Journal of Kidney Cancer and VHL 2015; 2(4): 153-162.
Revealing a Pre-neoplastic Renal Tubular Lesion by p-S6 Protein Immunohistochemistry after Rat Exposure to Aristolochic Acid
Alexandra Gruia1, Patrycja Gazinska2, Diana Herman1, Valentin Ordodi3, Calin Tatu3, Peter Mantle4
1Pathology Department, County Hospital Timisoara, Timisoara 300736, Romania; 2Breakthrough Breast Cancer Research Unit, Guy’s Hospital, King’s Health Partners AHSC, King’s College London School of Medicine, London SE1 9RT, UK; 3Department of Biology, University of Medicine and Pharmacy “Victor Babes”, Timisoara, Romania; 4Centre for Environmental Policy, Imperial College London, London SW7 2AZ, UK.
putative role of AA in tumours of the renal pelvis and urethra gained attention
in the 1990s during the local epidemic of nephropathy in Belgian women. This
was attributed to formulation error in a Chinese herbal slimming medication
involving accidental inclusion of Asian material in which AA was a
characteristic toxic alkaloid. Early
attempts to find an experimental animal model to mimic the development of renal
or urothelial tumours used female rabbits (7). Intraperitoneal delivery of a
mixture of AA I and II (0.1 mg/kg b.w.)
5 days a week to 12 animals for up to nearly 2 years caused marked glucosuria
and proteinuria and extensive renal tubular atrophy and interstitial fibrosis
in a context of reduced feed intake and poor concomitant growth. One rabbit
died, but three developed urinary tract tumours (one small in situ renal carcinoma, one tubulopapillary adenoma, and one
transitional cell carcinoma in mid-ureter together with an extensive peritoneal
papillary malignant mesothelioma.
Another report on rat tumourigenicity of AA, yielding kidney neoplasms
in all experimental individuals six months after sub-acute insult per os while not arising from
transitional cell epithelia, is that of Cui et al (8). If AA is an etiological
agent for renal or urothelial tumours in BEN, identification of early markers
of neoplastic transformation may aid in the diagnosis and management of
AA-induced renal/urothelial tumours. In this study, we tested the utility of
the ribosomal protein p-S6 (phospho S6 ribosomal protein) as a potential marker
for AA-induced renal tumours in rats treated with AA. Further, since our Balkan
1H NMR metabolomic study differentiated a BEN cohort in Romania from
one in Bulgaria (9), and that we are aware of ethnobotanical exposure to AAs,
we have included animals additionally given Pliocene lignite infusion and pilot
groups given chronic exposure to ethnobotanical extracts of A. clematitis.
on expression of ribosomal p-S6 protein in kidney of rats challenged with AA
has arisen from our previous experience with cancers in kidneys of rats given
chronic tolerable exposure to dietary OTA (10). That study revealed a
consistent specificity for the renal tumours attributable to that environmental
alkaloid in contrast to other neoplasms, e.g. testis tumours or subcutaneous
fibrosarcoma, of other causation. The OTA findings were reminiscent of those in
the spontaneous renal tumours of the Eker rat (11). The Eker strain is
heterozygous for a dominantly-inherited germline mutation in the Tsc 2 tumour
suppressor gene that is recognised as a valid model for human tuberous
sclerosis complex (12), with two genes (TSC1 and TSC 2) involved and tumours occurring
sometimes in kidney. Activation of the TSC 2 and folliculin genes in mice has
been associated with both renal tumour development and mammalian target of
rapamycin (mTOR) dysregulation (13). A few rare human cases of familial renal
cell carcinoma have been attributed to disruption of the TSC 2 gene (14). More
recently, Wilson et al (15) generated Tsc 1+/-mice with
predisposition to kidney cancer, and strong staining for p-S6 protein in those
tumours. These observations form a rationale for novel pilot exploration of
p-S6 as a marker for putative AA-induced renal/urothelial tumours or
Materials and methods
animal experiments complied with the European Convention for the Protection of
Vertebrate Animals used for Experimental and other Scientific Purposes
(Strasbourg, France, 1986). The University of Medicine and Pharmacy Timisoara
Ethical Committee approved the experimental protocols. Young female
Sprague-Dawley rats were caged in a day-night controlled room, properly
ventilated, and with ad libitum
access to food and water. Animals were observed daily and growth was monitored
by weight. Within available resources, several biochemical parameters were
monitored at intervals to detect any marked changes. For assessment of liver
and kidney function, aspartate aminotransferase (AST), alanine aminotransferase
(ALT), creatinine and urea were evaluated. Blood was collected from a femoral
vein, allowed to clot for 30 min, centrifuged and processed for serum
concentration according to Siemens Diagnostics ALT-AST kit, creatinine kit and
urea kit, respectively.
experiment tested aqueous extracts of A.
clematitis, using six rats (commencing 5 weeks old; 82-137 g) for six
months. A. clematitis plants had been
collected from wild habitats (Western and South-western Romania, May-June,
2006) and left to dry in a controlled humidity room. Dried leaves were powdered
with pestle and mortar. Two types of extract were prepared. For two rats, two
grams of leaves were Soxhlet-extracted in 200 mL of distilled water for 30 min.
For two other rats one gram of leaves was suspended in 200 mL of hot (80 oC)
distilled water and left to infuse for 30 min, in a manner reproducing the
ethnobotanical decoction preparation of A.
clematitis in some Romanian rural areas. Two more rats served as controls
and were given tap water.
A. clematitis leaf aqueous extracts were analyzed by
an HPLC method to establish AA concentrations. An analytical standard
containing a mixture of AA I and II was purchased from Sigma-Aldrich (St.
Louis, CO, USA). Acetonitrile (HPLC-grade) and HPLC grade water were also
obtained from Sigma-Aldrich. All experiments in this study were performed with
an Agilent 1100 HPLC system. An HP 1100 liquid chromatograph system (Agilent
Technologies, Santa Clara, CA, USA) consisting of a binary pump, a
thermostat-controlled column and UV detector plus on-line degasser was used.
Data were analyzed using the HP Chemstation System. The analytical column was a
Zorbax SB-C18 (5 μm, 4.6 x 250 mm) (Agilent Technologies). The eluents
consisted of HPLC grade water acidified with phosphoric acid 98 % to a pH value
of 3 (A) and acetonitrile (B). The initial condition was set at 20 % B with a
gradient to 70 % B in 25 min, then a linear gradient to 100 % B in 30 min. The
flow rate was 0.5 mL/min. All analyses were monitored at 390 nm. The column
temperature was set at 40 oC, and the sample injection volume was 20 μL. The
peak of AA I and II in samples was identified by comparing their retention time
values and UV spectra with those of the standard. The aqueous extracts obtained
by Soxhlet and hot water infusion were analyzed prior to the beginning of the
experiment and AA concentrations were determined. To calculate the
concentration of AA, a calibration curve was made based on standard AA
dilutions from 1 to 40 mg/100 mL. AA standard dilutions were prepared in
acetonitrile. On average, 2.96 mg AAI and II/100 mL was found in the Soxhlet
aqueous extract of A. clematitis
leaves, while the hot water infusion contained on average 2.5 mg/100 mL.
Predicted intake volume was 150 ml/kg/day (16), translating into approximately
900 and 750 μg AAs/day, respectively.
aqueous extracts obtained were filtered through 0.45 μm membrane filters prior
to use as drinking water. Extracts were freshly prepared, based on demand,
twice a week. Six months later all rats were euthanized for general autopsy,
but particularly studying urinary tracts. Kidneys and a liver sample were
collected and preserved in 4 % formalin solution for histology. Tissues were
embedded in wax blocks and sections (3 μm) stained with H & E by the
standard hospital pathology protocol for reviewing in Romania.
female Sprague-Dawley rats (5 weeks old, 100-125 g) were given AAI (purity
>97%; Sigma-Aldrich Corporation, St. Louis, MO, USA) by gavage at a dosage
of 50 mg/ kg b.w. Approximately 5.5 mg AAI sodium salt dissolved in 20% ethanol
in phosphate buffered saline was administered (1 ml) to each rat on three
consecutive days. Thereafter, animals were maintained on standard rat diet for
a further seven months at an average body weight of 255 g.
female Sprague-Dawley rats weighing 150-170 g were administered AA I in
solution in a Pliocene lignite aqueous extract. This extract was prepared from
coal samples collected from the Husnicioara open pit mine (South-western Romania)
in September 2011. A Soxhlet method was used for extraction, with 10 g of coal
and 250 mL bi-distilled water for 5 days; the aqueous extract had a brownish
colour and was used as a solvent for dissolving the AAI (purity >97%,
Sigma-Aldrich, St. Louis, MO, USA). The gavage solution (1 mL) delivered
approximately 5.5 mg AAI sodium salt/rat/day on three consecutive days.
Thereafter, animals were maintained in standard (control) conditions for six
months. Resource constraints focused all available animals in experiment 2 on
treated groups; general controls were those in experiment 1, noting that in (8)
22 control rats had been without disease throughout.
for p-S6 protein
protocol was as previously described (10) and included fresh H & E –
stained adjacent sections for direct comparison with those stained
immunohistochemically. In brief, immunostaining of 3 μm sections was with
Vectastain Elite ABC kit (PK-6101). In addition, avidin/biotin block
(Vectastain SP-2001) was applied prior to the primary antibody (polyclonal
phospho-S6 protein [Ser240/244, Cell Signalling #2215] in 1:200 dilution).
Sections were developed using DAB, counterstained in Gill ІІІ haematoxylin, dehydrated
and mounted with DPX. Immunostained, and standard haematoxylin and eosin (H
& E)-stained sections were scanned (Hamamatsu Nanozoomer) and stored on the
digital slide server (DSS) in ndpi
format for reviewing using Digital Images Hub (Slidepath system) for online
validation and record.
of the rats showed any adverse reaction to the treatments, no abnormalities
were evident at necropsy, and no H & E histopathology was evident in
longitudinal sections of kidney (as also for those stained in London; Figure 1 A), or in liver (not shown).
Concerning use for the first time of an immunohistochemical probe in AA
toxicology, a negative control for p-S6 protein antibodies (secondary antibody
only) showed no staining (Figure 1 B).
Consistent features in all control and treated rats were that in all kidneys
sectioned through the papilla that region was not stained (Figure 1 C), as in the negative control. There was no evidence of
proliferation of the transitional cell layer in the renal pelvis and there was
also no staining for p-S6 protein there. However, controls had variable diffuse
staining from inner medulla to cortex that seemed to be confined to vascular
elements but this did not involve glomeruli (Figure 1D); the reason for this distinction is unclear, but in BEN
renal atrophy glomeruli are generally well preserved. In the cortex of the
group receiving Soxhlet extract of leaves, diffuse staining of p-S6 was
observed. The group receiving hot water extract also showed patchy diffuse
staining for p-S6 protein in cortex and medulla, but there was no strong basis
for perceiving significant differences attributable to either treatment.
Overall, there was no macroscopic or microscopic evidence of tumour growth in
response to plant extracts, and the diffuse staining pattern of p-S6 was not
markedly different between groups.
given AA I alone were 100-125 g (mean 120 +/- 6.4 g). After one month the mean
was 186 g, reaching 255 g at seven months, all conforming to standard growth
data (17) and showing no persistent reaction to the treatment. Blood plasma
creatinine concentration increased from the puberty value at the start (0.33 +/- 0.06 mg/dL) to 0.45 +/- 0.05 mg/dL a month later and was sustained at 7 months (0.44 +/- 0.06
mg/dL). Plasma urea was 30 +/- 11 mg/dL at
the start, 28 +/- 10 mg/dL after one month and 33 +/- 10 mg/dL at 7 months, not
significantly different. Both hepatic enzymes declined slightly in
concentration over the period, but values remained within normal range. Animals
also given Pliocene lignite infusion as gavage were initially 150-170 g (mean
164 g). At term, six months later, their mean weight was 240 g, similarly
typical for this breed. Over the treatment period plasma creatinine (0.45 +/-
0.02 and 0.47 +/- 0.06 mg/dL) and urea (32 +/- 8 and 25 +/- 3 mg/dL) values were
maintained, AST was unchanged and ALT decreased only slightly, though within
the normal rat range. As in experiment 1, no significant histopathological
change was evident in initial H & E sections. In both experiments, ureters
and bladders were viewed at necropsy for gross change; none was found and so
tissues were not embedded for histology.
Figure 2. p-S6
staining in AA treated kidneys. A, AA I treated kidneys showing staining for
p-S6 protein principally in the papilla, contrasting with Experiment 1; B, detail in papilla (bar 100 μm).
Figure 3. Closely matching longitudinal sections through
renal papilla stained with haematoxylin and eosin (A) or for p-S6 protein (B)
correlating stained areas, probably vascular (note erythrocytes), between some
ducts and tubules. Bar 50 μm.
Figure 4. Seven months after oral gavage with AA. Adjacent sections in renal outer stripe of
the outer medulla stained with haematoxylin and eosin (A), or immunohistochemically for expression of p-S6 protein (B). Stained nephron fragment(s) are
interpreted as indicative of a pre-neoplastic lesion (bar 50 μm).
spite of six months of exposure to AAs in drinking water there was no
detectable adverse clinical or histopathological effect. The mild, diffuse
over-expression of ribosomal p-S6 protein in renal cortex of controls seems to
be at least partly an inconsequential feature of the local water supply or of
commercial rat diet, which had not been evident in previous UK studies in
London (10). However, the A. clematitis
infusions, prepared in distilled water, did not significantly affect the
intensity of immunohistochemical staining. Notably, the cumulative intake of AA
(~1.8 g/kg b.w.) was three-fold higher than that given during half the exposure
period (18) and which had caused extensive histopathological changes.
Therefore, the present pilot study suggests that, for environmental toxins, it
is futile to rely on convenient once-daily gavage or parenteral injection for
other than pharmacokinetic studies, because environmental AA seems to be rather
well-tolerated in rats if given slowly throughout each day. Humans may be
similar. In contemplating what might be necessary for A. clematitis to cause BEN and its tumours, the amount may exceed
any realistic human exposure.
semi-acute high-insult administration of AA I was adopted partly to mimic the
end-point outcome as described previously (8). Cui et al. (8) had used AA I
(> 95 % purity) isolated from Aristolochia
manshuriensis Kom, presumably not different from the commercial AA I used
here although there is recent concern (19) that highly mutagenic AA analogues
remain to be recognised in some species. Of the 14 rats used, 4 developed
unilateral kidney tumours, and the remaining animals showed pre-neoplastic
renal lesions. Lesions were small
nodules, the smallest of which were already 2-3 mm in diameter, while larger
ones sometimes extended through the renal capsule. We were unable to reproduce
these results, despite prolonging the experiments for seven months instead of
six. Clearly, our present concept of a pre-neoplastic lesion is very different
comparison can be found in the study of Schmeiser et al. (18) which used Wistar
males, gavaged daily at 10 mg AA І/kg b.w., sodium salt in water for 3 months.
The experiment terminated 7 months after starting, as in our present
experiment. Among 18 treated rats, most
gave squamous cell carcinomas in the forestomach and sometimes also in an ear
duct. Other tumours occurred in the small intestine and /or the pancreas. One
occurred in a kidney (not studied). Although the treatment had been widely
carcinogenic there were no transitional cell tumours. Similarly, even Ivic (2)
who first perceived a role for AA in the kidney atrophy of BEN, did not find
experimental urothelial tumours. The cumulative dose in (18) had been at least
that in experiment 2 here, although gavaged 5 days/week over a much longer
period. Clearly there is need for experiments on short and long-term exposure to
AA at a range of moderate doses, preferably given in feed (20) and based on US
National Toxicology Programme-like rigour (21), to establish the plausible risk
of renal cancer particularly in transitional cells of the kidney pelvis.
we did not observe renal tumours or lesions, we observed features of
pre-neoplastic transition in response to AA in some areas of the kidney. There
was a close match between p-S6 staining and morphological change as observed by
H & E staining. The principal value
of immunohistochemical staining for p-S6 protein hyper-expression has been the
highlighting, for the first time, of a small proliferation in the epithelium of
proximal straight tubule segments of nephron located in the OSOM, revealing and
matching what had not been recognised otherwise in a closely-adjacent
conventionally stained section. This histopathological change represents our
concept of a pre-neoplastic proliferation and is reminiscent of the periphery
of a matching small neoplastic lesion in a rat given protracted exposure to
dietary OTA (Figure 2A in 10). Our concept is also consistent with a literature
illustration (22). This lesion did not appear to be resolving seven months
after the AA insult and thus could have been a sufficient focus for subsequent
proliferation towards kidney cancer.
serial sectioning and immunohistochemical staining might have been expected to
reveal other examples, if resources had been available. The utility of p-S6 as
a marker of pre-neoplastic renal tubular lesions warrants further
investigation, but the important feature of the present findings is that of
AA-initiated potential carcinoma arising in renal parenchyma, as is the case
with another nephrotoxin OTA, but not by proliferation in the transitional cell
epithelium of the renal pelvis. To demonstrate the latter experimentally as
evidence for AA causing the urological tumours in BEN patients is
epidemiologically-desirable to satisfy classical (23) and modern (24, 25)
criteria. Currently, human AA exposure evidence for BEN is conflicted (15, 26,
27) and claims that AA is the disease determinant (28-30) appear premature.
Conflicts of Interest
authors declare that they have no competing interests.
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