Sara Panizo

RANKL Increases Vascular Smooth Muscle Cell Calcification Through a RANK-BMP4–Dependent Pathway

Vascular calcification commonly associated with several pathologies and it has been suggested to be similar to bone mineralization. The axis RANKL-OPG (receptor activator of nuclear factor &kgr;B ligand–osteoprotegerin) finely controls bone turnover. RANKL has been suggested to increase vascular calcification, but direct evidence is missing. Thus, in the present work, we assess the effect of RANKL in vascular smooth muscle cell (VSMC) calcification. VSMCs incubated with RANKL showed a dose-dependent increase in calcification, which was abolished by coincubation with OPG. To test whether the effect was mediated by signaling to its receptor, knockdown of RANK was accomplished by short hairpin (sh)RNA. Indeed, cells lacking RANK showed no increases in vascular calcification when incubated with RANKL. To further elucidate the mechanism by which RANK activation increases calcification, we blocked both nuclear factor (NF)-&kgr;B activation pathways. Only IKK&agr; inactivation inhibited calcification, pointing to an involvement of the alternative NF-&kgr;B activation pathway. Furthermore, RANKL addition increased bone morphogenetic protein (BMP)4 expression in VSMCs, and that increase disappeared in cells lacking RANK or IKK&agr;. The increase in calcification was also blunted by Noggin, pointing to a mediation of BMP4 in the calcification induced by RANKL. Furthermore, in an in vivo model, the increase in vascular calcium content was parallel to an increase in RANKL and BMP4 expression, which was localized in calcified areas. However, blood levels of the ratio RANKL/OPG did not change. We conclude that RANKL increases vascular smooth muscle cell calcification by binding to RANK and increasing BMP4 production through activation of the alternative NF-&kgr;B pathway.

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.

N-methyl-d-aspartate receptors are expressed in rat parathyroid gland and regulate PTH secretion

N-methyl-d-aspartate receptors (NMDAR) are tetrameric amino acid receptors which act as membrane calcium channels. The presence of the receptor has been detected in the principal organs responsible for calcium homeostasis (kidney and bone), pointing to a possible role in mineral metabolism. In the present work, the presence of the receptor was determined in normal parathyroid glands (PTG) by real-time PCR, immunoprecipitation, and immunohistrochemistry. Healthy animals showed a decrease in blood parathyroid hormone (PTH) levels 15 min after the treatment with NMDA. This effect was also observed in animals with high levels of PTH-induced EDTA injection, but not in uremic animals with secondary hyperparathyroidism (2HPT). Normal rat PTG incubated in media with low calcium concentration (0.8 mM CaCl2) showed a decrease in PTH release when NMDA was added to the media. This effect of NMDA was abolished when glands were coincubated with MK801 (a pharmacological blocker of the NMDA channel) or PD98059 (an inhibitor of the ERK-MAPK pathway). Glands obtained from animals with 2HPT showed no effect of NMDA in the in vitro release of PTH, together with a decrease in the expression of NMDAR1. In conclusion, NMDA receptor is present in PTG and is involved in the regulation of the PTH release. The mechanism by which NMDAR exerts its function is through the activation of the MAPK cascade. In uremic 2HPT animals the receptor expression is downregulated and the treatment with NMDA does not affect PTH secretion.

High phosphate diet increases arterial blood pressure via a parathyroid hormone mediated increase of renin.

Background: There is growing evidence suggesting that phosphate intake is associated with blood pressure levels. However, data from epidemiological studies show inconsistent results. Method and results: The present study was designed to evaluate the effect of high circulating phosphorus on arterial blood pressure of healthy rats and to elucidate the potential mechanism that stands behind this effect. Animals fed a high phosphate diet for 4 weeks showed an increase in blood pressure, which returned to normal values after the addition of a phosphate binder (lanthanum carbonate) to the diet. The expression of renin in the kidney was higher, alongside an increase in plasma renin activity, angiotensin II (Ang II) levels and left ventricular hypertrophy. The addition of the phosphate binder blunted the increase in renin and Ang II levels. The levels of parathyroid hormone (PTH) were also higher in animals fed a high phosphate diet, and decreased when the phosphate binder was present in the diet. However, blood P levels remained elevated. A second group of rats underwent parathyroidectomy and received a continuous infusion of physiological levels of PTH through an implanted mini-osmotic pump. Animals fed a high phosphate diet with continuous infusion of PTH did not show an increase in blood pressure, although blood P levels were elevated. Finally, unlike with verapamil, the addition of losartan to the drinking water reverted the increase in blood pressure in rats fed a high phosphate diet. Conclusion: The results of this study suggest that a high phosphate diet increases arterial blood pressure through an increase in renin mediated by PTH.

Sustained activation of renal N-methyl-d-aspartate receptors decreases vitamin D synthesis: a possible role for glutamate on the onset of secondary HPT

N-methyl-D-aspartate (NMDA) receptors (NMDAR) are tetrameric amino acid receptors that act as membrane calcium channels. The presence of the receptor has been detected in the principal organs responsible for calcium homeostasis (kidney, bone, and parathyroid gland), pointing to a possible role in mineral metabolism. The aim of this study was to test the effect of NMDAR activation in the kidney and on 1,25(OH)₂D₃ synthesis. We determined the presence of NMDAR subunits in HK-2 (human kidney cells) cells and proved its functionality. NMDA treatment for 4 days induced a decrease in 1α-hydroxylase levels and 1,25(OH)₂D₃ synthesis through the activation of the MAPK/ERK pathway in HK-2 cells. In vivo administration of NMDA for 4 days also caused a decrease in blood 1,25(OH)₂D₃ levels in healthy animals and an increase in blood PTH levels. This increase in PTH induced a decrease in the urinary excretion of calcium and an increase in urinary excretion of phosphorous and sodium as well as in diuresis. Bone turnover markers also increased. Animals with 5/6 nephrectomy showed low levels of renal 1α-hydroxylase as well as high levels of renal glutamate compared with healthy animals. In conclusion, NMDAR activation in the kidney causes a decrease in 1,25(OH)₂D₃ synthesis, which induces an increase on PTH synthesis and release. In animals with chronic kidney disease, high renal levels of glutamate could be involved in the downregulation of 1α-hydroxylase expression.

Assessment of the Potential Role of Active Vitamin D Treatment in Telomere Length: A Case–Control Study in Hemodialysis Patients

BACKGROUND Telomeres are special chromatin sequences located at the end of eukaryotic chromosomes, protecting these regions from recombination and degradation. Previous studies have reported a decrease in telomere length on white blood cells from hemodialysis (HD) patients, which suggests premature senescence. Active vitamin D treatment has been reported to have an effect on telomere length and beneficial effects on HD patients, but the mechanisms are unknown. OBJECTIVE Our aim was to assess the potential protective role of active vitamin D treatment on telomere length in peripheral mononuclear cells (PBMC) from HD patients. METHODS A retrospective case-control study of 62 stable HD patients and 60 healthy sex-matched controls was undertaken. Telomere length was measured in PBMC by Southern blot. After telomere length measurement, 5 control samples that did not reach quality-control standards were excluded. Standard epidemiological and biochemical parameters were recorded. Blood biochemistries were performed at the Biochemistry Department of the University Hospital Arnau de Vilanova in Lleida, Spain, using standard routine techniques. Differences in telomere length were analyzed using Students t test. Multiple regression analysis examined the independent contribution of the factors that significantly affected telomere length in the bivariate analysis. RESULTS HD patients presented shorter telomere length in PBMC, independent of age and sex (mean [SD] 8.8 [1.5] kbp vs 10.5 [2.9] kbp; P = 0.0001). Multivariate regression analysis of the HD subgroup suggested that patients under active vitamin D treatment have greater telomere length in PBMC than untreated patients (9.5 [0.2] kbp vs 8.4 [0.2] kbp; P = 0.003). CONCLUSIONS HD patients were observed to have decreased PBMC telomere length compared with healthy controls. HD patients treated with active vitamin D compounds had greater PBMC telomere length than untreated patients. Prospective studies are required to assess the potential role of active vitamin D treatment in PBMC telomere length.

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.

Vascular Calcification Induced by Chronic Kidney Disease Is Mediated by an Increase of 1α-Hydroxylase Expression in Vascular Smooth Muscle Cells

Vascular calcification (VC) is a complication of chronic kidney disease that predicts morbidity and mortality. Uremic serum promotes VC, but the mechanism involved is unknown. A role for 1,25(OH)2D3 in VC has been proposed, but the mechanism is unclear because both low and high levels have been shown to increase it. In this work we investigate the role of 1,25(OH)2D3 produced in vascular smooth muscle cells (VSMCs) in VC. Rats with subtotal nephrectomy and kidney recipient patients showed increased arterial expression of 1α‐hydroxylase in vivo. VSMCs exposed in vitro to serum obtained from uremic rats also showed increased 1α‐hydroxylase expression. Those increases were parallel to an increase in VC. After 6 days with high phosphate media, VSMCs overexpressing 1α‐hydroxylase show significantly higher calcium content and RUNX2 expression than control cells. 1α‐hydroxylase null mice (KO) with subtotal nephrectomy and treated with calcitriol (400 ng/kg) for 2 weeks showed significantly lower levels of vascular calcium content, Alizarin red staining, and RUNX2 expression than wild‐type (WT) littermates. Serum calcium, phosphorus, blood urea nitrogen (BUN), PTH, and 1,25(OH)2D3 levels were similar in both calcitriol‐treated groups. In vitro, WT VSMCs treated with uremic serum also showed a significant increase in 1α‐hydroxylase expression and higher calcification that was not observed in KO cells. We conclude that local activation of 1α‐hydroxylase in the artery mediates VC observed in uremia.