Epithelial Calcium Channel Research Articles

Expression of epithelial calcium transport system in rat cochlea and vestibular labyrinth

BackgroundThe low luminal Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca2+-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium.ResultsWe now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca2+-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)2vitamin D3 (VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system.ConclusionsThese findings demonstrate for the first time the expression of a complete Ca2+ absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.

Tamoxifen Inhibits TRPV6 Activity via Estrogen Receptor–Independent Pathways in TRPV6-Expressing MCF-7 Breast Cancer Cells

The epithelial calcium channel TRPV6 is upregulated in breast carcinoma compared with normal mammary gland tissue. The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy. Previously, we showed that tamoxifen inhibits calcium uptake in TRPV6-transfected Xenopus oocytes. In this study, we examined the effect of tamoxifen on TRPV6 function and intracellular calcium homeostasis in MCF-7 breast cancer cells transiently transfected with EYFP-C1-TRPV6. TRPV6 activity was measured with fluorescence microscopy using Fura-2. The basal calcium level was higher in transfected cells compared with nontransfected cells in calcium-containing solution but not in nominally calcium-free buffer. Basal influxes of calcium and barium were also increased. In transfected cells, 10 mumol/L tamoxifen reduced the basal intracellular calcium concentration to the basal calcium level of nontransfected cells. Tamoxifen decreased the transport rates of calcium and barium in transfected cells by 50%. This inhibitory effect was not blocked by the estrogen receptor antagonist, ICI 182,720. Similarly, a tamoxifen-induced inhibitory effect was also observed in MDA-MB-231 estrogen receptor-negative cells. The effect of tamoxifen was completely blocked by activation of protein kinase C. Inhibiting protein kinase C with calphostin C decreased TRPV6 activity but did not alter the effect of tamoxifen. These findings illustrate how tamoxifen might be effective in estrogen receptor-negative breast carcinomas and suggest that the therapeutic effect of tamoxifen and protein kinase C inhibitors used in breast cancer therapy might involve TRPV6-mediated calcium entry. This study highlights a possible role of TRPV6 as therapeutic target in breast cancer therapy.

Gitelman Syndrome

Gitelman Syndrome

The A563T variation of the renal epithelial calcium channel TRPV5 among African Americans enhances calcium influx

The transient receptor potential cation channel, subfamily V, member 5 (TRPV5) gene, which encodes the Ca(2+) channel in the apical membrane of distal convoluted tubule and connecting tubule of the kidney, exhibits an unusually high frequency of nonsynonymous single nucleotide polymorphisms (SNPs) among African Americans. To assess the functional impacts of the nonsynonymous SNP variations in TRPV5, these variants were analyzed with radiotracer (45)Ca(2+) influx assay and the voltage-clamp technique using Xenopus laevis oocytes. Among the variations tested, including A8V, R154H, A563T, and L712F, the latter two significantly increased TRPV5-mediated Ca(2+) influx. The A563T variant, which exists in African Americans with relative high frequency, exhibited increased Ca(2+) influx at extracellular Ca(2+) from 0.01 to 2 mM despite a lower expression level at the plasma membrane. This variant also exhibited a reduction in Na(+) current as a result of increased sensitivity to extracellular Mg(2+). By substituting threonine-563 (Thr(563)) with serine or valine residue, the bulky side chain of Thr(563) was shown to facilitate Ca(2+) transport, whereas the hydroxyl group of Thr(563) is likely related to Mg(2+) sensitivity. The A563T variant was capable of increasing TRPV5-mediated Ca(2+) influx, even when it was expressed under conditions mimicking heterozygous or compound state with other variants. In conclusion, the A563T variant of TRPV5 significantly increased Ca(2+) influx by affecting the Ca(2+) permeation pathway. Thus the A563T variation in TRPV5 may contribute to the superior ability of renal Ca(2+) conservation in African Americans.

TRPV5 gene polymorphisms in renal hypercalciuria

Kidney stone formation is a major socioeconomic problem in humans, involving pain, recurrent treatment and renal insufficiency. As most renal precipitates contain calcium as a major component, hypercalciuria is the main risk factor for renal stone formation. Different forms of hypercalciuria can be classified, which primarily arise from defects in the main organs involved in calcium homeostasis. A distinction can be made between renal, absorptive and resorptive hypercalciuria, originating from disturbed calcium handling in kidney, intestine and bone, respectively. A positive family history predisposes individuals to an increased risk of stone formation, which strongly indicates the involvement of genetic susceptibility factors. TRPV5 is the renal epithelial calcium channel that is the gatekeeper protein in active calcium reabsorption in the kidney. TRPV5 gene ablation in mice leads to severe hypercalciuria, implying that TRPV5 is an interesting candidate gene for renal hypercalciuria in humans. This study aims to identify and functionally characterize TRPV5 gene aberrations in patients with renal hypercalciuria. The TRPV5 coding region and intron-exon boundaries were screened for gene mutations in 20 subjects displaying renal hypercalciuria after which identified non-synonymous polymorphisms were functionally characterized by patch-clamp analysis. Wild-type and TRPV5 channels including polymorphisms were transiently expressed in human embryonic kidney (HEK) 293 cells and functionally characterized by path-clamp analysis. Genotyping TRPV5 in renal hypercalciuria patients revealed three non-synonymous and five synonymous polymorphisms. Electrophysiological characterization of the TRPV5 mutants did not reveal significant functional changes compared to wild-type TRPV5 channel recordings. In this specific patient cohort, our data do not support a primary role for TRPV5 in the pathogenesis of renal hypercalciuria. However, TRPV5 cannot be excluded as a candidate gene in hypercalciuria.

WNK3 positively regulates epithelial calcium channels TRPV5 and TRPV6 via a kinase-dependent pathway

WNK3, a member of the With No Lysine (K) family of protein serine/threonine kinases, was shown to regulate members of the SLC12A family of cation-chloride cotransporters and the renal outer medullary K+ channel ROMK and Cl(-) channel SLC26A9. To evaluate the effect of WNK3 on TRPV5, a renal epithelial Ca2+ channel that serves as a gatekeeper for active Ca2+ reabsorption, WNK3 and TRPV5 were coexpressed in Xenopus laevis oocytes and the function and expression of TRPV5 were subsequently examined. An 82.7 +/- 7.1% increase in TRPV5-mediated Ca2+ uptake was observed when WNK3 was coexpressed. A similar increase in TRPV5-mediated Na+ current was observed with the voltage-clamp technique. WNK3 also enhanced Ca2+ influx and Na+ current mediated by TRPV6, which is the closest homolog of TRPV5 that mediates active intestinal Ca2+ absorption. The kinase domain of WNK3 alone was sufficient to increase TRPV5-mediated Ca2+ transport, and the positive regulatory effect was abolished by the kinase-inactive D294A mutation in WNK3, indicating a kinase-dependent mechanism. The complexly glycosylated TRPV5 that appears at the plasma membrane was increased by WNK3. The exocytosis of TRPV5 was increased by WNK3, and the effect of WNK3 on TRPV5 was abolished by the microtubule inhibitor colchicine. The increased plasma membrane expression of TRPV5 was likely due to the enhanced delivery of mature TRPV5 to the plasma membrane from its intracellular pool via the secretory pathway. These results indicate that WNK3 is a positive regulator of the transcellular Ca2+ transport pathway.

WNK4 regulates the secretory pathway via which TRPV5 is targeted to the plasma membrane

TRPV5 and TRPV6 are two closely related epithelial calcium channels that mediate apical calcium entry in the transcellular calcium transport pathway. TRPV5, but not TRPV6, is enhanced by protein kinase WNK4 when expressed in Xenopus laevis oocytes. We report that the majority of human TRPV5 exogenously expressed in the Xenopus oocyte plasma membrane was complexly N-glycosylated whereas that for human TRPV6 was core-glycosylated. Unglycosylated N358Q mutants of TRPV5 and TRPV6 were able to be expressed in the plasma membrane albeit with decreased abilities in mediating calcium uptake. Syntaxin 6, a SNARE protein in the trans-Golgi network, blocked the complex glycosylation of TRPV5 and TRPV6, rendered the channels in core-glycosylated form. Blocking complex glycosylation of TRPV5 either by syntaxin 6 or by N358Q mutation abolished the enhancing effect of WNK4 on TRPV5. Thus the difference in membrane expression of TRPV5 and TRPV6 explains the selective effect of WNK4 on TRPV5, which is likely on the secretory pathway involving complex glycosylation of channel proteins.

Expression of calcium channel TRPV6 in ovine epithelial tissue

Functional studies indicate marked differences in the extent and site of gastrointestinal calcium absorption between monogastric and ruminant species. Given that the epithelial calcium channel TRPV6 has a pivotal role in active intestinal calcium transport, this study investigated TRPV6 expression in ovine duodenal, jejunal, ruminal and renal epithelial cells using RT-PCR, in situ hybridisation, Western blot analysis and immunohistochemistry. The expression patterns suggested that, in contrast to monogastric animals, the jejunum is more important than the duodenum for calcium absorption in sheep. Furthermore, the expression of TRPV6-specific transcripts in ruminal epithelium was much lower than expected and its sub-cellular localisation suggested that this ion channel does not contribute to calcium absorption from the forestomachs.

Rapid effects of 17β-estradiol on epithelial TRPV6 Ca 2+ channel in human T84 colonic cells

The control of calcium homeostasis is essential for cell survival and is of crucial importance for several physiological functions. The discovery of the epithelial calcium channel Transient Receptor Potential Vaniloid (TRPV6) in intestine has uncovered important Ca(2+) absorptive pathways involved in the regulation of whole body Ca(2+) homeostasis. The role of steroid hormone 17beta-estradiol (E(2)), in [Ca(2+)](i) regulation involving TRPV6 has been only limited at the protein expression levels in over-expressing heterologous systems. In the present study, using a combination of calcium-imaging, whole-cell patch-clamp techniques and siRNA technology to specifically knockdown TRPV6 protein expression, we were able to (i) show that TRPV6 is natively, rather than exogenously, expressed at mRNA and protein levels in human T84 colonic cells, (ii) characterize functional TRPV6 channels and (iii) demonstrate, for the first time, the rapid effects of E(2) in [Ca(2+)](i) regulation involving directly TRPV6 channels in T84 cells. Treatment with E(2) rapidly (<5 min) enhanced [Ca(2+)](i) and this increase was partially but significantly prevented when cells were pre-treated with ruthenium red and completely abolished in cells treated with siRNA specifically targeting TRPV6 protein expression. These results indicate that when cells are stimulated by E(2), Ca(2+) enters the cell through TRPV6 channels. TRPV6 channels in T84 cells contribute to the Ca(2+) entry/signalling pathway that is sensitive to 17beta-estradiol.

Active Intestinal Calcium Transport in the Absence of Transient Receptor Potential Vanilloid Type 6 and Calbindin-D9k

To study the role of the epithelial calcium channel transient receptor potential vanilloid type 6 (TRPV6) and the calcium-binding protein calbindin-D9k in intestinal calcium absorption, TRPV6 knockout (KO), calbindin-D9k KO, and TRPV6/calbindin-D(9k) double-KO (DKO) mice were generated. TRPV6 KO, calbindin-D9k KO, and TRPV6/calbindin-D9k DKO mice have serum calcium levels similar to those of wild-type (WT) mice ( approximately 10 mg Ca2+/dl). In the TRPV6 KO and the DKO mice, however, there is a 1.8-fold increase in serum PTH levels (P < 0.05 compared with WT). Active intestinal calcium transport was measured using the everted gut sac method. Under low dietary calcium conditions there was a 4.1-, 2.9-, and 3.9-fold increase in calcium transport in the duodenum of WT, TRPV6 KO, and calbindin-D9k KO mice, respectively (n = 8-22 per group; P > 0.1, WT vs. calbindin-D9k KO, and P < 0.05, WT vs. TRPV6 KO on the low-calcium diet). Duodenal calcium transport was increased 2.1-fold in the TRPV6/calbindin-D9k DKO mice fed the low-calcium diet (P < 0.05, WT vs. DKO). Active calcium transport was not stimulated by low dietary calcium in the ileum of the WT or KO mice. 1,25-Dihydroxyvitamin D3 administration to vitamin D-deficient null mutant and WT mice also resulted in a significant increase in duodenal calcium transport (1.4- to 2.0-fold, P < 0.05 compared with vitamin D-deficient mice). This study provides evidence for the first time using null mutant mice that significant active intestinal calcium transport occurs in the absence of TRPV6 and calbindin-D9k, thus challenging the dogma that TRPV6 and calbindin-D9k are essential for vitamin D-induced active intestinal calcium transport.

Gain-of-function haplotype in the epithelial calcium channel TRPV6 is a risk factor for renal calcium stone formation

The rate-limiting step of dietary calcium absorption in the intestine requires the brush border calcium entry channel TRPV6. The TRPV6 gene was completely sequenced in 170 renal calcium stone patients. The frequency of an ancestral TRPV6 haplotype consisting of three non-synonymous polymorphisms (C157R, M378V, M681T) was significantly higher (P = 0.039) in calcium stone formers (8.4%; derived = 502, ancestral = 46) compared to non-stone-forming individuals (5.4%; derived = 645, ancestral = 37). Mineral metabolism was investigated on four different calcium regimens: (i) free-choice diet, (ii) low calcium diet, (iii) fasting and (iv) after a 1 g oral calcium load. When patients homozygous for the derived haplotype were compared with heterozygous patients, no differences were found with respect to the plasma concentrations of 1,25-vitamin D, PTH and calcium, and the urinary excretion of calcium. In one stone-forming patient, the ancestral haplotype was found to be homozygous. This patient had absorptive hypercalciuria. We therefore expressed the ancestral protein (157R+378V+681T) in Xenopus oocytes and found a significantly enhanced calcium permeability when tested by a (45)Ca(2+) uptake assay (7.11 +/- 1.93 versus 3.61 +/- 1.01 pmol/min/oocyte for ancestral versus derived haplotype, P < 0.01). These results suggest that the ancestral gain-of-function haplotype in TRPV6 plays a role in calcium stone formation in certain forms of absorptive hypercalciuria.

In vitro study of the role of lenticular epithelial calcium channels and aquaporins in the development of cataract

The role of calcium and water channels (aquaporins) in the pathogenesis of cataract was studied in vitro. Aquaporin blockade caused opacity of the lens sooner than changes in calcium ion concentration in culture medium.

Coumestrol Decreases Intestinal Alkaline Phosphatase Activity in Post-delivery Mice but does not Affect Vitamin D Receptor and Calcium Channels in Post-delivery and Neonatal Mice

In this study, we investigated the effects of administration of coumestrol during pregnancy on calcium (Ca) metabolism in post-delivery maternal and neonatal mice. From 6.5 to 16.5 days post coitus (dpc), pregnant females were administered daily doses of coumestrol (200 microg/kg body weight/day). One day after parturition, blood samples and the kidneys, liver, jejunum and duodenum were obtained from each of maternal mouse, and blood samples and the kidneys and liver were obtained from neonatal mice. Coumestrol did not have any significant effect on the Ca and inorganic phosphorus concentrations in the sera of the maternal and neonatal mice. No notable effects of coumestrol were observed in relation to Vitamin D receptor expression in the maternal and neonatal mice by immunohistochemical analysis. Coumestrol did not affect the Vitamin D receptor and epithelial calcium channel and 2 mRNA levels in any of the organs investigated. Enzyme histochemical analysis showed that coumestrol decreased intestinal alkaline phosphatase activity in the maternal jejunum and duodenum. In the duodenum, coumestrol decreased expression of intestinal alkaline phosphatase, c-fos and vascular endothelial growth factor at the mRNA level. However, we did not observe any significant effects of coumestrol on the expression of these genes. In conclusion, coumestrol decreased intestinal alkaline phosphatase activity in the small intestines of maternal mice at the level used in the present study, and the mechanisms underlying this effect are different for the jejunum and duodenum.

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid-inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ-domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.

Vitamin D

Vitamin D maintains calcium homeostasis and is required for bone development and maintenance. Recent evidence has indicated an interrelationship between vitamin D and health beyond bone, including effects on cell proliferation and on the immune system. New developments in our lab related to the function and regulation of target proteins have provided novel insights into the mechanisms of vitamin D action. Studies in our lab have shown that the calcium-binding protein, calbindin, which has been reported to be a facilitator of calcium diffusion, also has an important role in protecting against apoptotic cell death in different tissues including protection against cytokine destruction of osteoblastic and pancreatic beta cells. These findings have important implications for the therapeutic intervention of many disorders including diabetes and osteoporosis. Recent studies in our laboratory of intestinal calcium absorption using calbindin-D(9k) null mutant mice as well as mice lacking the 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inducible epithelial calcium channel, TRPV6, provide evidence for the first time of calbindin-D(9k) and TRPV6 independent regulation of active calcium absorption. Besides calbindin, the other major target of 1,25(OH)(2)D(3) in intestine and kidney is 25(OH)D(3) 24 hydroxylase (24(OH)ase), which is involved in the catabolism of 1,25(OH)(2)D(3). In our laboratory we have identified various factors that cooperate with the vitamin D receptor in regulating 24(OH)ase expression including C/EBP beta, SWI/SNF (complexes that remodel chromatin using the energy of ATP hydrolysis) and the methyltransferases, CARM1 and G9a. Evidence is also presented for C/EBP beta as a nuclear coupling factor that coordinates regulation of osteopontin by 1,25(OH)(2)D(3) and PTH. Our findings define novel mechanisms that may be of fundamental importance in understanding how 1,25(OH)(2)D(3) mediates its multiple biological effects.

Regulation of the epithelial calcium channel TRPV5 by extracellular factors

Recent studies have greatly increased our knowledge concerning the regulation of renal calcium handling. This review focuses on newly identified calciotropic factors present in the pro-urine and the mechanisms by which they control the transient receptor potential channel vanilloid subtype 5 (TRPV5) which forms the gatekeeper of active renal calcium reabsorption. The antiaging hormone klotho regulates TRPV5 activity via a novel mechanism modifying its glycosylation status, thereby entrapping the channel at the cell surface. Functional characterization of tissue kallikrein knockout mice revealed that these animals exhibit a pronounced hypercalciuria, comparable to the calcium leak observed in TRPV5 knockout mice. Recently, it has been demonstrated that tissue kallikrein stimulates active calcium reabsorption via the bradykinin receptor type 2 pathway involving protein kinase C-dependent activation of TRPV5. Finally, the extracellular pH appears to act as a dynamic switch controlling cell surface expression of TRPV5. Unraveling the molecular mechanisms of TRPV5 channel regulation by the antiaging hormone klotho, tissue kallikrein and extracellular pH demonstrated the existence of novel regulatory mechanisms of active calcium reabsorption acting from the tubular lumen.

Interactions of waterborne and dietary cadmium on the expression of calcium transporters in the gills of rainbow trout: Influence of dietary calcium supplementation

Recent studies have shown that dietary Ca 2+ supplementation strongly inhibits uptake of Ca 2+ and Cd at the fish gill. To better understand the influence of dietary Ca 2+ on branchial Ca 2+ transport, we examined the expression of two trout gill calcium transporters during waterborne and dietary Cd exposure, at two different levels of dietary Ca 2+. Quantitative polymerase chain reaction (PCR) was used to monitor epithelial calcium channel (ECaC) and sodium–calcium exchange (NCX) mRNA levels following 7–28 days of exposure to these treatments. In brief, juvenile rainbow trout ( Oncorhynchus mykiss) were exposed to control, 3 μg/L waterborne Cd, 500 mg/kg dietary Cd, or a combined 3 μg/L waterborne plus 500 mg/kg dietary Cd exposure, supplemented with either 20 mg/g or 60 mg/g dietary calcium (Ca 2+). Two-way analysis of variance was used to discern the main effects of Cd exposure and dietary Ca 2+ supplementation on ECaC and NCX mRNA levels. We found that dietary Ca 2+ supplementation decreased significantly ECaC mRNA expression on days 14 and 21. In comparison, NCX mRNA levels were not influenced by dietary Ca 2+ supplementation, but rather were significantly inhibited in the combined waterborne and dietary Cd exposure on day 7 alone. Statistical analysis found no interactive effects between Cd exposure and dietary Ca 2+ exposure at any time point, except for day 28.This study provides evidence of the importance of nutritional status on the transcriptional regulation of ion transport at the fish gill. We discuss the importance of diet and nutritional status to the development of new regulatory approaches, such as the biotic ligand model, which currently do not account for the significance of diet on metal bioavailability in aquatic organisms.

Regulation of branchial zinc uptake by 1α,25-(OH) 2D 3 in rainbow trout and associated changes in expression of ZIP1 and ECaC

Zinc is a vital micronutrient to all organisms, but is also a toxicant to aquatic species. It is therefore of importance to determine the mechanisms by which zinc uptake is modulated. In the present study, we investigated the regulatory effects of the vitamin D metabolite, 1α,25-(OH) 2D 3, on branchial zinc influx in rainbow trout, Oncorhynchus mykiss. Our results showed that branchial zinc uptake in rainbow trout was stimulated 7 days after a single intraperitoneal injection of 1α,25-(OH) 2D 3 (0.01 μg/g fish). To understand the molecular components of zinc uptake regulation by 1α,25-(OH) 2D 3, a ZIP zinc transporter ( OmSLC39A1) and a partial vitamin D receptor ( OmVDR) were molecularly cloned from rainbow trout gill, and the transcriptional expression of OmSLC39A1, epithelial calcium channel ( OmECaC) and OmVDR genes in the gill was subsequently analyzed in response to 1α,25-(OH) 2D 3. OmECaC, OmSLC39A1 and OmVDR were all upregulated following treatment with 1α,25-(OH) 2D 3, but the effect was observed at different time points. OmECaC expression was significantly increased by 1α,25-(OH) 2D 3 on Days 3 and 5 after the injection, and expression of OmVDR was stimulated on Day 5. There was also an increased abundance of OmSLC39A1 mRNA on Day 7 following the injection with 1α,25-(OH) 2D 3, but given the late response the effect of 1α,25-(OH) 2D 3 on this gene might be indirect. The results from the present study provide strong evidence that administration of 1α,25-(OH) 2D 3 results in enhanced zinc uptake across rainbow trout gill and that this effect is associated with an increased expression of transporters that mediate zinc uptake. The implications of our findings, in terms of aquatic toxicology, are that vitamin D status influences zinc accumulation in gill and body of fish.

The effect of a low-protein diet in pregnancy on offspring renal calcium handling

Low birth weight humans and rats exposed to a low-protein diet in utero have reduced bone mineral content. Renal calcium loss during the period of rapid skeletal growth is associated with bone loss. Because young rats exposed to low protein display altered renal function, we tested the hypothesis that renal calcium excretion is perturbed in this model. Pregnant Wistar rats were fed isocalorific diets containing either 18% (control) or 9% (low) protein throughout gestation. Using standard renal clearance techniques, Western blotting for renal calcium transport proteins, and assays for Na(+)-K(+)-ATPase activity and serum calcitropic hormones, we characterized calcium handling in 4-wk-old male offspring. Histomorphometric analyses of femurs revealed a reduction in trabecular bone mass in low-protein rats. Renal calcium (control vs. low protein: 10.4 +/- 2.1 vs. 27.6 +/- 4.5 nmol x min(-1) x 100 g body wt(-1); P < 0.01) and sodium excretion were increased, but glomerular filtration rate was reduced in low-protein animals. Total plasma calcium was reduced in low-protein rats (P < 0.01), but ionized calcium, serum calcitropic hormone concentrations, and total body calcium did not differ. There was no significant change in plasma membrane Ca(2+)-ATPase pump, epithelial calcium channel, or calbindin-D(28K) expression in low-protein rat kidneys. However, Na(+)-K(+)-ATPase activity was 36% lower (P < 0.05) in low-protein rats. These data suggest that the hypercalciuria of low-protein rats arises through a reduction in passive calcium reabsorption in the proximal tubule rather than active distal tubule uptake. This may contribute to the reduction in bone mass observed in this model.

Marked Disturbance of Calcium Homeostasis in Mice With Targeted Disruption of the Trpv6 Calcium Channel Gene

We report the phenotype of mice with targeted disruption of the Trpv6 (Trpv6 KO) epithelial calcium channel. The mice exhibit disordered Ca(2+) homeostasis, including defective intestinal Ca(2+) absorption, increased urinary Ca(2+) excretion, decreased BMD, deficient weight gain, and reduced fertility. Although our Trpv6 KO affects the closely adjacent EphB6 gene, the phenotype reported here is not related to EphB6 dysfunction. The mechanisms underlying intestinal Ca(2+) absorption are crucial for overall Ca(2+) homeostasis, because diet is the only source of all new Ca(2+) in the body. Trpv6 encodes a Ca(2+)-permeable cation channel responsible for vitamin D-dependent intestinal Ca(2+) absorption. Trpv6 is expressed in the intestine and also in the skin, placenta, kidney, and exocrine organs. To determine the in vivo function of TRPV6, we generated mice with targeted disruption of the Trpv6 (Trpv6 KO) gene. Trpv6 KO mice are viable but exhibit disordered Ca(2+) homeostasis, including a 60% decrease in intestinal Ca(2+) absorption, deficient weight gain, decreased BMD, and reduced fertility. When kept on a regular (1% Ca(2+)) diet, Trpv6 KO mice have deficient intestinal Ca(2+) absorption, despite elevated levels of serum PTH (3.8-fold) and 1,25-dihydroxyvitamin D (2.4-fold). They also have decreased urinary osmolality and increased Ca(2+) excretion. Their serum Ca(2+) is normal, but when challenged with a low (0.25%) Ca(2+) diet, Trpv6 KO mice fail to further increase serum PTH and vitamin D, ultimately developing hypocalcemia. Trpv6 KO mice have normal urinary deoxypyridinoline excretion, although exhibiting a 9.3% reduction in femoral mineral density at 2 months of age, which is not restored by treatment for 1 month with a high (2%) Ca(2+) "rescue" diet. In addition to their deranged Ca(2+) homeostasis, the skin of Trpv6 KO mice has fewer and thinner layers of stratum corneum, decreased total Ca(2+) content, and loss of the normal Ca(2+) gradient. Twenty percent of all Trpv6 KO animals develop alopecia and dermatitis. Trpv6 KO mice exhibit an array of abnormalities in multiple tissues/organs. At least some of these are caused by tissue-specific mechanisms. In addition, the kidneys and bones of Trpv6 KO mice do not respond to their elevated levels of PTH and 1,25-dihydroxyvitamin D. These data indicate that the TRPV6 channel plays an important role in Ca(2+) homeostasis and in other tissues not directly involved in this process.