Sclerostin, an osteocyte-derived inhibitor of bone formation, is linked to mineral bone disorder. In order to validate its potential as a predictor of vascular calcification, we explored associations of circulating sclerostin with measures of calcification in 89 epigastric artery biopsies from patients with end-stage renal disease. Significantly higher sclerostin levels were found in the serum of patients with epigastric and coronary artery calcification (calcification score 100 or more). In Spearman's rank correlations, sclerostin levels significantly associated with age, intact parathyroid hormone, bone-specific alkaline phosphatase, and percent calcification. Multivariable regression showed that age, male gender, and sclerostin each significantly associated with the presence of medial vascular calcification. Receiver operating characteristic curve analysis showed that sclerostin (AUC 0.68) predicted vascular calcification. Vascular sclerostin mRNA and protein expressions were low or absent, and did not differ between calcified and non-calcified vessels, suggesting that the vasculature is not a major contributor to circulating levels. Thus, high serum sclerostin levels associate with the extent of vascular calcification as evaluated both by coronary artery CT and scoring of epigastric artery calcification. Among circulating biomarkers of mineral bone disorder, only sclerostin predicted vascular calcification.

Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification (VC) and requires carboxylation by vitamin K to exert calcification inhibition. Chronic kidney disease (CKD) patients undergo early vascular aging often involving extensive VC. The present cross-sectional study investigated the association between circulating dp-ucMGP levels, MGP expression in vascular tissue and MGP polymorphisms. In 141 CKD stage 5 patients, CAC score was significantly increased in the highest tertile of dp-ucMGP (p = 0.002), and a high medial VC score was associated with elevated dp-ucMGP levels. MGP vascular expression was associated with increased circulating dp-ucMGP and CAC scores. MGP SNP analysis revealed that patients homozygous for the C allele of the rs1800801 variant had a higher CAC score (median 15 [range 0-1312]) compared to patients carrying a T allele (median 0 [range 0-966] AU). These results indicate that plasma levels of dp-ucMGP are an independent predictor of increased VC in CKD5 patients and correlate with both higher CAC scores and degree of medial calcification. Additionally, high vascular expression of MGP was associated with higher CAC scores and plasma dp-ucMGP levels. Taken together, our results support that MGP is involved in the pathogenesis of VC. Matrix Gla protein (MGP) is a vitamin K dependent protein (VKDP) that is involved in the inhibition of vascular calcification (VC). MGP is small secretary protein (14 kD) that is primarily secreted by vascular smooth muscle cells (VSMCs) in the arterial wall 1. MGP contains five Glu residues that require carboxylation to become activated and to fulfill its calcification inhibitory function. This carboxylation step cannot take place in the absence of vitamin K, which has an unequivocal role in driving this post-translational step 2,3. Vitamin K is a co-factor for the enzyme γ-glutamyl carboxylase that converts glutamic acid (Glu) into γ-carboxyglutamic acid (Gla) residues 2. This conversion is critical for the activation of MGP. Additionally, there are three serine residues that need phosphorylation 4,5. The exact role of phosphorylation of MGP is still not known, but it is believed to play an important role in the regulation of secretion of the protein 3. Upon activation, MGP binds calcium-salts with high affinity, thereby affecting the calcification processes. The importance of MGP in the inhibition of calcification is illustrated by studies of MGP knockout mice, who die within two months after birth due to severe arterial calcification and rupture of the aorta 1. Chronic kidney disease (CKD) patients have an extremely high risk for developing vascular disease 4. VC, manifested both as medial and intimal calcification with distinct pathologies, is a common risk factor in CKD 5. Additionally, vitamin K deficiency is frequently encountered in CKD, which is associated with increased plasma levels of dephosphorylated uncarboxylated MGP (dp-ucMGP) plasma levels 6,7. Furthermore, increased plasma dp-ucMGP

Patients with chronic kidney disease (CKD) display a progeric vascular phenotype linked to apoptosis, cellular senescence and osteogenic transformation. This has proven intractable to modelling appropriately in model organisms. We have therefore investigated this directly in man, using for the first time validated cellular biomarkers of ageing (CDKN2A/p16INK4a, SA-β-Gal) in arterial biopsies from 61 CKD patients undergoing living donor renal transplantation. We demonstrate that in the uremic milieu, increased arterial expression of CDKN2A/p16INK4a associated with vascular progeria in CKD, independently of chronological age. The arterial expression of CDKN2A/p16INK4a was significantly higher in patients with coronary calcification (p=0.01) and associated cardiovascular disease (CVD) (p=0.004). The correlation between CDKN2A/p16INK4a and media calcification was statistically significant (p=0.0003) after correction for chronological age. We further employed correlate expression of matrix Gla protein (MGP) and runt-related transcription factor 2 (RUNX2) as additional pathognomonic markers. Higher expression of CDKN2A/p16INK4a, RUNX2 and MGP were observed in arteries with severe media calcification. The number of p16INK4a and SA-β-Gal positive cells was higher in biopsies with severe media calcification. A strong inverse correlation was observed between CDKN2A/p16INK4a expression and carboxylated osteocalcin levels. Thus, impaired vitamin K mediated carboxylation may contribute to premature vascular senescence.

Elevated CAC score is a mortality risk factor in ESRD independent of inflammation. Future studies should resolve if statins promote vascular calcification and inhibition of vitamin K synthesis in the uremic milieu.

IntroductionVascular calcification is a common, serious and elusive complication of end-stage renal disease (ESRD). As a pro-calcifying risk factor, non-thyroidal illness may promote vascular calcification through a systemic lowering of vascular calcification inhibitors such as matrix-gla protein (MGP) and Klotho.Methods and MaterialIn 97 ESRD patients eligible for living donor kidney transplantation, blood levels of thyroid hormones (fT3, fT4 and TSH), total uncarboxylated MGP (t-ucMGP), desphospho-uncarboxylated MGP (dp-ucMGP), descarboxyprothrombin (PIVKA-II), and soluble Klotho (sKlotho) were measured. The degree of coronary calcification and arterial stiffness were assessed by means of cardiac CT-scans and applanation tonometry, respectively.ResultsfT3 levels were inversely associated with coronary artery calcification (CAC) scores and measures of arterial stiffness, and positively with dp-ucMGP and sKlotho concentrations. Subfractions of MGP, PIVKA-II and sKlotho did not associate with CAC scores and arterial stiffness. fT4 and TSH levels were both inversely associated with CAC scores, but not with arterial stiffness.DiscussionThe positive associations between fT3 and dp-ucMGP and sKlotho suggest that synthesis of MGP and Klotho is influenced by thyroid hormones, and supports a link between non-thyroidal illness and alterations in calcification inhibitor levels. However, the absence of an association between serum calcification inhibitor levels and coronary calcification/arterial stiffness and the fact that MGP and Klotho undergo post-translational modifications underscore the complexity of this association. Further studies, measuring total levels of MGP and membrane bound Klotho, should examine this proposed pathway in further detail.

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Objectives. Low triiodothyronine levels, as part of the nonthyroidal illness syndrome, are common in dialysis patients and have repeatedly been shown to be associated with increased (cardiovascular) mortality rates. We hypothesized that increased vascular calcification may mediate this relationship.Methods. A total of 84 patients from the Stockholm region receiving maintenance peritoneal dialysis were included in the study. Serum concentrations of free triiodothyronine (fT3), thyroxine and thyroidstimulating hormone were measured. Coronary artery calcium (CAC) scores were assessed by cardiac computed tomography scans. Surrogates of arterial stiffness included aortic diastolic and systolic blood pressures, pulse pressure, augmentation pressure and Buckberg's subendocardial viability ratio measured by pulse waveform analyses. Patients were subsequently followed, and events of death and censoring were recorded. Thyroid hormone concentrations were associated with CAC scores, measures of arterial stiffness and allcause mortality. The associations between CAC scores and arterial stiffness surrogates and mortality were also determined to evaluate a possible causal pathway.Results. Both CAC scores and arterial stiffness surrogates were substantially higher in individuals with low fT3 levels. These associations persisted in multivariate logistic and linear regression analyses. During a median (interquartile range) follow-up of 32 (22-42) months, 24 patients died. Both fT3 levels below the median value [HR crude 4.1, 95% confidence interval (CI) 1.4-12.6] and CAC scores above the median value (HR crude 5.8, 95% CI 1.7-20.1) were strongly associated with mortality.Conclusions. In patients undergoing peritoneal dialysis, fT3 levels were strongly associated with arterial stiffness, coronary artery calcification and mortality. We speculate that the association between nonthyroidal illness and mortality may be partly mediated by acceleration of vascular calcification.