Epithelial calcium channels

TRPV6 (previously named ECaC2 or CaT1) and TRPV5 (previously named ECaC1 or CaT2) are the two epithelial Ca channels involved in Ca entry to the enterocytes. These channels are homologous members of the transient receptor potential (TRP) superfamily, belonging to the vanilloid subfamily (TRPV), which is different from the canonical (TRPC) and melastatin (TRPM) subfamilies⁽Reference van Abel, Hoenderop and Bindels²⁷⁾. TRPV6 and TRPV5 are co-expressed in the human kidney and intestine, but the first one is highly expressed in the intestine and the latter is the major isoform in the kidney. TRPV6 seems to be a major contributor to apical, intestinal Ca absorption, as suggested by a significant reduction in Ca absorption and serum Ca shown in TRPV6 knockout (KO) mice⁽Reference Bianco, Peng and Takanaga^28, Reference Cui, Li and Johnson²⁹⁾. Both channels are also expressed in the pancreas, prostate, and mammary, sweat and salivary glands⁽Reference van Abel, Hoenderop and Bindels²⁷⁾. They present a similar structure to other members of the TRP family: six transmembrane domains, a short hydrophobic stretch between segments 5 and 6 involved in the Ca pore and large intracellular N and C terminal tails. The intracellular segments contain phosphorylation sites, postsynaptic density protein motifs and ankyrin repeat domains; all of them are involved in the regulation of channel activity and trafficking⁽Reference Den Dekker, Hoenderop and Nilius³⁰⁾. It has been demonstrated that the tetrameric structure of TRPV6 and TRPV5 can be combined with each other to form different heterotetrameric channel complexes⁽Reference Hoenderop, Voets and Hoefs³¹⁾. Both channels have 75 % homology, share several properties, but have different N and C terminal tails. They are regulated by calcitriol, oestrogen and dietary Ca. Both are inactivated by intracellular Ca, but with a different kinetics. In addition, the affinity of TRPV5 for the inhibitor ruthenium red is 100-fold that of TRPV6⁽Reference Hoenderop, Vennekens and Müller³²⁾.

TRPV6 transcripts have been found in duodenum, but not in ileum, human biopsies. The duodenal expression of TRPV6 in men was detected to be vitamin D dependent, whereas in elderly women the TRPV6 and vitamin D receptor (VDR) expressions were low and not vitamin D dependent. This finding could explain, at least in part, the lower intestinal Ca absorption in elderly postmenopausal women⁽Reference Walters, Balesaria and Chavele³³⁾. In rats, the basal mRNA expression of TRPV6 has been found to be the highest in the duodenum, followed by the colon (46 % of duodenum), and negligible in the jejunum and ileum. The rank order of the basal levels of TRVP6 protein was duodenum > colon (72 % of duodenum) > ileum (25 % of duodenum)⁽Reference Chow, Quach and Vieth³⁴⁾.

Calbindins

These proteins appear to be responsible for carrying Ca²⁺ from the apical side of the enterocyte to the basal region of the cell. Calbindin (CB) CB_9k is present in the intestine of mammals and CB_28k in that from avian species⁽Reference Tolosa de Talamoni, Pérz and Alisio³⁵⁾. CB_9k has four α-helical regions forming an EF-hand pair consisting of a canonical and a non-canonical/pseudo EF-hand domain, which are joined by a linker region. These EF-hands organised in tandem domains are the physiological relevant structures and two Ca²⁺ ions bind with positive cooperativity⁽Reference Schwaller³⁶⁾. CB_28k has six EF-hand domains, four of which bind Ca²⁺ with medium/high affinity⁽Reference Cheung, Richards and Rogers³⁷⁾. EF-hand 2 is non-functional and under physiological conditions EF6 most probably is as well. The four medium/high-affinity sites⁽Reference Nägerl, Novo and Mody³⁸⁾ are considered Ca specific.

CB also buffer Ca²⁺ ions by keeping intracellular Ca²⁺ concentrations below 10^–7m, which contribute to the prevention of premature cell death by apoptosis. When there is a down-regulation of CB, an excess of Ca²⁺ is provoked that may trigger apoptosis in the epithelial cells⁽Reference Choi, Cho and Kim³⁹⁾. Furthermore, it has been reported that CB_28k also inhibits apoptosis in osteoblastic cells⁽Reference Bellido, Huening and Raval-Pandya⁴⁰⁾ and in germ cells from Robertsonian mice⁽Reference Merico, de Barboza and Vasco^41, Reference Rodriguez, Diaz de Barboza and Ponce⁴²⁾. In addition, it has been shown in kidney that CB_28k regulates the Ca²⁺ concentration in the vicinity of the TRPV5 pore by a direct association with the channel⁽Reference Lambers, Mahieu and Oancea⁴³⁾. This might occur in the intestine and in other tissues with important movements in intracellular Ca²⁺ concentrations.

Genetic studies have provided information that confused the understanding of CB on Ca homeostasis. Mice with ablation of the CB_28k gene do not exhibit calcemic abnormalities⁽Reference Airaksinen, Eilers and Garaschuk⁴⁴⁾. In CB_9k-null mutant mice as well as mice lacking the epithelial Ca channel TRPV6 it has been demonstrated that the regulation of active intestinal Ca absorption is independent of CB_9k and TRPV6. The authors think that in the KO mice there is compensation by another Ca²⁺ channel or protein and that other novel factors are involved in intestinal Ca absorption⁽Reference Christakos, Dhawan and Ajibade⁴⁵⁾. It has been found that an ablation of CB_9k alters the expression of paracellular tight junction genes. The compensatory expression of paracellular tight junction genes in the duodenum was associated with CB_9k, but not with CB_28kReference Hwang, Yang and Kang⁽⁴⁶⁾. This interaction between the transcellular and paracellular pathways might partially explain the variety of gut responses to absorb Ca under different pathophysiological conditions.

Calcium pump and Na⁺/Ca²⁺ exchanger

Plasma membrane Ca²⁺-ATPase (PMAC) 1 is an ATP-dependent transporter that pumps Ca out of the cytosol. This protein was detected in erythrocyte membranes and found to have a high Ca²⁺ affinity⁽Reference Schatzmann⁴⁷⁾. It presents four isoforms (PMCA1–4), which are divided into several subtypes by alternative splicing. PMCA1 is considered as the housekeeping isoform because its mRNA is in all tissues. The correlation between the regulation of Ca homeostasis by TRPV6 and PMCA1 and duodenal and renal function is not well known. Nevertheless, some reports have described a role for TRPV6 and PMCA1 in the uterus, duodenum, kidney and brain⁽Reference Barley, Howard and O’Callaghan^48–Reference Tribe, Moriarty and Poston⁵²⁾. PMCA has a relative molecular mass (M_r) of 130 kDa and a K _m for Ca²⁺ of 0·2 µm in the presence of calmodulin⁽Reference Ghijsen, De Jong and Van Os⁵³⁾. In the intestine, PMCA is located in the caveolae, which can exist in open and closed forms that control Ca²⁺ efflux from the cell⁽Reference Anderson⁵⁴⁾. The predominant form in the intestine is the isoform PMCA_1b. We have found that its expression and activity are higher in enterocytes from the villus tip than in those from the villus crypt, supporting the idea that mature enterocytes have the greatest capacity for transcellular Ca²⁺ movement⁽Reference Centeno, Díaz de Barboza and Marchionatti⁵⁵⁾.

Another novel protein seems to be crucial in the transcellular Ca²⁺ pathway. This is the protein 4.1R, which was also first identified in the erythrocyte membrane skeleton and is expressed in the epithelia of the intestine. So far, its physiological function remains unknown. Liu et al. ⁽Reference Liu, Weng and Chen⁵⁶⁾ have detected that 4.1R co-localises with PMCA_1b. These authors have shown that 4.1R KO mice exhibit deterioration in intestinal Ca absorption. In 4.1R KO mice, the expression of PMCA_1b in enterocytes was decreased. This finding that the deficiency in the adaptor protein 4.1R produces an impaired intestinal Ca absorption suggests that many yet to be defined molecules might also play important functions in epithelial Ca transport.

Apparently, the intestinal Na⁺/Ca²⁺ exchanger (NCX1) is responsible for about 20 % of Ca exit. However, this protein has received little attention and many recent reviews ignore it as another molecule involved in the Ca²⁺ exit from the intestine. The activity of the intestinal Na⁺/Ca²⁺ exchanger depends on the gradient created by Na⁺/K⁺-ATPase⁽Reference Ghijsen, De Jong and Van Os⁵⁷⁾. There are several isoforms that result from three different genes⁽Reference Philipson, Nicoll and Matsuoka⁵⁸⁾, but in the intestine NCX1 is present mainly in the enterocytes. It has been detected in rats⁽Reference Ghijsen, De Jong and Van Os⁵⁷⁾, mice⁽Reference Dong, Sellers and Smith⁵⁹⁾, chicks⁽Reference Centeno, Díaz de Barboza and Marchionatti⁵⁵⁾, horses⁽Reference Hwang, Jung and Yang⁶⁰⁾ and dogs⁽Reference Kim, Yang and Hwang⁶¹⁾, but not in rabbits⁽Reference Hoenderop, Hartog and Stuiver⁶²⁾. NCX1 has a stoichiometry of 3 Na⁺:1 Ca²⁺ and can function in either a forward mode (Ca²⁺ extrusion) or in a reversed mode (Ca²⁺ entry), depending on the Na⁺ and Ca gradients and the membrane potential⁽Reference Blaustein and Lederer⁶³⁾. We have found that the expression and activity of NCX1 are quite similar between mature and immature enterocytes from chick duodenum, but are slightly higher in the villus tip cells⁽Reference Centeno, Díaz de Barboza and Marchionatti⁵⁵⁾. Recently, it has been found that the gene expression of NCX1, PMCA_1b and CB_9k was down-regulated, whereas NCX1 expression was unchanged in the duodenum of a model of hypoxia in pregnant rats that shares clinical similarities with humans suffering from pre-eclampsia or other metabolic diseases. The authors have also found alterations in Ca²⁺ transporters from the placenta and kidney and showed that these changes caused Ca deficiencies associated with pre-eclampsia⁽Reference Yang, Lei and Cheng⁶⁴⁾. As they pointed out, it is quite possible that this study may contribute to a better understanding of the interrelationship between Ca imbalances and metabolic disturbances during pre-eclampsia pathogenesis.

Calcitriol

Calcitriol or 1,25-dihydroxycholecalciferol (1,25(OH)₂D₃) is the main stimulus for intestinal Ca absorption. It induces changes in the structure and function of intestinal epithelial cells, which enhance Ca transport across the intestine. Calcitriol acts through genomic and non-genomic pathways, after binding to a VDR. Most of the studies have been focused on the effect of calcitriol on the intestinal transcellular Ca pathway. It has been found that the expression or the activity of all molecules presumably involved in this route is increased by calcitriol in experimental animals and even in human subjects⁽Reference Bouillon, Lieben and Mathieu^69–Reference Balesaria, Sangha and Walters⁷²⁾. Recently, it has been shown in mice that a single administration of 1,25(OH)₂D₃ produced a 30-fold maximal increase in the ileal TRPV6 mRNA at 9 h. Multiple dosing of 1,25(OH)₂D₃ increased the ileal TRPV6 to 200- to 600-fold, being the highest changes observed at the 3rd and 4th doses. TRPV6 protein levels were increased 1·5-fold throughout the duodenum and ileum after the 3rd and 4th doses, while levels in the colon were increased after the 4th dose. These changes in protein correlate with a high TRPV6 mRNA induction in the ileum at the same period⁽Reference Chow, Quach and Vieth³⁴⁾. The 1,25(OH)₂D₃-enhanced Ca transport in mice was reported to be inhibited by FGF-23 as well as Ca transport in the colon cancer Caco-2 cells. Fibroblast growth factor-23 (FGF-23) produced an abolishment of the enhanced transcellular active Ca fluxes in both models. Despite the Arrhenius plot indicating that FGF-23 decreased the potential barrier of paracellular Ca movement, FGF-23 was found to modestly down-regulate the 1,25(OH)₂D₃-enhanced paracellular Ca transport⁽Reference Khuituan, Wongdee and Jantarajit⁷³⁾. VDR-null mice adapt to pregnancy by up-regulating duodenal TRPV6 and intestinal Ca absorption, which enables a rapid normalisation of bone mineral content. These mice lactate normally and fully restore bone mineral content after weaning. In other words, VDR seems not to be required for skeletal adaptation during pregnancy, lactation and after weaning⁽Reference Fudge and Kovacs⁷⁴⁾. In the elderly, there is an age-related decrease in Ca absorption and a higher Ca intake is needed. It seems that increasing Ca intake from dairy products and Ca-fortified foods is much better than supplements. The combination of vitamin D intake to 800 IU (20 μg) daily together with a total Ca intake of 1000 mg daily is a simple and inexpensive strategy that could reduce fractures in aged individuals by 30 %⁽Reference Gallagher⁷⁵⁾.

Two studies have demonstrated that calcitriol increases paracellular fluxes across the intestine, mainly in the jejunum and ileum⁽Reference Sheikh, Schiller and Fordtran^76, Reference Karbach⁷⁷⁾. Fujita et al. ⁽Reference Fujita, Sugimoto and Inatomi⁷⁸⁾ have demonstrated that 1,25(OH)₂D₃ significantly increases claudin 2 and claudin 12 mRNA levels in Caco-2 cells. They have also shown that mRNA and protein levels for these proteins were lower at 12 weeks in the jejunum of VDR KO mice in comparison with wild-type mice, and small interfering RNA against these claudins decreased Ca permeability in the Caco-2 cells.

An intestinal calcistat has been hypothesised in order to explain vitamin D deficiency (VDD) with and without the clinical disease. Not all individuals with varying degree of VDD present secondary hyperparathyroidism and decreased bone mineral density (BMD). The intestinal calcistat would control Ca absorption, independently of parathyroid hormone (PTH) levels. A protein called Ca receptor (CaR) would dampen the production of active vitamin D metabolites in intestinal cells and diminish transcellular Ca transport, but would increase the paracellular Ca pathway. This local adaptation would adjust the fractional Ca absorption (FCA) according to the body’s needs. When this local adaptation fails due to decreased Ca intake, decreased 25-hydroxyvitamin D₃ (25(OH)D₃), CaR mutation, the systemic adaptation comes into play. The systemic adaptations are an increase in PTH and in active vitamin D metabolites. A rise in PTH is the first indication of VDD with a decrease in BMD depending on the duration of VDD. Therefore, individuals with VDD with normal PTH and BMD should be called subclinical VDD⁽Reference Garg, Kalra and Mahalle⁷⁹⁾. The beneficial effect of vitamin D supplementation on this group of patients needs to be explored.

Parathyroid hormone

The action of PTH on intestinal Ca absorption occurs by the stimulation of renal CYP27B1 and, hence, increases 1,25(OH)₂D₃-dependent intestinal Ca absorption. Direct effects of PTH on Ca uptake by enterocytes from rat duodenum have been shown. PTH stimulates enterocyte Ca influx, which could be blocked by the Ca²⁺ channel antagonists verapamil and nitrendipine⁽Reference Picotto, Massheimer and Boland⁸⁰⁾. PTH/PTHrP receptors have been localised in intestinal epithelial cells along the villus⁽Reference Gentili, Morelli and de Boland⁸¹⁾. PTH promotes nuclear effects such as a regulation of gene transcription and cell proliferation of enterocytes⁽Reference Nemere and Larsson⁸²⁾. So far, a direct effect of PTH on the global process of intestinal Ca absorption has not been reported yet.

Thyroid hormones

Cross et al. ⁽Reference Cross, Debiec and Peterlik⁸³⁾ have demonstrated that thyroid hormone and vitamin D have a cooperative effect on intestinal Ca transport. They have also observed that thyroid hormones increase the genomic action of calcitriol in the intestine. Kumar et al. ⁽Reference Kumar and Prasad⁸⁴⁾ have reported that hyperthyroid rats show larger Ca uptake by brush-border membrane vesicles and Ca efflux from the basolateral membranes of enterocytes than hypothyroid rats. The authors have also found that the Ca²⁺-ATPase activity is not altered by thyroid hormones, while NCX1 activity is highly increased.

Growth hormone and insulin-like growth factor-1

Growth hormone (GH) can promote intestinal Ca absorption, which would occur indirectly mediated through an activation of renal CYP27B1 and the increase of serum 1,25(OH)₂D₃ concentration⁽Reference Zoidis, Gosteli-Peter and Ghirlanda-Keller⁸⁵⁾. Fleet et al. ⁽Reference Fleet, Bruns and Hock⁸⁶⁾ have shown that GH treatment increases intestinal Ca absorption and duodenal CB_9k levels in aged rats without increasing serum 1,25(OH)₂D₃ levels. The effect of GH on Ca absorption is mediated through insulin-like growth factor-1 (IGF-1) and there is evidence that this effect does not depend on vitamin D signalling. Intestinal Ca absorption in adult men has been shown to be positively correlated with IGF-1 and the age-related declines in IGF-1 have a negative impact on Ca absorption, which could not be explained by a decrease in serum 1,25(OH)₂D₃⁽Reference Fatayerji, Mawer and Eastell⁸⁷⁾. Since the vitamin D-independent mechanism by which the GH/IGF-1 axis may regulate intestinal Ca absorption is not clear, this issue needs to be investigated.

Oestrogen

Oestrogen corrects the decline in the efficiency of intestinal Ca absorption at the onset of menopause, as suggested by cell culture studies⁽Reference Cotter and Cashman⁸⁸⁾, but the mechanisms that underlie this effect remain unknown. Colin et al. ⁽Reference Colin, Van Den Bemd and Van Aken⁸⁹⁾ have shown that oestrogen acts independently of 1,25(OH)₂D₃ in the intestine, while others suggest that oestrogen alters intestinal Ca absorption through the vitamin D endocrine system⁽Reference Cotter and Cashman⁸⁸⁾. Most of the oestrogen studies have been done in ovariectomised (OVX) animals. This ablation decreases endogenous oestrogen, but not totally since the adrenal androgens can be aromatised to oestrogen⁽Reference Bouillon, Carmeliet and Van Cromphaut⁹⁰⁾. Oestrogen receptor (ER)α KO mice showed a decrease in duodenal TRPV6 mRNA expression, without changes in CB_9k, PMCA_1b and VDR levels. In contrast, ERβ KO mice did not alter the genes for intestinal Ca absorption. It seems that the genomic effects of oestrogen in mice are mainly mediated by ERα. This idea should not be extrapolated to humans because it has been shown that in normal colon and cancer colon cells the subtype β is the predominant form of the ER⁽Reference Campbell-Thompson, Lynch and Bhardwaj⁹¹⁾. In OVX rats treated with oestradiol, van Abel et al. ⁽Reference van Abel, Hoenderop and van der Kemp⁹²⁾ have found increased duodenal gene expression of TRPV5, TRPV6, CB_9k and PMCA_1b. They used CYP27B1 KO mice to analyse the calcitriol dependency of the stimulatory effects of oestradiol on intestinal Ca absorption and demonstrated that the oestradiol treatment increased mRNA levels of duodenal TRPV6.

The effect of two dietary phyto-oestrogens (coumestrol and apigenin) as well as ipriflavone, a synthetic phyto-oestrogen, on Ca absorption has been studied in the human Caco-2 cell line⁽Reference Cotter and Cashman⁹³⁾. A direct effect of these compounds on intestinal Ca absorption was not observed. These controversial results indicate that the mechanism(s) triggered by oestrogen in the intestine requires further investigation⁽Reference Park and Weaver⁹⁴⁾.

Glucocorticoids

Osteoporosis is one of the most important side effects after long-term glucocorticoid (GC) treatments. Despite a reduced intestinal Ca absorption being part of the pathogenesis of GC-induced osteoporosis⁽Reference Reid⁹⁵⁾, the mechanisms triggered by GC on the intestine are not clear. A short-term GC treatment in young animals does not affect the expression of genes involved in intestinal Ca absorption⁽Reference Van Cromphaut, Stockmans and Torrekens⁹⁶⁾, but a sustained dexamethasone suppresses mouse duodenal CB_9k expression through the GC receptor pathway⁽Reference Lee, Choi and Jeung⁹⁷⁾. It has been also reported that prednisolone for 10 d diminishes rat intestinal Ca absorption through a decreased expression of the active Ca transporters, which occurs independently of 1,25(OH)₂D₃⁽Reference Huybers, Naber and Bindels⁹⁸⁾.

Dietary calcium

Dietary Ca affects the composition of intestinal plasma membranes and alters intestinal Ca transport. All the genes involved in the transcellular pathway are enhanced by a low-Ca diet, which occurs by the activation of the vitamin D endocrine system⁽Reference Tolosa de Talamoni^19, Reference Centeno, Díaz de Barboza and Marchionatti^55, Reference Christakos, Dhawan and Liu^99, Reference Brown, Krits and Armbrecht¹⁰⁰⁾. The increase in the expression and activity of the intestinal Ca pump and NCX1 caused by a Ca-deficient diet occurs in both mature and immature enterocytes. However, VDR levels are decreased by low-Ca diets, independently of the degree of cell differentiation⁽Reference Centeno, Díaz de Barboza and Marchionatti⁵⁵⁾. We think that high levels of serum calcitriol provoked by low-Ca diets promote differentiation, which would produce cells more capable of expressing vitamin D-dependent genes required for Ca absorption. The activity of intestinal alkaline phosphatase (AP) is concomitantly increased by dietary Ca restriction, but a real role of this protein in intestinal Ca absorption cannot be discarded⁽Reference Centeno, Díaz de Barboza and Marchionatti⁵⁵⁾. Benn et al. ⁽Reference Benn, Ajibade and Porta¹⁰¹⁾ have found that a low-Ca diet increases intestinal Ca absorption in wild-type, TRPV6 KO and CB_9k KO mice. This study indicates that the active intestinal Ca absorption occurs in the absence of TRPV6 and CB_9k, which challenges the dogma that both proteins are necessary for vitamin D-induced active intestinal Ca transport. Probably, TRPV6 is not the rate-limiting factor in the transcellular pathway or another factor/s is/are involved in its absence to compensate partially its function.

We have also observed that a low-Ca diet increases the reactivity and availability of sulfhydryl groups from intestinal brush-border membrane proteins of chicks⁽Reference Tolosa de Talamoni, Mykkanen and Wasserman¹⁰²⁾. It is quite possible that the sulfhydryl status of the brush-border membrane proteins is involved in the vitamin D-dependent intestinal Ca absorption. With regard to the lipid composition, we have detected minor changes in the fatty acid content of the basolateral membrane, but the lipid fluidity of these membranes is highly increased by dietary Ca restriction⁽Reference Tolosa de Talamoni¹⁹⁾.

Recently, it has been reported that luminal Ca controls the intestinal Ca absorption through the modification of intestinal AP activity⁽Reference Brun, Brance and Rigalli¹⁰³⁾. Authors have found that luminal Ca concentration increases the activity of AP and simultaneously decreases percentage Ca absorption, acting as a minute-to-minute regulatory mechanism of Ca entry.

Some studies have found an inverse association between Ca intake and adiposity⁽Reference Zemel, Shi and Greer^104–Reference Huang and Qi¹⁰⁶⁾. However, not all studies have observed this relationship⁽Reference Barr¹⁰⁷⁾. The mechanisms underlying these responses are not quite clear.

Most patients with idiopathic hypercalciuria show an increased intestinal Ca absorption. This has been demonstrated in studies using radiolabelled Ca and comparing the intestinal Ca absorption in patients with idiopathic hypercalciuria with that in normal subjects⁽Reference Coe, Favus and Asplin¹⁰⁸⁾. One possible mechanism for the increased Ca absorption might be the highest levels of serum calcitriol found in these patients as compared with normal individuals⁽Reference Hoenderop, Nilius and Bindels⁶⁵⁾. Apparently, this response is independent of Ca intake and reflects an enhancement in active Ca transport by the intestine⁽Reference Lemann¹⁰⁹⁾.

With regard to the risk of kidney calculi formation in postmenopausal women, it is not clear if it is associated with Ca intake and vitamin D supplements. Haghighi et al. ⁽Reference Haghighi, Samimagham and Gohardehi¹¹⁰⁾ have shown that the administration of 1000 mg/d of dietary Ca and vitamin D had a weak association with the formation of kidney calculi (only 1·9 % of fifty-three patients). Jackson et al. ⁽Reference Jackson, LaCroix and Gass¹¹¹⁾ have reported that in a study with 36282 postmenopausal women treated with 1000 mg of Ca and 400 IU (10 μg) of vitamin D/d, a 17 % higher rate of kidney calculi was shown in the treated group after 7 years of follow-up. In contrast, another study contradicted this result, suggesting no association between Ca and vitamin D consumption and kidney calculi formation⁽Reference Diaz-Lopez and Cannata-Andia¹¹²⁾. A low-Ca diet enhances the absorption of oxalate in the gut from normal individuals and kidney stone formers leading to an increase in urinary oxalate excretion, which has an important role in Ca stone formation⁽Reference Curhan, Willett and Rimm^113, Reference Borghi, Schianchi and Meschi¹¹⁴⁾.

There is evidence that dietary Ca restriction is a risk factor for cancer incidence, particularly for colorectal cancer⁽Reference Park, Leitzmann and Subar¹¹⁵⁾. By contrast, a high intake of dairy products and Ca intake protects against the distal colon and rectal tumours as compared with the proximal colon and reduces the risk of colon cancer⁽Reference Tárraga López, Albero and Rodríguez-Montes¹¹⁶⁾. It is also quite possible that a low Ca intake contributes to the development of renal, gastric, pancreatic, ovarian, endometrial and lung cancer as well as multiple myeloma, but there is no conclusive evidence⁽Reference Peterlik, Grant and Cross¹¹⁷⁾.

Several studies have shown that a poor Ca intake is associated with an increased risk of breast cancer⁽Reference McCullough, Rodriguez and Diver^118–Reference Chen, Hu and Xie¹²⁰⁾. Vergne et al. ⁽Reference Vergne, Matta and Morales¹²¹⁾ have recently demonstrated that Ca intake is associated with a high DNA repair capacity level, which in turn decreases the development of breast cancer.

Observational studies have identified an inverse relationship between maternal Ca intake and the incidence of pre-eclampsia⁽Reference Belizan and Villar^122, Reference Belizan, Villar and Repke¹²³⁾. When Ca supplements are administered during pregnancy, the incidence of pre-eclampsia and its consequences are reduced, including severe maternal morbidity and death⁽Reference Camargo, Moraes and Souza¹²⁴⁾.

Several meta-analyses of randomised controlled trials have also indicated that Ca supplementation can lead to a small reduction in systolic and diastolic blood pressure; therefore, Ca supplements might be beneficial in patients with hypertension⁽Reference Griffith, Guyatt and Cook^125, Reference van Mierlo, Arends and Streppel¹²⁶⁾. Varenna et al. ⁽Reference Varenna, Manara and Galli¹²⁷⁾ have confirmed an association between hypertension and osteoporosis. There is a higher prevalence of hypertension in women with osteoporosis and a higher prevalence of osteoporosis in women with hypertension. The authors suggest that a low dairy Ca intake could be associated with an increased risk of both diseases and be a possible pathogenic link between the two conditions.

It is well known that a reduced intestinal Ca absorption is a risk factor for osteoporosis. There is evidence that the use of Ca supplements decreases bone turnover by about 20 %, and this is associated with a reduction in bone loss in postmenopausal women⁽Reference Reid, Mason and Horne¹²⁸⁾. Low Ca intake was significantly associated with low BMD and increased risk of osteoporosis. However, the association between Ca and BMD was not consistently linear, and a sufficient vitamin D level might compensate for the negative influence of low Ca intake on bone⁽Reference Kim, Choi and Lim¹²⁹⁾. Zhou et al. ⁽Reference Zhou, Langsetmo and Berger¹³⁰⁾ have observed that a higher cumulative Ca and vitamin D intake in adult women was associated with better bone health, as indicated by BMD at multiple sites.

Other ions

Lipids

At present, the effects of dietary lipids on intestinal Ca absorption are not clear. Hessov et al. ⁽Reference Hessov, Andersson and Isaksson¹⁵³⁾ have demonstrated that low-fat-diets increase intestinal Ca absorption in nine patients with fat malabsorption. Steatorrhoea has been associated with an inhibition of intestinal Ca absorption⁽Reference Haderslev, Jeppesen and Mortensen¹⁵⁴⁾. Jewell et al. ⁽Reference Jewell, Cusack and Cashman¹⁵⁵⁾ have found that both the paracellular Ca transport and transcellular Ca transport across monolayers of Caco-2 cells were significantly increased after exposure to conjugated linoleic acid. Murphy et al. ⁽Reference Murphy, Jewell and Hooiveld¹⁵⁶⁾ have found that zona occludens-1, Ocldn, and Cldn-4 were all up-regulated while Cldn-1 was down-regulated by trans-10, cis-12-conjugated linoleic acid, which explains the increase in the paracellular route. However, they did not find any effect on genes involved in the transcellular pathway, which should be further explored. Fatty acids directly contribute to increasing intestinal Ca absorption via a cation exchange mechanism between cellular H⁺ for luminal Ca²⁺ favoured by exchanger activity 2H⁺/Ca²⁺⁽Reference Coxam¹⁵⁷⁾. Fatty acids would increase the proliferation of colonic epithelial cells, producing a trophic effect on the mucosa; they contribute to increasing the absorptive surface⁽Reference Raschka and Daniel^158, Reference Lobo, Cocato and Jorgetti¹⁵⁹⁾.

Recently, in high-fat diet-fed mice, Xiao et al. ⁽Reference Xiao, Cui and Shi¹⁶⁰⁾ have found a marked decrease in the intestinal Ca absorption, which is accompanied by redox imbalance and increased duodenal oxidative damage, an effect that was avoided by lipoic acid or Ca supplementation. In addition, they have also shown that a high-fat diet down-regulates the gene expression of molecules involved in the transcellular pathway of intestinal Ca absorption, independently of calcitriol regulation. The authors think that the main cause for high-fat-diet-induced inhibitory intestinal Ca absorption is not Ca soap but duodenal oxidative stress.

Carbohydrates

Lactose is a disaccharide found in milk and dairy products that enhances Ca homeostasis, which should be beneficial for bone health⁽Reference Buchowski and Miller¹⁶¹⁾. It is well established that lactose stimulates the intestinal Ca absorption that seems to occur by passive transport in the small intestine⁽Reference Dupuis, Tardivel and Porembska¹⁶²⁾. Apparently, lactose promotes intestinal Ca absorption, independently of the vitamin D endocrine system. Natsuko et al. ⁽Reference Sogabe, Mizoi and Asahi¹⁶³⁾ have found that lactose alters intestinal AP in rats not only in a direct way, but also indirectly through a regulation of intestinal AP expression, mainly in the jejunum. Epilactose (a rare disaccharide in cows’ milk) has been demonstrated to increase Ca transport in everted small-intestinal sacs by promoting the generation of SCFA and other organic acids⁽Reference Nishimukai, Watanabe and Taguchi¹⁶⁴⁾. Later on, the same group of investigators has found that the epilactose-mediated promotion of intestinal Ca absorption involves the paracellular route in the rat small intestine through the induction of myosin regulatory light chain phosphorylation via myosin light chain kinase and Rho-associated kinase⁽Reference Suzuki, Nishimukai and Shinoki¹⁶⁵⁾. They have also shown that epilactose improves intestinal Ca absorption in gastrectomised rats. They think that the resulting SCFA production by intestinal microbes is responsible for this effect, as well as the increase in the caecal mucosa area and the soluble Ca concentration⁽Reference Suzuki, Nishimukai and Takechi¹⁶⁶⁾.

Some sugar alcohols such as erythritol, xylitol, sorbitol, maltitol, palatinit or lactitol have been found to enhance Ca transport from rat small and large intestine epithelium in vitro. Differences in Ca transport were shown in different segments of the intestine, but not between the sugar alcohols tested⁽Reference Mineo, Hara and Tomita¹⁶⁷⁾. Recently, Xiao et al. ⁽Reference Xiao, Li and Min¹⁶⁸⁾ have found that mannitol improves the absorption and retention of Ca²⁺ and Mg²⁺ in growing rats, an effect that occurs through the fermentation of mannitol in the caecum.

Prebiotics

The fortification of milk with milk Ca or Ca salts is among the strategies suggested to increase Ca intake or absorption or both, but the availability of Ca salts in milk has not been well characterised⁽Reference López-Huertas, Teucher and Boza¹⁶⁹⁾. In addition, several food ingredients such as fructo-oligosaccharides and caseinophosphopeptides (CPP) have been proposed as enhancers of the absorption of Ca from milk or other foods. Fructo-oligosaccharides belong to the group of non-digestible oligosaccharides (NDOs), which includes inulin, oligofructose and galacto-oligosaccharides. They can be digested by the colonic microflora, which produces SCFA that decreases intestinal pH and increases Ca solubility leading to an enhancement of paracellular and transcellular Ca transport⁽Reference Scholz-Ahrens, Schaafsma and van den Heuvel^170, Reference Suzuki and Hara¹⁷¹⁾. It has also been suggested that NDOs increase active Ca transport by the activation of CB_9k⁽Reference Takasaki, Inaba and Ohta¹⁷²⁾. Fukushima et al. ⁽Reference Fukushima, Aizaki and Sakuma¹⁷³⁾ have demonstrated in rats that fructo-oligosaccharide consumption increases the gene expression of TRPV6 and CB_9k through the SCFA formed in the fermentation. The fibres formed mainly by inulin have positive chronic effects on Ca metabolism related to changes in the intestine, resulting in improvement of bone health⁽Reference Legette, Lee and Martin¹⁷⁴⁾. In addition, galacto-oligosaccharides have also potential for improving mineral balance and bone properties⁽Reference Weaver, Martin and Nakatsu¹⁷⁵⁾. With regard to CPP, originating from casein digestion, it has been demonstrated that their addition to Ca-fortified milk increases intestinal Ca absorption in growing rats⁽Reference Tsuchita, Suzuki and Kuwata¹⁷⁶⁾, but the mechanisms involved in this response were not elucidated. Erba et al. ⁽Reference Erba, Ciappellano and Testolin¹⁷⁷⁾ have studied the influence of different four CPP/Ca ratios and three mineral concentrations on the amount of passive Ca absorbed across the everted distal small intestine of rats. The positive effect was dependent on the relative amount of both species in the intestinal lumen, the ratio 15 being the most efficient at increasing mineral transport. Nevertheless, in a randomised cross-over trial undertaken in fifteen adults, no effect of CPP was found on intestinal Ca absorption⁽Reference Teucher, Majsak-Newman and Dainty¹⁷⁸⁾. Cosentino et al. ⁽Reference Cosentino, Gravaghi and Donetti¹⁷⁹⁾ have demonstrated that both intestinal human HT-29 and Caco-2 cells have the ability to take up extracellular Ca under CPP stimulation. Recently, Colombini et al. ⁽Reference Colombini, Perego and Ardoino¹⁸⁰⁾ did not find effects of CPP on paracellular Ca absorption and on TRPV6 mRNA expression in intestinal human HT-29 and Caco-2 cell lines.

A recent study has shown that the daily intake of soluble maize fibre, a well-tolerated prebiotic fibre, increases short-term Ca absorption in adolescents consuming less than the recommended amounts of Ca⁽Reference Whisner, Martin and Nakatsu¹⁸¹⁾.

Probiotics

Probiotics are viable microbes that alter the microflora in a compartment of the host exerting beneficial health effects in this host⁽Reference Schrezenmeir and de Vrese¹⁸²⁾. Most probiotic products contain lactic acid-producing bacteria, which mainly belong to the genera Lactobacillus and Bifidobacterium. In growing rats, it has been demonstrated that probiotic yoghurt containing strains of Lactobacillus casei, L. reuteri and L. gasseri increase intestinal Ca absorption and bone mineral content⁽Reference Ghanem, Badawy and Abdel-Samam¹⁸³⁾. Besides, it has been observed that in Caco-2 cells, the probiotic L. salivarius causes an increase in Ca²⁺ uptake⁽Reference Gilman and Cashman¹⁸⁴⁾.

Synbiotics

Synbiotics are defined as products containing prebiotics and probiotics, in which the prebiotic compound favours the probiotic compound⁽Reference Schrezenmeir and de Vrese¹⁸²⁾. It has been shown in OVX rats that intestinal Ca absorption tended to be higher in the synbiotic group and was significantly higher in the prebiotic group in comparison with the control group⁽Reference Scholz-Ahrens, Ade and Marten¹⁸⁵⁾.

Proteins

Proteins are essential to bone, but the Ca-wasting effect of a high protein intake constitutes a point of debate. It has been known for many years that increasing dietary protein enhances urinary Ca either in human subjects or in rats⁽Reference Kerstetter, O’Brien and Insogna¹⁸⁶⁾. The idea was that the additional Ca excretion was of skeletal origin as a result of buffering in bones the metabolic acid load imposed by higher protein intake⁽Reference Johnson, Alcantara and Linkswiler¹⁸⁷⁾. The theory was that a high-protein diet, mainly meat, creates a higher acid load due to the high content of amino acids containing sulfur. This acid load cannot be neutralised by the kidneys and the body pulls Ca²⁺ from the skeleton to balance pH at the expense of bone, causing an enhancement of urinary Ca⁽Reference Darling, Millward and Torgerson¹⁸⁸⁾. However, clinical studies have demonstrated that a short-term high-protein diet (2·1 g/kg) significantly increases the intestinal Ca absorption as compared with a medium-protein diet (1 g/kg) and the increment in urinary Ca is quantitatively explained by an increase in intestinal Ca absorption efficiency⁽Reference Kerstetter, O’Brien and Caseria¹⁸⁹⁾. The transcellular route, the paracellular pathway or a combination of both mechanisms of intestinal Ca absorption might be involved in response to high dietary protein. By using duodenal brush-border membrane vesicles, Gaffney-Stomberg et al. ⁽Reference Gaffney-Stomberg, Sun and Cucchi¹⁹⁰⁾ have demonstrated that the transcellular component of Ca absorption was accelerated in rats fed a high-protein diet, which was due to an enhancement in maximum velocity, without affecting the Michaelis–Menten constant. However, they did not study whether the gene or protein expression of the molecules involved in the transcellular pathway was modified. They did not find increased bone resorption or changes in serum PTH and calcitriol levels.

Since more research is necessary to resolve the protein debate, it has been suggested not to reduce the protein intake below the dietary reference intake because it could be detrimental to bone health, especially in old individuals⁽Reference Skipper¹⁹¹⁾, and protein intakes and balance of different protein sources with a variety of different foods constitutes appropriate dietary advice⁽Reference Marcason¹⁹²⁾.

Black tea

Black tea (Camellia sinensis) is a medicinal plant with a rich flavonoid content and a plethora of health-promoting effects⁽Reference Das, Das and Mukherjee^193, Reference Sharma and Rao¹⁹⁴⁾. Das et al. ⁽Reference Das, Banerjee and Das¹⁹⁵⁾ have studied the ability of black tea extract as a suitable alternative adjunct for Ca supplementation in treating an OVX rat model of early osteoporosis. The results suggest that black tea could stimulate intestinal Ca absorption, which is associated with an increased activity of AP and Ca²⁺-ATPase. Black tea’s effectiveness in maintaining bone health was detected to be similar to 17β-oestradiol. Therefore, this study suggests that a simultaneous use of black tea is promising as a prospective candidate for adjunctive therapies for Ca supplementation in the early stage of menopausal bone changes.

Coffee

Coffee drinking is a popular habit worldwide. It is consumed in considerable amounts every day. Caffeine, a methylxanthine present in coffee, has been considered to be responsible for an increased risk of osteoporosis in coffee drinkers⁽Reference Hernandez-Avila, Colditz and Stampfer^196, Reference Welch, Bingham and Reeve¹⁹⁷⁾. At present, data are inconsistent⁽Reference Cooper, Atkinson and Wahner^198–Reference Hallström, Byberg and Glynn²⁰⁰⁾, hence, the effect of caffeine on intestinal Ca absorption is not well established. It has been demonstrated in rats that intestinal Ca absorption is stimulated by the increase in 1,25(OH)₂D₃ production after chronic administration of caffeine⁽Reference Yeh and Aloia²⁰¹⁾. In addition, urinary and faecal excretion is also increased. In postmenopausal osteoporotic women, a coffee intake in excess of 1000 ml could induce an extra Ca loss of 1·6 mmol Ca/d, while 1–2 cups of coffee/d would have little impact on Ca balance⁽Reference Hasling, Søndergaard and Charles²⁰²⁾. Metabolic balance studies show a weak negative effect of caffeine on the efficiency of intestinal Ca absorption. However, the effect of caffeine is small enough to be fully offset by 1–2 tablespoons (15–30 ml) of milk⁽Reference Heaney and Nordin¹³⁸⁾. A recent study in OVX rats has shown that low to moderate caffeine intake may exert some beneficial effects on the skeleton, increasing bone mineralisation, and improving the strength and structure of cancellous bone and the mechanical properties of compact bone; however, it did not cause any significant effect in rats with normal oestrogen levels⁽Reference Folwarczna, Pytlik and Zych²⁰³⁾. The continuous debate has weakened interest in the study of coffee as a risk factor for osteoporosis, which has been reinforced by the non-inclusion of coffee in the list of risk factors in the predictive scale for fracture informed by the WHO⁽Reference Cano-Marquina, Tarín and Cano²⁰⁴⁾. The understanding of physiological effects of coffee consumption is difficult because of the vast array of components included in the brewed product and the varied effects of each compound. Recently, an unfavourable effect of trigonelline, an alkaloid present in coffee, has been demonstrated on bone mechanical properties in oestrogen-deficient rats, but not in control rats⁽Reference Folwarczna, Zych and Nowińska²⁰⁵⁾. It is quite possible that the effects on intestinal Ca absorption and the Ca economy by caffeine or other bioactive compounds present in coffee depend on the amount and frequency of coffee intake. This is another issue that merits to be more investigated due to the considerable number of coffee drinkers and the rising life expectancy that will increase bone disorders in the next years.

Gene × diet interactions influence intestinal calcium absorption

Dietary Ca restriction increases the efficiency of intestinal Ca absorption, but the impact of genetics on this adaptive response is not clear. In humans, the efficiency of intestinal Ca absorption varies from 7 to 75 %⁽Reference Alevizaki, Ikkos and Singhelakis²³¹⁾. The large variation is probably owing to the influence of multiple physiological factors (for example, growth, pregnancy, lactation, ageing) and environmental variables (for example, dietary Ca intake, vitamin D). Little information is available for the impact of genetics on the efficiency of intestinal Ca absorption and the adaptive up-regulation of Ca absorption to a low dietary Ca intake. Two laboratories have studied the efficiency of intestinal Ca absorption in different mice and have found that it is higher in C3H/HeJ mice in comparison with C57BL/6J mice, which suggests that genetic background might influence this trait⁽Reference Chen and Kalu²³²⁾. In addition, racial differences in the ability of adolescent girls to increase Ca absorption efficiency during a low Ca intake also indicate that this adaptive response has a genetic component⁽Reference Wu, Martin and Braun^233, Reference Weaver, McCabe and McCabe²³⁴⁾ In agreement with this concept, adolescent black girls have been shown to exhibit higher intestinal Ca absorption as compared with white girls⁽Reference Bryant, Wastney and Martin²³⁵⁾, and this may contribute to the higher bone deposition found in black girls⁽Reference Braun, Palacios and Wigertz²³⁶⁾.

Replogle et al. ⁽Reference Replogle, Li and Wang²³⁷⁾ have examined eleven inbred lines of mice fed on defined diets containing either high or low Ca concentration from weaning to 12 weeks of age. The authors have shown that genetic variation and gene × diet interactions affect not only the active intestinal Ca absorption, but also its relationship to bone. These interactions are partially explained by variations in the traditional cellular mediators (i.e. TRPV6, CB_9k, PMCA_1b mRNA) and in the main hormonal regulator, 1,25(OH)₂D₃, of intestinal Ca absorption. This field is relatively new, hence many efforts are required to bring more light for the understanding of this knowledge.