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CLAUDIN-8 MODULATES PARACELLULAR PERMEABILITY TO ACIDIC AND BASIC IONS IN MDCK II CELLS
1
CLAUDIN-8 MODULATES PARACELLULAR PERMEABILITY TO ACIDIC AND
BASIC IONS IN MDCK II CELLS
Susanne Angelow
1,3
, Kwang-Jin Kim
2,3,4
and Alan S. L. Yu
1,3
Divisions of Nephrology
1
and Pulmonary and Critical Care Medicine
2
, Departments of Medicine and
Physiology and Biophysics
3
, and the Will Rogers Institute Pulmonary Research Center
4
, University of
Southern California Keck School of Medicine, Los Angeles, California 90033
Running title: Claudin-8 and H
+
permeability
Please address correspondence to:
Alan S. L. Yu
University of Southern California Keck School of Medicine
Division of Nephrology
2025 Zonal Avenue, RMR 406
Los Angeles, CA 90033
Tel: (323) 442 1331
Fax: (323) 442 3093
Email: alanyu@usc.edu

Physiology in Press; published online on December 1, 2005 as 10.1113/jphysiol.2005.099135 2
ABSTRACT
Renal net acid excretion requires tubular reabsorption of filtered bicarbonate, followed by secretion of
protons and ammonium in the collecting duct, generating steep transtubular gradients for these ions. To
prevent passive backleak of these ions, the tight junctions in the collecting duct must be highly
impermeable to these ions. We previously generated an MDCK II cell line with inducible expression of
claudin-8, a tight junction protein expressed in the collecting duct. In these cells, claudin-8 was shown to
function as a paracellular barrier to alkali metal and divalent cations. We have now used this model to
test the hypothesis that claudin-8 also functions as a paracellular barrier to acidic or basic ions involved
in renal acid excretion. We developed a series of precise and unbiased methods, based on a combination
of diffusion potential, short-circuit current, and pH-stat measurements, to estimate paracellular
permeability to protons, ammonium and bicarbonate in MDCK II cells. We found that under control
conditions (i.e. in the absence of claudin-8), these cells are highly permeable to the acidic and basic ions
tested. Interestingly, proton permeation exhibited an unusually low activation energy similar to that in
bulk solution. This suggests that paracellular proton transfer may occur by a Grotthuss mechanism,
implying that the paracellular pores are sufficiently wide that they can accommodate water molecules in
a freely mobile state. Induction of claudin-8 expression reduces permeability not only to protons, but
also to ammonium and bicarbonate. We conclude that claudin-8 likely functions to limit passive leak of
these three ions via paracellular routes, thereby playing a permissive role in urinary net acid excretion. 3
INTRODUCTION
The kidney plays an important role in acid-base balance by regulating urinary net acid excretion. This
involves two steps (Hamm, 2004). First, filtered HCO
3
-
is reabsorbed, mainly in the proximal tubule and
loop of Henle. Second, H
+
and NH
4
+
are actively secreted in the collecting duct. These active,
transcellular transport processes generate large transtubular concentration gradients. To prevent passive
backleak and dissipation of these gradients, the entire collecting duct, including the tight junctions, must
be highly impermeable to these ions. Defects in collecting duct ion permeability can impair acid
excretion and cause metabolic acidosis (Batlle & Flores, 1996; Zawadzki, 1998).
The rate-limiting step in paracellular permeability in renal tubular epithelium occurs at the tight
junction. Recent studies indicate that a family of transmembrane tight junction proteins known as
claudins form paracellular pores (Tsukita & Furuse, 2000; Schneeberger & Lynch, 2004; Van Itallie &
Anderson, 2004). Overexpression studies in epithelial cell lines have begun to delineate the permeability
properties of individual isoforms (McCarthy et al., 2000; Furuse et al., 2001; Van Itallie et al., 2001;
Amasheh et al., 2002; Van Itallie et al., 2003; Yu et al., 2003; Alexandre et al., 2005). Their selectivity
is in part determined by charged residues on their first extracellular domain (Colegio et al., 2002; Van
Itallie et al., 2003). By excluding certain solutes, claudins also form the paracellular barrier (Yu et al.,
2003).
Claudin-8 is expressed at the tight junction of the distal nephron, extending continuously from
the early distal convoluted tubule to the tip of the inner medullary collecting duct (Li et al., 2004). We
previously generated an MDCK II cell line with inducible expression of claudin-8 and used it to show
that claudin-8 acts as an important component in the formation of a significant barrier to monovalent
alkali metal cations, divalent cations, and organic cations (Yu et al., 2003). Because claudin-8 is
normally expressed in the entire collecting duct, we hypothesized that it might also be necessary for the
paracellular barrier to H
+
, NH
4
+
and/or HCO
3
-
, thereby playing a permissive role in renal acid excretion.
In this study, we developed methods to measure paracellular H
+
, NH
4
+
and HCO
3
-
permeability in 4
control MDCK II cells and investigated how these are affected by claudin-8 induction. Our results reveal
a strikingly different mechanism for paracellular H
+
transport compared to other monovalent cations. We
further show that claudin-8 also acts as a paracellular barrier to both NH
4
+
and HCO
3
-
, consistent with an
important role in net acid excretion.
METHODS
Cell culture
MDCK II TetOff claudin-8 NFL cells (Yu et al., 2003) were maintained in Dulbeccos modified Eagles
medium with 5% fetal bovine serum, and 20 ng/ml doxycycline for 2 days prior to plating onto 1 cm
2
Snapwell polyester filters (Corning Life Sciences, Corning, NY). To mitigate the potential effects of
differential growth rate, cell culture on filters was initiated by plating the cells at twice confluent density
(approximately 4 x 10
5
/cm
2
) and washing off excess cells after overnight incubation. The culture
medium was exchanged every three days. Because doxycycline in solution degraded rapidly, medium
containing doxycycline (made from frozen doxycycline stocks) was stored at 4°C in the dark, and used
within 7 days. To induce claudin-8 expression, doxycycline was omitted from the culture medium
starting from the day of plating (Dox-). In matching control plates, doxycycline was included in the
medium to suppress claudin-8 expression (Dox+). All studies were performed after 4-5 days in culture.
Ussing chamber setup
The solutions used are listed in Table 1. Filter inserts containing cell monolayers were gently rinsed two
times with buffered or unbuffered saline Ringer solution (solution A or C, respectively), mounted in one
of six Ussing chambers and allowed to stabilize for 15 to 30 min. The reservoir on each side of the
monolayer was filled with 4-6 mL fluid and continuously bubbled and stirred with either 100% O
2
or
95% O
2
/5% CO
2
(for HCO
3
-
-buffered solutions) gas lifts. The chambers were warmed (to 37°C, unless
otherwise indicated) by a water jacket fed from a circulating water bath. In studies to assess 5
temperature-dependence of permeability, the temperature in the Ussing chamber fluid was directly
monitored by immersion of a metal thermocouple probe (Physitemp Intruments, Clifton, NJ) and the
water bath temperature adjusted until the solution temperature was 37°C, 24°C, or 16°C. The coefficient
of variation in the temperature among the six chambers was 1%.
Voltage-sensing electrodes consisting of Ag/AgCl pellets and current-passing electrodes of Ag
wire were connected by agar bridges containing 3 M KCl and interfaced via headstage amplifiers to a
microcomputer-controlled voltage/current clamp (DM-MC6 and VCC-MC6, respectively; Physiologic
Instruments, San Diego, CA). Voltage-sensing electrodes were matched to within 1 mV asymmetry and
corrected by an offset-removal circuit. The fluid resistance was determined in the absence of a filter and
electrically compensated using the series compensation circuit on the clamp. The voltage was first
measured with blank filters in each combination of solutions to be used for the experiments. The values
obtained, which were generally less than 1 mV in magnitude, represent the difference in junction
potentials between the two voltage-sensing bridges, summed with any potential that might exist across
the blank filter membrane. These were subtracted from all subsequent measurements with filters
containing attached cell monolayers to determine transepithelial voltage. The spontaneous potential
difference in control MDCK II monolayers was generally less than 0.2 mV and was ignored.
Transepithelial voltages reported are referenced to the apical side. Short circuit current flowing in the
basolateral-to-apical direction was considered positive.
The total resistance between apical and basal side was determined at the start of the experiment
from the current evoked by a 5 mV bipolar voltage pulse. The background resistance determined with
blank filters was subtracted from the total resistance measured with filters containing attached cell
monolayers to determine the transepithelial resistance (TER) and hence conductance (TER
-1
). NaCl
dilution potentials were determined with 150 mM NaCl in one chamber and 75 mM NaCl in the other
(solutions C and F, respectively), and used to determine P
Na
and P
Cl
. CsCl biionic potentials were 6
determined with 1