review
http://www.kidney-international.org
& 2008 International Society of Nephrology
Aristolochic acid nephropathy: A worldwide problem
Frédéric D. Debelle1,2, Jean-Louis Vanherweghem1 and Joëlle L. Nortier1,2
1
Department of Nephrology, Dialysis and Renal Transplantation, Erasme Hospital, Brussels, Belgium and 2Experimental Nephrology
Unit, Faculty of Medicine, Université Libre de Bruxelles, Brussels, Belgium
Aristolochic acid nephropathy (AAN), a progressive renal
interstitial fibrosis frequently associated with urothelial
malignancies, was initially reported in a Belgian cohort of
more than 100 patients after the intake of slimming pills
containing a Chinese herb, Aristolochia fangchi. Although
botanicals known or suspected to contain aristolochic acid
(AA) were no longer permitted in many countries, several
AAN cases were regularly observed all around the world.
The incidence of AAN is probably much higher than initially
thought, especially in Asia and the Balkans. In Asian
countries, where traditional medicines are very popular, the
complexity of the pharmacopoeia represents a high risk for
AAN because of the frequent substitution of the botanical
products by AA-containing herbs. In the Balkan regions,
the exposure to AA found in flour obtained from wheat
contaminated with seeds of Aristolochia clematitis could be
responsible for the so-called Balkan-endemic nephropathy.
Finally, despite the Food and Drug Administration’s warnings
concerning the safety of botanical remedies containing
AA, these herbs are still sold via the Internet.
Kidney International (2008) 74, 158–169; doi:10.1038/ki.2008.129;
published online 16 April 2008
KEYWORDS: aristolochic acid; Aristolochia; renal interstitial fibrosis; urothelial
carcinoma; herbal remedies; Balkan-endemic nephropathy
Aristolochic acid nephropathy (AAN), a rapidly progressive
interstitial nephritis leading to end-stage renal disease and
urothelial malignancy, was originally reported in Belgium
in a group of patients who had ingested slimming pills
containing powdered root extracts of Chinese herbs (Figure
1a and b).1–4 This nephropathy, initially called Chinese-herb
nephropathy (CHN), appeared to be the dramatic consequence of a substitution of Stephania tetrandra by Aristolochia
fangchi rich in aristolochic acid (AA), because both herbs share
the same common name in Pin Yin (Han Fang Ji and Guang
Fang Ji), and one can be used instead of the other in traditional Chinese medicine irrespective of their botanical classification.1,3,5 After the publication of the index cases, new cases
of AAN were regularly reported, not only in Belgium but also
worldwide (Figure 1a).1–4,6–23 Actually, the true incidence of
AAN is largely unknown and probably underestimated, as
numerous ingredients known or suspected to contain AA are
used in traditional medicine in China, Japan, and India
(Figure 1c).24–26
Another reason to suspect a higher number of AAN cases
is based on the hypothesis that AA could be an environmental
cause of Balkan-endemic nephropathy (BEN), which is a
familial chronic tubulointerstitial disease characterized by an
insidious onset, a slow progression to end-stage renal disease,
and an increased frequency of urothelial cancer.23 This
nephropathy is endemic in Serbia, Bosnia, Croatia, Bulgaria,
and Romania (Figure 1d).27
Finally, despite the Food and Drug Administration’s
warnings regarding the safety of botanical remedies containing AA (known or suspected to contain AA), plants containing AA are still available via the Internet.28
AAN: THE BELGIAN OUTBREAK OF CHN
Correspondence: Joëlle L. Nortier, Nephrology Department, Erasme
Hospital, Route de Lennik, 808, Brussels B-1070, Belgium.
E-mails: jnortier@ulb.ac.be or Joelle.Nortier@erasme.ulb.ac.be
Received 3 December 2007; revised 24 January 2008; accepted 6
February 2008; published online 16 April 2008
158
In 1992, two young women with no previous history of renal
disease were admitted in our Nephrology department in
Brussels (Belgium) with severe interstitial nephritis that
progressed over a couple of months to end-stage renal
disease.1 One year before, these two patients had followed the
same weight-loss program in the same medical clinic in
Brussels. Interestingly, the composition of weight-reducing
pills was modified in June 1990 by introducing root extracts
from two Chinese herbs, labeled as S. tetrandra and Magnolia
officinalis.1 An epidemiological survey of the Nephrology
centers of Brussels revealed an unusual, increased incidence
of patients with ‘interstitial nephritis of unknown origin’
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FD Debelle et al.: Aristolochic acid nephropathy
admitted for dialysis in 1991 and 1992.1 This survey
identified seven other women who had followed the same
slimming regimen containing the Chinese herbs.1 Confronted to this rapidly progressive nephropathy probably
related to the ingestion of these two Chinese herbs, the
Belgian authorities decided to ban S. tetrandra and
M. officinalis from the Belgian market at the end of 1992.
Despite this safety measure, more than 100 cases of CHN
were reported in Belgium in 1998, 70% of them being in
end-stage renal disease.7
The time correlation between the introduction of the
Chinese herbs in weight-loss regimens and the appearance
of this rapidly progressive interstitial renal fibrosis focused
the search of the culprit on the Chinese herbs.1 Very soon,
the inadvertent replacement of Stephania by Aristolochia was
suspected. First, S. tetrandra (Pin Yin name: Han Fang Ji) and
A. fangchi (Pin Yin name: Guang Fang Ji) belong to the same
therapeutic ‘Fang Ji’ family in traditional Chinese medicine,
and the herbal ingredients are generally traded using their
common Pin Yin name. Second, the pathological aspect of
CHN is very similar to that of BEN, whose etiology is still
controversial, but some suggested that AAs containing
A. clematitis are the main culprit (see section below).1,2,29,30
The hypothesis of substitution was strengthened by the
phytochemical analyses of the S. tetrandra batches revealing
that most of them did not contain tetrandrine but AAs
(0.65±0.56 mg g1 of powder), the main compounds of the
Aristolochia sp.3 A survey including 71 CHN/AAN patients
Belgium: 128
Germany: 1
UK: 4
Korea: 1
France: 4
Japan: 6
USA: 2
Spain: 1
Taiwan: 33
China: 116
3
2
1
Slimming pill and crosssection of a root of
aristolochia
Extensive paucicellular interstitial
fibrosis and tubular atrophy
typically found in end-stage CHN
Autoradiographic pattern of
specific aristolochic acid-related
DNA adducts detected in human
renal tissue
Figure 1 | Aristolochic acid nephropathy: a worldwide problem. (a) Counting cases of CHN/AAN around the world reported in the
literature.1–4,6–23 (b) CHNA/AAN is a rapidly progressive interstitial nephritis leading to end-stage renal disease and urothelial malignancy,
which was originally reported in Belgium in the context of the intake of slimming pills containing powdered Chinese herbs (A. fangchi).
(c) The true incidence of AAN is largely unknown and probably underestimated, as numerous ingredients known or suspected to contain
AA are used in traditional medicines in India and Eastern Asia (see Tables 1–3 for more details). (d) Finally, another reason to suspect a
higher number of AAN cases is based on the hypothesis that the exposure to seeds of Aristolochia clematitis comingled with wheat grain
during the annual harvest could be responsible for BEN.
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A pharmacy selling traditional herbal remedies including Fang Chi and Mu Tong
Aristolochic acid containing
Aristolochia clematitis growing in a
field of wheat (Croatia)
By courtesy of Dr Bojan Jelaković
Figure 1 | Continued.
followed in our department demonstrated in a multiple
regression analysis that the cumulative dose of the so-called
Stephania (in fact, Aristolochia) was the only significant factor
predicting the slope of the inverse of plasma creatinine
levels.31 The causal role of AA was definitively confirmed
by the detection of AA DNA adducts in kidney removed
from CHN/AAN patients showing evidence of a previous
exposure to AA.32,33 In addition, the main histological and
functional features of CHN/AAN were successfully reproduced by administrating AA to New Zealand white rabbits or
male Wistar rats.34,35 Consequently, the term Chinese-herb
nephropathy has been progressively abandoned and replaced
by aristolochic acid nephropathy.
After the first report of cases in Belgium, other similar cases
were sporadically observed in France, Spain, Germany, United
Kingdom, and the United States, in the context of herbal
remedies for slimming purposes but also for all kinds of
indications such as eczema, hepatitis B, ‘liver enhancement’,
arthritis, rheumatism, and pain relief.6,8–10,13,15,17,20 As expected, numerous cases were also reported in Asian countries
where the complexities of the traditional pharmacopoeia
represent a high risk for AA exposure.12,14,16,19,21,22,36–40
Clinically, the initial presentation of CHN/AAN was
usually silent and the renal failure was discovered by routine
blood testing.41 However, few cases presenting with a Fanconi
syndrome or an acute renal failure due to tubular necrosis
were reported in the literature.11,12,15,19,21 Anemia was
present and was often more severe than might have been
anticipated from the degree of renal failure.42 In most
of the cases, urinary sediment was unremarkable and
dipstick analysis for albuminuria was negative.43 However,
urinary excretion of five low molecular weight proteins
(b2-microglobulin, cystatin C, Clara cell protein, retinal160
binding protein, and a1-microglobulin) was markedly
increased in five patients with CHN/AAN, and the urinary
low molecular weight protein/albumin ratio was higher than
in control patients with glomerular diseases.44 Moreover,
in 26 patients with CHN/AAN, levels of urinary neutral
endopeptidase, a 94 kDa ectoenzyme of the proximal tubule
brush border, were significantly decreased in those with
moderate renal failure and almost undetectable in those with
end-stage renal failure.45 NEP enzymuria positively correlated with individual creatinine clearance values (r ¼ 0.76;
P ¼ 0.0001) and negatively correlated with urinary low molecular weight protein levels (r ¼ 0.55; P ¼ 0.00001).45 An
in vitro study on the opossum kidney cell line demonstrated that AA intoxication led to a rapid and persistent
decrease in megalin expression in parallel with an inhibition
of receptor-mediated endocytosis of low molecular weight
protein.46 Taking together, these data indicated that proximal
tubular cells are the main target of the AA-containing
Chinese herb.
Macroscopically, the kidneys were shrunk, asymmetric in
about half of the cases with irregular outlines in one-third.43
Microscopically, an extensive interstitial fibrosis with atrophy
and loss of proximal tubules was the predominant lesion,
which was mainly located in the superficial cortex and
progressed toward the deep cortex (Figure 1b).2,29 The
glomeruli were relatively spared, although, in the later stage
of the disease, they displayed a mild collapse of the capillaries
and a wrinkling of the basement membrane. Although it was
not the general rule, an interstitial inflammatory infiltration
was retrieved in several renal biopsy specimens, suggesting an
immunological process as a possible pathophysiological
mechanism. This observation was the rational basis for a
pilot study with corticosteroids performed in 35 CHN/AAN
patients with chronic renal failure. Compared to an untreated
group, a significant reduction of the number of patients
reaching end-stage renal disease was observed after 1 year of
steroid therapy.47 Eight years later, in a larger group of CHN
patients, the steroid therapy was confirmed to slow down the
progression of renal failure.48
Finally, the finding of an endothelial wall thickening in
the interlobular and afferent arterioles suggested a possible
ischemic process induced by other substances concomitantly
administrated with the Chinese herb.49 The appetite suppressant (dex)fenfluramine, a serotonin agonist, could have
played a role in the development of CHN because serotonin
injection was experimentally shown to induce ischemic renal
lesions progressing to renal fibrosis.50 However, this hypothesis can be ruled out on the basis of reports of CHN cases
out of the context of a slimming regimen and the demonstration that dexfenfluramine did not enhance the nephrotoxicity of AA in a rat model of CHN/AAN.41,51 In the
same line, an extrarenal toxicity of Chinese herbs was also
suspected because 30–50% of the CHN patients displayed an
aortic insufficiency.43 However, the puzzling association of
valvular abnormalities with CHN appeared to be mainly
linked to the concomitant presence of anorectic drugs in the
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FD Debelle et al.: Aristolochic acid nephropathy
slimming pills, with the demonstration of a significant
dose–response relationship between the cumulative dose of
(dex)fenfluramine and the aortic regurgitation.52,53
AA CONTAINING HERBAL REMEDIES IN TRADITIONAL
MEDICINES
The uncontrolled use and the uncorrected identification of
medicinal herbs, which are usually considered by the general
population as inherently harmless, were at the heart of all
AAN cases encountered in the Western countries.54
On the other hand, in Asia, AAN was the dramatic
consequence of the complex pharmacopoeia of herbal
remedies used in traditional medicines and the lack of their
regulation as applied for conventional drugs.5 For example,
two series of 12 and 20 AAN cases, respectively, related to the
use of various herbal medications were reported in
Taiwan.14,16 The ingestion of AA-containing herbal remedies
used in traditional Sino-Japanese Kampo prescriptions
(vernacular names: Boui and Mokutsu) resulted in Fanconi
syndrome secondary to AAN in four Japanese patients.11,12 In
China, acute renal failure secondary to tubular necrosis was
observed in eight patients after the intake of Guanmutong
(Aristolochia manshuriensis Kom.), an AA-containing Chinese
herb widely used for the treatment of urinary and cardiovascular diseases.21 Two series of 58 and 51 AAN cases,
respectively, were reported in 2001 in China, most of them
after the intake of the liver tonic Longdan Xieganwan that
contained Caulis Aristolochia Manshuriensis.22 Five additional
AAN cases were also observed in Hong Kong, following
the substitution of the nontoxic Herba Solani Lyrati by the
AA-containing herb Aristolochia mollissimae.22
The preferential use of vernacular names in traditional
medicine terminology and the high risk for substitution
of the herbal products might explain the outbreak of AAN
in Asia (for more details, see Tables 1–3). However, one
can expect that the true incidence of AAN in Asia is
largely underestimated. Indeed, Akebia is commonly used in
Sino-Japanese prescriptions as well as Fang ji and Mu Tong
in traditional Chinese medicine.24,25 It is worthy to stress that
traditional medicine is still widely popular in China in 2007:
about 3000 hospitals provide traditional Chinese medicine
treatments to nearly 234 million patients each year.60 Despite
the fact that the AA-containing herbs were theoretically
banned in many countries around the world, including
Table 1 | Botanicals known or suspected to contain aristolochic acid and their vernacular names55–59
Botanical name
Common or other names
Aristolochia
Aristolochia
Aristolochia
Aristolochia
Aristolochia
Aristolochia
Aristolochia
Aristolochia, Guan Mu tong, Guang Mu tong
Oval leaf Dutchman’s pipe
Ukulwe
Birthwort
Ma Dou Ling (fruit), Bei Ma Dou Ling (root), Tian Xian Teng (herb)
Mil homens
Ma Dou Ling (fruit); Tian Xian Teng (herb), Qing Mu Xiang (root),
Sei-Mokkou (Japanese), Birthwort, Long birthwort, Slender Dutchman’s
pipe
Guang Fang ji (root), Fang ji, Fang chi, Mokuboi (Japanese), Kou-boui
(Japanese), Kwangbanggi (Korean)
Han Fang Ji
Indian birthwort (root), Yin Du Ma Dou Ling
Yellowmouth Dutchman’s pipe, Zhu Sha Lian
spp.
acuminata (Syn. Aristolochia tagala)
bracteata
clematitis
contorta
cymbifera
debilis (Syn. Aristolochia longa, A. recurvilabra, A. sinarum)
Aristolochia fangchi
Aristolochia heterophylla
Aristolochia indica
Aristolochia kaempferi (Syn. Aristolochia chrysops, A. feddei., A. heterophylla,
A. mollis, A. setchuenensis, A. shimadai, A. thibetica, Isotrema chrysops,
I. heterophylla, I. lasiops)
Aristolochia macrophylla (Syn. Aristolochia sipho)
Aristolochia manschuriensis (Syn. Hocquartia manshuriensis, Syn. Isotrema
manchuriensis)
Aristolochia maxima (Syn. Howardia hoffmannii)
Aristolochia mollissima
Aristolochia moupinensis
Aristolochia serpentaria (Syn. Aristolochia serpentaria)
Aristolochia triangularis
Aristolochia tuberosa
Aristolochia tubiflora
Aristolochia versicolar
Asarum canadense (Syn. Asarum acuminatum, A. ambiguum, A. canadense,
A. furcatum, A. medium, A. parvifolium, A. reflexum, A. rubrocinctum)
Asarum himalai(y)cum
Asarum splendens
Dutchman’s-pipe
Manchurian birthwort, Manchurian Dutchman’s pipe (stem) Guan
Mutong (stem), Kan-Mokutsu (Japanese), Mokuboi (Japanese),
Kwangbanggi (Korean)
Maxima Dutchman’s pipe, Da Ma Dou Ling
Wooly Dutchman’s pipe, Mian Mao Ma Dou Ling
Moupin Dutchman’s pipe, Huai Tong
Virginia snakeroot, Serpentaria, Virginia serpentary
Triangular Dutchman’s pipe, San Jiao Ma Dou Ling
Tuberous Dutchman’s pipe, Kuai Jing Ma Dou Ling
Tubeflower Dutchman’s pipe, Guan Hua Ma Dou Ling
Versicolorous Dutchman’s pipe, Bian Se Ma Dou Ling
Wild ginger, Indian ginger, Canada snakeroot, False coltsfoot, Colic root,
Heart snakeroot, Vermont snakeroot, Southern snakeroot, Jia Na Da
Xi Xin
Tanyou-saishin (Japanese)
Do-saishin (Japanese)
Other botanicals known or suspected to contain aristolochic acid are Aristolochia argentina, A. baetica. (Syn. A. bracteolata), A. chilensis, A. cinnabarina, A. elegans (Syn. A.
hassleriana), A. esperanzae, A. fimbriata, A. kwangsiensis (Syn. A. austroszechuanica), A. maurorum, A. rigida. A. rotunda, A. watsoni(i) (Syn. A. porphyrophylla), A. westlandi(i),
A. zollingeriana (Syn. A. kankauensis, A. roxburghiana, A. tagala, Hocquartia kankauensis), Bragantia wallichii.
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Table 2 | Botanicals that may be adulterated with aristolochic acid and their respective vernacular names55–59
Botanical name
Common or other names
Akebia spp.
Akebia, Mu tong, Ku mu tong, Zi mutong, Bai mu tong,
Mokutsu (Japanese), Mokt’ong (Korean)
Chocolate vine, Fiveleaf akebia, Bai Mu Tong (stem),
Mu Tong Gen (root), Yu zhi zi (seed), Mokutsu (Japanese)
Bai Mu Tong (stem), Bai Mu Tong Gen (root), Bai Mu Tong Zi (seed),
Three leaf akebia, Yu zhi zi, San Ye Mu Tong Zi (seed),
San Ye Mu Tong Gen (root)
Wild ginger (root, leaves)
Batei-saishin (Japanese)
Keirin-saishin (Japanese) Chinese wild ginger, Manchurian wild ginger,
Bei Xi Xin, Xin xin
Usuba-saishin (Japanese) Chinese wild ginger, Xi Xin, Hua Xi Xin,
Manchurian wild ginger, Siebold’s wild ginger
Clematis, Mufangji, Clematidis, Ireisen (Japanese), Wojoksum (Korean)
Armand’s clematis Chuan Mu tong (stem), Xiao mu tong,
Armand’s virgin bower
Chinese clematis, Wei Ling Xian
Chuan Mu Tong (stem)
Cocculus
Indian cockle, Yin Du Mu Fang Ji
Mu Fang Ji
Mu Fang Ji (root)
Akebia quinata (Syn. Rajania quinata)
Akebia trifoliata
Asarum caudatum
Asarum forbesii
Asarum heterotropoides (Syn. Asarum heterotropoides)
Asarum sieboldii (Syn. Asarum sieboldii, A. sieboldii var. seoulensis,
A. heterotropoides var. seoulense, A. sieboldii)
Clematis spp.
Clematis armandii (Syn. Clematis armandii fo. Farquhariana,
C. armandii var. biondiana, C. biondiana, C. ornithopus)
Clematis chinensis
Clematis montana (Syn. Clematis insulari-alpina)
Cocculus spp.
Cocculus indicus (Syn. Anamirta paniculata)
Cocculus orbiculatus (Syn. Cissampelos pareira)
Cocculus orbiculatus (Syn. Cocculus cuneatus, C. sarmentosus, C. sarmentosus
var. linearis, C. sarmentosus var. pauciflorus, C. sarmentosus var. stenophyllus,
C. thunbergii, C. trilobus, Menispermum orbiculatus, M. trilobum, Nephroia
sarmentosa)
Moku-boui (Japanese)
Cocculus palmatus
Cocculus palmatus (Syn. Jateorhiza miersii)
Cocculus pendulus (Syn. Cebatha pendula, Epibaterium pendulus,
Cocculus epibaterium)
Cocculus trilobus
Diploclisia chinensis
Saussurea lappa
Sinomenium acutum (Syn. Cocculus diversifolius var. cinereus,
C. heterophyllus, Menispermum acutum, Sinomenium
acutum var. cinereum, S. diversifolium)
Stephania spp.
Stephania tetrandra
Vladimiria souliei
Columba, Columbo
Colombo
Chui Mu Fang Ji
Mu Fang Ji (root)
Xiangfangchi
Mokkou (Japanese)
Orientvine, Xunfengteng, Dafengteng, Daqingmuxinag, Zhuigusan,
Da ye qingshener, Mufangji, Hanfangji, Tuteng, Zhuigufeng, Maofangji
Stephania
Fen fang ji, Fang ji (root), Han fang ji (root), Kanboi (Japanese),
Hanbanggi (Korean), Fun-boui (Japanese)
Sen-mokkou
Other botanicals that may be adulterated with aristolochic acid are: Clematis hexapetala, Clematis uncinata, (Syn. Clematis alsomitrifolia, C. chinensis var. uncinata, C. drakeana,
C. floribunda, C. gagnepainiana, C. leiocarpa, C. ovatifolia, C. uncinata var. biternata, C. uncinata var. coriacea, C. uncinata var. floribunda, C. uncinata var. ovatifolia, C. uncinata
var. taitongensis), Cocculus carolinus (Syn. Cebatha carolina, Epibaterium carolinum, Menispermum carolinum), Cocculus diversifolius (Syn. Cocculus madagascariensis), Cocculus
hirsutus (Syn. Cocculus villosus, Menispermum hirsutum), Cocculus laurifolius (Syn. Cinnamomum esquirolii), Cocculus leaebe, Cocculus madagascariensis (Syn. Cocculus
diversifolius), Cocculus thunbergii, Diploclisia affinis (Syn. Diploclisia chinensis, Cocculus affinis), Menispernum dauricum.
several in Asia, the risk of their mistaken use in traditional
Chinese medicine is still high and could lead to a major
concern for public health. Considering this fact, a reasonable
way to decrease this risk should be the systematic quality
control of herbal preparations by using reproducible and
accurate analytical methods, such as high-performance liquid
chromatography, liquid chromatography/mass spectrometry,
or capillary electrophoresis.61,62
Interestingly, Mani63 reported in a series of 2028 Indian
patients with chronic kidney disease that chronic interstitial
nephritis was a frequent cause (27.8%). We can speculate that
some of them are AAN, as Indian folk medicine used more
than 7500 plant species, including Aristolochia bracteata,
Aristolochia tagala, and Aristolochia indica.26 The identification of AA-related specific adducts on renal tissue could
confirm this hypothesis.32
162
AA-ASSOCIATED UROTHELIAL MALIGNANCIES
Clinical findings
The striking association between AA exposure and the
presence of urothelial abnormalities was described for the
first time by Cosyns et al.,29 who observed moderate atypia
and atypical hyperplasia of the urothelium in four pieces
of nephroureterectomies removed from three CHN/AAN
patients before or at the time of transplantation. In the same
time, two cases of urothelial carcinoma were reported among
the Belgian cohort of CHN/AAN patients.4,64 Considering
the high risk for urothelial malignancy related to AA exposure, the prophylactic bilateral removal of the native kidneys
and ureters was systematically proposed to CHN/AAN
patients treated by dialysis or renal transplantation.
It emerged that 40–45% of CHN/AAN patients displayed
multifocal high-grade transitional cell carcinomas, mainly in
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Table 3 | Products in which Mu Tong and Fang Ji are declared
as ingredients55,56
Name
Ba Zheng Wan
Chun Yang Zheng Ji Wan
Da Huang Qing Wei Wan
Dang Gui Si Ni Wan
Dao Chi Wan
Dieda Wan
Fu Ke Fen Quing Wan
Guan Xin Su He Wan
Ji Sheng Ju He Wan
Kat Kit Wan
Long Dan Xie Gan Wan
Quell Fire
Shi Xiang Fan Shen Wan
Xin Yi Wan
the upper urinary tract.33,65 The cumulative ingested dose
of Stephania (in fact, Aristolochia) was demonstrated to
be a significant risk for the development of urothelial
carcinomas.33
A further follow-up of these CHN/AAN patients was
performed in relation with the high risk of bladder carcinoma.
Prospective screening cystoscopies were proposed to all renaltransplanted CHN patients from our Nephrology department.
Among the 38 kidney recipients who accepted this follow-up
(cystoscopy and bladder biopsies every 6 months), bladder
urothelial carcinoma was diagnosed in 15 patients, 68–169
months after cessation of AA exposure (cumulative incidence
39.5%): eight urothelial carcinoma in situ, four noninvasive
low-grade papillary urothelial carcinoma, and three infiltrating
urothelial cancer. Out of 17 patients, 12 patients (71%) with a
previous history of upper tract urothelial carcinoma developed
bladder cancer during the follow-up, whereas this occurred
only in three out of 21 (14%) patients free of upper tract
urothelial carcinoma (Po0.01). Despite local and/or systemic
chemotherapy, three patients died and two radical cystectomies had to be performed.66
Almost all of the cases of urothelial cancers were detected
in CHN/AAN patients with end-stage renal disease. However,
the case of a generalized urinary tract cancer without a
significant renal failure after the intake of AA-containing
Chinese herbal remedies showed that a dissociation between
carcinogenicity and nephrotoxicity of AAs may occur.67
Other cases of urothelial carcinoma have been reported
outside Belgium: in Taiwan, United Kingdom, France,
and Hong Kong.14,16,20,36,68,69 Interestingly, some of these
cases, like those described by Laing, were reported after AA
had been banned in the respective countries.
Taking into account all cases of AAN and urothelial malignancies reported worldwide, the Food and Drug Administration issued warnings to healthcare professionals, industry
associations, and consumers regarding the safety of botanical
products and dietary complements containing AA. The FDA
recommended that all botanical remedies known or
suspected to contain AA be discarded.55,56 In 2002, the
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International Agency for Research on Cancer working group
concluded that there was sufficient evidence in humans for
the carcinogenicity of herbal remedies containing plant
species of the genus Aristolochia (Group 1).57
AA activation, DNA-adducts formation, and carcinogenesis
Aristolochic acids I (AAI) and II (AAII), two structurally
related nitrophenanthrene carboxylic acids, are the major
components of the AA mixture contained in the plant extract
of the Aristolochia species. Several enzymes have been
demonstrated to metabolize AAI and AAII to a cyclic
N-acylnitrenium ion with a delocalized positive charge able
to covalently bind to the exocyclic amino groups of purine
bases and to form DNA adducts.40 The 7-(deoxyadenosineN6-yl) aristolactam I, 7-(deoxyguanosine-N2-yl) aristolactam
II and 7-(deoxy-adenosin-N6-yl) aristolactam II are the main
DNA adducts retrieved in AAN patients (Figure 2).33
Based on studies characterizing and quantifying the DNA
adducts formed after modulation of metabolic pathways, the
nitroreduction of AAs was demonstrated to be a crucial step
leading to their ultimate DNA-binding species.40 In human
hepatic microsomes, the reductive activation of AA is mainly
mediated by cytochrome P450 (CYP) 1A2, and to a minor
extent by CYP1A1, whereas in human renal microsomes,
NADPH/CYP reductase is more effective in AA biotransformation.70,71 In addition, the NAD(P)H/quinine oxidoreductase and xanthine oxidase, two cytosolic enzymes found
in human livers and kidneys, catalyze the activation of AAI to
form DNA adducts.72 Several factors such as drugs, smoking
habit, environmental chemicals, and genetic polymorphisms
affect the expression levels and activities of these enzymes,
which could explain variations between individuals in the
susceptibility to AA toxicity.
The phase II metabolism of AA consists of the presence in
the urine and feces of AA metabolites in conjugated forms,
such as glucuronides, sulfate, or acetate esters.73,74 However,
the precise role of conjugation enzymes in AA activation
needs further investigation.75
The predominant 7-(deoxyadenosine-N6-yl) aristolactam I
in vivo, which is the most persistent DNA adduct detected in
the target tissue, is a mutagenic lesion leading to AT-TA
transversions. This specific mutation is retrieved at a high
frequency in codon 61 of the H-ras protooncogene in tumors
of rodents induced by AAI.76 In AAN patients, an overexpression of P53 protein was observed in urothelial atypia
and carcinomas, suggesting that the p53 gene is also mutated.65
Furthermore, DNA-binding studies demonstrated that AAI
and AAII preferentially react with purine bases in the human
p53 gene, and the adduct distribution was not random.77
A specific AAG-to-TAG mutation in codon 139 (Lys-Stop)
of exon 5 in p53 gene was detected in DNA isolated from
one AAN-associated urothelial carcinoma.78 These mutations
in the p53 gene probably trigger the tumorigenesis in AAN
patients in the same way as the mutations in codon 61 of
H-ras trigger the tumorigenesis in AA-intoxicated rodents. The
metabolic activation of AA and its mediated carcinogenesis
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FD Debelle et al.: Aristolochic acid nephropathy
Aristolochic acid I (AAI): R=OCH3
Aristolochic acid II (AAII): R=H
Aristolactam I (AAI): R=OCH3
Aristolactam II (AAII): R=H
O
COOH
O
O
N
NO2
O
O
O
O
NH
O
+
R
R
R
Aristolactam nitrenium ion
+DNA
O
O
O
R
R
N
dA-AAI
dA-AAII
NH
N
NH
N
C
O
N
O
O
H
O
O
N
N
H
NH
H
H
H
N
NH
O
O
O
H
H
O
NH
H
H
C
H
dG-AAI
dG-AAII
Figure 2 | Metabolic activation and DNA adduct formation of aristolochic acids I (AAI; R ¼ OCH3) and II (AAII; R ¼ H). The
7-(deoxyadenosine-N6-yl) aristolactam I and II and the 7-(deoxyguanosine-N2-yl) aristolactam I and II are formed after the reductive
metabolic activation mediated by cytosolic reductases (NAD(P)H/quinone oxidoreductase, xanthine oxidase) as well as enzymes in
hepatic (cytochrome P450 1A2 and 1A1, NADPH/CYP reductase) and renal microsomes (prostaglandin H synthase).
was the subject of an exhaustive and comprehensive review
recently published by Stiborova et al.75
AA TOXICITY: EXPERIMENTAL STUDIES
Carcinogenic aspects
In the 1980s, Mengs and colleagues devoted several experimental studies to the toxicity of AA, especially to their
carcinogenic properties. The administration by gavage of a
mixture of AA (77% AAI and 21% AAII) to male and female
Wistar rats at the dosage of 0.1, 1, and 10 mg per kg body
weight per day for 3–12 months led to the development of
forestomach carcinoma and urothelial dysplasia.79 Following
their initial observations, they examined the different steps of
appearance and development of the AA-induced carcinoma
in Wistar rats and NMRI mice.80,81
Just as CHN was frequently associated to urothelial
dysplasia and malignancies, injections of AA to New Zealand
white rabbits or male Wistar rats led to urothelial atypias.34,35
In the rat model, papillary urothelial carcinoma and fibrohistiocytic sarcoma at the injection site were also retrieved.35
Nephrotoxicity aspects and animal models for AAN
The acute toxicity of AA was evaluated in Wistar rats
and NMRI mice of both sexes (Table 4).82 The lethal dose
50 (LD50) ranged from 56 to 203 mg kg1 orally or 38
to 83 mg kg1 intravenously, depending on species and sex.
The histological evaluation revealed severe tubular necrosis,
atrophy of the lymphatic organs, and large areas of superficial
164
ulceration in the forestomach, followed by hyperplasia and
hyperkeratosis of the squamous epithelium. According to
the extensive tubular necrosis, the authors concluded that
the animals died as a result of acute renal failure, although
the renal functional parameters were not assessed.82 In a
subacute toxicity study, a daily oral administration of 25 mg
AA per kg body weight to male Wistar rats induced after
4 weeks a moderate renal tubular necrosis with a significant
glucosuria and proteinuria.83 However, neither the proximal
tubular atrophy nor the renal interstitial fibrosis, which are
the typical histological findings for CHN, were reported in
those studies.
Following the Belgian outbreak of CHN in 1993 and the
etiopathological hypothesis of AA, new experimental studies
were therefore undertaken. However, first attempts to experimentally reproduce CHN failed: two groups of seven Wistar
rats were orally given either pure AAs (10 mg per kg for
5 days a week for 3 months) or AA-containing herbal
powders mixed with fenfluramine. At the time of killing,
animals in both groups developed the expected tumors but
not renal tubulointerstitial fibrosis.89 On the contrary, typical
histological features of CHN/AAN, consisting of tubular
atrophy, interstitial fibrosis, and urothelial atypias, were
reproduced in 12 female New Zealand white rabbits after
17–21 months of intraperitoneal injections of 0.1 mg AA per
kg body weight, 5 days a week.34
In the same time, we developed a short-term model for
CHN by administrating subcutaneously 10 mg AA per kg body
Kidney International (2008) 74, 158–169
review
FD Debelle et al.: Aristolochic acid nephropathy
Table 4 | Most relevant studies investigating the renal effects of aristolochic acid in animal models
Species
Dosage
Duration
AA components
Renal findings
Rat/mice
38–86 mg kg1 IV
or 150–300 mg kg1
orally
0.2; 1.0; 5.0, or
25 mg kg1 day1 orally
10, 50, or 100 mg kg1
orally
0.1 mg kg1 day1,
5 days a week
1 or 10 mg kg1
day1 s.c.
Once
AAI (77%)/AAII (21%)
28 days
AAI (77%)/AAII (21%)
Once
AAI (77%)/AAII (21%)
17–21
months
35 days
AAI (44%)/AAII (56%)
AAI (40%)/AAII (60%)
Mice
5 mg kg1 day1 i.p
14 days
AAI (44%)/AAII (56%)
Rat
10 mg kg1 day1 s.c.
35 days
AAI (40%)/AAII (60%)
Mice
2.5 mg kg1 day1,
5 days a week
14 days
AAI (55%)/AAII (45%)
or AAI or AAII or
Aristolactam I or
AAIV
Rat
10 mg kg1 day1 s.c.
35 days
AAI (40%)/AAII (60%)
Severe PT cells necrosis. LD50 ranged from 56 to 203 mg kg1
orally or from 38 to 83 mg kg1 i.v., depending on species
and sex.
Proteinuria and glucosuria. PT cells atypia and mild necrosis at
highest dosage. Renal interstitial inflammatory cells infiltration.
At 100 mg kg1: m sCr and BUN, proteinuria, m gGT and NAG
enzymuria. m mitosis and necrosis of PT (pars recta)
m sCr, glucosuria and low molecular weight proteinuria.
Renal hypocellular interstitial fibrosis.
At 10 mg kg1 day1: glucosuria, proteinuria, k LAP enzymuria,
and m sCr on days 10 and 35. PT cells necrosis, mononuclear
cells infiltrates (day 10), proximal tubular atrophy and interstitial
fibrosis (day 35).
During the regeneration phase (day 28), circulating transgenederived HGF reduced AA-induced interstitial fibrosis, partially
through a k expression of TIMP-1 and m MMP-9 activity.
RAS blockade reduced ED-1 + macrophage infiltration but
not interstitial fibrosis induced by AA.
m sCr, glucosuria, proteinuria, proximal tubule injury,
mononuclear cells infiltration, and interstitial fibrosis.
Nephrotoxicity depending on strains (C3H/He4Balb/
c4C57Bl6) and AA components (AAI4AAII). No nephrotoxicity
of Aristolactam I and AAIV.
Defective activation of antioxidative enzymes and
mitochondrial damage. Impaired regeneration and apoptosis
of PT cells. Interstitial infiltration of monocytes/macrophages
and CD8+ lymphocytes. Loss of epithelial markers
concomitantly to de novo expression of mesenchymal cell
markers and disruption of TBM.
Rat
Rat
NZW
rabbit
Rat
Reference
82
83
84
34
35
85
86
87
88
Abbreviations: AA, aristolochic acid; BUN, blood urea nitrogen; gGT, gamma-glutamyltransferase; HGF, hepatocyte growth factor; i.p., intraperitoneal; i.v., intravenous;
LAP, leucine aminopeptidase; LD50, lethal dose 50; MMP, matrix metalloproteinase; NAG, N-acetyl-b-D-glucosaminidase; NZW, New Zeeland white; PT, proximal tubule;
RAS, renin–angiotensin system; s.c., subcuatenous; sCr, serum creatinine; TIMP, tissue inhibitor of metalloproteinase; TBM, tubular basement membrane.
wt per day to male Wistar rats.35 On day 35, AA-treated rats
displayed functional and histological renal impairment as a
significant increase of serum creatinine and foci of severe
proximal tubular atrophy surrounded by interstitial fibrosis.
Nephrotoxicity of different components of AA was also studied
in three strains of inbred male mice. The C3H/He mice
intraperitoneally injected with 2.5 mg of AA per kg body
weight, 5 days a week for 2 weeks, developed foci of proximal
tubule cell injury surrounded by mononuclear cell infiltration
on day 14.87 Two weeks later, signs of proximal tubule cell
proliferation were observed, whereas the inflammatory cell
infiltration became more severe and interstitial fibrosis
occurred.87 In a CH3/He mice model, AAI exhibited a higher
nephrotoxicity than AAII, which was also confirmed in in vitro
studies on the proximal tubular LLC-PK1 cell line.87,90,91
Pathogenesis of AAN
The pathophysiological mechanisms by which AA induces
renal interstitial fibrosis are still largely unknown. The treatment with an angiotensin-converting enzyme inhibitor±
angiotensin II receptor blocker did not modify the functional
and structural renal impairments in AA-treated Wistar rats,
suggesting that pathways leading to interstitial fibrosis seem
to be independent of the renin–angiotensin system in this
model.86
Kidney International (2008) 74, 158–169
An early phase of acute tubular necrosis preceding the
development of tubular atrophy and interstitial fibrosis was
also observed in several experimental studies.85,87,92 By using
a transgenic mice model, Okada et al.85 demonstrated that
hepatocyte growth factor did not interfere with the acute
phase but reduced the severity of interstitial fibrosis during
the tubular regeneration phase, partially through a decreased
expression of tissue inhibitor of metalloproteinase-1 and
increased matrix metalloproteinase-9 activity.
Pozdzik et al.88 recently showed in the rat AAN model
that AA tubulotoxicity resulted in defective activation of
antioxidative enzymes and mitochondrial damage. The
progressive tubular atrophy was related to impaired regeneration of proximal tubular epithelial cells and apoptosis
secondary to caspase-3 activation. The accumulation of
vimentin and a-smooth muscle actin-positive cells in the
interstitial areas expressing transforming growth factor-b
suggested an increase in resident peritubular fibroblasts and
their activation into myofibroblasts. These activated resident
fibroblasts are proposed as the main source of collagen
deposition during experimental AAN.88
As reported in several in vivo and in vitro studies, apoptosis is likely involved in the process of AA-induced proximal
tubular atrophy.85,90,93 In an in vitro study, LLC-PK1 cells
exposed to AA displayed a rapid increase in their intracellular
165
review
calcium content leading to endoplasmic reticulum and
mitochondrial stress, which in turn causes activation of the
caspase pathway and finally apoptosis.94 It should be noted
that the potential impact of the AA–DNA adducts formation
on the proximal tubular atrophy, for example, through a
defect of DNA repair, is largely unknown and deserves
further studies.
AAN AND BEN: THE TWO FACES OF JANUS?
Balkan-endemic nephropathy is characterized by chronic
interstitial fibrosis with slow progression to end-stage renal
disease and urothelial malignancy. It was first described about
50 years ago and affects residents of rural areas of Bulgaria,
Bosnia, Croatia, Romania, and Serbia along the Danube river
basin.95
As the etiology of BEN is currently unknown and different
diagnostic criteria have been used in various countries,
epidemiological data are difficult to compare. At least 25 000
individuals suffer from BEN or are suspected of having the
disease, whereas the total number of people at risk in these
countries may exceed 100 000. The significant epidemiologic
features of BEN include (1) its focal occurrence in certain
farming villages, with unaffected villages located in close
vicinity; (2) a familial but not inherited pattern of disease,
frequently affected members of the same household; (3)
occurrence only in individuals who are older than 18 years,
occurrence in o10% of households in endemic villages; and
(4) a strong association with upper urinary tract urothelial
carcinoma.27
A variety of environmental factors have been explored
during the past 50 years, including heavy metals, arsenic,
nitrogen species, silica, selenium deficiency, calcium and
magnesium deficiency, polycyclic aromatic hydrocarbons in
the water originating from Pliocene coal beds, viruses and
bacteria, and mycotoxins.96 Of these factors, ochratoxin A
(OTA) has been the most investigated target of research.97,98
Its presence in a variety of common foodstuffs, including
cereal grains, was recognized more than three decades ago.
OTA is classified by the International Agency for Research on
Cancer99 as a possible human carcinogen (Group 2B) on the
basis on sufficient evidence for carcinogenicity in experimental animals but inadequate evidence in humans. OTA was
shown to be a powerful rodent carcinogen, causing liver
tumors in mice and renal tumors in mice (males) and rats, in
particular adenomas and invasive carcinomas with elevated
DNA ploidy distribution.100,101
Nephrotoxicity of OTA is also well recognized in animals.
OTA induced the so-called porcine nephropathy after longterm exposure. Initially described in 1976 by Krogh, this renal
disease is characterized by lesions compatible with those
observed in chronic interstitial nephropathy, including
proximal tubule injury and interstitial fibrosis.95 However,
as recently well summarized by Mally et al.102, in all the
remaining animal species studied (rodents), the most
frequent histological observations are nuclear enlargement
and polyploidy of proximal tubule cells, reflecting nuclear
166
FD Debelle et al.: Aristolochic acid nephropathy
division without cytokinesis. No interstitial fibrosis has been
reported associated or not with proteinuria, glucosuria, and
increased creatininemia. Except in specific cases of acute
tubular necrosis, no similarity could be found in the tubulointerstitial compartment with histological lesions observed in
AAN or BEN but, contrasting with BEN, interstitial fibrosis
and tubular atrophy are not described.102 Moreover, kidney
tumors in OTA-exposed rats developed from the straight
segment of the proximal tubule. No upper urinary tract
carcinoma has been described, contrasting with its high
prevalence in AAN and BEN.
Some studies found higher exposure (as measured by
the intake of OTA and OTA levels in food stuff, blood, or
urine) in individuals from endemic villages as compared
with nonendemic villages (as reviewed by Stefanovic et al.;
Long and Voice; Pfohl-Leszkowicz and Manderville; and
Mally et al.).27,98,102,103 OTA–DNA-related adducts (but also
AA–DNA adducts) were detected in kidney tissue from
patients with urothelial carcinoma or ureteral stenosis living
in endemic areas and in approximately one-third of renal
tissue samples from patients suffering from BEN and
urothelial carcinoma.27,40,98 The long-term persistence of
such OTA–DNA adducts and even the methodological
procedures used to detect them are still a matter of debate
and may explain some discrepancy between data reported by
different research groups.97 Consequently, in the absence
of a specific mutation profile related to OTA and direct DNA
damage, some researchers like Turesky proposed an indirect
mechanism involving oxidative stress and OTA-mediated
cytotoxicity rather than direct genotoxic properties to explain
OTA carcinogenicity.104
The so-called ‘AA hypothesis’ in BEN was initially formulated by Ivic in 1970.105 He suggested a possible chronic
dietary intoxication from bread made from wheat flour
contaminated with seeds of Aristolochia clematidis. Aristolochia
is, indeed, a common weed in wheat fields in endemic areas,
and seeds are mixed with wheat grain during the annual
harvest. This hypothesis seemed to be totally forgotten until
the late 1990s.
In the first report of the Belgian CHN cases, the attention
was already pointed out to the clinical similarities between
this nephropathy and the Balkan endemic nephropathy.1
In 1994, Cosyns et al.29 underlined the hypocellular pattern
of interstitial fibrosis decreasing from outer to inner cortex,
which is a typical histopathological feature shared by both
nephropathies. More recently, by using an ultrasensitive,
quantitative 32P-postlabeling method, in conjunction with
HPLC and mass spectroscopic techniques, Grollman et al.23
reported that dA-aristolactam and dG-aristolactam adducts
were found in the DNA from the renal cortex of Croatian
patients with BEN and in urothelial tumors from residents
of BEN endemic villages. In addition, a predominance of
A:T-T:A p53 mutations was detected in urothelial cancers
from BEN patients (78%). This ‘signature’ mutation is rarely
observed in transitional cell cancers (less than 5%).77 Such
mutation profile has never been found for OTA.
Kidney International (2008) 74, 158–169
review
FD Debelle et al.: Aristolochic acid nephropathy
Renal interstitial fibrosis
and urothelial carcinoma
Similarities
Balkan endemic nephropathy
(1956)
Etiological factors:
ochratoxin A highly suspected,
aristolochic acid evoked
Similarities
Chinese herb nephropathy
(1992)
Etiological factors:
aristolochic acid highly
suspected, ochratoxin A evoked
Aristolochic acid Balkan endemic
nephropathy
(2007–)
Etiological factors:
aristolochic acid highly
suspected, ochratoxin A evoked
Aristolochic acid-related
specific DNA adducts in
renal tissue
Aristolochic acid nephropathy
(1996–)
Etiological factor:
role of aristolochic acid proven
Similarities
Renal interstitial fibrosis, urothelial carcinoma,
and aristolochic acid-related specific DNA adducts
Figure 3 | Chinese herb/AAN and Balkan endemic nephropathy: the two faces of Janus?
Laboratory experiments performed by introducing the
human p53 gene into mouse fibroblasts (Hupki cells) and
treating them with AA resulted in the observation of a similar
mutational A:T-T:A spectrum.106,107 It was found to be
similar to that reported in the H-ras gene of rodents treated
with AA.40
Taken together, these clinical, histopathological, epidemiological, and recent toxicological data provide evidence that
long-term exposure to AA may be the missing link to elucidate
the complex multifactorial etiology of BEN (Figure 3).108
CONCLUSION
Often considered harmless, the regular use of herbal products
may result in dramatic consequences as demonstrated by the
‘Chinese-herb nephropathy’ tragedy occurring in Belgium
in the 1990s. The replacement of one substance (Stephania)
by another more toxic compound (Aristolochia) was the
cause of the outbreak of progressive renal fibrosis and
urothelial carcinoma. Such substitution is probably responsible for a higher incidence of AAN than expected because
this practice is common and allowed in traditional medicines
that are based on a complex nomenclature using vernacular,
not botanical, names. In addition, these products are still
available legally in many countries and can be bought via the
Internet. Finally, AA is proposed as the environmental causal
factor for BEN affecting thousands of people living in the
Danube basin.
ACKNOWLEDGMENTS
This work was supported by grants from the Groupement pour
I’Etude, le Traitement et la Réhabilitation Sociale des Insuffisants
Kidney International (2008) 74, 158–169
Rénaux Chroniques, the Fonds de la Recherche Scientifique Médicale
(Belgium), and the Fondation Erasme (Erasme Hospital, Brussels,
Belgium). We are indebted to the medical, nursing, and technical
staffs of the nephrology and pathology departments for
continuous cooperation.
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