TLR2, TLR4 and the MYD88 Signaling Pathway Are Crucial for Neutrophil Migration in Acute Kidney Injury Induced by Sepsis

Abstract

The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2(-/-), TLR4(-/-) and MyD88(-/-) male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney tissue and blood samples were collected for analysis. The TLR2(-/-), TLR4(-/-) and MyD88(-/-) mice that were subjected to CLP had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice (WT). MyD88(-/-) mice were completely protected compared with the WT mice. We also observed reduced expression of proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1(+low) cells migration compared with the knockout mice and decreased in GR1(+high) cells migration into the peritoneal cavity. The TLR2(-/-), TLR4(-/-), and MyD88(-/-) mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular cells.

Full text

TLR2, TLR4 and the MYD88 Signaling Pathway Are
Crucial for Neutrophil Migration in Acute Kidney Injury
Induced by Sepsis
Angela Castoldi
1,2
,Ta
´rcio Teodoro Braga
2
, Matheus Correa-Costa
2
, Cristhiane Fa
´vero Aguiar
1
,E
ˆnio
Jose
´Bassi
2
, Reinaldo Correa-Silva
1
, Rosa Maria Elias
1
,Fa
´bia Salvador
1
, Pedro Manoel Moraes-Vieira
2
,
Marcos Anto
ˆnio Cenedeze
1
, Marlene Anto
ˆnia Reis
4
, Meire Ioshie Hiyane
2
,A
´lvaro Pacheco-Silva
1,3
,
Giselle Martins Gonc¸alves
2
, Niels Olsen Saraiva Ca
ˆmara
1,2
*
1Disciplina de Nefrologia, Departamento de Medicina, Universidade Federal de Sa
˜o Paulo, Sa
˜o Paulo, Brazil, 2Laborato
´rio de Imunobiologia de Transplantes,
Departamento de Imunologia, Universidade de Sa
˜o Paulo, Sa
˜o Paulo, Brazil, 3IIEP, Hospital Israelita Albert Einstein, Sa
˜o Paulo, Brazil, 4Patologia, Universidade Federal do
Tria
ˆngulo Mineiro, Uberaba, Brazil
Abstract
The aim of this study was to investigate the role of TLR2, TLR4 and MyD88 in sepsis-induced AKI. C57BL/6 TLR2
2/2
, TLR4
2/2
and MyD88
2/2
male mice were subjected to sepsis by cecal ligation and puncture (CLP). Twenty four hours later, kidney
tissue and blood samples were collected for analysis. The TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice that were subjected to CLP
had preserved renal morphology, and fewer areas of hypoxia and apoptosis compared with the wild-type C57BL/6 mice
(WT). MyD88
2/2
mice were completely protected compared with the WT mice. We also observed reduced expression of
proinflammatory cytokines in the kidneys of the knockout mice compared with those of the WT mice and subsequent
inhibition of increased vascular permeability in the kidneys of the knockout mice. The WT mice had increased GR1
+low
cells
migration compared with the knockout mice and decreased in GR1
+high
cells migration into the peritoneal cavity. The
TLR2
2/2
, TLR4
2/2
, and MyD88
2/2
mice had lower neutrophil infiltration in the kidneys. Depletion of neutrophils in the WT
mice led to protection of renal function and less inflammation in the kidneys of these mice. Innate immunity participates in
polymicrobial sepsis-induced AKI, mainly through the MyD88 pathway, by leading to an increased migration of neutrophils
to the kidney, increased production of proinflammatory cytokines, vascular permeability, hypoxia and apoptosis of tubular
cells.
Citation: Castoldi A, Braga TT, Correa-Costa M, Aguiar CF, Bassi E
ˆJ, et al. (2012) TLR2, TLR4 and the MYD88 Signaling Pathway Are Crucial for Neutrophil Migration
in Acute Kidney Injury Induced by Sepsis. PLoS ONE 7(5): e37584. doi:10.1371/journal.pone.0037584
Editor: Holger K. Eltzschig, University of Colorado Denver, United States of America
Received January 23, 2012; Accepted April 22, 2012; Published May 24, 2012
Copyright: ß2012 Castoldi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Brazilian Foundation – Fundac¸a
˜o de Apoio a
`Pesquisa do Estado de Sa
˜o Paulo (FAPESP), Grants Number: 07/07139-3),
International Associated Laboratory (CNPq- National Counsel of Technological and Scientific Development), and National Institute of Science and Technology
(INCT) Complex Fluids. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: niels@icb.usp.br
Introduction
Severe sepsis is the major cause of acute kidney injury (AKI) (2–
4) . Despite all efforts to better comprehend this pathology, little
progress has been achieved. This might be due to the fact that
most research groups have focused more on proving that AKI is
mainly caused by changes in kidney hemodynamics, while other
groups have shown the importance of non-hemodynamic factors
in the establishment of this disease, such as immunological factors
[1,2]. The kidney damage after sepsis is likely a result of these two
important contributions, starting with the recognition of bacterial
products by Toll-like receptors (TLRs), which recognize patho-
gens, such as PAMPs (pathogen associated molecular patterns) and are
responsible for the inflammatory cascade in sepsis.
Signal transduction through TLRs is partially mediated by a
TLR adapter protein MyD88 (Myeloid Differentiation Factor 88).
MyD88 can bind to the TIR domain and recruit signaling
proteins, leading to the activation of transcription factors, such as
NF-kB(Nuclear Factor- kappa B) and AP-1 (Activator Protein 1), and the
expression of genes related to the inflammatory response, such as
tubular necrosis factor TNF-a, interleukin- 6 and IL-1b.
Several studies have shown the importance of TLRs in the
development of sepsis, but few studies have correlated the
activation of TLRs with the development of AKI. During sepsis,
TLRs are activated by bacteria and endogenous ligands such as
HMGB-1 and HSP70, which are released during cellular stress,
and interact with the immune system in the extracellular
environment [3,4]. These alarmins exacerbate the kidney inflam-
matory process during sepsis, which results in greater damage and
necrosis.
Thus, we hypothesized that mice deficient in TLR2 and TLR4
and the adaptor protein MyD88 respond less effectively to
bacteria, leading to a milder inflammatory process and therefore
weaker protection of renal function compared with wild-type mice.
PLoS ONE | www.plosone.org 1 May 2012 | Volume 7 | Issue 5 | e37584

Materials and Methods
Mice
We used male C57BL/6 mice aged between 6 and 8 weeks,
weighing 20 g to 28 g, which were genetically deficient (
2/2
) for
TLR2, TLR4 and MyD88. The animals were provided by the
animal facility of the Center for Development of Experimental
Models for Medicine and Biology CEDEME-UNIFESP. The
animals were housed at the Nephrology Division, UNIFESP, in
cages containing a maximum of five animals, with the artificial
light/dark 12-hour cycle at a constant temperature of 22uC and
supplemented with water and food ad libitum.
Cecal ligation puncture (CLP) model
For the induction of sepsis, the animal’s cecum was punctured
twice with a 23-gauge needle, followed by light compression to
ensure the removal of intestinal contents. The animals were killed
24 hours after sepsis induction.
Assessment of renal function
Serum creatinine was measured by Jaffe´’s modified method.
Serum urea was measured using a Labtest Kit (Labtest, Minas
Gerais, Brazil).
RNA extraction and Real-Time PCR
Total RNA was isolated using the Trizol Reagent (Invitrogen,
Carlsbad, CA). First-strand cDNAs were synthesized using the
MML-V reverse transcriptase kit (Promega, Madison, Wisconsin,
USA). Reverse transcriptase polymerase chain reaction (PCR) was
performed using TaqMan probes. mRNA expression was
normalized to HPRT. The values were expressed relative to a
reference sample (the calibrator) and sham-operated samples. The
Ct (threshold cycle) for the target gene and for the internal control
were determined for each sample. Samples were run in triplicate.
The relative expression of each mRNA was calculated by 2
2DDCT
.
All experimental sample values are expressed as the n-fold
difference relative to the calibrator. The primers used are
described in Data S1 and S2.
Histology and quantification of ATN
The quantification of acute tubular necrosis (ATN) was
performed with H&E (Hematoxylin and Eosin) staining, and
analysis was performed using the computer program Image Pro
Plus for Windows (USA) coupled to an optical microscope
Olympus BX40F-3 (Olympus Optical Company, Japan) for
capturing and digitizing the images of the fields evaluated. For
the quantitative analysis, we used the computer program Image
Lab (Sa˜o Paulo, Brazil). Results were expressed as the percentage
of area affected (selected) in relation to the total area of the field
measured (0.073 cm
2
). We chose 20 fields randomly in an increase
of 20 times per slide. We studied five animals in each group. The
analysis was performed in such a way that the observer had no
access to material identification.
Flow cytometry
The kidneys were harvested, opened, macerated and sieved (70–
100 mm) with cold RPMI. The cells were resuspended in a solution
of DNAse/Collagenase (1 mg/2 mg per mL) and later separated
by Percoll gradient. The peritoneal lavage was collected after CLP
and centrifuged. Samples (,2 million cells) were then collected,
centrifuged and resuspended in 20 mL of FACS buffer. We then
labeled the cells with anti-GR1 APC and anti-F4/80 PERCP
monoclonal antibodies for the analysis of surface molecules and
anti-TNF-aPE for the analysis of the intracellular molecule (BD
Biosciences). The samples were analyzed using a FACSCanto
device using the FACSDIVA software (BD Biosciences) and then
analyzed with the software FlowJo (Tree Star, San Carlo, CA).
Background fluorescence was determined using unlabeled cells,
and compensation was performed using cells stained with APC,
PERCP and PE. We analyzed 1,000,000 events.
Assessment of renal microvascular protein leakage using
Evans blue dye
The microvascular leakage was assessed with Evans blue dye as
previously described [5,6]. The amount of Evans blue dye was
analyzed by measuring the absorbance at 620 nm. Results were
calculated from a standard curve of Evans blue dye and expressed
as micrograms of Evans blue dye per mg of kidney (wet weight).
Immunohistochemistry (IHC)
Pimonidazole (Chemicon International, Inc., CA, USA) was
administrated intraperitoneally at a dose of 60 mg/kg 1 hour
before sacrifice and detected by the Hypoxyprobe-1 Pab2627
(1:500) primary antibody as described previously [5]. Immuno-
histochemistry with cleaved caspase-3 antibody (diluted 1:1000
(Asp175), Cell Signaling Technology, Beverly, MA, USA) was also
performed as previously described [7]. Immunohistochemistry for
NF-kB p65 was also performed (#8242, diluted 1:100, Cell
Signaling Technology, Beverly, MA, USA). The presence of
primonidazole-HCL , cleaved caspase-3 and NF-kB p65 in renal
tissue was quantitated as a percentage in the cortex and medulla
using a computer program for image analysis (KS300, Zeiss
system). The average area of each sample was calculated for each
kidney.
Assessment of apoptosis
To detect apoptotic cells, the In situ Cell Death Detection Kit
TMR red (Roche Diagnostics GmbH, Mannheim, Germany) was
used (TUNEL technology).
Detection of Myeloperoxidase (MPO) in renal tissue
MPO in renal tissue was estimated as previously described by
Hillegass et al. [8]. The reading was performed in a spectropho-
tometer at a wavelength of 460 nM.
Western blotting analysis
Primary mouse IKKaantibody (SC-166231, Santa Cruz
Biotechnology, Inc) was used following manufacturer-recom-
mended dilutions, followed by a peroxidase-conjugated anti-mouse
IgG antibody (Jackson ImmunoResearch Laboratories, West-
Grove, USA). Mouse primary anti–b-tubulin or anti-b-actin
antibody (Sigma, St. Louis, USA) was also used to confirm and
estimate the loading and the transfer. We used the software
GeneSnap (Syngene, USA) and Gene Tools (Syngene, USA) to
analyze the bands.
Neutrophil depletion
Purified GR1 antibody RB6-8C5 (DNAX Research Institute,
Palo Alto, CA, USA) was obtained from a hybridoma culture
supernatant. To deplete the mice of neutrophils, a single dose of
0.25 mg was administered intraperitoneally 24 hours before sepsis.
Treatment with this dose of antibody induced severe neutropenia
for up to 5 days.
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Bacteria count in the peritoneal cavity
Quantitative bacterial culture was performed for peritoneal
colony-forming units (CFU) of control mice and 24 hours after
sepsis induced by CLP. The CFU were determined after serial
dilution, and culture medium agar was inoculated with 50
microliters of 1610
6
CFU and incubated in an oven at 37uC for
18 h.
CBA (Cytometric Bead Array)
Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cyto-
kine Kit (BD Biosciences) was performed to quantify IL-6, TNF-a
and IL-17 in the peritoneal fluid as described by manufacturer.
ELISA
To analyze the secretion of IL-1bin the peritoneal cavity after
sepsis, we used ELISA assay (R&D Systems, Minneapolis, MN,
USA).
Statistical analysis
The data are presented in graphs showing average and standard
deviation (SD) or median and lower and upper ranges (histomor-
phometric analysis). T tests, the Mann-Whitney test and ANOVA
on ranks tests were used to compare the data. The PCR results are
presented as a ratio of the calibrator gene HPRT and presented in
arbitrary units (AU). Differences were considered statistically
significant with p less than 0.05. To study survival, the animals
Figure 1. Effect of the absence of TLR2, TLR4 and MyD88 in the survival and in the development of acute Kidney Injury of animals
subjected to CLP. (a) Survival of WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice subjected to sepsis by CLP. Mice were evaluated twice each day until
the time 192 h after surgery. Results are representative experiment with 10 animals/group (WT vs MyD88
2/2
p,0.05). (b) Bacterial count in the
peritoneal cavity 24 hours after CLP . (c and d) Renal function of WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice 24 hours after CLP, assessed by levels of
serum creatinine and blood urea. (e) Gene expression of KIM-1 in kidney of control and WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice 24 hours after
CLP. The mRNA was normalized to HPRT expression and compared to normal group. Data shown as mean 6standard deviation (SD), *** p,0. 0001
vs WT; ** p,0.01 vs WT; *p,0.05 vs WT.
doi:10.1371/journal.pone.0037584.g001
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were monitored two times daily for 8 days (192 hours) after CLP.
The long-rank test was used for analysis of the survival curve. All
statistical analyses were performed with the aid of GraphPad
PRISMH.
Results
MyD88 knockout improves survival after sepsis-induced
AKI
Initially, we observed that there was an up-regulation of TLR2,
TLR4 and MyD88 in the WT mice that were subjected to sepsis.
Figure 2. Acute tubular necrosis in TLR2, TLR4 and MyD88 deficient mice subjected to CLP. Histology and quantification of ATN of
control(a) and WT(b), TLR2
2/2
(c), TLR4
2/2
(d) and MyD88
2/2
mice (e) 24 hours after CLP. Results of a representative experiment with 5 animals per
group. Data shown as mean 6standard deviation (SD), *** p,0.0001 vs WT.
doi:10.1371/journal.pone.0037584.g002
Figure 3. Endogenous ligands expression in kidney in animals subjected to CLP. mRNA expression of HMGB1 (a), HSP70 (b) in the kidney
of WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group.
Results of a representative experiment with 5 animals per group. Data presented as mean 6standard deviation (SD), * p,0.05 vs WT; ** p,0.01 vs
WT.
doi:10.1371/journal.pone.0037584.g003
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TLRs and Septic Renal Acute Injury
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We also observed that in the absence of TLR2, there is an over
expression of TLR4. Similarly, in the absence of TLR4, there was
an over expression of TLR2 (Figure S1).
To determine whether the absence of TLR2, TLR4 and
MyD88 affects the mortality in AKI induced by CLP, we
evaluated the survival of all mice for 192 hours after the induction
of sepsis. We observed that the MyD88
2/2
mice had higher
survival rates compared with other groups (p,0.05) (Figure 1a),
but the bacterial count in the peritoneal cavity was higher in the
MyD88
2/2
mice (Figure 1b).
Next, we observed that the MyD88
2/2
mice were completely
protected from renal dysfunction caused by sepsis, while the
TLR2
2/2
and TLR4
2/2
animals only seemed to improve but did
not reach statistical significance (Figure 1c and 1d). However,
we observed a significant increase in KIM-1 (kidney injury molecule-1)
gene expression in the WT mice and a decrease in the TLR2
2/2
and TLR4
2/2
mice and an even greater decrease in the
MyD88
2/2
mice (Figure 1e). Corroborating these data, the
renal histology showed that the WT mice developed ATN with
loss of brush borders and vacuolar degeneration 24 hours after
CLP, while the TLR2
2/2
and TLR4
2/2
mice presented with less
serious injury, demonstrating significant protection. Further, the
MyD88
2/2
mice showed no such histological changes (Figure 2),
showing a strong protective effect of the lack of MyD88 on AKI.
Sepsis induces expression of endogenous ligands in the
kidney
TLR activation in the kidney during sepsis can be elicited by
endogenous ligands called alarmins, such as HMGB1 and HSP70
[9]. We studied the mRNA levels of HMGB1 (Figure 3a) and
HSP70 (Figure 3b) in the kidney of the control, WT, TLR2
2/2
,
TLR4
2/2
and MyD88
2/2
mice 24 hours after CLP. We
observed that the WT, TLR2
2/2
, and TLR4
2/2
mice had
increased expression of these two alarmins in the kidney after
CLP. MyD88
2/2
mice showed a decrease in endogenous ligand
expression after CLP.
Figure 4. Expression of pro-inflammatory cytokines after CLP. mRNA expression of IL-1b(a), TNF-a(b), IL-6 (c), KC (d), IL-17 (e) and iNOS (f) in
the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment
with 5 animals per group. (g) Analysis of expression of a-IKK in kidney of WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice 24 hours after CLP. Results
representative of a experiment with two animals/group Data shown as mean 6standard deviation (SD), * p,0.05, ** p,0.01 and *** p,0.001 vs WT.
doi:10.1371/journal.pone.0037584.g004
Figure 5. NF-kB activity in the kidney of TLR2, TLR4 and MyD88 deficient mice subjected to CLP. (a) Histological analysis of NF-kB p65 by
immunohistochemistry of control (a), WT (b), TLR2
2/2
(c), TLR4
2/2
(d) and MyD88
2/2
(e) mice respectively, 24 hours after CLP . Results of a
representative experiment with five animals per group. (b) Score of NF-kB p65 in kidney represented in figure A. Data shown as mean 6standard
deviation (SD), *** p,0.01 vs WT.
doi:10.1371/journal.pone.0037584.g005
TLRs and Septic Renal Acute Injury
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TLR2, TLR4 and MyD88 deficiency is associated with
lower renal expression of pro-inflammatory cytokines
TLR2 and TLR4 activation via MyD88 leads to the nuclear
translocation of NF-kB and AP-1, resulting in the expression of
genes related to the inflammatory response. TNF-ais a major pro-
inflammatory molecule activated in experimental models of sepsis
and is considered one of the main mediators of AKI [10]. We
observed lower mRNA levels of IL1-b, IL-6 and TNF-ain
TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice compared with WT
(Figure 4a, b and c) 24 hours after CLP. Furthermore, we
observed an increased expression of IL-17 and KC in the WT
mice and reduced expression in the TLR2
2/2
and TLR4
2/2
mice, while in the MyD88
2/2
mice, the expression was almost
absent, suggesting that the absence of MyD88 causes reduced
migration of neutrophils into the kidney after CLP (Fig. 4 d and
e).
We also studied the gene expression of iNOS in kidney of these
mice, since iNOS is considered an important factor that
contributes to the pathogenesis of septic shock. We observed a
significant decrease of iNOS in the knockout mice, especially in
the MyD88
2/2
mice (Figure 4 f). We also observed that the
knockout mice subjected to CLP had lower activation of NF-kB
compared with the WT mice (p,0.05) (Figure 4g). The
significant decrease in inflammation in knockout mice seems to
be a consequence of decreased NF-kB activity in the kidney. We
observed by immunohistochemistry that the knockout mice were
significantly protected from the translocation of NF-kB p65 into
the nucleus compared to WT, thus culminating in decreased
transcription of inflammatory molecules (Figure 5a and b).
Figure 6. Renal vascular permeability in TLR2, TLR4 and MyD88
deficient mice subjected to CLP. Analysis of vascular permeability in
the kidney of the control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice
24 hours after CLP. Results are representative of an experiment with
five animals per group. Data shown as mean 6standard deviation (SD),
** p,0.01 vs WT and * p,0.05 vs. WT.
doi:10.1371/journal.pone.0037584.g006
Figure 7. Renal hypoxia in TLR2, TLR4 and MyD88 deficient mice subjected to CLP. (a) Histological analysis of hypoxia by
immunohistochemistry (IHC) of control (a), WT (b), TLR2
2/2
(c) , TLR4
2/2
(d) and MyD88
2/2
mice (e) respectively, 24 hours after CLP. Results of a
representative experiment with 5 animals per group. (b) Score of hypoxia level represented by iminohistochemistry in figure A. (c) Gene expression of
HIF-1ain the kidney 24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative
experiment with 5 animals per group. Data shown as mean 6standard deviation (SD), * p,0.05 vs WT and ** p,0.01 vs WT.
doi:10.1371/journal.pone.0037584.g007
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The absence of TLR2, TLR4 and MyD88 inhibits the
increase in renal vascular permeability after CLP
We observed that sepsis induced an increase in vascular
permeability in the kidneys of the WT mice (4.2060.33 mg/mg);
however, the TLR2
2/2
(2.9960.18 mg/mg), TLR4
2/2
(2.3160.31 mg/mg, p,0.01) and MyD88
2/2
mice
(2.3760.22 mg/mg, p,0.01) had lower vascular permeability
indexes compared with the WT septic mice, underscoring the
renal protection already observed (Figure 6).
Effects of sepsis on renal tubular hypoxia
We analyzed whether the absence of TLRs and consequent
decreased production of cytokines could lead to organ protection
through lower tubular hypoxia. The presence of hypoxia was
Figure 8. Apoptosis in the kidney of TLR2, TLR4 and MyD88 deficient mice subjected to CLP. (a) Histological analysis of apoptosis by
immunohistochemistry of cleaved caspase 3 of control (a), WT (b), TLR2
2/2
(c), TLR4
2/2
(d) and MyD88
2/2
(e) mice respectively, 24 hours after CLP .
Results of a representative experiment with 5 animals per group. (b) Histological analysis of apoptosis by immunofluorescence to TUNEL of control
(a), WT (b), TLR2
2/2
(c), TLR4
2/2
(d) and MyD88
2/2
mice (e) respectively, 24 hours after sepsis. (c) Score of cleaved caspase-3 in kidney represented
in figure A. (d) Score of TUNEL in kidney represented in figure B. (e) Gene expression of BCL-2 in the kidney 24 hours after CLP. The mRNA was
normalized to HPRT expression and compared to normal group. Results of a representative experiment with 5 animals/group. Data shown as mean 6
standard deviation (SD), * p,0.05 vs WT and ** p,0.01 vs WT.
doi:10.1371/journal.pone.0037584.g008
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Figure 9. Secretion of TNF-a, IL-1b, IL-6 and IL-17 in the peritoneal cavity after CLP. (a) Flow cytometry of the peritoneal cavity 24 hours
after CLP in control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice. (b) Frequency of TNF-a/F4/80+cells population in control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice 24 hours after CLP.(c) Analysis of TNF-a, (d) IL-6, (e) IL-17 by CBA in control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
. (f) Analysis of
IL-1bby ELISA in control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
. Data shown as mean 6standard deviation (SD). * p,0.05 vs. WT, ** p,0.01 vs. WT
and ***p,0.001 vs. WT.
doi:10.1371/journal.pone.0037584.g009
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observed in cortical tubules but not in the medullar areas of the
kidney. After CLP, we observed less areas of hypoxia in the
TLR2
2/2
, TLR4
2/2
and MyD88
2/2
kidneys when compared to
the control WT kidneys (Figure 7a and b). We also evaluated the
presence of renal hypoxia by HIF1-amRNA expression analysis
and observed increased expression of this gene induced by hypoxia
in the WT mice kidneys (Figure 7c).
Effects of sepsis on apoptosis in the kidney
Recent studies have shown that apoptosis may also be involved
in the pathogenesis of sepsis [11]. We observed less cleaved
caspase 3 in the tubular cells after CLP in the MyD88
2/2
mice
compared with those cells from the other mice (Figures 8a and
c). The TUNEL assay showed a similar result (Figures 8b and
d), but this method appeared to be more sensitive and showed that
the WT mice underwent more apoptosis than the knockout mice.
Interestingly, we observed that the MyD88
2/2
mice had higher
levels of the anti-apoptotic factor Bcl-2 than the WT mice
(Figure 8e).
Effects of TLR2, TLR4 and MyD88 knockout on secretion
of TNF-a, IL1-b, IL-6 and IL-17 in the peritoneal cavity
after CLP
We observed a significant increase in the production of TNF-a,
IL-1b, IL-6 and IL-17 from the peritoneal cavity in the WT mice
and a significant decrease in the knockout mice (Figure 9a, b, c,
d, e and f). These findings corroborate the gene expression of
these molecules in the kidneys of the septic mice.
Effect of TLR2, TLR4 and MyD88 deficiency on migration
of neutrophils to the peritoneal cavity after CLP
To investigate whether the absence of the receptors of innate
immunity contributes to the local inflammatory response in sepsis
through the migration of neutrophils to the peritoneal cavity, we
analyzed the expression of the surface molecule GR1 in the
Figure 10. Migration GR1
+
neutrophils into the peritoneal cavity after CLP. (a) Flow cytometry of the peritoneal cavity 24 hours after CLP in
control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice. (b, c) Frequency of GR1
low
and GR1
high
cells population in control, WT, TLR2
2/2
, TLR4
2/2
and
MyD88
2/2
mice 24 hours after CLP. Data shown as mean 6standard deviation (SD).* p,0.05 vs. WT and ** p,0.01 vs. WT.
doi:10.1371/journal.pone.0037584.g010
TLRs and Septic Renal Acute Injury
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peritoneal cavity after CLP. We observed significantly higher
migration of neutrophils (GR1
+low
) to the peritoneal cavity after
CLP in the WT mice compared with the TLR2
2/2
, TLR4
2/2
and MyD88
2/2
knockout mice (Figure 10 a and b). The
population with high expression of GR1
+
(GR1
+high
) was
significantly decreased in the peritoneal cavity of the WT mice
compared with the other knockouts (Figure 10a and c). The
population of GR1
+low
cells was smaller in the peritoneal cavity
after sepsis in the WT mice [12], but this population was also
smaller in the knockout mice, which had a higher migration of
GR1
+high
cells. The absence of innate immune receptors resulted
in decreased migration of the GR1
+low
population and increased
migration of the GR1
+high
population to the peritoneal cavity,
suggesting a role for these cells in the initial response to infection in
the absence of TLRs.
Effect of TLR2, TLR4 and MyD88 deficiency on neutrophil
migration to the kidney after CLP
To study whether the absence of TLRs and MyD88 leads to
reduced neutrophil migration to the kidney after CLP, we first
analyzed the expression of IL-17 and KC in the kidneys data
shown in figure 4. We also evaluated the presence of neutrophils in
the kidney by testing the myeloperoxidase activity. We observed
that WT animals had a significant increase in myeloperoxidase
activity compared with the control mice, and the MyD88
2/2
cells
had myeloperoxidase activity similar to that of normal controls
(Figure 11a). We also observed a significant decrease in
neutrophil migration to the kidneys of the TLR2
2/2
, TLR4
2/2
and especially MyD88
2/2
mice, as measured by GR1 by flow
cytometry of renal tissue (Figure 11b and c).
Neutrophil depletion in WT mice leads to amelioration of
AKI after sepsis
The decreased migration of neutrophils to the kidneys of the
knockout mice after sepsis led us to hypothesize that neutrophils
are also the main immune cells involved in the pathogenesis of
AKI secondary to sepsis. Based on this hypothesis, we depleted
neutrophils. We observed a significant decrease in spleen
neutrophils in mice treated with RB6-8C5, demonstrating that
depletion was achieved (Figure 12a). We found that the depleted
mice had a significant improvement in renal function (Figure 12b
and c). We also observed a significant decrease in the expression
of TNF-a, IL-6, IL-1b, IL-17, KC and iNOS 24 hours after CLP
(Figure 12d and e). These results suggested that an absence or
even decrease in neutrophils confers protection against sepsis-
induced AKI.
Discussion
Our data suggest that hypotension during sepsis leads to tissue
hypoxia, which could activate tubular and endothelial cells,
culminating in the release of cytokines and chemokines. This
release results in the increased vascular permeability that in turn
can lead to the necrosis or apoptosis of tubular cells. Indeed, we
observed tubular hypoxia as well as acute tubular necrosis and
apoptosis in the WT mice. Hypoxia is considered a critical factor
in organ dysfunction in sepsis [13]. Apart from hypotension, NF-
kB also regulates the expression of HIF1-a[14], which increases in
renal injury. The gene expression analysis of HIF1-aand
qualitative analysis of hypoxia in the kidneys suggested that the
TLRs and especially MyD88 are involved in renal injury. Some
studies have shown that even without lowering blood pressure, the
AKI induced by LPS or by other toxins results in the release of
Figure 11. Neutrophil infiltration in renal tissue 24 hours after CLP. Gene expression of KC (a), IL-17 (b) , iNOS (c) in kidney 24 hours after
CLP in WT, TLR2
2/2
, TLR4
2/2
and MyD882/2mice. The mRNA was normalized to HPRT expression and compared to normal group. (d) Assay of
myeloperoxidase activity in kidney of control, WT, TLR2
2/2
, TLR4
2/2
and MyD88
2/2
mice 24 hours after CLP. (e) GR1 expression in the kidney of mice
24 hours after CLP. (f) Results of a representative experiment with five animals/group. Data shown as mean 6standard deviation (SD).* p,0.05 vs WT
and ** p,0.01 vs WT. ND: not detected.
doi:10.1371/journal.pone.0037584.g011
TLRs and Septic Renal Acute Injury
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Figure 12. Partially neutrophils depletion in WT mice subjected to CLP. (a) Flow cytometry of spleen to determine the success of depletion
in control mice without depletion in mice only depleted and depleted mice subjected to sepsis (*p,0.05 vs control). (b, c) Kidney function of WT
mice and WT depleted mice subjected to CLP at time 24 hours, assessed by levels of serum creatinine and blood urea (*p,0.05 vs WT sepsis;
TLRs and Septic Renal Acute Injury
PLoS ONE | www.plosone.org 12 May 2012 | Volume 7 | Issue 5 | e37584

inflammatory mediators [15,16]. Changes in vascular permeability
are also important in the pathogenesis of sepsis-induced organ
injury after significant production of cytokines, which lead to
hemodynamic imbalance [17]. Renal vascular permeability
increased after CLP in the WT mice, whereas in the TLR2
2/2
,
TLR4
2/2
and MyD88
2/2
mice, vascular permeability was
similar to control levels. Increased vascular permeability can
cause a compression of peritubular capillaries [18], hemoconcen-
tration and decreased microvascular flow.
We found that the MyD88-deficient mice were fully protected
from damage caused by sepsis. The protection of renal function
was not observed in the TLR2
2/2
or TLR4
2/2
mice when we
analyzed the biochemical parameters, but there was a significant
improvement in ATN in these mice compared with WT by renal
histology, suggesting that TLR2 and TLR4 are required during
polymicrobial sepsis. However, the absence of ATN, the
significant decrease of the functional parameters and increased
expression of MyD88 in the WT mice after CLP suggested that the
mechanism of pathogen recognition in the kidney by both TLR2
and TLR4 occurs primarily through the adapter molecule
MyD88. Recently, in addition to histological and serum markers,
the molecule KIM-1 [19] was described as a marker of kidney
damage. Corroborating our findings of ATN, we also observed a
significant decrease of KIM-1 in the TLR2
2/2
and TLR4
2/2
and especially in the MyD88
2/2
mice. Thus, we showed that the
innate immunity receptors TLR2 and TLR4 and the adapter
protein MyD88 are important in the development of AKI
secondary to sepsis.
Wolfs and colleagues showed that TLR2 and TLR4 are
constitutively expressed predominantly in the renal epithelial cells
of distal and proximal tubules, the epithelium of Bowman’s
capsule and glomerular and endothelial cells, and the expression
increases during the inflammatory process in the presence of IFN-
cand TNF-a[20]. In kidney tissue after sepsis, TLR2 and TLR4
mRNA is highly expressed in the WT mice, which also have a six-
fold increased expression of MyD88. In the TLR2
2/2
mice, we
observed an overexpression of TLR4 compared with WT. It has
been suggested that TLR2 is also responsible for LPS recognition
[21], and in its absence, TLR4 is overexpressed. This may account
for the lack of AKI in the MyD88
2/2
mice.
TLRs may also be activated during sepsis by alarmins. Previous
studies showed increased levels of HMGB1 and HSP70 in
conditions of cell damage and inflammation [4,9]. We showed
that these ligands are highly expressed in the WT mice and have
decreased expression in the knockouts mice after sepsis. These
ligands can cause robust production of cytokines, and consequent-
ly the development of AKI in septic mice.
IL-1b, TNF-a, and IL-6 were decreased in the kidneys of the
TLR2
2/2
, TLR4
2/2
and especially in MyD88
2/2
mice as a
consequence of decreased NF-kB p65 translocation to the nucleus.
Previous studies with MyD88
2/2
mice showed improved renal
function of these animals after sepsis and also decreased serum
levels of TNF compared with controls [7]. Furthermore, studies
have shown that levels of TNF-a, IL-1b, and IL-6 are markedly
increased in patients with established sepsis [22]. In the kidney,
endotoxin causes mesangial cells to release TNF-a[23,24]. LPS
treatment in mice deficient in the TNF receptor caused lower
renal tubular apoptosis and infiltration of neutrophils compared
with WT mice [25,26].
Recently, the role of apoptosis in the pathogenesis of sepsis has
also been explored. Administration of the caspase inhibitors or
overexpression of the anti-apoptotic protein Bcl-2 significantly
improves the survival of CLP-induced septic mice [27,28,29]. Our
study showed that septic MyD88
2/2
mice have high expression of
the anti-apoptotic molecule BCL-2 in the kidney and absence of
apoptosis, indicating a possible mechanism that inhibits apoptosis
in these animals.
Sepsis results in decreased migration of neutrophils to the
infection site, hindering the removal of pathogens and initiating a
robust inflammatory response characterized by an inadequate
sequestration of neutrophils to organs [30]. We believe that in our
model, the renal dysfunction was due to an exaggerated response
to infectious stimuli and the activation and recruitment of
neutrophils to the kidney. Andonegui et al. demonstrated that
the absence of CD14 and TLR4 prevented neutrophil sequestra-
tion to the lungs after treatment with LPS [31], and Goseman et
al. also described the impairment of neutrophil migration to the
lungs of mice deficient in TLR4 [32]. Alves-Filho et al. showed
that deficiency in the migration of neutrophils to the infection after
CLP is associated with increased mortality [33] in TLR4 deficient
mice. Alves-Filho et al. also showed that TLR2 has a detrimental
role in polymicrobial sepsis and suggested that the inhibition of
TLR2 signaling may improve survival in sepsis [34].
We found increased TNF-asecretion by macrophages in the
peritoneal cavity in the WT mice compared with the knockouts
mice. The significant synthesis of TNF-ais responsible for the
severe sepsis and recruitment of neutrophils to the infectious focus.
Surprisingly, we found a significant decrease in the infiltration of
the GR1
+low
population, described as resident macrophages [35],
in the peritoneal cavity of knockout mice compared with WT
mice. Furthermore, we found a significant decrease in the
GR1
+high
population, which some authors describe as granulo-
cytes, in the peritoneal cavity of the WT mice. Miyazaki and
colleagues showed that 60 min post injection of LPS, the
proportion of GR1
+high
decreased and this process was depended
on TNF-a. Therefore, the decrease of GR1
+high
coincided with the
increase in cell accumulation in mesenteric lymph nodes [36]. This
may be related to the activation of the inflammatory response by
other pathways of pathogen recognition and the failure of
migration of neutrophils to the peritoneal cavity in the WT mice
after sepsis. This would also explain the migration of neutrophils in
the knockout mice.
In the kidneys of knockout mice with sepsis, especially the
MyD88 knockout mice, we found decreased expression of IL17,
KC, iNOS, and MPO activity. Furthermore, the expression of
GR1 decreased. We concluded that the migration of neutrophils to
the kidney after sepsis is dependent on the activation of TLR and
MyD88 and mostly decreases the release of pro-inflammatory
cytokines. In septic mice depleted of neutrophils, we observed a
significant improvement in renal function and decreased expres-
sion of TNF-a, IL-6, IL-1b, IL-17, KC and iNOS in the kidneys.
Experimental models using antagonists and knockout mice that
decrease the activity of neutrophils have shown improved organ
function [37,38,39].
The TLRs are important for tissue protection against bacteria,
but in a persistent infectious process, they can be harmful.
Persistent inflammation can lead to kidney damage and produc-
**p,0.01 vs WT sepsis). (d, e, f, g, h, i) Gene expression of TNF-a, IL-6, IL1-b, IL-17, KC and iNOS in the kidney of WT mice and WT depleted mice
24 hours after CLP. The mRNA was normalized to HPRT expression and compared to normal group. Results of a representative experiment with three
animals/group. Data shown as mean 6standard deviation (SD). *p,0.05 vs WT sepsis and **p,0.01 vs WT sepsis.
doi:10.1371/journal.pone.0037584.g012
TLRs and Septic Renal Acute Injury
PLoS ONE | www.plosone.org 13 May 2012 | Volume 7 | Issue 5 | e37584

tion of alarmins, which may exacerbate the TLR activation of the
inflammatory response and thus cause harmful positive feedback.
Supporting Information
Figure S1 mRNA expression of TLR2 (a), TLR4 (b),
MyD88 (c) in the kidney of WT, TLR2
2/2
, TLR4
2/2
and
MyD88
2/2
mice 24 hours after CLP. The mRNA was
normalized to HPRT expression. Results of a representative
experiment with 5 animals per group. Data shown as mean 6
standard deviation (SD), ND: Not Detected, *** p,0. 0001 vs
WT; ** p,0.01 vs WT.
(TIF)
Data S1 RT-PCR was performed using Taqman prim-
ers (Applied Biosystems, USA).
(TIF)
Data S2 RT-PCR was performed using Syber primers
(Applied Biosystems, USA).
(TIF)
Acknowledgments
The authors thank to Bernardo Paulo Albe for preparing the histology
slides and Claudia Silva Cunha for technical assistance.
Author Contributions
Conceived and designed the experiments: AC NOSC. Performed the
experiments: AC EJB RC-S. Analyzed the data: TTB EJB MAR.
Contributed reagents/materials/analysis tools: TTB MC-C CFA EJB
RC-S RME FS PMM-V MAC MAR MIH AP-S GMG. Wrote the paper:
AC. Co-orientation: GMG NOSC. Contributed to the preparation of the
paper: NOSC.
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