Avian and mammalian vitamin D-dependent calcium binding protein in reptilian nephron

A calcium binding protein (CaBP) with an apparent relative molecular weight of 28,000 was localized in the kidney of Anolis carolinensis with antisera directed against vitamin D-dependent CaBP from either rat kidney (RRCaBP) or chick intestine (CICaBP). When extracts of female saurian kidneys were fractionated by gel filtration on Sephadex G-100, both calcium binding activity, as measured by the chelex resin assay, and CaBP immunoreactivity, as measured by radioimmunoassay for RRCaBP, were observed near the 28,000-Da region similar to RRCaBP and CICaBP. Utilization of the immunoblot technique following SDS-polyacrylamide slab gel electrophoresis resulted in cross-reactivity for CaBP in the M_r 28,000 region for both rat and anolian kidneys. Immunoreactive CaBP was localized in the nephron using the unlabeled antibody peroxidase-antiperoxidase technique. Distal tubules (DT) gave a strong, specific reaction with either antiserum, but not all cells of the DT reacted with equal intensity. Cells in the transition zone between the DT and the terminal tubules, and cells in the collecting tubules, were occasionally positive. Renal corpuscles, proximal tubules, and thin segments gave no specific localization of CaBP. The sexual segment of male kidneys was also negative. The results indicate that a calcium binding protein with an apparent molecular weight of 28K is highly conserved during vertebrate evolution, and thus may have widespread but specific functional significance. The localization of CaBP to the DT suggests that this protein may be involved in the selective reabsorption and/or excretion of calcium.     

Vitamin D-dependent rat renal calcium-binding protein: development of a radioimmunoassay, tissue distribution, and immunologic identification

A sensitive double antibody RIA has been developed for the 28,000 mol wt rat renal vitamin D-dependent calcium-binding protein. Using this assay, concentrations of calcium-binding protein (CaBP) as low as 30 ng can be measured. The assay is precise (intraassay variability, 5.0%) and reproductible (interassay variability, 8.2%). Measurements of renal CaBP by RIA showed a good correlation with measurements of CaBP by the chelex resin assay and by polyacrylamide gel analysis by densitometric tracing using a purified CaBP marker. The concentration of CaBP in the vitamin D-replete rat kidney is 7.3 +/- 1.0 (mean +/- SEM) micrograms/mg protein. In vitamin D-deficient rats the level of renal CaBP is 2.6 +/- 0.3 micrograms/mg protein. Tissue distribution of immunoreactive rat renal CaBP showed the highest concentration of CaBP in the rat cerebellum (38.3 +/- 5.1 micrograms/mg protein). Lower concentrations of immunoreactive CaBP were detected in several other rat tissues. No immunoreactive CaBP was detected in rat or human serum. In necropsy human kidney and cerebellum, high levels of immunoreactive CaBP were also detected (1.5 +/- 0.1 and 27.3 +/- 2.1 micrograms/mg protein, respectively). When extracts of rat kidney and brain and human cerebellum and kidney were assayed at several dilutions, immunodisplacement curves parallel to that of pure renal CaBP were observed, indicating immunochemical similarity. Fractionation of extracts of rat cerebellum, human kidney, and human cerebellum on Sephadex G-100 revealed immunoreactivity and calcium-binding activity in the 28,000 mol wt region similar to rat kidney.     

Vitamin D-dependent calcium binding proteins in the kidney and intestine of the X-linked hypophosphatemic mouse: changes with age and responses to 1,25-dihydroxycholecalciferol

We have previously observed decreased intestinal 9 kilodalton (kd) vitamin D-dependent calcium binding protein (CaBP) and decreased calcium absorption in juvenile X-linked hypophosphatemic (Hyp) mice. The present studies were undertaken to examine whether the kidney CaBPs (9 kd and 28 kd) are also affected in young Hyp mice and to investigate the ability of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] to increase CaBP in the intestine and kidney. The 28 kd CaBP and the 9 kd CaBP were measured in the kidneys and the 9 kd CaBP in the intestines of normal and Hyp mice from 1 week to 40 weeks of age. At all times between 3 and 6 weeks, intestinal CaBP in Hyp mice was decreased by more than 50% (P less than 0.005-0.001) and no significant decrease was present in the adult Hyp mice (12 and 40 weeks of age). By contrast, both kidney CaBPs were decreased only slightly in young Hyp mice. Between 1 and 6 weeks of age, the 9 kd CaBP in Hyp mice was 82% +/- 4% of control (P less than 0.001) and the 28 kd protein was 89% +/- 3% of control (P less than 0.001). Minipumps containing 1,25-(OH)2D3 or vehicle were implanted in 4-week and 13-week-old Hyp mice for 3 days to provide a dose of 0.12 micrograms/kg mouse X day. The 9 kd CaBP was increased approximately 3-fold (P less than 0.001) by 1,25-(OH)2D3 in the intestines of Hyp mice at both ages. The 9 kd kidney CaBP in Hyp mice also was increased by 1,25-(OH)2D3 treatment at both ages, but only by 33-52%. The 28 kd CaBP in the kidney was not affected by 1,25-(OH)2D3 treatment of Hyp mice at either age. We conclude that (9 kd and 28 kd) CaBPs levels in both intestine and kidney are decreased in juvenile Hyp mice although to much different degrees. The administration of 1,25-(OH)2D3 to Hyp mice increases the 9 kd CaBP in both intestine and kidneys, whereas the renal 28 kd CaBP is unaffected.     

Dopamine D1-like receptor subtypes in the rat kidney: a microanatomical study

The microanatomical localization of dopamine D1A and D1B receptor subtypes was investigated in sections of rat kidney using immunohistochemicals techniques with antidopamine D1A and D1B receptor antibodies. Microanatomical analysis was limited to the various components of nephron. Dopamine D1A receptor immunoreactivity was found primarily in the epithelium of loop of nephron (loop of Henle) and of collecting tubules. A less intense immunoreactivity was observed within proximal and distal convoluted tubules as well as in juxtaglomerular complex. Dopamine D1B receptor immunoreactivity was found primarily in proximal and distal convoluted tubules and within the juxtaglomerular complex. A less intense immunoreactivity was observed in the epithelium of collecting tubules followed by the loop of nephron. The demonstration of the localization of dopamine D1A and D1B, receptor subtypes along the nephron may contribute to better define their significance in physiological and pathological conditions.     

Vitamin D-dependent calcium-binding protein of rat intestine: changes during postnatal development and sensitivity to 1,25-dihydroxycholecalciferol.

To study the role of the vitamin D-endocrine system during the perinatal period, we monitored vitamin D-dependent calcium-binding protein (CaBP) in rat intestine by radial immunodiffusion and polyacrylamide disc gel electrophoresis. Small amounts of CaBp were present 2 days before birth; these levels increased 74-fold by day 38 after birth. Approximately 80% of the increase in CaBP concentration occurred in a 5-day period at the time of weaning (days 17--22 after birth). Before this period, the concentration of CaBP was comparable to that found in rachitic (adult) rats. Administration of 1,25-dihydroxycholecalciferol to suckling rats on days 15 and/or 16 was followed by a premature increase in the amount of intestinal CaBP. These data demonstrate that although vitamin D-dependent CaBP is low in preweaned rats, the rat intestine is responsive to exogeneous 1,25-dihydroxyvitamin D3 at least as early as day 15 after birth. The close temporal correspondence between the increases in CaBP and previously reported changes in calcium transport and vitamin D metabolism suggest that the vitamin D-endocrine system plays a role in postnatal intestinal maturation and adaptation during the weaning period.     

Dopamine D1-Like Receptor Subtypes in the Rat Kidney: A Microanatomical Study

The microanatomical localization of dopamine D_1A and D_1B receptor subtypes was investigated in sections of rat kidney using immunohistochemicals techniques with anti-dopamine D_1A and D_1B receptor antibodies. Microanatomical analysis was limited to the various components of nephron. Dopamine D_1A receptor immunoreactivity was found primarily in the epithelium of loop of nephron (loop of Henle) and of collecting tubules. A less intense immunoreactivity was observed within proximal and distal convoluted tubules as well as in juxtaglomerular complex. Dopamine D_1B receptor immunoreactivity was found primarily in proximal and distal convoluted tubules and within the juxtaglomerular complex. A less intense immunoreactivity was observed in the epithelium of collecting tubules followed by the loop of nephron.

The demonstration of the localization of dopamine D_1A and D_1B receptor subtypes along the nephron may contribute to better define their significance in physiological and pathological conditions.     

Human growth hormone increases intestinal vitamin D-dependent calcium-binding protein in hypophysectomized rats

We examined the effects of hypophysectomy and pituitary hormone replacement on vitamin D-dependent calcium-binding protein (CaBP) in rat small intestine. The concentration of immunoreactive CaBP per mg intestinal protein was decreased by at least 56% in hypophysectomized rats compared to that in intact pair-fed controls. Alkaline phosphatase and total protein also were reduced by hypophysectomy, but pair-feeding produced comparable decreases. Daily injections of 2, 10, or 50 micrograms human GH (hGH) for 9 days produced a dose-dependent increase in CaBP. At the highest hGH dose (50 micrograms), the content of CaBP was increased 2- to 4-fold to intact levels. By comparison, the increases in total protein and alkaline phosphatase were small (25% to 40% and 80% to 90%, respectively). The induction of CaBP preceded the other protein responses; half-maximal increases in CaBP occurred after 2 days of hGH (50 micrograms/day) treatment before statistically significant changes in total protein or alkaline phosphatase activity. hGH was the most potent pituitary hormone tested; ovine TSH (25 mU/day) had no effect on CaBP, and ovine PRL (10 or 50 micrograms/day) increased CaBP by only 25-27% (P = 0.014). These studies indicate that the vitamin D-dependent intestinal CaBP in hypophysectomized rats is regulated by GH and provide further evidence that the pituitary may be involved in regulating vitamin D-dependent intestinal adaptations.     

Vitamin D-dependent calcium-binding protein in rat brain: biochemical and immunocytochemical characterization

Immunoreactive calcium-binding protein (CaBP) has been characterized in rat brain both biochemically and immunocytochemically. In this study antiserum to chick CaBP was used to characterize this protein and to describe its distribution in neurons and fibers of the rat fore- and midbrain. Immunostaining in neuronal elements was judged specific for this protein by the absence of staining in tissue sections after adsorption of the antiserum with either chick intestinal CaBP or the 28,000-dalton fraction from rat brain, but not with other molecular weight fractions with calcium-binding activity. Immunoreactive CaBP was found to have a widespread distribution throughout the central nervous system, and was present in most but not all major neuronal cell groups and fiber tracts. The protein was limited primarily to neuronal elements and some ependymal cells, and was absent in glia and blood vessels. The proportion of immunoreactivity in neuronal perikarya and fibers varied among nuclei and within a given structure at different rostral-caudal levels. Immunoreactivity was prominent in neocortex, hippocampal formation (primarily in CA1 and granular cells of the dentate gyrus), hypothalamus, and amygdala. These areas are responsible for the regulation of a variety of pituitary hormones, and several bind steroids. Immunoreactive CaBP was also a major constituent of nonlimbic system pathways. The widespread distribution of immunoreactive CaBP in the central nervous system suggests that CaBP and the vitamin D endocrine system may play a significant role in the regulation of mammalian brain function.     

The 28-kDa vitamin D-dependent calcium-binding protein has a six-domain structure.

Vitamin D-dependent 28-kDa calcium-binding protein (CaBP28) cDNA clones were isolated from a chicken intestinal library. The nucleotide sequence analysis of the CaBP28 cDNA shows an open reading frame of 786 nucleotides, coding for a 262-amino acid 30.167-kDa protein. Interestingly, the protein contains six repeats of a domain with the feature of a calcium-binding site. In two of the six domains, oxygen-containing amino acids important for the positioning of calcium are absent, suggesting that these two sites have lost their calcium-binding capability and might have adopted a new function in evolution. In the chicken intestine, three different sized species of CaBP28 mRNA (2.0, 2.8, and 3.1 kilobases) are detected. Primer extension and S1 nuclease mapping show that the three CaBP28 mRNA species share a common 5' end but differ in the length of their 3' noncoding sequence. A similar triplet of CaBP28 mRNAs is identified in the rat kidney by the chicken probe, showing an interspecies conservation of the CaBP28. In the rat intestine, however, no CaBP28 mRNA could be detected. Instead, a vitamin D-dependent 9-kDa CaBP (CaBP9) is expressed, with an mRNA size of approximately equal to 0.7 kilobase that does not cross-hybridize with the CaBP28 probe. This indicates that the CaBP28 and CaBP9 are the product of two independent genes.     

The distal nephron of rat kidney: a target site for glucagon.

Glucagon-sensitive adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activity was measured in nine different portions of the rat nephron. Each sample contained a single piece of tubule isolated by microdissection from collagenase-treated kidney tissue. As compared to basal activity, 1 microM porcine glucagon stimulated adenylate cyclase 60-fold in the medullary portion and 40-fold in the cortical portion of the thick ascending limb, 23-fold in the early distal convoluted tubule, 11-rold in the cortical collecting tubule, and 8-fold in the medullary collecting tubule. No stimulation was observed in proximaly tubules and thin segments of the loop of Henle. Half-maximal stimulations were obtained with about 10 nM glucagon in the responsive nephron portions.     

Membrane-associated vitamin D-induced calcium-binding protein (CaBP): quantification by a radioimmunoassay and evidence for a specific CaBP in purified intestinal brush borders

The vitamin D-induced intestinal calcium-binding protein (CaBP) was quantitated in membranous components of the intestinal mucosa by a specific and sensitive RIA. Inclusion of detergent (Triton X-100) in extraction buffer and in the RIA system was required to release and measure membrane-associated CaBP. Purified brush borders were shown to contain CaBP with a specific activity (micrograms per mg protein) about 12% of that in the total homogenate. By transferring proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose blots electrophoretically, CaBP was immunologically detected in brush borders from vitamin D3-treated chicks, but not in those from vitamin D3-deficient chicks. CaBP was also detected in isolated brush border membrane vesicles by the gel electrophoresis-blot transfer technique. Brush border CaBP was inaccessible to proteolytic hydrolysis by trypsin unless trypsinized in the presence of detergent. CaBP-binding substances were found to be present in purified brush borders, using the gel overlay technique. A specific binding protein with a mol wt in the range of 50,000-70,000 daltons was identified, as well as an avid CaBP binder at less than 14,000 mol wt. These observations provide evidence for the association of a significant fraction of total intestinal CaBP with brush borders in vivo, which might have physiological relevance.     

Evidence indicating that renal tubular metabolism of leptin is mediated by megalin but not by the leptin receptors

Leptin is secreted by adipocytes and is a circulating factor that regulates food intake and energy expenditure. Its serum level is elevated in patients with renal failure and has been suggested to be associated with malnutritional factors in these patients. Leptin has been suggested to be primarily metabolized by the kidneys, although the precise molecular mechanisms are not known. The purpose of this study was to determine the nephron segments and potential receptors involved in renal leptin metabolism. To determine the segment involved in leptin uptake, we performed histoautoradiography of kidney sections obtained from rats that had been injected iv with (125)I-leptin. The ability of megalin, a multiligand endocytic receptor in the proximal tubules, to bind and endocytose leptin was examined by ligand blotting analysis, quartz-crystal microbalance, and degradation assays using megalin-expressing rat yolk sac L2 cells. Immunohistochemistry was performed to localize leptin receptors (LEP-R) in the rat kidney using two antibodies that recognize different epitopes on the LEP-R proteins. Circulating (125)I-leptin was filtered by glomeruli and internalized by proximal convoluted tubules. Megalin bound leptin in the presence of Ca(2+) and mediated its cellular internalization and degradation. On immunohistochemistry, LEP-R were localized in the proximal straight tubules, loops of Henle, distal tubules, and collecting ducts. In conclusion, circulating leptin was filtered by glomeruli and taken up by proximal convoluted tubules, where megalin likely mediates its binding and uptake. The localization of LEP-R suggests that they are not primarily involved in leptin metabolism in the proximal tubules.     

Differential regulation by 1,25-dihydroxyvitamin D3 of calbindin-D9k and calbindin-D28k gene expression in mouse kidney

The mouse kidney is a unique tissue since both vitamin D-dependent calcium binding proteins (calbindin-D9k and calbindin-D28k) are present in the same cells of the distal convoluted tubule. We have used specific complementary DNAs to mouse calbindin-D9k and mouse calbindin-D28k and Northern and slot blot analyses in order to obtain a better understanding of the regulation of two different molecular expressions of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] action in the same cells. Both calbindins were found to be regulated developmentally in a similar manner (an increase in gene expression between birth and 1 week of age, coinciding with nephron differentiation, and a peak at 3 weeks of age). However, the time course of response of the messenger RNA of each calbindin to 1,25(OH)2D3 was markedly different. The peak of induction of renal calbindin-D28k mRNA was at 12 h after a single injection of 1,25(OH)2D3 (200 ng/100 g body wt) to vitamin D-deficient mice, and a decrease was observed at 24 h (similar to the time course of response of other steroid-regulated genes). Interestingly, unlike calbindin-D28k, a delayed response of renal calbindin-D9k mRNA to 1,25(OH)2D3 was observed (the peak of induction was at 24 h after 1,25(OH)2D3 administration). Both genes in mouse kidney did not respond to glucocorticoids, although a dose-dependent decrease (12-86%) of mouse intestinal calbindin-D9k mRNA was observed after dexamethasone treatment, suggesting tissue-specific multiple steroid interactions in the regulation of calbindin gene expression. The finding of a different time course of regulation of each calbindin by 1,25(OH)2D3 suggests that different factors may be regulating the expression of the two different calbindins in mouse kidney and that elucidation of these control mechanisms should provide new insight concerning 1,25(OH)2D3-regulated gene expression.     

Calcium-binding protein of chorioallantoic membrane: identification and development expression.

A calcium-binding protein (CaBP) which has a molecular weight of 100,000 and an isoelectric point of 8.0 has been isolated from the chorioallantoic membrane (CAM) of the chick embryo. Expression of the CaBP occurs simultaneously with the onset of calcium absorption from the egg shell by the CAM, and the temporal increase in CaBP activity is coincident with calcium deposition in the embryo. The CAM CaBP differs in properties from the CaBPs of the adult organs, including the vitamin-D-dependent protein of intestine.     

Molecular cloning of a vitamin D-dependent calcium-binding protein mRNA sequence from chick intestine.

We have constructed a recombinant cDNA library to facilitate study of the genomic actions of vitamin D3 and its hormonally active metabolite 1,25-dihydroxyvitamin D3 in initiation of the de novo biosynthesis of a 28,000-dalton vitamin D-dependent calcium binding protein (CaBP) present in chick intestine. The recombinant plasmids were prepared by the homopolymeric tailing and hybridization method using as a starting template poly(A)-enriched mRNA obtained from the intestinal mucosa of vitamin D3-replete (+D) chicks. Screening of 9,516 clones in this library was effected by using a comparative in situ colony hybridization technique with two [32P]cDNA probes; these probes were prepared from total poly(A)-RNA from chick intestinal mucosa of vitamin D-deficient (-D) chicks and a poly(A)-RNA specifically enriched for chick intestinal CaBP mRNA by immunoprecipitation of polysomes derived from vitamin D-replete (+D) chicks. We identified 26 clones that consistently displayed a significantly increased hybridization signal when comparing the -D vs. CaBP-enriched probe. Further evaluation of these clones by hybrid-selected translation showed the presence of CaBP-specific sequences. By "RNA gel" analysis of poly(A)-RNA, three independent mRNA species were found to hybridize to a CaBP clone; none of these RNA species were found in -D poly(A)-RNA. With this comparative colony hybridization procedure, we were able to identify CaBP-specific clones corresponding to a mRNA that is 0.1% of the total poly(A)-mRNA. The differential colony hybridization procedure using an enriched vs. a nonenriched probe should be of value in screening for other cDNA clones complementary to rare mRNA species.     

Distribution of vitamin D-dependent calcium-binding protein messenger ribonucleic acid in rat placenta and duodenum

The vitamin D-dependent calcium-binding protein (CaBP), cholecalcin or calbindin, is one of the best documented molecular expressions of 1,25-dihydroxyvitamin D, the hormonal metabolite of vitamin D. In this report, DNA/RNA hybridization assays have been used to examine cholecalcin (CaBP) mRNA production in the placenta and duodenum of 21-day pregnant rats. A cloned CaBP cDNA which codes for the rat intestinal 9000 mol wt cholecalcin (9KCaBP) was radiolabeled and used in hybridization assays to explore 1) the size and relative quantities of CaBP mRNA extractable from placenta and duodenum by molecular hybridization, and 2) the localization and quantification, by in situ hybridization histochemistry, of CaBP mRNA in specific cells in rat placenta and duodenum. Northern hybridization studies show that the [32P]cDNA sequence hybridizes to a single 500- to 600-nucleotide species in the placenta as in the duodenum and, therefore, demonstrate identical 9KCaBP mRNA processing in both tissues. Dot blot hybridization studies show that the concentration of 9KCaBP mRNA was greatest in the duodenum, while that of the inner (fetal) placenta was about 50% the duodenal level. Considerably less CaBP mRNA was found in the outer (maternal) placenta. The observed differences in 9KCaBP mRNA levels correlate well with the in vivo variations in 9KCaBP concentrations. In situ hybridization histochemistry using [3H]cDNA reveals that 9KCaBP mRNA visualized by silver grains was concentrated in the inner placenta over the cytoplasm of syncytial cells in the trophoblastic epithelium of the labyrinth and much less frequently in the cells of the outer placenta. In the duodenum, 9KCaBP mRNA was found only in the absorptive epithelial cells from the crypt region to the upper part of the villi. The silver grains were distributed throughout the cytoplasm of the columnar cells; they were densest in the perinuclear region and rarest in the nuclear region. The concentration was greater in the cells at the villous tips than in those of the crypts. This difference in 9KCaBP mRNA levels correlates well with the distribution of the protein itself along the villi. CaBP mRNA quantities detected by hybridization histochemistry showed greater labeling in the syncytial cells of the trophoblastic epithelium of the labyrinth than in the absorptive epithelial cells of the upper part of the villi (200% less), indicating accumulation of CaBP mRNA at a greater rate in the trophoblastic epithelium than in the absorptive epithelial cells. These results indicate that in the rat, 9KCaBP is synthesized in both the absorptive cells of the duodenum and the cells of the trophoblastic epithelium of the placenta.     

Effect of calbindin D 28K on sodium transport by the luminal membrane of the rabbit nephron.

We previously reported that in the rabbit, the vitamin D-dependent calcium binding protein 28K (CaBP 28K) increases calcium (Ca2+) transport in the distal tubule by opening a high affinity Ca2+ channel in the luminal membrane. Since Na+ and Ca2+ transports are interdependent in this membrane, we questioned whether the calbindin has any influence on Na+ transport. Luminal membranes from rabbit proximal and distal tubules were purified and 22Na uptake by the membrane vesicles was measured using the rapid filtration technique. The vesicles were loaded with 280 mM mannitol and 20 mM Tris-Hepes pH 7.4, with either 3 microM CaBP or the carrier. Incubation medium contained 1 mM 22NaCl, 278 mM mannitol, and 20 mM Tris-Hepes pH 7.4. The presence of 3 microM CaBP 28K in the distal luminal membrane vesicles increased the 0.5 mM Ca2+ uptake from 0.91 +/- 0.21 to 1.84 +/- 0.33 pmol/microg/10 s (P < 0.01) and decreased 1 mM Na+ uptake from 0.62 +/- 0.15 to 0.27 +/- 0.08 pmol/microg/10 s (P < 0.05). A similar decrease of Na+ uptake was observed in proximal luminal membrane experiments. The effect on Na+ uptake by the distal membrane was dose-dependent with a IC50 of 4.5 microM. Addition of 2 mM Ca2+ to the incubation medium decreased 1 mM Na + uptake from 0.62 +/- 0.15 to 0.49 +/- 0.12 pmol/microg/10 s (P < 0.05), but did not influence the effect of CaBP 28K on Na+ uptake. Experiments performed in the presence and absence of ethyl isopropyl amiloride (EIPA) suggest that the effect of calbindin involves the Na+/H+ exchanger activity.