59.3.2.1

The Paracellular Pathway of Dietary Calcium Absorption

Although paracellular Ca ^{2 +} transport is the major route of absorption in the intestine with high-calcium diets, its importance or regulation has not been investigated in great detail. It is generally believed that contrary to the transcellular pathways, paracellular Ca ^{2 +} transport is not tightly regulated. However, recent years have brought an increasing appreciation of the complexity and plasticity and dynamics of tight junction (TJ) assembly, and of the role of TJ components in paracellular Ca ^{2 +} permeability. Biochemical and physiological aspects of tight junctions are reviewed in more detail elsewhere in this book (T. Ma “Gut Barrier: Tight Junctions”). Kutuzova and Deluca identified a number of 1,25-(OH) ₂ D ₃ target genes whose protein products are localized in the vicinity of intestinal tight junctions and which could influence TJ integrity and permeability. These included not only transporters and channels but also several intra- and intercellular matrix-related proteins and G-proteins. There were also a number of 1,25-(OH) ₂ D ₃ downregulated genes encoding proteins that are involved in the regulation of tight junction permeability, including sodium-potassium ATPase, claudin 3, aquaporin 8, cadherin-17, and RhoA. The conclusion was drawn that 1,25-(OH) ₂ D ₃ may increase intestinal epithelial tight junction permeability or modulate their selectivity toward Ca ^{2 +} and other cations by regulating expression of proteins involved in tight junction formation. The increased tight junction permeability and/or selectivity, regulated by 1,25-(OH) ₂ D ₃ , could direct Ca ^{2 +} absorption through the tight junction-regulated paracellular pathway in the intestinal epithelium. This suggestion is in agreement with a recent study that demonstrated that 1,25-(OH) ₂ D ₃ stimulated an increase in tight junction conductance, and increased paracellular Ca ^{2 +} , Na ⁺ , Rb ⁺ , and mannitol transport in an enterocyte-like cell line (Caco-2). Under these conditions, no significant contribution of the Ca ^{2 +} -ATPase-mediated transcellular pathway to overall transepithelial Ca ^{2 +} transport was detected. More recently, Fujita et al. showed that 1,25-(OH) ₂ D ₃ upregulates claudin-2 and claudin-12 in intestinal epithelial cells via a VDR-dependent mechanism and that both claudins contribute to the vitamin D-induced increase in paracellular Ca ^{2 +} permeability. Claudin-16 (paracellin 1) has also been implicated in playing a significant role in paracellular permeability of Ca ^{2 +} and Mg ^{2 +,} and mutations within the claudin-16 gene have been linked with familial hypomagnesaemia with hypercalciurea and nephrocalcinosis (FHHNC). However, due to a limited pattern of expression (selective expression at tight junctions of renal epithelial cells of the thick ascending limb of the Henle loop), the role of claudin-16 in intestinal Ca ^{2 +} permeability is unlikely.

Additional evidence has accumulated demonstrating that 1,25-(OH) ₂ D ₃ enhanced both cell-mediated active and passive, paracellular Ca ^{2 +} and P _i transport in a Ussing chamber study with rat small intestine and for Ca ^{2 +} transport in Caco-2 cell monolayers. More recently, Tudpor et al. demonstrated nontranscriptionally mediated, short-term increase in paracellular Ca ^{2 +} transport across rat duodenum treated in vitro with 1,25-(OH) ₂ D ₃ . This solvent drag-induced increase in Ca ^{2 +} transport was dependent on phosphatidylinositol 3-kinase (PI3K), protein kinase C (PKC), and mitogen-activated protein (MAP) or extracellular signal-regulated (Erk) kinase (MEK) activity. Unlike the passive, Ca ^{2 +} gradient-directed transport, solvent drag-induced paracellular Ca ^{2 +} absorption resembles a secondary active transport in that it is dependent on the activity of Na ⁺ /K ⁺ -ATPase that creates a paracellular hyperosmotic gradient resulting from a sodium concentration ~ 15 mM higher than the surrounding milieu and is stimulated by high glucose level.

59.3.2.2

Facilitated Diffusion: Apical Ca ^{2 +} Entry

Early studies indicated that over a wide range of transepithelial Ca ^{2 +} transport rates, Ca ^{2 +} influx at the apical membrane is correlated in a 1:1 fashion with the apical to basolateral Ca ^{2 +} flux. Ca ^{2 +} entry was postulated to occur via Ca ^{2 +} -selective channels at the luminal membrane, under the influence of a steep inwardly directed electrochemical gradient. Initial studies demonstrated that 1,25(OH) ₂ D ₃ affected Ca ^{2 +} influx in enterocyte-like cells. This calciotropic hormone significantly increased radiolabled Ca ^{2 +} uptake within several minutes in a dose-dependent manner in rat, intestinal epithelial cells. These and other studies suggested that a Ca ^{2 +} channel was activated by 1,25(OH) ₂ D ₃ through a cAMP/PKA-dependent pathway in the mammalian intestine.

Further studies utilized functional expression cloning in an attempt to identify the apical calcium influx transporter/channel. The first such experiments were done by Hoenderop et al., who utilized a cDNA library constructed from poly(A) ⁺ RNA isolated from primary cultures of rabbit kidney cells. The cDNA clones were copied into cRNA, which was microinjected into Xenopus laevis oocytes, followed by screening for Ca ^{2 +} uptake activity in the presence of a cocktail of known voltage-gated Ca ^{2 +} channel inhibitors (the renal and intestinal Ca ^{2 +} channels were known to NOT be voltage-gated). After extensive screening procedures, a single transcript was isolated which encoded a novel epithelial Ca ^{2 +} channel (first called ECaC1 and recently renamed as transient receptor potential channel TRPV5; also known as ECaC and CaT2). When the TRPV5 channel was expressed in Xenopus oocytes and in HEK293 cells (human, embryonic kidney cells), calcium transport was inhibited by (in order of potency) La ^{3 +} > Cd ^{2 +} > Mn ^{2 +} , whereas Ba ^{2 +} and Sr ^{2 +} had no effect. Permeability to Na ⁺ was negligible in the presence of Ca ^{2 +} . Moreover, the TRPV5 channel was shown to be induced by 1,25(OH) ₂ D _3. Overall, these observations demonstrated that TRPV5 possesses all the expected characteristics of the calcium influx in Ca ^{2 +} -transporting epithelia. The TRPV5 transporter was subsequently determined to be expressed predominantly in the kidney and was conclusively shown to be responsible for renal Ca ^{2 +} reabsorption.

Subsequent studies by Hediger and coworkers applied the functional expression cloning technique to the search for the intestinal calcium entry channel, and they were able to identify an intestinal Ca ^{2 +} transporter, which they called CaT1. CaT1 shares 80% amino acid sequence identity to TRPV5. CaT1 has subsequently been renamed TRPV6 and is also known in the literature as ECaC2 and CaT-like. Electrophysiological studies, as described in detail below, demonstrated that the characteristics of TRPV6 are comparable to those measured for TRPV5, but the expression pattern of TRPV6 is more ubiquitous. Moreover, functional properties of both TRPV5 and TRPV6 are in congruence with the known properties of the putative epithelial calcium channels, which are responsible for intestinal and renal calcium (re)absorption. TRPV6 is currently thought to be the major player in active, intestinal calcium transport.

59.3.2.3

Facilitated Diffusion: Calcium Diffusion Across the Intestinal Epithelial Cell Cytoplasm

Epithelial cells involved in transcellular calcium transport are continuously challenged by substantial Ca ^{2 +} moving through the cytosol, while the cells simultaneously need to maintain very low levels of intracellular Ca ^{2 +} . Free ionized cytoplasmic Ca ^{2 +} concentrations have to be tightly controlled due to cytotoxic effects, and due to potential inhibitory effects on TRPV5 and TRPV6-mediated Ca ^{2 +} currents. Some of these effects are mediated by associated proteins like calmodulin and the protein kinase C substrate 80K-H. The facilitated Ca ^{2 +} diffusion model proposes binding of calcium to an intracellular buffering protein, which delivers the calcium to the basolateral membrane for export. Mathematical modeling predicts that intracellular diffusion of this protein/Ca ^{2 +} complex is the rate-limiting step in transepithelial calcium movement. Indeed, the 1,25(OH) ₂ D ₃ -dependent Ca ^{2 +} -binding protein calbindin-D _9k (CBD _9k ) has been detected in intestinal epithelial cells and has been proposed to deliver calcium to the basolateral membrane for export into the interstitial space. The expression level of calbindin-D _9k in the intestinal epithelium closely correlates with the efficiency of Ca ^{2 +} absorption, and therefore, this protein plays a central role in the facilitated diffusion model. Interestingly, calbindin D _9k may directly enhance plasma membrane Ca ^{2 +} -ATPase (PMCA) activity, which is the principle mechanism of calcium export across the basolateral membrane.

Calbindin-D _9k belongs to a group of intracellular proteins that bind Ca ^{2 +} with high affinity, which causes the protein to undergo structural changes due to electrostatic interactions. Another similar protein called calmodulin (CaM) is known to interact with and regulate the activity of voltage-gated Ca ^{2 +} channels in a Ca ^{2 +} -dependent fashion. The ubiquitously expressed CaM directly interacts with an “IQ motif” present in the carboxy-terminal of these channels, where it functions as a Ca ^{2 +} sensor. This IQ motif, however, is not present in TRPV6. It is unknown whether calbindin-D _9k can perform a similar Ca ^{2 +} sensor function for which a specific interaction with TRPV6 would be required. Furthermore, the striking colocalization of calbindin-D _9k with TRPV6 in the intestine suggests that a functional interaction between these two proteins may occur. Further experiments are needed to delineate whether the function of calbindin-D _9k is restricted to its buffering capacity to maintain low Ca ^{2 +} concentrations in the cell in close vicinity to the channel opening, or whether physical interaction between calbindin-D _9k and TRPV6 is needed to exert a direct regulatory function.

Despite the large body of literature on the role of calbindins D _9k acting both as a Ca ^{2 +} buffer and a ferry facilitating trans-cytoplasmic movement of Ca ^{2 +} to the basolateral site in the intestinal epithelial cells, its precise role in both functions remains controversial. Interestingly, TRPV6 ^−/− , CBD _9k ^−/− , or TRPV6 ^−/− /CBD _9k ^−/− double-knockout mice had normal plasma Ca ^{2 +} . In the same report by Benn et al., under low dietary calcium conditions, wild type, TRPV6 ^−/− and CBD _9k ^−/− mice showed a 4.1-, 2.9-, and 3.9-fold increase in duodenal calcium transport, respectively. A significant, 2.1-fold increase in duodenal calcium transport was also observed in TRPV6 ^−/− /CBD _9k ^−/− double-knockout mice fed a low-calcium diet. 1,25(OH) ₂ D ₃ administration to vitamin D-deficient null mutant and wild-type mice also resulted in a significant increase in duodenal calcium transport. These studies with genetically engineered mice challenge the dogma that TRPV6 and calbindin-D _9k are essential for vitamin D ₃ -induced active intestinal calcium transport and imply that either naturally or through compensatory mechanisms, active intestinal Ca ^{2 +} transport can occur in the absence of TRPV6, CBD _9k or both. Alternative transport models described below (vesicular transport model and Ca ^{2 +} tunneling through ER) may partially explain the lack of a significant phenotype in CBD _9k -deficient mice.

59.3.2.4

Facilitated Diffusion: Calcium Extrusion Across the Basolateral Membrane

Two transport proteins have been implicated in the cellular exit of Ca ^{2 +} . One of them is a bi-directional Na ⁺ /Ca ^{2 +} exchanger NCX1, which under physiological electrochemical gradients, is responsible for basolateral Ca ^{2 +} extrusion. It is widely expressed in absorptive epithelia, although it is considered to be of primary importance in the renal distal convoluted tubules, and of minor significance in the intestinal epithelium. The physiological importance of this exchanger is highlighted by the embryonic lethality of NCX1 null-knockout mice. In heterozygous mice, expression of NCX1 protein in the tubular epithelial cells and Ca ^{2 +} influx via NCX1 in renal tubules are markedly attenuated. The second transporter involved in basolateral Ca ^{2 +} extrusion is a P-type ATPase, a high-affinity Ca ^{2 +} efflux pump PMCA1b, which is abundantly expressed in the intestine. Calcium is expelled through a channel-like opening formed by the transmembrane elements, and phosphorylation is believed to bring about the necessary conformational change such that Ca ^{2 +} bound to the protein is propelled through the opening. Similar to NCX1, PMCA1b null knockout in mice is embryonically lethal.

Four PMCA isoforms have been identified to date and alternatively spliced transcripts have been detected ; PMCA1b is however the predominant isoform in the gut, where it is abundantly expressed in the small intestine. Several studies indicated that PMCA1b is positively regulated by 1,25(OH) ₂ D ₃ in the intestine to increase Ca ^{2 +} absorption. Northern blot analysis indicated that repletion of vitamin D-deficient chickens with 1,25(OH) ₂ D ₃ increases PMCA mRNA expression in the duodenum, jejunum, ileum, and colon. Additional studies by Johnson and Kumar demonstrated that 1,25(OH) ₂ D ₃ causes an increase in the abundance of PMCA protein and stimulates Ca ^{2 +} extrusion. However, plasma 1,25(OH) ₂ D ₃ was not found to be correlated with PMCA1 expression in humans. PMCA activation is dependent upon CaM and inhibition of CaM is, in turn, known to prevent PMCA stimulation. Although some data suggest that extrusion is not the rate-limiting step for Ca ^{2 +} absorption, the estimated V _max of PMCA1b has been reported to be in the range of 20–30 nM Ca/min/mg protein , which should be adequate to extrude Ca ^{2 +} even at the highest rates of Ca ^{2 +} transport.

59.3.2.5

Vesicular Transport Model

According to this model, apical formation of Ca ^{2 +} loaded vesicles is initiated upon TRPV-mediated Ca ^{2 +} uptake. These vesicles can then be transported vectorially by microtubules, or they can fuse with endoplasmic reticulum followed by passive diffusion within the ER lumen. Ca ^{2 +} is then released from the ER at the basolateral membrane through calcium release channels and extruded by NCX1 and PMCA1b (see below). Alternatively, these vesicles may fuse with lysosomes, which move laterally to eventually coalesce with the lateral membrane and release the content through exocytosis. This route is also stimulated by 1,25(OH) ₂ D ₃ . Net epithelial Ca ^{2 +} is inhibited by chloroquine, suggesting that this may be the main route of transcellular Ca ^{2 +} trafficking.

59.3.2.6

Tunneling Through the Endoplasmic Reticulum

This model of transcellular Ca ^{2 +} transport was demonstrated in pancreatic acinar cells. Although not confirmed experimentally, this route may also contribute to vectorial Ca ^{2 +} transport in intestinal epithelial cells. According to this model, Ca ^{2 +} enters the cell through channels as in the preceding models and is transported to the basolateral membrane through passive diffusion in the endoplasmic reticulum. This process involves active Ca ^{2 +} buffering rather than the passive buffering that has been described for facilitated diffusion. Ca ^{2 +} is extruded to the interstitium via basolaterally expressed Ca ^{2 +} -ATPases and Na ⁺ /Ca ^{2 +} exchangers. It has been hypothesized that the endoplasmic reticulum buds off transport vesicles filled with calcium. Fig. 59.2 depicts the three models of transcellular Ca ^{2 +} transport discussed above. Although still little is known about the relative contribution of each mechanism in the transepithelial Ca ^{2 +} transport in the intestine, it is likely that all three models function in a coordinated fashion to provide the necessary redundancy aimed at meeting the short- and long-term systemic Ca ^{2 +} requirements.

Model of transepithelial calcium transport in the duodenal epithelium. Three adjacent enterocytes are depicted along with the major proteins involved in transepithelial intestinal calcium transport. TRPV6 heterotetramers are responsible for brush-border calcium influx, then calcium interacts with the intracellular, buffering protein calbindin-D _9k . After release from calbindin-D _9k , calcium then exits enterocytes across the basolateral membrane via the plasma membrane Ca ^{2 +} -ATPase (PMCA1b). 1,25(OH) ₂ D ₃ is involved in regulating all three of the genes encoding these proteins, in response to body calcium needs. Transcellular and paracellular pathways are shown; paracellular ion movement occurs through tight junctions between adjacent enterocytes, which occurs with high calcium intake levels. Also shown on the apical membrane is the Ca _v 1.3 calcium channel, which may be involved in transcellular calcium transport during times of dietary sufficiency. Te lower enterocyte depicts a schematic representation of the vesicular Ca ^{2 +} transport model and Ca ^{2 +} tunneling through the ER.

(Adapted from Hoenderop JG, Nilius B, Bindels RJ. Calcium absorption across epithelia. Physiol Rev 2005; 85 (1):373–422.)

59.3.3.1

Dietary Calcium

The importance of adequate calcium intake has been best illustrated by studies utilizing VDR and 1α-hydroxylase-knockout mice. The skeletal phenotype of VDR-null mice can be completely rescued by feeding the animals a diet high in calcium, phosphorus, and lactose. Additionally, the abnormal phenotype of 1α-hydroxylase-knockout mice can be corrected by high dietary Ca ^{2 +} intake. The expression of calcium transport proteins was subsequently studied in these knockout models, so as to be able to distinguish the effects of hypocalcemia from those of vitamin D deficiency. Thus studies were initially performed in 1α-hydroxylase-ablated mice fed normal versus high Ca ^{2 +} rescue diets. It is known that under normal circumstances, increased plasma calcium levels act via a negative feedback loop to stimulate PTH secretion, which eventually suppresses 1α-hydroxylase activity, which in turn decreases renal 1,25(OH) ₂ D ₃ production and calcium transport in the intestine. This seems to be mediated by the expression of the intestinal calcium transport proteins TRPV6, calbindin-D _9k , and PMCA1b, which was normalized by feeding the knockout mice the high calcium rescue diet. Comparable observations were made in VDR-knockout mice where duodenal TRPV6 mRNA levels were induced by dietary calcium. Studies with both of these animal models revealed that calcium supplementation can increase transcriptional initiation of genes encoding calcium transporter proteins in the absence of circulating 1,25(OH) ₂ D ₃ , but the molecular mechanism of this vitamin D-independent calcium sensitive pathway remains elusive. It is, however, likely that in addition to the 1,25(OH) ₂ D ₃ response elements in the promoter regions of the TRVP6 and the calbindin-D _9k genes, calcium responsive cis -acting elements are also present. Several promoter elements have been proposed to function as calcium-sensitive transcriptional modulators, including the serum-responsive element in the cAMP/calcium responsive element. However, detailed promoter studies are necessary to characterize fully any putative calcium-responsive elements in the genes responsible for transepithelial calcium transport in the small intestine.

59.3.3.2

Intracellular Ca ^{2 +}

The Ca ^{2 +} concentration in the intestinal lumen can vary greatly, and the sudden appearance of high Ca ^{2 +} level in the lumen can be disastrous, as sustained increases in intracellular Ca ^{2 +} may cause cell death. Thus, Ca ^{2 +} -transporting cells express calbindins to buffer the increases in intracellular Ca ^{2 +} , resulting from Ca ^{2 +} entry through TRPV6 channels. Additionally, to avoid elevations in intracellular Ca ^{2 +} to toxic levels beyond the buffering capacity of the calbindins, TRPV6 exhibits Ca ^{2 +} -dependent inactivation. It has both a fast phase (within 50 ms) and a slow phase of inactivation (over ~ 1 s). The slow phase involves direct binding of calmodulin (CaM) to the TRPV6 C-terminal region. Ca ^{2 +} -dependent binding of CaM to human TRPV6 inactivates the channel, via a protein kinase C site present within the CaM-binding site. The phosphorylation of this protein kinase C site prevents CaM binding, thereby maintaining the activity of TRPV6, so as to allow more Ca ^{2 +} to enter the cell. The first intracellular loop of TRPV6 determines the fast inactivation, which may involve direct interaction of calcium with the channel on the cytoplasmic side of the membrane. In addition, H587, a positively charged amino acid residue in the last transmembrane segment of TRPV6, has also been identified as being involved in fast inactivation.

This feedback inhibition of TRPV6 activity accomplishes two objectives: (1) it protects the cell from Ca ^{2 +} overload and (2) it also limits the flow of Ca ^{2 +} into the cell. TRPV6 activity is inversely related to the intracellular Ca ^{2 +} level on its cytoplasmic side. Thus, the availability of calbindin-D _9k to buffer the local increase in Ca ^{2 +} near the apical membrane will reduce the Ca ^{2 +} -dependent feedback inhibition, and in turn increase the movement of Ca ^{2 +} into the cell. Therefore, a coordinated increase in the expression of both the apical channel TRPV6 and intracellular calbindin-D _9k is necessary to achieve maximal Ca ^{2 +} influx across the apical surface of intestinal epithelial cells. The observation of a lag period in the calbindin-D _9k response to 1,25(OH) ₂ D ₃ relative to that of TRPV6 suggests that the increase in calbindin-D _9k is an “amplifying” mechanism that increases Ca ^{2 +} influx, both by relieving feedback inhibition of the apical entry step and by facilitating diffusion of Ca ^{2 +} from the apical to the basolateral membrane. The time lapse in calbindin-D _9k production and response to 1,25(OH) ₂ D ₃ administration suggests that the increase in intracellular Ca ^{2 +} as a result of TRPV6 expression could contribute to calbindin induction.

59.3.3.3

1,25(OH) ₂ D ₃

The biochemistry, nutritional aspects, metabolism, and physiological and therapeutic potential of vitamin D and its analogs have been the subject of countless reviews and monographs. Most recent and most comprehensive reviews can be found in the recent edition of M.F. Holick’s “Vitamin D: Physiology, Molecular Biology and Clinical Applications” and in the chapter on vitamin D in this book by Wesley Pike and colleagues.

Vitamin D ₃ (cholecalciferol) is supplied from the diet, mostly from fortified dairy products and fish oils, or is synthesized in the skin as previtamin D ₃ from 7-dehydrocholesterol by ultraviolet irradiation. Previtamin D ₃ spontaneously, but slowly isomerizes to vitamin D ₃ (cholecalciferol), which is then transported in the blood by the vitamin D-binding protein (DBP) to the liver, where it is hydroxylated at C-25 by one or more cytochrome P450 vitamin D 25-hydroxylases (CYP2R1, CYP2D11, and CYP2D25), resulting in the formation of 25-hydroxyvitamin D ₃ (25(OH)D ₃ ). Due to its stability and ease of assay, 25(OH)D ₃ is almost exclusively used clinically as a measure of vitamin D status. 25(OH)D ₃ is transported by DBP to the kidney, where it is endocytosed in the proximal tubules and is further hydroxylated at position C-1 by 1α(OH)ase, resulting in the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D ₃ (1,25(OH) ₂ D ₃ ), which is responsible for most, if not all of the biologic effects of vitamin D. 24-Hydroxylase (CYP24, 24(OH)ase), a renal mitochondrial P450 enzyme, can hydroxylate both 25(OH)D ₃ and 1,25(OH) ₂ D ₃ , with preference for the latter. Catabolism of 1,25(OH) ₂ D ₃ to 1,24,25(OH) ₃ D ₃ decreases its biological activity, while hydroxylation of 25(OH)D ₃ to 24,25(OH) ₂ D ₃ leads to a reduction in the pool of 25(OH)D ₃ available for 1α-hydroxylation. Therefore, 24(OH)ase functions to inactivate vitamin D.

Vitamin D plays a key role in skeletal and mineral homeostasis. It is frequently viewed as a pro-bone anabolic hormone due to its positive effects on intestinal and renal Ca ^{2 +} and P _i (re)absorption and through its positive effects on osteoblast differentiation and bone matrix synthesis. Renal synthesis of 1,25(OH) ₂ D ₃ depends upon the Ca ^{2 +} status of the body. When Ca ^{2 +} intake is sufficient and the plasma Ca ^{2 +} concentration is normal, renal 1α-hydroxylase activity is low because there is no need for additional body Ca ^{2 +} . However, when body Ca ^{2 +} levels are low, dietary calcium absorption is low and the plasma Ca ^{2 +} concentration falls, the activity of this enzyme increases to produce the active metabolite 1,25(OH) ₂ D ₃ , to ensure that additional Ca ^{2 +} will be absorbed from the gastrointestinal tract. The molecular mechanism of 1,25(OH) ₂ D ₃ regulation of intestinal calcium transport likely involves the direct interaction of 1,25(OH) ₂ D ₃ with the nuclear vitamin D receptor (VDR). The genomic mechanism of action is similar to that of other steroid hormones, and is mediated by stereospecific interaction of 1,25(OH) ₂ D ₃ with the VDR, which heterodimerizes with the retinoid X receptor (RXR). The heterodimerized receptor then binds to a vitamin D-response element (VDRE) in the promoter of target genes, and transcriptional initiation occurs via an interaction of the VDR with coactivators and the transcriptional machinery. VDR is mainly expressed in epithelia that a play a role in Ca ^{2 +} (re)absorption, confirming the importance of the genomic actions of 1,25(OH) ₂ D ₃ for body calcium homeostasis. Although transcriptional mechanisms of vitamin D-mediated Ca ^{2 +} absorption received considerably more attention, posttranscriptional (nongenomic) mechanisms have also been reported as mediating Ca ^{2 +} apical entry, cytoplasmic diffusion, and paracellular transport.

While the majority of the discussion presented blow is related to the effects of the more biologically active 1,25(OH) ₂ D ₃ , it is not quite clear whether intestinal Ca ^{2 +} absorption is responsive solely to this metabolite, or if it can also be positively affected by an improved vitamin D status as measured by serum 25(OH)D ₃ independent of renal conversion to 1,25(OH) ₂ D ₃ . Heaney et al. found that Ca ^{2 +} absorption in healthy humans was increased by 25% after 4 weeks of treatment with 50 μg 25(OH)D per day without concomitant changes in serum 1,25(OH) ₂ D ₃ levels. These results could be explained by extrarenal expression of 1α hydroxylase (CYP27B1), which is also present in the intestinal mucosa. However, several other studies cast doubt on role for high vitamin D status as a regulator of intestinal calcium absorption by describing no significant association between 25(OH)D ₃ level and higher Ca ^{2 +} absorption efficiency, or showed only a very modest positive relationship.

Catabolic hydroxylation of 25(OH)D ₃ to 24,25(OH) ₂ D ₃ by 24(OH)ase has been typically viewed as leading to a reduction in the pool of 25(OH)D ₃ available for 1α-hydroxylation. However, 24,25(OH) ₂ D ₃ , made under conditions of vitamin D sufficiency, also acts as an endogenous inhibitor of both 1,25(OH) ₂ D ₃ and PTH-stimulated calcium transport. The mechanism of this phenomenon has been reviewed by Khanal and Nemere. 24,25(OH) ₂ D ₃ is believed to bind catalase and decrease its activity thus leading to elevated H ₂ O ₂ levels. Peroxide in turn downregulates the 1,25D ₃ -MARRS receptor (but not the VDR) as well as protein kinase C signaling pathway.

59.3.3.4

PTH and the Calcium-Sensing Receptor (CaSR)

The parathyroid glands play a critical role in maintaining extracellular calcium concentrations, via the ability to sense minute changes in blood calcium levels. The calcium-sensing receptor (CaR) in parathyroid chief cells is able to sense circulating calcium levels and subsequently to induce release of PTH when physiologically necessary. In response to low blood calcium, PTH is secreted into the circulation, which then activates the PTHR1 receptor predominantly in kidney and bone. This receptor, once activated by interaction with PTH, stimulates the activity of renal 1α- hydroxylase and suppresses renal 24-hydroxylase (CYP24) mRNA thereby indirectly leading to increased 1,25(OH) ₂ D ₃ -dependent intestinal calcium absorption. Furthermore, immunohistochemical analysis of rat duodenal sections demonstrated localization of the PTHR1 receptor in epithelial cells along the villus, with intense staining of brush-border and basolateral membranes and the cytoplasm. Direct effects of PTH on calcium transport by isolated rat duodenal enterocytes have been also reported. Initial studies utilizing perfusion of isolated duodenal loops showed that PTH administration increased calcium absorption. These findings were later confirmed in studies that demonstrated that PTH significantly stimulates enterocyte calcium uptake. However, despite these recent investigations, molecular mechanisms of direct PTH action in mediating intestinal calcium transport remain poorly understood. While serum PTH levels vary significantly throughout the course of a day (i.e., high in the morning, lower after consuming calcium-rich meals), Ca absorption efficiency has not been shown to follow a similar diurnal rhythm. To directly address the existence and physiological relevance of PTH signaling in intestinal Ca ^{2 +} absorption, a mouse model with targeted deletion of intestinal PTH receptor 1 is needed.

The CaSR thus plays a crucial role in the regulation of calcium homeostasis by sensing minute changes in circulating calcium levels. In the parathyroid gland, the CaSR allows parathyroid cells to detect changes in blood ionized calcium concentration, to modulate PTH secretion accordingly, and thus to maintain serum calcium levels within a narrow physiological range. CaSR forms a unique molecular target for drugs that can directly alter the activity of the receptor, and thereby modulate extracellular calcium balance. Recently, several compounds, including NPS R-467, have been identified that can activate the CaSR and suppress serum PTH and Ca ^{2 +} levels. However, the involvement of intestinal calcium transport in NPS R-467 mediated hypocalcemia is unclear. Thus, a recent investigation sought to determine the effect of this CaSR agonist on the expression of intestinal calcium transport proteins. These investigators infused mice with NPSR-467 continuously for 7 days and noted reduced serum PTH levels, which were accompanied by a significant decrease in serum calcium concentration. Molecular analysis of duodenal samples demonstrated downregulation of mRNA and protein expression levels of proteins involved in active transcellular calcium absorption. It is currently unknown, however, whether the effects of NPSR-467 could involve a direct action of CaSR in the intestine, or whether the acute hypocalcemic response to this and other similar compounds results from the inhibition of PTH secretion.

59.3.3.5

Estrogen

The intestine is a potential site for estrogen action and there is accumulating evidence that estrogen plays a physiological role in the regulation of intestinal calcium absorption. Estrogen receptors are present in the duodenum and colon; however, the underlying mechanism by which estrogen may regulate calcium absorption is still poorly understood. Additionally, it has not been conclusively established whether there is a direct effect of estrogen on intestinal calcium transport or whether it indirectly mediates this process via an effect on vitamin D metabolism.

It has been long known that in postmenopausal women, calcium balance falls significantly as a result of a reduction in calcium absorption and an increase in urinary calcium loss. Estrogen deficiency can reduce serum 1,25(OH) ₂ D ₃ level, but this can be reversed by estrogen repletion, which is accompanied by increased fractional calcium absorption. This suggested that estrogen depletion leads primarily to a disruption in vitamin D ₃ metabolism.

However, more recent findings suggested that TRPV6 expression was directly regulated by estrogen, as duodenal expression of TRPV6 mRNA in 1α-hydroxylase-null mice and ovariectomized rats is increased by 17β-estradiol administration. Also, another recent study demonstrated that intestinal calcium transport was stimulated by 17β-estradiol, independently of circulating 1,25(OH) ₂ D ₃ levels. It was additionally reported that duodenal TRPV6 expression is reduced in estrogen receptor α-knockout mice and enhanced by estrogen treatment, and further that the estrogen effect on intestinal calcium absorption is mediated by an estrogen receptor (ER). In this investigation, TRPV6 expression was also shown to be enhanced in pregnant VDR-knockout mice and in wild-type littermates. Furthermore, in lactating mice, duodenal TRPV6 expression levels increased 13-fold. Thus, it is clear that the expression of TRPV6 epithelial calcium channels is influenced by estrogen status. Estrogens, hormonal changes during pregnancy, and lactation have distinct vitamin D-independent effects on active duodenal calcium absorptive mechanisms, primarily via a major induction of TRPV6 mRNA expression. These estrogen effects seem to be mediated solely by estrogen receptor α. Interestingly, Weber et al. recently described an estrogen-responsive element in the promoter of the mouse TRPV6 gene ; however, detailed promoter analysis is necessary to identify the regulatory sites involved in estrogen-mediated regulation of TRPV6. Despite these recent investigations, it remains to be conclusively demonstrated that these changes have functional implications on intestinal Ca ^{2 +} absorption.

Overall, these data confirm that estrogen and 1,25(OH) ₂ D ₃ are independent, potent regulators of TRPV6 expression, which is involved in active intestinal calcium absorption. These data thus suggest that the function of estrogen in the maintenance of body calcium balance could be at least partially fulfilled by regulation of TRPV6 levels, thereby controlling intestinal calcium absorption to maintain normal serum Ca ^{2 +} levels, which are necessary for proper bone calcification.

59.3.3.6

Thyroid Hormone

The thyroid hormones, thyroxine (T ₄ ) and triiodothyronine (T ₃ ), function primarily as key regulators of metabolism. Several studies have suggested that thyroid dysfunction is also associated with disturbances of calcium and phosphate homeostasis. Hyperthyroidism has been associated with hypercalcemia and high bone turnover rates leading to osteopenia. Consistently, compared to healthy animals, Ca ^{2 +} uptake was higher in duodenal brush-border membrane vesicles (BBMV) isolated from hyperthyroid rats and lower in BBMV from hypothyroid rats. Thyroid hormones increase serum PTH and 1,25(OH) ₂ D ₃ levels; therefore, their effects on intestinal Ca ^{2 +} absorption may be indirect. T ₃ , however, directly affects the developing intestine. Cross and Peterlik showed that although T ₃ treatment alone had no effect upon jejunal calcium uptake, it augmented the effect of 1,25(OH) ₂ D ₃ on Ca ^{2 +} transport. These findings suggest that T ₃ may regulate VDR levels in the intestinal epithelium. This, however, has not been proven. Other mechanisms of thyroid hormone-induced sensitization to vitamin D ₃ were proposed by Schräder et al. who suggested that synergistic effects of T ₃ and 1,25(OH) ₂ D ₃ on calbindin D _9k gene transcription is mediated via the formation of vitamin D receptor (VDR)-thyroid hormone receptor (TR) heterodimers, which interacts with putative VDREs. However, this observation was not confirmed in another study where the authors showed the opposite effects and that thyroid hormone receptor (TR) expression can repress 1,25(OH) ₂ D ₃ -induced transcription predominantly by sequestering RXR.

59.3.3.7

Growth Hormone and IGF-1

Denis et al. demonstrated that intestinal calcium and phosphate transport is positively regulated in pigs by exogenous administration of porcine growth hormone (GH). These investigators treated growing pigs for two months with daily injections of growth hormone, and then performed a 10-day balance study at the end of this period. They found that all aspects of mineral homeostasis were stimulated, including circulating 1,25(OH) ₂ D ₃ levels (inc. by 40%), Ca ^{2 +} absorption and retention (inc. 70%), P _i absorption (inc. 33%) and retention (inc. 45%), and jejenal calbindin-D _9k levels (inc. 40%). It is not known however, if these physiological changes noted with exogenous growth hormone treatment are direct effects or if they are principally mediated by effects on vitamin D ₃ homeostasis.

During childhood and adolescence, GH and its physiologic mediator insulin-like growth factor I (IGF-1) have a major role in linear bone growth and accrual of bone mass. GH can also promote intestinal Ca ^{2 +} absorption either indirectly, through activation of renal 1α-OHase and increased 1,25(OH) ₂ D ₃ serum level ;, or directly by stimulating expression of duodenal calbindin D _9k levels without significantly affecting serum 1,25(OH) ₂ D ₃ levels. Moreover, GH treatment can prevent the decrease in VDR expression in ovariectomized rats, suggesting that GH may increase sensitivity to 1,25(OH) ₂ D _3.

At least a part of the effect of GH on Ca ^{2 +} absorption is believed to be mediated through IGF-1 via a vitamin D ₃ signaling-independent mechanism(s). In men, Ca ^{2 +} absorption was positively correlated with IGF-1 and that age-related correlation between declines in IGF-1 and Ca ^{2 +} absorption could not be explained simply by a reduction in serum 1,25(OH) ₂ D ₃ levels. However, the molecular mechanisms by which the GH/IGF-1 axis may influence the intestinal Ca ^{2 +} absorption independently of vitamin D ₃ remain unknown.

59.3.3.8

Klotho

In 1997, Kuro-o et al. described transgenic mice obtained by accidental mutational insertion, which exhibited symptoms typical of human aging: short lifespan, infertility, atherosclerosis, skin atrophy, severe osteoporosis, and emphysema. A then unknown gene was disrupted or mutated in its 5′-flanking promoter region by the random insertion of an exogenously introduced transgene. The coding region of this mutated gene termed αKlotho (frequently referred to as just Klotho, KL) was still preserved, but its expression was markedly reduced generating a mouse strain with a strong hypomorphic allele. The Klotho gene encodes a 130-kDa single pass transmembrane protein with β-glucuronidase activity, which is primarily expressed in epithelial cells of the renal distal tubules, the choroid plexus, and parathyroid gland. A soluble form can also be found in blood plasma and cerebrospinal fluid. αKlotho circulates as an endocrine substance with a multitude of effects, some of which are related to its role as a coreceptor for FGF23, a hormone originating primarily in osteoblasts, which is an intrinsic part of Ca ^{2 +} and P _i homeostases. Involvement of the Klotho gene in mineral homeostasis was suggested by recent genetic studies which showed that certain allelic variants of Klotho constitute one of the genetic factors influencing bone mineral density in male adults and in postmenopausal women. All this evidence implies that Klotho is a multifunctional protein that regulates phosphate/calcium metabolism as well as aging. Klotho has also been directly implicated in the regulation of transepithelial Ca ^{2 +} transport via modulation of apical entry as well as basolateral Ca ^{2 +} exit. Apical Ca ^{2 +} entry is partially dependent on TRPV5 and TRPV6 channel abundance at the epithelial cell surface. Shuttling of TRPV5 to and from a subapical pool is a key component of this regulation, and high Ca ^{2 +} influx may be obtained by prolonging the channel’s duration at the cell surface before inactivation or internalization. Klotho, through its glucuronidase activity, modulates N-glycosylation of TRPV5 at Asn358 by removal of the capping sialic acid, which enables physical interaction of galectin 1 with TRPV5 on the cell surface and promotes its apical retention. Reduced renal Klotho expression by colitis-associated proinflammatory cytokine IFNγ has been implicated in downstream endocytosis, ubiquitination, and degradation of TRPV5 and urinary Ca ^{2 +} loss. Activating effects of Klotho on TRPV6-mediated Ca ^{2 +} flux have also been shown, although the effects of Klotho on intestinal Ca ^{2 +} absorption has not been demonstrated to date.

βKlotho, encoded by a paralog KLB gene, regulates energy metabolism as the coreceptor for FGF15/19 and FGF21, analogous to the role of αKlotho for FGF23. Both βKlotho and FGF15/19 are expressed in the intestine and together with hepatic βKlotho have been implicated in the control of Cyp7a1 gene expression and bile acid synthesis.

59.3.3.9

Stanniocalcin

In mammals, early studies on stanniocalcin 1 (STC1) cloning and expression pattern suggested that this hypocalcemic factor is expressed in multiple organs including calcium-transporting epithelia, such as intestine, colon, kidney, and placenta. It is also released by the corpuscles of Stannius , specialized organs that are adjacent to and scattered throughout the kidney. Stanniocalcin acts locally in the gut to modulate calcium and phosphate absorption, and overexpression in mice results in high serum phosphate, dwarfism, and increased metabolic rate. In vivo studies using the rat kidney indicated that STC1 may regulate blood calcium by increasing reabsorption of phosphate, and in vitro experiments on mammalian intestine have shown that STC1 can reduce the movement of calcium while increasing absorption of phosphate across the gut. In both models, the predicted net effect would be a decrease in circulating levels of calcium, thereby acting as an antihypercalcemic factor. Furthermore, expression of stanniocalcin 1 is induced by 1,25(OH) ₂ D _3, thus suggesting its participation in a form of a negative feedback regulation. Indeed, overexpression of STC in Caco-2 cells downregulated TRPV5 and TRPV6 mRNA and protein expression, and the reverse trend was seen with siRNA-mediated STC1 knockdown, while PMCA1b, NCX1, or VDR remained unaffected. Few studies have addressed the expression or function of stanniocalcin 1 in mammals in vivo, and the expression of STC1 in the epithelial cells is questionable. Thus, further work is necessary before strong conclusions may be drawn regarding the involvement of this hormone in intestinal calcium transport.

59.4.2.1

Mechanisms and Sites of Phosphate Absorption

Intestinal phosphate absorption in mammals occurs primarily in the small intestine, with relative absorptive efficiency in duodenum being greater than jejunum, which is greater still than ileum. However, the bulk of phosphate absorption occurs in the jejunum, due to its longer length and increased transit time of the digesta through this gut segment. In experimental rodents, the contribution of proximal and distal small intestinal segments to P _i absorption varies by species. In mice, ileal P _i absorption is a significant contributor, whereas in rats maximal absorption occurs in the duodenum with very little absorption occurring in the ileum, which is similar to the pattern reported in humans. However, in both rodent species only the jejunum shows an increase in P _i absorption in response to 1,25(OH) ₂ D ₃ treatment.

Early studies of intestinal transport suggest that paracellular transport driven by a passive diffusional process predominates under standard dietary conditions. The 1980s brought a distinction between sodium-dependent and -independent components. Both of these mechanisms contribute to P _i transport across intestinal brush-border and basolateral membranes, with each of these membrane domains exhibiting significant differences in affinities and total transport capacity of the sodium-dependent transport process. Intestinal perfusion of the human jejunum demonstrates that active transport of phosphate is maximal at low phosphate intake levels and conversely, that nonsaturable, passive diffusion occurs with high phosphate concentrations. Due to the negative charges in paracellular intestinal phosphate transport channels, simple diffusion across the intestinal mucosa at low phosphate concentrations via this route is unlikely. The highly electronegative interior of the cell (~ 50 − 70 mV) results in a transmembrane potential difference between the interior of the cell and the intestinal lumen. This potential difference functions as a powerful barrier to the transport of negatively charged <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='H2PO4−’>H2PO4−H2PO4−
H 2 PO 4 −
or <SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='H2PO42−’>H2PO42−H2PO42−
H 2 PO 4 2 −
ions across the brush-border membrane. Whereas <SPAN role=presentation tabIndex=0 id=MathJax-Element-3-Frame class=MathJax style="POSITION: relative" data-mathml='H2PO4−’>H2PO4−H2PO4−
H 2 PO 4 −
ions, which can accumulate to high concentrations at acidic pH in proximal intestinal segments, are more readily transported against this electronegative gradient, <SPAN role=presentation tabIndex=0 id=MathJax-Element-4-Frame class=MathJax style="POSITION: relative" data-mathml='H2PO42−’>H2PO42−H2PO42−
H 2 PO 4 2 −
(the ionic species which predominates at the alkaline pH of the jejunum and ileum) requires an active, energy-dependent process to ensure adequate absorption. 1,25(OH) ₂ D ₃ stimulates active phosphate transport across both brush-border and basolateral membranes in the small intestine, and we are beginning to understand the molecular mechanisms involved (as discussed below). Furthermore, because the potential difference across the basolateral membrane of intestinal epithelial cells does not inhibit the transport of negatively charged, ionic phosphate molecules out of the cell into the interstitial space and because the P _i concentrations in the cell and plasma are ~ 2 and ~ 1.0 mM, respectively, efflux of phosphate across the basolateral membrane likely occurs passively down an electrochemical gradient. The exact mechanism(s) that control transepithelial movement of P _i , remain largely uncharacterized, since the overall regulation of this process is complicated by the fact that P _i becomes rapidly integrated into various metabolic processes once it enters enterocytes. As such, the rate and control of phosphate absorption are not only a function of the brush-border and basolateral membrane transport processes but it is also dependent on the rate of utilization and esterification of intracellular phosphate.

59.4.2.2

Na ⁺ -Dependent P _i Absorption: Type I NaP _i Cotransporters

Type I transporter group consists of sodium-phosphate cotransporter NPT1 (SLC17A1/NaP _i -1), NPT3 (SLC17A2), NPT4 (SLC17A3), and NPT5 (SLC17A4) that belong to the SLC17 transporter family, which also includes lysosomal acidic sugar transporter (sialin; SLC17A5), vesicular glutamate transporters (SLC17A7, SLC17A6, and SLC17A8, respectively), and a vesicular nucleotide transporter (SLC17A9). Although the first known member, NaP _i -1 was cloned as a P _i transporter, the SLC17 family is now recognized as multifunctional organic anion transporters, or as an anion:cation symporter family. As an example, expression of NPT5 has been identified in the intestine, but it was postulated to facilitate intestinal ureate excretion.

59.4.2.3

Na ⁺ -Dependent P _i Absorption: Type II NaP _i Cotransporters

Intestinal transport of dietary phosphate is dependent upon the activity of the type IIb, sodium-dependent phosphate cotransporter (NaP _i -IIb) ( Fig. 59.4 ). This protein is a member of the SLC34 family of solute carriers, which is comprised of three members: NaP _i -IIa ( SLC34A1 ), NaP _i -IIb ( SLC34A2 ), and NaP _i -IIc ( SLC34A3 ). All three have a preference for divalent ( <SPAN role=presentation tabIndex=0 id=MathJax-Element-5-Frame class=MathJax style="POSITION: relative" data-mathml='HPO42−’>HPO42−HPO42−
HPO 4 2 −
) phosphate and are inhibited by phosphonoformic acid (PFA); NaP _i -IIa and NaP _i -IIb are electrogenic and transport phosphate with a stoichiometry of 3:1 Na ⁺ 🙁 <SPAN role=presentation tabIndex=0 id=MathJax-Element-6-Frame class=MathJax style="POSITION: relative" data-mathml='HPO42−’>HPO42−HPO42−
HPO 4 2 −
) whereas NaP _i -IIc is electroneutral with a 2:1 Na ⁺ 🙁 <SPAN role=presentation tabIndex=0 id=MathJax-Element-7-Frame class=MathJax style="POSITION: relative" data-mathml='HPO42−’>HPO42−HPO42−
HPO 4 2 −
) stoichiometry. NaP _i -IIa is expressed in apical membranes of epithelial cells in the renal proximal tubules, in osteoclasts and also in neurons, while NaP _i -IIc expression is restricted to the renal epithelium. NaP-IIb ( SLC34A2 ) was originally identified based on EST clones derived from lung tissue and is highly expressed in lung, small intestine, and many other tissues. By immunofluorescence, NaP _i -IIb was localized to brush-border membranes of enterocytes along the entire villus length, although autoradiography studies showed that phosphate uptake is restricted to enterocytes in the mid- to upper regions of duodenal and jejunal villi. On western blots, fully glycosylated NaP _i -IIb is seen as a band of approximately 110 kDa in mice, however, smaller bands at 75–80 kDa have been reported in rat, brush-border membrane protein samples. Interestingly, in weanling animals, NaP _i -IIb is reported to be only partially glycosylated. Thus, the NaP _i -IIb cotransporter is strongly expressed in the small intestinal epithelium and has been long thought to be the major, active transport system for dietary phosphate. Null knockout of the mouse NaP _i -IIb resulted in early embryonic lethality, which was attributed its normally high expression in the extraembryonic tissues (labyrinthine zone, where embryonic and maternal circulations were in closest contact) and a defect in phosphate absorption from the maternal circulation. To address the role of NaP _i -IIb in postnatal life, Sabbagh et al. created an inducible global-knockout model of NaP _i -IIb by crossing SLC34A2 ^fl/fl or SLC34A2 ^+/fl mice with CMV-CreER ^T2 mice. In this study, inducible deletion of the SLC34A2 gene led to increased fecal phosphate excretion and hypophosphaturia. The latter was attributed to reduced serum levels of the phosphaturic hormone FGF23 and increased expression of the renal NaP _i -IIa protein. NaP _i -IIb-deficient mice fed low-phosphate diet absorbed approximately 50% less phosphate than their wild-type littermates after oral phosphate bolus, thus confirming a major role of this transporter in phosphate absorption. In vitro analysis of active phosphate transport in the isolated ileum (the major site of Na ⁺ -dependent P _i absorption in mice) demonstrated that NaP _i -IIb contributed > 90% of total active phosphate absorption. Consistent with this report, intestinal epithelial cell-specific deletion of SLC34A2 gene led to fecal wasting of P _i and complete absence of Na/P _i cotransport activity in the ileal brush-border membrane vesicles. Interestingly, blood P _i concentrations did not change in these mice, likely due to compensatory increase in renal P _i reabsorption via elevated NaP _i -IIa, as well as due to reduced plasma levels of intact FGF-23, which normally acts to inhibit NaP _i -IIa activity in the renal proximal tubules.

Only gold members can continue reading. Log In or Register to continue

Share this:

• Click to share on Twitter (Opens in new window)
• Click to share on Facebook (Opens in new window)

Related posts:

Gastrointestinal Hormones Cell Death Enteric Neural Regulation of Mucosal Secretion Enteric Nervous System Structure and Neurochemistry Related to Function and Neuropathology Bile Formation and the Enterohepatic Circulation The Vitamin D System: Biological and Molecular Actions in the Intestine and Colon

Stay updated, free articles. Join our Telegram channel

Join