Urinary Vitamin D Binding Protein and KIM-1 Are Potent New Biomarkers of Major Adverse Renal Events in Patients Undergoing Coronary Angiography.

BACKGROUND: Vitamin-D-binding protein (VDBP) is a low molecular weight protein that is filtered through the glomerulus as a 25-(OH) vitamin D 3/VDBP complex. In the normal kidney VDBP is reabsorbed and catabolized by proximal tubule epithelial cells reducing the urinary excretion to trace amounts. Acute tubular injury is expected to result in urinary VDBP loss. The purpose of our study was to explore the potential role of urinary VDBP as a biomarker of an acute renal damage. METHOD: We included 314 patients with diabetes mellitus or mild renal impairment undergoing coronary angiography and collected blood and urine before and 24 hours after the CM application. Patients were followed for 90 days for the composite endpoint major adverse renal events (MARE: need for dialysis, doubling of serum creatinine after 90 days, unplanned emergency rehospitalization or death). RESULTS: Increased urine VDBP concentration 24 hours after contrast media exposure was predictive for dialysis need (no dialysis: 113.06 ± 299.61 ng/ml, n = 303; need for dialysis: 613.07 ± 700.45 ng/ml, n = 11, Mean ± SD, p<0.001), death (no death during follow-up: 121.41 ± 324.45 ng/ml, n = 306; death during follow-up: 522.01 ± 521.86 ng/ml, n = 8; Mean ± SD, p<0.003) and MARE (no MARE: 112.08 ± 302.00 ng/ml, n = 298; MARE: 506.16 ± 624.61 ng/ml, n = 16, Mean ± SD, p<0.001) during the follow-up of 90 days after contrast media exposure. Correction of urine VDBP concentrations for creatinine excretion confirmed its predictive value and was consistent with increased levels of urinary Kidney Injury Molecule-1 (KIM-1) and baseline plasma creatinine in patients with above mentioned complications. The impact of urinary VDBP and KIM-1 on MARE was independent of known CIN risk factors such as anemia, preexisting renal failure, preexisting heart failure, and diabetes. CONCLUSIONS: Urinary VDBP is a promising novel biomarker of major contrast induced nephropathy-associated events 90 days after contrast media exposure.

Quantitation of 25-OH-Vitamin-D2 and 25-OH-Vitamin-D3 in Urine Using LC-MS/MS.

Patients with significant proteinuria represent a unique population with respect to vitamin D status due to the urinary losses of vitamin D-binding protein (DBP) to which >99 % of circulating 25-hydroxy vitamin D (25(OH)D) is bound. Low serum concentrations of 25(OH)D have been found in children and adults with nephrotic syndrome (NS). However, previously described assays developed to quantify the magnitude of urinary loss are technically challenging. This chapter describes a simple and sensitive method to quantify 25(OH)D2 and 25(OH)D3 in urine specimens in a single analytical LC-MS/MS analysis. This assay is more sensitive than previously described radioimmunoassays and offers the ability to quantitate both forms of 25-hydroxy vitamin D. The assay involves no chemical derivitization, has a linear measurement range of 20-1500 pg/mL and displays imprecision (CVs) below 7 % at various concentrations across the analytical measurement range.

Analysis of urinary vitamin D3 metabolites by liquid chromatography/tandem mass spectrometry with ESI-enhancing and stable isotope-coded derivatization.

The determination of the urinary vitamin D3 metabolites might prove helpful in the assessment of the vitamin D status. We developed a method for the determination of trace vitamin D3 metabolites, 25-hydroxyvitamin D3 [25(OH)D3] and 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], in urine using liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) combined with derivatization using an ESI-enhancing reagent, 4-(4'-dimethylaminophenyl)-1,2,4-triazoline-3,5-dione (DAPTAD), and its isotope-coded analogue, (2)H4-DAPTAD (d-DAPTAD). The urine samples were treated with ß-glucuronidase, purified with an Oasis hydrophilic-lipophilic balanced (HLB) cartridge, and then subjected to the derivatization. The DAPTAD derivatization enabled the highly sensitive detection (detection limit, 0.25 fmol on the column), and the use of d-DAPTAD significantly improved the assay precision [the intra- (n = 5) and inter-assay (n = 3) relative standard deviations did not exceed 9.5%]. The method was successfully applied to urine sample analyses and detected the increases of the urinary 25(OH)D3 and 24,25(OH)2D3 levels due to vitamin D3 administration.

Dietary resistant starch prevents urinary excretion of 25-hydroxycholecalciferol and vitamin D-binding protein in type 1 diabetic rats.

Diabetes is a rapidly growing epidemic affecting millions of Americans and has been implicated in a number of devastating secondary complications. We previously demonstrated that type 2 diabetic rats exhibit vitamin D deficiency due to aberrant megalin-mediated endocytosis and excessive urinary excretion of 25-hydroxycholecalciferol (25D3) and vitamin D-binding protein (DBP). Here, we examined whether a model of type 1 diabetes [T1D; streptozotocin (STZ)-treated Sprague-Dawley rats] would similarly excrete abnormally high concentrations of 25D3 and DBP due to renal damage and compromised expression of megalin and its endocytic partner, disabled-2 (Dab2). Moreover, we tested whether feeding diabetic rats starch that is resistant to digestion could alleviate these abnormalities. Control (n = 12) rats were fed a standard, semipurified diet (AIN-93G) containing 55% total dietary starch and STZ-treated rats were fed the AIN-93G diet (n = 12) or a diet containing 55% high-amylose maize that is partially resistant to digestion [20% total dietary resistant starch (RS); n = 12] for 2 and 5 wk. The RS diet attenuated weight loss and polyuria in STZ-treated rats. Histology and immunohistochemistry revealed that dietary RS also attenuated the loss of Dab2 expression in renal proximal tubules. Moreover, urinary concentrations of both 25D3 and DBP were elevated ∼10-fold in STZ-treated rats (5 wk post STZ injection), which was virtually prevented by the RS. We also observed a ∼1.5-fold increase in megalin mRNA expression in STZ-treated rats, which was attenuated by feeding rats the RS diet for 2 wk. Taken together, these studies indicate that consumption of low-glycemic carbohydrates can attenuate disruption of vitamin D homeostasis in T1D through the rescue of megalin-mediated endocytosis in the kidney.

Evidence in favor of a severely impaired net intestinal calcium absorption in patients with (early-stage) chronic kidney disease.

INTRODUCTION: Calcium and phosphorus are essential to many vital physiological processes. Little is known about the net and fractional intestinal absorption of calcium and phosphorus in patients with chronic kidney disease (CKD) and their clinical and hormonal determinants. METHODS: Blood and 24-hour urine samples were collected in 20 healthy volunteers (HV) and 72 stable CKD stage 1-4 patients and analyzed for parameters of mineral metabolism including calcidiol, calcitriol, and parathyroid hormone (PTH). Dietary intake was assessed by dietary history. RESULTS: The 24-hour urinary calcium excretion, as opposed to the phosphorus excretion, showed a stepwise decrease across CKD stages (median of 219, 84, 40, and 22 mg/day in HV and patients with CKD stages 1-2, 3 and 4, respectively). Younger age, high serum calcitriol, and high estimated GFR were associated with a high 24-hour urinary calcium excretion. High serum calcitriol levels and dietary phosphorus intake were associated with a high 24-hour urinary phosphorus excretion. The fractional intestinal calcium absorption, as estimated by the urinary-to-ingested calcium ratio, decreased across CKD stages. CONCLUSIONS: The 24-hour urinary excretion of calcium, as opposed to phosphorus, is markedly decreased in CKD, even in early-stage disease. This is partly explained by low calcitriol levels and older age. Assuming a neutral calcium balance at the time of urine collection, we infer that net intestinal calcium absorption may be severely impaired in CKD.

Changes in bone mineral density and bone metabolism markers in premenopausal women with multiple sclerosis and the relationship to clinical variables.

Bone mineral density (BMD) is affected in young adults with multiple sclerosis (MS), which leads to disabling disease. We aimed to show changes that were independent of immobilization by measuring BMD and laboratory markers of bone metabolism in mobile MS patients. We compared a total of 52 premenopausal female patients with relapsing-remitting multiple sclerosis (RRMS) to 41 women of similar age who had no risk factors for osteoporosis. The lumbar and femur BMD were measured using the dual energy X-ray absorptiometry (DXA) method. The urine concentration of serum 25-hydroxycholecalciferol (25-OH vit D(3)), and pyridinoline and deoxypyridinoline were also measured. The concentration of serum osteocalcin was measured to determine the speed of bone metabolism. The mean age of patients (+/- standard deviation [SD]) was 36.1+/-7.4. The average Expanded Disability Status Scale (EDSS) score was 2.2+/-1.8. The concentration of 25-OH vit D(3) and osteocalcin was lower, whereas the concentration of parathyroid hormone (PTH), alkaline phosphatase (ALP), pyridinoline and deoxypyridinoline was higher in the patient group. In the patient group, lumbar 2-4 BMD, T score and Z score and femur neck and trochantor BMD, T score and Z score were significantly lower than in the control group. There was a significant negative relationship between: the disease period and L 2-4 BMD, T score and Z scores; and the femoral neck BMD, T score and Z scores. There was a significant relationship between the total Functional Independence Measure score and the femoral neck, femoral trochanter BMD, T score, and Z score. There was a significant negative relationship between the average EDSS, L 2-4 and all the DXA measurements obtained from the femur. There was a significant relationship between the 25-OH vit D(3) concentration and L 2-4 T score and Z score from the DXA measurements obtained from the femur. There were no significant relationships between osteocalcin, pyridinoline, deoxypyridinoline levels and the BMD measurements. Therefore, the duration of the disease and decrease in functional capacity are the main factors that affect BMD in MS. Apart from the decrease in functional capacity, 25-OH vit D(3) deficiency and secondary PTH increase contribute to the BDM changes observed in MS.

Plasma 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol concentrations are decreased in hind limb unloaded Dahl salt-sensitive female rats.

Plasma 1,25-dihydroxyvitamin D (1,25-(OH)(2)D) concentration was shown to decrease during bed rest in several studies when baseline plasma 25-hydroxyvitamin D (25-OHD) concentration was sub-optimal. Dahl salt-sensitive female (S) rats, but not Dahl salt-resistant female (R) rats, demonstrated a 50% decrease in plasma 1,25-dihydroxycholecalciferol (1,25-(OH)(2)D(3)) concentration after 28 days of hind limb unloading (HU, disuse model) during low salt intake (0.3%). We tested the vitamin D endocrine system response of female S rats to hind limb unloading during high salt intake (2%, twice that of standard rat chow to mimic salt intake in the USA). Hind limb unloading resulted in lower plasma 25-OHD(3) concentrations in S-HU rats than in R-HU rats (P<0.05) and greater urinary loss of 25-OHD(3) by S-HU rats than by S rats (P<0.05). Plasma 1,25-(OH)(2)D(3) concentration of S-HU rats was half that of S rats, but was unchanged in R-HU rats. The association of low plasma 25-OHD concentration with decrease in plasma 1,25-(OH)(2)D concentration of hind limb unloaded rats and of bed rest participants (published studies) suggests that low vitamin D status might be a risk factor for decrease in plasma vitamin D hormone concentration during long-term immobilization or bed rest.

Fibroblast growth factor 23 is increased in calcium nephrolithiasis with hypophosphatemia and renal phosphate leak.

CONTEXT: Nephrolithiasis affects about 10% of the population in industrialized countries, with calcium salts composing more than 80% of renal stones. A significant percentage of patients with calcium nephrolithiasis and normal parathyroid function show hypophosphatemia and reduced renal phosphate reabsorption (i.e. a renal phosphate leak). OBJECTIVES: The objective of the study was to compare serum levels of fibroblast growth factor 23 (FGF23), a regulator of phosphate homeostasis, in 110 recurrent stone formers with or without renal phosphate leak, six patients affected by X-linked hypophosphatemic rickets, five patients affected by oncogenic osteomalacia, and 60 unrelated healthy controls. DESIGN: This was a prospective interventional study. METHODS: Renal phosphate leak was identified based on the occurrence of idiopathic hypophosphatemia [serum phosphate concentration < 2.50 mg/dl (<0.80 mmol/liter)] and reduced renal threshold phosphate concentration [<2.2 mg/liter (<0.70 mmol/liter)]. RESULTS: In 22 stone formers with renal phosphate leak, serum FGF23 concentration was significantly higher as compared with 88 stone formers without renal phosphate leak and with controls [83.3 (65.6-101.1) vs. 32.1 (26.8-37.4) and 24.5 (19.8-29.1) reference units (RU)/ml, respectively]. Stone formers with renal phosphate leak showed lower FGF23, compared with patients with oncogenic osteomalacia and X-linked hypophosphatemic rickets [572.3 (235.9-908.7) RU/ml]. Among stone formers and controls, serum FGF23 concentration displayed a strong inverse association with serum phosphate (r = -0.784, P = 0.009) and the rate of tubular phosphate reabsorption (r = -0.791, P = 0.008). CONCLUSIONS: In our study population, renal phosphate leak affected 20% of stone formers and was strongly associated with increased serum FGF23 concentration.

[Neonatal vitamin D status and calcium-phosphorus homeostasis in the third week of life]. / Zasoby ustrojowe witaminy D a homeostaza wapniowo-fosforowa u noworodków donoszonych w 3 tygodniu zycia.

AIM: Assessment of vitamin D status and calcium -phosphorus homeostasis in term newborns before routine supplementation. MATERIAL AND METHOD: Calcidiol (25OHD), calcium, phosphorus and alkaline phosphatase in serum and Ca (urine)/creatinine (urine) ratio (mg/mg), P (urine)/creatinine (urine) ratio (mg/mg) and tubular phosphate reabsorption rate (TRP= [1-(P(urine) / P(serum). creatinine serum/urine)].100%) in 3rd week of life in 56 appropriate for gestational age term neonates was measured. First group contains 35 newborns (62.5%) with normal 25OHD values and second group 21 newborns (37.5%) with hypovitaminosis D (25OHD < 11 ng/ml). RESULT: Mean 25OHD concentration was 15.23 ng/ml + 8.57 ng/ml. Maternal vitamin D supplementation (10 ug/day) for more than 4 months of pregnancy was similar in both groups (55.9% vs. 52.4%) (p>0.05). There were 51.43% breastfed newborns in group one and 85.71% in group two (p=0.009). Median 25OHD concentration in breastfed newborns was 11.2 ng/ml and 18.5 ng/ml in formula fed babies (p=0.017). There were no statistical differences between groups in calcium (2.44 vs. 2.41 mmol/l), phosphorus (2.27 vs. 2.22 mmol/l) and alkaline phosphatase (261 vs. 266 U/L) blood concentration and Ca (urine)/creatinine (urine) ratio (0,34 vs. 0,25mg/mg) and TRP (86% vs. 88%) (p>0.05). The P (urine) /creatinine (urine) ratio in the first group was 2.3mg/mg and 1.42 mg/mg in the second group (p=0.048). CONCLUSIONS: Neonatal vitamin D stores in the 3rd week of life are not more dependent on maternal vitamin D supplementation during pregnancy. Breastfed infants are at greater risk of hypovitaminosis D than formula fed infants, therefore earlier vitamin D supply should be considered. The hypovitaminosis D has no influence on basic parameters of Ca-P homeostasis in the 3rd week of life.

Mechanisms for the reduction of 24,25-dihydroxyvitamin D3 levels and bone mass in 24-hydroxylase transgenic rats.

24-Hydroxylase (CYP24) is an enzyme distributed in the target tissues of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Two functions for this enzyme have been reported: One is production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and the other is inactivation of 1alpha,25(OH)2D3. To elucidate other physiologic roles of CYP24 in vivo, we previously generated rats that constitutively express the CYP24 gene. These transgenic (Tg) rats developed unexpected phenotypes, such as low plasma levels of 24,25(OH)2D3, lipidemia, and albuminuria. In this study, we elucidated the mechanisms for inducing low plasma 24,25(OH)2D3 levels and bone loss. Tg rats excreted massive amounts of vitamin D binding protein (DBP), which coincided with the loss of albumin. In Tg rats, the renal expression pattern of megalin, which serves as an endocytotic receptor responsible for the reuptake of urinary proteins such as DBP and albumin, was identical to that of the wild-type rats. Excreted albumin appeared to compete for the binding and reabsorption of the DBP-25-hydroxyvitamin D3 [25(OH)D3] complex with megalin, resulting in a loss of 25(OH)D3 into the urine and subsequent reduction of plasma 24,25(OH)2D3. In this prominent rat model of nephritis, supplementation of 25(OH)D3 was effective in preventing bone loss in an early stage of renal insufficiency.

Cubilin dysfunction causes abnormal metabolism of the steroid hormone 25(OH) vitamin D(3).

Steroid hormones are central regulators of a variety of biological processes. According to the free hormone hypothesis, steroids enter target cells by passive diffusion. However, recently we demonstrated that 25(OH) vitamin D(3) complexed to its plasma carrier, the vitamin D-binding protein, enters renal proximal tubules by receptor-mediated endocytosis. Knockout mice lacking the endocytic receptor megalin lose 25(OH) vitamin D(3) in the urine and develop bone disease. Here, we report that cubilin, a membrane-associated protein colocalizing with megalin, facilitates the endocytic process by sequestering steroid-carrier complexes on the cellular surface before megalin-mediated internalization of the cubilin-bound ligand. Dogs with an inherited disorder affecting cubilin biosynthesis exhibit abnormal vitamin D metabolism. Similarly, human patients with mutations causing cubilin dysfunction exhibit urinary excretion of 25(OH) vitamin D(3). This observation identifies spontaneous mutations in an endocytic receptor pathway affecting cellular uptake and metabolism of a steroid hormone.

Persistent hypercalciuria and elevated 25-hydroxyvitamin D3 in children with infantile hypercalcaemia.

The aim of the study was to characterize abnormalities of calcium-phosphate and vitamin D3 metabolism in children with a past history of "mild" Lightwood-type idiopathic infantile hypercalcaemia. Seventeen seemingly healthy children aged 2-12 years, with long-term idiopathic hypercalcaemic syndrome since infancy were studied. Two reference groups were also included (vitamin D3 intoxication/healthy and Williams groups). Despite a long-term milk-restricted diet and a restricted vitamin D3 intake, urinary calcium excretion in the study group was 0.117 +/- 0.07 mumol/kg per 24 h. Compared with the reference groups (0.047 +/- 0.029 and 0.067 +/- 0.06 mumol/kg per 24 h, P < 0.05), there was significant hypercalciuria in the children with idiopathic hypercalcaemia since infancy. Serum concentrations of 25-hydroxyvitamin D3 in the study group were also elevated compared with the reference groups (57.4 +/- 15.5 vs. 34.6 +/- 9.3 and 22.7 +/- 10.5 ng/ml). 1,25-Dihydroxyvitamin D3 levels were at the upper limit of normal (45.9 +/- 13.1 vs. 35.0 +/- 8.1 and 30.0 +/- 13.7 pg/ml). Non-progressive, clinically silent nephrocalcinosis was visible on ultrasound examinations. The disturbances of vitamin D3 and calcium-phosphate metabolism persistent in the normocalcaemic phase of idiopathic infantile hypercalcaemia may be a primary metabolic defect of the condition. The mechanisms leading to elevation of metabolites of 1,25-dihydroxy- and 25-hydroxyvitamin D3 and the relationship between this and persistent hypercalciuria and nephrocalcinosis need pathophysiological explanation.

Demonstration of a difference in urinary calcium, not calcium absorption, in black and white adolescents.

Bone mineral density (BMD) of the forearm, lumbar spine, and femoral neck is greater in black than in white children. Studies were performed to determine whether differences in intestinal absorption of calcium or urinary calcium or both account for an assumed more positive calcium balance and greater bone mass in black children. Normal black and white boys and girls were admitted to a metabolic ward and given a constant daily diet containing 1000 mg calcium, 60% as calcium carbonate, for 2 1/2 days (study I) or 3 1/2 days (study II). Fasting blood and 24 h urine collections were obtained, and in study II, unidirectional fractional absorption of calcium (alpha) was determined with stable isotopes of calcium. It was found that (1) serum 25-hydroxyvitamin D (25-OHD) and urinary calcium were lower and serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] was higher in black than in white children, and (2) alpha was higher in boys than in girls with no racial difference, and (3) there were significant positive correlations between alpha and urinary calcium in the blacks and in the black and white children together. It is concluded that (1) alpha is higher in boys than in girls and (2) a lower urinary calcium, not increased intestinal absorption of calcium, is the means for a more positive calcium balance in blacks that accounts for the racial difference in BMD.

Families of children with idiopathic hypercalciuria. Evidence for the hormonal basis of familial hypercalciuria.

Six children with idiopathic hypercalciuria and their families were examined with an oral calcium loading test. Family members were divided into two clinical categories: group 1 consisted of the six index children and their parents and siblings with urolithiasis or unexplained hematuria; group 2 comprised the remaining parents and siblings without signs or symptoms associated with hypercalciuria. The results revealed that fasting urinary excretion of calcium was similar in both groups, but group 1 displayed a greater calciuric response to an oral calcium load. Serum concentrations of calcitriol (1,25-dihydroxyvitamin D3) and calcium were higher in group 1 than in group 2, while parathyroid activity was lower in group 1 patients. Urinary excretion of sodium, phosphorus, and magnesium, urine pH, serum levels of calcifediol (25-hydroxyvitamin D3) and phosphorus, and the renal tubular threshold for phosphate were not significantly different in the two groups. These findings suggest that idiopathic hypercalciuria may arise from a disturbance in the regulation of vitamin D metabolism that mediates enhanced intestinal absorption of calcium.

Renal calcium and magnesium handling in water immersion in nephrotic patients.

The effect of water immersion to the neck on renal calcium and magnesium handling was studied in 11 nephrotic patients. There was an increase in the urinary excretion of both calcium and magnesium on immersion, and a return towards preimmersion control values in the hour following immersion. Clearances of calcium and magnesium, and fractional excretion of calcium and of magnesium all increased significantly during water immersion, and decreased in the postimmersion hour. However, magnesium excretion was 10-50 times greater than calcium excretion. Fractional excretion of sodium was highly significantly related to fractional excretion of calcium (p less than 0.001) and magnesium (p less than 0.001). The relationship between fractional excretion of phosphate and fractional excretion of calcium was statistically significant (p less than 0.05), as was that between fractional excretion of phosphate and that of magnesium (p less than 0.01). Magnesium and calcium fractional excretions were significantly correlated (p less than 0.01). It was concluded that the increase in calcium and magnesium excretion on immersion is likely to be related to the sodium diuresis caused by central hypervolemia due to immersion.

Intestinal absorption and 25-hydroxylation of vitamin D in patients with primary biliary cirrhosis.

The absorption, metabolism, and excretion of vitamin D3 was studied in eight women with an established diagnosis of primary biliary cirrhosis (PBC), and the results were compared with those obtained from eight healthy women of a similar age. Four patients had hyperbilirubinemia, low serum calcium levels, and a reduced mineral content of the bone, whereas the other four were presymptomatic with respect to bone disease. Vitamin D absorption was studied after oral administration of tritiated vitamin D, and the appearance of serum radioactivity was recorded. After this, the liver 25-hydroxylation of vitamin D was studied by administering an intravenous dose of tritiated vitamin D and then chromatographing serum samples to determine the radioactivity of the 25-OH D fraction. All PBC patients had normal 25-hydroxylation capacity of the vitamin, and there was no difference in the urinary excretion of radioactivity. On the other hand, the intestinal absorption of vitamin D was severely impaired both in the symptomatic and asymptomatic patients. The absorption of the vitamin was negatively correlated to the amount of fecal fat, and the results suggest that low serum levels of 25-OH D in symptomatic PBC seem to be caused by the steatorrhea, whereas hepatic conversion of vitamin D into 25-OH D seems to be well preserved even in patients with hyperbilirubinemia and signs of osteomalacia. The absorption-metabolism test may be a valuable tool in the study of patients with cholestatic liver disease for determining the nature of the vitamin D deficiency and the logical form of substitution therapy.