Expression of the Na+-K+-2Cl(-)-cotransporter 2 in the normal and pressure-induced ischemic rat retina.

Abstract

To evaluate the expression of the Na(+)-K(+)-2Cl(-)-cotransporter 2 (NKCC2) in the ischemic rat retina.
Retinal ischemia was induced by pressures 90 to 120 mmHg, above systemic systolic pressure. Immunohistochemistry and western blot analysis were performed.
NKCC2 is expressed in the normal retina and its expression is increased by ischemia caused by intraocular pressure elevation. NKCC2 immunoreactivity was observed mainly in axon bundles of ganglion cells and horizontal cell processes in the retina. NKCC2 expression continuously increased with a peak value 3 days (to 415% of normal levels) after ischemic injury, and then gradually decreased to 314% of controls until 2 weeks post injury. The mean density of NKCC2-labeled ganglion cells per mm(2) changed from 1,255 ± 109 in normal retinas to 391 ± 49 and 185 ± 37 at 3 days and 2 weeks after ischemia, respectively (p < 0.05), implying cell death of ganglion cells labeled with NKCC2.
Taken together, these results suggest that NKCC2, which is expressed in retinal ganglion and horizontal cells, may contribute to cell death by ischemic injury in the retina, although the molecular mechanisms involved remain to be clarified.

Full text

Page 1

The Na+-K+-2Cl--cotransporter (NKCC) belongs to the
cation-chloride co-transporter family, which transports
Na+, K+, and Cl- ions across the plasma membrane of cells
with a stoichiometry of 1Na+ : 1K+ : 2Cl-. The NKCC plays
an important role in salt secretion and absorption, cell vol-
ume regulation, and maintenance of intracellular Cl- con-
centrations [1-3]. Recently, the NKCC has been reported to
be associated with the cell cycle, cellular proliferation, and
apoptotic cell death in ischemic tissues [4-7].
Two isoforms of the NKCC have been identified. The
NKCC1 isoform was initially discovered on the basolateral
membrane of the shark rectal gland and is expressed in a
wide variety of tissues, including epithelial cells and non-
epithelial cells. The NKCC2 isoform is expressed mainly
in the apical membrane of epithelial cells of the thick as-
cending limb of Henle’s loop in the kidney [1-3].
It has been reported that the NKCC is related with cell
damage in many ischemic models [8-11]. In cerebral isch-
emia, NKCC1 contributes to astrocyte swelling, causing
excitatory amino acid release, resulting in neuronal cell
death [8,9]. Thus, pharmacological inhibition of NKCC1
is neuroprotective in ischemic or traumatic injuries of the
brain. During heart ischemia, sodium accumulates outside
cells due to impairment of ion channels or transporters like
Na+-K+-ATPase, Na+-H+ exchanger, and Na+-Ca2+ exchang-
er and enters into cells through the NKCC [10]. Inhibition
of the NKCC can attenuate the ischemic rise in intracel-
lular sodium and maintain higher levels of high energy
phosphates, significantly improving functional recovery.
Previous studies of the retina demonstrated that the
NKCC is not expressed in retinal ganglion cells except
203
Original Article
Expression of the Na+-K+-2Cl--Cotransporter 2 in the Normal and
Pressure-Induced Ischemic Rat Retina

Do Hyun Kim1, Myung Douk Ahn2
1Yang-Ju St. Mary’s Eye Clinic, Yangju, Korea
2Department of Ophthalmology and Visual Science, Seoul St. Mary’s Hospital,
The Catholic University of Korea College of Medicine, Seoul, Korea
pISSN: 1011-8942 eISSN: 2092-9382
Korean J Ophthalmol 2012;26(3):203-211
http://dx.doi.org/10.3341/kjo.2012.26.3.203
© 2012 The Korean Ophthalmological Society
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses
/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Received: September 20, 2011 Accepted: November 22, 2011
Corresponding Author: Myung Douk Ahn, MD, PhD. Department of
Ophthalmology and Visual Science, Seoul St. Mary’s Hospital, The
Catholic University of Korea College of Medicine, #222 Banpo-daero,
Seocho-gu, Seoul 137-701, Korea. Tel: 82-2-2258-6195, Fax: 82-2-599-
7405, E-mail: mdahn@catholic.ac.kr
Purpose: To evaluate the expression of the Na+-K+-2Cl--cotransporter 2 (NKCC2) in the ischemic rat retina.
Methods: Retinal ischemia was induced by pressures 90 to 120 mmHg, above systemic systolic pressure. Im-
munohistochemistry and western blot analysis were performed.
Results: NKCC2 is expressed in the normal retina and its expression is increased by ischemia caused by intra-
ocular pressure elevation. NKCC2 immunoreactivity was observed mainly in axon bundles of ganglion cells
and horizontal cell processes in the retina. NKCC2 expression continuously increased with a peak value 3
days (to 415% of normal levels) after ischemic injury, and then gradually decreased to 314% of controls until 2
weeks post injury. The mean density of NKCC2-labeled ganglion cells per mm2 changed from 1,255 ± 109 in
normal retinas to 391 ± 49 and 185 ± 37 at 3 days and 2 weeks after ischemia, respectively (p < 0.05), imply-
ing cell death of ganglion cells labeled with NKCC2.
Conclusions: Taken together, these results suggest that NKCC2, which is expressed in retinal ganglion and
horizontal cells, may contribute to cell death by ischemic injury in the retina, although the molecular mecha-
nisms involved remain to be clarified.
Key Words: Ischemia, Na+-K+-2Cl--cotransporter 2, Retinal ganglion cells

Page 2

204
Korean J Ophthalmol Vol.26, No.3, 2012
during developing development [12,13]. However, the
presence of the NKCC2 and its role in the retina have not
yet been described. To address these issues, we analyzed
the expression of the NKCC2 in the retina and observed
expression change after retinal ischemia induced by intra-
ocular pressure elevation.
Materials and Methods
Induction of transient retinal ischemia
Thirty-five adult male albino Sprague-Dawley rats
weighing 200 to 250 g were used for this study. Animals
were anesthetized with 4% chloral hydrate (1 mL/100 g
body weight). The pupils were dilated with 1% tropicamide
eye drops. The intraocular pressure (IOP) was raised to 90
to 120 mmHg by cannulation of the anterior chamber with
a 30-gauge needle connected to a hydrostatic pressure de-
vice, and the elevated IOP was maintained for 60 minutes.
The applied pressure of 90 to 120 mmHg is in the range,
or slightly above, systemic systolic pressure in the rat.
Following removal of the cannula, recirculation began im-
mediately and the IOP decreased to normal values within 5
minutes. Animals were sacrificed by an overdose of chloral
hydrate at 12 hours, 24 hours, 3 days, 7 days, and 14 days
after reperfusion. The anterior segments of the eyeballs
were removed. For western blot analysis, retinal tissues
were quickly dissected on an ice-cold plate, frozen on dry
ice, and stored at -70°C. For immunocytochemistry and
quantitative analysis, the eyecups were fixed by immersion
in fixative (4% paraformaldehyde/0.2% picric acid in 0.1 M
phosphate buffer [PB, pH 7.4]), for 2 to 3 hours. Following
fixation, the retina was carefully dissected and transferred
to 30% sucrose in PB, for 24 hours at 4°C. They were then
frozen in liquid nitrogen, thawed, and rinsed in 0.01 M
phosphate buffered saline (PBS, pH 7.4). The central re-
gion near the optic disc was excised, dehydrated in a series
of graded alcohol, and embedded in wax.
Immunoblot analysis
Western blot analysis was performed on the retinal ex-
tracts, which were homogenized in 10 volumes of 20 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton
×-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl
sulfate (SDS), 0.02% sodium azide, 1 mM PMSF, and 5
µg/mL leupeptin. Protein concentration for each sample
was assayed by the Lowry method, in duplicate, and the
results were averaged. Duplicate sets of protein standards
containing 0, 1, 3, 5, 10, 20, 40, and 60 mg of bovine serum
albumin were assayed by the same method. The results
were averaged and graphed to produce a linear equa-
tion that was used to estimate the protein contents in the
retinal extracts. The optical density of each sample was
measured at 660 nm, using a spectrometer (Spectronic 20;
Bausch & Lomb, Rochester, NY, USA). Tissue samples
corresponding to 25 g of total protein were heated at 100°C
for 10 minutes with an equivalent volume of sample buf-
fer (containing 4% SDS and 10% mercaptoethanol) and
loaded onto 10% polyacrylamide gels. The proteins were
electrotransferred to a nitrocellulose membrane in Tris-
glycine-methanol buffer. The membrane was blocked for
1 hour at room temperature in a blocking solution contain-
ing 5% nonfat dry milk, 0.05% Tween-20, and PBS. The
membrane was then incubated for 15 hour at 4°C with a
goat polyclonal antibody directed against NKCC2 (1 : 1,000;
Chemicon, Temencula, CA, USA) in the blocking solution.
The membrane was rinsed with 0.05% Tween-20 in PBS
for three washes of 10 minutes and incubated for 1 hour
at room temperature in a 1 : 200 dilution of biotinylated
donkey anti-rabbit IgG (Vector Laboratories, Burlingame,
CA, USA). The blot was washed three times for 10 minutes
each and then processed for analysis using an enhanced
chemiluminescence detection kit (Amersham, Arlington
Heights, IL, USA).
Immunocytochemistry
For the immunocytochemistry of the NKCC2, 50-μm-
thick vibratome sections, prepared as described above,
were used. Endogenous peroxidase activity was blocked
by placing the sections in 1.4% H2O2 in methanol for 10
minutes. The sections were then incubated in 10% normal
serum (Vector Laboratories) in PBS for 1 hour at room
temperature in order to block nonspecific binding sites.
Sections were then incubated in a solution of antibodies
to the NKCC2 (1 : 1,000) for 1 day at 4°C. After washing
in PBS for 45 minutes (3 × 15 minutes), the retinal tissues
were incubated for 12 hours in biotinylated donkey anti-
rabbit IgG (1 : 50, Vector Laboratories) with 0.5% Triton
X-100 at 4°C, rinsed in PBS, and subsequently incubated in
Avidin-biotin-peroxidase complex (Vector Laboratories) in
PBS for 1 day at 4°C. Retinas were rinsed in two changes
of PBS and three changes of 0.05M Tris-HCl buffer (TB,
pH 7.4) for 5 minutes at room temperature, and were then
incubated in 0.05% 3,3-diaminobenzidine tetrahydrochlo-
ride in TB for 10 minutes. Hydrogen peroxide was added
to the incubation medium to a final concentration of 0.01%
H2O2, and the container was gently shaken as the reaction
proceeded. After 1.2 minutes of reaction, as determined
by the degree of staining, the reaction was stopped with
several washes of TB and PB. The stained tissues were
mounted onto gelatin-coated slides and coverslipped with
glycerol.
Confocal laser scanning microscopy
To identify whether cells demonstrating NKCC2 im-

Page 3

205
DH Kim, et al. Na+-K+-2Cl--Cotransporter 2 in Ischemic Rat Retina
munoreactivity were ganglion cells, double labeling tech-
niques using antibodies against the NKCC2 and Thy1.1, a
specific marker for ganglion cells, were performed. Sec-
tions were incubated overnight in monoclonal Thy1.1 anti-
body (1 : 500; Chemicon, Temencula, CA, USA) with 0.5%
Triton X-100 in 0.1M PB at 4°C. Sections were rinsed for
30 minutes with 0.01 M PBS, and the retinal tissues were
incubated for 2 hours in biotinylated donkey anti-mouse
IgG (1 : 50, Vector Laboratories), rinsed in PBS, and subse-
quently incubated for 2 hours in biotinylated donkey anti-
mouse FITC (1 : 50, Vector Laboratories). After rinsing for
30 minutes with 0.01 M PB and for 30 minutes with 0.01 M
PBS and with subsequent incubation for 1 hour in normal
donkey serum, the retina was incubated overnight in anti-
NKCC2 at 4°C. Sections were then rinsed for 30 minutes
with 0.01 M PBS and incubated for 2 hour in biotinylated
donkey anti-rabbit IgG (1 : 50, Vector Laboratories), rinsed
in PBS, and incubated for 2 hour in biotinylated Cy-3 (1 :
500, Vector Laboratories).
To identify whether cells demonstrating NKCC2 im-
munoreactivity were horizontal cells, double labeling tech-
niques using antibodies against the NKCC2 and calbindin,
a specific marker for horizontal cells, were performed. Af-
ter incubation overnight in monoclonal calbindin antibody
(1 : 3,000, Chemicon) with 0.5% Triton X-100 in 0.1 M PB
at 4°C, sections were incubated for 2 hours in biotinylated
donkey anti-mouse FITC (1 : 50, Vector Laboratories),
rinsed for 30 minutes with 0.01 M PB and 30 minutes with
0.01 M PBS, and then incubated for 2 hours in biotinylated
donkey anti-mouse IgG (1 : 50, Vector Laboratories) again.
We incubated retinal tissues overnight in biotinylated
donkey anti-mouse FITC (1 : 50, Vector Laboratories) and
rinsed for 30 minutes with 0.01 M PB and 30 minutes with
0.01 M PBS. In order to inhibit nonspecific reactions, sec-
tions were incubated in normal donkey serum for 1 hour.
After overnight incubation in anti-NKCC2 at 4°C, sections
were rinsed for 30 minutes with 0.01 M PBS. The retinal
tissues were incubated for 2 hours in biotinylated donkey
anti-rabbit IgG (1 : 50, Vector Laboratories) and then for 2
hours in biotinylated Cy-3 (1 : 500, Vector Laboratories).
Sections were analyzed using a Bio-Rad Radiance Plus
confocal scanning microscope (Bio-Rad, Hemel Hemp-
stead, UK), installed on a Nikon Eclipse E600 fluorescence
microscope (Nikon, Tokyo, Japan). FITC and Cy3 signals
were always detected separately. The FITC labeling was
excited using the 488 nm line of the Argon ion laser and
detected after passing an HQ513/30 (Bio-Rad) emission fil-
ter. For detection of the Cy3 signal, the 543 nm line of the
green HeNe laser was used in combination with the 605 /
32 (Bio-Rad) emission filter. Images were imported into
Adobe Photoshop ver. 7.0 (Adobe Systems, San Jose, CA,
USA) and printed on photo paper (Seiko Epson, Tokyo,
Japan). For presentation, all manipulations (brightness and
contrast only) were carried out equally for all images.
Whole-mount immunostaining and semiquantitative
analysis
For whole-mount immunostaining, the same immuno-
cytochemical procedures described above were used, but
with a longer incubation time. For semi-quantitative analy-
sis of post-ischemic changes in the NKCC2 immunoreac-
tive ganglion cells, the density (per unit area, 0.25 mm × 0.2
mm) of the labeled ganglion cells was counted in the mid
temporal region of the whole-mount retinas. The number
of labeled cells was divided by the unit area to obtain mean
densities of labeled cells per mm2. Data are given as mean
± standard deviation. Statistical significance was assessed
using one-way ANOVA.
Results
Western blot analysis of the Na+-K+-2Cl--cotransporter
2 levels
To evaluate the postlesional changes in the NKCC2
levels quantitatively, we performed an immunoblot analy-
sis on membranes prepared from control and ischemic
rat retina. As shown in Fig. 1, antibodies to the NKCC2
Fig. 1. Immunoblot analysis of the Na+-K+-2Cl--cotransporter 2
(NKCC2) protein levels in normal and ischemic rat retina. (A)
Immunoblot stained for the NKCC2 demonstrating a single band
at 120 to 130 kDa for all time points tested: normal retina (lane 1),
1 day (lane 2), 3 days (lane 3), 7 days (lane 4), and 14 days (lane
5) after ischemia/reperfusion. (B) Densitometric analysis of im-
munoblots as shown in (A). The NKCC2 level was upregulated
to approximately 115% of control by 1 day and to approximately
415% by 3 days. The NKCC2 levels decreased to 275% and 313%
at 7 days and 14 days of ischemia/reperfusion, respectively. As-
terisk represents a significant NKCC2 expression increase (p <
0.05, one way ANOVA).
A
Experimental time
120-130
kDa
Relative optical density (%)
600
500
400
300
200
100
0
Normal1 day 3 day1 wk2 wk
*
*
*
120-130
kDa
B

Page 4

206
Korean J Ophthalmol Vol.26, No.3, 2012
Fig. 2. Immunocytochemical staining for the Na+-K+-2Cl--cotransporter 2 (NKCC2) in normal and ischemic rat retina. Photomicrographs
show 50-μm-thick sections processed for immunocytochemistry. (A) Normal retina. (B,C,D,E) Ischemic retina 1, 3, 7, and 14 days after
ischemia/reperfusion, respectively. Labeling for the NKCC2 is present in both the ganglion and outer plexiform layers. After ischemia/
reperfusion, retinal ganglion cells decrease and marked axonal sprouting in the outer plexiform layer was observed. In the ganglion cell
layer, the ganglion cell axon bundles (asterisk) and ganglion cell body (arrow) are seen. Arrow head indicates the horizontal cell processes
in the outer plexiform layer. ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform
layer; GCL = ganglion cell layer. Scale bar = 50 μm.
A
*
*
D
B
E
C
ABC
DE
F
Fig. 3. Confocal micrographs taken from a vertical section of normal retina (A,B,C) and ischemic retina 2 weeks after injury (D,E,F).
Tissues were processed for Na+-K+-2Cl--cotransporter 2 (NKCC2) (A,D) and Thy1.1 (B,E) immunohistochemistry. The NKCC2 labeled
ganglion cells are immunofluorescent using Cy3-conjugated secondary antibody, and Thy1.1 was visualized using a FITC-conjugated
secondary antibody. The NKCC2 demonstrates good immunoreactivity, comparable to Thy1.1 (C,F) Merged picture shows that Thy1.1
immunoreactive profiles show NKCC2 immunoreactivity (yellow). ONL = outer nuclear layer; OPL = outer plexiform layer; INL = inner
nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Scale bar = 50 μm.

Page 5

207
DH Kim, et al. Na+-K+-2Cl--Cotransporter 2 in Ischemic Rat Retina
demonstrated a single band with a molecular mass of 100
to 150 kDa, the intensity of which increased up to 3 days
following ischemia/reperfusion and gradually declined
thereafter, in accordance with the immunocytochemi-
cal observations. Densitometric analysis revealed that the
NKCC2 level was upregulated to approximately 115% of
controls by 1 day and to approximately 415% by 3 days.
The NKCC2 levels decreased to 275% and 313% at 7 and
14 days post ischemia/reperfusion, respectively.
The Na+-K+-2Cl--cotransporter 2 is expressed in the
retinal ganglion cell layer and the outer plexiform layer
The thickness of the inner retina gradually decreased
with increasing time after ischemia, in agreement with
previous reports [14-16]. In normal rat retina immunos-
tained for the NKCC2, labeling was mainly observed in
the ganglion cell layer and outer plexiform layer (Fig. 2). In
the ganglion cell layer, axonal staining was stronger than
cell body staining. In the outer plexiform layer, labeled
profiles were horizontally oriented. After ischemia/reper-
fusion, the NKCC2 immunoreactivity increased compared
with controls.
The Na+-K+-2Cl--cotransporter 2 is expressed in retinal
ganglion cells and horizontal cell processes
To identify the nature of the labeled profiles in the
ganglion cell and outer plexiform layers, double label-
ing experiments using antisera against the NKCC2 were
performed with anti-Thy1.1 for the ganglion cell layer and
anti-calbindin for the outer plexiform layer. In the merged
picture (Fig. 3C and 3F), the NKCC2 labeled profiles in the
ganglion cell layer also demonstrated Thy1.1 immunore-
activity. Two weeks after injury, the ischemic retina (Fig.
3D-3F) demonstrated a decreased ganglion cell population
compared with the normal retina (Fig. 3A-3C). In the other
merged picture (Fig. 4C and 4F), the NKCC2 labeled pro-
files in the inner retina also demonstrated calbindin immu-
noreactivity. Two weeks after injury, the ischemic retina
ABC
DE
F
Fig. 4. Confocal micrographs taken from a vertical section of normal retina (A,B,C) and ischemic retina 2 weeks after injury (D,E,F).
Tissues were processed for Na+-K+-2Cl--cotransporter 2 (NKCC2) (A,D) and calbindin (B,E) immunohistochemistry. The NKCC2 labeled
ganglion cells are immunofluorescent using Cy3-conjugated secondary antibody and calbindin was visualized using a FITC-conjugated
secondary antibody. (C,F) Merged picture shows that calbindin immunoreactive profiles show NKCC2 immunoreactivity (yellow). After
ischemia/reperfusion, the NKCC2 immunoreactivity increased and the number of horizontal cells did not decrease. ONL = outer nuclear
layer; OPL = outer plexiform layer; INL = inner nuclear layer; IPL = inner plexiform layer; GCL = ganglion cell layer. Scale bar = 50 μm.