MicroRNA-145 and microRNA-486 are potential serum biomarkers for vascular calcification.

INTRODUCTION: MicroRNAs (miRs) regulate vascular calcification (VC), and their quantification may contribute to suspicion of the presence of VC. METHODS: The study was performed in four phases. Phase 1: miRs sequencing of rat calcified and non-calcified aortas. Phase 2: miRs with the highest rate of change, plus miR-145 [the most abundant miR in vascular smooth muscle cells (VSMCs)], were validated in aortas and serum from rats with and without VC. Phase 3: the selected miRs were analyzed in epigastric arteries from kidney donors and recipients, and serum samples from general population. Phase 4: VSMCs were exposed to different phosphorus concentrations, and miR-145 and miR-486 were overexpressed to investigate their role in VC. RESULTS: miR-145, miR-122-5p, miR-486 and miR-598-3p decreased in the rat calcified aortas, but only miR-145 and miR-486 were detected in serum. In human epigastric arteries, miR-145 and miR-486 were lower in kidney transplant recipients compared with donors. Both miRs inversely correlated with arterial calcium content and with VC (Kauppila index). In the general population, the severe VC was associated with the lowest serum levels of both miRs. The receiver operating characteristic curve showed that serum miR-145 was a good biomarker of VC. In VSMCs exposed to high phosphorus, calcium content, osteogenic markers (Runx2 and Osterix) increased, and the contractile marker (α-actin), miR-145 and miR-486 decreased. Overexpression of miR-145, and to a lesser extent miR-486, prevented the increase in calcium content induced by high phosphorus, the osteogenic differentiation and the loss of the contractile phenotype. CONCLUSION: miR-145 and miR-486 regulate the osteogenic differentiation of VSMCs, and their quantification in serum could serve as a marker of VC.

The receptor activator of nuclear factor κΒ ligand receptor leucine-rich repeat-containing G-protein-coupled receptor 4 contributes to parathyroid hormone-induced vascular calcification.

BACKGROUND: In chronic kidney disease, serum phosphorus (P) elevations stimulate parathyroid hormone (PTH) production, causing severe alterations in the bone-vasculature axis. PTH is the main regulator of the receptor activator of nuclear factor κB (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) system, which is essential for bone maintenance and also plays an important role in vascular smooth muscle cell (VSMC) calcification. The discovery of a new RANKL receptor, leucine-rich repeat-containing G-protein-coupled receptor 4 (LGR4), which is important for osteoblast differentiation but with an unknown role in vascular calcification (VC), led us to examine the contribution of LGR4 in high P/high PTH-driven VC. METHODS: In vivo studies were conducted in subtotally nephrectomized rats fed a normal or high P diet, with and without parathyroidectomy (PTX). PTX rats were supplemented with PTH(1-34) to achieve physiological serum PTH levels. In vitro studies were performed in rat aortic VSMCs cultured in control medium, calcifying medium (CM) or CM plus 10-7 versus 10-9 M PTH. RESULTS: Rats fed a high P diet had a significantly increased aortic calcium (Ca) content. Similarly, Ca deposition was higher in VSMCs exposed to CM. Both conditions were associated with increased RANKL and LGR4 and decreased OPG aorta expression and were exacerbated by high PTH. Silencing of LGR4 or parathyroid hormone receptor 1 (PTH1R) attenuated the high PTH-driven increases in Ca deposition. Furthermore, PTH1R silencing and pharmacological inhibition of protein kinase A (PKA), but not protein kinase C, prevented the increases in RANKL and LGR4 and decreased OPG. Treatment with PKA agonist corroborated that LGR4 regulation is a PTH/PKA-driven process. CONCLUSIONS: High PTH increases LGR4 and RANKL and decreases OPG expression in the aorta, thereby favouring VC. The hormone's direct pro-calcifying actions involve PTH1R binding and PKA activation.

Lamin A is involved in the development of vascular calcification induced by chronic kidney failure and phosphorus load.

Vascular calcification remains one of the main factors associated to morbidity and mortality in both ageing and chronic kidney disease. Both hyperphosphataemia, a well-known promoter of vascular calcification, and abnormal processing defects of lamin A/C have been associated to ageing. The main aim of this study was to analyse the effect of phosphorus load in the differential expression pattern of genes and proteins, particularly of lamin A/C, which are involved in phenotypic change of the vascular smooth muscle cells to osteoblast-like cells. The in vivo study of the calcified abdominal aortas from nephrectomized rats receiving a high phosphorus diet showed among others, a repression of muscle related proteins and overexpression of lamin A/C. Similar results were observed in vitro, where primary vascular smooth muscle cells cultured in calcifying medium showed increased expression of prelamin A and lamin A and abnormalities in the nuclear morphology. Co-immunoprecipitation assays showed novel and important physical interactions between lamin A and RUNX2 during the process of calcification. In fact, the knockdown of prelamin A and lamin A inhibited the increase of Runx2, osteocalcin and osteopontin gene expression, calcium deposition, nuclear abnormalities and the RUNX2 protein translocation into the nucleus of the cell. These in vivo and in vitro results highlight the important role played by lamin A in the process of vascular calcification.

MicroRNAs 29b, 133b, and 211 Regulate Vascular Smooth Muscle Calcification Mediated by High Phosphorus.

Vascular calcification is a frequent cause of morbidity and mortality in patients with CKD and the general population. The common association between vascular calcification and osteoporosis suggests a link between bone and vascular disorders. Because microRNAs (miRs) are involved in the transdifferentiation of vascular smooth muscle cells into osteoblast-like cells, we investigated whether miRs implicated in osteoblast differentiation and bone formation are involved in vascular calcification. Different levels of uremia, hyperphosphatemia, and aortic calcification were induced by feeding nephrectomized rats a normal or high-phosphorus diet for 12 or 20 weeks, at which times the levels of eight miRs (miR-29b, miR-125, miR-133b, miR-135, miR-141, miR-200a, miR-204, and miR-211) in the aorta were analyzed. Compared with controls and uremic rats fed a normal diet, uremic rats fed a high-phosphorous diet had lower levels of miR-133b and miR-211 and higher levels of miR-29b that correlated respectively with greater expression of osteogenic RUNX2 and with lower expression of several inhibitors of osteoblastic differentiation. Uremia per se mildly reduced miR-133b levels only. Similar results were obtained in two in vitro models of vascular calcification (uremic serum and high-calcium and -phosphorus medium), and experiments using antagomirs and mimics to modify miR-29b, miR-133b, and miR-211 expression levels in these models confirmed that these miRs regulate the calcification process. We conclude that miR-29b, miR-133b, and miR-211 have direct roles in the vascular smooth muscle calcification induced by high phosphorus and may be new therapeutic targets in the management of vascular calcification.

Improvement of mineral and bone metabolism markers is associated with better survival in haemodialysis patients: the COSMOS study.

BACKGROUND: Abnormalities in serum phosphorus, calcium and parathyroid hormone (PTH) have been associated with poor survival in haemodialysis patients. This COSMOS (Current management Of Secondary hyperparathyroidism: a Multicentre Observational Study) analysis assesses the association of high and low serum phosphorus, calcium and PTH with a relative risk of mortality. Furthermore, the impact of changes in these parameters on the relative risk of mortality throughout the 3-year follow-up has been investigated. METHODS: COSMOS is a 3-year, multicentre, open-cohort, prospective study carried out in 6797 adult chronic haemodialysis patients randomly selected from 20 European countries. RESULTS: Using Cox proportional hazard regression models and penalized splines analysis, it was found that both high and low serum phosphorus, calcium and PTH were associated with a higher risk of mortality. The serum values associated with the minimum relative risk of mortality were 4.4 mg/dL for serum phosphorus, 8.8 mg/dL for serum calcium and 398 pg/mL for serum PTH. The lowest mortality risk ranges obtained using as base the previous values were 3.6-5.2 mg/dL for serum phosphorus, 7.9-9.5 mg/dL for serum calcium and 168-674 pg/mL for serum PTH. Decreases in serum phosphorus and calcium and increases in serum PTH in patients with baseline values of >5.2 mg/dL (phosphorus), >9.5 mg/dL (calcium) and <168 pg/mL (PTH), respectively, were associated with improved survival. CONCLUSIONS: COSMOS provides evidence of the association of serum phosphorus, calcium and PTH and mortality, and suggests survival benefits of controlling chronic kidney disease-mineral and bone disorder biochemical parameters in CKD5D patients.

Natural antioxidants and vascular calcification: a possible benefit.

BACKGROUND: Several studies have demonstrated the impact of vascular calcification on morbidity and mortality both in the general and chronic kidney disease populations. The process of vascular calcification involves complex mechanisms including the overexpression of genes and proteins associated with mineralization and increments of reactive oxygen species (ROS). Taking into account previous findings, we decided to analyze in vitro the likely inhibitory effect of natural antioxidants in the process of vascular calcification. METHODS: Primary vascular smooth muscle cells (VSMCs) were cultured with either normal medium or normal medium supplemented with calcium and phosphorus (P + Ca) in combination with several antioxidants. Mineralization, intracellular reactive oxygen species levels and the protein expression of Cbfa1/RUNX2 and Mn-superoxide dismutase-2 (SOD-2) were investigated. RESULTS: Curcumin and silybin were the more effective, inhibiting both ROS increase and VSMC mineralization. Curcumin was able to prevent the increase in Cbfa1/RUNX2 expression, but did not modify SOD-2 expression in the VSMCs cultured with the P + Ca medium. CONCLUSIONS: These findings support the importance of performing further studies in this field, as some antioxidants might have potential benefits in the management of vascular calcification.

Calcium, phosphorus, PTH and death rates in a large sample of dialysis patients from Latin America. The CORES Study.

BACKGROUND: Mineral metabolism parameters may play a role in the survival of patients with chronic kidney disease (CKD). METHODS: In the CORES Study, we analysed the association between calcium, phosphorus and PTH and mortality (all-cause and cardiovascular) in 16 173 haemodialysis (HD) patients over 18 years from six Latin American countries, who underwent haemodialysis up to 54 months. Unadjusted, case-mix-adjusted and time-dependent multivariable-adjusted hazard ratio (HR) of death were calculated for categories of serum albumin-corrected calcium (Ca(Alb)), phosphorus and PTH using as 'reference values' the range in which the lowest death rate was observed. Age, gender, vitamin D treatment, diabetes, vintage, vascular access, weight, blood pressure and laboratory variables (serum albumin, haemoglobin, creatinine, ferritin and Kt/V) were used as confounding variables. RESULTS: Low (<9.5 mg/dL) and high (>10.5 mg/dL) Ca(Alb) increased the HR for all-cause mortality. Low (<9.0 mg/dL) Ca(Alb) increased the HR for cardiovascular mortality. High phosphorus (>5.5 mg/dL) increased the HR for both all-cause and cardiovascular mortality. Low phosphorus (<4.0 and <3.0 mg/dL) increased the HR for both all-cause and cardiovascular mortality. Furthermore, low (<150 pg/mL) and high (>500 and >300 pg/mL) PTH increased the HR for both all-cause and cardiovascular mortality. In addition, only phosphorus >6.0 mg/dL increased the HR for cardiovascular hospitalizations. No effect was observed with Ca(Alb) or PTH. CONCLUSIONS: In summary, in 16,173 HD patients, elevated and reduced serum levels of albumin-corrected calcium, phosphorus and PTH levels were associated with increments in all-cause mortality. Similar results were obtained when only cardiovascular mortality was analysed.

High phosphorus diet induces vascular calcification, a related decrease in bone mass and changes in the aortic gene expression.

In chronic kidney disease, hyperphosphatemia has been associated to vascular calcifications. Moreover, the rate and progression of vascular calcification have been related with the reduction of bone mass and osteoporotic fractures, hereby suggesting a strong link between vascular calcification and bone loss. Our aim was to prospectively study the effects of high phosphorus diet on bone mass, vascular calcification and gene expression profile of the arterial wall. A rat model of 7/8 nephrectomy fed with normal (0.6%) and moderately high (0.9%) phosphorus diet was used. Biochemical parameters, bone mineral density and vascular calcifications were assessed. A microarray analysis of the aortic tissue was also performed to investigate the gene expression profile. After 20 weeks, the rats fed with a high phosphorus diet showed a significant increase in serum phosphorus, PTH, and creatinine, together with aortic calcification and a decrease in bone mass. The histological analysis of the vascular calcifications showed areas with calcified tissue and the gene expression profile of this calcified tissue showed repression of muscle-related genes and overexpression of bone-related genes, among them, the secreted frizzled related proteins, well-known inhibitors of the Wnt pathway, involved in bone formation. The study demonstrated prospectively the inverse and direct relationship between vascular calcification and bone mass. In addition, the microarrays findings provide new information on the molecular mechanisms that may link this relationship.