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The Egyptian Journal of Hospital Medicine (Oct. 2015) Vol. 61, Page 575- 590
575
Received:9/01/2015 10.12816/0018762 DOI: Accepted:9/11/2015
Histological Study and DNA Changes in the Kidneys of Rat Fetuses
Maternally Treated with Clarithromycin
Asmaa M. Kandila, Gabri M. S. b, Abdel Razik H. Farragc,
Basma N. Hassanb, Doaa Ezz-Eldin I. S.b
a Pharmacology Department, National Organization for Drug Control and Research, Giza, Egypt.
b Zoology and Entomology Department, Faculty of Science, Helwan University, Cairo, Egypt.
C Pathology Department, Medical Research Division National Research Centre, Cairo, Egypt.
ABSTRACT
Introduction: macrolide antibiotics are a class of potent and well established antimicrobials that also
possess anti-inflammatory and/or immunomodulatory properties. Because of their size, lower levels of
macrolides are able to reach the developing fetuses.
Materials and method: the pregnant rats were orally administered with clarithromycin at early and
late gestational periods. The 20 day-old fetuses were dissected for excision of the kidney. Half of the
kidney was processed and stained with H & E, PAS, Masson’s trichrome and Feulgen techniques then
followed by morphometric measurements and statistical study. The other half of the kidney was
preserved for DNA fragmentation assay.Results: This study revealed that clarithromycin
administration to pregnant rats showed different histopathological, histochemical and DNA changes in
the kidneys of their fetuses.
Conclusion: Administration of the antimicrobial agent; clarithromycin at early and late gestational
periods exhibits nephrotoxicity in the developing fetuses.
Key words: Clarithromycin, Antimicrobial drug, Rat fetuses, Kidney.
INTRODUCTION
The correct choice of an antimicrobial
agent to treat urinary tract infections during
pregnancy is complex because it requires full
attention to maternal and fetal safety[1].
Antibiotic” earlier referred to a
compound or a substance produced by a
microorganism (i.e., a bacterial or a fungal
metabolite), which inhibits the growth of other
microorganisms. Today this designation
relates to any compound, natural,
semisynthetic or synthetic which exhibits such
inhibitory effect[2] .
Antibiotics can be classified based on the
cellular component or system they affect, in
addition to whether they induce cell death
(bactericidal drugs) or merely inhibit cell
growth(bacteriostatic drugs) [3] Clarithromycin
is a semisynthetic macrolide antibiotic which
exhibits broad-spectrum activity against gram-
positive and gram-negative aerobes. It is
known to have better oral bioavailability and
tissue penetration [4] . Clarithromycin acts by
binding to the peptidyl transferase region of
23S rRNA and inhibits bacterial protein
synthesis[5] .
Macrolides are bacteriostatic antibiotics
that inhibit protein biosynthesis via reversible
binding to the bacterial 50S ribosomal subunit.
Because of their size, lower levels of
macrolides are able to reach the fetus [6]. The
antibacterial spectrum of macrolides includes
predominantly gram-positive cocci, but also
chlamydia, mycoplasma and legionella,
campylobacter as well as coxiella, bartonella,
corynebacteria and several mycobacterium
species. Macrolides are suitable alternatives
for patients who are allergic to penicillin[7] .
MATERIALS AND METHODS:
1- Drug and dosage:
Clarithromycin is an antibacterial
agent, C38H69NO13, provided by Kahira
Pharmaceuticals and Chemical, Egypt, in
a commercial product called Klacid XL
as 14 film coated tablets. Each tablet
contains 500 mg of clarithromycin. The
drug was orally given once daily to
pregnant rats at different periods of
gestation by gastric tube at a dose of 45
mg/kg/day. The dose was equivalent to
therapeutic dose of human (500 mg/day)
and calculated according to interspecies
dosage conversion scheme of Paget and
Barnes[8]. The tablet was crushed to
powder and was dissolved in distilled
water. The given volume was adjusted so
that each 100g of the animal body weight
received 1ml solution containing the
required dose.
2- Experimental animals:
The present experimental study was
carried out on the adult female and male
Histological Study and DNA Changes in the Kidneys…
576
albino rats (Rattus norvegicus); weighing 140–
200 grams and they were obtained from the
National Organization for Drug Control and
Research (NODCAR, Cairo, Egypt). Male and
female rats were housed separately in metal
cages with wire-grid floors. The animals were
kept at standard housing facilities (25±2 ºC, 45
± 5% humidity and 12 hrs. light/dark cycles).
The animals were fed on a standard laboratory
chow and water ad libitum. The standard
guidelines of National Organization for Drug
Control and Research (NODCAR) were used
in handling animals.
3- Housing and mating:
Adult female rats in proestrus cycle were
caged overnight with males of proven fertility.
The day on which the sperm were found in the
vagina was designated as zero day of gestation
(GD) (Fig.1). A daily record of the weight of
the pregnant females was made. The abortion
was determined by the presence of blood
drops and sudden drop in weight of the
pregnant females[9] .
4- Experimental design:
The pregnant females were randomly
divided into four equal groups (n= 6, each) as
follow:
Group C1f : The pregnant rats were orally
received distilled water from the 1st to the 7th
day of gestation.
Group T1f : The pregnant rats were orally
administered with clarithromycin at a dose of
45 mg/kg/day from the 1st to the 7th day of
gestation.
Group C2f : The pregnant rats were orally
received distilled water from the 15th to the
19th day of gestation.
Group T2f : The pregnant rats were orally
administered with clarithromycin (45
mg/kg/day) from the 15th to the 19th day of
gestation.
5- Tissues sampling:
At 20th day of gestation, the pregnant
females were sacrificed by decapitation, the
peritoneal cavity was opened and fetuses were
separated. Kidneys of the fetuses were rapidly
excised then, half of them stored at -80°C for
isolation the genomic DNA and the other half
fixed immediately for the histopathological,
histochemical and histomorphometrical
investigations.
For histological and histochemical
examination:
The excised organ was fixed in 10%
neutral buffered formalin solution for about 24
hours, washed in running water, dehydrated in
ascending grades of ethyl alcohol, cleared in
xylene and impregnated in parablast for
blocking, serial sections of 5 μm thickness
were prepared and stained with: hematoxylin
and eosin, periodic acid schiff's, Masson’s
trichrome and Feulgen reaction.
DNA fragmentation assay:
DNA fragmentation determined via
agarose gel electrophoresis; genomic DNA,
was isolated from the fetuses' kidney tissue
according to Miller et al. [10] using DNA
Purification Kite Promega, Promega
Corporation, USA. Agarose gel
electrophoresis of DNA was done according to
the method of Sealey and Southern [11] .
Morphometric measurements:
Histomorphometric measurements were
performed using Leica Qwin 500 Image
Analyzer (LEICA Imaging systems Ltd,
Cambridge, London) in Pathology
Department, National Research Centre, Cairo.
The measurements included the glomerular
diameter in H&E stained sections and nuclear
area of the convoluted tubular cells in Feulgen
stained sections, the values were measured in
fifty randomly parameters per group at
magnification X 200 and X 800, respectively.
The mean grey of carbohydrate content and
DNA content and the mean area percent of
collagen content slides were measured within
10 non-overlapping fields/ section for each
animal, at X400 magnification.
Statistical analysis:
The morphometric results were expressed
as mean ± standard errors (SE). Statistical
analysis was carried out using the “prism
version 5” statistical software. Comparison
between different groups was done using one
way analysis of variance (ANOVA) followed
by Tukey test[12] . The results were considered
statistically significant when value was P<
0.05.
RESULTS
The kidney of 20-day old fetuses:
I. Histopathological investigations:
I.1. H&E stain:
a- Control fetuses:
Normal renal architecture of kidney
cortex of control fetuses showed: renal
corpuscles made up of glomeruli, Bowman’s
capsules and urinary spaces, proximal and
distal convoluted tubules (Fig. 2).
Asmaa M. Kandil et al
577
b-Treated fetuses:
Sections in the kidneys of 20-day old
fetuses maternally treated with clarithromycin
(group T1f) showed some histopathological
changes included enlarged glomeruli, some
necrotic tubules and other tubules with some
pyknotic nuclei ,some cytoplasmic
vacuolations was also seen (Fig. 3). Some
convoluted tubules showed cloudy swelling of
their lining cells with complete obstruction of
their lumen (Fig. 4). Congested blood
capillaries of the glomerular tufts and
hemorrhage in the interstitial space were
shown (Fig. 5).
Sections in the kidneys of 20-day old
fetuses maternally treated with clarithromycin
(group T2f) showed enlarged glomeruli and
degenerative changes in the epithelial cells
lining the renal tubules with cloudy swelling,
vacuolations, pyknosis, karyolysis and cells
detachment (Fig. 6). In addition, hemorrhagic
areas in the interstitial space and congestion
of blood capillaries of the glomerular tufts
were reported in the kidney of clarithromysin-
treated fetuses (Fig. 7).
I.2. Masson's trichrome stain:
a. Control fetuses:
Examination of sections in the renal
cortex of 20 day-old fetuses yielded from
control rats stained with Masson's trichrome
showed scanty collagen fibers in the capsular
wall, peritubular and intraglomerular
capillaries (Fig. 8).
b. Treated fetuses:
Sections in the kidneys of 20-day old
fetuses obtained from cleithromycin-treated
dams (group T1f) showed moderate increase in
the collagenous fibers in glomerular
capillaries, around the capsular wall and in the
interstitial tissues (Fig. 9).
Masson’s trichrome-stained sections in
the kidneys of 20-day old fetuses obtained
from clarithromycin-treated dams (group T2f)
showed severe increment in the collagenous
fibers in the tubulointerstitial tissues (Fig. 10).
II. Histochemical results:
II.1. Carbohydrate Content:
a. Control fetuses:
Examination of sections in the renal
cortex of 20 day-old fetuses yielded from the
control group and stained with PAS reaction
shows a positive PAS reaction in the brush
borders of the proximal convoluted tubules,
Bowman's capsule and the basement
membranes of the glomerular capillaries.
However, the tubular basement membranes as
well as the nuclei show PAS negative reaction
(Fig. 11).
b. Treated fetuses:
Section of the kidney of 20-day old
fetuses maternally treated with clarithromycin
(group T1f) showed a weak PAS reaction in
the capsular wall. Destructed brush borders of
the proximal convoluted tubules were shown
(Fig. 12).
Section of kidney of 20-day old treated
fetuses (group T2f) displayed weak PAS
reactivity of the brush borders of the tubular
cells(Fig. 13).
II.2. DNA Content:
a. Control fetuses:
The sections of kidneys of 20-day old
fetuses yielded from control group (Fig. 14)
showed normal distribution of DNA
molecules. Most of the nuclei are in the same
size and strongly stained magenta by using
Feulgen reaction. The cytoplasm of these cells
showed a negative staining reaction.
b. Treated fetuses:
DNA of sections of the kidneys of 20-
day old fetuses maternally treated with
clarithromycin (group T1f,) showed a weak
stain reaction in the karyolytic nuclei of
tubular cells. In addition, numerous pyknotic
nuclei were reported (Fig. 15).
The sections of kidneys of 20-day old
treated fetuses (group T2f ) showed a marked
decrease in DNA content in the karyolytic
nuclei. Moreover, numerous pyknotic nuclei
were densely stained (Fig. 16).
III. Histomorphometric analyses:
The renal sections of 20-day old fetuses
of different groups stained with Masson's
trichrome were subjected to image analysis for
determination of mean area percent of
collagen content. Moreover, renal sections
stained with PAS and Feulgen were subjected
to image analysis for determination of mean
grey of carbohydrate content and DNA
content, respectively. All data were
represented in Table 1 and Figures 17, 18 and
19. Sections of the kidneys of 20-day old
fetuses maternally treated with clarithromycin
(group T1f) showed a significant increase at
P<0.05 in mean area % of collagen contents
and the mean grey of carbohydrate content and
DNA content as compared to control group
(group C1f) (87.86, 13.78 and 4.3,
respectively).
Histological Study and DNA Changes in the Kidneys…
578
A significant increase P< 0.05 was
shown in the mean area % of collagen
contents and the mean grey of carbohydrate
content and DNA content in the kidneys of 20-
day old treated fetuses (group T2f) comparing
to its control (group C2f) (121.3, 14.43 and
6.22, respectively).
The kidney sections of 20-day old
fetuses of different groups were subjected to
histomorphometric measurements of the
glomerular diameter and the convoluted
tubules nuclear area. These data were
represented in Table 2 and Figures 20 and 21.
Sections of the kidneys of 20-day old
treated fetuses (group T1f) showed a non-
significant change of glomerular diameter and
significant decrease at P<0.05 in mean values
of nuclear area of CT, as compared to control
group (group C1f) and were recorded as -2.4
and -18.2 as a percentage change respectively.
A non-significant change in the mean
values of glomerular diameter (1.5) and a
significant decrease in the mean values of the
nuclear area of CT -18.6 were reported in the
kidneys of 20-day old fetuses maternally
treated with clarithromycin (group T2f) as
compared to the control group (group C2f) at
P<0.05.
IV. Molecular investigations:
IV.1. Determination of DNA purity:
The total genomic DNA isolated from
kidneys of 20-day old fetuses was evaluated
for purity and the results were summarized in
Table (3) and they showed the absence of
contamination in all samples.
IV.2- Agarose gel electrophoresis of DNA:
As shown in Figure (22), the renal
cells of control 20-day old fetuses showed
that, no smear (lane 2) was observed and the
fragmentation of the DNA remained negligible
while, DNA of the treated groups showed
some degree of DNA damage of smear
pattern.
The kidneys of 20-day old fetuses
maternally treated with clarithromycin (group
T1f) showed a moderate DNA fragmentation
(lane 3). Moreover, the kidneys of the other
treated group (T2f ) showed a strong DNA
damage of smear pattern (lane 4) compared to
the control group.
DISCUSSION
The kidney plays an important role in
the elimination of numerous hydrophilic
xenobiotics, including drugs, toxins, and
endogenous compounds [13].
Rat is commonly used as a model in
studies on embryology and reproduction
toxicology [14]. In the present study, the
histopathological examination showed that the
oral administration of the therapeutic dose of
clarithromycin extended release tablets once
daily in the different periods during pregnancy
in rats induced a nephrotoxicity in the
developing fetuses. This is in agreement with
the results of Chapelsky et al.[15] who found
that usual doses of clarithromycin (500 mg
orally every 12h for 13 doses) had significant
potential for causing nephrotoxicity and also,
Guay et al.[16] reported that clarithromycin had
low potential for ototoxicity, hepatotoxicity
and nephrotoxicity in studies involving rats,
dogs and primates.
The nephrotoxicity of clarithromycin
might be happened due to the induction of the
oxidative stress in the renal tissue. Olayinka
and Ore [15][17] found that oral administration
of clarithromycin has adverse effects on both
enzymic and non-enzymic antioxidant status
and induced oxidative stress, the macrolides
were extensively distributed in tissues and
obtain higher concentrations than
erythromycin in the bronchus, tonsils,
gastrointestinal tract, liver, kidney, spleen, and
bone [18], and also 30 to 40% of an oral dose of
clarithromycin was excreted in the urine via
glomerular filtration of the kidney [19] . Also
the sensitivity for toxic compounds is
increased during pregnancy [20]. Pregnancy
significantly alters the expression and activity
of drug metabolizing enzymes and these
changes are likely to have toxicological and
therapeutic implications [21].
Moreover, the macrolides accumulate in
the lysosomes of eucaryotic cells [22],
[23],causing metabolic alterations that can lead
to cell toxicity [24].
Clarithromycin is insoluble in water [25]
and as the passage of pharmacologic agents
across the placenta is influenced by solubility
as lipid soluble agents readily cross the
placenta, but water soluble compounds are less
readily transported. Drug exposure is
responsible for 2% of birth defects and is a
potentially avoidable risk [26].
Clarithromycin, a pregnancy category C
drug, has a higher placental passage rate than
other macrolide antibiotics and the mean
trans-placental transfer of clarithromycin was
Asmaa M. Kandil et al
579
6.1 % [27], thus causing nephrotoxicity to the
fetuses. Furthermore, during kidney
development, a large-scale proliferation has
been observed [28]. So, the renal development
is influenced by any insult disturbing the fine
balance in the interactions that form the
kidney [29].
Oxidative stress is a common
pathogenetic mechanism contributing to
initiation and progression of cell damage [30].
The oxidative stress refers to a cell’s state
characterized by excessive production of
reactive oxygen species (ROS) and/or reactive
nitrogen species (RNS) and/or a reduction in
antioxidant defenses responsible for their
metabolism [31], [32].
Damage induced by ROS includes
alterations of cellular macromolecules such as
membrane lipid, DNA, and/or protein. The
damage may alter cell function through
changes in intracellular calcium or
intracellular pH, and eventually can lead to
cell death [33], [34].
Premature neonates have an immature
antioxidant defense system and therefore are
highly susceptible to the deleterious effects of
reactive oxygen species (ROS) [35]. Tsunoda et
al. [36] demonstrated that oxidative stress
influences multiple physiological processes,
from oocyte maturation to fertilization and
embryo development. Increase in ROS is also
involved in defective embryo development
and retardation of embryo growth [37], which is
attributed to induced cell-membrane damage
and DNA damage [38].
Most drugs found to cause
nephrotoxicity and exert toxic effects by one
or more common pathogenic mechanisms.
These include altered intraglomerular
hemodynamics, tubular cell toxicity,
inflammation, crystal nephropathy,
rhabdomyolysis, and thrombotic microan-
giopathy [39]. The histopathological
examination of kidney of 20-day old fetuses
maternally treated with 45 mg/kg of
clarithromycin in present study showed that
clarithromycin induced nephrotoxicity as
manifested by the appearance of enlarged or
shrunken glomeruli with a wide urinary space
and some necrotic tubules with high
vacuolations, cells detachment, pyknotic and
karyolytic nuclei. Some convoluted tubules
showed cloudy swelling of their lining cells
with complete obstruction of their lumen.
Congested blood capillaries of the glomerular
tufts and hemorrhage in the interstitial space
were shown also. Similar results were
introduced by Lameire and Vanholder [40]
who reported that the histopathological
examination of kidneys suffering from
toxicological insult, the major changes appear
to be localized in the renal tubules, including
the degeneration or necrosis of tubular cells,
swelling of the epithelium and the detachment
of tubular epithelial cells from the basement
membrane. Renal tubular cells, in particular
proximal tubule cells, are vulnerable to the
toxic effects of drugs because their role in
concentrating and reabsorbing glomerular
filtrate exposes them to high levels of
circulating toxins [41]. Drugs that cause tubular
cell toxicity do so by impairing mitochondrial
function, interfering with tubular transport,
increasing oxidative stress, or forming free
radicals [42].
A significant increase in the mean value
of glomerular diameter of the kidneys of the
clarithromycin-treated fetuses at P<0.05 in
comparison with the control group was
showed in the present study. Also,
clarithromycin treatment induced
inflammatory infiltration in the interstitium
and this result does not agree with the result of
Özdemir et al. [43] who stated that
clarithromycin had a protective effect on
bowel injury owing to anti-inflammatory
effects. Tubulointerstitial inflammation may
result from leakage of filtered urine to the
interstitial space through capsular adhesions
[44]. The works of Kriz and LeHir [45] have
exquisitely shown the formation of bridges
between podocytes and the Bowman’s capsule
in sites of capsular adhesions and a remaining
narrow urinary space allows leakage of
ultrafiltrate to tubulointerstitial areas.
According to Cotran et al. [46] inflammation is
fundamentally a protective response whose
ultimate goal is to rid the organism of both the
initial cause of cell injury and the
consequences of such injury, the necrotic cells
and tissues. Inflammation serves destroy,
dilute, or wall off the injury agent.
In this study, the mean values of lumen
area of the convoluted tubules of the kidneys
of the clarithromycin-treated fetuses showed
significant decrease (P<0.05) in comparison
with the control group. This result appeared in
H&E stain as cloudy swelling of their lining
cells with small or complete obstruction of
their lumen. The morphology of necrosis is
Histological Study and DNA Changes in the Kidneys…
580
characterized by swelling and degeneration of
the entire cytoplasm [47]. de Wardener [48]
described the cloudy swelling mechanism and
stated that many disease processes may directly
inhibit the sodium pump of the cell. Sodium then
accumulates within the cell and subsequently the
water is drown into the cell causing this cloudy
swelling [49].
Vacuolations and deeply eosinophilic
cells in the epithelium lining the renal tubules
were seen in this work. Vacuolation and
deeply eosinophilic cells, representing necrotic
cells, in the epithelium lining the proximal
renal tubules are classic features of acute
tubular necrosis [50], [51]. Necrosis may occur as
a direct adverse effect of a metabolite or
xenobiotic on the tubules [52]. Necrotic cell
death begins with swelling of the cell and
mitochondrial contents, followed by rupture of
the cell membrane [53]. Necrosis can trigger an
inflammatory reaction in the surrounding
tissue as a result of the release of cytoplasmic
contents, many of which are proteolytic
enzymes [54].
Vacuolation is recognized as clear,
round spaces of variable size within the
cytoplasm. Clear vacuolation of variable
dimentions suggests hydropic change.
Smaller, more uniform translucent vacuoles
suggest fat or lipoprotein accumulation [55].
Hemorrhage in the interstitium was
shown in the present study in the kidneys of
clarithromycin-treated fetuses. This result
adrees with those of Olayinka and Ore [56] who
reported that foci of haemorrhages in the renal
cortex were observed after administration of
17.6 mg kg-1 clarithromycin. Hemorrhage
often accompanies acute injury and can occur
in the kidney as a primary lesion associated
with nephrotoxicants. Hemorrhage can occur
from inflammation, tubular necrosis, and
vascular injury or from the presence of calculi
or tumors [55].
Using Masson’s trichrome stain,
sections of the kidneys of the treated fetuses
showed fibrosis in glomeruli accompanied by
areas of tubulointerstitial fibrosis and around
the capsular wall. So, there is a significant
increase at P< 0.05 in the mean area % of
collagen contents in the kidneys of the treated
fetuses as compared to the control group.
Tubulointerstitial fibrosis is a common
feature of progressive renal injury in almost all
forms of renal diseases [57]. It has been shown
that tubulointerstitial injury is a more
consistent predictor of functional impairment
than glomerular damage [58].
The cortical interstitium is normally
composed of a network of fibroblasts and
dendritic cells with only a small number of
lymphocytes or macrophages [59]. In chronic
interstitial fibrosis, the interstitial cells have
myofibroblast characteristics which are
considered to be derived from the proliferation
and differentiation of the residual fibroblasts
[60]. In the present study clarithromycin
treatment induced glomerular degenerative
changes. Once hemodynamic or degenerative
changes in the glomerulus are initiated, a
complex sequence of events within the
mesangium and podocytes are initiated,
mediated by TGF-beta and CTGF, which
result in the stimulation of fibroblast
proliferation and collagen formation with the
eventual replacement of normal architecture
[45], [61].Oxygen and nitrogen free radicals are
highly reactive and are capable of damaging
macromolecules like carbohydrates [62]. So,
there was a weak PAS reaction in the
Bowman's capsular wall. Also, there were
destruction and thinning of brush border of the
proximal tubules. Lameire and Vanholder [40]
had reported that loss of the brush border was
appeared in kidneys suffering from
toxicological insult. A significant increase of
the mean grey of carbohydrate content at P<
0.05 was shown in renal sections of dams
treated with 45 mg/kg clarithromycin orally as
compared to control which confirmed the
decrement of polysaccharides content.
In this study, necrosis was assessed in
the kidneys of the treated fetuses with the
appearance of pyknosis or karyolysis. These
results were confirmed with measuring the
mean values of nuclear area of the convoluted
tubules that showed a significant decrease
(P<0.05) in comparison with the control
group. Also, the karyolysis was indicated by
the significant increase at P<0.05 in the mean
grey of DNA content as compared to control
group. The genomic DNA from renal cells of
20 day-old fetuses maternally treated with 45
mg/kg of clarithromycin showed a degree of
DNA damage of smear pattern which indicates
to the presence of necrosis. The degradation of
DNA due to the increased ROS [63] and RNS
(e.g. Peroxynitrite) which interact with cellular
macromolecules such as DNA and causes
chemical cleavage of DNA [64].
Asmaa M. Kandil et al
581
Pyknosis, or shrunken nuclei, is the
irreversible condensation of chromatin in the
nucleus of a cell undergoing necrosis [65], [66] or
apoptosis [67]. It is followed by karyorrhexis
which is the destructive fragmentation of the
nucleus of a dying cell [68] involving loss of
integrity of the nucleus. Karyolysis is the
complete dissolution of the chromatin of a
dying cell due to the enzymatic degradation by
endonucleases. The whole cell will eventually
stain uniformly with eosin after karyolysis in
which a Feulgen reaction was faint, ghost-like
image of the nuclei remained [66]. It is usually
preceded by karyorrhexis and occurs mainly
as a result of necrosis, while in apoptosis after
karyorrhexis the nucleus usually dissolves into
apoptotic bodies [69], [70]. Finally, the above-
stated findings lead to the conclusion that the
presence of the histopathological changes
revealed nephrotoxicity in the fetuses
maternally treated with clarithromycin. So,
clarithromycin is not safe for pregnant
mothers and their fetuses and it should not be
used except under strict conditions in
medication.
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Fig. 1: Estrus stage of vaginal smear of rat with spermatozoa.
Fig. 2: Photomicrograph of a section in the cortex of kidney of 20 day-old fetus yielded
from a control dam (C1f) showing spherical renal corpuscles with evident Bowman’s spaces
(asterisk), capillary tufts "glomerulus" (G) and Bowman’s capsule (white arrow). Normal
histological structures of proximal (yellow arrow) and distal (black arrow) convoluted
tubules are recorded.(H&E, ×400).
Fig. 3: Photomicrograph of a section in the cortex of kidney of 20 day-old fetus yielded
from a clarithromycin-treated dam (T1f) showing enlarged glomerulus (G) and some
necrotic tubules with high vacuolations (white arrow) and pyknotic nuclei (yellow arrow).
(H&E, ×400).
Asmaa M. Kandil et al
585
Fig. 4: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T1f) showing cloudy swelling of convoluted tubule cells with
complete obstruction of their lumen in some tubules (arrow). (H&E, ×400)
Fig. 5: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a clarithromycin-
treated dam (T1f) showing congested blood capillaries of the glomerular tufts (white arrow) and
hemorrhage in the interstitial space (yellow arrow). (H&E ×400).
Fig. 6: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T2f) showing enlarged glomerulus (G) and degenerative
changes in the epithelial cells lining the renal tubules with cloudy swelling (black thick
arrow), vacuolations (black thin arrow), pyknosis (yellow arrow), karyolysis (white arrow)
and cell detachment (blue arrow). (H&E, ×400).
Fig. 7: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T2f) showing congested blood capillaries of the glomerular
tufts (white arrow) and hemorrhage in the interstitial space (yellow arrow).(H&E, ×400).
Histological Study and DNA Changes in the Kidneys…
586
Fig. 8: Photomicrograph of a section in the cortex of the kidney of 20 day-old fetus yielded
from a control dam (C1f) showing a scanty collagen fibers around capsular wall (white
arrow), intraglomerular capillaries (yellow arrow) and in the interstitial tissues (blue arrow).
(MT, ×400)
Fig. 9: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T1f) showing a moderate increase of the collagen fibers
distributed in glomerular capillaries (yellow arrow), around the capsular wall (white arrow)
and in the interstitial tissues (blue arrow). (MT, ×400)
Fig. 10: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T2f) showing a severe increment of collagenous fibers in the
tubulointerstitial tissues (blue arrow). (MT, ×400)
Asmaa M. Kandil et al
587
Fig. 11: Photomicrograph of a section in the cortex of kidney of 20 day-old fetus yielded from a
control dam (C1f) showing positive PAS reaction in the brush borders (white arrow) of the proximal
convoluted tubules, Bowman's capsule (yellow arrow) and the glomerular capillaries (blue arrow). The
tubular basement membranes show a negative reaction (black thick arrow). (PAS & H ×400)
Fig. 12: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T1f) showing a weak PAS reaction in the capsular wall (white arrow)and
in the brush borders (blue arrow) are shown. (PAS & H, ×400
Fig. 13: Photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T2f) showing a weak PAS reaction in the brush borders
(yellow arrow) of the proximal convoluted tubules.(PAS & H, ×400)
Histological Study and DNA Changes in the Kidneys…
588
Fig. 14: Photomicrograph of a section in the cortex of kidney of 20 day-old fetus yielded from a control
dam (C1f) showing the normal distribution of DNA content in the nuclei of renal tubules. (F, ×1000)
Fig. 15: A photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T1f)showing a pyknotic (white arrow) and karyolytic nuclei
(yellow arrow). (F, ×1000)
Fig. 16: A photomicrograph of a section in the kidney of 20 day-old fetus yielded from a
clarithromycin-treated dam (T2f) displaying intensive stain in the pyknotic (white arrow)
and weak stain in the karyolytic nuclei (yellow arrow). (F, ×1000)
Asmaa M. Kandil et al
589
.
.
Fig. 18: Histogram showing the
mean grey of PAS +ve reaction
in the kidney of 20-day old fetuses
of different groups.
Fig. 19: Histogram showing the mean
grey of Feulgen +ve reaction in the kidney
of 20-day old fetuses of different groups.
Fig. 17: Histogram showing the
mean area % of collagen content in
the kidney of 20-day old fetuses of
different groups
Fig. 20: Histogram showing glomerular
diameter in the kidney of 20-day old
fetuses of different groups.
groups.
Fig. 21: Histogram showing the nuclear
area of CT in the kidney of 20-day old
fetuses of different groups.
Fig. 22: Agarose gel electrophoresis of
genomic DNA isolated from kidney of
20-day old fetuses of different groups
Histological Study and DNA Changes in the Kidneys…
590
Table (1): Image analysis of mean area % of collagen content, and the mean grey of carbohydrate
content and DNA content of kidneys of 20-day old fetuses of different groups.
Parameters
Groups
Collagen content
PAS +ve Rx
Feulgen +ve Rx
Group C1f
4.499±0.6485
158.1±4.088
179.1 ± 0.6843
Group T1f
8.452±1.296*
(87.86)
179.9 ±4.596**
(13.78)
186.8 ± 1.014***
(4.3)
Group C2f
4.912±0.8863
159.4±3.993
178.6±0.8724
Group T2f
10.87±0.7522**
(121.3)
182.4 ±3.532**
(14.43)
189.7 ± 0.8284***
(6.22)
The number of animals were 6 in each group. Data are expressed as mean ± SE.
*: Significant change at p < 0.05 with respect to corresponding control group (Group C1f or C2f).
( ): % difference with respect to control value.
Table (2): Histomorphometrical measurements of the glomerular diameter, and the nuclear area of
convoluted tubules (CT) in kidneys of 20-day old fetuses of different groups.
Parameters
Groups
Glomerular diameter
CT nuclear area
Group C1f
31.55± 0.71
100.5± 3.71
Group T1f
30.78± 0.77
(-2.4)
82.22± 3.84**
(-18.2)
Group C2f
29.46±0.63
99.99±3.721
Group T2f
29.89± 0.62
(1.5)
81.38± 3.464**
(-18.6)
The number of animals were 6 in each group. Data are expressed as mean ± SE.
*: Significant change at P < 0.05 with respect to corresponding control group (Group C1f or C2f).
( ): % difference with respect to control value.
Table (3): The purity of total genomic DNA isolated from kidneys of 20-day old fetuses of different
groups.
parameters
Groups
A260
A280
DNA Purity
(A260/A280)
Group C
0.17
0.185
1.8
Group T1f
0.22
0.11
2
Group T2f
0.13
0.07
1.83
