Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

The role of TRPV6 in breast carcinogenesis

TRPV6 is an endothelial calcium entry channel that is strongly expressed in breast adenocarcinoma tissue. In this study, we further confirmed this observation by analysis of breast cancer tissues, which indicated that TRPV6 mRNA expression was up-regulated between 2-fold and 15-fold compared with the average in normal breast tissue. Whereas TRPV6 is expressed in the cancer tissue, its role as a calcium channel in breast carcinogenesis is poorly understood. Therefore, we investigated how TRPV6 affects the viability, apoptosis, and calcium transport in the breast cancer cell line T47D. Hormones can also affect the tumor development; hence, we determined the effects of estradiol, progesterone, and 1,25-vitamin D on TRPV6 transcription. Interestingly, the estrogen receptor antagonist tamoxifen reduced expression of TRPV6 and is able to inhibit its calcium transport activity (IC(50), 7.5 micromol/L). The in vitro model showed that TRPV6 can be regulated by estrogen, progesterone, tamoxifen, and 1,25-vitamin D and has a large influence on breast cancer cell proliferation. Moreover, the effect of tamoxifen on cell viability was enhanced when TRPV6 expression was silenced with small interfering RNA. TRPV6 may be a novel target for the development of calcium channel inhibitors to treat breast adenocarcinoma expressing TRPV6.

PTH-related protein is released into the mother's bloodstream during location: evidence for beneficial effects on maternal calcium-phosphate metabolism

Recent studies have indicated that parathyroid hormone-related protein (PTHrP) may have important actions in lactation, affecting the mammary gland, and also calcium metabolism in the newborn and the mother. However, there are as yet no longitudinal studies to support the notion of an endocrine role of this peptide during nursing. We studied a group of 12 nursing mothers, mean age 32 years, after they had been nursing for an average of 7 weeks (B) and also 4 months after stopping nursing (A). It was assumed that changes occurring between A and B correspond to the effect of lactation. Blood was assayed for prolactin (PRL), PTHrP (two-site immunoradiometric assay with sheep antibody against PTHrP(1-40), and goat antibody against PTHrP(60-72), detection limit 0.3 pmol/l), intact PTH (iPTH), ionized calcium (Ca2+), 25-hydroxyvitamin D3 (25(OH)D3) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), alkaline phosphatase (alkP), as well as for creatinine (Cr), protein, phosphorus (P), and total calcium (Ca). Fasting 2-h urine samples were analyzed for Ca excretion (CaE) and renal phosphate threshold (TmP/GFR). PRL was significantly higher during lactation than after weaning (39 +/- 10 vs. 13 +/- 9 micrograms/l; p = 0.018) and so was PTHrP (2.8 +/- 0.35 vs. 0.52 +/- 0.04 pmol/l; p = 0.002), values during lactation being above the normal limit (1.3 pmol/l) in all 12 mothers. There was a significant correlation between PRL and PTHrP during lactation (r = 0.8, p = 0.002). Whole blood Ca2+ did not significantly change from A (1.20 +/- 0.02 mmol/l) to B (1.22 +/- 0.02, mmol/l), whereas total Ca corrected for protein (2.18 +/- 0.02 mmol/l) or uncorrected (2.18 +/- 0.02 mmol/l) significantly rose during lactation (2.31 +/- 0.02 mmol/l, p = 0.003 and 2.37 +/- 0.03 mmol/l, p = 0.002, respectively). Conversely, iPTH decreased during lactation (3.47 +/- 0.38 vs. 2.11 +/- 0.35 pmol/l, A vs. B, p = 0.02). Serum-levels of 25(OH)D3 and 1,25(OH)2D3 did not significantly change from A to B (23 +/- 2.3 vs. 24 +/- 1.9 ng/ml and 29.5 +/- 6.0 vs. 21.9 +/- 1.8 pg/ml, respectively). Both TmP/GFR and P were higher during lactation than after weaning (1.15 +/- 0.03 vs. 0.86 +/- 0.05 mmol/l GF, p = 0.003 and 1.25 +/- 0.03 vs. 0.96 +/- 0.05 mmol/l, p = 0.002, respectively) as was alkP (74.0 +/- 7.1 vs. 52.6 +/- 6.9 U/l, p = 0.003). CaE did not differ between A and B (0.015 +/- 0.003 vs. 0.017 +/- 0.003 mmol/l GF, A vs. B, NS). We conclude that lactation is accompanied by an increase in serum PRL. This is associated with a release of PTHrP into the maternal blood circulation. A rise in total plasma Ca ensues, probably in part by increased bone turnover as suggested by the elevation of alkP. PTH secretion falls, with a subsequent rise of TmP/GFR and plasma P despite high plasma levels of PTHrP.

Fibroblast growth factor 23 and markers of mineral metabolism in individuals with preserved renal function

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate homeostasis. Circulating FGF23 is elevated in chronic kidney disease (CKD) and independently associated with poor renal and cardiovascular outcomes and mortality. Because the study of FGF23 in individuals with normal renal function has received little attention, we examined in a large, population based study of 1128 participants the associations of FGF23 with markers of mineral metabolism and renal function. The median estimated glomerular filtration rate (eGFR) of the cohort was 105 ml/min per 1.73 m2, and the median plasma FGF23 was 78.5 RU/ml. FGF23 increased and plasma 1,25-dihydroxyvitamin D3 decreased significantly below an eGFR threshold of 102 and 99 ml/min per 1.73 m2, respectively. In contrast, plasma parathyroid hormone increased continuously with decreasing eGFR and was first significantly elevated at an eGFR of 126 ml/min per 1.73 m2. On multivariable analysis adjusting for sex, age, body mass index, and GFR, FGF23 was negatively associated with 1,25-dihydroxyvitamin D3, and urinary absolute and fractional calcium excretion but not with serum calcium or parathyroid hormone. We found a positive association of FGF23 with plasma phosphate, but no association with urinary absolute or fractional phosphate excretion and, unexpectedly, a positive association with tubular maximum phosphate reabsorption/GFR. Thus, in the absence of CKD, parathyroid hormone increases earlier than FGF23 when the eGFR decreases. The increase in FGF23 occurs at a higher eGFR threshold than previously reported and is closely associated with a decrease in 1,25-dihydroxyvitamin D3. We speculate that the main demonstrable effect of FGF23 in the setting of preserved renal function is suppression of 1,25-dihydroxyvitamin D3 rather than stimulation of renal phosphate excretion.

Comprehensive analysis of lymph node stroma-expressed Ig superfamily members reveals redundant and nonredundant roles for ICAM-1, ICAM-2, and VCAM-1 in lymphocyte homing

Although it is well established that stromal intercellular adhesion molecule-1 (ICAM-1), ICAM-2, and vascular cell adhesion molecule-1 (VCAM-1) mediate lymphocyte recruitment into peripheral lymph nodes (PLNs), their precise contributions to the individual steps of the lymphocyte homing cascade are not known. Here, we provide in vivo evidence for a selective function for ICAM-1 > ICAM-2 > VCAM-1 in lymphocyte arrest within noninflamed PLN microvessels. Blocking all 3 CAMs completely inhibited lymphocyte adhesion within PLN high endothelial venules (HEVs). Post-arrest extravasation of T cells was a 3-step process, with optional ICAM-1-dependent intraluminal crawling followed by rapid ICAM-1- or ICAM-2-independent diapedesis and perivascular trapping. Parenchymal motility of lymphocytes was modestly reduced in the absence of ICAM-1, while ICAM-2 and alpha4-integrin ligands were not required for B-cell motility within follicles. Our findings highlight nonredundant functions for stromal Ig family CAMs in shear-resistant lymphocyte adhesion in steady-state HEVs, a unique role for ICAM-1 in intraluminal lymphocyte crawling but redundant roles for ICAM-1 and ICAM-2 in lymphocyte diapedesis and interstitial motility.

Effects of dietary supplementation with synthetic vitamin D-3 and 25-hydroxycholecalciferol on blood calcium and phosphate levels and performance in laying hens

We investigated the effects of different dietary vitamin D regimen on selected blood parameters in laying hens. Supplementation with vitamin D-3 only was compared with a combination of vitamin D-3 and its metabolite 25-hydroxy-cholecalciferol (25(OH)D-3). Blood concentrations of total calcium, phosphate and 25 (OH)D-3 were determined. Four thousand one-day-old LSL chicks were split in two treatment groups and distributed to eight pens. The control group was given a commercial animal diet containing 2800 IU synthetic vitamin D-3 in the starter feed and 2000 IU synthetic vitamin D-3 in the pullet feed. The experimental group was fed the same commercial diet in which half the synthetic vitamin D-3 content had been substituted with 25(OH)D-3 (Hy center dot D (R)). At 18 weeks of age, pullets were transferred to the layer house. At the ages of 11, 18 and 34 weeks, between 120 and 160 blood samples were collected from both the control and the experimental groups, respectively. The experimental group had higher levels of 25 (OH)D-3 than the control group at all three ages. Serum calcium levels did not differ between the treatment groups at any age. With the onset of laying, calcium levels rose significantly. Whereas blood serum concentration at 18 weeks was 3 mmol/L in both treatment groups, it increased to 8.32 mmol/L in the control group and to 8.66 mmol/L in the experimental group at week 34. At weeks 11 and 34, phosphate was significantly lower in the experimental group. In conclusion, HyD (R) significantly affected serum phosphate and 25(OH)D-3 levels. No effects of (25(OH)D-3 supplementation on performance, shell quality and fractures of keelbones were found.

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C. Methodology/Principal Findings Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D3 (25[OH]D3) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D3<20 ng/mL) during all seasons, but 25(OH)D3 serum levels were not associated with treatment outcome. Conclusions/Significance Our study suggests a role of bioactive vitamin D (1,25[OH]2D3, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D3 is not a suitable predictor of treatment outcome.

The prevalence of vitamin D inadequacy amongst women with osteoporosis: an international epidemiological investigation

INTRODUCTION: Vitamin D is essential for calcium metabolism as well as for fracture prevention, and a recent review suggested that the optimal serum 25(OH)D lies in the region of 50-80 nmol L-1 (20-32 ng mL-1). A high prevalence of inadequacy has been reported in many studies but the prevalence of inadequacy amongst women with osteoporosis in different regions of the world has not been well characterized. SETTING AND SUBJECTS: A multinational study of 18 countries at various latitudes (range 64N-38S) was conducted in 2004 and 2005 to determine the average levels of serum 25(OH)D and the prevalence of vitamin D inadequacy. A total of 2606 postmenopausal women with osteoporosis (low bone mineral density, history of fragility fracture) seeking routine medical care were enrolled and serum 25(OH)D levels were measured at a single laboratory visit. RESULTS: Mean serum 25(OH)D level was 26.8 ng mL-1 (SE 0.3) and ranged from 7 to 243 ng mL-1. Regional mean values were highest in Latin America (29.6 ng mL-1, SE 0.6) and lowest in the Middle East (20.4 ng mL-1, SE 0.5). Overall, 64% of women had serum levels<30 ng mL-1. Serum parathyroid hormone reached a nadir at serum 25(OH)D levels>35 ng mL-1. In nonequatorial countries, women recruited during the winter months had somewhat lower serum 25(OH)D levels than those recruited during the summer months in some, but not all, countries. CONCLUSIONS: Low levels of serum 25(OH)D are common amongst women with osteoporosis. The results underscore the value of assuring vitamin D adequacy in these women.

Effect of disodium monofluorophosphate, calcium and vitamin D supplementation on bone mineral density in patients chronically treated with glucocorticosteroids: a prospective, randomized, double-blind study

To study the effect of fluoride on bone mineral density (BMD) in patients treated chronically with glucocorticosteroids, 15 subjects (renal grafted, n = 12; skin disease, n = 1; broncho pulmonary disorder, n = 1; Crohn's disease, n = 1) were prospectively studied in a double-blinded manner and randomly allocated either to group 1 (n = 8) receiving 13.2 mg/day fluoride given as disodium monofluorophosphate (MFP) supplemented with calcium (1,000 mg/day) and 25-hydroxyvitamin D (calcifediol) (50 micrograms/day), or to group 2 (n = 7) receiving Cas+ calcifediol alone. An additional group of 14 renal transplant patients treated chronically with glucocorticosteroids but exempt of specific therapeutic intervention for bone disease was set up as historical controls. BMD was measured by dual-energy X-ray absorptiometry (DXA, Hologic QDR 1000) performed at months 0, 6 and 12 for groups 1 and 2 (lumbar spine, total upper femur, diaphysis and epiphysis of distal tibia), or 11-31 months apart with calculation of linear yearly changes for the historical cohort. Lumbar BMD tended to rise in groups 1 and 2, and to fall in group 3, the change reaching statistical significance (p < 0.05) in group 1, thus leading to a significant difference between groups 1 and 3 (p < 0.05). At upper femur, tibial diaphysis and tibial epiphysis, no significant change in BMD occurred in any of the groups. In conclusion, lumbar BMD rises more after a mild dosis of fluoride given as MFP and combined to calcium and calcifediol than on Ca+ calcifediol alone, without changes in BMD at the upper femur or distal tibia.

Genetic Analyses Reveal a Role for Vitamin D Insufficiency in HCV-Associated Hepatocellular Carcinoma Development

Background Vitamin D insufficiency has been associated with the occurrence of various types of cancer, but causal relationships remain elusive. We therefore aimed to determine the relationship between genetic determinants of vitamin D serum levels and the risk of developing hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC). Methodology/Principal Findings Associations between CYP2R1, GC, and DHCR7 genotypes that are determinants of reduced 25-hydroxyvitamin D (25[OH]D3) serum levels and the risk of HCV-related HCC development were investigated for 1279 chronic hepatitis C patients with HCC and 4325 without HCC, respectively. The well-known associations between CYP2R1 (rs1993116, rs10741657), GC (rs2282679), and DHCR7 (rs7944926, rs12785878) genotypes and 25(OH)D3 serum levels were also apparent in patients with chronic hepatitis C. The same genotypes of these single nucleotide polymorphisms (SNPs) that are associated with reduced 25(OH)D3 serum levels were found to be associated with HCV-related HCC (P = 0.07 [OR = 1.13, 95% CI = 0.99–1.28] for CYP2R1, P = 0.007 [OR = 1.56, 95% CI = 1.12–2.15] for GC, P = 0.003 [OR = 1.42, 95% CI = 1.13–1.78] for DHCR7; ORs for risk genotypes). In contrast, no association between these genetic variations and liver fibrosis progression rate (P>0.2 for each SNP) or outcome of standard therapy with pegylated interferon-α and ribavirin (P>0.2 for each SNP) was observed, suggesting a specific influence of the genetic determinants of 25(OH)D3 serum levels on hepatocarcinogenesis. Conclusions/Significance Our data suggest a relatively weak but functionally relevant role for vitamin D in the prevention of HCV-related hepatocarcinogenesis.

Vitamin D time profile based on the contribution of non-genetic and genetic factors in HIV-infected individuals of European ancestry

BACKGROUND Vitamin D deficiency is prevalent in HIV-infected individuals and vitamin D supplementation is proposed according to standard care. This study aimed at characterizing the kinetics of 25(OH)D in a cohort of HIV-infected individuals of European ancestry to better define the influence of genetic and non-genetic factors on 25(OH)D levels. These data were used for the optimization of vitamin D supplementation in order to reach therapeutic targets. METHODS 1,397 25(OH)D plasma levels and relevant clinical information were collected in 664 participants during medical routine follow up visits. They were genotyped for 7 SNPs in 4 genes known to be associated with 25(OH)D levels. 25(OH)D concentrations were analyzed using a population pharmacokinetic approach. The percentage of individuals with 25(OH)D concentrations within the recommended range of 20-40ng/ml during 12 months of follow up and several dosage regimens were evaluated by simulation. RESULTS A one-compartment model with linear absorption and elimination was used to describe 25(OH)D pharmacokinetics, while integrating endogenous baseline plasma concentrations. Covariate analyses confirmed the effect of seasonality, body mass index, smoking habits, the analytical method, darunavir/r and the genetic variant in GC (rs2282679) on 25(OH)D concentrations. 11% of the interindividual variability in 25(OH)D levels was explained by seasonality and other non-genetic covariates and 1% by genetics. The optimal supplementation for severe vitamin D deficient patients was 300000 IU two times per year. CONCLUSIONS This analysis allowed identifying factors associated with 25(OH)D plasma levels in HIV-infected individuals. Improvement of dosage regimen and timing of vitamin D supplementation is proposed based on those results.

Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case-control study using centralized ultrasensitive 454 pyrosequencing.

OBJECTIVES It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. METHODS This Europe-wide case-control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%-25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. RESULTS Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35-5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76-6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12-5.18, P = 0.024). A dose-effect relationship between virological failure and mutational load was found. CONCLUSIONS Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART.

Vitamin D status among children and adolescents on anticonvulsant drugs in southern Switzerland.

INTRODUCTION It is recognised that vitamin D status is often inadequate (<50 nmol/l) in epileptic children, mainly because some anticonvulsant drugs induce the enzymes responsible for its metabolism. The purpose of the present study was to address vitamin D status among children and adolescents treated with anticonvulsant drugs and control subjects who reside in southern Switzerland, a high solar radiation region. METHODS Between January and May 2013, total serum 25-hydroxyvitamin D was assessed by liquid chromatography-tandem mass spectrometry in 58 children and adolescents with epilepsy and 29 controls residing in southern Switzerland. Dark-skinned individuals, females wearing dress styles covering practically the whole body and subjects with body mass index ≥85th percentile for age and sex were excluded. RESULTS Concentration of serum 25-hydroxyvitamin D was similar in epilepsy patients (48 [37-62] nmol/l; median and interquartile range) and controls (53 [47-64] nmol/l). An inadequate serum 25-hydroxyvitamin D concentration was common both among patients (55%) and control subjects (34%). Serum 25-hydroxyvitamin D was significantly lower among patients treated with anticonvulsant drugs that induce the metabolism of vitamin D (30 [21-51] nmol/l) than among the remaining patients (51 [40-65] nmol/l) and controls. CONCLUSIONS The present study indicates a relevant tendency towards inadequate vitamin D status among children with and without anticonvulsant drug management who reside in southern Switzerland. This tendency is more prominent in patients treated with anticonvulsant drugs that induce the metabolism of 25-hydroxyvitamin D.

Vitamin D and central hypersensitivity in patients with chronic pain.

BACKGROUND Low vitamin D is implicated in various chronic pain conditions with, however, inconclusive findings. Vitamin D might play an important role in mechanisms being involved in central processing of evoked pain stimuli but less so for spontaneous clinical pain. OBJECTIVE This study aims to examine the relation between low serum levels of 25-hydroxyvitamin D3 (25-OH D) and mechanical pain sensitivity. DESIGN We studied 174 patients (mean age 48 years, 53% women) with chronic pain. A standardized pain provocation test was applied, and pain intensity was rated on a numerical analogue scale (0-10). The widespread pain index and symptom severity score (including fatigue, waking unrefreshed, and cognitive symptoms) following the 2010 American College of Rheumatology preliminary diagnostic criteria for fibromyalgia were also assessed. Serum 25-OH D levels were measured with a chemiluminescent immunoassay. RESULTS Vitamin deficiency (25-OH D < 50 nmol/L) was present in 71% of chronic pain patients; another 21% had insufficient vitamin D (25-OH D < 75 nmol/L). After adjustment for demographic and clinical variables, there was a mean ± standard error of the mean increase in pain intensity of 0.61 ± 0.25 for each 25 nmol/L decrease in 25-OH D (P = 0.011). Lower 25-OH D levels were also related to greater symptom severity (r = -0.21, P = 0.008) but not to the widespread pain index (P = 0.83) and fibromyalgia (P = 0.51). CONCLUSIONS The findings suggest a role of low vitamin D levels for heightened central sensitivity, particularly augmented pain processing upon mechanical stimulation in chronic pain patients. Vitamin D seems comparably less important for self-reports of spontaneous chronic pain.