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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.

Background and Aims Bone fragility fractures (bone fractures) are extremely frequent in haemodialysis (HD) patients. Serum phosphate (P) has been suggested as a risk factor for bone fracture, nonetheless, evidence is poor. The aim of this study was to assess the association between incidence of bone fractures and serum phosphate (P), calcium (Ca) and parathyroid hormone (PTH) in patients from the COSMOS study. Method COSMOS is an observational, prospective, open cohort study with 3 years of follow-up including 6797 haemodialysis patients from 227 centres randomly selected from 20 European countries. At baseline, demographics, comorbidities, treatments, serum biochemical parameters of the previous six months and bone fractures of the previous 12 months were collected. Every 6 months, all these variables, outcomes, and incident bone fractures, were collected. Patients who had at least one bone fracture during follow-up were compared with those who had not. Multivariate binary logistic regression and Poisson regression were used to assess the association between incidence of bone fractures and serum P, Ca and PTH. Time to fracture (and possible re-fracture) was also evaluated using Cox regression and Cox regression for recurrent events. All the results were adjusted by 23 variables including Ca, P, PTH, albumin and haemoglobin (full adjusted model). Results Analysis included 6274 patients who had follow-up data and non-missing information regarding bone fractures at baseline and during follow-up; 252 patients (4.0%), suffered at least one incident bone fracture. The fractured patients were older (68.1±12.9 vs. 63.8±14.5 years, p Multivariate binary logistic regression showed that “well known risk factors for bone fracture”, such as a previous bone fracture (OR: 7.78[95%CI:4.83-12.55], p 800 pg/mL (OR: 1.60[95%CI: 1.01-2.55], p=0.047), were associated with a higher incidence of follow-up bone fractures. In addition to the “well known bone risk factors for fracture”, serum P > 6.1 mg/dL (OR: 1.50[95% CI: 1.07-2.11], p=0.02) and serum Ca > 9.7 mg/dL (OR: 1.42[95%CI: 1.01-2.03], p=0.043), were also associated with a higher incidence of bone fractures in the full-adjusted model. These findings were partly consistent with Poisson regression for serum P >6.1 mg/dL (IRR: 1.46[95% CI: 1.00-2.13], p=0.0085) and serum Ca > 9.7 mg/dL (IRR: 1.49[95%CI: 1.06-2.08], p=0.0047), but not for PTH. In addition, Cox regression analysis showed association between bone fractures and serum P >6.1 mg/dl (HR: 1.61 [95%CI: 1.16-2.24], p=0.0049) and 1.60 [95%CI: 1.14-2.24] (p=0.0066) for simple and recurrent events respectively, but no association was found with serum Ca and PTH in the 2 full-adjusted models for the Cox regression analyses. Conclusion In COSMOS, a large haemodialysis patient’s cohort with 3 years follow up, serum P was the only biochemical parameter that remained as a significant risk factor for bone fractures after 4 statistic approaches. Thus, for the first time, in a large scale well controlled study, high serum P has been identified as a new independent potential risk factor for incident bone fractures in haemodialysis patients.

Background and Aims The stratification of early cardiovascular (CV) risk is a major unmet need in chronic kidney disease (CKD). Since angiogenic T-cells are emerging mediators of vascular homeostasis and have been linked to CV disease (CVD), this study evaluated the frequency of circulating angiogenic T-cells in CKD patients undergoing peritoneal dialysis (CKD-5D) and their potential as predictive biomarkers of CVD. Method In 30 peritoneal dialysis patients and 16 matched healthy controls (HC), we quantified: Vascular calcification (by Kauppila Index); left carotid intima media thickness (lc-IMT by ultrasound), carotid adventitial vasa vasorum density (an early marker of atherosclerosis stage, by Superb Microvascular Ultrasound Image), pulse wave velocity (a marker of vascular stiffness and dysfunction, using the Complior Analyzer), angiogenic T cell frequency in blood (by Flow cytometry) and the traditional 10-year CVD risk (using the SCORE chart). Results As expected, CKD-5D patients exhibited higher Kauppila index (p0.05) and did not correlate with Kauppila (r=-0.350, p=0.080) or lcIMT (r=-0.154, p=0.600), but strongly associated negatively with both pulse wave velocity (r=-0.734, p Conclusion Angiogenic-T cell frequency was decreased in CKD-5D patients and could be considered as biomarkers of vascular dysfunction and early atherosclerosis stage.

Background and Aims Diabetes is the most common cause of chronic kidney disease (CKD) stage 5 and a relevant risk factor for mortality. The available CKD-MBD guidelines do not make differences between diabetics and non-diabetics regarding the optimal targets of the serum bone and mineral biochemical parameters. Thus, the objective of this analysis of COSMOS was to compare the association between the main serum bone and mineral biochemical parameters (calcium-Ca, phosphorus-P and parathyroid hormone-PTH) and mortality in diabetic and non-diabetic patients in haemodialysis. Method COSMOS is a 3-year, observational, prospective, open-cohort study that includes 6797 patients from 227 dialysis centres that were randomly selected from 20 European countries. Cox proportional hazard regression models with penalized splines smoothing were used to analyse the association between relative risk of mortality vs. Ca, P or PTH. Hazard ratios were adjusted by demographics, comorbidities, treatments and biochemical parameters. Interaction analysis was used to analyse if diabetes can influence the association between relative risk of mortality and Ca, P and PTH. Results The present analysis includes 6306 patients with follow-up data and accurate information on diabetes status. The mortality rate in COSMOS was 13.3 deaths per 100 patient-years (17.8 in diabetics and 11.4 in non-diabetics). At baseline, diabetic patients were older (66.7 ± 11.7 vs. 62.8 ± 15.3 years, p Conclusion These results suggest for the first time a differential effect of diabetes on the association between relative risk of mortality and serum PTH. High serum PTH predicts death only in diabetics. These findings could have relevant implications for the diagnosis and treatment of CKD-MBD in diabetic and non-diabetic patients in haemodialysis.

Background Uraemic cardiomyopathy, a process mainly associated with increased myocardial fibrosis, is the leading cause of death in chronic kidney disease patients and can be prevented by vitamin D receptor activators (VDRAs). Since some microRNAs (miRNAs) have emerged as regulators of the fibrotic process, we aimed to analyse the role of specific miRNAs in VDRA prevention of myocardial fibrosis as well as their potential use as biomarkers. Methods Wistar rats were nephrectomized and treated intraperitoneally with equivalent doses of two VDRAs: calcitriol and paricalcitol. Biochemical parameters, cardiac fibrosis, miRNA (miR-29b, miR-30c and miR-133b) levels in the heart and serum and expression of their target genes collagen I (COL1A1), matrix metalloproteinase 2 (MMP-2) and connective tissue growth factor (CTGF) in the heart were evaluated. Results Both VDRAs attenuated cardiac fibrosis, achieving a statistically significant difference in the paricalcitol-treated group. Increases in RNA and protein levels of COL1A1, MMP-2 and CTGF and reduced expression of miR-29b and miR-30c, known regulators of these pro-fibrotic genes, were observed in the heart of chronic renal failure (CRF) rats and were attenuated by both VDRAs. In serum, significant increases in miR-29b, miR-30c and miR-133b levels were observed in CRF rats, which were prevented by VDRA use. Moreover, vitamin D response elements were identified in the three miRNA promoters. Conclusions VDRAs, particularly paricalcitol, attenuated cardiac fibrosis acting on COL1A1, MMP-2 and CTGF expression, partly through regulation of miR-29b and miR-30c. These miRNAs and miR-133b could be useful serum biomarkers for cardiac fibrosis and also potential new therapeutic targets.

Vascular calcification, bone loss and increased fracture risk are age-associated disorders. Several epidemiological studies have suggested a relationship between vascular calcification, impaired bone metabolism and increased mortality. So far, this relationship had been under-estimated as osteoporosis and vascular calcification have been considered non-modifiable disorders of aging. Recent data suggest that this association is not simply an artefact of age, stressing that the co-incidence of vascular calcification with low bone activity and osteoporosis could be biologically linked. During the development of vascular calcification, the transition of vascular smooth muscle cells towards an osteoblast-like phenotype promotes the release of the vesicular structures and mineralization within these structures is promoted by several players, including those related to mineral metabolism, like phosphorus, calcium or parathyroid hormone, which influence either the supersaturation within the structure or the expression of osteogenic factors. However, an intriguing question is whether the presence of vascular calcification impacts bone metabolism, thus demonstrating true crosstalk between these tissues. Evidence is now emerging, suggesting that some inhibitors of the Wnt pathway, such as secreted frizzled Proteins 2 and 4 and Dickkopf related protein-1 (DKK-1), may play a role linking vascular calcification and bone loss. An additional important question to answer, from the patient’s perspective, is whether or not progression of vascular calcification can be prevented or restricted and whether altering this progression we can efficiently impact patients’ outcomes. Much evidence suggests that the control of the chronic kidney disease–mineral and bone disorder components, particularly serum phosphorus, are the main targets to maintain normal bone turnover and protect against vascular calcification.

Background The aim of this study was to investigate whether nanomolar concentrations of lanthanum could influence the calcium-sensing receptor (CaSR) response. Methods Embryonic kidney (HEK-293) cells transiently transfected with the human CaSR were used to test the ability of lanthanum to activate the CaSR, either alone or in combination with calcium. CaSR activation was measured by flow cytometry. Parathyroid glands from 4-month-old male Wistar rats with normal renal function (n = 60) were also cultured ex vivo with different concentrations of lanthanum to measure parathyroid hormone (PTH) secreted to the medium and PTH mRNA. Results The maximal CaSR activation induced by 1 muM lanthanum chloride (LaCl(3)) was similar to that induced by 16 mM calcium chloride (CaCl(2) 16 mM: 294 +/- 14%; LaCl(3) 1 muM: 303 +/- 11%). Lanthanum half effective concentration (EC(50)) was 77.28 nM, lower than the 2.30 mM obtained for calcium, supporting the concept that this metal is a strong agonist of the CaSR. Moreover, lanthanum was also able to enhance CaSR sensitivity to calcium. The presence of 1 nM LaCl(3) significantly left-shifted the CaSR response curve, changing the EC(50) value for calcium from 2.30 mM (calcium alone) to 1.26 mM (calcium + 1 nM lanthanum). The parathyroid glands cultured with lanthanum showed a trend to secrete less PTH compared to the control glands: 1.51 +/- 0.23 (control), 0.91 +/- 0.17 (La 100 nM) and 1.04 +/- 0.18 (La 400 nM) [(pg/h)/(pg/h), mean +/- SEM] (ANOVA P = 0.0145). A similar trend was also observed in PTH synthesis measured by PTH mRNA levels. Conclusions These in vitro findings demonstrate that lanthanum, at nanomolar concentrations, is an agonist of the CaSR able to activate it in the absence of calcium. In addition, it can also enhance CaSR sensitivity to calcium, modulating PTH synthesis and secretion.

Background. Vascular calcifications and the bone fractures caused by abnormal bone fragility, also called osteoporotic fractures, are frequent complications associated with chronic kidney diseases (CKD). The aim of this study was to investigate the association between vascular calcifications, osteoporotic bone fractures and survival in haemodialysis (HD) patients. Methods. A total of 193 HD patients were followed up to 2 years. Vascular calcifications and osteoporotic vertebral fractures (quoted just as vertebral fractures in the text) were assessed by thoracic, lumbar spine, pelvic and hand X-rays and graded according to their severity. Clinical, biochemical and therapeutic data gathered during the total time spent on HD were collected. Results. The prevalence of aortic calcifications was higher in HD patients than in a random-based general population (79% versus 37.5%, P < 0.001). Total time on any renal replacement therapy (RRT) and diabetes were positively associated with a higher prevalence of vascular calcifications. In addition to these factors, time on HD was also positively associated with the severity of vascular calcifications, and higher haemoglobin levels were associated with a lower prevalence of severe vascular calcifications in large and medium calibre arteries. The prevalence of vertebral fractures in HD patients was similar to that of the general population (26.5% versus 24.1%). Age and time on HD showed a positive and statistically significant association with the prevalence of vertebral fractures. Vascular calcifications in the medium calibre arteries were associated with a higher rate of prevalent vertebral fractures. In women, severe vascular calcifications and vertebral fractures were positively associated with mortality [RR = 3.2 (1.0–10.0) and RR = 4.8 (1.7–13.4), respectively]. Conclusions. Positive associations between vascular calcifications, vertebral fractures and mortality have been found in patients on HD.