Free Radical Biology and Medicine 131 (2019) 162–172

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Free Radical Biology and Medicine

journal homepage: www.elsevier.com/locate/freeradbiomed

Original article

Peroxiredoxin 3 deficiency accelerates chronic kidney injury in mice T

through interactions between macrophages and tubular epithelial cells
⁎
Inah Hwang1, Md Jamal Uddin1, Gayoung Lee1, Songling Jiang, Eun Seon Pak, Hunjoo Ha
Graduate School of Pharmaceutical Sciences, College of Pharmacy, Ewha Womans University, Seoul, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: Chronic kidney disease (CKD) has become epidemic worldwide. Mitochondrial reactive oxygen species (ROS)-
Prx3 deficiency induced oxidative stress is an important mediator of CKD, and Prx3 plays a critical role in maintenance of
Oxidative stress mitochondrial ROS. The present study examined the role of Prx3 in the context of fibrosis, a common feature of
Macrophage activation CKD, using Prx3 KO mice under obstructive and diabetic stress. Prx3 deficiency accelerated fibrosis and in-
CKD
flammation accompanied by mitochondrial oxidative stress in obstructed and diabetic kidneys as well as in
proximal tubular epithelial (mProx) cells. In addition, Prx3 deficiency induced Raw264.7 macrophages activa-
tion, leading to upregulation of proinflammatory cytokines. Conditioned media from LPS-stimulated Prx3 de-
ficient macrophages accelerated proinflammatory and profibrotic cytokines in mProx cells. Interestingly, Prx3
deficiency induced most inflammatory and fibrotic cytokines at basal condition in both tissues and cells. Taken
together, these results demonstrate that Prx3 deficiency can accelerate CKD through interactions between
macrophages and tubular epithelial cells.

1. Introduction capacity of mitochondria is critical for balancing mitochondrial redox

Chronic kidney disease (CKD) has become epidemic worldwide. It is
an independent risk factor for cardiovascular morbidity and mortality
[1]. Diabetic nephropathy (DN) is the leading cause of CKD and end-
stage kidney disease (ESKD) [2].
Renal interstitial fibrosis is characterized by excessive extracellular
matrix (ECM) accumulation, epithelial-mesenchymal transition (EMT),
tubular atrophy, and nephron loss. It is an inevitable common feature of
CKD regardless of primary etiology [3,4]. While the contribution of
EMT to renal fibrosis in vivo remains controversial [5–8], excessive
deposition of ECM in the tubulointerstitium is correlated with the de-
cline in kidney function [9,10].
The pathological role of macrophages in the development and
progression of fibrosis has been reported [11–13]. Damaged tubular
cells can secrete chemokines to rapidly recruit macrophages that ac-
tively participate in inflammation through secreting proinflammatory
cytokines such as TNFα and IL-1β [14]. Systemic macrophage depletion
can reduce inflammation and fibrosis in CKD [15,16].
Mitochondrial ROS plays a crucial role in the development and
progression of CKD [17–19] as in other tissue injuries. Mitochondrial
dysfunction resulting from mitochondrial ROS is linked to macrophage
activation [20] and inflammation [21,22]. Thus, the antioxidant
state which is linked to maintenance of organ homeostasis.
Peroxiredoxins (Prxs) are a family of cellular thiol peroxidases that
scavenge peroxides. Among six distinct members, Prx3 is a unique an-
tioxidant exclusively localized in the mitochondria to maintain mi-
tochondrial redox state [23,24]. Prx3 is abundant in the kidney prox-
imal and distal tubular epithelial cells in rats [25]. Higher Prx3
expression is associated with better renal function recovery after acute
kidney injury [26]. Overexpression of Prx3 has protective effects in
neurons [27], infarcted myocardium [28], and ischemia-reperfused
kidneys [29]. However, deficiency of Prx3 increases sensitivity to lung
inflammation [30], muscular disorders [31], adipocytes dysfunctions
[32], and liver injury [33]. Prx3 deficient macrophages can increase
intra cellular ROS production and TNFα secretion in response to LPS
[34] while Prx3 depletion or inactivation decreases mitochondrial
membrane potential, leading to mitochondrial dysfunction [35,36].
However, the functional role of Prx3 in CKD has not been reported yet.
Therefore, the objective of the present study was to investigate
whether endogenous Prx3 might play a role in the development and
progression of CKD using Prx3 knockout (KO) mice under streptozo-
tocin (STZ)-induced diabetic as well as unilateral ureteral obstruction
(UUO) stress [11,37,38], a standard model for progressive tubu-
lointerstitial fibrosis. To dissect the role of Prx3 in tubular epithelial

⁎
Correspondence to: College of Pharmacy, Ewha Womans University, 52 Ewhayeodae-gil, Seodaemun-gu, Seoul 120-750, Republic of Korea.
E-mail address: hha@ewha.ac.kr (H. Ha).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.freeradbiomed.2018.12.002
Received 28 November 2018; Accepted 3 December 2018
Available online 04 December 2018
0891-5849/ © 2018 Elsevier Inc. All rights reserved.
I. Hwang et al. Free Radical Biology and Medicine 131 (2019) 162–172

cells and macrophages, we also employed in vitro study using condi- Table 1
tioned media from Prx3 deficient macrophages. Primers used for real time RT-PCR analysis.
Gene Primer sequences (mouse)
2. Materials and methods
Arg1 Forward 5′- TGGCTTGCGAGACGTAGAC- 3′
Reverse 5′- CGTCAGGTGAATCGGCCTTT- 3′
2.1. Materials
COX2 Forward 5'-CCT GCT GCC CGA CAC CTT CAA- 3'
Reverse 5'-CAG CAA CCC GGC CAG CAA TCT- 3'
Reagents used for immunohistochemical staining were purchased CTGF Forward 5'-CAC AGA GTG GAG CGC CTG TTC- 3'
from DAKO A/S (Glostrup, Denmark). Anti-E-cadherin from BD Reverse 5'-GAT GCA CTT TTT GCC CTT CTT AAT G- 3'
Biosciences (San Jose, CA, USA), anti-Prx3 from Young In Frontier Col1α1 Forward 5'-CGGATAGCAGATTGAGAACATCCG- 3'
Reverse 5'-CGGCTGAGTACGGAACACCAC- 3'
(Seoul, Korea), and other antibodies from Santa Cruz Biotechnology
E-cadherin Forward 5'-AGC GTG TGT GAC TGT GAA GG- 3'
Inc. (Santa Cruz, CA, USA) were obtained. FBS and FCS were purchased Reverse 5'-GCT GGC TCA AGT CAA AGT CC- 3'
from GIBCO BRL (Gaithersburg, MD, USA) while mitoSOX™ and F4/80 Forward 5'- CTGTAACCGGATGGCAAACT- 3'
CMH2DCF-DA were obtained from Molecular Probes (Carlsbad, CA, Reverse 5'- ATGGCCAAGGCAAGACATAC- 3'
FN Forward 5'-TACCAAGGTCAATCCACACCCC- 3'
USA). PRDX3 siRNA and negative siRNA were synthesized by Bioneer
Reverse 5'-CAGATGGCAAAAGAAAGCAGAGG- 3'
(Daejeon, Korea). Other chemicals and tissue culture plates were ob- IL-6 Forward 5'-AGTTGCCTTCTTGGGACTGA- 3'
tained from Sigma-Aldrich Company (St. Louis, MO, USA) and Thermo Reverse 5'-TCCACGATTTCCCAGAGAAC- 3'
Scientific (Barrington, IL, USA), respectively, unless otherwise stated. ICAM-1 Forward 5'-CTT CCG ACT AGG GTC CTG AA- 3'
Reverse 5'-CTT CAG AGG CAG GAA ACA GG- 3'
IL-10 Forward 5′- GCTCTTACTGACTGGCATGA- 3′
2.2. Animals
Reverse 5′- CGCAGCTCTAGCATGTG- 3′
MCP1 Forward 5'-CTTCTGGGCCTGCTGTTCA- 3'
For experimental diabetes, male wild-type (WT) and Prx3 knockout Reverse 5'-CCAGCCTACTCATTGGGATCA- 3'
(Prx3 KO) C57BL/6 J mice [32] were used in this study. Eight-week-old Prx3 Forward 5'-GCGGCTGCGGGAAGGTTGCT- 3'
mice were divided into four groups: control, WT diabetic (WT+DM), Reverse 5'-TGCTGGGTGACAGCAGGGGT- 3'
TNFα Forward 5'-CGTCAGCCGATTTGCTATCT- 3'
Prx3 KO, and Prx3 KO+DM. Animals were fed with a standard chow Reverse 5'-CGGACTCCGCAAAGTCTAAG- 3'
diet and water ad libitum. Diabetes was induced by intraperitoneal TGFβ1 Forward 5'-CAGGAGCGCACAATCATGTT- 3'
injection of 50 mg/kg STZ for 5 days [39]. Age-matched control mice Reverse 5'-CTTTAGGAAGGACCTGGGTT- 3'
were injected with an equivalent volume of sodium citrate buffer αSMA Forward 5'-GTCCCAGACATCAGGGAGTAA- 3'
Reverse 5'-TCGGATACTTCAGCGTCAGGA- 3'
(100 mM sodium citrate, 100 mM citric acid, pH 4.5). Mice were sa-
18S Forward 5'-CGAAAGCATTTGCCAAGAAT- 3'
crificed after anesthesia with 16.5% urethane (10 mL/kg). After per- Reverse 5'-AGTCGGCATCGTTTATGGTC- 3'
fusion through heart by diethylpyrocarbonate-phosphate, left kidneys
were rinsed and then stored at −80 °C for further analysis. Right kid-
neys were fixed with 2% periodate-lysine paraformaldehyde via heart Shizuoka, Japan) antibodies. A Zeiss microscope equipped with an Axio
perfusion and kept at room temperature for further analysis. Cam HRC digital camera and Axio Cam software (Carl Zeiss, Thorn-
For experimental fibrosis, eight-week-old male WT and Prx3 KO also wood, NY, USA) was used to capture images. Staining intensities were
were used. UUO was performed under pentobarbital-induced an- quantified using Image-Pro Plus 4.5 software (Media 149 Cybernetics,
esthesia [40]. After flank incision, the left ureter was ligated with silk Silver Springs, MD, USA) as described previously [42].
(6/0) at two locations and cut between ligatures to prevent urinary
tract infection. The right side was subjected to sham surgery and the
2.5. Electron microscopy
kidney was used as sham control. Mice were sacrificed after 14 days of
UUO and their kidneys were analyzed. All animal experiments were
WT and Prx3 KO mice kidneys were sliced and treated with or
approved by Ewha Womans University's Institutional Animal Care and
without H2O2. After H2O2 stimulation, tissue for electron microscopy
Use Committee (approval No. 2010–7-6).
(EM) was fixed with 1% glutaraldehyde and 1% osmium tetroxide at
4 °C for 1 h. After washing with 0.1 M phosphate buffer, sections were
2.3. Measurement of plasma creatinine
dehydrated in a graded series of ethanol and embedded in epoxy resin
(Epon 812; Sigma-Aldrich, USA). Ultrathin sections were cut, counter-
Blood was collected in heparin-coated syringe before mice were
stained with 1% uranyl acetate, and photographed with a transmission
sacrificed. After centrifugation, plasma was collected. Creatinine level
electron microscope (H-7650; Hitachi, Japan) [43].
in the plasma was measured using a Detect X Serum Creatinine
Detection Kit (Arbor Assays, Ann Arbor, MI, USA).
2.6. Cell culture
2.4. Histology and immunohistochemistry
Immortalized murine proximal tubular epithelial (mProx) cells de-
For each mouse, paraffin-embedded kidney sections were stained rived from microdissected proximal tubular segments of C57BL6/J
with PAS reagent. Quantitative analysis of glomerular volume, tuft adult mouse kidneys were obtained from Dr. Sugaya at St. Marianna
area, and fractional mesangial area was performed as described pre- University School of Medicine, Kanagawa, Japan. These mProx cells
viously [41]. To examine collagen matrix, paraffin-embedded sections were maintained in Dulbecco's Modified Eagle's Medium (DMEM)
were stained with Masson's Trichome stain kit (HT15, Sigma-Aldrich). supplemented with 10% fetal calf serum, 100 U/mL penicillin, 100 μg/
For immunohistochemistry, we used anti-nephrin (1:100; Progen bio- mL streptomycin, and 44 mM NaHCO3 under 5% CO2 environment at
technik GmbH, Heidelberg, Germany), anti-F4/80 (1:200; Santa Cruz 37 °C. Raw264.7 macrophages were purchased from American Type
Biotechnology), anti-neutrophil gelatinase-associated lipocalin (NGAL) Culture Collection (Manassas, VA, USA) and maintained per company's
(1:100; Abcam, Cambridge, MA, USA), anti-collagen IV (1:200; instructions. Sub-confluent mProx and Raw264.7 cells were knocked
Southern Biotechnology Associates, Birmingham, AL, USA), anti-ni- down using PRDX3 siRNA or Prx3 KD (sense 5’- UGA CAA AGC CAA
trotyrosine (NT, 1:100; Santa Cruz Biotechnology), anti-8-Oxo-2'- UGA AUU U-3’, antisense 5’-AAA UUC AUU GGC UUU GUC A-3’), and
deoxyguanosine (8-oxo-dG, 1:200, Trevigen Inc., Gaithersburg, MD, Lipofectamine™ RNAiMAX (Life Technologies, Carlsbad, CA, USA).
USA), and anti-4-hydroxynonenal (4-HNE, 1:200, Nikken SEIL Co., After 24 h of transfection, cultured cells were growth-arrested with a

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Fig. 1. Effects of Prx3 deficiency on kidney morphology and injury in diabetic mice. Diabetic kidneys were embedded in paraffin and cut into 3 µm sections which
were then stained with PAS reagent (A). After PAS staining, (B) glomerular volume, (C) mesangial area, and (D) tuft area were analyzed using Image-Pro Plus 4.5.1,
Original magnification: 630 × ; scale bar: 10 µm. Paraffin embedded diabetic kidneys were stained with (E and G) NGAL (Original magnification: 100 ×; scale bar:
100 µm) and (F and H) nephrin (Original magnification: 630 ×; scale bar: 10 µm). (I) Plasma creatinine level was measured using a Detect X Serum Creatinine
Detection Kit (Arbor Assays, Ann Arbor, MI, USA). DM: STZ-induced diabetic mice. Data are presented as means ± SE of 4–5 mice/group; *p < 0.05 vs. WT,
†p < 0.05 vs. WT+DM, and #p < 0.05 vs. Prx3 KO.

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Fig. 2. Prx3 deficiency accelerates fibrosis in diabetic and UUO mouse model. Diabetic kidneys were embedded in paraffin and cut into 3 µm sections which were
then stained with collagen IV (A) and masson's trichrome (B). Original magnification: 200 × ; scale bar: 100 µm. (C-D) mRNA expression of TGFβ1, CTGF, E-
cadherin, αSMA, FN, and Col1α1. (E) Protein expression of Prx3, PAI-1, SNAI-1, E-cadherin, αSMA, vimentin, FN and collagen1, (F) Quantification of protein density
from C using ImageJ software. mRNA levels were determined using real-time RT-PCR and protein levels were measured using western blotting system. Data are
presented as mean ± SE of 4–5 mice/group; *p < 0.05 vs. WT, †p < 0.05 vs. WT+UUO, and #p < 0.05 vs. Prx3 KO.

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low-serum DMEM medium. Raw264.7 were cultured with LPS 100 ng/
mL for 6 h and replenished with DMEM. After 18 h of incubation,
conditioned media was collected and centrifuged. After serum starva-
tion, mProx cells were incubated with conditioned media of Raw264.7
cells for 24 h. To prevent glucose starvation, 22.5 mM glucose was
added to conditioned media.
2.7. Real-time RT-PCR analysis
Total RNA was extracted using Trizol reagent (Life Technologies)
and used for cDNA synthesis. mRNA expression level was measured by
real-time PCR using an ABI7300 system (Applied Biosystems, Carlsbad,
CA, USA). PCR was performed in 20 μL reaction volume containing
cDNA transcripts, primer pairs, and SYBR Green PCR Master Mix
(Applied Biosystems) as described previously [42]. Quantifications
were normalized to levels of 18 S rRNA. PCR primer sequences are
listed in Table 1.
2.8. Western blot analysis
Cells or tissues were analyzed using standard western blotting
analysis [42]. Protein samples were analyzed using Prx3 (1:2000, Ab-
Frontier, Seoul, Korea), PAI-1 (1:2000, Santa-Cruz Biotechnology),
SNAI-1 (1:500, Santa-Cruz Biotechnology), E-cadherin (1:3000, BD
Biosciences), vimentin (1:2500, Santa Cruz Biotechnology), αSMA
(1:1000, Sigma-Aldrich), fibronectin (FN) (1:2000, Santa-Cruz Bio-
technology), collagen 1 (Col1) (1:1000, Southern Biotech), ICAM-
1(1:2000, Santa-Cruz Biotechnology), iNOS (1:2000, Santa-Cruz Bio-
technology), p-NF-κB (1:5000, Cell Signaling Technology, Danvers, MA,
USA), NF-κB (1:4000, Cell Signaling Technology), and β-actin (1:1000,
Cell Signaling Technology) primary antibodies.
2.9. Detection of ROS
Kidney tissues from control or STZ-induced diabetic Prx3 KO mice
were embedded as paraffin block. Dihydroethidium (DHE) (5 μM,
Molecular Probes) was applied to these paraffin embedded kidney
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Free Radical Biology and Medicine 131 (2019) 162–172
Fig. 3. Prx3 deficiency accelerates fibrotic responses
in tubular epithelial cells. mProx cells were transfected
with scramble siRNA or Prx3 KD (Prx3 siRNA) for
24 h. After transfection, cells were treated with TGFβ1
or LPS for 6 or 24 h. mRNA levels of Prx3 and E-cad-
herin (A), and TGFβ1, FN, and αSMA (B) were mea-
sured after treated with or without TGFβ1 for 6 h. (C-
D) Protein expression levels of E-cadherin, αSMA and
FN were measured after treated with or without
TGFβ1 for 24 h. mRNA levels were analyzed using
real-time RT-PCR and protein levels were analyzed
using western blotting system. Data are presented as
mean ± SE of four experiments; *p < 0.05 vs.
scramble, †p < 0.05 vs. scramble+TGFβ1, and
#p < 0.05 vs. Prx3 KD.
segments (10 µm) for 15 min at 37 °C to reveal the presence of ROS with
red fluorescence at 561 nm followed by DAPI staining. Images were
taken using a Zeiss ApoTome Axiovert 200 M microscope (Carl Zeiss
Microscopy GmbH, 07745 Jena, Germany).
Mitochondrial ROS and cellular H2O2 were measured using
MitoSOX™ and CMH2-DCF-DA, respectively. Briefly, after stimulation,
mProx cells were washed and loaded with 2.5 μM MitoSOX for 10 min
or 5 μM CMH2-DCF-DA for 25 min. Unbound dye was then removed.
Fluorescence was analyzed using a flow cytometer (BD Biosciences) and
visualized by confocal microscopy (Carl Zeiss).
2.10. Statistical analysis
All results are expressed as mean ± standard error (SE) with N as
the number of experiments. Statistical significance of differences among
groups was compared by analysis of variance (ANOVA) and subsequent
Fisher post-hoc analysis. Statistical significance was set at p < 0.05.
3. Results
3.1. Prx3 deficiency accelerates kidney injury
PAS staining showed that histopathological parameters such as
glomerular volume, fractional mesangial area, and tuft area were sig-
nificantly increased in diabetic WT mice compared to those in the
control. However, these parameters were accelerated in diabetic Prx3
KO mice compared to those in diabetic WT (Fig. 1A-D). These were also
significantly increased in Prx3 KO control mice (Fig. 1A-D). NGAL ex-
pression was increased in diabetic WT mice. It was further increased in
diabetic Prx3 KO mice (Fig. 1E and G). On the other hand, nephrin
expression was significantly decreased in diabetic mice. It was further
decreased in diabetic Prx3 KO mice (Fig. 1F and H). Plasma creatinine
was increased in diabetic WT mice. It seemed to have a further increase
in diabetic Prx3 KO mice (Fig. 1I). In addition, kidney NGAL and KIM-1
mRNA levels were increased in UUO WT mice. They were further in-
creased in UUO Prx3 KO mice (Supplementary Fig. 1A-B). These data
suggest that Prx3 deficiency can accelerate kidney injury.
I. Hwang et al. Free Radical Biology and Medicine 131 (2019) 162–172

Fig. 4. Prx3 deficiency accelerates inflammation. (A) Diabetic kidneys were embedded in paraffin and cut into 3 µm sections which were then stained with F4/80
(Original magnification: 200 ×; scale bar: 100 µm). (B) Staining intensities were then quantified using Image-Pro Plus 4.5 software. Data are presented as
means ± SE of 4–5 mice/group; *p < 0.05 vs. WT, †p < 0.05 vs. WT+DM, and #p < 0.05 vs. Prx3 KO. In addition, at 14 days after UUO, kidney tissues were
subjected for analysis. (C-F) mRNA expression levels of F4/80, TNFα, MCP1, IL-10, and Arg1 mRNAs were analyzed using real-time RT-PCR. Data are presented as
mean ± SE of 4–5 mice/group; *p < 0.05 vs. WT, †p < 0.05 vs. WT+UUO, and #p < 0.05 vs. Prx3 KO. Furthermore, mProx cells were transfected with scramble
siRNA or Prx3 KD (Prx3 siRNA) for 24 h. After transfection, cells were treated with LPS for 6 h. (G) mRNA levels of TNFα, MCP1, COX2, and ICAM-1 were measured
after treated with or without LPS for 6 h. (H and I) Protein expression levels of ICAM-1 and iNOS were measured after treated with or without LPS for 24 h. mRNA
levels were analyzed using real-time RT-PCR and protein levels were analyzed using western blotting system. Data are presented as mean ± SE of four experiments;
*p < 0.05 vs. scramble, †p < 0.05 vs. scramble+LPS, and #p < 0.05 vs. Prx3 KD.

3.2. Prx3 deficiency accelerates kidney fibrosis
Collagen deposition in the kidney was increased in diabetic WT
mice. It was further increased in diabetic Prx3 KO mice (Fig. 2A and B).
We also employed UUO model to elucidate the role of Prx3 in the
progression of renal fibrosis. At 14 days after UUO, TGFβ1 and CTGF
mRNA levels were significantly increased in obstructed WT kidneys
(Fig. 2C). Accordingly, FN and Col1 mRNA and protein levels were
higher in obstructed WT kidneys than those in sham WT kidneys
(Fig. 2D-F). Markers of EMT (decrease of E-cadherin and increase of
αSMA, SNAI-1, and vimentin) showed significant alterations in ob-
structed WT kidneys compared to those in sham WT kidneys (Fig. 2C, E,
and F). Prx3 deficiency accelerated renal interstitial fibrosis as mea-
sured by profibrotic markers (TGFβ1, CTGF, and PAI-1), ECM accu-
mulation (FN and Col1), and EMT (SNAI-1 and αSMA) expression in
kidneys of obstructed Prx3 KO mice (Fig. 2C-F). Interestingly, most
profibrotic genes were altered even in kidneys of sham operated Prx3
KO mice (Fig. 2C and D).
To confirm these in vivo results, we transfected mProx cells with
scramble or Prx3 KD (Prx3 siRNA) and stimulated with or without
TGFβ1. As expected, mRNA and protein expression levels of TGFβ1,
αSMA, and FN were significantly increased in TGFβ1-stimulated Prx3
KD compared to those in scramble and Prx3 KD cells (Fig. 3B-D). In
addition, mRNA and protein expression levels of E-cadherin were de-
creased in TGFβ1-stimulated Prx3 KD as compared to those in scramble
and Prx3 KD cells (Fig. 3A-D). These results suggest that Prx3 deficiency
can accelerate renal fibrosis.

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Fig. 5. Prx3 deficiency enhances mitochondrial oxidative stress. Paraffin embedded kidney sections were stained with (A and B) NT, (C and D) 8-oxo-dG, and (E and
F) 4-HNE. Original magnification: 100 × ; scale bar: 50 µm. (G) Paraffin embedded kidney sections were stained with DHE at 5 μM. Original magnification: 100 × ;
scale bar: 50 µm. (H-I) In addition, mProx cells were transfected with scramble siRNA or Prx3 KD for 24 h. After transfection, cells were stimulated with TGFβ1 for
6 h. (H) After stimulation, cells were incubated with 2.5 μM MitoSOX for 10 min and fluorescence was visualized by confocal microscopy. Original magnification:
200 × ; scale bar: 100 µm. (I) After stimulation, cells were incubated with 5 μM CMH2-DCF-DA for 25 min and analyzed using a flow cytometer. Data are presented as
mean ± SE of four experiments; *p < 0.05 vs. scramble, †p < 0.05 vs. scramble+TGFβ1, and #p < 0.05 vs. Prx3 KD. (J) WT and Prx3 KO mice kidneys were
treated with or without 1 mM H2O2 in DMEM for 1 h (slice thickness, 1–2 mm) and images were taken using electron microscopy. Morphology of mitochondria (H2O2
treated KO kidneys had many scattered and rounded mitochondria with many vacuoles and apoptotic or necrotic nuclei, while WT mice kidney had elliptical and
dense mitochondria along with healthy nuclei and less vacuoles), scale bar: 2 µm. Data are presented as mean ± SE of 4–5 mice/group; *p < 0.05 vs. WT+H2O2.

3.3. Prx3 deficiency accelerates kidney inflammation has an important role in maintenance of redox state in the kidney.

Expression of F4/80 as measured by immunostaining was sig-
nificantly increased in the tubular interstitium of diabetic WT mice
(Fig. 4A and B). This was accelerated in diabetic Prx3 KO mice com-
pared to that in diabetic WT mice (Fig. 4A and B). In UUO Prx3 KO
mice, mRNA expression levels of inflammatory cytokines such as F4/
80, TNFα, and MCP1 were significantly increased compared to those in
obstructed WT mice (Fig. 4C-D). In addition, markers of M2 macro-
phages in kidney such as IL-10 and Arg1 (Fig. 4E and F) in UUO Prx3
KO mice were decreased compared to those in UUO WT mice, although
they showed no significant difference between Prx3 KO and WT mice.
We found that MCP1 mRNA expression levels were significantly in-
creased even in kidneys of sham operated Prx3 KO mice (Fig. 4D).
In order to confirm the effect of Prx3 deficiency on inflammatory
cytokines, we transfected mProx cells with scramble or Prx3 KD and
stimulated them with or without LPS. Results showed that mRNA ex-
pression levels of different proinflammatory cytokines such as TNFα,
MCP1, COX2, and ICAM-1 were significantly increased in LPS-stimu-
lated scramble cells compared to those in the control. However, they
were accelerated in Prx3 KD cells compared to those in LPS-stimulated
scrambled cells (Fig. 4G). In addition, TNFα and COX2 mRNA expres-
sion levels were significantly increased even in Prx3 KD mProx cells
(Fig. 4G). In line with mRNA expression results, protein expression le-
vels of ICAM-1 and iNOS were also significantly increased in LPS-
treated Prx3 deficient cells (Fig. 4H and I). Together, these results in-
dicate that Prx3 deficiency may accelerate macrophage infiltration and
inflammation in kidney injury.
3.4. Prx3 deficiency enhances kidney mitochondrial oxidative stress
Markers of oxidative stress such as NT, 8-oxo-dG, and 4-HNE were
significantly increased in diabetic WT kidneys. They were accelerated
by Prx3 deficiency (Fig. 5A–F). Further, kidney ROS as measured by
DHE staining was increased in diabetic WT mice. It was accelerated by
Prx3 deficiency (Fig. 5G). We next measured mitochondrial ROS using
MitoSOX to evaluate whether Prx3 deficiency affected mitochondrial
ROS. In mProx cells, TGFβ1 treatment increased MitoSOX intensity
while Prx3 siRNA significantly accelerated such effect as compared
with scramble control (Fig. 5H). Prx3 deficiency significantly increased
mitochondrial ROS even at basal condition (Fig. 5H). Similarly, TGFβ1
treatment increased intracellular ROS levels while Prx3 siRNA sig-
nificantly accelerated such effect as compared with scramble control in
mProx cells (Fig. 5I). These results depict that Prx3 plays an important
role in the regulation of mitochondrial ROS-mediated oxidative stress.
In H2O2-treated kidneys taken from Prx3 KO mice, electron micro-
scopy showed that there were many apoptotic or necrotic nuclei and the
cytoplasm was occupied by many rounded and scattered mitochondria
as well as many vacuoles as compared to H2O2-treated kidneys of WT
mice (Fig. 5J). Interestingly, these abnormalities were also observed in
Prx3 KO mice at basal (Fig. 5J) while elliptical and dense mitochondria
along with healthy nuclei and less vacuole were found in kidneys of WT
mice at basal (Fig. 5J). Gene expression of mitochondrial DNA (mtDNA)
and its regulators such as PGC1α and Nrf1 were significantly reduced in
H2O2-treated kidneys of Prx3 KO mice compared to those in WT mice
(Supplementary Fig. 2). These results suggest that mitochondrial Prx3
3.5. Prx3 deficiency activates macrophages and increases proinflammatory
and profibrotic cytokines
To evaluate whether activated macrophages accelerated renal in-
terstitial fibrosis in Prx3 KO mice, we employed in vitro study using
Prx3 deficient macrophages. In Raw264.7 macrophages, LPS-stimulated
p-NF-κB expression was accelerated by Prx3 deficiency (Fig. 6A). In
Raw264.7 macrophages, LPS-induced mitochondrial or intracellular
ROS was significantly higher in Prx3 deficient cells than that in control
cells (Fig. 6B and C). In addition, Prx3 deficiency accelerated mRNA
expression levels of TNFα, COX2, and IL6 in LPS-stimulated macro-
phages (Fig. 6D-E). Prx3 deficiency significantly increased TNFα, COX2
and IL6 mRNA expression level in macrophages even under control
conditions (Fig. 6D-E). These data indicate that Prx3 deficiency can
accelerate activation of macrophages.
To further investigate the involvement of macrophages, Prx3 defi-
cient Raw 264.7 cells were treated with LPS for 6 h. Prx3 deficient
mProx cells were then stimulated with conditioned media (CM1,
scramble siRNA; CM2, Prx3 siRNA) from Raw 264.7 cells for 24 h.
Results showed that mRNA levels of TNFα, COX2, MCP1, and ICAM-1
were significantly upregulated in Prx3 deficient mProx cells incubated
in CM1. They were further increased by treatment with CM2 (Fig. 6F).
These data indicated that macrophage adhesion and inflammatory re-
sponses were higher in Prx3-deficient tubular epithelial cells than those
in controls. We also observed that TGFβ1 and fibronectin mRNA ex-
pression levels were increased in Prx3 deficient mProx cells incubated
with CM1. They were accelerated by treatment with CM2 (Fig. 6F).
Interestingly, we also observed increases in mRNA levels of ICAM-1 and
TGFβ1 in the scramble mProx cells incubated in CM2 (Fig. 6F). These
results indicate that Prx3-deficiency may induce macrophage activation
which may secrete cytokines, leading to promotion of fibrosis in tubular
epithelial cells.
4. Discussion
The present study provided experimental evidences that Prx3 defi-
ciency could accelerate CKD including fibrosis and inflammation uti-
lizing UUO and diabetic kidney disease mouse models. Prx3 deficiency
activated macrophages and accelerated fibrosis and inflammation in
proximal tubular epithelial cells at basal condition as well as under
inflammatory stimulation. We found that Prx3 deficiency accelerated
oxidative stress and inflammation in obstructive and diabetic kidney
injury in vivo and mProx cells in vitro. Consistently, previous studies
have demonstrated that Prx3 deficiency is involved in various pathol-
ogies such as lung inflammation, muscular disorders, adipocyte dys-
functions, and liver injury [30–33]. Its overexpression has protective
effects against neuronal toxicity, myocardial infarction, and renal
ischemia-reperfusion injury [27–29].
In our study, antioxidant Prx3 was decreased in kidneys under stress
stimuli. Prx3 deficiency increased ROS not only after treating with
TGFβ1 and LPS, but also at basal condition in tubular epithelial cells
and Raw267.4 macrophages, respectively. Furthermore, kidneys of
Prx3 KO mice showed abnormal morphology of mitochondria and de-
creased expressions of mitochondrial biogenesis markers after

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Fig. 6. Prx3 deficiency activates Raw264.7 macrophages and increases proinflammatory and profibrotic cytokines. Raw264.7 cells were transfected with scramble
siRNA or Prx3 KD (Prx3 siRNA) for 24 h. After transfection, cells were stimulated with LPS (0, 10 and 100 ng/mL) for 6 h. (A) Phosphorylated-NF-κB measured by
western blotting. (B-C) After transfection, Raw264.7 cells were stimulated with LPS (100 ng/mL) for 6 h. (B) Mitochondrial ROS was measured using MitoSOX, and
(C) intracellular ROS was measured using CMH2-DCF-DA. Data are presented as mean ± SE of four experiments; *p < 0.05 vs. control. (D-E) mRNA expression
levels of Prx3, TNFα, COX2, and IL6 (100 ng/mL LPS) measured by real-time RT-PCR. Data are presented as mean ± SE of four experiments; *p < 0.05 vs. scramble,
†p < 0.05 vs. scramble+LPS, and #p < 0.05 vs. Prx3 KD. Furthermore, mProx cells were transfected with scramble or Prx3 KD (Prx3 siRNA) for 24 h. After
transfection, cells were stimulated with conditioned medium (CM1, scramble and CM2, Prx3 KD) collected from Raw264.7 cells for 24 h. (F) mRNA levels of TNFα,
COX2, MCP1, ICAM-1, TGFβ1, and FN were measured by real-time RT-PCR. Data are presented as mean ± SE of four experiments; *p < 0.05 vs. CM1 scramble,
#p < 0.05 vs. respective scramble, and † indicates difference between CM1 and CM2 in Prx3 KO.

stimulation with H2O2 or at basal condition. However, mitochondrial
dysfunction due to overproduction of ROS-induced oxidative stress is
linked to inflammation [21,22] and kidney fibrosis [44]. The present
data suggest an important role of Prx3 in maintenance of mitochondrial
redox state, leading to kidney homeostasis.
EMT of tubular epithelial cells is characterized by loss of epithelial
cell characteristics and gain of ECM-producing myofibroblast char-
acteristics. It is an important mechanism involved in tubulointerstitial
fibrosis [45,46], although the role of EMT in renal fibrosis in vivo re-
mains controversial [5–8]. Higher expression of Prx3 is found to be co-
related with lower interstitial fibrosis, tubular injury, and inflammation
in kidneys from patients with acute tubular necrosis [26]. The role of
Prx3 in CKD, however, has not been demonstrated. In the present study,
for the first time, we showed that Prx3 deficiency accelerated renal
interstitial fibrosis as measured by markers of profibrotic cytokines,
ECM accumulation, and EMT in Prx3 KO kidney tissues or proximal

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Fig. 7. Suggested scheme for effects of Prx3 deficiency on CKD. Diabetes or
UUO stress triggers mitochondrial ROS leading to EMT as well as upregulation
of ECM and cytokine synthesis in proximal tubular cells. These stress stimuli
also trigger mitochondrial ROS in macrophages, leading to upregulation of
proinflammatory cytokines. The secreted cytokines from epithelial cells help
the macrophages to produce more ROS leading to acceleration of inflammation
and fibrosis in kidney through activation of different signaling molecules. Prx3
deficiency-mediated acceleration of stress-induced mitochondrial ROS leads to
increased kidney inflammation and fibrosis through interactions between
macrophages and tubular epithelial cells.
tubular cells.
Increased macrophage infiltration is closely associated with CKD.
While macrophage activation contributing to fibroblast activation and
myofibroblast generation [11,47] is another mechanism involved in
kidney fibrosis, Prx3 deficiency also contributes to macrophage acti-
vation and inflammation through exposure to oxidative stress in mouse
bone marrow [34]. Recently, antifibrotic effects of macrophages on
renal fibrosis have been reported [48,49]. Macrophages can also reduce
renal fibrosis through phagocytic clearance [48]. Furthermore, M1
macrophages, but not M2 macrophages, can induce kidney injury
[15,50,51]. Consistently, in our study, markers of M1 macrophages
were significantly increased in diabetic or UUO WT mice. They were
accelerated in diabetic or UUO Prx3 KO mice. On the other hand,
markers of M2 macrophages were significantly decreased in diabetic or
UUO Prx3 KO mice compared to those in diabetic or UUO WT. Along
with fibrotic markers, inflammation markers were accelerated in Prx3
deficient mProx cells. Interestingly, mRNA levels of MCP1 in Prx3 KO
mice as well as TNFα and COX2 in Prx3 KD mProx cells were increased
at basal condition. These data indicate that Prx3 may play an important
role in inhibition of macrophage infiltration which either directly or
indirectly induces tubular dysfunction in CKD. NF-κB signaling is re-
sponsible for kidney injury in mice [52]. Thus, we measured expression
levels of NF-κB in Raw264.7 macrophages in order to know the status of
macrophage activation. Interestingly, Prx3 deficiency accelerated ex-
pression of proinflammatory cytokines such as TNFα, COX2, and IL6 in
Raw264.7 macrophages in addition to activation of NF-κB. To further
study the direct role of macrophages in tubular damage in the kidney,
we used conditioned media from Prx3 deficient Raw264.7 macrophages
to treat Prx3 deficient mProx cells. Our data showed that conditioned
medium from Prx3 deficient Raw264.7 macrophages accelerated Prx3
KD-induced inflammatory and fibrotic responses in mProx cells. These
results suggest that Prx3-deficiency can induce macrophage activation
which then secretes cytokines, thus promoting fibrosis in tubular epi-
thelial cells.
In conclusion, our findings provide evidence that Prx3 deficiency
accelerates CKD including fibrosis, oxidative stress, and inflammation
accompanied by mitochondrial oxidative stress and macrophages acti-
vation. In addition, Prx3 deficiency induced most of inflammatory and
fibrotic cytokines at basal condition in both tissues and cells. These data
suggest that Prx3 may control CKD in mice by modulating interactions
between macrophages and tubular epithelial cells (Fig. 7). Therefore,
Prx3 might be an important target for treating progressive CKD.
171
Free Radical Biology and Medicine 131 (2019) 162–172
Grants
This work was supported by a National Research Foundation grant
(No. 2016R1A2B4006575) and Korean Research Fellowship Program
(No. 2015H1D3A1062189), Republic of Korea.
Disclosures
None of the authors have any competing interest to disclose.
CRediT authorship contribution statement
Inah Hwang: Conceptualization, Data curation, Formal analysis,
Investigation, Methodology, Project administration, Software,
Visualization. Md Jamal Uddin: Formal analysis, Funding
acquisition, Software, Supervision, Writing - original draft, Writing -
review & editing. Gayoung Lee: Data curation, Methodology, Software,
Visualization. Songling Jiang: Data curation, Methodology, Software,
Visualization. Eun Seon Pak: Data curation, Methodology, Software,
Visualization. Hunjoo Ha: Conceptualization, Funding acquisition,
Investigation, Project administration, Resources, Supervision,
Validation, Writing - review & editing.
Acknowledgements
We acknowledge Professor KH Han at Department of Anatomy,
College of Medicine, Ewha Womans University for his help in the in-
terpretation of EM images.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.freeradbiomed.2018.12.002.
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