Serum Calcification Propensity Is Increased in Myocardial Infarction and Hints at a Pathophysiological Role Independent of Classical Cardiovascular Risk Factors.

BACKGROUND: Vascular calcification is associated with increased mortality in patients with cardiovascular disease. Secondary calciprotein particles are believed to play a causal role in the pathophysiology of vascular calcification. The maturation time (T50) of calciprotein particles provides a measure of serum calcification propensity. We compared T50 between patients with ST-segment-elevated myocardial infarction and control subjects and studied the association of T50 with cardiovascular risk factors and outcome. METHODS: T50 was measured by nephelometry in 347 patients from the GIPS-III trial (Metabolic Modulation With Metformin to Reduce Heart Failure After Acute Myocardial Infarction: Glycometabolic Intervention as Adjunct to Primary Coronary Intervention in ST Elevation Myocardial Infarction: a Randomized Controlled Trial) and in 254 matched general population controls from PREVEND (Prevention of Renal and Vascular End-Stage Disease). We also assessed the association between T50 and left ventricular ejection fraction, as well as infarct size, the incidence of ischemia-driven reintervention during 5 years of follow-up, and serum nitrite as a marker of endothelial dysfunction. RESULTS: Patients with ST-segment-elevated myocardial infarction had a significantly lower T50 (ie, higher serum calcification propensity) compared with controls (T50: 289±63 versus 338±56 minutes; P<0.001). In patients with ST-segment-elevated myocardial infarction, lower T50 was associated with female sex, lower systolic blood pressure, lower total cholesterol, lower LDL (low-density lipoprotein) cholesterol, lower triglycerides, and higher HDL (high-density lipoprotein) cholesterol but not with circulating nitrite or nitrate. Ischemia-driven reintervention was associated with higher LDL (P=0.03) and had a significant interaction term for T50 and sex (P=0.005), indicating a correlation between ischemia-driven reintervention and T50 above the median in men and below the median in women, between 150 days and 5 years of follow-up. CONCLUSIONS: Serum calcification propensity is increased in patients with ST-segment-elevated myocardial infarction compared with the general population, and its contribution is more pronounced in women than in men. Its lack of/inverse association with nitrite and blood pressure confirms T50 to be orthogonal to traditional cardiovascular disease risk factors. Lower T50 was associated with a more favorable serum lipid profile, suggesting the involvement of divergent pathways of calcification stress and lipid stress in the pathophysiology of myocardial infarction.

Augmentative effects of leukemia inhibitory factor reveal a critical role for TYK2 signaling in vascular calcification.

Medial vascular calcification in chronic kidney disease (CKD) involves pro-inflammatory pathways induced by hyperphosphatemia. Several interleukin 6 family members have been associated with pro-calcific effects in vascular smooth muscle cells (VSMCs) and are considered as therapeutic targets. Therefore, we investigated the role of leukemia inhibitory factor (LIF) during VSMC calcification. LIF expression was found to be increased following phosphate exposure of VSMCs. LIF supplementation aggravated, while silencing of endogenous LIF or LIF receptor (LIFR) ameliorated the pro-calcific effects of phosphate in VSMCs. The soluble LIFR mediated antagonistic effects towards LIF and reduced VSMC calcification. Mechanistically, LIF induced phosphorylation of the non-receptor tyrosine-protein kinase 2 (TYK2) and signal transducer and activator of transcription-3 (STAT3) in VSMCs. TYK2 inhibition by deucravacitinib, a selective, allosteric oral immunosuppressant used in psoriasis treatment, not only blunted the effects of LIF, but also interfered with the pro-calcific effects induced by phosphate. Conversely, TYK2 overexpression aggravated VSMC calcification. Ex vivo calcification of mouse aortic rings was ameliorated by Tyk2 pharmacological inhibition and genetic deficiency. Cholecalciferol-induced vascular calcification in mice was improved by Tyk2 inhibition and in the Tyk2-deficient mice. Similarly, calcification was ameliorated in Abcc6/Tyk2-deficient mice after adenine/high phosphorus-induced CKD. Thus, our observations indicate a role for LIF in CKD-associated vascular calcification. Hence, the effects of LIF identify a central pro-calcific role of TYK2 signaling, which may be a future target to reduce the burden of vascular calcification in CKD.

Serum T50 predicts cardiovascular mortality in individuals with type 2 diabetes: A prospective cohort study.

BACKGROUND AND AIMS: Individuals with type 2 diabetes (T2D) have a higher risk of cardiovascular disease, compared with those without T2D. The serum T50 test captures the transformation time of calciprotein particles in serum. We aimed to assess whether serum T50 predicts cardiovascular mortality in T2D patients, independent of traditional risk factors. METHODS: We analyzed 621 individuals with T2D in this prospective cohort study. Cox regression models were performed to test the association between serum T50 and cardiovascular and all-cause mortality. Causes of death were categorized according to ICD-10 codes. Risk prediction improvement was assessed by comparing Harrell's C for models without and with T50. RESULTS: The mean age was 64.2 ± 9.8 years, and 61% were male. The average serum T50 time was 323 ± 63 min. Higher age, alcohol use, high-sensitive C-reactive protein, and plasma phosphate were associated with lower serum T50 levels. Higher plasma triglycerides, venous bicarbonate, sodium, magnesium, and alanine aminotransferase were associated with higher serum T50 levels. After a follow-up of 7.5[5.4-10.7] years, each 60 min decrease in serum T50 was associated with an increased risk of cardiovascular (fully adjusted HR 1.32, 95% CI 1.08-1.50, and p = 0.01) and all-cause mortality (HR 1.15, 95%CI 1.00-1.38, and p = 0.04). Results were consistent in sensitivity analyses after exclusion of individuals with estimated glomerular filtration rate <45 or <60 mL/min/1.73 m2 and higher plasma phosphate levels. CONCLUSIONS: Serum T50 improves prediction of cardiovascular and all-cause mortality risk in individuals with T2D. Serum T50 may be useful for risk stratification and to guide therapeutic strategies aiming to reduce cardiovascular mortality in T2D.

The Effect of Magnesium Supplementation on Vascular Calcification in CKD: A Randomized Clinical Trial (MAGiCAL-CKD).

SIGNIFICANCE STATEMENT: Magnesium prevents vascular calcification in animals with CKD. In addition, lower serum magnesium is associated with higher risk of cardiovascular events in CKD. In a randomized, double-blinded, placebo-controlled trial, the authors investigated the effects of magnesium supplementation versus placebo on vascular calcification in patients with predialysis CKD. Despite significant increases in plasma magnesium among study participants who received magnesium compared with those who received placebo, magnesium supplementation did not slow the progression of vascular calcification in study participants. In addition, the findings showed a higher incidence of serious adverse events in the group treated with magnesium. Magnesium supplementation alone was not sufficient to delay progression of vascular calcification, and other therapeutic strategies might be necessary to reduce the risk of cardiovascular disease in CKD. BACKGROUND: Elevated levels of serum magnesium are associated with lower risk of cardiovascular events in patients with CKD. Magnesium also prevents vascular calcification in animal models of CKD. METHODS: To investigate whether oral magnesium supplementation would slow the progression of vascular calcification in CKD, we conducted a randomized, double-blinded, placebo-controlled, parallel-group, clinical trial. We enrolled 148 subjects with an eGFR between 15 and 45 ml/min and randomly assigned them to receive oral magnesium hydroxide 15 mmol twice daily or matching placebo for 12 months. The primary end point was the between-groups difference in coronary artery calcification (CAC) score after 12 months adjusted for baseline CAC score, age, and diabetes mellitus. RESULTS: A total of 75 subjects received magnesium and 73 received placebo. Median eGFR was 25 ml/min at baseline, and median baseline CAC scores were 413 and 274 in the magnesium and placebo groups, respectively. Despite plasma magnesium increasing significantly during the trial in the magnesium group, the baseline-adjusted CAC scores did not differ significantly between the two groups after 12 months. Prespecified subgroup analyses according to CAC>0 at baseline, diabetes mellitus, or tertiles of serum calcification propensity did not significantly alter the main results. Among subjects who experienced gastrointestinal adverse effects, 35 were in the group receiving magnesium treatment versus nine in the placebo group. Five deaths and six cardiovascular events occurred in the magnesium group compared with two deaths and no cardiovascular events in the placebo group. CONCLUSIONS: Magnesium supplementation for 12 months did not slow the progression of vascular calcification in CKD, despite a significant increase in plasma magnesium. CLINICAL TRIALS REGISTRATION: www.clinicaltrials.gov ( NCT02542319 ).

Solving the insoluble: calciprotein particles mediate bulk mineral transport.

Protein mineral complexes, or calciprotein particles, are formed by calcium, phosphate, and the plasma protein fetuin-A. Crystalline calciprotein particles cause soft tissue calcification, oxidative stress, and inflammation, all well-known complications in chronic kidney disease. The T50 calcification propensity test measures how long it takes for amorphous calciprotein particles to crystallize. A study in this volume demonstrates remarkably low calcification propensity in cord blood, despite high mineral concentration. This hints to previously unidentified calcification inhibitors.

Effect of vitamin K supplementation on serum calcification propensity and arterial stiffness in vitamin K-deficient kidney transplant recipients: A double-blind, randomized, placebo-controlled clinical trial.

Vitamin K deficiency is common among kidney transplant recipients (KTRs) and likely contributes to progressive vascular calcification and stiffness. In this single-center, randomized, double-blind, placebo-controlled trial, we aimed to investigate the effects of vitamin K supplementation on the primary end point, serum calcification propensity (calciprotein particle maturation time, T50), and secondary end points arterial stiffness (pulse wave velocity [PWV]) and vitamin K status in 40 vitamin K-deficient KTRs (plasma dephosphorylated uncarboxylated matrix Gla protein [dp-ucMGP] ≥500 pmol/L). Participants (35% female; age, 57 ± 13 years) were randomized 1:1 to vitamin K2 (menaquinone-7, 360 µg/day) or placebo for 12 weeks. Vitamin K supplementation had no effect on calcification propensity (change in T50 vs baseline +2.3 ± 27.4 minutes) compared with placebo (+0.8 ± 34.4 minutes; Pbetween group = .88) but prevented progression of PWV (change vs baseline -0.06 ± 0.26 m/s) compared with placebo (+0.27 ± 0.43 m/s; Pbetween group = .010). Vitamin K supplementation strongly improved vitamin K status (change in dp-ucMGP vs baseline -385 [-631 to -269] pmol/L) compared with placebo (+39 [-188 to +183] pmol/L; Pbetween group < .001), although most patients remained vitamin K-deficient. In conclusion, vitamin K supplementation did not alter serum calcification propensity but prevented progression of arterial stiffness, suggesting that vitamin K has vascular effects independent of calciprotein particles. These results set the stage for longer-term intervention studies with vitamin K supplementation in KTRs. TRIAL REGISTRY: EU Clinical Trials Register (EudraCT Number: 2019-004906-88) and the Dutch Trial Register (NTR number: NL7687).

Effect of the phosphate binder sucroferric oxyhydroxide in dialysis patients on endogenous calciprotein particles, inflammation, and vascular cells.

BACKGROUND: Calciprotein particles (CPPs), colloidal mineral-protein nanoparticles, have emerged as potential mediators of phosphate toxicity in dialysis patients, with putative links to vascular calcification, endothelial dysfunction and inflammation. We hypothesized that phosphate binder therapy with sucroferric oxyhydroxide (SO) would reduce endogenous CPP levels and attenuate pro-calcific and pro-inflammatory effects of patient serum towards human vascular cells in vitro. METHODS: This secondary analysis of a randomised controlled crossover study compared the effect of 2-week phosphate binder washout with high-dose (2000 mg/day) and low-dose (250 mg/day) SO therapy in 28 haemodialysis patients on serum CPP levels, inflammatory cytokine/chemokine arrays and human aortic smooth muscle cell (HASMC) and coronary artery endothelial cell (HCAEC) bioassays. RESULTS: In our cohort (75% male, 62 ± 12 years) high-dose SO reduced primary (amorphous) and secondary (crystalline) CPP levels {-62% [95% confidence interval (CI) -76 to -44], P < .0001 and -38% [-62 to -0.14], P < .001, respectively} compared with washout. Nine of 14 plasma cytokines/chemokines significantly decreased with high-dose SO, with consistent reductions in interleukin-6 (IL-6) and IL-8. Exposure of HASMC and HCAEC cultures to serum of SO-treated patients reduced calcification and markers of activation (IL-6, IL-8 and vascular cell adhesion protein 1) compared with washout. Serum-induced HASMC calcification and HCAEC activation was ameliorated by removal of the CPP-containing fraction from patient sera. Effects of CPP removal were confirmed in an independent cohort of chronic kidney disease patients. CONCLUSIONS: High-dose SO reduced endogenous CPP formation in dialysis patients and yielded serum with attenuated pro-calcific and inflammatory effects in vitro.

The Swiss Kidney Stone Cohort: A Longitudinal, Multicentric, Observational Cohort to Study Course and Causes of Kidney Stone Disease in Switzerland.

BACKGROUND: Kidney stone disease has a high prevalence worldwide of approximately 10% of the population and is characterized by a high recurrence rate. Kidney stone disease results from a combination of genetic, environmental, and lifestyle risk factors, and the dissection of these factors is complex. METHODS: The Swiss Kidney Stone Cohort (SKSC) is an investigator-initiated prospective, multicentric longitudinal, observational study in patients with kidney stones followed with regular visits over a period of 3 years after inclusion. Ongoing follow-ups by biannual telephone interviews will provide long-term outcome data. SKSC comprises 782 adult patients (age >18 years) with either recurrent stones or a single stone event with at least one risk factor for recurrence. In addition, a control cohort of 207 individuals without kidney stone history and absence of kidney stones on a low-dose CT scan at enrolment has also been recruited. SKSC includes extensive collections of clinical data, biochemical data in blood and 24-h urine samples, and genetic data. Biosamples are stored at a dedicated biobank. Information on diet and dietary habits was collected through food frequency questionnaires and standardized recall interviews by trained dieticians with the Globodiet software. CONCLUSION: SKSC provides a unique opportunity and resource to further study cause and course of kidney disease in a large population with data and samples collected of a homogeneous collective of patients throughout the whole Swiss population.

Protective effects of spironolactone on vascular calcification in chronic kidney disease.

BACKGROUND: Vascular calcification is common in chronic kidney disease (CKD) and associated with increased cardiovascular mortality. Aldosterone has been implicated as an augmenting factor in the progression of vascular calcification. The present study further explored putative beneficial effects of aldosterone inhibition by the mineralocorticoid receptor antagonist spironolactone on vascular calcification in CKD. METHODS: Serum calcification propensity was determined in serum samples from the MiREnDa trial, a prospective, randomized controlled clinical trial to investigate efficacy and safety of spironolactone in maintenance hemodialysis patients. Experiments were conducted in mice with subtotal nephrectomy and cholecalciferol treatment, and in calcifying primary human aortic smooth muscle cells (HAoSMCs). RESULTS: Serum calcification propensity was improved by spironolactone treatment in patients on hemodialysis from the MiREnDa trial. In mouse models and HAoSMCs, spironolactone treatment ameliorated vascular calcification and expression of osteogenic markers. CONCLUSIONS: These observations support a putative benefit of spironolactone treatment in CKD-associated vascular calcification. Further research is required to investigate possible improvements in cardiovascular outcomes by spironolactone and whether the benefits outweigh the risks in patients with CKD.

The effect of increasing dialysate magnesium on calciprotein particles, inflammation and bone markers: post hoc analysis from a randomized controlled clinical trial.

BACKGROUND: The formation of calciprotein particles (CPPs) may be an important component of the humoral defences against ectopic calcification. Although magnesium (Mg) has been shown to delay the transition of amorphous calcium-/phosphate-containing primary CPP (CPP-1) to crystalline apatite-containing secondary CPP (CPP-2) ex vivo, effects on the endogenous CPP pool are unknown. METHODS: We used post hoc analyses from a randomized double-blind parallel-group controlled clinical trial of 28 days treatment with high dialysate Mg of 2.0 mEq/L versus standard dialysate Mg of 1.0 mEq/L in 57 subjects undergoing maintenance hemodialysis for end-stage kidney disease. CPP load, markers of systemic inflammation and bone turnover were measured at baseline and follow-up. RESULTS: After 28 days of treatment with high dialysate Mg, serum total CPP (-52%), CPP-1 (-42%) and CPP-2 (-68%) were lower in the high Mg group (all P < 0.001) but were unchanged in the standard dialysate Mg group. Tumour necrosis factor-α (-20%) and interleukin-6 (-22%) were also reduced with high dialysate Mg treatment (both P < 0.01). High dialysate Mg resulted in higher levels of bone-specific alkaline phosphatase (a marker of bone formation) (+17%) but lower levels of tartrate-resistant acid phosphatase 5 b (a marker of bone resorption; -33%) (both P < 0.01). Inflammatory cytokines and bone turnover markers were unchanged in the standard dialysate Mg group over the same period. CONCLUSIONS: In this exploratory analysis, increasing dialysate Mg was associated with reduced CPP load and systemic inflammation and divergent changes in markers of bone formation and resorption.

Serum Calcification Propensity and the Risk of Cardiovascular and All-Cause Mortality in the General Population: The PREVEND Study.

OBJECTIVE: Vascular calcification contributes to the cause of cardiovascular disease. The calciprotein particle maturation time (T50) in serum, a measure of calcification propensity, has been linked with adverse outcomes in patients with chronic kidney disease, but its role in the general population is unclear. We investigated whether serum T50 is associated with cardiovascular mortality in a large general population-based cohort. Approach and Results: The relationship between serum T50 and cardiovascular mortality was studied in 6231 participants of the PREVEND (Prevention of Renal and Vascular End-Stage Disease) cohort. All-cause mortality was the secondary outcome. Mean (±SD) age was 53±12 years, 50% were male, and mean serum T50 was 329±58 minutes. A shorter serum T50 is indicative of a higher calcification propensity. Serum T50 was inversely associated with circulating phosphate, age, estimated glomerular filtration rate, and alcohol consumption, whereas plasma magnesium was positively associated with serum T50 (P<0.001, total multivariable model R2=0.281). During median (interquartile range) follow-up for 8.3 (7.8-8.9) years, 364 patients died (5.8%), of whom 95 (26.1%) died from a cardiovascular cause. In multivariable Cox proportional hazard models, each 60 minutes decrease in serum T50 was independently associated with a higher risk of cardiovascular mortality (fully adjusted hazard ratio [95% CI], 1.22 [1.04-1.36], P=0.021). This association was modified by diabetes mellitus; stratified analysis indicated a more pronounced association in individuals with diabetes mellitus. CONCLUSIONS: Serum T50 is independently associated with an increased risk of cardiovascular mortality in the general population and thus may be an early and potentially modifiable risk marker for cardiovascular mortality.

Serum free thiols predict cardiovascular events and all-cause mortality in the general population: a prospective cohort study.

BACKGROUND: Serum free thiols (R-SH, sulfhydryl groups) reliably reflect systemic oxidative stress. Since serum free thiols are rapidly oxidized by reactive species, systemic oxidative stress is generally associated with reduced serum free thiol levels. Free thiols associate with favorable disease outcomes in many patient cohorts, and the current hypothesis is that oxidative stress might also play an important role in cardiovascular disease. In this study, we aimed to establish the role of serum free thiols in the general population by investigating their relationship with the risk of cardiovascular (CV) events and all-cause mortality. METHODS: Participants (n = 5955) of the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) cohort study from the general population were included. At baseline, serum levels of free thiols were quantified and adjusted to total protein levels. Protein-adjusted serum free thiol levels were studied for their associations with clinical and biochemical parameters, as well as with the risk of CV events and all-cause mortality. RESULTS: The mean protein-adjusted serum free thiol level was 5.05 ± 1.02 µmol/g of protein. Protein-adjusted serum free thiols significantly predicted the risk of CV events, even after adjustment for potential confounding factors (hazard ratio [HR] per doubling 0.68 [95% confidence interval [CI] 0.47-1.00], P = 0.048). Similarly, protein-adjusted serum free thiols were significantly predictive of the risk of all-cause mortality (HR per doubling 0.66 [95% CI 0.44-1.00], P = 0.050). Stratified analyses revealed lower HRs for subjects with a lower body mass index (BMI), without hypertension, and without diabetes. Conversely, HRs were lower in subjects with albuminuria. CONCLUSIONS: In this large population-based cohort study, serum free thiols significantly predicted the risk of CV events and all-cause mortality. Our results highlight the potential significance and clinical applicability of serum free thiols since they are amendable to therapeutic intervention.

Oxidative stress is associated with suspected non-alcoholic fatty liver disease and all-cause mortality in the general population.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive lipid accumulation, inflammation and an imbalanced redox homeostasis. We hypothesized that systemic free thiol levels, as a proxy of systemic oxidative stress, are associated with NAFLD. METHODS: Protein-adjusted serum free thiol concentrations were determined in participants from the Prevention of Renal and Vascular End-Stage Disease (PREVEND) cohort study (n = 5562). Suspected NAFLD was defined by the Fatty Liver Index (FLI ≥ 60) and Hepatic Steatosis Index (HSI > 36). RESULTS: Protein-adjusted serum free thiols were significantly reduced in subjects with FLI ≥ 60 (n = 1651). In multivariable logistic regression analyses, protein-adjusted serum free thiols were associated with NAFLD (FLI ≥ 60) (OR per doubling of concentration: 0.78 [95% CI 0.64-0.96], P = .016) even when adjusted for potential confounding factors, including systolic blood pressure, diabetes, current smoking, use of alcohol and total cholesterol (OR 0.80 [95% CI 0.65-0.99], P = .04). This association lost its significance (OR 0.94 [95% CI 0.73-1.21], P = .65) after additional adjustment for high-sensitive C-reactive protein. Stratified analyses showed significantly differential associations of protein-adjusted serum free thiol concentrations with suspected NAFLD for gender (P < .02), hypertension (P < .001) and hypercholesterolemia (P < .003). Longitudinally, protein-adjusted serum free thiols were significantly associated with the risk of all-cause mortality in subjects with NAFLD (FLI ≥ 60) (HR 0.27 [95% CI 0.17-0.45], P < .001). CONCLUSION: Protein-adjusted serum free thiol levels are reduced and significantly associated with all-cause mortality in subjects with suspected NAFLD. Quantification of free thiols may be a promising, minimally invasive strategy to improve detection of NAFLD and associated risk of all-cause mortality in the general population.

Dialysis initiation improves calcification propensity.

BACKGROUND: Cardiovascular morbidity and mortality is high in patients starting dialysis and could be related to modifications of calcification inducers and inhibitors by dialysis, promoting cardiovascular events. The impact of dialysis initiation on serum calcification propensity evolution and arterial stiffness is unknown. We therefore prospectively determined the evolution of the one-half maximal transition time (T50) value and its main determinants as well as pulse wave velocity over the first 3 months of dialysis initiation. METHODS: We analysed the evolution of T50, fetuin-A and mineral metabolism parameters before dialysis initiation (M0) and monthly until Month 3 (M3) in incident patients starting haemodialysis (HD) or peritoneal dialysis (PD) in two tertiary Swiss university hospitals. Arterial stiffness was assessed by pulse tonometry at M0 and M3 and biological parameters were compared between M0 and M3 and before/after HD. Linear mixed models were used to assess parameter evolution over time, taking into account repeated measures and other influencing variables. RESULTS: Forty-six patients on HD and 12 on PD were followed. Among them, 45 were male (78%) with a median age of 67 years (25th-75th quartile range 54-77). T50 significantly increased between M0 and M3 from 183 (120-266) to 246 min (175-330) (P < 0.001). Fetuin-A, calcium and magnesium also increased while phosphate decreased. Factors associated with T50 changes over time were fetuin-A, phosphate and magnesium (P < 0.001). Fetuin-A changes were associated with inflammation-related factors (albumin, C-reactive protein) but not calcium and phosphate levels. Arterial stiffness was not significantly modified over 3 months. PD and HD initiation showed similar trends. CONCLUSIONS: Dialysis initiation significantly improves calcification propensity and fetuin-A levels. These modifications do not explain the high mortality related to dialysis initiation. The clinical relevance of using T50 values to initiate dialysis awaits further studies.

Calciprotein particle inhibition explains magnesium-mediated protection against vascular calcification.

BACKGROUND: Phosphate (Pi) toxicity is a strong determinant of vascular calcification development in chronic kidney disease (CKD). Magnesium (Mg2+) may improve cardiovascular risk via vascular calcification. The mechanism by which Mg2+ counteracts vascular calcification remains incompletely described. Here we investigated the effects of Mg2+ on Pi and secondary crystalline calciprotein particles (CPP2)-induced calcification and crystal maturation. METHODS: Vascular smooth muscle cells (VSMCs) were treated with high Pi or CPP2 and supplemented with Mg2+ to study cellular calcification. The effect of Mg2+ on CPP maturation, morphology and composition was studied by medium absorbance, electron microscopy and energy dispersive spectroscopy. To translate our findings to CKD patients, the effects of Mg2+ on calcification propensity (T50) were measured in sera from CKD patients and healthy controls. RESULTS: Mg2+ supplementation prevented Pi-induced calcification in VSMCs. Mg2+ dose-dependently delayed the maturation of primary CPP1 to CPP2 in vitro. Mg2+ did not prevent calcification and associated gene and protein expression when added to already formed CPP2. Confirmatory experiments in human serum demonstrated that the addition of 0.2 mmol/L Mg2+ increased T50 from healthy controls by 51 ± 15 min (P < 0.05) and CKD patients by 44 ± 13 min (P < 0.05). Each further 0.2 mmol/L addition of Mg2+ led to further increases in both groups. CONCLUSIONS: Our results demonstrate that crystalline CPP2 mediates Pi-induced calcification in VSMCs. In vitro, Mg2+ delays crystalline CPP2 formation and thereby prevents Pi-induced calcification.

Safety and Tolerability of Sodium Thiosulfate in Patients with an Acute Coronary Syndrome Undergoing Coronary Angiography: A Dose-Escalation Safety Pilot Study (SAFE-ACS).

BACKGROUND: In animal studies, hydrogen sulfide (H2S) has been shown to protect the heart from ischemia-reperfusion injury. This study evaluates the safety and tolerability of the H2S donor sodium thiosulfate (STS) in patients with acute coronary syndrome (ACS). METHODS: Eighteen patients, undergoing coronary angiography for ACS, received STS intravenously immediately after arrival at the catheterization laboratory according to a "3 + 3 dose-escalation design" with fixed dosing endpoint (0, 2.5, 5, 10, 12.5, and 15 grams). This first dose STS was combined with verapamil and nitroglycerin required for transradial procedures. A second dose STS was administered 6 hours later. Primary endpoint was dose-limiting toxicity, defined as significant hemodynamic instability or death up to 24 hours or before discharge from the coronary care unit. Secondary outcomes included the occurrence of anaphylaxis, nausea, vomiting, and systolic blood pressure (SBP) course. RESULTS: Sixteen patients received two dosages of STS and two patients one dosage. None of the patients reached the primary endpoint, nor experienced a serious adverse event. We observed a clinically well-tolerated decline in SBP 1 hour after administration of the first STS dose and concomitant verapamil/nitroglycerin. SBP for all patients together reduced 16.8 (8.1-25.5) mmHg (P = 0.0008). No significant decline in SBP occurred after the second dose. Mild nausea was observed in one patient. CONCLUSION: This is the first report on sodium thiosulfate administration in patients with acute coronary syndromes. Our data suggest that sodium thiosulfate was well tolerated in this setting. The potential benefit of this intervention has to be examined in larger studies.

Urinary sulfate excretion and risk of late graft failure in renal transplant recipients - a prospective cohort study.

Hydrogen sulfide (H2 S), produced from metabolism of dietary sulfur-containing amino acids, is allegedly a renoprotective compound. Twenty-four-hour urinary sulfate excretion (USE) may reflect H2 S bioavailability. We aimed to investigate the association of USE with graft failure in a large prospective cohort of renal transplant recipients (RTR). We included 704 stable RTR, recruited at least 1 year after transplantation. We applied log-rank testing and Cox regression analyses to study association of USE, measured from baseline 24 h urine samples, with graft failure. Median age was 55 [45-63] years (57% male, eGFR was 45 ± 19 ml/min/1.73 m2 ). Median USE was 17.1 [13.1-21.1] mmol/24 h. Over median follow-up of 5.3 [4.5-6.0] years, 84 RTR experienced graft failure. RTR in the lowest sex-specific tertile of USE experienced a higher rate of graft failure during follow-up than RTR in the middle and highest sex-specific tertiles (18%, 13%, and 5%, respectively, log-rank P < 0.001). In Cox regression analyses, USE was inversely associated with graft failure [HR per 10 mmol/24 h: 0.37 (0.24-0.55), P < 0.001]. The association remained independent of adjustment for potential confounders, including age, sex, eGFR, proteinuria, time between transplantation and baseline, BMI, smoking, and high sensitivity C-reactive protein [HR per 10 mmol/24 h: 0.51 (0.31-0.82), P = 0.01]. In conclusion, this study demonstrates a significant inverse association of USE with graft failure in RTR, suggesting high H2 S bioavailability as a novel, potentially modifiable factor for prevention of graft failure in RTR.

Ossified blood vessels in primary familial brain calcification elicit a neurotoxic astrocyte response.

Brain calcifications are commonly detected in aged individuals and accompany numerous brain diseases, but their functional importance is not understood. In cases of primary familial brain calcification, an autosomally inherited neuropsychiatric disorder, the presence of bilateral brain calcifications in the absence of secondary causes of brain calcification is a diagnostic criterion. To date, mutations in five genes including solute carrier 20 member 2 (SLC20A2), xenotropic and polytropic retrovirus receptor 1 (XPR1), myogenesis regulating glycosidase (MYORG), platelet-derived growth factor B (PDGFB) and platelet-derived growth factor receptor ß (PDGFRB), are considered causal. Previously, we have reported that mutations in PDGFB in humans are associated with primary familial brain calcification, and mice hypomorphic for PDGFB (Pdgfbret/ret) present with brain vessel calcifications in the deep regions of the brain that increase with age, mimicking the pathology observed in human mutation carriers. In this study, we characterize the cellular environment surrounding calcifications in Pdgfbret/ret animals and show that cells around vessel-associated calcifications express markers for osteoblasts, osteoclasts and osteocytes, and that bone matrix proteins are present in vessel-associated calcifications. Additionally, we also demonstrate the osteogenic environment around brain calcifications in genetically confirmed primary familial brain calcification cases. We show that calcifications cause oxidative stress in astrocytes and evoke expression of neurotoxic astrocyte markers. Similar to previously reported human primary familial brain calcification cases, we describe high interindividual variation in calcification load in Pdgfbret/ret animals, as assessed by ex vivo and in vivo quantification of calcifications. We also report that serum of Pdgfbret/ret animals does not differ in calcification propensity from control animals and that vessel calcification occurs only in the brains of Pdgfbret/ret animals. Notably, ossification of vessels and astrocytic neurotoxic response is associated with specific behavioural and cognitive alterations, some of which are associated with primary familial brain calcification in a subset of patients.

Signaling pathways involved in vascular smooth muscle cell calcification during hyperphosphatemia.

Medial vascular calcification has emerged as a putative key factor contributing to the excessive cardiovascular mortality of patients with chronic kidney disease (CKD). Hyperphosphatemia is considered a decisive determinant of vascular calcification in CKD. A critical role in initiation and progression of vascular calcification during elevated phosphate conditions is attributed to vascular smooth muscle cells (VSMCs), which are able to change their phenotype into osteo-/chondroblasts-like cells. These transdifferentiated VSMCs actively promote calcification in the medial layer of the arteries by producing a local pro-calcifying environment as well as nidus sites for precipitation of calcium and phosphate and growth of calcium phosphate crystals. Elevated extracellular phosphate induces osteo-/chondrogenic transdifferentiation of VSMCs through complex intracellular signaling pathways, which are still incompletely understood. The present review addresses critical intracellular pathways controlling osteo-/chondrogenic transdifferentiation of VSMCs and, thus, vascular calcification during hyperphosphatemia. Elucidating these pathways holds a significant promise to open novel therapeutic opportunities counteracting the progression of vascular calcification in CKD.

Serum Calcification Propensity and Coronary Artery Calcification Among Patients With CKD: The CRIC (Chronic Renal Insufficiency Cohort) Study.

RATIONALE & OBJECTIVE: Coronary artery calcification (CAC) is prevalent among patients with chronic kidney disease (CKD) and increases risks for cardiovascular disease events and mortality. We hypothesized that a novel serum measure of calcification propensity is associated with CAC among patients with CKD stages 2 to 4. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: Participants from the Chronic Renal Insufficiency Cohort (CRIC) Study with baseline (n=1,274) and follow-up (n=780) CAC measurements. PREDICTORS: Calcification propensity, quantified as transformation time (T50) from primary to secondary calciprotein particles, with lower T50 corresponding to higher calcification propensity. Covariates included age, sex, race/ethnicity, clinical site, estimated glomerular filtration rate, proteinuria, diabetes, systolic blood pressure, number of antihypertensive medications, current smoking, history of cardiovascular disease, total cholesterol level, and use of statin medications. OUTCOMES: CAC prevalence, severity, incidence, and progression. ANALYTICAL APPROACH: Multivariable-adjusted generalized linear models. RESULTS: At baseline, 824 (65%) participants had prevalent CAC. After multivariable adjustment, T50 was not associated with CAC prevalence but was significantly associated with greater CAC severity among participants with prevalent CAC: 1-SD lower T50 was associated with 21% (95% CI, 6%-38%) greater CAC severity. Among 780 participants followed up an average of 3 years later, 65 (20%) without baseline CAC developed incident CAC, while 89 (19%) with baseline CAC had progression, defined as annual increase≥100 Agatston units. After multivariable adjustment, T50 was not associated with incident CAC but was significantly associated with CAC progression: 1-SD lower T50 was associated with 28% (95% CI, 7%-53%) higher risk for CAC progression. LIMITATIONS: Potential selection bias in follow-up analyses; inability to distinguish intimal from medial calcification. CONCLUSIONS: Among patients with CKD stages 2 to 4, higher serum calcification propensity is associated with more severe CAC and CAC progression.