho activities and vascular calcification. Extensive vascular calcification is observed in Fgf23-KO mice by 6 weeks of age (48), interestingly, expression of the sodium-P cotransporter NaPi2a by their proximal tubular epithelial cells is upregulated. Moreover, the bone mineral density (BMD) of Fgf23-KO mice is strikingly reduced. Human studies have also shown an association between reduced BMD and vascular calcification (49), and low BMD has been suggested to independently predict coronary artery disease in women, with a higher odds ratio than traditional risk factors (50). In view of the phenotypes of both Fgf23- and Kloth-KO mice, it seems likely that in vivo dysregulation of the FGF-23-Klotho axis can lead to vascular calcification, possibly by affecting mineral ion metabolism (51).
INHIBITORS OF VASCULAR CALCIFICATION
Fetuin-A is principally synthesized in the liver, and circulating concentrations fall during the cellular immunity phase of inflammation (52). Fetuin-A is an extracellular calcium-regulatory protein that functions as a potent inhibitor of Ca-P precipitation (53), inhibits calcification by binding hydroxyapatite (54), and it protects VSMCs from the detrimental effects of Ca overload and subsequent calcification (55). Fetuin-A inhibits VSMC apoptosis by interfering with death-signalling pathways: (i) it is internalized by VSMCs, concentrated in intracellular vesicles, and secreted via vesicle release from apoptotic and viable VSMCs; (ii) the presence of fetuin-A in vesicles abrogates their ability to nucleate basic calcium phosphate; and (iii) fetuin-A promotes phagocytosis of vesicles by VSMCs. These observations provide evidence that the uptake of serum fetuin-A by VSMCs is a key event in the inhibition of vesicle-mediated VSMC calcification (55). In vitro, fetuin-A has been found to antagonize the antiproliferative action of transforming growth factor-β1 (TGF-β1) and block osteogenesis and Ca-containing matrix deposition in dexamethasone-treated rat bone marrow cells (53). In addition, fetuin-A-knockout mice develop extensive ectopic calcifications in the myocardium, kidney, lung, tongue and skin (53). Ketteler et al. (53) showed that a group of CKD patients who had lower serum fetuin-A levels had a higher mortality rate from CV events, suggesting that fetuin-A is involved in preventing the accelerated extra-skeletal calcification.
Matrix Gla protein (MGP) is a small ubiquitous matrix protein that was initially isolated from bone (56), and it is a key regulator of vascular calcification. To achieve full biological activity, MGP needs to be activated, and its activation depends on the availability of vitamin K (57). MGP inhibits the calcification of cartilage and blood vessels (58). MGP exerts its effects on vascular calcification directly by inhibiting Ca crystal formation, and indirectly, by influencing transcription factors that inhibit VSMC differentiation to the osteoblast-like phenotype (59). MGP also appears to be an important factor in ensuring proper differentiation of VSMCs (58). A decline in glomerular filtration rate has been shown to result in a decreased uncarboxylated MGP level which is associated with vascular calcification and atherosclerosis (60).
The receptor activator of nuclear factor κ-light-chain-enhancer of activated B-cells (RANK), and RANK ligand (RANKL), and osteoprotegerin (OPG) may be involved in regulating vascular calcification. RANKL actions are blocked by OPG that functions as a decoy receptor to prevent RANKL/RANK interactions (61). This system may play a role in bone-vascular calcification imbalance and could be a marker of the extent of vascular calcification. In a recent study, Morena et al. (62) found that, in CKD patients, coronary arterial calcification (CAC) is strongly associated with plasma OPG values. The OPG levels >757.7 pg/mL were predictive of the presence of CAC in CKD patients. These findings are consistent with those described in our previous report (63). The mechanism of the relationship between OPG levels and CAC is unknown. Understanding the functional significance of circulating OPG has been complicated by a number of factors including the relative contribution of various tissue sources and the presence of multiple comorbidities. OPG has been found to be a protective factor against vascular Ca deposition in animal models (64). Surprisingly, higher OPG levels have been reported in patients with vascular damage, suggesting that an increase in OPG level may represent a compensatory self-defense mechanism against factors that promote vascular calcification, atherosclerosis, and other forms of vascular damage (65).
ASSESSMENT OF VASCULAR CALCIFICATION
A number of non-invasive imaging techniques are available to screen for the presence of vascular calcification: plain X-rays for macroscopic calcification of the aorta and peripheral arteries; two-dimensional ultrasound for calcification of the carotid arteries, femoral arteries, and aorta; and echocardiography for assessment of valvular calcification; and CT constitutes the gold standard for quantification of coronary artery and aorta calcification.
Electron-beam CT (EBCT) and the newer multi-detector CT (MDCT) are highly sensitive methods of accurately and quantitatively assessing vascular calcification, especially CAC, which use an electrocardiographic trigger that enables imaging of the heart in diastole, thereby avoiding motion artifacts (66). These methods could be successfully used to study vascular calcifications, progressive vascular calcification, and the impact of therapy on vascular calcification (67). EBCT is not available in many hospitals, whereas almost every hospital has an MDCT and, with software adjustments to allow gated imaging, the newer faster CT can assess vascular calcification. However, there have been conflicting results in regard to the correlation between the severity of CAC measured by EBCT and subsequent clinical cardiac events in dialysis patients (68,69). The conflicting results can be explained by the fact that the arterial calcification score generated by CT scanning is a composite of both medial and intimal calcification, and the fact that it is a limitation of these CT-based imaging techniques. Unlike EBCT, MDCT can also be used to assess vascular calcification in the aorta (70,71). Conventional CT scan may be used to evaluate non-coronary vascular calcification, especially aortic calcification. The proportion of the aortic circumference that is calcified can be used as an aortic calcification index (ACI). The conventional CT method seems to be simple, relatively inexpensive, and useful for an initial diagnosis of vascular calcification. Taniwaki et al. (72) used this method to quantify aortic calcification in diabetic hemodialysis (HD) patients. Again, the ACI can not be used to quantify the medial/intimal distribution of vascular calcification.
Plain lateral-abdominal radiography is a valuable and inexpensive tool for detection of vascular calcification in CKD patients, but it is semi-quantitative, and subtle changes in the evolution of vascular calcification may be missed. Lateral abdominal radiograph can be used as an alternative to CT (73). The pattern of vascular calcification seen on plain radiographs may yield some information about the localization of the calcification within the arterial wall (intima vs. media). Kauppila et al. (74) used lateral lumbar films to detect the presence of calcification in the abdominal aortic wall, in the region corresponding to the position of the first to the fourth lumbar vertebrae. This semi-quantitative method is a more widely available and less expensive method for studying calcification and can be used for cardiovascular risk stratification.
A simple method has been introduced to evaluate the extent of aortic arch calcification (AoAC) by plain chest X-radiography in HD patients (75). Aortic arch calcification score (AoACS) was calculated as a percentage of the calcified part of aortic arch. The mean AoACS was 5.0 ± 4.5% ranging from 0 to 15%. Age and dialysis vintage were higher in the patients with AoAC than in patients without AoAC. The AoACSs were strongly correlated with the aortic arch calcification volume estimated by MDCT. We suggest that screening HD patients for AoAC is a cost-effective, efficient way to identify patients at the highest risk of CV events and of enabling treatment by anticalcific strategies.
TREATMENT OF VASCULAR CALCIFICATION
Hyperphosphatemia contributes to SHPT, CV mortality and all-cause mortality. The P binders that are currently used to manage hyperphosphatemia include sevelamer, lanthanum, and the calcium-based phosphate binders (CBPBs) CaCO3 and Ca acetate. Sevelamer is an aluminum- and Ca-free P binder that does not promote hypercalcemia, allows better serum P control than CBPBs, suppresses the progression of aortic calcification in HD patients, and has a favorable effect on the lipid profile, because it reduces low-density lipoprotein (LDL)-cholesterol and increases high-density lipoprotein (HDL)-cholesterol (76). In a comparative study of 200 HD patients, Chertow et al. (77) demonstrated that sevelamer had attenuated the progression of coronary and aortic calcification better than CBPBs after 1 year of treatment. These findings were confirmed by Cozzolino et al. (78), who showed that treatment with sevelamer, rather than treatment with CaCO3, was associated with less vascular calcification within the myocardium, aorta, and kidney. The probable mechanism consists of a strong P-binding capacity of sevelamer in the intestine, without excessive Ca loading. In contrast, the Renagel in New Dialysis study in patients with baseline CAC scores of 30 or higher, there was no significant difference in the rate of progression of calcification at any point up to 18 months of follow-up between a group of patients treated with sevelamer and a group treated with CBPBs. (79). In vitro studies have shown that acetylated LDL promotes VSMC calcification, whereas HDL inhibits it (80). In human studies, sevelamer has been shown consistently to reduce LDL and often to increase HDL levels. The improved lipid profile may play a role in the lower degree of vascular calcification observed after sevelamer therapy since intensive LDL-cholesterol-lowering therapy with atorvastatin in the Calcium Acetate Renagel Evaluation-2 study disclosed similar progression of CAC in the group of HD patients treated with Ca acetate and those treated with sevelamer (81).
The Ca-sensing receptor (CaR) is a G protein-coupled cell surface receptor that senses extracellular Ca ions and enables cells to respond to small changes in the extracellular Ca ion concentration (82). Arterial CaR expression has been shown to be significantly lower in patients with end-stage renal disease than in the general population (83). These findings are in line with those published by Alam et al. who detected low levels of CaR immunoreactivity in atherosclerotic, calcified human arteries in comparison with noncalcified arteries (84). These findings suggest that a close relationship between CaR and vascular calcification may exist locally in the vessel wall. Ivanovski et al. (85) provided evidence of direct inhibition of P-induced human VSMC calcification in vitro by R-568 via local activation of the CaR. Lopez et al. (86) investigated the effect of the calcimimetic R-568 alone and in combination with calcitriol on the development of vascular calcification and calcification of other soft tissue in a rat model of uremia-associated SHPT. They showed that the R-568 reduced PTH levels without inducing vascular calcification, attenuated calcitriol-induced calcifying effects on vascular tissue, and decreased mortality associated with administration of calcitriol. They concluded that R-568 reduces elevated PTH levels in uremic rats without inducing vascular calcification and prevents calcitriol-induced vascular calcification.
Bisphosphonates may have a future role in the management of vascular calcification, because they have been shown to reduce vascular calcification in experimental models. Tamura et al. (87) showed that etidronate reduced calcitriol-induced aortic calcification in 5/6 nephrectomized rats. They demonstrated that low-dose etidronate (2 mg/kg of body weight) was ineffective but that calcification was inhibited by a dose of 5–10 mg/kg of body weight. In another study using bovine aortic smooth muscle cells, pamidronate inhibited arterial calcification (88). In HD patients, etidronate has been found to reduce and even reverse the progression of CAC in some of HD patients (8,89), but the mechanism by which it does so is unclear. Bisphosphonates inhibit bone resorption, with reduced efflux of calcium and phosphate, limiting their availability for deposition in the vasculature, or may influence the activity of the sodium/phosphate co-transporter in VSMCs (90).