Original Article

Anti-high Mobility Group Box 1 Antibody
Ameliorates Albuminuria in MRL/lpr
Lupus-Prone Mice
Haruki Watanabe,1 Katsue S. Watanabe,1 Keyue Liu,2 Sumie Hiramatsu,1 Sonia Zeggar,1 Eri Katsuyama,1Noriko Tatebe,1 Akiya Akahoshi,3 Fumiaki Takenaka,3 Takahisa Hanada,3 Masaru Akehi,3 Takanori Sasaki,3Ken-ei Sada,1 Eiji Matsuura,3 Masahiro Nishibori,2 and Jun Wada1
1Department of Nephrology, Rheumatology, Endocrinology and Metabolism, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,

Okayama 700-8558, Japan; 2Department of Pharmacology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558,
Japan; 3Collaborative Research Center for OMIC, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558, JapanWe evaluated the efﬁcacy of a neutralizing anti-high mobility
group box 1 (HMGB1) monoclonal antibody in MRL/lpr
lupus-prone mice. The anti-HMGB1 monoclonal antibody(5 mg/kg weight) or class-matched control immunoglobulin
G2a (IgG2a) was administered intravenously twice a week for
4–15 weeks. Urine albumin was monitored, and histologicalevaluation of the kidneys was conducted at 16 weeks. Lymphadenopathies were evaluated by 1-(20 -deoxy-20 -[18F]ﬂuoro-b-D-arabinofuranosyl)cytosine ([18F]FAC) positron emission tomography/computed tomography (PET/CT) at 12 weeks. Following
4-week treatment, [18F]FAC-PET/CT showed similar accumulation in cervical and axillary lymph nodes at 12 weeks ofage. However, anti-HMGB1 monoclonal antibody sufﬁciently
inhibited the increase in albuminuria compared to an isotype
control following 15-week treatment. Complement deposition
was also improved; however, there were no signiﬁcant differences in IgG deposition and renal pathological scores between
the two groups. Anti-double-stranded DNA (dsDNA) antibody
titers and cytokine and chemokine levels were also unaltered.
Although there were no signiﬁcant differences in glomerular
macrophage inﬁltration, neutrophil inﬁltration was signiﬁcantly decreased by the anti-HMGB1 monoclonal antibody.
Antagonizing HMGB1 treatment suppressed HMGB1 translocation from nuclei in the kidney and suppressed neutrophil
extracellular traps. The anti-HMGB1 monoclonal antibody
demonstrated therapeutic potential against albuminuria inlupus nephritis by inhibiting neutrophil recruitment andneutrophil extracellular traps.

emerged, randomized controlled trials have failed to show their effectiveness against LN.1
Impaired clearance of apoptotic cells in lupus patients has been reported;3 the consequently released DNA and nuclear proteins activate
the innate immunity. High mobility group box 1 (HMGB1) is a ubiquitous non-histone nuclear protein that regulates transcription, repair,and recombination by exerting effects on chromosomal architecture.4,5
In addition, HMGB1 can actively or passively translocate from thenucleus to the extracellular space under inﬂammation conditions;
extracellular HMGB1 exerts pleiotropic effects as an alarmin-mediating inﬂammation through toll-like receptors (TLRs), receptor foradvanced glycation end products (RAGE), and cytosolic DNA/RNA
sensors or by promoting apoptosis and autophagy.4–7 The redox statusof HMGB1 distinguishes its cytokine-inducing and chemokine activity.8,9 HMGB1 has also been shown to promote type 1 interferon(IFN) production through the TLR9 and RAGE pathways in response
to DNA.10 Type 1 IFN is known as a crucial cytokine in lupus;11 therefore, these data suggest that HMGB1 is involved in lupus pathogenesis.
Indeed, elevated concentrations of HMGB1 are observed in the sera,
urine, kidney tissues, and skin lesions of patients with lupus.12–14
The relationship of HMGB1 with other autoimmune diseases, such as
rheumatoid arthritis (RA), has also been suggested. HMGB1 is morestrongly expressed in the synovial ﬂuid of RA patients than in that ofosteoarthritis patients, inducing the release of proinﬂammatory cytokines from synovial ﬂuid macrophages.15 In addition, HMGB1 boosts
proinﬂammatory cytokine and matrix metalloproteinase production
in synovial ﬁbroblasts from RA patients.16

INTRODUCTION
Lupus nephritis (LN) is a refractory complication of systemic lupus
erythematosus (SLE), which causes end-stage renal disease, resulting
in lower survival rates and quality of life.1 The pathogenesis of LN has
yet to be fully elucidated; however, it is assumed that innate and adaptive immunity abnormalities are associated with chronic systemic
inﬂammation.2 Although novel biologics targeting B or T cells have

Received 20 March 2017; accepted 21 May 2017;
http://dx.doi.org/10.1016/j.omtm.2017.05.006.
Correspondence: Katsue S. Watanabe, MD, PhD, Department of Nephrology,
Rheumatology, Endocrinology and Metabolism, Okayama University Graduate
School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho,
Kita-ku, Okayama 700-8558, Japan.
E-mail: kwatanabe@md.okayama-u.ac.jp

Molecular Therapy: Methods & Clinical Development Vol. 6 September 2017 ª 2017 The Author(s).
This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

31

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Table 1. Organ Weights

Total body weightaHMGB1

Control

36.8 ± 0.8

36.1 ± 0.8

signiﬁcant differences (Figures 2B and 2C). In addition, chemokine
levels were also unaltered (Figure 2B). Moreover, antagonizing
HMGB1 treatment did not result in a signiﬁcant reduction in plasma
HMGB1 levels (Figure 2D).

Right kidney

0.20 ± 0.009

0.20 ± 0.009

Left kidney

0.19 ± 0.008

0.20 ± 0.008

Urinary Albumin Excretion and Renal Pathological Evaluation

Spleen0.44 ± 0.05

0.37 ± 0.05
0.08 ± 0.005

The anti-HMGB1 mAb sufﬁciently inhibited the increase in albuminuria compared to the increase observed for an isotype control at
16 weeks (p = 0.008, Figure 3A). Consistent with albuminuria,
glomerular complement deposition also improved (Figure 3B). However, there were no signiﬁcant differences in immunoglobulin G (IgG)
deposition and renal pathological scores (activity index) between thetwo groups (Figures 3C and 3D).

Thymus0.07 ± 0.005

Peritoneal lymph nodes

1.9 ± 0.15

1.7 ± 0.15

Cervical lymph nodes

0.83 ± 0.11

0.74 ± 0.11

Axillary lymph nodes

0.65 ± 0.07

0.59 ± 0.07

Total lymph nodesa

3.37 ± 0.3

3.06 ± 0.3

Data presented are the mean ± SE of 19 mice per group. Weight values are in grams.
aTotal lymph nodes, the combination of peritoneal, cervical, and axillary lymph nodes.

Thus, HMGB1 plays a pivotal role in the pathogenesis of autoimmune
diseases and may constitute a potential therapeutic target. Interestingly, HMGB1 inhibition by a speciﬁc antibody has been shown to
alleviate lupus-like disease in BXSB mice.17 In contrast, a different
study has recently reported that treatment with an anti-HMGB1
monoclonal antibody (mAb) does not affect lupus activities in
MRL/lpr mice.18 To elucidate this discrepancy, we examined the efﬁcacy of anti-HMGB1 mAb to determine whether it ameliorates lupus
activities, including nephritis and serological abnormalities, in MRL/lpr lupus-prone mice. Our mAb recognizes the C-terminal sequenceof the HMGB1 molecule and can neutralize the intercellular adhesion
molecule 1 (ICAM1)-inducing activity of HMGB1 in vitro;19 moreover, therapeutic effects against brain stroke, atherosclerosis, and viral
infections have also been reported.19–21

RESULTS
Organ Weights and Lymphoid Tissue Functions

There were no signiﬁcant differences in organ weight at 16 weeks(Table 1) following 12 weeks of treatment. Next, we evaluated thelymphoid organs using 1-(20 -deoxy-20 -[18F]ﬂuoro-b-D-arabinofuranosyl)cytosine ([18F]FAC) positron emission tomography/computed
tomography (PET/CT) at an early stage. FAC accumulates in T cells;
therefore, this imaging analysis enabled us to evaluate not only thesizes of organs, but also their functions.22 [18F]FAC PET/CT imaging
analysis at 12 weeks was similar in the cervical and axillary lymph
nodes of the two groups (Figure 1A). In addition, the amount ofincorporated probe was also similar in the cervical lymph nodes
and spleen (Figure 1B). These results indicate that lymphoid tissue
weight and functions, especially of those of the T cells, were unaltered.
Serological Abnormalities and the Effects of theAnti-HMGB1 mAb

As shown in Figure 2A, anti-HMGB1 mAb treatment did not result inany signiﬁcant reduction in anti-double-stranded DNA (dsDNA)
antibody titers at 16 weeks. Although the plasma levels of a numberof cytokines, such as interleukin-1b (IL-1b), IFN-g, and tumor
necrosis factor alpha (TNF-a), tended to decrease, there were no32

Glomerular Cell Infiltration

To investigate the mechanism underlying the favorable renal effects
of anti-HMGB1 mAb without serological improvement, we focusedon glomerular inﬂammatory cells. No signiﬁcant differences in theglomerular inﬁltration of F4/80-positive macrophages were apparent,
whereas the inﬁltration of Ly-6G-positive neutrophils was signiﬁcantly suppressed (p = 0.034, Figures 3E and 3F).
Renal mRNA Expression

We hypothesized that certain types of in situ cytokines or chemokines
may drive neutrophil inﬁltration; however, renal mRNA expression
was not suppressed by anti-HMGB1 mAb treatment (Figure S1).
The expression of HMGB1 and its receptors was also similar (Figure S1). Although HMGB1 is an important factor in innate immunity,
the expression of inﬂammasome-related genes was also unaltered(Figure S1).
Effect on HMGB1 Translocation and Neutrophil Extracellular
Traps

Next, we evaluated whether the anti-HMGB1 mAb suppresses
HMGB1 translocation from the nucleus in the kidney to determine
the mechanism by which inﬂammation is prevented. Immunoﬂuorescence staining revealed that treatment with this antibody suppressedtranslocation compared to the control antibody (p = 0.035, Figure 4A). However, the HMGB1 translocation appeared to be extensive in both treated mice groups compared to non-treated control
mice (Figure 4A).
Neutrophil extracellular traps (NETs) were originally identiﬁed as asystem for degrading virulence factors and killing bacteria via neutrophils;23 however, recent studies have suggested their involvement inautoimmunity, including lupus and vasculitis.24,25 Therefore, we
also evaluated the change of NETs in the kidney. Immunoﬂuorescence staining revealed that the anti-HMGB1 mAb successfully suppressed the manifestation of NETs in the glomerulus (p = 0.022,
Figure 4B).

DISCUSSION
Our data demonstrate that the anti-HMGB1 mAb ameliorates
albuminuria through inhibition of neutrophil inﬁltration, HMGB1

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Figure 1. Accumulation of FAC in Lymphoid Tissues(A) PET/CT images. (B) Incorporated probe in lymphoid tissues. Units signify % injected dose/gram tissue (%ID/g). Fivemice per group were examined. LN, lymph node.

translocation, and NETs compared to control IgG treatment, without
serological improvement.
With respect to the mechanism of action of the anti-HMGB mAb, weinitially examined its inhibitory effect against neutrophil inﬁltrationin glomeruli. Neutrophils are the ﬁrst line of defense against bacterial
and fungal infections;26 they are rapidly recruited from the bloodstream into sites of inﬂammation. Neutrophils induce responses ininﬂamed tissues to destroy the microorganisms through chemokine
and cytokine release, phagocytosis and reactive oxygen species formation, and degranulation and NETs during infection.27 In addition tothese primary effectors against microbial pathogens, neutrophils arealso central mediators of sterile inﬂammatory injury, suggesting
they constitute a key player in autoimmune diseases.28,29 Neutrophils
are indeed activated in SLE, and increased numbers of abnormal neutrophils or low-density granulocytes in the circulation have been
observed.11,30 Cell death by necrosis or abnormal apoptosis releases
multiple endogenous pro-inﬂammatory damage-associated molecular patterns, including proteins, nucleic acids, and lipid mediators,
and then leads to neutrophil recruitment.28,31 Anti-HMGB1 mAb
could interact with these dangerous molecules by inhibiting
HMGB1 translocation and interrupting neutrophil recruitment for
local inﬂammation. Furthermore, Huebener et al.32 recently reported
that HMGB1 triggers inﬁltration of neutrophils toward acetaminophen-induced liver necrosis through its receptor RAGE, which may
further conﬁrm our ﬁndings.
The second mechanism explaining the efﬁcacy of the anti-HMGB
mAb is its inhibitory effects against NET formation in glomeruli.
Increased formation of NETs due to impairment in their degradation
has been reported in lupus.33 NETs promote type I IFN production
and therefore may be involved in pathogenesis.34 Peptidylarginine
deiminase (PAD) 4 plays a fundamental role in NET formation,
including hypercitrullination of histone H3.35 Inhibition of PAD4
disrupts NET formation and shows therapeutic potential for lupus

activities in various mouse models.36,37 Thus,
NETs may constitute a promising target for mitigating SLE. An interaction between NETs and
HMGB1 has also been reported. Tadie et al.38
demonstrated that HMGB1 enhanced the formation of NETs through TLR4 and detected extensive
citrullinated H3 in HMGB1-treated neutrophils,
which were signiﬁcantly suppressed by neutralizing antibodies against HMGB1. Another study
revealed that HMGB1 and histones released byinjured hepatocytes during ischemia/reperfusion
injury stimulated NET formation through TLR4
and TLR9-MyD88 signaling pathways.39 MRL/lpr mice also exhibit a vasculitis phenotype, and an in vitro study
has revealed the linkage between HMGB1 and NETs in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).40
HMGB1 can potentiate ANCA-induced NET formation through
TLR2, TLR4, and RAGE and may be involved in the pathogenesis
of AAV.41 On the basis of these previous ﬁndings, anti-HMGB1mAb may suppress NETs by inhibiting neutrophil inﬁltration as
well as by direct inhibition through HMGB1 receptors.
The effects of antagonizing HMGB1 treatment for arthritis have beenreported as being generally beneﬁcial.42,43 Moreover, Zhang et al.17
demonstrated the effectiveness of the anti-HMGB1 antibody forlupus in BXSB mice; they peritoneally injected 30 mg of antiHMGB1 antibody every week from 16 to 26 weeks. Although the antibody dose seems considerably low, they showed attenuated proteinuria, glomerulonephritis, anti-dsDNA, and serum cytokine levels.
However, a recent study has reported that the anti-HMGB1 antibody
did not affect lupus activities in MRL/lpr mice.18 Although thisprevious study used the same mouse model, the treatment protocol was different from our protocol, i.e., an antibody dose of
100 mg/mouse/week was used in contrast to our dose of approximately 150–400 mg/mouse/week, with peritoneal administration
and a treatment period of 7–17 weeks. Therefore, the total administered dosage was smaller and the maximum blood concentration
might be lower than that of our protocol. Moreover, the antibody
used in the previous study recognizes the A-box,7 which is different
from the antibody used in our study raised against the c-tail epitope.
The A-box domain antagonizes inﬂammation; therefore, A-box inhibition might exacerbate the disease.5 Collectively, their treatment
might not be sufﬁcient to result in any advantages. In contrast,
BXSB mice exhibit TLR pathway abnormalities; therefore, the former
study could demonstrate favorable results despite the lower treatment
dose than the other protocol. Our treatment protocol was determinedbased on previous studies.19–21 Moreover, we ﬁrst tried subcutaneous
injection using the same dosage (5 mg/kg) as a preliminary

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Figure 2. Effects of Anti-HMGB1 Monoclonal Antibody on Autoantibody Production and Inflammation in MRL/lpr Mice(A) Anti-dsDNA antibody (n = 19, p = 0.2). (B) Various cytokines and chemokines evaluated by Bio-Plex (n = 6 [MIP1a: n = 8, MCP1 and TNF-a: n = 11], pg/mL). (C) IFNa (n = 6,
p = 0.76). (D) HMGB1 (n = 7, p = 0.47). Anti-dsDNA antibody, IFNa, and HMGB1 were evaluated by ELISA. MIP, macrophage inflammatory protein; MCP1, monocyte
chemoattractant protein-1.

experiment; however, we were not able to ﬁnd any beneﬁcial effects
for MRL/lpr mice (data not shown). Although our results could notshow absolute efﬁcacy (organomegaly and serological abnormalities
did not improve), increased dose and higher blood concentrationcompared to the previous study might contribute to the efﬁcacy fornephritis.18 However, HMGB1 was actively translocated from thenucleus in MRL/lpr mice, as determined by immunoﬂuorescence;
therefore, the dose might be insufﬁcient to antagonize the target inthis mouse model. The efﬁcacy of the antagonizing HMGB1 strategy
for stroke has been established in animal experiments; however,clinical trials examining such treatments for stroke and autoimmune
diseases have yet to be conducted according to ClinicalTrials.gov.
Interestingly, there is an HMGB1 antagonist in Chinese herbal
medicine, glycyrrhizin, which is approved for clinical practice. The
efﬁcacy of glycyrrhizin against various inﬂammatory conditions
has been demonstrated,44–46 and animal experiments have shownthat glycyrrhizin attenuates lupus nephritis.47 Therefore, glycyrrhizin
might mark the ﬁrst step for clinical application of a treatment
strategy that antagonizes HMGB1.
Our anti-HMGB1 mAb failed to improve lymphadenopathy; nevertheless, it demonstrated therapeutic potential against albuminuria
in lupus nephritis by inhibiting neutrophil recruitment and NETs
without altering autoantibody production. It might be effective especially for membranous lupus nephritis. Although our results demonstrated modest efﬁcacy in MRL/lpr mice, it may be dependent on theseverity of the disease model in mice and epitope recognition of the34

antibody. Optimization of dosages and epitope recognition of
HMGB1 antibody is required in future translational research inhumans.

MATERIALS AND METHODS
Animals

4-week-old female MRL/MpJ-Faslpr/J (MRL/lpr) mice (Jackson Laboratory) were used for animal experiments. Littermates were divided
into two groups and then administered with the anti-HMGB1 mAb(5 mg/kg weight; n = 19) or the class-matched control, IgG2a against
keyhole limpet hemocyanin (5 mg/kg weight; n = 19), intravenously
twice a week from 4 to 15 weeks of age. 24-hr urine samples werecollected every 2 weeks from 6 to 16 weeks of age, whereas sera samples were collected every 4 weeks from 8 to 16 weeks of age. Mice weresacriﬁced at 16 weeks of age and used for the following experiments.
For imaging studies, MRL/lpr mice were treated similarly (ﬁve mice
per group) from 8 to 12 weeks, then sedated and injected with [18F]
FAC. PET/CT imaging was performed as previously described, with
modiﬁcations.22 In brief, 2-[18F]ﬂuoro-1,3,5-tri-O-benzoyl-D-arabinofuranose was prepared by nucleophilic substitution of 2-O-(triﬂuoromethanesulfonyl)-1,3,5-tri-O-benzoyl-a-D-ribofuranose, withreactor-produced [18F]ﬂuoride. This 18F-labeled sugar derivative
was reacted with a freshly prepared trimethylsilyl-protected derivative of cytosine in dichloroethane. The intermediate analog wasthen treated with a solution of sodium methoxide in methanol
to remove the protecting groups. Following neutralization with

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Figure 3. Urinary Albumin Excretion and Renal
Pathological Evaluation(A) Transitions of urine albumin/Cr ratio (model-based
adjusted predictions with 95% confidence intervals; n = 19;
one value in each group at 6 weeks was missing). At
16 weeks, the 95% confidence intervals of the prediction were
not overlapping between the two groups. (B and C) Glomerular depositions of complement (B) ( 200) and IgG (C)( 200); ten glomeruli were analyzed in each kidney (n = 8). (D)PAS staining of kidney tissues ( 200) and activity indexes;
ten glomeruli or tubular regions were analyzed in each kidney(n = 8). (E and F) Glomerular macrophage (E) ( 200, n = 8) andneutrophil infiltration (F) ( 200, n = 7). The number of F4/80positive or Ly-6G-positive cells was calculated in ten glomeruli
per animal, and the mean number of positive cells perglomerulus was used for estimation. Cr, creatinine.

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Figure 4. HMGB1 Translocation and NETs
(A) HMGB1 staining of kidneys. HMGB1-positive cells were counted in three to four low-power fields ( 200) per animal, and the mean number was used for estimation (n = 8).
(B) NETs in kidneys. The number of NETs was calculated in ten glomeruli per animal, and the mean values per glomerulus were used for estimation (n = 7).

hydrochloric acid, the crude product was puriﬁed by semi-preparative
high-pressure liquid chromatography, resulting in [18F]FAC with99% radiochemical purity. PET/CT images were analyzed with
PMOD software (PMOD Japan). In addition, the amount of incorporated probe was measured by scintillation counting with the PerkinElmer MicroBeta (PerkinElmer).
These methods were carried out in accordance with the approved
guidelines. All experimental protocols were approved by the Animal
Care and Use Committee of the Department of Animal Resources,
Advanced Science Research Center, Okayama University (OKU2013112).
Measurement of Urinary Albumin Excretions, Anti-dsDNA
Antibodies, HMGB1, Cytokines, and Chemokines

Urinary albumin excretions were measured using the latex agglutination immuno-assay (SHIMA Laboratories) and standardized by urine
creatinine concentration. Anti-dsDNA IgG titers, HMGB1 level, andIFNa were measured with mouse anti-dsDNA ELISA kits (Shibayagi), HMGB1 ELISA kits (Shino-Test), and mouse IFNa ELISA
kits (Cloud-Clone), respectively. Cytokine and chemokine analyses(except for IFNa) were performed using the cytokine mouse 23plex Bio-Plex kit (Bio-Rad Laboratories) with a Bio-Plex 200 Luminex
machine according to the manufacturer’s protocol.
Histopathology and Scoring

Kidney specimens were ﬁxed with 10% buffered formalin andembedded in parafﬁn. Serial 2-mm sections were stained with H&E
or Periodic acid-Schiff (PAS) for histological examination by light36

microscopy. Activity and chronicity indices were scored according
to previously described criteria.48 Ten renal glomeruli and ten
selected tubular areas were examined in each mouse by the CMIC
Bioresearch Center.
Immunofluorescence Staining

Kidney tissues (n = 8) were embedded in an optimum cutting temperature compound (Sakura Finetek Japan) and immediately frozen in
acetone cooled on dry ice. Frozen sections (4-mm thick) were stainedwith ﬂuorescein-conjugated anti-mouse IgG (1:50; MP Biomedicals)
or anti-mouse C3 (1:50; MP Biomedicals). The brightness of each image ﬁle was analyzed using Lumina Vision software (Mitani), as previously described.49 Brieﬂy, image ﬁles were inverted and opened ingray scale mode. IgG or C3 indices were calculated using the following
formula: [X (density)  positive area]/glomerular total area, where
staining density is indicated by a number from 0 to 256 in gray scale.
For HMGB1 staining, the kidney sections (n = 8) were washed with
PBS containing 0.5% Tween 20 (Sigma-Aldrich) and immersed in
10% normal goat serum (Nichirei) containing 1% BSA for 30 min
to block nonspeciﬁc binding. The sections were incubated overnightwith a rabbit anti-HMGB1 Ab (Abcam) in combination with a rat
anti-type IV collagen alpha 5 chain mAb (Chondrex) as the primary
antibodies at 4 C, and then with Alexa-568-labeled anti-rat IgG (Invitrogen) and Alexa-488-labeled anti-rabbit IgG (Invitrogen) as thesecondary antibodies at room temperature (25 C) for 1 hr. Following
staining with DAPI, the slides were mounted using ﬂuorescence
mounting medium (Dako) and observed under an LSM 780 confocal
imaging system (Carl Zeiss). HMGB1-positive cells were counted in

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three to four low-power ﬁelds (200  magniﬁcation) per animal, andthe mean number was used for estimation.
For neutrophil inﬁltration, the kidney sections (n = 7) were washedwith PBS and similarly blocked. The sections were incubated overnight with a rabbit anti-Ly-6G antibody (Clone: 1A8; BioLegend) as
the primary antibody at 4 C, and then with Alexa-568-labeled antirat IgG as the secondary antibody at room temperature for 1 hr.
Nuclear staining and mounting were conducted as described above.
The number of Ly-6G-positive cells was calculated in ten glomeruli
per animal, and the mean number of positive cells per glomerulus
was used for estimation.
The nuclei, Ly-6G, and citrulline R2 + R8 + R17 (cit-histone H3) were
stained on 4-mm kidney sections (n = 7) with DAPI and the anti-Ly6G Ab, followed by Alexa-Fluor-568-labeled anti-goat IgG and anticit-histone H3 Ab (Abcam), and followed by Alexa-Fluor-488-labeledanti-rabbit IgG. NETs (both cit-histone-H3- and Ly6G-positive sites)
were counted in ten glomeruli per animal, and the mean number ofsites per glomerulus was used for the estimation.

determined at 0.05/23 or 0.05/15 using the Bonferroni correction toadjust for multiple testing. These statistical analyses were performed
using the JMP Statistical Package for Windows software, version 9.0.2(SAS Institute). To evaluate the effects on albuminuria, we estimatedthe coefﬁcient and 95% conﬁdence intervals (CIs) for local progression using multilevel linear regression analysis (MLA). In MLA, a
two-level data structure was employed: level 1 was urine-albumin/creatinine (u-alb/Cr) measurement and level 2 was individual mouse.
U-alb/Cr at six time points (6, 8, 10, 12, 14, and 16 weeks) was
clustered within the mice. Individual mice were added to the MLA
model with a random intercept. We adjusted for the group (antiHMGB1 versus control) time points (as a dummy variable) and interaction term (group  time points) (Supplemental Information).
Model-based adjusted predictions with 95% conﬁdence intervals
were compared. MLA was performed using Stata version 14.2 (StataCorp LP).

SUPPLEMENTAL INFORMATION
Supplemental Information includes one ﬁgure and one table and canbe found with this article online at http://dx.doi.org/10.1016/j.omtm.
2017.05.006.

Immunoperoxidase Staining

Immunoperoxidase staining was performed as previously described.50 In brief, macrophage inﬁltration was analyzed using a
monoclonal murine monocyte/macrophage antibody (F4/80; Abcam), followed by an HRP-conjugated goat anti-rat IgG antibody(Merck KGaA). The number of F4/80-stained cells was evaluatedsimilarly to the neutrophil counts.
Real-Time RT-PCR

Total RNA was extracted from kidney tissues using the RNeasy Mini
Kit (QIAGEN) according to the manufacturer’s instructions andquantitated using a NanoDrop 2000c spectrophotometer (Thermo
Scientiﬁc). Total RNA was reverse transcribed using the HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems).
Quantitative real-time PCR was performed using the Step One Plus
Real-Time PCR System (Applied Biosystems) with TaqMan Array
96-Well Fast Plates containing speciﬁc primers and Universal
Master Mix II (Life Technologies) to evaluate the gene expressionof IL-1b (Mm00434228_m1), IL-2 (Mm00434256_m1), IL-6
(Mm00446190_m1), IL-10 (Mm00439614_m1), IL-18 (Mm00434225_
m1), TNF-a (Mm00443258_m1), IFNa (Mm04207507_gH), ICAM1(Mm00516023_m1), C-X-C motif chemokine 5 (Mm00436451_g1),
HMGB1 (Mm00849805_gH), RAGE (Mm01134790_g1), TLR4
(Mm00445273_m1), TLR7 (Mm00446590_m1), TLR9 (Mm00446193_
m1), and NACHT, LRR, and PYD domains containing protein 3(Mm00840904_m1). The relative mRNA abundance was standardized using GAPDH and 18S mRNA as the invariant control.
Statistical Analysis

Data were compared using the Student’s t test or Mann-Whitney U
test, depending on data distribution. The tests were two-tailed, andthe threshold for signiﬁcance was p < 0.05. When analyzing 23-plex
Bio-Plex kit or TaqMan Array results, statistical signiﬁcance was

AUTHOR CONTRIBUTIONS
Conceptualization, H.W., K.S.W., and J.W.; Methodology, H.W.,
K.S.W., K.L., S.H., E.K., S.K., E.M., M.N., and J.W.; Formal Analysis,
H.W.; Investigation, H.W., K.S.W., K.L., S.Z., N.T., A.A., F.T., T.H.,
M.A., T.S., and J.W.; Data Curation, H.W.; Writing – Original Draft,
H.W.; Writing – Review and Editing, K.S.W. and J.W.; Visualization,
H.W.; Supervision, K.S., E.M., M.N., and J.W.; Project Administration, J.W.; Funding Acquisition, H.W. and K.S.W.

CONFLICTS OF INTEREST
J.W. receives grant support from Astellas, Bayer, Chugai, Daiichi Sankyo, Kissei, Kyowa Hakko Kirin, MSD, Otsuka, Teijin, Torii, Pﬁzer,
Takeda, and Taisho Toyama.

ACKNOWLEDGMENTS
We wish to thank Dr. Toshiharu Mitsuhashi at the Center for Innovative Clinical Medicine of Okayama University Hospital for themultilevel analysis and all technical staff at our department for theirassistance. This work was supported by JSPS KAKENHI grant
numbers JP16K19600 and JP25860810.

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