130 • Pathogenic mechanisms of redox/inflammatory responses to endurance exercise

changes of thioredoxin, oxidative stress markers, inflammation andmuscle/renal damage following intensive endurance exercise
Kaoru sugama1, 2, Katsuhiko suzuki3, 4, Kayo yoshitani5, Koso shiraishi5,
shigeki Miura6, hiroshi yoshioka6, yuichi Mori6, takashi Kometani7

Institute for Nanoscience & Nanotechnology, Waseda University, Tokyo, Japan.
Division of Cell Regeneration and Transplantation, Department of Functional Morphology, Nihon University School of Medicine, Tokyo, Japan.
3 Faculty of Sport Sciences, Waseda University, Saitama, Japan.
4 Cooperative Major in Advanced Health Science, Tokyo University of Agriculture and Technology/ Waseda University,
Tokyo, Japan.
5 Health Science Laboratory, Ezaki Glico Co., Ltd, Osaka, Japan.
6 Mebiol Inc., Kanagawa, Japan.
7 Department of Food Science and Nutrition, Faculty of Agriculture, Kinki University, Nara, Japan.

1

2

aBstRact
Thioredoxin (TRX) is a 12 kDa protein that is induced byoxidative stress, scavenges reactive oxygen species (ROS) andmodulates chemotaxis. Furthermore it is thought to play aprotective role in renal ischemia/reperfusion injury. Complement 5a (C5a) is a chemotactic factor of neutrophils and is
produced after ischemia/reperfusion injury in the kidney. BothTRX and C5a increase after endurance exercise. Therefore, it
may be possible that TRX has an association with C5a inrenal disorders and/or renal protection caused by endurance
exercise. Accordingly, the aim of this study was to investigate
relationships among the changes of urine levels of TRX, C5a
and acute kidney injury (AKI) caused by ischemia/reperfusion, inflammatory responses, and oxidative stress following
intensive endurance exercise. Also, we applied a newly-developed measurement system of neutrophil migratory activity
and ROS-production by use of ex vivo hydrogel methodology
with an extracellular matrix to investigate the mechanisms ofmuscle damage. Fourteen male triathletes participated in aduathlon race consisting of 5 km of running, 40 km of cycling
and 5 km of running were recruited to the study. Venous bloodand urine samples were collected before, immediately following, 1.5 h and 3 h after the race. Plasma, serum and urine
were analyzed using enzyme-linked immunosorbent assays, afree radical analytical system, and the ex vivo neutrophil
functional measurement system. These data were analyzed byAddress for correspondence:
Katsuhiko Suzuki, MD, PhD.
Faculty of Sport Sciences, Waseda University,
2-579-15, Mikajima, Tokorozawa, Saitama 359-1192, Japan.
Tel: +81-4-2947-6898, Fax: +81-4-2947-6898
Email: katsu.suzu@waseda.jp
EIR 21 2015

assigning participants to damaged and minor-damage groups
by the presence and absence of renal tubular epithelial cells
in the urinary sediments. We found strong associations among
urinary TRX, C5a, interleukin (IL)-2, IL-4, IL-8, IL-10, interferon (IFN)-γ and monocyte chemotactic protein (MCP)-1.
From the data it might be inferred that urinary TRX, MCP-1
and β-N-acetyl-D-glucosaminidase (NAG) were associatedwith renal tubular injury. Furthermore, TRX may be influenced by levels of IL-10, regulate chemotactic activity of C5a
and IL-8, and control inflammatory progress by C5a and IL-8.
In the longer duration group (minor-damage group), circulating neutrophil count, plasma concentration of myeloperoxidase (MPO) and serum concentration of myoglobin weremarkedly increased. In the higher intensity group (damaged
group), neutrophil activation and degranulation of MPO
might be inhibited, because not only was ROS production
observed to be higher, but also antioxidant capacity and antiinflammatory cytokines were increased. Critically, the newlydeveloped ex vivo methodology corroborated the neutrophil
activation levels in the two groups of participants.
Key words: TRX, C5a, ROS, antioxidant, anti-inflammation,acute kidney injury (AKI)

intRODUctiOnEndurance exercise not only promotes the generation of reactive oxygen species (ROS), mainly as a result of increased
oxygen utilization, ischemia-reperfusion and leukocyte activation, but also consumes endogenous antioxidants (2, 3, 54).
This unbalanced state induces oxidative stress and cellular tissue damage in the body. Oxidative stress-induced injury andinflammation are important considerations for athletes.
Recently, it has been reported that aerobic exercise interventions can have a positive effect on chronic renal failure. In
the patients with chronic kidney diseases including thoseundertaking dialysis therapies or in receipt of kidney transplant, it was demonstrated that aerobic exercise reduced
oxidative stress and improved quality of life (18, 27). For

Pathogenic mechanisms of redox/inflammatory responses to endurance exercise • 131
these reasons, exercise is recommended for these patients(26). The underlying mechanisms for the direct improvementof renal functions due to aerobic exercise have not yet been
identified. However, it has been suggested that moderate exercise may promote nitric oxide production, and inhibit therenin-angiotensin-aldosterone system, or improve renal bloodflow by modifying dyslipidemia and intraglomerular pressure(29, 33, 48). However, the acute effects of exercise are different from the chronic effects of exercise training and therefore
these exercise modes should be considered separately (33). In
acute exercise, it is known that deterioration of glomerular filtration rate and oliguria are induced following endurance
exercise (69), and that intensive endurance exercise may
cause rhabdomyolysis-induced acute renal failure (10, 52).
Rhabdomyolysis may manifest in acute renal failure due toacute tubular necrosis leading to deterioration of glomerular
filtration rate. This is induced by glomerulus degeneration andreduced renal blood flow leading to a reduced supply of oxygen and energy, thereby resulting in ischemic vascularendothelial cell damage (6). In animal models of acute renal
failure, ROS and lipid peroxides increase, whereas scavengers
of ROS such as glutathione and superoxide dismutase (SOD)
are decreased in renal tissue (51). It was reported that ROS,
such as superoxide anion and hydroxyl radical, contributed tothe onset of acute renal failure through reduced renal bloodflow and disorder of tubular cells (55). Thus, acute endurance
exercise-induced oxidative stress may cause renal failure, andtherefore it is important to evaluate oxidative stress in thiscontext.
Currently, oxidative stress is evaluated using metabolicoxidation end products, because the real time evaluation ofthis phenomenon is difficult. Recently, it was reported thatthioredoxin (TRX) is secreted by renal tubular cells due toischemia and oxidative stress (30). Furthermore, its usefulness
as a specific biomarker of acute kidney injury (AKI) causedby ischemia and oxidative stress is being established (30).
TRX is a small (12 kDa) multifunctional protein that contains
a redox-active dithiol/disulfide in the conserved active site
sequence: -Cys-Gly-Pro-Cys- (20). TRX is stress-inducible,
and it protects cells from various types of stress (45, 46). TRX
functions as an antioxidant, as an inhibitor of chemotaxis, andas a redox-regulating protein in signal transduction (12, 21,
22, 57). TRX eliminates hydrogen peroxide and acts as a radical scavenger, as demonstrated where recombinant TRX has aprotective activity against hydrogen peroxide cytotoxicity(23, 38, 45). It is reported that elevated serum TRX in various
diseases is associated with increased oxidative stress (25, 32,
40, 46, 47, 60, 61, 83). Moreover, serum levels and expression
levels of TRX increase following endurance exercise (36, 70,85). Recently, it was reported that the urine level of TRX is a
specific marker for ischemia and oxidative stress-induced
acute renal failure, because it is secreted from renal tubularepithelial cells in response to ischemia/reperfusion injury inrenal tissue (30).
Another protein associated with AKI is complement 5a(C5a) (1). C5a is a multifunctional proinflammatory mediator,
and a chemotactic factor, which increases the permeability ofblood vessels and promotes the migration of leukocytes
towards inflammatory sites and their generation of ROS (14,35). It is reported that C5a increases and causes inflammation
following marathon race (8). C5a is an important pathogenic

factor in renal ischemia/reperfusion injury (1). The role of
C5a in the tubulointerstitial component is demonstrated in anexperimental model of progressive glomerulonephritis (80).
Indeed, C5a receptor activation in glomerular mesangial cells
has been shown to induce proliferation, produce cytokinesand growth factors, as well as upregulate certain transcription
factors and early response genes (81). The terminal complement complex in plasma and urine, and the anaphylatoxin C5a
in plasma and urine, might have potential as an early and reliable marker for acute renal allograft rejection (44). In thisregard, urinary C5a level is positively correlated with theseverity of renal injury, which highlights the important role of
C5a in renal damage of human anti-glomerular basement
membrane disease (14, 35).
Given the stresses experienced by endurance athletes, thefirst aim of this study was to investigate relationships among
urine levels of TRX, C5a and AKI caused by ischemia/reperfusion and oxidative stress following intensive endurance
exercise. Furthermore, in our previous study, we reported urinary excretion of interleukin (IL)-2, IL-4, IL-8, IL-10, interferon (IFN)-γ and monocyte chemotactic protein (MCP)-1 instressed athletes suffering from renal tubular epithelial damage. The damaged kidney might be responsible, at least inpart, for the kinetics of some cytokines after endurance exercise (59). Therefore, the second aim was to clarify associations between urine levels of TRX or C5a and those of IL-2,
IL-4, IL-8, IL-10, IFN-γ, MCP-1 as well as urine albumin(ALB) and serum creatinine (Cr) as renal function markers.
The final aim was to determine oxidative stress responses in
the circulation after exercise. Here, we applied a newly-developed system of measurement for neutrophil migratory activity
and ROS-production. This system uses ex vivo hydrogel
methodology with an extracellular matrix as a means to investigate the mechanisms of tissue damage (28).

MethODs

subjects
Fourteen male triathletes [age 28.7 ± 7.9 (mean ± SD) yr andbody mass 63.2 ± 6.0 kg], volunteered to participate in thisstudy. The subjects were seven professional triathletes andseven amateur triathletes. They completed a medical questionnaire and gave written informed consent prior to the study.
None of the athletes had been ill in the previous month. The
experimental procedure was approved by the institutional
ethics committee of Waseda University.
Renal tubular epithelial cells and renal tubular epithelial
casts were observed in the urinary sediments of seven subjects, among the fastest eight subjects for race time (59). In
this study, according to the values of serum Cr in the AKI
diagnosis criteria such as “Risk, Injury, Failure, Loss, End
Stage Kidney Disease (ESKD): RIFLE criteria” (4) and“acute kidney injury network: AKIN” (34, 37), AKI following
endurance exercise showed “Risk” or “StageI”at 0 h and 1.5 hafter the race in the seven subjects with the existence of renal
tubular epithelial cells in the urinary sediments. Immediately
after exercise in the other seven subjects, there was no evidence of renal tubular epithelial cells in the urinary sediments.
After this, the athletes were analysed as two subgroups thatwere divided according to the existence (damaged group,
EIR 21 2015

132 • Pathogenic mechanisms of redox/inflammatory responses to endurance exercisen=7) or non-existence (minor-damage group, n=7) based on
the levels of renal tubular epithelial cells in the urinary sediments (59).

Duathlon race
The present investigation was conducted in an official
duathlon race held on the road course of Miyako Island, Okinawa, Japan as described previously (59). Briefly, the race
consisted of 5 km of running, 40 km of cycling, and 5 km of
running, and began at 14:00. The weather was fair, and theambient temperature was 24.6 °C.

Research design
All participants agreed to avoid the use of vitamin/mineral
supplements, herbs and medications from the previous day
until after the last sampling point. All participants ate an
identical breakfast at 08:30. The breakfast contained 574
kcal, with 22.1 g protein, 13.7 g fat and 88.8 g carbohydrate. The pre-race blood and urine samples (Pre) were collected at 10:30 while the participants were at rest. The athletes did not exercise for approximately 18 h before the prerace blood and urine sampling. The post-race blood andurine samples were collected immediately (0 h), 1.5 h and 3
h after the race. Peripheral blood samples were drawn byantecubital venipuncture with the participants in the sitting
position. Urine samples were collected into designated vessels. They ate lunch at 11:00. The lunch contained 211 kcal,
with 9.3 g protein, 2.4 g fat and 38.6 g carbohydrate. All
participants drank the same quantity of fluid during exercise. After a warm-up, they each drank 600 ml of fluid
before the race. During the race, they each drank 1400 mlof fluid. Therefore, the total fluid intake for each individual
was 2000 ml. They each drank 1500 ml of water until 3 hafter the race.

serum, plasma, urine sampling, urinary sediments andbiochemical parameters
Approximately 7 ml of blood was drawn by a standard
venipuncture technique from the antecubital vein using vacutainers containing no additive or sodium heparin and disodium EDTA as an anticoagulant to obtain serum and plasma
samples, respectively. Collected blood samples containing no
additives were allowed to clot at room temperature for 1 hour
before centrifugation at 1000 × g for 10 min for serum preparation, whereas blood samples containing disodium EDTA
were centrifuged immediately for plasma preparation. Plasma
was stored at -80 °C until the day of analysis. Serum concentrations of Cr, myoglobin (Mb), uric acid (UA), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine
aminotransferase (ALT) and plasma concentration of lactate
were measured using an automated analyzer (Model 747-400,
Hitachi, Tokyo, Japan) (59).
Urine sample volume was measured and then approximately 8 ml was stored at 4 °C without centrifugation until analysis of sediments. Remaining urine samples were centrifuged
immediately at 1000 × g for 10 min to remove sediments, andthe supernatants were stored at -80 °C until the day of analysis. Urinary concentrations of Cr, ALB, UA, Mb and β-Nacetyl-D-glucosaminidase (NAG) activity were measured
using an automated analyzer (Model 747-400, Hitachi, Tokyo,
Japan) (59). The urinary data are reported as the gross amountEIR 21 2015

per minute (urinary excretion rate) as described previously(59).

MPO, tRx, c5a, cytokines and chemokines
Myeloperoxidase (MPO), IL-1 receptor antagonist (IL-1ra),IL-6, IL-8 and IL-10 were measured in plasma, and TRX,
C5a, IL-2, IL-4, IL-8, IL-10, IFN-γ and MCP-1 were measured in urine samples with enzyme-linked immunosorbent
assay (ELISA) kits according to the manufacturer’s instructions (59). The following kits were used for all measurements:
MPO (Hbt ELISA test, Hycult biotechnology, Uden, The
Netherlands), TRX (TRX ELISA Kit, Redox Bio Science,
Kyoto, Japan), IL-6 (Quantikine HS, R&D Systems, Inc.,
Minneapolis, MN), IL-1ra and MCP-1 (Quantikine, R&D
Systems, Inc.), C5a, IL-2, IL-4, IL-8, IL-10 and IFN-γ(OptEIA, Beckton Dickinson Biosciences, San Diego, CA,
USA) (59). For all assays, the absorbance was measured spectrophotometrically on a microplate reader (VERSAmax,
Molecular Devices, Sunnyvale, CA, USA) and the concentration of each cytokine was calculated by comparison with astandard curve established in the same measurement. The urinary data are reported as the gross amount per minute (urinary
excretion rate).

neutrophil function
Neutrophil function was measured using modified Mebiol(scaffold-thermoreversible galation polymer: S-TGP) gel(Mebiol Co., Hiratsuka, Kanagawa, Japan) and luminol as
described previously (17, 28, 68). Peripheral blood samples
were drawn in a 2 ml sodium-heparin tubes (Venoject II,
Terumo Co., Tokyo, Japan). The blood samples were mixed
with 2.5 mM luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione; Sigma Aldrich, MO, USA) at a ratio of 1:1.
Then, 150 μl luminol-blood samples were layered on 50 μl STGP gel prepared in a tube at 37°C, and was promptly measured by relative light unit (RLU) using a luminometer (GeneLight 55; Microtec Co., Ltd, Funabashi, Chiba, Japan). The
samples were incubated at 37°C, and the production of ROS
from neutrophils was monitored in a kinetic mode for 60 min.
After measurement of luminol-dependent chemiluminescence (LmCL) for 60 min, luminol-blood samples were
removed. The tubes with 50 μl S-TGP gel in which neutrophils migrated were washed three times with PBS at 37°C.
Then, the tubes with gel were cooled on ice and mixed wellfollowing addition of 50 μl Turk solution (Wako, Osaka,
Japan). The suspension obtained in this way was set on the CChip (Disposable haemocytometer, Neubauer improved,
DHC-No.1, Digital Bio, Seoul, Korea), and the migratory cellnumber was counted microscopically. Migrating neutrophils
were calculated by a 20 times multiplication of the counted
cell number (28).
Oxidative stress markers
To analyze the plasma levels of reactive oxygen metabolites,
and antioxidant capacity, diacron reactive oxygen metabolites(d-ROMs), biological antioxidant potential (BAP), and total
antioxidant capacity (OXY-absorbent test) were performed
respectively using the Free Radical Analytical System(Diacron, Grosseto, Italy) according to the manufacturer’s
instructions. The d-ROMs test provides a measure for theoxidative stress of blood samples by evaluating the level ofPathogenic mechanisms of redox/inflammatory responses to endurance exercise • 133
reactive oxygen metabolites particular to hydroperoxides. This
assay is based on the capability of N,N-dimethyl-paraphenylen-diamine (DMPD) to give a stable, colored solution
when it is transformed into its radical cation (DMPD+). The
assay was performed in a 5 ml plastic tube by adding 20 μl of
DMPD (final concentration 1mM) and 10 μl of plasma sample
to 2 ml of 0.1 M acetate buffer, pH 4.8. The formation of thecolored DMPD+ was monitored by reading the absorbance at
505 nm. The amount of the colored DMPD+ is related to theoxidative stress of the plasma and can be expressed in terms ofhydrogen peroxide equivalents, with 1 U. CARR (Carratelliunit) corresponding to 0.8 mg/l hydrogen peroxide (11,74). The
BAP assay is a photometric test that determines the serum concentration of antioxidants capable of reducing the iron from theferric to the ferrous form. A plasma aliquot (10 μl) was dissolved in 1 ml of colored solution obtained by mixing 50 μl of
ferric ions (FeCl3; ferric chloride) with a chromogenic substrate(a sulfur-derived compound). Following 5 min incubation, theintensity of the color change was assessed spectrophotometrically at 505 nm. The amount of reduced ferric ions was calculated and the BAP unit was expressed as μM (71). The OXYabsorbent test allows assessment of the antioxidant power ofplasma by measuring the ability of such barrier to oppose thelarge oxidant action of hypochlorous acid (HOCl). HOCl is
used as an indicator because it is one of the strongest ROS produced by leukocytes. In the OXY-absorbent test, 1 ml of R1
reagent (HOCl solution) was put into an empty cuvette, towhich 10 μl of previously diluted sample (plasma or serum)
was added and mixed. The solution was incubated at 37°C for
10 min, before addition of 10 μl of reagent R2 (chromogen).
The cuvette contents were mixed and the absorbance measured
spectrophotometrically at 546 nm. The results were expressed
as μM of HOCl adsorbed by 1 ml of sample (μM HOCl/ml).

statistical analyses
Data were presented as mean ± standard deviation (SD). Statistical validation was assessed using Friedman’s test. If significance was detected, the Scheffe method was used for multiple comparisons. Associations among measured variables
were determined by Spearman’s rank correlation coefficient
analysis. Statistical findings were deemed to be significant
where the probability of events occurring at random was lessthan 5% (p< 0.05).

between the two groups. Serum OXY increased significantly
immediately after exercise compared with pre exercise in thedamaged group only (0 h: 1.2-fold).

Urinary tRx and c5a
As shown in Figure 1, the excretion rate of TRX in the minordamage group did not significantly change, while in the damaged group, TRX was significantly increased at 1.5 h (20.1fold) after the race and then decreased 3 h (4.9-fold) postexercise. There was no significant change in the excretion rateof C5a for the minor-damage group. The excretion rate of C5a
increased significantly 3 h (31.8-fold) after exercise in thedamaged group when compared to pre-exercise values.

associations between urinary nag, tRx, alB and McP-1
As shown in Table 2, the urinary excretion rate of NAG, as amarker of renal tubular epithelial cell injury, was positively
correlated with that of TRX, ALB and MCP-1 in the damaged
group. In the minor-damage group, the urinary excretion rateof NAG 0 h after the race was positively correlated with thatof ALB immediately after exercise only.

Relations among urinary tRx, c5a, renal function
makers and cytokines
As shown in Table 3, the area under the curve (AUC) for pre-,0 h, 1.5 h and 3 h of urinary excretion rate of TRX was positively correlated with that of ALB in the damaged group. Furthermore, there was a trend for a positive correlation withserum Cr concentrations, whereas there was a trend for TRX
to be negatively correlated with AUC of urinary excretion rateof C5a, IL-2 and negatively correlated with AUC of urinary
excretion rate of IL-4, IL-8, IL-10 and IFN-γ. There was atrend for the AUC of urinary excretion rate of C5a to be nega-

ResUlts

Markers of renal function in urine and plasma lactate
The degree of renal damage, as measured by creatinine clearance, urinary excretion rates of ALB and NAG, was higher inthe damaged group than those in the minor-damage group(59). As shown in Figure 2, plasma lactate concentrations
increased significantly after the race compared with the prerace values in both groups, but were higher in the damaged
group (3.4-fold) than those in the minor-damage group (2.7fold).

Oxidative stress and renal function parameters in the circulation and ex vivo
As shown in Table 1 and Figure 2, many biochemical variables were affected by the exercise intervention and varied!
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EIR 21 2015

134 • Pathogenic mechanisms of redox/inflammatory responses to endurance exercise
Ta b l e 1 . C h a n g e s o f l e u k o c y t e s , c y t o k i n e s a n d b i o c h e m i c a l v a r i a b l e s f o l l o w i n g t h e d u a t h l o n r a c e .
Renaltubularepithelial

Unit

Pre

0h

1.5 h

3h

cells
128.9±32.5

112.6±25.3**

52.9±15.8

168.7±31.2

143.6±39.6

137.3±43.5**

11.2±5.2

1.0±0.9

3.4±1.7

4.4±1.3**

Pre-0 h**10.8±5.1

2.1±1.1

5.5±2.4

4.9±1.9**Pre-0 h*20.0±9.6

3.2±2.6

11.0±3.2

15.2±9.2**

21.0±10.9

6.6±4.513.8±9.6

29.7±31.5*

Pre-0 h†5.0±2.0

115.5±69.0

198.1±154.8

41.4±36.8*

Pre-1.5 h*

4.6±2.3

129.7±111.2

44.9±47.4

19.9±27.3**

4.5±0.3

5.2±0.4

4.9±0.2

4.8±0.3**

Pre-0 h**(-)

4.5±0.2

4.9±0.2

4.7±0.3

4.7±0.3**

Pre-0 h**(+)

6.6±2.4

2.5±1.88.7±3.6

8.1±2.7**

5.9±3.6

5.1±1.8

6.8±2.6

4.9±1.2NSNS

1.7±0.8

0.5±0.3

2.5±1.2

12.2±15.7**

0 h-3 h**(-)

2.7±1.9

2.4±3.2

2.8±2.3

2.6±1.5

NS

NS

(+)

188.0±36.8

266.6±31.9250.3±35.2

243.1±45.6

**

(-)

174.9±38.6

249.7±38.0

235.0±41.5

231.7±45.9

**

(+)

32.3±11.8

41.0±14.938.9±13.6

38.9±12.0**(-)

26.9±8.733.4±10.3

33.1±10.1

35.6±11.6**(+)

21.9±10.6

26.0±12.3

24.4±11.3

24.1±10.6**(-)

20.1±4.922.6±5.0

21.6±5.1

22.1±5.3*(+)

17.2±11.1

49.8±23.1

35.6±11.3

24.3±11.5**(-)!102/µl(+)µg/min(+)

ng/min(-)(+)
ALB-U #

µg/min(-)

ALB-P

NAG-U #(+)

g/dl

mU/min(-)
MCP-1-U #Pre-0 h**127.1±32.6

(-)

Mb-U

Scheffe test

41.9±8.9

n=7

UA-U

-man
test

(+)
n=7
Blood leukocytes

Fried

(+)

LDH-S

pg/min

IU/l

AST-S

IU/l

ALT-S

IU/l

Pre-1.5 h*
Pre-0 h**Pre-1.5 h*

Pre-0 h**0 h-1.5 h*

Pre-0 h**0 h-3 h*0 h-1.5 h*
0 h-3 h†Pre-0 h**Pre-1.5 h*
Pre-0 h**Pre-3 h†Pre-0 h**Pre-3 h*Pre-0 h†Pre-3 h**Pre-0 h**Pre-0 h*Pre-3 h†Pre-0 h**IL-8-P #IL-10-P #0 h-3 h*

pg/ml
(-)
(+)

pg/ml

(-)

Pre-1.5 h*

Pre-0 h*16.1±10.4

40.1±17.3

30.1±14.1

19.9±11.3**

5.1±11.6

25.6±42.2

21.8±54.4

20.6±52.3**

Pre-0 h*1.0±0.3

6.6±11.6

1.2±0.4

0.8±0.4**

0 h-3 h**0 h-3 h*Values: means 㼼 SD (n=7). Statistics: ** p < 0.01, * p < 0.05, † p < 0.1 䍺 not significance (NS).
-P: Data are plasma concentrations.
-S: Data are serum concentrations.
-U: Data are the gross amount in the volume of urinary excretion per one minute (urinary excretion rate).
The pre-exercise (pre), immediately post-exercise (0 h), 1.5 hours post-exercise (1.5 h) and 3 hours post-exercise (3 h) are sampling points.
(+): damaged group; renal tubular epithelial cells existed in the urinary sediments.
(-): minor-damage group; renal tubular epithelial cells did not exist in the urinary sediments.
#: Data modified from Figure 1 of the reference No. 59.

Abbreviations: uric acid (UA), myoglobin (Mb), albumin. (ALB), "-N-acetyl-D-glucosaminidase (NAG), monocyte chemotactic protein (MCP)-1,
lactate dehydrogenese (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukin (IL).

EIR 21 2015

Pathogenic mechanisms of redox/inflammatory responses to endurance exercise • 135

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DiscUssiOn

It is known that blood flow is redistributed during endurance
exercise. In this study, we provide evidence that the presenceof renal epithelial cells in urine may be induced byischemia/reperfusion caused by a reduction in renal blood
flow. We have already investigated AKI caused by endurance
exercise and the possible associations between AKI and theincreases in urinary levels of IL-2, IL-4, IL-8, IL-10, IFN-γ
and MCP-1 (59). This study further analyzed the associations
among AKI, cytokines, inflammation and oxidative stresswith a special focus on TRX and C5a. In particular, the excre-

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tion rate of TRX increased significantly at 1.5 h (20.1-fold)from pre-exercise in the damaged group only (Figure 1), andthe excretion rates of NAG (3 h) were positively correlatedwith those of TRX (3 h) (Table 2). These findings suggested
that TRX was related to renal tubular injury. Therefore, it
might be possible that the excretion rate of TRX increased in
response to oxidative stress as a result of renal tubular injury
following intensive endurance exercise.
In a previous study on murine kidney, transgenic hTRX
was predominantly observed in the outer medulla after renal
ischemia/reperfusion. Thereafter, the immunoreactivity for
hTRX was revealed in the intraluminal region of the renal
tubule, coinciding with a decrease in TRX protein in the kidneys and an increase in urine. Interestingly, TRX protein concentration did not change in the blood, and expression of TRX
mRNA did not reveal localization or change in abundance
after renal ischemia/reperfusion. Therefore, it is suggested
that urinary TRX protein is derived from proximal tubule cortical region of the kidney (30). In this study, however, it might
EIR 21 2015

138 • Pathogenic mechanisms of redox/inflammatory responses to endurance exercise
be possible that blood-derived TRX was mixed with TRX
from proximal tubule cortical region, because the excretion
rates of TRX at the same time points after the race were positively correlated with the excretion rates of ALB at pre, 0 h,
1.5 h and 3 h after exercise and TRX is small protein of 12
kDa. Whereas urinary excretion rates of ALB at the same time
points after the race tended to be positively correlated with theexcretion rates of NAG (as a generally accepted marker ofrenal tubular injury) at pre, 0 h, 1.5 h and 3 h after the race,the correlations between urinary excretion rates of TRX andNAG at 3 h after exercise were significant (Table 2). Serum
NAG protein is not excreted into urine because its molecularweight is 130-140 kDa. Hence, it might be possible that urinary excretion of TRX was derived from both blood and kidney. In this study, renal ischemia/reperfusion of subjects was
induced by endurance exercise, but in the previous work,
bilateral renal arteries were clipped for 30 min and thenreleased (30). The inconsistency of the results might bederived from the difference of the above induction methods forischemia and exercise. It is reported that chemokines such as
MCP-1 are key modulators in renal ischemia/reperfusion
injury, and urinary chemokines are good markers of clinicaldiseases and AKI (78, 79). In the damaged group, urinary
excretion rates of MCP-1 significantly increased (Table 1), andtended to be positively correlated with the excretion rates of
TRX and NAG (Table 2). This suggests that TRX, MCP-1 and
NAG may be associated with renal tubular injury.
In the present study, the excretion rates of C5a in the damaged group and the minor-damage group significantly increased
following intensive endurance exercise (Figure 1). Urinary
excretion rate of C5a was positively correlated with that of IL-2,
IL-4, IL-8, IL-10 and IFN-γ after intensive endurance exercise(Table 3). It is suggested that the chemotactic factors C5a andIL-8 increased after reperfusion, making inflammatory cells
infiltrate into tubular epithelium or glomerular capillary (13, 14,
15, 35, 39, 42, 76, 77 84). On the other hand, it may also be suggested that IL-10 increased to suppress progressive inflammation, and/or to repair damaged tissues (58). Therefore, it might
be possible the excretion rates of C5a, IL-8 and IL-10 reflect
inflammatory levels in the renal tubular injury. In contrast, inthe damaged group, the excretion rates of TRX after intensive
exercise were negatively correlated with the excretion rates ofC5a, IL-2, IL-4, IL-8, IL-10 and IFN-γ following intensive
exercise (Table 3). In the damaged group, it may be possiblethat levels of TRX were influenced by levels of IL-10 as an antiinflammatory cytokine, and TRX regulates chemotactic activity
C5a and IL-8, or TRX controls inflammatory progress by C5a
and IL-8, because TRX functions as an antioxidant, as a chemotaxis inhibitor and as a redox-regulating protein in the signaltransduction (12, 21, 22, 57).
We examined systemic oxidative stress and inflammationinduced by endurance exercise within two groups (damaged
group and minor-damage group) based on urinary measures of
AKI. When evaluating the circulating oxidative stress andinflammatory state in these same groups, lactate levels wereincreased significantly in both (0 h: damaged group 3.4-fold;minor-damage group 2.7-fold). Lactate-related factors such aslactate threshold (LT) and onset of plasma lactate accumulation (OPLA) are critical for setting exercise intensity (16, 72).
Since this study was carried out in an actual competition race,
we could not examine LT and OPLA, but the athletes’ lactate
EIR 21 2015

levels suggested a difference of exercise intensity between thetwo participant groups.
Endurance exercise increases the circulating number ofleukocytes, especially neutrophils, which exhibit the greatest
change in cell count and function (7, 41, 49, 50 62-65, 82).
Moreover, IL-6, IL-8 and M-CSF responses are positively
correlated with the delayed-onset neutrophil mobilizationfrom the bone marrow reserve after exercise (64, 82), particularly when the duration is over 2 h. In this study, leukocytes
and neutrophil counts in the minor-damage group (whose
race time was over 2 h) tended to be greater than the damaged group (Figure 1 and Table 1). MPO catalyses the conversion of hydrogen peroxide into hypochlorous acid in neutrophils and macrophages (75). MPO is located in the primary (azurophilic) granules (5) and is a marker of neutrophil
activation after exercise (7, 50). MPO produces a large
amount of ROS and induces oxidative damage to proteins,
lipids and DNA (43). MPO increases depending on exercise
intensity (49, 53). These findings suggest that the intensity
was also higher in the minor-damage group, but MPO
increased depending on exercise duration rather than intensity in case of such a long-duration exercise. In the damaged
group we found serum concentrations of d-ROMs as anoxidative stress marker, and BAP and OXY as antioxidant
capacities tended to be higher than those in the minor-damage group immediately after the race. It was suggested that
acute endurance exercise-induced oxygen consumption in
many organs in the damaged group was greater and produced ROS, because the intensity of the damaged group was
higher compared with that in the minor-damage group. Previous studies showed that scavengers such as enzymatic
activities of plasma SOD and catalase (a scavenger for H2O2
) and plasma concentration of vitamin C (ascorbate: a scavenger for O2－,•OH, 1O2 and other oxidants) for toxic ROS
might be induced in response to intensive exercise (67).
Moreover, it was reported that free radical scavengers prevent not only oxidation of molecules in the body but alsoadhesion of neutrophils to the endothelial lining and inhibiting neutrophil infiltration (19, 57). It was also reported thatanti-inflammatory cytokines prevent inflammatory tissue
damage (31, 66, 73). In particular, the anti-inflammatory
cytokine IL-10 is an immunosuppressive cytokine that
inhibits both proinflammatory cytokine production and ROS
production by activated neutrophils (31). Furthermore, antiinflammatory cytokines and free radical scavengers work tocounteract oxidative tissue damage by ROS (9, 24, 56). In
this study, the plasma concentration of IL-10 significantly
increased only in the damaged group (Table 1). Plasma IL1ra concentrations increased significantly after exercise inboth groups (59) and were significantly correlated with exvivo neutrophil migratory activity and ROS-production in
the damaged group only. Accordingly, increased antioxidant
capacity and anti-inflammatory cytokines in the damaged
group might inhibit neutrophil activation as compared withthose in the minor-damage group. We found also that serum
Mb concentrations in the minor-damage group (0 h: 6.2-fold,
1.5 h: 7.0-fold) were higher than those in the damaged group(0 h: 4.8-fold, 1.5 h: 4.2-fold) after the race. These results
might suggest that muscle damage increased due to ROS
from activated neutrophils in the minor-damage group,
whereas in the damaged group, muscle damage was prevent-Pathogenic mechanisms of redox/inflammatory responses to endurance exercise • 139
ed by elevated antioxidant capacity and anti-inflammatory
cytokines.
Kanda et al. reported enhanced neutrophil migration and
ROS production after one-leg calf-raise exercise through theuse of a newly-developed ex vivo methodology in imitation oftissue damage (28). In this study, we assessed ROS production
by neutrophils that migrated into the hydrogel. We found thatboth neutrophil migration and ROS production increased after
exercise in both groups. The ROS production immediately
after the race in the damaged group was lower than that in theminor-damage group, but serum concentration of OXY andplasma concentration of IL-10 were higher than those in theminor-damage group. These results suggest that neutrophil
activation was suppressed by antioxidant and anti-inflammatory cytokines immediately following intensive endurance
exercise.
In conclusion, we infer that the excretion rates of TRX,
MCP-1 and NAG were associated with renal tubular injury. It
might be possible that the excretion rates of C5a, IL-8 and IL10 reflect inflammatory levels in renal tubular injury, where
the excretion rate of C5a was strongly associated with that of
IL-2, IL-4, IL-8, IL-10 and IFN-γ. In the damaged group, theexcretion rates of TRX after exercise were negatively correlated with the excretion rates of C5a, IL-2, IL-4, IL-8, IL-10
and IFN-γ following exercise. Therefore, in the damaged
group, it could be inferred that levels of TRX were influenced
by levels of IL-10 as an anti-inflammatory cytokine, and that
TRX regulates chemotactic activity of C5a and IL-8, or that
TRX controls inflammatory progress by C5a and IL-8,
because TRX functions as an antioxidant, as a chemotaxis
inhibitor and as a redox-regulating protein in the signal transduction. Clarification of these pathways might be valuable in
the assessment of AKI risk following intensive endurance
exercise.
In the circulation of the damaged group ROS production
was found to be higher than the minor damage group, whileantioxidant capacity and anti-inflammatory cytokines
increased immediately after intensive endurance exercise.
From these data it might be inferred that neutrophil activationand efflux of MPO were inhibited. Therefore, we suggest thatdamage to muscle and other tissues are likely to be lower inthis group. On the other hand, results from the longer durationgroup (minor-damage group) showed neutrophil count andefflux of MPO in the circulation were higher when comparedto the damaged group. Furthermore, both variables were significantly correlated with neutrophil count and plasma concentration of MPO immediately following intensive
endurance exercise. In combination, these results suggest thatmuscle is likely to be damaged by activated neutrophils to agreater extent than in the damaged group. This inference wasfurther supported by the results from our application of the
newly-developed ex vivo method that estimated the functional
impact of activated neutrophils.
In the present study, we confirmed that intensive endurance
exercise caused “Risk” or “stage I” in the AKI diagnosis criteria such as RIFLE and AKIN, suggesting that not only blood
but also urine analyses are important for estimating tissue
damage. The relationships among the variables in the urine
and circulation, and further delineation of their clinical significance must be revealed in future research.

acKnOwleDgeMents

The authors thank Dr. Cecilia Shing and Dr. James Broadbent
for English editing. This study was partly supported by grants
from the Ministry of Education, Culture, Sports Science andTechnology of Japan, the Grant-in-Aid for the Scientific
Research (A) 23240097.

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