Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways
Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways
Jian-bo Yu 0 1
Jia Shi☯ 0 1
Yuan Zhang☯ 0 1
Li-rong Gong 0 1
Shu-an Dong 0 1
Xin-shun Cao 0 1
Li-li Wu 0 1
Li-na Wu 0 1
0 Department of Anesthesiology, Tianjin Nankai Hospital, Tianjin Medical University , Tianjin , China
1 Editor: Benedetta Bussolati , Center for Molecular Biotechnology, ITALY
Electroacupuncture at select acupoints have been verified to protect against organ dysfunctions during endotoxic shock. And, heme oxygenase (HO)-1 as a phase II enzyme and antioxidant contributed to the protection of kidney in septic shock rats. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway mediated the activation of NF-E2 related factor-2 (Nrf2), which was involved in HO-1 induction. To understand the efficacy of electroacupuncture stimulation in ameliorating acute kidney injury (AKI) through the PI3K/Akt/Nrf2 pathway and subsequent HO-1 upregulation, a dose of LPS 5mg/kg was administered intravenously to replicate the rabbit model of AKI induced by endotoxic shock. Electroacupuncture pretreatment was handled bilaterally at Zusanli and Neiguan acupoints for five consecutive days while sham electroacupuncture at non-acupoints as control. Results displayed that electroacupuncture stimulation significantly alleviated the morphologic renal damage, attenuated renal tubular apoptosis, suppressed the elevated biochemical indicators of AKI caused by LPS, enhanced the expressions of phospho-Akt, HO-1protein, Nrf2 total and nucleoprotein, and highlighted the proportions of Nrf2 nucleoprotein as a parallel. Furthermore, partial protective effects of elecroacupuncture were counteracted by preconditioning with wortmannin (the selective PI3K inhibitor), indicating a direct involvement of PI3K/Akt pathway. Inconsistently, wortmannin pretreatment made little difference to the expressions of HO-1, Nrf2 nucleoprotein and total protein, which indicated that PI3K/Akt may be not the only pathway responsible for electroacupuncture-afforded protection against LPS-induced AKI. These findings provide new insights into the potential future clinical applications of electroacupuncture for AKI induced by endotoxic shock instead of traditional remedies.
Funding: Financial support for this research was
provided by grant No. 81372096 from the National
Natural Science Foundation of China, Beijing, China,
and grant No. 12ZCZDSY013300 from the Key
Projects in the Tianjin Science & Technology Pillar
Program, Tianjin, China. The funders had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
Septic shock is the most common contributing factor to acute kidney injury (AKI) in critically
patients, and even progress into acute renal failure . The combination of acute renal failure
and sepsis accounts for more than 70% mortality, despite multifarious potential interventions
. Lipopolysacchride (LPS) is a primary initiator of inflammatory and hemodynamic
perturbations of sepsis and involved in experimental AKI studies . There is experimental evidence
that endotoxin-related increase in tumor necrosis factor-α (TNF-α) and oxygen radicals are
major causes of AKI during endotoxic shock [4, 5]. Heme oxygenase (HO)-1, as the rate
limiting enzyme of heme degradation, was first recognized in preserving renal function and
reducing mortality . Our prior studies definitely elucidated the protective role of HO-1 on the
kidney in endotoxic shock rats . The biological effect of HO-1 attributed to the degradation
of heme and the products of enzymatic reaction including biliverdin and carbon monoxide,
which exerted antioxidant, antiproliferative and also anti-inflammatory properties .
Acupuncture represents a potentially valuable adjunct to prevent clinical disorders in China
and other Asian countries for the past 3000 years . It was proven that electrical stimulation
of the vagus nerve could modulate systemic inflammatory responses to endotoxin, inhibit the
synthesis of TNF and prevent the development of shock . In addition, electroacupuncture
was found to produce neuro-protective effect by enhancing the activities of antioxidant
enzymes and reducing the extent of lipid peroxidation . Traditionally, Zusanli (ST36) and
Neiguan (PC6), two specific points, were considered of choice for good health care. Recent
studies have shown that ST36 acupuncture pretreatment exerted protective effects against
sepsis-induced kidney injury . Besides, Liu et al indicated that electroacupuncture at PC6
retrieved blood pressure and improved endotoxic shock in rats . However, little was known
about its mechanisms of modulation.
Several transcriptional factors including NF-E2 related factor-2(Nrf2), activator protein
(AP)-1 and nuclear factor-κβ (NF-κβ) have been implicated in the regulation of HO-1
expression . Among these, Nrf2 played a crucial role in mounting the innate immune response
and maintaining redox homeostasis, which determined survival during endotoxic shock .
Moreover, only phosphatidylinositol 3-kinase (PI3K)-related signal pathway controlled the
activation of Nrf2 was involved in the induction of HO-1 [16, 17]. Meanwhile, PI3K/Akt
pathway was thought to be pivotal in the maintenance of homeostasis and the integrity of the
immune response during sepsis . Our preliminary studies have confirmed that
electroacupuncture exerted anti-inflammatory or antioxidant capacity to reverse lung injury
induced by endotoxic shock through upregulation of HO-1 and activation of Nrf2/ARE
pathway . Nevertheless, the role of HO-1 in protective effect of electroacupuncture against
endotoxic shock induced AKI and the regulatory mechnisms have been unconfirmed
Based on these previous data, we hypothesized that electroacupuncture stimulation at
bilateral ST36 and PC6 acupoints could attenuate AKI evoked by endotoxic shock in rabbits via the
induction of HO-1 through modulating PI3K/Akt/Nrf2 pathway.
Materials and Methods
For the current study, two-month-old male New England white rabbits weighing 1.5–2.0kg
were obtained from Laboratory Animal Center of Nankai Clinical Institution of Tianjin
Medical University. All animal experiments were carried out in strict accordance with the
recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes
of Health. The protocols were approved by the Committee on the Ethics of Animal
Experiments of Tianjin Nankai Hospital (NKH-20131107). The rabbits were housed at 18~22°C, and
acclimatized to a 12/12-h light-dark regimen. Food and water were supplied ad libitum for a
5-day period before the experiment procedures. The night before surgery rabbits were denied
access to food but had access to water. All operations were performed under the anesthesia of
urethane, and all efforts were made to minimize the suffering of the animals.
Two pairs of stainless steel needles (diameter 0.3mm) were inserted perpendicularly to a depth
of 5mm and then held in place. The selected acupoints were Zusanli(ST36), located between
the tibia and fibular approximately 5 mm lateral to the anterior tubercle of the tibia, and
Neiguan (PC6), located between the radius and ulna probably one-sixth the length of the line from
the wrist to elbow [12,20]. A series of nonacupoints located 5mm lateral to ST36 or PC6
original location served as controls. As described in detail previously [19, 21], stimulation
(alternating disperse-dense wave of 2Hz and 15Hz, once a day) were delivered using an electrical
stimulation device (HANS G6805-1A, Huayi Co, Shanghai, China) for 15 minutes. Current
intensity ( 1.0mA) was adjusted to elicit slight twitches of the limbs. Electroacupuncture was
initiated for a five-consecutive-day before the experiment and throughout the operating steps
for 6h during the experimental day . For acupuncture sessions, the rabbits were lightly
immobilized using hands to minimize stress. Acupuncture points were identified by an
experienced acupuncturist (ZY), who was not involved in data analysis.
Rabbits were sedated with 5mg/kg of 20% urethane via the marginal ear vein. Anesthesia was
maintained with continuous infusion of ketamine at 3mg/kg/h throughout the experiment.
Briefly, tracheotomy was performed aseptically so as to facilitate spontaneous respiration. The
animals were placed on a heating pad under a radiant heat lamp to keep the body temperature
at 37.7–40.3°C. A PE-50 catheter was inserted through the right internal carotid artery to
monitor arterial pressure continuously by using Hellige monitor instrument (Germany). The
internal jugular vein was cannulated with a 24-g catheter for drugs administration. A retention
catheter was inserted into the bladder via urethra for urine collection. Lactated Ringer’s
solution (8ml/kg/h) was infused intravenously for the duration of each experiment.
Sixty rabbits were randomly assigned to six groups (n = 10/group): group ELW, W, C, EL,
SEL, and L (Fig 1). Rabbits subjected to 0.5ml (5mg/kg) LPS (L2630, sigma, USA) in group
ELW, EL, SEL and L to mimic endotoxic shock-induced acute kidney injury . To serve as
controls, the rabbits in group C received an equal amount of normal saline. Electroacupuncture
treatment was applied bilaterally at ST36 and PC6 points in group EL and ELW, while group
SEL acupunctured at non-acupoints as described above. To block PI3K/Akt signaling pathway,
wortmannin (the selective PI3K inhibitor, 0.6mg/kg; Selleck, USA) was administered in group
ELW intravenously 30min before LPS injection . Meanwhile, its drug vehicle, dimethyl
sulfoxide (DMSO, 0.08ml/kg) was given in other groups instead, and W group rabbits were
accepted wortmannin alone. Mean arterial pressure (MAP) was monitored consecutively after
LPS administration. MAP did not decrease within 2h or rabbits died within 6h were deemed as
the exclusion criteria of current study .
The overall health status of all rabbits enrolled in current study were monitored every 30
min~1h for indicators of distress following the injection of LPS. In particular, as with the
Fig 1. Schematic diagram of depicting the experimental protocols. “EA” presented electroacupuncture at bilaterally Zusanli and Neiguan acupoints;
“SEA” presented sham electroacupuncture at non-acupoints; “none” means no electroacupuncture pretreatment for five days. “surgery” means the
experimental operation of rabbits. “. “Brace” means electroacupuncture or sham electroacupuncture throughout the operation of rabbits.
suggestions of Nemzek et al, rabbits were euthanized by CO2 asphyxiation either at the end of
the experiment, namely at 6h after LPS administration, or upon signs of imminent death
including inability to ambulate, labored breathing and cyanosis, prolonged hypothermia, and
unconsciousness to external stimuli [25, 26]. The blood sample was drawn from the right
internal carotid artery into EDTA tubes immediately. Then, the blood specimen was centrifuged at
3000 rpm for 15min at 4°C and the supernatant was frozen at -80°C for subsequent analysis. In
addition, urine samples from the bladder were extracted. After the rabbits were euthanized, the
kidneys were harvested immediately and quickly perfused with phosphate-buffered saline
(PBS) to remove blood. The left kidney tissues were snap frozen in liquid nitrogen and stored
at -80°C for later analyses. Portions of the remnant kidney tissues were fixed in 4%
formaldehyde for histopathological analysis.
Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents in the renal
tissues were respectively quantified by spectrophotometry  and by means of Loewenberg
, both of which were expressed as per unit of protein determined by the Lowry method
. Besides, Plasma levels of blood urea nitrogen (BUN) and creatinine (Cr) were evaluated
by a Hitachi 7060 Fully Automated Biochemistry Analyzer (Tokyo, Japan). Urine
concentrations of N-acetyl-glucosaminidase (NAG) were determined by para nitrophenol colorimetry
(Kit supplied by Shanghai Debo Biotechnology limited company, Shanghai, China). In
addition, tumor necrosis factor-alpha (TNF-a) and interleukin-10 (IL-10) levels in plasma were
measured with commercially available enzyme-linked immunosorbent assay kits (R&D
systems, Minneapolis, MN, USA). All the procedures were performed according to manufacturer
Briefly, formalin-fixed paraffin-embedded kidney were sectioned at 4μm and stained with
hematoxylin and eosin for general histological assessment. The semiquantitative analysis of the
kidney slides were conducted by a trained histopathologist with no knowledge of treatment
allocation. Each successive field was assessed individually for severity of tubulointerstitial
lesions and graded as follows [30, 31]: 0, normal; 1, areas of tubular epithelial cell swelling,
vacuolar degeneration, necrosis and desquamation involving <25% of cortical tubules; 2, similar
changes involving >25% but <50% of cortical tubules; 3, similar changes involving >50%
but<75% of cortical tubules; 4, similar changes involving>75% of cortical tubules. At least 10
different fields of each slice were visualized by microscopy (×400). The mean scores of all fields
were taken as renal injury scores of above kidney sections
Paraffin-embedded renal sections (4μm) were stained with the terminal deoxynucleotidyl
transferase mediated deoxyuridin triphosphate nick-end labeling (TUNEL) assay using an in
situ apoptosis detection kit (Roche, Germany) according to the manufacturer’s instructions.
Brown labeled TUNEL positive cells were counted in ten randomly selected fields from each
slides at a magnification of ×200, and three different kidney sections were observed for per
animals (n = 10/group) by light microscopy. The TUNEL-positive cells for each field were
quantified as the percentage of total renal cells counted by a blind observer .
Tissue samples were homogenized in lysis buffer solution (13.2 mmol/L Tris-HCl, 5.5%
glycerol, 0.44% SDS and 10% β-mercaptoethanol). After the supernatants were collected, the
protein extracts were obtained by using the total and nuclear protein isolation kit (Thermo, USA).
Protein levels were detected by the Bicichoninic acid (BCA) assay kit (Thermo, USA). Equal
amounts of separated protein (50μg) were fractionated by 12% SDS-PAGE and transferred to a
PVDF membrane (Bio-Rad, USA). Blots were incubated overnight with at 4°C with polyclonal
rabbit antibodies against phosphor(Ser 473)-specific Akt (1:300, Bioss Inc, USA), HO-1(1:800,
Abcam, UK) and Nrf2 (1:300, Biorbyt, UK). Then, blots were washed triple for 10 min with
TBS-0.05% Tween 20, and incubated with 1:3000 dilution of the horseradish peroxidase
(HRP)-conjugated goat anti-rabbit IgG (Biorbyt, UK) for 1h at 37°C. Antigenic detection was
visualized by enhanced chemiluminesence (Bio-Rad, USA) as described by Wang et al , and
the relative density of bands was quantified by densitometry (Molecular Analyst
Imageanalysis Software, Bio-Rad, USA). The densitometer values of measured proteins were
normalized to β-actin used as loading controls.
To estimate the intracellular distribution of Nrf2 nucleoprotein, immunoflurescence analysis
was perfomed as previously described . Kidney tissues were embedded in paraffin,
sectioned at 4μm, and then dewaxed in xylene and rehydrated with graded ethanol solutions.
After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2
primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK). Following washing triple
in phosphate-buffered saline (PBS), slides were counterstained with 4’,
6-diamidino-2-phenylindole (DAPI) (Roche, Shanghai, China) to visualize the nuclei. The images were captured by
a fluorescent microscope (Olympus U-25ND25, Tokyo, Japan) coupled to a digitial camera.
The overlay color of blue staining in nucleus, accompanied with green staining both in
cytoplasm and nucleus was considered to be positive. A semiquantitative numeric scores were
determined for kidney samples based on the percentage of positive protein in five fields of each
slices at a 400 multiple signal magnification using Image-pro plus software (Image pro plus 6.0,
media Cybernetics, USA) [32, 33].
Data are expressed as means ± SD. For signal comparisons, renal injury scores were analyzed
by the non-parametric unpaired Mann-Whitney test. Besides, parametric data were analyzed
for statistical significance by means of one-way ANOVA, followed by the Bonferroni-Dunn
method for multiple comparisons. The analyses were carried out with GraphPad Prism 5.0
(GraphPad software, La Jolla, CA, USA). P values < 0.05 were considered to indicate statistical
As shown in Fig 2, LPS potently decreased the activities of SOD, while increased the contents
of MDA compared to group C (all P<0.01). By contrast, electroacupuncture treatment
significantly counteracted the alterations induced by LPS (P<0.01) (augment 25.4% for SOD,
attenuation 20.2% for MDA). No significant influences in above parameters were discovered when
compared group SEL with group L (P>0.05). Additionally, administration of wortmannin in
group ELW could reverse the protective effects of electroacupuncture at ST36 and PC6
Plasma TNF-a and IL-10
Plasma levels of TNF-α and IL-10 at 6h after LPS injection were shown in Fig 2.
Electroacupuncture stimulation obviously attenuated TNF-a by 29.9% and enhanced IL-10 by 32.6% in
group EL compared with group L or SEL (P<0.01). However, above protective effects were
neutralized to some extent by the selective PI3K inhibitor, wortmannin (P<0.05). There were
no significant differences between group L and group SEL (P>0.05).
Plasma BUN, Cr and urine NAG
Data in Table 1 revealed that rabbits from group L, EL, SEL and ELW possessed higher levels
of BUN, Cr and NAG than group C or W (P<0.01). Besides, as markers of renal function, both
BUN and Cr, as well as NAG were distinctly reduced in electroacupuncture treatment group
compared with group L (P<0.01). Wortmannin pretreatment notably restrained the levels of
these indicators in group ELW (P<0.05). We did not find a significant difference in the levels
of BUN, Cr and NAG between group SEL and group L (P>0.05).
Fig 2. Analysis of SOD activities (A), MDA contents (B) in the renal tissue, as well as plasma IL-10 (C)
and TNF-α levels (D) in each groups. Electroacupuncture at bilaterally Zusanli and Neiguan acupoints
significantly increased SOD activities, decreased MDA contents accompanied with increased levels of IL-10
and decreased TNF-α levels. Pretreatment with wortmannin significantly restrained the above effects of
electroacupuncture in group ELW. Values were presented as mean ± SD (n = 10). The data were analyzed
using one-way ANOVA and the Bonferroni test for multiple comparisons. Significant differences were
indicated with an asterisk: *P<0.05, **P<0.01.
Abbreviations: BUN, blood urea nitrogen; Cr, creatinine; NAG, N-acetyl-glucosaminidase. Values were
+P<0.01 versus group L;
#P<0.05 versus group EL).
As shown in Fig 3, no abnormal structures were visible in group C and group W. In
comparison, kidney tissues from LPS group had extensive cellular vacuolization, swelling and
desquamation in proximal tubules, interstitial edema and neutrophil infiltration. There was a distinct
reduction in the extent of morphological damage in rabbits treated with electroacupuncture at
ST36 and PC6. However, preprocessing with wortmannin suppressed the efficacy of
electroacupuncture treatment. The results, presented as the scores of acute kidney injury, were
summarized in Table 2. Consistently, the levels of kidney injury scores in group EL were lower than
group L, and higher than group ELW (P<0.05). Meanwhile, similar results were observed in
group SEL and group L (P>0.05).
To assess whether electroacupuncture stimulation decreased the amount of renal tubular cell
death in LPS-induced AKI, and the proportion of apoptotic cells in renal slides were
determined by TUNEL staining (Fig 4). An elevated number of TUNEL-positive tubular cells were
observed in rabbits subjected to LPS than that of controls (P<0.05). In contrast to group L,
electroacupuncture preconditioning caused a significant decrease in the number of
TUNELpositive cells in group EL, while wortmannin pretreatment in group ELW blocked the
protective effect of electroacupuncture stimulation (P<0.05). Additionally, the percentage of
TUNEL-positive cells in group SEL resembled that of group L (P>0.05).
Fig 3. Microphotographs of histopathologic changes of kidney sections stained with hematoxylin and
eosin. (original magnification×400): (W, C) Normal morphology of kidneys; (L, SEL) Rabbits treated with LPS
or sham electroacupuncture showing infiltration of inflammatory cells, tubular epithelial cell vacuolization,
swelling and desquamation. (EL) Marked attenuation of tubular damages were displayed in treatment with
electroacupuncture at ST36 and PC6 acupoints. (ELW) Preprocessing with wortmannin suppressed the
protective efficacy of electroacupuncture to some extent. Black arrows: the destruction of renal capsule and
capsular space in group ELW while infiltration of inflammatory cells in group EL or group SEL.
+P<0.05 versus group L;
#P<0.05 versus group EL).
Fig 4. TUNEL in situ assay of tubular cell death in the renal tissues. (original magnification×200): (A) Representative photomicrographs of TUNEL
stained rabbit kidneys corresponding to group ELW, W, C, EL, SEL and group L. White arrows: normal cells; Black arrows: the TUNEL-positive renal tubular
cells. (B) Quantification of the number of TUNEL-positive cells using semi-quantitative analysis of the positive staining from ten randomly selected fields. The
data were expressed as mean ± SD (n = 10; **P<0.01, using one-way ANOVA and the Bonferroni test for multiple comparisons).
Exposure to LPS notably increased the expressions of p-Akt protein, HO-1 protein, Nrf2 total
and nucleoprotein compared with group C (P<0.05), illustrated in Fig 5. Besides,
electroacupuncture treatment markedly upregulated the levels of above mentioned proteins in contrast to
group L or group SEL (P<0.05). In wortmannin-treated rabbits, phosphorylation of Akt was
substantially decreased (P<0.05), while HO-1 protein, Nrf2 total and nucleoprotein were
slightly decreased compared with that in group EL (P>0.05). However, there were no
Fig 5. Western blot analysis of phospho-Akt protein (A), HO-1 protein (B), Nrf2 total (C) and nucleoprotein (D) expressions in the renal tissue of six
groups. β-actin was monitored as the internal standard to ensure similar gel loading of the starting materials in each sample. Blot images were cropped for
comparison (E). Electroacupuncture treatment significantly increased the levels of phospho-Akt protein, HO-1 protein, Nrf2 total and nucleoprotein compared
with group L or group SEL. In wortmannin-treated rabbits, phosphorylation of Akt was significantly decreased while HO-1 protein, Nrf2 total and nucleoprotein
was slightly decreased compared with group EL. All values were expressed as mean ±SD (n = 10; *P<0.05; **P<0.01, using one-way ANOVA and the
Bonferroni test for multiple comparisons). The blots were representative of three independent experiments.
significant differences between group SEL and group L in terms of the measured protein
Renal distribution ratio of Nrf2 nucleoprotein expression
The translocation of Nrf2 from cytoplasm to nucleus by electroacupuncture could be visualized
by Immunofluorescence analysis (Fig 6). After 6h of LPS administration, nuclear localization
of Nrf2 was apparent compared to the negligible distribution in sham operation group
(P<0.01). In addition, electroacupuncture treatment at ST36 and PC6 acupoints dramatically
increased the nuclear accumulation of Nrf2 protein contrast with group L or group SEL
(P<0.01). Moreover, Wortmannin was scarcely effective at inhibiting
electroacupunctureinduced nuclear translocation of Nrf2, yet wortmannin alone had no effect. Sham
electroacupuncture group exhibited indistinguishable expression of Nrf2 nucleoprotein compared with
Data from current study revealed that electroacupuncture stimulation at bilateral ST36 and
PC6 acupoints dramatically alleviated acute kidney injury during endotoxic shock in rabbits,
coincident with the expressions of HO-1 protein, Nrf2 total and nucleoprotein as well as
phospho-Akt protein. Furthermore, electroacupuncture treatment significantly inhibited
LPSFig 6. Immunofluorescence assays of Nrf2 nucleoprotein by fluorescence microscope. (original magnification×400): (A) The pictures of
immunofluorescence staining. Green stands for Nrf2-FITC stained sections, while blue stands for images of DAPI stained nuclei. The overlay color of blue
staining in nucleus, accompanied with green staining both in cytoplasm and nucleus was considered to be positive. (B) Quantification of nuclear localization
of Nrf2 protein among six groups. Electroacupuncture protocols dramatically increased translocation of Nrf2 from cytoplasm into nucleus compared with
group L or SEL. To some degree, pretreatment with wortmannin counteracted nuclear accumulation of Nrf2 protein, while wortmannin alone had no effects.
Data were representative of three independent experiments. Values were expressed as mean ± SD (n = 10; **P<0.01, using one-way ANOVA and the
Bonferroni test for multiple comparisons).
induced tubular cell apoptosis, increased SOD activities, decreased MDA contents in renal
tisits anti-oxidative and anti-inflammatory efficacy. However, blockade of PI3K/Akt pathway by
the administration of wortmannin subtracted the above-mentioned results in
electroacupuncture treated animals. In summary, the protective effects of electroacupuncture at ST36 and PC6
were mainly confirmed against renal injury in lipopolysaccharide-stimulated rabbits via
induction of HO-1 through the upstream PI3K/Akt/Nrf2 pathway.
Acute renal injury in endotoxemia patients is an independent predictor of mortality .
Despite recent advances in renal replacement therapy, the mortality of patients sustained
sepsis-induced kidney injury remained unchanged . However, the etiology of AKI during
septic shock may be multifactorial. The interplay of inflammation and oxidative stress was put
forward to explain the clinical phenotype of endotoxic shock-induced AKI, reflected by the
higher levels of BUN and Cr in plasma . In present study, the scores of acute renal injury,
the number of TUNEL-positive cells, as well as plasma levels of TNF-α and renal contents of
MDA were significantly increased in LPS induced groups. Meanwhile, urinary NAG, as a
sensitive marker of renal tubular damage , was increased as well. Concordant with previous
studies [7, 38], we established the experimental model of injured kidney induced by endotoxic
shock by systemic LPS exposure.
To our knowledge, the application time and frequency of electroacupuncture stimulation
seems critical for producing a prophylactic effect . Hence, acupuncture was delivered
5 days before LPS injection with the disperse-dense wave (2Hz or 15Hz) and the intensity of
less than 1.0 mA to exert renoprotection effects in current study [21, 40]. From another aspect,
HO-1 with the subsequent metabolites of heme catabolism possessed intriguing signaling
properties affecting numerous critical cellular functions, including inflammation, oxidative
stress, cellular proliferation, and cellular survival . Data from this study verified the direct
involvement of HO-1 in mitigating LPS-induced acute kidney injury by electroacupuncture
treatment at ST36 and PC6 acupoints.
In response to oxidative stress or inflammatory, Nrf2, as a bZIP transcription factor,
dissociated from its cytosolic inhibitor, kelch-like ECH-associated protein (Keap1), and translocated
to the nucleus, dimerized with a cis-element referred to as antioxidant response element (ARE)
and ultimately transcribed its target genes . Activation of Nrf2/ARE pathway has been
reported to account for the therapeutic effects of HO-1 induction against sepsis . Besides,
as a conserved family of signal transduction, phosphoinositide 3-kinases (PI3K) are involved in
nuclear translocation of Nrf2 via actin cytoskeletal changes . Kuwana H et al. indicated
, PI3K/Akt pathway lessened cisplatin-induced acute kidney injury and played a critical
role in the maintenance of renal function. Consequently, upregulated of phospho-Akt protein,
HO-1 protein, Nrf2 total and nucleoprotein expressions by electroacupuncture treatment was
displayed in renal tissue, followed with the increments of Nrf2 protein accumulated in the
nucleus by immunofluorescence staining. In contrast, wortmannin, the selective PI3K
inhibitor, moderately eliminated above beneficial effects of electroacupuncture, indicating the
protection against acute kidney injury during endotoxic shock was mediated through PI3K/Akt/
There were some limitations to this study that we would like to acknowledge. Firstly,
Coincident with previous studies , both the levels of Nrf2 total protein and nucleoprotein were
significantly upregulated by electroacupuncture treatment, which called for further
exploration. Secondly, urine NAG excretion was used as a marker of renal tubular damage in current
study, however, needed to be further evaluated in clinical settings . Hence, it is desirable to
develop novel biomarkers for early diagnosis of acute kidney injury, especially in septic
patients. Finally, wortmannin pretreatment made a little difference to the expressions of HO-1,
Nrf2 nucleoprotein and total protein, which were not in consistent with levels of p-Akt protein.
The contradictory results indicated that PI3K/Akt may be not the only pathway responsible for
electroacupuncture-afforded protection against LPS-induced AKI. Besides, the P38 MAPK,
ERK, and PKC signaling pathways have been reported to mediate the phosphorylation of Nrf2
and the expression of HO-1 . Therefore, to identify which signal cascade facilitated the
activation of Nrf2/ARE by electroacupuncture and the interplay between these cascades and PI3K/
Akt pathway still required further investigation.
Based on the data from current study, we concluded that treatment with electroacupuncture
at ST36 and PC6 acupoints could ameliorate acute kidney injury induced by LPS in rabbits, the
underlying mechanism of which involved the upregulation of HO-1 through the modulation of
PI3K/Akt/Nrf2 pathway. This present finding aspires to lay a foundation for potential future
clinical applications of electroacupuncture for acute kidney injury induced by endotoxic shock.
We thank Dr. Jian-bo Wang (Washington University School of Medicine) for his technical
support and valuable advice.
Conceived and designed the experiments: JBY LRG. Performed the experiments: JS YZ SAD
XSC LLW LNW. Analyzed the data: JS SAD. Contributed reagents/materials/analysis tools:
JBY YZ. Wrote the paper: JBY JS. Technical support and valuable advice: JBY.
induced acute kidney injury. Anesthesiology. 2012; 117: 126–136. doi: 10.1097/ALN.
0b013e31825b57c9 PMID: 22634873
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