Renal Calcium-binding Protein Research Articles

Intestinal Vitamin D Receptor Is Required for Normal Calcium and Bone Metabolism in Mice

Vitamin D receptor (VDR)-knockout mice develop severe hypocalcemia and rickets, accompanied by disruption of active intestinal calcium absorption. To specifically study the effects of VDR in intestinal calcium absorption, we investigated whether restoration of intestinal VDR is sufficient to recover the phenotype of VDR-knockout mice. We generated mice with intestine-specific transgenic expression of human VDR and crossed them to VDR knockout mice. The intestine, kidney, and bone phenotypes of the VDR- knockout mice with intestine-specific expression of human VDR (knockout/transgenic [KO/TG]) were analyzed. Transgenic expression of VDR in the intestine of VDR-knockout mice normalized duodenal vitamin D-regulated calcium absorption as well as vitamin D-regulated calcium binding protein D9k and TRPV6 gene expression in the duodenum and proximal colon. As a result, animal growth and the serum levels of calcium and parathyroid hormone were normalized in KO/TG mice. Other phenotypes were revealed when calcium metabolism was normalized in KO/TG mice: serum 1,25 dihydroxyvitamin D levels were higher in KO/TG mice than normal mice owing to reduced renal expression of the vitamin D-degrading enzyme CYP24, urinary calcium excretion was higher and associated with lower renal calcium binding protein D9k and calcium binding protein D28k than normal mice, and bone density and volume increased in KO/TG compared with normal mice owing to increased mineral apposition rate and osteoblast number. Intestinal VDR and vitamin D-regulated intestinal calcium absorption are critical for controlling whole-body calcium metabolism in growing mice. Normalizing intestinal calcium absorption and metabolism reveals essential roles for VDR in control of bone formation and renal control of serum 1,25(OH)2D and urinary calcium excretion.

Effects of methylprednisolone and uremia on renal and intestinal calbindin-D in the rat

The effects of glucocorticoids on renal and intestinal calcium binding protein (calbindin-D 28K and calbindin-D 9K) were examined in normal and uremic rats. Chronic uremic rats and normal controls were treated with either methylprednisolone (MP) 1.3 mg/kg/d or isotonic saline given as a continuous intraperitoneal infusion for 1 week before sacrifice. Renal calbindin-D 28K was measured by rocket immunoelectrophoresis and intestinal calbindin-D 9K was measured by an enzymelinked immunoadsorbent assay. Methylprednisolone treatment of chronic uremic rats increased plasma phosphate levels ( P < 0.05), but plasma calcium and 1, 25-dihydroxyvitamin D 3 were unchanged in all groups. MP treatment did not affect the renal calbindin-D 28K in either normal or uremic rats. In normal rats, MP treatment reduced intestinal calbindin-D 9K by 28% when compared to placebo ( P < 0.05). In contrast, chronic uremia increased renal calbindin-D 28K by 51% and 38% ( P < 0.001) in placebo and MP treated uremic rats, respectively, while intestinal calbindin-D 9K was unchanged. Thus, MP treatment and chronic uremia induced different changes in renal and intestinal calbindin-D of the rat suggesting that different mechanisms are involved in the regulation of these vitamin D dependent proteins.

Intestinal and renal calcium-binding protein in rats with experimental short bowel syndrome

Intestinal calcium-binding protein (calbindin-D9K) and renal calcium-binding protein (calbindin-D28K) levels were measured by enzyme-linked immunoadsorbent assays in rats with short bowel syndrome induced by resection of about 85% of the small intestine. Rats with short bowel syndrome had significantly lower mucosal concentrations of calbindinD9K (P < 0.001) and a parallel reduction of both intestinal calcium absorption (P < 0.001) andp-1,25-dihydroxyvitamin D (P < 0.01) in spite of a general hypertrophy of the duodenal mucosa. Interestingly, the renal concentration of calbindin-D28K was significantly elevated (P < 0.05) in rats with short bowel syndrome, a change apparently related to factors independent of vitamin D.

Renal and intestinal calcium-binding proteins in normal and uraemic rats

The effect of chronic uraemia on the concentrations of the 28 kDa renal and 9 kDa intestinal calcium-binding proteins (calbindin-D28K and calbindin-D9K) was investigated in rats. Calbindin-D9K was measured by a competitive enzyme-linked immunoadsorbent assay and calbindin-D28K by rocket immunoelectrophoresis. Chronic uraemia was induced by 5/6 nephrectomy and the results were compared to sham-operated animals. Rats were fed on a diet containing 0.9% calcium and 1.2% phosphorous. Plasma creatinine and plasma urea were elevated in the nephrectomized rats (p less than 0.001), while plasma-1,25-dihydroxycalciferol vitamin D and fractional calcium absorption were unchanged. Plasma parathyroid hormone was significantly elevated in the uraemic rats. The concentration of calbindin-D28K in renal tissue was increased (p less than 0.001) in rats with chronic uraemia and a direct correlation was found between renal calbindin-D28K and plasma urea (p less than 0.05). Intestinal calbindin-D9K correlated inversely with plasma creatinine (p less than 0.05), but the mean level of calbindin-D9K was unchanged in this model of moderate chronic uraemia. Thus, different regulatory mechanisms control levels of calbindin-D9K and calbindin-D28K.

CaBPr facilitates intracellular diffusion for Ca pumping in distal convoluted tubule.

The system of renal Ca transport in the rat is modeled in terms of two classes of processes: a nonsaturable flux that predominates in the proximal tubule, and an active, vitamin D-dependent flux with major expression in the distal convoluted tubule. There transport is against an electrochemical gradient, with much of the efflux probably mediated by the Ca/Mg-ATPase. Calculations of the rate of free Ca diffusion in tubular cells indicate that an unaided flux would be only one-seventy-seventh of that found experimentally. It is suggested that the vitamin D-induced renal calcium binding protein, CaBPr, Mr approximately 28,000, in raising total cellular calcium by three orders of magnitude, increases the transcellular Ca flux and thus the free intracellular Ca ion concentration at the basolateral pole, allowing the Ca/Mg-ATPase to function near its maximum. Analysis of the rate of nonsaturable Ca flux throughout the kidney tubule suggests a paracellular pathway via bulk flow, following water that is driven osmotically. Evaluation of whole animal data in terms of these two classes of calcium fluxes indicates that our model is consistent with experimental observations and assigns a functional role to active calcium transport.

Effect of variations in dietary calcium on renal and intestinal calcium-binding proteins.

This study was designed to determine if protein-induced calciuria was related to alterations in the intestinal and renal calcium-binding proteins (CaBP). Weanling Sprague-Dawley rats were fed diets containing low, normal, or high Ca and protein diets for 2 wk. Twenty-four-h urine and fecal samples were collected before the termination of the study. Plasma, kidney, duodenum, ileum, and femur samples were obtained for selected mineral and CaBP analyses. Growth was significantly depressed on the low protein diets and this was independent of Ca levels. Hypercalcemia, hypercalciuria, and increased renal CaBP were associated with the high Ca intakes but not with the high protein diets. It is suggested that in conditions where Ca intakes are high, the renal CaBP has a role in Ca excretion and responds to changes in Ca concentrations occurring in the distal tubule. No loss of femur Ca was seen in rats on the high protein diets.

1,25-Dihydroxyvitamin D3 induces the synthesis of vitamin D-dependent calcium-binding protein in cultured chick kidney cells.

A 28,000 mol wt vitamin D-dependent calcium-binding protein (CaBP), first isolated from avian intestine, has recently been shown to exist in kidney and other tissues. To study the mechanism regulating the production of renal CaBP, we used primary cultures of chick kidney cells as an in vitro model. Renal cortical tubules isolated from vitamin D-deficient chicks were grown in serum-free, hormone-supplemented medium. Confluent cells were epithelioid and expressed CaBP, as demonstrated by immunocytochemistry and a specific RIA. 1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] increased cellular CaBP content in a dose-dependent manner (10(-10)-10(-7) M) from basal concentrations of 50-240 ng/mg protein to maximal concentrations of 600-1200 ng/mg protein 48 h after dosing. Cycloheximide (2 microM) inhibited 1,25-(OH)2D3 induction of CaBP, indicating that the mechanism requires new protein synthesis. Western blotting of cell extracts confirmed the identity of the inducible protein as the 28,000 mol wt CaBP. 25-Hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were effective but somewhat less potent inducers of CaBP, whereas vitamin D3 was without significant effect. The activities of 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 are attributed to their rapid conversion by kidney cells to 1 alpha-hydroxylated metabolites. The dose-response profiles for two additional bioresponses in these cells, namely induction of 24-hydroxylase and inhibition of 1 alpha-hydroxylase activity, were comparable to those for CaBP induction. To our knowledge, this is the first description of a cell culture system that exhibits 1,25-(OH)2D3-inducible CaBP in vitro. This model should permit the study of certain aspects of the physiology of 1,25-(OH)2D3 and CaBP in kidney that are not possible in vivo.

Calcium‐binding protein (28,000 Mr calbindin‐D28k) in kidneys of the bullfrog Rana catesbeiana during metamorphosis

A protein of approximately 28,000 relative molecular mass (Mr) cross-reacting with antiserum against the 28,000-Mr rat renal calcium-binding protein (calbindin-D28k) has been localized in the kidney of a salientian amphibian, Rana catesbeiana. Cells reactive for calbindin-D28k were found in the distal tubule at all stages of metamorphosis by the unlabeled antibody peroxidase-antiperoxidase technique. Adult kidneys appeared to have more calbindin-D28k-positive cells. The renal corpuscle, neck, and proximal tubule were negative. An immunoreactive 28,000-Mr band that comigrated with the band of calbindin-D28k was visualized by the immunoblot technique. The finding of the 28,000-Mr calbindin-D in the anamniotic kidney demonstrates that this calcium-binding protein (CaBP) is phylogenetically older than our previous studies of higher vertebrates had revealed (Rhoten et al., 1985). Although the function of calbindin-D28k in the distal nephron is unknown, this CaBP can now be presumed to have functional significance in the mesonephric as well as the metanephric kidney.

Specific in vitro activation of Ca,Mg-ATPase by vitamin d-dependent rat renal calcium binding protein (calbindin D 28K)

The vitamin D-dependent, calcium-binding protein from rat kidney, calbindin D28k (renal CaBP) specifically stimulates Ca,Mg-ATPase activity of human erythrocyte plasma membranes in a dose-dependent, calcium-sensitive manner. This stimulation was about two-fold compared to a three-fold stimulation by calmodulin. The effect was specific since other calcium-binding proteins and low molecular weight proteins did not stimulate Ca,Mg-ATPase activity. Renal CaBP did not stimulate cyclic nucleotide phosphodiesterase at concentrations greater than those which stimulated Ca,Mg-ATPase activity. This is the first report of a specific in vitro effect of renal CaBP on an enzyme system.

Vitamin D-dependent calcium-binding protein of mouse yolk sac. Biochemical and immunochemical properties and responses to 1,25-dihydroxycholecalciferol.

The present studies were performed to further characterize a mouse yolk sac protein which is similar or identical to the vitamin D-dependent intestinal calcium-binding protein (CaBP). Yolk sac protein and purified rat intestinal CaBP displayed full identity upon immunodiffusion (Ouchterlony) using antiserum to the rat intestinal CaBP. Immunoreactive CaBP in yolk sac homogenates eluted from gel permeation columns with the low molecular weight peak of 45Ca2+ binding (Chelex assay), and the electrophoretic mobility of the protein was markedly increased by EDTA. On days 11-13 of gestation, the concentrations of immunoreactive CaBP in yolk sac were 4-5-fold higher than in placenta; by days 16-17, the concentrations in yolk sac and placenta were similar. Incubation of yolk sac with [3H]leucine demonstrated synthesis of immunoprecipitable [3H]CaBP. A single band of 3H-labeled protein was seen on sodium dodecyl sulfate gel electrophoresis of the immunoprecipitate. This protein co-migrated with radioactive placental CaBP with an apparent Mr of 10,050. Addition of 1,25-dihydroxycholecalciferol (calcitriol) to organ culture media with or without serum increased the amount and concentration of CaBP in yolk sac (p less than 0.001) at 48 h. CaBP synthesis in yolk sac appeared to be independent of calcitriol concentrations in the maternal circulation since injection of the hormone into the maternal compartment produced no change in yolk sac CaBP despite increases of maternal intestinal and renal CaBP. These studies demonstrate that yolk sac immunoreactive CaBP is synthesized in yolk sac and has an apparent molecular size and calcium-binding properties characteristic of mammalian vitamin D-dependent calcium-binding proteins. The in vitro response of yolk sac CaBP to calcitriol is the first evidence of a vitamin D effect on the fetal membranes and suggests one function for calcitriol receptors in these tissues.

Immunocytochemical Localization and Immunological Characterization of Vitamin D-Dependent Calcium-Binding Protein in the Bullfrog Cerebellum

Antiserum prepared against rat renal calcium-binding protein (CaBP) was used with the unlabeled antibody peroxidase-antiperoxidase (PAP) technique to localize the 28,000 molecular weight CaBP in the cerebellum of the bullfrog, Rana catesbeiana. Whole brains of premetamorphic tadpoles and adults were fixed in Bouin's solution for 2 or 24 h and embedded in paraffin. 8-microns parasagittal sections were prepared and treated by the PAP method. Purkinje cells of the cerebellum in tadpoles and adults were specifically stained for CaBP. In the premetamorphic corpus cerebelli, the stained Purkinje cells corresponded to the precociously developed Purkinje cells described previously. In the auricular lobe region of the cerebellum mature Purkinje cells were stained. In addition, smaller stained cells were seen. The latter were presumed to be immature Purkinje cells that would mature at the time of metamorphosis. Immunoblot procedure demonstrated cross-reactivity for the ranid brains in the 28,000 molecular weight region. This immunoreactive band comigrated with the immunoreactive band observed with purified rat renal CaBP. Although the exact functional significance of CaBP is unknown at this time, our immunocytochemical and immunological findings indicate that CaBP is an excellent marker for studies of Purkinje cell maturation.

Cell-Free Translational Analysis of Messenger Ribonucleic Acid Coding for Vitamin D-Dependent Rat Renal Calcium-Binding Protein*

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been found to exert its effects in a manner entirely analogous to that of other steroid hormones and is known to induce the synthesis of a calcium-binding protein (CaBP). The effects of 1,25-(OH)2D3 and dietary alteration on genomic expression in rat kidney were studied by in vitro translation of poly(A+)-containing RNA and by immunoprecipitation. Poly(A+)RNA from rat kidneys was translated in a rabbit reticulocyte lysate system in the presence of [35S]methionine, and the renal CaBP mRNA translation product was identified and quantitated by specific immunoprecipitation. Total translation products and specific immunoprecipitable products were visualized on sodium dodecyl sulfate-gels, followed by fluorography. After the addition of affinity purified rat renal CaBP antiserum to the 35S-labeled translation products, only one protein band, electrophoretically indistinguishable from that of purified renal CaBP (mol wt, 28,000), was observed. When 10 micrograms purified renal CaBP were added to the translation product before addition of the antiserum, immunoprecipitation of the 35S-labeled 28,000 mol wt protein was not observed. A comparison of the peptides produced after limited digestion with trypsin of 125I-labeled CaBP and [3H]tyrosine-labeled translation product indicated a good coincidence of peaks from purified 125I-labeled CaBP and the immunoprecipitated translation product, suggesting that the immunoprecipitated translation product is indeed vitamin D-dependent renal CaBP. When 100 ng 1,25-(OH)2D3 were injected for 7 days to 8-week-old vitamin D-deficient rats, there was a 4-fold increase in CaBP mRNA levels in the kidney (quantitated by densitometry of immunoprecipitates analyzed on sodium dodecyl sulfate-polyacrylamide gels). This increase in mRNA was accompanied by a corresponding increase in the concentration of renal CaBP, as measured by RIA, thus establishing a bridge between CaBP and the putative transcriptional effect of 1,25-(OH)2D3 in rat kidney. Similarly, both the concentration of renal CaBP and renal CaBP mRNA levels increased 4-fold in rats fed low phosphorus diets, increased 2-fold in rats fed low calcium diets, and decreased 67% in rats fed low sodium diets, providing evidence that the nutritional induction or decrease in renal CaBP is accompanied by a corresponding alteration in the concentration of its specific translatable mRNA. These results are consistent with a transcriptional control mechanism for the induction of renal CaBP.

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) ( M r 28,000), two other kidney proteins of M r 16,500 and M r 18,000 were significantly enriched in their contents in the vitamin D-replete rat. Partial characterization of the M r 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 M r 16,500 and M r 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 D 3 was increased synthesis of the M r 28,000 protein (3.2- to 4.6-fold increase compared to -D rats, P < 0.001). Synthesis of a M r 54,500 protein increased by 1.3- to 1.5-fold ( P < 0.05) and [ 35S]methionine incorporation into a M r 66,000 protein decreased by 1.2- to 1.3-fold ( P < 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 M r 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) 2D 3 can influence the expression as well as the suppression of other specific kidney proteins.

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.

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.

Vitamin D-dependent calcium-binding protein in rat kidney. Purification and physiocochemical and immunological characterization.

A vitamin D-dependent Mr = 28,000 calcium-binding protein (CaBP) has been isolated from rat kidney. Rat renal CaBP was purified from heat-treated post-mitochondrial supernatants by gel filtration on Sephadex G-100 followed by preparative gel electrophoresis. The specific properties and characteristics of the protein were examined. Rat renal CaBP was found to have a pI of 4.8 and showed increased electrophoretic mobility during polyacrylamide gel electrophoresis in the presence of 1 mM EDTA. Amino acid analysis of renal CaBP revealed a high content of glutamic and aspartic acids and a low level of methionine, histidine, cysteine, and tyrosine, similar but not identical to chick intestinal CaBP. Circular dichroism studies indicated that the alpha-helical content of renal CaBP was of the order of 31% and was changed to a minor degree by the addition of calcium. A study of the thermal stability indicated that renal CaBP is heat-stable up to 75 degrees C. Binding studies utilizing the technique of equilibrium dialysis established a dissociation constant of 2.1 X 10(-6) M and binding sites with a capacity of approximately 4 mol Ca2+/mol of CaBP. Immunologically, using Ouchterlony immunodiffusion, a precipitin line which joined with total coalescence with that due to purified renal CaBP was observed with extracts of rat brain and kidney but not with extracts of rat parathyroid, skeletal muscle, myocardium, bone, pancreas, intestine, and liver. In summary, these studies present the first purification and characterization of vitamin D-dependent rat renal calcium-binding protein. The antibody as well as the protein will be useful for the development of a radioimmunoassay and for the determination of the molecular mechanisms of induction of renal calcium-binding protein.

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.

Calmodulin and rat vitamin D-dependent calcium-binding proteins: Biochemical and immunochemical comparison

Purified vitamin D-dependent rat intestinal ( M r 10,000) and rat renal ( M r 28,000) calcium-binding proteins (CaBPs) have been compared to vertebrate calmodulin, and the vitamin D-dependent CaBPs have been found to be distinct from calmodulin by biochemical and immunochemical criteria. Rat renal and rat intestinal CaBPs do not stimulate 3′,5′-cyclic nucleotide phosphodiesterase, do not compete with iodinated calmodulin for binding to phenothiazine-Sepharose conjugates, do not cross-react immunochemically, and do not contain N ϵ-trimethyllysine. In addition, although calmodulin exhibits a characteristic calcium-dependent mobility shift on polyacrylamide gels in the presence of sodium dodecyl sulfate, a similar mobility shift is not observed for the vitamin D-dependent CaBPs. Immunocytochemically, calmodulin has a widespread localization in the kidney, whereas CaBP is present specifically in the distal tubules of the kidney. These localizations suggest a specialized role for CaBP in the kidney. Thus, although the vitamin D-dependent CaBPs and calmodulin are similar in that they are small, acidic, calcium-binding proteins, these two classes of proteins are biochemically and immunochemically distinct.

Evidence that sodium deprivation influences vitamin D dependent rat renal calcium binding protein

In order to provide some insight concerning the role of renal calcium binding protein (CaBP) in the functioning of the mammalian kidney, the response of renal CaBP to dietary alterations was examined. Three week old rats were fed diets deficient in calcium, phosphorous or sodium supplemented with vitamin D for a four week period. The specific activity of renal CaBP (as measured by the chelex resin assay; Ca 2+ bound protein/Ca 2+ bound resin per mg protein) in the 28,000 M r region was found to increase four fold in rats fed the low phosphorus diet and two fold rats fed the low calcium diet when compared to rats fed the control diet. Renal CaBP/mg protein from rats fed the low sodium diet decreased 50% from the control values. Changes in renal CaBP were confirmed by polyacrylamide gel analysis of the 28,000 M r fraction by densitometric tracing using a purified CaBP marker. The greater response to dietary phosphorus restriction suggests that renal CaBP may be regulated by a mechanism different from that of intestinal CaBP. The decrease in renal CaBP in rats fed the low sodium diet suggests for the first time that sodium is required for vitamin D dependent distal tubular calcium transport processes.

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

Vitamin D 3 and its biologically active metabolite 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D 3-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 D 3 or 1,25(OH) 2D 3 to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h −1 ( t 1 2 = 29 h ) and that for renal CaBP was 0.019 h −1 ( t 1 2 = 36 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) 2D 3 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) 2D 3. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D 3-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) 2D 3 to each target organ.