Activation of M1 macrophages in sepsis-induced acute kidney injury in response to heparin-binding protein
Activation of M1 macrophages in sepsis- induced acute kidney injury in response to heparin-binding protein
Li Xing 0 1
Lu Zhongqian 0 1
Song Chunmei 0
Chen Pingfa 0 1
He Lei 0 1
Jin Qin 0 1
Mu Genhua 0 1
Deng Yijun 0 1
0 Editor: Partha Mukhopadhyay, National Institutes of Health , UNITED STATES
1 Department of ICU, Yancheng City No.1 People's Hospital , Yancheng , China , 2 Nursing College of Nantong University , Nantong , China
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
High levels of HBP were obviously detected 24 h after sepsis-induced AKI. Heparin inhibited
HBP expression during sepsis-induced AKI. The suppression of HBP expression by heparin
injection after the establishment of sepsis-induced AKI resulted in a reduction in renal injury
severity accompanied with a significant repression of M1 macrophage activation and
expression of TNF-α and IL-6.
HBP plays an important role in the initial inflammatory reaction associated with
sepsisinduced AKI, presumably by activating M1 macrophages and suppressing TNF-α and IL-6
Acute kidney injury (AKI) is a common complication in critically ill patients and is associated
with increased morbidity and mortality [
]. Sepsis is the most common cause of AKI [
Animal models of sepsis have been developed and demonstrate that the pathogenesis of AKI is
caused by inflammatory cell infiltration, renal endothelial cell dysfunction, intrarenal
hemodynamic alterations, and renal cell apoptosis in the kidney [
]. Sepsis-induced AKI is caused by a
combination of multiple mechanisms, including inadequate vascular leakage/perfusion, local
tubular inflammation and cell cycle arrest [
]. Of these factors, significant tissue inflammation
in the kidney appears to be a critical mediator of sepsis-induced AKI [
]. Recent studies have
indicated that innate immunity and inflammatory signaling pathways are involved in the
pathogenesis of septic AKI, and these processes are initiated a few hours after injury by the infiltration
of immune cells from the kidney and are of central importance to kidney regeneration [
Macrophages, the most common type of leukocytes involved in the process of renal injury,
play different roles at different stages of injury [
]. Because of differences in the immune
microenvironment, macrophages can be classified into several phenotypes and functional
subclasses that exhibit different functions. In the early stage of sepsis, macrophages undergo M1
differentiation, resulting in the production of inflammatory mediators and AKI [
macrophages upregulate the expression of pro-inflammatory mediators, including inducible nitric
oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α), and increase the production
of reactive oxygen and nitrogen species [
]. In contrast, anti-inflammatory M2 macrophages
upregulate the expression of arginase-1 (Arg-1), scavenger and mannose receptors, and the
intracellular protein found in inflammatory zone 1 (FIZZ1). iNOS expression has been used as
a marker of M1 responses, whereas Arg-1 and FIZZ1 are classical inducers of M2 gene
Heparin-binding protein (HBP), also known as CAP37, is a promising biomarker for
predicting the development and prognosis of severe sepsis and septic shock and has recently been
proposed to be involved in the pathophysiology of AKI [
]. HBP acts as a chemoattractant
for neutrophils, T cells and monocytes and enhances monocyte cytokine release, phagocytosis
and adhesion to the endothelium . HBP induces inflammation and capillary leakage in the
kidney, as demonstrated by the findings of Fisher et al [
]. However, whether HBP induces
macrophages to promote inflammation is unknown. To date, there have been no studies on
the association of HBP with M1 macrophages in sepsis-induced AKI.
To further improve our understanding of inflammatory processes associated with
sepsisinduced AKI mouse, we used a sepsis-induced AKI mouse model to investigate the in vivo
expression of HBP. Meanwhile, the suppression of HBP expression by heparin injection after
the establishment of sepsis-induced AKI resulted in a reduction in renal injury severity
accompanied with a significant repression of M1 macrophage activation and expression of
Materials and methods
Six- to eight-week-old wild-type male C57BL6 mice were housed in specific pathogen-free
cages at 23±2ÊC and 60±10% humidity, with a 12-hour light/12-hour dark cycle and free access
to food and water. All animal procedures were performed in compliance with the Institute
of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals of the
National Institutes of Health and were approved by the Institutional Animal Care and Use
Committee of Nanjing Medical University.
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Septic animal models
CLP was performed using a previously described method [
], with slight modifications.
Briefly, a 4±0 silk ligature was placed 15 mm from the cecal tip after laparotomy under
isoflurane anesthesia. The cecum was punctured twice with an 18-gauge needle and gently squeezed
to express a small amount of fecal material before being returned to the central abdominal
cavity. In sham-operated animals, the cecum was located but neither ligated nor punctured. The
abdominal incision was closed in two layers with 6±0 nylon sutures. After surgery, animals
were fluid resuscitated with 40 ml/kg of subcutaneously administered sterile saline and given
free access to water but not food. During the surgical procedure, body temperature was
maintained at approximately 37ÊC. Animals were sacrificed by cervical dislocation after 72 h, blood
was collected, and kidney samples were harvested and stored at -80ÊC until further analysis.
Unfractionated heparin (UFH) preparation and administration
Unfractionated heparin (UFH) and phosphate-buffered saline liposomes were prepared
according to previously described methods [
]. mices received an intraperitoneal bolus of
0.4 U/g body weight of UFH or phosphate-buffered saline liposomes (Formumax, USA) 12 h
prior to sepsis-induced AKI, as previously reported.
Blood chemistry assay
After blood collection, the concentrations of blood urea nitrogen (BUN) and serum creatinine
(Scr) were immediately analyzed using a Roche Diagnostic analyzer (Roche, Indianapolis, IN).
The Scr content was determined using a creatinine (serum) colorimetric assay kit (Cayman
Chem, Ann Arbor, MI).
Renal histology analysis
Tissues were fixed with 10% formalin and embedded in paraffin. Four-micrometer sections
were stained with periodic acid-Schiff (PAS) reagent. Histological changes in the cortex and
the outer stripe of the outer medulla (OSOM) were assessed by quantitative measurements of
tissue damage. As tubular damage was mainly vacuolization, the damage was defined as
tubular vacuolar degeneration. The degree of kidney damage was estimated in 200X magnification
images of more than 100 randomly selected tubules for each animal using the following
criteria: 0, normal; 1, area of damage <25% of tubules; 2, damage in 25% to 50% of tubules; 3,
damage in 50% to 75% of tubules; and 4, 75% to 100% of tubules were affected. The histological
analysis was performed by two pathologists graded sections in a blind fashion to avoid bias.
RNA was isolated from snap-frozen kidneys stored at 80ÊC using standard procedures
(RNeasy, QIAGEN). RNA was treated with DNase1 (Invitrogen), and reverse transcription
was performed using 0.5 g of total RNA (iScript, Bio-Rad). PCR was performed on 1/20 of the
RT product using the following primer pairs: iNOS forward 5’-GAATTCCCAGCTCATCCG
GT-3’ and reverse, 5’- GGTGCCCATGTACCAAC CGGT-3’; Arg-1 forward, 5’-CCGCAG
CATTAAGGAAAGC-3’ and reverse, 5’-CCC GTGGTCTCTCACATTG-3’; HBP forward,
5’-ACAACCTCA ACGTCATCCTG G-3’ and reverse, 5’-
GTCTTCATTGAGGGCGTTGC3’; and β-actin forward, 5’-CAGTAACAGTCCGCCTAGAA-3’ and reverse, 5’-GATTACTG
CTCTGGCT CCTA-3’. The cycling conditions were a melting temperature (Tm) of 55ÊC for
30 cycles, and PCR products were visualized with ethidium bromide staining after separation
on 2% agarose gels and photographed.
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Renal tissue sections were subjected to immunohistochemical staining for F4/80 and Fi67. For
immunohistochemical staining, 3-mm renal sections were deparaffinized and rehydrated in a
graded alcohol series. The sections were immersed in 3% hydrogen peroxide for 10 min to
block endogenous peroxidase activity and then incubated in buffered normal horse serum to
block non-specific binding. Prior to immunochemistry, sections were subjected to antigen
retrieval by immersion in 0.1 mol/L citrate buffer (pH 6.0) for 25 minutes, followed by heating
in an electrical pressure cooker for 5 minutes. Sections were incubated with mouse
monoclonal anti-CD68 antibody (1:100, Abcam, USA) primary antibodies overnight at 4ÊC. Control
experiments omitted either the primary or secondary antibody. On the next day, sections were
incubated with a horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rat anti-mouse
secondary antibody (Beijing Zhongshan Biotechnology Co., Beijing, China) for 1 h at room
temperature. Then, 3, 3-diaminobenzidine tetrahydrochloride (DAB, Beijing Zhongshan
Biotechnology Co., Beijing, China) was applied to the slides to develop a brown color.
Counterstaining was performed with hematoxylin, and photomicrographs were captured with an
Western blot analysis
Whole-cell extracts were prepared. Protein samples were resolved on 10% SDS-PAGE gels,
transferred onto PVDF membranes, and blocked with 5% skim milk. Subsequently, the
primary antibody incubation was performed overnight at 4ÊC, followed by an incubation with an
HRP-conjugated secondary antibody conjugated for 1.5 h. Protein bands were detected using
an enhanced chemiluminescence (ECL) detection system (Pierce, Rockford, USA).
The results are presented as the means±SEM. The Wilcoxon test was used to analyze
nonparametric data. After the normal distribution of the data was assessed with the
KolmogorovSmirnov test, statistical comparisons between experimental groups were evaluated using
Student's t test and one-way ANOVA with SPSS 20.0 software (SPSS Inc., Chicago, IL, USA); a P
value <0.05 was considered significant.
Expression of HBP in the kidney following sepsis-induced AKI
To detect the renal HBP production during different sepsis-induced AKI, mice were subjected
to cecal ligation puncture (CLP) surgery and sacrificed at various times (Fig 1). Active renal
HBP production remained low in sham-operated animals. However, renal HBP production
significantly increased at 24 h in response to sepsis-induced AKI and then subsequently
decreased. To obtain more detailed evidence, we quantitatively assessed renal HBP expression
using real-time PCR analysis. High levels of HBP were detected 24 h post-injury, which
decreased over the following days.
Heparin downregulated HBP expression during sepsis-induced AKI
Heparin is a multifunctional negatively charged glycosaminoglycan that binds to HBP. Mice
were given an intraperitoneal bolus of heparin 12 h prior to the induction of sepsis-induced
AKI. We performed western blotting in mouse renal tissues to determine the expression of
HBP at 24 h (Fig 2). The heparin+CLP group exhibited a significant downregulation of TNF-α
expression than did the CLP and control (Ctl) groups.
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Fig 1. HBP mRNA levels following sepsis-induced AKI in mices. Mices were subjected to CLP surgery. Renal tissue
sections were collected from the CLP group at various times (0, 6, 12, 24, 36 and 48h). Levels of the HBP mRNA were
measured using RT-PCR (A) and quantified by densitometry (B). The data are presented as the means±standard errors
of the means (SEM); aP<0.05 compared with the sham group; bP<0.05 compared with the 36h group (n = 6 mices/
HBP increased renal damage and dysfunction during sepsis-induced AKI
Histological examinations and tubular damage scoring were performed on samples 24 h after
sepsis-induced renal injury (Fig 3). Widespread damage was observed in the form of a dilated,
flattened, and swollen epithelium and the loss of proximal tubular epithelial cells, along with
luminal cast formation. The damage was more severe in the CLP group and the Ctl group than
in the heparin+CLP group. The heparin+CLP group exhibited a significant reduction in blood
urea nitrogen (BUN) and serum creatinine (Scr) levels than did the CLP and Ctl groups,
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Fig 2. Effect of heparin on HBP protein levels with sepsis-induced AKI in mices. Mices were subjected to CLP
surgery. Twenty-four hours prior to the onset of CLP, mices were treated with heparin. Levels of the HBP protein in
the kidney were determined by Western blotting (A) and quantified by densitometry (B). Protein levels were
normalized to β-actin. Graphs represent means±SEM; aP<0.05 compared with the sham group; bP<0.05 compared
with the heparin+CLP group (n = 6 mices/group).
indicating that HBP increased renal damage and renal dysfunction during sepsis-induced
HBP increased macrophage infiltration during sepsis-induced AKI
Renal cortical tissue sections were stained with a specific antibodies against the mouse
macrophage marker CD68 for the assessment of the degree of macrophage infiltration during renal
injury after the CLP procedure (Fig 4). Sham-operated mice exhibited minimal interstitial
macrophage staining. However, mice that were subjected to CLP surgery exhibited significant
macrophage accumulation after 24 h. The degree of sepsis-induced macrophage infiltration
was significantly reduced in the presence of heparin. Thus, HBP induced macrophage
activation and infiltration after sepsis-induced AKI.
HBP promoted M1 differentiation following sepsis-induced AKI
The expression of iNOS mRNA is higher in M1 macrophages. However, the expression of
Arg-1 and FIZZ1 mRNAs is increased in M2 macrophages. We subjected mouse renal tissue
sections to real-time PCR analysis to determine the expression of markers of M1/M2
macrophages at different time points after CLP surgery (Fig 5). Higher levels of iNOS mRNA were
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Fig 3. Effect of HBP on histology and renal function in mices with sepsis-induced AKI. (A) Photomicrographs of
H&E-stained kidney sections (200x). All fields were chosen from the cortex and outer medulla. (B) Semi-quantitative
scoring of histological injury. (C and D) BUN and Scr levels were measured to determine renal function. The data are
presented as the means±SEM; aP<0.05 compared with the sham group; bP<0.05 compared with the heparin+CLP
group (n = 6 mices/group).
detected in the CLP and Ctl groups than in the heparin+CLP group. However, FIZZ1 and
Arg-1 mRNA expression remained low in all three groups, indicating that HBP promoted M1
differentiation following sepsis-induced AKI.
HBP increased TNF-α and IL-6 secretion during sepsis-induced AKI
Because the highest total number of M1 macrophages was observed at 24 h post-surgery, we
subsequently examined TNF-α and IL-6 expression at that time point (Fig 6). Western blots of
Fig 4. Effect of HBP on macrophage infiltmiceionwith sepsis-induced AKI in mices. (A) Expression of the CD68
protein was detected by immunohistochemical staining (200x). (B)Number of CD68-positive cells per HPF. The data
are presented as the means±SEM; aP<0.05 compared with the sham group; bP<0.05 compared with the heparin+CLP
group (n = 6 mices/group).
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Fig 5. Effect of HBP on M1 macrophage differentiation with sepsis-induced AKI in mices. Levels of the iNOS and
Arg-1 mRNA were measured using RT-PCR (A) and quantified by densitometry (B). The data are presented as the
means±standard errors of the means (SEM); aP<0.05 compared with the sham group; bP<0.05 compared with the
heparin+CLP group (n = 6 mices/group).
whole kidney homogenates demonstrated a marked increase in total TNF-α and IL-6 protein
concentrations in the CLP and Ctl groups than in the heparin+CLP group.
Sepsis-induced AKI is a common condition associated with high morbidity and mortality
]. Macrophages are pleiotropic cells of the innate immune system, with roles spanning host
defense, cytotoxicity, clearance of apoptotic cells and promotion of tissue repair. Macrophages
are also known to be important mediators of renal injury in other experimental models of renal
disease including transplantation, obstruction and glomerulonephritis . In response to
certain stimuli, macrophages can be released into the circulation from the bone marrow to migrate
into target tissues and differentiate into resident macrophages. Inflammation is closely related
to the activation of macrophages±M1 macrophages exhibit pro-inflammatory activities, while
M2 macrophages are involved in inflammation resolution [
]. According to their potential
mechanisms, M1 macrophages are pivotal in antigen presentation, pro-inflammatory cytokine
secretion, and phagocytic activity.
The plausibility of HBP as a marker of septic organ dysfunction can be supported by its early
release in response to infection and its powerful effects on immune cells and endothelial cells,
which may act as causative factors in sepsis [
]. Heparin is a multifunctional negatively
charged glycosaminoglycan that binds to HBP. Studies have suggested heparin blocks
HBPinduced IL-6 production, representing a mechanism by which heparin inhibits HBP likely by
blocking GAG-binding sites on HBP . Evidence from recent studies has also shown that
HBP activation was suppressed by heparin [
]. In this study, mice received heparin injection
12 h after the establishment of sepsis-induced AKI. HBP expression remained low after heparin
injection during sepsis-induced AKI, suggesting that heparin may inhibit HBP expression.
Over the last decade, many studies have confirmed that HBP can act as a chemotactic signal
for macrophages and promote the accumulation of macrophages at infected sites [
]. HBP in
endothelial tissues can induce mononuclear cells to accumulate in infected sites through
calcium-dependent channels. HBP can also activate mononuclear cells and increase phagocytosis
of macrophages [
]. In the present study, HBP expression was particularly increased 24 h
post-injury and decreased over the following hours. This is in agreement with findings that
HBP expression is particularly increased during the early phase of sepsis-induced AKI when
the predominant inflammatory responses occur. Our previous study has demonstrated the
presence of a substantial number of M1 macrophages 24 h after sepsis-induced AKI [
findings enabled us to identify a potentially novel relation between macrophages and HBP.
Thus, we can speculate that HBP may play a significant role in sepsis-induced AKI by
activating M1 macrophages.
Previous studies have shown that HBP induces renal tubular cell inflammation and loss of
renal endothelial cells in mice, which represent symptoms of kidney damage [
]. In this
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Fig 6. Effect of HBP on TNF-α and IL-6 protein levels with sepsis-induced AKI in mices. Levels of the TNF-α and
IL-6 protein in the kidney were determined by Western blotting (A)(C) and quantified by densitometry (B)(D).
Protein levels were normalized to β-actin. Graphs represent means±SEM; aP<0.05 compared with the sham group;
bP<0.05 compared with the heparin+CLP group (n = 6 mices/group).
study, HBP increased renal tubular damage and renal dysfunctions and played an important
role in the initial inflammatory reactions associated with renal injury. Administration of
heparin after the establishment of sepsis-induced AKI reduced renal damage at 24 h after
sepsisinduced AKI. Interestingly, there was also a simultaneous significant decrease in macrophage
infiltration, indicating that heparin likely reduces tubular injury and macrophage infiltration
by inhibiting HBP.
M1 macrophages promote the process of inflammation [
]. The main focus of our study
was to investigate the potential role of HBP in the activation of M1 macrophages during
sepsis-induced AKI. We sought to determine which subtype of macrophages, M1 or M2, was
predominantly present in mouse renal tissue sections at 24 h after sepsis-induced AKI. Real-time
PCR analysis showed increased iNOS expression in the kidneys of the four groups of mice but
no increase in Arg-1 or FIZZ1 expression, suggesting that HBP activated M1 macrophages in
M1 macrophages secrete high levels of pro-inflammatory cytokines (e.g., TNF-α, IL-6,
IL1β), and the generation reactive nitrogen and oxygen intermediates [
]. In the present study,
we observed increased levels of TNF-α and IL-6 in the kidneys of mice subjected to CLP
surgery, with less pronounced expression in the presence of heparin. Based on these results, the
pro-inflammatory effects of HBP on sepsis-induced AKI is likely related to the activation of
Based on our findings, HBP plays an important role in the initial inflammatory reaction
associated with sepsis-induced AKI, presumably by activating M1 macrophages and
suppressing TNF-α and IL-6 secretion. Therefore, strategies that limit early macrophage infiltration or
activation may represent a novel approach in the prevention or treatment of AKI in septic
patients. However, the signaling pathways involved in the mechanism of activation of M1
macrophages need further investigation. Thus, our study contributes to a better understanding of
the complex events involved in sepsis-induced AKI, which is key for the development of more
effective therapeutic strategies.
S1 Dataset. Dataset showing the data for Fig 3C (Scr) and 3D (BUN).
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S1 Fig. HBP mRNA levels following sepsis-induced AKI in mices. Levels of the HBP mRNA
were measured using RT-PCR (0, 6, 12, 24, 36 and 48h).
S2 Fig. HBP mRNA levels following sepsis-induced AKI in mices. Levels of the HBP mRNA
were measured using Western blotting (0, 6, 12, 24, 36 and 48h).
S3 Fig. Twenty-four hours prior to the onset of CLP, mices were treated with heparin.
Levels of the HBP protein in the kidney were determined by Western blotting.
S4 Fig. Photomicrographs of H&E-stained kidney sections (200x).
S5 Fig. Expression of the CD68 protein was detected by immunohistochemical staining
S6 Fig. Levels of the iNOS and Arg-1 mRNA were measured using RT-PCR.
S7 Fig. Levels of the TNF-α protein in the kidney were determined by Western blotting.
S8 Fig. Levels of the IL-6 protein in the kidney were determined by Western blotting.
Conceptualization: Li Xing, Mu Genhua.
Data curation: Li Xing, Chen Pingfa, He Lei, Jin Qin, Mu Genhua.
Formal analysis: Li Xing, Song Chunmei.
Funding acquisition: Li Xing, Lu Zhongqian, Deng Yijun.
Methodology: Li Xing, Lu Zhongqian, Song Chunmei.
Project administration: Li Xing, Lu Zhongqian, Deng Yijun.
Resources: Li Xing, Song Chunmei, Chen Pingfa, Deng Yijun.
Software: Li Xing, Song Chunmei.
Supervision: Li Xing.
Validation: Li Xing, Chen Pingfa, Jin Qin.
Visualization: Li Xing.
Writing ± original draft: Li Xing.
Writing ± review & editing: Li Xing, Chen Pingfa, He Lei, Deng Yijun.
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