Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice
Aim: Polychemotherapy (PCT), widely used for the antitumor treatment has a pronounced toxic effect on the organism, and its cytostatic effect sometimes is canceled by multidrug resistance of a neoplasia. Comprehension of the nature and development of pathological changes caused by the PCT during the...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice / E.V. Zonov, E.I. Voronina, M.A. Zenkova, T.A. Ageeva, E.I. Ryabchikova // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 30-36. — Бібліогр.: 30 назв. — англ. |
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irk-123456789-1391292018-06-20T03:06:27Z Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice Zonov, E.V. Voronina, E.I. Zenkova, M.A. Ageeva, T.A. Ryabchikova, E.I. Original contributions Aim: Polychemotherapy (PCT), widely used for the antitumor treatment has a pronounced toxic effect on the organism, and its cytostatic effect sometimes is canceled by multidrug resistance of a neoplasia. Comprehension of the nature and development of pathological changes caused by the PCT during the treatment of cancer is very important to improve the efficiency of the therapy and to clarify the mechanisms of tumor-host interactions. This study was aimed to examine PCT impact on kidney cells and tissues in mice with transplanted resistant lymphosacroma (RLS) and to analyze morphology of metastases of the tumor in kidney during PCT. Materials and Methods: Male mice CBA/LacSto (55 animals) were intramuscularly implanted in the right hind paw by 105 cells/ml of tumor RLS (a diffuse large B-cell lymphosarcoma) with multi-drug resistance (MDR) phenotype. Mice received combination of cyclophosphamide (50 mg/kg), oncovin (0.1 mg/kg), hydroxydaunorubicin (4 mg/kg), and prednisone (5 mg/kg) accordingly to CHOP scheme each 7 days after inoculation of the tumor. The kidneys were sampled on days 1, 3 and 7 after each series of injection of PCT preparations and processed for light and electron microscopy, immunohistochemical analysis of Ki-67 and Apaf-1 proteins also was performed. Results: Tumor RLS produced metastases comprised of small cells in the kidneys of mice after 8 days post inoculation. Application of PCT resulted in destruction of small-cell metastases and development of many large-cell metastases in kidney. Application of PCT induced the development of prominent damage of nephron cells, primarily in S3 segments of proximal tubules. Even one series of PCT caused reduction of basal plasma folds in these cells and alteration of mitochondria. Damage of proximal tubules and involvement of distal tubules, renal bodies and interstitial tissue in the pathologic process, increased during the experiment. This work presents the description of morphological changes in kidney as well as of the tumor metastases under PCT influence. Conclusion: The obtained data should be considered while designing of remedies for recovery of internal organs functions after antitumor PCT. 2013 Article Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice / E.V. Zonov, E.I. Voronina, M.A. Zenkova, T.A. Ageeva, E.I. Ryabchikova // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 30-36. — Бібліогр.: 30 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/139129 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Original contributions Original contributions |
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Original contributions Original contributions Zonov, E.V. Voronina, E.I. Zenkova, M.A. Ageeva, T.A. Ryabchikova, E.I. Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice Experimental Oncology |
| description |
Aim: Polychemotherapy (PCT), widely used for the antitumor treatment has a pronounced toxic effect on the organism, and its cytostatic effect sometimes is canceled by multidrug resistance of a neoplasia. Comprehension of the nature and development of pathological changes caused by the PCT during the treatment of cancer is very important to improve the efficiency of the therapy and to clarify the mechanisms of tumor-host interactions. This study was aimed to examine PCT impact on kidney cells and tissues in mice with transplanted resistant lymphosacroma (RLS) and to analyze morphology of metastases of the tumor in kidney during PCT. Materials and Methods: Male mice CBA/LacSto (55 animals) were intramuscularly implanted in the right hind paw by 105 cells/ml of tumor RLS (a diffuse large B-cell lymphosarcoma) with multi-drug resistance (MDR) phenotype. Mice received combination of cyclophosphamide (50 mg/kg), oncovin (0.1 mg/kg), hydroxydaunorubicin (4 mg/kg), and prednisone (5 mg/kg) accordingly to CHOP scheme each 7 days after inoculation of the tumor. The kidneys were sampled on days 1, 3 and 7 after each series of injection of PCT preparations and processed for light and electron microscopy, immunohistochemical analysis of Ki-67 and Apaf-1 proteins also was performed. Results: Tumor RLS produced metastases comprised of small cells in the kidneys of mice after 8 days post inoculation. Application of PCT resulted in destruction of small-cell metastases and development of many large-cell metastases in kidney. Application of PCT induced the development of prominent damage of nephron cells, primarily in S3 segments of proximal tubules. Even one series of PCT caused reduction of basal plasma folds in these cells and alteration of mitochondria. Damage of proximal tubules and involvement of distal tubules, renal bodies and interstitial tissue in the pathologic process, increased during the experiment. This work presents the description of morphological changes in kidney as well as of the tumor metastases under PCT influence. Conclusion: The obtained data should be considered while designing of remedies for recovery of internal organs functions after antitumor PCT. |
| format |
Article |
| author |
Zonov, E.V. Voronina, E.I. Zenkova, M.A. Ageeva, T.A. Ryabchikova, E.I. |
| author_facet |
Zonov, E.V. Voronina, E.I. Zenkova, M.A. Ageeva, T.A. Ryabchikova, E.I. |
| author_sort |
Zonov, E.V. |
| title |
Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice |
| title_short |
Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice |
| title_full |
Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice |
| title_fullStr |
Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice |
| title_full_unstemmed |
Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice |
| title_sort |
influence of polychemotherapy on the morphology of metastases and kidney of resistant rls-bearing mice |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2013 |
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Original contributions |
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http://dspace.nbuv.gov.ua/handle/123456789/139129 |
| citation_txt |
Influence of polychemotherapy on the morphology of metastases and kidney of resistant RLS-bearing mice / E.V. Zonov, E.I. Voronina, M.A. Zenkova, T.A. Ageeva, E.I. Ryabchikova // Experimental Oncology. — 2013. — Т. 35, № 1. — С. 30-36. — Бібліогр.: 30 назв. — англ. |
| series |
Experimental Oncology |
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| fulltext |
30 Experimental Oncology 35, 30–36, 2013 (March)
INFLUENCE OF POLYCHEMOTHERAPY ON THE MORPHOLOGY
OF METASTASES AND KIDNEY OF RESISTANT RLS-BEARING MICE
E.V. Zonov1,2, E.I. Voronina3, M.A. Zenkova1, T.A. Ageeva3, E.I. Ryabchikova1,*
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy
of Sciences, Novosibirsk 630090, Russia
2Novosibirsk State University, Novosibirsk 630090, Russia
3Novosibirsk State Medical University, Novosibirsk 630091, Russia
Aim: Polychemotherapy (PCT), widely used for the antitumor treatment has a pronounced toxic effect on the organism, and its cyto-
static effect sometimes is canceled by multidrug resistance of a neoplasia. Comprehension of the nature and development of patho-
logical changes caused by the PCT during the treatment of cancer is very important to improve the efficiency of the therapy and
to clarify the mechanisms of tumor-host interactions. This study was aimed to examine PCT impact on kidney cells and tissues in mice
with transplanted resistant lymphosacroma (RLS) and to analyze morphology of metastases of the tumor in kidney during PCT. Ma-
terials and Methods: Male mice CBA/LacSto (55 animals) were intramuscularly implanted in the right hind paw by 105 cells/ml of tu-
mor RLS (a diffuse large B-cell lymphosarcoma) with multi-drug resistance (MDR) phenotype. Mice received combination of cyclo-
phosphamide (50 mg/kg), oncovin (0.1 mg/kg), hydroxydaunorubicin (4 mg/kg), and prednisone (5 mg/kg) accordingly to CHOP
scheme each 7 days after inoculation of the tumor. The kidneys were sampled on days 1, 3 and 7 after each series of injection of PCT
preparations and processed for light and electron microscopy, immunohistochemical analysis of Ki-67 and Apaf-1 proteins also was
performed. Results: Tumor RLS produced metastases comprised of small cells in the kidneys of mice after 8 days post inoculation.
Application of PCT resulted in destruction of small-cell metastases and development of many large-cell metastases in kidney. Applica-
tion of PCT induced the development of prominent damage of nephron cells, primarily in S3 segments of proximal tubules. Even one
series of PCT caused reduction of basal plasma folds in these cells and alteration of mitochondria. Damage of proximal tubules and
involvement of distal tubules, renal bodies and interstitial tissue in the pathologic process, increased during the experiment. This work
presents the description of morphological changes in kidney as well as of the tumor metastases under PCT influence. Conclusion: The
obtained data should be considered while designing of remedies for recovery of internal organs functions after antitumor PCT.
Key Words: MDR-phenotype lymphosarcoma, mice, metastasis, kidney, polychemotherapy.
Polychemotherapy (PCT) is one of the main mo-
dalities for treatment of human tumors, however high
toxicity limits its application [1, 2]. Kidneys and liver,
which are responsible for detoxification of the organ-
ism, are most affected by PCT [3, 4], and need special
treatment to improve their functions. The development
of pathological changes in these organs during PCT was
not examined; in publications we can see just recorded
facts of pathological changes in organs at the moment
of patient’s death [5, 6]. Meantime, knowledge what
is the nature of pathological changes caused by PCT
and how they progress during the courses of PCT is very
important for understanding of their mechanisms.
It is necessary to trace the effect of repeated injec-
tions of PCT preparations on the visceral organs in the
dynamics of disease. Studies of this kind could be per-
formed in appropriate animal experimental models. One
of such models is diffuse large-B-cell lymphosarcoma
called “resistant lymphosarcoma” (RLS) in CBA mice.
The tumor has multi-drug resistance (MDR) phenotype,
which is due to overexpression of genes mdrlb and bcl-
2, reduced expression of gene p53 and also to low con-
centration of ceramide in tumor cells. These features
provide “evacuation” of drugs out of the cell and also
impede initiation of apoptosis in tumor RLS [7, 8]. Such
type of MDR is observed also in humans [9, 10], what
makes this experimental model particularly valuable.
Goal of our study was to examine PCT impact
on kidneys of mice inoculated with tumor RLS and
to analyze morphology of metastases of the tumor
in this organ during PCT treatment. We used CHOP
chemotherapy scheme (cyclophosphamide, hydroxy-
daunorubicin, oncovin, prednisone), which is widely
used in clinic of lymphomas [2]. We tried to be as close
as possible to way of human treatment and applied the
same timing and doses of the preparations at mice
inoculated with tumor RLS.
MATERIALS AND METHODS
Mice. Male mice of CBA/LacSto line (further CBA)
weighing 22–25 g were kept under natural lighting
in cages (10 animals in each) and had free access
to commercial food and water. Housing and care of the
animals, as well as the experiments, corresponded
to article 11 of Helsinki declaration of second Medi-
cal Association (1964), “International guidelines for
conducting biomedical researches using animals”
(1985) and “Rules of laboratory practice in Russian
Federation”(Order of Department of Health № 267,
19.06.2003).
Tumor. RLS is a diffuse large B-cell lymphosarcoma,
which was obtained in Institute of Cytology and Genetics
of SB RAS and maintained in ascities form [11]. The tumor
Received: December 19, 2012.
*Correspondence: Fax: +7 383 363 51 53
E-mail: lenryab@yandex.ru
Abbreviations used: CHOP — cyclophosphamide, hydroxydaunoru-
bicin, oncovin, prednisone; MDR — multi-drug resistance; PCT —
polychemotherapy; RLS — resistant lymphosarcoma.
Exp Oncol 2013
35, 1, 30–36
Experimental Oncology 35, 30–36, 2013 (March) 31
RLS possesses MDR phenotype and is cross-resistant
to cytostatic preparations such as adriablastin, vinblastin,
doxorubicin and citarabin [7]. We applied experimental
protocol of lymphosarcoma modeling in mice devel-
oped earlier [4]. Briefly, tumor cells in ascities fluid were
washed by saline and diluted with saline to concentration
105 cells/ml. Mice (55 animals) received 0.2 ml of cell sus-
pension by intramuscular injection in the right hind paw.
Drugs. Cyclophosphamide (Mediafarm, Russia), Hy-
droxydaunorubicin (Mediafarm, Russia), Oncovin (Lens-
Farm, Russia) and Prednisone (Ferain, Russia) were
dissolved in sterile saline and injected at doses of 50; 4;
0.1 and 5 mg/kg, respectively. The same concentrations
are used in humans receiving PCT of lymphosarcomas
under the scheme CHOP [4].
Experimental model. Mice received standard
combination of PCT preparations under the scheme
CHOP each 7 days after inoculation of the tumor: cy-
clophosphamide, oncovin and prednisone were injected
intraperitoneally at intervals of 20–25 min. The volume
of each injection was 0.2 or 0.25 ml. The hydroxydauno-
rubicin was injected into tail vein in a volume of 0.1 ml.
Mice were sacrificed by cervical dislocation, cavities
were opened and visceral organs visually examined. The
kidneys were collected from experimental animals after
1, 3 and 7 days after each injection of PCT preparations.
Kidneys were cut into two halves along the long axis and
placed into 4% paraphormaldehyde in Hank’s balanced
solution for 24–48 h. Five intact mice also were used
to obtain “normal” kidneys for morphological study.
Histopathological and immunohistochemi-
cal studies. The samples for light microscopy were
routinely processed using Zeiss STP 120–1 (Zeiss,
Germany) machine and embedded in paraffin. Sagittal
sections (3–4 μm) of the whole kidney were prepared
and routinely stained with hematoxylin and eosin. Par-
affin sections for immunohistochemical analysis were
mounted on polylysine-coated slides (Thermo Scientific,
USA). Reactions with antibodies to proteins Ki-67 and
Apaf-1 were performed according to manufacturer’s pro-
tocols (Abcam, Great Britain). Protein Ki-67 is a nuclear
protein and serves as a marker of proliferating cells.
The protein reveals itself in late G1-phase, S-, G2- and
M-phases of cell cycle and is absent in cells, which are
in G0-phase. The protein is located in perinuclear region,
in cell nucleus, and also on chromosomes’ surface [12,
13]. Protein Apaf-1 (apoptotic peptidase activation fac-
tor 1) is an apoptosis marker and is located in cytosol.
Apaf-1 is activated by cytochrome-c and takes a part
in formation of apoptosome and in activation of cas-
pase-9 [14]. Reaction product was visualized by detec-
tion system (Novocastra, Great Britain) and AEC chro-
mogen (Sigma, USA), staining of sections was finished
with Erlich’s hematoxylin. Immunohistochemical analysis
of kidneys of mice received the treatment was carried out
on 1st and 7th days after each injection of the preparations.
Paraffin sections were examined in light micro-
scopes Zeiss Axioscope 40 with digital camera Ax-
ioCam MRc (Zeiss, Germany) and Leica DM 2500 with
digital camera Leica DFC420 C (Leica, Germany).
Electron microscopy. Fixed kidneys were sliced
into 1–2 mm thick strips, and postfixed in 1% osmium
tetroxide solution, routinely processed and embedded
into a mixture of epon-araldite (SPI, USA). The same
protocol was applied for processing of tumor RLS
cells from ascities fluid which was used for the tumor
incoculation. Semithin sections were prepared from
hard blocks and stained with Azur 2. The sections were
examined in light microscope and areas for ultrathin
sectioning were selected. Ultrathin and semithin sec-
tions were cut on ultramicrotome Ultracut-6 (Reichert-
Young, Austria), routinely contrasted by uranylacetate
and lead citrate (SPI, USA), and examined in transmis-
sion electron microscope JEM 1400 (Jeol, Japan). The
images were collected by side-mounted digital camera
Veleta (SIS, Germany).
RESULTS AND DISCUSSION
RLS tumor cells of ascitic fluid that was used for
the inoculation of mice have been examined by means
of electron microscopy. Cell population mainly com-
prised of small cells having diameter of 4–6 μm.
Remaining cells varied in size and reached 15 μm in di-
ameter. Both small and large cells had roundish shape
and rare short protrusions on the surface (Fig. 1). Endo-
cytosis vesicles were rare finding. Large nucleus of oval
or beanlike shape had high content of euchromatin and
prominent well-structured nucleoli with large granular
zone. Volume of cytoplasm and number of cell organ-
oids varied in different cells depending on their sizes.
Cytoplasm had average electron density, grainy struc-
ture and few cisterns of rough endoplasmic reticulum.
Short oval mitochondria (about 0.5 μm in length) having
matrix of average electron density and rare cristae were
accumulated near the nucleus. The Golgi complex was
small, sparse “coated” and smooth-membrane vesicles
were seen around short flattened cisterns showing
low activity of intracellular membrane transport. Mul-
tivesicular bodies and lysosomes were extremely rare
in tumor RLS cells. Many large cells contained relatively
big lipid droplets (Fig. 1, b). In general, cells of tumor
RLS in ascitic fluid showed poor signs of membrane-
associated synthesis and transport against active
production of ribosomes in large nucleoli. Probably
the metabolism in ascitic tumor cells mainly occurred
in non-membrane compartments.
Histological examination of kidneys in mice inocu-
lated with tumor RLS found metastasis foci in pelvis area
and in cortex (Fig. 2, a, b), while in kidney medulla the
metastases were not detected during whole experiment.
The observed distribution of metastases corresponded
with location of lymphosarcoma metastases in kidneys
of sick humans [6, 15]. Metastasizing lymphosarcoma
cells spread mainly by hematogenic way [16], so absence
of metastases in kidney medulla is probably related to low
blood flow in this part of the organ.
The kidneys of mice on day 8 after inoculation of the
tumor RLS showed 6–8 metastases (40–60 μm in size)
in kidney cortex per sagittal section. Number of me-
tastases in renal pelvis area was 4–6 metastases
32 Experimental Oncology 35, 30–36, 2013 (March)
per section. Sizes of these metastases varied from
50 to 100 μm after 14 days post inoculation of the
tumor RLS. All the metastases comprised of identical
cells having size 4–6 μm. These cells had roundish
nucleus edged by small ring of weakly basophilic cy-
toplasm (Fig. 2, a, b). The reaction with antibodies for
Ki-67 protein was negative showing arrest of mitotic
division of tumor cells. Reaction for Apaf-1 protein also
was negative, indicating the absence of apoptosis.
The morphology of metastases in kidney cortex and
in renal pelvis area changed after two series of PCT
injections (15 days post inoculation of the tumor). In kid-
ney cortex number of metastases consisting of small
cells decreased, and their size diminished to 20–50 μm.
Size of metastases in renal pelvis area also decreased
(to 50–70 μm), number of cell elements lessened, and
they were separated by layers of connective tissue.
Metastases located in renal pelvis area contained cells
positively stained for anti-Apaf-1 protein that indicate
apoptosis (Fig. 2, c). Reaction with antibodies for Ki-
67 protein was negative in all of the metastases that
point to suppression of tumor RLS cells proliferation.
At the same time with regression of small cell metas-
tases we found large tumor cells of 12–18 μm in size
(some of them exceed 30 μm) in kidney parenchyma.
On paraffin sections these cells had dark polymorphous
nucleus with barely discernible borders and huge ho-
mogeneous basophilic cytoplasm (Fig. 2, d).
Next time point of the experiment, 21 days post
inoculation of the tumor RLS, showed absence of small
cell metastases in mice kidneys, instead of them large
cell metastases of 50–80 μm size were observed in kid-
ney cortex. “Explosive” growth of metastases in kidney
cortex was observed in period of days 22–29 after
tumor RLS inoculation (3 and 4 series of PCT injec-
tions): their number was 16–22 per sagittal section
and size was 30–130 μm (Fig. 2, e). The metastases
in this period consisted only of large cells, which dem-
onstrated proliferative activity (positive reaction with
anti-Ki-67 antibodies) despite injections of cytostatics.
No signs of apoptosis were observed.
Electron-microscopic study of large tumor RLS cells
revealed polymorphous nuclei with matrix of average
density, small clumps of heterochromatin and promi-
nent large nucleolus (Fig. 2 e, f). The cells contained
grainy cytoplasm, long cisterns of rough endoplasmic
reticulum, numerous free ribosomes, well-developed
cisterns of Golgi complex, multivesicular bodies, elec-
tron dense lipid droplets, “coated” vesicles. Number
of mitochondria varied from a few to some tens per
cell section. The structure of large cells in kidney me-
tastases indicated active metabolic processes both
in membrane- and non-membrane compartments.
Thus, injections of cytostatic preparations under
the scheme CHOP to mice resulted in disappearance
of metastases consisting of small (4–5 μm) cells and
development of metastases formed by large tumor
cells (12–18 μm). Earlier studies showed that a sus-
pension of cells of ascitic tumor RLS contained 10%
cells with MDR phenotype [10], thereby we inoculated
mice with a mixture of 90% sensitive to PCT cells, and
10% — resistant. We propose that large cells which
appeared in kidneys during the PCT and formed me-
tastases represent a progeny of MDR phenotype cells
injected in mice. Apparently PCT serves as selective
factor for these cells which can proliferate in tumor
node and then migrate to kidneys. Presence of pro-
liferative activity in large-cell metastases illustrates
their resistance to PCT. In this way tumor RLS has
heterogeneous cellular structure and contains at least
two kinds of cells. Presence of large tumor cells, which
preserve ability to proliferate and metastasize during
PCT, supports the concept of “stem” tumor cells, ex-
istence of which was shown in glioblastoma, benign
papilloma and intestinal adenoma [17–19].
Histological structure of the kidneys in intact CBA
mice corresponded to those described for this animal
species [20, 21]. Light microscopy of kidneys on day 8 af-
ter tumor RLS inoculation in mice revealed pathological
changes which were not directly related with metastases.
The tissues neighboring with the metastases maintained
their “normal” histological structure. The injury was
a b
Fig. 1. Small (a) and large (b) cells of tumor RLS in ascities fluid of CBA mice. 1 — nucleus, 2 — lipid droplet, arrows show the
nucleoli. Ultrathin sections, transmission electron microscopy (x 40000)
Experimental Oncology 35, 30–36, 2013 (March) 33
a b
c
e
d
f g
Fig. 2. Metastases (shown with arrows) of tumor RLS in renal pelvis area (HEx40) (a) and kidney cortex (HEx100) (b) of mice re-
ceived PCT under scheme CHOP, 8 days after tumor inoculation (1 series of PCT injections). Asterisks show kidney tubules. Positive
reaction with anti-Apaf-1 antibodies (shown by arrows) in pelvis area (x40) (c) of a mouse, 21 days post inoculation of the tumor
RLS, 2 series of PCT injections. Large tumor cell (shown by arrows) in kidney tissue (HEx100) (d), 15 days post inoculation of the
tumor, 2 series of PCT injections; asterisk indicates lumen of blood vessel. Metastasis (shown by arrows) in kidney cortex (HEx40)
(e) 28 days after tumor inoculation, 3 series of PCT injections; asterisks show kidney tubules. Large tumor cells in mice kidney (f,
g), 28 days after tumor inoculation, 3 series of PCT injections. 1 — nucleus, 2 — nucleolus, arrows show endoplasmic reticulum
on fig. f, and cell borders — on fig. g. Ultrathin sections, transmission electron microscopy (x 50000, x 15000)
34 Experimental Oncology 35, 30–36, 2013 (March)
observed in proximal tubules of outer strip, where S3-
segments of the tubules are located, and involved about
5% of area in kidney sections. Proximal tubules showed
widening of lumen and cell debris in the lumens; some
foci composed of necrotic epitheliocytes and epithelio-
cytes with homogeneous bright eosinofilic cytoplasm
(2–3 cells in field of view at a 400x magnification) also
were observed (Fig. 3, a). Eosinophilic cells showed
high electron density of cytoplasm and hardly discerned
organoids in ultrathin sections. Similar eosinophilic cells
in proximal tubules of rodents were described in different
studies and are considered as a sign of necrotic changes
of the epitheliocytes [22, 23].
Electron microscopy found decrease of cytoplasm
density in proximal tubule cells in area of outer strip af-
ter a day post first injections of PCT preparations; many
of the cells showed visible reduction of folds in basal
plasmalemma, while number of apical microvilli visually
did not change (Fig. 3, b). Alteration of plasmalemma
organization indicates the impairment of ion transport
and perhaps is related with overlapping impacts of tumor
process and cytostatic preparations, particularly cyclo-
phosphamide. Cyclophosphamide and its metabolites
cause dose-dependent rise of expression of aquaporines
1 and 7 (AQP1 and AQP7) in kidney tubules of rodents
and thereby disturb water-ion transport. It is interesting
that expression of AQP7 reveals itself only in cells of S3-
segment of proximal tubules [24, 25]. Another factor
of alteration of the cells of S3-segment by various anti-
tumor preparations is high activity of glutathion-synthase
[26]. Thus, selective alteration of cells in S3-segment
of mice kidney found in this study corresponds to known
higher sensitivity of the S3-segments to damaging fac-
tors in comparison with other parts of nephron [24].
Toxic effect of PCT also touched mitochondria
of epithelium in proximal tubules which changed their
shape to roundish. Similar change of mitochondria
shape was reported after injections of cisplatin, which
has nephrotoxic effect [27]. Thereby, single applica-
tion of preparations under scheme CHOP caused
distinct damage to epithelium of proximal tubules
and first of all to cells of S3-segment in RLS-bearing
mice. First injection of PCT preparations also affected
interstitial tissue of kidneys which showed prominent
changes: swelling of collagen fibers and break of their
characteristic striation, increase of electron density
of intercellular matrix (Fig. 3, c). These changes were
present in kidney cortex during whole experiment and
were observed in all mice treated with PCT.
Morphology of renal bodies looked unaltered in this
period, some of the bodies showed widening of filtra-
tion space in comparison with that in non-damaged
areas (7.26 and 2.1 μm correspondingly) what points
to malfunction of primary urine outflow. Morphology
of medulla and distal tubules did not visually differ from
those in intact mice.
After second injection of PCT preparations (15th day
post inoculation of the tumor) pathologically changed
area of kidney cortex grew, as well as number and area
of necroses in proximal tubules. “Sludge” of eryth-
rocytes and change of blood plasma which became
electron dense were observed in blood vessels of kid-
neys signaling about alteration of organ blood supply.
Morphology of other structures of kidney remained visu-
ally unchanged. Ultrastructural examination of kidneys
showed disappearance of basal folds of plasmalemma
in cells of proximal tubules in all mice, indicating a break
of cell functions. Basal folds normally occupy large areas
in proximal tubule cells providing essential square for
water-ion transport. How can they disappear? We found
myelin-like structures which looked similar to a ball
of string on the cut in these cells (Fig. 3, d). We suppose
that these structures represent final stage of basal folds
degradation. Basal folds altered by cytostatics would
be internalized by cells for further destruction and new
portions of membrane components would be inserted
into basal plasmalemma. Absence of signs of plasma-
lemma “recovery” represents another evidence of dra-
matic alteration of epithelial cells in proximal tubules
in mice received PCT. The disappearance of basal folds
in epithelium of proximal tubules under the PCT or single
cytostatics influence was not reported yet.
Pathologic changes in nephrons grew a week after
3rd injection of PCT preparations (28th day after tumor
inoculation) and involved distal and proximal tubules
in kidney cortex. Epithelial cells of distal tubules showed
decreased electron density of cytoplasm and electron-
dense lipid droplets (Fig. 3, e). Epitheliocytes of proximal
tubules contained myelin-like structures in cytoplasm
and no basal folds. These cells significantly decreased
in the height, number of apical microvilli markedly re-
duced, and electron-dense lipid droplets accumulated
in cytoplasm (Fig. 3, b). Similar changes were described
in kidneys of rats injected with doxorubicin [28, 29].
Pathological changes in kidneys increased after four
series of injections of PCT preparations (29th day post
inoculation of the tumor). Lesion of proximal tubules
extended to about 50% of kidney section, necroses
spread to considerable part of kidney cortex. Small cavi-
ties appeared in kidney interstitial tissue looking similar
to those described in rodents after single injection
of doxorubicin [28, 29]. It should be noted that devel-
opment of pathological changes of epithelium in distal
tubules “falls behind” the damage of proximal tubules.
We observed widening of filtration space and lumens
of capillaries in renal bodies (Fig. 3, f), some capillaries
contained clumps of electron dense plasma and cell
detritus. Widening of capillary lumen can be a con-
sequence of malfunction of blood supply and of toxic
impact of doxorubicin upon endothelial functions [30].
In contrast to tubular epithelium, podocytes remained
unaltered against the background of PCT, however signs
of activation of synthetic processes in these cells were
observed. Increase of number of granular endoplasmic
reticulum cisterns and length of cisterns of Golgi complex
was seen in these cells. Number of “coated” vesicles and
multivesicular bodies grew showing activation of clathrin-
dependent endocytosis. Similar changes in kidneys
of mice were caused by single injection of doxorubicin
[29]. So, podocytes possess relative resistance to impact
Experimental Oncology 35, 30–36, 2013 (March) 35
of chemotherapeutic drugs, which caused activation
of metabolic processes in these cells.
Our study was the first attempt to visualize and
describe changes which develop in kidneys during
PCT treatment of lymphosarcoma. Toxic impact of PCT
is well known [1, 2], and we tried to trace development
of pathological changes caused by PCT in mice bear-
ing the tumor RLS. The obtained results showed that
a b
c d
e f
Fig. 3. Outer strip area of kidney (HEx100) (a) in a mouse received PCT under scheme CHOP, 8 days after tumor RLS inoculation
(1 series of PCT injections). Arrows show bright eosinofilic cells, asterisks show lumen of kidney tubules. Cells of proximal tubule
(x 20000) (b), 28 days after tumor RLS inoculation, 3 series of PCT injections. 1 — nucleus, 2 — microvilli, 3 — cytoplasm, 4 — roundish
mitochondria, arrows show electron-dense lipid droplets. Longitudinal (1) and transverse (2) sections of collagen fibers in interstitial
tissue of mouse kidney (x100000) (c), 8 days after tumor RLS inoculation, 1 series of PCT injections. Arrows show electron-dense
intercellular matrix. Apical area of proximal tubule cell (x80000) (d), 21 days after tumor inoculation, 2 series of PCT injections.
1 — microvilli, arrows and box show myelin-like structures. Cells of distal tubule (x30000) (e), 21 days after tumor RLS inoculation,
2 series of PCT injections. 1 — nucleus, 2 — cell debris in tubule’s lumen, arrows show basal folders of plasma membrane. Kidney
glomeruli (x15000) (f), 28 days after tumor RLS inoculation, 3 series of PCT injections. 1 — widened capillary, 2 — filtration space,
3 — mesangial tissue, 4 — nucleus of podocyte, 5 — cell debris, 6 — neutrophil. Ultrathin sections, transmission electron microscopy
36 Experimental Oncology 35, 30–36, 2013 (March)
PCT leads to prominent structural damage of nephron
cells, primarily in proximal tubules, which are respon-
sible for transport of water, ions, metabolites and xe-
nobiotics through epithelial barrier. Electron microcopy
demonstrated that PCT impact was primarily directed
to plasmalemma and mitochondria — the structures
responsible for water-ion transport. Pathological
changes found in mice obviously reflect mechanisms
of PCT toxic impact and should be considered while
designing of remedies for recovery of visceral organs
after PCT of oncologic diseases.
ACKNOWLEDGEMENTS
This work was supported by a grant from “OPTEC”
(Russia) company.
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