467

INVITED REVIEW
Paracellular calcium transport across renal and intestinal
epithelia1
R. Todd Alexander, Juraj Rievaj, and Henrik Dimke
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Abstract: Calcium (Ca2+) is a key constituent in a myriad of physiological processes from intracellular signalling to the miner-
alization of bone. As a consequence, Ca2+ is maintained within narrow limits when circulating in plasma. This is accomplished
via regulated interplay between intestinal absorption, renal tubular reabsorption, and exchange with bone. Many studies have
focused on the highly regulated active transcellular transport pathways for Ca2+ from the duodenum of the intestine and the
distal nephron of the kidney. However, comparatively little work has examined the molecular constituents creating the
paracellular shunt across intestinal and renal epithelium, the transport pathway responsible for the majority of transepithelial
Ca2+ flux. More specifically, passive paracellular Ca2+ absorption occurs across the majority of the intestine in addition to the
renal proximal tubule and thick ascending limb of Henle's loop. Importantly, recent studies demonstrated that Ca2+ transport
through the paracellular shunt is significantly regulated. Therefore, we have summarized the evidence for different modes of
paracellular Ca2+ flux across renal and intestinal epithelia and highlighted recent molecular insights into both the mechanism
of secondarily active paracellular Ca2+ movement and the identity of claudins that permit the passage of Ca2+ through the tight
junction of these epithelia.

Key words: kidney, NHE3, claudins, solvent drag.

Résumé : Le calcium (Ca2+) est un important constituant d’une myriade de processus physiologiques allant de la signalisation
For personal use only.

intracellulaire à la minéralisation de l’os. Conséquemment, le Ca2+ est maintenu à l’intérieur de limites étroites lorsqu’il circule
dans le plasma. Cela s’accomplit grâce à l’influence réciproque régulée entre l’absorption intestinale, la réabsorption rénale et
l’échange avec l’os. Plusieurs études se sont concentrées sur la voie hautement régulée du transport actif trans-cellulaire du Ca2+
du duodénum et du néphron distal. Cependant, en comparaison, peu d’études ont examiné les constituants moléculaires qui
créent une dérivation para-cellulaire à travers l’épithélium intestinal et rénal, la voie de transport responsable de la majorité du
flux de Ca2+ trans-épithélial. Plus spécifiquement, l’absorption para-cellulaire passive de Ca2+ survient le long de la plus grande
partie de l’intestin en plus du tubule proximal du rein et de la branche ascendante large de l’anse de Henle. Fait important, des
études récentes démontrent que le transport de Ca2+ à travers la dérivation para-cellulaire est sujet à une importante régulation.
Les auteurs ont donc fait la synthèse des indices qui suggèrent qu’il existe différents modes de flux para-cellulaires de Ca2+ à
travers l’épithélium du rein et de l’intestin, et souligné les récentes percées sur les plans du mécanisme moléculaire du
mouvement para-cellulaire de Ca2+ secondairement actif et de l’identité des claudines qui permettent le passage du Ca2+ à travers
les jonctions serrées de ces épithéliums. [Traduit par la Rédaction]

Mots-clés : rein, NHE3, claudines, déplacement de solvant.

Introduction
Calcium (Ca2+)participates in a diverse array of physiological
processes, including neural transmission, muscle contraction,
blood coagulation, intracellular signal transduction as a second
messenger, and the mineralization of bone as part of the hy-
droxylapatite crystal. Consequently, the concentration of free
ionized Ca2+ is tightly regulated in plasma. Hypocalcemic or hy-
percalcemic states can cause severe neurological and cardiovascu-
lar sequelae. In fact, disturbances in Ca2+ balance may in some
instances produce tetany, carpopedal spasms, life-threatening
arrhythmias, coma, or cardiac arrest (Bilezikian 1993; Guise and
Mundy 1995). Globally, Ca2+ homeostasis is maintained via regu-
lated interplay between 3 organ systems: intestinal absorption of
Ca2+ from the diet, storage, and exchange of Ca2+ with bone, and
changes in the renal reabsorptive capacity for Ca2+. These pro-
cesses effectively stabilize systemic Ca2+ concentrations within
normal limits. Parathyroid glands play a central role in sensing
changes in serum Ca2+ concentrations and respond by amending
secretion of parathyroid hormone (PTH). Increased intestinal ab-
sorption or augmented resorption from bone increases serum
Ca2+ typically leading to increased levels of Ca2+ in the urine be-
cause of compensatory reductions in renal transport capacity (Pak
et al. 1975). Similarly, dysregulation of select renal transporters
within the kidney results in hypercalciuria. Importantly, hyper-
calciuria remains the single greatest risk factor for developing a
Ca2+-based kidney stone (Levy et al. 1995), a disease with high and

Received 9 May 2014. Revision received 30 August 2014. Accepted 14 September 2014.
R.T. Alexander. Department of Pediatrics, The University of Alberta, 4-585 Edmonton Clinic Health Academy, 11405 – 87 Ave, Edmonton, AB T6G 2R7,
Canada; Membrane Protein Disease Research Group, The University of Alberta, Edmonton, AB T6G 2H7, Canada.
J. Rievaj. Membrane Protein Disease Research Group, The University of Alberta, Edmonton, AB T6G 2H7, Canada; Department of Physiology, The
University of Alberta, Edmonton, AB T6G 2H7, Canada.
H. Dimke. Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark.
Corresponding author: R. Todd Alexander (e-mail: todd2@ualberta.ca).
1This review is part of a Special Issue commemorating The Canadian Society for Molecular Biosciences 57th Annual Meeting – Membrane Proteins in Health

and Disease, held in Banff, Alberta, 9–13 April 2014.

Biochem. Cell Biol. 92: 467–480 (2014) dx.doi.org/10.1139/bcb-2014-0061 Published at www.nrcresearchpress.com/bcb on 17 September 2014.
 468 Biochem. Cell Biol. Vol. 92, 2014

Fig. 1. Modes of transepithelial Ca2+ flux. Ca2+ is absorbed down its electrochemical gradient via passive diffusion (top tight junction) or with
water following the osmotic gradient created by solute absorption (often sodium, bottom junction). Transcellular Ca2+ flux (bottom cell)
occurs via apical entry through a channel down the electrochemical gradient. Ca2+ then moves to the basolateral membrane bound to a
calcium-binding protein and then is extruded across the basolateral membrane via a calcium pump or in exchange for sodium.

VTE

Paracellular Diffusion
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[Ca2+] [Ca2+]

Na+ Na+

Na+

Ca2+
For personal use only.

H2O H2O Solvent Drag

Ca2+ Ca2+
Calb
Na+
Ca2+
Ca2+ Ca 2+
Ca2+

Transcellular Flux

Lumen

increasing prevalence (Stamatelou et al. 2003; Coe et al. 2005).
Therefore, it is important to establish the physiological regulation
of these epithelial Ca2+ transport pathways and the molecular
constituents driving these processes in an effort to treat or pre-
vent these diseases.
Movement of Ca2+ from the lumen of the intestine or renal
tubule into the blood can proceed via 1 of 2 pathways: transcellu-
lar (i.e., the ion moves through the cell) or paracellular (i.e., the
ion is transported between epithelial cells; Fig. 1) (Dimke et al.
2010, 2011). Transcellular Ca2+ absorption has been reported from
the duodenum, jejunum, cecum, and proximal colon in the intes-
tine as well as the renal distal convoluted and connecting tubules
(Table 1). This process occurs via apical influx of Ca2+ through
selective Ca2+ channels, largely predicted to be of the transient
receptor potential of the vanilloid subtype (either TRPV5 or 6).
Ca2+ is then shuttled to the basolateral membrane typically bound
to a Ca2+ binding protein, such as a calbindin, and then secreted
across the basolateral membrane via a Ca2+-dependent ATPase or
in exchange for Na+ by a Na+/Ca2+ exchanger (Hoenderop et al.
2005; Dimke et al. 2011). Transcellular Ca2+ absorption from the
renal tubule and the intestine is subject to intense regulation,
which has been the focus of a large amount of work and the topic
of a number of reviews (Hoenderop et al. 2005; Dimke et al. 2010,
2011).
Paracellular Ca2+ flux purportedly accounts for the majority of
Ca2+ absorption from the intestine and the renal tubule. Although
once thought to be a static unregulated process, emerging evi-
dence suggests otherwise. Similar to ion channels, tight junctions
are characterized by differing size and charge selectivity, which
controls the permeation of ions. Recent evidence has greatly
expanded our knowledge about the constituents of the tight
junction, which is composed of a family of proteins called the
claudins. These proteins determine the paracellular permeability
characteristics of a given epithelial type (Simon et al. 1999; Wilcox
et al. 2001; Konrad et al. 2006; Hou et al. 2008b; Markov et al. 2010)
and regulation of these pore-forming claudins can dramatically

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 Alexander et al.
Table 1. Type of transintestinal Ca2+ absorptiona reported by segment.
Transcellular Paracellular
Segment flux flux, passive
Duodenum Yes Yes
Jejunum Yesc Yes
Ileum No Yes
Cecum and proximal colon Yes Yes
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Distal colon Yes Yes
aTranscellular Ca2+ flux is always absorption, the direction of passive paracellular flux is dependent on the electrochemical gradient, and the direction of
secondarily active paracellular Ca2+ flux is segment dependent.
bThere is some debate as to whether there is secondarily active Ca2+ absorption from the duodenum. Decreased flux after the apical removal of glucose supports
absorption; however, both voltage clamp experiments and analysis of concentration dependence supports a slight asymmetry mediating secretion.
cTranscellular flux has been observed across the proximal jejunum of rats.
affect the paracellular permeability characteristics of the afore-
mentioned epithelia (Gong et al. 2012; Dimke et al. 2013; Gong and
Hou 2014). This review aims to summarize current knowledge of
Ca2+ transport occurring via the paracellular pathway in intestine
and kidney. In particular, we highlight the molecular constitu-
ents involved in setting the driving forces and permeability char-
acteristics that allow passive paracellular Ca2+ flux across renal
For personal use only.
and intestinal epithelia. Moreover, we describe how regulated
changes in specific tight junction proteins alter paracellular Ca2+
absorption from these epithelia.
Intestine
Daily dietary intake of Ca2+ in the Western diet should be ap-
proximately 1 g (Bailey et al. 2010), although a minority of this
(20%–40% of intake) is actually absorbed (Brine and Johnston 1955;
Nordin et al. 1979). Uptake of dietary Ca2+ from the intestinal
lumen can occur via active transcellular or passive paracellular
transport (Bronner 1998). Active transcellular Ca2+ flux is satura-
ble and predominates when dietary Ca2+ is low. This process
permits the uptake of Ca2+ into the enterocyte when low intralu-
minal Ca2+ concentrations are present (Bronner and Pansu 1999).
In contrast, absorption of Ca2+ via the passive paracellular path-
way requires a sufficiently high luminal Ca2+ concentration and,
hence, adequate dietary Ca2+ intake (Bronner and Pansu 1999); the
molecular details of both pathways are depicted in Fig. 2. The
small intestine is thought to be responsible for most Ca2+ uptake,
with the colon absorbing less than 10% under normal conditions
(Marcus and Lengemann 1962a, 1962b; Cramer 1965). The relative
amounts of Ca2+ taken up paracellularly in different intestinal
segments relate to several variables. The most important are the
intraluminal Ca2+ concentration, the solubility of Ca2+, the per-
meability of the tight junction, and sojourn time (Duflos et al.
1995). Sojourn time is the time that the intestinal content spends
transiting each segment. Combined, these factors determine the
paracellular transport rate of Ca2+ across a given intestinal seg-
ment. The sojourn time varies considerably between individual
segments, with very short transit times in the duodenum and
much longer times in the distal small intestine, cecum, and colon
(Marcus and Lengemann 1962b; Duflos et al. 1995). In rats fed a
high Ca2+ diet, sojourn times were measured to be 3 min in duo-
denum, 43 min in jejunum, 141 min in ileum, 92 min in cecum,
and 92 min in colon (Bronner and Pansu 1999). The dissimilarity
between sojourn times in segments of the intestine is reflected in
the total Ca2+ absorption measured, with 7%–8% of Ca2+ uptake
occurring from the duodenum, 4%–17% from the jejunum, and
65%–88% from the ileum (Cramer and Copp 1959; Marcus and
469
Paracellular flux,
secondarily active References
Yes, secretion b (Charoenphandhu et al. 2001, 2006; Karbach 1992;
Pansu et al. 1983; Tudpor et al. 2008)
Yes, secretion (Karbach 1992; Karbach and Feldmeier 1993;
Pansu et al. 1983; Rievaj et al. 2013;
Walling and Kimberg 1973)
Yes, secretion (Hu et al. 1993; Karbach 1992; Karbach and Feldmeier 1993;
Nellans and Kimberg 1979; Pansu et al. 1983;
Walling and Kimberg 1973)
Yes, absorption (Karbach 1992; Karbach and Feldmeier 1993;
Karbach and Rummel 1987; Kraidith et al. 2009;
Nellans and Goldsmith 1981; Rievaj et al. 2013)
Yes, secretion (Karbach et al. 1986)
Lengemann 1962a, 1962b; Cramer 1965; Wasserman 2004). Some
of these studies measured the disappearance of radiotracers from
the intestinal lumen and did not consider Ca2+ flux in the opposite
direction (i.e., secretion). Consequently, they likely underestimated
the relative contribution of more distal segments; radiotracer ab-
sorption from distal segments may be falsely underrepresented
due to proximal secretion. Significant distal intestinal Ca2+ ab-
sorption has also been observed in humans (Birge et al. 1969).
Passive paracellular intestinal Ca2+ absorption
Most intestinal Ca2+ absorption, on a normal Ca2+-containing
diet, is reported to occur via passive, or secondarily active, para-
cellular transport (Bronner 2003). This assumption is largely
based on in vivo experiments employing the ligated loop tech-
nique. The relationship between luminal Ca2+ concentration and
rate of Ca2+ absorption obtained by this method can be conceptu-
alized as the sum of 2 processes: a saturable component, which is
observed in the duodenum and to a lesser extent the jejunum, and
an unsaturable process with linear dependency observed in all
segments of the small intestine (Pansu et al. 1983). The saturable
process is assumed to represent active transcellular absorption,
whereas the unsaturable process is interpreted as passive paracel-
lular absorption (Bronner et al. 1986; Bronner 2003). However, this
interpretation is limited by a paucity of data supporting the as-
sumption that the entire unsaturable component occurs in a
paracellular fashion; that is, one may envisage transcellular
mechanisms that may not display saturable kinetics under the
experimental conditions (McCormick 2002; Kellett 2011). Further,
as the relative permeability for Ca2+ (per unit of intestinal length)
is similar for all segments of the small intestine (Pansu et al. 1983),
the amount and direction of passive paracellular Ca2+ flux would
be strongly dependent on the electrochemical gradient for Ca2+
across intestinal epithelium. The potential difference across the
whole length of the intestine is lumen negative (Geall and
Summerskill 1969). Measurements of the transepithelial potential
difference in vivo across the small intestine, regardless of seg-
ment, found a difference of approximately –5 mV (lumen nega-
tive), whereas a transepithelial voltage potential of approximately
–20 mV was measured across the large intestine (although mea-
surements of up to –60 mV have been reported) (Geall et al. 1969;
Geall and Summerskill 1969). Similar values are reported from
Ussing chamber studies, although these reports typically do not
exceed –20 mV even for large bowel preparations (Clarke 2009).
Consequently, the energy for paracellular Ca2+ absorption must
be derived from the chemical concentration gradient.
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 470 Biochem. Cell Biol. Vol. 92, 2014

Fig. 2. Intestinal transepithelial Ca2+ flux. The vectorial movement of Na+ creates an osmotic gradient for water reabsorption from the
intestine, which can either create a concentration gradient for Ca2+ to diffuse down (top junction) or drive paracellular Ca2+ flux via
convection/solvent drag (bottom junction). There is evidence supporting a role for NHE3 in paracellular Ca2+ flux across some intestinal
segments and sodium-glucose transport has also been implicated (Charoenphandhu et al. 2001). The claudins (CLDN) implicated in creating
paracellular Ca2+ permeability are depicted. In the duodenum and cecum, there is significant transcellular Ca2+ absorption. The players here
include apical entry via TRPV6, shuttling to the basolateral membrane via calbindin-D9K (Calb-D9K), and then efflux across the basolateral
membrane via the calcium pump, PMCA1b. Note, we have depicted a generic intestinal epithelium; however, in different parts of the
intestine, different pathways will function to a greater or lesser extent (please refer to the text and Table 1 for details).

paracellular diffusion
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CLDN CLDN
2 15?
Ca2+ Ca2+
CLDN CLDN
12 2
Glucose

SGLT1
+
Na Na+ a+
Na NaK
ATPase

Na+ K+
a+
Na
NHE3
H+
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CLDN CLDN
2+ 2 15?
Ca H2O H2O solvent drag
CLDN CLDN
12 2

Ca2+
Calb
D9K Ca2+
Ca2+
TRPV6 Ca2+ Ca2+ PMCA1b

transcellular flux

Employing the Nernst equation and assuming a free Ca2+ con-
centration in the blood of 1.2 mmol/L, one can calculate that the
concentration of free Ca2+ at the surface of the small bowel re-
quired to overcome a –5 mV transepithelial potential difference
must be at least 1.74 mmol/L. Yet, to our knowledge, the free Ca2+
concentration on the apical surface of intestinal epithelium has
not been investigated. The free Ca2+ concentration in the whole
lumen of different intestinal segments was found to be dependent
on the Ca2+ content of ingested food. When food with a “low” Ca2+
content (0.15% w/w) was ingested, the intraluminal free Ca2+ con-
centration was always observed lower than the concentration in
blood throughout the intestine (Sernka and Borle 1969). However,
when ingested Ca2+ content was increased to 1%–1.5%, an intralu-
minal concentration of 1.5–8 mmol/L was observed. For food
containing approximately 3% Ca2+, the intraluminal free Ca2+ con-
centration in the small intestine increased along its course and
peaked at approximately 10–45 mmol/L by the terminal ileum
(Cramer 1965; Duflos et al. 1995). This increase in the luminal Ca2+
concentration along the course of the intestine occurs because
of Na+ and, consequently, osmotically driven water absorption
(Cramer 1964, 1965). This secondary concentration gradient is one
purported mechanism connecting paracellular Ca2+ flux to water
absorption. The second is solvent drag, which is the movement
of Ca2+ together with paracellular water flow via convection
(Diamond and Bossert 1967; Pappenheimer and Reiss 1987; Larsen
et al. 2000). Distinguishing between a secondary concentration
gradient and solvent drag is not easily accomplished because both
are considered secondarily active processes and are, in principle,
able to move Ca2+ ions against the observed electrochemical
gradient. Further, the value of the transintestinal potential
difference and luminal concentration of Ca2+ detailed previously

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 Alexander et al.
are influenced by the methods employed and model systems stud-
ied and, therefore, should be considered estimates.
Evidence in support of secondary active paracellular
intestinal Ca2+ absorption
Evidence for the existence of secondary active paracellular Ca2+
flux is largely derived from Ussing chamber studies. This in vitro
technique benefits from being able to control both the electrical
and chemical transepithelial gradients. The relationship between
extracellular Ca2+ concentration in the apical compartment and
rate of Ca2+ flux from apical to basolateral side of the epithelium
reveals a saturable and a linear unsaturable component to total
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Ca2+ flux, likely reflecting transcellular and paracellular transport
routes, respectively. The unsaturable component contributes sig-
nificantly to total Ca2+ flux in all parts of the small and large
intestines (Karbach et al. 1986; Nellans 1990; Karbach 1992; Karbach
and Feldmeier 1993). These data obtained in Ussings chambers are
comparable to results obtained by the ligated loop technique de-
scribed previously. Ussing chamber experiments also permit the
measurement of Ca2+ flux in the presence of divergent voltage
clamps, permitting the separation of Ca2+ flux into either voltage-
dependent (presumably paracellular) or voltage-independent (pre-
sumably transcellular flux) components (Frizzell and Schultz 1972).
A detailed discussion of the merits of these different techniques is
beyond the scope of this review.
Importantly, fluxes assumed to be “paracellular” by the afore-
mentioned techniques are not always equivalent when measured
in opposite directions (i.e., apical to basolateral vs. basolateral to
apical) even in the absence of an electrochemical gradient. This
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difference favors paracellular Ca2+ absorption (i.e., higher apical
to basolateral than basolateral to apical flux) from the cecum
and ascending colon and paracellular Ca2+ secretion from all
segments of the small intestine and the descending colon. This net
paracellular Ca2+ movement has been attributed to solvent drag
(Nellans 1990; Karbach and Feldmeier 1993; Rievaj et al. 2013).
Because water flux is assumed (but rarely measured under the
same experimental conditions) to be in the apical to basolateral
direction, the term “anomalous solvent drag” was coined to ex-
plain paracellular Ca2+ secretion from the small intestine (Nellans
and Kimberg 1979).
However, data from Ussing chamber studies measure much
higher fluxes in the basolateral to apical direction than what is
generally assumed from in vivo studies. This led to the reporting
of net Ca2+ secretion in the jejunum and no net transport from the
duodenum when symmetrical Ca2+ concentrations were em-
ployed in Ussing chamber experiments under voltage clamp (i.e.,
in the absence of an electrochemical gradient) (Walling and
Kimberg 1973, 1975; Karbach and Feldmeier 1993; Rievaj et al.
2013). Although net Ca2+ secretion has also been observed during
in vivo studies, this always occurred down its electrochemical
gradient (Krawitt and Schedl 1968; Sernka and Borle 1969). We can
only speculate about the reasons for the discrepancy in measure-
ment between methodologies, with possible explanations includ-
ing selective damage to water absorptive cells during prolonged in
vitro measurements (Inagaki et al. 2005; Inagaki-Tachibana et al.
2008a, 2008b), the presence of substances in vivo which prevent
anomalous solvent drag such as bile salts (Hu et al. 1993), or the
presence of greater unstirred layers on the basolateral side of
epithelia leading to underestimates of apical to basolateral flux
in Ussing chamber studies, especially when performed on un-
stripped epithelium.
The other way to estimate the involvement of secondarily active
paracellular Ca2+ absorption is by altering the driving force of
water movement. All intestinal segments demonstrate both water
absorptive and water secretive properties. Under physiological
conditions, water absorption prevails. Water can move across an
epithelium via either a transcellular or paracellular pathway. Al-
though both mechanisms of water flux could generate a second-
471
ary concentration gradient for Ca2+ absorption, paracellular water
movement is necessary for solvent drag. However, the relative
contribution of each pathway to water absorption is unknown.
Although several aquaporins have been identified in the epithelia
of small and large intestines (Laforenza 2012), their role in trans-
epithelial water flux has not been examined with the exception of
aquaporin-4. This water channel plays a minor role in colonic
water absorption, as evinced by a knockout animal that has
mildly increased water content in defecated stool (Wang et al.
2000). Similarly, little is known about paracellular intestinal wa-
ter movement. Although the tight junction protein claudin-2
(Cldn2) has been implicated in mediating paracellular water flux
across a renal epithelial cell line (Rosenthal et al. 2010) and Cldn2 is
expressed in mouse intestine (Fujita et al. 2008), Cldn2 knockout
mice do not display a phenotype consistent with decreased intes-
tinal water absorption (Tamura et al. 2011).
Regardless of the pathway, water movement passively follows
the movement of ions (Curran and Macintosh 1962). Conse-
quently, water secretion has been attributed to electrogenic
anion secretion through apical anion channels, whereas water
absorption is coupled to epithelial Na+ uptake. The mecha-
nisms mediating Na+ uptake can be divided into electroneutral
and electrogenic transport. Electrogenic transport occurs via api-
cal Na+ channels expressed mainly in the distal colon (Garty and
Palmer 1997) and Na+/nutrient-linked cotransporters, in particu-
lar Na+-dependent glucose cotransporters (SGLTs) expressed in the
small intestine. Electroneutral Na+ absorption occurs through the
apically expressed Na+/H+ exchangers, where NHE3 appears to
play the predominant role in Na+ absorption from both the small
and large intestine (Schultheis et al. 1998; Gawenis et al. 2002). Na+
efflux occurs across the basolateral membrane via the Na+/K+
ATPase for both pathways. The exact mechanism coupling water
flux to sodium transport is still debated. Among multiple differ-
ent theories, the revised standing gradient model (Diamond and
Bossert 1967) and the Na+ recirculation theory, which is summa-
rized in detail in Larsen and Mobjerg (2006) and Larsen et al.
(2007), are probably the most developed. Both theories are based
on the observation that Na+/K+ ATPase is predominantly localized
to the lateral membrane of intestinal epithelial cells (DiBona and
Mills 1979). This lateral localization of the Na+/K+ ATPase and the
vastly different resistance between the tight junction and the
basal junction would generate a hyperosmotic and hyperbaric
lateral intercellular space, which would provide the conditions
for fluid uptake in the absence of an electrochemical gradient or
even against a concentration gradient. Additionally, the Na+ recir-
culation theory assumes influx of Na+ from the basolateral mem-
brane and hence “recirculation” of Na+. Although this would be
energetically unfavourable for a tissue, there is experimental
evidence consistent with the existence of this phenomenon
(Nedergaard et al. 1999).
Given the dependence of secondarily active paracellular intes-
tinal Ca2+ absorption on water flux, which is driven by intestinal
Na+ absorption, it can be hypothesized that molecules implicit to
intestinal Na+ absorption would also play a role in intestinal Ca2+
absorption. This was tested by removal of glucose from the muco-
sal side of intestinal epithelium, thereby inhibiting Na+-coupled
glucose transport by depriving the cotransporter of substrate
(Charoenphandhu et al. 2001). However, this method has been
criticized due to its effect on the polarity of the apical membrane
(Kellett 2011). To test whether electroneutral NHE3-mediated Na+
flux contributes to intestinal Ca2+ uptake, we measured intestinal
45Ca2+ uptake after gastric gavage in wild-type and Nhe3 knockout
mice (Pan et al. 2012). Importantly, Ca2+ uptake from the intestine
was reduced in Nhe3−/− mice. Direct measurements of Ca2+ flux
across duodenum and cecum (the 2 parts of intestine with known
net Ca2+ absorption) in Ussing chambers confirmed decreased lu-
minal to basolateral transepithelial Ca2+ flux (Pan et al. 2012;
Rievaj et al. 2013), confirming that NHE3 contributes to paracellu-
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 472
lar Ca2+ uptake. Finally, to avoid potential compensatory changes
resulting from an altered Ca2+ balance in Nhe3−/− mice, we con-
firmed the findings using a NHE3 inhibitor in cecum of wild-type
mice (Rievaj et al. 2013). Together these data support a role for
NHE3 in transepithelial intestinal Ca2+ absorption, likely via driv-
ing paracellular Ca2+ flux either via convection or by allowing
water removal and thereby generating a favourable concentration
gradient for Ca2+ to follow.
Molecules setting the paracellular permselectivity across
intestinal epithelia
For paracellular ion flux to occur, there must be both a driving
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force and a Ca2+-permeable pore. The discovery and investigation
of a family of tight junction proteins, termed claudins, has con-
tributed significantly to our understanding of paracellular pores.
Claudins are 4-pass transmembrane proteins with both the amino
and carboxy termini resident in the cytosol. They form homo-
meric and heteromeric interactions with one another in the same
tight junction and in the tight junction of the adjacent epithelial
cell. Although the structure of this complex is poorly understood,
it clearly plays a role in determining the permeability of the tight
junction (for recent reviews see Gunzel and Fromm (2012) and
Gunzel and Yu (2013)).
Evidence supports a role for Cldn2 and Cldn12 in creating
cation-permeable paracellular pores, hence permitting Ca2+ flux
between intestinal epithelial cells (Fujita et al. 2008). Cldn2, when
overexpressed in epithelial cell culture models, creates a paracel-
lular cation-selective pore permeable to Ca2+ (Furuse et al. 2001;
Amasheh et al. 2002; Fujita et al. 2008; Yu et al. 2009, 2010). Exam-
For personal use only.
ination of the Cldn2 knockout mouse confirmed that it forms a
paracellular cation-selective pore across the intestine and renal
proximal tubule (Muto et al. 2010; Tamura et al. 2011). However,
direct Ca2+ flux studies have not been reported from Cldn2−/− mice
to date. Overexpression and siRNA-mediated knockdown of Cldn12
in intestinal epithelial cells demonstrate that this claudin also
creates a cation-selective pore permeable to Ca2+ (Fujita et al.
2008). However, the phenotype of a Cldn12 knockout animal has
not been reported. Overexpression studies also demonstrate the
ability for Cldn15 to form a paracellular cation-selective pore
(Colegio et al. 2002; Van Itallie et al. 2003). Interestingly, deletion
of this gene in mice leads to the development of megaintestine
(Tamura et al. 2011). The knockout mice also display decreased
intestinal Na+ and water absorption, which appears exacerbated
in the Cldn2 and Cldn15 double knockout (Wada et al. 2013). Given
these findings, it is tempting to speculate that Cldn15 also contrib-
utes to the formation of cation-selective paracellular pores
permeable to Ca2+ along the course of the intestine. Again, direct
measurements of Ca2+ flux across the intestine of Cldn15 knockout
mice have not been reported.
The localization of Cldn2, Cldn12, and Cldn15 has been exam-
ined along the course of the mouse intestine (Fujita et al. 2006,
2008). Consistent with a role in paracellular transport, Cldn2 and
Cldn12 expression is greatest in the distal segments of the small
bowel. This expression pattern differs slightly in rats; however,
significant expression of both isoforms is seen in the distal small
bowel (Markov et al. 2010). Expression of Cldn15 in mice is greater
in more proximal intestinal segments, although there is signifi-
cant expression along the course of the bowel consistent with a
possible role in paracellular Ca2+ reabsorption (Fujita et al. 2006).
The expression of CLDN12 and CLDN15 has been observed along the
course of small and large bowel in humans, although CLDN2 has
only been reported in the small bowel as summarized in Lu et al.
(2013).
Regulation of paracellular Ca2+ absorption in intestine
The calciotropic hormone 1,25-dihydroxyvitamin D3 acts to in-
crease intestinal Ca2+ absorption. Much research has focused on
how 1,25-dihydroxyvitamin D3 increases the expression and activ-
Biochem. Cell Biol. Vol. 92, 2014
ity of channels and transporters that govern transcellular Ca2+
reabsorption as recently reviewed in Fleet and Schoch (2010) and
Christakos (2012). A role for 1,25-dihydroxyvitamin D3 in regulat-
ing paracellular Ca2+ flux has been debated; however, recent evi-
dence is consistent with 1,25-dihydroxyvitamin D3 also increasing
paracellular Ca2+ flux (Wasserman 2004; Fujita et al. 2008). In-line
with a role for 1,25-dihydroxyvitamin D3 in altering paracellular
Ca2+ flux, the expression of Cldn2 and Cldn12 was reduced in
vitamin D–receptor knockout mice (Fujita et al. 2008). Moreover,
treatment of the intestinal Caco-2 cell line with 1,25-dihydroxyvitamin
D3 increased Cldn2 and Cldn12 expression, and increased paracel-
lular Ca2+ flux (Fujita et al. 2008). Further, it has been speculated
that increased 1,25-dihydroxyvitamin D3–responsive paracellular
Ca2+ flux compensates, at least in part, for decreased transepithe-
lial Ca2+ flux in TRPV6 and calbindin-D9K double knockout mice
(Christakos et al. 2010). Although this discussion is consistent with
the possibility of claudin pores contributing to regulated paracel-
lular Ca2+ absorption from the intestine, much work still needs to
be done to firmly establish this. This includes direct measure-
ments of Ca2+ flux across the intestine of claudin-specific knock-
out animals.
Active transcellular Ca2+ absorption occurs from both the
duodenum and proximal large bowel
Active transcellular Ca2+ absorption is predominantly studied
in duodenum, whereas active paracellular Ca2+ absorption has
only been observed in the cecum and proximal colon. The cecum
and proximal colon are also sites of significant transcellular Ca2+
absorption. As in the duodenum, the same transcellular Ca2+ ab-
sorption machinery is present and measurements in Ussing
chambers have confirmed both significant transcellular and
secondarily active paracellular Ca2+ flux across these segments,
in rodents at least (Nellans and Goldsmith 1981; Karbach and
Feldmeier 1993; Kraidith et al. 2009; Zhang et al. 2010; Rievaj et al.
2013). Consistent with a role of proximal large bowel in Ca2+ ho-
meostasis, resection of the cecum in rats leads to decreased bone
mineral density secondary to enhanced trabecular bone resorp-
tion (Charoenphandhu et al. 2012; Jongwattanapisan et al. 2012).
These results are not likely directly translatable to humans given
the relatively larger size of the cecum in rodents vs. the cecum in
humans (Casteleyn et al. 2010). The role of the proximal large
bowel in Ca2+ homeostasis in humans has been relatively unex-
plored; we are aware of only a single study directly confirming
active transcellular flux from this segment after vitamin D3 ad-
ministration (Grinstead et al. 1984). Kinetic studies suggest that
under physiological conditions, there is a small but measurable
contribution of colon/cecum to total Ca2+ absorption (Barger-Lux
et al. 1989). Thus, the large intestine seems to provide a reserve,
which can be used in situations when absorption from the small
intestine is inadequate (Hylander et al. 1980, 1990; Abrams et al.
2007). Whether increased Ca2+ absorption from the cecum/colon
plays a role in situations of increased Ca2+ requirements, such as
childhood growth, pregnancy, lactation, or when Ca2+ intake is
reduced, remains to be determined. Given the frequency of co-
lonic resection, it would be important to determine the effect of
this procedure on Ca2+ homeostasis in humans.
Kidney
Ca2+ filtered by the kidney is reclaimed by the nephron to main-
tain serum levels within normal limits. Like the intestine, the
majority of Ca2+ is reabsorbed via passive paracellular processes.
The bulk, approximately 70% of filtered Ca2+, is reabsorbed in this
fashion from the proximal tubule and approximately 25% via pas-
sive paracellular fluxes from the thick ascending limb. The re-
mainder of Ca2+ reabsorption occurs via active transcellular flux
from the distal convoluted and connecting tubules. This latter
process appears to occur via an analogous process in the duode-
num and cecum. Here, TRPV5 channels mediate apical uptake of
Published by NRC Research Press
 Alexander et al.
Fig. 3. Proximal tubular Ca2+ reabsorption. The majority of Ca2+ flux across the proximal tubule occurs in a paracellular fashion, driven by
the transepithelial flux of sodium (Na+), which in turn drives water (H2O) flux. Water removal from the proximal tubular lumen could act to
concentrate Ca2+ providing a concentration gradient (bottom junction) or paracellular water movement may drive Ca2+ flux via convection
(i.e., solvent drag) (top junction). The epithelial Na+/H+ exchanger (NHE3) is the primary Na+ influx pathway, whose activity is driven by the
Na+/K+-ATPase (NaK ATPase). Note that two-thirds of water flux across the proximal tubule occurs in a transcellular fashion mediated by
aquaporin-1 pores (not depicted here). There is also significant paracellular Na+ flux.
CLDN
2
2+
Ca H O H2O
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2
CLDN
2
Na+
NHE3
H+
CLDN
2
Ca2+ Ca2+
CLDN
For personal use only.
the ion, with calbindin-D28K buffering intracellular Ca2+ and ex-
trusion across the basolateral membrane occurs via the plasma
membrane Ca2+-dependent ATPase and the Na+/Ca2+ exchanger
(NCX) (Hoenderop et al. 2005; Dimke et al. 2010, 2011). The remain-
der of the review will focus on the mechanisms mediating Ca2+
flux across the proximal tubule and thick ascending limb.
Proximal tubule
Most (approximately 55%–60%) Ca2+ filtered by the glomerulus
is reabsorbed by the proximal convoluted tubule (Duarte and
Watson 1967; Suki 1979). A further 10% of filtered Ca2+ is reab-
sorbed from the straight portion of the proximal tubule (Suki
1979; Seldin 1999). Therefore, most (i.e., at least two-thirds) of Ca2+
in the ultrafiltrate is reclaimed by the proximal tubule. Evidence
from micropuncture studies performed on multiple species is
consistent with Ca2+ uptake from this nephron segment occur-
ring in a passive paracellular fashion (Duarte and Watson 1967;
Morel et al. 1969; Sutton and Dirks 1975). The ratio of Ca2+ concen-
tration in tubular fluid to the ultrafiltrate, [(TF/UF)Ca], is consis-
tently reported to be between 1.0 and 1.2. This ratio was also
consistently similar, if not identical, to the ratio for Na+, inferring
that active Na+ reabsorption provides the driving force for Ca2+
reabsorption. Consistent with this, attempts to uncouple proxi-
mal Na+ reabsorption from Ca2+ reabsorption by the administra-
tion of parathyroid hormone, furosemide, hydrochlorothiazide,
or acetazolamide were unsuccessful (Agus et al. 1973; Beck and
Goldberg 1973; Edwards et al. 1973; Sutton and Dirks 1975). That
Ca2+ reabsorption from the proximal tubule occurs in a paracel-
lular fashion is consistent with microperfusion studies of the
proximal tubule in the absence of an electrochemical gradient,
which demonstrate predominant passive paracellular flux (Ng
et al. 1984). These authors did detect a small (<10%) active trans-
cellular component as well. Consistent with this, the paracellular
shunt in the proximal tubule is very permeable to Ca2+, thereby
facilitating transport of the ion (Murayama et al. 1972; Sutton and
473
Na+
Na+
NaK
ATPase
K+
Dirks 1975). Recent studies have begun to dissect out the molecu-
lar components mediating passive paracellular flux of Ca2+ across
the proximal tubule and their findings are outlined subsequently.
The generation and characterization of Nhe3 knockout mice
have implicated this transporter in the bulk of Na+ and, conse-
quently, osmotically driven water flux across the proximal tubule
(and bowel as described previously) (Schultheis et al. 1998; Lorenz
et al. 1999). Both microperfusion and micropuncture studies dem-
onstrate significantly reduced Na+ and water reabsorption from
the proximal tubule of Nhe3 knockout mice relative to wild-type
littermates (Schultheis et al. 1998). These data and the known
dependence of proximal tubular Ca2+ reabsorption on Na+ and
water reabsorption led us to examine whether NHE3 drives prox-
imal paracellular Ca2+ reabsorption. To this end, we looked at
paracellular Ca2+ flux across a proximal tubular cell culture model
where NHE3 was overexpressed and found that this doubled Ca2+
flux (Pan et al. 2012). That NHE3 activity per se was required for
this effect was confirmed by inhibiting Na+/H+ exchange with the
removal of Na+. Examination of the Nhe3 null mouse revealed
significantly increased fractional excretion of Ca2+ consistent
with decreased proximal Ca2+ reabsorption (Pan et al. 2012), al-
though specific studies of Ca2+ flux across the proximal tubule
were not made. The proposed role of NHE3 in proximal tubular
Ca2+ reabsorption is depicted in Fig. 3. Ultimately, increased renal
excretion of Ca2+ and decreased intestinal Ca2+ uptake led to
poorly mineralized bones in the knockout animals (Pan et al.
2012).
As observed in the intestine, passive paracellular Ca2+ flux de-
pends not only on a driving force, but also on a “leaky” tight
junction permeable to Ca2+. Given the known role and function of
the claudin family of tight junction proteins, some combination
of claudins in the proximal tubule likely confer the leaky perme-
ability characteristics of this nephron segment permitting Ca2+
flux. Thus far, only Cldn2, Cldn10a, and Cldn17 have been identi-
Published by NRC Research Press
 474
fied in the proximal tubule of adult animals (Enck et al. 2001;
Kiuchi-Saishin et al. 2002; Gunzel et al. 2009b; Krug et al. 2012).
Cldn2 has been studied extensively in cell culture models, where
it consistently forms a cation-selective pore (Furuse et al. 2001;
Amasheh et al. 2002; Yu et al. 2009). Moreover, detailed microper-
fusion studies of proximal tubules from Cldn2 knockout mice con-
firm the role of Cldn2 in generating a cation-selective pore across
this tissue in vivo (Muto et al. 2010). Although direct measure-
ments of Ca2+ flux across the proximal tubule of Cldn2 knockout
mice have not been made, the lack of expression of this claudin
from more distal segments in kidney combined with data confirm-
ing its selective permeability to cations and the presence of hyper-
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calciuria in Cldn2 knockout mice (Muto et al. 2010) implicates this
isoform in mediating paracellular Ca2+ flux across the proximal tu-
bule. However, it should be noted that in Cldn2-deficient mice, the
altered structure of the tight junction causes reduced reabsorption
of Na+, Cl–, and water (Muto et al. 2010). Consequently it cannot be
excluded that urinary Ca2+ loss in these animals is due to reduced
solvent drag and (or) diminished gradient formation because these
cannot be uncoupled. In fact, changes in Na+ and water transport
from the PT correlate with changes in Ca2+ transport (Agus et al. 1973;
Beck and Goldberg 1973; Edwards et al. 1973; Sutton and Dirks 1975).
In cell culture, Cldn10a and Cldn17 form anion-selective pores
(Van Itallie et al. 2006; Krug et al. 2012). Consequently, they are
unlikely to form Ca2+-selective pores in the proximal tubule. How-
ever, altering the relative expression of these isoforms in propor-
tion to the amount of Cldn2 or another cation-selective isoform
may regulate paracellular Ca2+ flux across the proximal tubule. A
detailed examination of the expression of all claudins in the prox-
For personal use only.
imal tubule has not been completed. Consequently, it is possible
that other cation-selective claudins may be expressed in this seg-
ment and contribute to the formation of a paracellular Ca2+ pore.
Cldn12 expression in the proximal tubule has not been examined;
however, given that it is expressed in a proximal tubule cell cul-
ture model (Borovac et al. 2012) and its purported role in paracel-
lular Ca2+ flux across the intestine (Fujita et al. 2008), it is
tempting to speculate that it may perform a similar function in
the proximal tubule.
Early postnatally, the proximal tubule undergoes developmen-
tal changes and the ability to reabsorb a number of substrates,
including Ca2+, increases with age (Lelievre-Pegorier et al. 1983).
There is increasing NHE3 expression coincident with increased
Ca2+ reabsorption (Becker et al. 2007), perhaps contributing to an
increased driving force. However, there are also changes in prox-
imal tubule claudin expression. Specifically, there is significant
Cldn6 and Cldn9 expression postnatally, which decreases with age
and is absent in adult animals (Abuazza et al. 2006). Both of these
claudins form cation barriers (Sas et al. 2008). Perhaps their re-
duced expression explains some of the increased ability of the
proximal tubule to reabsorb Ca2+ with age. It is important to note
that proximal tubule Mg2+ reabsorption decreases concurrently
during development, so the age-dependent alterations in perme-
ability characteristics likely reflect a more complicated change
within the paracellular pore (Lelievre-Pegorier et al. 1983).
Thick ascending limb of Henle's loop
The thick ascending limb of Henle's loop (TAL) plays an impor-
tant role in the reabsorption of filtered Ca2+. Detailed micropunc-
ture measurements estimate that 20%–25% of Ca2+ in the filtrate is
reclaimed by the TAL (Lassiter et al. 1963; Suki 1979). Ca2+ reab-
sorption across the TAL epithelium occurs mainly by passive para-
cellular transport. This is largely dependent on a lumen-positive
transepithelial voltage gradient acting to drive Ca2+ through the
paracellular shunt. This lumen-positive transepithelial voltage
gradient is generated by 2 interdependent mechanisms as out-
lined in Fig. 4. NKCC2-dependent furosemide-sensitive cotrans-
port drives 1 Na+, 1 K+, and 2 Cl– ions across the apical membrane.
This inward transport of monovalent ions is reliant on the apical
Biochem. Cell Biol. Vol. 92, 2014
secretion of K+ by renal outer medullary potassium (ROMK) chan-
nels and its subsequent recycling because K+ is limiting. The K+
efflux back into the lumen provides part of the lumen-positive
potential difference. The inwardly transported Na+ and the 2 Cl–
ions exit basolaterally via the Na+/K+-ATPase and CLC-Kb channels,
respectively. Collectively, these transport processes create a
lumen-positive voltage gradient of approximately 5–10 mV
(Greger and Schlatter 1983; Greger and Velazquez 1987). In addi-
tion to this, significant paracellular backflux of Na+ is thought to
occur when the transepithelial Na+ concentration is reversed be-
cause filtrate ascends toward the top of the TAL (i.e., when the
luminal Na+ concentration is lower than that of the interstitium).
This backflux of interstitial Na+ generates a diffusion potential
thought to amplify the voltage gradient to reach values approach-
ing +30 mV (Rocha and Kokko 1973; Greger 1981; Mandon et al.
1993; Hou et al. 2005). Consequently, the cortical segment of the
TAL appears to be the main site of paracellular Ca2+ transport
(Suki et al. 1980; Di Stefano et al. 1990; De Rouffignac et al. 1991).
The lumen-positive transepithelial voltage gradient is critical in
establishing the driving force for Ca2+ transport across the TAL.
Furosemide, a pharmacological blocker of NKCC2, effectively dis-
sipates the lumen-positive potential difference across the seg-
ment, which potently inhibits transepithelial Ca2+ transport
across the TAL segment (Burg 1976; Quamme 1981; Good et al.
1984; Di Stefano et al. 1993; Deschenes et al. 2001).
The paracellular pathway permitting transport of Ca2+ across
the epithelium of the cortical TAL shows cation selectivity (Greger
1981; Di Stefano et al. 1988). The selectivity of this shunt is likely
determined by the combined interactions between specific clau-
dins. Cldn16 (previously referred to as paracellin-1) was the first
claudin identified to play a role in determining the selectivity
characteristics of the TAL tight junction. The CLDN16 gene was
identified as a causative gene responsible for the phenotype of
familial hypomagnesemia with hypercalciuria and nephrocalci-
nosis (FHHNC) (Simon et al. 1999). Individuals with FHHNC pres-
ent with severe hypomagnesemia due to renal wasting, which is
not surprising because the TAL also takes up a large portion
(⬃60%) of filtered Mg2+ via the paracellular pathway (de
Rouffignac et al. 1983; Simon et al. 1999). In-line with a common
paracellular shunt for Ca2+ and Mg2+ in the TAL, FHHNC patients
also display hypercalciuria and nephrocalcinosis, which likely
promotes renal insufficiency. However, FHHNC patients tend to
maintain serum Ca2+ within normal limits (Michelis et al. 1972;
Praga et al. 1995). Another group of FHHNC patients have been
described with a similar phenotype, but present with more severe
ocular symptoms (Konrad et al. 2006). Mutations in the CLDN19
gene were shown to be the molecular cause of their disease.
Importantly, both Cldn16 and Cldn19 localize to the TAL and
distal convoluted tubule segments of the kidney (Simon et al.
1999; Kiuchi-Saishin et al. 2002; Konrad et al. 2006). The majority
of data on the ion selectivity of Cldn16 and Cldn19 are derived
from cell model experiments. It should be noted that data ob-
tained from these in vitro studies utilizing different cell model
systems to express Cldn16 and Cldn19 differ substantially, as re-
viewed in detail by Gunzel and Yu (2008). These differences may
be due to several factors, but are most likely explained by the cell
line utilized. Different cell types form monolayers with widely
varying transepithelial resistances and differing cation/anion se-
lectivity in their basal state (Ikari et al. 2004; Hou et al. 2005,
Kausalya et al. 2006; Angelow et al. 2007; 2008a, 2008b; Gunzel
et al. 2009a). Such variability is due, in part, to the endogenous
expression of various claudin genes. Importantly, transfection of
an exogenous claudin can affect the expression of endogenous
claudins (Gunzel et al. 2009b; Dimke et al. 2013). This may help
explain the conflicting data obtained when evaluating the effect
of overexpression of Cldn16 or Cldn19 on divalent cation perme-
ation (Ikari et al. 2004; Hou et al. 2005, 2008a, 2008b; Kausalya
et al. 2006; Angelow et al. 2007; Gunzel et al. 2009a). In general, in
Published by NRC Research Press
 Alexander et al. 475

Fig. 4. The Ca2+-sensing receptor claudin-14 axis. Top panel: Thick ascending limb Ca2+ reabsorption under conditions of normocalcemia.
Asymmetrical ion fluxes across the TAL generates a lumen-positive potential difference which drives paracellular Ca2+ flux. The paracellular
pore is created by claudin-16 (CLDN16) and claudin-19 (CLDN19). Bottom panel: When hypercalcemia ensues, more Ca2+ is available to activate
the Ca2+-sensing receptor (CaSR), which relays cellular signalling–increasing claudin-14 (CLDN14) expression. CLDN14 then localizes to the tight
junction in the TAL, where it blocks paracellular Ca2+ flux, thereby promoting calciuria. Importantly, CLDN14 also prevents the backflux of Na+.
NKCC2, furosemide-sensitive Na+, K+, 2Cl– cotransporter; ROMK, renal outer medulla K+ channel; ClC-Kb, Cl– channel Kb; NaK ATPase,
Na+/K+-ATPase.

Normocalcemia
CLDN CLDN
16 19
Na+
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CLDN CLDN
Na+
16 19
Na+
NaK
ROMK
K+ ATPase
K+
CLCKB
Na+ Cl-
NKCC2
K+
2Cl- CaSR

CLDN CLDN
2+ 16 19
Ca
For personal use only.

CLDN CLDN

+ 16 19
Vte

Hypercalcemia
CLDN CLDN CLDN
16 19
2+ 14
Ca
CLDN CLDN CLDN Na+
14 16 19 +
Na
NaK
ATPase
ROMK
K+ K+

CLCKB
Na+ Cl-
NKCC2
K+
2Cl- CaSR Ca2+
CLDN CLDN CLDN
Ca2+ 14 16 19

CLDN CLDN CLDN

14 16 19
+ Vte

vitro overexpression studies found that divalent cation perme-
ation was not as severely disturbed as would be expected from
mutations causing FHHNC. Studies done utilizing the proximal
tubular LLC-PK1 line found that Cldn16 expression increases the
permeability for cations, especially for Na+ (Hou et al. 2005). In
contrast, the overexpression of Cldn19 leads to a reduction in Cl–
permeation, while leaving cation permeability unaltered (Hou
et al. 2008a). In LLC-PK1 cells, combined expression of the 2 clau-
dins promotes the formation of a cation-selective pore, with sim-
ilar permeability characteristics to that of the TAL (Greger 1981; Di
Stefano et al. 1988; Hou et al. 2008a). Based on these observations,
it is likely that FHHNC mutations reduce either cation selectivity
or increase anion permeation of the paracellular shunt in the TAL.
Both types of mutations would ultimately dissipate the backflux
of Na+ and, hence, prevent augmentation of the lumen positive
diffusion voltage, thereby reducing the driving force for divalent
cation flux (Hou et al. 2005, 2008a). Critical to the validity of this
permeability model is the observation that FHHNC mutations in

Published by NRC Research Press

 476
Cldn16 or Cldn19, largely abolish the cation selectivity of Cldn16
or reduce the ability of Cldn19 to block anion permeation (Hou
et al. 2005, 2008a).
Two transgenic mice models of Cldn16 deficiency have been
generated, one utilized RNA interference to knockdown Cldn16
gene expression (Hou et al. 2007), whereas the other employed
standard targeted transgenic strategies for genomic deletion of
the gene (Will et al. 2010). Although variability exists between the
2 animal models, both display hypercalciuria and hypomag-
nesemia. It is of relevance to note that electrophysiological mea-
surements in microperfused tubules differ substantially between
the models. Knockdown of Cldn16 shows reduced cation selectivity
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of the paracellular shunt in the TAL segment (i.e., reduced PNa/PCl),
whereas the permeability characteristics between different cat-
ions remain unaffected (Hou et al. 2007). This is in contrast to
Cldn16 knockout mice, which have an intact PNa/PCl ratio, but ex-
hibit decreased PCa/PNa and PMg/PNa permeability ratios (Will et al.
2010). In essence, the Cldn16 knockout mice have a reduced per-
meability to divalent cations over that of Na+, but in contrast to
the Cldn16 knockdown mice, display no change in permeability of
Na+ over that of Cl–. This could indicate that changes in these
claudins not only affect backflux of Na+ via the paracellular shunt
in TAL, but may also directly affect the permeation of divalent
cations under certain conditions. In fact, the exact composition of
claudins within an epithelium is critical in determining the per-
meability characteristics of the epithelium. With the realization
that the claudin composition of the TAL is difficult to mimic
within cell lines and that changes in the composition leads to
changes in the abundance of other claudins (Dimke et al. 2013;
For personal use only.
Gunzel et al. 2009b), more studies are needed to definitively
answer how the interaction between the different claudins in the
TAL sets the permselectivity of the paracellular shunt in this seg-
ment.
Although it is likely that several other claudins in addition to
Cldn16 and Cldn19 are expressed in the TAL, only the functions of
Cldn10 and Cldn14 have been established (Breiderhoff et al. 2012;
Gong et al. 2012; Dimke et al. 2013; Gong and Hou 2014). Deletion
of Cldn10 specifically from the TAL produces an interesting pheno-
type. The mice develop hypermagnesemia and increased Mg2+
reabsorption, whereas Ca2+ balance is less affected. The mice
show lower serum Ca2+ concentrations and slightly reduced tubu-
lar excretion of Ca2+. Notably, the mice develop nephrocalcinosis
in the form of extensive Ca2+ deposits in the renal medulla
(Breiderhoff et al. 2012). In isolated microperfused TAL segments,
a lack of Cldn10 promotes an increase in the lumen-positive volt-
age gradient, whereas the short circuit current remains unaf-
fected, suggesting that only paracellular permeability is affected.
In fact, a 4-fold decrease was observed in the permeability of Na+
over that of Cl– (PNa/PCl). However, the permeability of divalent
cations over that of Na+ was markedly increased, suggesting a
functional change within the permeability filter of the shunt
(Breiderhoff et al. 2012). The reduced shunt permeability to Na+
may be related to a 20-fold increase in the expression of the pore-
blocking Cldn14; however, this does not adequately explain the
increased permeability to divalent cations over Na+ because
Cldn14 blocks both divalent and monovalent cation flux (Gong
et al. 2012; Dimke et al. 2013; Gong and Hou 2014). The regulation
and function of Cldn14 is described in detail in the subsequent
sections.
Paracellular transport of divalent ions across the TAL is under
intense hormonal regulation. Thus, these mechanisms act to reg-
ulate paracellular flux via acute and chronic pathways. Early stud-
ies demonstrated that acute application of PTH and other
calciotropic hormones elicited a cAMP-dependent increase in elec-
trolyte transport across the TAL (Di Stefano et al. 1990; Wittner
et al. 1993). Notably, it was reported that PTH produced an acute
change in the permeability of the paracellular pathway because
the increased transport of divalent ions surpassed the change
Biochem. Cell Biol. Vol. 92, 2014
in the lumen-positive voltage (Wittner et al. 1993). A phos-
phorylation site in Cldn16 exists at Ser217. In Madin-Darby canine
kidney cells (MDCK), this site is a substrate for protein kinase A
(PKA). The phosphorylated form of Cldn16 localizes to the tight
junction, whereas the dephosphorylated form is targeted for lys-
osomal degradation (Ikari et al. 2006). Thus, changes in cellular
cAMP production within the TAL may rapidly affect the localiza-
tion and expression of CLDN16. Another key regulator of cAMP
production in the TAL is the Ca2+-sensing receptor (CaSR), which
can effectively inhibit up to 90% of hormone-stimulated cAMP
production (Desfleurs et al. 1998). The protein is located in the
basolateral membrane, where it responds to changes in systemic
Ca2+ concentrations and relays the signal to amend transport
across the TAL (Vargas-Poussou et al. 2002; Egbuna and Brown
2008). Detailed physiological measurements of Ca2+ transport
across microperfused TAL tubules demonstrate that CaSR stimu-
lation specifically inhibits Ca2+ flux across the segment, without
affecting the absorption of Na+ or Cl– and consequently the
lumen-positive voltage (Desfleurs et al. 1998; Motoyama and
Friedman 2002; Loupy et al. 2012). In Cldn16-transfected MDCK
cells, CaSR activation decreases the amount of phosphorylated
Cldn16, diverting the protein to the lysosomal compartment,
likely due to reduced cAMP levels and decreased PKA activity
(Ikari et al. 2008). Whether this occurs in vivo remains to be de-
termined. Other posttranslational mechanisms likely exist to
acutely regulate TAL claudins and the paracellular permeability of
the segment.
Using thyroidectomized and parathyroidectomized rats re-
supplemented with l-thyroxine and PTH, Loupy et al. (2012) were
able to show that chronic CaSR antagonism lead to significantly
higher renal tubular Ca2+ reabsorption, which was not dependent
on circulating PTH levels. The effects were likely of renal origin
because CaSR antagonism had the same effect on systemic Ca2+
concentrations after pretreatment with bisphosphonates. Simi-
larly, selective ablation of the Casr gene in kidney using the Six2-
CRE driver line also showed significantly lower urinary excretion
of Ca2+ (Toka et al. 2012). Thus, CaSR expression in kidney plays an
important role in regulating the urinary excretion of Ca2+. CaSR
activation has recently been shown to greatly stimulate the ex-
pression of Cldn14 within the TAL (Gong et al. 2012; Dimke et al.
2013; Gong and Hou 2014). A potential role for Cldn14 in regulat-
ing Ca2+ balance was originally indicated following the results of
a large genome-wide association study. Here it was found that
single nucleotide polymorphisms (SNPs) in the human CLDN14
gene associated with kidney stones and lower bone mineral den-
sity (Thorleifsson et al. 2009). However, deleterious mutations in
the human CLDN14 gene were not associated with changes in Ca2+
balance; rather, these individuals presented with nonsyndromic
deafness (Wilcox et al. 2001). Thus, the mechanism responsible for
the generation of kidney stones by SNPs in the CLDN14 gene re-
mained elusive. Cldn14 was subsequently shown to localize to
the TAL and respond to changes in serum Ca2+ (Gong et al.
2012). In fact, both increased dietary Ca2+ intake and 1,25-
dihydroxyvitamin D3 stimulated hypercalcemia markedly increased
expression of Cldn14 in kidney (Gong et al. 2012; Dimke et al. 2013).
This regulation was found to be dependent on the CaSR (Gong
et al. 2012; Dimke et al. 2013). We found that chronic activation of
the CaSR using the calcimimetic, Cinacalcet, led to a 40-fold in-
crease in Cldn14 gene expression (Dimke et al. 2013). However, the
effect of pharmacological activation of the CaSR is rapid, with
3-fold increased Cldn14 expression observed as early as 2 h after
oral gavage (Gong and Hou 2014). Moreover, after genetic ablation
of the Casr in kidney, the expression of cldn14 was significantly
reduced (Toka et al. 2012). In renal cell culture models, Cldn14
physically interacts with Cldn16 (Gong et al. 2012). In LLC-PK1 cells,
opossum kidney cells, and MDCK cells, Cldn14/CLDN14 overexpres-
sion provided specific permeability characteristics to the monolayer.
A decrease was observed in both the permeability of Na+ over that of
Published by NRC Research Press
 Alexander et al.
Cl– (PNa/PCl) as well as the permeability of Ca2+ (PCa), suggesting that
Cldn14 acts as a nonselective cation barrier (Ben-Yosef et al. 2003;
Gong et al. 2012; Dimke et al. 2013). Based on these permeability
characteristics, it seems that Cldn14 functions to reduce both the
backflux of Na+ and hence the lumen-positive voltage gradient in the
cortical TAL, in addition to blocking permeation of Ca2+ directly
(Fig. 4). The regulation of Cldn14 by the CaSR might be critical in
conditions of hypercalcemia because the luminal Ca2+ concentration
in the TAL increases substantially and would otherwise allow unin-
tended paracellular reabsorption of Ca2+, based on changes in the
chemical gradient (Dimke et al. 2013). Genetic models of altered
Cldn14 expression support these observations. In fact, when Cldn14-
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by San Francisco (UCSF) on 12/02/14
deficient mice are placed on a high Ca2+ diet, the fractional excretion
of divalent cations are significantly reduced, in-line with a defect in
blocking Ca2+ permeation of the shunt across the TAL (Gong et al.
2012). Recently, a new mouse model was generated that overex-
pressed the Cldn14 protein specifically in the TAL driven by the
Tamm-Horsfall promoter. In-line with previous publications, overex-
pression of Cldn14 in the TAL markedly increased the fractional ex-
cretion of divalent cations (Gong and Hou 2014).
Summary/future directions
Given that altered Ca2+ homeostasis can lead to cardiovascular
and neurological complications in addition to changes in the den-
sity of bone, understanding how Ca2+ is absorbed from the gut and
renal tubule is prerequisite to adequately treating and preventing
these sequelae. A common manifestation of altered epithelial
Ca2+ handling is hypercalciuria and kidney stone formation.
Hypercalciuria may be caused by increased intestinal absorption
For personal use only.
of Ca2+, an augmented release of Ca2+ from bone, by dysregulation
of renal transport pathways, or a combination thereof. We have
recently acquired a preliminary understanding of how Ca2+ is
reabsorbed from the TAL in normal physiological conditions;
however, the extent to which this mechanism is perturbed in
kidney stone formers remains to be completely defined. Given the
genome-wide association study (GWAS) implicating CLDN14 in kid-
ney stone formation, it seems reasonable to assume that those
individuals somehow inappropriately upregulate CLDN14 expres-
sion. There is also much work to be done on the proximal tubule.
Specifically, the claudins involved in Ca2+ reabsorption need to be
identified and their potential regulation understood. To this end,
micropuncture and (or) microperfusion studies examining the
flux of Ca2+ across the proximal tubule in claudin-specific knock-
out mice will be beneficial. Further, given the lack of direct mea-
surements of Ca2+ flux across the intestine of Cldn2 and Cldn15
knockout mice, this seems a logical area to explore in the short
term. Moreover, the generation and characterization of a Cldn12
knockout animal will provide clarity into its role in intestinal and
renal tubular Ca2+ reabsorption. Finally, there remain many ques-
tions with respect to paracellular Ca2+ flux across the bowel. Is
there significant backflux (i.e., serosal to mucosal Ca2+ movement)
as part of the recirculation of Ca2+ across intestinal epithelium
(e.g., between meals or when fasting)? If yes, what is the role of the
cecum/colon in this process? How exactly is the paracellular path-
way regulated? Is there any transcellular Ca2+ absorption from the
ileum? What is the role of solvent drag in paracellular Ca2+ move-
ment in vivo? What is the effect of proximal large bowel resection/
disease on Ca2+ homeostasis in humans? Answers to these questions
will aid in maintaining a positive Ca2+ balance in both health and
disease.
Acknowledgements
The laboratory of R.T. Alexander is supported by funding from
the Kidney Foundation of Canada, the Women and Children's
Health Research Institute, and the Canadian Institute of Health
Research (CIHR). A Clinician Scientist Award from CIHR and an
Alberta Innovates Health Solutions Clinical Investigator Award
477
supports Dr. R.T. Alexander. The laboratory of H. Dimke is funded
by the Novo Nordisk Foundation, the Carlsberg Foundation, and
the A.P. Møller Foundation for the Advancement of Medical
Science.
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