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Despite the presence of vitamin D receptor (VDR) in endothelial cells, the effect of vitamin D on endothelial function is unknown. An unbalanced production of vasoactive endothelial factors such as nitric oxide (NO) or endothelin-1 (ET-1) results in endothelial dysfunction, which can alter the normal cardiovascular function. Present experiments were devoted to assess the effect of active vitamin D (calcitriol) on the synthesis of endothelial vasoactive factors. The results were that, in cells, calcitriol increased ET-1 and NO productions, which were measured by ELISA and fluorimetric assay, respectively. Calcitriol also increased endothelin-converting enzyme-1 (ECE-1) and endothelial-nitric oxide synthase (eNOS) activities, their mRNA (qPCR), their protein expressions (Western-blot), and their promoter activities (transfection assays). Calcitriol did not change prepro-ET-1 mRNA. The effect was specific to VDR activation because when VDR was silenced by siRNA, the observed effects disappeared. Mechanisms involved in each upregulation differed. ECE-1 upregulation depended on AP-1 activation, whereas eNOS upregulation depended directly on VDR activation. To evaluate the in vivo consequences of acute calcitriol treatment, normal Wistar rats were treated with a single ip injection of 400 ng/kg calcitriol and euthanized 24 h later. Results confirmed those observed in cells, that production and expression of both factors were increased by calcitriol. Besides, calcitriol-treated rats showed a slight rise in mean blood pressure, which decreased when pretreated with FR-901533, an ECE-1 antagonist. We conclude that calcitriol increases the synthesis of both ET-1 and NO in endothelial cells. However, the ET-1 upregulation seems to be biologically more relevant, as animals acutely treated with calcitriol show slight increases in blood pressure.

Both maternal and offspring-derived factors contribute to lifelong growth and bone mass accrual, although the specific role of maternal deficiencies in the growth and bone mass of offspring is poorly understood. In the present study, we have shown that vitamin B12 (B12) deficiency in a murine genetic model results in severe postweaning growth retardation and osteoporosis, and the severity and time of onset of this phenotype in the offspring depends on the maternal genotype. Using integrated physiological and metabolomic analysis, we determined that B12 deficiency in the offspring decreases liver taurine production and associates with abrogation of a growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. Taurine increased GH-dependent IGF1 synthesis in the liver, which subsequently enhanced osteoblast function, and in B12-deficient offspring, oral administration of taurine rescued their growth retardation and osteoporosis phenotypes. These results identify B12 as an essential vitamin that positively regulates postweaning growth and bone formation through taurine synthesis and suggests potential therapies to increase bone mass.

Activation of the insulin growth factor receptor-1 signaling pathways has been largely related to the aging process. Amadori products are produced in pathological conditions such as diabetes and aging, and are potentially involved in diabetic nephropathy or age-associated decline of renal function. We hypothesize that Amadori products induce senescence in primary human mesangial cells through the activation of IGF-1 receptor and investigate, in the present work, the intracellular mechanism involved after this activation. We treated cultured human mesangial cells with glycated albumin, one of the most abundant Amadori product, and senescence was assessed by determining the senescence associated β-galactosidase activity and the expression of the cell cycle regulators p53 and p21. We demonstrated that prolonged exposition (more than 24h) to glycated albumin induced senescence and, in parallel, incremented the release of IGF-1 and the activation of the IGF-1 receptor. Inhibition of the IGF-1 activation prevented the GA induced senescence. Activation of IGF-1R, after GA addition, promoted a reduction in the catalase content through the constitutive activation of Ras and erk1/2 proteins which were, in turn, responsible of the observed GA-induced senescence. In conclusion, we propose that the Amadori product, glycated albumin, promotes premature cell senescence in mesangial cells through the activation of the IGF-1 receptor and the subsequent reduction in the antioxidant enzyme catalase.

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.

Estrogen deficiency is the main factor implicated in bone loss in postmenopausal osteoporosis.1 As a consequence of the lack of estrogens, bone turnover increases, leading to an imbalance between bone formation and bone resorption, favoring the latter.2,3 This imbalance affects calcium–phosphate metabolism and may increase serum parathyroid hormone (PTH) levels.4 Estrogen replacement therapy prevents bone loss and fractures,5,6 acting directly on bone cells through their specific estrogen receptors (ERs): α and β.7,8 In addition, in postmenopausal women, estrogens can also reduce PTH serum levels4,9 through an as of yet poorly understood mechanism. A possible direct effect of estrogens reducing PTH acting through ERα and ERβ located in the parathyroid cells has been suggested, but the existence of ERα and ERβ in parathyroid tissue is still a controversial issue.10–13 Estrogens may also decrease PTH secretion by acting on other factors such as calcium,14,15 1,25(OH)2 D3 (calcitriol),15 and phosphorus,16,17 among others. Recently, fibroblast growth factor 23 (FGF23), involved in phosphorus and vitamin D metabolism,18 has been suggested to influence PTH synthesis and secretion.19 In women with chronic kidney disease (CKD), little is known about the role that estrogen deficiency plays in the pathogenesis and progression of bone disease.20,21 Understanding the mechanism through which estrogens act on PTH is also a subject of interest in these patients, because of the high prevalence of secondary parathyroid disorders.22 Because several aspects of the effects of estrogens on PTH remain unclear, the objective of this study was to investigate the factors and mechanisms involved in the likely effect of estrogens on the parathyroid gland.

This paper reviews the pathogenesis of hyperphosphataemia and its role in the regulation of parathyroid hormone synthesis and parathyroid cell proliferation in chronic kidney disease. The association between hyperphosphaemia and vascular calcification, and the interventions that can be used to control plasma phosphate are also discussed.

Bone loss and increased fractures are common complications in chronic kidney disease. Because Wnt pathway activation is essential for normal bone mineralization, we assessed whether Wnt inhibition contributes to high-phosphorus-induced mineralization defects in uremic rats. By week 20 after 7/8 nephrectomy, rats fed a high-phosphorus diet had the expected high serum creatinine, phosphorus, parathyroid hormone, and fibroblast growth factor 23 (FGF23) levels and low serum calcium. There was a 15% reduction in tibial mineral density and a doubling of bone cortical porosity compared to uremic rats fed a normal-phosphorus diet. The decreases in tibial mineral density were preceded by time-dependent increments in gene expression of bone formation (Osteocalcin and Runx2) and resorption (Cathepsin K) markers, which paralleled elevations in gene expression of the Wnt inhibitors Sfrp1 and Dkk1 in bone. Similar elevations of Wnt inhibitors plus an increased phospho-β-catenin/β-catenin ratio occurred upon exposure of the osteoblast cell line UMR106-01 either to uremic serum or to the combination of parathyroid hormone, FGF23, and soluble Klotho, at levels present in uremic serum. Strikingly, while osteoblast exposure to parathyroid hormone suppressed the expression of Wnt inhibitors, FGF23 directly inhibited the osteoblastic Wnt pathway through a soluble Klotho/MAPK–mediated process that required Dkk1 induction. Thus, the induction of Dkk1 by FGF23/soluble Klotho in osteoblasts inactivates Wnt/β-catenin signaling. This provides a novel autocrine/paracrine mechanism for the adverse impact of high FGF23 levels on bone in chronic kidney disease.

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.

Two matrix Gla protein (MGP) polymorphisms were associated with progression of aortic calcification and femoral neck bone loss in men. All these findings were also functionally corroborated in two vascular and bone in vitro systems indicating that MGP genetic variations can be partly responsible of higher risk of bone loss and vascular calcification.MGP plays an important role in bone and vascular mineralization as confirmed by MGP-deficient murine model. We therefore aimed to find a genetic association among -138TC, -7GA, and Thr83Ala MGP single-nucleotide polymorphisms (SNPs), bone loss, and progression of aortic calcification in a randomly selected general population of 296 individuals who participated in the European Vertebral Osteoporosis Study.To evaluate the rate of change in bone mineral density (BMD) and the progression of aortic calcification, dual X-ray absorptiometry and lateral spine X-rays were performed at baseline and after 4 years of follow-up. Genotyping for the three polymorphisms was carried out using polymerase chain reaction and restriction fragment length analysis. In addition, functional studies of MGP-7GA and Thr83Ala SNPs were performed on transiently transfected osteoblast-like UMR-106 and vascular smooth muscle A7r5 cells.The proportion of men who had lost BMD in the femoral neck was higher among homozygous -7AA and 83Ala-Ala (p = 0.039 and p = 0.009, respectively), and also featured a higher risk of progression of aortic calcifications (OR = 5.6, 95% CI = 1.2-27.8 and OR = 6.8, 95% CI = 1.4-32.3, respectively). No effect was observed in women. The MGP-7A allele produced a reduction in luciferase activity compared to MGP-7G: 47% less in vascular cells and 34% less in bone cells (p = 0.001 and 0.012, respectively). In vascular cells under calcifying conditions, the MGP 83Thr allele showed a slightly higher, although not significant, inhibition than the MGP 83 Ala allele in calcium content suggesting functional differences between both variants.These results suggest that MGP genetic variations could predict a higher risk of bone loss and progression of vascular calcification in men.

The current regulation of parathyroid hormone (PTH) and the development of parathyroid disorders in chronic kidney disease involve complex mechanisms. Factors such as calcium, phosphorous, calcitriol, vitamin D receptor, calcium-sensing receptor and fibroblast growth factor 23 play a key role in the regulatory process in the pathogenesis of secondary hyperparathyroidism.This review provides an analysis of published results related to the different models and approaches used to study the mechanisms involved in the pathogenesis of secondary hyperparathyroidism. The review includes clinical studies, animal and ex vivo/in vitro models which have been extensively used in this area.Readers will have an overview of the main findings and progress achieved in the knowledge of the parathyroid function combining the results obtained from the different models used to understand the parathyroid gland regulation.Each of the available models used to study the complex system of parathyroid regulation has advantages and limitations; therefore, it is necessary to combine the information obtained from more than one model in order to have a more complete knowledge of the mechanisms involved in PTH regulation.

Vascular calcification plays a major role in cardiovascular disease, which is one of the main causes of mortality in chronic kidney disease patients. Vascular calcification is determined by prevalent traditional and uraemia-related (non-traditional) risk factors. It occurs mainly in the arteries, which are classified into three types according to their size and structural characteristics. In addition, vascular calcification has been associated with bone loss and fractures in chronic kidney disease patients and the general population, stressing the fact that both disorders can share pathogenetic pathways. The strategies to control vascular calcification involve several measures, chief among them the control of hyperphosphataemia. Furthermore, it has been recently described that strategies that reduce bone resorption and increase bone mineralization may decrease the risk of vascular calcifications; however, this approach still remains controversial. The mechanisms involved in vascular calcification are complex and not yet fully understood. Phosphorus plays a major role, while other factors related to bone formation have been recently identified.

At present, new compounds are available to treat secondary hyperparathyroidism, namely calcimimetics, novel phosphorus binders and also novel vitamin D receptor activators. Calcimimetics increase the sensitivity of the parathyroid gland to calcium through spatial configurational changes of the calcium-sensing receptor. In addition, experimental studies have demonstrated that calcimimetics also upregulate both the calcium-sensing receptor and the vitamin D receptor. They are efficacious in children, though the experience in paediatric chronic kidney disease is still limited. Sevelamer, lanthanum carbonate and magnesium iron hydroxycarbonate are novel phosphorus binders available on the market. Several studies have demonstrated their efficacy and safety up to 6 years, though costs are the main limitation for a wider use. Since almost all the experience available on the new phosphorus binders comes from its use in adults, studies on children are needed in order to confirm the efficacy and safety of these products. Other new salts and polymers are also being developed. New vitamin D receptor activators, such as paricalcitol, are as effective at suppressing parathyroid hormone (PTH) as the traditional vitamin D receptor activators used for the past two decades, but they have a better and safer profile, showing fewer calcaemic and phosphoraemic effects while preserving the desirable effects of the vitamin D receptor activators on the cardiovascular system, hypertension, inflammation and fibrosis. Their use in children with chronic kidney disease has revealed similar responses to those of adults. The novel compounds discussed in this review should facilitate and improve the management of mineral and bone disorders in children with chronic kidney disease.

OBJECTIVES Inherited and acquired risk factors contribute to the development of the atherosclerotic lesion and its most common clinical manifestation, myocardial infarction (MI). Multiple studies have suggested a role for matrix metalloproteinases (MMPs) in atherosclerosis, and several functional polymorphisms in the MMP-1 gene have been linked to the risk of MI. The aim of this study was to evaluate the association between MMP-1 promoter polymorphisms and early MI in a Spanish cohort. METHODS We carried out a case-control study with 261 unrelated patients who had suffered an MI before 55 years of age and 194 healthy controls, all male and smokers. The genotypes for the three MMP-1 promoter polymorphisms -1607 1G/2G, -519 A/G, and -340 T/C were determined through PCR-restriction fragment length polymorphism. Allelic, genotypic, and haplotypic frequencies were statistically compared between groups. RESULTS Frequencies of the three polymorphisms did not differ between patients and controls. The -1607 1G/2G and -519 A/G variants were in linkage disequilibrium. Analysis of the haplotype frequencies showed significant associations of the 2G(-1607)-G(-519)-T(-340) (odds ratio = 2.40; 95% confidence interval = 1.27-4.55; P

Background The regulatory mechanisms of parathyroid hormone (PTH) synthesis are complex, involving calcium, calcitriol, the calcium-sensing receptor (CaR) and the vitamin D receptor (VDR). In this study, the effects of calcium and calcitriol on the simultaneous expression of CaR and VDR mRNA and protein levels were assessed in parathyroid glands cultured in vitro. Methods Parathyroid glands (N = 424) were removed and cultured for 24 h to study the effect of calcium on the CaR, VDR and PTH. In addition, the effect of calcitriol at low calcium concentrations (0.6 mM) on CaR and VDR levels was studied after 48 h of incubation. CaR, VDR and PTH mRNAs were measured by quantitative real-time PCR (qRT-PCR), and CaR and VDR protein levels were measured by immunohistochemistry. Results PTH gene expression was reduced by high calcium concentration. No differences were found in the CaR mRNA levels among the different calcium concentrations tested (0.6 mM calcium: 100%; 1.2 mM calcium: 120%; 2.0 mM calcium: 112%; median values), but VDR gene expression rose when calcium increased (0.6 mM calcium: 100%; 1.2 mM calcium: 164%; 2.0 mM calcium: 195%; median values). Calcitriol increased both CaR (control: 100%; 10(-8) M calcitriol: 196%; median values) and VDR genes expression (control: 100%; 10(-8) M calcitriol: 176%; median values). The same findings were corroborated at protein levels for both CaR and VDR. Conclusions In parathyroid glands cultured in vitro, calcium up-regulates VDR but not CaR. Conversely, calcitriol up-regulates both VDR and CaR mRNAs and protein levels, even at low calcium concentrations.

Introduction The human genome is peppered with mobile repetitive elements called long interspersed nuclear element–1 (L1) retrotransposons. Propagating through RNA and cDNA intermediates, these molecular parasites copy and insert themselves throughout the genome, with potentially disruptive effects on neighboring genes or regulatory sequences. In the germ line, unique sequence downstream of L1 elements can also be retrotransposed if transcription continues beyond the repeat, a process known as 3′ transduction. There has been growing interest in retrotransposition and 3′ transduction as a possible source of somatic mutations during tumorigenesis. Rationale To explore whether 3′ transductions are frequent in cancer, we developed a bioinformatic algorithm for identifying somatically acquired retrotranspositions in cancer genomes. We applied our algorithm to 290 cancer samples from 244 patients across 12 tumor types. The unique downstream sequence mobilized with 3′ transductions effectively fingerprints the L1 source element, providing insights into the activity of individual L1 loci across the genome. Results Across the 290 samples, we identified 2756 somatic L1 retrotranspositions. Tumors from 53% of patients had at least one such event, with colorectal and lung cancers being most frequently affected (93% and 75% of patients, respectively). Somatic 3′ transductions comprised 24% of events, half of which represented mobilizations of unique sequence alone, without any accompanying L1 sequence. Overall, 95% of 3′ transductions identified derived from only 72 germline L1 source elements, with as few as four loci accounting for 50% of events. In a given sample, the same source element could generate 50 or more somatic transductions, scattered extensively across the genome. About 5% of somatic transductions arose from L1 source elements that were themselves somatic retrotranspositions. In three of the cases in which we sequenced more than one sample from a patient’s tumor, we were able to place 3′ transductions on the phylogenetic tree. We found that the activity of individual source elements fluctuated during tumor evolution, with different subclones exhibiting much variability in which elements were “on” and which were “off.” The ability to identify the individual L1 source elements active in a given tumor enabled us to study the promoter methylation of those elements specifically. We found that 3′ transduction activity in a patient’s tumor was always associated with hypomethylation of that element. Overall, 2.3% of transductions distributed exons or entire genes to other sites in the genome, and many more mobilized deoxyribonuclease I (DNAse-I) hypersensitive sites or transcription factor binding sites identified by the ENCODE project. Occasionally, somatic L1 insertions inserted near coding sequence and redistributed these exons elsewhere in the genome. However, we found no general effects of retrotranspositions on transcription levels of genes at the insertion points and no evidence for aberrant RNA species resulting from somatically acquired transposable elements. Indeed, as with germline retrotranspositions, somatic insertions exhibited a strong enrichment in heterochromatic, gene-poor regions of the genome. Conclusion Somatic 3′ transduction occurs frequently in human tumors, and in some cases transduction events can scatter exons, genes, and regulatory elements widely across the genome. Dissemination of these sequences appears to be due to a small number of highly active L1 elements, whose activity can wax and wane during tumor evolution. The majority of the retrotransposition events are likely to be harmless “passenger” mutations.