Vitamin K deficiency in CKD patients: a modifiable risk factor for vascular calcification?

http://www.kidney-international.org & 2009 International Society of Nephrology deficiency in CKD patients: a modifiable risk factor for vascular calcification? Thilo Krueger 1, Ralf Westenfeld 2, Markus Ketteler 3, Leon J. Schurgers 4 and Jürgen Floege 1 1 Division of Nephrology and Clinical Immunology, University Hospital, RWTH University of Aachen, Germany; 2 Division of Cardiology, University Hospital, RWTH University of Aachen, Aachen, Germany; 3 Division of Nephrology, Hospital Coburg, Coburg, Germany and 4 Cardiovascular Research Institute and VitaK, University of Maastricht, Maastricht, The Netherlands The purpose of this review is to summarize currently available evidence implicating vitamin K in the pathogenesis of vascular calcification (VC), in particular arterial medial calcification. In doing so, we try to provide a rationale for an interventional clinical study testing whether vitamin K supplementation can retard VC or even affect cardiovascular mortality in chronic kidney disease patients. Additionally, we wish to give an overview of the current literature indicating potential adverse effects of long-term vitamin K antagonists in this population. Kidney International (2009) 76, 18 22; doi:10.1038/ki.2009.126; published online 22 April 2009 KEYWORDS: dialysis; Matrix Gla protein; vascular calcification; vitamin K Cardiovascular disease underlies 50% of all deaths in chronic kidney disease (CKD) patients. Vascular calcifications (VCs), that is, atherosclerotic intima calcifications and calcification of media, are major contributors to mortality. Although atherosclerotic lesions are cholesterol- and lipid-associated, medial calcification is associated with diabetes, uremia, and disturbed mineral metabolism. Medial calcifications often underlie VC in CKD patients and non-invasive distinction between intimal and medial calcification is difficult and both may occur simultaneously. 1 It is assumed that medial calcification increases vascular stiffness resulting in left ventricular hypertrophy, a major contributor to mortality. Accordingly, any detection of VC has been associated with cardiovascular mortality. 2 4 Numerous factors influence VC development. 5 This review focuses on the major local calcification inhibitor in the arterial media, the matrix-gla protein (MGP), a vitamin K-dependent protein. Accumulating evidence suggests that diet-induced vitamin K deficiency or its antagonism may induce VC. supplementation is a safe approach but whether it can reduce CKD complications, will require an interventional trial. Moreover, these data imply that vitamin K antagonists in CKD patients should only be given after a thorough assessment of its benefits. Correspondence: Thilo Krueger, Division of Nephrology, University Hospital, RWTH Aachen, Pauwelsstr 30, Aachen 52074, Germany. E-mail: tkrueger@ukaachen.de Received 28 October 2008; revised 2 March 2009; accepted 10 March 2009; published online 22 April 2009 Matrix Gla protein: vitamin K-dependent inhibition of calcification Matrix-Gla protein, an approximately 10 kda protein, is produced and secreted by osteoclasts, chondrocytes, and vascular smooth muscle cells (VSMCs) of the arterial media. MGP needs to undergo post-translational g-glutamyl carboxylation of five glutamate residues to achieve full biological activity. This activation depends on the availability of vitamin K and thereby resembles the biology of the coagulation factors II, VII, IX, X and proteins C and S. During vitamin K deficiency MPG is not activated, under-carboxylated MGP (ucmgp) is formed. replenishment then leads to the bioactive fully carboxylated MGP (cmgp). Complete absence of MGP in MGP / mice led to disturbed bone mineralization and the animals all died at about 8 weeks after birth because of the rupture of a severely 18 Kidney International (2009) 76, 18 22

T Krueger et al.: deficiency in CKD patients mini review calcified aorta. 6 In humans, a loss-of-function mutation within the MGP gene is responsible for the Keutel syndrome, which among others is characterized by abnormal cartilage calcification and accelerated calcification of the aorta as well as coronary and cerebral arteries. 7 To exert its anticalcification actions, MGP needs to be expressed locally in the vessel wall. Thus, the lethal phenotype of MGP / mice was rescued by re-expressing MGP in arterial VSMCs, but not by an increase in circulating MPG after hepatic overexpression. 8 The mechanisms by which MGP prevents arterial calcification are unclear. One of the potential modes of action is a direct inhibition of calcium precipitation and crystallization. This is probably why MGP is enriched in socalled matrix vesicles, which are released by cells such as VSMC. These vesicles are thought to counterbalance potentially harmful high intracellular/intravesicular calcium concentrations. 9 Another anti-calcification mechanism is MGP s antagonism of the bone formation inducing bone morphogenetic protein-2. 10 The development of conformation-specific antibodies against the two MGP subtypes led to studies in rodents and humans, showing that the inactive form, ucmgp, is detected in areas of VC, whereas bioactive cmgp is localized to intact vessel walls 11 (Figure 1). In rodent studies, vitamin K depletion by coumarin resulted in VC of the tunica media, 12 that is, the major site of MGP expression, and this could be prevented by the coadministration of vitamin K 2, 13 but not vitamin K 1. 14 Growth arrest-specific 6 protein: another vitamin K-dependent calcification inhibitor? Gas6, the product of the gene growth arrest-specific 6 (GAS6), like MGP, depends on carboxylation of glutamate residues to achieve bioactivity. In its active form, Gas6 prevents apoptosis of VSMC. 15 This is relevant in the context of VC, as apoptotic VSMC can serve as a nidus for calcium phosphate precipitation. After warfarin administration, Gas6 could no longer phosphorylate its receptor Axl, thus blocking the signal cascade of Gas6, 16 upregulating apoptosis in VSMC, 17 and increasing calcification of VSMC in vitro. 18 a b However, the extent to which reduced Gas6 bioactivity contributes to VC in vivo is currently unknown. Besides its effects on apoptosis, Gas6 also affects inflammation and thrombogenesis (reviewed in 19 ). : the basics was discovered in the 1930s, when chicken on a fat-free diet developed hemorrhages. Administration of vitamin K was then shown to cure bleeding complications in patients with hepatic and biliary diseases. The group of K vitamins comprises phylloquinone (vitamin K 1 ) and several menaquinones (vitamin K 2 ). The synthetic forms, menadion (vitamin K 3 ) and esterified menadion (vitamin K 4 ), have no importance for normal human food. 1 is bound to membranes of the chloroplasts of green leafy vegetables. In contrast, vitamin K 2 is relatively rare in Western diets. The main source of dietary vitamin K 2 is fermented food such as cheese and in particular the Japanese natto, a fermentation product from soy beans. The mammalian intestinal flora and/or intestinal enterocytes are also able to produce vitamin K 2, but the extent to which this contributes to the daily intake is unknown. 20,21 The individual denomination of the several isoforms of menaquinones (MK) is dependent on the number of isoprenoid residues in the side chain. In human food those having 7, 8, or 9 isoprenoid groups are most common. On account of the increased hydrophobic properties of longer side chains, the various isoforms exhibit different half-lives. For example, MK-4 with four isoprenoid groups has a halflife time of 1 h and MK-7 of around 3 days. In mammals, vitamin K serves as a redox partner in cellular metabolism pathways of g-glutamyl carboxylations, and a recycling mechanism helps to reduce the daily requirement of vitamin K. The vitamin K cycle can be effectively inhibited by coumarins such as warfarin, primarily known as potent coagulation inhibitors (Figure 2). Quinone reductase hydroquinone Protein Warfarin γ-glutamyl carboxylase Figure 1 Human vascular calcification and ucmgp. (a) Human artery after von Kossa staining showing a medial lesion with vascular calcification. (b) Immunohistochemical detection of undercarboxylated MGP colocalizing with the medial calcification identified in (a). Original magnification 10. Vitamin K-epoxide reductase epoxide Figure 2 Schematic illustration of the vitamin K cycle (modified from Stafford 43 ). γ-carboxylated protein Kidney International (2009) 76, 18 22 19

T Krueger et al.: deficiency in CKD patients Both vitamin K 1 and vitamin K 2 catalyze the g-glutamyl carboxylation of all vitamin K-dependent proteins, including coagulation factors, osteocalcin, and MGP. Osteocalcin, also called bone Gla protein, is exclusively synthesized by osteoblasts and odontoblasts and is a regulator of bone formation. Differences in vitamin K 1 and K 2 bioactivities result from differences in enzyme affinity and tissue distribution: K 2 vitamins have a higher affinity for the enzyme g-glutamyl carboxylase, which implies that lower concentrations of vitamin K 2 are needed to obtain a similar activity of the g-glutamyl carboxylase compared with vitamin K 1. In terms of tissue distribution, vitamin K 1 predominantly accumulates in the liver, where it catalyzes the g-glutamyl carboxylation of the coagulation factors. 2 has a more widespread tissue distribution and is also involved in the carboxylation of MGP and osteocalcin. The recommended daily allowance of total vitamin K is 1 mg/kg per d; the estimated daily intake in a Western diet ranges from 60 to 200 mg, of which vitamin K 1 constitutes the major part. However, only 10% of K 1 is absorbed from food given its tight binding to the chloroplast membranes. There are no exact data on the amount of vitamin K 2 intake, but it is assumed that around 10% of the total vitamin K intake is vitamin K 2. 13 The mean daily dose is sufficient to maintain a normal coagulation status, but is it unclear whether the dose is sufficient to enable extrahepatic protein carboxylation. Determination of the ratio of extrahepatic carboxylated versus under-carboxylated vitamin K-dependent proteins, such as osteocalcin or MGP, could serve as a biomarker to assess whether vitamin K intake is sufficient. Indeed, with a Western diet, the carboxylation grade of serum osteocalcin is only about 70%, indicating suboptimal vitamin K 2 concentrations in extrahepatic organs. 11,22 The percentage of circulating ucmgp in the healthy population is around 40%, again pointing to a relative vitamin K deficiency (Schurgers, unpublished data). Does vitamin K deficiency play a role in human disease? In postmenopausal women with osteoporosis vitamin K levels were lower in affected patients than in healthy controls. 23 Vice versa, in 72,000 women, the risk of hip fractures was significantly lower in patients consuming higher amounts of vitamin K. 24 In double-blind, placebocontrolled clinical trials, bone loss was reduced by 35% with vitamin K 1 over a 3-year period 25 and by 100% with vitamin K 2. 26 These studies are of relevance for the present discussion given that increasing degrees of osteoporosis are associated with increasing VC. 27 However, this area is controversial, as recent data do not confirm such an association. 28 In the Rotterdam trial in 4800 elderly patients, low vitamin K 2 intake was associated with a higher incidence of severe aortic calcification and increased mortality. 29 In a clinical trial in 100 postmenopausal women, the supplementation of vitamin K 1 for 3 years improved vascular compliance, distensibility, and intima media thickness compared with placebo. 30 However, a specific link between low vitamin K 2 intake leading to accelerated VC through an increase in ucmgp has not been proven so far. Blockade of the vitamin K metabolism with vitamin K antagonists affects skeletal and vascular health as well. In males receiving long-term warfarin, the odds ratio for fractures was 30% higher in comparison with patients without warfarin. 31 Furthermore, both the prevalence and extent of aortic valve and coronary calcifications increased significantly in patients on coumarin. 32 In hemodialysis patients as well long-term use of warfarin was associated with more severe aortic valve calcification. 33 Finally, epidemiological studies have identified the treatment with warfarin as a risk factor for calciphylaxis, a severe complication in CKD patients, which is characterized by extensive VC. 34 levels, MGP bioactivity, and cardiovascular calcification in CKD patients On account of its lipophilic properties and incorporation into lipoproteins, vitamin K is not supposed to be dialysed. Earlier reports of a vitamin K deficiency in dialysis patients 35 therefore point to a diminished dietary intake. Thus, as discussed above, a differential assessment of carboxylated versus under-carboxylated vitamin K-dependent proteins may yield better insight into the vitamin K status. Indeed, increased under-carboxylated osteocalcin levels were detected in dialysis patients compared with healthy controls, 36 pointing to subclinical vitamin K deficiency. Data on ucmgp levels in the circulation of CKD patients are more difficult to interpret, which is in line with the experimental observations cited above, 8 in which local vascular but not systemically overexpressed MGP is protected from VC. In patients, low rather than high circulating ucmgp was a powerful predictor of coronary artery calcification 37 and ucmgp was decreased rather than increased within the serum of CKD patients. 38 One explanation for these unexpected findings may be an accumulation of ucmgp within the calcified vessel wall resulting in low circulating levels. 38 Alternatively, uncarboxylated Gla proteins are secreted less from the cells, possibly to avoid inactive proteins to enter the tissues. Moreover, besides carboxylation, altered phosphorylation of MGP may, at least in part, influence MGP levels. Three serine residues of MGP are supposed to be phosphorylated, which seems to be important for the secretion of this protein. As a consequence, phosphorylated and dephosphorylated MGP are also detectable, probably resembling immature and mature forms of MGP. Recent findings from Schurgers and colleagues show that the serum levels of phosphorylated ucmgp are inversely associated with calcification scores in patients and thus may reflect the degree of existing VC. 37,39 On the other hand, dephosphorylated ucmgp is set free more easily in the circulation even in the presence of extensive VC and thus may allow better assessment of the vitamin K status. 37,40 Taken together, the assessment of MGP may be more complex because of its several modifications and needs further investigation to specify the relevance of each form. 20 Kidney International (2009) 76, 18 22

T Krueger et al.: deficiency in CKD patients mini review supplementation as a prophylaxis against VC? At present only scant data are available to answer the above question. The answer clearly awaits interventional outcome studies in CKD patients. The rationale for such studies, that is, to place CKD patients with their excessive cardiovascular risk on is given below. In a study in 190 women, the incidence of fractures was significantly reduced in those supplemented with 45 mg vitamin K 2 per day. 41 In another Japanese study, dialysis patients received 45 mg per day vitamin K 2 over a 12-month period, which resulted in increased serum levels of carboxylated osteocalcin within 1 month. 42 In rodent studies, the therapeutic administration of vitamin K 2 resulted in reduced VC (own observations and Schurgers et al. 13 ), but confirmation in humans is lacking. However, in a pilot study in 50 dialysis patients we have recently shown that the daily administration of 360 mg vitamin K 2 resulted in a significant increase of cmgp as well as osteocalcin (Westenfeld R et al.: ASN Renal Week 2008, Abstract Book). With regard to the safety of vitamin K 2 supplements, no relevant side effects have been described so far. In particular, no thromboembolic events or shunt dysfunctions were reported. 41,42 Moreover, vitamin K substitution has been carried out in neonates in Asia and Europe for years with no side effects being observed. On account of its availability for extrahepatic tissues, vitamin K 2 should be regarded as the isoform being optimal for supplementation. The optimal dose of vitamin K 2 remains to be tested. Isoforms with longer side chains, for example, MK-7, may be the best choice given their half-life. Schurgers et al. 13 showed that 146 mg MK-7 daily significantly increased serum levels of vitamin K 2 after 14 days. CONCLUSION supplementation in the form of vitamin K 2 isoforms may be an easy intervention to counteract the relative vitamin K deficiency observed in particular in dialysis patients. Although such an intervention has been shown to be safe, the optimal daily dosage and the clinical long-term benefits, in particular reduced VC and improved bone mineral density or fracture rates, need to be determined in clinical studies. Vice versa, the above findings indicate that warfarin may not be neutral in the context of VC and larger trials could elucidate the role of warfarin, especially in CKD patients with their high risk for VC. In addition, whether vitamin K 2 supplementation is safe and efficacious in preventing VC progress in patients with an absolute indication for long-term vitamin K antagonist therapy remains to be tested. Disclosure All the authors declared no competing interests. 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