Blockade of the renal tubular effects of vitamin D by cycloheximide in the rat

To further characterize the mechanisms by which 25(OH)vitamin D₃ (25(OH)D₃) and 1.25(OH)₂ vitamin D₃ (1,25(OH)₂D₃) suppress the phosphaturic action of parathyroid hormone (PTH) we have studied the effects of cycloheximide (cyclohex), a protein synthesis inhibitor, on the interaction between PTH and vitamin D metabolites in parathyroidectomized (PTX) rats, both in vivo and in vitro experiments. In clearance studies PTX PTH-infused rats were pretreated with cyclohex 2 h before the administration of vitamin D. In control, PTX PTH-infused rats not pretreated with cyclohex, the administration of 25(OH)D₃ and 1,25(OH)₂D₃ was associated with a fall in fractional excretion of phosphate (CP/CIN) from 0.30±0.05 to 0.16±0.02 and from 0.31±0.05 to 0.13±0.01 (P<0.005) respectively. Cyclohex-pretreated PTX PTH-infused rats failed to respond to both 25(OH)D₃ and 1,25(OH)₂D₃, and CP/CIN, which rose after PTH, remained 0.32±0.05 and 0.29±0.03 respectively. In vitro, both 25(OH)D₃ and 1,25(OH)₂D₃ inhibited the PTH-induced activation of adenylate cyclase in the renal isolated membrane fractions. Pretreatment with cyclohex abolished this effect of vitamin D metabolites. These results show that cyclohex blocks the antiphosphaturic effects of both 25(OH)D₃ and 1,25(OH)₂D₃ but does not alter the response to PTH. These findings are consistent with the possibility that the acute renal action of vitamin D depends on de novo synthesis of protein.An abstract of this work appeared in Clinical Research, 28 (2) A 387, 1980.     

Long-term therapy with cyclosporin A does not influence serum concentrations of vitamin D metabolites in patients with multiple sclerosis

Summary Animal studies have shown that cyclosporin A (CyA) stimulates renal 25-hydroxyvitamin D₃ [25(OH)D₃]-1-hydroxylase activity; in contrast, studies in renal transplant recipients indirectly suggest that CyA reduces 1,25-dihydroxyvitamin D₃ [1,25 (OH)₂D₃] production. To clarify the effect of CyA on vitamin D metabolite concentrations, we measured parameters of calcium metabolism in 37 CyA-treated patients (median trough whole blood levels 171–222 ng/ml) with multiple sclerosis and initially normal kidney function. The patients participated in a randomized double-blind study to assess the efficacy of CyA in multiple sclerosis. An age- and sex-matched control group (n = 39) received azathioprine (Aza). Measurements were made at the end of a 2-year treatment period. The 1,25(OH)₂D₃ serum concentrations were not significantly different between the two groups, although they were numerically lower in CyA-treated patients [median (range), 28.4 pg/ml (7.8–85.9) vs 41.0 pg/ml (9.2–105.1) in Aza-treated patients]. The 25(OH)D₃ levels were comparable in both groups. There was no correlation between the 25(OH)D₃ and 1,25(OH)₂D₃ concentrations. The renal function in both groups was stable in the last 6 months of the study. At the end of the study period, the endogenous creatinine clearance was significantly lower in the CyA-treated group (85 ± 17 ml/min versus 99 ± 22 in the Aza-treated group, P < 0.05). The carboxyterminal parathyroid hormone (C-PTH) was within the normal range in both groups, although CyA-treated patients had significantly higher concentrations (P<0.01). The urinary excretion of mineral ions, cations and protein was similar in both groups. Our data suggest that long-term treatment with CyA does not cause clinically important alterations of vitamin D metabolism in humans. Subtle differences in the concentrations of 1,25(OH)₂D₃ and C-PTH between CyA- and Aza-treated patients result presumably from a slight impairment of renal function through CyA.Abbreviations CyA cyclosporin A - Aza azathioprine - 25(OH)D₃ 25-hydroxyvitamin D₃ - 1,25(OH)₂D₃ 1,25-dihydroxyvitamin D₃ - PTH parathyroid hormone - C-PTH carboxyterminal-PTH - AP alkaline phosphatase - Ccr endogenous creatinine clearance - gamma-GT gamma-glutamyltransferase     

Regulation of Matrix Vesicle Metabolism by Vitamin D Metabolites

Matrix vesicles are membrane organelles found in the extracellular matrix of calcifying cells. Vitamin D-responsive alkaline phosphatase specific activity has been localized to matrix vesicles in chondrocyte and osteoblast cultures. The effect of hormone is both metabolite and cell specific. Alkaline phosphatase in matrix vesicles produced by resting zone chondrocytes is stimulated by 24, 25(OH)₂D₃ whereas alkaline phosphatase in matrix vesicles produced by growth zone chondrocytes is responsive to 1,25(OH)₂D₃. However, mesenchymal cell cultures, which exhibit a chondrogenic phenotype when exposed to bone inductive proteins in vitro, produce vesicles with alkaline phosphatase activity that is unaffected by either 1,25(OH)₂D₃ or 24, 25(OH)₂D₃. Incorporation and release of arachidonic acid into phosphatidylethanolamine is also differentially regulated by 1,25(OH)₂D₃ and 24, 25(OH)₂D₃ in chondrocytes. These data suggest that vitamin D metabolites may regulate endochondral ossification by altering matrix vesicle enzyme activities, perhaps through changes in membrane phospholipid metabolism.     

Regulation of Na-dependent phosphate influx across the mucosal border of duodenum by 1,25-dihydroxycholecalciferol

Animals treated with disodium ethane-1-hydroxy-1,1-diphosphonate (EHDP), at doses which decrease the renal production and/or the plasma levels of 1,25-dihydroxycholecalciferol [1,25(OH)₂D₃], display a reduced net absorption of phosphate. In this study we investigated whether EHDP-treatment and administration of 1,25(OH)₂D₃ to EHDP-treated animals affected the phosphate influx across the mucosal border of rabbit duodenum. The initial rate of phosphate influx into mucosal cells was measured in isolated intestine. In control, untreated rabbits, the phosphate influx shows a saturable, Na-dependent component and a diffusional, Na-independent uptake. In tissue from rabbits treated for 3 days with EHDP, the phosphate influx was found to be strongly reduced. EHDP-treatment decreased the Na-dependent, carrier mediated phosphate influx in duodenum. Administration of 1,25(OH)₂D₃ to EHDP-treated animals reversed the reduced phosphate influx. These effects were mainly apparent through changes in theJ _mc ^max of the phosphate influx, which was decreased from 211±38.7 nmole/cm² h in controls to 42.1±18.1 nmole/cm² h in the EHDP-treated group and increased to 413±43.6 nmole/cm² h by 1,25(OH)₂D₃. The treatment did not appear to affect the diffusional, Na-independent phosphate influx. EHDP-treatment did not affect the influx of alanine in this segment suggesting that EHDP-treatment affects only 1,25(OH)₂D₃-dependent transport mechanisms. The results suggest that 1,25(OH)₂D₃ modulates the number of carrier sites available at the mucosal membrane for Na-dependent phosphate entry.     

The Synergistic Effect of TGFβ and 24, 25-(OH)2D3 on Resting Zone Chondrocytes is Metabolite-Specific and Mediated by PKC

Transforming growth factor-β (TGFβ) and 24, 25-(OH)₂D₃ play a major role in chondrocyte maturation during endochondral bone formation. TGFβ and vitamin D metabolites when added separately to resting zone (RC) or growth zone (GC) chondrocyte cultures, activate protein kinase C (PKC). The present study determined whether there is an additive or synergistic effect of vitamin D₃ metabolites and TGFβ on alkaline phosphatase and PKC specific activities and whether this effect is cell maturation-dependent. GC and RC chondrocytes were isolated from rat costochondral cartilage and cultured to fourth passage. The cells were incubated with vitamin D₃ metabolites and TGFβ alone or in combination, and the specific activity of alkaline phosphatase, as well as the specific activity of PKC, were measured. The addition of 24, 25-(OH)₂D₃ with TGFP to RC cells caused a synergistic effect on alkaline phosphatase activity; this result was not found if the vitamin D₃ metabolite was replaced by l, 25-(OH)₂D₃. The addition of l, 25-(OH)₂D₃ or 24, 25-(OH)₂D₃ with TGFβ on GC cells had no synergistic or additive effect. The addition of 24, 25-(OH)₂D₃ and TGFβ for 12 hours caused a synergistic effect on PKC activity; this effect was also observed if TGFβ was added first for 12 hours and 24, 25-(OH)₂D₃ for the last 90 min. However, the addition of 24, 25-(OH)₂D₃ for 90 min followed by the addition of TGFβ for an additional 10.5 hours had no synergistic effect. This study indicates that TGFβ and 24, 25-(OH)₂D₃ have a synergistic effect on chondrocyte differentiation as well as on PKC activity, suggesting that the synergistic effect of 24, 25-(OH)₂D₃ and TGFβ on chondrocyte differentiation may be mediated through activation of PKC. The synergistic effect of 24, 25-(OH)₂D₃ and TGFβ was cell maturation dependent.     

Rapid stimulation of Na+/H+ exchange by 1,25-dihydroxyvitamin D3; interaction with parathyroid-hormone-dependent inhibition

We have examined the rapid effect of 1,25-dihydroxyvitamin-D₃ [1,25(OH)₂D₃] on apical Na⁺/H⁺ exchange activity in opossum kidney (OK) cells and in MCT cells (a culture of simian-virus-40-immortalized mouse cortical tubule cells) grown on filter support. Addition of 1,25(OH)₂D₃ (10 nM) for 1 min increased apical Na⁺/H⁺ exchange activity [recovery from an acid load; measured by 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein] in OK cells (by 56%) and in MCT cells (by 36%). The cellular mechanisms involved in 1,25(OH)₂D₃-dependent stimulation of Na⁺/H⁺ exchange were analysed in OK cells; stimulation of Na⁺/ H⁺ exchange by 1,25(OH)₂D₃ was not prevented by actinomycin D. Applying parathyroid hormone (PTH) reduced Na⁺/H⁺ exchange activity in OK cells (by 34% at 10 nM, 5 min); 1,25(OH)₂D₃ reversed PTH-induced inhibition, either when PTH was added prior to 1,25(OH)₂D₃ or when the two agonists were applied together. 1,25(OH)₂D₃ had no effect on basal OK cell cAMP content or on [Ca²⁺]_i (fura-2). 1,25(OH)₂D₃ attenuated PTH-induced cAMP accumulation and had no effect on the PTH-dependent increase in [Ca²⁺]_i. These data suggest a regulatory control (stimulation) of proximal tubular brush-border Na⁺/H⁺ exchange by 1,25(OH)₂D₃. This effect is non-genomic and might in part be explained by a release from cAMP-dependent control of transport activity.     

Evidence for interference of vitamin D with PTH/PTHrP receptor expression in opossum kidney cells

 Vitamin D counters the phosphaturic action of parathyroid hormone (PTH) in rats in vivo. The present study was undertaken to examine this interaction using monolayers of Opossum kidney (OK) cells. ³²P uptake, cAMP generation, PTH/PTHrP receptor mRNA expression and intracellular Ca²⁺ [Ca²⁺]_i were measured in (1) control cells, (2) cells exposed to PTH, (3) cells pretreated with 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], and (4) 1,25(OH)₂D₃-pretreated cells exposed to PTH. ³²P uptakes were in (1) 5.00±0.20 (mean ±SE), in (2) 2.30±0.14 (P<0.001 versus group 1), in (3) 4.80±0.24 (P NS versus group 1) and in (4) 3.70±0.20 (P<0.001 versus group 2) nmol P_i/(mg·prot 10 mm). cAMP levels were in (1) 10±3, in (2) 210±8, in (3) 12±4, and in (4) 122±12 pmol cAMP/mg protein (P<0.001 versus group 2). PTH/PTHrP receptor mRNA expression was in relative units: (1) 100±0, (2) 99.5±6.2, (3) 68.7±2.6 (P<0.001 versus group 1), and (4) 34.8±3.3 (P<0.001 versus group 1). In groups 2 and 4 PTH induced equal transient increments in [Ca²⁺]_i. These experiments demonstrate that the effect of vitamin D on phosphate transport is associated with a commensurate diminution in PTH/PTHrP receptor gene expression and PTH-induced cAMP formation but not with Ca²⁺ transients. Vitamin D per se does not affect ³²P uptake or cAMP generation while it slightly decreased PTH/PTHrP receptor gene expression. These observations demonstrate that: (1) 1.25(OH)₂D₃ directly antagonizes the effects of PTH on ³²P uptake in OK cells, (2) this effect is mediated via inhibition of PTH-induced activation of AC/cAMP system, (3) the diminution in PTH-induced cAMP formation may stem at least in part from a decrease in the expression of PTH/PTHrP receptor mRNA. Received: 2 December 1997 / Received after revision: 19 January 1998 / Accepted: 28 January 1998     

Plasma 1,25 Dihydroxy Vitamin D<Subscript>3</Subscript> Level and Expression of Vitamin D Receptor and Cathelicidin in Pulmonary Tuberculosis

Introduction  Vitamin D₃, which exerts its effect through vitamin D receptor (VDR), is known for its potent immunomodulatory activities. Associations between low serum vitamin D₃ levels and increased risk of tuberculosis have been reported. Study Subjects and Methods  Plasma 1,25 dihydroxy vitamin D₃ levels (1,25(OH)₂ D₃) and ex vivo levels of VDR protein from peripheral blood mononuclear cells were studied in 65 pulmonary tuberculosis (PTB) patients and 60 normal healthy subjects (NHS) using enzyme-linked immunosorbent assay-based methods. Using real-time polymerase chain reaction (PCR), induction of VDR, cathelicidin, and CYP27B1 mRNA were studied in live Mycobacterium tuberculosis-stimulated macrophage cultures treated with or without 1,25 dihydroxy vitamin D₃. VDR and CYP27B1 (-1077 A/T) gene polymorphisms were studied using PCR-based methods. Results  1,25(OH)₂ D₃ were significantly increased (p = 0.0004), while ex vivo levels of VDR protein were significantly decreased in PTB patients (p = 0.017) as compared to NHS. 1,25(OH)₂ D₃ levels were not different between variant genotypes of CYP27B1. A trend towards decreased levels of VDR protein was observed among NHS with BsmI BB and TaqI tt genotypes compared to NHS with other genotypes. Relative quantification of mRNA using real-time PCR revealed increased VDR mRNA expression in live M. tuberculosis-stimulated culture in PTB patients (p < 0.01) than normal healthy subjects. Cathelicidin mRNA expression was significantly increased in vitamin D₃-treated cultures compared to unstimulated and M. tuberculosis-stimulated culture in both patients (p < 0.001) and NHS (p < 0.05). Conclusions  The present study suggests that PTB patients may have increased 1,25(OH)₂ D₃ levels, and this might lead to downregulation of VDR expression. Decreased VDR levels could result in defective VDR signaling. Moreover, addition of 1,25(OH)₂ D₃ might lead to increased expression of cathelicidin which could enhance the immunity against tuberculosis.     

1,25-Dihydroxyvitamin D3 inhibits proliferation but not the suppressive function of regulatory T cells in the absence of antigen-presenting cells

Vitamin D₃ is known to induce regulatory T (Treg) cells by rendering antigen-presenting cells tolerogenic, its direct effect on human naturally occurring Treg cells is unclear. Here, we investigated if and how 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] can directly affect the proliferation and function of human naturally occurring Treg cells in vitro. First, we demonstrated that these Treg cells express vitamin D receptors that were up-regulated following anti-CD3/CD28-bead stimulation. 1,25(OH)₂D₃ inhibited proliferation of Treg cells even when exogenous interleukin-2 was provided. Treg cells were more susceptible to the inhibitory effect of 1,25(OH)₂D₃ than conventional T cells_. 1,25(OH)₂D₃ neither affected the anergic state nor the suppressive function of Treg cells but induced a subtle increase in interleukin-10-secreting cells. The cell-division-inhibiting effect of 1,25(OH)₂D₃ on Treg cells was also demonstrated in vivo by supplementing vitamin D-deficient HIV-1-infected patients with 2000 IU cholecalciferol (vitamin D₃). Increased serum 1,25(OH)₂D₃ levels were associated with a drop in the number and percentage of Treg cells, which may be attributed to a decrease in the proliferating Foxp3⁺ Treg cell population. In conclusion, 1,25(OH)₂D₃ directly affects Treg cell growth and promotes interleukin-10 production without apparent effects on activation status and suppressive phenotype whereas in vivo, high serum 1,25(OH)₂D₃ levels are associated with reduced Treg cell proliferation and a reduced number of Treg cells.     

1,25-dihydroxyvitamin D3 enhances the ability of transferred CD4+ CD25+ cells to modulate T helper type 2-driven asthmatic responses

The severity of allergic diseases may be modified by vitamin D. However, the immune pathways modulated by the active form of vitamin D, 1,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃], are yet to be fully elucidated. In this study, naturally occurring CD4⁺ CD25⁺ cells from the skin‐draining lymph nodes (SDLN) of mice treated with topical 1,25(OH)₂D₃ had an increased ability to suppress T helper type 2 (Th2) ‐skewed immune responses. CD4⁺ CD25⁺ cells transferred from mice treated with topical 1,25(OH)₂D₃ into ovalbumin (OVA) ‐sensitized mice challenged intranasally with OVA 18 hr later, significantly suppressed the capacity of airway‐draining lymph node (ADLN) cells to proliferate and secrete cytokines in response to further OVA stimulation ex vivo. The CD4⁺ CD25⁺ cells from 1,25(OH)₂D₃‐treated mice also reduced airway hyperresponsiveness and the proportions of neutrophils and eosinophils in bronchoalveolar lavage fluid (BALF). To test the effect of 1,25(OH)₂D₃ on cells able to respond to a specific antigen, CD4⁺ CD25⁺ cells were purified from the SDLN of OVA‐T‐cell receptor (TCR) transgenic mice treated 4 days earlier with topical 1,25(OH)₂D₃. CD4⁺ CD25⁺ cells from OVA‐TCR mice treated with 1,25(OH)₂D₃ were able to alter BALF cell content and suppress ADLN responses to a similar degree to those cells from non‐transgenic mice, suggesting that the effect of 1,25(OH)₂D₃ was not related to TCR signalling. In summary, topical 1,25(OH)₂D₃ increased the regulatory capacity of CD4⁺ CD25⁺ cells from the SDLN to suppress Th2‐mediated allergic airway disease. This work highlights how local 1,25(OH)₂D₃ production by lung epithelial cells may modulate the suppressive activity of local regulatory T cells.     

Subacute effects of thiazide administration on renal hemodynamics and calcium metabolism

Summary To elucidate the renal effects of thiazides as a function of sodium intake, 8 healthy volunteers without renal disease were studied at baseline and 1 day as well as 4 days after the administration of 100 mg hydrochlorothiazide/day. The subjects were compared on two different dietary sodium intakes (120 mmol/day and 220 mmol/day). Measurements comprised inulin clearance (Cin) and paraaminohippurate clearance (Cpah) by infusion clearance technique, total and ionised calcium, immunoreactive parathyroid hormone (1,84 iPTH), 1.25 (OH)₂ vitamin D₃, and indices of hemoconcentration. Acute administration of hydrochlorothiazide (HCTZ) caused no change in Cin (before 111 ± 3 ml/min 1.73 m² ; 24 h after, 107 ± 2 ml/min 1.73 m²) or Cpah (before, 579 ± 9 ml/min 1.73 M²; after, 584 ± 12 ml/min 1.73 m²), while a significant (P < 0.01) decrease was noted on the 4th day after 100 mg HCTZ/day and normal sodium intake. No significant change of creatinine clearance (Ccr) was seen with either manouever. Renal hemodynamic changes after HCTZ administration were marginal when hemoconcentration was prevented by a high salt intake. Acute administration (1 h) of HCTZ caused suppression of 1,84 iPTH (before, 2.3 ±0.5 pmol/l; after, 1.9 ± 0.2 pmol/l; P < 0.01), but after 4 days a lower ionised calcium (baseline, 1.25 ± 0.01 mmol/l; day 5, 1.20 ± 0.02 mmol/l; P < 0.01) was noticed in parallel with hemoconcentration, metabolic alkalosis, and reduced 1,25 (OH)₂ vitamin D₃ concentrations. The level of 1,84 iPTH was elevated. We conclude that (i) hydrochlorothiazide does not affect the renal hemodynamics if hemoconcentration is avoided and (ii) hydrochlorothiazide acutely lowers PTH, while subacutely metabolic alkalosis and decreased ionised calcium may occur with concomitant increase in 1,84 iPTH and decrease in 1,25 (OH)₂ vitamin D₃ concentrations unless hemoconcentration is prevented.Abbreviations GFR glomerular filtration rate - PTH parathyroid hormone - iPTH immunoreactive PTH - PAH paraaminohippurate - HCTZ hydrochlorothiazide - FF filtration fraction - cAMP cyclic adenosine monophosphate - Cin inulin clearance - Cpah PAH clearance - Ccr creatinine clearance - CV coefficient of variation - HPLC high performance liquid chromatography - PRA plasma renia activity     

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)₂D₃, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)₂D₃ treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (R_a 0.54 and 4.92 μm) in the presence of control media or media containing 1,25-(OH)₂D₃ or 1,25-(OH)₂D₃ plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A₂ inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)₂D₃ inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)₂D₃ restored cell number to that seen in the control cultures. Inhibition of phospholipase A₂ in the presence of 1,25-(OH)₂D₃ caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)₂D₃ on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)₂D₃ treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)₂D₃ displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A₂ also inhibited the 1,25-(OH)₂D₃-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)₂D₃ effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)₂D₃ mediate their effects through phospholipase A₂, which catalyzes one of the rate-limiting steps in prostaglandin E₂ production. Further downstream, prostaglandin E₂ activates protein kinase A.     

Vitamin D status is not associated with inflammatory cytokine levels during experimental human endotoxaemia

Vitamin D has been shown to modulate innate immune responses in vitro and ex vivo; however, human in‐vivo data are lacking. At high latitudes, seasonal vitamin D deficiency is common due to alternating ultraviolet (UV)‐B radiation exposure. In the present study, we investigated whether levels of 25 hydroxyvitamin D₃ [25(OH)D₃] and its active metabolite 1,25 dihydroxyvitamin D₃ [1,25(OH)₂D₃] are subject to seasonal variation and whether plasma levels of these vitamin D metabolites correlate with the in‐vivo cytokine response during experimental human endotoxaemia [administration of lipopolysaccharide (LPS) in healthy volunteers]. Plasma levels of 25(OH)D₃ and 1,25(OH)₂D₃ were determined in samples obtained just prior to administration of an intravenous bolus of 2 ng/kg LPS (derived from Escherichia coli O:113) in 112 healthy male volunteers. In the same subjects, plasma levels of the inflammatory cytokines tumour necrosis factor (TNF)‐α, interleukin (IL)‐6 and IL‐10 were analysed serially after endotoxin administration. Plasma levels of 1,25(OH)₂D₃, but not 25(OH)D₃, were subject to significant seasonal variation, with lower levels in autumn and winter. 25(OH)D₃ and 1,25(OH)₂D₃ levels did not correlate with plasma cytokine responses. Furthermore, 25(OH)D₃ deficient subjects (< 50 nmol/l) displayed an identical cytokine response compared with sufficient subjects. In conclusion, plasma levels of vitamin D are not correlated with the LPS‐induced TNF, IL‐6 and IL‐10 cytokine response in humans in vivo. These findings question the direct role of vitamin D in modulation of the innate immune response.     

Measurements of the criticalPO2 of renal mitochondria

Summary The criticalPO₂ of isolated mitochondria from the cortex and outer medullary region of the rat kidney was polarographically measured using the O₂-platinum-electrode. With succinate for substrate at 37°C and 25°C mean criticalPO₂-values of 1.54 Torr (SD±0.58) and 1.01 Torr (SD±0.46) were found resp. Using malate for substrate the corresponding mean values were 0.92 Torr (SD±0.28 and 0.65 Torr (SD±0.25). A positive linear relationship between O₂-uptake and criticalPO₂ was observed. The results are compared with data on the O₂-consumption of rat and dog renal cortex in situ and withPO₂-values measured in the same organs. On the basis of the results here presented with regard to the lowestPO₂-values found in the renal cortex of the dog an additional explanation of the flow limitation of the renal O₂-consumption is developed.This work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.     

Disturbed calcium metabolism in subjects with elevated diastolic blood pressure

Summary Essential hypertension has been associated with disturbed calcium metabolism, but the available data are controversial. We measured parameters of calcium metabolism in groups of untreated male subjects (n = 78) with elevated diastolic blood pressure (101 ± 6 mmHg, mean ± SD) and age-matched male subjects (n=79) with low diastolic blood pressure (62 ± 4 mmHg). The participants of the study were drawn from a random population sample. Subjects with high diastolic blood pressure had significantly higher carboxy-terminal parathyroid hormone (PTH) plasma concentrations than controls with low diastolic blood pressure (median 114 vs. 43 pmol/l, P < 0.01). The 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D concentrations were comparable in both groups. Individuals with high diastolic blood pressure had significantly lower total serum calcium (2.41 ± 0.10 vs. 2.47 ± 0.10 mmol/l, mean ± SD; P < 0.01). PTH concentrations were correlated with diastolic pressure (r = –0.39, P < 0.001). The data are compatible with increased parathyroid activity despite unchanged concentrations of vitamin D metabolites in human hypertension.Abbreviations PTH parathyroid hormone - C-PTH carboxy-terminal parathyroid hormone - 1,25(OH)₂D 1,25-di-hydroxyvitamin D - 25(OH)D 25-hydroxyvitamin D     

25-hydroxyvitamin D, 24, 25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D in human cerebrospinal fluid

Summary Samples of CSF and plasma were obtained simultaneously from 46 adult patients who had no endocrine disorders and were undergoing routine diagnostic lumbar puncture because of suspected or proved prolapse of a disc. Concentrations of 25-OHD, 24,25(OH)₂D and 1,25(OH)₂D were measured. The samples were purified by column chromatography and fractionated by HPLC. In the appropriate fractions the vitamin D metabolites were measured by PBA, and cytoreceptor assay. The results were as follows (median, range in brackets): 25-OHD in CSF 8.3 ng/ml (2.0–24.8), in plasma 14.5 ng/ml (7.0–36.0). 24,25(OH)₂D in CSF 1.8 ng/ml (0.3–4.6) and 2.5 ng/ml (0.4–4.7) in plasma. 1.25(OH)₂ D in CSF 25.0 pg/ml (2.2–39.0) and 31.0 pg/ml (10.1–55.0) in plasma. The correlations between plasma and CSF concentrations were as follows: 25-OHDr=0.479 (P<0.001); 24,25(OH)₂Dr=0.815 (P<0.001) and for 1.25(OH)₂Dr=0.497 (P<0.001).Our findings showed vitamin D metabolites to be present in human CSF.Abbreviations Ca Calcium - CSF Cerebrospinal fluid - Vitamin D₃ Cholecalciferol - CPM Counts per min - 24, 25 (OH)₂D 24, 25-dihydroxyvitamin D₃ - 1,25(OH)₂D 1,25-dihydroxyvitamin D₃ - Vitamin D₂ Ergocalciferol - HPLC High-pressure liquid chromatography - 25OHD 25-hydroxyvitamin D₃ - PTH Parathyroid hormone - PBA Protein binding assay - RIA Radioimmunoassay - D-CaBP Vitamin D dependent calcium-binding protein     

Phosphate fluxes in isolated enterocytes from vitamin D replete and vitamin D deficient rats — early effects of calcitriol

In the present work we studied rapid in vitro effects of calcitriol (1,25(OH)₂ vitamin D₃) on the intestinal transport of inorganic phosphate (P_i). Enterocytes from vitamin D replete (D⁺) as well as vitamin D depleted (D^–) rats were isolated mechanically from the duodeno-jejunum. In this model, P_i uptake was a temperature and Na⁺-dependent phenomenon. The in vitro-addition of calcitriol (1 pM) resulted in a significant enhancement of initial P_i uptake rate by enterocytes from D⁺ (P<0.01) and D^– (P<0.05) rats. This effect which was Na⁺-dependent, was observed within the time of 20 min, but not before. A similar effect on P_i uptake rates of D⁺ or D^– enterocytes could be elicited by the in vitro addition of the methyl ester of cis-vaccinic acid (MCVA) which is thought to increase membrane fluidity by modifying the lipid composition of the cell membrane. The stimulatory effect of calcitriol on P_i uptake rate was blunted in the presence of the methyl ester of transvaccinic acid (MTVA) thought to decrease membrane fluidity. Enterocyte P_i efflux rate constant (^o KP_i) remained unchanged in the presence of calcitriol (1 pM). In conclusion, the study demonstrates a rapid in vitro effect of calcitriol on P_i uptake by isolated enterocytes from D⁺ and D^– rats. It suggests, but does not prove, that the hormone may act via an action independent of genomic nuclear activation.     

A computerized histomorphometric study of the effects of intoxication with vitamin D3 or 1,25 (OH)2D3 on growth and dentin production of impeded and unimpeded rat incisors

Summary The effects of Vitamin D₃ and 1,25(OH)₂D₃ intoxication on growth and dentinogenesis were studied in impeded and unimpeded rat incisors. Animals were intoxicated either with Vitamin D₃ (2 mg/kg/day) or 1,25(OH)₂D₃ (400 ng/kg/day) for 19 days. Undecalcified transverse sections were obtained from all lower incisors. The outlines of all dental tissues were traced and fed into a computer which depicted the changes in all variables measured (circumference and cross-sectional area of tooth, pulp and dentin, width of dentin, CEJ [cemento-enamel junction] diameter and maximal labiolingual diameter). Eruption rates, which had been recorded previously every 48 h, were used to compute the physiological age of each tooth site, so that only areas of similar age be compared. Growth. External dimensions of all intoxicated teeth decreased as the experiment progressed in contrast with control incisors which continued to increase in size. This effect was more pronounced in unimpeded incisors and may be due to the inhibitory influence of 1,25(OH)₂D₃ on cellular proliferation at the apical progenitor end. Dentinogenesis. 1,25(OH)₂D₃ intoxication had no significant effect on the rate of eruption and dentin formation in bothimpeded andunimpeded incisors. The same was true for impeded incisors intoxicated with Vitamin D₃ but, both eruption and dentin production rates were impaired in Vitamin D₃ unimpeded incisors.     

Role of 1,25-Dihydroxy Vitamin D3 and Parathyroid Hormone in Urinary Calcium Excretion in Calcium Stone Formers

Purpose

To find out the possible role of 1,25(OH)₂ vitamin D₃ [1,25(OH)₂D₃] and parathyroid hormone (PTH) as intrinsic factors in urinary calcium stone formers (SFs), we investigated their relationship with serum and urinary biochemical parameters.

Materials and Methods

A total of 326 calcium SFs (male: 204, female: 122) were enrolled and underwent outpatient metabolic evaluations including 1,25(OH)₂D₃ and PTH as well as serum and 24-hour urinary biochemical parameters. As control, 163 age- and sex-matched (2:1) individuals (non-SFs) who have never urinary stone episode were included.

Results

1,25(OH)₂D₃ level was positively correlated with urinary calcium excretion (r=0.347, p<0.001). The hypercalciuric group and recurrent SFs had higher serum 1,25(OH)₂D₃ levels than the normocalciuric group (p<0.001) and first SFs (p=0.050). In the adjusted multiple linear regression analysis, serum 1,25(OH)₂D₃ level (β=0.259, p<0.001) and serum PTH level (β=-0.160, p<0.001) were significantly correlated with urinary calcium excretion. The patients in highest tertile of 1,25(OH)₂D₃ had a more than 3.1 fold risk of hypercalciuria than those in the lowest tertile (odds ratio=3.14, 95% confidence interval: 1.431-6.888, p=0.004). No correlation was observed between PTH and 1,25(OH)₂D₃ (R=0.005, p=0.929) in calcium SFs, while a negative correlation was found in controls (R=-0.269, p=0.001).

Conclusion

1,25(OH)₂D₃ was closely correlated with urinary calcium excretion, and high 1,25(OH)₂D₃ levels were detected in the hypercalciuric group and in recurrent SFs. However, 1,25(OH)₂D₃ was not correlated with PTH in calcium SFs. These findings suggest that 1,25(OH)₂D₃ might be important intrinsic factor for altered calcium regulation in SFs.     

Decreased immunosuppressive actions of 1α, 25-dihydroxyvitamin D3 in patients with immune thrombocytopenia

Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease. 1α, 25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] and vitamin D receptor (VDR) play important immune-suppressive roles in immune system. It has been reported that serum 1,25(OH)₂D₃ were lower in ITP patients. In this study, we evaluated local 1,25(OH)₂D₃ level and VDR mRNA expression further, and determined whether 1,25(OH)₂D₃/VDR were correlated with T cell dysfunction in ITP patients. We found that 1,25(OH)₂D₃/VDR levels were decreased in active ITP patients, and 1,25(OH)₂D₃ had significant anti-inflammatory effects on ITP patients, including both anti-proliferation of peripheral blood mononuclear cells (PBMCs) and reversing the abnormal T cells polarization. 1,25(OH)₂D₃ inhibited the differentiation of T helper (Th)1 and Tc1 cells but induced the differentiation of Th2, Tc2 and T regulatory (Treg) cells in ITP patients. However, the percentage of Th17 cells were not affected obviously with 1,25(OH)₂D₃. In addition, 1,25(OH)₂D₃ also suppressed pro-inflammatory cytokines (INF-γ and IL-17A) but promoted anti-inflammatory cytokine (IL-10) secretion in ITP patients. In conclusion, decreased 1,25(OH)₂D₃/VDR might participate in the pathogenesis of ITP, and appropriate supplement of 1,25(OH)₂D₃ may be a promising treatment.