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Magnesium as a Calcification Inhibitor
ACKD
Magnesium as a Calcification Inhibitor Lucie Henaut and Ziad A. Massy Vascular calcification (VC) is associated with elevated cardiovascular mortality rates in patients with CKD. Recent clinical studies of patients with advanced CKD have observed an association between low serum magnesium (Mg) levels on one hand and elevated VC and cardiovascular mortality on the other. These findings have stimulated interest in understanding Mg’s impact on CKD in general and the associated VC in particular. In vitro and preclinical in vivo data indicate that Mg has the potential to protect vascular smooth muscle cells against calcification via several different molecular mechanisms. Accordingly, data from pilot interventional studies in the clinic suggest that oral Mg supplementation reduces VC in patients with CKD. The present review provides an overview of our current understanding of the impact of Mg on the development of VC in patients with CKD. Q 2017 by the National Kidney Foundation, Inc. All rights reserved. Key Words: Chronic kidney disease, Magnesium, Vascular calcification
INTRODUCTION Cardiovascular disease is the leading cause of death in patients with CKD. In particular, vascular calcification (VC) is more prevalent and much more severe in this setting than in the general population.1 This degenerative process is characterized by the accumulation of calcium (Ca) and phosphate salts within the cardiovascular system (notably in the intimal and medial layers of vessels and in cardiac valves) and is associated with elevated cardiovascular mortality.2 Intimal and medial calcification notably contribute to cardiovascular disease and excess cardiovascular mortality in patients with CKD. Intimal VC (a consequence of atherosclerosis) may favor coronary ischemic events, whereas medial VC (which develops along the elastic lamina) increases vessel stiffness, arterial pulse wave velocity, systolic blood pressure, and pulse pressure and thus favors the development of cardiac failure, left ventricular hypertrophy, and diastolic dysfunction. Calcification of cardiac valves is also common in ESRD and is associated with a worse prognosis. Over the last few decades, studies of CKD patients have shown that when VC is accompanied by vessel stiffness, arterial hypertension, left ventricular hypertrophy, and cardiomyopathy, the severity of the condition is a strong, independent predictor of death. It is widely admitted that VC is irreversible once established; none of the treatments tested to date can unambiguously prevent or reverse VC. Accordingly, understanding the pathophysiologic mechanisms involved in the development of VC is of critical importance for identifying the most effective means of prevention and treatment. It has been suggested that along with traditional cardiovascular risk factors, nontraditional risk factors (such as those related to uremic status and disturbed CKD mineral and bone disorder [CKD-MBD]) may explain the elevated prevalence of VC and cardiovascular disease in a CKD setting. CKD-MBD is a systemic disorder characterized by persistently elevated parathyroid hormone (PTH) levels, bone abnormalities, extraskeletal calcification, and a broad spectrum of mineral metabolism disorders (including hypocalcemia, hyperphosphatemia, and hypermagnesemia). Over the last few decades, studies of CKDMBD have focused on the perturbation of Ca/phosphate homeostasis. However, increasing attention had been paid to the impact of magnesium (Mg) homeostasis on Adv Chronic Kidney Dis. 2018;25(3):281-290
CKD-MBD, in view of (1) the metal’s important role in the pathophysiology of the cardiovascular system, and (2) the reported association between Mg disorders and elevated cardiovascular morbidity/mortality. In fact, Mg is involved in the regulation of vascular tone, cardiac rhythm, and platelet-activated thrombosis. The maintenance of normal serum Mg levels (0.75-1 mmol/L) is determined by dietary ingestion and absorption, bone and soft tissue deposition and efflux, and kidney excretion. In CKD, urinary Mg excretion is normal or sometimes even elevated as long as the glomerular filtration rate is above 30 mL/min. Below that value, the kidney excretion of Mg may not be able to balance the dietary intake Mg—which then becomes a major determinant of serum and wholebody Mg levels.3 Although hypermagnesemia in CKD patients is usually mild and asymptomatic (,1.5 mmol/L), severe, symptomatic hypermagnesemia can be induced by exogenous Mg administration. Indeed, administration of Mg-containing drugs (eg, antacids and laxatives) and high-Mg concentrations in dialysate may provoke severe, symptomatic and sometimes even fatal hypermagnesemia. Conversely, the excessive use of diuretics, reduced gastrointestinal uptake (due to acidosis, and poor nutrition and absorption), and a low Mg concentration in dialysate may lead to abnormally low serum Mg levels.3,4 The results of many recent clinical studies show that low serum Mg levels are associated with increased VC and elevated cardiovascular mortality in patients with ESRD. These From the INSERM U1088, CURS, Amiens, France; Division of Nephrology, Ambroise Pare University Hospital, APHP, Boulogne-Billancourt/Paris, France; and INSERM U1018, Team 5, CESP, UVSQ, Villejuif, France. Financial Disclosure: Z.A.M. reports grants for CKD REIN and other research projects from Amgen, Baxter, Fresenius Medical Care, GlaxoSmithKline, Merck Sharp and Dohme-Chibret, Sanofi-Genzyme, Lilly, Otsuka, and the French government as well as personal fees and grants to charities from Amgen, Bayer, and Sanofi-Genzyme. These sources of funding are not necessarily related to the content of the present manuscript. Also see Acknowledgment(s) on page 288. Address correspondence to Ziad A. Massy, Division of Nephrology, Ambroise Pare University Hospital, 9 Avenue Charles de Gaulle, F-92104 Boulogne Billancourt cedex, France. E-mail: [email protected] Ó 2017 by the National Kidney Foundation, Inc. All rights reserved. 1548-5595/$36.00 https://doi.org/10.1053/j.ackd.2017.12.001
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findings have stimulated interest in understanding Mg’s role in CKD in general and the associated VC in particular. The present review provides an overview of our current understanding of the impact of Mg on the development of VC in patients with CKD.
elevated number of Ca/P nanocrystals formed in response to CKD-related loss of circulating inhibitors and hyperphosphatemia may directly promote the osteo/chondrogenic conversion of vascular cells.7 These Ca/P nanocrystals can also be taken up via endocytosis into VSMCs, where subsequent lysosomal degradation inMOLECULAR MECHANISMS OF VASCULAR creases Ca and Pi levels within the intracellular milieu. In an attempt to compensate for this increase, VSMCs CALCIFICATION IN CKD VC is a complex process that involves not only the preciprelease matrix vesicles loaded with Ca/P products. Once this compensatory mechanism is overwhelmed, the intraitation of minerals (due to supersaturated phosphate and cellular Ca/Pi overload triggers apoptosis, resulting in the Ca concentrations in the extracellular milieu, that is, an inorganic component) but also tightly regulated, cellformation of Ca/P-containing apoptotic bodies in the VSMCs. This phenomenon can be accentuated by addimediated process including apoptosis, osteochondrogenic tional Pi uptake through the sodium-dependent phosdifferentiation, matrix vesicle release, and elastin degradaphate transporter Pit-1. The apoptotic bodies and matrix tion (ie, a cellular component).5 Despite the significant vesicles ultimately contribute to a positive feedback loop recent growth in our knowledge of VC, the order of by releasing nanocrystals appearance and time course into the surrounding milieu of these 2 steps in vivo is still CLINICAL SUMMARY subject to debate. and thus amplifying the calcification process. Under normal physiolog Vascular calcification (VC) is a major contributor to ical conditions, blood vessels cardiovascular disease and excess cardiovascular and cardiac valves are proMAGNESIUM AS A mortality in patients with CKD. tected from supersaturated CALCIFICATION Epidemiologic studies indicate a possible link between low concentrations of serum Ca INHIBITOR IN CKD serum magnesium (Mg) levels and the development of VC and phosphate by a number in both the general population and patients with CKD. Lessons Learned from of active inhibitors. Pyro Data from in vitro studies and animal models suggest that In Vitro Studies phosphate, adenosine, maMg supplementation protects vascular cells against In early reports, Posner and trix Gla protein (MGP), calcification. fetuin-A, bone morphogeassociates demonstrated that Mg could directly innetic protein-7 (BMP7) and The results of pilot clinical studies suggest that oral Mg terfere with the process other factors have been supplementation reduces cardiovascular calcification. shown to prevent the transwhereby Ca and P crystallize into hydroxyapatite; the reformation of soluble, amorsearchers found that the addition of Mg to metastable phous calcium phosphate (Ca/P) complexes into harmful, stable hydroxyapatite crystals. In the CKD popuCa/P solutions inhibited the formation of Ca/P apatite and Ca-acidic phospholipid–phosphate complexes.8,9 lation, a decrease in levels of active inhibitors and a This inhibition stabilized amorphous Ca/P and favored simultaneous increase in levels of active inducers the formation of the Mg-containing crystal whitlockite of calcification (including hyperphosphatemia, hypercal[(Ca,Mg)3(PO4)2]. Hydroxyapatite had long been thought cemia, inflammatory cytokines, oxidative stress, uremic to be the sole mineral phase present in human uremic toxins, and advanced glycation end products) are responVC.10 However, a recent synchrotron study revealed the cosible for the extremely high prevalence of intimal and localization of whitlockite with hydroxyapatite in the armedial VC and valvular calcification. Hyperphosphatemia teries of patients with moderately impaired kidney (a typical manifestation of CKD-MBD) is the calcification function (CKD Stages 2-4).11 This observation suggests inducer most strongly associated with VC in CKD. Within that Mg (like the calcification inhibitors fetuin-A, MGP, the vascular system, elevated levels of inorganic phosand osteopontin) is closely associated with microcalcificaphate (Pi) and extracellular Ca and the deposition of amortion. phous Ca/P lead to the conversion of vascular smooth In a series of in vitro experiments, the use of MgCl2 muscle cells (VSMCs), resident fibroblasts, and quiescent reportedly prevented Pi-induced calcification and the ostevalve interstitial cells into osteochondrogenic cells. During ogenic differentiation of human, bovine, and rodent this conversion, the vascular cells downregulate the VSMCs by increasing or restoring the activity of the Mg expression of VC-inhibiting genes (such as MGP and transporter TRPM7. These effects were associated with BMP7) and start to express bone-promoting genes (such greater expression of anticalcification proteins (including as BMP2, RUNX2, and alkaline phosphatase) that act to osteopontin, BMP7, and MGP) and a decrease in VSMC transform amorphous Ca/P deposits into a wellapoptosis. MgCl2 was also shown to reduce Ca entry organized, calcified, crystalline structure.6 This phenomeinto VSMCs; this is of particular interest because elevated non is associated with (1) the secretion of a procalcifying Ca is known to upregulate Pit-1, which in turn increases Pi matrix rich in type I collagen and (2) the production of matrix metalloproteinases 2 and 9 (known to promote elastin uptake.12 According to recent studies, the Mg-associated inhibition of VSMC calcification is mainly mediated by indegradation and the subsequent release of highly procalcihibition of the Pi-induced Wnt/b-catenin signaling fying elastin peptides). According to a recent report, the Adv Chronic Kidney Dis. 2018;25(3):281-290
Magnesium and Vascular Calcification
pathway.13 In line with these data, Herencia and associates14 recently demonstrated that angiotensin II was able to prevent Pi-induced calcification and the osteogenic differentiation of human VSMCs by stimulating Mg influx and the subsequent inhibition of the canonical Wnt/b-catenin signaling pathway. It is noteworthy that addition of MgCl2 to calcified VSMC cultures decreased the Ca content, downregulates RunX2 and Osterix mRNA expression, and upregulates MGP and OPG mRNA expression.13 This effect was not observed when 2aminoethoxy-diphenylborate (an inhibitor of cellular Mg transport via TRPM7) is added to the culture medium— suggesting that calcification is not only halted but also even decreases through a cell-mediated process following addition of Mg. More recently, MgCl2 was reported to locally activate the calcium-sensing receptor (CaSR) expressed by VSMCs and thus inhibit Pi-induced VSMC calcification and osteogenic differentiation.15 The increased CaSR expression associated with this effect is of particular interest, since loss of a functional CaSR had previously been reported in calcified VSMCs. These data are in agreement with other reports in which vascular CaSR activation protects against VC.16 Our group recently investigated the role of microRNAs in the Mg-mediated protection against VC.17 Exposure to MgCl2 inhibited the Pi-induced decrease in miR-30b, miR-133a, and miR-143 and modulated the latter’s known targets (Runx2, Smad1, and Osterix, respectively). These data prompted a potential mechanistic hypothesis for the Mg-induced downregulation of osteogenic markers during the course of Pi-induced VC. MgCl2 supplementation also reportedly downregulates the osteogenic reprogramming of VSMCs induced by hydroxyapatite particles— confirming that Mg’s prime effect does not consist in preventing the initial Ca/P particle from forming.15 On these lines, we recently looked at whether the effects on live VSMCs mediated in vitro by MgCl2 also occur during passive Ca/P deposition on fixed VSMCs. We found that MgCl2 failed to prevent Pi-induced Ca/P deposition on fixed cells, which suggests that cells must be alive if Mg ions are to exert a protective effect.18 In an attempt to characterize this process in more detail, we recently used micro-Fourier transform infrared spectroscopy, field effect scanning electron microscopy, and energy dispersive Xray spectrometry to investigate Mg’s involvement in crystal formation in in vitro cultures of primary human aortic VSMCs. As expected, only Ca/P apatite was detected in cells incubated with elevated Ca 3 Pi medium. Supplementation with MgCl2 did not alter the crystals’ composition or structure. However, a qualitative analysis suggested that 5 mM Mg reduced the number and intensity of the Ca/P apatite micro-Fourier transform infrared spectroscopy spots. Hence, our results suggest that Mg has an active, cellular role in attenuating VC rather than a physicochemical effect on the growth, composition or structure of Ca/P apatite crystals. Our data differ from those gathered in the synchrotron study by Fischer and associates19 of whether moderately impaired kidney function (CKD Stages 2-4) might affect the physiochemical composition and/or the formation of whitlockite in arterial Adv Chronic Kidney Dis. 2018;25(3):281-290
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atherosclerotic microcalcifications. The researchers found that arterial microcalcification in atherosclerotic patients with normal or reduced kidney function consisted of Ca/ P (either as amorphous deposits or as apatite) and whitlockite. Although the proportion of patients with a given type of mineral phase in their microcalcifications did not differ from one group to another, the proportion of whitlockite in the microcalcifications was significantly greater in atherosclerotic individuals with normal kidney function. In the study by Fischer and associates, the serum Mg level was not significantly associated with the glomerular filtration rate at the time of surgery carotid stenosis. Hence, these data indicate that CKD affects the local Mg homeostasis and thus contributes to the physicochemical composition of microcalcifications in atherosclerotic patients. Mg’s putative effects on VSMC calcification are presented in Figure 1. Lessons Learned from In Vivo Studies In studies of several different non-CKD animal models, Mg has repeatedly been shown to prevent the progression of VC (Table 1). For example, the effects of dietary Mg have been extensively explored in the Abcc62/2 mouse model of Pseudoxanthoma pseudoxanthoma elasticum.20-22 elasticum is an autosomal recessive multisystem disorder characterized by the mineralization of ectopic connective tissue. The clinical manifestations of pseudoxanthoma elasticum primarily affect the skin, eyes, and cardiovascular system. In the Abcc62/2 mouse, increasing the dietary intake of Mg significantly reduced the calcification of kidney blood vessels and connective tissue,21,22 while restriction of dietary Mg intake accelerated ectopic mineralization.20 Treatment of Abcc62/2 mice with Mg carbonate–containing phosphate binder prevented the onset and the progression of calcification, whereas treatment with lanthanum carbonate had no effect.23 In the inbred DBA/2 mouse model, the combination of a low Mg intake and a high Pi intake was associated with severe calcification of cardiac and kidney tissues, while the combination of a high Mg intake and a low Pi intake partially prevented cardiac calcification.24 In a rat model in which the juxtacardial aorta was grafted to the infrarenal aorta, dietary supplementation with a combination of Mg, alkali citrate, and bases prevented the development of medial calcification within the graft.25 In Sprague–Dawley rats supplemented with vitamin D3 and nicotine, administration of Mg sulfate decreased the aortic alkaline phosphatase activity and the severity of aortic calcification.26 Therefore, increasing the dietary Mg intake appears to protect against VC in non-CKD animals. In male Sprague-Dawley rats with adenine-induced CKD, Mg supplementation attenuated the severity of calcitriol-induced calcification in the abdominal aorta (by 51%), the iliac artery (by 44%), and the carotid artery (by 46%). These effects were associated with a 20%-50% increase in vascular Mg content and a reduction in calcitriol-induced vascular TRPM7 deficiency. Therefore, the use of Mg supplementation to modify calcitriol’s adverse effects may be a plausible treatment approach in
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Amorphous Ca/P High Ca
High Pi
Magnesium HA crystals Ca-channels
TRPM7
mir30b mir133a mir143
CaSR
Pit1/2
Wnt-β-catenin
Loss of inhibitors Osteogenic differentiation Apoptosis
Vascular calcification
VSMC
Figure 1. Cellular and molecular mechanisms by which Mg prevents VSMC calcification in vitro. Within the vascular system, elevated levels of inorganic phosphate (Pi) and extracellular calcium (Ca) and the deposition of amorphous calcium phosphate lead to vascular smooth muscle cells (VSMCs) osteogenic conversion. During this process, VSMC downregulates the expression of VC-inhibiting genes (such as MGP and BMP7) and starts to express bone-promoting genes (such as BMP2, RUNX2, and alkaline phosphatase) that act to transform amorphous calcium phosphate deposits into a wellorganized, calcified, crystalline structure. Mg enters the cell via TRPM7 and restores the balance between expression of calcification promoters and inhibitors. These effects are mediated through the blockade of Pi-induced Wnt/b-catenin signaling pathway. Exposure to Mg also inhibits Pi-induced decrease in miR-30b, miR-133a, and miR-143, and subsequent VSMC osteogenic transition. Besides, Mg may prevent VC through local activation of the calcium-sensing receptor (CaSR) expressed by VSMC. Moreover, exposure to Mg reduces VSMC apoptosis and subsequent release of procalcific apoptotic bodies. Mg also functions as a Ca-channel antagonist and thus inhibits the entry of Ca into the cells. Some reports show that Mg may interfere with the process of vascular calcification by inhibiting transformation of amorphous Ca/P to apatite. However, this fact remains a matter of debate. Dotted lines: further studies are needed to fully demonstrate the concept. Abbreviations: Ca/P, calcium phosphate; HA, hydroxyapatite; Mg, magnesium.
the growing proportion of CKD patients receiving this active metabolite of vitamin D.27 In male Wistar Kyoto rats with adenine-induced CKD, treatment with either a phosphate binder containing Mg carbonate and calcium acetate (CaMg) or sevelamer effectively controlled serum phosphate levels and reduced aortic calcification. In this model, treatment with CaMg displayed no additional effects on VC, relative to sevelamer alone.28 A synchrotron-based X-ray diffraction analysis of aortic calcifications in CaMg2, sevelamer- and vehicle-treated animals showed that the mineral precipitates in the various groups contained Ca hydroxyapatite alone. These data are inconsistent with the previous observation by Verberckmoes and associates29 of whitlockite in aortic calcifications in male Wistar Kyoto rats with adenine-induced CKD. In the latter study, calcitriol supplementation was associated with increased deposition of whitlockite in the aorta. The researchers suggested that this deposition resulted from the calcitriol-stimulated intestinal absorption of Mg. Changes in Mg status in animal models have also been shown to promote various systemic disorders, known as
classical inducers of VC. Among them, Mg deficiency was reported to promote systemic inflammation,30,31 to influence vitamin D metabolism,32 and to trigger disturbances in mineral and bone metabolism.33-35 Whether the protective effects of Mg on VC are the result of local actions on cardiovascular tissues or of indirect, systemic actions cannot be determined in the in vivo setting and needs further investigations. Lessons Learned from Observational and Epidemiological Studies The possible clinical relationship between serum Mg levels and VC has been assessed in several observational studies (Table 2). In an early observational study of 44 patients with ESRD treated with continuous ambulatory peritoneal dialysis, Meema and associates36 reported that the mean serum Mg concentration was lower in individuals with X-ray evidence of peripheral arterial calcification (ie, in the hands, ankles, and feet) than in individuals lacking such evidence. No intergroup differences were found for other serum biochemistry parameters with known or putative roles in VC (Ca, phosphate, total alkaline Adv Chronic Kidney Dis. 2018;25(3):281-290
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Table 1. Animal Models Used to Investigate the Influence of Mg on VC Animal’s CKD Status Non-CKD animals
Animal Model
Mg Treatment
Ref 20
Mg carbonate-enriched diet (1.1%)
[Mineralization of the dermal sheath of vibrissae, cartilage, kidney, heart, arteries YKidney blood vessel calcification YMineralization of vibrissae connective tissue YMineralization of vibrissae
DAB/2 mice
High Pi and low dietary Mg (0.02%) High Pi and high dietary Mg (0.08%)
[Cardiac calcification YCardiac calcification
24
Lewis Rats (VC induced by transplantation of juxtacardial aorta to the infrarenal aorta)
4.3 g/L Mg citrate (29 mmol/L)
YAortic graft calcification
25
SD rats supplemented with vitamin D3 and nicotine
Administration of low or high doses of Mg sulfate
YAortic alkaline phosphatase activity YAortic calcification
26
SD rats with adenineinduced CKD and treated with calcitriol
High dietary Mg (0.2%)
YCalcification in aorta, iliac, and carotid arteries [Mg vascular content [Vascular TRPM7
27
WKY rats with adenineinduced CKD
Ca acetate and Mg carbonate containing phosphate binder (375 mg/kg or 750 mg/kg)
YAortic calcification No difference in osteochondrogenic gene expression
28
Abcc62/2 mice (PXE)
Low dietary Mg (0.04%)
High dietary Mg (0.2%) High dietary Mg (1.1%)
CKD animals
Vascular Effects
21 22
23
Abbreviations: Ca, calcium; Mg, magnesium; Pi, inorganic phosphate; VC, vascular calcification.
phosphatases, and intact PTH). It is noteworthy that after 5 years of follow-up, a marked regression of arterial calcifications was observed in 3 patients. The mean serum Mg level was higher in these patients than in 17 individuals who had never developed arterial calcification. However, these differences were not statistically significant, given the small number of observations. The observational study by Ishimura and associates37 of 390 nondiabetic Japanese patients on maintenance hemodialysis found significantly lower serum Mg concentrations in individuals with phalangeal vessel calcification than in those without. A multivariate logistic regression analysis revealed that the serum Mg concentration was a significant, independent factor for the presence of VC (odds ratio [95% confidence interval] for a 1 mg/dL increase in serum Mg: 0.28 [0.09-0.92]; P ¼ 0.036) after adjustment for age, gender, hemodialysis duration, serum Ca, phosphate, and intact PTH concentrations. In both observational studies, the investigators only applied a semiquantitative X-ray assessment of the small hand arteries; this prevented a precise analysis of the possible relationship between serum Mg concentrations and the extent of arterial calcification. However, these data suggest that (1) higher serum Mg concentrations may protect against the development of VC in patients on dialysis, and thus, (2) the Mg concentration in dialysis fluid should perhaps be increased accordingly. A cross-sectional analysis of the Framingham Heart Study of the general population demonstrated a significant association between Mg intake and coronary artery calcification (CAC).41 Furthermore, the cross-sectional Adv Chronic Kidney Dis. 2018;25(3):281-290
study by Lee and associates42 of 34,553 Korean participants with normal kidney function observed an association between low serum Mg levels and CAC in participants at a low risk of cardiovascular disease. In order to better characterize the relationship between serum Mg levels and CAC in patients with CKD, Molnar and associates38 recently performed a cross-sectional study of the degree of abdominal aortic calcification (AAC) seen on a lateral lumbar spine X-ray (a validated surrogate marker for CAC) in 80 prevalent peritoneal dialysis patients. The mean 6 standard deviation serum Mg level was 0.8 6 0.2 mmol/L, and the mean (range) AAC score was 8.9 (0-24). A higher serum Mg level was associated with a lower AAC score (R2 ¼ 0.06, unstandardized coefficient B ¼ 27.81; P ¼ 0.03); this was also the case after adjustment for important confounding variables associated with VC, such as age, serum phosphate and PTH levels, and diabetes (adjusted R2 ¼ 0.36, serum Mg and AAC score B ¼ 211.44; P ¼ 0.001). Moreover, a 0.1 mmol/L increase in serum Mg was independently associated with a 1.1point decrease in the AAC score—suggesting that Mg might inhibit VC. Mitral valve calcification is a common complication in hemodialysis patients.46 In an early cross-sectional study, Tzanakis and associates39 investigated the association between serum Mg levels and mitral valve calcification in 56 patients on chronic hemodialysis. Twenty-three of the patients (41%) presented mitral annular calcification. Although no differences in serum phosphate, Ca, or intact PTH levels were found when comparing patients with or
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Table 2. Observational and Interventional Studies Having Investigated the Influence of Mg on VC Type of Clinical Study Observational, epidemiological studies
CKD patients
Non-CKD patients
Interventional studies
CKD patients
Patient’s CKD Status
n
Mg Level/Treatment
Vascular Effects
Ref
ESRD 1 PD
44
Low serum Mg level (2.69 mg/dL ¼ 1.106 mmol/L)
[Peripheral arterial calcification
36
HD without diabetes
390
Low serum Mg level (2.69 mg/dL ¼ 1.106 mmol/L)
[Phalangeal vessel calcification
37
PD
80
Low serum Mg level (,0.74 mmol/L)
[AAC
38
HD
56
Low serum Mg level (1.23 mmol/L)
[Mitral valve calcification
39
Mild-to-moderate CKD and diabetes
150
Low serum Mg level
[Mitral valve calcification
40
Framingham Heart Study cohort
2695
50-mg/d increment in total Mg intake
YCAC YAAC
41
Korean population
34,553
Low serum Mg level (l , 1.9 mg/dL ¼ 0.781 mmol/L)
[CAC
42
HD
1
Low Mg in dialysate
[Joint swelling and pain
43
HD
7
Mg carbonate (86 mg elemental Mg/100 mg elemental Ca)
YCAC
44
HD
72
Mg carbonate 1 Ca acetate (435 mg of Ca acetate containing 110 mg of elemental Ca, combined with 235 mg of Mg carbonate containing 60 mg of elemental Mg)
YArterial calcification
45
Abbreviations: AAC, abdominal aortic calcification; Ca, calcium; CAC, coronary artery calcification; HD, hemodialysis; Mg, magnesium; PD, peritoneal dialysis; VC, vascular calcification.
without mitral annular calcification, serum Mg levels were significantly lower in those with mitral annular calcification than in those without. Patients with a serum Mg level ,1.23 mmol/L presented an increased risk of developing mitral valve calcification. By applying a multiple logistical regression analysis, the researchers showed that serum Mg levels predicted the presence of mitral annular calcification with an accuracy of 86% (after controlling for patient age and biochemical parameters other than Mg). More recently, Silva and associates40 investigated the association between serum Mg and FGF-23 levels and mitral valve calcification in 150 diabetic patients with mild-to-moderate CKD. The presence and extent of calcification were assessed using echocardiography. Patients with Wilkins grade 4 calcification presented higher serum phosphorus, PTH, and FGF-23 levels, a lower eGFR, and a lower serum Mg level. In a multivariate linear regression, the eGFR and serum Ca, FGF-23, and Mg levels were found to be independent predictors of mitral valve calcification. Taken as a whole, these data indicate that the serum Mg level may be a clinically relevant marker or predictor of mitral valve calcification and thus of VC.
Lessons Learned from Interventional Studies Although observational studies have provided evidence of a correlation between high serum Mg levels and low VC in patients with CKD, few interventional studies have evaluated the impact of Mg supplementation on the progression of VC in this setting. The first clue came from an early case study in which the clinical symptoms of soft tissue calcification (such as joint swelling or pain) disappeared after treatment with a dialysate containing a high Mg concentration.43 The symptoms reappeared when the Mg concentration in the dialysate was subsequently reduced and were again relieved when high-Mg dialysate was reintroduced. More recently, Spiegel and Farmer44 performed an openlabel, prospective pilot study to evaluate the effects of long-term administration of Mg carbonate (provided as a phosphate binder) on the change over time in CAC scores (as assessed by electron beam computed tomography) in 7 hemodialysis patients. All patients had baseline CAC scores over 30. The change in the CAC score from baseline was recorded after 6, 12, and 18 months of follow-up. In fact, the CAC scores did not rise markedly throughout the study; the median percentage change in the CAC score Adv Chronic Kidney Dis. 2018;25(3):281-290
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Table 3. Animal and Clinical Studies Having Investigated the Influence of Mg on Vascular Abnormalities Type of Study Animal models
Observational, epidemiological studies
Characteristics
Vascular Effects
Reference
Swiss OF1 mice
Severe Mg deficient diet
[Aortic wall thinning [MMP2/MMP9 [Elastic fiber degradation [Collagen abnormalities
65
Inbred mice
Low intracellular Mg levels
[Systolic blood pressure [Impaired endothelial function [Outward hypertrophic remodeling of arteries [Vascular inflammation
48
LDL receptor–deficient male mice
Increased Mg sulfate intake (50 g/L in drink)
[Mg plasma levels YAortic sinus atherosclerosis
49
Mice on atherogenic diet (10% linoleic acid)
Increased Mg intake
YSerum total cholesterol and lipid peroxides No difference in serum phospholipid, triglyceride, and HDL cholesterol levels. YAortic cholesterol
50
New Zealand white Rabbits and a high cholesterol diet
Increased dietary intake of Mg aspartate hydrochloride
YSerum cholesterol and triglycerides YAtherosclerotic process
51
Abcc62/2 mice (PXE)
Increased dietary Mg
YIntima media thickness
52
CKD patients
Low serum Mg level Low serum Mg level (0.62-0.89 mmol/L) Low serum Mg level (1.8 mg/dL ¼ 0.74 mmol/L) Low serum Mg level (,2.05 mg/dL ¼ 0.843 mmol/L) High serum Mg level (.1.78 mg/dL ¼ 1.732 mmol/L) Low serum Mg levels Low dietary Mg Low serum Mg level (,2.2 mg/dL ¼ 0.904 mmol/L)
[IMT [PWV
40,53-55
[PP
56
[FMD [Cardiovascular mortality YPWV
57
[Coronary heart disease
59,60
[IMT [Risk of at least 2 carotid plaques
61
Mg citrate (610 mg every other d) Mg oxide (440 mg 3 times a wk, vs placebo)
YBilateral carotid IMT
62
YCarotid IMT No effects on FMD
63
Mg oxide (350 mg/d vs placebo)
No effects on FMD/RHI No effects on plasma markers of endothelial function
64
Non-CKD patients
Interventional studies
Mg Treatment
CKD patients
Non-CKD patients
55
58
Abbreviations: FMD, flow-mediated dilatation; HDL, high-density lipoprotein; IMT, intima media thickness; LDL, low-density lipoprotein; Mg, magnesium; PP, pulse pressure; PWV, pulse wave velocity; RHI, reactive hyperemia index.
(vs baseline) was 7.99%, 3.93%, and 8.01% at 6, 12, and 18 months, respectively. Furthermore, the CAC score’s direction of change was not significantly modified during the 18-month follow-up period (P ¼ 0.0737 in a paired test). These data are of interest because in other studies of patients with CKD, the annual percentage change in CAC ranged from 5% to 33% for sevelamer and 25%75% for a Ca-containing phosphate binder. Hence, these data indicate that Mg carbonate might slow CAC progression more than other phosphate binders do. However, the Adv Chronic Kidney Dis. 2018;25(3):281-290
small sample size and the lack of a control group in the study by Spiegel and associates prevent firm conclusions from being drawn. In a recent, prospective, double-blind controlled trial, Tzanakis and associates45 studied the change over time in arterial calcifications in hemodialysis patients receiving either Mg carbonate plus Ca acetate as a phosphate binder (the Mg group) or Ca acetate alone (Ca group). The presence and progression of arterial calcification were quantified by using the simple VC score to rate plain X-rays of the femur, pelvis, hands, and
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abdomen. The severity of arterial calcification decreased in 4 of the 32 patients (15.6%) in the Mg group and in none of the 27 patients (0%) in the Ca group (P ¼ 0.040). A multivariate logistic regression analysis revealed that serum Mg was the only independent predictor for the absence of progression (ie, a decrease or stability) of arterial calcification (P ¼ 0.047). Again, the small sample size prevents definitive conclusions from being drawn. In this context, the ongoing multicenter, double-blind, placebo-controlled randomized MAGiCAL-CKD trial is assessing whether oral Mg supplementation (ie, treatment with either slowrelease Mg hydroxide 30 mmol/d or matching placebo in a 1:1 ratio) can prevent the progression of CAC in subjects with predialysis CKD.47 If successful, the MAGiCAL-CKD trial might pave the way for larger randomized clinical trials of Mg supplementation, the use of Mg-based phosphate binders, or the use of hemodialysis/peritoneal dialysis solutions with a higher Mg concentration. Such studies would be of particular interest in patients on peritoneal dialysis, who are at highest risk for hypomagnesemia because the most commonly used peritoneal dialysis solutions have a low Mg concentration. CONCLUSION Evidence from in vitro experiments, animal models, and clinical studies indicate that low Mg levels are associated with VC. Furthermore, data from animal models and clinical studies show that Mg also protects against atherosclerosis, elastin/collagen degradation, vascular inflammation, and endothelial dysfunction (Table 3); these actions might account for a beneficial effect on VC. Nevertheless, the preliminary nature of interventional studies means that hard evidence is still lacking. Indeed, small interventional studies have suggested that oral Mg supplementation may slow the progression of CAC, peripheral arterial calcification, and mitral annular calcification in patients with CKD. However, data from well-designed, randomized, controlled trials will be needed to confirm a possible clinical benefit of Mg supplementation on VC and to demonstrate the safety and tolerability of long-term oral Mg supplementation in a CKD setting. In case of positive outcomes, this may lead to the introduction of Mg in the clinic as a cost-effective and safe nutritional strategy to prevent VC progression in dialysis patients. ACKNOWLEDGMENTS The authors are grateful to the Picardie Regional Council and the RHU project “STOP-AS” (Search Treatment and improve Outcome of Patients with Aortic Stenosis) for funding L.H.
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41. Hruby A, O’Donnell CJ, Jacques PF, Meigs JB, Hoffmann U, McKeown NM. Magnesium intake is inversely associated with coronary artery calcification: the Framingham Heart Study. JACC Cardiovasc Imaging. 2014;7(1):59-69. 42. Lee SY, Hyun YY, Lee KB, Kim H. Low serum magnesium is associated with coronary artery calcification in a Korean population at low risk for cardiovascular disease. Nutr Metab Cardiovasc Dis. 2015;25(11):1056-1061. 43. Izawa H, Imura M, Kuroda M, Takeda R. Proceedings: effect of magnesium on secondary hyperparathyroidism in chronic hemodialysis: a case with soft tissue calcification improved by high Mg dialysate. Calcif Tissue Res. 1974;15(2):162. 44. Spiegel DM, Farmer B. Long-term effects of magnesium carbonate on coronary artery calcification and bone mineral density in hemodialysis patients: a pilot study. Hemodial Int. 2009;13(4):453459. 45. Tzanakis IP, Stamataki EE, Papadaki AN, Giannakis N, Damianakis NE, Oreopoulos DG. Magnesium retards the progress of the arterial calcifications in hemodialysis patients: a pilot study. Int Urol Nephrol. 2014;46(11):2199-2205. 46. Mazzaferro S, Coen G, Bandini S, et al. Role of ageing, chronic renal failure and dialysis in the calcification of mitral annulus. Nephrol Dial Transplant. 1993;8(4):335-340. 47. Bressendorff I, Hansen D, Schou M, Kragelund C, Brandi L. The effect of magnesium supplementation on vascular calcification in chronic kidney disease-a randomised clinical trial (MAGiCALCKD): essential study design and rationale. BMJ Open. 2017;7(6):e016795. 48. Paravicini TM, Yogi A, Mazur A, Touyz RM. Dysregulation of vascular TRPM7 and annexin-1 is associated with endothelial dysfunction in inherited hypomagnesemia. Hypertension. 2009;53(2):423-429. 49. Sherer Y, Shaish A, Levkovitz H, et al. Magnesium fortification of drinking water suppresses atherogenesis in male LDL-receptordeficient mice. Pathobiology. 1999;67(4):207-213. 50. Yamaguchi Y, Kitagawa S, Kunitomo M, Fujiwara M. Preventive effects of magnesium on raised serum lipid peroxide levels and aortic cholesterol deposition in mice fed an atherogenic diet. Magnes Res. 1994;7(1):31-37. 51. Altura BT, Brust M, Bloom S, Barbour RL, Stempak JG, Altura BM. Magnesium dietary intake modulates blood lipid levels and atherogenesis. Proc Natl Acad Sci U S A. 1990;87(5):1840-1844. 52. Kupetsky EA, Rincon F, Uitto J. Rate of change of carotid intimamedia thickness with magnesium administration in Abcc6⁻/⁻ mice. Clin Transl Sci. 2013;6(6):485-486. 53. Tzanakis I, Virvidakis K, Tsomi A, et al. Intra- and extracellular magnesium levels and atheromatosis in haemodialysis patients. Magnes Res. 2004;17(2):102-108. 54. Zaher MM, Abdel-Salam M, Abdel-Salam R, Sabour R, Morsy AA, Gamal D. Serum magnesium level and vascular stiffness in children with chronic kidney disease on regular hemodialysis. Saudi J Kidney Dis Transpl. 2016;27(2):233-240. 55. Salem S, Bruck H, Bahlmann FH, et al. Relationship between magnesium and clinical biomarkers on inhibition of vascular calcification. Am J Nephrol. 2012;35(1):31-39. 56. Fragoso A, Silva AP, Gundlach K, B€ uchel J, Neves PL. Magnesium and FGF-23 are independent predictors of pulse pressure in predialysis diabetic chronic kidney disease patients. Clin Kidney J. 2014;7(2):161-166. 57. Kanbay M, Yilmaz MI, Apetrii M, et al. Relationship between serum magnesium levels and cardiovascular events in chronic kidney disease patients. Am J Nephrol. 2012;36(3):228-237. 58. Van Laecke S, Marechal C, Verbeke F, et al. The relation between hypomagnesaemia and vascular stiffness in renal transplant recipients. Nephrol Dial Transpl. 2011;26(7):2362-2369. 59. Liao F, Folsom AR, Brancati FL. Is low magnesium concentration a risk factor for coronary heart disease? The Atherosclerosis
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Risk in Communities (ARIC) Study. Am Heart J. 1998;136(3): 480-490. 60. Ma J, Folsom AR, Melnick SL, et al. Associations of serum and dietary magnesium with cardiovascular disease, hypertension, diabetes, insulin, and carotid arterial wall thickness: the ARIC study. Atherosclerosis Risk in Communities Study. J Clin Epidemiol. 1995;48(7):927-940. 61. Hashimoto T, Hara A, Ohkubo T, et al. Serum magnesium, ambulatory blood pressure, and carotid artery alteration: the Ohasama study. Am J Hypertens. 2010;23(12):1292-1298. 62. Turgut F, Kanbay M, Metin MR, Uz E, Akcay A, Covic A. Magnesium supplementation helps to improve carotid intima media thickness in patients on hemodialysis. Int Urol Nephrol. 2008;40(4):1075-1082.
63. Mortazavi M, Moeinzadeh F, Saadatnia M, Shahidi S, McGee JC, Minagar A. Effect of magnesium supplementation on carotid intima-media thickness and flow-mediated dilatation among hemodialysis patients: a double-blind, randomized, placebo-controlled trial. Eur Neurol. 2013;69(5):309-316. 64. Joris PJ, Plat J, Bakker SJ, Mensink RP. Effects of long-term magnesium supplementation on endothelial function and cardiometabolic risk markers: a randomized controlled trial in overweight/obese adults. Sci Rep. 2017;7(1):106. 65. Pages N, Gogly B, Godeau G, Igondjo-Tchen S, Maurois P, Durlach J, et al. Structural alterations of the vascular wall in magnesiumdeficient mice. A possible role of gelatinases A (MMP-2) and B (MMP-9). Magnes Res. 2003;16(1):43-48.
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Magnesium as a Calcification Inhibitor Lucie Henaut and Ziad A. Massy Vascular calcification (VC) is associated with elevated cardiovascular mortality rates in patients with CKD. Recent clinical studies of patients with advanced CKD have observed an association between low serum magnesium (Mg) levels on one hand and elevated VC and cardiovascular mortality on the other. These findings have stimulated interest in understanding Mg’s impact on CKD in general and the associated VC in particular. In vitro and preclinical in vivo data indicate that Mg has the potential to protect vascular smooth muscle cells against calcification via several different molecular mechanisms. Accordingly, data from pilot interventional studies in the clinic suggest that oral Mg supplementation reduces VC in patients with CKD. The present review provides an overview of our current understanding of the impact of Mg on the development of VC in patients with CKD. Q 2017 by the National Kidney Foundation, Inc. All rights reserved. Key Words: Chronic kidney disease, Magnesium, Vascular calcification
INTRODUCTION Cardiovascular disease is the leading cause of death in patients with CKD. In particular, vascular calcification (VC) is more prevalent and much more severe in this setting than in the general population.1 This degenerative process is characterized by the accumulation of calcium (Ca) and phosphate salts within the cardiovascular system (notably in the intimal and medial layers of vessels and in cardiac valves) and is associated with elevated cardiovascular mortality.2 Intimal and medial calcification notably contribute to cardiovascular disease and excess cardiovascular mortality in patients with CKD. Intimal VC (a consequence of atherosclerosis) may favor coronary ischemic events, whereas medial VC (which develops along the elastic lamina) increases vessel stiffness, arterial pulse wave velocity, systolic blood pressure, and pulse pressure and thus favors the development of cardiac failure, left ventricular hypertrophy, and diastolic dysfunction. Calcification of cardiac valves is also common in ESRD and is associated with a worse prognosis. Over the last few decades, studies of CKD patients have shown that when VC is accompanied by vessel stiffness, arterial hypertension, left ventricular hypertrophy, and cardiomyopathy, the severity of the condition is a strong, independent predictor of death. It is widely admitted that VC is irreversible once established; none of the treatments tested to date can unambiguously prevent or reverse VC. Accordingly, understanding the pathophysiologic mechanisms involved in the development of VC is of critical importance for identifying the most effective means of prevention and treatment. It has been suggested that along with traditional cardiovascular risk factors, nontraditional risk factors (such as those related to uremic status and disturbed CKD mineral and bone disorder [CKD-MBD]) may explain the elevated prevalence of VC and cardiovascular disease in a CKD setting. CKD-MBD is a systemic disorder characterized by persistently elevated parathyroid hormone (PTH) levels, bone abnormalities, extraskeletal calcification, and a broad spectrum of mineral metabolism disorders (including hypocalcemia, hyperphosphatemia, and hypermagnesemia). Over the last few decades, studies of CKDMBD have focused on the perturbation of Ca/phosphate homeostasis. However, increasing attention had been paid to the impact of magnesium (Mg) homeostasis on Adv Chronic Kidney Dis. 2018;25(3):281-290
CKD-MBD, in view of (1) the metal’s important role in the pathophysiology of the cardiovascular system, and (2) the reported association between Mg disorders and elevated cardiovascular morbidity/mortality. In fact, Mg is involved in the regulation of vascular tone, cardiac rhythm, and platelet-activated thrombosis. The maintenance of normal serum Mg levels (0.75-1 mmol/L) is determined by dietary ingestion and absorption, bone and soft tissue deposition and efflux, and kidney excretion. In CKD, urinary Mg excretion is normal or sometimes even elevated as long as the glomerular filtration rate is above 30 mL/min. Below that value, the kidney excretion of Mg may not be able to balance the dietary intake Mg—which then becomes a major determinant of serum and wholebody Mg levels.3 Although hypermagnesemia in CKD patients is usually mild and asymptomatic (,1.5 mmol/L), severe, symptomatic hypermagnesemia can be induced by exogenous Mg administration. Indeed, administration of Mg-containing drugs (eg, antacids and laxatives) and high-Mg concentrations in dialysate may provoke severe, symptomatic and sometimes even fatal hypermagnesemia. Conversely, the excessive use of diuretics, reduced gastrointestinal uptake (due to acidosis, and poor nutrition and absorption), and a low Mg concentration in dialysate may lead to abnormally low serum Mg levels.3,4 The results of many recent clinical studies show that low serum Mg levels are associated with increased VC and elevated cardiovascular mortality in patients with ESRD. These From the INSERM U1088, CURS, Amiens, France; Division of Nephrology, Ambroise Pare University Hospital, APHP, Boulogne-Billancourt/Paris, France; and INSERM U1018, Team 5, CESP, UVSQ, Villejuif, France. Financial Disclosure: Z.A.M. reports grants for CKD REIN and other research projects from Amgen, Baxter, Fresenius Medical Care, GlaxoSmithKline, Merck Sharp and Dohme-Chibret, Sanofi-Genzyme, Lilly, Otsuka, and the French government as well as personal fees and grants to charities from Amgen, Bayer, and Sanofi-Genzyme. These sources of funding are not necessarily related to the content of the present manuscript. Also see Acknowledgment(s) on page 288. Address correspondence to Ziad A. Massy, Division of Nephrology, Ambroise Pare University Hospital, 9 Avenue Charles de Gaulle, F-92104 Boulogne Billancourt cedex, France. E-mail: [email protected] Ó 2017 by the National Kidney Foundation, Inc. All rights reserved. 1548-5595/$36.00 https://doi.org/10.1053/j.ackd.2017.12.001
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findings have stimulated interest in understanding Mg’s role in CKD in general and the associated VC in particular. The present review provides an overview of our current understanding of the impact of Mg on the development of VC in patients with CKD.
elevated number of Ca/P nanocrystals formed in response to CKD-related loss of circulating inhibitors and hyperphosphatemia may directly promote the osteo/chondrogenic conversion of vascular cells.7 These Ca/P nanocrystals can also be taken up via endocytosis into VSMCs, where subsequent lysosomal degradation inMOLECULAR MECHANISMS OF VASCULAR creases Ca and Pi levels within the intracellular milieu. In an attempt to compensate for this increase, VSMCs CALCIFICATION IN CKD VC is a complex process that involves not only the preciprelease matrix vesicles loaded with Ca/P products. Once this compensatory mechanism is overwhelmed, the intraitation of minerals (due to supersaturated phosphate and cellular Ca/Pi overload triggers apoptosis, resulting in the Ca concentrations in the extracellular milieu, that is, an inorganic component) but also tightly regulated, cellformation of Ca/P-containing apoptotic bodies in the VSMCs. This phenomenon can be accentuated by addimediated process including apoptosis, osteochondrogenic tional Pi uptake through the sodium-dependent phosdifferentiation, matrix vesicle release, and elastin degradaphate transporter Pit-1. The apoptotic bodies and matrix tion (ie, a cellular component).5 Despite the significant vesicles ultimately contribute to a positive feedback loop recent growth in our knowledge of VC, the order of by releasing nanocrystals appearance and time course into the surrounding milieu of these 2 steps in vivo is still CLINICAL SUMMARY subject to debate. and thus amplifying the calcification process. Under normal physiolog Vascular calcification (VC) is a major contributor to ical conditions, blood vessels cardiovascular disease and excess cardiovascular and cardiac valves are proMAGNESIUM AS A mortality in patients with CKD. tected from supersaturated CALCIFICATION Epidemiologic studies indicate a possible link between low concentrations of serum Ca INHIBITOR IN CKD serum magnesium (Mg) levels and the development of VC and phosphate by a number in both the general population and patients with CKD. Lessons Learned from of active inhibitors. Pyro Data from in vitro studies and animal models suggest that In Vitro Studies phosphate, adenosine, maMg supplementation protects vascular cells against In early reports, Posner and trix Gla protein (MGP), calcification. fetuin-A, bone morphogeassociates demonstrated that Mg could directly innetic protein-7 (BMP7) and The results of pilot clinical studies suggest that oral Mg terfere with the process other factors have been supplementation reduces cardiovascular calcification. shown to prevent the transwhereby Ca and P crystallize into hydroxyapatite; the reformation of soluble, amorsearchers found that the addition of Mg to metastable phous calcium phosphate (Ca/P) complexes into harmful, stable hydroxyapatite crystals. In the CKD popuCa/P solutions inhibited the formation of Ca/P apatite and Ca-acidic phospholipid–phosphate complexes.8,9 lation, a decrease in levels of active inhibitors and a This inhibition stabilized amorphous Ca/P and favored simultaneous increase in levels of active inducers the formation of the Mg-containing crystal whitlockite of calcification (including hyperphosphatemia, hypercal[(Ca,Mg)3(PO4)2]. Hydroxyapatite had long been thought cemia, inflammatory cytokines, oxidative stress, uremic to be the sole mineral phase present in human uremic toxins, and advanced glycation end products) are responVC.10 However, a recent synchrotron study revealed the cosible for the extremely high prevalence of intimal and localization of whitlockite with hydroxyapatite in the armedial VC and valvular calcification. Hyperphosphatemia teries of patients with moderately impaired kidney (a typical manifestation of CKD-MBD) is the calcification function (CKD Stages 2-4).11 This observation suggests inducer most strongly associated with VC in CKD. Within that Mg (like the calcification inhibitors fetuin-A, MGP, the vascular system, elevated levels of inorganic phosand osteopontin) is closely associated with microcalcificaphate (Pi) and extracellular Ca and the deposition of amortion. phous Ca/P lead to the conversion of vascular smooth In a series of in vitro experiments, the use of MgCl2 muscle cells (VSMCs), resident fibroblasts, and quiescent reportedly prevented Pi-induced calcification and the ostevalve interstitial cells into osteochondrogenic cells. During ogenic differentiation of human, bovine, and rodent this conversion, the vascular cells downregulate the VSMCs by increasing or restoring the activity of the Mg expression of VC-inhibiting genes (such as MGP and transporter TRPM7. These effects were associated with BMP7) and start to express bone-promoting genes (such greater expression of anticalcification proteins (including as BMP2, RUNX2, and alkaline phosphatase) that act to osteopontin, BMP7, and MGP) and a decrease in VSMC transform amorphous Ca/P deposits into a wellapoptosis. MgCl2 was also shown to reduce Ca entry organized, calcified, crystalline structure.6 This phenomeinto VSMCs; this is of particular interest because elevated non is associated with (1) the secretion of a procalcifying Ca is known to upregulate Pit-1, which in turn increases Pi matrix rich in type I collagen and (2) the production of matrix metalloproteinases 2 and 9 (known to promote elastin uptake.12 According to recent studies, the Mg-associated inhibition of VSMC calcification is mainly mediated by indegradation and the subsequent release of highly procalcihibition of the Pi-induced Wnt/b-catenin signaling fying elastin peptides). According to a recent report, the Adv Chronic Kidney Dis. 2018;25(3):281-290
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pathway.13 In line with these data, Herencia and associates14 recently demonstrated that angiotensin II was able to prevent Pi-induced calcification and the osteogenic differentiation of human VSMCs by stimulating Mg influx and the subsequent inhibition of the canonical Wnt/b-catenin signaling pathway. It is noteworthy that addition of MgCl2 to calcified VSMC cultures decreased the Ca content, downregulates RunX2 and Osterix mRNA expression, and upregulates MGP and OPG mRNA expression.13 This effect was not observed when 2aminoethoxy-diphenylborate (an inhibitor of cellular Mg transport via TRPM7) is added to the culture medium— suggesting that calcification is not only halted but also even decreases through a cell-mediated process following addition of Mg. More recently, MgCl2 was reported to locally activate the calcium-sensing receptor (CaSR) expressed by VSMCs and thus inhibit Pi-induced VSMC calcification and osteogenic differentiation.15 The increased CaSR expression associated with this effect is of particular interest, since loss of a functional CaSR had previously been reported in calcified VSMCs. These data are in agreement with other reports in which vascular CaSR activation protects against VC.16 Our group recently investigated the role of microRNAs in the Mg-mediated protection against VC.17 Exposure to MgCl2 inhibited the Pi-induced decrease in miR-30b, miR-133a, and miR-143 and modulated the latter’s known targets (Runx2, Smad1, and Osterix, respectively). These data prompted a potential mechanistic hypothesis for the Mg-induced downregulation of osteogenic markers during the course of Pi-induced VC. MgCl2 supplementation also reportedly downregulates the osteogenic reprogramming of VSMCs induced by hydroxyapatite particles— confirming that Mg’s prime effect does not consist in preventing the initial Ca/P particle from forming.15 On these lines, we recently looked at whether the effects on live VSMCs mediated in vitro by MgCl2 also occur during passive Ca/P deposition on fixed VSMCs. We found that MgCl2 failed to prevent Pi-induced Ca/P deposition on fixed cells, which suggests that cells must be alive if Mg ions are to exert a protective effect.18 In an attempt to characterize this process in more detail, we recently used micro-Fourier transform infrared spectroscopy, field effect scanning electron microscopy, and energy dispersive Xray spectrometry to investigate Mg’s involvement in crystal formation in in vitro cultures of primary human aortic VSMCs. As expected, only Ca/P apatite was detected in cells incubated with elevated Ca 3 Pi medium. Supplementation with MgCl2 did not alter the crystals’ composition or structure. However, a qualitative analysis suggested that 5 mM Mg reduced the number and intensity of the Ca/P apatite micro-Fourier transform infrared spectroscopy spots. Hence, our results suggest that Mg has an active, cellular role in attenuating VC rather than a physicochemical effect on the growth, composition or structure of Ca/P apatite crystals. Our data differ from those gathered in the synchrotron study by Fischer and associates19 of whether moderately impaired kidney function (CKD Stages 2-4) might affect the physiochemical composition and/or the formation of whitlockite in arterial Adv Chronic Kidney Dis. 2018;25(3):281-290
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atherosclerotic microcalcifications. The researchers found that arterial microcalcification in atherosclerotic patients with normal or reduced kidney function consisted of Ca/ P (either as amorphous deposits or as apatite) and whitlockite. Although the proportion of patients with a given type of mineral phase in their microcalcifications did not differ from one group to another, the proportion of whitlockite in the microcalcifications was significantly greater in atherosclerotic individuals with normal kidney function. In the study by Fischer and associates, the serum Mg level was not significantly associated with the glomerular filtration rate at the time of surgery carotid stenosis. Hence, these data indicate that CKD affects the local Mg homeostasis and thus contributes to the physicochemical composition of microcalcifications in atherosclerotic patients. Mg’s putative effects on VSMC calcification are presented in Figure 1. Lessons Learned from In Vivo Studies In studies of several different non-CKD animal models, Mg has repeatedly been shown to prevent the progression of VC (Table 1). For example, the effects of dietary Mg have been extensively explored in the Abcc62/2 mouse model of Pseudoxanthoma pseudoxanthoma elasticum.20-22 elasticum is an autosomal recessive multisystem disorder characterized by the mineralization of ectopic connective tissue. The clinical manifestations of pseudoxanthoma elasticum primarily affect the skin, eyes, and cardiovascular system. In the Abcc62/2 mouse, increasing the dietary intake of Mg significantly reduced the calcification of kidney blood vessels and connective tissue,21,22 while restriction of dietary Mg intake accelerated ectopic mineralization.20 Treatment of Abcc62/2 mice with Mg carbonate–containing phosphate binder prevented the onset and the progression of calcification, whereas treatment with lanthanum carbonate had no effect.23 In the inbred DBA/2 mouse model, the combination of a low Mg intake and a high Pi intake was associated with severe calcification of cardiac and kidney tissues, while the combination of a high Mg intake and a low Pi intake partially prevented cardiac calcification.24 In a rat model in which the juxtacardial aorta was grafted to the infrarenal aorta, dietary supplementation with a combination of Mg, alkali citrate, and bases prevented the development of medial calcification within the graft.25 In Sprague–Dawley rats supplemented with vitamin D3 and nicotine, administration of Mg sulfate decreased the aortic alkaline phosphatase activity and the severity of aortic calcification.26 Therefore, increasing the dietary Mg intake appears to protect against VC in non-CKD animals. In male Sprague-Dawley rats with adenine-induced CKD, Mg supplementation attenuated the severity of calcitriol-induced calcification in the abdominal aorta (by 51%), the iliac artery (by 44%), and the carotid artery (by 46%). These effects were associated with a 20%-50% increase in vascular Mg content and a reduction in calcitriol-induced vascular TRPM7 deficiency. Therefore, the use of Mg supplementation to modify calcitriol’s adverse effects may be a plausible treatment approach in
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Amorphous Ca/P High Ca
High Pi
Magnesium HA crystals Ca-channels
TRPM7
mir30b mir133a mir143
CaSR
Pit1/2
Wnt-β-catenin
Loss of inhibitors Osteogenic differentiation Apoptosis
Vascular calcification
VSMC
Figure 1. Cellular and molecular mechanisms by which Mg prevents VSMC calcification in vitro. Within the vascular system, elevated levels of inorganic phosphate (Pi) and extracellular calcium (Ca) and the deposition of amorphous calcium phosphate lead to vascular smooth muscle cells (VSMCs) osteogenic conversion. During this process, VSMC downregulates the expression of VC-inhibiting genes (such as MGP and BMP7) and starts to express bone-promoting genes (such as BMP2, RUNX2, and alkaline phosphatase) that act to transform amorphous calcium phosphate deposits into a wellorganized, calcified, crystalline structure. Mg enters the cell via TRPM7 and restores the balance between expression of calcification promoters and inhibitors. These effects are mediated through the blockade of Pi-induced Wnt/b-catenin signaling pathway. Exposure to Mg also inhibits Pi-induced decrease in miR-30b, miR-133a, and miR-143, and subsequent VSMC osteogenic transition. Besides, Mg may prevent VC through local activation of the calcium-sensing receptor (CaSR) expressed by VSMC. Moreover, exposure to Mg reduces VSMC apoptosis and subsequent release of procalcific apoptotic bodies. Mg also functions as a Ca-channel antagonist and thus inhibits the entry of Ca into the cells. Some reports show that Mg may interfere with the process of vascular calcification by inhibiting transformation of amorphous Ca/P to apatite. However, this fact remains a matter of debate. Dotted lines: further studies are needed to fully demonstrate the concept. Abbreviations: Ca/P, calcium phosphate; HA, hydroxyapatite; Mg, magnesium.
the growing proportion of CKD patients receiving this active metabolite of vitamin D.27 In male Wistar Kyoto rats with adenine-induced CKD, treatment with either a phosphate binder containing Mg carbonate and calcium acetate (CaMg) or sevelamer effectively controlled serum phosphate levels and reduced aortic calcification. In this model, treatment with CaMg displayed no additional effects on VC, relative to sevelamer alone.28 A synchrotron-based X-ray diffraction analysis of aortic calcifications in CaMg2, sevelamer- and vehicle-treated animals showed that the mineral precipitates in the various groups contained Ca hydroxyapatite alone. These data are inconsistent with the previous observation by Verberckmoes and associates29 of whitlockite in aortic calcifications in male Wistar Kyoto rats with adenine-induced CKD. In the latter study, calcitriol supplementation was associated with increased deposition of whitlockite in the aorta. The researchers suggested that this deposition resulted from the calcitriol-stimulated intestinal absorption of Mg. Changes in Mg status in animal models have also been shown to promote various systemic disorders, known as
classical inducers of VC. Among them, Mg deficiency was reported to promote systemic inflammation,30,31 to influence vitamin D metabolism,32 and to trigger disturbances in mineral and bone metabolism.33-35 Whether the protective effects of Mg on VC are the result of local actions on cardiovascular tissues or of indirect, systemic actions cannot be determined in the in vivo setting and needs further investigations. Lessons Learned from Observational and Epidemiological Studies The possible clinical relationship between serum Mg levels and VC has been assessed in several observational studies (Table 2). In an early observational study of 44 patients with ESRD treated with continuous ambulatory peritoneal dialysis, Meema and associates36 reported that the mean serum Mg concentration was lower in individuals with X-ray evidence of peripheral arterial calcification (ie, in the hands, ankles, and feet) than in individuals lacking such evidence. No intergroup differences were found for other serum biochemistry parameters with known or putative roles in VC (Ca, phosphate, total alkaline Adv Chronic Kidney Dis. 2018;25(3):281-290
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Table 1. Animal Models Used to Investigate the Influence of Mg on VC Animal’s CKD Status Non-CKD animals
Animal Model
Mg Treatment
Ref 20
Mg carbonate-enriched diet (1.1%)
[Mineralization of the dermal sheath of vibrissae, cartilage, kidney, heart, arteries YKidney blood vessel calcification YMineralization of vibrissae connective tissue YMineralization of vibrissae
DAB/2 mice
High Pi and low dietary Mg (0.02%) High Pi and high dietary Mg (0.08%)
[Cardiac calcification YCardiac calcification
24
Lewis Rats (VC induced by transplantation of juxtacardial aorta to the infrarenal aorta)
4.3 g/L Mg citrate (29 mmol/L)
YAortic graft calcification
25
SD rats supplemented with vitamin D3 and nicotine
Administration of low or high doses of Mg sulfate
YAortic alkaline phosphatase activity YAortic calcification
26
SD rats with adenineinduced CKD and treated with calcitriol
High dietary Mg (0.2%)
YCalcification in aorta, iliac, and carotid arteries [Mg vascular content [Vascular TRPM7
27
WKY rats with adenineinduced CKD
Ca acetate and Mg carbonate containing phosphate binder (375 mg/kg or 750 mg/kg)
YAortic calcification No difference in osteochondrogenic gene expression
28
Abcc62/2 mice (PXE)
Low dietary Mg (0.04%)
High dietary Mg (0.2%) High dietary Mg (1.1%)
CKD animals
Vascular Effects
21 22
23
Abbreviations: Ca, calcium; Mg, magnesium; Pi, inorganic phosphate; VC, vascular calcification.
phosphatases, and intact PTH). It is noteworthy that after 5 years of follow-up, a marked regression of arterial calcifications was observed in 3 patients. The mean serum Mg level was higher in these patients than in 17 individuals who had never developed arterial calcification. However, these differences were not statistically significant, given the small number of observations. The observational study by Ishimura and associates37 of 390 nondiabetic Japanese patients on maintenance hemodialysis found significantly lower serum Mg concentrations in individuals with phalangeal vessel calcification than in those without. A multivariate logistic regression analysis revealed that the serum Mg concentration was a significant, independent factor for the presence of VC (odds ratio [95% confidence interval] for a 1 mg/dL increase in serum Mg: 0.28 [0.09-0.92]; P ¼ 0.036) after adjustment for age, gender, hemodialysis duration, serum Ca, phosphate, and intact PTH concentrations. In both observational studies, the investigators only applied a semiquantitative X-ray assessment of the small hand arteries; this prevented a precise analysis of the possible relationship between serum Mg concentrations and the extent of arterial calcification. However, these data suggest that (1) higher serum Mg concentrations may protect against the development of VC in patients on dialysis, and thus, (2) the Mg concentration in dialysis fluid should perhaps be increased accordingly. A cross-sectional analysis of the Framingham Heart Study of the general population demonstrated a significant association between Mg intake and coronary artery calcification (CAC).41 Furthermore, the cross-sectional Adv Chronic Kidney Dis. 2018;25(3):281-290
study by Lee and associates42 of 34,553 Korean participants with normal kidney function observed an association between low serum Mg levels and CAC in participants at a low risk of cardiovascular disease. In order to better characterize the relationship between serum Mg levels and CAC in patients with CKD, Molnar and associates38 recently performed a cross-sectional study of the degree of abdominal aortic calcification (AAC) seen on a lateral lumbar spine X-ray (a validated surrogate marker for CAC) in 80 prevalent peritoneal dialysis patients. The mean 6 standard deviation serum Mg level was 0.8 6 0.2 mmol/L, and the mean (range) AAC score was 8.9 (0-24). A higher serum Mg level was associated with a lower AAC score (R2 ¼ 0.06, unstandardized coefficient B ¼ 27.81; P ¼ 0.03); this was also the case after adjustment for important confounding variables associated with VC, such as age, serum phosphate and PTH levels, and diabetes (adjusted R2 ¼ 0.36, serum Mg and AAC score B ¼ 211.44; P ¼ 0.001). Moreover, a 0.1 mmol/L increase in serum Mg was independently associated with a 1.1point decrease in the AAC score—suggesting that Mg might inhibit VC. Mitral valve calcification is a common complication in hemodialysis patients.46 In an early cross-sectional study, Tzanakis and associates39 investigated the association between serum Mg levels and mitral valve calcification in 56 patients on chronic hemodialysis. Twenty-three of the patients (41%) presented mitral annular calcification. Although no differences in serum phosphate, Ca, or intact PTH levels were found when comparing patients with or
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Table 2. Observational and Interventional Studies Having Investigated the Influence of Mg on VC Type of Clinical Study Observational, epidemiological studies
CKD patients
Non-CKD patients
Interventional studies
CKD patients
Patient’s CKD Status
n
Mg Level/Treatment
Vascular Effects
Ref
ESRD 1 PD
44
Low serum Mg level (2.69 mg/dL ¼ 1.106 mmol/L)
[Peripheral arterial calcification
36
HD without diabetes
390
Low serum Mg level (2.69 mg/dL ¼ 1.106 mmol/L)
[Phalangeal vessel calcification
37
PD
80
Low serum Mg level (,0.74 mmol/L)
[AAC
38
HD
56
Low serum Mg level (1.23 mmol/L)
[Mitral valve calcification
39
Mild-to-moderate CKD and diabetes
150
Low serum Mg level
[Mitral valve calcification
40
Framingham Heart Study cohort
2695
50-mg/d increment in total Mg intake
YCAC YAAC
41
Korean population
34,553
Low serum Mg level (l , 1.9 mg/dL ¼ 0.781 mmol/L)
[CAC
42
HD
1
Low Mg in dialysate
[Joint swelling and pain
43
HD
7
Mg carbonate (86 mg elemental Mg/100 mg elemental Ca)
YCAC
44
HD
72
Mg carbonate 1 Ca acetate (435 mg of Ca acetate containing 110 mg of elemental Ca, combined with 235 mg of Mg carbonate containing 60 mg of elemental Mg)
YArterial calcification
45
Abbreviations: AAC, abdominal aortic calcification; Ca, calcium; CAC, coronary artery calcification; HD, hemodialysis; Mg, magnesium; PD, peritoneal dialysis; VC, vascular calcification.
without mitral annular calcification, serum Mg levels were significantly lower in those with mitral annular calcification than in those without. Patients with a serum Mg level ,1.23 mmol/L presented an increased risk of developing mitral valve calcification. By applying a multiple logistical regression analysis, the researchers showed that serum Mg levels predicted the presence of mitral annular calcification with an accuracy of 86% (after controlling for patient age and biochemical parameters other than Mg). More recently, Silva and associates40 investigated the association between serum Mg and FGF-23 levels and mitral valve calcification in 150 diabetic patients with mild-to-moderate CKD. The presence and extent of calcification were assessed using echocardiography. Patients with Wilkins grade 4 calcification presented higher serum phosphorus, PTH, and FGF-23 levels, a lower eGFR, and a lower serum Mg level. In a multivariate linear regression, the eGFR and serum Ca, FGF-23, and Mg levels were found to be independent predictors of mitral valve calcification. Taken as a whole, these data indicate that the serum Mg level may be a clinically relevant marker or predictor of mitral valve calcification and thus of VC.
Lessons Learned from Interventional Studies Although observational studies have provided evidence of a correlation between high serum Mg levels and low VC in patients with CKD, few interventional studies have evaluated the impact of Mg supplementation on the progression of VC in this setting. The first clue came from an early case study in which the clinical symptoms of soft tissue calcification (such as joint swelling or pain) disappeared after treatment with a dialysate containing a high Mg concentration.43 The symptoms reappeared when the Mg concentration in the dialysate was subsequently reduced and were again relieved when high-Mg dialysate was reintroduced. More recently, Spiegel and Farmer44 performed an openlabel, prospective pilot study to evaluate the effects of long-term administration of Mg carbonate (provided as a phosphate binder) on the change over time in CAC scores (as assessed by electron beam computed tomography) in 7 hemodialysis patients. All patients had baseline CAC scores over 30. The change in the CAC score from baseline was recorded after 6, 12, and 18 months of follow-up. In fact, the CAC scores did not rise markedly throughout the study; the median percentage change in the CAC score Adv Chronic Kidney Dis. 2018;25(3):281-290
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Table 3. Animal and Clinical Studies Having Investigated the Influence of Mg on Vascular Abnormalities Type of Study Animal models
Observational, epidemiological studies
Characteristics
Vascular Effects
Reference
Swiss OF1 mice
Severe Mg deficient diet
[Aortic wall thinning [MMP2/MMP9 [Elastic fiber degradation [Collagen abnormalities
65
Inbred mice
Low intracellular Mg levels
[Systolic blood pressure [Impaired endothelial function [Outward hypertrophic remodeling of arteries [Vascular inflammation
48
LDL receptor–deficient male mice
Increased Mg sulfate intake (50 g/L in drink)
[Mg plasma levels YAortic sinus atherosclerosis
49
Mice on atherogenic diet (10% linoleic acid)
Increased Mg intake
YSerum total cholesterol and lipid peroxides No difference in serum phospholipid, triglyceride, and HDL cholesterol levels. YAortic cholesterol
50
New Zealand white Rabbits and a high cholesterol diet
Increased dietary intake of Mg aspartate hydrochloride
YSerum cholesterol and triglycerides YAtherosclerotic process
51
Abcc62/2 mice (PXE)
Increased dietary Mg
YIntima media thickness
52
CKD patients
Low serum Mg level Low serum Mg level (0.62-0.89 mmol/L) Low serum Mg level (1.8 mg/dL ¼ 0.74 mmol/L) Low serum Mg level (,2.05 mg/dL ¼ 0.843 mmol/L) High serum Mg level (.1.78 mg/dL ¼ 1.732 mmol/L) Low serum Mg levels Low dietary Mg Low serum Mg level (,2.2 mg/dL ¼ 0.904 mmol/L)
[IMT [PWV
40,53-55
[PP
56
[FMD [Cardiovascular mortality YPWV
57
[Coronary heart disease
59,60
[IMT [Risk of at least 2 carotid plaques
61
Mg citrate (610 mg every other d) Mg oxide (440 mg 3 times a wk, vs placebo)
YBilateral carotid IMT
62
YCarotid IMT No effects on FMD
63
Mg oxide (350 mg/d vs placebo)
No effects on FMD/RHI No effects on plasma markers of endothelial function
64
Non-CKD patients
Interventional studies
Mg Treatment
CKD patients
Non-CKD patients
55
58
Abbreviations: FMD, flow-mediated dilatation; HDL, high-density lipoprotein; IMT, intima media thickness; LDL, low-density lipoprotein; Mg, magnesium; PP, pulse pressure; PWV, pulse wave velocity; RHI, reactive hyperemia index.
(vs baseline) was 7.99%, 3.93%, and 8.01% at 6, 12, and 18 months, respectively. Furthermore, the CAC score’s direction of change was not significantly modified during the 18-month follow-up period (P ¼ 0.0737 in a paired test). These data are of interest because in other studies of patients with CKD, the annual percentage change in CAC ranged from 5% to 33% for sevelamer and 25%75% for a Ca-containing phosphate binder. Hence, these data indicate that Mg carbonate might slow CAC progression more than other phosphate binders do. However, the Adv Chronic Kidney Dis. 2018;25(3):281-290
small sample size and the lack of a control group in the study by Spiegel and associates prevent firm conclusions from being drawn. In a recent, prospective, double-blind controlled trial, Tzanakis and associates45 studied the change over time in arterial calcifications in hemodialysis patients receiving either Mg carbonate plus Ca acetate as a phosphate binder (the Mg group) or Ca acetate alone (Ca group). The presence and progression of arterial calcification were quantified by using the simple VC score to rate plain X-rays of the femur, pelvis, hands, and
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abdomen. The severity of arterial calcification decreased in 4 of the 32 patients (15.6%) in the Mg group and in none of the 27 patients (0%) in the Ca group (P ¼ 0.040). A multivariate logistic regression analysis revealed that serum Mg was the only independent predictor for the absence of progression (ie, a decrease or stability) of arterial calcification (P ¼ 0.047). Again, the small sample size prevents definitive conclusions from being drawn. In this context, the ongoing multicenter, double-blind, placebo-controlled randomized MAGiCAL-CKD trial is assessing whether oral Mg supplementation (ie, treatment with either slowrelease Mg hydroxide 30 mmol/d or matching placebo in a 1:1 ratio) can prevent the progression of CAC in subjects with predialysis CKD.47 If successful, the MAGiCAL-CKD trial might pave the way for larger randomized clinical trials of Mg supplementation, the use of Mg-based phosphate binders, or the use of hemodialysis/peritoneal dialysis solutions with a higher Mg concentration. Such studies would be of particular interest in patients on peritoneal dialysis, who are at highest risk for hypomagnesemia because the most commonly used peritoneal dialysis solutions have a low Mg concentration. CONCLUSION Evidence from in vitro experiments, animal models, and clinical studies indicate that low Mg levels are associated with VC. Furthermore, data from animal models and clinical studies show that Mg also protects against atherosclerosis, elastin/collagen degradation, vascular inflammation, and endothelial dysfunction (Table 3); these actions might account for a beneficial effect on VC. Nevertheless, the preliminary nature of interventional studies means that hard evidence is still lacking. Indeed, small interventional studies have suggested that oral Mg supplementation may slow the progression of CAC, peripheral arterial calcification, and mitral annular calcification in patients with CKD. However, data from well-designed, randomized, controlled trials will be needed to confirm a possible clinical benefit of Mg supplementation on VC and to demonstrate the safety and tolerability of long-term oral Mg supplementation in a CKD setting. In case of positive outcomes, this may lead to the introduction of Mg in the clinic as a cost-effective and safe nutritional strategy to prevent VC progression in dialysis patients. ACKNOWLEDGMENTS The authors are grateful to the Picardie Regional Council and the RHU project “STOP-AS” (Search Treatment and improve Outcome of Patients with Aortic Stenosis) for funding L.H.
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