Microbial Pathogenesis 186 (2024) 106482

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Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Progression of renal damage and tubular regeneration in pregnant and

non-pregnant adult female rats inoculated with a sublethal dose of Shiga
toxin 2
Lilian K. Fischer Sigel a, b, Daiana S. Sánchez a, b, Flavia Sacerdoti b, c, Elsa Zotta b, c, d,
Claudia Silberstein a, b, *
a
Universidad de Buenos Aires (UBA), Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas, Laboratorio de Fisiología Renal, Buenos Aires, Argentina
b
Universidad de Buenos Aires – Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET), Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO
Houssay), Buenos Aires, Argentina
c
Universidad de Buenos Aires, Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas, Laboratorio de Fisiopatogenia, Buenos Aires, Argentina
d
Universidad de Buenos Aires, Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas. Laboratorio de Patología, and Facultad de Farmacia y Bioquímica,
Cátedra de Fisiopatología, Buenos Aires, Argentina

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Shiga toxin-producing Escherichia coli is the main cause of post-diarrheal hemolytic uremic syn­
Shiga toxin type 2 drome (HUS) which produces acute kidney injury mainly in children, although it can also affect adults. The
Sublethal hemolytic uremic syndrome rat kidneys are the organs most affected by Shiga toxin type 2 (Stx2) in patients with HUS. However, previous
model
studies in pregnant rats showed that a sublethal dose of Stx2 causes severe damage in the uteroplacental unit and
Pregnancy
Renal injury
induces abortion, whereas produces mild to moderate renal damage. The aim of the present work was to study
Renal tubular regeneration the progression of renal injury caused by a sublethal dose of Stx2, as well as renal recovery, in pregnant and non-
pregnant rats, and to investigate whether pregnancy physiology may affect renal damage progression mediated
by Stx2.
Methods: Renal function and histopathology was evaluated in pregnant rats intraperitoneally injected with a
sublethal dose of Stx2 (0.5 ng/g bwt) at the early stage of gestation (day 8 of gestation), and results in these rats
were compared over time with those observed in non-pregnant female rats injected with the same Stx2 dose.
Hence, progression of cell proliferation and dedifferentiation in renal tubular epithelia was also investigated.
Results: The sublethal dose of Stx2 induced abortion in pregnant rats as well as a significant more extended
functional and histological renal injury in non-pregnant rats than in pregnant rats. Stx2 also caused decreased
ability to concentrate urine in non-pregnant rats compared to their controls. However, renal water handling in
pregnant rats was not altered by Stx2, and was significantly different than in non-pregnant rats. The greatest
renal injury in both pregnant and non-pregnant rats was observed at 4 days post-Stx2 injection, and coincided
with a significant increase in tubular epithelial proliferation. Expression of mesenchymal marker vimentin in
tubular epithelia was consistent with the level of tubular damage, being higher in non-pregnant rats than in
pregnant rats. Recovery from Stx2-induced kidney injury was faster in pregnant rats than in non-pregnant rats.
Conclusions: Adaptive mechanisms developed during pregnancy such as changes in water handle and renal he­
modynamic may contribute to lessen the Stx2-induced renal injury, perhaps at the expense of fetal loss.

1. Introduction characterized by hemolytic anemia, thrombocytopenia, and acute kid­

ney injury [1,2]. STEC-induced HUS mostly affects children, mainly
Shiga toxin (Stx)-producing Escherichia coli (STEC) is the main cause under 5 years, in many parts of the world with a case mortality <4 % [3].
of post-diarrheal hemolytic uremic syndrome (HUS), which is Approximately 30 % of the affected children who survive the disease are

* Corresponding author. Universidad de Buenos Aires (UBA), Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas, and UBA-CONICET IFIBIO-
Houssay, Laboratorio de Fisiología Renal. Paraguay 2155, piso 4, Buenos Aires Argentina.
E-mail address: csilber@fmed.uba.ar (C. Silberstein).

https://doi.org/10.1016/j.micpath.2023.106482
Received 8 September 2023; Received in revised form 13 November 2023; Accepted 29 November 2023
Available online 10 December 2023
0882-4010/© 2023 Elsevier Ltd. All rights reserved.
L.K. Fischer Sigel et al.
at risk of developing chronic renal injury and long-term renal sequels,
which can lead for the requirement of a renal transplant [4–6]. The
long-term prognosis in patients with HUS has been associated with the
severity of renal damage during the acute phase [4,7]. Supportive care is
applied to reduce mortality in patients with HUS since there is no spe­
cific therapy for this disease [5]. STEC infections not only affects chil­
dren but also adults, as occurred during the outbreak of STEC-HUS in
Germany, in 2011, in which the majority of affected patients were
adults, and women were overrepresented [8]. Moreover, several cases of
neonatal HUS caused by STEC transmission from mother to the newborn
during delivery, as well as cases of STEC-mediated HUS during preg­
nancy, were reported [9–13]. Nevertheless, so far, there are no reports
about Stx effects during human pregnancy or described pregnancy
complications associated with STEC infection.
Shiga toxin type 1 and 2 (Stx1 and Stx2, respectively) and/or their
subtypes (Stx1a, Stx1c, Stx2a, Stx2b, Stx2c, Stx2d and Stx2f) are the
main virulent factors produced by STEC, being Stx2a-producing STEC
the most virulent strains that cause HUS [3,14]. The toxin is composed
of an A subunit and a pentamer of B subunits, which binds to the
glycolipid globotriaosylceramide (Gb3) located on the plasma mem­
brane of target cells [15,16]. Stx2 is endocytosed and retrograde
transported to the endoplasmic reticulum [17,18]. The internalized Stx
causes cell death by inducing protein synthesis inhibition and apoptosis
[19,20].
The kidneys are the organs most affected by Stx2 in patients with
HUS associated with diarrhea due to the high expression of Gb3 receptor
in the surface of human renal cells [21–24]. We have previously
demonstrated the cytotoxic action of Stx2 in human renal tubular
epithelial cells [24–26] and in experimental models in rats [27,28].
Around 63–70 % of the affected children who survive the acute phase
of the disease achieve full recovery without renal sequelae [4,7,29]. The
renal tubular epithelium is almost mitotically inactive, but after stimu­
lation or moderate injury the tubular epithelial cells acquire a regen­
erative ability [30]. Different works suggest that the response to renal
tubular damage involve the dedifferentiation of epithelial cells that ac­
quire a mesenchymal phenotype. This process involves cell proliferation
and eventually redifferentiation into functional epithelial cells, thus
restoring tissue integrity [31,32]. These processes have been detected
through changes in the expression of mesenchymal markers [32,33],
proliferation markers [32,34], and in the expression or activation of
intracellular signal pathway components, among other factors [34]. The
absence or reduction of epithelial regeneration could predispose to form
tubulointerstitial scars and to develop chronic renal disease.
The main route of STEC infection in patients with HUS is through the
ingestion of undercooked meat and contaminated vegetables and bev­
erages [3]. However, the presence of Stx2 and other STEC virulence
factors has also been detected in the endocervix of asymptomatic
pregnant women, which could be another previously unexplored route
of infection [35]. Besides, the human placenta also expresses high levels
of Gb3 and is sensitive to Stx [36]. Infections associated with STEC could
be a cause of fetal morbidity and mortality in pregnant women. How­
ever, a single case of STEC infection was reported during the third
trimester of pregnancy with the birth of a normal, full-term baby [37].
On the other hand, studies carried out in pregnant rats and mice injected
with Stx2 before or after placental development demonstrated the in­
duction of abortion and premature labor [38–40].
In an experimental model developed in pregnant rats injected with a
filtrated bacterial supernatant containing Stx2, in the late stage of
gestation (14–16 days of gestation (dg)), morphological and histological
damages were observed in the uteroplacental unit. However, the authors
reported that those rats did not develop renal injury such as what
occurred in non-pregnant rats injected with equivalent doses of Stx2
[38]. On the other hand, pregnant rats intraperitoneally (i.p.) injected
with a sublethal dose of purified Stx2 in an early stage of pregnancy (8
dg) produced placental hypoxia, inflammation, and abortion, as well as
renal tubular necrosis and moderate increase of plasma creatinine levels
2
Microbial Pathogenesis 186 (2024) 106482
[39,41].
The aim of the present work was to investigate whether the hemo­
dynamic and adaptive changes during pregnancy may modify the renal
damage induced by Stx2, and the renal tubular epithelia regeneration.
Therefore, an exhaustive analysis of renal functional and histopatho­
logical evolution as well as of tubular epithelia proliferation and
dedifferentiation was carried out in the experimental model [41] of
pregnant rats i.p. inoculated with a sublethal dose of Stx2 at the early
stage of gestation (8 dg), equivalent to the first trimester of pregnancy in
humans, and results were compared with those observed in
non-pregnant female rats i.p. injected with the same Stx2 dose.
2. Materials and methods
2.1. Reagents
Shiga Toxin 2a (Stx2a) purified was purchased to Phoenix Lab, Tuft
Medical Center, Boston, MA.
Antibodies: Mouse IgG anti-Ki67 monoclonal antibody (BD Bio­
sciences, San Diego, CA, USA), rabbit anti-Ki67 polyclonal antibody
(Abcam, USA), mouse anti-vimentin monoclonal antibody (Biolegends,
San Diego, CA, USA), rabbit anti-neutrophil gelatinase-associated lip­
ocalin (NGAL) polyclonal antibody (Invitrogen, Thermo Fisher Scienti­
fic, USA), rabbit anti-rat AQP4 IgG antibody (Alpha Diagnostic, USA),
rabbit anti-NKCC2 polyclonal antibody (Milipore, CA, USA). Alexa fluor
488-goat-conjugated anti-mouse IgG, and Alexa Fluor TM 555 goat
conjugated anti rabbit IgG (Invitrogen, Waltham, MA, USA), and Cy3-
conjugated goat anti-rabbit (Jackson Immuno Research Lab, PA, USA)
secondary antibodies.
2.2. Animal model and experimental design
Female Sprague-Dawley rats (200–250 g), were acquired from the
animal facility at the School of Pharmacy and Biochemistry. Protocols
for animal studies were approved by the Institutional Committee for the
Care and Use of Laboratory Animals, Buenos Aires University (CICUAL,
protocol N◦ 2238-2016 and N◦ 639–2020). The animals were housed
under controlled conditions of dark-light (12:12 h) cycle and tempera­
ture (22–24 ◦ C), and received water and food ad libitum.
Randomly, half of female rats were kept virgins and the rest were
mated with Sprague-Dawley males [39]. The first day of gestation was
determined when sperm was observed in the vagina smear.
Pregnant and non-pregnant rats were randomly divided into two
groups each. Pregnant Sprague-Dawley rats, at eighth day of gestation,
were intraperitoneally (i.p.) injected with a single sublethal dose of Stx2
(0.5 ng Stx2/g bwt) (PS rats) dissolved in PBS (phosphate buffered sa­
line). Non-pregnant virgin rats were also i.p. injected with the same dose
of Stx2 (NPS rats). Control pregnant (PC rats) and non-pregnant (NPC
rats) rats were i.p. injected with the same volume (1 μL/g) of PBS. Rats
were individually housed, and water drinking, food intake, and body
weight (bwt) were daily monitored. Data of bwt are presented as Δbwt
= bwt at n dpi minus bwt at 0 dpi. The health status of the animals was
observed daily. Delivery time as well as pregnancy loss, evidenced as
vaginal bleeding, were evaluated in pregnant rats. Rats were euthanized
at 1, 2, 4, 8, and 30 days post injection (dpi), using a carbon dioxide CO2
chamber.
2.3. Biochemical analysis, renal function and water balance
Urine was collected during 24 h in metabolic cages in which rats
were with water access ad libitum but without food. Water balance was
calculated as urinary flow to water intake ratio.
Blood samples were obtained by cardiac puncture prior to sacrifice.
Serum and urine creatinine and urea concentrations were measured
using commercial kits (Wiener Lab, Argentina). To evaluate the
glomerular filtration rate (GFR), the creatinine clearance (CCreat) was
L.K. Fischer Sigel et al.
calculated as urine creatinine concentration × urinary flow/serum
creatinine concentration.
Sodium concentration was measured in serum and urine samples by
flame photometry. The sodium fractional excretion (FENa) was calcu­
lated as the ratio of Na+ excretion rate to its filtered rate.
Serum and urine osmolality were measured using a vapor pressure
osmometer (VAPRO 5520, Wescor Inc., Logan, UT, USA). The transport
of free water (Tc) reabsorbed in the collecting duct per unit time was
calculated as the Osmolal clearance (COsm) minus the urinary flow.
Fractional excretion (FEsol) of total solutes was calculated as COsm to
CCreat ratio.
2.4. Tissue process for histopathology
Anesthetized rats were immediately fixed by intravascular perfusion
with 10 % formalin in a saline solution of 10 mmol/L sodium phosphate
buffer, 0.9 % NaCl, pH 7.4 (PBS). The kidneys were removed and
immerse in the same fixing solution. Tissue samples were then dehy­
drated and embedded in paraffin. Renal sections of 3 μm were depar­
affinized, hydrated and stained with periodic acid-Schiff (PAS).
To evaluate tubular damage extent in cortical renal sections, the
percentage of necrosis, brush border loss in proximal tubules, epithelial
flattening, tubular nuclei loss, presence of debris and nuclei in lumen,
were evaluated per tubular area analyzed by Image J (NIH). The per­
centage of tubules with nuclei in lumen was calculated in kidney med­
ullary sections. For histopathological analysis, 10 to 15 images of
cortical and medullary fields per kidney were capture under light mi­
croscopy from sections of six rats per group. Histopathological evalua­
tion was performed independently by two trained investigators without
knowledge of the experimental group.
2.5. Immunofluorescence
For indirect immunofluorescence, histological sections were depar­
affinized and hydrated followed by antigen retrieval in 100 mmol/L
citrate buffer, pH: 6.0 (98 ◦ C, for 1 h), and permeabilized in 0.1 % Triton
X-100 in PBS. Renal sections were blocked with 5 % fetal bovine serum
(FBS) in PBS and incubated overnight with mouse anti-Ki67 (1:50), or
mouse anti-vimentin (1:100) primary antibodies, in a humidified
chamber at 4 ◦ C. Sections were then incubated with Alexafluor-488 goat
anti-mouse (1:200) secondary antibody, for 1 h. To count the total
number of cells per field, nuclei were stained with Hoechst (1 μg/mL).
The percent of Ki67 or vimentin positive tubular epithelial cells to total
cells per field, was calculated from renal medullary and cortical sections.
For NGAL detection, renal sections were incubated with rabbit anti-
NGAL (1:100) primary antibody followed by Alexa Fluor TM 555 goat
conjugated anti rabbit IgG (1:500) secondary antibody.
For double-fluorescence staining, sections were labeled with a mouse
anti-vimentin (1:100) primary antibody and Alexafluor488-conjugated
goat anti-mouse (1:200) secondary antibody, followed by incubations
with a rabbit anti-Ki67 (1:500) primary antibody and Cy3-conjugated
goat anti-rabbit (1:800) secondary antibody.
To localize Ki67 and vimentin positive cells, double-fluorescence
were performed by incubating the sections with mouse anti-Ki67 or
anti-vimentin antibodies followed by rabbit anti-AQP4 (1:100) or rabbit
anti-NKCC2 (1:100) primary antibodies, and corresponding secondary
antibodies.
Fluorescence was observed with a Nikon Eclipse E− 2000 fluores­
cence microscope. In each section, 10 to 15 images of renal cortex and
medulla were capture with a digital camera Nikon E4300 and processed
and analyzed using the Image J (NIH) software.
2.6. TUNEL assay
To evaluate apoptosis, renal sections were subject to antigen
retrieval and permeabilized, as explained above. TUNEL assay was then
3
Microbial Pathogenesis 186 (2024) 106482
performed using the In situ cell death detection kit (Roche, USA), and
following the recommended manufacture proceedings. The analysis of
TUNEL in renal sections was performed under fluorescence microscope.
Microphotographs of renal sections were taken from 3 rats per group,
and analyzed using the Image J (NIH) software.
2.7. Statistical analysis
Results are reported as mean ± standard error of the mean (SEM).
Mann Whitney test was used for non-parametric data for individual
comparisons between two groups. One-way ANOVA and ANOVA for
repeated measures, followed by Tukey post hoc test, and Kruskal-Wallis
test for non-parametric data, followed by Dunn’s post hoc test, were
used to calculate statistical significance for multiple comparisons. A p-
value <0.05 was considered significant. The statistical analysis was
performed using GraphPad Prism 6 software.
3. Results
3.1. General parameters
The effects of Stx2 sublethal (0.5 ng/g bwt) dose on general pa­
rameters were studied and compared between pregnant and non-
pregnant rats and their respective control groups. Graphics in Fig. 1a
and b, show the time course of food intake and body weight, respec­
tively, from one to eight days after Stx2 or PBS i.p. injection in rats.
NPC rats maintained their food intake and Δbwt over time. Due to
pregnancy, PC rats maintained a higher food intake and significantly
increased their Δbwt over time, compared to PS and non-pregnant rats,
until delivery. Post-Stx2 injection, NPS rats, showed lower food intake
and Δbwt with respect to NPC, and started to recover Δbwt at about 6–8
dpi.
Delivery of PC rats took place between days 22 and 24 of gestation,
which corresponded to 14–16 dpi, therefore, these rats immediately
decreased their food intake and started to lose weight (Fig. 1c and d).
Otherwise, PS rats showed vaginal bleeding and pregnancy loss around
the 14th day of gestation, corresponding to the 6th day post-Stx2 in­
jection. None of PS rats delivered any pup at term. Besides, fetus
resorption at the implantation sites were observed in the uterus of PS
rats at 30 dpi. At three weeks post-injection, none of the groups showed
significant differences in food intake and Δbwt among them (Fig. 1c and
d).
Signs of piloerection were observed in both NPS and PS groups from
1 to 5 dpi. None of pregnant and non-pregnant rats injected with the
sublethal dose of Stx2 died during the study.
3.2. Renal functional parameters
At 4 dpi, NPS rats showed a significant increase in serum creatinine
and urea levels, compared to the other groups of rats (Fig. 2a and b,
Table 1). Serum creatinine levels of PS rats, slightly and significantly
increased with respect to PC and NPC rats at 4 days post-Stx2 injection
(Fig. 2a). However, serum urea values of PS rats remained within the
normal range (Fig. 2b and Table 1).
GFR, measured by CCreat was significantly decreased in NPS rats
compared to NPC and PC rats (Fig. 2c). In PC rats, CCreat tends to be
higher than in NPC rats. But, the inoculation of pregnant rats with Stx2,
significantly decreased their CCreat compared to PC rats at 4 dpi.
At 8 dpi, CCrreat and serum urea of NPS rats recomposed to normal
levels, while serum creatinine remained significantly higher than in the
rest of the groups, despite having decreased with respect to values at 4
dpi (Table 1). At 8 dpi, CCreat and serum creatinine and urea of PS rats
showed normal values like in PC rats (Table 1).
Prior to 4 dpi, serum creatinine and urea as well as CCreat remained
within normal values in the four groups of rats (data not shown). Hence,
at 30 dpi, no significant differences were observed in these parameters
L.K. Fischer Sigel et al. Microbial Pathogenesis 186 (2024) 106482

Fig. 1. Time course of food intake and Δbody weight (Δbwt). Time course of food intake (a and c) and Δbwt (b and d) per day, were evaluated in pregnant and
non-pregnant rats i.p. injected with sublethal dose of Stx2 (NPS and PS) or PBS (NPC and PC). Each point of the curves represents the mean ± SEM, n = 6–18 rats.
ANOVA for repeated measures followed by Tukey post hoc test indicates significant differences: *p < 0.05 and ***p < 0.001 vs NPC; ###p < 0.001 vs. PC; and &p <
0.05 vs PS.

Fig. 2. Renal function. Graphics show serum creatinine (a) and serum urea (b) levels, and creatinine clearance (c) from pregnant and non-pregnant rats i.p. injected
with Stx2 (PS and NPS) or PBS (PC and NPC), at 4dpi. Data are expressed as mean ± SEM, n = 6–9 rats. One way ANOVA followed by Tukey post hoc test indicates
significant differences: *p < 0.05, ***p < 0.001vs NPC; #p < 0.05, ###p < 0.001 vs PC; and &p < 0.05 vs PS.

among the four groups of animals (Table 1), demonstrating that GFR
returned to normal values in pregnant and non-pregnant rats injected
with Stx2.
3.3. Water intake, urinary flow and water balance
Water intake, urinary flow and water balance were analyzed in all
groups of rats at 4, 8, and 30 dpi (Fig. 3a and b, and Table 1). At 4 days
post-Stx2 injection, NPS rats significantly increased water intake and
urinary flow, compared to NPC. Despite the injection with Stx2, the
urinary flow of PS rats was not significantly different from that of PC
rats. Although not significant, water intake and urinary flow of both PC
and PS rats were higher than those of NPC rats, but lower than those of
NPS rats, at 4 dpi.
The high diuresis of NPS rats correlates with the significant decrease
of the urinary osmolality and the transport of free water reabsorbed in
the collecting ducts (Tc), compared to NPC rats at 4 dpi (Fig. 3c and d).
These results indicate that the sublethal dose of Stx2 impaired the urine
concentration in female non-pregnant rats. However, neither urinary
osmolality nor Tc of PS rats were significantly different from those of PC
rats. These parameters in PC and PS rats were lower than those of NPC
rats and non-significantly different than those of NPS rats.
At 8 and 30 dpi, non-significant differences were observed in water
intake, diuresis, urinary osmolality and Tc among groups (Table 1).
Variations of water intake were proportional to those of urinary flow,
therefore, no significant differences in water balance were observed
among the groups of rats at all times studied (Table 1).
Serum osmolality and Osmolal clearance remained without signifi­
cant changes along time in all groups of rats, and without showing
significant differences among groups (data not shown).
3.4. Solute balance and renal sodium handling
Fractional excretion of total solutes (FEsol), was significantly

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L.K. Fischer Sigel et al. Microbial Pathogenesis 186 (2024) 106482

Table 1
Renal parameters evaluated in serum and urine of pregnant (PS) and non-pregnant (NPS) rats at 4, 8 and 30 days post-Stx2 intraperitoneal injection (dpi). Control
pregnant (PC) and non-pregnant (NPC) rats were injected with eluent. CCreat: Creatinine Clearance. One way ANOVA followed by Tukey post hoc test indicates sig­
nificant differences: *p < 0.05, ***p < 0.001vs NPC; #p < 0.05,###p < 0.001 vs PC; and &p < 0.05 vs PS.
NPC NPS PC PS
#&
4 dpi Serum Creatinine (mg/dl) 0.6 ± 0.05 1.1 ± 0.09*** 0.7 ± 0.04 0.9 ± 0.05#
Serum Urea (mg/dl) 33.3 ± 5.26 61.4 ± 5.8***# & 35.4 ± 2.24 37.7 ± 3.04
CCreat (ml/min) 1.3 ± 0.17 0.7 ± 0.09*### 1.9 ± 0.28 0.8 ± 0.15###
Water Intake (ml/d × 100 g bwt) 8.0 ± 1.98 16.1 ± 1.26* 11.8 ± 1.78 11.5 ± 1.58*
Urinary Flow (ml/d × 100 g bwt) 6.6 ± 1.01 16.1 ± 1.09*** 10.8 ± 1.44* 11.6 ± 1.38*
Water Balance 1.1 ± 0.08 0.9 ± 0.06 0.9 ± 0.04 1.0 ± 0.09
Urinary Osmolality (mOsmol/Kg) 584 ± 74.9 307 ± 19.5*** 440 ± 65.2* 413 ± 42.7*
Tc (ml/d × 100 g bwt) 8.2 ± 1.53 0.4 ± 1.13*** 3.5 ± 2.02* 4.2 ± 1.48*

8 dpi Serum Creatinine (mg/dl) 0.7 ± 0.04 0.9 ± 0.07*& 0.7 ± 0.04 0.8 ± 0.05
Serum Urea (mg/dl) 37.4 ± 3.80 38.9 ± 2.23 37.6 ± 1.81 42.5 ± 4.91
CCreat (ml/min) 1.4 ± 0.32 1.3 ± 0.32 1.3 ± 0.25 1.6 ± 0.30
Water Intake (ml/d × 100 g bwt) 9.5 ± 2.01 12.1 ± 3.01 7.3 ± 1.17 10.5 ± 0.92
Urinary Flow (ml/d × 100 g bwt) 7.8 ± 2.90 12.9 ± 1.02* 9.6 ± 2.23 12.3 ± 1.18
Water Balance 1.3 ± 0.23 1.0 ± 0.11 1.6 ± 0.16 1.2 ± 0.11
Urinary Osmolality (mOsmol/Kg) 475 ± 32.6 467 ± 46.3 422 ± 58.8 416 ± 19.7
Tc (ml/d × 100 g bwt) 4.6 ± 0.65 3.0 ± 0.74 7.2 ± 2.78 4.4 ± 0.47

30 dpi Serum Creatinine (mg/dl) 0.6 ± 0.08 0.7 ± 0.11 0.5 ± 0.03 0.6 ± 0.01
Serum Urea (mg/dl) 39.2 ± 2.76 38.7 ± 3.73 40.1 ± 1.74 38.3 ± 2.70
CCreat (ml/min) 1.0 ± 0.13 1.2 ± 0.16 1.0 ± 0.11 1.2 ± 0.20
Water Intake (ml/d × 100 g bwt) 8.3 ± 1.15 9.5 ± 1.60 6.9 ± 1.80 8.4 ± 1.86
Urinary Flow (ml/d × 100 g bwt) 8.9 ± 1.07 10.1 ± 1.70 7.1 ± 0.74 8.2 ± 1.83
Water Balance 1.1 ± 0.11 1.2 ± 0.13 0.9 ± 0.16 1.0 ± 0.09
Urinary Osmolality (mOsmol/Kg) 472 ± 48.6 451 ± 50.6 440 ± 49.5 476 ± 60.2
Tc (ml/d × 100 g bwt) 3.9 ± 0.89 6.4 ± 1.95 5.8 ± 1.59 5.0 ± 1.28

Fig. 3. Water handling. Graphics show water intake (a), urinary flow (b), urinary osmolality (c), and Tc (d), from pregnant and non-pregnant rats i.p. injected with
Stx2 (PS and NPS) or PBS (PC and NPC), at 4dpi. Data are expressed as mean ± SEM, n = 6–7 rats. One way ANOVA followed by Tukey post hoc test indicates
significant differences. *p < 0.05 and ***p < 0.001 vs NPC.

increased in NPS and PS rats with respect to NPC and PC rats at 4 dpi Renal sodium handling was evaluated in the four groups of rats at 4
(Table 2). NPS rats presented a higher but not significant FEsol than PS dpi. As shown in Table 2, NPS rats significantly increased sodium
rats. These results demonstrated that Stx2 caused a decrease in solute excretion rate (ERNa) and sodium fractional excretion (FENa) compared
reabsorption along renal tubules at 4 dpi. to NPC rats. Hence, FENa in NPS rats was significantly higher than in PS

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L.K. Fischer Sigel et al. Microbial Pathogenesis 186 (2024) 106482

Table 2 histomorphology was observed in kidneys of NPC and PC rats at all times
Renal solute and sodium handle. Excretion rate of solutes (ERsol), sodium evaluated. Mild to moderate renal injury with focal tubular necrosis
(ERNa), and urea (ERUrea), as well as fractional excretion of solutes (FEsol), so­ including brush border loss in proximal tubules, epithelial flattening,
dium (FENa) and urea (FEUrea) were measured in pregnant (PS) and non- tubular nuclei loss, and presence of debris and nuclei in tubular lumen,
pregnant (NPS) rats at 4, 8 and 30 days post-Stx2 intraperitoneal injection was observed in renal cortical sections of both NPS and PS, at 4 and 8
(dpi). Control pregnant (PC) and non-pregnant (NPC) rats were injected with
dpi. However, glomeruli of NPS and PS rats were not affected by Stx2.
eluent. One way ANOVA followed by Tukey post hoc test indicates significant
Tubular damage extent, calculated from renal cortical sections of the
differences: *p < 0.05, **p < 0.01, ***p < 0.001vs NPC; #p < 0.05 vs PC; and &p
< 0.05 vs PS. four groups of rats (Fig. 4b), demonstrated that NPS rats showed sig­
nificant higher extent of cortical tubular damage than PS rats at 4 and 8
NPC NPS PC PS
dpi. However, at 30 dpi, both NPS and PS showed a conserved renal
ERsol (mmol/d) 9.8 ± 0.83 11.2 ± 1.03 11.4 ± 0.82 12.4 ± 0.61 cortical tubular morphology as in NPC and PC control groups.
FEsol (%) 1.8 ± 0.25 4,3 ± 0.76*** 1.4 ± 0.16 3.0 ± .88#*
A significant increase in the percentage of tubules with nuclei lost in
ERNa (mmol/d) 0.4 ± 0.08 0.8 ± 0.17* 0.6 ± 0.15* 0.8 ± 0.02*
FENa (%) 0.2 ± 0.03 0.9 ± 0.23**& 0.2 ± 0.01 0.3 ± 0.05
the tubular lumen was observed in the renal medulla of NPS and PS rats
ERUrea (mmol/d) 4.0 ± 0.30 3.8 ± 0.39 5.5 ± 0.54 6.2 ± 1.53 compared to NPC and PC rats at 4 dpi, being significantly higher in NPS
FEUrea (%) 44.7 ± 8.9 31.8 ± 9.9 43.7 ± 12.1 38.9 ± 11.3 than in PS rats (Fig. 4c and d). At 8 dpi, the percentage of tubules with
nuclei lost in lumen remained increased in only NPS rats and, at 30 dpi,
returned to low values as in the others groups of rats (Fig. 4d).
rats.
Representative micrographs in Fig. 5a and b, show tubular apoptosis
Both PC and PS rats showed higher ERNa than NPC rats. No signifi­
(green nuclei), evaluated by Tunel assay, in cortical and medullary renal
cant differences was observed in ERNa between PC and PS groups
fields of both NPS and PS, at 4 dpi. Almost no positive labeling for Tunel
(Table 2).
was observed in renal sections of NPC and PC rats.
Neither ERurea nor FEurea were significantly different among groups
The immunofluorescence for NGAL (Fig. 5c) corroborated the his­
at 4 dpi (Table 2).
tological observations in renal sections. Positive staining for NGAL was
observed on the apical side of renal tubules in NPS and PS rats, mainly in
3.5. Histomorphology and tunel the medulla, at 4 dpi. A significantly higher percent of NGAL-positive
tubules was observed in NPS compared with PS renal sections
As shown in representative micrographs in Fig. 4a, conserved renal (Fig. 5d). Control pregnant and non-pregnant rats did not show NGAL

Fig. 4. Renal histomorphology. a: Representative microphotographs show renal cortical sections, stained with PAS, from pregnant and non-pregnant rats i.p.
injected with Stx2 (PS and NPS) or PBS (PC and NPC) at 4, 8 and 30 days post injection. Scale bar = 50 μm. Asterisks (*) indicate tubular damage with loss of brush
border, debris and nuclei in the tubular lumen, and loss of tubular cells. b: The graphic represents the percentage of cortical tubular damage per tubular area related
to control non-treated rats representing 1 %. c: Representative microphotographs show renal medullary sections from the same rats, stained with PAS. Asterisks (*)
indicate nuclei loss in the tubular lumen. Scale bar = 50 μm. d: The graphic represents the percentage of tubules with nuclei loss in tubular lumen. Graphics bars
represent the mean ± SEM, n = 4–6 rats. Kruskal-Wallis test followed by Dunn’s post hoc test indicates significant differences. *p < 0.05, **p < 0.01, and ***p <
0.001 vs NPC; #p < p < 0.05, and ###p < 0.001 vs PC; &p < 0.05, and &&&p < 0.001 vs NPC.

6
L.K. Fischer Sigel et al.
Fig. 5. Tunel assay and NGAL immunofluorescence in renal sections. Representative microphotographs show apoptotic cells (green nuclei), evaluated by Tunel
assay, in renal cortical (a) and medullary (b) sections from pregnant and non-pregnant rats at 4 days post-Stx2 (PS and NPS) or PBS (PC and NPC) injection. Scale bar
= 50 μm. Microphotographs in c show NGAL immunofluorescence in renal sections of NPC, NPS, PC and PS rats. The graphic in d shows the percentage of NGAL-
positive renal tubules per total tubules per field in the four groups of rats at 4 days post-injection. Mann Whitney test was used to indicate significant differences,
***p < 0.001 NPS vs PS.
staining in tubules.
3.6. Tubular proliferation and vimentin expression
Representative microphotographs in Fig. 6a and graphic in Fig. 6b,
show that those rats injected with a sublethal dose of Stx2 significantly
increased Ki67 expression in nuclei of renal medullary tubular epithelial
cells in both NPS and PS rats with respect to NPC and PC rats, at 4 dpi,
demonstrating an enhance of tubular cell proliferation. The expression
of Ki67 remained significantly higher in NPS rats than in PS rats at 8 dpi.
At 30 dpi, the percentage of Ki67 in NPS and PS rats significantly
decreased to basal levels, showing no significant differences respect to
control rats and among groups. The percentage of Ki67 in renal medulla
sections at 1 and 2 dpi (data not shown), as well as in cortical sections at
4, 8 and 30 dpi, was scarce in all groups of rats (Fig Suppl 1).
Fig. 6c and d, show representative micrographs of double immuno­
fluorescence for Ki67 with AQP4 and NKCC2, respectively, in PS and
NPS rats. About 80 % of Ki67 positive tubular epithelial cells were also
positive for AQP4 in their basolateral membranes, indicating that the
increase of Ki67 was mostly localized in medullary collecting ducts. Less
than 20 % of Ki67 positive tubular cells were positive for NKCC2 in their
apical surface, indicating that some few epithelial cells of the Henle’s
loop thick ascending limb also may proliferate in response to Stx2
damage in rats.
The expression of the mesenchymal marker vimentin was also
evaluated in kidney sections. In renal cortex of all groups of rats,
vimentin was strongly expressed in the glomerular and extra-glomerular
vasculature, but not in tubular epithelial cells, showing no significant
differences among the different treatments (Figure Suppl 2). In renal
medulla of NPC and PC rats, vimentin was expressed only in the vascular
endothelia (Fig. 7a). However, at 4 days post-Stx2 injection, NPS
showed vimentin expression mainly in the basolateral membrane of
some medullary epithelial cells, which was maintained at 8 dpi (Fig. 7b
and c). Pregnant PS rats also showed vimentin positive tubular epithelial
cells in the renal medulla, but in a significant lower percent than in NPS
kidneys, at both 4 and 8 dpi.
Double-immunofluorescence for vimentin and AQP4 assessed in
renal sections of NPS and PS rats, demonstrated that tubular vimentin is
mostly co-localized with AQP4 in the basolateral membrane of the
medullary collecting duct epithelial cells (Fig. 7b). It is remarkable that,
7
Microbial Pathogenesis 186 (2024) 106482
at 8 days post-Stx2 injection, a high and significant amount of epithelial
cells around the lumen of many collecting ducts were observed in renal
medullary sections of NPS rats (Fig. 7b).
Double-immunofluorescence for vimentin and Ki67 (Fig. 7d) in renal
medullary sections of NPS and PS rats, showed that most of vimentin
tubular expression was localized in Ki67 positive cells.
At 30 dpi, vimentin was no longer expressed in renal tubular
epithelia of both NPS and PS rat kidneys (data not shown).
4. Discussion
In this study, we have evaluated the progression of renal injury
caused by the treatment with a sublethal dose of Stx2, as well as renal
recovery, in two experimental models developed in pregnant rats and in
female non-pregnant rats of the same age. For this purpose, pregnant
Sprague-Dawley rats were i.p. inoculated with a sublethal dose of Stx2 at
the early stage of gestation (8 dg), and results were compared with those
obtained in non-pregnant female rats injected with the same Stx2 dose.
Previous studies carried out in Stx2-injected pregnant rats were focus
on the uteroplacental unit’s morphological and histological damages,
and on pregnancy and fetus complications, while some few renal studies
were performed in the short term post-Stx2 injection [38,39,41].
Therefore, in the present work, more exhaustive studies on renal his­
tology and function were addressed. Evolution of renal injury and
tubular repair was evaluated from 1 to 30 dpi, which allowed us to track
the pathology, and analyze whether pregnancy may lessen or protect
from Stx2-induced renal damage in rats, and its correlation with renal
water handling. Moreover, given that most of the patients with HUS
recover after the prodromal stage of the disease, it was of our interest to
study sublethal models of HUS in rats to investigate renal tubular
mechanisms involved in the epithelial regeneration after Stx2-induced
damage.
The sublethal dose of Stx2 produced mild to moderate acute kidney
injury in both pregnant and non-pregnant rats, being significantly more
severe in NPS than in PS rats. Hence, differences in the way Stx2 affects
renal parameters between both experimental models were analyzed.
In PS rats, Stx2 impeded the normal evolution of pregnancy and
caused the loss of the fetuses at 6 dpi, as previously reported in the same
experimental model [39]. In previous studies, morphological and his­
tological damages in the uteroplacental unit with fetal resorption, in
L.K. Fischer Sigel et al. Microbial Pathogenesis 186 (2024) 106482

Fig. 6. Evaluation of renal tubular cell proliferation using Ki67 marker. a: Microphotographs show immunofluorescence for Ki67 (green nuclei) merged with
Hoechst (blue nuclei) staining in renal medullary sections of pregnant and non-pregnant rats, at 4, 8 and 30 days post-Stx2 or PBS injection. Scale bar = 100 μm. b:
The graphic represents the percentage of Ki67 positive cells with respect to the total cells stained with Hoechst. Results are expressed as mean ± SEM, n = 3–8 rats.
Kruskal-Wallis test followed by Dunn’s post hoc test indicates significant differences. ***p < 0.001 vs NPC; ###p < 0.001 vs PC; and &p < 0.05 vs NPS. c: Double
immune fluorescence for Ki67 (green nuclei) and AQP4 (red) indicates that most of Ki67 positive nuclei are localized in collecting duct principal cells. d: Double
immune fluorescence for Ki67 (green) and NKCC2 (red) cotransporter indicates thick ascending limb of Henle’s loop localization. Scale bar = 50 μm.

coincidence with a significant decrease in serum progesterone levels,
were observed in pregnant rats at 6 days post-Stx2 injection compared
with control pregnant rats [39,41]. It was reported that in rodents,
during the early phases of pregnancy, progesterone synthesis in decidual
tissue is essential for successful implantation of the embryo and main­
tenance of pregnancy [42].
A significant decrease in the GFR was observed in both PS and NPS
rats, compared with PC and NPC rats. However, renal histological ob­
servations at 4 and 8 dpi, revealed that glomeruli in the PS and NPS
groups exhibited a preserved appearance with no damage. Findings from
previous studies in the same rat model showed some positive Stx2
staining in glomeruli of pregnant rats at 6 h post-Stx2 sublethal dose
injection, and only some glomerular epithelial adherences but without
glomerular endothelial damage at 6 dpi [39]. One plausible explanation
for these results could be the epithelial cell desquamation, as evidenced
by Stx2-induced loss of cell nuclei in the lumen of medullary tubules.
These cells may obstruct the tubules consequently increasing the hy­
drostatic pressure in the glomeruli resulting in a mild reduction in GFR,
along with the increase in serum creatinine and urea levels.
Although the increased serum creatinine levels in NPS rats were
slightly but significantly higher than in PS rats, CCreat in NPS tended to
be lower, albeit not significantly different than that in PS rats, possibly
due to data variability influencing clearance calculation. Instead, serum
urea levels in NPS rats increased nearly double than those in PS and
control rats. These results may be due to the tubular damage caused by
Stx2, which was significantly greater in NPS rats than in PS rats at 4 dpi,
and that could have affected the mechanisms of tubular urea reabsorp­
tion and secretion in the injured epithelia. However, further studies are
necessary to corroborate these mechanisms.
Therefore, Stx2-induced histological damage was observed only in
renal tubules, which was corroborated by the increase of tubular
apoptosis and NGAL staining. NGAL have been frequently used in
experimental and clinical studies as a urine biomarker for acute kidney
injury, including in HUS disease [43]. Other studies proved that there
are differences in Gb3 renal expression and localization in rats and mice
with respect to human kidney and, therefore, in the location of the renal
injury. In this regard, Gb3 localization in renal tubules but not in
glomeruli of a murine model, i.p. injected with Stx2, was demonstrated
[21].
In our studies, some PC rats showed a higher CCreat than NPC rats at 4
dpi, possibly due to hyperfiltration characteristic during pregnancy
[44]. During pregnancy, a reduced vascular reaction to vasoconstrictors
results in systemic and intrarenal vasodilatation which leads to the in­
crease of renal plasma flow and GFR [45]. It has been reported that
vasodilatation starts in early pregnancy, and slowly decrease towards
term pregnancy both in humans and animals [44]. In rats, these effects
seems to be dependent on up regulation of the nitric oxide system and
not on sympathetic tone or vasopressin [44,46].

8
L.K. Fischer Sigel et al. Microbial Pathogenesis 186 (2024) 106482

Fig. 7. Vimentin expression. a: Microphotographs show Vimentin positive cells (green) merged with cell nuclei stained with Hoechst (blue) in renal medullary
sections of pregnant and non-pregnant rats, at 4 days post injection (dpi) with Stx2 (PS and NPS) or PBS (PC and NPC) injection. b: Double immunofluorescence for
Vimentin (green) and aquaporin 4 (AQP4) (red), and Vimentin - AQP4 merge (yellow), in renal medullary sections of PS and NPS rats, at 8 days post-Stx2 injection.
Scale bar = 50 μm. c: The graphic represents the percentage of Vimentin positive cells with respect to the total cells stained with Hoechst at 4 and 8 dpi. Results are
expressed as mean ± SEM, n = 10–15 fields from 3 rats. Kruskal-Wallis followed by Dunn’s post hoc test indicates significant differences. ***p < 0.001 NPS vs PS at 4
dpi; ##p < 0.01vs PS at 4 dpi; and &&p < 0.01 vs NPS at 8 dpi. d: Double immune fluorescence for Vimentin (green) and Ki67 (red). Scale bar = 50 μm.

In the present work, all animals were kept under conditions of water
intake ad libitum. Water balance was maintained with no significant
differences among groups and no signs of dehydration were shown.
Nevertheless, transient impairment in the ability to concentrate the
urine at 4 days post-Stx2-injection, which was subsequently reversed at
8 dpi, was observed in only NPS rats. These results corroborate those
reported by other groups in rat and murine models [21,47]. Sugatani
et al. [47], showed that non-lethal doses of Stx2 induced morphological
changes in renal tubules associated with polyuria with elevated urinary
aquaporin 2 (AQP2) levels and reduction in AQP2 expression in renal
plasma membranes.
Noteworthy, in our study renal water handling in pregnant rats was
significantly different than in non-pregnant rats. Control PC rats pro­
duced significant more dilute and abundant urine than NPC rats. Taking
into account that, unlike what occurred in non-pregnant rats, no sig­
nificant differences were observed in urinary flow and osmolality, and in
Tc, between PS and PC rats at 4 dpi, it is possible that intrarenal vaso­
dilatation and the increased renal plasma flow during pregnancy may
prevent or mask the Stx2 effects on urinary concentration mechanism.
These adaptive changes during pregnancy could produce similar effects
as the intravenous volume expansion, a supportive treatment used
during the early phase of the illness to avoid dehydration and which has
been demonstrated to ameliorate the renal injury progression and
decrease the need for dialysis in patients with HUS [5]. Other works also
reported that pregnant Sprague-Dawley rats showed an altered ability
for elaborating concentrated urine compared to virgin rats [48,49].
Besides, other studies showed that angiotensin-(1–7) (ANG-(1–7)), an
active vasodilator of the renin-angiotensin system, rises progressively
during human pregnancy and also with gestation in rats [50]. A study in
pregnant rats, treated with an inhibitor of ANG-(1–7), proposed that the
rise of renal ANG-(1–7) during pregnancy produce increased urinary
flow with decreased urinary osmolality [49].
In addition, the present work shows that Stx2 also caused a signifi­
cant decrease in tubular solutes reabsorption in both NPS and PS groups.
Sodium reabsorption was mainly altered in NPS rats. Hence, same as
observed for renal water regulation, no significant differences in renal
sodium handle was observed between PS and PC groups.
Regarding the evolution of Stx2-renal effects, in both PS and NPS
rats, the greatest functional and histological renal injury was observed at
4 dpi. Only NPS rats showed kidney damage, although less severe, up to
8 dpi, and then evolved to full recovery. In fact, at 30 dpi all rats injected
with Stx2 showed a conserved renal histology and recovered the ability
to concentrate urine. Therefore, tubular epithelial proliferation and
epithelial to mesenchymal transformation was evaluated.
A correlation in time between the extension of tubular damage and
the expression of the proliferation marker Ki67 and the mesenchymal
marker vimentin was observed. Although Stx2 caused renal tubular
damage in both cortex and medulla, double immunolabeling with AQP4
demonstrated that the medullary collecting duct principal cells showed
the highest expression of Ki67 and vimentin in both PS and NPS rats,
which coincided with NGAL tubular expression. Therefore, in rats,
medullary collecting ducts were the epithelia most affected in the kidney
that proliferated and dedifferentiated after Stx2-damage. This results
may be due to Gb3 expression levels in the collecting ducts, which make
these tubules more sensitive to Stx2, and also to the great ability of
tubular segments to proliferate and regenerate for moderate damage. In
fact, studies in mice intravenously injected with Stx2, showed renal
tubular apoptosis and polyuria, and Gb3 expression in some AQP1
positive tubular proximal tubules as well as in the apical membranes of
collecting duct tubular epithelial cells in co-localization with AQP2
[21]. Another study showed cortical renal tubular repair, evaluated by
Ki67, only in the subacute model but not in the acute HUS model in mice

9
L.K. Fischer Sigel et al.
[51]. Moreover, kidney biopsies from STEC-infected patients, showed
significantly increased apoptosis with consecutively increased Ki67
expression in the cortical tubular epithelium [34].
In our studies, transient vimentin tubular expression appeared only
in Stx2-injected rats, and was consistent with the extension of tubular
damage, being higher in non-pregnant rats than in pregnant rats. In this
regard, other studies reported that vimentin-positive tubular cells were
not detectable in healthy rat kidneys, whereas they appeared upon
unilateral ureteral obstruction [33]. Another work supports that re­
covery after acute kidney injury involves hypertrophy of remnant
tubular cells and limited progenitor-driven regeneration that can be
potentiated pharmacologically [52]. Our results demonstrate that
despite the damage caused by Stx2, tubular epithelial cells were able to
dedifferentiate and regenerate, possibly replacing the damaged cells and
thus recovering the renal ability to concentrate the urine, which was
observed at 30 dpi.
4.1. Conclusions
Our studies demonstrated that the sublethal dose of Stx2 induced a
significant more extended renal damage in non-pregnant rats than in
pregnant rats. Moreover, Stx2-induced tubular mild to moderate dam­
age allowed the triggering of tubular epithelial cell regeneration, ac­
cording on the severity and the extension of the damage. Hence, control
rats in the early/middle stage of pregnancy showed a different renal
water handle than non-pregnant rats, evidenced by the increased
diuresis and decreased urine osmolality, which was not significantly
modified by Stx2.
It is worth asking whether the adaptive mechanisms that lead to
systemic and intrarenal vasodilatation during pregnancy could protect
the kidney from Stx2. It could be argued that during pregnancy gener­
alized vasodilatation and volume expansion, induced by the increase of
female hormones and nitric oxide, which lead to increased renal plasma
flow and renal tubular flow, may result in more efficient removal of Stx2
by the kidneys in rats. In addition to adaptive mechanisms during
pregnancy, placenta recruitment of Stx2 might cause systemic protec­
tion during pregnancy in rats, as proposed in previous studies [39,53].
Future studies are necessary to investigate the factors that regulate the
adaptive mechanisms during pregnancy that lead to protection from
renal damage caused by Stx2, and that could be useful for the treatment
of acute renal injury in patients with HUS.
Financial support
This work was supported by grants to C. Silberstein from the Uni­
versity of Buenos Aires (UBACYT20020160100078BA), and to C Sil­
berstein, and E Zotta from the National Scientific and Technical
Research Council (CONICET: PUE 0041), and the National Agency for
Promotion of Science and Technology (ANPCYT-PICT 2016-0292).
Credit author statement
Daiana S. Sánchez: Methodology, Investigation. Flavia Sacerdoti:
Writing – review & editing, Methodology. Lilian K. Fischer Sigel: Writing
– review & editing, Methodology, Investigation, Formal analysis. Elsa
Zotta: Writing – review & editing, Funding acquisition, Formal analysis.
Claudia Silberstein: Writing – review & editing, Writing – original draft,
Supervision, Investigation, Funding acquisition, Formal analysis,
Conceptualization
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
10
Microbial Pathogenesis 186 (2024) 106482
Acknowledgments
We are grateful to Agostina Presta (Universidad de Buenos Aires
(UBA), Facultad de Medicina, Dto. de Ciencias Fisiológicas, IFIBIO-
Houssay) and Alicia Aráoz (UBA, Facultad de Medicina, Dto. de Bio­
logía Celular e Histología) for the technical assistance in the histological
processing of tissues. We also want to thank Lucía Garbini (UBA, Fac­
ultad de Medicina, Dto. de Ciencias Fisiológicas, IFIBIO-Houssay) for the
technical assistance with rats in the animal facility.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.micpath.2023.106482.
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