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NDT Advance Access originally published online on May 7, 2008
Nephrology Dialysis Transplantation 2008 23(10):3263-3271; doi:10.1093/ndt/gfn226
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


The circulating calcification inhibitors, fetuin-A and osteoprotegerin, but not Matrix Gla protein, are associated with vascular stiffness and calcification in children on dialysis

Rukshana C. Shroff1,2, Vanita Shah3, Melanie P. Hiorns4, Michael Schoppet5, Lorenz C. Hofbauer6, Gerhard Hawa7, Leon J. Schurgers8, Atul Singhal9, Ian Merryweather9, Paul Brogan10, Catherine Shanahan11, John Deanfield2 and Lesley Rees1

1 Nephrourology 2 Vascular Physiology 3 Infectious Disease 4 Radiology Departments, Great Ormond Street Hospital & UCL Institute of Child Health, London, UK 5 Department of Internal Medicine and Cardiology, Philipps-University, Marburg 6 Division of Endocrinology and Metabolic Bone Disease, Technical University of Dresden, Dresden, Germany 7 Biomedica Medizinprodukte, Vienna, Austria 8 CARIM and VitaK, University of Maastricht, Maastricht, The Netherlands 9 Childhood Nutrition Research Centre 10 Department of Rheumatology, Great Ormond Street Hospital & UCL Institute of Child Health, London, UK 11 Cardiovascular Division, King's College London, UK

Rukshana C. Shroff, Research Registrar, Nephrourology Unit, Institute of Child Health, 30, Guilford Street, London WC1N 1EH, UK. Tel: +44-020-7405-9200, ext 0065; Fax: +44-020-7813-8532; E-mail: ShrofR{at}gosh.nhs.uk



   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Background. Vascular calcification occurs in the majority of patients with chronic kidney disease, but a subset of patients does not develop calcification despite exposure to a similar uraemic environment. Physiological inhibitors of calcification, fetuin-A, osteoprotegerin (OPG) and undercarboxylated-matrix Gla protein (uc-MGP) may play a role in preventing the development and progression of ectopic calcification, but there are scarce and conflicting data from clinical studies.

Methods. We measured fetuin-A, OPG and uc-MGP in 61 children on dialysis and studied their associations with clinical, biochemical and vascular measures.

Results. Fetuin-A and OPG were higher and uc-MGP lower in dialysis patients than controls. In controls, fetuin-A and OPG increased with age. Fetuin-A showed an inverse correlation with dialysis vintage (P = 0.0013), time-averaged serum phosphate (P = 0.03) and hs-CRP (P = 0.001). Aortic pulse wave velocity (PWV) and augmentation index showed a negative correlation with fetuin-A while a positive correlation was seen with PWV and OPG. Patients with calcification had lower fetuin-A and higher OPG than those without calcification. On multiple linear regression analysis Fetuin-A independently predicted aortic PWV (P = 0.004, ß = –0.45, model R2 = 48%) and fetuin-A and OPG predicted cardiac calcification (P = 0.02, ß = –0.29 and P = 0.014, ß = 0.33, respectively, model R2 = 32%).

Conclusions. This is the first study to define normal levels of the calcification inhibitors in children and show that fetuin-A and OPG are associated with increased vascular stiffness and calcification in children on dialysis. Higher levels of fetuin-A in children suggest a possible protective upregulation of fetuin-A in the early stages of exposure to the pro-calcific and pro-inflammatory uraemic environment.

Keywords: dialysis; fetuin-A; matrix Gla protein; osteoprotegerin; vascular calcification



   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Cardiovascular disease is the most common cause of death in patients with chronic kidney disease (CKD) [1]. Structural and functional abnormalities and calcification in the large vessels begin as early as the first decade of life in patients with CKD [2,3] contributing to a 30-fold higher mortality than the age-matched population [1]. In vitro studies and animal experiments have shown that ectopic calcification is a highly regulated, cell-mediated process that involves a balance between inducers and inhibitors of calcification [4].

In CKD pro-calcific stimuli such as increased calcium (Ca) and phosphate (PO4) [2,5–8] and intact parathyroid hormone (iPTH) levels [2], and, potentially, treatment with Ca-based PO4 binders [2,6–9] and vitamin D [2,8,10] can promote vascular and soft-tissue calcification. Yet, some patients with CKD do not develop calcification despite exposure to the same uraemic milieu [9]. In fact, all extracellular fluid contains Ca and PO4 in concentrations exceeding their solubility product for spontaneous precipitation [11], suggesting that under normal conditions inhibitors of calcification prevent the development and progression of vascular calcification.

Fetuin-A ({alpha}2-Heremans-Schmid protein) is the prototypic calcification inhibitor [11,12] that contributes to ~50% of the calcification inhibitory capacity of human plasma [13]. Osteoprotegerin (OPG) is a soluble decoy receptor for the receptor activator of nuclear factor-{kappa}B ligand (RANKL), which stimulates osteoclastic bone resorption [14]. Serum OPG levels are associated with the progression of carotid atherosclerosis [15] and coronary calcification [16]. Matrix Gla ({gamma}-carboxyglutamic acid) protein (MGP) is expressed in the media of arteries where it acts as a local inhibitor of Ca–PO4 precipitation [17]. Circulating fetuin-A, OPG and MGP levels have been linked with cardiovascular mortality [18–25] in adults with CKD, but there is a complex and poorly understood relationship between these physiological calcification inhibitors at different stages of CKD and also conflicting data on their impact on vascular measures.

Circulating inhibitors of calcification have not been studied in children with CKD, nor have their normal levels been defined in the healthy childhood population. In this study we describe serum fetuin-A, OPG and undercarboxylated-matrix Gla protein (uc-MGP) levels in a cohort of healthy children and dialysis patients. Children provide an ideal opportunity to study uraemic influences on the arterial wall, as they rarely have confounding pro-atherosclerotic risk factors such as diabetes and dyslipidaemia that are prevalent in adults, and also allow us to study the earliest events in the calcification process. We hypothesize that normal levels of the circulating calcification inhibitors protect against vessel stiffness and calcification in children on dialysis.



   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Study design
From January 2005 to December 2006, 61 consecutive children (5–18 years) who had been on dialysis for ≥3 months were recruited. Serum levels of the calcification inhibitors, fetuin-A, OPG and uc-MGP, as well as hs-CRP were measured and related to carotid artery intima media thickness (cIMT), aortic PWV and calcification score on multi-slice CT scan. Patients were compared with 75 healthy children: 55 children formed part of a larger study investigating nutritional parameters in healthy children and 20 underwent routine corrective surgery for external auricular malformations or squints in our hospital. Controls were age and gender matched with the dialysis cohort and confirmed to have no known medical illnesses, family history of heart disease or active infections at the time of the study. As vascular scans could only be obtained in 18 of the healthy children, the vascular measures in our dialysis cohort were compared with 40 age-matched controls who participated in a previous study from our group [2]. Informed written consent was obtained from all parents or caregivers, and children where appropriate. The study was approved by the local research ethics committee.

Biochemical measures
Blood samples were taken before a mid-week session of haemodialysis (HD) or immediately after a peritoneal dialysis (PD) session. Serum Ca, PO4 and iPTH levels and the dosage of elemental Ca intake from PO4 binders and vitamin D therapy were recorded at monthly intervals from the start of CKD stage IV and expressed as mean time-integrated values. In controls, a single blood test at the time of the scans was performed.

  1. Serum fetuin-A was measured using a human fetuin-A enzyme-linked immunosorbent assay (ELISA) kit (Epitope Diagnostics Inc., San Diego, CA, USA). The assay utilizes the two-site ‘sandwich’ technique with two selected polyclonal antibodies that bind to different epitopes of fetuin-A. The intra-assay and inter-assay coefficient of variation were < 5.5% and < 6.8%, respectively. The reference range for healthy adults quoted by the manufacturer was 0.5–1.0 g/L, and the minimum sensitivity of the assay was 5.0 ng/mL.
  2. OPG serum concentrations were analysed using an ELISA system (Biomedica, Vienna, Austria) that detects both free and complexed OPG. In brief, a monoclonal IgG antibody was used as capture antibody and a biotin-labelled polyclonal antihuman OPG antibody as detection antibody. The intra-assay and inter-assay variabilities were 9% and 10%, respectively. RANKL levels were determined by ELISA (Biomedica) based on microtitre plates coated with OPG. Soluble RANKL (sRANKL) from the sample binds to the coated OPG and is detected by a biotin-labelled polyclonal anti-human sRANKL antibody. The intra-assay and inter-assay variations ranged from 3 to 5% and 6 to 9%, respectively.
  3. uc-MGP was measured by a novel ELISA technique developed at the vitamin K-research institute (VitaK BV, Maastricht, The Netherlands). In brief, the moAb uc-MGP was used in this competitive ELISA to capture either native MGP in the sample or biotinylated tracer. The uc-MGP concentration was calculated using a calibration curve of synthetic full-length uc-MGP. The intra- and inter-assay variabilities were 7.2% and 11.4%, respectively.
  4. hs-CRP levels were measured by an ELISA (Biomedica) according to the manufacturer's instructions.

Vascular measures

  1. cIMT was measured by B-mode ultrasound of both common carotid arteries using a 12-MHz linear-array transducer (Vivid 7, GE Medical, Horton, Norway). Longitudinal 2D images of the vessel were acquired on the R wave of the ECG, frozen in diastole and analysed off-line. The cIMT was calculated as the distance between the leading edge of the lumen–intima interface and the media–adventitia interface on the far wall of the artery.
  2. Applanation tonometry for carotid–femoral PWV and carotid pulse wave analysis was performed with a micromanometer (SPC-301, Millar Instruments, Houston, TX, USA) using proprietary software (SphygmoCor version 7.0, Scanmed, Gloucestershire, UK). PWV was not measured in the controls, as permission was not granted for femoral artery measures. The peripheral pressure–pulse waveform from the right carotid artery was acquired using the same equipment as described above and the average of waveforms recorded over 10 s was used to calculate a carotid augmentation index.
  3. The calcification score was measured by 16-slice spiral CT scans (Somatom Sensation 16, Siemens Medical Solutions Inc, Erlangen, Germany) using the standard Ca scoring protocol. Prospective ECG triggering was performed to obtain image acquisition in diastole, using 60% of the RR interval for image reconstruction. The Agatston score [26] for each main epicardial coronary artery, cardiac valves and aorta and an overall score for each patient were determined. In the control group, CT was not undertaken because of radiation concerns.

Statistical analysis
Results are presented as mean ± SD unless otherwise indicated. Spearman's (non-parametric) correlations were used to test for associations between the calcification inhibitor levels and selected clinical, anthropometric, biochemical and vascular measures. Comparisons between patient and control groups were made using one-way analysis of variance (ANOVA). Two separate stepwise multiple linear regression analyses were performed to test the associations between the calcification inhibitors against the outcome variables PWV and calcification score. Variables with P < 0.1 on univariate analyses (age, time in CKD stage IV, dialysis vintage, mean time-integrated Ca x PO4 product PTH and serum albumin and dosage of alphacalcidol) were entered into the multiple regression models. Given the interaction between serum PO4 and Ca x PO4 (P = 0.02), we have excluded serum PO4 from the regression models despite its significant association with fetuin-A levels on univariate analysis so as to avoid collinearity. All the three calcification inhibitors under study (fetuin-A, OPG and uc-MGP) were entered into both the regression models even though uc-MGP did not show any significant associations with vascular measures on univariate analyses. Also, given the known fluctuations in biochemical measures and calcification inhibitor levels [27–29] following a single HD session, and to address the issue of pooling all dialysis (HD and PD) patients for analyses, the dialysis modality was entered into both regression models despite non-significance on univariate analyses. P < 0.05 was considered statistically significant. Statistical analyses were performed using SPSS version 14.0 (SPSS Inc., Chicago, IL, USA).



   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The clinical and biochemical characteristics of the patient and control groups are shown in Table 1. Of the 61 patients (37 boys), 39 had renal dysplasia, 9 inherited nephropathies, 5 cystic kidney disease, 4 primary renal tubular disorders, 3 renovascular disorders and 1 Wilm's tumour. None of the patients had diabetes, dyslipidaemias or active infections at the time of the study, and none were on coumadin therapy.


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  		Table 1 Demographic, clinical, anthropometric and biochemical characteristics of patients and controls

 
Levels of fetuin-A, OPG and uc-MGP in healthy children
The mean fetuin-A levels in the healthy controls were 0.41 ± 0.13 g/L, and lower than that reported in adults (0.5–1.0 g/L). Fetuin-A showed a linear increase with age (P < 0.0001, r2 = 0.55, Figure 1A), but was independent of gender. In children aged 12–18 years, fetuin-A levels were lower in those ≥ 50th percentile for age-appropriate height as compared to children below this percentile (0.45 ± 0.1 versus 0.6 ± 0.2 g/L, P = 0.03). In this cohort with a normal biochemical profile and no evidence of inflammation, fetuin-A did not show any associations with serum Ca, PO4 or PTH levels, fasting glucose, triglyceride or hs-CRP. In the 18 controls who underwent vascular scans, no associations were seen with calcification inhibitor levels.


Figure 1
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  		Fig. 1 Circulating serum fetuin-A and osteoprotegerin (OPG) levels showed a linear increase with age in healthy controls, P < 0.0001, r2 = 0.55 (A) and P = 0.004, r2 = 0.34 (B), respectively.

 
The mean OPG levels in healthy children were 5.2 ± 1.2 pmol/L. OPG levels also showed a linear increase with age (P = 0.004, r2 = 0.34, Figure 1B), but no correlation was found between OPG and any anthropometric, biochemical or vascular measure. The mean uc-MGP levels in the healthy controls were 527 ± 185 µM, and were independent of age and unrelated to any other measured parameters.

Fetuin-A levels in dialysis patients (Table Table 2)
Unlike the levels seen in adult CKD patients, fetuin-A levels in children on dialysis were significantly higher as compared to controls (0.84 ± 0.3 versus 0.41 ± 0.13 g/L, P < 0.0001, Figure 2A). The correlation between age and fetuin-A in healthy controls was not seen in the dialysis population (P = 0.33). Fetuin-A showed an inverse correlation with time on dialysis (P = 0.0013, r2 = 0.14, Figure 2B). Fetuin-A was lower in HD (n = 18) as compared with PD patients (0.69 ± 0.4 versus 1.11 ± 0.2 g/L, P = 0.03), but this significance was lost after correction for the time on dialysis.


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  		Table 2 Associations between the calcification inhibitors and clinical, anthropometric, biochemical and vascular measuresa

 

Figure 2
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  		Fig. 2 Serum fetuin-A levels in children on dialysis were significantly higher as compared to controls, 0.84 ± 0.3 versus 0.41 ± 0.13 g/L, P < 0.0001 (A), and showed an inverse correlation with time on dialysis, P = 0.0013, r2 = 0.14 (B).

 
Fetuin-A showed an inverse correlation with the mean time-integrated serum PO4 levels (P = 0.03, r2 = 0.19) and Ca x PO4 product (P < 0.0001, r2 = 0.24). Fetuin-A levels showed a strong negative correlation with hs-CRP (P = 0.001, r2 = 0.42). Fetuin-A was associated with vessel stiffness: both the aortic PWV and the aortic augmentation index showed an inverse correlation with fetuin-A levels [P = 0.016, r2 = 0.19 (Figure 3A) and P = 0.03, r2 = 0.11, respectively]. Fetuin-A levels were significantly lower in children with coronary or valvular calcification (n = 14) on CT scan than in those without calcification (0.64 ± 0.2 versus 0.89 ± 0.4 g/L, P = 0.007, Figure 3B). For every 0.1 g/L increase in serum fetuin-A, there was a 5% decrease in risk of calcification (95% CI 0.84–0.91, P = 0.013).


Figure 3
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  		Fig. 3 The aortic pulse wave velocity (PWV) showed an inverse correlation with serum fetuin-A levels, P = 0.016, r2 = 0.19 (A). Fetuin-A levels were significantly lower in children with coronary or valvular calcification (n = 14) on CT scan than in those without calcification, 0.64 ± 0.2 versus 0.89 ± 0.4 g/L, P = 0.007 (B).

 
OPG levels in dialysis patients (Table Table 2)
OPG levels were significantly higher in dialysis patients as compared to healthy controls (6.7 ± 2.2 versus 5.2 ± 1.2 pmol/L, P < 0.0001, Figure 4). A linear relationship was seen between OPG and iPTH levels (P = 0.01, r2 = 0.35). OPG levels were higher in HD compared to PD patients (8.9 ± 1.6 versus 6.0 ± 0.9 pmol/L, P = 0.02), but this reflected the higher PTH levels in the HD patients. OPG levels were associated with vessel stiffness and calcification. The aortic PWV increased with increasing OPG levels (P = 0.03, r2 = 0.18, Figure 5A), and children with calcification had significantly higher OPG levels than those without calcification (8.1 ± 1.6 versus 6.3 ± 2.2 pmol/L, P = 0.005, Figure 5B). There was no correlation between RANKL or OPG/RANKL and any clinical, biochemical or vascular measure.


Figure 4
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  		Fig. 4 Serum osteoprotegerin (OPG) levels were significantly higher in dialysis patients as compared to healthy controls, 6.7 ± 2.2 versus 5.2 ± 1.2 pmol/L, P < 0.0001.

 

Figure 5
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  		Fig. 5 The aortic pulse wave velocity (PWV) was positively associated with increasing serum osteoprotegerin (OPG) levels, P = 0.03, r2 = 0.18 (A). Children with calcification had significantly higher OPG levels than those without calcification, 8.1 ± 1.6 versus 6.3 ± 2.2 pmol/L, P = 0.0026 (B).

 
uc-MGP levels in dialysis patients (Table Table 2)
uc-MGP levels in children on dialysis were significantly lower as compared to controls (232 ± 116 versus 527 ± 185 µM, P < 0.001, Figure 6), but no further associations with uc-MGP levels were found.


Figure 6
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  		Fig. 6 Serum levels of undercarboxylated-matrix Gla protein (uc-MGP) were lower in children on dialysis as compared to controls, 232 ± 116 versus 527 ± 185 µM, P < 0.001.

 
Correlations between the calcification inhibitors
We were unable to find any correlations between serum levels of fetuin-A, OPG and uc-MGP in the overall cohort, but in the patients with calcification, an association was seen between fetuin-A and OPG levels (P = 0.04, r = 0.09). There was no correlation with uc-MGP and fetuin-A or OPG.

Associations between the calcification inhibitor levels and determinants of vascular measures
On stepwise multiple linear regression analyses fetuin-A levels independently predicted aortic PWV (P = 0.004, ß = – 0.45, model R2 = 48%) and fetuin-A and OPG predicted cardiac calcification (P = 0.02, ß = – 0.29 and P = 0.014, ß = 0.33, model R2 = 32%, respectively).



   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This is the first study to describe circulating levels of the calcification inhibitors fetuin-A, OPG and uc-MGP in a paediatric population and shows that children on dialysis have a significant perturbation of these levels that is associated with increased vascular stiffness and calcification.

In healthy children, both fetuin-A and OPG levels increased with age, presumably because both these proteins are expressed in bone [30] and play a role in skeletal mineralization [31]. Also, in healthy controls, fetuin-A levels were lower in taller children as compared to their age-related peers, suggesting that fetuin-A may be consumed in the course of active skeletal mineralization, as shown in an animal model [31]. We were unable to find a correlation between fetuin-A and features of bone turnover including height, alkaline phosphatase or PTH levels in dialysis patients; however, as the PTH levels in our cohort were lower than that recommended by the K/DOQI guidelines [32], a high-turnover bone state is unlikely.

Although several studies have reported that adults on dialysis have significantly lower fetuin-A levels than controls [20,22,33–36], we found higher levels of fetuin-A in paediatric dialysis patients as compared to healthy age-matched controls. Only children with evidence of calcification on CT scan had reduced fetuin-A, but even this group had higher levels than the controls. Nevertheless, with increasing dialysis vintage and hs-CRP, fetuin-A levels decreased. Our findings are supported by reports showing that adults with early CKD do not have a reduction in fetuin-A [37] and that there is no change in fetuin-A levels in dialysis patients with low levels of inflammatory activity [38]. Fetuin-A is a negative acute phase reactant [39], and in the pro-inflammatory dialysis environment its production may be reduced. In addition, the pro-calcific uraemic milieu may consume circulating fetuin-A: in vitro studies have shown that fetuin-A contributes to ~50% of the calcification inhibitory capacity of human plasma [13], and by ‘shielding’ mechanisms prevents further crystal growth [12]. Taken together, this suggests that a protective mechanism allows an upregulation of fetuin-A in the early stages of CKD and dialysis, and only severe or prolonged exposure to a pro-inflammatory and/or pro-calcific environment eventually leads to low levels due to reduced production and/or increased consumption.

However, it may also be that patients with calcification have genetically lower fetuin-A levels that predispose them to calcification. Genetic polymorphism studies have given conflicting results [33,40], but the association between reduced fetuin-A levels and cardiovascular [19–22] and even all-cause mortality [19] in four large studies suggests that fetuin-A is likely to have a causal effect on vascular calcification. Moreover, low circulating fetuin-A levels are associated with high serum PO4 levels even in the general population [40] and have been associated with valvular calcification in patients with normal renal function [41]. In our study, fetuin-A was a significant and independent predictor of vascular stiffness and calcification irrespective of dialysis vintage, Ca x PO4 levels or hs-CRP, implying that genetic polymorphisms may indeed play a role in an individual patient's susceptibility to calcify, possibly by modulating the magnitude of change in fetuin-A production in response to a pro-inflammatory or pro-calcific environment.

We found an independent association between fetuin-A levels and aortic PWV and calcification, and there are few and conflicting reports in the literature on this. While a Japanese study has shown that fetuin-A levels predict carotid artery stiffness even in healthy subjects [42], Hermans et al. could not find an independent association between PWV or augmentation index and fetuin-A in dialysis patients [38]. The above dialysis cohort in fact had normal fetuin-A levels and a low level of inflammatory activity as compared to controls, and may not be a representative population of dialysis patients. Animal studies have shown that fetuin-A knock-out mice develop widespread soft-tissue and myocardial calcification, whereas their large arteries are spared [43]. In these animals the myocardial Ca content can increase up to 60-fold, initiating a profound pro-fibrotic response with the ‘myocardial stiffness’ leading to cardiac fibrosis, diastolic dysfunction, reduced cardiac output and an impaired tolerance to ischaemia [43]. Indeed, fetuin-A levels are an independent predictor of death in non-renal failure patients following electrocardiogram changes of ST-elevation and acute myocardial infarction [44].

In adults, serum OPG levels are associated with the progression of carotid atherosclerosis [45] and coronary calcification [16], and there are two reports, one in HD patients [46], linking serum OPG levels with vascular stiffness [47]. However, the vessel wall is a site of abundant OPG production, and mouse genetic studies have in fact suggested a vasculo-protective role for OPG [48–50]: targeted deletion of the OPG gene leads to calcification of the aorta and renal arteries [48], transgenic overexpression in OPG-null mice leads to rescue of the phenotype of vascular calcification [49], and finally, vascular calcification induced by warfarin or pharmacological doses of vitamin D can be inhibited by simultaneous application of OPG [50]. Thus, changes in OPG levels may be either cause or consequence of vascular calcification, and high circulating OPG levels in patients with cardiovascular disease could also mean that raised OPG levels detrimentally affect vascular homeostasis.

Although uc-MGP levels were significantly lower in dialysis patients than healthy controls, we were unable to find any correlations with uc-MGP and clinical or vascular measures. In vitro work has shown that in vessels with intimal and/or medial calcification, uc-MGP is localized around areas of calcification, whereas in healthy arteries active or {gamma}-carboxylated MGP is present, with no uc-MGP [25,51]. It is possible that uc-MGP levels were so low that correlations with biochemical or vascular parameters is no longer possible. Also, we have not measured total MGP levels, and the low circulating uc-MGP may represent a lack of MGP production or reduced vitamin K-dependent {gamma}-carboxylation of the uc-MGP to its active form. As MGP levels can potentially be modulated by dietary supplementation of vitamin K [52], the role of circulating MGP, if any, needs to be further explored.

There is clearly a complex relationship between the calcification inhibitory proteins and vascular measures in different clinical settings and even at the different stages of uraemic vasculopathy that are impossible to discern given the cross-sectional nature of this study and indeed all the other published work in this field. The results of the present study warrant further investigations of circulating inhibitor levels as well as their genetic polymorphisms to elucidate the potential clinical utility of these biomarkers. As cardiovascular morbidity is known to begin in the early stages of CKD, future studies should assess serial measures of the circulating inhibitors from CKD stages II–III.

In conclusion, we have shown for the first time that fetuin-A and OPG impact on vascular stiffness and calcification in children on dialysis. Paediatric dialysis patients have an upregulation of fetuin-A, possibly as a protective response to the pro-calcific and pro-inflammatory uraemic environment. While further longitudinal studies in both adult and paediatric CKD patients are required to fully characterize these circulating biomarkers, they may prove to be a useful and convenient measure of an individual patient's susceptibility to calcify.



   Acknowledgments
 
This study was funded by the Kidney Research Aid Fund and funding for CT scans was obtained from the Eyck and Strutt scholarship from the British Medical Association. This work was presented as an oral communication at the American Society of Nephrology meeting in San Francisco in November 2007.

Conflict of interest statement. G. Hawa is full time employee of Biomedica Medizinprodukte, Vienna, Austria. The remainder of the authors do not have any conflict of interest.



   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Received for publication: 23. 1.08
Accepted in revised form: 2. 4.08


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