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.     

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.     

Effect of vitamin D deficiency on the composition and in vitro labeling of rat kidney proteins

Densitometric analysis of single-dimension gels consistently demonstrated that, in addition to rat renal calcium binding protein (CaBP) (Mr 28,000), two other kidney proteins of Mr 16,500 and Mr 18,000 were significantly enriched in their contents in the vitamin D-replete rat. Partial characterization of the Mr 18,000 and 16,500 proteins revealed that these proteins were heat-stable and distinct from calmodulin, as determined by their inability to undergo the calcium-dependent mobility shift in sodium dodecyl sulfate gels which is characteristic of calmodulin. The Mr 16,500 and Mr 18,000 kidney proteins did not cross-react with rat renal or rat intestinal CaBP antisera, as assessed by radioimmunoassay and Western blot analysis. A comparison of peptide maps of tryptic digests of these proteins and purified rat renal CaBP, as analyzed by high-pressure liquid chromatography, revealed no apparent homology. Protein synthesis studies using [35S]methionine and short-term tissue culture of kidney cortex fragments indicated that the most pronounced effect of vitamin D or 1,25 dihydroxyvitamin D3 was increased synthesis of the Mr 28,000 protein (3.2- to 4.6-fold increase compared to -D rats, P less than 0.001). Synthesis of a Mr 54,500 protein increased by 1.3- to 1.5-fold (P less than 0.05) and [35S]methionine incorporation into a Mr 66,000 protein decreased by 1.2- to 1.3-fold (P less than 0.05) in +D rats. This study represents the first detailed characterization of the effects of vitamin D on the composition and synthesis of rat kidney proteins. The data indicate that the most significant effect of vitamin D on kidney proteins is increased synthesis of the Mr 28,000 CaBP, suggesting that a major role of vitamin D in renal function is regulation of calcium transport at the distal tubule. However, dietary vitamin D or 1,25(OH)2D3 can influence the expression as well as the suppression of other specific kidney proteins.     

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.     

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)     

Increased level of calcitonin mRNA after 1,25-dihydroxyvitamin D3 injection in the rat

Vitamin D metabolites are able to change plasma calcitonin (CT) levels, but nothing is known about a possible effect at the CT gene level. Here we have investigated the acute effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the CT biosynthetic activity of thyroid glands from adult rats. Plasma CT levels were significantly increased (X2) 1 and 2 h after 1,25-(OH)2D3 injection in the face of unchanged plasma calcium values. The thyroidal CT content also was unchanged. A 2-fold increase in CT mRNA level measured by dot-blot hybridization occurred 1 and 2 h after 1,25-(OH)2D3 administration. Expression of CT gene products was examined in the rabbit reticulocyte lysate cell-free translation assay. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A single precursor of Mr approximately equal to 15 000 could be specifically immunoprecipitated with CT antisera. A 3-4-fold rise in translatable CT mRNA activity was observed 1 and 2 h after 1,25-(OH)2D3 injection. Thus, parallel changes in CT mRNA level, CT mRNA activity and plasma CT levels were observed in adult female rats after administration of 1,25-(OH)2D3. These findings demonstrate for the first time that 1,25-(OH)2D3 enhanced CT gene expression in the face of unchanged plasma calcium levels.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

The effect of active vitamin D3 analogs and dexamethasone on the expression of osteocalcin gene in rat tibiae in vivo.

We tested the effects of 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3), 2 beta-(3-hydroxypropoxy)-1 alpha,25-dihydroxyvitamin D3 (ED-71) and dexamethasone on osteocalcin mRNA levels in rat tibiae in vivo. Northern blot analysis showed that both 1,25-(OH)2D3 and ED-71 caused an increase in osteocalcin mRNA levels in bone: 1,25-(OH)2D3 induced a transient increase in the mRNA levels followed by a decrease in the control level by 12 h post administration. In contrast, ED-71 caused a persistent increase in osteocalcin mRNA level for seven days post administration. Serum osteocalcin levels paralleled the osteocalcin mRNA level in bone in both groups. Dexamethasone caused a marked reduction in both osteocalcin mRNA and serum osteocalcin levels. Suppressive effect of dexamethasone on osteocalcin expression was persistent for seven days at higher dose. Our results represent the first demonstration of the effect of active vitamin D and corticosteroid on the expression of osteocalcin mRNA in bone in vivo.     

In situ detection of vitamin D-induced calcium-binding protein (9-kDa CaBP) messenger RNA in rat duodenum

We have previously described the molecular cloning of a cDNA fragment synthesized from rat duodenal mRNA coding for a 9000-dalton vitamin D-induced calcium-binding protein (9-kDa CaBP) (3). We now report the use of this cloned cDNA to study the cytological distribution of 9-kDa CaBP mRNA in rat duodenum by in situ hybridization. Tissue sections, fixed in ethanol:acetic acid, were hybridized to the 3H-cDNA probe and processed for autoradiography. The specificity of the CaBP mRNA-DNA hybrid formation was checked using 3H-labeled plasmid pBR322 DNA as a control probe. 9k-Da CaBP mRNA, visualized by silver grains, was found only in the absorptive epithelial cells, and the concentration was greater in the cells at the villous tips than in those of the crypts. The 9k-Da CaBP mRNA was observed mainly in the cytoplasm of the columnar cells and less frequently in the nucleus. Labeling was not seen in the brush border and goblet cells. The submucosa, with Brunner's glands and muscularis, also showed no specific 9-kDa CaBP mRNA concentration. This demonstration of 9-kDa CaBP gene activity in the columnar cells of the rat duodenum illustrates the usefulness of in situ hybridization for characterization of specific cells involved in the expression of 1,25(OH)2 D3 activity.     

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.