Regulation of kidney calcium-binding protein in the bird (Gallus domesticus)

The possible involvement of plasma calcium and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in the regulation of the concentration of kidney calcium-binding protein (CaBP) was investigated. Chicks were fed diets varying in Ca2+ and P, with or without vitamin D. CaBP and 1,25(OH)2D3 were determined by competitive binding assays. A significant correlation between plasma and kidney 1,25(OH)2D3 was found, the linear regression equation of best-fit was plasma 1,25(OH)2D3 = 0.14 + 1.56 kidney 1,25(OH)2D3. In the vitamin D-fed chicks, kidney CaBP varied independently of the circulating or organ level of 1,25(OH)2D3 (P greater than 0.05), but was lower in the vitamin D-deficient than in the vitamin D-fed birds. A significant correlation was observed between kidney CaBP and plasma calcium (Cap). The regression equations were CaBP = Cap/(85.57-4.00 Cap) (R = 0.845) and CaBP = 0.0558 + 0.0404 Cap (R = 0.749), for vitamin D-treated and vitamin D-deficient chicks, respectively. The results suggest that the concentration of kidney CaBP is modulated by plasma calcium, but one or more of the vitamin D metabolites may be required for its synthesis.     

Acute actions of 1,25-dihydroxyvitamin D3 upon chick pancreatic calbindin-D28K

We have compared the relative responsiveness of pancreatic, intestinal and renal tissue calbindin-D28K protein content to the stimulatory actions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in vitamin D-deficient (-D) chicks. Tissue concentrations of calbindin-D28K were undetectable in the -D chick intestine but present, albeit at low concentrations (less than 1 microgram CaBP/mg protein) in the -D kidney and pancreas. Intestinal, pancreatic and renal calbindin-D28K content was stimulated 318, 9.8 and 2.9 fold respectively, 48 hours after -D chicks received a single dose of 1,25(OH)2D3 [6.5 nmol/animal]. The pancreatic calbindin-D28K content could be significantly stimulated as early as 5 hours after 1,25(OH)2D3 administrations in vivo. These findings support the contention that the pancreas is a target for vitamin D, and is consistent with the view that calbindin-D28K plays a role in normal pancreatic functions.     

Vitamin D-dependent calcium-binding protein synthesis by chick kidney and duodenal polysomes

The hormonally active form of vitamin D, 1,25-dihydroxy vitamin D₃, is known to induce in the intestine and kidney of chicks the synthesis of a calcium-binding protein (CaBP). Here we report a correlation between the tissue levels of CaBP and the levels of apparent messenger RNA in total polysomes as determined by the vitamin D and dietary calcium status. Polysomes from pooled duodenal mucosa and kidney were prepared by the Mg²⁺ precipitation method. After translation in a heterologous, rabbit nuclease-treated reticulocyte system, the immunoprecipitated pellet of CaBP was dissolved and the proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gels. When 13 nmol of D₃ was given to 4-week-old rachitic chicks which were sacrificed 48 h later, it was found that the duodenum had eightfold more apparent mRNA for CaBP in the polysomes than the kidney. This was also reflected in the values of CaBP/mg protein in these tissues (duodenum, 7 μg/mg vs kidney, 0.9 μ/mg). Also, after giving D₃, there was a twofold increase in both apparent mRNA levels in the polysomes and in CaBP levels in the duodena of chicks which were raised on low-calcium diets versus chicks raised on high-calcium diets. While apparent mRNA for CaBP was present in polysomes from rachitic chick kidney, it was not detectable in the duodenum. From these studies it appears that the induction of CaBP by 1,25(OH)₂D₃ in both the intestine and kidney is determined by similar control mechanisms.     

Modulation of rat calbindin-D28 gene expression by 1,25-dihydroxyvitamin D3 and dietary alteration

We have used a specific cDNA to the mammalian 28,000 Mr vitamin D-dependent calcium binding protein (calbindin-D28k) to study the regulation of the expression of this mRNA in rat kidney and brain. The effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and dietary alteration on genomic expression were characterized by both Northern and slot blot analysis. Administration of 1,25-(OH)2D3 for 7 days (25 ng/day) to vitamin D-deficient rats resulted in a marked increase in renal calbindin-DmRNA, renal calbindin, and serum calcium. When vitamin D-deficient rats were supplemented for 10 days with calcium (3% calcium gluconate in the water, 2% calcium in the diet) serum calcium levels were similar to the levels observed in the 1,25-(OH)2D3-treated rats. However, in the calcium-supplemented rats the levels of renal calbindin and renal calbindin mRNA were similar to the levels observed in the vitamin D-deficient rats, suggesting that calcium alone without vitamin D does not regulate renal calbindin gene expression in vivo. In dietary alteration studies in vitamin D-replete rats, renal calbindin protein and mRNA increased 2.5-fold in rats fed diets low in phosphate providing evidence that in the rat the nutritional induction of calbindin is accompanied by a corresponding alteration in the concentration of its specific mRNA. Under low dietary calcium conditions, the levels of renal calbindin protein and mRNA were similar to the levels observed in control rats, although 1,25-(OH)2D3 serum levels were markedly elevated, suggesting that factors in addition to 1,25-(OH)2D3 can modulate renal calbindin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25-Dihydroxyvitamin D3 regulates tubulin expression in chick intestine

As in many other cell types, autoregulation of tubulin synthesis is evident in the intestinal epithelium of normal (vitamin D-replete) chicks: Suppression of protein (tubulin) synthesis by cycloheximide administration in vivo resulted within 30 min in a two-fold increase in RNA hybridizing with an alpha-tubulin probe. Vitamin D status revealed an additional regulatory component. alpha-Tubulin mRNA was elevated in vitamin D-deficient (-D) chicks and those treated with 1,25(OH)2D3 for 1-10 h prior to sacrifice, but declined precipitously 15-20 h after hormone, and in normal birds. These results suggested hormonally increased tubulin levels which in turn suppressed cellular alpha-tubulin mRNA. Analyses of total tubulin levels by [3H]-colchicine binding revealed low levels of the protein(s) in -D chicks, increased levels at 1-15 h after 1,25(OH)2D3, and maximum binding at 20 h after hormone and in normal birds.     

Effect of hormones and development on the expression of the rat 1,25-dihydroxyvitamin D3 receptor gene. Comparison with calbindin gene expression

We have used specific cDNAs to the rat vitamin D receptor (VDR) and to the mammalian vitamin D-dependent calcium-binding proteins (calbindin-D9k in intestine and calbindin-D28k in kidney) in order to obtain a better understanding of the regulation of the VDR gene and its relationship to calbindin gene expression. Hormonal regulation and development expression of the rat VDR gene were characterized by both Northern and slot blot analyses. Administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 25 ng/day for 7 days) to vitamin D-deficient rats resulted in an increase in calbindin mRNA in intestine and kidney but no change in VDR mRNA in these tissues. Vitamin D-deficient rats responded to dexamethasone treatment (100 micrograms/100 g of body weight/day for 4 days) with a 2.5-fold increase in intestinal VDR mRNA which was accompanied by a 4-fold decrease in intestinal calbindin-D9k mRNA. Developmental studies indicated a pronounced increase in renal VDR mRNA and calbindin-D28k mRNA between birth and 1 week of age. In the intestine, an induction of VDR and calbindin-D9k gene expression was observed at a later time, during the 3rd postnatal week (the period of increased duodenal active transport of calcium). Taken collectively, our data indicate that in the adult rat, target tissue response to hormone is not modified by a corresponding alteration in new receptor synthesis. However, developmental studies indicate that the induction of 1,25(OH)2D3 receptor mRNA is correlated with the induction of calbindin gene expression. Our results also demonstrate that glucocorticoid administration can result in an alteration in intestinal calbindin and VDR gene expression.     

Analysis of rat vitamin D-dependent calbindin-D28k gene expression

We report the use of a cloned cDNA for mammalian calbindin-D28k (28-kDa vitamin D-dependent calcium-binding protein) to study the expression of the rat calbindin gene. Tissue distribution studies, using Northern analysis, indicated that calbindin-D28k-mRNA is detected in rat kidney and brain but is not detected in rat intestine, testes, bone, pancreas, liver, lung, or skeletal muscle. Both rat kidney and brain contain three RNA species (1.9, 2.8, and 3.2 kilobase pairs). The regulation of the gene was characterized by both Northern and slot blot analysis. Hormonal regulation, developmental expression of calbindin-D28k-mRNA, and the effect of dietary alteration were examined. In the kidney all three species of mRNA were dependent on the presence of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) for their induction. The time course of induction of renal calbindin-D28k-mRNA indicated that a significant increase in calbindin-D-mRNA was detectable as early as 2 h following a single injection of 1,25-(OH)2D3 (200 ng/100 g of body weight), reaching a maximum at 12 h. Unlike the kidney high levels of calbindin-D28k-mRNA were observed in the brain of vitamin D-deficient rats. The concentration of calbindin-D28k-mRNA in brain was unchanged after 1,25-(OH)2D3 administration. Developmental studies indicated that calbindin-D-mRNA in rat kidney and brain is present prior to birth but is developmentally regulated in a tissue-specific manner. The most pronounced changes in the abundance of renal calbindin-D28k-mRNA occur between birth and 1 week of age. Unlike the kidney a large increase in brain calbindin-D28k-mRNA occurs at a later time, between 1 and 2 weeks of age (the period of major synapse formation). In dietary alteration studies results of Northern blot analysis indicate that low dietary phosphorus results in increased calbindin-D-mRNA in kidney but not in brain. These studies represent the first analysis of the rat calbindin-D28k gene and its regulation in vivo. Our findings suggest that in rat kidney and brain there are significant differences both in the expression of the gene for calbindin-D28k and its regulation by 1,25-(OH)2D3.     

Development of 1,25-dihydroxycholecalciferol receptor in the duodenal cytosol of chick embryo.

The development of 1,25-(OH)2D3 receptor in the duodenal cytosol of chick embryo was studied by the sucrose density gradient analysis. The binding profile for 1,25-(OH)2D3 in the cytosol of vitamin D-deficient chick duodenum on the sucrose density gradient revealed 3 binding components, and the sedimentation constant was estimated as 2.5, 3.5 and 5.5S respectively. The 3.5S binding component has high affinity and low capacity for 1,25-(OH)2D3 and is thought to be 1,25-(OH)2D3 receptor. During the development of chick embryo, the 3.5S binding component was not detected in 13-day embryonic duodenum, it appeared on 15th day of incubation and then gradually increased to the level of vitamin D-deficient chick on 19th day of incubation. The 5.5S binding component was specific for 25-OH-D3 and it was found even in 13-day embryo, but it did not show any significant change during development. On the other hand, the 2.5S component was not specific for either 1,25-(OH)2D3 or 25-OH-D3. However, it was main binding component in early stages of development and decreased during development. From these results, it is suggested that the receptor for 1,25-(OH)2D3 is available a few days before hatching and the inability to produce CaBP in the duodenum of chick embryo could not be ascribed to the absence of the receptor.     

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.     

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.     

Immunochemical studies on the putative plasmalemmal receptor for 1, 25-dihydroxyvitamin D(3). III. Vitamin D status

The effect of vitamin D status on levels of the putative 1, 25(OH)(2)D(3) membrane receptor (pmVDR) was studied in chick intestine, kidney, and brain. Western analyses and assays for specific [(3)H]1,25(OH)(2)D(3) binding indicated that, in intestine, pmVDR levels were greatest in -D chicks relative to +1,25D and +D animals (P < 0.05). In kidney, protein levels and specific binding followed the order +D > +1,25D, -D. In brain, vitamin D status did not affect protein levels or specific binding levels. In tissue from normal chicks, both protein and specific binding followed the order of intestine > kidney > brain membranes. Intestinal cells were further evaluated for the effect of 1,25(OH)(2)D(3) on selected "rapid responses." Extrusion of (45)Ca in response to 130 pM 1, 25(OH)(2)D(3) in vitro was greater in cells from -D chicks than from +1,25D or normal birds. Analyses of signal transduction events revealed diminished hormone-induced intracellular calcium oscillations (as assessed by fura-2 fluorescence), and lack of steroid-enhanced protein kinase (PK) A activity in intestinal epithelial cells from -D chicks relative to +D chicks. PK C activation by 130 pM 1,25(OH)(2)D(3) was approximately twofold in cells from +D or -D chicks. The combined results indicate that vitamin D status differentially affects the pmVDR in intestine, kidney, and brain. In intestine, vitamin D deficiency differentially affects (45)Ca handling, intracellular calcium oscillations, PK A and PK C activities in response to 1,25(OH)(2)D(3).     

1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K)

A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport. Endocytic internalization of Ca2+, fusion of the vesicles with lysosomes, and exocytosis at the basal lateral membrane complete the transport process.     

Diabetes and renal calcium binding protein in the rat

Renal calcium binding protein (CaBP), a vitamin D-dependent protein of 28,000 Mr, may be involved in calcium transport by cells of the renal tubule. The streptozotocin-diabetic rat is hypercalciuric and shows markedly decreased concentration of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] in serum and of CaBP in small intestine. To examine the relationship of renal CaBP in diabetes to 1,25-(OH)2D3 and urinary calcium excretion, renal CaBP, serum 1,25-(OH)2D3, and urinary calcium were measured in control, diabetic, and insulin-treated diabetic rats. Treatment of the diabetic rat with insulin decreased urinary calcium excretion and elevated 1,25-(OH)2D3 toward normal. Renal CaBP was found to be the same in controls and diabetics despite a tenfold difference in concentration of 1,25-(OH)2D3 in serum, and to be unaffected by insulin treatment, which elevated 1,25-(OH)2D3 by a factor of 7 above untreated diabetics. It is concluded that in the diabetic rat either (1) the threshold concentration of 1,25-(OH)2D3 for inducing synthesis of renal CaBP is set at a much lower level than that for intestinal CaBP, or (2) since both 1,25-(OH)2D3 and renal CaBP are produced in the kidney, 1,25-(OH)2D3 exerts a paracrine effect on renal CaBP production because of its high local concentration. The increased urinary calcium excretion in the untreated streptozotocin-diabetic rat is not secondary to an alteration in renal CaBP.     

Determination of the rates of synthesis and degradation of vitamin D-dependent chick intestinal and renal calcium-binding proteins

Vitamin D₃ and its biologically active metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D₃-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D₃ or 1,25(OH)₂D₃ to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 29}\text{h}$) and that for renal CaBP was 0.019 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 36}\text{h}$) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH)₂D₃ to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH)₂D₃. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D₃-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH)₂D₃ to each target organ.