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http://www.kidney-international.org & 2014 International Society of Nephrology
see commentary on page 232
Impaired vitamin K recycling in uremia is rescued by vitamin K supplementation Nadine Kaesler1, Elke Magdeleyns2, Marjolein Herfs2, Thomas Schettgen3, Vincent Brandenburg4, Danilo Fliser5, Cees Vermeer2, Ju¨rgen Floege1, Georg Schlieper1,6 and Thilo Kru¨ger1,6 1
Department of Nephrology, RWTH University Hospital Aachen, Aachen, Germany; 2VitaK BV, University of Maastricht, Maastricht, The Netherlands; 3Department of Environmental and Social Medicine, University Hospital of the RWTH Aachen, Aachen, Germany; 4 Department of Cardiology, RWTH University Hospital Aachen, Aachen, Germany and 5Department of Internal Medicine IV, Saarland University Medical Centre, Homburg, Germany
In chronic kidney disease, vitamin K–dependent proteins, including the calcification inhibitor matrix Gla protein, are largely uncarboxylated indicating that functional vitamin K deficiency may contribute to uremic vascular calcification. Since the effects of uremia on the vitamin K cycle are unknown, we investigated the influence of uremia and vitamin K supplementation on the activity of the vitamin K cycle and extraosseous calcification. Uremia was induced in rats by an adenine-supplemented diet and vitamin K1 or K2 was administered over 4 and 7 weeks. After 4 weeks of adenine diet, the activity of the vitamin K cycle enzyme c-carboxylase but not the activities of DT-diaphorase or vitamin K epoxide reductase were reduced. Serum levels of undercarboxylated matrix Gla protein increased, indicating functional vitamin K deficiency. There was no light microscopy–detectable calcification at this stage but chemically determined aortic and renal calcium content was increased. Vitamin K treatment reduced aortic and renal calcium content after 4 weeks. Seven weeks of uremia induced overt calcification in the aorta, heart, and kidneys; however, addition of vitamin K restored intrarenal c-carboxylase activity and overstimulated it in the liver along with reducing heart and kidney calcification. Thus, uremic vitamin K deficiency may partially result from a reduction of the c-carboxylase activity which possibly contributes to calcification. Pharmacological vitamin K supplementation restored the vitamin K cycle and slowed development of soft tissue calcification in experimental uremia. Kidney International (2014) 86, 286–293; doi:10.1038/ki.2013.530; published online 15 January 2014 KEYWORDS: gamma-carboxylase; nephropathy; vascular calcification; vitamin K
Correspondence: Thilo Kru¨ger, Division of Nephrology and Clinical Immunology, University Clinic of the RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail: [email protected] 6
All the authors declared no competing interests.
Received 3 January 2013; revised 26 October 2013; accepted 31 October 2013; published online 15 January 2014 286
Vascular calcification is common in chronic kidney disease (CKD). In hemodialysis patients, coronary artery calcification is associated with ischemic cardiovascular disease1 and arterial media calcification is highly correlated with mortality in these patients.2 Possible contributors to the development of vascular calcification in CKD are an increased calcium phosphate product, parathyroid hormone, as well as reduced levels of inhibitors of vascular calcification such as fetuin-A and insufficient activity of matrix Gla protein (MGP).3 MGP is a potent vessel wall–based inhibitor of arterial calcification. MGP knockout mice develop spontaneous calcification of arteries.4 The mechanism by which MGP inhibits vascular calcification may involve bone morphogenetic protein-2 antagonism and a direct calcium-complexing effect.5 MGP is expressed predominantly by vascular smooth muscle cells in the arterial media and chondrocytes. It contains five glutamic acid residues that can be g-carboxylated (Glu-Gla) by the vitamin K–dependent g-carboxylase. This step in which undercarboxylated MGP (ucMGP) changes into carboxylated MGP is essential for the protein to gain full bioactivity.6 The Gla residues are essential for calcium binding.7 The g-glutamyl carboxylase (GGCX; Enzyme Commission number (EC) 4.1.1.90) requires reduced vitamin KH2 as a cofactor. The latter is provided by and recycled in the so-called vitamin K cycle. This cycle is formed by vitamin K epoxide reductase (VKOR; EC 1.1.4.1), GGCX and DT-diaphorase (NAD(P)H-quinone-oxidoreductase, EC 1.6.5.2).8,9 VKOR is inhibited by coumarins such as warfarin, a widely used anticoagulant. Data are accumulating that treatment with coumarins augments vascular calcification in rats and humans,10–13 but with also some conflicting results in humans.14,15 In turn, a high intake of vitamin K2 (MK4) was capable of regressing warfarininduced medial calcification in Wistar rats.16 Hemodialysis patients exhibit markedly increased levels of undercarboxylated proteins such as MGP, osteocalcin and PIVKA (prothrombin induced by vitamin K absence).17,18 Moreover, low levels of carboxylated MGP predict mortality in such patients.19 The origin of the functional vitamin K Kidney International (2014) 86, 286–293
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N Kaesler et al.: c-Carboxylase activity in uremic calcification
RESULTS Experimental groups
Two different degrees of kidney disease were induced by adenine feeding for either 4 weeks (a model of mild kidney failure without overt vascular calcification, diets a–d) or 7 weeks (severe kidney failure including tissue calcification, diets e–i, Figure 1). The 7-week approach included a 2-week interphase off adenine in weeks 5 and 6 for recovery. The vitamin K2 diet contained 100 (diets c, d, and g) or 500 mg/ kg MK4 (diet h); vitamin K1 was added at 100 mg/kg (diet i). In the severe model, adenine diet was combined with low protein to increase the penetrance of uremic calcification as described previously.21 All animals survived the 4-week treatment. Seven weeks of adenine resulted in three deaths; necropsy failed to show any obvious reason. The average food intake was 23 (±8) g/day in the healthy groups and 13 (±7) g/day in the adenine groups. Adenine-fed rats lost B16% of body weight that was similar in all adenine-fed groups. Serum parameters
After 4 weeks of adenine treatment, serum creatinine increased sevenfold and urea sevenfold and hyperphosphatemia developed (Supplementary Table S1 online). Adenine diet combined with low-protein diet did not affect creatinine and phosphate serum levels after 4 weeks compared with adenine diet with normal protein (data not shown), but at 7 weeks of treatment, creatinine levels were increased 10-fold (Supplementary Table S1 online). Serum phosphate levels in the vitamin K2 high-dose group (500 mg/kg þ adenine) were significantly lower than in CKD (after 7 weeks of adenine based on low protein, Supplementary Table S2 online). Serum calcium was unchanged in all groups at every time point (Supplementary Table S1 online). Compared with corresponding controls, significantly higher levels of serum ucMGP were measured after 4 weeks of CKD (group b, Figure 2) but not after 7 weeks (not shown). Systolic blood pressure levels measured at the beginning and the end of each treatment period were similar in all groups (not shown). Kidney International (2014) 86, 286–293
Standard diet (SD) Adenine diet (AD) AD + Vit K2 (100 mg/kg) SD + Vit K2 (100 mg/kg) Standard diet (SD; low protein*) AD*
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7
Figure 1 | Overview of the different treatment groups. Seven weeks of treatment contained a 2-week interphase without adenine supplementation. *Protein content was lowered to 2.5% in the 7-week groups (see Supplemetary Table S2 online). All diets contained 5 mg/kg vitamin (Vit) K1 (except diet in group i).
*
15,000 ucMGP (nmol/l)
deficiency in CKD is only partially understood. Reduced vitamin K intake has been described in dialysis patients,20 but we reasoned that this cannot fully explain the marked functional vitamin K deficiency. The aims of this study were, first, to assess the activity of the enzymes of the vitamin K cycle in short-term experimental uremia and, second, to investigate whether vitamin K supplementation with both vitamin K1 or vitamin K2 improves soft tissue calcification and modulates enzyme activities in long-term kidney failure. Finally, we attempted to characterize the origin of altered GGCX activity in uremia by in vitro coincubation with three major uremic toxins: urea, indoxyl sulfate, and p-cresol.
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Figure 2 | Undercarboxylated matrix Gla protein (ucMGP) measured in serum after 4 weeks of treatment. *Po0.05.
Tissue content of vitamin K and ucMGP
Hepatic vitamin K1 content was significantly increased in rats receiving 100 mg/kg vitamin K1 (group i, Figure 3a), whereas vitamin K2 (MK4) content was not affected in rats receiving 500 mg/kg vitamin K2 (group h, Figure 3b). Intrarenal vitamin K levels were not affected by either supplement (not shown). Aortic quantification of ucMGP immunohistology showed significantly increased positive areas only in CKD animals without vitamin K supplementation after 7 weeks (group f, Figure 4). Treatment with vitamin K abrogated this significant increase. Vascular calcification colocalized with ucMGP staining (Supplementary Figure S1 online). Enzyme activities
An overview of the changes in vitamin K cycle enzyme activity is displayed in Table 1. 287
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b 1500 ** 400
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+ 2.5 5 – 7
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+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 3 | Hepatic vitamin K content. (a) Vitamin K1 measured in liver tissue after 7 weeks of diet; (b) MK4 measured in liver tissue after 7 weeks of diet. **Po0.01.
Table 1 | Overview of the changes in enzyme activities in the vitamin K cycle (GGCX, DT-diaphorase, and VKOR) compared with corresponding control group (a for a–e; e for f–i)
ucMGP (positive area %)
5 4 3
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0 Adenine – Protein (%) 2.5 K1 (mg/kg) 5 K2 (mg/kg) – Weeks 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 4 | Quantification of undercarboxylated matrix Gla protein (ucMGP) staining in aortas after 7 weeks of treatment. *Po0.05.
(a) Compared with non-CKD controls (groups a, d, and e), GGCX was significantly less active in the uremic groups after 4 (groups b and c) and 7 weeks (groups f and g) weeks in kidney tissues (Figure 5a and b) but not in liver tissue (Figure 5c and d). High-dose vitamin K1 or K2 significantly increased GGCX activity in kidney tissue and exceeded baseline values in liver tissue after 7 weeks (Figure 5b and d). Reduced GGCX activity was also found in aortic tissue after 7 weeks of adenine diet (significant in t-test) that was restored by high intake of vitamin K1 or K2 (Figure 6). After 4 weeks of adenine diet, no influence of vitamin K treatment was detected in aortic tissue (not shown). Urea can modify molecular enzyme activities by carbamylation of lysine residues. In vitro coincubation of 50 mmol/l urea with microsomes from healthy animals did not lead to a reduction of GGCX activity (data not shown). No changes in enzyme activity were found after in vitro incubation with p-cresol or indoxyl sulfate. Adenine itself did not show any effect on GGCX activity (not shown). (b) VKOR activity did not differ significantly between the groups and ranged from 0.4 to 2.6 mmol/g/min in kidney 288
a (healthy control) b (CKD) c (CKD þ K2) d (healthy þ K2) e (healthy LP ctrl.) f (CKD) g (CKD þ K2) h (CKD þ high K2) i (CKD þ K1)
DT-diaphorase
Kidney Liver Aorta Kidney 2 k k 2 2 kk kk k k
2 (k) (k) 2 m (k) (k) mm mm
2 nm nm nm 2 k k 2 2
2 m m 2 2 2 m 2 2
VKOR
Liver Kidney Liver 2 m 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
Abbreviations: CKD, chronic kidney disease; ctrl., control; GGCX, g-glutamyl carboxylase; LP, low protein; nm, not measurable; VKOR, vitamin K epoxide reductase. Arrows depict changes in enzyme activity.
tissue and from 0.4 to 7.1 mmol/g/min in liver tissue (Table 1 and Supplementary Table S2 online). (c) DT-diaphorase was significantly more active in kidney and liver tissue in the 4-week CKD model (group b) (3.6fold and 2.7-fold, respectively, Figure 7a and c). There was a significant correlation between decreased GGCX and increased DT-diaphorase activities for kidney tissue after 4 weeks (R2 ¼ 0.34, P ¼ 0.013). Treatment with adenine over 7 weeks increased DT-diaphorase activity nonsignificantly in kidneys but reached significance after 100 mg/kg vitamin K2 treatment compared with controls (Figure 7b). DT-diaphorase activity in the liver was not changed in all 7-week treatment groups (Figure 7d). GGCX gene expression
The GGCX gene expression assessed by reverse transcriptase–PCR in liver tissue was similar in all groups (data not shown). Calcium measurements
After 4 weeks of treatment, adenine treatment increased calcium content significantly in aortas and kidneys compared with healthy controls (group a, Supplementary Figure S2I þ V Kidney International (2014) 86, 286–293
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Kidney
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0 – 17.35 5 – 4
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+ 17.35 5 100 4
– 17.35 5 100 4
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 5 | The c-glutamyl carboxylase (GGCX) activity. In kidneys after 4 (a) and 7 (b) weeks and in liver after 4 (c) and 7 (d) weeks (mean±s.d.). *Po0.05 and **Po0.01.
*
GGCX (nmol/g/min) aorta
1000
Quantification of calcified areas by von Kossa staining in aortas showed no statistical significance between all groups. Overt vascular calcification was present after 7 weeks of adenine treatment in 7 of 10 animals but the remainder exhibited almost no calcification. Quantification of stained areas of aortic specimens showed a mean 7.7-fold increase after 7 weeks of CKD compared with the status after 4 weeks (Supplementary Figure S3I þ II online). We did not detect significant changes in the calcium content in pulmonary tissue (not shown).
*
100
10
1 Adenine Protein (%) k1 (mg/kg) k2 (mg/kg) Weeks
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 6 | The c-glutamyl carboxylase (GGCX) activity in aortas after 7 weeks (mean±s.d.) of treatment. *Po0.05.
online). Seven weeks of adenine treatment (group f) significantly increased the calcium content in heart, kidney, and aorta (the latter significant in t-test, Supplementary Figure S2II, IV þ IV online). The 4-week vitamin K2 treatment reduced calcium deposition nonsignificantly in the aorta and significantly in kidneys of uremic animals (group c). After 7 weeks, only high-dose vitamin K treatment reduced calcium content in tissues reaching significance in heart and kidneys for vitamin K2 and vitamin K1 in kidneys alone (groups h and i, Supplementary Figure S2 online). Kidney International (2014) 86, 286–293
DISCUSSION
High serum levels of undercarboxylated proteins such as MGP demonstrate that the large majority of uremic patients are more or less vitamin K deficient.17,18 Here we used a rat model of CKD to investigate the effect of uremia on the vitamin K cycle and subsequently uremic calcification. Furthermore, we assessed in a two-step approach whether correcting vitamin K deficiency in this setting rescues the uremic alterations and whether differences exist between treatment with vitamin K1 or K2. In the first step, after 4 weeks of kidney failure, we measured the activity of the enzymes of the vitamin K cycle, that is, at a time point when the animals did not exhibit overt calcifications. Here, vitamin K deficiency was already present as evidenced by increased ucMGP serum levels. Our first major finding was that the activity of GGCX was already reduced at this stage of CKD, possibly contributing to the subsequent development of vascular calcification found at 289
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DT-diaph. (nmol/g/min)
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+ 17.35 5 100 4
– 17.35 5 100 4
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 7 | The DT-diaphorase activity. In kidneys after 4 (a) and 7 (b) weeks and in liver after 4 (c) and 7 (d) weeks (mean±s.d.). *Po0.05.
week 7. As the VKOR activity was not reduced, and the DTdiaphorase was even more active, this suggests that in uremia GGCX is the rate-limiting enzyme in the vitamin K cycle and a potential major contributor to diminished vitamin K availability in tissues (Table 1). The increased DT-diaphorase activity may be a compensatory mechanism intended to provide higher intracellular levels of the reduced form of vitamin K, as suggested by the significant correlation between GGCX and DT-diaphorase activities after 4 weeks in kidney tissue. Why GGCX activities were higher in the control group after 7 weeks compared with 4 weeks remains unknown. An influence of low-protein diet—the only significant difference between these two groups—on GGCX activity has not been reported so far. In addition, low dietary vitamin K intake has been noted in dialysis patients20 augmenting vitamin K deficiency in uremia. Indeed, dietary vitamin K2 supplementation (100 mg/kg, group c) lowered serum levels of ucMGP in this study. These experimental observations parallel those made recently in hemodialysis patients where ucMGP serum levels were elevated and could be markedly reduced by dietary vitamin K2 supplementation.17 Our 4-week CKD model contributes to the understanding of ucMGP action in the early stage. Vitamin K treatment resulted in significantly lower calcium tissue content in kidneys with a parallel trend in the aorta, strengthening the rationale for our planned vitamin K interventional trial VitaVasK targeting cardiovascular calcification in hemodialysis patients (www. clinicaltrials.gov NCT01742273). To further analyze the influence of vitamin K treatment on calcifications, we also studied rats after 7 weeks of kidney 290
failure, when overt vascular calcification was established. Calcification was evident in von Kossa–stained aortic specimens. The activity of GGCX was still reduced after 7 weeks of adenine. Our second major finding was that high dietary vitamin K2 (500 mg/kg) or K1 (100 mg/kg) restored and even overstimulated the activity of GGCX in the liver. In addition, this also significantly reduced calcification of the kidneys and heart. In aortic tissue, we failed to reach significant differences in calcium contents that might be because of the high interindividual variability in the extent of aortic calcification in this model. In the heart and kidneys, lower dosages of vitamin K1 (100 mg/kg) than vitamin K2 (500 mg/kg) were comparably effective in preventing calcification. Although prior observations favor vitamin K2 above K1 in acting on MGP and vascular calcification,22 our data suggest that vitamin K1 is also active in these processes. Vitamin K1 can be converted to K2 as proposed before,23 but increased hepatic and renal vitamin K2 tissue concentrations were not detected following vitamin K1 supplementation that suggests that the effects detected may be because of the vitamin K1 itself. However, we cannot fully exclude, for example, aortic vitamin K1 to K2 conversion. Why in our study the supplementation of 100 mg/kg vitamin K1 was even more potent than 100 mg/ kg vitamin K2 in increasing the GGCX activity and reducing calcifications needs further investigation. Our data are consistent with supplementary vitamin K2 reducing vascular calcification in a warfarin16 and the adenine model (here vitamin K1).24 These experimental data are also supported by a positive correlation between the progression of arterial calcification and low vitamin K1 serum level of patients.25 Kidney International (2014) 86, 286–293
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Compared with the daily dietary recommendation for humans (1 mg/kg body weight), the dosage of vitamin K supplements was markedly higher in our rat experiments (up to 15 mg/kg body weight). Such high dosages were chosen given that there is no evidence of toxicity from even very high vitamin K doses in humans26 and based on dosages in prior experimental studies.16 Unexpectedly, after 7 weeks of adenine treatment, ucMGP levels were on average 41% lower than after 4 weeks of adenine treatment. One explanation is the accumulation of ucMGP in calcified areas in uremia as we found an increased ucMGP staining in group f. In addition, transdifferentiation of MGP-producing vascular smooth muscle cells into an MGP-negative osteoblastic phenotype as published before27 might further reduce ucMGP serum levels. We and others have observed that MGP-synthesizing vascular smooth muscle cells undergo apoptotic processes in calcification.28,29 Finally, we investigated potential mechanisms underlying the low GGCX activity in CKD. The reduced activity was not because of a lower GGCX mRNA expression, although vitamin K is known to enter the nucleus and to influence gene expression, for example, of bone markers.30 To assess post-transcriptional alterations, we focused on carbamylation of this enzyme by the elevated urea concentrations in CKD. As urea levels rise in CKD, it is spontaneously transformed into cyanate that is known to irreversibly carbamylate lysine residues. Reversible modifications occur at tyrosine, serine, threonine, or cysteine residues, resulting in modification of the activities of enzymes.31 However, in this study, in vitro urea concentrations similar to uremic serum levels failed to affect GGCX enzyme activity. Two further uremic toxins tested in vitro, p-cresol and indoxyl sulfate, did not result in altered GGCX activity either. Adenine itself as a suppressor of GGCX activity was excluded. Therefore, the mechanism of reduced g-carboxylase activity presently remains unknown. A limitation of the study besides the supraphysiological vitamin K dosage is the translation of rodent data to CKD patients. Hyperparathyroidism and disturbed bone and mineral homeostasis occur in the adenine model and likely contributed to the development of vascular calcification. The relative contribution of reduced GGCX activity and thus vitamin K deficiency to this process is unknown at present. The effect of kidney disease on GGCX activity needs to be confirmed in human tissues as well. Furthermore, the agent decreasing GGCX activity needs to be identified. Finally, results of the ucMGP assay obtained here cannot be compared with human results as the assay for yet unknown reasons yields different findings in both species. In summary, we found reduced activity of GGCX in uremia in several tissues as a potential contributor to the development of vascular calcification even before overt calcification became present. This possibly contributes to the observed functional vitamin K deficiency in dialysis patients. Reduced GGCX activity might represent a key mechanism explaining increased ucMGP serum levels and excess cardiovascular calcification in CKD in rats and Kidney International (2014) 86, 286–293
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humans. Vitamin K1 and vitamin K2 were able to increase GGCX activity and at least partially reduced extraosseous calcium content in the adenine nephropathy model. Thus, dietary vitamin K supplementation in CKD not only reverses the low intake, but also rescues endogenous vitamin K recycling. As a consequence, the supplementation represents a promising therapeutic approach to the massively increased extraosseous calcification in CKD. MATERIALS AND METHODS Animals and diets Ten male Wistar rats (initial weight 365±18 g; Charles River, Sulzfeld, Germany) in each group were fed the diets shown in Figure 1 (all diets were from Altromin, Lage, Germany; Supplementary Table S2 online). Food and water were administered ad libitum. The rats were housed in a 12-h day and night cycle. Animals were killed under isoflurane anesthesia by heart puncture. Before harvesting, organs were perfused with 20 ml ice-cold phosphate-buffered saline. All animals were treated in accordance with the requirements of the Federation of the European Laboratory Animals Science Associations. Biochemistry Serum was collected by tail vein puncture or finally by puncture of the left ventricle and spun after sedimentation at 2000g for 10 min. Urine was collected for 24 h before killing. Serum and urine parameters were measured by clinical routine laboratory diagnostics. Total ucMGP was measured in serum by competitive enzymelinked immunosorbent assay by a monoclonal antibody (MGP sequence 35-49; VitaK BV, Maastricht, The Netherlands).32 Blood pressure was measured noninvasively by determining the tail blood volume pressure by an occlusion tail cuff on a Kent Scientific CODA system (Kent Scientific, Torrington, CT) at weeks 0, 2, 4, and 7. Vitamin K measurements and ucMGP staining Vitamin K was extracted from snap-frozen tissues by hexane followed by cleaning using a Sep-Pak plus silica cartridge (Waters, Eschborn, Germany). Vitamin K was eluted with 3% diethylether in hexane that was evaporated in a argon stream and diluted in isopropanol for injection in high-pressure liquid chromatography (HPLC; Merck Hitachi, Tokyo, Japan). Vitamin K1 was separated in reversed-phase HPLC (with a Max RP C12 column; Phenomenex, Aschaffenburg, Germany). Isocratic methanol at pH 5 was used as a mobile phase. Vitamin K1 and MK4 were detected at 246 nm. Vitamin K was quantified by external standards. UcMGP in aortic tissues was visualized by incubation with the anti-GluMGP antibody (VitaK BV) as described previously.33 Preparation of microsomes Microsomes were isolated as described earlier.34 Briefly, the postmicrosomal fraction was gained by centrifugation at 10,000 g of homogenized tissue in 300 mmol/l sucrose buffer, followed by ultracentrifugation at 100,000 g for 1 h. Microsomes were stored at 80 1C. Protein content was determined by the Pierce Bicinchoninic Acid method as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA).35 291
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Enzyme activities (a) The g-carboxylase activity was measured by carboxylation of FLEFLK-FITC followed by reversed-phase HPLC coupled with fluorescence detection.34 Vitamin K1 was reduced to KH2 by incubation in a mixture of 20 mmol/l dithiothreitol, 50 mmol/l NaCl, and 2 mmol/l Tris at 37 1C for 12 h in the dark. For a selective influence of uremic toxins or adenine on the g-carboxylase activity, microsomes from healthy rats were first incubated with urea (50 and 500 mmol/l) or indoxyl sulfate (250 and 500 mmol/l) or p-cresol (100 mmol/l) at room temperature. Adenine was used at 0, 2.5, 5, 10, and 22 mmol/l. Urea was obtained from Bio-Rad (Bio-Rad, Hercules, CA). Vitamin K1, dicoumarol, indoxyl sulfate, p-cresol, and isopropanol were purchased from Sigma Aldrich (Munich, Germany). NADPH, MTT, and glucose-6-phosphate were from Carl Roth (Karlsruhe, Germany); glucose-6-phosphate-dehydrogenase, AgNO3, NaOH, Na2CO3 were from Merck (Darmstadt, Germany), and all other chemicals were from AppliChem (Darmstadt, Germany). (b) VKOR activity was measured by conversion of vitamin K1 2,3epoxide (K14O; stable for at least 6 months at 80 1C (99% purity)) to vitamin K1.36 The reaction mixture, with a total volume of 200 ml, contained 500 mg microsomal protein, 8 ml 5 mmol/l vitamin K epoxide, and the reaction was started by the addition of 5 ml 200 mmol/l dithiothreitol. The reaction was stopped after 60 min at 30 1C in the dark by 500 ml 0.05 M AgNO3 in isopropanol. The amount of vitamin K was quantified by using the HPLC setup. For epoxidation, vitamin K1 was diluted in hexane and mixed with 0.5 M NaOH, 0.2 M Na2CO3, and 5% H2O2 for 12 h at 37 1C in the dark. Vitamin K14O quality was monitored by HPLC measurements. (c) DT-diaphorase activity was analyzed in the kidney and liver by the standard assay37 with NADPH as electron donor and menadione as electron acceptor. A total volume of 150 ml reaction mixture contained 7.5 ml 0.5 M Tris-Cl (pH 7.4), 100 mg bovine serum albumin, 1 ml 1.5% Tween-20, 0.1 ml 7.5 FAD, 1 ml 150 mmol/l glucose-6-phosphate, 90 ml 50 mmol/l NADPH, 300 U yeast glucose-6-phosphate-dehydrogenase, and 45 mg MTT. The assay was carried out in the presence and absence of 3 mmol/l dicoumarol and 50 mmol/l menadione with 250 mg microsomal protein. The reaction was stopped after 5 min at 20 1C by a 3 mmol/l dicoumarol and 5 mmol/l KCl. The DTdiaphorase activity was calculated as the specific activity inhibited by dicoumarol. Reduced MTT as the formazan dye was detected at 610 nm on a Tecan sunrise microplate absorbance reader (Tecan, Mennedorf, Switzerland).
Reverse transcriptase–PCR RNA was harvested from RNA later (Qiagen, Hilden, Germany) stabilized tissue using Qiagen RNeasy. Purity and RNA concentration were analyzed with the Agilent RNA 6000 Nano Kit (Agilent, Bo¨blingen, Germany). The reverse transcriptase was performed by the Reverse Transcriptase Core Kit (Eurogentec, Cologne, Germany). The qPCR Core Kit for SYBR Green I (Eurogentec) was applied for a two-step quantification on an Applied Biosystems 7500 Real-Time PCR TagMan system. The GGCX TaqMan gene expression assay (RN00582138) was from Applied Biosystems (Life Technologies, Darmstadt, Germany). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers used were the following: sense: 50 -ACAA GATGGTGAAGGTCGGTA-30 , antisense: 50 -AGAAGGCAGCCCT GGTAACC-30 , probe: 50 -CGGATTTGGCCGTATCGGACGC-30 . 292
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Calcium determination The extent of vascular and tissue calcium content was measured colorimetrically. Therefore, tissues were lyophilized on a Christ Loc 1mALPHA 1-4 (Martin Christ, Osterode am Harz, Germany) coupled with a vacuum hybrid pump RL 6 (Vacubrand, Wertheim, Germany). Calcium was eluted in 10% formic acid and quantified by the cresolphthalein method (Randox Laboratories, Crumlin, UK). Absorption was measured at 550 nm on a Tecan sunrise microplate absorbance reader. Localization of the calcification was visualized by von Kossa staining as previously described.38 In brief, sections were immersed in 1% aqueous AgNO3 solution for 5 min. After washing, samples were incubated in a solution of 5% NaCO3 and 9.25% formalin for 1 min. After a second rinse, sections were developed using sodium thiosulfate (5% in water) for 5 min and counterstained in 0.1% safranin-O followed by a final rinse using tap water. Statistical analysis The difference between the treatment groups was assessed by oneway analysis of variance followed by Tukey’s multiple comparison test (if not stated elsewhere). Equal variances were tested with Bartlett’s method. D’Agostino and Pearson normality test was performed to check for Gaussian distribution. Statistical significance was defined as Po0.05. DISCLOSURE
All the authors declared no competing interests. ACKNOWLEDGMENTS
We thank Katrin Haerthe for the excellent work in animal keeping and in vitro experiments. Special thanks go to Paul Brenchley for very helpful and inspiring discussion of these experiments. SUPPLEMENTARY MATERIAL Table S1. Final biochemical results at the end of the experiment (groups a-d 4 weeks; groups e-i 7 weeks); Groups referring to Figure 1. Table S2. Composition of diets and treatment duration; Groups referring to Figure 1. Table S3. VKOR activity in kidney and liver (mean±s.d.). Figure S1. Von Kossa and ucMGP staining in rat aortic tissue (100 x); early calcification after 4 weeks of adenine and overt calcification after 7 weeks of adenine diet. Figure S2. Calcium content in tissues (mean±s.d.). Calcium content in aorta (I after 4 weeks; II after 7 weeks), heart (III after 4 weeks; IV after 7 weeks) and kidney heart (V after 4 weeks; VI after 7 weeks). Figure S3. Quantification of von Kossa staining in aortic tissue (mean±s.d.). Supplementary material is linked to the online version of the paper at http://www.nature.com/ki REFERENCES 1.
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Raggi P, Boulay A, Scott CT et al. Cardiac calcification in adult hemodialysis patients. A link between end-stage renal disease and cardiovascular disease? J Am Coll Cardiol 2002; 39: 695–701. London GM, Guerin AP, Marchais SJ et al. Arterial media calcification in end-stage renal disease: impact on all-cause and cardiovascular mortality. Nephrol Dial Transplant 2003; 18: 1731–1740. Ketteler M, Schlieper G, Floege J. Calcification and cardiovascular health: new insights into an old phenomenon. Hypertension 2006; 47: 1027–1034. Luo G, Ducy P, McKee MD et al. Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 1997; 386: 78–81. Zebboudj AF, Imura M, Bostrom K. Matrix GLA Protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem 2002; 277: 4388–4394. Kidney International (2014) 86, 286–293
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Al Rajabi A, Booth SL, Peterson JW et al. Deuterium-labeled phylloquinone has tissue-specific conversion to menaquinone-4 among Fischer 344 male rats. J Nutr 2012; 142: 841–845. McCabe KM, Booth SL, Fu X et al. Dietary vitamin K and therapeutic warfarin alter the susceptibility to vascular calcification in experimental chronic kidney disease. Kidney Int 2013; 83: 835–844. Shea MK, Booth SL, Miller ME et al. Association between circulating vitamin K1 and coronary calcium progression in community-dwelling adults: the Multi-Ethnic Study of Atherosclerosis. Am J Clin Nutr 2013; 98: 197–208. Truong J, Booth SL. Emerging issues in vitamin K research. JEBCAM 2011; 26: 73–79. Montezano AC, Zimmerman D, Yusuf H et al. Vascular smooth muscle cell differentiation to an osteogenic phenotype involves TRPM7 modulation by magnesium. Hypertension 2010; 56: 453–362. Kru¨ger T, Oelenberg S, Kaesler N et al. Warfarin induces cardiovascular damage in mice. Arterioscler Thromb Vasc Biol 2013; 33: 2618–2624. Shroff RC, McNair R, Skepper JN et al. Chronic mineral dysregulation promotes vascular smooth muscle cell adaption and extracellular matrix calcification. J Am Soc Nephrol 2010; 21: 103–112. Suhara Y, Watanabe M, Motoyoshi S et al. Synthesis of new vitamin K analogues as steroid and xenobiotic receptor (SXR) agonists: insights into the biological role of the side chain part of vitamin K. J Med Chem 2011; 54: 4918–4922. Kraus LM, Kraus AP. Carbamoylation of amino acids and proteins in uremia. Kidney Int 2001; 59: 102–107. Cranenburg E, Brandenburg VM, Vermeer C et al. Uncarboxylated matrix Gla protein (ucMGP) is associated with coronary artery calcification in haemodialysis patients. Thromb Haemost 2009; 101: 359–366. Schurgers LJ, Teunissen KJ, Knapen MH et al. Novel conformation-specific antibodies against matrix gamma-carboxyglutamic acid (Gla) protein: undercarboxylated matrix Gla protein as marker for vascular calcification. Arterioscler Thromb Vasc Biol 2005; 25: 1629–1633. Kaesler N, Schettgen T, Mutucumarana VP et al. A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity. Anal Biochem 2012; 421: 411–416. Smith PK, Krohn RI, Hermanson GT et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985; 150: 76–85. Chu P, Huang TY, Williams J et al. Purified vitamin K epoxide reductase alone is sufficient for conversion of vitamin K epoxide to vitamin K and vitamin K to vitamin KH2. Proc Natl Acad Sci USA 2006; 103: 19308–19313. Prochaska HJ, Santamaria AB. Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers. Anal Biochem 1988; 169: 328–336. Westenfeld R, Schaefer C, Smeets R et al. Fetuin-A (AHSG) prevents extraosseous calcification induced by uraemia and phosphate challenge in mice. Nephrol Dial Transplant 2007; 22: 1537–1546.
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see commentary on page 232
Impaired vitamin K recycling in uremia is rescued by vitamin K supplementation Nadine Kaesler1, Elke Magdeleyns2, Marjolein Herfs2, Thomas Schettgen3, Vincent Brandenburg4, Danilo Fliser5, Cees Vermeer2, Ju¨rgen Floege1, Georg Schlieper1,6 and Thilo Kru¨ger1,6 1
Department of Nephrology, RWTH University Hospital Aachen, Aachen, Germany; 2VitaK BV, University of Maastricht, Maastricht, The Netherlands; 3Department of Environmental and Social Medicine, University Hospital of the RWTH Aachen, Aachen, Germany; 4 Department of Cardiology, RWTH University Hospital Aachen, Aachen, Germany and 5Department of Internal Medicine IV, Saarland University Medical Centre, Homburg, Germany
In chronic kidney disease, vitamin K–dependent proteins, including the calcification inhibitor matrix Gla protein, are largely uncarboxylated indicating that functional vitamin K deficiency may contribute to uremic vascular calcification. Since the effects of uremia on the vitamin K cycle are unknown, we investigated the influence of uremia and vitamin K supplementation on the activity of the vitamin K cycle and extraosseous calcification. Uremia was induced in rats by an adenine-supplemented diet and vitamin K1 or K2 was administered over 4 and 7 weeks. After 4 weeks of adenine diet, the activity of the vitamin K cycle enzyme c-carboxylase but not the activities of DT-diaphorase or vitamin K epoxide reductase were reduced. Serum levels of undercarboxylated matrix Gla protein increased, indicating functional vitamin K deficiency. There was no light microscopy–detectable calcification at this stage but chemically determined aortic and renal calcium content was increased. Vitamin K treatment reduced aortic and renal calcium content after 4 weeks. Seven weeks of uremia induced overt calcification in the aorta, heart, and kidneys; however, addition of vitamin K restored intrarenal c-carboxylase activity and overstimulated it in the liver along with reducing heart and kidney calcification. Thus, uremic vitamin K deficiency may partially result from a reduction of the c-carboxylase activity which possibly contributes to calcification. Pharmacological vitamin K supplementation restored the vitamin K cycle and slowed development of soft tissue calcification in experimental uremia. Kidney International (2014) 86, 286–293; doi:10.1038/ki.2013.530; published online 15 January 2014 KEYWORDS: gamma-carboxylase; nephropathy; vascular calcification; vitamin K
Correspondence: Thilo Kru¨ger, Division of Nephrology and Clinical Immunology, University Clinic of the RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany. E-mail: [email protected] 6
All the authors declared no competing interests.
Received 3 January 2013; revised 26 October 2013; accepted 31 October 2013; published online 15 January 2014 286
Vascular calcification is common in chronic kidney disease (CKD). In hemodialysis patients, coronary artery calcification is associated with ischemic cardiovascular disease1 and arterial media calcification is highly correlated with mortality in these patients.2 Possible contributors to the development of vascular calcification in CKD are an increased calcium phosphate product, parathyroid hormone, as well as reduced levels of inhibitors of vascular calcification such as fetuin-A and insufficient activity of matrix Gla protein (MGP).3 MGP is a potent vessel wall–based inhibitor of arterial calcification. MGP knockout mice develop spontaneous calcification of arteries.4 The mechanism by which MGP inhibits vascular calcification may involve bone morphogenetic protein-2 antagonism and a direct calcium-complexing effect.5 MGP is expressed predominantly by vascular smooth muscle cells in the arterial media and chondrocytes. It contains five glutamic acid residues that can be g-carboxylated (Glu-Gla) by the vitamin K–dependent g-carboxylase. This step in which undercarboxylated MGP (ucMGP) changes into carboxylated MGP is essential for the protein to gain full bioactivity.6 The Gla residues are essential for calcium binding.7 The g-glutamyl carboxylase (GGCX; Enzyme Commission number (EC) 4.1.1.90) requires reduced vitamin KH2 as a cofactor. The latter is provided by and recycled in the so-called vitamin K cycle. This cycle is formed by vitamin K epoxide reductase (VKOR; EC 1.1.4.1), GGCX and DT-diaphorase (NAD(P)H-quinone-oxidoreductase, EC 1.6.5.2).8,9 VKOR is inhibited by coumarins such as warfarin, a widely used anticoagulant. Data are accumulating that treatment with coumarins augments vascular calcification in rats and humans,10–13 but with also some conflicting results in humans.14,15 In turn, a high intake of vitamin K2 (MK4) was capable of regressing warfarininduced medial calcification in Wistar rats.16 Hemodialysis patients exhibit markedly increased levels of undercarboxylated proteins such as MGP, osteocalcin and PIVKA (prothrombin induced by vitamin K absence).17,18 Moreover, low levels of carboxylated MGP predict mortality in such patients.19 The origin of the functional vitamin K Kidney International (2014) 86, 286–293
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RESULTS Experimental groups
Two different degrees of kidney disease were induced by adenine feeding for either 4 weeks (a model of mild kidney failure without overt vascular calcification, diets a–d) or 7 weeks (severe kidney failure including tissue calcification, diets e–i, Figure 1). The 7-week approach included a 2-week interphase off adenine in weeks 5 and 6 for recovery. The vitamin K2 diet contained 100 (diets c, d, and g) or 500 mg/ kg MK4 (diet h); vitamin K1 was added at 100 mg/kg (diet i). In the severe model, adenine diet was combined with low protein to increase the penetrance of uremic calcification as described previously.21 All animals survived the 4-week treatment. Seven weeks of adenine resulted in three deaths; necropsy failed to show any obvious reason. The average food intake was 23 (±8) g/day in the healthy groups and 13 (±7) g/day in the adenine groups. Adenine-fed rats lost B16% of body weight that was similar in all adenine-fed groups. Serum parameters
After 4 weeks of adenine treatment, serum creatinine increased sevenfold and urea sevenfold and hyperphosphatemia developed (Supplementary Table S1 online). Adenine diet combined with low-protein diet did not affect creatinine and phosphate serum levels after 4 weeks compared with adenine diet with normal protein (data not shown), but at 7 weeks of treatment, creatinine levels were increased 10-fold (Supplementary Table S1 online). Serum phosphate levels in the vitamin K2 high-dose group (500 mg/kg þ adenine) were significantly lower than in CKD (after 7 weeks of adenine based on low protein, Supplementary Table S2 online). Serum calcium was unchanged in all groups at every time point (Supplementary Table S1 online). Compared with corresponding controls, significantly higher levels of serum ucMGP were measured after 4 weeks of CKD (group b, Figure 2) but not after 7 weeks (not shown). Systolic blood pressure levels measured at the beginning and the end of each treatment period were similar in all groups (not shown). Kidney International (2014) 86, 286–293
Standard diet (SD) Adenine diet (AD) AD + Vit K2 (100 mg/kg) SD + Vit K2 (100 mg/kg) Standard diet (SD; low protein*) AD*
SD*
AD*
AD + Vit K2 (100 mg/kg)*
SD + Vit K2 (100)*
AD + Vit K2 100*
AD + Vit K2 (500 mg/kg)*
SD + Vit K2 (500)*
AD + Vit K2 500*
AD + Vit K1 (100 mg/kg)*
SD + Vit K1 (100)*
AD + Vit K1 100*
0
4 Weeks
6
7
Figure 1 | Overview of the different treatment groups. Seven weeks of treatment contained a 2-week interphase without adenine supplementation. *Protein content was lowered to 2.5% in the 7-week groups (see Supplemetary Table S2 online). All diets contained 5 mg/kg vitamin (Vit) K1 (except diet in group i).
*
15,000 ucMGP (nmol/l)
deficiency in CKD is only partially understood. Reduced vitamin K intake has been described in dialysis patients,20 but we reasoned that this cannot fully explain the marked functional vitamin K deficiency. The aims of this study were, first, to assess the activity of the enzymes of the vitamin K cycle in short-term experimental uremia and, second, to investigate whether vitamin K supplementation with both vitamin K1 or vitamin K2 improves soft tissue calcification and modulates enzyme activities in long-term kidney failure. Finally, we attempted to characterize the origin of altered GGCX activity in uremia by in vitro coincubation with three major uremic toxins: urea, indoxyl sulfate, and p-cresol.
*
12,500 10,000 7500 5000 2500
0 Adenine
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+
+
–
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5
5
5
5
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–
–
100
100
Weeks
4
4
4
4
Figure 2 | Undercarboxylated matrix Gla protein (ucMGP) measured in serum after 4 weeks of treatment. *Po0.05.
Tissue content of vitamin K and ucMGP
Hepatic vitamin K1 content was significantly increased in rats receiving 100 mg/kg vitamin K1 (group i, Figure 3a), whereas vitamin K2 (MK4) content was not affected in rats receiving 500 mg/kg vitamin K2 (group h, Figure 3b). Intrarenal vitamin K levels were not affected by either supplement (not shown). Aortic quantification of ucMGP immunohistology showed significantly increased positive areas only in CKD animals without vitamin K supplementation after 7 weeks (group f, Figure 4). Treatment with vitamin K abrogated this significant increase. Vascular calcification colocalized with ucMGP staining (Supplementary Figure S1 online). Enzyme activities
An overview of the changes in vitamin K cycle enzyme activity is displayed in Table 1. 287
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b 1500 ** 400
**
**
MK4 (ng/g) liver
Vit K1 (ng/g) liver
a **
200
0 Adenine – Protein (%) 2.5 K1 (mg/kg) 5 K2 (mg/kg) – 7 Weeks
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500 0
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 3 | Hepatic vitamin K content. (a) Vitamin K1 measured in liver tissue after 7 weeks of diet; (b) MK4 measured in liver tissue after 7 weeks of diet. **Po0.01.
Table 1 | Overview of the changes in enzyme activities in the vitamin K cycle (GGCX, DT-diaphorase, and VKOR) compared with corresponding control group (a for a–e; e for f–i)
ucMGP (positive area %)
5 4 3
*
GGCX Group
2 1
0 Adenine – Protein (%) 2.5 K1 (mg/kg) 5 K2 (mg/kg) – Weeks 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 4 | Quantification of undercarboxylated matrix Gla protein (ucMGP) staining in aortas after 7 weeks of treatment. *Po0.05.
(a) Compared with non-CKD controls (groups a, d, and e), GGCX was significantly less active in the uremic groups after 4 (groups b and c) and 7 weeks (groups f and g) weeks in kidney tissues (Figure 5a and b) but not in liver tissue (Figure 5c and d). High-dose vitamin K1 or K2 significantly increased GGCX activity in kidney tissue and exceeded baseline values in liver tissue after 7 weeks (Figure 5b and d). Reduced GGCX activity was also found in aortic tissue after 7 weeks of adenine diet (significant in t-test) that was restored by high intake of vitamin K1 or K2 (Figure 6). After 4 weeks of adenine diet, no influence of vitamin K treatment was detected in aortic tissue (not shown). Urea can modify molecular enzyme activities by carbamylation of lysine residues. In vitro coincubation of 50 mmol/l urea with microsomes from healthy animals did not lead to a reduction of GGCX activity (data not shown). No changes in enzyme activity were found after in vitro incubation with p-cresol or indoxyl sulfate. Adenine itself did not show any effect on GGCX activity (not shown). (b) VKOR activity did not differ significantly between the groups and ranged from 0.4 to 2.6 mmol/g/min in kidney 288
a (healthy control) b (CKD) c (CKD þ K2) d (healthy þ K2) e (healthy LP ctrl.) f (CKD) g (CKD þ K2) h (CKD þ high K2) i (CKD þ K1)
DT-diaphorase
Kidney Liver Aorta Kidney 2 k k 2 2 kk kk k k
2 (k) (k) 2 m (k) (k) mm mm
2 nm nm nm 2 k k 2 2
2 m m 2 2 2 m 2 2
VKOR
Liver Kidney Liver 2 m 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
2 2 2 2 2 2 2 2 2
Abbreviations: CKD, chronic kidney disease; ctrl., control; GGCX, g-glutamyl carboxylase; LP, low protein; nm, not measurable; VKOR, vitamin K epoxide reductase. Arrows depict changes in enzyme activity.
tissue and from 0.4 to 7.1 mmol/g/min in liver tissue (Table 1 and Supplementary Table S2 online). (c) DT-diaphorase was significantly more active in kidney and liver tissue in the 4-week CKD model (group b) (3.6fold and 2.7-fold, respectively, Figure 7a and c). There was a significant correlation between decreased GGCX and increased DT-diaphorase activities for kidney tissue after 4 weeks (R2 ¼ 0.34, P ¼ 0.013). Treatment with adenine over 7 weeks increased DT-diaphorase activity nonsignificantly in kidneys but reached significance after 100 mg/kg vitamin K2 treatment compared with controls (Figure 7b). DT-diaphorase activity in the liver was not changed in all 7-week treatment groups (Figure 7d). GGCX gene expression
The GGCX gene expression assessed by reverse transcriptase–PCR in liver tissue was similar in all groups (data not shown). Calcium measurements
After 4 weeks of treatment, adenine treatment increased calcium content significantly in aortas and kidneys compared with healthy controls (group a, Supplementary Figure S2I þ V Kidney International (2014) 86, 286–293
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GGCX (nmol/g/min) kidney
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*
** ** **
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** **
** **
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0 Adenine Protein (%) K1 (mg/kg) K2 (mg/kg) Weeks
*
**
0 – 17.35 5 – 4
+ 17.35 5 – 4
+ 17.35 5 100 4
– 17.35 5 100 4
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 5 | The c-glutamyl carboxylase (GGCX) activity. In kidneys after 4 (a) and 7 (b) weeks and in liver after 4 (c) and 7 (d) weeks (mean±s.d.). *Po0.05 and **Po0.01.
*
GGCX (nmol/g/min) aorta
1000
Quantification of calcified areas by von Kossa staining in aortas showed no statistical significance between all groups. Overt vascular calcification was present after 7 weeks of adenine treatment in 7 of 10 animals but the remainder exhibited almost no calcification. Quantification of stained areas of aortic specimens showed a mean 7.7-fold increase after 7 weeks of CKD compared with the status after 4 weeks (Supplementary Figure S3I þ II online). We did not detect significant changes in the calcium content in pulmonary tissue (not shown).
*
100
10
1 Adenine Protein (%) k1 (mg/kg) k2 (mg/kg) Weeks
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 6 | The c-glutamyl carboxylase (GGCX) activity in aortas after 7 weeks (mean±s.d.) of treatment. *Po0.05.
online). Seven weeks of adenine treatment (group f) significantly increased the calcium content in heart, kidney, and aorta (the latter significant in t-test, Supplementary Figure S2II, IV þ IV online). The 4-week vitamin K2 treatment reduced calcium deposition nonsignificantly in the aorta and significantly in kidneys of uremic animals (group c). After 7 weeks, only high-dose vitamin K treatment reduced calcium content in tissues reaching significance in heart and kidneys for vitamin K2 and vitamin K1 in kidneys alone (groups h and i, Supplementary Figure S2 online). Kidney International (2014) 86, 286–293
DISCUSSION
High serum levels of undercarboxylated proteins such as MGP demonstrate that the large majority of uremic patients are more or less vitamin K deficient.17,18 Here we used a rat model of CKD to investigate the effect of uremia on the vitamin K cycle and subsequently uremic calcification. Furthermore, we assessed in a two-step approach whether correcting vitamin K deficiency in this setting rescues the uremic alterations and whether differences exist between treatment with vitamin K1 or K2. In the first step, after 4 weeks of kidney failure, we measured the activity of the enzymes of the vitamin K cycle, that is, at a time point when the animals did not exhibit overt calcifications. Here, vitamin K deficiency was already present as evidenced by increased ucMGP serum levels. Our first major finding was that the activity of GGCX was already reduced at this stage of CKD, possibly contributing to the subsequent development of vascular calcification found at 289
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Kidney
DT-diaph. (nmol/g/min)
a
b
* *
6000 4000
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0 Adenine Protein (%) K1 (mg/kg) K2 (mg/kg) Weeks
0 – 17.35 5 – 4
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+ 17.35 5 100 4
– 17.35 5 100 4
– 2.5 5 – 7
+ 2.5 5 – 7
+ 2.5 5 100 7
+ 2.5 5 500 7
+ 2.5 100 – 7
Figure 7 | The DT-diaphorase activity. In kidneys after 4 (a) and 7 (b) weeks and in liver after 4 (c) and 7 (d) weeks (mean±s.d.). *Po0.05.
week 7. As the VKOR activity was not reduced, and the DTdiaphorase was even more active, this suggests that in uremia GGCX is the rate-limiting enzyme in the vitamin K cycle and a potential major contributor to diminished vitamin K availability in tissues (Table 1). The increased DT-diaphorase activity may be a compensatory mechanism intended to provide higher intracellular levels of the reduced form of vitamin K, as suggested by the significant correlation between GGCX and DT-diaphorase activities after 4 weeks in kidney tissue. Why GGCX activities were higher in the control group after 7 weeks compared with 4 weeks remains unknown. An influence of low-protein diet—the only significant difference between these two groups—on GGCX activity has not been reported so far. In addition, low dietary vitamin K intake has been noted in dialysis patients20 augmenting vitamin K deficiency in uremia. Indeed, dietary vitamin K2 supplementation (100 mg/kg, group c) lowered serum levels of ucMGP in this study. These experimental observations parallel those made recently in hemodialysis patients where ucMGP serum levels were elevated and could be markedly reduced by dietary vitamin K2 supplementation.17 Our 4-week CKD model contributes to the understanding of ucMGP action in the early stage. Vitamin K treatment resulted in significantly lower calcium tissue content in kidneys with a parallel trend in the aorta, strengthening the rationale for our planned vitamin K interventional trial VitaVasK targeting cardiovascular calcification in hemodialysis patients (www. clinicaltrials.gov NCT01742273). To further analyze the influence of vitamin K treatment on calcifications, we also studied rats after 7 weeks of kidney 290
failure, when overt vascular calcification was established. Calcification was evident in von Kossa–stained aortic specimens. The activity of GGCX was still reduced after 7 weeks of adenine. Our second major finding was that high dietary vitamin K2 (500 mg/kg) or K1 (100 mg/kg) restored and even overstimulated the activity of GGCX in the liver. In addition, this also significantly reduced calcification of the kidneys and heart. In aortic tissue, we failed to reach significant differences in calcium contents that might be because of the high interindividual variability in the extent of aortic calcification in this model. In the heart and kidneys, lower dosages of vitamin K1 (100 mg/kg) than vitamin K2 (500 mg/kg) were comparably effective in preventing calcification. Although prior observations favor vitamin K2 above K1 in acting on MGP and vascular calcification,22 our data suggest that vitamin K1 is also active in these processes. Vitamin K1 can be converted to K2 as proposed before,23 but increased hepatic and renal vitamin K2 tissue concentrations were not detected following vitamin K1 supplementation that suggests that the effects detected may be because of the vitamin K1 itself. However, we cannot fully exclude, for example, aortic vitamin K1 to K2 conversion. Why in our study the supplementation of 100 mg/kg vitamin K1 was even more potent than 100 mg/ kg vitamin K2 in increasing the GGCX activity and reducing calcifications needs further investigation. Our data are consistent with supplementary vitamin K2 reducing vascular calcification in a warfarin16 and the adenine model (here vitamin K1).24 These experimental data are also supported by a positive correlation between the progression of arterial calcification and low vitamin K1 serum level of patients.25 Kidney International (2014) 86, 286–293
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Compared with the daily dietary recommendation for humans (1 mg/kg body weight), the dosage of vitamin K supplements was markedly higher in our rat experiments (up to 15 mg/kg body weight). Such high dosages were chosen given that there is no evidence of toxicity from even very high vitamin K doses in humans26 and based on dosages in prior experimental studies.16 Unexpectedly, after 7 weeks of adenine treatment, ucMGP levels were on average 41% lower than after 4 weeks of adenine treatment. One explanation is the accumulation of ucMGP in calcified areas in uremia as we found an increased ucMGP staining in group f. In addition, transdifferentiation of MGP-producing vascular smooth muscle cells into an MGP-negative osteoblastic phenotype as published before27 might further reduce ucMGP serum levels. We and others have observed that MGP-synthesizing vascular smooth muscle cells undergo apoptotic processes in calcification.28,29 Finally, we investigated potential mechanisms underlying the low GGCX activity in CKD. The reduced activity was not because of a lower GGCX mRNA expression, although vitamin K is known to enter the nucleus and to influence gene expression, for example, of bone markers.30 To assess post-transcriptional alterations, we focused on carbamylation of this enzyme by the elevated urea concentrations in CKD. As urea levels rise in CKD, it is spontaneously transformed into cyanate that is known to irreversibly carbamylate lysine residues. Reversible modifications occur at tyrosine, serine, threonine, or cysteine residues, resulting in modification of the activities of enzymes.31 However, in this study, in vitro urea concentrations similar to uremic serum levels failed to affect GGCX enzyme activity. Two further uremic toxins tested in vitro, p-cresol and indoxyl sulfate, did not result in altered GGCX activity either. Adenine itself as a suppressor of GGCX activity was excluded. Therefore, the mechanism of reduced g-carboxylase activity presently remains unknown. A limitation of the study besides the supraphysiological vitamin K dosage is the translation of rodent data to CKD patients. Hyperparathyroidism and disturbed bone and mineral homeostasis occur in the adenine model and likely contributed to the development of vascular calcification. The relative contribution of reduced GGCX activity and thus vitamin K deficiency to this process is unknown at present. The effect of kidney disease on GGCX activity needs to be confirmed in human tissues as well. Furthermore, the agent decreasing GGCX activity needs to be identified. Finally, results of the ucMGP assay obtained here cannot be compared with human results as the assay for yet unknown reasons yields different findings in both species. In summary, we found reduced activity of GGCX in uremia in several tissues as a potential contributor to the development of vascular calcification even before overt calcification became present. This possibly contributes to the observed functional vitamin K deficiency in dialysis patients. Reduced GGCX activity might represent a key mechanism explaining increased ucMGP serum levels and excess cardiovascular calcification in CKD in rats and Kidney International (2014) 86, 286–293
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humans. Vitamin K1 and vitamin K2 were able to increase GGCX activity and at least partially reduced extraosseous calcium content in the adenine nephropathy model. Thus, dietary vitamin K supplementation in CKD not only reverses the low intake, but also rescues endogenous vitamin K recycling. As a consequence, the supplementation represents a promising therapeutic approach to the massively increased extraosseous calcification in CKD. MATERIALS AND METHODS Animals and diets Ten male Wistar rats (initial weight 365±18 g; Charles River, Sulzfeld, Germany) in each group were fed the diets shown in Figure 1 (all diets were from Altromin, Lage, Germany; Supplementary Table S2 online). Food and water were administered ad libitum. The rats were housed in a 12-h day and night cycle. Animals were killed under isoflurane anesthesia by heart puncture. Before harvesting, organs were perfused with 20 ml ice-cold phosphate-buffered saline. All animals were treated in accordance with the requirements of the Federation of the European Laboratory Animals Science Associations. Biochemistry Serum was collected by tail vein puncture or finally by puncture of the left ventricle and spun after sedimentation at 2000g for 10 min. Urine was collected for 24 h before killing. Serum and urine parameters were measured by clinical routine laboratory diagnostics. Total ucMGP was measured in serum by competitive enzymelinked immunosorbent assay by a monoclonal antibody (MGP sequence 35-49; VitaK BV, Maastricht, The Netherlands).32 Blood pressure was measured noninvasively by determining the tail blood volume pressure by an occlusion tail cuff on a Kent Scientific CODA system (Kent Scientific, Torrington, CT) at weeks 0, 2, 4, and 7. Vitamin K measurements and ucMGP staining Vitamin K was extracted from snap-frozen tissues by hexane followed by cleaning using a Sep-Pak plus silica cartridge (Waters, Eschborn, Germany). Vitamin K was eluted with 3% diethylether in hexane that was evaporated in a argon stream and diluted in isopropanol for injection in high-pressure liquid chromatography (HPLC; Merck Hitachi, Tokyo, Japan). Vitamin K1 was separated in reversed-phase HPLC (with a Max RP C12 column; Phenomenex, Aschaffenburg, Germany). Isocratic methanol at pH 5 was used as a mobile phase. Vitamin K1 and MK4 were detected at 246 nm. Vitamin K was quantified by external standards. UcMGP in aortic tissues was visualized by incubation with the anti-GluMGP antibody (VitaK BV) as described previously.33 Preparation of microsomes Microsomes were isolated as described earlier.34 Briefly, the postmicrosomal fraction was gained by centrifugation at 10,000 g of homogenized tissue in 300 mmol/l sucrose buffer, followed by ultracentrifugation at 100,000 g for 1 h. Microsomes were stored at 80 1C. Protein content was determined by the Pierce Bicinchoninic Acid method as described by the manufacturer (Thermo Fisher Scientific, Waltham, MA).35 291
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Enzyme activities (a) The g-carboxylase activity was measured by carboxylation of FLEFLK-FITC followed by reversed-phase HPLC coupled with fluorescence detection.34 Vitamin K1 was reduced to KH2 by incubation in a mixture of 20 mmol/l dithiothreitol, 50 mmol/l NaCl, and 2 mmol/l Tris at 37 1C for 12 h in the dark. For a selective influence of uremic toxins or adenine on the g-carboxylase activity, microsomes from healthy rats were first incubated with urea (50 and 500 mmol/l) or indoxyl sulfate (250 and 500 mmol/l) or p-cresol (100 mmol/l) at room temperature. Adenine was used at 0, 2.5, 5, 10, and 22 mmol/l. Urea was obtained from Bio-Rad (Bio-Rad, Hercules, CA). Vitamin K1, dicoumarol, indoxyl sulfate, p-cresol, and isopropanol were purchased from Sigma Aldrich (Munich, Germany). NADPH, MTT, and glucose-6-phosphate were from Carl Roth (Karlsruhe, Germany); glucose-6-phosphate-dehydrogenase, AgNO3, NaOH, Na2CO3 were from Merck (Darmstadt, Germany), and all other chemicals were from AppliChem (Darmstadt, Germany). (b) VKOR activity was measured by conversion of vitamin K1 2,3epoxide (K14O; stable for at least 6 months at 80 1C (99% purity)) to vitamin K1.36 The reaction mixture, with a total volume of 200 ml, contained 500 mg microsomal protein, 8 ml 5 mmol/l vitamin K epoxide, and the reaction was started by the addition of 5 ml 200 mmol/l dithiothreitol. The reaction was stopped after 60 min at 30 1C in the dark by 500 ml 0.05 M AgNO3 in isopropanol. The amount of vitamin K was quantified by using the HPLC setup. For epoxidation, vitamin K1 was diluted in hexane and mixed with 0.5 M NaOH, 0.2 M Na2CO3, and 5% H2O2 for 12 h at 37 1C in the dark. Vitamin K14O quality was monitored by HPLC measurements. (c) DT-diaphorase activity was analyzed in the kidney and liver by the standard assay37 with NADPH as electron donor and menadione as electron acceptor. A total volume of 150 ml reaction mixture contained 7.5 ml 0.5 M Tris-Cl (pH 7.4), 100 mg bovine serum albumin, 1 ml 1.5% Tween-20, 0.1 ml 7.5 FAD, 1 ml 150 mmol/l glucose-6-phosphate, 90 ml 50 mmol/l NADPH, 300 U yeast glucose-6-phosphate-dehydrogenase, and 45 mg MTT. The assay was carried out in the presence and absence of 3 mmol/l dicoumarol and 50 mmol/l menadione with 250 mg microsomal protein. The reaction was stopped after 5 min at 20 1C by a 3 mmol/l dicoumarol and 5 mmol/l KCl. The DTdiaphorase activity was calculated as the specific activity inhibited by dicoumarol. Reduced MTT as the formazan dye was detected at 610 nm on a Tecan sunrise microplate absorbance reader (Tecan, Mennedorf, Switzerland).
Reverse transcriptase–PCR RNA was harvested from RNA later (Qiagen, Hilden, Germany) stabilized tissue using Qiagen RNeasy. Purity and RNA concentration were analyzed with the Agilent RNA 6000 Nano Kit (Agilent, Bo¨blingen, Germany). The reverse transcriptase was performed by the Reverse Transcriptase Core Kit (Eurogentec, Cologne, Germany). The qPCR Core Kit for SYBR Green I (Eurogentec) was applied for a two-step quantification on an Applied Biosystems 7500 Real-Time PCR TagMan system. The GGCX TaqMan gene expression assay (RN00582138) was from Applied Biosystems (Life Technologies, Darmstadt, Germany). The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers used were the following: sense: 50 -ACAA GATGGTGAAGGTCGGTA-30 , antisense: 50 -AGAAGGCAGCCCT GGTAACC-30 , probe: 50 -CGGATTTGGCCGTATCGGACGC-30 . 292
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Calcium determination The extent of vascular and tissue calcium content was measured colorimetrically. Therefore, tissues were lyophilized on a Christ Loc 1mALPHA 1-4 (Martin Christ, Osterode am Harz, Germany) coupled with a vacuum hybrid pump RL 6 (Vacubrand, Wertheim, Germany). Calcium was eluted in 10% formic acid and quantified by the cresolphthalein method (Randox Laboratories, Crumlin, UK). Absorption was measured at 550 nm on a Tecan sunrise microplate absorbance reader. Localization of the calcification was visualized by von Kossa staining as previously described.38 In brief, sections were immersed in 1% aqueous AgNO3 solution for 5 min. After washing, samples were incubated in a solution of 5% NaCO3 and 9.25% formalin for 1 min. After a second rinse, sections were developed using sodium thiosulfate (5% in water) for 5 min and counterstained in 0.1% safranin-O followed by a final rinse using tap water. Statistical analysis The difference between the treatment groups was assessed by oneway analysis of variance followed by Tukey’s multiple comparison test (if not stated elsewhere). Equal variances were tested with Bartlett’s method. D’Agostino and Pearson normality test was performed to check for Gaussian distribution. Statistical significance was defined as Po0.05. DISCLOSURE
All the authors declared no competing interests. ACKNOWLEDGMENTS
We thank Katrin Haerthe for the excellent work in animal keeping and in vitro experiments. Special thanks go to Paul Brenchley for very helpful and inspiring discussion of these experiments. SUPPLEMENTARY MATERIAL Table S1. Final biochemical results at the end of the experiment (groups a-d 4 weeks; groups e-i 7 weeks); Groups referring to Figure 1. Table S2. Composition of diets and treatment duration; Groups referring to Figure 1. Table S3. VKOR activity in kidney and liver (mean±s.d.). Figure S1. Von Kossa and ucMGP staining in rat aortic tissue (100 x); early calcification after 4 weeks of adenine and overt calcification after 7 weeks of adenine diet. Figure S2. Calcium content in tissues (mean±s.d.). Calcium content in aorta (I after 4 weeks; II after 7 weeks), heart (III after 4 weeks; IV after 7 weeks) and kidney heart (V after 4 weeks; VI after 7 weeks). Figure S3. Quantification of von Kossa staining in aortic tissue (mean±s.d.). Supplementary material is linked to the online version of the paper at http://www.nature.com/ki REFERENCES 1.
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