Intestinal Calcium Absorption

Kannikar Wongdee,1,2 Krittikan Chanpaisaeng,2,3 Jarinthorn Teerapornpuntakit,2,4 and

Narattaphol Charoenphandhu*2,5,6,7

ABSTRACT
In this article, we focus on mammalian calcium absorption across the intestinal epithelium in
normal physiology. Intestinal calcium transport is essential for supplying calcium for metabolism
and bone mineralization. Dietary calcium is transported across the mucosal epithelia via saturable
transcellular and nonsaturable paracellular pathways, both of which are under the regulation of
1,25-dihydroxyvitamin D3 and several other endocrine and paracrine factors, such as parathyroid
hormone, prolactin, 17β-estradiol, calcitonin, and fibroblast growth factor-23. Calcium absorption
occurs in several segments of the small and large intestine with varying rates and capacities.
Segmental heterogeneity also includes differential expression of calcium transporters/carriers
(e.g., transient receptor potential cation channel and calbindin-D9k ) and the presence of favorable
factors (e.g., pH, luminal contents, and gut motility). Other proteins and transporters (e.g., plasma
membrane vitamin D receptor and voltage-dependent calcium channels), as well as vesicular
calcium transport that probably contributes to intestinal calcium absorption, are also discussed.
© 2021 American Physiological Society. Compr Physiol 11:1-27, 2021.

Didactic Synopsis • Neural regulation of intestinal calcium absorption by

Major Teaching Points
• Polarity is an important aspect of epithelia. Epithelial
cells including intestinal enterocytes have two domains,
that is, apical and basolateral domains. Apical domain is
exposed to lumen or external environment and respon-
sible for absorption and protection. Basolateral domain
is associated with neighboring epithelial cells and the
basement membrane.
• Calcium (Ca2+ —as free-ionized calcium) traverses the
intestinal epithelia via transcellular and paracellular
pathways.
• Transcellular Ca2+ transport is a three-step process
consisting of TRPV6-mediated apical calcium uptake,
cytoplasmic diffusion of Ca2+ -laden calbindin-D9k ,
and PMCA1b - and NCX1-mediated basolateral calcium
extrusion.
• Paracellular Ca2+ transport is determined by tight junc-
tion permselectivity and the expression of tight junction
proteins, for example, claudin-2, -12, or -15 as well as per-
ijunctional actomyosin ring remodeling.
enteric nervous system is plausible but direct evidence to
support this notion is scant and inconclusive.
Introduction
Calcium absorption from the intestine is the sole supply of
calcium for various body functions including bone miner-
alization, neural transmission, cardiomyocyte contraction,
hormonal secretion, and blood coagulation. Daily dietary
calcium requirements vary according to demographics, for
example, age, gender, ethnicity, and physiological conditions
*Correspondence to: naratt@narattsys.com
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 Functional Ingredients and Food Innovation Research Group,
National Center for Genetic Engineering and Biotechnology (BIOTEC),
National Science and Technology Development Agency (NSTDA),
Pathum Thani, Thailand
4 Department of Physiology, Faculty of Medical Science, Naresuan
University, Phitsanulok, Thailand
5 Department of Physiology, Faculty of Science, Mahidol University,

• The calciotropic hormone 1,25-dihydroxyvitamin D3 (an Bangkok, Thailand

6 Institute of Molecular Biosciences, Mahidol University, Nakhon
active form of vitamin D) is a potent positive regulator of
Pathom, Thailand
transcellular and paracellular calcium absorption. 7 The Academy of Science, The Royal Society of Thailand, Bangkok,

• A number of humoral factors (e.g., fibroblast growth Thailand

factor-23 and calcitonin) and certain luminal factors Published online, May 2021 (comprehensivephysiology.com)
(e.g., iron and alkaline pH in the bulk phase of intestinal DOI:10.1002/cphy.c200014
lumen) can reduce intestinal calcium absorption. Copyright © American Physiological Society.

Volume 11, May 2021 1

Intestinal Calcium Absorption
such as pregnancy and lactation, and exposure to sunlight
or ultraviolet B radiation (for reviews, please see Refs. 42,
201, 230). In adults, it ranges from 1000 to 1300 mg elemen-
tal calcium per day (42). In rodents, females normally have
higher rates of intestinal calcium absorption than males (212).
Higher calcium demand is apparent in certain conditions, for
example, bone calcium accretion during adolescence, placen-
tal calcium transfer during pregnancy, and milk production
in lactating women (36, 157, 235). On the other hand, the
rate of intestinal calcium absorption declines with age due in
part to downregulation of nuclear vitamin D receptor (VDR)
expression and the decrease in responsiveness of target tissues
to 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3 ] (82).
Regarding calcium absorption, the gastrointestinal mucosal
epithelia exhibit varying degrees of heterogeneity. Most mam-
mals including humans, rats, mice, horses, and ruminants
absorb dietary calcium predominantly in the small intestine
in a vitamin D-dependent manner (69, 173, 238). However,
each intestinal segment utilizes different cellular mechanisms
to absorb calcium. For example, the proximal small intestine
of the rat—duodenum and proximal jejunum—is capable of
transporting calcium via both transcellular and paracellular
pathways, whereas the paracellular route is more important
in the distal small intestine (121). In addition, the intestine
also shows heterogeneity along the crypt-villus axis, that
is, calcium secretion predominantly takes place in the crypt
region, whereas calcium absorption mainly occurs in the
upper two-thirds of the villus which corresponds to the abun-
dant expression of calcium-binding protein calbindin-D9k
(also known as S100 calcium-binding protein G or S100G) at
the villous tip (100).
In this article, we aim to elaborate on the cellular and
molecular mechanisms of calcium transport across the mam-
malian intestinal epithelium as well as its local and systemic
regulation by paracrine and endocrine factors. Dietary nutri-
ents and luminal factors affecting calcium absorption are also
discussed.
Segmental Heterogeneity of Calcium
Absorption
Calcium absorption occurs throughout the gastrointestinal
tract yet there is heterogeneity in the efficiency of intestinal
calcium absorption and transport pathways used in each
segment. The efficiency of calcium absorption partly depends
on luminal calcium concentration that is mostly attributed to
the dietary intake. Calcium is transported across the epithe-
lium through two primary mechanisms, that is, transcellular
[saturable, 1,25(OH)2 D3 -, and energy-dependent] and para-
cellular (non-saturable, concentration-dependent) transport.
The cellular machinery for these two transport pathways
is discussed in the section titled “Cellular Mechanisms of
Gastrointestinal Calcium Absorption”. When calcium intake
is adequate or high, the efficiency of calcium absorption in
any given segment is determined by the mechanism used for
2 Volume 11, May 2021
Comprehensive Physiology
absorption, intestinal sojourn time, and solubility of dietary
calcium. The following section will address how these factors
affect the amount and efficiency of calcium absorption in a
given segment (Figures 1 and 2).
Stomach
Calcium absorption is generally absent in the stomach (155).
However, the stomach plays a role in providing an acidic
environment for calcium solubilization (for review, please
see Ref. 127). Previous preclinical and clinical observations
consistently showed that gastrectomy resulted in impaired
calcium absorption and low bone mass (23, 41, 153). Stomach
acid produced by gastric parietal cells increases dissolution
of calcium salts and allows calcium ions to form complexes
with other food constituents. As a result, soluble calcium can
be absorbed when it passes into the intestine. A variety of
transporters and gastrin receptors in the gastric mucosa are
involved in maintaining normal gastric acidification. Animals
with genetic disruption of genes expressing proteins involved
in the stomach acid production, for example, Cckbr, Tcirg1,
Snx10, had defect in gastric acid secretion, hypocalcemia,
and osteopathy (83, 205, 252). These findings implicate the
importance of normal stomach acid secretion in calcium
homeostasis. In support of this, extensive investigations in
individuals with decreased gastric acidity (e.g., elderly and
atrophic gastritis patients) revealed that insoluble calcium
salts had reduced calcium solubility in these subjects (243).
Taken together, these data strongly suggest that gastric acidi-
fication is indispensable for intestinal calcium absorption and
helps maintain normocalcemia.
Duodenum
When acidic chyme enters the duodenum, it will be neu-
tralized by bicarbonate and alkaline mucus secreted from
Brunner’s glands—also known as the submucosal duodenal
glands. Because of a high amount of free-ionized calcium,
the immediate change of pH in the intestinal environment
does not impact the amount of soluble calcium, and calcium
solubility is highest in the duodenum as compared to other
distal intestinal segments (60, 73). In addition, food in a liquid
form yields a higher amount of available calcium in a shorter
time compared to the solid food (155). However, with only
2 to 3 min sojourn time of food in the duodenum (60), the
amount of calcium absorbed in the duodenum accounts for
approximately 8% of the total intestinal calcium absorption
(44, 60, 159).
Both active transcellular and paracellular transport mech-
anisms take place in the duodenum (Figure 3B). When
individuals receive inadequate calcium intake, transcellular
transport predominates in the duodenum to increase the
efficiency of calcium absorption. It is well established that
1,25(OH)2 D3 increases transcellular absorption against a
chemical gradient (26, 71, 115, 116, 183) by upregulat-
ing calcium-selective transporters [e.g., transient receptor
Comprehensive Physiology
Duodenum
Jnet +++ Percent absorbed 8%
Colon
Jnet ++ Percent absorbed 15%
Cecum
Jnet ++++ Percent absorbed 10%
Producing acidic environment
from microfloral fermentation
Figure 1 Segmental heterogeneity of calcium absorption. Key features including magnitude of net absorptive calcium flux
rate (Jnet = Jmucosa-to-serosa − Jserosa-to-mucosa ) and percentage of total absorbed calcium under normal (calcium- and vitamin D-
replete) conditions are presented for each intestinal segment. The number of “+” signs represents the amount of net calcium
flux compared to cecum which has the highest net flux marked with “++++”.
potential cation channel, subfamily V, member 6 (TRPV6)
and a lesser extent TRPV5] and calcium-binding proteins
(e.g., calbindin-D9k in mammals and calbindin-D28k in
birds). Because this transport mechanism can be saturated,
consequent absorption across the enterocytes decreases with
increased calcium intake (183). On the other hand, the para-
cellular transport of calcium is nonsaturable and is driven by
the electrochemical gradient of calcium and solvent drags
(3, 116, 238). Accordingly, our investigation of rat duodenum
showed that when luminal concentration of free-ionized
calcium is greater than 5 mmol/L, the paracellular transport
serves as the major pathway (up to 10:1) as compared to the
transcellular transport (31).
Jejunum
Calcium ions that enter the jejunum are progressively exposed
to higher pH as they approach the distal part of jejunum (pH
∼6.6–8), resulting in an increase in insoluble calcium in
the lumen (60). In rats fed high-calcium diets, the sojourn
time in the entire jejunum is 40 to 45 min (20, 60), which is
20 times longer than the time spent in the duodenum. Thus,
the absorbable amount of dietary calcium is higher than
in the duodenum (17% vs. 7%) (44). The jejunal calcium
transport occurs transcellularly and paracellularly. In the
rat jejunum, paracellular transport is responsible for more
than 80% of calcium absorption (183). Interestingly, there is
evidence suggesting that 1,25(OH)2 D3 treatment upregulated
Volume 11, May 2021
Intestinal Calcium Absorption
Stomach
• Producing acidic environment
for calcium solubilization
• JCa, detectable but unclear
physiological significance
(probably negligible)
Jejunum
Jnet + Percent absorbed 17%
Ileum
Percent absorbed
Jnet +
65%–88%
Rectum
Unclear physiological significance
(probably negligible)
the expression of the tight junction proteins claudin-2 and
-12 in the jejunum. Specifically, the mRNA and protein
levels of these claudins decreased significantly (3–4 fold) in
the jejunum of VDR knockout mice compared to wild-type
mice (79). The reduced expression of these proteins was also
observed in the duodenum, ileum, and colon of the VDR
knockout animals, but to a lesser extent than in the jejunum
(79). Claudin-2 is a tight junction protein in intestinal epithe-
lial cells that possesses paracellular cation-selectivity (162,
218). A recent study using claudin-2 knockout animals
showed that the absence of this protein led to a decrease in
paracellular calcium absorption (46), thus emphasizing the
importance of claudin-2 in this transport pathway.
While paracellular calcium flux takes place throughout the
jejunum, small saturable component of calcium absorption
(∼15%) exists mostly in the proximal jejunum (183). Steady
state perfusion studies in healthy adults with exogenous
1,25(OH)2 D3 administration showed an increase in jejunal
calcium absorption (131). However, recent reports showed
the expression of TRPV6 was much lower in the jejunum
of adult mice compared to newborns (13, 195). Therefore,
transcellular calcium absorption occurs in the jejunum is
postulated to play a role in ensuring a positive calcium
balance in postnatal development when the transcellular
mechanism in the duodenum is not fully developed. Using
the Ussing chamber technique, Beggs et al. (13) measured
transcellular calcium flux across jejunum of TRPV6 mutant
and Cav 1.3 (L-type calcium channel) knockout mice in
3
Intestinal Calcium Absorption Comprehensive Physiology

)
in
(m

9k
e

-D
tim

in
y
lit

nd

5
Intestinal segments

1b
rn

-2

-1

-1
bi

A
PV
u

bi

n
lu

R
jo

ld

ld

ld
al

PM

VD
TR
So
So

C
Stomach 240–300 ++++ – N/A N/A N/A N/A N/A +

++++ ++++ ++++ + ++ +++ ++++

Duodenum 3 ++ ++ +++ +++ ++ ++ +++

++, – – + + + + ++++
Jejunum 40–45 ++
++ ++ ++ ++ +++ +++

– – + + ++ (S) ++ ++++
Ileum ~130 +
++ + ++ ++ ++ ++

++++, ++ + +++ (S) + + + ++

Proxmimal

Cecum 92 +
colon

++++ ++++ +++ ++ ++ ++

+ + +++ +++ + + ++
Proximal
50 + ++ +++ +++ ++++ ++ ++
colon
Cecum

+ + ++ ++ ++ + ++
Distal colon 42 +
+++ +++ ++ +++ ++ ++

(20, 60) (60) (185, 195) (195) (195) (195) (195) (195) (53)
References
(219) (219) (219) (219) (219) (219)

Figure 2 Intestinal segment-specific sojourn time, calcium solubility, and expression profile of genes/proteins related to
calcium transport. The number of “+” signifies the relative abundance of a feature for a given intestinal segment. A “–”
indicates that an mRNA expression is absent in a segment. The sojourn time in the table represents time of chyme. The
lower lines of each segment (red text) represent distribution of mRNA expression of genes related to calcium transport in
female rats refer to our previous study (219). N/A, not available; S, sexual disparity in the expression profile; Cldn, claudin;
PMCA1b , plasma membrane Ca2+ ATPase-1b; TRPV6, transient receptor potential cation channel, subfamily V, member 6;
VDR, vitamin D receptor.

suckling period. They found a significant increase in TRPV6-
and Cav 1.3-mediated transcellular calcium absorption in the
jejunum of 2-week-old suckling mice, whereas these effects
disappeared in the 2-month-old mice. Furthermore, in vivo
perfusion studies (1.25 mmol/L luminal calcium) showed the
activity of Cav 1.3 in the proximal jejunum to mid-ileum. This
suggests that when luminal calcium is high, Cav 1.3-mediated
pathway may enhance calcium absorption in addition to the
paracellular pathway (159); however, its exact contribution
requires further studies.
Despite a relatively high mucosa-to-serosa calcium flux,
the serosa-to-mucosa calcium flux in the jejunum is twice as
much leading to net ionized calcium secretion into the intesti-
nal lumen (Figure 3B) (116, 195). It is believed that this secre-
tory process may account for the endogenous fecal calcium
observed in calcium balance studies (232). Taken together, it
is likely that the calcium absorbed from the jejunum does not
contribute much to the plasma calcium pool.
Ileum
Among different segments of the small intestine, the ileum is
where most of ionized calcium is absorbed (155). Although
it has a more basic environment, that is, pH > 8, that reduces
calcium solubility and limits the amount of calcium available
for absorption, the total amount of calcium absorbed is
not reduced (60). This is because when a small amount of
ionized calcium in the lumen is absorbed, more insoluble
calcium will be ionized and become available for absorption.
Because the food bolus spends the longest transit time in
the ileum (121.5 min) (60), the slow, yet steady process
of paracellular absorption yields a relatively large amount
of absorbable calcium (65%–88%) (44, 155). Duflos et al.
(60) have shown that, in young adult rats fed 1.5% dietary
calcium, the sojourn time of the duodenum, mid-jejunum
and distal ileum were 2.5, 12, and 58 min, respectively.
Therefore, a larger amount of calcium is absorbed in the
ileum even though the “rate” of calcium transport in the
ileum is much slower than in the cecum and duodenum
(117). Similarly, in healthy premenopausal women, the
mouth-to-cecum transit time appeared to be positively cor-
related with the efficiency of calcium absorption (12). The
ileal mucosa-to-serosa calcium flux mostly occurs through
the paracellular pathway and exhibits a limited capacity
for active transcellular absorption (183). Claudin-2 and -12

4 Volume 11, May 2021

Comprehensive Physiology Intestinal Calcium Absorption

(A) (B)

Lumen Villous tip

c
Calbindin-D9k expression
Villi

a –4 mV

Lacteal
0 mV

e Blood
vessels
f

b Basement
Crypt membrane
Goblet cell
d

a = net Ca2+ absorption c = Transcellular transport

b = net Ca2+ secretion d = Paracellular transport
e = Voltage-dependent Ca2+ secretion
(almost negligible)
f = Unstirred water layer/acid microclimate

Figure 3 Diagrams of intestinal villi. (A) Longitudinal section of villous showing sites of net calcium absorption at
villous (a) and net calcium secretion at crypts of intestine (b). The heterogeneity of calbindin-D9k (S100 calcium-binding
protein G, S100G) expression and the primary direction of calcium flux are presented along the crypt-villous axis.
(B) Transverse section of villous tip featuring transcellular (c) and paracellular calcium absorption (d), secretion (e),
and the existence of unstirred water layer and acid microclimate (f).

which are tight junction proteins involved in the paracellular
transport are highly expressed in the ileum compared to other
segments (78). On the other hand, the expression of TRPV6
and calbindin-D9k is virtually absent and the expression of
plasma membrane Ca2+ ATPase-1b (PMCA1b ) was very low
in the ileum of mice and rats (21, 99, 183, 185, 195). In
addition, studies in animals given 1,25(OH)2 D3 12 h prior
to calcium absorption test using everted gut sac or in situ
intestinal loop techniques showed that ileal calcium absorp-
tion was not changed (14, 15, 116, 183). Despite evidence
illustrating the absence of transcellular calcium transport in
the ileum, another line of evidence has suggested that the
ileum probably has the capacity to actively transport calcium
(234). When stimulated with 1,25(OH)2 D3 , the mRNA and
protein expression of calbindin-D9k and PMCA1b in the ileum
of aged rats were upregulated (8, 9). The explanation for this
discrepancy could be the temporal responsive nature of the
ileum to 1,25(OH)2 D3 treatment and very low concentration
of calbindin-D9k (protein level in ileum vs. duodenum at
16 h post-treatment: ∼2.7 vs. 45 μg/mg) and basolateral
calcium pump protein in the ileum (8). Though transcellular
calcium transport proteins are present in the ileum, more
studies are needed to determine whether this pathway in the
ileum significantly contributes to the total intestinal calcium
absorption.
Similar to the jejunum, calcium secretion in the ileum
was also demonstrated in Ussing chamber experiments over
a range of 0.125 to 11.2 mmol/L calcium concentration in
the bathing solution (119, 165, 232). The serosa-to-mucosa
flux was proposed to be generated by hydrostatic driving
force resulting in paracellular calcium movement (14, 119,
165). By using in situ loop study in female rats, Krishnamra
et al. (132) demonstrated calcium secretion along the small
and large intestine and found that the calcium secretion was
directly proportional to the plasma calcium concentration.
For example, calcium secretion in hypercalcemic rats was
increased while secretion was decreased in hypocalcemic rats.
Cecum
It has been suggested that the transcellular mechanism of
calcium transport is prominent in the cecum. Findings sup-
porting this notion showed that rat and mouse cecum exhibited
high mRNA expression levels (second to duodenum) of many
transporters, for example, TRPV6 and PMCA1b , while other
intestinal segments had extremely low expression levels
of these proteins (195, 219). Furthermore, Dhawan et al.
(53) showed that the transgenic expression of human VDR
exclusively in ileum, cecum, and colon (distal intestine) could
rescue global VDR knockout mice from hypocalcemia and

Volume 11, May 2021 5

Intestinal Calcium Absorption
abnormal bone mineralization. With 1,25(OH)2 D3 injection,
the mRNA expression level of TRPV6 and Cyp24A1—the
genes known to be regulated by 1,25(OH)2 D3 —was highest
in the cecum (53). Other studies showed significant response
in cecal calcium flux and expression of transcellular calcium
transport proteins in response to vitamin D treatment (117,
187). These findings highlight the importance of vitamin
D-dependent transcellular calcium transport in the cecum and
its significance in whole-body calcium homeostasis.
When bidirectional calcium fluxes were measured using
Ussing chamber technique, cecum showed a significantly
high net calcium absorption. This supports the importance
of this segment in intestinal calcium absorption in mice
(195) and rats (34, 117, 164). However, some studies showed
different or opposite findings. For example, Brommage et al.
(19) measured intestinal calcium absorption in cecectomized
rats receiving intraperitoneal 1,25(OH)2 D3 injection. They
found that fractional calcium absorption of 47 Ca/47 Sc in the
control and cecectomized rats was not significantly differ-
ent. Thus, physiological significance of the cecal calcium
absorption remains unclear and requires further investiga-
tion. Indeed, the overall calcium absorption might exhibit
redundancy or compensatory response—that is, other intesti-
nal segments can compensate for the loss of cecal calcium
uptake.
Colon
The large intestine has been shown to account for less than
10% of total intestinal calcium absorption (20, 44), with
calcium transport mostly occurs in the ascending colon and
not at all in the transverse colon (20). By the time, the food
bolus arrives at the colon, only a small amount of calcium is
still available for absorption. Duflos et al. (60) studied cal-
cium absorption along the entire intestine of 8-week-old male
Sprague Dawley rats and reported that approximately 15%
of the total soluble calcium was found in the large intestine.
Since a quarter of intestinal sojourn time is spent in the colon
(92 min vs. 371 min total time) (20), an appreciable amount
of calcium is absorbed at the colon via both transcellular and
paracellular pathways. During high calcium intake, the colon
participates in calcium absorption via paracellular pathway
(118, 129, 187, 195).
Transport proteins involved in the transcellular (TRPV6,
calbindin-D9k , and PMCA1b ) and paracellular (claudin-2,
-12, and -15) pathways are expressed in both proximal and
distal colon (20, 195). In response to vitamin D2 treatment
(2 doses of 20,000 IU), the colon of vitamin D-deficient
mice showed high mucosa-to-serosa flux and low serosa-
to-mucosa transport resulting in net calcium absorption
(187). Transcellular calcium transport is suggested to occur
specifically in the descending colon (64). However, due to a
multiple-fold decrease in expression of transcellular transport
proteins as compared to the duodenum, the active pathway
in the colon is postulated to contribute much less to overall
calcium absorption.
6 Volume 11, May 2021
Comprehensive Physiology
Paracellular calcium absorption is proposed to be the major
calcium transport pathway in the colon. By using in situ loop
technique, Krishnamra et al. (132) showed that calcium
absorption in the colon of normocalcemic and hypercalcemic
rats was similar as opposed to the reduction in absorption in
other intestinal segments of hypercalcemic rats. Interestingly,
only thyroparathyroidectomized (TPTX)-induced hypocal-
cemic rats showed a significantly lower rate of calcium
absorption in the colon (132). Such a reduction of calcium
absorption in the colon of TPTX-induced hypocalcemia was
hypothesized to be due to a decrease in circulating level of
parathyroid hormone (PTH) or a decrease in 1,25(OH)2 D3
after TPTX (132).
In addition, Jongwattanapisan et al. (111) demonstrated
the compensatory colonic calcium hyperabsorption in
cecectomized rats. Therein, a calcium balance study was
demonstrated in 8-week-old cecectomized female rats.
While calcium loss was presented in the first week after the
operation, the colonic calcium absorption was significantly
elevated. These data suggest that there is a tight regulation
in the intestinal tract to ensure overall preservation of body
calcium. Furthermore, colonic perfusion studies in healthy
human showed that exogenous 1,25(OH)2 D3 administration
increased net colonic calcium movement from zero toward a
positive net absorption (92). These studies illustrate that what
we observed in preclinical studies can likely be extrapolated
to humans (111, 187). However, it could be said that in normal
conditions, the colon probably contributes very little to the
overall calcium economy but appears to have the machinery
to compensate should the need arise.
Cellular Mechanisms of Gastrointestinal
Calcium Absorption
Transcellular and paracellular pathways are two primary
cellular mechanisms of calcium transport. In the past, these
two systems were generally viewed as separate entities,
with the former mainly responsible for calcium transport in
times of calcium deficiency. Until recently, new evidence
suggests that transcellular and paracellular transport systems
may cooperate to maintain calcium homeostasis. Whereas
the paracellular calcium transport is predominant during
high calcium intake when luminal calcium concentration is
much greater than plasma ionized calcium, the transcellular
calcium transport becomes important during high calcium
demand (e.g., pregnancy and lactation) or low calcium intake.
The cellular machinery for these two transport pathways is
discussed below.
Transcellular calcium transport
Free-ionized calcium cannot freely traverse the cell mem-
brane of the intestinal enterocytes. Therefore, they require
specific transporters or calcium channels on both apical
Comprehensive Physiology Intestinal Calcium Absorption

Apical Ca2+ K+ Basolateral

NCKX 4Na+
Trifluoperazine
PMCA1b Vanadate

Ca2+ Ca2+
Ruthenium red TRPV6
Calbindin-D9k
Ca2+
Ca2+ KB-R7943
CAM 3Na+ NCX1
TRPV5
S100G
3Na+

NKA
2K+

Ouabain

Figure 4 Mechanism of transcellular calcium transport across the enterocyte. Calcium is trans-
ported across apical membrane through transient receptor potential cation channel, subfamily
V, member 6 (TRPV6), and, to a lesser extent, TRPV5, facilitated diffusion through the cytosol,
and active basolateral extrusion by the plasma membrane Ca2+ ATPase-1b (PMCA1b ) and other
transporters [e.g., Na+ /Ca2+ exchanger 1 (NCX1) and K+ -dependent Na+ /Ca2+ exchanger
(NCKX)]. Some inhibitors of each calcium transporter are also indicated. Calcium-binding pro-
teins including calbindin-D9k (CaBP-9k) and calmodulin (CAM) may play a role in a buffering
system to prevent intracellular calcium overload as well as translocate calcium ions from the
mucosa to the serosa. The stoichiometry for NCX1 is 4 Na+ to 1-2 Ca2+ moved per transport
cycle; however, an average stoichiometry is 3 Na+ :1 Ca2+ . The stoichiometry of NKA is 3 Na+ :2
K+ . TRPV6, NKA, and NCX1 activities are inhibited by ruthenium red, ouabain, and KB-R7943,
respectively. PMCA1b activity is inhibited by trifluoperazine and vanadate.

and basolateral membranes to facilitate calcium movement
from the intestinal mucosa to the serosal side. This calcium
transport is a vitamin D-regulated process (26, 81, 98) that
consists of three major steps: (i) apical calcium entry that
is mainly facilitated by TRPV6; (ii) intracellular calcium
translocation mediated by calcium-binding proteins; and (iii)
basolateral calcium extrusion (Figure 4).
Apical calcium entry
TRPV channels are the main regulators for apical calcium
entry. This subfamily of ion channels was first discov-
ered in an extensive screening of cloned DNA from apical
calcium transporters in rat duodenum using the model of
Xenopus laevis oocytes (124). Though all six members of
TRPV subfamily are capable of aiding calcium flux, only
TRPV5 (also known as CaT2 or ECAC1) and TRPV6 (also
known as CaT1 or ECAC2) are highly selective to calcium
(PCa / PNa > 100) (99). Both TRPV5 and TRPV6 are epithelial
calcium channels that mainly facilitate calcium reabsorption
in the renal tubules and calcium absorption in the intestine,
respectively (94, 158). In the duodenum where mostly active
calcium transport takes place, both proteins were expressed in
humans, whereas only TRPV6 was predominantly expressed
in the mouse and rat (50). In laying hens, the TRPV6 pro-
tein expression was also abundant in the proximal small
intestine—duodenum and jejunum—particularly in the apical
membrane of villous cells, but not in the intestinal crypts and
goblet cells (251). Because of a much higher expression of
TRPV6 than TRPV5 in the duodenum, TRPV5 is not critical
to the intestinal calcium absorption (169).
Under normal conditions, TRPV6 plays a critical role in
apical calcium entry. Since TRPV6 fully functions under a
hyperpolarized potential (120), the electrochemical gradient
in the duodenum favors mucosa-to-serosa calcium ion flux.
Consistently, the inducible transgenic TRPV6 expression
in the duodenum of the VDR knockout mice increased
calcium absorption and bone density (45). Moreover, when
human intestinal-like Caco-2 cells were examined using
chromatin immunoprecipitation, five putative vitamin D
response elements (VDRE) were identified upstream to
TRPV6 coding region, and mutagenesis of these VDRE
abolished the response to 1,25(OH)2 D3 (156). By binding
to VDRE and subsequently upregulating VDR-mediated
transcription, both exogenous and endogenously produced
1,25(OH)2 D3 lead to upregulation of genes involved in active
calcium absorption, for example, TRPV6, and calbindin-D9k
(8, 213, 214). Multiple studies exemplified the importance
of intestinal VDR in calcium homeostasis. For example,
a transgenic intestinal-specific expression of VDR in the
VDR knockout mice could restore their calcium absorp-
tion to normal (248). Not only does 1,25(OH)2 D3 directly
upregulates intestinal calcium transporters, but it could also
indirectly modulates other factors that promote calcium
absorption such as mucosal morphology and increased sur-
face area (49, 204). Specifically, in VDR knockout mice, the

Volume 11, May 2021 7

Intestinal Calcium Absorption Comprehensive Physiology

apical membrane of duodenal enterocytes exhibited elonga-
tion of microvilli—perhaps as a compensatory mechanism
to improve calcium absorption rate—with no change in
duodenal villous height or crypt depth (133). These data
strongly support the importance of VDR and subsequently
the critical role of 1,25(OH)2 D3 in the regulation of calcium
absorption.
Despite the strong evidence supporting TRPV6 as a pri-
mary transporter in transcellular calcium transport, another
line of research suggests that the transcellular calcium trans-
port mechanism exhibits redundancy since the absence of
certain transporters—for example, in TRPV6 knockout mice
and TRPV5/TRPV6 double knockdown Caco-2 cells—did
not completely abolish calcium transport (134, 135, 163).
Furthermore, Benn et al. (15) explored whether TRPV6 was
indispensable to active transcellular calcium transport by
using wild-type and TRPV6 knockout mice fed either a low
(0.02%) or high (1%) calcium diet and the everted gut sac
assay. Under high calcium conditions where active calcium
transport was limited, wild-type and TRPV6 knockout mice
showed similarly low transcellular calcium flux (15). In
contrast, TRPV6 knockout mice under low dietary calcium
conditions had a significant decrease in intestinal calcium
transport when compared to wild-type mice. Despite this
decrease, TRPV6 knockout mice fed the low calcium diet
still demonstrated a significant 2.9-fold increase in transcellu-
lar calcium transport as compared to TRPV6 knockout mice
fed the high calcium diet (15). Moreover, active intestinal
calcium absorption of TRPV6 knockout mice could still be
increased by 1,25(OH)2 D3 injection (15, 135). These findings
have challenged the dogma that TRPV6 is the only trans-
porter necessary for 1,25(OH)2 D3 -mediated apical calcium
flux and imply that other unidentified mechanisms for active
transcellular transport may exist.
Another transport protein Cav 1.3 encoded by CACNA1D
gene has been proposed to be involved in active transcel-
lular calcium absorption. In contrast to TRPV6 which
normally opens at hyperpolarized potential of less than
−50 mV, Cav 1.3—as a voltage-dependent L-type calcium
channel—has relatively low threshold and opens at sub-
threshold potential or slightly depolarized potential of
approximately −40 mV (the resting potential of the apical
membrane of enterocytes is approximately −47 mV) (120).
The nifedipine/verapamil-sensitive Cav 1.3-mediated calcium
absorption was shown to be coupled with transcellular glu-
cose absorption (151), thus being consistent with the previous
finding that luminal glucose was apparently a potent enhancer
of transcellular calcium absorption (24). During the epithelial
glucose uptake in the duodenum and proximal jejunum
via Na+ -dependent glucose transporter (SGLT)-1, sodium
entry induces slight depolarization of the apical membrane,
thereby triggering Cav 1.3 opening (Figure 5) (120, 238).
Interestingly, the Cav 1.3-mediated calcium transport led to
terminal web myosin II phosphorylation, which, in turn,
initiated insertion of glucose transporter isoform GLUT2
into the apical membrane for additional glucose uptake in
rats (151).
While this line of research supports the role of Cav 1.3 in
calcium absorption and its potential coupling with glucose,
Reyes-Fernandez and Fleet (193) did not observe the effect of
glucose (25 mmol/L) in enhancing calcium absorption or any
changes in the mRNA expression levels of Cav 1.3 in duode-
num, jejunum, or ileum of 9-week-old male C57BL/6J mice
fed either a high or low calcium diet for 1 week. Furthermore,
Li et al. (143) reported that the mRNA expression of Cav 1.3

Apical Depolarization Basolateral

Na+
ΔVt
NKA
PMCA1b
Na+
SGLT1 K+
Opening at subthreshold Glu Ca2+
potential (~ –40 mV)
Dihydropyridines Cav1.3 Calbindin-D9k
Ca2+
Ca2+
TRPV6 Na+ NCX1
CAM
Ca2+
TRPV5 Na+

NKA
Resting potential K+
–47 mV

Figure 5 Effect of apical membrane potential changes on transcellular intestinal calcium

absorption. During glucose uptake via Na+ -dependent glucose transporter (SGLT)-1, sodium
entry induces slight depolarization of the apical membrane, thereby triggering Cav 1.3 open-
ing at slight depolarized potential of approximately −40 mV as compared with −47 mV resting
potential of the apical membrane. Cav 1.3 activity is inhibited by dihydropyridine. PMCA1b ,
plasma membrane Ca2+ ATPase-1b; NCX1, Na+ /Ca2+ exchanger 1; NKA, Na+ /K+ -ATPase;
calbindin-D9 k (S100 calcium-binding protein G, S100G); CAM, calmodulin.

8 Volume 11, May 2021

Comprehensive Physiology
in male wild-type mice was greater than those in female. By
using Cav 1.3 null mutant mice, they found that only bone
parameters of male mice, but not female mice, were nega-
tively affected by loss of Cav 1.3 when compared to wild-type
mice (143). Taken together, Cav 1.3 may contribute to intesti-
nal calcium absorption in a sex-specific manner, which could
also be governed by species specificity. Therefore, future
research examining the effect of Cav 1.3 in both genders of
rats, mice, or humans is required to resolve this question.
Although the physiological significance of Cav -mediated
calcium absorption under normal conditions—particularly in
vitamin D-replete conditions—remains unclear, Cav appears
to be the salient calcium transporter during pregnancy and
lactation when prolactin becomes a major calcium-regulating
hormone (32, 163). A study in Caco-2 monolayer further
showed that prolactin directly enhanced the Cav -mediated
calcium transport via PI3K and PKC𝜁 pathways (221).
Furthermore, pre-weaning suckling and post-weaning mice
differentially exhibited the TRPV6- and Cav 1.3-mediated cal-
cium transport mechanisms in different segments of the small
intestine. Specifically, in 2-week-old, pre-weaning suck-
ling mice, Cav 1.3-mediated calcium uptake in the jejunum
and ileum was most prominent, whereas in 2-month-old,
post-weaning mice, the TRPV6-mediated calcium uptake in
the duodenum was the main pathway for intestinal calcium
absorption (13). The impaired bone mineralization observed
in CACNA1D knockout pups supported that Cav 1.3 was
essential for maintaining positive calcium balance and bone
calcium accretion in the pre-weaning period (13).
Some evidence suggests that Cav may play a role in
calcium absorption in patients with diabetes mellitus, liver
cirrhosis, and inflammatory bowel diseases (IBD) who
have high plasma levels of advanced oxidation protein
products. By using cultured Caco-2 cells in the presence
of advanced oxidation protein products, Wu et al. showed
that the mRNA and protein expression levels of all calcium
transport proteins [i.e., TRPV6, calbindin-D9k , PMCA1b ,
and Na+ /Ca2+ exchanger 1 (NCX1)], except Cav 1.3, were
downregulated (245). These findings suggest that the Cav 1.3-
mediated calcium absorption was spared and could be used
along with other alternative intracellular calcium translo-
cation pathways (e.g., calcium shunting or tunneling) to
facilitate intestinal calcium flux in these conditions. Although
one study reported downregulation of Cav 1.3 in the colon of
patients with severe IBD and in mice with trinitrobenzene
sulfonic acid-induced colitis (192), these observations do
not completely rule out the plausible involvement of Cav in
intestinal calcium absorption. Thus, more investigations on
Cav 1.3 in the intestine of rodents and humans of different age
groups and under physiological and pathological conditions
are still warranted.
Intracellular calcium translocation
The intracellular calcium concentration must be maintained
below 10−7 mol/L in order to facilitate intracellular signal
Volume 11, May 2021
Intestinal Calcium Absorption
transduction while preventing cell apoptosis. Once a bulk
of ionized calcium enters the enterocytes, cells must have
mechanisms for buffering the extra calcium as well as translo-
cating these calcium ions from the mucosa to the serosal side.
The intracellular calcium translocation is mediated primarily
by calcium-binding proteins. Other mechanisms including
intraorganellar tunneling, vesicular calcium transport, and
free-ionized calcium diffusion may also be used and are
discussed in the following section.
Calbindin-D9k is the primary calcium-binding protein in
the enterocytes with small high-affinity EF-hand domains
that can bind 2 moles of calcium per mole of protein (207).
An increase in the concentration of this cytosolic calcium-
binding protein was detected at weaning in mice or rats along
with increase in the nonsaturable transcellular pathway of
absorption (224). Calbindin-D9k is prominently expressed in
the small intestine and is accumulated in the villi of duodenal
enterocytes, especially at the villous tip (Figure 3A) (100).
It has been very well established that calbindin-D9k is
regulated at the transcriptional level by 1,25(OH)2 D3 medi-
ated through vitamin D signaling (48, 71, 198). VDRE were
identified in the 5′ -flanking regions of calbindin-D9k gene
in humans and rats (48, 109). Fleet and colleagues (72)
tested whether the intestinal-specific overexpression of VDR
(HV2 mice) would improve transcellular calcium transport in
mice fed low calcium diets. The investigators observed that
1,25(OH)2 D3 injection significantly increased calbindin-D9k
in duodenum of wild-type mice with no significant increase
in HV2 mice (72). This confirms that the transcription of
calbindin-D9k is under the regulation of vitamin D.
Another line of research indicated that intestinal expression
of calbindin-D9k could be modulated by other factors besides
1,25(OH)2 D3 . Wang and co-workers, by using the Caco-2
clone TC7 cells, reported that calbindin-D9k gene was upreg-
ulated more strongly by differentiation of the enterocytes
than 1,25(OH)2 D3 (>100-fold vs. 2-fold) (233). Deletion-
mutation studies showed that the promoter sequences for
Cdx-2 (a homeobox protein) and hepatocyte nuclear factor
(HNF)-1 had a strong impact on calbindin-D9k gene expres-
sion during differentiation (233). In addition, an increase in
calcium influx via TRPV6 could also upregulate the mRNA
and protein levels of calbindin-D9k even in mice lacking VDR
(45). These findings suggested that while 1,25(OH)2 D3 could
promote the transcription of calbindin-D9k , other factors
(e.g., increased calcium flux, Cdx-2, and HNF-1) are also
important for basal calbindin-D9k expression.
Even though calbindin-D9k is believed to be an important
player in transcellular pathway, the level of calbindin-D9k
mRNA does not always correspond with the degree of active
calcium absorption (139). When compared with wild-type
mice, calbindin-D9k knockout mice showed similar serum
calcium and no change in calcium absorption either with or
without 1,25(OH)2 D3 treatment (2, 15, 134). Lee et al. (139)
demonstrated mRNA expression of TRPV6 and PMCA1b in
the duodenum of calbindin-D9k knockout mice were upreg-
ulated compared to wild-type mice, suggesting alternative
9
Intestinal Calcium Absorption Comprehensive Physiology

Apical Basolateral
Vesicular transport Ca2+
2+
Ca
Cai Free diffusion PMCA1b
2+
Ca
TRPV6 IP3R
STIM1 Ca2+
ER
Ca2+

Unidentified Tunneling RyR

Ca2+ channel SERCA

Figure 6 Model of vesicular and reticular transport of calcium. Diagram shows

vesicular calcium transport and calcium tunneling through the meshwork of endoplas-
mic reticulum (ER). Calcium enters the cell through TRPV6 or other calcium channels.
Intracellular calcium pool (Cai ) in the apical compartment is moved into the vesi-
cles or pumped into ER lumen by sarco-endoplasmic reticulum Ca2+ -ATPase (SERCA).
Calcium diffuses along the ER tunnel to the basolateral side and released into the
cytoplasm by 1,4,5-trisphosphate receptor (IP3 R) and ryanodine receptor (RyR) and
extrudes by the plasma membrane Ca2+ ATPase-1b (PMCA1b ). In cellular calcium
depleted condition, a calcium sensor stromal interacting molecule 1 (STIM1) is translo-
cated to the apical membrane to induce electrogenic calcium influx. The pathways
with red texts (i.e., STIM1, SERCA, ER, IP3R, and RyR) are hypothetical from indirect
evidence and need more investigations to confirm their physiological significance.
TRPV6, transient receptor potential cation channel, subfamily V, member 6.

compensatory mechanisms to maintain normal transcellular
calcium transport. Lastly, genetic inactivation of calbindin-
D9k did not change the serum calcium level (2, 139). These
data suggest that calbindin-D9k may not be the only calcium-
binding protein responsible for the intracellular calcium
translocation.
In addition to calbindin-D9k , several other calcium-binding
proteins, such as parvalbumin, calmodulin, and soluble
resistance-related calcium-binding protein (i.e., sorcin) might
have a role in translocating calcium in the cytoplasm (244).
A DNA microarray analysis in Caco-2 cells showed that
sorcin expression was upregulated by 1,25(OH)2 D3 (244).
In addition, parvalbumin, calmodulin, and sorcin may also
bind to and directly regulate the activities of the basolateral
PMCA1b and NCX1 (17, 253). Therefore, these proteins may
modulate the amount of calcium being translocated across
the cell in correspondence to the rate of basolateral calcium
extrusion.
Recently, new evidence has shown that transcellular and
paracellular transport systems might work cooperatively.
In a study of calbindin-D9k knockout mice, Hwang et al.
(105) showed that, under normal calcium diet, the expression
of tight junction genes occludin, claudin-2, and claudin-15
in the duodenum was significantly increased compared to
wild-type mice. On the contrary, the calbindin-D9k knock-
out mice under low calcium and low vitamin D diet had
reduced expression of occludin, zonula occludens (ZO)-1,
and claudin-15 as compared to wild-type animals (105). The
adaptation to the absence of calbindin-D9k was also reflected
in an increased expression of the tight junction proteins in the
kidney suggesting a compensatory increase in renal paracel-
lular calcium reabsorption (104). Further research is required
to gain insight into a coordination between transcellular and
paracellular calcium transport pathways.
Besides the passive buffering of cytoplasmic calcium by
calbindin-D9k and other calcium-binding proteins (Figure 6),
other mechanisms of intracellular calcium mobilization
have been reported. Endoplasmic reticulum (ER) has been
hypothesized to play an important role in the active buffering,
presumably as a part of the store-operated calcium entry via
TRPV6 (186). The ER lumen stretches between the apical and
basolateral membrane and the sarco-endoplasmic reticulum
Ca2+ -ATPase (SERCA) is localized to the apical compart-
ment where it pumps cytoplasmic calcium into the ER lumen.
Thus, calcium can diffuse along the ER tunnel network to the
basolateral region where inositol 1,4,5-trisphosphate (IP3)
and ryanodine receptors serve to release calcium into the
cytoplasm. Onodera et al. (179) reported in a study of rat
colonic epithelium and human colonic HT29/B6 cell line
that stromal interacting molecule 1 (STIM1) and Orai1—a
calcium sensor and a calcium release-activated calcium
channel, respectively—were crucial for the apical calcium
uptake into the colonic cells. When calcium store is depleted,
STIM1 is translocated to the apical membrane (179) where
it forms complexes with Orai1 and induces electrogenic
calcium influx. TRPV6 and reverse-mode NCX1 might also
contribute to calcium entry during calcium store depletion

10 Volume 11, May 2021

Comprehensive Physiology
(179, 186). Nevertheless, it remains unclear whether intraor-
ganellar tunneling of calcium significantly contributes to the
overall intestinal calcium absorption or is merely a part of
intracellular calcium signaling for enhancing transport of
other ions such as chloride and potassium.
Various intracellular membrane-bound vesicles, such as
lysosome and endocytic vesicles, have also been postulated to
contribute to a bulk transport of calcium and some other tran-
sition elements (e.g., iron and copper) across the cytoplasm
(160, 167). Lysosome-like vesicles near the apical mem-
brane also accumulate some other ions, including aluminum,
gadolinium, and terbium (1). Calcium probably enters the
absorptive cells during endocytosis, which is partially con-
trolled by 1,25(OH)2 D3 through its membrane receptor
known as 1,25(OH)2 D3 -MARRS (membrane-associated,
rapid response, steroid-binding) protein (166, 167). Alter-
natively, calcium may first traverse the apical membrane
via TRPV6 or other calcium channels before being moved
into the vesicles by unidentified transporter(s). The amount
of accumulated calcium ions is dependent on vesicular
acidic pH; therefore, a disruption of vesicular acidification
by chloroquine halts the process (167). Calbindin in the
vesicles probably helps buffer ionized calcium and facilitate
accumulation of calcium (137). Although passive diffusion
of ionized calcium in the apical-to-basolateral direction is
possible especially during 1,25(OH)2 D3 -stimulated con-
ditions with steep calcium gradients, its diffusion rate is
much slower (∼1/70) than facilitated diffusion mediated by
Ca2+ -laden proteins (e.g., calbindin-D9k and parvalbumin)
(65, 66). Indeed, an increase in cytosolic ionized calcium
during the enhanced calcium absorption probably signals
most calcium transporters particularly PMCA1b to accelerate
calcium efflux into the extracellular space (223).
Since most acidified lysosome-like vesicles have a limited
volume, other metals particularly iron, have been hypothe-
sized to compete with calcium for uptake into the vesicles
(130). In other words, iron-bound transferrin may occupy
space in the lysosome, leaving less space for calcium accu-
mulation. This phenomenon partially explains how high iron
intake inhibits intestinal calcium absorption (141).
Calcium transport across the basolateral membrane
The basolateral calcium efflux is a cellular energy-dependent
process. Several transporters, particularly P-type primary
active transporter ATP2B1 (i.e., PMCA1b ) and secondary
active transporter SLC8A1 (i.e., NCX1), predominantly move
cytoplasmic calcium across the basolateral membrane of the
intestinal absorptive cells (69, 98). Many other transporters
such as ATP2B4 and K+ -dependent Na+ /Ca2+ exchanger
(SLC24 or NCKX) may also contribute to calcium extrusion
(5, 101).
PMCA1b is the major calcium transporter at the baso-
lateral membrane as its activity contributes up to 80% of
the basolateral calcium efflux (54, 86). PMCA1b is potently
Volume 11, May 2021
Intestinal Calcium Absorption
activated by serine/threonine phosphorylation, cytosolic cal-
cium, calmodulin, and calbindin-D9k while it can be inhibited
by vanadate (209). Trifluoperazine—an antipsychotic drug
that acts as a calmodulin inhibitor—is capable of blocking
PMCA1b -mediated calcium efflux (114) and preventing
PMCA1b activation in rat duodenum and descending colon
(31, 63). Unlike wild-type mice, mice with intestine-specific
deletion of Pmca1 did not show an increase in active calcium
absorption in response to intraperitoneal administration of
1,25(OH)2 D3 . Moreover, these mice also showed marked
reduction in growth and bone mineralization (202). Recently,
4.1R protein is suggested to be crucial for the PMCA1b
activity. Liu et al. (148) demonstrated that the 4.1R protein
co-localized with PMCA1b and the 4.1R knockout mice had
complex phenotypes that include hypocalcemia, elevated
serum 1,25(OH)2 D3, and PTH, impaired calcium absorption
in the small intestine, downregulation of PMCA1b expression
and osteopenia. Thereby, this suggests that 4.1R protein may
be an interacting protein required for action of PMCA1b .
Collectively, these findings indicate that pharmacological
inhibition and genetic deficiency of PMCA1b , as well as
4.1R protein, can have a consequence in calcium and bone
homeostasis.
The NCX1-mediated calcium transport is responsible for
approximately 20% of basolateral calcium flux (86, 98).
Nevertheless, both PMCA1b and NCX1 work together in
an interdependent manner. A complete inhibition of NCX1
by small molecules such as KB-R7943 led to cessation
of PMCA1b activity and vice versa (57). It is plausible
that blockade of one basolateral transporter culminates in
increased cytoplasmic calcium, which halts the entire process
of transcellular calcium transport. This hypothesis is in line
with the observations that elevated cytoplasmic calcium
levels in the vicinity of TRPV6 and Cav calcium channels
led to inactivation of these channels, thereby decreasing
apical calcium influx (51, 120, 138, 170). However, whether
compensatory upregulation of PMCA1b in nonfunctional
NCX1 condition exists requires further investigations. Under
normal conditions, the electrogenic NCX1 in the duodenal
enterocytes extrudes calcium with a stoichiometry of 3 Na+ :1
Ca2+ (forward mode) and by coupling with Na+ /K+ -ATPase,
sodium concentration gradient across the basolateral mem-
brane could be maintained (98). A change in sodium gradient
may trigger NCX1-induced calcium entry (reverse mode).
Dong et al. (55) have shown that a reverse-mode NCX1 was
essential for the duodenal HCO3 − secretion, which required
an increase in intracellular calcium.
NCKX is a plasma membrane transporter that appears to
mediate Ca2+ extrusion along with K+ and the Na+ comes
into the cell. Among five isoforms of NCKX (i.e., NCKX1–5)
(4, 5), the NCKX1 is the first isoform that was initially discov-
ered in the outer segment of retinal rod (25, 206). Recently,
Yang et al. (250) reported that male NCKX3 knockout mice
had a significantly decreased mRNA expression of TRPV6
and calbindin-D9k in the duodenal mucosal cells despite hav-
ing normal serum calcium levels (250). However, the NCKX3
11
Intestinal Calcium Absorption
knockout mice had a significant increase of plasma PTH and
a decrease in bone mineral content (250), suggesting that cal-
cium might have been drawn from the bone to compensate
the decreased extracellular calcium pool. These data implicate
NCKX3 as another basolateral transporter involved in intesti-
nal calcium absorption; however, their physiological signifi-
cance remains to be investigated.
Lastly, calcium-rich vesicles localized at the basolateral
compartment of the enterocytes are able to fuse with the
basolateral membrane for calcium extrusion (137). However,
under stimulated conditions with increased cytoplasmic
calcium, some vesicular cation-selective channels, such as
two-pore channels (TPCs), can induce calcium release into
the cytoplasm close to the basolateral membrane (255). TPCs
also help maintain proper vesicular osmolality, volume, and
endocytic vesicle trafficking (75). Meanwhile, an increase
in cytoplasmic calcium near basolateral membrane induces
basolateral calcium extrusion via PMCA1b and NCX1.
Other transporters associated with calcium transport
Several investigations have suggested that a number of trans-
porters, for example, Na+ /H+ exchanger 3 (NHE3), Na+ /K+ -
ATPase, and cystic fibrosis transmembrane conductance
regulator (CFTR), contribute to intestinal calcium absorption.
For example, NHE3 knockout mice exhibited the impairment
in calcium, sodium, and water absorption across the intestine,
particularly in the cecum (195) where the rate of active
transcellular calcium transport is much higher than other
intestinal segments (111, 117). We previously reported that
100 nM tenapanor—a potent NHE3 inhibitor—completely
abolished hepcidin-induced duodenal calcium absorption in
hemizygous β-globin knockout thalassemic mice (27) as well
as leucine- and leucine tripeptide-induced calcium transport
in the rat duodenum (220).
NHE3 has been postulated to profoundly affect pericellu-
lar and intracellular microenvironment required for calcium
absorption. Since intracellular acidic pH can reduce transcel-
lular calcium transport (35), NHE3 inhibition that leads to
H+ accumulation in the cytoplasm is expected to disrupt cal-
cium transport process. Apical Na+ uptake by NHE3 is also
crucial for a number of processes pertaining to paracellular
calcium transport (222, 240). For instance, high Na+ uptake
and accumulation in the paracellular space are able to increase
solvent drag or convective water flux that favors paracellular
calcium movement in an ATP-dependent manner (3). Further-
more, because of its activity, the apical H+ efflux is increased.
NHE3 is probably important for maintaining high calcium
solubility in the acid microclimate near the mucosal surface
(172, 181, 195, 222). Failure to maintain this luminal acidic
environment close to the microvilli might render calcium salt
to precipitate into insoluble complexes, thus compromising
calcium absorption.
NHE3 also couples or directly interacts with other transport
systems, for example, SGLT1 and CFTR. Coordination of
SGLT1-NHE3 system ensures continuous uptake of sodium
12
Comprehensive Physiology
(96, 225), thereby strengthening solvent drag-induced cal-
cium transport. The apical glucose uptake together with
Na+ influx is likely to modulate the transcellular calcium
transport, presumably by inducing the apical membrane
depolarization that opens Cav 1.3 and providing substrates
for ATP production from oxidation of glucose (120). As far
as the coordination between NHE3 and CFTR is concerned,
the underlying mechanism of how NHE3-CFTR system
modulates calcium absorption remains unclear
An appropriate sodium gradient across the basolateral
membrane is tightly controlled by Na+ /K+ ATPase, which
is expressed predominantly in the lateral membrane and
to a lesser extent in the basal membrane (6). This gradient
provides a driving force for NCX1 as well as NHE1. NHE1 is
a salient intracellular pH regulator that maintains H+ gradient
and can modulate the activity of various membrane proteins
including protein 4.1R, NCX1, and perhaps PMCA1b (145,
174). For example, NHE1 is required for an interaction
between NCX1 and its modulator calmodulin (145). More
investigations are required to gain more understanding in
the interplay among these plasma membrane proteins and
transporters.
Paracellular calcium transport
It was previously believed that the paracellular space—also
known as lateral intercellular space—is merely a simple con-
duit in which water and ions can diffuse passively along the
electrochemical gradient. Although the paracellular calcium
flux is nonsaturable and independent of the amount of cal-
cium intake (182), several lines of evidence have confirmed
that fluid and ion movement across this space is actively
controlled by the absorptive cells as well as systemic and
local mediators such as 1,25(OH)2 D3 and cytokines produced
by immune cells (22, 95, 116, 221). Paracellular transport
pathway in human intestinal epithelium-like Caco-2 mono-
layer also showed rectification since under the voltage-clamp
experiments, the apical-to-basolateral calcium flux was much
greater than flux in the opposite direction (221).
Generally, paracellular calcium diffusion is dependent
on voltage gradient or transepithelial potential difference
(PD), and its concentration gradient. Owing to electrogenic
transport of cations—notably sodium, the lumen compart-
ment of all intestinal segments has negative potential as
compared to the plasma compartment (30, 115). Therefore,
the voltage gradient often favors calcium transport in the
basolateral-to-apical direction, that is, calcium secretion into
the lumen (115, 116). Nevertheless, the voltage-dependent
calcium transport in the rat duodenum is considered negli-
gible because of a relatively small PD of approximately 3 to
4 mV (30).
The calcium concentration gradient is considerable in the
proximal part of the small intestine, that is, duodenum and
proximal jejunum. Dietary calcium salts, as well as many
other minerals, become ionized in the acidic gastric juice
before they pass into the duodenum (60, 149). Luminal
Volume 11, May 2021
Comprehensive Physiology
Apical Tight junction Basolateral
Occludin
E-cadherin
Claudins
Standing osmotic gradient
Ca2+ Ca2+
K+
Na+ NKA
ZO-1
Actin cytoskeleton
Luminal [Ca2+] Plasma ionized [Ca2+]
>5 mM 1.25 mM
Figure 7 Paracellular intestinal calcium transport. Although calcium
concentration gradient is a driving force for paracellular calcium trans-
port, the tight junction (i.e., claudins, occludin, ZO-1) acts as a barrier
to restricts paracellular ion movement in a charge- and size-selective
manner. Calcium and small water-soluble molecules move along the
stream of water in apical to basolateral direction (solvent drag-induced
calcium absorption). Elevation of NKA activity increases the driving
force for the solvent drag-induced calcium transport. ZO-1, zonula
occludens-1.
calcium concentration may be as high as approximately 3 to
10 mmol/L versus 1.2 to 1.3 mmol/L free-ionized calcium
in the interstitium, and it is certainly elevated after oral cal-
cium supplementation or ingestion of calcium-rich diet (60).
Experiments using ex vivo rat duodenum and epithelium-like
Caco-2 monolayer showed a significant increase in paracel-
lular calcium flux when the luminal calcium concentrations
exceeded 5 mmol/L (31, 108). In the distal small intestine
like ileum, the calcium uptake appeared to be 1,25(OH)2 D3 -
independent (184). Pansu et al. (184) determined calcium
absorption in duodenum and ileum of vitamin D-deficient
rats before and after intraperitoneal 1,25(OH)2 D3 injection.
Treatment of 1,25(OH)2 D3 induced transcellular calcium
absorption only in the duodenum, but not the ileum.
The intestinal absorptive cells regulate the rate of para-
cellular calcium flux by various mechanisms. In mice, the
tight junction width between adjacent duodenal epithelial
cells is 16 to 18 nm, similar to that found in humans (133).
Perijunctional localization of polymeric F-actin filaments
and contraction of actomyosin ring complex near the tight
junction alter its size-selective property and also enhance dif-
fusion rate of calcium (128, 226). In addition, the absorptive
cells are able to change the expression of claudins and some
other transmembrane tight junction proteins, for example,
occludin, to modulate size and charge selectivity of the tight
junction (Figure 7) (7, 74).
In humans and rodents, the claudin family of transmem-
brane tight junction proteins consists of >20 members, which
Volume 11, May 2021
Intestinal Calcium Absorption
can form heteromers to produce an anastomosing meshwork
of tight junction ring (7). The extracellular loops with charged
amino acids protrude into the paracellular space to create
channel-like pores with different permselectivity (39). For
example, claudin-2, -12, and -15 contain negatively charged
amino acids in their extracellular loops, which attract cations
including Ca2+ and Na+ (78, 228).
The expression of intestinal claudins can be regulated by
vitamin D signaling and 1,25(OH)2 D3 . Fujita et al. (79) have
demonstrated that VDR knockout mice had a reduced expres-
sion of claudin-2 and -12 in the jejunum, ileum, and colon,
whereas 1,25(OH)2 D3 markedly upregulated claudin-2 and
-12 protein expression in Caco-2 cells. Consistently, by using
TRPV6/calbindin-D9k double knockout, Christakos et al.
(38) reported that 1,25(OH)2 D3 increased the expression of
claudin-2 and -12 while downregulated the expression of
cadherin-17. Even though calbindin-D9k knockout mice did
not show reduced intestinal calcium absorption, the mRNA
expression levels of claudin-2 and -15 were upregulated,
presumably to allow more calcium entry via paracellular
pathway (105). Furthermore, siRNA knockdown of claudin-
15 expression in Caco-2 monolayer was found to diminish
prolactin-induced paracellular calcium transport (32), while
lactating rats with calcium hyperabsorption exhibited upreg-
ulation of claudin-15 protein expression in the duodenum
(237). Thus, intestinal claudins play an important role in
promoting paracellular calcium transport and may serve as a
compensatory mechanism when transcellular calcium trans-
port is compromised or when there is an increased calcium
demand.
Recently, a rare missense mutation in CLDN2 gene
encoding claudin-2 was identified in an Iranian family with
azoospermia, renal stones, and hypercalciuria, the latter of
which resulted from a decrease in the claudin-2-mediated
paracellular calcium reabsorption in the proximal renal
tubule (10, 46). Claudin-2 knockout mice also manifested
a marked reduction in the colonic paracellular permeability
to calcium; however, a calcium balance study in claudin-2
knockout mice revealed greater net calcium absorption than
in wild-type animals (46). While the loss of claudin-2 led to
reduced colonic permeability to calcium along with decreased
passive calcium secretion in the colon, calcium secretion in
duodenum and ileum were similar between the genotypes,
thus leading to greater net calcium absorption in claudin-2
knockout mice (46). In addition, Gloux et al. (90) showed that
the mRNA level of claudin-2 in the duodenum and jejunum of
laying hens was significantly increased, presumably to meet
an increased calcium demand for eggshell biomineralization
(∼2 g/day) while minimizing resorption of medullary bone
(91). Collectively, claudin-2 is one of the major tight junction
proteins that form calcium-selective paracellular pores in
human, rodent, and possibly in avian.
Moreover, the intestinal absorptive cells can promote
paracellular calcium flux by increasing solvent drag. In a
leaky epithelium with transepithelial resistance (TER) of
≤100 Ω⋅cm2 like the intestinal epithelium, a significant
13
Intestinal Calcium Absorption
volume of water can flow through the paracellular pathway.
Calcium and small water-soluble molecules dissolved in
luminal fluid thus move along the stream of water in the
apical-to-basolateral direction—a phenomenon known as
the solvent drag-induced calcium absorption (28). Since the
paracellular water flow is dependent on sodium accumulation
and hyperosmotic milieu in the paracellular space distal to
the tight junction, the intestinal absorptive cells may increase
the activity of Na+ /K+ -ATPase lining the lateral membrane
to transport more sodium. Consequently, this increases the
driving force for the solvent drag-induced calcium transport
(28, 98). Under normal conditions, solvent drag-induced
calcium transport contributes up to 75% to 80% of the total
calcium absorption across the proximal small intestine, where
a great deal of glucose and sodium is absorbed.
Systemic Controls of Mucosal Calcium
Uptake
Endocrine regulation
Canonical parathyroid gland-kidney-intestine axis
Mucosal calcium uptake is controlled by several factors such
as endocrine and luminal factors. PTH and 1,25(OH)2 D3
are the two most important hormones that maintain cal-
cium homeostasis by exerting their actions on three organs,
that is, bone, kidney, and intestine (for reviews, please see
(69, 127). As depicted in Figure 8, the canonical hormonal
axis, a decrease in plasma ionized calcium is detected by
calcium-sensing receptor (CaSR)—a member of C fam-
ily G protein-coupled receptor—in the parathyroid chief
cells, which, in turn, release PTH to indirectly promote
25-hydroxyvitamin D3 1α-hydroxylase (CYP27B1) in the
proximal renal tubular cells (127). CYP27B1 catalyzes the
hydroxylation of 25(OH)D3 to 1,25(OH)2 D3 . This active form
of vitamin D is a potent calciotropic hormone that directly
stimulates the intestinal calcium absorption (for review,
please see Ref. 238). In humans and rodents, the small and
large intestine abundantly express nuclear VDR and are well
responsive to 1,25(OH)2 D3 . The binding of 1,25(OH)2 D3 to
the VDR response element promotes expression of calcium
transport machinery including TRPV5, TRPV6, calbindin-
D9k , NCX1, PMCA1b , claudin-2, and -12. Furthermore, the
1,25(OH)2 D3 -mediated induction of TRPV6 expression has
been shown to precede an increase in duodenal calcium
absorption in mice (214). Therefore, the VDR-mediated
signaling could enhance both transcellular and paracellular
calcium transport (79, 238). The expression of TRPV5,
TRPV6, and calbindin-D9k in the duodenal epithelial cells
of rats injected subcutaneously with 2 μg/kg 1,25(OH)2 D3
were markedly elevated as early as 3 h post-injection (122).
Since the enterocytes also produce CYP27B1 and CYP24A1,
the latter of which is a mitochondrial monooxygenase for
1,25(OH)2 D3 catabolism (178, 203), they probably respond
to 1,25(OH)2 D3 and, in turn, modulate calcium transporters.
14
Comprehensive Physiology
However, direct evidence supporting the local conversion of
vitamin D is still needed.
Based on the transcaltachia model—a model of rapid
stimulation of calcium transport by hormones—in mammal
and avian, treating the perfused duodenum from chicks
with PTH and 1,25(OH)2 D3 directly enhanced the vectorial
vesicular calcium transport (137, 168). Indeed, 1,25(OH)2 D3
is able to induce a rapid response and enhance vesicular
calcium transport within a few minutes (68). The underly-
ing cellular mechanism of this rapid response is not well
understood but it has been postulated to be mediated by
1,25(OH)2 D3 -MARRS protein or VDR, the latter of which
is recruited to the caveolin-1-rich plasma membrane or
caveolae (102). Moreover, Sterling and Nemere (215) further
demonstrated that both PTH 1-34 (an active peptide fragment
of PTH with 34 amino acids) and 25(OH)D3 were capable
of enhancing vesicular calcium transport in isolated chick
duodenal epithelial cells. It is likely that the rapid action
of 1,25(OH)2 D3 is mediated by nongenomic mechanisms
involving phospholipase C (e.g., PLC-γ), c-Src kinase, and
ERK1/2 (184).
Paracellular calcium transport is markedly decreased in
vitamin D deficiency, in part by the downregulated expres-
sion of claudin-2 and -12 (79). VDR knockout mice also
manifested a decrease in the expression level of claudin-2
in the duodenal mucosa although the mucosal permeability
to FITC-D 4000 (a large-size paracellular marker) remained
unaltered (133). Besides the effect on the intestine, Kladnit-
sky et al. (125) used a luciferase reporter assay in HEK 293
and OK (opossum kidney) cells to show that 1,25(OH)2 D3
suppressed the human CLDN16 promoter activity and this
is consistent with a decrease in renal CLDN16 expression
observed in mice received parenteral 1,25(OH)2 D3 . These
findings reveal the suppressive role of this hormone on
CLDN16 at the transcriptional level. Since 1,25(OH)2 D3
decreases renal claudin 16 transcription and claudin-16
promotes calcium and magnesium reabsorption in the thick
ascending limb of Henle’s loop (TAL), it is tempting to
speculate that an acute 1,25(OH)2 D3 administration and
the resultant intestinal hyperabsorption of calcium is prob-
ably associated with calciuria. In contrast to the effect of
1,25(OH)2 D3 in mediating transcellular calcium reabsorption
in the distal convoluted tubule, 1,25(OH)2 D3 possibly inhibits
paracellular calcium reabsorption in the TAL—through the
downregulation of claudin-16—in response to intestinal
hyperabsorption of calcium. This further suggests that
1,25(OH)2 D3 also helps maintain a balance between the
intestinal calcium/magnesium absorption and renal excretion.
Roles of bone-intestinal axis and other endocrine
factors
The bone-intestinal axis for regulating mineral metabolism
has recently been reported. The very first example is the
role of osteoblast- and osteocyte-derived fibroblast growth
factor (FGF)-23 in the regulation of intestinal phosphate
Volume 11, May 2021
Comprehensive Physiology Intestinal Calcium Absorption

Ca2+ deposition in bone

Calcitonin (inhibition of osteoclastic
bone resorption)

Thyroid gland
(C cell)

Rising Ca2+ levels in blood

Blood Ca2+ levels decrease
Increase Ca2+ excretion

Homeostasis
Normal blood Ca2+ level

mM mg/dL
Total calcium 2.0–2.5 8–10
Ionized calcium 1.2–1.3 4.5–5.5

Rising Ca2+ levels in blood

Bone Ca2+ release Blood Ca2+ levels decrease

Increase Ca2+ absorption

1,25(OH)2D3 PTH

PTH

Cyp27B1
ER
Mitochondria
CaSR Ca2+
Proximal tubular cell
Parathyroid chief cell

Figure 8 Systemic control of mammalian calcium homeostasis by three organs (i.e., bone, kidney, and intestine).
The details are fully explained in the section titled “Systemic Controls Mucosal Calcium Absorption”. Cyp27B1, 25-
hydroxyvitamin D3 1α-hydroxylase; PTH, parathyroid hormone.

absorption. Since FGF-23 is a phosphaturic and hypophos-
phatemic hormone, its actions are mainly to counterbalance
the effects of 1,25(OH)2 D3 on intestinal phosphate absorp-
tion and directly reduce phosphate reabsorption in the kidney
(191). Recently, intravenous FGF-23 injection has been
reported to inhibit 1,25(OH)2 D3 -induced intestinal calcium
transport in rodents (122), indicating that FGF-23 is also
a calcium-regulating hormone. Several circulating factors,
for example, plasma ionized calcium, inorganic phosphate,
and 1,25(OH)2 D3 , have been reported to upregulate FGF-23
production and secretion (190, 191). FGF-23 also increases
CYP24A1 activity and decreases CYP27B1 activity in the
kidney and some other extrarenal cells (e.g., monocytes),
thereby reducing 1,25(OH)2 D3 actions (11).
In addition to FGF-23, several other hormones have been
reported to participate in the systemic regulation of calcium
absorption such as calcitonin and prolactin. Calcitonin is
a major calcium-regulating hormone that reduces plasma
calcium concentration. The primary site of calcitonin produc-
tion is parafollicular cells of the thyroid gland. After being
released into the circulation, calcitonin binds the osteoclasts
to inhibit bone resorption (76, 77). Although known for

Volume 11, May 2021 15

Intestinal Calcium Absorption
this mechanism in bone, the role of calcitonin in intestinal
calcium absorption is still controversial and most studies
were performed in animals (216). Some in vitro evidence
revealed inhibitory effect of calcitonin on intestinal calcium
absorption, but animal studies showed otherwise. Employing
isolated small intestine from vitamin D-replete rats, Olson
and colleagues (177) reported the immediate reduction of
intestinal calcium absorption after acute infusion of low
dose of calcitonin (10 mU) while a high dose (500 mU)
enhanced calcium absorption. However, these effects were
absent in the intestine from vitamin D-deficient rats (177).
On the other hand, physiological and pharmacological doses
of calcitonin did not alter intestinal calcium absorption in
thyroparathyroidectomized rat, a calcitonin-deficient model,
(43) and a 12-day infusion of 200-mU calcitonin in these
rats conversely increased plasma calcium by 50%, which
was likely resulted from an increase in intestinal calcium
absorption (107). Based on these data, the effect of calcitonin
on intestinal calcium absorption seems to be dose- and vita-
min D-dependent. Although current evidence suggests the
involvement of this hormone in intestinal calcium transport,
inconsistency observed in the findings emphasizes the need
to investigate the exact roles and underlying mechanisms of
calcitonin in well-controlled mammalian models and humans.
Prolactin and 17β-estradiol are potent stimulators of
intestinal calcium absorption (28, 163, 227). Both hormones
are able to directly upregulate the expression of transcellular
calcium transporters, such as TRPV6 (163, 227). Meanwhile,
17β-estradiol increased VDR expression in the rat duodenum,
thus augmenting 1,25(OH)2 D3 responsiveness (147). As a
lactogenic hormone, prolactin has been shown to enhance
intestinal calcium absorption during pregnancy and lactation,
which are reproductive periods with high calcium demand
for fetal bone development and lactogenesis (32). Prolactin
not only stimulates transcellular calcium transport but also
markedly increases paracellular calcium flux, in part by
modulating the expression and function of tight junction
protein claudin-15 (32, 241). The activity of transcellular
calcium-transporting proteins (e.g., calbindin-D9k , PMCA1b ,
and Cav 1.3) is also dependent on prolactin (163, 221, 242).
A transcriptome analysis of the duodenal epithelial cells from
pituitary-grafted hyperprolactinemic rats further revealed
a number of upregulated calcium channel transcripts, for
example, Cacnb1, Cacna1h, and Cacna1g (37) although
whether these calcium channels contribute to the duodenal
calcium absorption remains unclear. These data support
the importance of prolactin and 17β-estradiol in regulating
intestinal calcium absorption.
Hormones that promote skeletal growth and development
[e.g., growth hormone (GH), insulin-like growth factor
1, placental lactogen, and thyroxine] have been reported
to increase calcium absorption across the small intestinal
mucosa (70, 154, 180, 254). In aged female rats (16 months
old), 12-day GH injection was found to increase the duodenal
calbindin-D9k levels, and a combination of GH and PTH
treatment showed an additive effect (70). Interestingly, these
16
Comprehensive Physiology
hormones appear to contribute to calcium homeostasis by
synchronizing the intestinal calcium supply with the rate of
bone calcium accretion.
In summary, several hormones either produced from the
canonical parathyroid gland-kidney-intestine axis or those
produced from other tissues like FGF-23, prolactin, 17β-
estradiol, and GH are involved in the regulation of intestinal
calcium absorption (Figure 9). The multitude of hormonal
regulators ensures that the plasma ionized calcium level
is strictly maintained within the physiologic range while
adequately supplying calcium to meet certain needs (e.g.,
growth, lactation) at different stages of life.
Local Controls of Mucosal Calcium
Uptake
Possible roles of FGF-23 and CaSR
As mentioned earlier, FGF-23 is well known as a bone-
derived phosphate-regulating systemic factor that promotes
phosphate excretion in the kidney and increases 1,25(OH)2 D3
catabolism (93, 113, 229). To prevent hyperphosphatemia
that causes ectopic calcification (110), the increased circu-
lating level of phosphate stimulates secretion of PTH and
FGF-23, which, in turn, enhances phosphate excretion. In
regard to direct actions of FGF-23 on physiological renal
phosphate excretion, circulating FGF-23 binds to a recep-
tor complex of fibroblast growth factor receptor (FGFR)
and α-Klotho in the renal tubular epithelial cells. There
are four major FGFR isoforms, that is, FGFR1-4. Under
physiological conditions, FGFR1 appears to be the main
FGF-23 receptor that works with membrane-associated
Klotho and activates a signaling cascade involving ERK1/2
and serum/glucocorticoid-regulated kinase-1 (SGK1) (194).
This signaling cascade results in phosphorylation of the
scaffolding protein Na+ /H+ exchange regulatory cofactor
(NHERF)-1 and internalization of sodium/phosphate cotrans-
porter (NaPi)-2a and -2c in proximal renal tubules and reduces
the tubular phosphate reabsorption (62, 229). FGF-23 also
indirectly decreases phosphate absorption by suppressing
1,25(OH)2 D3 synthesis. In the same time, FGF-23 promotes
1,25(OH)2 D3 catabolism by upregulating CYP24A1. This
reduces the circulating active 1,25(OH)2 D3 levels and, in
turn, decreasing intestinal phosphate uptake (113, 126, 211,
229).
Besides being produced by bone cells, FGF-23 transcript
has been shown to express in cells of other tissues such
as kidney, brain, lung, liver, and spleen (122, 126). Thus,
it may also act as a local factor to control certain local
metabolism. Several studies showed that FGF-23 and FGFRs
were expressed in small and large intestinal enterocytes (161,
217, 239, 242). Specifically, by using immunohistochemistry
in the duodenum of female rats, Khuituan et al. reported
that FGFR1-4, but not α- and β-Klotho, were expressed in
the duodenal enterocytes (122). Although the expression
Volume 11, May 2021
Comprehensive Physiology Intestinal Calcium Absorption

Neurocrine factors

VIP
Substance P
Dopamine
Serotonin

Enteric VIPergic neuron

Luminal factors
Ca2+
Stimulators TTX sensitive

Amino acids
Glucose/Galactose
SCFAs
Paracrine
Prebiotics Cldn-2, -3, -12
Inhibitors Ca2+
NKA Neuroendocrine
2+ 3+
Fe and Fe PMCA1b cell
Zinc Na+
Copper Ca2+ K+ Ca2+ Endocrine factors
TRPV6
Ca2+ Stimulatory Inhibitory
Ca2+
TRPV5 Calbindin-D9k Na + NCX1 1,25(OH)2D3 FGF-23
Prolactin Calcitonin
Ca2+ Na+
Cav1.3 Estrogen Corticosteroids
NKA GH
K+ IGF-1
1,25(OH)2D3-dependent Thyroid hormone

Prolactin-dependent
Apical Basolateral

Figure 9 Extrinsic (humoral and neural) and intrinsic (luminal) regulators of intestinal calcium absorption. The luminal and endocrine
factors that act as stimulator and inhibitor on intestinal calcium absorption showing in the green and orange boxes, respectively. Although
the presented luminal and endocrine factors are known to modulate calcium absorption, the involvement of neurocrine factors (marked as
italic text), or factors released from neuroendocrine cells in the enteric nervous system is in the emerging area of interest and needs more
investigations to confirm the physiological significance. Cldn, claudin; FGF-23, fibroblast growth factor-23; GH, growth hormone; IGF-1,
insulin-like growth factor-1; SCFAs, short-chain fatty acids; TTX, tetrodotoxin; VIP, vasoactive intestinal peptide; NCX1, Na+ /Ca2+ exchanger
1; NKA, Na+ /K+ -ATPase; PMCA1b , plasma membrane Ca2+ ATPase-1b; calbindin-D9k (S100 calcium-binding protein G, S100G); TRPV,
transient receptor potential cation channel, subfamily V.

of membrane-associated Klotho has not been reported in
the intestinal enterocytes, a body of research supports the
role of FGF-23 in regulating intestinal calcium absorption
through both systemic and local (paracrine) mechanisms
(for its systemic actions, please see section titled “Roles of
Bone-Intestinal Axis and Other Endocrine Factors”). It is
noteworthy that soluble Klotho in the circulation probably
contributes to FGF-23 signaling in the intestine.
An investigation in Caco-2 cell culture further sug-
gested that intestinal epithelial cells could secrete FGF-23
peptide into the apical and basolateral compartments in
response to 1,25(OH)2 D3 and apical exposure to high cal-
cium concentration, which were conditions that mimicked
1,25(OH)2 D3 -induced calcium hyperabsorption and high-
calcium intake, respectively (199, 239). Functional studies
in the duodenum of female rats confirmed that FGF-23
exerted a direct action on the intestine by directly inhibiting
the 1,25(OH)2 D3 -mediated transcellular and paracellular
calcium absorption (122, 123). Although the exact molecular
mechanism and signaling pathway(s) by which FGF-23
reduces calcium transport remains elusive, several signaling
proteins, such as MAPK/ERK, p38 MAPK, and protein
kinase C might be involved in the process (122, 123).
High calcium and 1,25(OH)2 D3 exposures appear to be the
driving force for FGF-23 secretion and the resultant suppres-
sion of calcium transport. The pathway in the enterocytes may
involve the CaSR, which plays a role in monitoring calcium
transport across the enterocytes (239). By using Caco-2 cells,
Wongdee et al. (239) reported that CaSR inhibitors were
capable of suppressing the 1,25(OH)2 D3 -induced upregula-
tion of FGF-23. Furthermore, similar to apical high-calcium
exposure, the apical CaSR activation by cinacalcet and
AC-265347 diminished the 1,25(OH)2 D3 -mediated transcel-
lular calcium transport presumably via the upregulation of

Volume 11, May 2021 17

Intestinal Calcium Absorption Comprehensive Physiology

(A) Apical Basolateral

Transcellular
1,25(OH)2D3
FGF-23
FGF-23

Paracellular

CaSR
FGF-23
Activators FGF-23
CaSR
2+
Ca
TRPV6
Transcellular

(B)

SCFAs

PMCA1b
Luminal pH 2
TRPV6
Ca2+ Ca2+
Calcium complexes Calbindin-D9k
[e.g., CaHPO4, Ca(H2PO4)2]
Na+ NCX1

Na+
1 Free Ca2+ NKA
K+
1 SCFAs enhance free ionized calcium
2 SCFAs increase expression of TRPV6

Figure 10 The emerging topic of intestinal calcium absorption. (A) A schematic diagram
shows possible mechanism of fibroblast growth factor (FGF)-23 and calcium-sensing recep-
tor (CaSR) on local negative feedback regulation of calcium absorption. (B) A schematic
diagram showing a possible mechanism of how short-chain fatty acids (SCFAs) enhances
transcellular calcium transport in the intestine. Hypothetical pathways or pathways with
indirect evidence are presented as dashed lines. NCX1, Na+ /Ca2+ exchanger 1; NKA,
Na+ /K+ -ATPase; PMCA1b , plasma membrane Ca2+ ATPase-1b; calbindin-D9k (S100
calcium-binding protein G, S100G); TRPV, transient receptor potential cation channel,
subfamily V.

FGF-23. These findings corroborated the involvement of
FGF-23 and CaSR in an ultrashort negative feedback loop for
preventing excessive calcium absorption, particularly during
prolonged 1,25(OH)2 D3 treatment and high-calcium intake
(Figure 10A) (239).
Another mechanism that has been proposed as a safeguard
to prevent serum calcium overload is basolateral CaSR sig-
naling. CaSR is indeed expressed throughout the intestine
in both the apical and basolateral membrane of enterocytes.
Lee et al. (140) applied CaSR activator cinacalcet to either
apical or basolateral hemichamber of the Ussing chamber
and demonstrated that only activation of basolateral CaSR
led to a decrease in transcellular calcium absorption (140).
They further tested the effects of basolateral cinacalcet
treatment in the proximal colon of nonfunctional TRPV6
(TRPV6D541A ) mice and found that the net calcium flux
was unchanged compared with pretreatment state, thereby
indicating that the CaSR-mediated reduction of calcium
absorption may work through attenuation of TRPV6 expres-
sion on the apical membrane or TRPV6 function (140).
Taken together, these data support that CaSR in the apical
and/or basolateral membrane could play a role in regulating
calcium homeostasis but may act through different molecular
pathways.
Factors Affecting Calcium Absorption
Besides being controlled by hormones, the intestinal calcium
absorption is also affected by the local environment in the gut
lumen such as the presence of luminal nutrients, amino acids,
sugars, or microbiota-derived metabolites.

18 Volume 11, May 2021

Comprehensive Physiology
Luminal pH and unstirred water layer
An in vitro digestion study revealed that various forms of
calcium sources, for example, calcium carbonate, calcium
phosphate, and calcium citrate malate, are mostly soluble
under gastric pH, but become insoluble (∼20%–36% bioac-
cessibility) under small intestinal pH (149). The apical
domain of enterocytes is seemingly close to luminal fluid,
which contains variety of nutrients and minerals. However,
there is a thin layer of gel-like fluid covering the microvilli
that blocks the enterocytes from exposing directly to the lumi-
nal fluid. This layer is called unstirred water layer that helps
maintain particular physicochemical profiles of ion and water
(97, 142). The unstirred water layer can create an anomalous
ion gradient that makes the diffusion rate unpredictable, thus
becoming a pre-epithelial diffusion barrier. Bulk flow of
luminal fluid over more viscous fluid of the unstirred water
layer might create Saffman-Taylor instability—also known
as viscous fingering—at the interface. This unstable interface
allows luminal nutrients to penetrate deep into the unstirred
water layer similar to the way that H+ penetrates through the
mucus gel layer produced by gastric mucin (16). Furthermore,
the fluid layer near the microvilli is an acid microclimate,
which is maintained by H+ efflux into a mucin-rich milieu
by NHE3, thereby providing H+ gradient or a source of H+
for protonation of certain anions and solubility of minerals
including calcium (222). The unstirred water layer in the
small intestine has approximately 500 to 800 μm thickness
with constant weak acid microenvironment (pH 6–7). This
layer is comprised of water (∼95%) and other solutes, for
example, mucin or glycans, secretory proteins, ions, extracel-
lular part of transporters, and receptors (97, 210). Neurogenic
chloride secretion, locomotion activity, or gut motility can
reduce the thickness of unstirred water layer to as thin as thin
as <300 μm and this thinner layer is expected to enhance
absorption of nutrients and minerals (40, 142).
Luminal nutrients
Generally, Na+ -coupled nutrient cotransport induces myosin
light chain kinase-mediated remodeling of perijunctional
actomyosin ring complex (95). This phenomenon can
increase the paracellular permeability to small ions including
Na+ and Ca2+ . Indeed, phosphorylation of myosin II reg-
ulatory light chain and ZO-1 reorganization are crucial for
the overall barrier function of tight junction, thus increasing
paracellular transport of many molecules and ions (95).
Luminal nutrients can also modulate intestinal calcium
transport (Figure 9). A number of l-amino acids, especially
leucine, proline, hydroxyproline, isoleucine, alanine, and
lysine, are capable of enhancing intestinal calcium trans-
port (220). Dipeptides and tripeptides—as substrates of
H+ -dependent peptide transporter (PEPT)-1—also directly
stimulate calcium absorption in NHE3-dependent fashion
(220). Furthermore, certain small peptides, such as peptides
from tilapia bone collagen hydrolysate, were found to exhibit
calcium-chelating capacity and increase calcium uptake
Volume 11, May 2021
Intestinal Calcium Absorption
across Caco-2 monolayer (146), suggesting that these bio-
logically active peptides may directly enhance calcium
transporter activity or act as slow-release calcium complexes
and/or prevent precipitation of ionized calcium into an
insoluble form.
In addition to small peptides and amino acids, monosac-
charides have ability to modulate calcium absorption. For
example, the SGLT-1 substrates such as glucose and galactose
exhibit a stimulatory effect on intestinal calcium absorption.
On the other hand, fructose has an inhibitory effect on
calcium absorption (59, 200). High fructose intake has been
shown to indirectly diminish the intestinal calcium absorp-
tion in growing and lactating rats by increasing 1,25(OH)2 D3
catabolism and decreasing 1,25(OH)2 D3 synthesis (58, 59).
In vitro and in vivo studies in growing rats revealed that fruc-
tose diminished intestinal calcium absorption, by reducing
circulating 1,25(OH)2 D3 levels, TRPV6, and calbindin-D9K
expression, thereby decreasing transcellular calcium transport
(59). The underlying mechanism of fructose-induced reduc-
tion of 1,25(OH)2 D3 might be associated with FGF-23—a
regulatory hormone of 1,25(OH)2 D3 production. Douard
et al. (59) found that fructose increased circulating FGF-23
levels, which inhibited CYP27B1 and stimulated CYP24A1
mRNA expression. As a consequence, this fructose-mediated
elevation of FGF-23 reduced 1,25(OH)2 D3 production.
Several minerals, such as nonheme iron and zinc, can
also indirectly hinder the transepithelial calcium transport
(67, 130). In alkalinized luminal fluid, zinc has a tendency
to precipitate with calcium and phosphate into an insoluble
complex. The formation of these complexes is experimentally
prevented by casein phosphopeptides (67). High iron intake
and certain conditions with duodenal iron hyperabsorption,
for example, β-thalassemia, are associated with a reduction
in duodenal calcium transport (130). However, investigations
pertaining to the effects of transition metals (e.g., copper)
on intestinal calcium absorption are scant. A study in Ross
308 chicks has suggested that copper nanocolloid feeding
and copper accumulation in the small intestinal cells led to
a significant decrease in calcium absorption; however, the
underlying cellular mechanism remains elusive (175). Since
the intestinal oxidative stress caused by reactive oxygen
species (ROS) often results in the downregulation of cal-
bindins, PMCA1b and/or NCX1 as well as inhibition of
calcium absorption (196, 197, 247), it is possible that iron
and copper diminish intestinal calcium absorption, in part, by
inducing ROS production.
Microbiota-derived metabolites
Microbiota produce large quantities of small acidic molecules,
which increase the solubility of calcium complexes and thus
directly stimulates calcium absorption (Figure 9) (47). Since
only ionized calcium is readily absorbed by the intestinal
absorptive cells or easily diffuses across the paracellular
space, the efficient sites of absorption as indicated by the
relatively high transport rate are segments with acidic pH, that
19
Intestinal Calcium Absorption
is, duodenum and proximal large intestine (60), the latter of
which (precisely cecum and proximal colon) is the major site
of microfloral fermentation. Gut microbiota produces several
metabolites from digested foods and prebiotics. Among these
molecules, short-chain fatty acids (SCFAs) are known to
modulate intestinal calcium absorption (47). It has long been
demonstrated that SCFAs, for example, acetate, propionate,
and butyrate, were capable of increasing calcium absorption
in the rat colon (150). Consistently, a randomized controlled
trial study has also suggested the use of soluble corn fiber to
increase calcium absorption in female adolescents. Soluble
corn fiber is a corn-derived nondigestible carbohydrate that
promotes SCFAs production and expansion of gut micro-
biota such as Bifidobacterium and Parabacteroides (236),
thus suggesting that the gut microbiota may play a role in
calcium homeostasis and bone health. At the cellular level
(Figure 10B), SCFAs (∼2 mmol/L) were reported to induce
SCFA response element and transcription of TRPV6 gene in
Caco-2 cells (80). However, future research is needed to iden-
tify the exact mechanisms of action of SCFAs on intestinal
calcium absorption, calcium homeostasis, and bone strength.
Prebiotics, probiotics, and synbiotics have become impor-
tant ingredients for nutraceutical supplements for promotion
of calcium absorption and bone health, especially in certain
subgroups of individuals with osteopathy, such as insulin
resistance, diabetes mellitus, or dyslipidemia. For example,
the probiotics Lactobacillus paracasei HII01, prebiotics
xylooligosaccharide, and synbiotics mixture of both have
been shown to augment osteoblastic bone formation in obese,
insulin-resistant rats (61). In addition, one of the widely used
nutraceutical ingredients is fructooligosaccharide, which has
been reported to increase calcium and magnesium absorption
across the colonic and rectal epithelia in rats (176). Unlike
fructose, a monosaccharide, that is readily absorbed in the
small intestine, fructooligosaccharide is indigestible and
its absorption is negligible. Thus it travels along the small
intestine as an intact molecule to the colon where it serves as
substrate for bacterial fermentation in the colon and stimulates
the growth of bacterial microflora. As a result, SCFAs are
produced from the fermentation process and these SCFAs are
likely to contribute to enhance bone formation and calcium
absorption as discussed in the aforementioned paragraph.
Emerging Areas of Interest
Neural control of calcium absorption
Since enteric nervous system (ENS) is formed by submucosal
and myenteric plexuses and consists of many secretomotor
neurons, these neurons can directly innervate the enterocytes
and control epithelial transport of major ions, especially Na+
and Cl− (40). Several neurotransmitters and neurohormones
such as acetylcholine, serotonin, nitric oxide, bradykinin,
and vasoactive intestinal peptide (VIP) have been shown to
regulate intestinal chloride secretion (Figure 9) (40, 189).
In addition, activation of 5-HT4 receptor by serotonin
20
Comprehensive Physiology
was found to inhibit NHE3 activity in Caco-2 cells (88).
Although there is no direct evidence of neural regulation
of intestinal calcium transport, the enteric neurons may
indirectly modulate calcium absorption through alteration
of sodium absorption or NHE3 activity. Changes in the
intestinal HCO3 − secretion by enteric neurons might alter
the luminal pH, thereby affecting calcium solubility (29,
136). Moreover, the submucosal secretomotor neurons also
produce bradykinin and arachidonic acid metabolites, which
are known to induce neurogenic chloride secretion across the
small intestinal epithelium (189). In the kidney, bradykinin
activated TRPV5 for tubular calcium reabsorption (89), but
whether it is able to activate the intestinal TRPV5 or TRPV6
remains to be investigated.
VIP is another important neurotransmitter that directly
modulates paracellular calcium transport. A study in Caco-2
and HT29-Cl.19A monolayers suggested that direct exposure
to VIP on the basolateral side markedly increased transep-
ithelial resistance while decreasing paracellular calcium
transport (18). In the feline gallbladder mucosa, intravenous
VIP infusion led to net secretion of calcium into the lumen,
whereas sympathetic stimulation enhanced net calcium
absorption (171). It is noteworthy that direct innervation
of enteric VIPergic neurons to the mouse ileal mucosa has
been shown to be essential for production of new goblet cells
(208). These goblet cells secrete mucin into unstirred water
layer, thus probably modulating diffusion of luminal ions
near the brush border.
Substance P, a neurotransmitter in the ENS, possibly
contributes to the regulation of calcium permeability despite
having minimal effect on net calcium absorption across
the rat ileum (231). Furthermore, dopamine was found to
reduce calcium secretion across the rabbit ileum, thereby
decreasing total ileal calcium content and perhaps mitigat-
ing intestinal calcium wasting (56). We also observed that
tetrodotoxin—a potent sodium channel blocker and inhibitor
of neural transmission—significantly diminished the rat duo-
denal calcium transport (Charoenphandhu and Thammayon,
unpublished observations). Taken together, it is tempting to
speculate that neural control of calcium transporter activity
exists in the intestine.
Gut-derived serotonin
ENS and some special cell types in the intestine have potential
to secrete local regulatory factors to control calcium transport
such as gut-derived serotonin. Approximately 90% of total
serotonin in our body is produced by cells in the gastrointesti-
nal tract, for example, the duodenal enterochromaffin cells.
The remaining serotonin which is synthesized by serotoner-
gic neurons in brain stem does not cross blood-brain barrier
and thus has no role in regulating intestinal calcium transport.
Normally, the gut-derived serotonin has a role in controlling
gut motility and fluid and electrolyte transport (47, 249).
Tryptophan hydroxylase (TPH) is the rate-limiting enzyme
essential for serotonin biosynthesis. There are two isoforms of
Volume 11, May 2021
Comprehensive Physiology
this enzyme. TPH1 is mainly synthesized in enterochromaffin
cells, while TPH2 is produced in neurons (144). Serotonin
exerts its effects via serotonin receptors and reuptake into
the cells via serotonin reuptake transporters (SERTs) that
are expressed apically on the intestinal epithelial cells (85).
The activity of SERTs is dependent on extracellular sodium
and chloride and is inhibited by selective serotonin reuptake
inhibitor (SSRI) such as fluoxetine (52). Enterochromaffin-
derived serotonin has reported to stimulate peristaltic reflexes
and modify intestinal motility in normal and pathological
conditions (84). In contrast to enterochromaffin cells, sero-
tonergic neurons appear to be crucial for controlling gut
motility. An experiment in TPH2 null mice showed longer
total gastrointestinal transit time than in wild-type controls,
especially in the colon (144).
Although the effects of gut-derived serotonin on gut motil-
ity are extensively studied, evidence on the direct effects of
serotonin on intestinal calcium absorption is still scant. Some
investigations have indirectly suggested responsiveness of
the intestine to serotonin. Gill et al. (87) reported that, among
different intestinal segments, SERT showed the highest
expression level in the ileum, while duodenum, jejunum,
and colon had lower mRNA levels, respectively. Moreover,
increases in extracellular serotonin induced by fluoxetine
treatment has been shown to upregulate the expression of
transcellular calcium transporters (e.g., TRPV6, Cav 1.3,
and NCX1) in male rats subjected to restraint stress. These
data suggest potential regulation of gut-derived serotonin on
intestinal calcium absorption (33).
Stanniocalcin (STC)
In bony fish, the intestinal calcium transport is under the
control of STC1. STC1 is a glycosylated peptide hormone
that first discovered in the corpuscles of Stannius—a small
endocrine gland in the kidney of teleostean and holostean
fish. STC1 was discovered to have similar function to calci-
tonin which appears to prevent hypercalcemia by preventing
calcium entry into the gill and intestine of fish (106, 112).
In mammals, human recombinant STC has been reported to
decrease calcium absorption across the rat duodenum (152).
This observed phenomenon might be due to a downregulation
of TRPV5 and TRPV6 expression, but not NCX1, PMCA1b ,
or VDR expression (246). Hung et al. (103) demonstrated
that the expression of STC1 in renal proximal tubular cells
of rats was positively regulated by 1,25(OH)2 D3 and PTH,
presumably as part of a negative feedback loop.
Conclusions and Perspectives
Intestinal calcium absorption is a complex cellular energy-
dependent process that more than 20 protein families, for
example, TRPV5/6, Cav 1.3, calbindins, PMCAs, NCX1, and
claudins, are involved in the mechanisms of transcellular
and paracellular calcium transport. While transcellular active
Volume 11, May 2021
Intestinal Calcium Absorption
calcium transport has been well established especially in
humans and rodents, the cellular and molecular mechanisms
of paracellular calcium transport remain unclear and are
mostly based on in vitro studies. For example, it is unclear
whether the calcium-selective pores formed by claudin het-
erodimers really exist in the intestine. More investigations
are required to demonstrate the underlying mechanisms of
factors affecting paracellular calcium absorption. These fac-
tors include unstirred water layer, extracellular pH, luminal
and pericellular calcium, other nutrients as well as endocrine
and paracrine factors. Although the PTH-vitamin D system is
the most important regulator of calcium absorption, the role
of several other hormones including prolactin, GH, estro-
gen, and thyroid hormone could predominate under specific
conditions. For instance, prolactin is required to promote
systemic responses to meet a huge demand of calcium for
lactogenesis (37).
The information pertaining to negative regulators or
counterbalancing factors is scant. Local and systemic FGF-
23 system, as well as CaSR, expressed in the enterocytes
probably help prevent calcium hyperabsorption as part of
a negative feedback loop, but little is known on how these
mediators regulate the function of calcium transporters in the
absorptive villous epithelial cells. Since the rate of calcium
absorption is dependent on nutrient contents in meals, such
as sodium, amino acids, oligopeptides, prebiotic derivatives,
and small acidic molecules (60, 80, 95, 240), the cellular
mechanisms of calcium absorption must be different among
different human populations and species in different habitats
with a wide variety of diets. However, intestinal calcium
absorption of some species (e.g., Cryptomys damarensis and
Heterocephalus glaber) has been reported to be independent
of vitamin D (188). Therefore, there are other calciotropic
factors yet to be identified.
Acknowledgements
The authors thank Professor Nateetip Krishnamra for valu-
able comments on this article and Thitapha Kiattisirichai for
artwork. Our research was supported by grants from Mahi-
dol University – Multidisciplinary Research Cluster Grant
(to NC), the Thailand Research Fund (TRF) through the TRF
Senior Research Scholar Grant (RTA6080007 to NC), TRF
International Research Network Program (IRN60W0001 to
KW and NC), TRF – Office of the Higher Education Com-
mission Research Grant for New Scholar (MRG6280198 to
JT), Faculty of Allied Health Sciences, Burapha University
(to KW), National Research Council of Thailand (NRCT, to
NC), and Faculty of Science, Mahidol University (CIF and
CNI grant, to NC).
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