Biological aging [1
] and diseases like chronic kidney disease (CKD) [2
] or diabetes mellitus [3
] are associated with an increased incidence of cardiovascular calcification, an independent cardiovascular risk factor accompanied by enhanced morbidity and mortality [4
For decades vascular calcification (VC) was thought to be the consequence of passive precipitation of calcium (Ca) and phosphate (P) ions resulting from a supersaturated Ca × P product [5
]. Today cardiovascular calcification is appreciated as an actively regulated, cell-mediated process characterized by the interaction of inductive and inhibitory proteins [6
]. According to the anatomical localization, VC can be classified as intimal and medial calcification, although a clear-cut distinction is almost impossible in clinical practice [7
]. Intimal calcification is linked to atherosclerosis and characterized by inflammatory accumulation of oxidized lipids [8
]. Medial calcification develops independently of inflammation and lipid deposition along the elastic fibers. It is a typical consequence of aging and found in patients suffering from chronic kidney disease or diabetes mellitus [9
]. Despite differences in etiology, the underlying pathophysiological mechanism of intimal and medial calcification is similar [6
The vitamin K-dependent matrix Gla-protein (MGP) is a potent inhibitor of arterial calcification [12
]. MGP was presented first in 1983 by Price and colleagues as a 14kD protein purified from bovine bone matrix [13
]. That the function of MGP was mainly vascular became clear from MGP-deficient mice, all of which died within a few months of birth due to blood vessel rupture as a result of VC [14
]. Likewise, humans with a hereditary dysfunctional MGP (Keutel syndrome) suffer from widely distributed extraosseous calcifications [15
]. There are two posttranslational modifications in MGP: gamma-glutamate carboxylation and serine phosphorylation [16
]. While the function of posttranslational MGP phosphorylation is not completely understood, Murshed and colleagues showed that MGP has to be carboxylated to prevent VC [17
]. Diminished MGP carboxylation is associated with an increased tendency of calcification of the vasculature [18
]. Analogous to vitamin K-dependent blood clotting factors (Factors II, VII, IX, X, and Protein C, S, and Z), the biological activity of MGP depends on the presence of vitamin K as cofactor [19
]. Current medical treatment with vitamin K antagonists may, besides providing effective anticoagulation, also increase the risk for VC [20
Patients with CKD are characterized by widespread extraosseous calcification. We as well as others have shown that CKD patients display vitamin K insufficiency, associated with elevated plasma concentrations of inactive MGP [21
]. Increased plasma levels of inactive ucMGP are paralleled by enhanced morbidity and mortality in patients suffering from CKD, aortic stenosis, or congestive heart failure [22
]. Supplementation with vitamin K results in a dose- and time-dependent decrease of ucMGP plasma levels [23
]. So far, no trials reported “hard outcomes” such as VC in the context of CKD and vitamin K supplementation.
Fat-soluble vitamin K is an essential micronutrient [28
]. There are two forms of vitamin K in nature: phylloquinone (vitamin K1
) and the menaquinones (vitamin K2
; MK-n). VK1
is tightly bound to the chloroplast membrane of plants [29
]. Menaquinones differ in side chains of varying length. They are described as MK-n, in which n denotes the number of unsaturated isoprenoid residues. MK-7 is produced by bacteria and is present in fermented foods such as cheese or sauerkraut [30
]. In this study we used MK-7 because of its long half-life and good bioavailability. Two independent observational studies [31
] described a protective cardiovascular effect of nutritional intake of menaquinones, whereas no effect was found for phylloquinone. This discrepancy in function was ascribed to the better availability and transport of long-chain menaquinones such as MK-7 as compared to VK1
. However, in animal models of warfarin-induced vascular calcification, the short chain menaquinone MK-4 was tested, which displayed greater potency in the inhibition of vascular calcification [33
]. The MK-7 supplementation dose of 100 μg/g diet used in this present study was based on these MK-4 studies [33
The aim of this study is to evaluate the impact of high-dose MK-7 supplementation on the development of cardiovascular calcification and the impact on cardiovascular function in a murine model of chronic kidney disease characterized by enhanced extraosseous calcification.
2. Materials and Methods
2.1. Animals and Diets
The animal study protocol was authorized by the responsible governmental office called LANUV (“Landesamt für Natur, Umwelt und Verbraucherschutz Nordrhein-Westfalen”; file reference 87-51.04.2010.A275). All experiments were executed according to the German animal welfare act in close cooperation with veterinaries of the Heinrich Heine University. We used 42 male Wistar rats aged 12 weeks with a body weight about 300 g at the beginning of the study protocol. Rats were kept in a climate-controlled room (22–24 °C, relative humidity 60%–80%) with a 12-hour light, 12-hour dark cycle. Food and water were given ad libitum.
Animals were divided into an interventional group undergoing 5/6 nephrectomy, receiving a high phosphate diet and into a control group undergoing sham operations. Half of each group received a high MK-7 diet, so that we distinguished between four different treatment groups receiving different diets (sniff-Spezialdiäten GmbH) (Table 1
Pure synthetic MK-7 was provided by NattoPharma ASA (Hovik, Norway).
|Co (n = 10)||Co-K2 (n = 10)|
|Standard diet:||MK-7-supplemented standard diet:|
|0.36% Phosphate||0.36% Phosphate|
|0.6% Calcium||0.6% Calcium|
|0.5 µg/g VK1||0.5 µg/g VK1|
|0 MK-7||100 µg/g MK-7|
|Intervention (5/6-Nephrectomy + high phosphate diet)|
|CKD (n = 11)||CKD-K2 (n = 11)|
|High phosphate diet:||Phosphate- and MK-7-rich diet:|
|1.2% Phosphate||1.2% Phosphate|
|0.6% Calcium||0.6% Calcium|
|0.5 µg/g VK1||0.5 µg/g VK1|
|0 MK-7||100 µg/g MK-7|
2.2. Study Design
All animals took part in a three-month study protocol. On the first day we measured blood pressure using a tail-cuff system. Blood samples were taken by retro orbital bleeding. Body weight was taken twice weekly. 5/6 nephrectomy was performed according to a surgical technique initially described by Perez-Ruiz [36
]. Briefly, we performed right-sided nephrectomy and one week later following recovery from the initial surgery, rats underwent functional 2/3 nephrectomy of the remaining left kidney by careful ligation of the renal parenchyma. Controls underwent a similar two-step laparotomy exposing the kidneys but without nephrectomy. After three and eight weeks we repeated the measurements from the preoperative day. At the end of the study after three months we collected 24-hour urine samples from six animals in each group in metabolic cages. Animals were sacrificed under anesthesia by puncture of the vena cava inferior. Blood was collected for serum analyses. Heart, aorta, and kidney tissues were collected for further analyses.
Echocardiography was performed as described previously [33
]. Briefly, rats were anesthetized using Isoflurane and two-dimensional and M-mode measurements were accomplished using Vivid i, GE Healthcare (GE Healthcare, Buckinghamshire, England) with a 12 MHz probe. Animals were placed in the supine-lateral position with ECGs were obtained throughout the procedure. Parasternal long-axis and short-axis views of the left ventricle (LV) were obtained, ensuring that the mitral and aortic valves and apex were well visualized. Area fraction and wall area were determined by planimetry of end-diastolic and systolic volumes in parasternal short axis. Measurements of LV end-diastolic and end-systolic dimensions were obtained in M-mode at mid-papillary level from more than three beats and fractional shortening (FS) was calculated as FS (%) = ((LVIDd – LVIDs)/LVIDd) × 100, where LVID is LV internal diameter, s is systole, and d is diastole. Diastole is defined as the maximum measurable area; systole is defined as the minimum measurable area. Doppler flow spectrum of the ascending aorta was recorded from the suprasternal view. Peak velocity was measured, and the waveform was also traced to obtain a velocity time-integral calculation and peak gradient.
2.4. Blood/Urine Analyses
Blood and urine analyses were performed by Animal Blood Counter (Scil Animal Care Company GmbH, Viernheim, Germany) and in the Institute of Clinical Chemistry of University Hospital Düsseldorf.
Tissues were perfused with cold phosphate-buffered saline (PBS) solution by cannulation of left ventricle. Afterwards tissues were embedded in TissueTek (Sakura Finetek Europe B.V., Alphen aan den Rijn, the Netherlands) on dry ice for cryofixation, paraffin embedded, or placed in frozen nitrogen. Different histological and immunohistological stainings were performed and analyzed with a Leica DM4000 M RL microscope mounted with a Leica DFC 425C camera (Leica Mikrosysteme GmbH, Wetzlar, Germany). Quantitative measurements were performed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
Real-Time PCR mRNA was extracted using the commercial kits RNAlater and RNeasy (Qiagen, Hilden, Germany) with proteinase K digestion before RNA extraction to maximize mRNA yield. Integrity and amount of mRNA were analyzed by capillary electrophoresis (Agilent Bioanalyzer 2100; Agilent Technologies, Böblingen, Germany). Reverse transcription and real-time PCR were performed with Applied Biosystems 7500 Fast Real Time PCR System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The expression level in untreated mice was arbitrarily assigned the value 1.0, and all other expression values were expressed as fold changes thereof. Values were analyzed using REST software tool (Quiagen, Hilden, Germany).
We performed ANOVA with Bonferroni’s post hoc analysis using GraphPad Prism 5 software (GraphPad, San Diego, CA, USA) to estimate the overall differences between experimental groups. Confidence intervals over 95% were regarded as significant.