Response of renal calcium-binding protein. Independence of kidney vitamin D hydroxylation.

Dietary calcium and dietary phosphorus restriction were studied in chicks fed either cholecalciferol or 1alpha-hydroxycholecalciferol. Intestinal calcium absorption and calcium-binding protein of 1alpha-hydroxycholecalciferol-treated chicks remained unchanged under dietary calcium restriction, but increased under dietary phosphorus restriction. Kidney calcium-binding protein was not altered by dietary caclium restriction in chidks treated with either cholecalciferol or 1alpha-hydroxycholecalciferol, but increased under dietary phosphorus restriction independent of the vitamin D source. In contrast to the intestine, calcium-binding activity of the kidney was found to be poorly related to the calcium-binding protein concentration. It is suggested that kidney calcium-binding protein is regulated by a mechanism different from that of intestinal calcium-binding protein, and that its concentration in renal tissue is related to renal caclium excretion or plasma calcium level.     

Skin calcium-binding protein: effect of vitamin D deficiency and vitamin D treatment

The amount of skin calcium-binding protein, evaluated using a sensitive radioimmunoassay and indirect immunofluorescence, was decreased in vitamin-D deficient rats and increased after one week vitamin D3 or 1 alpha-hydroxyvitamin D3 treatment. In vitamin D replete and in vitamin D-deficient animals, skin calcium-binding protein was not sensitive to changes in dietary and/or serum calcium concentrations. These results indicate that this protein is different from other calcium-binding proteins such as parvalbumin and calmodulin which are not vitamin D-dependent, and also different from intestinal calcium-binding protein which, in D replete animals, is sensitive to changes in dietary and serum calcium concentrations. Skin calcium-binding protein may, therefore, represent a new class of vitamin D-dependent protein.     

Multiple forms of vitamin D-dependent calcium-binding protein in rat kidney

The technique of two-dimensional electrophoresis was used in combination with a highly sensitive silver stain to study vitamin D-dependent calcium-binding protein (CaBP) in rat kidney. Rat renal CaBP was shown to co-migrate almost exactly with CaBP purified from chick intestine suggesting evolutionary conservation of this protein. In some cases rat renal CaBP appeared not as a single polypeptide, but rather as a cluster of 4 polypeptides. Formation of the satellite cluster of CaBP in response to high doses of 1,25-dihydroxyvitamin D3 occurred in young rats which had been maintained on a vitamin D-deficient diet for 2 weeks, as well as in older rats which had been maintained on the same diet for 5 months. The 4 forms of CaBP were not the result of various states of Ca2+ binding, but rather the result of an enzymatic reaction. This was shown by 3 experiments. 1) Adding excess EGTA to samples containing the 4 satellite forms did not change the two-dimensional electrophoretogram. 2) Incubation of purified chick intestinal CaBP with kidney cytosols from D-deficient rats brought about the formation of the satellite CaBP forms from the chick protein. However, purified chick CaBP was unchanged by incubation in buffer alone for up to 2 h at 37 degrees C. 3) Placing rat kidney cytosols in a boiling water bath for 10 min inactivated the factor which generated the satellite forms as would be expected for an enzyme. The physiological significance of these forms of CaBP is as yet unknown.     

Induction of calcium-binding protein in organ-cultured chick intestine by fluoro analogs of vitamin D3

Vitamin D-like steroids added to the culture medium induce a specific calcium-binding protein (CaBP) in embryonic chick duodenum maintained in organ culture. This system provides a biologically relevant assay, i.e., a physiological response in a principle target organ, for the study of the relative biopotency of vitamin D metabolites and analogs. A number of fluoro analogs of vitamin D₃ (D₃) and its metabolites were assayed in the present study. Analogs fluorinated in the lα position (1α-F-D₃) or in both the 1α and 25 positions (1α,25-F₂-D₃) were markedly more potent than vitamin D₃ itself although 1α,25-F₂-D₃ was only $\text{1}\text{7}\text{th}$ as potent as 1α-F-D₃. The 25-fluoro analog (25-F-D₃) was a very weak inducer; only $\text{1}\text{45}\text{th}$ as potent as vitamin D₃. The 25-fluoro analog of 1α-hydroxyvitamin D₃ (1α-OH-25-F-D₃) was less potent than its nonfluorinated counterpart. Although 25-fluorination reduced biopotency in all other analogs tested, 24R-OH-25-F-D₃ was about 15 times more potent than 24R,25-(OH)₂-D₃. Of considerable interest was the effect of difluorination at the 24-carbon position: both 24,24-F₂-25-OH-D₃ and 24,24-F₂-1α,25-(OH)₂-D₃ were about four times as potent as their nonfluorinated counterparts. The 24,24-F₂-1α,25-(OH)₂-D₃ is, therefore, the most potent vitamin D₃ analog yet tested in this system i.e., it is four times more potent than the most potent naturally occurring vitamin D₃ metabolite, 1α,25-(OH)₂-D₃.     

Intestinal vitamin D-induced calcium-binding protein: time-course of immunocytological localization following 1,25-dihydroxyvitamin D3

The vitamin D-induced calcium-binding protein (CaBP) was localized in histological sections of chick duodenum using the peroxidase-antiperoxidase immunocytochemical technique. The time-course of appearance of CaBP in rachitic chicks was investigated from 0 to 120 hr after stimulation by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). CaBP was not routinely detected at 0 hr after 1,25(OH)2D3 administration. CaBP was first noted in some, but not all, of the samples taken 2 hr following 1,25(OH)2D3 and was detected in all 2 1/2 hr samples. The number of CaBP-containing absorptive cells and the apparent CaBP concentration both increased to a maximum at about 16-24 hr. At later times, as CaBP free cells migrated up the villi, the CaBP-containing cells decreased in number, but even at 120 hr post 1,25(OH)2D3 dose there were significant numbers of CaBP-containing cells present. The relationships between time-course of CaBP location on intestinal villi, enterocyte migration rates, and the time-course of 1,25(OH)2D3 stimulated intestinal calcium transport are discussed.     

Immunocytochemical localization of rat intestinal vitamin D-dependent calcium-binding protein

Vitamin D-dependent calcium-binding protein (CaBP) was localized in intestinal tissue sections obtained from rats raised under three different nutritional conditions: a normal vitamin D-replete diet, a vitamin D-free diet followed by supplementation with vitamin D3, or a vitamin D-free diet without additional supplementation. An indirect immunoperoxidase technique, with immunocontrols, was used to visualize the specific sites of CaBP. CaBP was visualized only in the cytoplasm of absorptive cells. In the duodenum of animals raised on a normal diet, CaBP was present in absorptive cells from the upper crypt region to the villus tips. In the jejunum, many fewer absorptive cells contained CaBP, while in the ileum only random absorptive cells near the villus tips contained CaBP. In rats raised on a vitamin D-deficient diet then supplemented with vitamin D3, CaBP was present in cells at the full depth of the crypts and in absorptive cells along the total villus length in the duodenum. Rats raised on the same deficient diet but without supplementation with additional vitamin D exhibited no CaBP in crypt cells nor in absorptive cells more than half way up the villi. Absorptive cells higher on the villi contained immunoreactive CaBP but the intensity of immunostaining and number of CaBP-containing cells was markedly reduced compared to the vitamin D-supplemented group.     

Immunocytochemical demonstration of two vitamin D-dependent calcium-binding proteins in mammalian kidney

Distribution of vitamin D-dependent calcium-binding proteins (CaBPs) were studied in four mammalian species using monospecific antibodies raised against chick duodenal CaBP (D-CaBP), human cerebellar CaBP (L-CaBP), and rat duodenal CaBP (S-CaBP). The immunoperoxidase technique of unlabelled antibodies was employed. The distribution of D-CaBP/L-CaBP was identical in all the species studied except for the monkey. In the rat, pig, and human nephrons, D-CaBP/L-CaBP was seen in the cytoplasm of the cells of the distal convoluted tubules, initial segments of the collecting ducts and interspersed cells of the collecting ducts. Proximal convoluted tubules, glomeruli and maculae densae were negative. In the monkey, in addition to the cells of the distal convoluted tubules, the cells along the entire length of the collecting ducts were also strongly positive. S-CaBP was found to be species-specific, and hence positive results were obtained only in the rat nephron. The strongest positive reaction for S-CaBP was seen in the cells of the distal convoluted tubules. These same cells were also positive for D-CaBP/L-CaBP. S-CaBP was also detected in the cells of the thick ascending limb of the loop of Henle, along the entire length of the collecting ducts and in smaller amounts in cells of the macula densa. Intracellularly the S-CaBP was present only in the apical cytoplasm of positive cells. D-CaBP/L-CaBP stained the entire cytoplasm but the staining in the apical cytoplasm was denser.     

Immunohistochemical localization of vitamin D-dependent calcium-binding protein in duodenum,kidney, uterus and cerebellum of chickens

Summary Calcium-binding protein (CaBP) has been localized with the immunoperoxidase method using antiserum against purified chick duodenal CaBP. Different preparative procedures were employed to investigate the experimental conditions possibly responsible for the contradictory reports in the literature of the precise cellular localization of CaBP. Freeze substitution, frozen sections followed by fixation and coagulant and non-coagulant fixatives were used with appropriate control sections to demonstrate that the true localization of CaBP in the chick duodenum is in the absorptive cell cytoplasm. The goblet cell localization reported in the literature seems to be a diffusion artifact due to inadequate fixation. CaBP was also localized in several other tissues. In the hen uterus, the tubular glands beneath the surface epithelium showed intense reaction. In the kidney, CaBP was present in the cells of the straight and convoluted segments of distal tubules. The cortex of the chick cerebellum showed the CaBP in Purkinje cells. The entire dendritic trees contained the reaction product. No other neurons in the molecular or the granular layer were stained. In the deep cerebellar nuclei, all neurons were negative and these were outlined by deeply staining axons of the Purkinje cells and their synaptic endings.     

The ontogenesis of calcium-binding protein in fetal rat kidney

Summary The presence of calcium-binding protein (CaBP) in the nucleus and cytoplasm of epithelial cells of renal tubules varies with the physiologic state. As part of a study to determine whether or not intranuclear CaBP precedes intracytoplasmic CaBP in the same cell, we used peroxidase-labeled antibody against human renal CaBP to localize CaBP in fetal rat kidney tubules. This paper reports examination of kidneys from rats on each day of gestation from the 10th to term (21 days) and on each of the first seven postnatal days. CaBP was first detected in fetal rat kidneys on the 19th gestational day. The histochemical staining reaction that revealed the CaBP was less intense than that produced in kidneys from adult animals, but its distribution was like that in adults, with some cells having no CaBP, others having it in the cytoplasm only, in the nucleus only, or in both. By the seventh postnatal day the staining reaction was similar to the adult patterns in all respects. Send offprint request to:commander,Department of Comparative Medicine, Letterman Army Institute of Research, Attn. : Research Librarian, Presidio of San Francisco, CA 94129, USAIn conducting the research described in this report, the investigators adhered to the Guide for Laboratory Animal Facilities and Care, as promulgated by the Committee on the Guide for Laboratory Animal Resources, National Academy of Sciences-National Research CouncilThe opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense     

Side-chain hydroxylation of vitamin D3 and its physiological implications

Evidence is accumulating that, in vivo and in vitro, both 25-OH-D3 and 1,25-(OH)2D3 undergo side-chain modification leading to side-chain cleaved metabolites lacking the 24, 25, 26, and 27 carbons. The enzymes involved are D-dependent and are located in the kidney, bone, intestine, and perhaps other sites. We speculate that the extra-renal side-chain pathway may be primarily for target organ destruction of 1,25-(OH)2D3, whereas the renal pathway may be primarily for destruction of 25-OH-D3 formed in large amounts in hypervitaminosis D.     

Amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum

The amino acid sequence of vitamin D-dependent calcium-binding protein from bovine cerebellum has been determined. It is composed of 260 amino acid residues and its N-terminus is acetylated. The molecular mass is calculated to be 29 851 Da. The presence of six calcium-binding sites (I-VI) has been proposed, two of them (sites II and VI) have lost their calcium-binding function through amino acid replacements, and the other four are able to bind calcium. Six calcium-binding domains are supposed to be derived from two gene duplications of the two ancestral calcium-binding domains. In comparison with the sequence of chick intestinal calcium-binding protein deduced from a cDNA sequence [(1985) Nucleic Acids Res. 13, 8867-8881], the bovine calcium-binding protein is two amino acid residues shorter at the N-terminus and the other parts show 78.5% identity.     

Suppressed expression of calcium-binding protein regucalcin mRNA in the renal cortex of rats with chemically induced kidney damage

The alteration of Ca²⁺-binding protein regucalcin mRNA expression in the kidney cortex of rats administered cisplatin and cephaloridine, which can induce kidney damage, was investigated. Cisplatin (0.25, 0.5 and 1.0 mg/100 g body weight) or cephaloridine (25, 50 and 100 mg/100 g) was intraperitoneally administered in rats, and 1, 2 and 3 days later they were sacrificed. The alteration in serum findings after the administration of cisplatin (1.0 mg/100 g) or cephaloridine (50 and 100 mg/100 g) demonstrated chemically induced kidney damage; blood urea nitrogen (BUN) concentration increased markedly and serum inorganic phosphorus or calcium concentration decreased significantly. Moreover, the administration of cisplatin (1.0 mg/100 g) or cephaloridine (100 mg/100 g) caused a remarkable increase of calcium content in the kidney cortex of rats, indicating kidney damage. The expression of regucalcin mRNA in the kidney cortex was markedly reduced by the administration of cisplatin or cephaloridine in rats, when the mRNA levels were analyzed by Northern blotting using rat liver regucalcin cDNA (0.9 kb). The mRNA decreases were seen with the used lowest dose of cisplatin or cephaloridine. The present study clearly demonstrates that the mRNA expression of Ca²⁺-binding protein regucalcin in the kidney cortex of rats is decreased by chemically induced kidney damage.     

Regulation of renal vitamin D hydroxylase activity in vitamin D deficient rats

The regulation of renal mitochondrial 1-hydroxylase activity in chronic vitamin D deficiency was studied in male rats. These rats were born of mothers who had been raised from weaning (21 days) on a vitamin D deficient diet and who had no detectable serum 1,25-dihydroxycholecalciferol (1,25-(OH)2D) at the time their offspring were weaned (28 days). In the pups, renal mitochondrial 1-hydroxylase activity was undetectable before the 3rd week of life even though the animals were severely hypocalcemic from birth. The 1-hydroxylase activity first became detectable at 26 days of age, rapidly reached a maximum at day 34, then decreased to become undetectable again by 65 days. Throughout this time serum calcium concentration was less than 5.0 mg/dL and serum parathyroid hormone (PTH) concentration, measured by a midmolecule radioimmunoassay, was two- to five-fold greater than that found in vitamin D replete rats. 1-Hydroxylase activity could be restored in the +65-day-old animals by administration of a single dose of 2.5 micrograms vitamin D3. Enzyme activity was detected within 24 h, was maximal at 72 h, and returned to undetectable levels by 96 h after administration of the vitamin. Serum 1,25-(OH)2D which was undetectable before administration of the vitamin D3, was 108 and 458 pg/mL at 16 and 40 h, respectively, after the injection. The serum concentration of this metabolite then decreased progressively to 80 pg/mL by 6 days. 24-Hydroxylase activity first became detectable 48 h after vitamin D administration, increased to a maximum at 96 h, and thereafter decreased to become undetectable by 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on the binding of lanthanides and calcium to vitamin D-dependent chick intestinal calcium-binding protein. Implications regarding calcium-binding protein function

The binding of calcium and terbium to purified chick vitamin D-dependent intestinal calcium-binding protein was studied by terbium fluorescence, circular dichroism, and intrinsic protein fluorescence techniques. Calcium-binding protein bound, with high affinity, at least 3 mol of terbium/mol of protein; numerous low affinity terbium-binding sites were also noted. The three highest affinity sites were resolved into one very high affinity site (site A) and two other sites (sites B and C) with slightly lower affinity. Resonance energy transfer from tryptophan residues to terbium occurred only with site A. This site was filled before sites B and C. Competition experiments in which calcium was used to displace terbium bound to the protein showed that larger amounts of calcium were needed to displace terbium from site A than from sites B and C. Energy transfer from terbium to holmium indicated that the terbium-binding sites (B and C) were located close to each other (about 7-12 A) but were distant (greater than 12 A) from site A. The addition of EDTA to calcium-binding protein resulted in a 25% decrease in intrinsic protein fluorescence, suggesting a conformational change in the protein. The titration of EDTA-treated calcium-binding protein with calcium resulted in recovery of intrinsic protein fluorescence. A reversible calcium-dependent change in the ellipticity of calcium-binding protein in circular dichroism experiments was also seen. These observed properties suggest that vitamin D-dependent chick intestinal calcium-binding protein behaves in a manner similar to other well-known calcium-binding regulatory proteins.     

Association of vitamin D binding protein polymorphism with long-term kidney allograft survival in Hispanic kidney transplant recipients

Polymorphism of genes encoding components of the vitamin D pathway including vitamin D receptor (VDR) and vitamin D binding protein (VDBP), have been widely explored due to the complex role played by vitamin D in renal transplant outcomes. In this study, we investigated whether polymorphisms of genes encoding VDR and VDBP were associated with allograft survival or acute rejection (AR) among a Hispanic kidney transplant population. A total of 502 Hispanic renal allograft recipients at the St. Vincent Medical Center between 2001 and 2010 were genotyped for four different single nucleotide polymorphisms of VDR: FokI C>T (rs2228570), BsmI G>A (rs1544410), ApaI T>G (rs7975232), and TaqI T>C (rs731236). We also performed genotyping for one common polymorphism in the VDBP gene (rs4588). Survival was significantly improved for patients who were homozygous GG for the rs4588 G>T allele in the VDBP gene (GG vs. GT + TT, OR = 0.63, p = 0.02) while GT genotype was associated with a higher risk of graft loss (GT vs. GG + TT, OR = 1.67, p = 0.01). We found no association for polymorphic markers in VDR with allograft survival and AR. The frequency of the haplotype GTCG (in the order of VDR FokI C>T, BsmI G>A, ApaI T>G, and TaqI T>C), was significantly different in the patients with graft rejection compared to the control (p = 0.007) while ACCA haplotype was found to be associated with graft loss (p = 0.02). Hence, the VDBP G>T polymorphism (rs4588) and two haplotypes (GTCG and ACCA) of VDR appear to be associated with renal allograft outcomes among Hispanic allograft recipients.     

The lack of relationships between vitamin D3 metabolites and calcium-binding protein in the eggshell gland of laying birds

The relationship of the metabolism of vitamin D3 and calcium-binding protein (CaBP) to calcium transport by the eggshell gland (ESG) was assessed in chickens. Plasma or ESG 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) and ESG CaBP were no different between periods of ESG inactivity and of shell calcification. A severe dietary calcium deficiency resulted in increased kidney 25-hydroxycholecalciferol-1-hydroxylase activity (542%), plasma and ESG 1,25(OH)2D3 concentrations (193 and 274%, respectively), but in decreased ESG CaBP (34%), associated with the production of poorly calcified eggs. Significant correlations were found between 25 hydroxycholecalciferol-1-hydroxylase, plasma 1,25(OH)2D3 and ESG 1,25(OH)2D3, but not between ESG 1,25(OH)2D3 and CaBP. Hens with a low shell density had a significantly lower (55%) ESG CaBP than those with high shell density, without any significant change in ESG 1,25(OH)2D3. Significant correlations were found between ESG CaBP and shell calcium. Total receptors for 1,25(OH)2D3 were lower in ESG than in the intestine. The results suggest that CaBP level and calcium transport in the ESG are not regulated by 1,25(OH)2D3.