Renal chymase-dependent pathway for angiotensin II formation mediated acute kidney injury in a mouse model of aristolochic acid I-induced acute nephropathy
Renal chymase-dependent pathway for angiotensin II formation mediated acute kidney injury in a mouse model of aristolochic acid I-induced acute nephropathy
Wen-Yeh Hsieh 0 1
Teng-Hsiang Chang 1
Hui-Fang Chang 1
Wan-Hsuan Chuang 1
Li-Che Lu 1
Chung-Wei Yang 1
Chih-Sheng LinID 1
Chia-Chu ChangID 1
0 Division of Pulmonary Medicine, Department of Internal Medicine, Hsinchu Mackay Memorial Hospital , Hsinchu, Taiwan , 2 Division of Nephrology, Department of Internal Medicine, Changhua Christian Hospital , Changhua, Taiwan , 3 Department of Biological Science and Technology, National Chiao Tung University , Hsinchu, Taiwan , 4 Division of Endocrinology, Department of Internal Medicine, Hsinchu Mackay Memorial Hospital , Hsinchu, Taiwan , 5 Division of Nephrology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital , Taipei, Taiwan , 6 Division of Nephrology, Department of Internal Medicine, National Taiwan University Hospital Hsinchu Branch , Hsinchu, Taiwan, 7 Center for Intelligent Drug Systems and Smart Bio-devices (IDS2B) , National Chiao Tung University , Hsinchu, Taiwan , 8 School of Medicine, Chung- Shan Medical University , Taichung, Taiwan , 9 Department of Environmental and Precision Medicine Laboratory, Changhua Christian Hospital , Changhua, Taiwan , 10 Department of Nutrition, Hungkuang University , Taichung, Taiwan , 11 Department of Internal Medicine, Kuang Tien General Hospital , Taichung , Taiwan
1 Editor: Michael Bader, Max Delbruck Centrum fur Molekulare Medizin Berlin Buch , GERMANY
Angiotensin-converting enzyme (ACE) is the primary enzyme that converts angiotensin I (Ang I) to angiotensin II (Ang II) in the renin-angiotensin system (RAS). However, chymase hydrates Ang I to Ang II independently of ACE in some kidney diseases, and it may play an important role. The present study investigated whether chymase played a crucial role in aristolochic acid I (AAI)-induced nephropathy. C57BL/6 mice were treated with AAI via intraperitoneal injection for an accumulated AAI dosage of 45 mg/kg body weight (BW) (15 mg/kg BW per day for 3 days). The animals were sacrificed after acute kidney injury development, and blood, urine and kidneys were harvested for biochemical and molecular assays. Mice exhibited increased serum creatinine, BUN and urinary protein after the AAI challenge. Significant infiltrating inflammatory cells and tubular atrophy were observed in the kidneys, and high immunocytokine levels were detected. Renal RAS-related enzyme activities were measured, and a significantly increased chymase activity and slightly decreased ACE activity
Data Availability Statement: All relevant data are
within the manuscript and its Supporting
Funding: This work was supported by the grants of
MOST 104-2313-B-009-001-MY3 and MOST
1072313-B-009-002-MY3 from the Ministry of Science
and Technology (MOST), Taiwan. This work was
also financially supported by the "Center For
Intelligent Drug Systems and Smart Bio-devices
(IDS2B)" from The Featured Areas Research Center
were observed in the AAI-treated mice. The renal Ang II level reflected the altered profile of
RAS enzymes and was significantly increased in AAI-treated mice. Treatment of
AAIinduced nephropathic mice with an ACE inhibitor (ACEI) or chymase inhibitor (CI;
chymostatin) reduced renal Ang II levels. The combination of ACEI and CI (ACEI+CI) treatment
significantly reversed the AAI-induced changes of Ang II levels and kidney inflammation and
injuries. AAI treatment significantly increased renal p-MEK without increasing p-STAT3 and
p-Smad3 levels, and p-MEK/p-ERK1/2 signalling pathway was significantly activated. CI
Program within the framework of the Higher
Education Sprout Project of the National Chiao
Tung University and Ministry of Education (MOE),
Taiwan. 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.
and ACEI+CI treatments reduced this AAI-activated signaling pathway. AAI-induced
nephropathy progression was significantly mitigated with CI and ACEI+CI treatment. This
study elucidates the role of RAS in the pathogenesis of AAI-induced nephropathy.
Aristolochic acid nephropathy (AAN) is a rapidly progressive interstitial nephritis that leads to
urothelial malignancy, end-stage renal disease and irreversible kidney failure. AAN was
originally reported in Belgium in a group of patients who ingested slimming pills that contained
powdered root extracts of Chinese herbs [
]. The incidence of AAN is likely high because of
the presence of aristolochic acid (AA) in herbal remedies and lack of awareness of the disease
. AA is derived from the Aristolochia species, and it is the active principle agent in slimming
pills. AA is a mixture of structurally related nitro-phenanthrene carboxylic acids, which are
primarily composed of aristolochic acid I (AAI) and aristolochic acid II (AAII) [
these, AAI has been proven as the major factor of the nephrotoxicity associated with
AAIinduced nephropathy [
]. Renal histology in the chronic pathology of AAN reveals the
formation of tubulointerstitial fibrosis (TIF) and tubular atrophy [
]. Renal microvasculature
injury and an imbalance of endothelial vasoactive agents may lead to fibrosis in AAN [
Sclerosis of glomeruli are also observed [
]. Animal models of AAN are widely used in
investigations of renal toxicity of Aristolochia and Asarum genus herbs [
], and exhibit similar
pathological characteristics as human chronic kidney diseases. Animal models of AAN have
been used for the two past decades to examine the underlying molecular and cellular
mechanisms involved in AAN pathogenesis . AAI-induced rodent models of acute or chronic
kidney injury/disease models are well-established [
]; however, information on the disease
mechanisms of AAI-induced acute kidney injuries related to the dysregulation or imbalance of
the renin-angiotensin system (RAS) are not known.
Angiotensin-converting enzyme (ACE) is the primary and classical enzyme that converts
angiotensin I (Ang I) to angiotensin II (Ang II) in the renin-angiotensin system of
cardiovascular and renal systems [
]. Unbalanced RAS and an abnormally activated ACE/Ang II axis
are the primary effectors that contribute to the onset and progression of renal damage [
Abnormally excessive local Ang II may also directly contribute to the acceleration of renal
damage via sustaining cell growth, inflammation, and fibrosis [
]. Clinical therapy for renal
diseases generally includes angiotensin-converting enzyme inhibitor (ACEI) and Ang II
receptor blockers (ARBs) to decrease ACE/Ang II activation and ameliorate disease development.
However, the results of these treatments vary depending on the person and disease [
The treatment outcome of the combination of ACEIs and ARBs is controversial [
Therefore, the need for new therapeutic targets is fueled by the failure of traditional RAS
blockade, such as the direct renin inhibitor aliskiren and chymase inhibitors.
Chymase is a serine protease that primarily converts Ang I to Ang II via an
ACE-independent pathway [
]. The evidence has suggested that chymase is an alternative pathway of ACE
conversion and Ang II formation in tissues [
], and it exhibits a catalytic efficiency 20-fold
greater than ACE [
]. Chymase is weakly expressed in glomeruli and vascular smooth muscle
cells in normal human kidney, and it is markedly upregulated in diabetic kidneys [
chymase ameliorated renal TIF in unilateral ureteral obstruction [
]. In diabetic nephropathy
rodent models, chymase inhibition protected diabetic rats from renal lesions [
chymase released TGF-?1 from the extracellular matrix (ECM) via specific proteolytic cleavage of
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the latent TGF-? binding protein to increase cellular inflammation [
]. Many interventions
that inhibit RAS activity are renoprotective and may retard the progression of chronic
nephropathies, but several studies suggested that chymase played an important role in some
renal diseases [
]. Chymase plays a pivotal role in the pathogenesis of renal disease and
kidney injury. Therefore, the present study examined the effects of the inhibition of
chymaseinduced Ang II formation in acute kidney injury in mice with AAI-induced nephropathy.
Methods and materials
An animal model of acute nephropathy was performed in C57BL/6 mice. Mice were treated
with AAI via intraperitoneal (i.p.) injection, and the accumulated AAI dosages was 45 mg/kg
BW (body weight) (15 mg/kg BW per day for 3 days). Animals were sacrificed 24 hours after
acute nephropathy development, and blood and kidneys were harvested for further
biochemical and molecular assays. Blood and tissue samples were immediately isolated after
anesthetization to reduce the physiological effects of the anesthesia, including RAS elements.
Mice were randomly divided into five groups (n = 9 for each group) and received the
following treatments: (1) Control group?mice treated with saline; (2) AAI group?mice
treated with i.p. AAI (15 mg/kg BW per day for 3 days); (3) AAI/ACEI group?mice treated
with i.p. AAI + i.p. Captopril (10 mg/kg BW per day for 3 days); (4) AAI/CI (chymase
inhibitor) group?mice treated with i.p. AAI + i.p. Chymostatin (10 mg/kg BW per day for 3
days); and (5) AAI/ACEI+CI group?mice treated with i.p. AAI + i.p. Captopril (10 mg/kg
BW per day for 3 days) + i.p. Chymostatin (10 mg/kg BW per day for 3 days). AAI
(#A5512), Captopril (ACEI; #C4042) and Chymostatin (#C7268) were purchased from
Sigma-Aldrich (St. Louis, MO, USA).
All mice were maintained in a specific pathogen-free facility with free access to food and
water. Animal care and all experimental procedures were performed in accordance with the
Guideline for Animal Use Protocol National Chiao Tung University with approval of the
Institutional Animal Care and Use Committee (permission number: NCTU-IACUC-103013).
Mice were routinely checked twice daily for signs of illness. Mice that were likely to reach the
humane endpoint prior to the next monitoring event (i.e., 20% body weight loss, hair removal
or behavioral disorder) were immediately euthanized using CO2. None of the mice were found
dead. Forty-five mice were humanely euthanized via exsanguination under anesthesia for
tissue collection, and every effort was made to minimize distress and suffering. All animal care
workers had over 2 years of experience working with laboratory animals.
Sample preparation and Western blot
Kidney samples for use in biochemical and molecular analyses were prepared as described
]. Organ samples were collected after animal sacrifice and homogenized 3 times in
the lysis buffer PRO-PREPTM Protein Extraction Solution (iNtRON Biotechnology,
KyungkiDo, Korea). Samples were centrifuged at 13,000 x g for 10 min for separation of supernatants
and pellets. The total amount of protein in the homogeneous extract was measured using the
Bradford dye binding assay (Bio-Rad Laboratories, Hercules, CA, USA). The supernatants
were aliquoted and stored at -80?C until further use.
Kidney homogenates with an equivalent protein content of 25 ?g protein were
electrophoresed on 12% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes
(Immobilon-PTM; Millipore, Bedford, MA, USA). Primary antibodies against phosphor-STAT3
(pSTAT3), p-ERK1/2, p-JNK, p-p38, p-Smad, p-MEK and ?-actin were obtained from Genetex
(Irvine, CA, USA) or Cell Signaling Technology (Beverly, MA, USA). Chemiluminescence
substrates were visualized using enhanced chemiluminescence detection and a luminescence
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image system (LAS-3000; Fuji Film, Stamford, CT, USA). Bands on the images were detected
at the anticipated location based on size. Band intensity was quantified using Scion Image
software (Scion, Frederick, MD, USA). The levels of p-STAT3, p-ERK1/2, p-JNK, p-p38, p-Smad,
p-MEK, p-ERK1/2 and p-STAT3 were normalized to ?-actin.
Measurements of ACE and ACE2 activity
Renal ACE and angiotensin-converting enzyme 2 (ACE2) activities were assayed using the
fluorogenic substrates Mca-YVADAPK and Mca-APK-Dnp (AnaSpec, San Jose, CA, USA),
respectively, according to our previous report [
]. The assay was performed in a microquartz
cuvette using 20 ?L of diluted kidney homogenates and 2 ?L of the fluorogenic substrates
(stock concentration: 4 mM ACE substrate/1.5 mM ACE2 substrate) in ACE or ACE2 assay
buffer (a total of reaction volume is 300 ?L). The reaction was measured for 15 sec every 45 sec
for 1 hour using a fluorescence reader at 330 nm/390 nm. Each sample was detected in
duplicate and normalized to a positive control in the same plate. All samples were fitted and plotted
using Grafit v. 4.0 (Sigma-Aldrich, St. Louis, MO, USA). Samples were also incubated with the
above-mentioned reaction mixture in the presence of 1 ?M captopril (Sigma-Aldrich; a
specific ACE inhibitor) or 1 ?M DX600 (AnaSpec; a specific ACE2 inhibitor) for the ACE and
ACE2 activity assays, respectively.
Measurements of chymase activity
Chymase activity was detected using the gold nanoparticles (AuNPs)-peptide probe developed
by our laboratory (FITC-Acp-DRVYIHPFHLDDDDDC-AuNPs) [
]. A total volume
250 ?L, including AuNPs-peptide probe (125 ?L), reactive buffer (pH 8, TTC buffer) and
kidney tissue homogenous extract, was incubated in a micro-quartz cuvette at 37?C for 15 min.
The fluorescence intensity was recorded and analyzed at 515 nm at an excitation wavelength
of 495 nm using a fluorescence spectrophotometer. The specific chymase inhibitor
chymostatin (Sigma-Aldrich) was used in parallel samples to determine the accurate activity of chymase
in kidney tissue.
Renal Ang II and inflamatory cytokine measurements
The concentrations of renal Ang II and inflamatory cytokines in mouse kidneys were
determined using mouse Ang II (Biocodon; Broadmoore, KS, USA), IL-6, TNF-? and TGF-?1
ELISA kits (Abcam, Cambridge, MA, USA). The levels in each samples were determined in
the same volumes in duplicate. Measurements were performed according to the
manufacturer?s instructions. Briefly, tissue homogenates were incubated in 96-well ELISA plates with
primary antibodies. Biotinylated antibodies were added, and the plates were washed and
reacted with HRP-conjugated streptavidin. Tetramethylbenzidine (TMB) one-step substrate
tablets were used for the detection of targets (Ang II, IL-6, TNF-?, and TGF-?1), and the
results were measured at 450 nm using a micro-plate reader (Thermo Scientific, Waltham,
MA, USA). The intensity of the color developed was inversely proportional to the target
concentration in the samples.
Kidney tissues were collected from the experimental mice, soaked in 10% formalin overnight,
embedded in paraffin, and cut into 6-?m-thick sections on acid-pretreated slides for
hematoxylin/eosin (H&E) staining to investigate leukocyte infiltration, which indicated inflammation,
and periodic acid?Schiff (PAS) stain to examine lesions of tubular atrophy. The stained
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pathological sections were photographed using a digital camera mounted on a microscope. A
computerized microscope equipped with a high-resolution video camera (BX 51; Olympus,
Tokyo, Japan) was used for morphometric analysis.
Animal body weights were recorded daily or weekly during the experiments. Urine samples
were collected weekly using metabolic cages (Tecniplast, Buguggiate, Italy) for 24 hr and stored
at -30?C after centrifugation at 3,000 rpm for 15 min. Urine and plasma levels of creatinine,
albumin and BUN were measured using a clinical chemical analyzer (#DRI-CHEM 3500;
Fujifilm Medical, Tokyo, Japan) to evaluate mouse renal functions.
All values are expressed as the means ? standard deviation (SD). One-way analysis of variance
(ANOVA) tests were performed to evaluate differences between multiple groups. Significant
data were further examined using post hoc tests to determine the significance between groups.
A value of p < 0.05 was considered statistically significant.
Physiological changes of mice with AAI-induced kidney injury
Body weights of C57BL/6 mice were recorded daily during AAI treatment. The body weights of
Control group mice treated with PBS exhibited no significant changes. However, body weights
decreased significantly after 3 days of AAI treatment, and significantly recovered after ACEI, CI
and ACEI+CI treatment compared to the AAI treatment group (Fig 1A). The decrease in body
weight was the result of a significant decrease in food and water intake after AAI treatment.
This physiological change reflects the effect of AAI treatment on organ damage.
Biochemical analysis revealed significantly increased serum BUN and creatinine
concentrations in mice treated with AAI for 3 days during the progression of acute kidney injury. The
levels of serum BUN and creatinine were lower in the groups treated with AAI and ACEI, CI
and ACEI+CI treatment than the AAI group (Fig 1B). Urine protein and albumin levels were
also significantly elevated in mice after AAI treatment. The concentrations of urine protein
and albumin decreased significantly after CI and ACEI+CI treatment compared to the AAI
group (Fig 1C). Increased serum BUN and creatinine and proteinuria are related to kidney
dysfunction, and our results indicated that AAI caused severe renal impairment in the
Pathological findings in kidney
Analysis of renal immunocytokine expression confirmed that AAI treatment induced renal
inflammation in mice. The levels of renal IL-6, TGF-?1 and TNF-? in mice treated with AAI
were approximately 4.1- (p < 0.01), 2.7- (p < 0.01) and 3.0-fold (p < 0.01) higher than the
control mice, respectively (Fig 2). The inhibition of Ang II generation also efficiently reduced the
expression and secretion of renal immunocytokines. The effect of chymase inhibition was
larger than ACE inhibition.
Pathological staining of kidney tissues revealed significant interstitial infiltration of
inflammatory cells and severe tubular atrophy in the AAI group compared to the Control group.
These results indicate the AAI induced acute nephropathy in mice. Kidney tissues from the
AAI-treated mice that received ACEI, CI and ACEI+CI treatments exhibited significantly
reduced inflammatory cells and lesions of tubular atrophy. These results indicate a dramatic
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Fig 1. Body weights, serum and renal biochemical determinations in mice with AAI-induced acute nephropathy. Animal model
of nephropathy was performed in C57BL/6 mice. The mice were treated with AAI via intraperitoneal (i.p.) injection, and the
accumulated AAI dosage was 45 mg/kg BW (15 mg/kg BW per day for 3 days). Five groups of 9 mice per group were used. Mouse
body weights were recorded daily (A), changes in serum BUN and creatinine (B) levels, and urine protein and urine albumin (C)
levels were determined. All values are expressed as the means ? SD of each group (n = 9). indicates p < 0.01 compared to the
Control group; ? and ?? indicate p < 0.05 and p < 0.01 compared to the AAI group, respectively.
PLOS ONE | https://doi.org/10.1371/journal.pone.0210656
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Fig 2. Renal inflammatory cytokine determinations in mice with AAI-induced acute nephropathy. Changes in renal IL-6 (A), TGF-?1 (B)
and TNF-? (C) levels were determined. All values are expressed as the means ? SD from each group (n = 9). indicates p < 0.01 compared to
the Control group; ? and ?? indicate p < 0.05 and p < 0.01 compared to the AAI group, respectively.
amelioration of acute kidney injury in mice treated with ACEI and/or CI treatments compared
to AAI-treated mice (Fig 3).
Renal RAS-related enzyme activities and Ang II levels
Renal chymase level was significantly increased 2.4-fold after AAI treatment (p < 0.01). ACE2
level was significantly decreased 0.6-fold (p < 0.01), and renal ACE activity was slightly
decreased compared to the Control group (Fig 4). The levels of renal ACE were significantly
decreased in ACEI and ACEI+CI treatment groups (Fig 4A), and chymase activities were
significantly reduced in AAI-treated mice treated with CI and ACEI+CI treatments (Fig 4B).
Renal ACE2 levels were not significantly altered after ACEI, CI and ACEI+CI treatments (Fig
4C). These results demonstrated that the inhibition of Ang II production did not affect renal
ACE2 expression. Renal Ang II levels reflect changes in the profile of these RAS enzymes and
were significantly increased in AAI-treated mice (i.e., AAI group), the AAI/ACEI and AAI/CI
groups compared to the Control group. Ang II levels decreased significantly after CI and ACEI
+CI treatment in AAI-treated mice (p < 0.01). Notably, no significant difference in Ang II
levels was observed between the AAI/ACEI+CI and Control groups (Fig 5).
Molecular expression in the involved signaling pathways
The levels of renal p-MEK, p-STAT3 and p-Smad3 expression increased significantly after
AAI treatment by 2.06- (p < 0.01), 1.26- (p < 0.05) and 1.25-fold (p < 0.01), respectively (Fig
6). Notably, CI and ACEI+CI treatment significantly decreased AAI-induced p-MEK
expression (Fig 6A). However, the inhibition of Ang II generation did not reduce AAI-induced
pSTAT3 and p-Smad3 expression (Fig 6B and 6C). The expression levels of p-ERK, p-JNK and
p-38 in the MAPK signaling pathway were also significantly increased in AAI-treated mice by
2.44- (p < 0.01), 1.28- (p < 0.05) and 1.58-fold (p < 0.01), respectively (Fig 7). The levels of
AAI-induced p-ERK expression were significantly reduced after ACEI, CI and ACEI+CI
treatments of AAI-treated mice (Fig 7A). However, Ang II blocker treatment only slightly reduced
AAI-induced p-JNK and p-38 levels (Fig 7B and 7C).
The present study induced acute kidney injury in mice using a high dosage of i.p. AAI, and the
biochemical, pathological and immunological findings demonstrated that AAI-related acute
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Fig 3. Pathological features of AAI-induced kidney injury revealed by light microscopy. The upper panels show H&E staining and leukocyte
infiltration indicative of the inflammation induced by AAI treatment and the alleviation of this response by ACEI, CI and the combination of ACEI
and CI (ACEI + CI) treatments. The lower panels show PAS staining and the lesions of tubular atrophy induced by AAI treatment, and the
alleviation of lesions by ACEI, CI and ACEI + CI treatments.
nephropathy was induced. The AAI-induced acute nephropathy was associated with
inflammation, RAS dysregulation and abnormally increased renal Ang II. This study is the first
demonstration that modulation of Ang II signaling, especially chymase inhibition, alleviated
AAIinduced kidney injury.
AAI is nephrotoxic and causes upper urinary tract urothelial carcinoma and kidney fibrosis
]. AAI-induced acute kidney injury in a mouse model was recently identified by impaired
expression of profibrotic genes and proteins associated with their downstream target genes in
Fig 4. Changes in RAS enzyme activity in kidneys of mice with AAI-induced acute nephropathy. Renal ACE (A), chymase (B) and ACE2 (C)
activities were determined, and the related activity levels are shown as levels relative to the Control group. All values are expressed as the
means ? SD from each group (n = 9). and indicate p < 0.05 and p < 0.01 compared to the Control group, respectively; ?? indicates p < 0.01
compared to the AAI group.
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Fig 5. Changes in Angiotensin II (Ang II) in kidneys of mice with AAI-induced acute nephropathy. Renal Ang II
levels were determined, and the related activity levels are shown as the levels relative to the Control group. All values
are expressed as the means ? SD from each group (n = 9). and indicate p < 0.05 and p < 0.01 compared to the
Control group, respectively; ?? indicates p < 0.01 compared to the AAI group.
renal tissues [
]. Generally, dosage of 1 to 5 mg/kg BW of AAI administration is used to
induce chronic kidney disease . A dosage of AAI > 10 mg/kg BW [
] and a single AAI
dose of 10, 20 and 30 mg/kg BW [
] were used in mouse models of acute kidney injury. LD50
values in mice after oral and intravenous (i.v.) administration of AA are 60?106 mg/kg BW
(survival time of 1?15 days) and 38?70 mg/kg BW (survival time of 1?13 days), and female
mice are more tolerable to acute AA toxicity than male mice [
]. The effects of AAI-induced
kidney toxicity in experimental mice are variable and depend on mouse species, age and
gender. Our study used female C57BL/6 mice, and AAI was injected i.p. Therefore, an LD50 > 70
mg/kg BW was expected. Our preliminary results revealed that mice did not die until the 6th
day after 45 mg/kg BW AAI treatment. Therefore, an accumulated AAI dosage of 45 mg/kg
BW (15 mg/kg BW per day for 3 days) was administered to induce acute kidney injury in
Ang II in RAS is a key factor in the inflammatory and fibrotic responses in kidney diseases
]. Abnormally increased Ang II may be involved in the renal fibrotic process because of
its behavior as a proinflammatory cytokine. Adequate Ang II regulation may prevent kidney
inflammatory injury and fibrosis progression . Chymase is the main ACE-independent
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Fig 6. The level of inflammatory signaling pathways in the kidneys of mice with AAI-induced acute nephropathy. The expression of p-MEK
(A), p-STAT3 (B) and p-Smad3 (C) in kidney tissues was determined using Western blotting and representative results of Western blotting are
shown (D). All values are expressed as the means ? SD from each group (n = 9). and indicate p < 0.05 and p < 0.01 compared to the Control
group, respectively; ?? indicates p < 0.01 compared to the AAI group.
pathway of Ang II generation in mice, and it is involved in blood pressure regulation during
ACE inhibitor therapy [
]. Chymase activity is associated with glomerulosclerosis and renal
interstitial fibrosis in animal models [
]. Animal studies suggest that chymase inhibition is
beneficial for the treatment of hypertensive, diabetic and inflammatory nephropathies [
Chymase is primarily released during tissue injury or inflammation, and it promotes tissue
]. Renal ACE activity in the AAI group was not significantly altered, and renal
chymase activity was markedly increased in our study. The levels of renal Ang II indicated
changes in the profile of these RAS enzymes, and a noticeable increase in renal Ang II was
observed in AAI-induced acute nephropathy. The combination of ACEI and CI treatment
significantly reduced Ang II levels and alleviated acute kidney injury in AAI-treated mice. These
results suggest that the chymase-dependent Ang II axis is a critical pathway in AAI-induced
Our results demonstrated significantly higher levels of renal p-MEK, p-STAT3 and
Smad3 in AAI-treated mice. However, only the inhibition of Ang II generation with ACEI, CI,
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Fig 7. The MAPK signaling pathways in the kidneys of mice with AAI-induced acute nephropathy. The expression
of p-REK1/2 (A), p-JNK (B) and p-38 (C) in kidney tissue was determined using Western blotting, and representative
results of Western blotting are shown (D). All values are expressed as the means ? SD from each group (n = 9). and
indicate p < 0.05 and p < 0.01 compared to the Control group, respectively; ? and ?? indicates p < 0.05 and
p < 0.01 compared to the AAI group, respectively.
and ACEI+CI significantly mitigated AAI-induced p-MEK activation. MEK is the most
representative molecule in the MAPK signaling pathway, and it is activated by the oxidative and
inflammatory stresses induced by excess Ang II [
]. Inflammation triggered by IL-6 and
TGF-? induces p-STAT3 and p-Smad3, respectively [
]. TGF-? leads to kidney tissue
fibrosis via inhibition of matrix degradation and stimulation of myofibroblast activation.
Smad3 is a key factor in this process because deletion of Smad3 protects against AAN [
results explain why the inhibition of Ang II production significantly reduced active p-MEK
but minimally reduced AAI-induced p-STAT3 and p-Smad3 levels. These results confirm the
multiple mechanisms of tissue damage induced by AAI treatment [
mechanisms of AA-induced cytotoxicity are well defined, such as increased oxygen stress,
inflammation, fibrosis, and the formation of DNA adducts [
]. The damage mechanism also includes an
imbalance in RAS, which was demonstrated in our study. Therefore, these results explain why
treatments with ACEI, CI and ACEI/CI ameliorated, but did not completely prevent, the toxic
effects of AAI.
ERK, JNK and p38 are three major members of the MAPK signaling pathway [
However, Ang II blockade with ACEI, CI, and especially ACEI+CI only significantly mitigated
active p-ERK in AAI-treated mice. This result suggests that AAI-induced acute nephropathy
involved the p-ERK signaling pathway, which was associated with abnormally excessive Ang II
formation. Numerous stimuli activate the ERK signaling pathway, which is correlated with the
regulation of survival, differentiation, proliferation, mitosis, and apoptosis [
]. p-ERK plays a
crucial role in the oxidation-dependent axis that results in kidney impairment [
], and it is
activated in proliferative glomerulonephritis [
] and unilateral ureteral obstruction-induced
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renal fibrosis in rats [
]. p-MEK is an upstream regulator in the ERK signaling pathway
. Therefore, we demonstrated that AAI-induced acute kidney injury was associated with
an imbalance of the RAS axis, which generated excess Ang II and p-MEK/p-ERK signaling
activation. We observed a dramatic elevation of the p-MEK/p-ERK1/2 signaling pathway in
the AAI group, which may be related to renal Ang II levels, and the combination treatment of
ACEI and CI reflected the reduction of the AAI-activated signaling pathway and the changed
profile of renal Ang II levels. This hypothesis is consistent with the previous studies. Qin et al.
] reported that Ang II deteriorated renal interstitial fibrosis via the induction of oxidative
stress and the ERK/MAPK signaling pathway. Liu et al. [
] indicated that Ang-II exacerbated
glomerulosclerosis via enhancing p-ERK in chronic kidney disease.
The experimental mice treated with a high dose of AAI exhibited renal inflammation and
acute nephropathy. RAS dysregulation was associated with AAI-induced nephropathic
progression and abnormally increased renal Ang II. Our results suggest that chymase plays a role
in pathogenesis, and the AAI-induced chymase-Ang II axis exacerbated kidney injuries via the
p-MEK/p-ERK1/2 signaling pathway. Therefore, the lowering of Ang II levels using inhibitors
and specific chymase inhibition of the Ang II-generating pathways may effectively mitigate
AAI-induced acute kidney injury. The present study elucidates the role of RAS in the
pathogenesis of AAI-induced acute nephropathy. However, further research is needed to confirm
whether the ACEI and CI combination treatment is a more potent renoprotective therapy for
AAI-induced acute nephropathy.
This work was supported by the grants of MOST 104-2313-B-009-001-MY3 and MOST
1072313-B-009-002-MY3 from the Ministry of Science and Technology (MOST), Taiwan. This
work was also financially supported by the "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 of the National Chiao Tung University and
Ministry of Education (MOE), Taiwan.
Conceptualization: Chia-Chu Chang.
Investigation: Li-Che Lu.
Methodology: Li-Che Lu, Chung-Wei Yang.
Project administration: Chih-Sheng Lin.
Supervision: Chih-Sheng Lin, Chia-Chu Chang.
Validation: Wen-Yeh Hsieh, Teng-Hsiang Chang.
Visualization: Wen-Yeh Hsieh.
Data curation: Hui-Fang Chang, Wan-Hsuan Chuang, Li-Che Lu, Chung-Wei Yang.
Writing ? original draft: Wen-Yeh Hsieh, Teng-Hsiang Chang, Hui-Fang Chang,
Writing ? review & editing: Chih-Sheng Lin, Chia-Chu Chang.
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