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Introduction
Tubular dilatation, vacuolation and deeply eosi-
nophilic cells, representing necrotic cells, in the
epithelium lining the proximal renal tubules are
classic features of Acute Tubular Necrosis (ATN)
[1]. Although tubular cell necrosis is not a con-
stant feature in the clinical setting of this condi-
tion, which is now termed Acute Kidney Injury
(AKI) [2], it is frequently observed in post mor-
tem examinations of children and adult patients
where it is interpreted as part of the hemody-
namic failure that culminates in death.
In our daily practice in perinatal pathology, we
frequently identify the above described changes
in non-macerated fetuses. We are not aware
that these features have been described in the
English language medical literature.
C9 is part of the membrane attack complex
(C5b-9) of the complement system and is a reli-
able marker of complement deposition which
occurs in necrotic cells of different tissues and
organs [3-6]. Therefore, immunostaining with
anti-C9 antibody is recognized as highly specific
and its use has proven to be useful in demon-
strating necrosis in different tissues, such as
acute myocardial infarction [4] and hypoxic-
ischemic brain lesions [6].
The aim of our study was to describe the occur-
rence of epithelial cell necrosis in the proximal
renal tubules of 30 consecutive autopsies of the
second trimester fetuses, confirmed with C9
immunostaining, and discuss its potential links
to ATN.
Material and methods
The reports, slides and tissue blocks of 30 con-
secutive post mortem examinations of non-
macerated fetuses performed in a 12 months
period with appropriate consent for research
were retrieved from the files. The main criterion
for inclusion was a non-macerated fetus. Induc-
tion of labour was the method used in cases of
termination of pregnancy.
Haematoxylin and Eosin (H&E)-stained slides
containing both kidneys were reviewed using a
Nikon Eclipse 80i light microscope with
Int J Clin Exp Pathol 2012;5(4):326-330
www.ijcep.com
/ISSN: 1936-2625/IJCEP1201007
Original Article
Necrotic epithelial cells in proximal renal tubules of 2
nd
trimester fetuses: is this “acute tubular necrosis”?
Luiz Cesar Peres, Chitra Sethuraman, Mudher Al-Adnani, Marta Cecilia Cohen
Department of Histopathology, Sheffield Children’s Hospital NHS Foundation Trust, Sheffield, S10 2TH,
United Kingdom
Received January 20, 2012; accepted March 30, 2012; Epub April 16, 2012; Published May 30, 2012
Abstract: The aim of this study is to describe the occurrence of necrotic tubular cells in kidneys of non-macerated
fetuses. Methods: Description of histology and immunostaining results using C9 immunostain of proximal tubular
epithelium of kidneys from 30 consecutive non-macerated fetuses’ autopsies. Results: the gestational age ranged
from 13 to 22 weeks. The mean gestational age was 18.6 weeks; the cause of death was acute chorioamnionitis in
13 cases (43.3%), termination of pregnancy for fetal anomalies in 13 (43.3%) and other causes in 4 (13.3%). Histol-
ogy of the kidneys revealed vacuolation of proximal tubule epithelial cells (100%), dilatation of tubules (93.4%) and
tubular cell necrosis (53.4%). C9 immunostaining was positive in 24 cases (80%) and was seen in all gestational
ages. Conclusions: These results indicate that tubular cell necrosis is not an uncommon finding in the kidneys of 2
nd
trimester fetuses and may represent acute tubular necrosis (ATN). C9 is a helpful marker in confirming this diagnosis.
Future studies may further explore this preliminary observation.
Keywords: Fetus, C9, autopsy, hypoxic-ischemic lesion, kidney, tubular necrosis
Tubular cell necrosis in fetuses
327 Int J Clin Exp Pathol 2012;5(4):326-330
attached Nikon DS-Fi1 digital camera. The pres-
ence or absence of the following changes in the
proximal tubules was recorded: deeply eosino-
philic epithelial cells (interpreted as necrotic
cells), vacuolation of epithelial cells and dilata-
tion of tubular lumen.
Immunostaining for C9 was performed on for-
malin-fixed, paraffin-embedded sections of rep-
resentative sections of the kidney. Sections
were cut at a thickness of 4 µm, mounted onto
coated Dako IHC Flex slides (Dako Cambridge
House, St Thomas Place, Ely, Cambridgeshire,
U.K., CB7 4EX) and stored overnight at 37
o
C.
Slides were deparaffinized, rehydrated via xy-
lene and graded alcohols, followed by heat-
induced epitope retrieval. Pre-treatment was
performed using Dako PT-Link at pH 6.0 for 20
min at 97
o
C. Endogenous peroxide was blocked
with Envision tm Peroxide-blocking reagent
(SM801) for 5 min. Primary anti-C9 antibody
was applied to the slides for 60 min at room
temperature, then incubated with Envision™
Flex
+
Mouse for 15 min. Subsequent steps were
carried out on a Dako Autostainer Link 48 im-
munostainer. Slides were incubated with anti-
C9 antibody (Abcam plc, Cambridge, U.K.) at a
dilution of 1:50 for 60 min at room temperature
using Dako Envision™ Flex
+
Plus. The tissues
were then treated with Envision™ Flex
+
mouse
linker for 15 min and subsequently with Envi-
sion™ Flex/HRP for 30 min. DAB was applied for
10 min and the slides were counterstained with
Hematoxylin for 5 min. Acute myocardial infarc-
tion was used as a positive control. The same
tissue was used as negative control by omitting
the primary antibody.
C9 was considered positive when crispy, brown
DAB pigment was identified on tubular epithelial
cells in dilated tubules.
The scores for all findings, whether histological
or immunostaining, were defined as 0= absent;
1= occasional (finding noted in a few cells and
in focal areas); 2= moderate (intermediate be-
tween occasional and diffuse); 3= diffuse
(finding in multiple cells and in multiple areas).
The study was approved by South Yorkshire Eth-
ics Committee REC reference number 09/
H1308/132 and funded by Sheffield Children’s
Hospital Charity CA090018.
Results
The mean gestational age of the 30 cases stud-
ied was 18.6 weeks, ranging from 13 to 22
weeks. The cause of death was acute chorioam-
nionitis in 13 cases (43.3%), termination of
pregnancy for fetal anomalies in 13 (43.3%)
and miscarriage due to different causes in 4
(13.3%) (2 cases of small placenta, one twin
fetus with associated hydatidiform mole in the
other placenta and one case of unknown
cause). None of the cases had any macroscopic
abnormality in the kidneys, ureters or the lower
urinary tract.
Histological evaluation revealed adequate matu-
ration for gestational age and a range of
changes summarised in Table 1. The most con-
stant finding (present in all cases) was vacuola-
tion of epithelial cells from proximal tubules
(Figure 1B), which was frequently diffuse. Wide-
spread dilatation of proximal tubules was an-
other common finding, seen in 93.4% of cases
(Figure 1B). Intense cytoplasmic eosinophilia,
indicative of cell necrosis (Figure 1C), was pre-
sent in 16 (53.4%) cases.
Immunostaining for C9 was positive in 24 cases
(80%), of moderate intensity in 5 cases (16.6%)
and occasional in 19 cases (63.3%), with no
case showing diffuse and intense positivity.
Positive C9 immunostain was most of the times
Table 1. Histological and C9 immunostaining findings in the renal tubules from fresh, non-macerated
fetuses submitted to post mortem examination.
Finding Score (%)
0 1 2 3
Vacuolation 0 7 (23.3) 4 (13.3) 19 (36.3)
Dilatation 2 (6.66) 4 (13.3) 6 (20) 18 (60)
Necrosis 14 (43.6) 6 (20) 6 (20) 4 (13.3)
C9 positivity 6 (20) 19 (63.3) 5 (16.6) 0
Scores: 0= absent; 1= occasional (finding noted in a few cells and in focal areas); 2= moderate (intermediate between occasional
and diffuse); 3= diffuse (finding in multiple cells and in multiple areas)
Tubular cell necrosis in fetuses
328 Int J Clin Exp Pathol 2012;5(4):326-330
noted on the cells which were eosinophilic on
histological examination, confirming their ne-
crotic nature, but it was also noted on vacuo-
lated cells, and therefore it was more sensitive
in identifying necrotic cells than histology. Posi-
tive C9 immunostain was noted in all gesta-
tional ages, the youngest being 13 weeks
(Figure 1D). There was no difference regarding
positivity for C9 and the cause of death among
the three groups.
Discussion
Tubular dilatation, tubular epithelial cell vacuo-
lation, representing tubular cell oedema, and
necrosis of epithelial cells are histological fea-
tures observed in ATN [1]. These features were
present in a significant proportion of our cases
and are not the result of autolysis. Although ATN
is frequently seen in post mortem examinations
of neonates, children and adults, mainly in the
context of hypoxic-ischemic conditions [7, 8], we
have been unable to identify any previous ob-
servation of the occurrence of tubular cell ne-
crosis in the non-viable human fetus. This is
mainly due to the rapid autolysis of tubular
epithelial cells after death [9]. In our study, all
cases were non-macerated in order to avoid
false positive or false negative results.
C9 immunostaining proved to be more sensitive
in the identification of necrotic tubular cells
than using H&E-stained sections alone. This is
in keeping with previous studies which demon-
strated that C9 identifies necrotic cells in acute
myocardial infarction [4], hypoxic-ischemic brain
Figure 1. All pictures correspond to kidneys of 13-week fetuses. A. fetus with no acute tubular necrosis for compari-
son. Note the small size of the tubules, narrow lumen and preserved tubular cells (H&E, x60). B to D is from a case
with acute tubular necrosis. B. Histology of kidney depicting dilated tubules and vacuolated tubular cells (H&E, x60).
C. In another area the dilated proximal tubules contain eosinophilic tubular cells indicative of necrosis (H&E, x60). D.
Immunostain with C9 of this kidney reveals strongly positive cells in the dilated tubules, confirming the necrosis (C9
immunostain, x60).
Tubular cell necrosis in fetuses
329 Int J Clin Exp Pathol 2012;5(4):326-330
lesions [6], and laminar necrosis of the placen-
tal membranes [10].
Tubular dilatation and tubular cell vacuolation
represent cell oedema and therefore are re-
versible changes, whereas tubular cell necrosis,
whether identified with histology or C9 immu-
nostain, is not and from the morphological point
of view are necessary for the diagnosis of ATN.
According to this, 80% of the cases presented
ATN, which is explained by the acute mode of
death.
One interesting finding was C9 immunostaining
positivity at all gestational ages studied. C9
positivity has been previously demonstrated in
the serum of fetuses with gestational age over
18 weeks [11] and it has been assumed that it
is not seen in very low gestational age. Our find-
ings contradict this view, as we found C9 ex-
pression even at 13 weeks (Figure 1D), demon-
strating that even in such a very low gestational
age both the mechanisms responsible for tubu-
lar cell necrosis and C9 deposition are opera-
tive.
Although we did not restrict gestational age, all
our cases were at or below 22 weeks and this
can be explained by the main inclusion criteria
of non-macerated fetuses.
It is well recognised that acute chorioamnionitis
is a trigger of spontaneous abortion in this age
group and fetuses are often non-macerated
[12]. Acute chorioamnionitis can cause ATN
through secondary fetal sepsis, which induces
the release of inflammatory cytokines, in-
creased blood vessels permeability and acido-
sis.
The processes of miscarriage and termination
of pregnancy involve the separation of the pla-
centa with resultant reduction and loss of blood
supply to the fetus, inducing hypoxic-ischemic
lesion.
Due to the widespread use of ultrasound scan
in early pregnancy, most of the major congenital
abnormalities can be detected and termination
of pregnancy is offered to the parents. Termina-
tion of pregnancy in our cases was done by in-
duction of labour and not feticide as this is the
usual procedure in this gestational age period
[13]. This explains why these fetuses are born
non-macerated and may undergo a hypoxic
event prior to death.
Hypoxia initially causes increased cardiac out-
put and heart rate to maintain effective circula-
tion. Then it leads to redistribution of the blood
flow to fetal organs. The brain, lungs and adre-
nals are the preferentially perfused organs,
whereas blood flow to the gut, spleen, kidneys
and limbs is reduced [7]. Previous studies have
indicated that irreversibility of “fetal shock” is
caused by the development of coagulation dis-
orders and fibrin deposits in fetal organs, fall in
blood pressure, fetal acidosis and increased
permeability of the blood vessels [14].
Intravascular volume contraction or decreased
effective blood volume that reduces blood flow
to the kidneys is regarded as a “pre-renal”
cause of renal failure, which is reversible when
the circulatory changes are restored to normal.
When sufficiently prolonged, pre-renal injury can
result in AKI due to hypoxic-ischemic ATN [15].
The evolution of pre-renal injury to ATN is not
sudden, and several compensatory mecha-
nisms maintain renal perfusion when renal
hemodynamics are not optimal. With increasing
intensity of renal insults, and importantly, as
they are multiplied, tubular injury develops, as
has been documented by urinary detection of
biomarkers such as cytokines (IL-6, IL-8, IL-18),
NGAL, NAG, and KIM-1 [2].
The mechanism of hypoxic/ischemic AKI may
involve disturbed vascular tone associated with
endothelin or Nitric Oxide release, depletion of
ATP, interference with the cytoskeleton and
heat shock protein [15].
In summary, tubular epithelial cell necrosis is
not an uncommon finding in post mortem ex-
amination of second trimester fetuses and its
identification and nature can be demonstrated
by C9 immunostain. This finding may indicate
the occurrence of a terminal hypoxic/ischemic
event. Further research is needed to explore
this initial observation.
Address correspondence to: L. Cesar Peres, MD, PhD,
Department of Histopathology, Sheffield Children’s
Hospital NHS Foundation Trust, Western Bank, Shef-
field, U.K., S1 2EH. Tel: (+44 114) 226-0738, Fax:
(+44 114) 271-7365, E-mail: ce-
sar.peres@sch.nhs.uk; l.cesar.peres@gmail.com
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