Role of Kir4.1 Channels in Aminoglycoside-Induced Ototoxicity of Hair Cells

Abstract

The Kir4.1 channel, an inwardly rectifying potassium ion (K+) channel, is located in the hair cells of the organ of Corti as well as the intermediate cells of the stria vascularis. The Kir4.1 channel has a crucial role in the generation of endolymphatic potential and maintenance of the resting membrane potential. However, the role and functions of the Kir4.1 channel in the progenitor remain undescribed. To observe the role of Kir4.1 in the progenitor treated with the one-shot ototoxic drugs (kanamycin and furosemide), we set the proper condition in culturing Immortomouse-derived HEI-OC1 cells to express the potassium-related channels well. And also, that was reproduced in mice experiments to show the important role of Kir4.1 in the survival of hair cells after treating the ototoxicity drugs. In our results, when kanamycin and furosemide drugs were cotreated with HEI-OC1 cells, the Kir4.1 channel did not change, but the expression levels of the NKCC1 cotransporter and KCNQ4 channel are decreased. This shows that inward and outward channels were blocked by the two drugs (kanamycin and furosemide). However, noteworthy here is that the expression level of Kir4.1 channel increased when kanamycin was treated alone. This shows that Kir4.1, an inwardly rectifying potassium channel, acts as an outward channel in place of the corresponding channel when the KCNQ4 channel, an outward channel, is blocked. These results suggest that the Kir4.1 channel has a role in maintaining K+ homeostasis in supporting cells, with K+ concentration compensator when the NKCC1 cotransporter and Kv7.4 (KCNQ4) channels are deficient.

Full text

Research Article
Role of Kir4.1 Channels in Aminoglycoside-Induced
Ototoxicity of Hair Cells
Jin Sil Choi,
1,2
Ye Ji Ahn,
1,2
SuHoon Lee,
1,2
Dong Jun Park,
1,2
JeongEun Park,
1,2
Sun Mok Ha,
1,2
and Young Joon Seo
1,2
1
Research Institute of Hearing Enhancement, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea
2
Department of Otorhinolaryngology, Yonsei University Wonju College of Medicine, Wonju, Republic of Korea
Correspondence should be addressed to Young Joon Seo; okas2000@hanmail.net
Received 12 September 2022; Revised 27 October 2023; Accepted 14 November 2023; Published 16 December 2023
Academic Editor: Arif Jamal Siddiqui
Copyright © 2023 Jin Sil Choi et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The Kir4.1 channel, an inwardly rectifying potassium ion (K
+
) channel, is located in the hair cells of the organ of Corti as well as
the intermediate cells of the stria vascularis. The Kir4.1 channel has a crucial role in the generation of endolymphatic potential and
maintenance of the resting membrane potential. However, the role and functions of the Kir4.1 channel in the progenitor remain
undescribed. To observe the role of Kir4.1 in the progenitor treated with the one-shot ototoxic drugs (kanamycin and furosemide),
we set the proper condition in culturing Immortomouse-derived HEI-OC1 cells to express the potassium-related channels well.
And also, that was reproduced in mice experiments to show the important role of Kir4.1 in the survival of hair cells after
treating the ototoxicity drugs. In our results, when kanamycin and furosemide drugs were cotreated with HEI-OC1 cells, the
Kir4.1 channel did not change, but the expression levels of the NKCC1 cotransporter and KCNQ4 channel are decreased. This
shows that inward and outward channels were blocked by the two drugs (kanamycin and furosemide). However, noteworthy
here is that the expression level of Kir4.1 channel increased when kanamycin was treated alone. This shows that Kir4.1, an
inwardly rectifying potassium channel, acts as an outward channel in place of the corresponding channel when the KCNQ4
channel, an outward channel, is blocked. These results suggest that the Kir4.1 channel has a role in maintaining K
+
homeostasis in supporting cells, with K
+
concentration compensator when the NKCC1 cotransporter and Kv7.4 (KCNQ4)
channels are deficient.
1. Introduction
Ototoxicity is the cellular degeneration of cochlear and/or
vestibular tissues. The cellular deterioration leads to func-
tional deterioration [1]. Aminoglycoside antibiotics, loop
diuretics, or antineoplastic agents including cisplatin have
ototoxic properties. Ototoxicity is considered an otologic
emergency because there is a low chance of recovery from
functional damage when the appropriate treatment is not
provided promptly. Cisplatin ototoxicity occurs in 23% to
50% in adults and up to 60% in children [2], while ototoxic-
ity due to aminoglycosides and furosemide occurs in an esti-
mated 25% and 6% to 7% of cases, respectively [3].
Aminoglycoside antibiotics disrupt cross-links between
adjacent lipopolysaccharide molecules in the outer mem-
brane for gram-negative bacteria and passively enter the
periplasmic space [4]. Although little is known about the cel-
lular and molecular mechanisms of drug ototoxicity, amino-
glycosides can cause vestibular toxicity, which includes
disequilibrium and dizziness, and cochlear toxicity, which
can include hearing loss and/or tinnitus. Gentamicin enters
the inner ear fluids from the strial capillaries through the
strial marginal cells [5]. Both endocytosis and the mechan-
oelectrical transducer channel located at the top of hair cell
stereocilia have been proposed to mediate the uptake of ami-
noglycosides into sensory hair cells [6–9]. The ototoxic drug
affects the ion channels of the stria vascularis and causes hair
cell death because it affects the endolymphatic potential [10].
Hair cell mechanoreceptors rely on gradients of potassium
ion (K
+
) with a unique organization in the inner ear. K
+
Hindawi
BioMed Research International
Volume 2023, Article ID 4191999, 12 pages
https://doi.org/10.1155/2023/4191999

passively flows into the cell and circulates in the inner ear,
where it may be important in maintaining the endolym-
phatic potential of cochlea [11].
Among the ion channels in the inner ear, K
+
inwardly
rectifying 4.1 (Kir4.1) channel is an important conduit for
K
+
to the endolymphatic space for generation of the endo-
lymphatic potential. Kir4.1 is an important mechanism for
the delivery of K
+
to the K
+
-secretory strial marginal cells
in the stria vascularis. The Kir4.1 channel is also present in
hair cells, particularly in the basal side of the hair cell, where
it may be important in the K
+
transport of potassium [11,
12]. Weak Kir4.1 labeling of Deiters’cells surrounding the
outer hair cell layer has been described [13, 14]. This specific
location implies that Kir4.1 in the organ of Corti may also
play a role in K
+
absorption for the recycling of K
+
in the
cochlea [15].
The Kir4.1 channel mutations to human diseases are
EAST syndrome (Bockenhauer et al., 2009) [16] and SeS-
AME syndrome (Scholl et al., 2009) [17] describing epilepsy,
ataxia, sensorineural deafness, tubulopathy (EAST syn-
drome) and seizures, sensorineural deafness, ataxia mental
retardation, and electrolyte imbalance (SeSAME syndrome)
caused by mutations in KCNJ10 gene encoding Kir4.1 chan-
nels [16, 17]. Other syndromes are also directly related to
Kir4.1 dysfunction. These are Rett’s syndrome (Olsen et al.,
2015; Nwaobi et al., 2016) [18, 19] and Alper’s syndrome
(Smith et al., 2023) [20]. There are diseases linked to the
downregulation of Kir4.1 channels such as diabetes (Pan-
nicke et al., 2006; Rivera-Aponte et al., 2015) [21, 22] and
Huntington’s disease (Proft and Weiss, 2014) [23].
In our previous study, kanamycin and furosemide have
each been used in single application regimens in ototoxic
drug-induced hearing loss animal models [24]. We thought
that the combined use of kanamycin and furosemide could
directly affect Kir4.1. We explored the importance of the
Kir4.1 channel in this combination treatment using HEI-
OC1 cells grown in a condition in which Kir4.1 is highly
expressed. The cochlear cell line (progenitor HEI-OC1) cells
are multipotent progenitors called “House Ear Institute-
Organ of Corti 1 (HEI-OC1)”cells, which are used for
in vitro screening of ototoxic drugs. These cells are common
progenitors for auditory (receptor and sensory) cells and for
supportive cells (Kalinec et al., 2016) that are susceptible for
commonly used drugs such as cisplatin [25].
2. Materials and Methods
2.1. HEI-OC1 Cell Culture. HEI-OC1 cells provided by Kali-
nec et al., House Ear Institute (Los Angeles, CA, USA), were
cultured in high-glucose Dulbecco’s modified Eagle’s
medium (Gibco BRL, Gaithersburg, MD, USA) supple-
mented with 10% fetal bovine serum (Gibco BRL, Gaithers-
burg, MD, USA) and 50 U/ml gamma-interferon (Genzyme,
Cambridge, MA, USA) without any antibiotics in a permis-
sive condition (33
°
C, 10% CO
2
). For the nonpermissive con-
dition, cells were first cultured in the permissive condition
for a certain period and then transferred to 39
°
C, 5% CO
2
[26]. Growth curves were performed to identify the growth
patterns of cells in each condition. Cells cultured at 33
°
C
in a 10% CO
2
atmosphere were recorded on day 0 after cell
counting before seeding. Cells were recovered daily from the
cell dish and enumerated. This was done for up to 14 days.
In the cells shifted to 39
°
C, incubation at 33
°
C was done
for 5 days (which was determined to be optimal for differen-
tiation in preliminary experiments), and cells were enumer-
ated just prior to being shifted to the higher temperature
condition. Following the shift, cells were enumerated daily
for 14 days.
2.2. Preparation of Ototoxic Drugs. As explained above, HEI-
OC1 cells were cultured in permissive conditions (33
°
C, 10%
CO
2
) until day 5 and then transferred to the nonpermissive
condition (39
°
C, 5% CO
2
).
Four drug treatment groups were used: untreated (con-
trol), kanamycin, kanamycin and furosemide (both from
Cayman Chemical, Ann Arbor, MI, USA), and furosemide.
In preliminary experiments, the CCK assay was performed
as described next to confirm if the treatment concentration
affected viability. Almost no cell death was observed with
1 mM kanamycin or 50 μM furosemide. Subsequent experi-
ments used 100 μM kanamycin and 10 μM furosemide to
ensure no deleterious effects on cell viability. High-glucose
DMEM supplemented with 0.5% FBS was used.
2.3. CCK Assay. After the treatment of HEI-OC1 cells with
the aforementioned concentrations of kanamycin and furo-
semide, the EZ-3000 CCK assay (Dogen, Seoul, Korea) was
performed. After culturing the cells for 4 days at 39
°
C,
Trypsin-EDTA (Gibco BRL, Gaithersburg, MD, USA) was
used to remove the cells, which were seeded (2×10
4cells/
ml) in wells of a 96-well dish. The total volume of each well
was 100 μl, and there were five wells of each of the four
aforementioned groups, for a total of 20 wells. After 24
hours, the medium in each well was replaced with the
drug-containing medium. CCK was measured from 24 to
168 hours by the addition of 10 μl CCK solution to each well,
incubation for 1 hour, and measurement of the absorbance
was measured to determine cytotoxicity.
2.4. Reverse Transcriptase Polymerase Chain Reaction (RT-
PCR) and Quantitative Real-Time (qRT)-PCR. One milliliter
of TRIZOL (Invitrogen, Carlsbad, CA, USA) was added to
cells (5×10
6cells/ml), distributed by vortexing, and incu-
bated for 5 minutes at room temperature (20
°
C-25
°
C). Two
hundred microliters of chloroform was added, mixed by vor-
texing for at least 30 seconds, and incubated at room tem-
perature for 10 minutes. The sample was recovered by
centrifugation at 12,000 rpm for 15 minutes, and the super-
natant was transferred to a new tube. An equal volume of
2-propanol was added and vortexed for at least 30 seconds.
The sample was incubated at room temperature for 10
minutes and recovered by centrifugation at 11,000 rpm for
10 minutes. The supernatant was discarded, and the pellet
was stored on ice. Distilled water (30-50 μl) was added, and
the RNA was dissolved at 55
°
C for 10 minutes using a heat
block. The extracted RNA was quantified, and cDNA
(TOYOBO, Osaka, Japan) was synthesized with 1 μgof
RNA. 4xDN was added and incubated at 37
°
C for 5 minutes.
2 BioMed Research International

Reverse transcriptase (5x) was added and incubated at 37
°
C
for 15 minutes followed by 98
°
C for 5 minutes. The synthe-
sized cDNA was diluted 1/5 and used for PCR. To screen the
Kir4.1 ion channels in HEI-OC1 cells, RT-PCR was per-
formed. Template (4 μl), primer (1 μl), and Hot Taq DNA
polymerase (10 μl) (Komabiotech, Seoul, Korea) were added
to 5 μl of distilled water, and PCR was performed with one
cycle of initial denaturation at 95
°
C for 15 minutes; 35 cycles
of denaturation (94
°
C, 30 seconds), annealing (51-58
°
C, 30
seconds), and extension (72
°
C, 1 minutes); and one cycle
of a final extension (72
°
C, 10 minutes). We confirmed
Kir4.1, Na-K-2Cl cotransporter-1 (NKCC1), potassium
voltage-gated channel subfamily Q member 4 (KCNQ4),
Kcnmb1, ClcnkA, ClcnkB, Scnn1A, Scnn1G, Gja1, Gjb6,
Slc26A4, Atp2B1, Atp6V0A4, and β-actin by RT-PCR [27]
(Supplement Table 1). After PCR was completed,
electrophoresis was performed for 15 minutes with a 2%
agarose gel mixed with loading dye. The resolved band was
confirmed using a Bioanalytical Imaging system. The RNA
levels of Kir4.1, NKCC1, and KCNQ4 were confirmed by
qRT-PCR by adding 14 μl of template, 3.5 μl of primer, and
17.5 μl of SYBR (Applied Biosystems, Foster, CA, USA) to
make a total volume of 35 μl. Each well of a 384-well plate
was loaded with 10 μl. Each sample was loaded in triplicate.
Real-time PCR was performed using one cycle of 95
°
C for
10 minutes; 40 cycles of 95
°
C for 15 seconds, 54
°
C for 1
minute, and 72
°
C for 30 seconds; and one final cycle of
95
°
C for 15 seconds, 60
°
C. After the PCR was completed, a
graph was created using the GraphPad Prism program.
2.5. Western Blot Analysis. Pro-prep solution (200 μl)
(Intron Biotechnology, Seongnam, Korea) was added to a
pellet-shaped cell and vortexed to create a homogenous sus-
pension. Each sample was kept on ice for 30 minutes and
centrifuged at 13,000 rpm for 20 minutes. The supernatant
was transferred to a new tube. The extracted proteins were
quantitated using the Bradford assay, and the defined quan-
tity of total protein was used for a western blot assay. SDS-
PAGE (10%) was performed at 0.02 A for 2.5 hours. The
resolved proteins were transferred to a PVDF membrane at
100 V for 1 hour. After transfer, proteins were confirmed
by the Ponceau S stain and blocked with 5% skim milk for
1 hour at room temperature. Primary antibodies were
against Kir4.1, NKCC1 (ab59791; Abcam, Cambridge, UK),
KCNQ4 (ab65797, Abcam, Cambridge, UK), and β-actin
(Santa Cruz Biotechnology, Dallas, TX, USA). Each primary
antibody was diluted 1 : 1,000 in 2.5% skim milk and incu-
bated overnight at 4
°
C. The secondary antibody was diluted
1 : 10,000 in 1% skim milk and incubated for 1 hour at room
temperature. After washing five times with 1x Tris-buffered
saline Tween 20 (TBST) for 5 minutes, ECL solution (Milli-
pore, USA) was added for 1 minute, and the band was con-
firmed with the ChemiDoc gel imaging system (Bio-Rad,
Hercules, CA, USA).
2.6. Immunofluorescence. Immunofluorescence was deter-
mined in vitro and ex vivo. In vitro, cells were cultured at
33
°
Cor39
°
C for 3 or 7 days. Cells were then seeded
(4×10
4cells/ml) in 4-well dishes. After culturing for 24
hours, each sample was washed with 1 ml PBS, and 500 μl
of 4% paraformaldehyde was added to each well and fixation
proceeded for 1 hour. The paraformaldehyde was removed,
and each sample was washed three times for 5 minutes each
in PBS. After the final wash, 500 μl of 0.1% Triton X-100 was
added and incubated for 15 minutes at room temperature.
Triton X-100 was removed, and samples were washed three
times for 5 minutes each in PBS. The primary antibody
(Kir4.1, APC-035; Alomone labs, Jerusalem, Israel) diluted
1 : 100 in 5% normal goat serum (NGS) was added, and the
cells were incubated for 1 hour at room temperature. PBS
(500 μl) was then added to each well and washed three times
(5 minutes per wash). The secondary antibody (goat anti-
rabbit IgG H&L, ab150077; Abcam, Cambridge, UK) was
diluted 1 : 200 in 5% normal goat serum (NGS) and incu-
bated for 1 hour at room temperature. Each sample was then
mounted using mounting solution and stained with 4′,6-
diamidino-2-phenylindole (DAPI), and a cover glass was
added. The solution was dried for 24 hours in a dark room
and examined using confocal microscopy.
2.7. Small Interfering RNA (siRNA) Transfection. After cul-
turing at permissive conditions of 33
°
C and 10% CO
2
, the
cells were transferred to 39
°
C. After 2 days, the cells were
detached with Trypsin-EDTA and seeded in a 60 mm dish
at a density of 3×10
5cells/ml. After 24 hours, Kir4.1 siRNA
(sc-42625, Santa Cruz Biotechnology, Dallas, TX, USA) was
added (40 pmol). After 6 hours, 1 ml of medium containing
2X FBS was added to each well and incubated for 48 hours.
The cells were subjected to PCR and western blotting as
described above. When drug treatment (100 μM kanamycin
or 10 μM furosemide) was performed within the Kir4.1
inhibited condition, the drug was added 48 hours after the
siRNA treatment (39
°
C, day 5) and maintained for 24 hours.
2.8. Animal Experiments and Paraffin Section. The animal
study protocol was approved by the Institutional Animal
Care and Use Committee of Yonsei University, Korea
(YWC-180703-1), and the procedures followed were in
accordance with the institutional guidelines. Animals were
purchased through the “Daehan Bio”company according
to institutional guidelines. Animals were cared for at a
temperature of 18-26
°
C, and the humidity was maintained
at 40-60%.
In addition, the bedding was changed twice a week, and
the cage and water bottle were sterilized before use.
To alleviate the suffering of animals through an experi-
mental process, mice were anesthetized with a mixture of
ketamine and Rompun.
In the drug-induced ototoxicity animal model, 550 mg/
kg kanamycin and 130 mg/kg furosemide were injected as
previously described [24]. Ototoxicity was evident in mice
as decreased audiometry from the third day after the Audi-
tory Brainstem Response test (data not shown). The mice
exposed to kanamycin or furosemide were sacrificed on
day 14. Cardiac perfusion was performed with 1×PBS
(50 ml) and fixed with 50 ml of 4% paraformaldehyde. Only
the cochlea was obtained from each mouse, and 4% PFA was
added at 4
°
C overnight. Twenty-four hours later, the
3BioMed Research International

decalcification process was performed with Calci-Clear
Rapid (Chayon Laboratories, INC, Seoul, Korea), followed
by dehydration with 30% sucrose. The cochlea were paraffin
embedded and sections were obtained at a thickness of 5 μm.
Sections were deparaffinized by dipping for 5 minutes in
Neo-clean, and dehydration was performed using 100%,
95%, 90%, and 70% ethanol. The slides were washed with
1×PBS for 5 minutes, and immunofluorescence was per-
formed as described above. In ex vivo samples, the cochlea
were obtained from sacrificed C57BL/6 mice (6 weeks of
age, male). After the bulla was removed from the cochlea,
a hole was made in the apex portion of the cochlea to
remove the bone. In the cochlear turn, the stria vascularis
and Reissner membrane were removed to leave only the
organ of Corti. The sensory epithelium was placed on a
one-well slide dish and immunofluorescence was performed.
2.9. Explant Models. We performed a cochlear surface prep
to check the surface of the cochlear. Mice were anesthetized
with ketamine and Rompun to relieve pain. The anesthetized
mouse was euthanized by performing cervical dislocation,
and the cochlear was harvested.
Cochlear performed surface prep immediately after har-
vesting to keep it as fresh as possible. We used H buffer for
cochlear surface prep, and H buffer was made with 1/10
HEPES (Gibco BRL, Cat. 15630080, Gaithersburg, MD,
USA) and 10 mM HBSS (Stemcell, NC9 162583, Vancouver,
Canada) in D.W. After that, it was cultured for 24 hours in
explant culture media, and the composition was DMEM
F12 media (Gibco BRL, Cat. 11320033, Gaithersburg, MD,
USA), 10% FBS (Gibco BRL, Gaithersburg, MD, USA),
ampicillin 10 ug/mL, 1% N2 supplement (Gibco BRL, Cat.
17502048, Gaithersburg, MD, USA), and 1% B27 (Gibco
BRL, Cat. 17504044, Gaithersburg, MD, USA).
2.10. Statistical Analysis. Statistical analysis was performed
using SPSS statistical package version 21.0 (SPSS Inc., USA).
Descriptive results of continuous variables are expressed as
the mean ± standard deviation (SD) for normally distributed
variables. Means were compared by 2-way analysis of
variance. The level of statistical significance was set to 0.05.
3. Results
3.1. Screening of Ion Channels in HEI-OC1 Cells. Ion chan-
nels are expressed in hair cells. HEI-OC1 is an immortalized
organ of the Corti-derived epithelial cell line. In this study,
we screened for representative ion channels in HEI-OC1
cells. Sixteen ion channels were identified by RT-PCR using
the primer sequences summarized in Supplementary
Table 1. The control used mouse kidney and compared
the expression levels in permissive and nonpermissive
conditions. Most of the channels were highly expressed at
39
°
C. In particular, the expression of the potassium-related
channels Kir4.1, NKCC1, and KCNQ4 was increased in
the differentiation conditions (Figure 1(a)). When the
Kir4.1
NKCC1
KCNQ4
Kcnmb1
ClcnkA
ClcnkB
Scnn1A
Scnn1B
Scnn1G
Gja1
Gja6
Slc26A4
Atp2B1
Atp6V1B1
Atp6V0A4
33°C39°CCtrl
�-Actin
(a)
33 39 33 39 33 39
0
2
4
6
8
∗∗∗ ∗∗
∗
Kir4.1 NKCC1 KCNQ4
Relative expression
(b)
Figure 1: Screening of hair cell markers in HEI-OC1 cells and the expressions of the main potassium channels. HEI-OC1 cells were cultured
at 33
°
C followed by 39
°
C, each for 5 days. Expression of marker genes in hair cells was confirmed and expression of main potassium
channels was confirmed. (a) RT-PCR results of hair cell markers in HEI-OC1 cells. RT-PCR was performed on day 5 of incubation at
33
°
C and 39
°
C. Kidney tissue was used as a control. (b) Expression of marker genes and main potassium channels were confirmed in day
5 HEI-OC1 cells at 33
°
C and 39
°
C.
4 BioMed Research International

expression levels of the main potassium channels Kir4.1,
NKCC1, and KCNQ4 were obtained by qRT-PCR, the
expression of all three channels was greater in the
nonpermissive condition than in the permissive condition
(Figure 1(b)). Based on these results, the 39
°
C condition
was used to achieve the increased expression of ion channels.
3.2. Changes in Expression of Kir4.1 in HEI-OC1 Cells at
Different Conditions. Growth curves of HEI-OC1 revealed
that the permissive condition was significantly increased
from day 5 compared to the nonpermissive condition. In
the latter, proliferation was slightly prolonged to 14 days
(Figure 2(a)). Kir4.1 was highly expressed at 39
°
C compared
to 33
°
C, which was confirmed at the protein level as well as
at the RNA level (Figures 2(b) and 2(c)). Immunofluores-
cence revealed that the expression was significantly increased
at 39
°
C compared with 33
°
C, and the expression level was
further increased at day 7 than at day 3 (Figure 2(d)). Micros-
copy examination revealed the increased expression of Kir4.1
throughout the cytoplasm (Figure 2(e)).
3.3. Role of Kir4.1 in HEI-OC1 Cells Treated with Kanamycin
and/or Furosemide. Ototoxic drugs affect the stria vascularis
and the endolymphatic potential. To confirm whether the
drugs could directly affect the ion channels of hair cells,
the doses of the ototoxic drugs kanamycin and furosemide
that were previously established in the mouse model were
converted into treatments of HEI-OC1 cells. Kanamycin
was applied at 100 μM and furosemide at 10 μM. Both con-
centrations were less than the concentrations that did not
produce cytotoxicity in the CCK assay after 14 days
(Figures 3(a) and 3(b)). Individual treatment with kanamy-
cin or furosemide increased the response of Kir4.1. However,
when the two drugs were administered together, a significant
reduction in Kir4.1 expression was observed compared to the
control (Figure 4(a)). NKCC1 showed a similar pattern to
that of control in the presence of kanamycin, but expression
was decreased in the presence of furosemide alone or the
combination of kanamycin and furosemide (Figure 4(b)).
Compared to control, the expression of KCNQ4 was
decreased in the presence of kanamycin alone or in combina-
tion with furosemide. However, KCNQ4 expression was
increased in the presence of furosemide alone compared to
control (Figure 4(c)). Kanamycin blocks the KCNQ4 chan-
nel, and furosemide blocks the NKCC1 cotransporter. Thus,
the results supported the anticipated result of a complemen-
tary role of K
+
channels with ototoxic drugs. To confirm that
Kir4.1 plays an important role in HEI-OC1 cells, Kir4.1 was
0 3 5 7 9 14
0
5.0×106
1.0×107
1.5×107
(Day)
(a)
(b)
(c)
Cell growth curve
Cell counting
39°C, 5% CO2
33°C, 10% CO2
KIR4.1 DAPI
(d)
(e)
Merge
33-3 day
33-7 day
39-3 day
39-7 day
33-3 day
39-7 day
3 5 7 9 14 3 5 7 9 14
0
2
4
6
(Day)
33 39
∗∗
∗
Relative expression
(Day)
33 39
Kir4.1
3579 14 3579 14
�-Actin
(d)
Figure 2: Characteristics of HEI-OC1 cells and the expression of Kir4.1. Cell growth curve and Kir4.1 expression levels were observed to
determine the experimental condition in HEI-OC1 cells. (a) Cell growth curves were obtained at 33
°
C and 39
°
C for 14 days in HEI-OC1
cells. (b) The change in the expression level of Kir4.1 was confirmed by qRT-PCR. (c) Expression of Kir4.1 at 33
°
C and 39
°
C was
confirmed at the protein level by western blot. (d, e) Immunofluorescence assay was performed to confirm the changes in the expression
level of Kir4.1 on day 3 and day 7 of cells at 33
°
C and 39
°
C, respectively. Expression of Kir4.1 was enhanced at 39
°
C compared to 33
°
C.
High magnification microscopy revealed that the expression was mainly at the cell membrane surface.
5BioMed Research International

completely inhibited by the transfection with 40 pmol siRNA
(Figure 5(a)). Cell death did not occur in this concentration
(Figure 5(c)). When treated with both 40 pmol of siRNA
and ototoxic drug, Kir4.1 was completely inhibited
(Figure 5(b)). In the absence of ototoxic drug, there was no
effect on cell death, while cell death occurred in the presence
of ototoxic drug (Figure 5(d)). The observations indicated the
significant impact of the reduction of Kir4.1 on the survival
of hair cells.
3.4. Changes of Kir4.1 in the Mouse Model of Ototoxicity.
Kir4.1 expression in the cochlea of the mouse ototoxic hear-
ing loss model was confirmed upon treatment with the
kanamycin-furosemide combination. Immunofluorescence
assay results revealed the strong expression of Kir4.1 in the
stria vascularis and outer hair cells in control mice. In con-
trast, mice with ototoxicity-related hearing loss displayed
decreased expression of Kir4.1 in the stria vascularis as well
as in outer hair cells (Figure 6). Figure 7 shows the
NT 1 1 2.5 2.5 5 5 10 10 F 50 uM
0.0
0.1
0.2
0.3
+
F 50 uM
+
F 50 uM
+
F 50 uM
+
F 50 uM
∗∗∗∗ ∗∗ ∗∗∗
Absorbance (450 nM)
100 93.9 87.5 86.2 73.5 70.7 63.8 60 56.7 93.1
(a)
Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7
0
200
400
600
800
No treat
Kan 100 uM
Kan-Furo
Furo 10 uM
Absorbance (% of control)
(b)
Figure 3: Concentration conditions of ototoxic drug treatment in HEI-OC1 cells and in the CCK viability assay. Drug concentrations that
lead to cell death and cell damage were observed up to day 7 to establish the ototoxic drug dose. (a) Measurement of concentrations of
kanamycin and furosemide to determine safe treatment dose. (b) CCK assays performed after 7 days with kanamycin 100 μM and
furosemide 10 μMat39
°
C and 5% CO
2
.
Kir4.1
NT Kan 10 uM Kan-Furo Furo 1 uM
0.0
0.5
1.0
1.5
2.0
2.5
Relative expression
(a)
NKCC1
NT Kan100 uM Kan-Furo Furo10 uM
0.0
0.5
1.0
1.5
∗∗ ∗∗
Relative expression
(b)
NT Kan100 uM Kan-Furo Furo10 uM
0
2
4
6
8
10
KCNQ4
∗∗
Relative expression
(c)
Kir4.1
�-Actin
NKCC1
KCNQ4
NT Kan K-F Furo
(d)
Figure 4: Association of potassium channels Kir4.1, NKCC1, and KCNQ4 in HEI-OC1 cells. Western blot and qRT-PCR results to
determine the association of Kir4.1, NKCC1, and KCNQ4 in HEI-OC1 cells. (a) Treatment of HEI-OC1 cells with either kanamycin or
furosemide increased the expression of Kir4.1. (b) When furosemide was treated in HEI-OC1 cells, NKCC1 was blocked, and kanamycin
was not affected. (c) When kanamycin was treated in HEI-OC1 cells, KCNQ4 was blocked, and there was no specificeffect upon
furosemide treatment. (d) Changes in expression levels of potassium channels in ototoxic drug-treated HEI-OC1 cells confirmed by
western blotting.
6 BioMed Research International

destruction of outer hair cells in ototoxic hearing loss model
and decreased expression of Kir4.1. NKCC1 and KCNQ4
expressions were confirmed in the mouse model using the
kanamycin-furosemide combination. In the immunofluores-
cence assay, NKCC1 and KCNQ4 were highly expressed in
the stria vascularis and outer hair cells of the control mice,
but the expressions in both locations were reduced in mice
treated with the drug combination (Figure 8).
4. Discussion
The presence of K
+
in the endolymphatic circulation of the
inner ear is important, since K
+
is the major charge carrier
for sensory transduction [26, 27]. The present data affirm
the importance of the Kir4.1 in hair cells, one of the ion
channels that is influential in maintaining the EP of the
cochlea. Kir4.1 is also expressed in hair cells in addition to
the stria vascularis in the inner ear. The changes in the
correlation of Kir4.1 channels with the NKCC1 cotranspor-
ter and KCNQ4 channels during exposure to ototoxic
drugs (kanamycin and/or furosemide) were demonstrated
(Figure 9). Endolymphatic K
+
flows into the sensory hair cells
via the apical transduction channel and is released from the
hair cells via basolateral K
+
channels including KCNQ4
[11]. We previously described that a single application of
kanamycin induced hearing loss in a mouse model incorpo-
rating furosemide [24]. The mechanism of ototoxicity was
not definitely determined. Kanamycin inhibits the outward
channel KCNQ4. When kanamycin was applied, Kir4.1
expression increased, which maintained the K
+
concentra-
tion inside the cells. The Kir4.1 channel seems to have a role
as an outward channel for the compensation of the K
+
osmotic gradient. Although the Kir4.1 channel is an inwardly
rectifying K
+
channel, it can have an outward role in some
cases [14, 28, 29]. Kanamycin treatment did not change the
expression of NKCC1 from that of the untreated group. This
is because the Kir4.1 channel has an outward role in place of
KCNQ4, suggesting that the expression of the NKCC1
inward channel is similar to that of the untreated group. Dur-
ing treatment with furosemide, which blocks NKCC1, the
expression of KCNQ4 increased. This is because K
+
is
released through the KCNQ4 channel, so the Kir4.1 channel
only acts as an inward channel [29]. We suggest that the
Kir4.1 channel has a key compensatory role in K
+
circulation
in hair cells when the NKCC1 cotransporter or KCNQ4
channel is injured.
NT
NT 20 pm 40 pm
20 pmol
18S (151pb
KIR4.1 (411bp)
40 pmol
0.0
0.5
1.0
1.5
Relative expression
(a)
NT NT Kan100 uM Kan-Furo Furo10 uM
0.0
0.5
1.0
1.5
siRNA
Relative expression
(b)
24 hr 48 hr
0
50
100
150
200
No treat
20 pmol
40 pmol
Absorbance (% of control)
(c)
24 hr 48 hr 72 hr
0
20
40
60
80
100
Absorbance (% of control)
No treat
Kan100 uM
Kan-Furo
Furo10 uM
(d)
Figure 5: Changes in HEI-OC1 cells upon Kir4.1 channel inhibition. (a) qRT-PCR results of HEI-OC1 cells cultured 39
°
C for 3 days
following Kir4.1 inhibition. (b) Expression of Kir4.1 with ototoxic drug treatment in HEI-OC1 cells cultured at 39
°
C for 3 days following
siRNA transfection. (c) CCK assay of cell viability following transfection with Kir4.1 siRNA. (d) CCK assay results of Kir4.1 channel
inhibition HEI-OC1 cells after 72 h of treatment with ototoxic drugs.
7BioMed Research International

Supportive cells are in fact glia cells. That is why Kir4.1
(the glia channel (Pooplalasundaram et al., 2000) [30]) is
expressed in the glia-neuronal progenitors HEI-OC1. Such
progenitors express the potassium inwardly rectifying chan-
nel Kir4.1 encoded by KCNJ10 gene which is the major
channel that helps to keep healthy membrane potential in
glial cells and supports not only K-buffering but also, most
importantly, glutamate buffering by glia cells (Kucheryavykh
et al., 2007) [31]. Glutamate buffering is extremely impor-
tant to avoid the toxicity of glutamate excess.
The conditions for HEI-OC1 cell culture to assess
cytotoxicity might be better at 33
°
C and 10% CO
2
(the
Control
Kir4.1 DAPI Merge
Ototoxicity
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 6: Changes in Kir4.1 expression in control and ototoxic mice. (a–d) Kir4.1 expression in control mice. (a) Expression of Kir4.1 in
stria vascularis (StV) and outer hair cells (OHC) of control mice. (b) Pattern of staining with 4′,6-diamidino-2-phenylindole (DAPI). (c)
Merged image. (d) High expression of Kir4.1 in OHC. (e–h) Kir4.1 expression in ototoxic mice. (e) Expression of Kir4.1 in StV and
OHC of ototoxic mice. (f) Pattern of DAPI staining. (g) Merged image. (h) Organ of Corti displayed decreased intensity expression of
Kir4.1 in OHC.
Kir4.1 DAPI Merge
ControlOtotoxicity
(a) (b) (c)
(d) (e) (f)
OHC
IHC
OHC
IHC
Figure 7: Changes in expression of Kir4.1 ex vivo. Kir4.1 expression in outer hair cells and inner hair cells (OHC) in cochlear explants of
control and ototoxic mice. (a–c) Explant of the control mouse. (a) Kir4.1 is strongly expressed in OHC. (b) Upon DAPI staining, cell loss
was observed. (d–f) Explant of ototoxic mouse. (d) The expression of Kir4.1 in OHC was decreased compared to the control. (e) Upon DAPI
staining, cell loss was observed in OHC.
8 BioMed Research International

permissive condition for proliferation) than at 39
°
C and 5%
CO
2
(the nonpermissive condition for differentiation) [25].
In the nonpermissive condition, cells display similar charac-
teristics of adult mouse hair cells in the inner ear. However,
most researchers have selected the permissive condition
because it permits an examination of whether cell death
occurred due to spontaneous apoptosis or drug-related cyto-
toxicity. The present study developed the culture condition
for the ototoxicity experiments during 5 days of growth at
33
°
C followed by 5 days at 39
°
C. The ion channels we ana-
lyzed in HEI-OC1 cells displayed different characteristics
in the permissive and the nonpermissive conditions. In one
study, Kir4.1 channels could induce cell maturation charac-
terized by a shift of the cells from G2/M phase to the G0/G1
phase, which involved membrane hyperpolarization [32].
We presently utilized the nonpermissive condition for the
experiments examining the ion channels of hair cells that
were similar to adult differentiated hair cells. The K
+
NKCC1KCNQ4
Control
(a) (b) (c) (d)
(e) (f) (g) (h)
Ototoxicity
Figure 8: Changes of NKCC1 and KCNQ4 expression in control and ototoxic mice. The changes of NKCC1 and KCNQ4 expression in the
outer hair cells (OHC), stria vascularis (StV), and spiral ganglion (SG) of control mice and ototoxic mice. (a–d) NKCC1 expression in
control and ototoxic mice. (a) StV, OHC, and SG displayed strong expression of NKCC1. (b) Magnified image of the organ of Corti
revealing the strong expression of NKCC1 in OHC. (c) NKCC1 expression of StV, OHC, and SG are decreased compared to control. (d)
Magnified image of the organ of Corti revealing the significant decrease of NKCC1 in OHC. (e–h) KCNQ4 expression in an ototoxic
mouse. (e) OHC and SG display strong expression of KCNQ4. (f ) Magnified image of the organ of Corti revealing high-intensity
fluorescence of KCNQ4 expressed in the cell body part of the OHC. (g) KCNQ4 expression in OHC and SG is decreased compared with
control. (h) Magnified image of the organ of Corti revealing the significant decrease in fluorescence intensity of KCNQ4 in OHC.
NKCC1
KCNQ4
Kir4.1
(a)
Kir4.1
KCNQ4 Kanamycin
NKCC1 Furosemide
(b)
Figure 9: Correlation of Kir4.1, NKCC1, and KCNQ4 in HEI-OC1 cells. Correlation between the main potassium channels Kir4.1, NKCC1,
and KCNQ4. When NKCC1 is blocked, the Kir4.1 channel has compensatory action and allows increased influx of potassium into cells.
Kir4.1 is normally an inwardly rectifying K
+
potassium channel. However, when the KCNQ4 channel is blocked, Kir4.1 channel allows
efflux of K
+
instead of through the KCNQ4 channel.
9BioMed Research International

channels of the hair cells were typically more strongly
expressed at 39
°
Cthanat33
°
C. When cultured at 33
°
C for
less than 5 days and then transferred to 39
°
C, Kir4.1 was
not expressed constantly at 39
°
C. For this reason, we cul-
tured the cells at 33
°
C for a certain period, shifted the cells
to 39
°
C, and continued the cell culture.
The inward rectifier K
+
channel family has more than
20 members. They comprise seven subtypes (designated
Kir1.x–7.x) [33]. Kir4.1 in the cochlea is expressed only in
the intermediate cells of the stria vascularis in the cochlear
lateral wall [12, 34, 35]. Liu et al. [14] studied the Kir4.1 tran-
scriptome, which reflects the genes that are being actively
expressed in a cell, in the inner hair cells and outer hair cells
of CBA/J mice. Kir4.1 had a relatively higher level of expres-
sion in both inner and outer hair cells. Jin et al. reported [36]
that in guinea pig cochlea, KCNJ10 immunoreactivity was
detected in strial intermediate cells, Deiters’cells, pillar cells
in the organ of Corti, and spiral ganglion satellite cells. The
observations are consistent with the view that although the
role of Kir4.1 in the lateral wall of the cochlea is to facilitate
the entry of K
+
into the scala media, Kir4.1 of hair cells could
have an important role in recycling K
+
from the hair cells to
the perilymphatic space [15]. To explore this possibility, we
studied the presence of Kir4.1 in HEI-OC1 cells and found
that the combination of kanamycin and furosemide directly
affected ion channels in hair cells and caused ototoxicity.
We screened ion channels that are commonly expressed in
the kidney and inner ear. K
+
channels were thought to be
important in maintaining the endocochlear potential of the
cochlea. Therefore, three K
+
channels (Kir4.1, NKCC1, and
KCNQ4) among the channels screened in HEI-OC1 cells
were predicted to be mainly responsive to ototoxic drugs.
We targeted these three channels. The ion and water trans-
port functions in the inner ear help maintain the proper
endolymph K
+
concentration required for hair cell function.
NKCC1 is also a K
+
cotransporter protein that plays an
important role in K
+
recycling [11]. The survival of the outer
hair cell critically depends on a specificK
+
conductance
mediated by KCNQ4 (Kv7.4) channels [37, 38]. The amino-
glycoside antibiotics gentamicin and neomycin inhibit
KCNQ4 channels in cochlear outer hair cells by depleting
phosphatidylinositol [4, 5] bisphosphate [39]. Therefore,
the Kir4.1, NKCC1, and KCNQ4 K
+
channels were predicted
to respond to ototoxic drugs. Hence, we targeted these three
channels.
The single injection of the combination of kanamycin
and furosemide is a novel technique for inducing ototoxicity
in several models [24, 40, 41]. Abbas and Rivolta [41] used
400 to 500 mg/kg kanamycin, followed by an intraperitoneal
injection of furosemide 100 mg/kg after 20 to 30 min in
gerbils. Ju et al. [24] used various doses of kanamycin
(420–600 mg/kg) with 130 mg/kg furosemide in C57BL/6
mice. Knowledge of the mechanism of ototoxicity induced
by the combination of kanamycin and furosemide is impor-
tant for an understanding of the maintenance of EP. How-
ever, the mechanism remains unclear. Presently, kanamycin
(<1 mM) and furosemide (50 μM) did not kill HEI-OC1 cells.
The concentrations were determined based on experiments
in mice. We think this was because of ion channel compensa-
tion. After treatment with siRNA to reduce KCNJ10 gene
and, thus, disable the most important channel, cytotoxicity
was confirmed by immediate response to the dose of ototoxic
drugs. This suggests that the combined ototoxic drug directly
affects hair cells and that Kir4.1 plays an important role in the
compensation. When mice were treated with kanamycin and
furosemide without any suppression of Kir4.1, hair cell death
and hearing loss were observed. In vivo kanamycin and furo-
semide may also affect Kir4.1 in the stria vascularis, resulting
in disruption of the balance of ion channels in the hair cells.
This study was limited as the use of HEI-OC1 cells does
not reflect the complex structures of cochlea in animals.
However, the use of HEI-OC1 cells did identify the impor-
tance of Kir4.1, suggesting a robust method for the ototoxic-
ity of drugs. Kir4.1 was sufficiently expressed in the culture
condition for ototoxicity experiments by a regimen involv-
ing cell growth for 5 days at 33
°
C followed by a shift to
39
°
C for 5 days. Kir4.1 in HEI-OC1 cells has a role in com-
pensating for the balance of potassium transport with
NKCC1 and KCNQ4. With the inhibition of Kir4.1, kana-
mycin 100 μM and furosemide 10 μM were lethal to cells.
Although the expression of Kir4.1 was decreased mainly in
the lateral wall of the cochlea in mice after injection of kana-
mycin and furosemide, Kir4.1 in outer hair cells was directly
injured by the drugs. The decrease of Kir4.1 in hair cells ren-
dered the cells vulnerable to death and hearing loss. Thus, if
the decrease in Kir4.1 in hair cells can be prevented, it may
be possible to prevent cytotoxicity caused by ototoxic drugs.
This will require further study.
Abbreviations
HEI-OC1 cells: House ear institute-organ of Corti 1 cells
Kir4.1: Inwardly-rectifying potassium channel
RT-PCR: Reverse transcriptase PCR
qRT-PCR: Quantitative real-time PCR
Kan: Kanamycin
Furo: Furosemide
StV: Stria vascularis
OC: Organ of Corti
SG: Spiral ganglion
OHC: Outer hair cell
IHC: Inner hair cell
RM: Reissner membrane.
Data Availability
All data generated or analyzed during this study are included
in this published article.
Conflicts of Interest
The authors declare that there is no conflict of interest
regarding the publication of this article.
Acknowledgments
This work was supported by the National Research Founda-
tion of Korea (NRF) grant funded by the Korea government
10 BioMed Research International

(MSIT) (No. NRF-2020R1A2C100978914) and the Korean
Fund for Regenerative Medicine funded by the Ministry of
Science and ICT and the Ministry of Health and Welfare
(21C0721L1, Republic of Korea).
Supplementary Materials
Supplementary Table 1: primer sequences. (Supplementary
Materials)
References
[1] P. Ganesan, J. Schmiedge, V. Manchaiah, S. Swapna,
S. Dhandayutham, and P. P. Kothandaraman, “Ototoxicity: a
challenge in diagnosis and treatment,”Journal of Audiology
and Otology, vol. 22, no. 2, pp. 59–68, 2018.
[2] E. A. Neuwelt, K. Gilmer Knight, and D. Kraemer, “Ototoxic-
ity in children receiving platinum chemotherapy: underesti-
mating a commonly occurring toxicity that may impact
academic and social development,”Journal of Clinical Oncol-
ogy, vol. 23, Supplement 16, pp. 8548–8548, 2005.
[3] L. P. Rybak, C. Whitworth, and V. Scott, “Comparative acute
ototoxicity of loop diuretic compounds,”European Archives
of Otorhinolaryngology, vol. 248, no. 6, pp. 353–357, 1991.
[4] L. E. Bryan and H. M. Van Den Elzen, “Effects of membrane-
energy mutations and cations on streptomycin and gentamicin
accumulation by bacteria: a model for entry of streptomycin
and gentamicin in susceptible and resistant bacteria,”Antimi-
crobial Agents and Chemotherapy, vol. 12, no. 2, pp. 163–177,
1977.
[5] Q. Wang and P. S. Steyger, “Trafficking of systemic fluorescent
gentamicin into the cochlea and hair cells,”Journal of the Asso-
ciation for Research in Otolaryngology, vol. 10, no. 2, pp. 205–
219, 2009.
[6] E. Hashino and M. Shero, “Endocytosis of aminoglycoside
antibiotics in sensory hair cells,”Brain Research, vol. 704,
no. 1, pp. 135–140, 1995.
[7] G. P. Richardson, A. Forge, C. J. Kros, J. Fleming, S. D. Brown,
and K. P. Steel, “Myosin VIIA is required for aminoglycoside
accumulation in cochlear hair cells,”Journal of Neuroscience,
vol. 17, no. 24, pp. 9506–9519, 1997.
[8] W. Marcotti, S. M. van Netten, and C. J. Kros, “The aminogly-
coside antibiotic dihydrostreptomycin rapidly enters mouse
outer hair cells through the mechano-electrical transducer
channels,”The Journal of Physiology, vol. 567, no. 2, pp. 505–
521, 2005.
[9] J. R. Waguespack and A. J. Ricci, “Aminoglycoside ototoxicity:
permeant drugs cause permanent hair cell loss,”The Journal of
Physiology, vol. 567, no. 2, pp. 359-360, 2005.
[10] D. Dalian, L. Hong, Q. Weidong et al., “Ototoxic effects and
mechanisms of loop diuretics,”Journal of Otology, vol. 11,
no. 4, pp. 145–156, 2016.
[11] A. A. Zdebik, P. Wangemann, and T. J. Jentsch, “Potassium
ion movement in the inner ear: insights from genetic disease
and mouse models,”Physiology, vol. 24, no. 5, pp. 307–316,
2009.
[12] J. Chen and H. B. Zhao, “The role of an inwardly rectifying
K+ channel (Kir4.1) in the inner ear and hearing loss,”
Neuroscience, vol. 265, pp. 137–146, 2014.
[13] N. Rozengurt, I. Lopez, C. S. Chiu, P. Kofuji, H. A. Lester, and
C. Neusch, “Time course of inner ear degeneration and deaf-
ness in mice lacking the Kir4. 1 potassium channel subunit,”
Hearing Research, vol. 177, no. 1-2, pp. 71–80, 2003.
[14] H. Liu, J. L. Pecka, Q. Zhang, G. A. Soukup, K. W. Beisel, and
D. Z. Z. He, “Characterization of transcriptomes of cochlear
inner and outer hair cells,”Journal of Neuroscience, vol. 34,
no. 33, pp. 11085–11095, 2014.
[15] P. Wangemann, “K
+
cycling and the endocochlear potential,”
Hearing Research, vol. 165, no. 1-2, pp. 1–9, 2002.
[16] D. Bockenhauer, S. Feather, H. C. Stanescu et al., “Epilepsy,
ataxia, sensorineural deafness, tubulopathy, and KCNJ10
mutations,”New England Journal of Medicine, vol. 360,
no. 19, pp. 1960–1970, 2009.
[17] U. I. Scholl, M. Choi, T. Liu et al., “Seizures, sensorineural
deafness, ataxia, mental retardation, and electrolyte imbalance
(SeSAME syndrome) caused by mutations in KCNJ10,”Pro-
ceedings of the National Academy of Sciences, vol. 106, no. 14,
pp. 5842–5847, 2009.
[18] K. C. Patterson, V. E. Hawkins, K. M. Arps, D. K. Mulkey, and
M. L. Olsen, “MeCP2 deficiency results in robust Rett-like
behavioural and motor deficits in male and female rats,”
Human Molecular Genetics, vol. 25, pp. 3303–3320, 2016.
[19] S. E. Nwaobi, V. A. Cuddapah, K. C. Patterson, A. C. Randolph,
and M. L. Olsen, “The role of glial-specificKir4.1innormal
and pathological states of the CNS,”Acta Neuropathologica,
vol. 132, no. 1, pp. 1–21, 2016.
[20] L. A. Smith, C. Chen, N. Z. Lax, R. W. Taylor, D. Erskine, and
R. McFarland, “Astrocytic pathology in Alpers’syndrome,”
Acta Neuropathologica Communications, vol. 11, no. 1,
pp. 1–3, 2023.
[21] T. Pannicke, I. Landiev, A. Wurm et al., “Diabetes alters
osmotic swelling characteristics and membrane conductance
of glial cells in rat retina,”Diabetes, vol. 55, no. 3, pp. 633–
639, 2006.
[22] D. E. Rivera-Aponte, M. P. Méndez-González, A. F. Rivera-
Pagán et al., “Hyperglycemia reduces functional expression
of astrocytic Kir4. 1 channels and glial glutamate uptake,”Neu-
roscience, vol. 310, pp. 216–223, 2015.
[23] J. Proft and N. Weiss, “Rectifying rectifier channels in
Huntington disease,”Communicative & Integrative Biology,
vol. 7, no. 4, article e29410, 2014.
[24] H. M. Ju, S. H. Lee, J. S. Choi, and Y. J. Seo, “A Simple Model
for Inducing Optimal Increase of _SDF-1_ with Aminoglyco-
side Ototoxicity,”BioMed Research International, vol. 2017,
Article ID 4630241, 9 pages, 2017.
[25] G. Kalinec, P. Thein, C. Park, and F. Kalinec, “HEI-OC1 cells
as a model for investigating drug cytotoxicity,”Hearing
Research, vol. 335, pp. 105–117, 2016.
[26] G. M. Kalinec, P. Webster, D. J. Lim, and F. Kalinec, “A
cochlear cell line as an in vitro system for drug ototoxicity
screening,”Audiology and Neurotology, vol. 8, no. 4, pp. 177–
189, 2003.
[27] P. Wangemann, K. Nakaya, T. Wu et al., “Loss of cochlear
HCO3- secretion causes deafness via endolymphatic acidifica-
tion and inhibition of Ca2+ reabsorption in a Pendred syn-
drome mouse model,”American Journal of Physiology. Renal
Physiology, vol. 292, no. 5, pp. F1345–F1353, 2007.
[28] P. Wangemann, “
K⁺
cycling and its regulation in the
cochlea and the vestibular labyrinth,”Audiology and Neurotol-
ogy, vol. 7, no. 4, pp. 199–205, 2002.
[29] H. Hibino, Y. Horio, A. Inanobe et al., “An ATP-dependent
inwardly rectifying potassium channel, KAB-2 (Kir4. 1), in
11BioMed Research International

cochlear stria vascularis of inner ear: its specific subcellular
localization and correlation with the formation of endoco-
chlear potential,”The Journal of Neuroscience, vol. 17, no. 12,
pp. 4711–4721, 1997.
[30] P. Kofuji, P. Ceelen, K. R. Zahs, L. W. Surbeck, H. A. Lester,
and E. A. Newman, “Genetic inactivation of an inwardly recti-
fying potassium channel (Kir4. 1 subunit) in mice: phenotypic
impact in retina,”Journal of Neuroscience, vol. 20, no. 15,
pp. 5733–5740, 2000.
[31] Y. V. Kucheryavykh, L. Y. Kucheryavykh, C. G. Nichols et al.,
“Downregulation of Kir4. 1 inward rectifying potassium chan-
nel subunits by RNAi impairs potassium transfer and gluta-
mate uptake by cultured cortical astrocytes,”Glia, vol. 55,
no. 3, pp. 274–281, 2007.
[32] H. Higashimori and H. Sontheimer, “Role of Kir4. 1 channels
in growth control of glia,”Glia, vol. 55, no. 16, pp. 1668–1679,
2007.
[33] F. Reimann and F. M. Ashcroft, “Inwardly rectifying potas-
sium channels,”Current Opinion in Cell Biology, vol. 11,
no. 4, pp. 503–508, 1999.
[34] Y. P. Liu and H. B. Zhao, “Cellular characterization of Con-
nexin26 and Connnexin30 expression in the cochlear lateral
wall,”Cell and Tissue Research, vol. 333, no. 3, pp. 395–403,
2008.
[35] X. H. Wang, M. Streeter, Y. P. Liu, and H. B. Zhao, “Identifica-
tion and characterization of pannexin expression in the mam-
malian cochlea,”Journal of Comparative Neurology, vol. 512,
no. 3, pp. 336–346, 2009.
[36] Z. Jin, D. Wei, and L. Järlebark, “Developmental expression
and localization of KCNJ10 K+ channels in the guinea pig
inner ear,”Neuroreport, vol. 17, no. 5, pp. 475–479, 2006.
[37] R. Nouvian, J. Ruel, J. Wang, M. J. Guitton, R. Pujol, and J. L.
Puel, “Degeneration of sensory outer hair cells following phar-
macological blockade of cochlear KCNQ channels in the adult
guinea pig,”European Journal of Neuroscience, vol. 17, no. 12,
pp. 2553–2562, 2003.
[38] T. Kharkovets, K. Dedek, H. Maier et al., “Mice with altered
KCNQ4 K+ channels implicate sensory outer hair cells in
human progressive deafness,”The EMBO Journal, vol. 25,
no. 3, pp. 642–652, 2006.
[39] M. G. Leitner, C. R. Halaszovich, and D. Oliver, “Aminoglyco-
sides inhibit KCNQ4 channels in cochlear outer hair cells via
depletion of phosphatidylinositol(4,5)bisphosphate,”Molecu-
lar Pharmacology, vol. 79, no. 1, pp. 51–60, 2011.
[40] T. Nakagawa, T. S. Kim, N. Murai et al., “A novel technique for
inducing local inner ear damage,”Hearing Research, vol. 176,
no. 1-2, pp. 122–127, 2003.
[41] L. Abbas and M. N. Rivolta, “Aminoglycoside ototoxicity and
hair cell ablation in the adult gerbil: a simple model to study
hair cell loss and regeneration,”Hearing Research, vol. 325,
pp. 12–26, 2015.
12 BioMed Research International