Hypoxic mesenchymal stem cells ameliorate acute kidney ischemiareperfusion injury via enhancing renal tubular autophagy

Abstract for Hypoxic mesenchymal stem cells ameliorate acute kidney ischemiareperfusion injury via enhancing renal tubular autophagy

Background:

Hypoxic mesenchymal stem cells ameliorate acute kidney ischemiareperfusion injury via enhancing renal tubular autophagy Acute kidney injury (AKI) is an emerging global healthcare issue without effective therapy yet. Autophagy
recycles damaged organelles and helps maintain tissue homeostasis in acute renal ischemia-reperfusion (I/R) injury.
Hypoxic mesenchymal stem cells (HMSCs) represent an innovative cell-based therapy in AKI. Moreover, the conditioned
medium of HMSCs (HMSC-CM) rich in beneficial trophic factors may serve as a cell-free alternative therapy. Nonetheless,
whether HMSCs or HMSC-CM mitigate renal I/R injury via modulating tubular autophagy remains unclear.

Methods:

Renal I/R injury was induced by clamping of the left renal artery with right nephrectomy in male Sprague-
Dawley rats. The rats were injected with either PBS, HMSCs, or HMSC-CM immediately after the surgery and sacrificed 48
h later. Renal tubular NRK-52E cells subjected to hypoxia-reoxygenation (H/R) injury were co-cultured with HMSCs or
treated with HMSC-CM to assess the regulatory effects of HSMCs on tubular autophagy and apoptosis. The association of
tubular autophagy gene expression and renal recovery was also investigated in patients with ischemic AKI.

Result:

HMSCs had a superior anti-oxidative effect in I/R-injured rat kidneys as compared to normoxia-cultured
mesenchymal stem cells. HMSCs further attenuated renal macrophage infiltration and inflammation, reduced tubular
apoptosis, enhanced tubular proliferation, and improved kidney function decline in rats with renal I/R injury. Moreover,
HMSCs suppressed superoxide formation, reduced DNA damage and lipid peroxidation, and increased anti-oxidants
expression in renal tubular epithelial cells during I/R injury. Co-culture of HMSCs with H/R-injured NRK-52E cells also
lessened tubular cell death. Mechanistically, HMSCs downregulated the expression of pro-inflammatory interleukin-1β,
proapoptotic Bax, and caspase 3. Notably, HMSCs also upregulated the expression of autophagy-related LC3B, Atg5 and
Beclin 1 in renal tubular cells both in vivo and in vitro. Addition of 3-methyladenine suppressed the activity
of autophagy and abrogated the renoprotective effects of HMSCs. The renoprotective effect of tubular
autophagy was further validated in patients with ischemic AKI. AKI patients with higher renal LC3B expression
were associated with better renal recovery.

Conclusion: The present study describes that the enhancing effect of MSCs, and especially of HMCSs, on
tissue autophagy can be applied to suppress renal tubular apoptosis and attenuate renal impairment during
renal I/R injury in the rat. Our findings provide further mechanistic support to HMSCs therapy and its
investigation in clinical trials of ischemic AKI.
Keywords: Acute kidney injury, Autophagy, Hypoxic mesenchymal stem cells, Ischemia-reperfusion injury
Introduction
Acute kidney injury (AKI), namely an abrupt decrease in
kidney function, is a frequent clinical syndrome with
incidence rates of approximately 7–18% of all hospital
inpatients and 20–200 per million population in the
community [1]. AKI is associated with increased medical
expenditure, higher morbidity, and considerable mortality
[2, 3]. Renal ischemia-reperfusion (I/R) injury is a
common cause of AKI and occurs in various clinical settings
including shock, major surgeries, trauma, and sepsis
[4, 5]. The interruption of renal blood flow (ischemia)
and subsequent reperfusion result in a mismatch of local
tissue oxygen supply and demand as well as imbalance
of nutrient delivery and waste removal in the cells of the
kidney [5]. This imbalance initiates renal cell death, oxidative
stress, inflammation, and subsequent organ dysfunction
[5, 6]. The extensive molecular degeneration
caused by oxidative stress further raises the levels of
DNA oxidation and lipid peroxidation byproducts which
are implicated in the progression of acute renal cell injury
[7]. The proximal tubule is the main injured site
during ischemia in mammalian kidneys, and the death of
injured proximal tubular epithelial cells constitutes the
primary feature of renal I/R injury and the subsequent
loss of renal function [8]. During the I/R injury,
damaged renal tubular epithelial cells also generate proinflammatory
and chemotactic cytokines, thereby further
aggravating the extent of renal inflammation and injury
[5, 6, 8]. Although great efforts have been made to
improve the treatment of AKI, there are still no proven
effective therapeutic agents found to be protective in
AKI [3]. Therefore, to develop an innovative therapeutic
approach for ischemic AKI remains an unmet medical
need.
Autophagy is a highly conserved lysosomal degradation
and recycling process of damaged or superfluous
organelles and macromolecules [9]. Autophagy
prevents cell damage and promotes cell survival in response
to energy shortage and cytotoxic insults [9].
Dysregulated autophagy contributes to the pathogenesis
of various human pathologies, ranging from cancer,
neurodegenerative disorders to kidney diseases [9,
10]. Recently, autophagy has been implicated in the
pathophysiology of AKI [10]. Proximal tubule-specific deletion
of autophagy-related gene 5 (Atg5) in mice increases
the susceptibility to I/R-induced renal injury, and worsens
tubular apoptosis and renal impairment [11]. Mice with targeted
deletion of another autophagy associated gene Atg7
are also more sensitive to renal I/R injury than their wildtype
littermates [12]. Collectively, autophagy plays a renoprotective
role during renal I/R injury and strategies to enhance
the autophagy may be a novel therapeutic approach
to treat AKI.
Mesenchymal stem cells (MSCs) represent a new frontier
of therapeutic strategies to repair various kinds of
kidney injury for their anti-oxidative, anti-inflammatory
and tissue repair properties [13–18]. The therapeutic effects
of the MSCs can be mediated by direct “homing”
to the injured organs and transdifferentiation into injured
cells or by secretion of beneficial factors that exert
an indirect paracrine/endocrine effect on damaged cells
and tissue [16, 17]. Although MSCs have demonstrated
a renoprotective effect in AKI, detailed mechanism by
which MSCs lessen renal I/R injury is still incompletely
understood [16, 17]. Many challenges still exist while
applying the MSCs into clinical AKI patients because
the efficacy of MSCs is influenced by several important
aspects including administration routes and culture conditions
[19–21]. Direct intra-arterial delivery that bypasses
the lung can increase the number of accumulated
MSCs in the I/R-injured kidneys [22], but whether this
increased retention of MSCs confers a better renoprotection
remains unclear. Furthermore, MSCs lose the
proliferative and trophic potency over time during culture
[23]. Preconditioning of the MSCs by hypoxia before
the therapeutic application increases their secretion
of beneficial trophic factors and augments the antioxidative
property [20, 24–27]. Our previous works have
found that hypoxic mesenchymal stem cells (HMSCs)
can circumvent replicative senescence, enhance the proliferation
rate and differentiation potential, and secrete
more anti-inflammatory cytokines in vitro [28, 29].
Moreover, the hypoxic culture also increases the engraftment
of MSCs, enhances angiogenesis, and prevents
limb amputation in mice with hindlimb ischemia as
compared to normoxic MSCs [30], suggesting that the
hypoxia-preconditioning may be also a promising
approach for better therapeutic benefits of MSCs in ischemic
AKI. Recently, MSCs are reported to enhance
the autophagy, promote β-amyloid clearance and increase
the viability of co-cultured β-amyloid-treated
neuronal cells [31]. Moreover, administration of MSCs
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 2 of 22
also increases the autophagy level and decrease pulmonary
cell death in mice with acute lung injury and cocultured
pulmonary micro-vascular endothelial cells
[32]. Till now, whether MSCs mitigate renal I/R injury
via modulating tubular autophagy has not yet been
elucidated.
The present study aimed to investigate whether
hypoxic culture, homing/paracrine actions, and different
administration routes influence the renoprotective efficacy
of MSCs in ischemic AKI. We found that HMSCs
had a superior anti-oxidative effect than normoxiacultured
MSCs in renal I/R injury. We further demonstrated
that intra-renal arterial administration of HMSCs
attenuated inflammation and tubular cell apoptosis and
kidney function decline in renal I/R injury through
upregulation of renal tubular autophagy. The protective
effect of tubular autophagy in I/R injury was further
confirmed in patients with AKI. This observation
provides mechanistic support of HMSCs therapy in renal
I/R injury through modulating tubular autophagy and
helps apply HMSCs in treating AKI patients.
Materials and methods
Isolation and culture of HMSCs
MSCs were isolated from 7-week-old male Sprague-
Dawley rats as described previously [33]. Briefly, the
ends of each tibia and femur were removed to expose
the marrow. Thereafter, the bones were inserted into
adapted centrifuge tubes and centrifuged at 400 g for 1
min to collect the marrow. The cell pellet of the marrow
was then resuspended in 3 mL of complete medium [α-
MEM (α-minimal essential medium; Gibco, Gaithersburg,
MD), supplemented with 16.6% fetal bovine serum
(FBS, ThermoFisher, Waltham, MA), 100 units/mL penicillin,
100 μg/mL streptomycin, and 2 mM L-glutamine],
filtered through a 70-μm nylon mesh filter, and seeded
in dishes. After 24 h, non-adherent cells were washed
away with phosphate-buffered saline (PBS), and 10 mL
of fresh complete medium was added. After reaching
subconfluence around 2 weeks later, adherent cells were
washed with PBS and detached by incubation in 4 mL of
0.25% trypsin/1 mM ethylenediaminetetraacetic acid
(Invitrogen, Carlsbad, CA) at 37 °C for 2 min. The cells
were then expanded by plating at 100 cells/cm2 and
grown in the complete medium which was refreshed
twice per week.
The normoxia-cultured MSCs were expanded at 37 °C
in a humidified incubator (model 3130; ThermoFisher)
containing 74% N2, 5% CO2, and 21% O2. For hypoxic
culture, HMSCs were cultured in a gas mixture composed
of 94% N2, 5% CO2, and 1% O2. The hypoxic gas
mixture was maintained by a compact gas oxygen controller
(ProOx P110, BioSpherix, Lacona, NY) supplied
with N2 gas. If the O2 concentration rose above the
desired level, N2 gas was automatically delivered into the
system to displace the excess O2.
For collection of condition medium (CM), HMSCs
were plated at 5000 cells/cm2 and incubated in the
complete medium for one day. Afterwards, the cells were
washed three times with PBS, and the complete medium
was replaced with serum-free α-MEM to generate
HMSC-CM. The HMSC-CM was collected after 48 h of
culture, centrifuged at 2000 rpm for 10 min, and passed
through a 0.2-μm filter. The HMSC-CM was further
concentrated using centrifugal filter units with 5 kDa
cut-off (Millipore, Billerica, MA).
Characterization of HMSCs
To analyze the expression levels of stem cell surface
marker proteins, HMSCs were harvested by TryPLE
Select (ThermoFisher). Afterwards, 1 × 106 cells were
incubated with either fluorescein isothiocyanate (FITC)-
or phycoerythrin-conjugated antibodies against mouse
CD11b (BioLegend, San Diego, CA), CD29 (BioLegend),
CD44 (BioLegend), CD45 (BioLegend), CD73 (Bioss,
Woburn, MA), CD90 (BioLegend) and CD105 (Bioss).
To analyze CD31 expression, the HMSCs were stained
with rabbit anti-mouse anti-CD31 antibody (Santa Cruz
Biotechnology, Santa Cruz, CA) followed by FITCconjugated
goat anti-rabbit secondary antibody (Jackson
ImmunoResearch, West Grove, PA). Ten thousand labeled
cells were acquired and analyzed using a FACS
Canto II flow cytometer (BD Biosciences, San Diego, CA).
Differentiation of MSCs was induced as previously
described with slight modification [33]. For in vitro
differentiation into osteoblasts and adipocytes, cells were
seeded at a density of 1 × 104/cm2 and induced in osteoblast
induction medium [α-MEM supplemented with
10% FBS, 10−8 M dexamethasone (Sigma-Aldrich, St
Louis, MO), 10 mM β-glycerol phosphate (Sigma-Aldrich)
and 50 μM ascorbate-2-phosphate (Sigma-Aldrich)]
and adipocyte induction medium [α-MEM
supplemented with 10% FBS, 10−7 M dexamethasone, 50
μM indomethacin (Sigma-Aldrich), 0.45 mM 3-isobutyl-
1-methyl-xanthine (Sigma-Aldrich) and 10 μg/mL insulin
(Sigma-Aldrich)] for 2 weeks. For in vitro differentiation
into chondrocytes, 5 × 107 cells were centrifuged
(300×g, 4 °C) to form a high-density micromass culture
pellet. The pellet was then continuously cultured in
chondrocyte induction medium [serum-free α-MEM
supplemented with 10−7 M dexamethasone, 1% (vol/vol)
insulin-transferrin-selenium premix (Corning Inc., Corning,
NY), 50 μM ascorbate-2-phosphate, 40 μg/mL (wt/
vol) proline (Sigma-Aldrich), and 10 ng/mL (wt/vol)
transforming growth factor-β1 (R&D systems, Minneapolis,
MN)] for 3 weeks. After the appearance of morphological
features of differentiation, cells treated in the
induction medium of osteoblasts and adipocytes were
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 3 of 22
stained with alizarin red S (Sigma-Aldrich) and oil red O
(Sigma-Aldrich), respectively. The cell pellet cultured in
the chondrocyte induction medium was prepared in paraffin
sections, stained with Alcian blue (Millipore), and
counterstained with nuclear fast red (Sigma-Aldrich).
Animal model of renal I/R injury
Adult Sprague-Dawley rats (male, 8 weeks old, approximately
300 g) were purchased from the Laboratory Animal
Center of the National Yang-Ming University (Taipei,
Taiwan) and raised in a sound-attenuated, temperaturecontrolled
(22 ± 1 °C) room with a 12-h light/dark cycle.
Standard rodent chow and drinking water were supplied
ad libitum. The renal I/R injury model was performed as
previously described [34, 35]. Briefly, for induction of total
ischemia in the kidney, the left renal artery was clamped
with a nontraumatic vascular clamp for 45 min. Afterward,
reperfusion was initiated by releasing the clamp, and the
right kidney was removed simultaneously. The intraoperative
body temperature, cardiac function, and respiratory
rates and pattern of the rats were monitored closely. The
respiratory rates and pattern were assessed by visual observation.
The cardiac function was assessed by monitoring
heart rate, mucous membrane color, and capillary refill
time. The core temperature was measured by a rectal
thermometer. During the surgery, the rats were kept on a
heating pad to maintain the core temperature between
36.5 and 37 °C. After surgery, either PBS, MSCs (5 × 105
cells) or HMSCs (5 × 105 cells) were administered via
intra-renal arterial (IA) or intraperitoneal routes (IP). To
investigate the paracrine effect, concentrated HMSC-CM
was injected intraperitoneally. Sham-operated animals
underwent similar operative procedures without receiving
clamping of the left renal artery and right nephrectomy.
The rats were humanely euthanized by decapitation under
anesthesia 48 h later to collect the blood and kidney tissue
samples for further analyses. Serum levels of blood urea nitrogen
(BUN) and creatinine were determined by an Olympus
AU-2700 autoanalyzer (Olympus Ltd, Tokyo, Japan).
Because estimating the effect size of stem cell therapy is
difficult, the conventional sample size calculation cannot
be applied. We instead utilized the “resource equation”
method, which is commonly used in experimental animal
studies [36]. All experimental procedures conformed to the
Guide for the Care and Use of Laboratory Animals
published by the National Institutes of Health. The study
was approved by the Institutional Animal Care and Use
Committee of the National Yang-Ming University and the
Taipei Veterans General Hospital under the license numbers
of 991261 and 2017-075, respectively.
Tracking analyses of HMSCs in vivo
The HMSCs were labeled with a commercial PKH67
staining kit (PKH67GL-1KT, Sigma-Aldrich), according
to the manufacturer’s instructions. Briefly, after washed
by PBS, HMSCs were mixed with PKH67 solution and
incubated for 5 min at room temperature. Unbound
PKH67 molecules were quenched by adding 2 mL of
10% bovine serum albumin, and then the cell suspension
was centrifuged. The supernatant was discarded and the
PKH67-labeled HMSCs were resuspended in PBS for
further use. Immediately after the renal I/R injury, the
PKH67-labelded HMSCs were administered via either
IA or IP route. The I/R-injured rats were sacrificed 48 h
later and the kidneys were harvested. After embedded in
the optimal cutting temperature compound, the kidney
tissues were cut into 4-μm sections, fixed by 4% paraformaldehyde
for 15 min at room temperature, and then
counterstained with 4′, 6-diamidino-2-phenylindole
(DAPI, Sigma-Aldrich). The emission of PKH67 was detected
by using the standard filter setup for FITC.
Histology analysis and immunohistochemical staining
The kidney tissue was fixed with 4% phosphate-buffered
formalin, embedded in paraffin block, and cut into 4-μm
sections. Sections were stained with periodic acid-Schiff
(PAS) as previously described [34]. Tubular injury (denudation
of tubular cells, loss of brush border, flattening
of tubular cells, formation of intratubular casts) was
scored from 0 to 4 (0, no changes; 1, changes affecting <
25%; 2, changes affecting 25 to 50%; 3, changes affecting
50 to 75%; 4, changes affecting 75 to 100% of the section).
Twenty randomly selected non-overlapping highpower
fields (× 40 objective) were scored for each rat
and the average for each group were then analyzed [34].
All scoring was performed in a blinded fashion.
Immunohistochemical staining was performed on
formalin-fixed paraffin-embedded sections of kidneys as
previously [37]. Briefly, after deparaffinization by xylene
and rehydration by graded alcohol, consecutive 4-μm
sections of kidneys were subjected to heat antigen retrieval
in a microwave oven (650 W, 12 min) in 10 mM sodium
citrate buffer (pH 6.0). Afterwards, endogenous peroxidase
activity was quenched by 3% hydrogen peroxide for 10
min. Thereafter, tissue sections were incubated with the
primary antibodies at 4 °C overnight and then with secondary
antibody (Envision+Dual Link System-HRP, Dako,
Glostrup, Denmark) for 30 min at room temperature. Signals
were developed with diaminobenzidine and counterstained
with Gill’s hematoxylin. Twenty randomly selected
non-overlapping high-power fields (× 40 objective) at the
renal cortex were evaluated for each mouse. Analysis of
the diaminobenzidine-positive area was carried out using
Image J with the “Threshold Colour” plug-in (version
1.16, https://imagejdocu.tudor.lu/plugin/color/threshold_
colour/start#threshold_colour) as previously reported
[37]. Details of the antibodies used in the immunohistochemistry
were listed in the Supplemental Table S1.
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 4 of 22
Renal reactive oxygen species (ROS) detection
ROS formation in the I/R-injured kidneys was determined
by in vivo detection of 2-methyl-6-(p-methoxyphenyl)-
3,7-dihydroimidazo [1,2-alpha]pyrazin-3-one
(MCLA)-enhanced chemiluminescence from the kidney
surface and by ex vivo detection of lucigenin-enhanced
chemiluminescence from the kidney homogenate as described
previously with slight modifications [34, 38].
Both MCLA-enhanced and lucigenin-enhanced chemiluminescence
are widely used as an indicator of superoxide
production and have been utilized to estimate
superoxide generation in tissue homogenates [38–40].
For in vivo detection of superoxide, the rats were anesthetized,
kept on a heating pad to maintain the body
temperature between 36.5 and 37 °C, and put in a dark
box with a shielding plate. Only the renal window was
left unshielded to detect the photons from the exposed
kidney surface. MCLA (0.2 mg/mL/h, Cat. no. 87787,
Sigma-Aldrich) was continuously infused via the right
femoral vein and the PBS, MSCs, or HMSCs were administered
via an intra-renal arterial catheter through
the left femoral artery of I/R-injured rats. MCLAenhanced
chemiluminescence signals from the I/R-injured
or sham-operated kidneys were measured continuously in
a Chemiluminescence Analyzing System (CLD-110; Tohoku
Electronic Industrial Co., Sendai, Japan). For ex vivo
detection of renal ROS, the renal tissue samples were first
homogenized with PBS (50 mg/mL) and put in a completely
dark chamber of the Chemiluminescence Analyzing
System (CLD-110). After a 60-s determination of
background luminescence level, the superoxide levels in
the kidney homogenates were measured by adding 0.5 mL
of 0.1 mM lucigenin solution (pH 7.4, Sigma-Aldrich,
M8010) to the kidney homogenate solution (0.2 mL). The
lucigenin-enhanced chemiluminescence was then monitored
continuously for an additional 540 s. The total
amount of chemiluminescence was calculated by integrating
of the area under the 540-s chemiluminescence curve
after subtracting the background luminescence level. The
amount of ROS was expressed as chemiluminescence
counts/10 s.
In situ detection of superoxide by dihydroethidium (DHE)
staining
DHE staining was utilized to analyze the location of ROS
production as described previously with modifications
[34]. Briefly, 4-μm formalin-fixed paraffin-embedded
sections of kidneys were deparaffinized, rehydrated, and
subjected to heat antigen retrieval in a microwave oven
(650 W, 12 min) in 10 mM sodium citrate buffer (pH 6.0).
Endogenous peroxidase activity was quenched by 3%
hydrogen peroxide for 10 min. Afterward, the section was
incubated with mouse anti-rat CD68 antibody (GeneTex,
Irvine, CA) at 4 °C overnight. Then, the section was
incubated with FITC-conjugated secondary antibody
(Jackson ImmunoResearch) at room temperature for 2 h.
The section was stained with DHE (100 μM; Sigma-
Aldrich) at room temperature for 15 min and counterstained
with DAPI (Sigma-Aldrich).
Terminal deoxynucleotidyl transferase-mediated
digoxigenin-deoxyuridine triphosphate nick-end labeling
(TUNEL) assay
To detect the apoptosis in the kidney, TUNEL assay was
conducted using the Apoptag Peroxidase In Situ Apoptosis
Detection kit (Millipore) according to the manufacturer’s
instruction as previously described [34]. Apoptotic cells
were visualized by diaminobenzidine (Dako). The presence
of nuclear brown staining (TUNEL-positive) indicated
apoptotic cells. Negative controls were stained with omission
of the terminal deoxynucleotidyl transferase.
Immunoblotting
Protein of kidney tissue and cells was extracted by the
RIPA buffer containing 0.1% (v/v) protease inhibitor
(Pierce) and the western blot analysis was performed as
previously [37, 41]. Briefly, 50 μg of protein extract was
loaded to 12% SDS-PAGE gels and transferred to polyvinylidene
fluoride membranes. The membranes were incubated
at 4 °C overnight with primary antibodies and
then amplified by species-directed secondary antibodies.
Signals were developed with a West Femto Chemiluminescent
Substrate kit (Thermo Scientific, Hudson, NH).
Bands were visualized and quantified using a ChemiDoc-
It Imaging system (UVP, Cambridge, UK). Data were
normalized to β-actin expression. Details of the antibodies
used in the immunohistochemistry were listed in
the Supplemental Table S1.
Cell culture and in vitro hypoxia-reoxygenation model
Rat proximal renal tubular epithelial NRK-52E cells were
obtained from the Food Industry Research and Development
Institute (Hsinchu, Taiwan) and grown in Dulbecco’s
modified Eagle’s medium (DMEM) containing 4
mM L-glutamine, 1.5 g/L sodium bicarbonate, 4.5 g/L
glucose (Gibco), supplemented with 10% fetal bovine
serum (ThermoFisher). The cells were incubated at 37
°C in a 5% CO2 atmosphere and passaged twice a week.
In vitro hypoxia-reoxygenation model was performed as
previously described [34]. Briefly, NRK-52E cells were
plated in 35-mm dishes (Corning Inc.) at a density of 1
× 106 cells/dish for one day. For hypoxia experiments,
the cells were placed in a hypoxia C-chamber (Bio-
Spherix) inside a standard CO2 incubator (model 3130;
ThermoFisher) with a compact gas oxygen controller
(ProOx P110; BioSpherix) to maintain oxygen concentration
at 1% by introducing a gas mixture of 95% N2
and 5% CO2. After exposure to hypoxia for 24 h, the
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 5 of 22
cells were returned to normoxic conditions (21% O2/
74% N2/5% CO2) for reoxygenation for 6 h. Control cells
were incubated in a regular cell culture incubator with
21% O2 constantly. At the end of the study, normoxiacultured
and hypoxia-reoxygenation (H/R)-injured NRK-
52E cells were harvested with indicated buffers to collect
cell lysates for biochemical analyses.
To investigate the effect of HMSCs on H/R, NRK-52E
cells (5 × 105) in a six-well plate were washed by PBS
and then co-cultured with or without HMSCs (co-culture
ratio 1:1) by Transwell (8 μm, Millipore) during H/
R. For HMSC-CM treatment experiment, NRK-52E was
treated with one-fold HMSC-CM after subjected to H/R.
To suppress autophagy, 3-methyladenine (3-MA, 30
mM, Cayman Chemicals, Ann Arbor, MI) was added
into the culture medium of NRK-52E cells in the indicated
experiments.
Cell viability assay
NRK-52E cells were seeded into 24-well plates and cell
viability was measured by the colorimetric 3-[4,5-dimethylthiazol-
2-yl]-2,5-diphenyltetrazolium bromide (MTT)
assay as previously described [34]. After the NRK-52E
cells were subjected to an H/R injury, 10 μl of MTT solution
(5 mg/ml, Sigma-Aldrich) was added to each well and
incubated for 1 h at 37 °C. Wells were emptied and blue
formazan crystals were dissolved with 100 μl of MTT
solubilization solution (10% Triton X-100, 0.1 N HCl in 2-
propanol). The optical density was then analyzed at 570
nm using a TiterTek Multiskan microplate reader (Flow
Laboratories, McLean, VA).
Analysis of autophagy flux
Normoxia-cultured and H/R-injured NRK-52E cells were
co-cultured with HMSCs in the presence or absence of 3-
MA. Autophagic flux was measured by a Premo Autophagy
Tandem Sensor RFP-GFP-LC3B kit (Life Technologies,
Rockville, MD) according to the manufacturer’s
instructions. Fluorescence images were acquired after 48 h
of incubation and imaged using a ×63 objective on a confocal
fluorescence microscope (FV10i, Olympus). The
LC3B puncta of ten non-overlapping images were quantified
using the Image J software.
Analysis of autophagy-related gene expression in human
samples
Human kidney specimens were obtained from diagnostic
renal biopsies for patients with unexplained acute kidney
injury in the Taipei Veterans General Hospital, Taiwan,
from October, 2018, to October, 2019. Patients with
pathological diagnosis of ischemic acute tubular injury
were then enrolled in the study (n = 35). The renal biopsy
specimen was microdissected and the glomeruli
was removed. Subsequently, the total RNA from the
kidney tissue was extracted by a NEBNext UltraRNA
Library Prep Kit (New England Biolabs, Ipswich, MA).
The RNA with a RIN ≥ 7.0 were used for further RNA
sequencing. The cDNA libraries were constructed,
pooled, and sequenced on an Illumina NovaSeq 6000
platform with 150 bp pair-ended reads. Quality control
of the RNA-sequencing reads was through FastQC (version
0.11.8) and only reads with Phred quality scores ≥
30 were retained. Thereafter, adaptors and lower-quality
bases were trimmed by Trimmomatic (version 0.39) and
the trimmed reads were then aligned to the human reference
genome (GRCh38/hg38) with HISAT2 (version
2.2.1). The read counts of each sample were obtained
using featureCounts (version 2.0.1). Autophagy-related
gene expression was estimated as transcripts per million
(TPM) [42, 43]. Baseline demographics, comorbidity,
and laboratory data were recorded at the time of biopsy.
Thereafter, the patients were followed periodically until
February, 2021 to assess the recovery of renal function,
which was defined as the occurrence of a glomerular filtration
rate greater than or equal to the baseline value
during the follow-up period. The institutional review
board of the Taipei Veterans General Hospital approved
the study under the license number 2018-06-
008B and 2018-09-004C. All enrolled participants gave
their informed consents.
Statistical analysis
Categorical variables were expressed with numbers and
percentages, and the numerical variables were shown as
mean ± SEM for experimental data and as median plus
interquartile range for clinical data. Between-group comparisons
were determined by the Pearson’s chi-squared
test, Fisher’s exact test, Student’s t test, Wilcoxon rank
sum test, or one-way ANOVA with post hoc Tukey’s
test where appropriate. Correlation between the
autophagy-related gene and anti-oxidant genes was analyzed
by the Pearson’s correlation analysis. A two-tailed
p value < 0.05 was considered statistically significant. All
analyses were performed with the R software (version
4.0.4, R Foundation for Statistical Computing, Vienna,
Austria).
Results
Characterization of HMSCs
To study the therapeutic roles of HMSCs on renal I/R
injury, we first characterized the properties of the
HMSCs. The HMSCs were isolated from the bone
marrow of adult Sprague-Dawley rats, cultured at low
density in a hypoxic atmosphere, and differentiated as
previously described [33]. The rat HMSCs positively
expressed stem cell markers including CD29, CD44,
CD73, CD90, and CD105, but did not express
hematopoietic cell markers, such as CD11b, CD31, and
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 6 of 22
CD45 (Fig. 1a). The HMSCs were also able to differentiate
into osteoblastic, chondrocytic, and adipocytic
lineages (Fig. 1b).
HMSCs confer a superior anti-oxidative effect in rats with
I/R injury
Given that oxidative stress plays central roles in the progression
of acute renal I/R injury [7, 44], next, we compare
the anti-oxidative effects of HMSCs and normoxic
MSCs. The rat model of renal I/R injury was established
by clamping of the left renal artery for 45 min and right
nephrectomy, followed by 48 h of reperfusion as previously
described [34]. Either PBS, normoxic MSCs, or
HMSCs were injected via the left renal artery immediately
after the release of clamping. Because hypoxic
MSCs would encounter a physiologically normoxic
condition (21% O2) after intra-renal arterial (IA) or
intraperitoneal (IP) administration into the rats with I/R
injury, the beneficial gene expression while the HMSCs
were transferred from the hypoxic to normoxic condition
was first analyzed. We found that the hypoxiaderived
upregulation of beneficial genes in HMSCs such
as heparin-binding epidermal growth factor-like growth
factor (Hbegf) can last for 48 h after the HMSCs were
transferred to a normoxic atmosphere (Supplemental
Figure S1). To further confirm that the HMSCs actually
retained in the I/R-injured kidneys, the distribution of
PKH67-labeled HMSCs was tracked after IA or IP administration.
The green PKH67-labeled HMSCs can be
identified in the renal tubules 48 h after the administration
both in the IA and IP groups (Fig. 1c). The in vivo
analyses of oxidative stress showed that I/R injury induced
an abrupt increase of superoxide immediately
after reperfusion, while administration of normoxic
MSCs or HMSCs attenuated the increment of superoxide
(Fig. 2a). Notably, HMSCs, but not normoxic
MSCs, further suppressed the total oxidative content in
the I/R-injured kidney homogenate as compared to PBS
control (Fig. 2b). I/R injury resulted in severe renal tubular
injury including diffuse denudation of tubular cells,
tubular dilatation, and intratubular cast formation in the
PBS group as compared to the sham-operated group.
Intra-renal arterial administration of HMSCs decreased
the I/R injury-induced renal tubular injury and the
elevated blood urea nitrogen levels to a greater degree as
compared to MSCs treatment (Fig. 2c–f). These results
indicate that HMSCs have better anti-oxidative and
renoprotective effects for renal I/R injury than normoxic
MSCs.
Effect of HMSCs by different administration routes on
renal I/R injury
Substantially different outcomes have been observed
when the same stem cell therapy is administered by
different routes [21]. To further compare the efficacy between
the local and systemic delivery modes, HMSCs
were administered either intra-arterially through the left
renal artery (IA) or via the intraperitoneal route (IP) immediately
after I/R injury. I/R injury resulted in severe
renal tubular injury including sloughing and flattening of
tubular cells, tubular dilatation, and cast formation in
the PBS group as compared to the sham-operated group.
I/R injury also led to excessive ROS production and elevated
serum BUN and creatinine levels in the PBS
group. Both IA and IP administration of HMSCs limited
the tubular injury and ameliorated renal function
decline. Notably, only IA administration of HMSCs
suppressed the I/R injury-induced excessive ROS as
compared to the PBS group (Fig. 3a–e).
To further clarify whether the beneficial effects of
HMSCs in renal I/R injury were through the direct
homing or indirect paracrine effect, rats subjected to I/R
injury were intraperitoneally injected with different concentration
of HMSC-conditioned medium (HMSC-CM).
There was a dose-response relationship among HMSCCM
treatment groups and 100-fold concentrated HMSCCM
significantly reduced renal I/R injury (Fig. 3f–j). These
results suggest that HMSCs provide a renoprotective
effect via suppressing excess oxidative stress in renal I/R
injury.
HMSCs enhanced anti-oxidant expression and inhibited
superoxide generation in rats with I/R injury
Hypoxic culture during in vitro expansion has been
shown to enhance the anti-oxidative properties of the
MSCs [18]. Nonetheless, whether HMSCs ameliorate
oxidative injury by increasing the endogenous antioxidant
responses in renal I/R injury remains unclear.
To further decipher the anti-oxidative effect of HMSCs,
we then determined the production of superoxide in I/
R-injured rats treated with HMSCs or HMSC-CM. DHE
staining showed that the superoxide in the I/R-injured
rat kidneys was predominantly generated from the renal
tubular epithelial cells. Administration of HMSCs and
HMSC-CM attenuated the formation of superoxide in
the kidneys (Fig. 4a, c). We also determined the therapeutic
potential of HMSCs and HMSC-CM on the DNA
oxidation and lipid peroxidation products. I/R injury significantly
increased the expression of 8-hydroxy-2-deoxyguanosine
(8OHdG) and 4-hydroxynonenal (4-HNE) in
renal tubular epithelial cells. Administration of HMSCs
and HMSC-CM reduced these biomolecular damages induced
by I/R injury (Fig. 4b, d). Additionally, I/R injury
upregulated the expression of anti-oxidative nuclear factor
erythroid 2–related factor 2 (Nrf-2) expression in I/
R-injured rat kidneys as compared to the PBS group
(Fig. 4b, d). Notably, IA administration of HMSCs not
only further significantly enhanced the Nrf-2 expression
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 7 of 22
Fig. 1 (See legend on next page.)
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 8 of 22
but also increased heme oxygenase-1 (HO-1) and
superoxide dismutase 1 (SOD1) expression (Fig. 4e–i).
These data indicated that IA administration of HMSCs
significantly suppressed superoxide formation, reduced
DNA damage and lipid peroxidation, and enhanced antioxidants
expression in renal tubular epithelial cells during
I/R injury.
HMSCs suppress inflammation and apoptosis in I/Rinjured
rat kidneys
The damaged renal tubular epithelial cells are not
merely passive victims of I/R injury but also active
attackers that generate chemotactic cytokines to attract
inflammatory cells and perpetuate renal injury [5].
Macrophages are recruited early in the renal I/R injury
and contribute to the ensuing interstitial inflammation
and apoptosis of tubular cells [45]. Moreover, different
macrophage subpopulations are recently found to direct
the extent of tubular injury and repair [6]. Therefore, we
next determined the effects of HMSCs and HMSC-CM
treatment on the expression of inflammation markers
and apoptosis in I/R-injured rat kidneys. I/R injury significantly
increased the number of ED1-positive macrophages
in the renal interstitium. Meanwhile, I/R injury
also increased TUNEL-positive apoptotic renal tubular
cells. Both IA and IP injection of HMSCs markedly suppressed
ED1-positive macrophages and apoptotic tubular
cells in I/R-injured kidneys. The reduction of macrophage
infiltration and tubular apoptosis was lower in the
IA group and that in the IP group. Moreover, I/R injury
increased the interstitial infiltration of inducible nitric
oxide synthase-positive M1 macrophage but not arginase
1-positive M2 macrophages. Administration of HMSCs
or HMSC-CM significantly downregulated the M1 macrophage
infiltration but upregulated M2 macrophages infiltration.
Notably, HMSCs also augmented the expression
of PCNA-positive renal tubular cells in rat I/R-injured kidney
tissue (Fig. 5a–f). Moreover, the levels of apoptosisrelated
proteins in the I/R-injured kidneys were also
examined. I/R injury increased the expression of activated
Bax, caspase 3, caspase 1, and interleukin-1β but decreased
the expression of Bcl-2. Both HMSCs and HMSCCM
treatment suppressed the expression of activated
caspase 3 and interleukin-1β and decreased the Bax/Bcl-2
ratio in the I/R-injured kidneys. Nonetheless, only IA
injection of HMSCs downregulated the expression of
caspase 1 (Fig. 5g–l). These findings indicate that IA
injection of the HMSCs has more pronounced antiinflammatory,
anti-apoptotic, and pro-regenerative effects
in renal I/R injury.
HMSCs enhance autophagy expression in I/R-injured rat
kidneys
Mounting evidence indicates that autophagy is essential
to maintain the function of renal proximal tubules
during ischemic injury [10]. To further investigate
whether HMSCs ameliorate renal I/R injury by modulating
autophagy, we then determined the expression of
autophagy in rat kidneys with or without I/R injury.
Immunohistochemistry showed that the expression
levels of autophagy-related protein including LC3B,
Atg5, and Beclin 1 were increased in the renal tubular
cells of the I/R-injured rat kidneys by IA administration
of HMSCs (Fig. 6a, b). The western blot analysis also
confirmed that IA administration of HMSCs significantly
increased the expression of LC3B, Atg5, and Beclin 1.
Consistently, the level of autophagy adaptor protein p62,
which inversely correlates with autophagy activity, was
also downregulated in the I/R-injured rat kidneys by IA
injection of HMSCs (Fig. 6c, d). These results indicated
that HMSCs enhance renal tubular autophagy in rats
with renal I/R injury.
HMSCs ameliorate H/R-injured renal tubular cells through
upregulating autophagy
To further confirm that HMSCs improve renal I/R
injury via upregulating autophagy, we conducted an
in vitro H/R injury model to simulate rats with I/R injury
as previously described [34]. After being subjected
to hypoxia for 24 h with subsequent reoxygenation for 6
h, rat renal proximal tubular NRK52E cells exhibited a
significant lowering survival rate as compared to the
normoxia-cultured group. Interestingly, co-culture with
HMSCs or treatment with HMSC-CM both attenuated
H/R injury-related death of NRK-52E cells. Nonetheless,
addition of 3-methyladenine, an autophagy inhibitor,
significantly abolished the beneficial effect of HMSCs
and HMSC-CM (Fig. 7a). RFP-GFP-LC3B analysis of the
autophagic flux showed that H/R injury induced the
autophagy of NRK-52E cells and co-culture of HMSCs
further promoted the autophagy flux. The expression of
RFP-GFP-LC3B puncta was the most prominent in the
(See figure on previous page.)
Fig. 1 Characteristics and tracking of hypoxic rat mesenchymal stem cells (HMSCs) in ischemia-reperfusion injured rat kidneys. a HMSCs from the
bone marrow of adult Sprague-Dawley rats were analyzed for the expression of surface markers (red lines) by flow cytometry. Gray shaded areas
indicate unstained controls. b HMSCs had adipogenic, chondrogenic, and osteogenic differentiation abilities. Scale = 100 μm. c The rats with
renal ischemia-reperfusion injury were administered with PKH67-labeled HMSCs or phosphate-buffered saline (PBS) via intra-renal arterial (IA) or
intraperitoneal (IP) routes and sacrificed 48 h later. Fluorescence microscopy analysis revealed that PKH67-labeled HMSCs (arrowheads) retained
the I/R-injured rat kidneys. n = 2 per group. Scale = 50 μm
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 9 of 22
Fig. 2 (See legend on next page.)
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 10 of 22
HMSC co-culture group. However, addition of 3-MA
decreased the expression of RFP-GFP-LC3B puncta
(Fig. 7b–c). Western blotting also showed that coculture
with HMSCs upregulated the expression of
LC3B, Atg5, and Beclin 1, and downregulated p62 expression
in H/R-injured NRK-52E cells. Moreover, coculture
of HMSCs also increased the expression of antioxidants
including Nrf-2, HO-1, SOD1, and catalase in
H/R-injured NRK-52E cells (Supplemental Figure S2).
Addition of 3-MA decreased the expression of autophagy
proteins (Fig. 7d–e). Taken together, these data
corroborated that HMSCs attenuate renal I/R injury
through upregulating tubular autophagy in vitro.
Increased autophagy level correlates with higher renal
recovery in patients with AKI
To validate the beneficial effect of autophagy in renal I/R
injury, we explore the role of LC3B expression in patients
with ischemic AKI. The mRNA expression of LC3B was
determined in the microdissected renal tubules of kidney
biopsy specimens from patients with ischemic AKI. Patients
with categorized into mild (injured tubules < 50%, n = 20)
and severe (injured tubules ≥ 50%, n = 15) ischemic AKI
groups according to the extent of tubular injury in the
histological examination (Fig. 8a–b). We found that the severe
AKI group had significant lower baseline glomerular
filtration rate but higher extent of interstitial fibrosis and inflammation
(Fig. 8c and Table 1). Interestingly, the LC3B
expression was higher in the severe AKI group as compared
to the mild AKI group (Fig. 8d). The renal LC3B mRNA
expression was inversely correlated with baseline renal
function but positively correlated with Nrf2 mRNA expression
(Fig. 8e–f). There was no significant correlation
between LC3B with HO-1, SOD1, catalase, or CD68 expression
(Supplemental Figure S3). To further evaluate the
prognostic role of LC3B expression in patients with AKI.
Notably, multivariable regression analyses showed that a
higher LC3B level in the biopsied kidney was associated
with a greater chance of renal recovery (odds ratio of 3.93
for every 10 TPM increase of LC3B mRNA, Table 2). These
clinical data further verify our experimental results that increased
autophagy protect against renal I/R injury.
Discussion
While MSCs have been reported to attenuate acute renal
I/R injury, the detailed therapeutic mechanisms of the
MSCs in this major clinical disease remains incompletely
understood. In this report, we have demonstrated that
hypoxia-preconditioned HMSCs had a greater antioxidative
effect than normoxia-cultured MSCs in rats with
renal I/R injury. We further found that HMSCs decreased
renal interstitial inflammation, facilitated an M1-to-M2
macrophage transition, promoted renal tubular cell survival,
and attenuated renal impairment in renal I/R injury.
Mechanistically, we have discovered that HMSCs upregulated
the autophagy expression both in the rats with renal
I/R injury and in the renal proximal tubular cells with H/
R injury. Notably, inhibition of the autophagy abrogated
the renoprotective effect from HMSCs. The protective effect
of tubular autophagy was also validated in clinical
AKI patients. To the best of our knowledge, this is the first
study to demonstrate that HMSCs attenuate renal I/R injury
through enhancing tubular autophagy.
The most striking results of our study were that
HMSCs attenuated renal I/R-related AKI through upregulating
autophagy in renal tubular cells both in vivo and
in vitro. Autophagy has been shown to exert protective
roles in renal I/R injury [10, 11]. Nonetheless, current
pharmacological inducers of autophagy like rapamycin
and everolimus are limited to certain toxicities [10]. Although
HMSCs play a renoprotective effect in AKI, it remains
unclear that whether HMSCs lessen renal I/R
injury through modulating the autophagy of renal tubular
epithelial cells. We found that HMSCs upregulated
the autophagy-related protein expression both in I/R-injured
rat kidneys and H/R-injured renal tubular epithelial
cells. After addition of 3-MA, the autophagy of renal
tubular cells was inhibited and protective effect of
HMSCs was then abrogated. Our data indicated that
HMSCs attenuated I/R-induced renal tubular death
(See figure on previous page.)
Fig. 2 Effect of hypoxic rat mesenchymal stem cells (HMSCs) on oxidative stress induced by renal ischemia-reperfusion (I/R) injury. a In vivo
detection of reactive oxygen species (ROS) in the rat kidneys subjected to sham-operation (Sham), ischemia-reperfusion surgery followed by intrarenal
arterial injection (IA) of either phosphate-buffered saline (I/R+PBS), normoxia-cultured mesenchymal stem cells (I/R+MSC, 5 × 105 cells per
rat), or HMSCs (I/R+HMSC, 5 × 105 cells per rat) was measured immediately after reperfusion. The amount of ROS was expressed as
chemiluminescence counts/10 s. Please see the “Material and methods” for details. b Quantification of in vivo detection of the reactive oxygen
species in the rat kidneys of Sham, I/R+PBS, I/R+MSC, and I/R+HMSC groups. **p < 0.01 by one-way ANOVA with Tukey’s post hoc comparison.
ns, nonsignificant; n = 3 per group. c Representative periodic acid-Schiff stained images of kidney sections from rats subjected to sham operation
(Sham group), renal ischemia for 45 min followed by 48 h of reperfusion and treated with either PBS, MSCs, or HMSCs at the onset of reperfusion.
The I/R injury induced diffuse denudation of the renal tubular cells with loss of brush border (arrows) and flattening of tubular cells with
intratubular cast formation (yellow arrowheads) in the I/R+PBS group. The extent of acute tubular injury (arrows) were decreased in the I/R+IAMSC
and I/R+IA-HMSC groups. Scale = 100 μm. d Quantification of the renal tubular injury. **p < 0.01, ***p < 0.001 by one-way ANOVA with
Tukey’s post hoc comparison, n = 5 per group. e, f Serum blood urea nitrogen and creatinine levels in the Sham, I/R+PBS, I/R+IA-MSC, and I/
R+IA-HMSC groups at 48 h after reperfusion. **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison, n = 5 per group
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 11 of 22
through enhancing the autophagy in renal tubular cells.
In accordance with our findings, Shin et al. recently
found that administration of MSCs increase autophagosome
and beclin-1 expression in the hippocampus of β-
amyloid-treated mice and in the β-amyloid-treated neuronal
cells, thereby promoting β-amyloid clearance and
decreasing neuronal cell death [31]. Li et al. also showed
that MSCs exert an autophagy-inducing effect in cocultured
pulmonary endothelial cells and attenuate acute
lung injury [32]. Taken together, our novel finding
that upregulation of renal tubular autophagy by
HMSCs to repair renal I/R injury increases the understanding
of the complex therapeutic mechanisms of
HMSCs in AKI. Our study not only makes a conceptual
breakthrough for understanding how HMSCs
modulate renal tubular autophagy in I/R injury but
also provides bench evidence to apply HMSCs to
treat patients with ischemic AKI.
Fig. 3 Hypoxic rat mesenchymal stem cells (HMSCs) and HMSC-conditioned medium (CM) suppressed oxidative stress, tubular injury, and renal
dysfunction in ischemia-reperfusion (I/R)-injured rat kidneys. a Representative periodic acid-Schiff stained photomicrographs showed that I/R
injury resulted in severe renal tubular injury such as tubular cell sloughing and flattening (arrows) as well as casts formation (yellow arrowheads)
in phosphate-buffered saline (PBS)-treated groups as compared to the sham-operated (Sham) group. Scale = 100 μm. b Quantification of the
tubular injury. Intra-renal arterial (IA-HMSC) or intraperitoneal (IP-HMSC) administration of HMSCs significantly reduced tubular injury. n = 6 per
group. c Quantification of in vivo detection of the reactive oxygen species in the rat kidneys. I/R injury induced an increase of reactive oxygen
species in the rat kidneys as compared to the Sham group. The I/R injury-induced oxidative stress was attenuated in the IA-HMSC group. n = 3–4
per group. d, e I/R injury caused a renal function decline with elevated serum blood urea nitrogen (BUN) and creatinine levels. Both intra-arterial
and intraperitoneal administration of HMSCs improved the renal function decline significantly. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way
ANOVA with Tukey’s post hoc comparison. ns, nonsignificant; n = 6 per group. f Representative periodic acid-Schiff stained photomicrographs
showed that the extent of sloughing and necrosis of the renal tubular cells due to I/R injury was gradually reduced by the HMSC-CM. CM25,
CM50, and CM100 denote 25-fold, 50-fold, and 100-fold concentrated HMSC-CM, respectively. g Quantification of the tubular injury. n = 6 per
group. h Quantification of in vivo detection of the reactive oxygen species in the rat kidneys. n = 3–5 per group. i, j Serum BUN and creatinine
levels in the rats of renal I/R injury with treatment of PBS or HMSC-CM. Only 100-fold concentrated HMSC-CM (CM100) ameliorated oxidative
stress, tubular injury and renal dysfunction. *p < 0.05 by one-way ANOVA with Tukey’s post hoc comparison, n = 6 per group
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 12 of 22
Fig. 4 Hypoxic rat mesenchymal stem cells (HMSCs) decreased oxidative damage and increased anti-oxidant response in the ischemia-reperfusion (I/
R)-injured rat kidneys. a, c Immunofluorescence staining for superoxide by dihydroethidium (DHE) staining and macrophage by anti-CD68 staining in
renal tissues of the sham-operated rats (Sham), I/R-injured rats with PBS administration (PBS), I/R-injured rats with intra-renal arterial administration of
HMSCs (IA-HMSCs), I/R-injured rats with intraperitoneal administration of HMSCs (IP-HMSCs), and I/R-injured rats with intraperitoneal administration of
100-fold concentrated HMSCs-conditioned medium (HMSC-CM). DHE staining was predominantly at the renal tubular cells and at some macrophages
(arrowheads). 4′,6-Diamidino-2-phenylindole (DAPI) represented nuclear staining. Scale bar = 50 μm. **p < 0.01, ***p < 0.001 by one-way ANOVA with
Tukey’s post hoc comparison, n = 4 for the sham group; n = 5 for other groups. b, d Immunohistochemical staining for 8-hydroxy-2-deoxyguanosine
(8OHdG), 4-hydroxynonenal (4-HNE), and nuclear factor erythroid 2–related factor 2 (Nrf-2) in I/R-injured rat kidneys. Renal I/R injury increased the
expression of 8OHdG, 4-HNE, and Nrf-2 in tubular epithelial cells. IA and IP administration of the HMSCs as well as intraperitoneal injection of HMSCCM
decreased the numbers of 8OHdG and 4-HNE-positive tubular cells but further upregulated the number of Nrf-2-positive tubular cells. Scale = 100
μm. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. ns, nonsignificant. n = 4 for the shamgroup; n = 5 for
other groups. e–i Western blot analyses of anti-oxidant proteins in I/R-injured rat kidneys. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with
Tukey’s post hoc comparison. ns, nonsignificant. n = 6 per group
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 13 of 22
Fig. 5 (See legend on next page.)
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 14 of 22
Oxidative stress is one of the major pathogenic mechanism
of the I/R-related AKI [46–48]. Following renal ischemia,
shortage of the oxygen and nutrients supply
leads to tubular epithelial cell injury [5]. The injured epithelial
cells and subsequent recruited pro-inflammatory
macrophages produce large amount of ROS [47]. These
highly reactive oxidizing molecules further contribute to
tubular cell death through scavenging vasodilatory nitric
oxide, destabilizing cytoskeleton, and increasing DNA
oxidation and lipid peroxidation of plasma membrane
[47]. ROS also perpetuate renal inflammation and trigger
the expression of pro-inflammatory cytokines and chemokines
[48]. Conversely, endogenous defense system
like Nrf2, the downstream detoxifying enzymes, and
other stress response proteins are activated during renal
I/R injury to maintain cellular redox homeostasis [7, 49].
Increasing evidence suggests that oxidative stress and
autophagy are intricately connected [48]. Mild oxidative
stress induces cell repairing mechanisms such as autophagy
[48]. Nonetheless, excessive production of ROS overwhelms
the protective autophagy mechanism and leads
to oxidative damage and ultimately cell demise [48]. Till
now, the inter-connection between oxidative stress and
autophagic processes in kidney diseases has not been investigated
in detail. Our study revealed that I/R injury in
rat kidneys induces a surge of ROS. DHE staining for
characterization of in situ ROS further showed that the
superoxide was predominantly from renal tubular epithelial
cells in the renal I/R injury. The biomolecular
damages from oxidative stress like 8OHdG and 4HNE
were also increased in the I/R-injured renal tubular epithelial
cells. Administration of HMSCs and HMSC-CM
reduced the superoxide production, DNA oxidation, and
lipid peroxidation in renal tubular cells of I/R-injured rat
kidneys. Moreover, we also found that HMSC administration
upregulated stress response proteins and detoxifying
enzymes such as Nrf2, HO-1, SOD1, and catalase.
Meanwhile, HMSC administration also enhances tubular
autophagy, decreases tubular cell death, and attenuates
renal function impairment. The autophagy adaptor p62
is recently found to interact with Keap1, the Nrf2 inhibitor,
thus stabilizing and increased transcriptional activity
of Nrf2 to prevent tissue damage and eventually to repair
lesion and avoid cancerous lesion formation [50]. In
accord with this, we found that HMSCs treatment simultaneously
upregulated the autophagy pathway and
Nrf2 expression in tubular epithelial cells of I/R-injured
rat kidneys, suggesting these two systems may synergistically
ameliorate renal injury. Clearly, the detailed interaction
between autophagy and Nrf2 signaling promoted
by HMSCs needs further investigation. Collectively,
HMSCs treatment limits the extent of oxidative stress
and activates tubular autophagy, both of which contributing
to the protection of kidneys.
MSCs represent an appealing therapeutic approach for
kidney diseases and their therapeutic efficacy is determined
by the differentiation, trophic and immunomodulatory
properties [51]. One critical aspect for the efficacy
of MSCs is the delivery method and the optimal cell delivery
technique would provide the greatest regenerative
benefits and the least side effects [52]. In systemic administration
through intravenous or intraperitoneal injection,
most of the stem cells are trapped in the lung,
liver, or spleen [53], and only few cells reach the injured
site through homing effect [54, 55]. By contrast, direct
intra-arterial injection of MSCs into the injured kidney
would ensure the delivery. Our study also compared effect
of locally delivery by intra-arterial route and systemic
delivery by intraperitoneal route. We found that
administration of HMSCs through intra-arterial route
had better anti-oxidative and renoprotective effects in
rats with I/R injury. By contrast, Moustafa et al. recently
found that there was no difference between intraarterial,
intravenous or subcapsular injection of 5 × 106
MSCs in cisplatin-induced AKI [56]. The discrepancy
between Moustafa’s study and our report may be explained
by the different dosage of stem cells and
methods to induce AKI. Our previous study discloses
(See figure on previous page.)
Fig. 5 Hypoxic rat mesenchymal stem cells (HMSCs) modulated macrophage phenotypes, reduced tubular apoptosis, and enhanced tubular
proliferation in the ischemia-reperfusion (I/R)-injured rat kidneys. a–f Representative immunohistochemical photomicrographs showed that the
phosphate-buffered saline (PBS) group had more infiltrated ED1-positive and inducible nitric oxide synthase (iNOS)-positive macrophages, and terminal
deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic tubular cells as compared to the sham-operated (Sham) group. Intrarenal
arterial (IA) and intraperitoneal (IP) administration of the HMSCs as well as intraperitoneal injection of the 100-fold concentrated HMSCconditioned
medium (CM) decreased the numbers of infiltrated iNOS-positive macrophages and TUNEL-positive apoptotic tubular cells. By contrast,
renal I/R injury did not increase the number of arginase 1 (Arg1)-positive macrophages. IP administration of HMSCs as well as HMSC-CM significantly
increased the number of Arg1-positive macrophages in I/R-injured rat kidneys. Only the IA-HMSC group had an increased number of proliferating cell
nuclear antigen (PCNA)-positive tubular cells. Scale = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc
comparison, ns, nonsignificant, n = 4–5 for the shamgroup, n = 5–6 for other groups. g–l Western blot analyses showed that the I/R+PBS group had a
higher ratio of Bax/Bcl-2 expressions and increased levels of active caspase 3 (CPP32), interleukin-1β (IL-1β) as well as caspase 1 in the I/R-injured rat
kidneys. IA-HMSC, IP-HMSC, and HMSC-CM groups reduced the I/R-injury-induced elevation of Bax/Bcl2 ratio, CPP32, and IL-1β levels. Only IA-HMSC
group supressed the I/R-injured-induced expression of caspase 1. *p < 0.05, ***p < 0.001 as compared to the sham-operated (sham) group; #p < 0.05,

p < 0.01, ###p < 0.001 as compared to the I/R+PBS group, by one-way ANOVA with Tukey’s post hoc comparison, n = 6 per group

Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 15 of 22
Fig. 6 Hypoxic rat mesenchymal stem cells (HMSCs) upregulated autophagy in the tubular epithelial cells of the ischemia-reperfusion (I/R)-injured
rat kidneys. a, b Representative immunohistochemical photomicrographs showed that intra-renal arterial (IA) administration of HMSCs
upregulated autophagy-related protein expression including LC3B, Atg5, and Beclin 1 but downregulated the autophagy adaptor protein p62
expression in the renal tubular epithelial cells of I/R-injured rat kidneys. IP, intraperitoneal administration; CM, 100-fold concentrated conditioned
medium. Scale = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison, n = 6 per group. c, d
Western blot analyses showed that the I/R injury markedly increased the autophagy adaptor protein p62 expression, which was suppressed in the
I/R+IA-HMSC, I/R+IP-HMSC, and I/R+HMSC-CM groups. The levels of LC3B, Atg5, and Beclin 1 were mildly increased in the I/R+PBS group but
markedly increased in the I/R+HMSC group. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. ns,
nonsignificant. n = 6 per group
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 16 of 22
that the best renoprotective effect of stem cells in renal
I/R injury is achieved by administering 5 × 106 stem cells
because too many cells delivered intra-arterially also impede
the renal perfusion [35]. Moreover, the present
study also determined the effect of HMSC-CM to investigate
the paracrine effect. Interestingly, the beneficial
effect of HMSC-CM in renal I/R injury was only observed
in the 100-fold concentrated group. Furthermore,
the anti-inflammatory, anti-apoptotic, and regenerative
effects of the HMSC-CM group were less prominent as
compared to the IA administration of HMSC group. In
line with our findings, a recent pilot clinical trial shows
Fig. 7 Hypoxic rat mesenchymal stem cells (HMSCs) ameliorated hypoxia-reoxygenation (H/R) injured renal tubular cells through enhancing
autophagy. a H/R injury (hypoxia for 24 h followed by reoxygenation for 6 h) significantly decreased cell survival in NRK-52E cells determined by
an MTT assay. Either co-culture with HMSCs or addition of HMSC-conditioned medium (CM) decreased H/R-injury-induced cell death. Addition of
3-methyl adenine (3-MA), an autophagy inhibitor, abolished the protective effects of HSMCs and HMSC-CM. *p < 0.05, **p < 0.01, ***p < 0.001 by
one-way ANOVA with Tukey’s post hoc comparison. $p < 0.05 vs H/R group; #p < 0.05 vs H/R+HMSC group; §p < 0.05 vs H/R+CM group. n = 3
per group. b, c Autophagic flux was assayed by a Premo Autophagy Tandem Sensor RFP-GFP-LC3B kit. Scale = 10 μm. Confocal microscopy
showed that the LC3B puncta (yellow color) in the NRK-52E were increased by the co-culture with HMSCs or treatment with HMSC-CM. Addition
of 3-MA decreased the formation of LC3B puncta. *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. $p <
0.05 vs H/R group; #p < 0.05 vs H/R+HMSC group; §p < 0.05 vs H/R+CM group. n = 3 per group. d, e Western blot analyses showed that the H/R
injury markedly increased the autophagy adaptor protein p62 expression in NRK-52E cells, which was suppressed in the H/R+HMSC and H/R+CM
groups. The levels of LC3B, Atg5, and Beclin 1 were significantly increased in the H/R+HMSC group. Addition of 3-MA suppressed the HMSCupregulated
expression of LC3B, Atg5, and Beclin 1. *p < 0.05, **p < 0.01, **p < 0.001 by one-way ANOVA with Tukey’s post hoc comparison. #p < 0.05 vs H/R+HMSC group by Student’s t test. n = 3 per group Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 17 of 22 that intra-arterial infusion of autologous MSCs in patients with renovascular disease improves renal tissue oxygenation and cortical blood flow [57]. Therefore, the present study indicates that intra-arterial administration of HMSCs in I/R-related AKI is an effective and feasible delivery method. Replicative exhaustion, early senescence, decreased differentiation potential, and impaired immunosuppressive ability remain major challenges of MSC therapy in clinical application [17]. Expansion of MSCs in the hypoxic atmosphere not only inhibits senescence, increases the proliferation rate, and improves differentiation ability but also enhances the paracrine effects through upregulation of various secretory trophic factors [26, 27]. Growing evidence suggests that hypoxiapreconditioning potentiates the therapeutic effects of MSC in renal I/R injury by enhancing the secretion of trophic factors like vascular endothelial growth factor, insulin-like growth factor-1, and hepatocyte growth factor [58]. The present study found that IA administration of HMSCs ameliorated I/R injury-induced renal impairment as compared that normoxic MSCs. We also demonstrated that the hypoxic culture increased the gene expression of hepatocyte growth factor and Hbegf as compared to normoxic culture. Notably, the hypoxia-derived upregulation of beneficial genes in HMSCs such as Hbegf lasted for 48 h after the HMSCs were transferred to a normoxic atmosphere. HBEGF has been implicated in angiogenesis [59] and enhances the proliferation of MSCs [60]. Exogenous HBEGF also accelerates renal recovery from acute ischemic injury Fig. 8 Tubular autophagy is associated with better renal recovery in patients with ischemic acute kidney injury (AKI). a, b Representative periodic acid- Schiff stained photomicrographs indicated the extent of acute tubular injury including loss of brush border, dilation of tubular lumens, and flattening or vacuolization of epithelial cells (arrows) in patients with mild (injured tubules < 50%) or severe (injured tubules ≥ 50%) ischemic AKI. Scale = 100 μm. c Baseline renal function in patients with mild (n = 20) and severe (n = 15, injured tubules ≥ 50%) ischemic AKI, compared by the unpaired t test. d Expression of LC3B mRNA levels (transcripts per million, TPM) in patients with mild and severe AKI, compared by the Wilcoxon rank sum test. e, f Correlation between renal LC3B expression with baseline renal function and Nrf2 in AKI patients by Pearson’s correlation test Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 18 of 22 by activating the epidermal growth factor receptor [61]. Altogether, our data indicate the hypoxia-derived upregulation of beneficial genes in MSCs can be maintained even after the cells were transferred from the hypoxic to normoxic condition to confer therapeutic effects. Currently, MSCs-based therapy has been investigated in many clinical untreatable diseases [62]. While MSCs therapy is generally considered safe, there is still a concern regarding the tumorigenic risk after stem cell transplantation [62]. MSCs may promote tumor growth in the animal models of various solid malignancies by the secretion of proangiogenic factors [62]. By contrast, several studies have shown that MSCs can inhibit tumor progression and metastasis by inhibiting angiogenesis, downregulating Akt and Wnt signaling, and inducing apoptosis or cell cycle arrest [63]. A recent meta-analysis including 36 studies indicates that there is no association between MSCs therapy and tumor formation [64]. Administration of MSCs to patients of kidney diseases has also exhibited good safety and tolerability without tumor formation in clinical trials [17]. Moreover, there is also a dispute about the effect of hypoxia-preconditioning on the genetic stability of MSCs. A previous report demonstrated that low oxygen levels downregulated the expression of DNA repair proteins of MSCs [65]. However, latter studies have shown that a culture of MSCs at low oxygen tension improves genetic stability [66], and a long-term hypoxic culture may be more suitable for bone marrow-derived MSCs [67]. Till now, there is no unambiguous answer regarding the potential of MSCs in tumorigenesis. Use of MSCs expressing normal karyotype and genetic integrity help prevent tumorigenic transformation [27]. Long-term follow-up studies are still warranted to fully address the safety issue of MSCs-based therapy. Conclusions In summary, our findings demonstrate that HMSCs exert anti-oxidative, anti-inflammatory, and anti-apoptotic effect in I/R-related AKI. We further found that HMSCs upregulated renal tubular autophagy, thereby attenuating renal tubular death, suppressing interstitial inflammation, and decreasing renal function impairment. The present study highlights the importance of modulation of tubular autophagy in repairing I/R injury, provides further mechanistic support for the reparative effects of HMSCs and facilitates the utilization of HMSCs in treating patients with ischemic AKI. Table 1 Baseline characteristics of patients with mild or severe ischemic acute kidney injury Baseline characteristics Mild AKI# (n = 20) Severe AKI# (n = 15) p value†
Demographics
Age, years 54.0 (26.3) 66.7 (12.4) 0.013c
Male 12 (60.0) 11 (73.3) 0.64a
Smoking 3 (15.0) 3 (20.0) 1.00b
Diabetes 3 (15.0) 4 (26.7) 0.43b
Hypertension 11 (55.0) 9 (60.0) 1.00b
Laboratory and histological data
Serum creatinine, mg/dL 0.90 (0.47) 3.56 (4.01) < 0.001c
Glomerular filtration rate, mL/min 89.2 (43.7) 16.7 (25.7) < 0.001c
Interstitial fibrosis, % 10 (10) 30 (10) < 0.001c
Interstitial inflammation, % 10 (7.5) 30 (20) < 0.001c
LC3B mRNA levels, TPM 104 (25.1) 120 (19.9) 0.006c
*Values for categorical and continuous variables are expressed as numbers (percentages) and median (interquartile range), respectively

Patients were stratified into mild (injured tubules < 50%) or severe (injured tubules ≥ 50%) ischemic acute kidney injury according to the percentage of injured

tubules in the pathology of renal biopsy specimens
†Between-group comparisons by the aPearson’s χ2 test, bFisher’s exact test, or cWilcoxon rank sum test where appropriate
Table 2 Renal LC3B expression and recovery of renal function in patients with ischemic acute kidney injurya
Odds ratio (95% CI)c p valueb
Renal LC3B expression
(per 10 TPM increase)b
Model 1: unadjusted 2.88 (1.50~7.46) 0.007
Model 2: adjusted for age and gender 4.40 (1.80~16.82) 0.007
Model 3: adjusted for age, gender and baseline renal function 3.94 (1.50~17.37) 0.024
Model 4: adjusted for age, gender, baseline renal function,
interstitial fibrosis, and inflammation
3.93 (1.40~21.55) 0.041
aRecovery of renal function was defined as the occurrence of a glomerular filtration rate greater than or equal to the baseline value during the follow-up period
bThe LC3B mRNA levels in the renal biopsy specimens were expressed by TPM
cOdds ratio and 95% confidence interval (CI) for recovery of renal function were calculated by multivariable-adjusted logistic regression models
Tseng et al. Stem Cell Research & Therapy (2021) 12:367 Page 19 of 22
Abbreviations
3-MA: 3-Methyladenine; 4-HNE: 4-Hydroxynonenal; 8OHdG: 8-Hydroxy-2-
deoxyguanosine; AKI: Acute kidney injury; Atg: Autophagy-related gene;
BUN: Blood urea nitrogen; CM: Conditioned medium; DAPI: 4′,6-Diamidino-2-
phenylindole; DHE: Dihydroethidium; DMEM: Dulbecco’s modified Eagle’s
medium; FBS: Fetal bovine serum; FITC: Fluorescein isothiocyanate; H/
R: Hypoxia-reoxygenation; Hbegf: Heparin-binding epidermal growth factorlike
growth factor; SC: Hypoxic mesenchymal stem cell; HMSC-CM: Hypoxic
mesenchymal stem cell-conditioned medium; HO-1: Heme oxygenase-1; I/
R: Ischemia-reperfusion; IA: Intra-renal arterial; IP: Intraperitoneal; MCLA: 2-
Methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo [1,2-alpha]pyrazin-3-one;
MEM: Minimal essential medium; MSC: Mesenchymal stem cell; MTT: 3-[4,5-
Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Nrf2: Nuclear factor
erythroid 2–related factor 2; PAS: Periodic acid-Schiff; PBS: Phosphatebuffered
saline; ROS: Reactive oxygen species; SOD1: Superoxide dismutase 1;
TUNEL: Terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine
triphosphate nick-end labeling
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s13287-021-02374-x.
Additional file 1: Supplemental Materials and Methods. Figure S1.
Upregulation of beneficial genes of mesenchymal stem cells (MSCs) by
hypoxic culture. Figure S2. Hypoxic rat mesenchymal stem cells (HMSCs)
increased antioxidant response protein in hypoxia-reoxygenation (H/R)-injured
renal tubular epithelial cells. Figure S3. Correlation between renal
mRNA levels of LC3B with endogenous antioxidants and macrophage in
patients with ischemic acute kidney injury. Supplementary Table S1.
Primary and secondary antibodies list. Supplementary Table S2. Primer
sequences and probe numbers in qPCR experiments.
Acknowledgements
The authors thank the Division of Experimental Surgery, Department of
Surgery, the Clinical Research Core Laboratory, and the Medical Science &
Technology Building of Taipei Veterans General Hospital for technical
assistance and provision of the experimental space and facilities. They also
thank the National Center for High-performance Computing (NCHC) of
Taiwan for providing computational and storage resources.
Authors’ contributions
WCT and DCT conceived and designed the study. WCT and PYL performed
the experiments. WCT analyzed and interpreted the data. PYL, MTT, FPC, NJC,
CTC, SCH, and DCT interpreted the data. WCT drafted the article. PYL, MTT,
FPC, NJC, CTC, SCH, and DCT revised the article critically for important
intellectual content. The authors read and approved the final manuscript.
Funding
This work was supported in part by the Ministry of Science and Technology,
Taiwan [grant number MOST 106-2314-B-010-039-MY3, MOST 107-2314-B-
075-064-MY3, MOST 109-2314-B-010-056-MY3, MOST 109-2314-B-075-097-
MY3]; the Taipei Veterans General Hospital [grant number VN100-002, V107-
B-037, V107C-127, V108-C-175, V108-C-103, V108-D42-001-MY3, V109C-101,
V109C-114, V110C-127]; Foundation for Poison Control, Ministry of Education’s
Aim for the Top University Plan in the National Yang-Ming University, Taiwan;
and Center For Intelligent Drug Systems and Smart Bio-devices (IDS2B) from
The Featured Areas Research Center Program within the framework of the
Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.
The funding sources had no role in the study design, conduct, or reporting.
Availability of data and materials
The datasets used and/or analyzed during the current study are available
from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
All animal experiments conformed to the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of Health and
approved by the Institutional Animal Care and Use Committee of the
National Yang-Ming University and the Taipei Veterans General Hospital
under the license numbers of 991261 and 2017-075, respectively. All enrolled
patients in this study gave their informed consents and human samples in
this study were collected under protocols that were reviewed and approved
by the institutional review board of the Taipei Veterans General Hospital
under the license number 2018-06-008B and 2018-09-004C.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1Division of Nephrology, Department of Medicine, Taipei Veterans General
Hospital, 201, Section 2, Shih-Pai Road, Taipei 11217, Taiwan. 2Faculty of
Medicine, School of Medicine, National Yang-Ming University, Taipei, Taiwan.
3Institute of Clinical Medicine, School of Medicine, National Yang-Ming
University, Taipei, Taiwan. 4Center for Intelligent Drug Systems and Smart
Bio-devices (IDS2B), National Chiao-Tung University, Hsinchu, Taiwan.
5Institute of Clinical Medicine, School of Medicine, National Yang Ming Chiao
Tung University, Taipei, Taiwan. 6Holistic Education Center, Tzu Chi University
of Science and Technology, Hualien, Taiwan. 7Department of Pathology and
Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
8Institute of Microbiology and Immunology, School of Medicine, National
Yang-Ming University, Taipei, Taiwan. 9Department of Life Science, School of
Life Science, National Taiwan Normal University, Taipei, Taiwan. 10Integrative
Stem Cell Center, Department of Orthopedics, and Institute of New Drug
Development, New Drug Development Center, China Medical University,
Taichung, Taiwan. 11Institute of Biomedical Sciences, Academia Sinica, 128,
Section 2, Academia Road, Taipei 11529, Taiwan. 12Department and Institute
of Physiology, School of Medicine, National Yang-Ming University, Taipei,
Taiwan. 13Department of Biological Science and Technology, College of
Biological Science and Technology, National Chiao-Tung University, Hsinchu,
Taiwan.
Received: 23 March 2021 Accepted: 9 May 2021
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