Ergosta-4,6,8(14),22-tetraen-3-one isolated from Polyporus umbellatus prevents early renal injury in aristolochic acid-induced nephropathy rats.

Abstract

Aristolochic acid (AA) nephropathy, first reported as Chinese herbs nephropathy, is a rapidly progressive tubulointerstitial nephropathy that results in severe anemia, interstitial fibrosis and end-stage renal disease. Tubulointerstitial injury was studied in a rat model of AA nephropathy to determine whether ergosta-4,6,8(14),22-tetraen-3-one (ergone) treatment prevents early renal injury in rats with aristolochic acid I-induced nephropathy.
Early renal injury via renal interstitial fibrosis was induced in rats by administration of aristolochic acid I (AAI) solution intragastrically for 8 weeks. Ninety-six rats were randomly divided into four groups (n = 24/group): (1) control (2) AAI (3) AAI + ergone (10 mg/kg) and (4) AAI + ergone (20 mg/kg). Blood and urine samples were collected and rat were sacrificed for histological assessment of the kidneys on at the end of weeks 2, 4, 6 and 8.
AAI caused progressive elevation of blood urea nitrogen, creatinine, potassium, sodium, chlorine, proteinuria and urinary N-acetyl-β-D-glucosaminidase (NAG). Ergone suppressed elevation of blood urea, nitrogen, creatinine, proteinuria and urinary NAG to some degree, but the AAI-ergone-treated group did not differ from AAI-treated group for body weight, serum potassium, sodium and chlorine. The progress of the lesions in the kidney after AAI administration was also observed by histopathological examinations, but kidneys from rats of AAI-ergone-treated group displayed fewer lesions.
Ergone treatment conferred protection against early renal injury in a rat model of AA nephropathy. Early administration of ergone may prevent the progression of renal injury and the subsequent renal fibrosis in AA nephropathy.

Full text

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Ergosta-4,6,8(14),22-tetraen-3-one isolated from Polyporus
umbellatus prevents early renal injury in aristolochic
acid-induced nephropathy ratsjphp_13611581..1586
Ying-Yong Zhaoa, Li Zhangc, Jia-Rong Maob, Xiao-Hong Chengb,
Rui-Chao Lina,d, Yongmin Zhangeand Wen-Ji Suna
aBiomedicine Key Laboratory of Shaanxi Province, The College of Life Sciences, Northwest University,
bKidney Disease Center of Affiliated Hospital, Shanxi Provincial Academy of Traditional Chinese
Medicine,cDepartment of Nephrology, Xi’an No. 4 Hospital,dNational Institutes for Food and Drug
Control, State Food and Drug Administration, Beijing, China andeUniversité Pierre and Marie Curie,
Institut Parisien de Chimie Moléculaire, UMR CNRS 7201, 4 Place Jussieu, Paris, France
Abstract
Objectives Aristolochic acid (AA) nephropathy, first reported as Chinese herbs nephropa-
thy, is a rapidly progressive tubulointerstitial nephropathy that results in severe anemia,
interstitial fibrosis and end-stage renal disease. Tubulointerstitial injury was studied in a rat
model ofAAnephropathy to determine whether ergosta-4,6,8(14),22-tetraen-3-one (ergone)
treatment prevents early renal injury in rats with aristolochic acid I-induced nephropathy.
Methods Early renal injury via renal interstitial fibrosis was induced in rats by adminis-
tration of aristolochic acid I (AAI) solution intragastrically for 8 weeks. Ninety-six rats were
randomly divided into four groups (n = 24/group): (1) control (2) AAI (3) AAI + ergone
(10 mg/kg) and (4) AAI + ergone (20 mg/kg). Blood and urine samples were collected and
rat were sacrificed for histological assessment of the kidneys on at the end of weeks 2, 4, 6
and 8.
Key findings AAI caused progressive elevation of blood urea nitrogen, creatinine, potas-
sium, sodium, chlorine, proteinuria and urinary N-acetyl-b-D-glucosaminidase (NAG).
Ergone suppressed elevation of blood urea, nitrogen, creatinine, proteinuria and urinary
NAG to some degree, but the AAI–ergone-treated group did not differ from AAI-treated
group for body weight, serum potassium, sodium and chlorine. The progress of the lesions
in the kidney afterAAI administration was also observed by histopathological examinations,
but kidneys from rats of AAI–ergone-treated group displayed fewer lesions.
Conclusions Ergone treatment conferred protection against early renal injury in a rat
model of AA nephropathy. Early administration of ergone may prevent the progression of
renal injury and the subsequent renal fibrosis in AA nephropathy.
Keywords aristolochic acid I; early renal injury; ergosta-4,6,8(14),22-tetraen-3-one; renal
interstitial fibrosis
Introduction
Chronic renal failure (CRF) is one of the most important public health problems, with
increasing rates of incidence and prevalence.[1]Although the number of patients with end-
stage renal disease has increased rapidly over the last three decades and, prior to the advent
of dialysis, some treatments – including dietary control, drug therapy with a spherical
carbonaceous absorbents, and antihypertensive drugs, including ACEIs (angiotensin con-
verting enzyme inhibitors) and ARBs (angiotensin receptor blockades) – have shown some
inhibition of the progression of CRF, these treatments are still not effective enough to curtail
this increase. The development of new effective drugs that prevent the progression of CRF
is therefore an urgent requirement. In the present article, we show the first indication that
ergosta-4,6,8(14),22-tetraen-3-one (ergone) from a medical fungus Polyporus umbellatus
could be such a candidate.
The fungus Polyporus umbellatus has commonly been used in Chinese traditional medi-
cine for hundreds of years for treatment of various renal diseases. It possesses a relative
diuretic effect and the ability to reinforce the functioning of water pathways capable of drain
dampness, and is described as Shennong’s Herbal Classics. It is also described as promoting
Research Paper
JPP 2011, 63: 1581–1586
© 2011 The Authors
JPP © 2011 Royal
Pharmaceutical Society
Received December 9, 2010
Accepted August 29, 2011
DOI
10.1111/j.2042-7158.2011.01361.x
ISSN 0022-3573
Correspondence: Ying-Yong
Zhao, Biomedicine Key
Laboratory of Shaanxi Province,
The College of Life Sciences,
Northwest University, No.229
Taibai North Road, Xi’an,
Shaanxi 710069, China.
E-mail: zhaoyybr@163.com;
zyy@nwu.edu.cn
1581

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urination and leaving out dampness, the problems caused by
stagnance of dampness such as edema, scanty urine, vaginal
discharge and cloudy painful urinary dysfunction.[2]Ergone is
an anti-aldosteronic diuretic steroid, which is one of the main
components in Polyporus umbellatus.[3–5]It also exists widely
in other medicinal fungi, lichens and plants such as Cordyceps
sinensis, Vietnamese xylaria and Zopfiella longicaudata.[6–8]
We have recently reported that ergone also has diuretic and
cytotoxic activity,[9–11]consistent with previous reports.[12,13]
Our pharmacokinetic experiments have demonstrated that
ergone levels are higher in faeces than in urine. Almost 57%
of the loading dose is cumulative in the faeces within 24 h
after oral administration, but ergone could be undetected in
the urine.[14–16]
In recent years, many countries, including the USAand the
UK, have realized that some Chinese herbs contain kidney-
toxic aristolochic acid (AA) and have banned the use of
Chinese traditional medicines containingAAin medical prac-
tice. Besides carcinogenic and mutagenic properties, AA can
cause kidney damage and ultimately lead to renal interstitial
fibrosis.[17–23]The mechanisms that lead to kidney damage by
AA to date are not well understood, but AA-induced renal
interstitial fibrosis in rats is a good model to use to study the
pathogenesis of the condition.[24–26]Here we report the find-
ings of an experimental study on ergone chemoprevention of
early renal injury induced by an aristolochic acid I (AAI) from
Caulis aristolochiae manshuriensis (Chinese: Guan-Mu-
Tong).
Materials and Methods
Chemicals and animals
Ergone was synthesized by the author (Ying-Yong Zhao) and
its purity (?98.5%) was determined by HPLC.AAI (batch no.
110746–200406, purity 99.0%) was obtained from the
National Institutes for Food and Drug Control (Beijing,
China).
The study was conducted in accordance with the Regula-
tions of Experimental Animal Administration issued by the
State Committee of Science and Technology of People’s
Republic of China. All procedures and the care of the rats
were in accordance with institutional guidelines for animal
use in research. Male Sprague–Dawley rats were obtained
from the Central Animal Breeding House of Xi’an Jiaotong
University (Xi’an, China).They were maintained at a constant
humidity (c. 60%) and temperature (c. 23°C) with a light/dark
cycle of 12 h.
Experimental design using aristolochic acid
I-induced nephropathy rats
Male rats underwent an adaptation period of several days,
during which they were fed a commercial feed. The rats,
weighing 190–210 g, were divided into four groups (n = 24/
group) after measuring body weight. Groups 2, 3 and 4 then
gavaged with 70 mg/kg body weight of AAI dissolved in 1%
(w/v) gum acacia solution, which produced experimental
renal failure in the animals after 8 weeks. Group 1 was given
with an equal volume of gum acacia solution. During the
period of gastric gavage of AAI, after 3 h, groups 3 and 4
were gavaged with 10 and 20 mg/kg body weight of ergone
dissolved in 1% (w/v) gum acacia solution for 8 weeks,
while groups 1 and 2 were similarly given an equal volume
of gum acacia solution. Body weight was measured daily
for all rats. At the end of weeks 2, 4, 6 and 8, six individual
rats of each group selected randomly were placed in
metabolic cages to obtain 24-h urine collections for the
measurement of proteinuria concentrations. They were then
decapitated. Trunk blood was collected and used for deter-
mination of serum creatinine (Scr) and blood urea nitrogen
(BUN) concentrations. Bilateral kidneys were removed.
The left kidney was stored in 10% formaldehyde solution.
Specimens were embedded in paraffin and cut transversely
into 5-mm-thick slices on a microtome (Leica Corp., Nuss-
loch, Germany). The right kidney was quickly frozen for
molecular studies.
Renal function and proteinuria
The rats were placed in metabolic cages for assessment of
proteinuria. The concentrations of Scr, BUN and urinary
protein were measured with an Olympus AU640 automatic
analyser and all reagents were from Olympus Diagnos-
tics.[27,28]The concentrations of serum potassium, sodium and
chlorine were measured with an Easylyte plus Analyzer
(Medica,America).[29]The concentrations of urinary N-acetyl-
b-d-glucosaminidase (NAG) were measured spectrophoto-
metrically.
Histopathological analysis
The kidneys were fixed in 10% buffered formalin and embed-
ded in paraffin. Sections were stained with haematoxylin and
eosin (HE) and periodic acid Schiff (PAS) reagents.
Statistical analysis
Statistical significance was determined by two-way ANOVA
followed by the Duncan test to determine differences between
treatment groups and by paired or unpaired t-tests as appro-
priate; P < 0.05 was considered significant.
Results
Body weight, renal function and urinary
protein excretion
Table 1 shows pathophysiologic parameters in the four groups
of rats. Table 1 shows body weight over the 8-week study
period. Whereas body weight steadily increased in control
rats, it increased only slightly in both AAI-treated and AAI–
ergone-treated rats. In the AAI-treated group, body weights
were significantly decreased compared to the control group.
Body weights in the AAI–ergone-treated rats were slightly
greater than those treated with AAI alone over the 8-week
study period, but did not arrive at statistical significance
(Table 1). The normal levels of Scr and BUN in rats are
59.1 mmol/l and 5.5 mmol/l (n = 6), respectively. The Scr
level in only the AAI-treated rats markedly increased beyond
the normal level. The administration of ergone at doses of 10
and 20 mg/kg significantly reduced Scr levels (Table 1). Simi-
larly, the BUN level in rats treated with AAI alone increased
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to c. 28.6 mmol/l. The administration of ergone at doses of 10
or 20 mg/kg significantly reduced BUN levels (Table 1).
Serum potassium, sodium and chlorine concentrations in
the AAI–ergone-treated rats were slightly lower than those
treated with AAI alone over the 8-week study period, but
did not arrive at statistical significance (Table 1). Urinary
protein excretion was not significantly different between
AAI-treated (15.6 ? 4.6 mg/day) and AAI–ergone-treated
(13.2 ? 3.8 mg/day for 10 mg/kg and 12.3 ? 4.0 mg/day for
20 mg/kg oral administration) groups at the end of week 2.
However, urinary protein excretion in theAAI–ergone-treated
rats was markedly decreased compared with AAI-treated rats
at the end of weeks 4, 6 and 8 (Table 1). In AAI–ergone
treated rats, urinary NAG excretion was 4.56 ? 1.51 mg/day
for 10 mg/kg and 4.25 ? 1.02 mg/day for 20 mg/kg oral
administration, values that were not significantly different
from those in rats given AAI alone (5.15 ? 2.09 mg/day) at
the end of week 2, but urinary protein excretion in the AAI–
ergone-treated rats was markedly decreased as compared with
onlyAAI-treated rats at the end of weeks 4, 6 and 8 (Table 1).
Histopathological changes
Figure 1 shows histopathological changes in the four groups
of rats at the end of week 6. Kidney tissues were stained with
HE or with PAS. No significant abnormalities were observed
in the renal tissue samples obtained over the 8-week study
period from control rats (Figure 1 (H1) and (P1)). In
contrast, the progress of the lesions in the kidney after AAI
Table 1
ergone (10 or 20 mg/kg)
Body weight, serum and urinary parameters in control rats, rats treated with aristolochic acid I (AAI) alone and rats treated with AAI plus
Parameters AAI administration period
2 weeks4 weeks6 weeks 8weeks
Body weight (g)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
Scr (mmol/l)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
BUN (mmol/l)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
Potassium (mmol/l)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
Sodium (mmol/l)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
Chlorine (mmol/l)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
Proteinuria (mg/day)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
NAG (mg/day)
Control
AAI
AAI + ergone 10 mg/kg
AAI + ergone 20 mg/kg
262 ? 20
202 ? 16b
203 ? 21d
196 ? 23d
328 ? 21
187 ? 23b
195 ? 23d
197 ? 19d
382 ? 21
189 ? 23b
217 ? 18d
221 ? 20d
439 ? 23
209 ? 19b
225 ? 26d
232 ? 23d
59.55 ? 3.84
69.75 ? 4.80b
67.23 ? 3.25
68.09 ? 4.05
60.74 ? 3.23
87.02 ? 3.56b
69.57 ? 4.27d
68.72 ? 3.95d
57.24 ? 3.05
95.56 ? 4.07b
79.08 ? 3.95d
78.56 ? 4.56d
58.72 ? 5.45
104.95 ? 5.38b
85.56 ? 4.38d
83.25 ? 3.96d
5.50 ? 0.65
8.29 ? 0.92b
6.20 ? 1.12c
6.71 ? 0.83c
5.29 ? 0.72
14.71 ? 1.29b
10.76 ? 2.32d
9.75 ? 3.02d
5.65 ? 0.59
23.01 ? 2.45b
16.48 ? 2.45d
15.09 ? 2.29d
5.76 ? 0.91
28.62 ? 3.08b
20.63 ? 1.92d
18.62 ? 3.48d
5.40 ? 0.51
5.49 ? 1.02
5.32 ? 0.97
5.42 ? 0.68
5.31 ? 0.98
7.25 ? 1.21b
6.14 ? 1.23
6.12 ? 1.54
5.50 ? 1.54
7.38 ? 1.12b
6.25 ? 1.08
5.98 ? 1.12
5.41 ? 0.64
7.63 ? 0.78b
6.91 ? 0.87
6.45 ? 0.98c
139 ? 13
138 ? 15
137 ? 12
140 ? 16
151 ? 14
154 ? 18
153 ? 16
154 ? 17
147 ? 10
151 ? 12
150 ? 14
153 ? 13
148 ? 15
148 ? 13
148 ? 15
146 ? 14
101 ? 10
110 ? 8
108 ? 12
110 ? 10
102 ? 13
115 ? 12
113 ? 10
111 ? 13
99 ? 9
117 ? 15a
115 ? 15
112 ? 12
101 ? 15
121 ? 9a
120 ? 14
117 ? 16
5.4 ? 1.2
15.6 ? 1.5b
13.2 ? 0.9
12.3 ? 1.3
5.4 ? 4.6
20.7 ? 3.9b
14.9 ? 3.5c
12.9 ? 4.2d
5.8 ? 3.8
22.1 ? 4.3b
17.1 ? 4.4c
15.7 ? 3.8d
5.7 ? 4.0
24.5 ? 3.2b
19.3 ? 4.3c
18.1 ? 3.7d
2.51 ? 0.91
5.15 ? 0.56b
4.56 ? 1.09
4.25 ? 0.85
2.57 ? 2.09
7.54 ? 1.94b
5.02 ? 1.81c
5.32 ? 1.94c
2.61 ? 1.51
9.11 ? 1.26b
7.09 ? 1.31c
6.76 ? 1.65c
2.54 ? 1.02
10.68 ? 1.41b
8.21 ? 1.53c
8.01 ? 1.38c
Values are mean ? SD. The number of animals in each group was 6.aP < 0.05,bP < 0.01 vs control by ANOVA;cP < 0.05,dP < 0.01 vs AAI by
ANOVA.
Ergone prevents early renal injury in ratsYing-Yong Zhao et al.
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administration was observed. Slight granular degeneration of
the tubular epithelium in the kidney was seen at the end of
week 4 after AAI administration. Lesions of granular degen-
eration, vacuolar degeneration and lymphocytic infiltrate were
observed at the end of week 6 after AAI administration
(Figure 1 (H2) and (P2)). Lesions characterized by tubular
dilation along with discrete tubular necrosis were seen in the
kidney at the end of week 8 (not shown). Multiple foci of
tubular necrosis and atrophy, as well as lymphocytic innltrate
free of polynuclear neutrophils, were present in the deep
cortex (not shown). Simultaneously, kidneys from rats of
AAI–ergone-treated group displayed fewer lesions at ergone
doses of 10 mg/kg (Figure 1 (H3) and (P3)) and 20 mg/kg
(Figure 1 (H4) and (P4)).
Discussion
Our present study indicates that AAI induces a kidney injury
with the general characteristics of the progress of renal inter-
stitial fibrosis and that ergone has to some degree a preventa-
tive effect on the development of early renal injury. In recent
years, kidney injuries caused by Chinese herbal medicines
have been increasingly reported, most of which are related to
Caulis aristolochiae manshuriensis.[30,31]Although the toxic
component in Caulis aristolochiae manshuriensis has been
identified asAA, the mechanisms and pathological features in
this toxicity and how to prevent kidney injury from such a
herb are only poorly understood. Our study indicates that
blood (BUN, Scr) and urine (proteinuria, urinary NAG)
Figure 1
Kidney tissues were stained with HE (H1–H4) or PAS (P1–P4). No morphological changes were noted for control rats (H1 and P1) at the end of week
6. Typical lesions, consisting of granular degeneration (arrows), vacuolar degeneration (arrowheads) and lymphocytic infiltrate (asterisk), were
observed in bothAAI- (H2, P2) andAAI–ergone-treated rats (H3, H4, P3 and P4) at the end of week 6. Paraffin-embedded sections (5 mm) were stained
with haematoxylin and eosin (H1–H4), or with periodic acid Schiff (P1–P4). Magnification: ¥200.
Kidney histological features of rat with AAI oral administration. Mice were sacrificed at the indicated time after AAI administration.
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biochemistry indices have significant differences between the
AAI–ergone-treated group and the AAI-treated group, sug-
gesting that ergone may prevent early renal injury to a certain
degree. HE (H2–H4), or PAS (P2–P4) staining shows obvious
differences in the degree of early renal injury. Many studies
have demonstrated that the renin–angiotensin–aldosterone
system is implicated as a major mechanism in sustaining renal
disease.[32,33]Recent studies in humans and experimental
models have shown that aldosterone plays a pivotal role in the
pathophysiology of renal injury. Aldosterone causes inflam-
matory response, endothelial dysfunction and fibrosis by
increasing plasminogen activator inhibitor-1 (PAI-1) and
TGF-b1 expression and by stimulating production of reactive
oxygen species.[34]Ergone is an anti-aldosteronic com-
pound.[12]In our and other studies on the association of aldos-
terone with renal fibrosis, a similar phenomenon was
observed, although the detailed mechanisms are unclear.[34]
Conclusions
In conclusion, a rat model of AA nephropathy may be a
powerful tool for studying the progression of tubulointerstitial
nephropathy at the early stage. Long-term treatment with
ergone prevented early tubular damage and thereby inhibited
the progression of interstitial fibrosis in a rat model of AA
nephropathy. These results suggest that early treatment with
ergone, successfully used in an animal model, represents a
potential new treatment to limit renal tissue damage in AA
nephropathy in humans.
Declarations
Conflict of interest
TheAuthor(s) declare(s) that they have no conflicts of interest
to disclose.
Funding
This study was supported in part by grants from the National
Scientific Foundation of China project No. 81001622 and No.
2011ZX09401-308-34 from the Ministry of Science and
Technology of the People’s Republic of China and Scientific
Research Program. It was also funded by the Shaanxi Provin-
cial Education Department (No. 11JK0678).
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