Renal calbindin-D28k Research Articles

Low systemic exposure and calcemic effect of calcipotriol/betamethasone ointment in rats with imiquimod-induced psoriasis-like dermatitis

Vitamin D3 (VD3) analogues-containing ointments are known to occasionally cause hypercalcemia in psoriasis patients, and the frequency of hypercalcemia is suggested to vary based on the VD3 analogue used. In this study, to address the differences in calcemic effects of VD3-containing ointments, the calcemic effects of marketed VD3-containing ointments, including calcipotriol (Cal), maxacalcitol (Max), tacalcitol (Tac), calcipotriol/betamethasone dipropionate (Cal/BDP) and maxacalcitol/betamethasone butyrate propionate (Max/BBP) ointments, were evaluated in a rat model of imiquimod-induced dermatitis. The topical application of Tac, Max and Max/BBP ointments, but not Cal and Cal/BDP ointments, to the imiquimod-induced skin lesions significantly induced an increase in the serum calcium level compared with the vaseline-treated group. Calcemic effect of VD3 analogues in rats treated with VD3-containing ointments was analyzed by evaluating the expression of vitamin D receptor target genes, such as Cyp24a1, Trpv5 and CalbindinD28k, in the intestine and kidney. Real-time reverse transcription PCR (RT-PCR) analysis showed that the renal and intestinal Cyp24a1 expressions in the Cal- and Cal/BDP-treated groups were significantly lower than those in the Tac-, Max- and Max/BBP-treated groups, suggesting that systemic exposure of VD3 analogues in the Cal- and Cal/BDP-treated groups were lower than those in the other ointment-treated groups. In addition, the renal Trpv5 and CalbindinD28k expressions, calcium-transporting genes, were increased in the Max- and Max/BBP-treated groups compared with the Cal- and Cal/BDP-treated groups. Thus, because of the low systemic exposure of VD3 analogues, Cal and Cal/BDP ointments have lower calcemic effect than the other VD3-containing ointments in rats with psoriasis-like dermatitis.

Effects of a high-sodium diet on renal tubule Ca2+transporter and claudin expression in Wistar-Kyoto rats

BackgroundUrinary Ca2+ excretion increases with dietary NaCl. NaCl-induced calciuria may be associated with hypertension, urinary stone formation and osteoporosis, but its mechanism and long-term effects are not fully understood. This study examined alterations in the expressions of renal Ca2+ transporters, channels and claudins upon salt loading to better understand the mechanism of salt-induced urinary Ca2+ loss.MethodsEight-week old Wistar-Kyoto rats were fed either 0.3% or 8% NaCl diet for 8 weeks. Renal cortical expressions of Na+/Ca2+ exchanger 1 (NCX1), Ca2+ pump (PCMA1b), Ca2+ channel (TRPV5), calbindin-D28k, and claudins (CLDN-2, -7, -8, -16 and −19) were analyzed by quantitative PCR, western blot and/or immunohistochemistry.ResultsFractional excretion of Ca2+ increased 6.0 fold with high-salt diet. Renal cortical claudin-2 protein decreased by approximately 20% with decreased immunological staining on tissue sections. Claudin-16 and −19 expressions were not altered. Renal cortical TRPV5, calbindin-D28k and NCX1 expressions increased 1.6, 1.5 and 1.2 fold, respectively.ConclusionsChronic high-salt diet decreased claudin-2 protein and increased renal TRPV5, calbindin-D28k, and NCX1. Salt loading is known to reduce the proximal tubular reabsorption of both Na+ and Ca2+. The reduction in claudin-2 protein expression may be partly responsible for the reduced Ca2+ reabsorption in this segment. The concerted upregulation of more distal Ca2+-transporting molecules may be a physiological response to curtail the loss of Ca2+, although the magnitude of compensation does not seem adequate to bring the urinary Ca2+ excretion down to that of the normal-diet group.

Characterization of vitamin D-deficient klotho-/- mice: do increased levels of serum 1,25(OH)2D3 cause disturbed calcium and phosphate homeostasis in klotho-/- mice?

Klotho(-/-) mice display disturbed Ca(2+) and vitamin D homeostasis. Renal cytochrome p450 27b1 (Cyp27b1), the enzyme that catalyzes the hydrolysis to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), is increased in klotho(-/-) mice, and a 1,25(OH)(2)D(3)-deficient diet partially normalized Ca(2+) homeostasis in these klotho(-/-) mice. The aim of the present study was to further delineate the interplay between 1,25(OH)(2)D(3) and klotho and their relative contribution to the Ca(2+) homeostasis of klotho(-/-) mice. Double-klotho(-/-)/Cyp27b1(-/-) mice were generated and mice aged 8-12 weeks were housed in metabolic cages to collect 24-h urine. Blood samples were taken and the animals were sacrificed, and the kidney and duodenum tissues were sampled for RNA extraction. The bone was fixed in 10% v/v formalin and analysed by microcomputed tomography (μCT) scans. Klotho(-/-)/Cyp27b1(-/-) mice, like Cyp27b1(-/-) mice, displayed significantly decreased serum total calcium concentrations compared with wild-type mice (1.44 ± 0.03 and 2.25 ± 0.02 mM) along with normal urinary total calcium excretion. Hyperphosphataemia of klotho(-/-) mice normalized to wild-type levels in klotho(-/-)/Cyp27b1(-/-) mice. The mRNA levels of duodenal transient receptor potential vanilloid subtype 6 (TRPV6) and calcium-binding protein-D(9K), and renal calbindin-D(28K) and NCX1 were significantly reduced in the double knockouts compared with wild-type or klotho(-/-) mice. Elevated TRPV5 protein levels in klotho(-/-) mice normalized to wild type in klotho(-/-)/Cyp27b1(-/-) mice, but were decreased in Cyp27b1(-/-) mice. μCT scans showed that klotho(-/-)/Cyp27b1(-/-) mice, as Cyp27b1(-/-) mice, display significant bone hypomineralization and severely decreased bone mass. Klotho(-/-) mice show a reduced bone mass and increased trabecular numbers. Klotho(-/-)/Cyp27b1(-/-) mice resemble Cyp27b1(-/-) mice. Since 1,25(OH)(2)D(3) is absent in these mice, our results imply that Ca(2+) homeostasis in klotho(-/-) mice is affected by their excessive 1,25(OH)(2)D(3) levels.

Soy isoflavones increase bone mineral density without altering markers of whole body vitamin D or calcium metabolism in mice

The soy isoflavone genistein enhances the biological activity of the vitamin D (VD) metabolite 1,25 dihydroxyvitamin D (1,25D) in cultured cells by inhibiting CYP24‐mediated degradation of the hormone. We examined if similar effects can be recapitulated in vivo to improve calcium and bone metabolism. Weanling male B6D2F1 mice were fed AIN‐93G diets containing 50 or 400 IU VD with, or without, Novasoy® 400 soy isoflavone concentrate (added to provide 250 mg genistein/kg diet) until 13 weeks of age. Bone mineral density (BMD) was measured by DEXA. Serum VD metabolites were measured by RIA. Gene expression in duodenum and kidney was measured by RT‐PCR. Higher dietary VD intake increased serum 25 hydroxyvitamin D (25D) from levels viewed as adequate (61 ± 6 nM) to optimal (164 ± 10 nM) for human health but this had no impact on either BMD or molecular indices of calcium metabolism. In contrast, isoflavones increased both serum 25D and femur BMD by 15% (p < 0.01). However, neither serum 1,25D nor tissue gene expression (e.g. duodenal calbindin D9K, TRPV6; renal calbindin D9K or D28K, TRPV5) was influenced by isoflavones. Our data show that the beneficial effect of soy isoflavones on bone that we observed is independent of their ability to modulate whole body VD or calcium metabolism and suggest a direct effect of isoflavones on bone formation or turnover. We thank ADM for Novasoy® 400. Funding: Purdue‐UAB Botanicals Center Pilot Grant.Grant Funding Source: Purdue‐UAB Botanicals Center Pilot Grant

Dietary Calcium and 1,25-Dihydroxyvitamin D3 Regulate Transcription of Calcium Transporter Genes in Calbindin-D9k Knockout Mice

The effect(s) of oral calcium and vitamin D(3) were examined on the expression of duodenal and renal active calcium transport genes, i.e., calbindin-D9k (CaBP-9k) and calbindin-D28k (CaBP-28k), transient receptor potential cation channels (TRPV5 and TRPV6), Na(+)/Ca(2+) exchanger 1 (NCX1) and plasma membrane calcium ATPase 1b (PMCA1b), in CaBP-9k KO mice. Wild-type (WT) and KO mice were provided with calcium and vitamin D(3)-deficient diets for 10 weeks. The deficient diet significantly decreased body weights compared with the normal diet groups. The serum calcium concentration of the WT mice was decreased by the deficient diet but was unchanged in the KO mice. The deficient diet significantly increased duodenal transcription of CaBP-9k and TRPV6 in the WT mice, but no alteration was observed in the KO mice. In the kidney, the deficient diet significantly increased renal transcripts of CaBP-9k, TRPV6, PMCA1b, CaBP-28k and TRPV5 in the WT mice but did not alter calcium-relating genes in the KO mice. Two potential mediators of calcium-processing genes, vitamin D receptor (VDR) and parathyroid hormone receptor (PTHR), have been suggested to be useful for elucidating these differential regulations in the calcium-related genes of the KO mice. Expression of VDR was not significantly affected by diet or the KO mutation. Renal PTHR mRNA levels were reduced by the diet, and reduced expression was also seen in the KO mice given the normal diet. Taken together, these results suggest that the active calcium transporting genes in KO mice may have resistance to the deficiency diet of calcium and vitamin D(3).

Murine TNFΔARE Crohnʼs disease model displays diminished expression of intestinal Ca2+ transporters

Patients suffering from Crohn's disease (CD) show increased incidence of low bone mineral density. Investigating this complication is difficult because the exact etiology of CD remains elusive. Mice carrying a deletion in the tumor necrosis factor (TNF) AU-rich elements (ARE) are reported as a model for human CD and are characterized by elevated TNF-alpha levels and inflammations in the terminal ileum. To evaluate whether these mice have a Ca(2+) handling problem, this study analyzed the Ca(2+) homeostasis in heterozygous TNF(DeltaARE) mice (TNF(DeltaARE/+)) in comparison to wildtype littermates. Beside serum Ca(2+) and vitamin D levels, the expression of Ca(2+) transporters was analyzed in intestine, kidney and bone using quantitative real-time PCR, Western blot and immunohistochemistry. Bone scans were performed to measure bone parameters. Ca(2+) transporters in duodenum (TRPV6, calbindin-D(9K), PMCA1b) and kidney (TRPV5, calbindin-D(28K), NCX1) showed significantly reduced mRNA expression levels in TNP(DeltaARE/+) mice, except for renal TRPV5. In bone, only calbindin-D(9K) mRNA displayed a significant down-regulation. These findings were supported by declined duodenal calbindin-D(9K) and renal calbindin-D(28K) protein values. Likely, this down-regulation of Ca(2+) transporters in TNP(DeltaARE/+) mice is mediated by the 58 +/- 9% reduction in serum 1,25(OH)(2)D(3) levels. Diminished expression of Ca(2+) transporters combined with unchanged serum Ca(2+) levels assumes Ca(2+) loss from bone to compensate for the body's overall Ca(2+) shortage. Indeed, microcomputed tomography scanning demonstrated reduced trabecular and corticol bone thickness and volume in TNF(DeltaARE/+) mice. This finding is further supported by increased total deoxypyridinoline in serum. Our results imply that TNF(DeltaARE/+) mice have a disturbed Ca(2+) homeostasis characterized by reduced duodenal and renal Ca(2+) transporters, diminished 1,25(OH)(2)D(3) levels, and increased bone resorption associated with profound bone abnormalities.

Acid-Base Status Determines the Renal Expression of Ca2+ and Mg2+ Transport Proteins

Chronic metabolic acidosis results in renal Ca2+ and Mg2+ wasting, whereas chronic metabolic alkalosis is known to exert the reverse effects. It was hypothesized that these adaptations are mediated at least in part by the renal Ca2+ and Mg2+ transport proteins. The aim of this study, therefore, was to determine the effect of systemic acid-base status on renal expression of the epithelial Ca2+ channel TRPV5, the Ca2+-binding protein calbindin-D28K, and the epithelial Mg2+ channel TRPM6 in relation to Ca2+ and Mg2+ excretion. Chronic metabolic acidosis that was induced by NH4Cl loading or administration of the carbonic anhydrase inhibitor acetazolamide for 6 d enhanced calciuresis accompanied by decreased renal TRPV5 and calbindin-D28K mRNA and protein abundance in wild-type mice. In contrast, metabolic acidosis did not affect Ca2+ excretion in TRPV5 knockout (TRPV5-/-) mice, in which active Ca2+ reabsorption is effectively abolished. This demonstrates that downregulation of renal Ca2+ transport proteins is responsible for the hypercalciuria. Conversely, chronic metabolic alkalosis that was induced by NaHCO3 administration for 6 d increased the expression of Ca2+ transport proteins accompanied by diminished urine Ca2+ excretion in wild-type mice. However, this Ca2+-sparing action persisted in TRPV5-/- mice, suggesting that additional mechanisms apart from upregulation of active Ca2+ transport contribute to the hypocalciuria. Furthermore, chronic metabolic acidosis decreased renal TRPM6 expression, increased Mg2+ excretion, and decreased serum Mg2+ concentration, whereas chronic metabolic alkalosis resulted in the exact opposite effects. In conclusion, these data suggest that regulation of Ca2+ and Mg2+ transport proteins contributes importantly to the effects of acid-base status on renal divalent handling.

Hypervitaminosis D Mediates Compensatory Ca2+ Hyperabsorption in TRPV5 Knockout Mice

Vitamin D plays an important role in Ca(2+) homeostasis by controlling Ca(2+) (re)absorption in intestine, kidney, and bone. The epithelial Ca(2+) channel TRPV5 mediates the Ca(2+) entry step in active Ca(2+) reabsorption. TRPV5 knockout (TRPV5(-/-)) mice show impaired Ca(2+) reabsorption, hypercalciuria, hypervitaminosis D, and intestinal hyperabsorption of Ca(2+). Moreover, these mice demonstrate upregulation of intestinal TRPV6 and calbindin-D(9K) expression compared with wild-type mice. For addressing the role of the observed hypervitaminosis D in the maintenance of Ca(2+) homeostasis and the regulation of expression levels of the Ca(2+) transport proteins in kidney and intestine, TRPV5/25-hydroxyvitamin-D(3)-1alpha-hydroxylase double knockout (TRPV5(-/-)/1alpha-OHase(-/-)) mice, which show undetectable serum 1,25(OH)(2)D(3) levels, were generated. TRPV5(-/-)/1alpha-OHase(-/-) mice displayed a significant hypocalcemia compared with wild-type mice (1.10 +/- 0.02 and 2.54 +/- 0.01 mM, respectively; P < 0.05). mRNA levels of renal calbindin-D(28K) (7 +/- 2%), calbindin-D(9K) (32 +/- 4%), Na(+)/Ca(2+) exchanger (12 +/- 2%), and intestinal TRPV6 (40 +/- 8%) and calbindin-D(9K) (26 +/- 4%) expression levels were decreased compared with wild-type mice. Hyperparathyroidism and rickets were present in TRPV5(-/-)/1alpha-OHase(-/-) mice, more pronounced than observed in single TRPV5 or 1alpha-OHase knockout mice. It is interesting that a renal Ca(2+) leak, as demonstrated in TRPV5(-/-) mice, persisted in TRPV5(-/-)/1alpha-OHase(-/-) mice, but a compensatory upregulation of intestinal Ca(2+) transporters was abolished. In conclusion, the elevation of serum 1,25(OH)(2)D(3) levels in TRPV5(-/-) mice is responsible for the upregulation of intestinal Ca(2+) transporters and Ca(2+) hyperabsorption. Hypervitaminosis D, therefore, is of crucial importance to maintain normocalcemia in impaired Ca(2+) reabsorption in TRPV5(-/-) mice.

Proteomic Identification and Immunolocalization of Increased Renal Calbindin-D28k Expression in OVE26 Diabetic Mice

Diabetic nephropathy is a common diabetic complication that is associated with alterations in the expression of several renal proteins and abnormal calcium homeostasis. We performed proteomic analysis to screen for global changes of renal protein expression in diabetic kidney. Proteins extracted from the whole kidney of 120-day-old OVE26 (a transgenic model of Type 1 diabetes) and FVB (non-diabetic background strain) mice were separated by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and visualized by SYPRO Ruby staining (n = 5 in each group). Quantitative intensity analysis revealed 41 differentially expressed proteins, of which 30 were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) followed by peptide mass fingerprinting. One of the altered proteins with the greatest magnitude of change was the calcium-binding protein, calbindin-D28k, whose expression was increased 6.7-fold in diabetic kidney. We confirmed the increase in calbindin-D28k expression in diabetic kidney by Western blot analysis. Immunohistochemical study demonstrated that calbindin-D28k expression was markedly increased in tubular epithelial cells of distal convoluted tubules (DCT), collecting ducts (CD), and proximal convoluted tubules (PCT) in diabetic kidney. Calbindin-D28k plays a critical role in maintaining calcium homeostasis. The elevation in renal calbindin-D28k expression in our model may indicate a compensatory mechanism to overcome hypercalciuria in diabetes.

Cyclosporine A-induced hypercalciuria in calbindin-D28k knockout and wild-type mice

It is known that cyclosporine A (CsA) treatment induces high bone-turnover osteopenia and hypercalciuria. It has been proposed that down-regulation of renal calbindin-D28k by CsA results in renal calcium wasting. We investigated the role of the kidney and bone in CsA-induced hypercalciuria in calbindin-D28k knockout (KO) and wild-type (WT) mice. Two sets of experiments were performed. In experiment 1, KO and WT mice were treated with CsA 20 mg/kg/day intraperitoneally (IP) for 7 days. In experiment 2, to eliminate the CsA effect on bone resorption, pamidronate (APD) 2.5 mg/kg IP was given every 4 days with the first dose given 4 days prior to the 7-day course of CsA. Serum levels of creatinine, calcium, and osteocalcin, as well as renal calcium excretion were measured to assess CsA's effects on calcium homeostasis. Effects of CsA on the expression of calbindin-D28k, and two calcium channels in the apical membrane of the distal tubule, epithelial calcium channel (ECaC) and alpha1G-subunit of a voltage-dependent Ca channel (alpha1G), in the kidney were examined by semiquantitative reverse transcription polymerase chain reaction (RT-PCR). KO mice had a threefold increase in renal calcium excretion when compared with WT mice at the baseline. This difference disappeared when calcium load was reduced by overnight fasting. After the CsA treatment, both WT and KO mice had a significant increase of renal calcium excretion (urine Ca/Cr ratio in WT, 0.11 +/- 0.01 to 1.29 +/- 0.17; in KO, 0.39 +/- 0.04 to 1.18 +/- 0.13; both P < 0.01). CsA treatment decreased renal calbindin-D28k mRNA by 61%, but did not affect the expression of ECaC and alpha1G. Baseline serum osteocalcin level of KO mice was significantly lower than that of WT mice. After CsA treatment, both groups had a 50% increase in the serum osteocalcin level, indicating increased bone turnover. When mice were treated with both CsA and APD, the increase in serum osteocalcin level was prevented, and renal calcium excretion was significantly lower than that in mice treated with CsA alone. However, there was still a significant increase in the urine Ca/Cr ratio in WT and KO mice compared with pretreatment levels (urine Ca/Cr in WT, 0.11 +/- 0.01 to 0.76 +/- 0.05, P < 0.01; in KO, 0.39 +/- 0.05 to 0.79 +/- 0.06; P < 0.01). Calbindin-D28k KO mice have diet-dependent hypercalciuria and a lower bone turnover rate. CsA treatment suppresses the expression of calbindin-D28k in mice, but has no effects on ECaC and alpha1G gene expression at the mRNA level. The pathogenesis of CsA-induced hypercalciuria involves both down-regulation of calbindin-D28k with subsequent impaired renal calcium reabsorption and CsA-induced high turnover bone disease. Additionally, our results suggest that mechanism(s) independent of calbindin-D28k within the kidney also may contribute to the CsA-induced calcium leak.

Separate effects of 1,25-dihydroxyvitamin D and calcium on renal calbindin-D28k and intestinal calbindin-D9k.

The effects of normal, low and high plasma concentrations of 1,25-(OH)2vitamin D (1,25-(OH)2D) combined with normal, low and high concentrations of plasma calcium on renal calbindin-D28k and intestinal calbindin-D9k were examined in rats. We found that the expression of renal calbindin-D28k was significantly (P<0.05) increased by high levels of 1,25-(OH)2D, but was not affected by a 50% reduction in 1,25-(OH)2D. In contrast the intestinal calbindin-D9k responded significantly (P<0.005) to both high and low 1,25-(OH)2D levels. The effect of 1,25-(OH)2D on intestinal calbindin-D9k was modulated by plasma calcium concentrations. Increased plasma calcium levels did not affect the renal calbindin-D28k concentrations, but suppressed intestinal calbindin-D9k (P<0.05). The effect of calcium was not mediated by calciotropic hormones. This suggests the existence of a calcium-sensing mechanism in the proximal duodenum. It is concluded that intestinal calbindin-D9k is more sensitive than renal calbindin-D28k to changes in 1,25-(OH)2D and that intestinal calbindin-D9k in contrast to renal calbindin-D28k is sensitive to changes in plasma calcium concentrations.

4. Regulation of renal calbindin‐D28k

Pharmacology & ToxicologyVolume 87, Issue s3 p. 15-15 Free Access 4. Regulation of renal calbindin-D28k First published: 03 November 2003 https://doi.org/10.1034/j.1600-0773.87.s3.6.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume87, Issues3September 2000Pages 15-15 RelatedInformation

Calbindin-D28k Is Expressed in Osteoblastic Cells and Suppresses Their Apoptosis by Inhibiting Caspase-3 Activity

The rate of osteoblast apoptosis is a critical determinant of the rate of bone formation. Because the calcium-binding protein calbindin-D(28k) has anti-apoptotic properties in neuronal cells and lymphocytes, we searched for the presence of this protein in osteoblastic cells and investigated whether it can modify their response to proapoptotic signals. Calbindin-D(28K) was expressed at low levels in several osteoblastic cell lines and at high levels in primary cultures of murine osteoblastic cells. Transient transfection of rat calbindin-D(28k) cDNA blocked tumor necrosis factor alpha (TNFalpha)-induced apoptosis in osteoblastic MC3T3-E1 cells, as determined by cell viability and nuclear morphology of cells cotransfected with the green fluorescent protein targeted to the nucleus, whereas transfection of the empty vector had no effect. Calbindin-D(28k) levels in several stably transfected MC3T3-E1 lines were directly related to protection from TNFalpha-induced apoptosis. Purified rat calbindin-D(28k) markedly reduced the activity of caspase-3, a critical molecule for the degradation phase of apoptosis, in a cell-free assay. In addition, cell extracts from MC3T3-E1 cells expressing high levels of calbindin-D(28k) decreased caspase-3 activity, compared with extracts from vector-transfected cells. This effect was apparently unrelated to the calcium binding properties of calbindin, as chelation of calcium by EGTA or addition of other calcium-binding proteins such as calbindin-D(9k), S100, calmodulin, and osteocalcin, did not affect caspase-3 activity. Last, calbindin-D(28k) interacts with the active form of caspase-3 as demonstrated by a GST pull-down assay. These results demonstrate that calbindin-D(28k) is a biosynthetic product of osteoblasts with a role in the regulation of apoptosis. They also reveal that the antiapoptotic properties of calbindin-D(28k) may result not only from calcium buffering but also from the ability of the protein to interact with and to inhibit caspase-3 activity, a property that is independent of its calcium binding capability.

Altered calbindin mRNA expression and calcium regulating hormones in rat diabetic pregnancy.

Offspring of rats with diabetes mellitus are at risk of reduced calcium and bone mineral content. Altered expression of the maternal calcium binding proteins, calbindin-D(9K) and calbindin-D(28K), which are involved in renal and placental calcium transport, may underlie these problems.We have investigated the effect of diabetes on circulating concentrations of regulatory hormones with respect to calbindin-D mRNA concentrations. Three rat groups were studied; control (CP), streptozotocin-induced diabetic (DP), and insulin-treated diabetic (DPI) pregnant rats. Calbindin-D(9K) and calbindin-D(28K) mRNA abundance in placenta and maternal kidney were measured at days 7, 15, 18 and 21 of gestation, together with serum or plasma concentrations of 1,25 dihydroxyvitamin D(3) (1, 25(OH)(2)D(3)), parathyroid hormone (PTH), PTH-related protein (PTHrP), calcitonin, oestradiol and IGF-I. An increase in placental calbindin-D(9K) mRNA abundance between days 18 and 21 in CP and DPI rats was severely blunted in the DP rats. In contrast, renal calbindin-D(28K) mRNA abundance was greater at days 7, 15 and 18 in DP compared with CP rats, as was calbindin-D(9K) at day 18. Calcitonin concentrations showed no differences between the groups, and both PTH and IGF-I were reduced over the first half of gestation, unlike the calbindins. In contrast, the concentrations of PTHrP and 1,25(OH)(2)D(3) were reduced at term in the DP group compared with the other two groups. Plasma oestradiol concentrations were lower in DP than in CP rats at days 7, 15 and 18, and most striking was the absence in DP rats of the peak of oestradiol seen at day 18 in CP rats. Despite the similarity between changes in placental calbindin mRNA and 1,25(OH)(2)D(3), previous work has shown placental calbindin-D(9K) regulation to be vitamin-D-independent. These studies produce suggestive evidence, therefore, that PTHrP and oestradiol may be involved in the altered calbindin-D expression by kidney and placenta in rat diabetic pregnancy.

Evidence for the impairment of the vitamin D activation pathway by cyclosporine A

Cyclosporine A (CsA) is a potent immunosuppressant with the drawback of renal side effects. We reported that CsA markedly decreases calcium-binding protein calbindin-D28k mRNA levels in rat kidneys, and showed that this decrease is associated with its adverse renal effects. The transcription of the calbindin-D28k gene is activated via the vitamin D pathway. In this work, the potential CsA-mediated impairment of the vitamin D pathway was investigated. Wistar rats were treated for 12 days with 50 mg/kg/day CsA or for 20 days with 50 mg/kg/day of the non-immunosuppressant and non-nephrotoxic SDZ PSC 833, which had been previously shown not to affect calbindin-D28k mRNA levels. The expression of the three vitamin D-regulated genes calbindin-D28k, 1,25-dihydroxyvitamin D 3-24-hydroxylase (24-OHase), and vitamin D receptor (VDR) were quantified in rat kidney homogenates by real-time reverse transcription–polymerase chain reaction. Plasma parathyroid hormone (PTH) as well as plasma and kidney 1,25 dihydroxyvitamin D 3 (calcitriol) levels were monitored in all animals. CsA induced a 85% decrease in calbindin-D28k mRNA levels as well as a 40% and 69% decrease in VDR and 24-OHase mRNA levels, respectively. Plasma and kidney 1,25 dihydroxyvitamin D 3 as well as plasma PTH levels were increased by CsA, but not by SDZ PSC 833. The treatment with SDZ PSC 833 did not affect calbindin-D28k or VDR expression, but did cause a 73% decrease in 24-OHase mRNA levels. Taken together, these results indicate an association between CsA-mediated down-regulation of rat renal calbindin-D28k mRNA and the decrease in other 1,25 dihydroxyvitamin D 3-regulated genes, suggesting an impairment of the vitamin D pathway by CsA which may be related to its adverse renal side effects and its immunosuppressive activity.

Capacity of 1,25-Dihydroxyvitamin D to Stimulate Expression of Calbindin D Changes with Age in the Rat

Studies in rats and humans have shown that there is an age-related decline in the stimulation of intestinal Ca transport by 1,25-dihydroxyvitamin D3[1,25(OH)2D], the active metabolite of vitamin D. The calbindins are a family of vitamin D-dependent calcium-binding proteins found in the intestine (calbindin D-9k) and kidney (calbindin D-28k) and are thought to play a role in calcium transport and homeostasis. The purpose of this study was to determine if the capacity of 1,25(OH)2D to stimulate the expression of calbindin protein and mRNA changes with age. Young (2 months), adult (12 months), and old (22 months) male F344 rats were made deficient in 1,25(OH)2D by being fed a strontium-containing diet and then being given a single ip injection of 1,25(OH)2D. Calbindin protein levels were measured by immunological techniques using specific antisera, and mRNA levels were measured by Northern and dot blots. The maximal amount of calbindin D-9k protein induced by 1,25(OH)2D declined with age in the duodenum but not in the ileum. In time-course studies, there was a delay in calbindin D-9k induction in the duodenum but not in the ileum of adult rats compared to young rats. In contrast to protein induction, maximal calbindin D-9k mRNA levels in response to 1,25(OH)2D were greater in the adult animal and showed no time lag compared to those in the young animal. In the kidney, maximal levels of renal calbindin D-28k protein and mRNA did not change with age, but there was delayed induction in the adult. These studies demonstrate that there is an age-related decrease in the induction of calbindin protein in response to 1,25(OH)2D in the duodenum, but not in the ileum or kidney. This decline may be due to decreased translation of calbindin D-9k mRNA into protein in the duodenum with age.

Estrogen responsiveness of renal calbindin-D28k gene expression in rat kidney.

In women, calcium excretion in the urine rises after menopause and falls with estrogen replacement therapy. The amount of calcium lost in the urine following estrogen therapy is less than should occur based on changes in serum calcium and the amount of calcium filtered by the kidney. This suggests there may be a direct effect of estrogen therapy to increase renal calcium reabsorption. Calbindin D28k is a putative calcium ferry protein located in the distal renal tubules which has been shown to increase transcellular calcium transport. We proposed that estrogen loss after menopause may diminish gene expression of renal calbindin D28k and subsequently diminish renal calcium reabsorption. We used the ovariectomized rat model of estrogen deficiency to investigate changes at the messenger RNA level of calbindin D28k in ovariectomized rats (OVX), sham ovariectomized rats (S-OVX), and estrogen treated ovariectomized rats (E-OVX). We have demonstrated that ovariectomy in rats diminishes the gene expression of renal calbindin D28k. The mRNA levels were approximately three times lower in OVX rats than S-OVX rats. Administration of 17 beta estradiol to OVX rats produced a significant increase in mRNA level to greater than the S-OVX rats by 4 h. Measurement of serum 1,25 dihydroxyvitamin D3 showed lower levels in OVX rats than S-OVX rats but no significant change in E-OVX animals. In conclusion, our results indicate that estrogen increases renal calbindin D28k mRNA levels, by a mechanism independent of changes in 1,25 dihydroxyvitamin D3. This may result in increased expression of calbindin D28k protein which may have a role in reducing renal calcium excretion.

Urinary calcium excretion and renal calbindin-D28k.

The present investigation examined the possible influence of urinary calcium excretion on the concentration of renal calbindin-D28k. Thiazide diuretics stimulate calcium transport across the epithelial cells of the distal tubule, which express calbindin-D28k in high concentrations. Calbindin-D28k is assumed to facilitate transcellular Ca diffusion. Reduced urine calcium excretion and increased urine output were induced in Wistar rats by infusion of bendroflume-thiazide 1 mg/kg/day. The two control groups had infusions of either furosemide 20 mg/kg/day or vehicle, n = 8 in each group. Urinary Ca excretion was reduced to 10% in the thiazide group and increased by 50% in the furosemide group. Renal concentrations of calbindin-D28 showed no difference between vehicle, thiazide- and furosemide-treated rats. No differences in plasma concentrations of calcium, magnesium, phosphorus, urea, PTH, calcitonin and 1,25-(OH)2D were found between the groups. The present study describes that urine calcium excretion selectively can be manipulated without accompanying changes in renal calbindin-D28k concentrations. The data, therefore, suggest that urinary calcium excretion is not a significant determinator of cytosolic concentrations of renal calbindin-D28k.

Effect of vitamin D metabolites and analogs on renal and intestinal calbindin-D in the rat.

The effects on renal and intestinal calbindin-D of vitamin D3 metabolites and synthetic 20-epi-vitamin D3 analogs with different calcemic actions were examined in Wistar rats. The compounds were administered intraperitoneally once daily for 5 days. The dosages of the metabolites were 1,25-(OH)2D3 0.01, 0.05, 0.1, and 0.4 microg/kg x d, 24,25-(OH)2D3 0.1, 1 and 10 microg/kg x d, and 25-(OH)D3 10 and 400 microg/kg x d. The dosage of the synthetic analogs were MC903 0. 1, 10, and 100 microg/kg x d, EB1213 0.1 and 10 microg/kg x d, KH1060 0.1 and 0.4 microg/kg x d, and GS1725 0.01 and 0.1 microg/kg x d. Two control groups had either vehicle alone or no treatment. N = 8 in each group. 1,25-(OH)2D3 increased renal and intestinal calbindin-D levels, induced hypercalcemia, and suppressed plasma PTH and magnesium concentrations. 24,25-(OH)2D3 increased intestinal calbindin-D9k and plasma calcium, but had no effect on renal calbindin-D28k, plasma PTH, and magnesium. The dosage of 24, 25-(OH)2D3 that was required to increase plasma calcium was larger than the dosage required to increase intestinal calbindin-D9k. 25-(OH)D3 did not change the calcium metabolic parameters. MC903, a low calcemic analog with a relative high affinity for the vitamin D receptor and a short half-life, increased renal calbindin-D28k without increasing ionized calcium or intestinal calbindin-D9k. EB1213, an analog with a reduced calcemic action and short half-life, increased renal calbindin-D28k and ionized calcium without increasing intestinal calbindin-D9k. The effect of the high calcemic vitamin D analogs KH1060 and GS1725 on calbindin-D was directly related to their calcemic activity. In conclusion, these results demonstrate that 24,25-(OH)2D3 increases intestinal calbindin-D9k, but has no effect on renal calbindin-D28k, that low calcemic analogs may increase renal calbindin-D28k without increasing intestinal calbindin-D9k, and that the effect of high calcemic analogs on calbindin-D is directly related to their calcemic activity.

Effect of parathyroid hormone on renal calbindin-D28k.

The present investigation was conducted to examine the effects of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP) on renal calbindin-D28k in rats. Four groups of studies were performed: (1) parathyroidectomy (PTX) or a sham operation followed by infusion of 1,25-dihydroxyvitamin D (1,25[OH]2D) or vehicle; (2) infusions of PTH(1-34), PTH(1-84), 1,25(OH)2D, or vehicle; (3) infusion of PTHrP(1-34), PTHrP (1-86), PTH(1-34), or vehicle; and (4) injections of calcium or vehicle. PTX reduced renal calbindin-D28k levels even when plasma concentrations of 1,25(OH)2D were kept constant by infusion of 1,25(OH)2D. Infusions of PTH(1-34), PTH(1-84), and 1,25(OH)2D all increased renal calbindin-D28k and plasma calcium, whereas PTHrP(1-34) and PTHrP(1-86) increased renal calbindin-D28k before an increase of plasma calcium took place. Hypercalcemia induced by the injection of calcium did not affect the levels of renal calbindin-D28k. The present data suggest that PTH and PTHrP exert a direct effect on renal calbindin-D28k, which is not mediated by changes of 1,25(OH)2D or calcium.