by K Wongdee · 2019 · Cited by 18 — For example, luminal iron, circulating fibroblast growth factor (FGF)-23, and stanniocalcin can decrease calcium absorption, thereby preventing excessive

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The Journal of Physiological Sciences (2019) 69:683Œ696 REVIEW Factors inhibiting intestinal calcium absorption: hormones and˜luminal factors that˜prevent excessive calcium uptake Kannikar˜Wongdee 1,2˜· Mayuree˜Rodrat 2,3˜· Jarinthorn˜Teerapornpuntakit 2,4˜· Nateetip˜Krishnamra 2,3˜· Narattaphol˜Charoenphandhu 2,3,5,6Received: 4 March 2019 / Accepted: 9 June 2019 / Published online: 20 June 2019 © The Physiological Society of Japan and Springer Japan KK, part of Springer Nature 2019 Abstract Besides the two canonical calciotropic hormones, namely parathyroid hormone and 1,25-dihydroxyvitamin D [1,25(OH) 2D3], there are several other endocrine and paracrine factors, such as prolactin, estrogen, and insulin-like growth factor that have been known to directly stimulate intestinal calcium absorption. Generally, to maintain an optimal plasma calcium level, these positive regulators enhance calcium absorption, which is indirectly counterbalanced by a long-loop negative feedback mechanism, i.e., through calcium-sensing receptor in the parathyroid chief cells. However, several lines of recent evidence have revealed the presence of calcium absorption inhibitors present in the intestinal lumen and extracellular ˜uid in close vicinity to enterocytes, which could also directly compromise calcium absorption. For example, luminal iron, circulating ˚broblast growth factor (FGF)-23, and stanniocalcin can decrease calcium absorption, thereby preventing excessive calcium uptake under certain conditions. Interestingly, the intestinal epithelial cells themselves could lower their rate of calcium uptake after exposure to high luminal calcium concentration, suggesting a presence of an ultra-short negative feedback loop independent of systemic hormones. The existence of neural regulation is also plausible but this requires more supporting evidence. In the present review, we elaborate on the physiological signi˚cance of these negative feedback regulators of calcium absorption, and provide evidence to show how our body can e˛ciently restrict a ˜ood of calcium in˜ux in order to maintain calcium homeostasis. Keywords Calcium absorption˝· Calcium-sensing receptor (CaSR)˝· Fibroblast growth factor (FGF)-23˝· Iron transport˝· Parathyroid hormone (PTH)˝· Vitamin D Introduction Besides being the major inorganic component in bone, cal -cium is an essential element that has roles in several func – tions, e.g., neurotransmitter release, muscle contraction, blood coagulation, and intracellular signal transduction. Ninety-nine percent of body calcium is stored in bone in the form of hydroxyapatite crystal [Ca 10 (PO 4)6(OH) 2], while the remaining 1% is distributed in the plasma, interstitium, intracellular ˜uid, and within the cells in mitochondria and endoplasmic reticulum. The intracellular calcium is main – tained at concentration as low as 0.1˝µM, which is lower than the extracellular calcium concentration (free ionized cal – cium of 1.1Œ1.3˝mM) by ~ 1000-fold. An excess of calcium in either the intracellular or extracellular ˜uid is extremely dangerous. Because free ionized calcium is toxic to the cell, a prolonged rise in the intracellular calcium can lead to cell death by activating various enzymes, such as protein kinase * Narattaphol Charoenphandhu 1 Faculty of˝Allied Health Sciences, Burapha University, Chonburi, Thailand 2 Center of˝Calcium and˝Bone Research (COCAB), Faculty of˝Science, Mahidol University, Bangkok, Thailand 3 Department of˝Physiology, Faculty of˝Science, Mahidol University, Rama VI Road, Bangkok˝10400, Thailand 4 Department of˝Physiology, Faculty of˝Medical Science, Naresuan University, Phitsanulok, Thailand 5 Institute of˝Molecular Biosciences, Mahidol University, Nakhon˝Pathom, Thailand 6 The Academy of˝Science, The Royal Society of˝Thailand, Dusit, Bangkok, Thailand

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684 The Journal of Physiological Sciences (2019) 69:683Œ696 C, caspases, phospholipases, proteases, and endonucleases as well as apoptotic process [ 59]. Therefore, the intestine, which is the only site for calcium entry into the body, must have mechanisms to regulate calcium uptake. Besides, the intestinal epithelial cells themselves also need to tightly control their intracellular calcium concentration to prevent super˜uous calcium uptake, which may damage themselves as well as other cells in the body [ 57, 59, 106].Since the intestine is the only route for calcium uptake, it is subjected to local and systemic regulation, which helps protect against inadequate as well as exces – sive absorption of calcium. Both stimuli and inhibitors of calcium absorption have been described, with the plasma calcium-PTH-vitamin D feedback loop as the most prominent feedback regulation. Although local hormones, secretory factors, and some components in the ingested foods, e.g., iron, phytate, oxalate, and tannin (Fig.˝ 1), can inhibit calcium absorption, the physiological significance of these substances is not fully understood. The present article thus focuses on the regulatory roles of these intestine- and nutrient- derived factors and their feedback mechanisms in the suppression of intestinal calcium absorption. It is gen – erally accepted that both stimulatory and inhibitory regulators of calcium absorption are humoral factors with autocrine, paracrine, and endocrine functions, as depicted in Fig.˝ 1. Apparently, the possible role of neural regulation may also exist and may be analogous to the splanchnic nerve regulation of calcium uptake across the gallbladder mucosa [ 73]. The overview of factors controlling intestinal calcium transport is sum – marized in Table˝ 1.Fig. 1 Intrinsic (humoral and neural) and extrinsic (luminal) regulators of intestinal calcium absorption (please see text for details)

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685The Journal of Physiological Sciences (2019) 69:683Œ696 Mechanisms of˜intestinal calcium absorption in˜mammalsIn mammals, calcium is absorbed through the intestinal epithelial cells via two major pathways, i.e., transcellular and paracellular pathways [ 24]. The relative contribution of transcellular and paracellular calcium absorption depends on several factors including the amount of calcium intake, solubility and chyme alkalinity, bioavailability, and segment transit time [ 52]. Although both calcium transport mecha – nisms are found along the entire length of the intestine in humans and rodents, the vitamin D-dependent transcellu – lar calcium transport is predominant in the proximal small intestine, particularly the duodenum, and is of importance in low-calcium intake conditions [ 3, 7, 103]. A regular diet without dairy products is generally considered as a low-nor -mal calcium diet, which requires an active calcium transport mechanism. The uphill transcellular active transport is composed of three steps, i.e., (1) apical vitamin D-dependent calcium entry via the transient receptor potential cation channel, subfamily V, member 6 (TRPV6), and to a lesser extent Table 1 Summary of possible factors a˙ecting calcium absorption across the intestinal epithelium CaBP-D 9k calbindin-D9k, CaSR calcium-sensing receptor, Cav1.3 L-type voltage-gated calcium channel, FGF-23 ˚broblast growth factor 23, ENS enteric nervous system, N/A not available, NCX1 sodium calcium exchanger 1, TJ tight junction, VIP vasoactive intestinal peptide Factors Transcellular Paracellular Net Ca2+ absorption References Extrinsic /luminal˝Ca2+ (long-term low luminal Ca2+ or ˝˝˝low calcium diet) Increase expression of TRPV5/6, CaBP-D 9k, NCX1, PMCA 1b,Increase activity of PMCA 1b and NCX1 N/A[4, 8, 10] ˝Ca2+ (prolong exposure to high luminal ˝˝Ca2+)Possibly by increase FGF-23 expression (Ca2+ > 30˝mM) and inhibit transcellular transport by unknown mechanism N/A[84] ˝Iron N/AN/A[58] ˝Glucose/galactose (SGLT1 substrates) Decrease Cav1.3 activityIncrease solvent drag-induced paracellular calcium ˜ow [52, 91] ˝Amino acids N/AIncrease NHE activity [96]Increase nutrient-induced paracel -lular calcium ˜ow ˝Fructose Decrease expression of TRPV6 and CaBP-D 9kN/A[27, 28]Intrinsic ˝Hormone ˝˝1,25(OH)2D3Increase expression of TRPV5/6, CaBP-D 9k, NCX1, PMCA 1bStimulate nongenomic signaling pathways involving PI3K, PKC, and MEK to enhance calcium transport [32, 40]Increase expression of claudin-2, 12 ˝˝PTHIncrease calcium uptake (Unknown mechanism) N/A[71] ˝˝FGF-23 Decrease 1,25(OH) 2D3-enhanced expressions of TRPV5/6, CaBP- D9kDecrease 1,25(OH) 2D3-enhanced paracellular calcium transport and calcium permeability [53, 54] Paracrine ˝˝FGF-23 Increase CaSR activity N/A[84] ˝˝Stanniocalcin-1 Decrease expression of TRPV5/6, CaBP-D 9kN/A[107]No change in the expression of NCX1, PMCA 1bNeural (ENS) ˝˝VIPN/AN/A(Indirect evidence) [73]

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686 The Journal of Physiological Sciences (2019) 69:683Œ696 TRPV5, (2) cytoplasmic calcium translocation (bound to calbindin-D9k), and (3) basolateral calcium extrusion via plasma membrane Ca2+-ATPase 1b (PMCA 1b) and Na +/Ca2+-exchanger 1 (NCX1) [ 13, 24]. Regarding calcium entry into the enterocytes, besides TRPV5/6 channels, the apical voltage-dependent L-type calcium channel subtype 1.3 (Cav1.3) is also involved in calcium uptake, particularly in the presence of depolarizing nutrients in the lumen, such as glucose, galactose, and some amino acids [ 52, 68, 96].Once calcium enters the cells, it is bu˙ered by the cyto -plasmic calcium-binding protein, calbindin-D 9k [24, 86]. Other calcium-binding proteins such as calmodulin may also contribute, albeit to a lesser extent, to the intracellular cal – cium translocation [ 24]. Finally, calcium is extruded across the basolateral membrane through several transporters, e.g., PMCA 1b, NCX1 coupled to Na +/K+-ATPase, and Na +/Ca2+/K+-exchanger (NCKX), the latter of which extrudes one K+ and one Ca2+ with in˜ux of four sodium ions to maintain low intracellular calcium concentration [ 52, 57, 88].The second major route is the paracellular pathway, which allows calcium to traverse the lateral intercellular (paracel – lular) space. Calcium transport via this route can be both cellular energy-dependent active or gradient-dependent pas – sive transport. The paracellular active transport is generally mediated by solvent drag, which is induced by sodium eˆux by Na +/K+-ATPase that creates hyperosmotic environment in the paracellular space. This subsequently induces water di˙usion in a lumen-to-plasma direction, dragging along with it small permeable molecules, including calcium [ 6, 24, 52].Regarding the paracellular passive calcium transport driven by the concentration gradient of calcium between the lumen and plasma, it occurs more readily in the condition of high-calcium intakeŠgenerally considered signi˚cant when luminal calcium > 5˝mMŠand can occur throughout the entire length of the intestine [ 24 , 30 ]. Paracellular transport is also determined notably by the size- and charge-selective properties of the tight junction, which creates speci˚c bar -riers for ions, such as calcium, sodium, and chloride [ 112]. Claudins is the major family of the tight junction-associated proteins responsible for the paracellular size- and charge- selective properties. Claudin-2, -12, and -15 in particular have been shown to have signi˚cant roles in the regulation of paracellular calcium transport across the intestinal epi – thelia [ 14, 24, 32]. Claudin-2 has negatively charged amino acids (e.g., aspartate) in the extracellular domains, which protrude into the paracellular space to form a tight junction pore. With these negative charges, claudin-2 is permeable to cations [21, 110]. In the presence of upregulated claudin-2 expression, the luminal calcium is expected to easily di˙use across the paracellular space in both lumen-to-plasma and plasma-to-lumen directions. When the luminal concentra – tion is greater than the plasma ionized calcium level (e.g., luminal calcium > 5˝mM), calcium prefers to di˙use into the body. On the other hand, an extremely low calcium con – centration in the lumen may aggravate calcium secretion, which probably results in net calcium loss during claudin-2 overexpression. However, further experiment is required to con˚rm the latter hypothesis. Possible feedback loops for˜intestinal calcium absorption To maintain calcium homeostasis, the amount of calcium absorbed by the intestine is ˚ne-tuned to match the body calcium requirement by several factors, including hor -mones and luminal nutrients [ 57]. Since calcium absorp – tion is relatively low under normal conditions (~ 25Œ35% of total calcium intake) as compared with other minerals (such as magnesium and phosphorus), most regulatory factors are stimulators for enhancing calcium absorption. However, little is known regarding how calcium absorption is regulated when faced with excessive calcium intake and a potential risk of calcium toxicity. Normally, the level of plasma calcium is considered as a component of the nega – tive feedback regulation to suppress parathyroid hormone (PTH) release and production of 1,25-dihydroxycholecal – ciferol [1,25(OH) 2D3], thereby slowing down the intestinal calcium absorption as discussed below. Roles of˜hormonesThe classical hormones involved in the positive regu – lation of intestinal calcium absorption are PTH and 1,25(OH)2D3, the latter of which is synthesized in the kidney by the renal proximal tubule and which directly enhances the intestinal calcium absorption through active transcellular and passive paracellular pathways in both genomic and non-genomic fashion (for review, please see Ref. [ 57]). For the active transcellular pathway, 1,25(OH)2D3 exerts genomic actions by binding to vita -min D receptor (VDR), thus increasing the expression of calcium transport machinery, i.e., TRPV6, calbindin- D9k, PMCA 1b, and NCX1 [ 11, 20, 24, 104]. In addition, 1,25(OH)2D3 probably exerts a non-genomic action to rapidly enhance calcium transport by binding to the plasma membrane receptor 1,25(OH) 2D3-MARRS (mem-brane-associated, rapid response steroid-binding) pro -tein [72]. In brief, the non-genomic action occurs when 1,25(OH)2D3 binds to the membrane-bound 1,25(OH) 2D3-MARRS instead of the intracellular/nuclear VDR, which is a transcription factor [ 29]. This membrane-bound receptor can activate certain second messengers, includ – ing phospholipase A2 (PLA2) and protein kinase C (PKC) [ 26, 87]. Regarding the paracellular absorption,

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687The Journal of Physiological Sciences (2019) 69:683Œ696 1,25(OH)2D3 enhances calcium transport through both passive diffusion and solvent drag mechanisms, in part by modifying the charge- and size-selective properties of the tight junction proteins, particularly claudin-2 and -12 [ 32].PTH is widely known as a hypercalcemic and hypophosphatemic hormone upstream to 1,25(OH) 2D3. It elevates the plasma calcium levels by enhancing osteo – clastic bone resorption, renal calcium reabsorption, and increasing intestinal calcium uptake [ 37]. However, the role of PTH on intestinal calcium absorption is normally indirect through renal 1,25(OH) 2D3 production. Speci˚ -cally, PTH stimulates the transcription of the CYP27B1 gene for renal enzyme 1ˇ-hydroxylase, and suppresses CYP24A1 for renal 24-hydroxylase, an enzyme respon – sible for 1,25(OH) 2D3 degradation [ 111], thus elevating the plasma level of 1,25(OH) 2D3. For the direct action of PTH on the intestinal calcium absorption, it was reported that N-terminal fragment 1Œ34 of PTH could stimulate calcium transport in perfused duodenal loops from nor -mal chicks transcaltachia (rapid, non-genomic)Ša rapid hormonal stimulation of intestinal calcium absorption [ 71, 75]. Moreover, the presence of PTH receptor 1 (PTH1R) in the basolateral membrane of rat intestinal epithelial cells suggests possible direct action of PTH in the intestine. However, this study did not demonstrate the regulation of PTH on intestinal calcium absorption [ 36]. Indeed, direct exposure to PTH not only increases calcium absorption but also promotes transport of other ions, e.g., potassium, chloride, and bicarbonate, across the intestinal epithelium. There was evidence that PTH regulation of bicarbonate secretion is not an adaptive mechanism but is more like transcaltachia (rapid, non-genomic). These studies used Caco-2 cells, which may re˜ect a colonic phenotype unless the results are con˚rmed in small intestine [ 16, 61]. Up till now, the physiological signi˚cance of the PTH-regulated ion transport in the gut is not clearly known. Another hormone that was recently shown to have a calcium-regulating function is the bone-derived ˚broblast growth factor (FGF)-23 [ 53, 54, 79]. FGF-23 was gener -ally recognized as a phosphaturic hormone produced and secreted from osteocytes and osteoblasts. Its secretion is enhanced by high serum phosphate, 1,25(OH) 2D3, and perhaps calcium [ 64, 79, 100]. The systemic actions of FGF-23 are to increase urinary phosphate excretion and suppress 1,25(OH) 2D3 production. FGF-23 suppresses CYP27B1 gene expression and stimulates CYP24A1 gene expression, leading to a decreased circulating 1,25(OH) 2D3 [78, 89]. Furthermore, FGF-23 has recently been shown to act directly on the intestine to abolish the 1,25(OH) 2D3-stimulated intestinal calcium absorption [ 53 , 54]. The cel -lular mechanism of FGF-23 as a local inhibitor of calcium absorption is elaborated in fiintrinsic inhibitory factorsfl. Roles of˜calcium-sensing receptor (CaSR) Parathyroid chief cells, renal tubular cells, and osteoblasts are known to express CaSR for sensing pericellular calcium. Normally, CaSR also senses other positively charged ions (e.g., Mg2+, Cd2+, Ba2+, La3+ and Gd3+), cationic molecules (e.g., ˘-glutathione), and amino acids and polyamines (e.g., tryptophan, L-phenylalanine, spermidine, and spermine) [ 12, 22]. Thus, an increase in plasma calcium level is detect – able by parathyroid chief cells, resulting in an inhibition of PTH release and decrease in calcium absorption (Fig.˝ 2a). However, up until now, the role of CaSR in the intestinal epithelial cells has been elusive, and whether it can directly modulate intestinal calcium absorption independent of PTH is largely unknown. Indeed, CaSR may potentially play a role in local regulation of intestinal calcium absorption since it is abundantly expressed in both apical and baso – lateral membranes of the enterocytes [ 17]. However, most studies on the intestinal CaSR have been performed in the large intestine rather than the small intestine where calcium absorption is most active [ 5, 18, 41]. Direct activation of this receptor in the apical membrane can increase calcium absorption in the large intestine [ 18, 50], while reducing calcium uptake in the small intestinal-like Caco-2 monolayer (Fig.˝ 2b). Rodrat et˝al. have reported that CaSR probably sensed high apical calcium which in turn increased FGF-23 expression to suppress calcium transport [ 84]. The roles of intestinal CaSR were also studied in Casr intestinal-speci˚c knockout mice. Although Casr-de˚cient mice manifested an impaired intestinal integrity, altered composition of the gut microbiota and in˜ammation [ 19, 77], the function related to the regulation of calcium transport was not determined. Similarly, the renal tubular epithelia also use local CaSR to modulate calcium reabsorption. Activation of CaSR on the luminal side of renal proximal tubular cells is capable of stimulating Na +/H+-exchanger 3 (NHE3), thereby enhanc -ing the paracellular calcium transport via a solvent drag- mediated mechanism (Fig.˝ 2c) [101]. On the other hand, the basolateral CaSR plays a role in the inhibition of the para – cellular calcium reabsorption in the thick ascending limb of Henle™s loop independent of PTH action [ 98].Stimulation of˜intestinal calcium absorption by˜luminal nutrients It has also been reported that a number of nutrients, includ -ing amino acids, oligosaccharides, disaccharides, and mono – saccharides (glucose and galactose), can stimulate calcium absorption [ 91, 92, 96, 109], however, it remains largely unknown how this process is counterbalanced. Generally, there are at least three possible mechanisms to explain how the enterocytes enhance calcium transport in response to

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688 The Journal of Physiological Sciences (2019) 69:683Œ696 luminal nutrients, i.e., nutrient-induced paracellular calcium ˜ow, Cav1.3 activation, and NHE3 activation. Solvent drag and˜nutrient-induced paracellular calcium ˚ow Glucose and/or galactose enter the intestinal cells via api -cal sodium-dependent glucose transporter-1 (SGLT1), after which sodium is pumped out into the paracellular space by Na +/K+-ATPase especially those at the lateral membrane. As explained earlier, the increase in the para – cellular osmolality together with relative permeability of tight junction to water drives paracellular water ˜ow from luminal to the blood side, bringing calcium with it. Therefore, this calcium transport is referred to as the sol – vent drag-induced calcium absorption [ 52, 91]. In addi-tion, the fermented dairy products and incomplete digested carbohydrate, e.g., short-chain fatty acid, oligosaccharides, and polysaccharides, can also enhance calcium absorption in the small and large intestine in rats by increasing tight junction permeability to calcium [ 80, 108]. Although the solvent drag was not directly determined in most studies, the solvent drag-induced paracellular calcium transport should contribute to the process since the uptake of these small organic molecules is often sodium-dependent. How -ever, the direct e˙ect of microbiota-derived organic mol – ecules on the transcellular calcium transporters cannot be ruled out and requires future investigation. Fig. 2 Responses of calcium- sensing receptor (CaSR) to extracellular calcium in a para – thyroid chief cells, b intestinal epithelial cells, and c renal tubular epithelial cells (please see text for details) ABC

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690 The Journal of Physiological Sciences (2019) 69:683Œ696 paracellular space. In other words, the intestinal epithelial cells are able to closely monitor ionized calcium levels at both entry and exit points by using CaSR which in turn acti – vates secondary negative regulators, e.g., FGF-23, to slow down calcium absorption, thus preventing excessive calcium uptake into the body [ 53 , 54 , 84 ]. Detail of the inhibitory e˙ect of FGF23 is further described in the following section. Iron It has long been known that high oral calcium intake inter -feres with heme and nonheme iron absorption [ 63, 76], but whether high iron intake causes a reduction in calcium absorption is still uncertain. A recent investigation in iron hyperabsorptivhalassemic mice showed that duodenal calcium absorption had an inverse correlation with tran -sepithelial iron transport [ 58]. Interestingly, after injection of hepcidinŠa liver-derived inhibitory factor of intestinal iron absorption [ 38]Šthe thalassemia-induced impair -ment of calcium absorption was alleviated [ 58 ]. However, the exact cellular and molecular mechanisms of iron-asso – ciated impairment of calcium absorption require further investigation. Sugars Although some monosaccharides, particularly SGLT1 sub -strates (e.g., glucose and galactose) stimulate intestinal cal – cium absorption, a ketonic monosaccharide fructose found in fruits, vegetables, and grains can produce an inhibitory e˙ect [28, 85]. Both in˝vitro and in˝vivo studies revealed that fructose diminished intestinal calcium absorption, in part by reducing circulating 1,25(OH) 2D3 levels, TRPV6 and calbindin-D9K expression, and thus transcellular calcium transport [ 28]. Furthermore, high fructose intake was found to diminish calcium absorption in growing and lactating rats through disrupting 1,25(OH) 2D3 metabolism, i.e., enhanc -ing 1,25(OH) 2D3 catabolism and impairing 1,25(OH) 2D3 synthesis [ 27, 28].Natural substances and˜byproducts of˜nutrient digestion A number of naturally occurring substances in many green leafy vegetables (e.g., spinach, beet greens, and tea), for example, oxalate, phytate, and tannin, can block the intesti – nal calcium absorption by binding to calcium, thereby ren – dering calcium insoluble and unavailable for absorption [ 2, 69]. However, their physiological signi˚cance and the ˚nal e˙ect on calcium absorption remain controversial. For exam -ple, calcium bioavailability of green leafy vegetables such as centella, quinoa, and roselle, all of which contain high amounts of inhibitory factors, was not changed by cook -ing process or phytase digestion [ 2, 69]. On the other hand, Nigerian children fed phytase-treated or untreated meal showed no di˙erence in their fractional calcium absorption [95]. Nevertheless, a correlation analysis on calcium and inhibitory factors, including oxalate, tannin, phytate, and dietary ˚ber revealed that oxalate had the greatest negative e˙ect on calcium bioavailability [ 2].Besides nutrient and non-nutrient substances, other lumi -nal compounds can inhibit calcium absorption, for instance, lithocholic acid (LCA), deoxycholic acid or its salt, sodium deoxycholate (NaDOC). LCA and NaDOC are secondary bile acids that are formed by enzymes of the intestinal ˜ora. A high concentration of NaDOC not only damages liver tissue and promotes colon cancer [ 92], but also impairs intestinal calcium absorption [ 46, 66, 83]. Determination of calcium absorption in chick duodenum revealed that NaDOC suppressed calcium absorption by downregulating transcellular calcium transport machinery, i.e., PMCA 1b, calbindin-D 28k , and NCX1. Such negative e˙ects might be a consequence of oxidative stress as suggested by concurrent increases reactive oxygen species (ROS) production, glu – tathione reduction, and mitochondrial swelling. Indeed, ROS has been shown to inhibit calcium absorption [ 83]. More studies are required to show if NaDOC-induced impaired intestinal calcium absorption would be of clinical concern on bone health. Intrinsic inhibitory factors Intestinal calcium absorption can be a˙ected by humoral agents acting from the serosal side. Although calcitonin is known as a hypocalcemic hormone, it does not have any direct action on the intestinal epithelial cells. It induces hypocalcemia mainly by inhibiting osteoclast-mediated bone resorption and enhancing renal calcium excretion [ 57 ]. So the importance of calcitonin in the day-to-day calcium homeostasis is negligible. Recently, bone-derived FGF-23 was proposed as a novel inhibitory regulator of intestinal calcium absorption [ 53, 54, 105].FGF-23In general, calcium in the plasma is the major factor mod -erate the intestinal calcium absorption through the long loop negative feedback by inhibiting PTH production and release. Speci˚cally, a slight increase in the plasma-free ion – ized calcium leads to activation of CaSR in the parathyroid chief cells [ 82], thereby reducing PTH release. A reduc – tion in serum PTH results in decreases in the circulating 1,25(OH)2D3 level and ˚nally the intestinal calcium absorp -tion. However, some factors can also reduce the plasma cal -cium by acting at the absorption site. As mentioned earlier, FGF-23 is primarily known as a bone-derived phosphaturic hormone that mainly enhances

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691The Journal of Physiological Sciences (2019) 69:683Œ696 renal phosphate excretion and 1,25(OH) 2D3 catabolism [ 39, 51]. Besides being produced by bone cells, it is also abun -dantly expressed in other cells, such as kidney, brain, lung, liver, spleen, and intestinal enterocytes [ 53, 56]. Normally, an increase in serum phosphate stimulates the secretion of PTH and FGF-23, both of which enhance urinary phosphate excretion to prevent undesirable clinical problems due to hyperphosphatemic spikes, such as ectopic calci˚cation [ 43, 49]. FGF-23 enhances phosphate excretion directly through inactivation of sodium/phosphate cotransporter (NaPi)-2a and -2c in the proximal renal tubules and indirectly by sup – pressing 1,25(OH) 2D3 synthesis and promoting 1,25(OH) 2D3 conversion to inactive 24,25(OH) 2D3, thereby reducing the circulating 1,25(OH) 2D3 levels and also intestinal phosphate [51, 56, 90]. These complete the negative feedback loop for phosphorus homeostasis. However, an experiment in VDR knockout mice sug -gested that elevation of plasma calcium was a potent stimu – lator of FGF-23 production in a VDR-independent manner [90]. Later evidence supported the notion that FGF-23 not only controls phosphorus metabolism but also acts as a cal – cium-regulating hormone that directly controls the intestinal calcium absorption through both systemic and local (parac – rine) mechanisms. The systemic e˙ect was demonstrated by complete abolishment of 1,25(OH) 2D3-enhanced duodenal calcium transport by intravenous FGF-23 injection [ 53]. Fur -thermore, the ˚nding that FGF-23 in the intestine, ex˝vivo studies in murine duodenum demonstrated that FGF-23 directly diminished the 1,25(OH) 2D3-enhanced transcellular and paracellular calcium absorption in ex˝vivo murine duo – denum evinced that FGF-23 could exert direct action on the intestine [ 53, 54]. Moreover, other studies reported that FGF receptors (FGFRs) express in both small and large intestinal cells [70, 93, 105]. These ˚ndings con˚rmed that FGF-23 may have direct actions on the intestinal cells. Although there were a number of reports in Fgfr1, Fgfr3, and Fgfr4 mice [33, 34], the intestinal calcium ˜ux in FGFRs knockout animals has never been investigated. Interestingly, FGF-23 not only elicits action from the basolateral side but also exerts a negative feedback regu – lation on calcium transport across the Caco-2 monolayer from the apical side [ 84 ], consistent with the presence of FGF receptors in both apical and basolateral membranes of the duodenal enterocytes [ 53]. The expression of FGF-23 protein in the enterocytes is upregulated by 1,25(OH) 2D3 as well as high apical calcium in a concentration-dependent manner [84]. As mentioned earlier, CaSR on both apical and basolateral membrane of the enterocytes have a role in the monitoring of calcium transport across the entero – cytes. It was interesting to note that activation of CaSR led to FGF-23 production and suppression of calcium transport. Although the exact molecular mechanism and signaling pathway by which FGF-23 reduces the calcium transport remains elusive, several signaling proteins, such as MAPK/ ERK, p38 MAPK, and PKC might be involved in the process [53, 54].FGF-23 production is not only stimulated by 1,25(OH)2D3, it is also enhanced by other calciotropic hor -mones, such as prolactin [ 105]. In mammalian, prolactin is known as a milk-producing hormone that acts as a major calcium-regulating hormone during pregnancy and lactation, the reproductive periods of high calcium demand [ 15]. Since prolactin markedly stimulates the transcellular and solvent drag-induced paracellular calcium absorption across the intestinal epithelium [ 48, 91], the local production of FGF- 23 may be a counterregulatory mechanism to restrict exces – sive calcium absorption during these reproductive periods. Stanniocalcin Previously recognized as a hypocalcemic hormone from the corpuscles of Stannius in osteichthyan ˚sh, stanniocalcin-1 was also identi˚ed in the kidney of several mammal species, including rodent, cow, and human [ 44, 45, 99]. Regulation of stanniocalcin production and release in mammals remain elusive, but 1,25(OH) 2D3 may be a part of its regulatory loop [ 44], similar to the negative feedback loop of FGF- 23. In opossum kidney proximal tubular cells, 1,25(OH) 2D3 exposure was found to enhance stanniocalcin expression in a dose-dependent manner [42, 44]. Once released from the kidney, the circulating stanniocalcin is thought to acti – vate its receptor on the basolateral membrane, but not the apical membrane, of enterocytes, to enhance the intestinal phosphate absorption, while suppressing calcium absorp -tion [45, 65]. In the gut, stanniocalcin has been reported to express in stomach, small intestine, and colon of neonatal and mature rats [ 55]. Functional study by Madsen and col – leagues reported the negative e˙ect of stanniocalcin-1 on net calcium absorption across the swine duodenum, consistent with its hypocalcemic action observed in ˚sh [ 65].Furthermore, stanniocalcin-1 not only functions as an endocrine factor but is also produced locally in the intestine to control calcium absorption. Xiang et˝al. [ 107] demon- strated in intestinal epithelium-like Caco-2 cells that over -expression of stanniocalcin-1 could downregulate TRPV5 and TRPV6 protein expression, suggesting that stannioc – alcin-1Šas a paracrine/autocrine factorŠmight directly and locally fine-tune calcium flux across the intestinal epithelium. Exposure to recombinant stanniocalcin-1 also directly suppressed TRPV5 and TRPV6 expression in stan – niocalcin-1-knockdown Caco-2 cell without having an e˙ect on PMCA 1b, NCX1, or VDR expression [ 107]. Although stanniocalcin-2 with ~ 80% homology to stanniocalcin-1 has been identi˚ed in mammals, it is unclear whether this peptide can locally modulate intestinal calcium absorption.

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