New cannabinoid receptor antagonists as pharmacological tool.

Synthesis and pharmacological evaluation of a new series of cannabinoid receptor antagonists of indazole ether derivatives have been performed. Pharmacological evaluation includes radioligand binding assays with [3H]-CP55940 for CB1 and CB2 receptors and functional activity for cannabinoid receptors on isolated tissue. In addition, functional activity of the two synthetic cannabinoids antagonists 18 (PGN36) and 17 (PGN38) were carried out in the osteoblastic cell line MC3T3-E1 that is able to express CB2R upon osteogenic conditions. Both antagonists abolished the increase in collagen type I gene expression by the well-known inducer of bone activity, the HU308 agonist. The results of pharmacological tests have revealed that four of these derivatives behave as CB2R cannabinoid antagonists. In particular, the compounds 17 (PGN38) and 18 (PGN36) highlight as promising candidates as pharmacological tools.

Direct inhibition of osteoblastic Wnt pathway by fibroblast growth factor 23 contributes to bone loss in chronic kidney disease.

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-ß-catenin/ß-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/ß-catenin signaling. This provides a novel autocrine/paracrine mechanism for the adverse impact of high FGF23 levels on bone in chronic kidney disease.

Long-term response of cultured rat parathyroid glands to calcium and calcitriol: the effect of cryopreservation.

AIM: This study aimed to investigate the longest period in which parathyroid glands cultured in vitro maintained their viability and functionality in order to study the response of the glands to factors that exert their main action in the long term (1-7 days). METHODS: Rat parathyroid glands from 104 Wistar rats were used. Cell viability was measured by flow cytometry for 7 days. Parathyroid tissue functionality was determined by parathyroid hormone (PTH) secretion in basal conditions and in the response of the glands to calcium and calcitriol. Calcium sensing receptor (CaR) synthesis was determined measuring protein levels by immunohistochemistry. Parathyroid glands were cryopreserved to study them in the same way as fresh tissue. RESULTS: Intact parathyroid glands maintained their cell viability >80% until the 6th day in culture, while the functional capacity was limited to 4 days: PTH release was stable for 4 days, whilst from the 5th day onwards, PTH secretion reduced to undetectable levels. Parathyroid glands responded accurately when calcium was reduced in the culture medium; a mean increase >50% in PTH secretion was observed. No differences were observed in CaR levels before and after the culture period. PTH synthesis and secretion inhibition was observed when the parathyroid glands were cultured with calcitriol; this inhibition achieved 90% after 4 days in culture. Cryopreserved parathyroid glands maintained their viability, but partially lost their functionality, as they were unable to respond to calcium. CONCLUSIONS: Intact parathyroid glands cultured in vitro maintained their functionality and their capacity to respond to their effectors for longer periods than in previously developed studies. It seems that part of this capacity is lost after cryopreservation. Nevertheless, this long-term culture model can be useful to study the response of the parathyroid glands.

Hyperphosphatemia induces cellular senescence in human aorta smooth muscle cells through integrin linked kinase (ILK) up-regulation.

Aging is conditioned by genetic and environmental factors. Hyperphosphatemia is related to some pathologies, affecting to vascular cells behavior. This work analyze whether high concentration of extracellular phosphate induces vascular smooth muscle cells senescence, exploring the intracellular mechanisms and highlighting the in vivo relevance of this phenomenon. Human aortic smooth muscle cells treated with ß-Glycerophosphate (BGP, 10mM) suffered cellular senescence by increasing p53, p21 and p16 expression and the senescence associated ß-galactosidase activity. In parallel, BGP induced ILK overexpression, dependent on the IGF-1 receptor activation, and oxidative stress. Down-regulating ILK expression prevented BGP-induced senescence and oxidative stress. Aortic rings from young rats treated with 10mM BGP for 48h, showed increased p53, p16 and ILK expression and SA-ß-gal activity. Seven/eight nephrectomized rats feeding a hyperphosphatemic diet and fifteenth- month old mice showed hyperphosphatemia and aortic ILK, p53 and p16 expression. In conclusion, we demonstrated that high extracellular concentration of phosphate induced senescence in cultured smooth muscle through the activation of IGF-1 receptor and ILK overexpression and provided solid evidences for the in vivo relevance of these results since aged animals showed high levels of serum phosphate linked to increased expression of ILK and senescence genes.

Response of parathyroid glands to calcitriol in culture: Is this response mediated by the genetic polymorphisms in vitamin D receptor?

BACKGROUND: Recently, we have developed a model of parathyroid tissue culture that allows the study of the response of the parathyroid glands to long-term effectors, such as calcitriol, and that is also useful to study the likely effect of the genetic polymorphisms in the functionality of the glands. The aim of this study was to evaluate the response to calcitriol of cultured parathyroid tissue from patients with secondary hyperparathyroidism (HPT) and the possible effect of vitamin D receptor (VDR) gene polymorphisms on this response. METHODS: Parathyroid glands (N = 37) from 34 parathyroidectomized patients (17 men, 17 women) were used. Several gland fragments were cultured for 60 hours in the presence of calcitriol 10(-9) mol/L or 10(-8) mol/L. DNA from each fragment was extracted to normalize the hormone secretion levels and to genotype the restriction sites ApaI, BsmI, TaqI, and FokI in the VDR gene. RESULTS: The percentages of secretion observed in the response to calcitriol were: 69%+/- 28% (range, 3-100) and 46%+/- 19% (range, 8-78) for calcitriol 10(-9) mol/L and 10(-8) mol/L, respectively (P = 0.004). None of the polymorphisms showed statistical differences in response to calcitriol with any of the concentrations used. CONCLUSION: Parathyroid glands cultured in vitro from patients with secondary HPT are able to respond to calcitriol decreasing PTH synthesis. These results, however, do not support the current hypothesis that VDR polymorphisms are involved in the modulation of the parathyroid gland response.

Influence of polymorphisms in VDR and COLIA1 genes on the risk of osteoporotic fractures in aged men.

BACKGROUND: Osteoporosis in chronic renal failure is a common finding caused by several factors, including age. In the last decade, the likely effect of genetic markers related with the appearance and evolution of osteoporosis has been mainly studied in women, with no categorical results. The aim of this study was to assess the influence of polymorphisms of the vitamin D receptor (VDR) and COLIA1 genes on the risk of osteoporotic fractures in men older than 50 years. METHODS: The study population comprised 156 men, aged 64 +/- 9 (50-86), randomly selected from the population list of Oviedo, Spain. Prevalent vertebral fractures and incident non-vertebral fractures were identified, as well as several genetic polymorphisms. Prevalent vertebral fractures were considered according to the Genant grade 2 classifications. The analyzed genetic polymorphisms were located on restriction sites BsmI (B,b), ApaI (A,a), and TaqI (T,t) in the VDR and on Sp1 (S,s) in COLIA1. RESULTS: Although none of the VDR gene polymorphisms separately analyzed showed any differences between fractured and non-fractured men, the utilization of haplotypes could be employed in order to find osteoporotic fractures in men. By contrast, the COLIA1 polymorphism was associated with osteoporotic fractures. The percentage of prevalent vertebral fractures was significantly higher in the "ss" genotype with respect to the other genotypes. These results show that in men, the "ss" genotype of COLIA1 polymorphism could be the best osteoporotic fracture risk genetic predictor, independent of bone mass values.

Effect of VDR gene polymorphisms on osteocalcin secretion in calcitriol-stimulated human osteoblasts.

BACKGROUND: The impact of vitamin D receptor (VDR) gene polymorphisms in bone metabolism remains controversial. Some authors have found a beneficial effect of some VDR gene polymorphisms, while others found no differences, or even a lower bone mass in subjects with the same type of polymorphisms. The aim of this study was to assess if the VDR gene polymorphisms could have an effect on the calcitriol-stimulated osteocalcin in human osteoblasts. METHODS: Osteoblasts were obtained from human femoral necks replaced because of osteoarthritis. Bones were cut into pieces of 1 to 2 mm and placed in a nylon mesh. After the migration of osteoblasts, the pieces were collected and cultured with different concentrations of calcitriol (10(-8), 10(-9), and 10(-1)0 mol/L). After 48 hours of incubation with calcitriol, the osteocalcin secreted into the medium (corrected by either total proteins or total DNA content) was measured. The DNA was extracted from the osteoblasts, amplified by polymerase chain reaction (PCR), and analyzed for target sequences sites of the BsmI, ApaI, TaqI, and FokI restriction enzymes. RESULTS: The response observed in osteocalcin secretion in the bb or TT genotypes doubled the response observed in the BB or tt genotypes (calcitriol 10(-8) and 10(-9) mol/L). A slight trend was also observed with the aa genotype. Men showed higher levels of osteocalcin secretion than women. Age did not show any influence in osteocalcin secretion. CONCLUSION: VDR alleles and gender demonstrated an effect on the osteocalcin secretion. BB or tt genotypes, and also the "A" allele, showed the lowest calcitriol-stimulated osteocalcin secretion.

Effect of aluminium on calcium-sensing receptor expression, proliferation, and apoptosis of parathyroid glands from rats with chronic renal failure.

BACKGROUND: To assess the effect of aluminium on the calcium-sensing receptor expression, proliferation, and apoptosis in parathyroid glands from rats with chronic renal failure, 2(1/2)-month-old male Wistar rats were 7/8 nephrectomized. METHODS: Eight weeks after surgery the rats were divided into two groups, one receiving intraperitoneal AlCl3 for 8 weeks and the other receiving intraperitoneal placebo. Serum Al, Ca, P, creatinine, and PTH were measured. Parathyroid glands were removed, formaldehyde-fixed, and paraffin-embedded. Calcium-sensing receptor and proliferation were detected by immunohistochemistry and apoptosis by TUNEL and propidium iodide uptake. RESULTS: At the end of the study, despite higher levels of serum P in the aluminium group (6.27 +/- 0.63 vs. 5.56 +/- 0.58 mg/dL; P = 0.045), serum PTH was lower (89.6 +/- 57.7 vs. 183.1 +/- 123.8 pg/mL; P = 0.059). No significant differences were found in the calcium-sensing receptor expression between groups (aluminium: 27.1 +/- 7.6; placebo: 25.4 +/- 3.5 RU). Rats receiving aluminium showed a significantly lower cell proliferation rate than the control rats (0.54 +/- 0.69 vs. 4.43 +/- 3.10 cells/mm2; P = 0.003). No apoptotic events were detected. CONCLUSION: Aluminium was able to reduce the cell proliferation of the parathyroid glands. Due to the low apoptosis rate, however, it was not possible to find any change. Aluminium had no effect on the calcium-sensing receptor expression.

Compliance and echographic carotid plaque evolution in patients with acute ischemic stroke treated with atorvastatin according to usual care.

The aim of the paper is to assess the compliance and efficacy of atorvastatin treatment according to usual care on carotid atheromatous plaque and lipids during follow-up for one year in patients with acute ischemic stroke. Sixty-six patients were included. Morphological plaque characteristics were described and a complete blood analysis was performed at admission and at one year. Nineteen patients stopped treatment. Lipid reduction was significant in triglycerides, cholesterol and LDL cholesterol, with an increase in HDL cholesterol. Morphological characteristics of carotid plaque were not modified. In conclusion, an irregular compliance has been seen in patients with acute ischemic stroke treated with atorvastatin according to usual care. Nearly one-third of our patients stopped the treatment before the course of a year. The effect on lipids and its pleiotropic effect on atheromatous carotid artery disease might support the long-term use of atorvastatin, regardless of the dose, in patients with acute ischemic stroke.