Natalia Carrillo-López

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.

Indirect Regulation of PTH by Estrogens May Require FGF23

The mechanisms by which estrogens modulate PTH are controversial, including whether or not estrogen receptors (ERs) are present in the parathyroid glands. To explore these mechanisms, we combined a rat model of CKD with ovariectomy and exogenous administration of estrogens. We found that estrogen treatment significantly decreased PTH mRNA and serum levels. We did not observe ERalpha or ERbeta mRNA or protein in the parathyroids, suggesting an indirect action of estrogens on PTH regulation. Estrogen treatment significantly decreased serum 1,25(OH)(2) vitamin D(3) and phosphorus levels. In addition, estrogens significantly increased fibroblast growth factor 23 (FGF23) mRNA and serum levels. In vitro, estrogens led to transcriptional and translational upregulation of FGF23 in osteoblast-like cells in a time- and concentration-dependent manner. These results suggest that estrogens regulate PTH indirectly, possibly through FGF23.

Simultaneous changes in the calcium-sensing receptor and the vitamin D receptor under the influence of calcium and calcitriol

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.

Vitamin D receptor activation, left ventricular hypertrophy and myocardial fibrosis

BACKGROUND Left ventricular hypertrophy (LVH), a common complication in chronic kidney disease (CKD), is associated with high cardiovascular mortality. The aim of this experimental study was to analyze the effect of different vitamin D receptor activators (VDRAs) on both LVH and myocardial fibrosis in chronic renal failure (CRF). METHODS Male Wistar rats with CRF, carried out by 7/8 nephrectomy, were treated intraperitoneally with equivalent doses of VDRAs (calcitriol, paricalcitol and alfacalcidol, 5 days per week) during 4 weeks. A placebo group (CRF + vehicle) and a Sham group with normal renal function served as controls. Biochemical, morphological, functional and molecular parameters associated with LVH were evaluated, as well as cardiac fibrosis, collagen I, transforming growth factor β1 (TGFβ1) and matrix metalloproteinase-1 (MMP1) expression. RESULTS All VDRAs treatment prevented LVH, with values of cardiomyocyte size, LV wall and septum thickness and heart-body weight ratio similar to those observed in the Sham group. At molecular levels, all VDRAs attenuated atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) expression compared with CRF + vehicle. The phosphorylation of ERK1/2, a signal for activating growth, was stimulated in the CRF + vehicle group; VDRAs use prevented this activation. Paricalcitol was the only VDRA used that maintained in the normal range all parameters associated with myocardial fibrosis (total collagen, collagen I, TGFβ1 and MMP1). CONCLUSIONS Our findings demonstrated that the three VDRAs used induced similar changes in bone metabolic parameters and LVH. In addition, paricalcitol was the only VDRA which showed a relevant beneficial effect in the reduction of myocardial fibrosis, a key factor in the myocardial dysfunction in CKD patients.

Lanthanum activates calcium-sensing receptor and enhances sensitivity to calcium

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.

Differential effects of 17β-estradiol and raloxifene on bone and lipid metabolism in rats with chronic kidney disease and estrogen insufficiency

Objective:The aim of this study was to evaluate the effect of 17&bgr;-estradiol, raloxifene, and 1-&agr;,25-dihydroxycholecalciferol or calcitriol on bone and lipid metabolism in chronic kidney disease and estrogen insufficiency. Methods:Six-month-old female Sprague-Dawley rats (n = 48) were ovariectomized and nephrectomized (seven eighths). One week after surgery, the rats were divided into six groups and treated with (1) placebo, (2) 17&bgr;-estradiol 10 &mgr;g kg−1 day−1, (3) raloxifene 1 mg kg−1 day−1, (4) calcitriol 10 ng kg−1 day−1, (5) 17&bgr;-estradiol + calcitriol, and (6) raloxifene + calcitriol. A group of untreated animals with chronic kidney disease and normal ovarian function was used as a control group (n = 5). The rats were killed after 8 weeks of treatment. Blood samples were drawn for serum analyses; the right tibia was removed to perform histomorphometric analyses, uteri were used as tissue markers of estrogen replacement, and paraffin-embedded sections of the uterus and the fourth breast were used for histopathologic evaluation. Results:Raloxifene, alone or combined with calcitriol, and 17&bgr;-estradiol combined with calcitriol significantly diminished total cholesterol level compared with placebo. Qualitative histological and histomorphometric analyses showed that both the single treatments and their combinations were able to increase the trabecular connectivity compared with placebo. The less beneficial results were obtained with 17&bgr;-estradiol alone, whereas the more beneficial results were obtained with the combined treatments, particularly with raloxifene and calcitriol. Conclusions:In summary, this experimental study demonstrates the advantages of replacing both hormonal deficiencies together. The combination of calcitriol and raloxifene, a selective estrogen receptor modulator, showed a better lipid, uterus, and bone profile.

PATHOGENESIS OF BONE AND MINERAL RELATED DISORDERS IN CHRONIC KIDNEY DISEASE: KEY ROLE OF HYPERPHOSPHATEMIA

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.

Estrogens and bone disease in chronic kidney disease: role of FGF23.

Purpose of reviewTo describe the direct and indirect effects of estrogen on bone with special emphasis on the analysis of the recent findings related to the putative role of FGF23 in the estrogen-dependent parathyroid hormone (PTH) suppression. Recent findingsEstrogens act directly on bone cells, downregulating osteoclast precursors and differentiation, increasing osteoclast apoptosis and stimulating osteoblast proliferation and differentiation. However, estrogens can also act indirectly on bone, modulating the calcium–phosphorus–vitamin D–PTH axis. It has been recently demonstrated that estrogens suppress PTH synthesis and secretion in a dose-dependent manner and reduce serum calcitriol and phosphorus levels by an indirect mechanism. In-vivo and in-vitro experiments demonstrated that FGF23 positively correlated, also in a dose-dependent manner, with the dose of estrogens and with the observed changes in calcitriol and phosphorus. SummaryThese new findings support the importance of the indirect effects of estrogens on bone, suggesting a role for FGF23 in the regulation of PTH by estrogens.

Regulation of miR-29b and miR-30c by vitamin D receptor activators contributes to attenuate uraemia-induced cardiac fibrosis

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.

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.