Effect of Intravenous 25OHD Supplementation on Bone Turnover and Inflammation in Prolonged Critically Ill Patients.

Critically ill patients have low circulating 25-hydroxyvitamin D (25OHD), vitamin D binding protein (DBP), and 1,25-dihydroxyvitamin D [1,25(OH)2D]. Low 25OHD is associated with poor outcomes, possibly explained by its effect on bone and immunity. In this prospective, randomized double-blind, placebo-controlled study, we investigated the feasibility of normalizing 25OHD in prolonged (>10 days) critically ill patients and the effects thereof on 1,25(OH)2D, bone metabolism, and innate immunity. Twenty-four patients were included and compared with 24 matched healthy subjects. Patients were randomized to either intravenous bolus of 200 µg 25OHD followed by daily infusion of 15 µg 25OHD for 10 days, or to placebo. Parameters of vitamin D, bone and mineral metabolism, and innate immune function were measured. As safety endpoints, ICU length of stay and mortality were registered. Infusion of 25OHD resulted in a sustained increase of serum 25OHD (from median baseline 9.2 -16.1 ng/ml at day 10), which, however, remained below normal levels. There was no increase in serum 1,25(OH)2D but a slight increase in serum 24,25(OH)2D. Mineral homeostasis, innate immunity and clinical safety endpoints were unaffected. Thus, intravenous 25OHD administration during critical illness increased serum 25OHD concentrations, though less than expected from data in healthy subjects, which suggests illness-induced alterations in 25OHD metabolism and/or increased 25OHD distribution volume. The increased serum 25OHD concentrations were not followed by a rise in 1,25(OH)2D nor were bone metabolism or innate immunity affected, which suggests that low 25OHD and 1,25OHD levels are part of the adaptive response to critical illness.

Impact of a VAP bundle in Belgian intensive care units.

BACKGROUND: In order to decrease the incidence of ventilator-associated pneumonia (VAP) in Belgium, a national campaign for implementing a VAP bundle involving assessment of sedation, cuff pressure control, oral care with chlorhexidine and semirecumbent position, was launched in 2011-2012. This report will document the impact of this campaign. METHODS: On 1 day, once a year from 2010 till 2016, except in 2012, Belgian ICUs were questioned about their ventilated patients. For each of these, data about the application of the bundle and the possible treatment for VAP were recorded. RESULTS: Between 36.6 and 54.8% of the 120 Belgian ICUs participated in the successive surveys. While the characteristics of ventilated patients remained similar throughout the years, the percentage of ventilated patients and especially the duration of ventilation significantly decreased before and after the national VAP bundle campaign. Ventilator care also profoundly changed: Controlling cuff pressure, head positioning above 30° were obtained in more than 90% of cases. Oral care was more frequently performed within a day, using more concentrated solutions of chlorhexidine. Subglottic suctioning also was used but in only 24.7% of the cases in the last years. Regarding the prevalence of VAP, it significantly decreased from 28% of ventilated patients in 2010 to 10.1% in 2016 (p ≤ 0.0001). CONCLUSION: Although a causal relationship cannot be inferred from these data, the successive surveys revealed a potential impact of the VAP bundle campaign on both the respiratory care of ventilated patients and the prevalence of VAP in Belgian ICUs encouraging them to follow the guidelines.

Introducing TOPMAST, the first double-blind randomized clinical trial specifically dedicated to perioperative maintenance fluid therapy in adults.

BACKGROUND: Although prescribed to every patient undergoing surgery, maintenance fluid therapy is a poorly researched part of perioperative fluid therapy. The tonicity of the chosen solutions, could be an important cause of morbidity, with hyponatremia being a potential side effect of hypotonic solutions, where isotonic solution could lead to fluid overload. METHODS: The TOPMAST-trial is an ongoing prospective single-center double-blind randomized trial comparing an isotonic and a hypotonic maintenance fluid strategy during and after surgery in patients undergoing different types of major thoracic surgery. Patients receive NaCl 0.9% in glucose 5% with an added 40 mmol L-1 of potassium chloride or a premixed solution containing 54 mmol L-1 sodium, 55 mmol L-1 chloride and 26 mmol of potassium at a rate of 27 mL per kg of body weight per day. The primary hypothesis is that isotonic maintenance solutions cause a more positive perioperative fluid balance than hypotonic fluids. Different secondary safety endpoints will be explored, especially the effect of the study treatments on the occurrence electrolyte disturbances (e.g. hyponatremia, hyperchloremia) and a set of clinical endpoints. Efficacy endpoints include the need for resuscitation fluids and assessment of renal and hormonal adaptive mechanisms. An anticipated 68 patients will be included between March 2017 and January 2018. DISCUSSION: The study will provide the most comprehensive evaluation of clinically important outcomes associated with the choice of perioperative maintenance fluid therapy.

Feasibility of diaphragm pacing in patients after bilateral lung transplantation.

Recent animal studies and intraoperative studies in humans suggested that phrenic nerve stimulation could attenuate ventilator-induced diaphragm dysfunction. The purpose of the present study is to examine the safety and feasibility of diaphragm pacing during the weaning process after bilateral lung transplantation. Four patients, suffering from chronic pulmonary disease, were included, and diaphragm pacing was evaluated after lung transplantation. Implantation of electrodes at the end of the lung transplant procedure was possible in three of the four patients. In all implanted patients, stimulation of the diaphragm could trigger the ventilator. Implanted electrodes were completely removed by percutaneous retraction after up to 7 days of pacing. Adverse events related to pacing included occurrence of pain. Diaphragm pacing with temporary electrodes, inserted during surgery, is feasible and is able to trigger the ventilator in patients after bilateral lung transplantation. The use of intradiaphragmatic electrodes creates the additional opportunity to monitor the evolution of diaphragm electromyography during the postoperative weaning process.

Critical illness-induced bone loss is related to deficient autophagy and histone hypomethylation.

BACKGROUND: Survivors of critical illness are at increased risk of fractures. This may be due to increased osteoclast formation during critical illness, leading to trabecular bone loss. Such bone loss has also been observed in Paget's disease, and has been related to deficient autophagy. Deficient autophagy has also been documented in vital organs and skeletal muscle of critically ill patients. The objective of this study was to investigate whether deficient autophagy can be linked to critical illness-induced bone loss. METHODS: Osteoclasts grown in vitro and their precursor cells isolated from peripheral blood of critically ill patients and from matched healthy volunteers were analysed for the expression of autophagy genes (SQSTM1, Atg3 and Atg7), and proteins (p62, Atg-5, and microtubule-associated protein light chain 3-II (LC3-II)) and for autophagy and epigenetic signalling factors via PCR arrays and were treated with the autophagy inducer rapamycin. The effect of rapamycin was also investigated at the tissue level in an in vivo rabbit model of critical illness. RESULTS: Many more osteoclasts formed in vitro from the blood precursor cells isolated from critically ill patients, which accumulated p62, and displayed reduced expression of Atg5, Atg7, and LC3-II compared to healthy controls, suggesting deficient autophagy, whilst addition of rapamycin reduced osteoclast formation. PCR arrays revealed a down-regulation of histone methyltransferases coupled with an up-regulation of negative regulators of autophagy. Critically ill rabbits displayed a reduction in trabecular and cortical bone, which was rescued with rapamycin. CONCLUSIONS: Deficient autophagy in osteoclasts and their blood precursor cells at least partially explained aberrant osteoclast formation during critical illness and was linked to global histone hypomethylation. Treatment with the autophagy activator Rapamycin reduced patient osteoclast formation in vitro and reduced the amount of bone loss in critically ill rabbits in vivo. These findings may help to develop novel therapeutic targets to prevent critical illness-induced bone loss.

Acute outcomes and 1-year mortality of intensive care unit-acquired weakness. A cohort study and propensity-matched analysis.

RATIONALE: Intensive care unit (ICU)-acquired weakness is a frequent complication of critical illness. It is unclear whether it is a marker or mediator of poor outcomes. OBJECTIVES: To determine acute outcomes, 1-year mortality, and costs of ICU-acquired weakness among long-stay (≥8 d) ICU patients and to assess the impact of recovery of weakness at ICU discharge. METHODS: Data were prospectively collected during a randomized controlled trial. Impact of weakness on outcomes and costs was analyzed with a one-to-one propensity-score-matching for baseline characteristics, illness severity, and risk factor exposure before assessment. Among weak patients, impact of persistent weakness at ICU discharge on risk of death after 1 year was examined with multivariable Cox proportional hazards analysis. MEASUREMENTS AND MAIN RESULTS: A total of 78.6% were admitted to the surgical ICU; 227 of 415 (55%) long-stay assessable ICU patients were weak; 122 weak patients were matched to 122 not-weak patients. As compared with matched not-weak patients, weak patients had a lower likelihood for live weaning from mechanical ventilation (hazard ratio [HR], 0.709 [0.549-0.888]; P = 0.009), live ICU (HR, 0.698 [0.553-0.861]; P = 0.008) and hospital discharge (HR, 0.680 [0.514-0.871]; P = 0.007). In-hospital costs per patient (+30.5%, +5,443 Euro per patient; P = 0.04) and 1-year mortality (30.6% vs. 17.2%; P = 0.015) were also higher. The 105 of 227 (46%) weak patients not matchable to not-weak patients had even worse prognosis and higher costs. The 1-year risk of death was further increased if weakness persisted and was more severe as compared with recovery of weakness at ICU discharge (P < 0.001). CONCLUSIONS: After careful matching the data suggest that ICU-acquired weakness worsens acute morbidity and increases healthcare-related costs and 1-year mortality. Persistence and severity of weakness at ICU discharge further increased 1-year mortality. Clinical trial registered with www.clinicaltrials.gov (NCT 00512122).

Humanized culture of periosteal progenitors in allogeneic serum enhances osteogenic differentiation and in vivo bone formation.

The translation of stem cell-based regenerative solutions from the laboratory to the clinic is often hindered by the culture conditions used to expand cell populations. Although fetal bovine serum (FBS) is widely used, regulatory bodies and safety concerns encourage alternative, xeno-free culturing practices. In an attempt to apply this approach to a bone-forming combination product of human periosteal progenitors (human periosteum derived cells) on a clinically used calcium phosphate carrier, FBS was substituted for human allogeneic serum (hAS) during cell expansion. It was found that cell proliferation was increased in hAS along with an apparent commitment to the osteogenic lineage, indicated by enhanced Runx2 expression, as well as alkaline phosphatase activity and matrix mineralization. Following analysis of signaling pathways, it was found that interferon-mediated signaling was downregulated, whereas JAK-STAT signaling was upregulated. STAT3 phosphorylation was enhanced in hAS-cultured human periosteum derived cells, inhibition of which ablated the proliferative effect of hAS. Furthermore, following in vivo implantation of hAS-cultured cells on NuOss scaffolds, enhanced bone formation was observed compared with FBS (71% increase, p < .001). Interestingly, the de novo-formed bone appeared to have a higher ratio of immature regions to mature regions, indicating that after 8 weeks implantation, tissue-formation processes were continuing. Integration of the implant with the environment appeared to be altered, with a decrease in calcium phosphate grain size and surface area, indicative of accelerated resorption. This study highlights the advantages of using humanized culture conditions for the expansion of human periosteal progenitors intended for bone regeneration.

Effect of tolerating macronutrient deficit on the development of intensive-care unit acquired weakness: a subanalysis of the EPaNIC trial.

BACKGROUND: Patients who are critically ill can develop so-called intensive-care unit acquired weakness, which delays rehabilitation. Reduced muscle mass, quality, or both might have a role. The Early Parenteral Nutrition Completing Enteral Nutrition in Adult Critically Ill Patients (EPaNIC) trial (registered with ClinicalTrials.gov, number NCT00512122) showed that tolerating macronutrient deficit for 1 week in intensive-care units (late parenteral nutrition [PN]) accelerated recovery compared with early PN. The role of weakness was unclear. Our aim was to assess whether late PN and early PN differentially affect muscle weakness and autophagic quality control of myofibres. METHODS: In this prospectively planned subanalysis of the EPaNIC trial, weakness (MRC sum score) was assessed in 600 awake, cooperative patients. Skeletal muscle biopsies, harvested from 122 patients 8 days after randomisation and from 20 matched healthy controls, were studied for autophagy and atrophy. We determined the significance of differences with Mann-Whitney U, Median, Kruskal-Wallis, or χ(2) (exact) tests, as appropriate. FINDINGS: With late PN, 105 (34%) of 305 patients had weakness on first assessment (median day 9 post-randomisation) compared with 127 (43%) of 295 patients given early PN (absolute difference -9%, 95% CI -16 to -1; p=0·030). Weakness recovered faster with late PN than with early PN (p=0·021). Myofibre cross-sectional area was less and density was lower in critically ill patients than in healthy controls, similarly with early PN and late PN. The LC3 (microtubule-associated protein light chain 3) II to LC3I ratio, related to autophagosome formation, was higher in patients given late PN than early PN (p=0·026), reaching values almost double those in the healthy control group (p=0·0016), and coinciding with less ubiquitin staining (p=0·019). A higher LC3II to LC3I ratio was independently associated with less weakness (p=0·047). Expression of mRNA encoding contractile myofibrillary proteins was lower and E3-ligase expression higher in muscle biopsies from patients than in control participants (p≤0·0006), but was unaffected by nutrition. INTERPRETATION: Tolerating a substantial macronutrient deficit early during critical illness did not affect muscle wasting, but allowed more efficient activation of autophagic quality control of myofibres and reduced weakness. FUNDING: UZ Leuven, Research Foundation-Flanders, the Flemish Government, and the European Research Council.

Does the surface-treated AN69 membrane prolong filter survival in CRRT without anticoagulation?

PURPOSE: The need for continuous anticoagulation remains a significant drawback in continuous renal replacement therapy (CRRT), especially in patients with increased bleeding risk. Polyethyleneimine treatment of the AN69 membrane (AN69ST) reduces thrombogenicity through decreased contact activation and promotion of heparin binding. The aim of this study is to evaluate whether this membrane prolongs filter survival in CRRT without anticoagulation. METHODS: A single-center, prospective, randomized, double-blind controlled trial with cross-over design comparing filter survival with the AN69ST membrane and the original AN69 membrane in 39 patients treated with continuous venovenous hemofiltraton (CVVH) without additional heparin. RESULTS: Filter survival with the AN69ST membrane (n = 75) was 14.2 ± 8.2 h, which is not significantly different from the 13.3 ± 10.3 h for the original AN69 membrane (n = 76; p = 0.59). Limiting the analysis to those treatments that were interrupted for filter clotting yielded similar results: 14.4 ± 8.2 h for the AN69 ST membrane (n = 62) versus 14.1 ± 7.5 h for the original AN69 membrane (n = 56) (p = 0.93). CONCLUSIONS: Compared with the original AN69 membrane, the surface-treated AN69ST membrane does not prolong filter survival during CVVH without systemic anticoagulation and with the CRRT settings used in this study.

Critical illness-related bone loss is associated with osteoclastic and angiogenic abnormalities.

Critically ill patients are at increased risk of fractures during rehabilitation, and can experience impaired healing of traumatic and surgical bone fractures. In addition, markers of bone resorption are markedly increased in critically ill patients, while markers of bone formation are decreased. In the current study, we have directly investigated the effect of critical illness on bone metabolism and repair. In a human in vitro model of critical illness, Fluorescence-activated cell sorting (FACS) analysis revealed an increase in circulating CD14+/CD11b+ osteoclast precursors in critically ill patient peripheral blood compared to healthy controls. In addition, the formation of osteoclasts was increased in patient peripheral blood mononuclear cell (PBMC) cultures compared to healthy controls, both in the presence and absence of osteoclastogenic factors receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). Culturing PBMCs with 10% critically ill patient serum further increased osteoclast formation and activity in patient PBMCs only, and neutralization studies revealed that immunoglobulin G (IgG) antibody signaling through the immunoreceptor Fc receptor common γ chain III (FcRγIII) played an important role. When analyzing bone formation, no differences in osteogenic differentiation were observed using human periosteal-derived cells (hPDCs) treated with patient serum in vitro, but a decrease in the expression of vascular endothelial growth factor receptor 1 (VEGF-R1) suggested impaired vascularization. This was confirmed using serum-treated hPDCs implanted onto calcium phosphate scaffolds in a murine in vivo model of bone formation, where decreased vascularization and increased osteoclast activity led to a decrease in bone formation in scaffolds with patient serum-treated hPDCs. Together, these findings may help to define novel therapeutic targets to prevent bone loss and optimize fracture healing in critically ill patients.

Early versus late parenteral nutrition in critically ill adults.

BACKGROUND: Controversy exists about the timing of the initiation of parenteral nutrition in critically ill adults in whom caloric targets cannot be met by enteral nutrition alone. METHODS: In this randomized, multicenter trial, we compared early initiation of parenteral nutrition (European guidelines) with late initiation (American and Canadian guidelines) in adults in the intensive care unit (ICU) to supplement insufficient enteral nutrition. In 2312 patients, parenteral nutrition was initiated within 48 hours after ICU admission (early-initiation group), whereas in 2328 patients, parenteral nutrition was not initiated before day 8 (late-initiation group). A protocol for the early initiation of enteral nutrition was applied to both groups, and insulin was infused to achieve normoglycemia. RESULTS: Patients in the late-initiation group had a relative increase of 6.3% in the likelihood of being discharged alive earlier from the ICU (hazard ratio, 1.06; 95% confidence interval [CI], 1.00 to 1.13; P=0.04) and from the hospital (hazard ratio, 1.06; 95% CI, 1.00 to 1.13; P=0.04), without evidence of decreased functional status at hospital discharge. Rates of death in the ICU and in the hospital and rates of survival at 90 days were similar in the two groups. Patients in the late-initiation group, as compared with the early-initiation group, had fewer ICU infections (22.8% vs. 26.2%, P=0.008) and a lower incidence of cholestasis (P<0.001). The late-initiation group had a relative reduction of 9.7% in the proportion of patients requiring more than 2 days of mechanical ventilation (P=0.006), a median reduction of 3 days in the duration of renal-replacement therapy (P=0.008), and a mean reduction in health care costs of €1,110 (about $1,600) (P=0.04). CONCLUSIONS: Late initiation of parenteral nutrition was associated with faster recovery and fewer complications, as compared with early initiation. (Funded by the Methusalem program of the Flemish government and others; EPaNIC ClinicalTrials.gov number, NCT00512122.).

Intensive insulin therapy for patients in paediatric intensive care: a prospective, randomised controlled study.

BACKGROUND: Critically ill infants and children often develop hyperglycaemia, which is associated with adverse outcome; however, whether lowering blood glucose concentrations to age-adjusted normal fasting values improves outcome is unknown. We investigated the effect of targeting age-adjusted normoglycaemia with insulin infusion in critically ill infants and children on outcome. METHODS: In a prospective, randomised controlled study, we enrolled 700 critically ill patients, 317 infants (aged <1 year) and 383 children (aged >or=1 year), who were admitted to the paediatric intensive care unit (PICU) of the University Hospital of Leuven, Belgium. Patients were randomly assigned by blinded envelopes to target blood glucose concentrations of 2.8-4.4 mmol/L in infants and 3.9-5.6 mmol/L in children with insulin infusion throughout PICU stay (intensive group [n=349]), or to insulin infusion only to prevent blood glucose from exceeding 11.9 mmol/L (conventional group [n=351]). Patients and laboratory staff were blinded to treatment allocation. Primary endpoints were duration of PICU stay and inflammation. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00214916. FINDINGS: Mean blood glucose concentrations were lower in the intensive group than in the conventional group (infants: 4.8 [SD 1.2] mmol/L vs 6.4 [1.2] mmol/L, p<0.0001; children: 5.3 [1.1] mmol/L vs 8.2 [3.3] mmol/L, p<0.0001). Hypoglycaemia (defined as blood glucose median) stay in PICU was 132 (38%) in the intensive group versus 165 (47%) in the conventional group (p=0.013). Nine (3%) patients died in the intensively treated group versus 20 (6%) in the conventional group (p=0.038). INTERPRETATION: Targeting of blood glucose concentrations to age-adjusted normal fasting concentrations improved short-term outcome of patients in PICU. The effect on long-term survival, morbidity, and neurocognitive development needs to be investigated. FUNDING: Research Foundation (Belgium); Research Fund of the University of Leuven (Belgium) and the EU Information Society Technologies Integrated project "CLINICIP"; and Institute for Science and Technology (Belgium).

Duodenal calcium absorption in dexamethasone-treated mice: functional and molecular aspects.

Reduced intestinal calcium absorption may be part of the pathogenesis of glucocorticoid-induced osteoporosis. 1,25(OH)2D3 is the major regulator of the expression of the active duodenal calcium absorption genes: TRPV6 (influx), calbindin-D9K (intracellular transfer) and PMCA1b (extrusion). We investigated the influence of dexamethasone (5 days: 2 mg/kg bw) on calcium absorption in vivo and on the expression of intestinal and renal calcium transporters in calcium-deprived mice. Total and free 1,25(OH)2D3-concentrations were halved, in line with decreased 25(OH)D3-1-alpha-hydroxylase and increased 24-hydroxylase expression. Nevertheless, no difference in duodenal or renal calcium transporter expression pattern could be detected between vehicle and dexamethasone-treated mice. Accordingly, dexamethasone did not affect in vivo calcium absorption. By contrast, increased calcemia and collagen C-terminal telopeptide levels reflected increased bone resorption. Decreased osteocalcin levels suggested impaired bone formation. Hence, short-term glucocorticoid excess in young animals affected bone metabolism without detectable changes in intestinal or renal calcium handling.

Vitamin D resistance.

Vitamin D is a secosteroid of nutritional origin but can also be generated in the skin by ultraviolet light. After two hydroxylations 1,25-(OH)2 vitamin D avidly binds and activates the vitamin D receptor (VDR), a nuclear transcription factor, hereby regulating a large number of genes. The generation of VDR deficient mice has expanded the knowledge on vitamin D from a calcium-regulating hormone to a humoral factor with extensive actions. The effects of the vitamin D system on calcium and bone homeostasis are largely mediated by promoting active intestinal calcium transport via the induction of the epithelial calcium channel TRPV6. Although VDR is redundant in bone, it may regulate the differentiation and function of several bone cells. In skin, VDR expression in keratinocytes is essential in a ligand-independent manner for the maintenance of the normal hair cycle. Therefore, VDR but not vitamin D deficiency results in alopecia. Moreover, 1,25-(OH)2 vitamin D impairs the proliferation not only of keratinocytes but also of many cell types by regulating the expression of cell cycle genes, leading to a G1 cell cycle arrest. In addition, VDR inactivation in mice results in high renin hypertension, cardiac hypertrophy and thrombogenesis. Finally, a dual effect of vitamin D was observed in the immune system where it stimulates the innate immune system while tapering down excessive activation of the acquired immune system. Taken together, the vitamin D endocrine system not only regulates calcium homeostasis but affects several systems mainly by altering gene expression but also by ligand-independent actions.

Disorders of calcium homeostasis.

To ensure a multitude of essential cellular functions, the extracellular concentration of calcium is maintained within a narrow physiological range. This depends on integrated regulation of calcium fluxes with respect to the intestine, kidneys and bone. The precise regulation of serum calcium is controlled by calcium itself, through a calcium receptor and several hormones, the most important of which are parathyroid hormone and 1,25(OH)(2) vitamin D. This balance can be disturbed by mutations in the calcium-sensing receptor, inappropriately high or low levels of parathyroid hormone, resistance to parathyroid hormone effects, insufficient intake or production of 1,25(OH)(2) vitamin D and inactivation of the vitamin D receptor. Mineral homeostasis is moreover influenced by many other systemic factors (e.g. sex steroid, thyroid and glucocorticoid hormones) or humoral factors (e.g. cytokines and growth factors). A specific example is the major abnormalities of mineral homeostasis in case of malignancy by excessive production of parathyroid hormone-related peptide resulting in hypercalcaemia. Several new drugs have been developed based on factors in this axis, including calcimimetics, calcilytics, vitamin D analogues and parathyroid hormone-related peptide inhibitors.

Pregnancy in mice lacking the vitamin D receptor: normal maternal skeletal response, but fetal hypomineralization rescued by maternal calcium supplementation.

Fetal mineralization appears to be driven by the pregnancy-induced stimulation of intestinal Ca absorption. We thus hypothesized that mineralization would be impaired in fetuses of mice that lack the vitamin D receptor (VDR). Here we report on the maternal response to pregnancy, and the fetal mineralization, in mice with a homozygous disruption of the VDR gene (VDR-/-) mated with wild-type (wt) males. We found that VDR-/- mice show mild hypocalcemia, clear rickets and osteomalacia on bone histomorphometry, lower cortical bone density on quantitative tomography, and reduced concentrations of calbindin-D9k (CaBP-D9k) in duodenal mucosa and kidney. The skeletal response to pregnancy was comparable in wt and VDR-/- mice; duodenal CaBP-D9k concentrations increased during pregnancy in VDR-/- as in wt mice, but remained 40% lower than in wt mice. We confirmed our hypothesis that mineralization is defective in d18.5 VDR+/- fetuses of VDR-/- mice, both by whole-body Ca determination and histomorphometric evaluation; the number of osteoclastic cells in bone was increased. The fetuses were hypercalcemic and had a 5-fold increase in circulating 1,25(OH)2D3. We then studied pregnancies in VDR-/- females, mated with wt males, fed a high Ca/P/lactose rescue diet during pregnancy. The rescue diet normalized the mineralization, the number of osteoclastic cells, and plasma Ca and 1,25(OH)2D3 concentrations in the fetuses. We interpret the data as evidence that, to ensure normal fetal mineralization, the maternal VDR-dependent intestinal Ca absorption can be substituted by passive Ca absorption entrained by a higher Ca intake. Alternatively or additionally, elevated 1,25(OH)2D3 in utero may disturb bone development.