Exhaustive exercise, renal damage and cytokine release • 29

Urinary excretion of cytokines versus their plasma
levels after endurance exercise

Kaoru Sugama1, Katsuhiko Suzuki1,2*, Kayo Yoshitani3, Koso Shiraishi3,
Takashi Kometani3
1 Cooperative Major in Advanced Health Science, Tokyo University ofAgriculture and Technology/ Waseda University, Japan.

2 Faculty of Sport Sciences, Waseda University, Japan (*Equal contribution to3

this study with the first author).
Health Science Laboratory, Ezaki Glico Co., Ltd, Japan.

ABSTRACT

It has been consistently shown that circulating levels of interleukin (IL)-6, IL-8,
IL-1 receptor antagonist (IL-1ra) and IL-10 increase remarkably following endurance exercise longer than 2 h such as marathon and triathlon races. However,no studies have compared changes in the plasma and urinary levels of these cytokines after endurance exercise, including the recovery period. In the present
study, we investigated kinetic changes in the urinary excretion of cytokines following endurance exercise up to 3 h after exercise to evaluate the magnitude ofchange in comparison to the plasma levels and to explore the possible biologicalsignificance and the mechanisms of cytokine dynamics following exercise. Fourteen male athletes participated in a duathlon race consisting of 5 km of running,
40 km of cycling, and 5 km of running. Venous blood and urine samples werecollected before, immediately after, 1.5 h and 3 h after the race. Plasma and urine
were analyzed using enzyme-linked immunosorbent assays (ELISA). Plasma concentrations of IL-1β, IL-1ra, IL-6, IL-8, IL-10 and monocyte chemotactic protein(MCP)-1 increased significantly after the race, whereas tumour necrosis factor(TNF)-α, IL-2, IL-4, IL-12 and interferon (IFN)-γ did not change significantly.
Urinary concentrations of IL-1β, IL-1ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IFNγ and MCP-1 increased significantly after the race. When the urine concentrations were adjusted by creatinine concentration, urine volume and sampling time,the increases of IL-2, IL-4, IL-8, IL-10, IFN-γ and MCP-1 were evident and thesewere notably present in urine of the stressed athletes suffering from renal tubularepithelial damage. The present study provides new evidence that the kinetics andmagnitude of changes in urinary cytokine concentrations differ from plasma cytokine concentrations following endurance exercise, especially, in the recovery
Address 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 19 2013

30 • Exhaustive exercise, renal damage and cytokine release
period several hours after exercise, and that the damaged kidney might be responsible at least in part for the kinetics of some cytokines. Urinary cytokines may
be sensitive biomarkers of the impact of exhaustive exercise workload on renal
damage and inflammation in the recovery period after endurance exercise.

Key words: exertion, anti-inflammation, chemokine, urinalysis, acute kidney
injury (AKI)INTRODUCTION

Cytokines are potent intercellular signalling molecules that usually act within thelocal tissues in an autocrine or paracrine manner. However, systemic spillover ofpro-inflammatory cytokines such as tumour necrosis factor (TNF)-α, interleukin(IL)-1β and IL-6 from damaged tissues can occur in response to a variety of serious insults such as severe trauma, burns, hemorrhagic shock, sepsis andischaemia-reperfusion injuries, which are integrated as a systemic inflammatory
response syndrome (SIRS) and multiple organ failure syndrome characterized byhypercytokinaemia (1, 19, 46). For example, in a sepsis model, TNF-α is the firstcytokine released systemically, and peaks within 2 h after the onset of sepsis, followed shortly thereafter by peaks of IL-1, and then IL-6 within 4 h after the disease onset (1, 37). These pro-inflammatory cytokines induce pyrogenesis andpromote subsequent acute inflammatory responses such as neutrophilia, lymphocytopenia by inducing IL-8, interferon (IFN)-γ and monocyte chemotactic protein(MCP)-1 (19, 37). In contrast to these pro-inflammatory cytokines, anti-inflammatory cytokines such as IL-1 receptor antagonist (IL-1ra), IL-4 and IL-10 may
prevent inflammatory tissue damage but cause immunosuppressive states of the
body (10, 16, 46, 52).
The plasma concentrations of the classical pro-inflammatory cytokines,
TNF-α and IL-1β, in general, do not increase immediately after exercise (4, 9, 14,
24, 33-35, 45-49). In contrast, IL-6 is the first cytokine present in the circulation
during exercise (9, 14, 17, 24-26, 28, 37, 39, 46). The level of circulating IL-6
increases up to 100-fold, depending on the intensity and duration of endurance
exercise, and declines in the post-exercise period (28-30, 32-35, 45-49). Contracting skeletal muscle is the main source of IL-6 in the circulation. In response to
exercise, an increase in the IL-6 mRNA content in the contracting skeletal muscle
is detectable after 30 min of exercise, and up to 100-fold increases of the IL-6
mRNA content occur at the end of the exercise bout (17, 25, 33-35). In addition, it
is demonstrated that both intramuscular IL-6 mRNA expression and protein
release are enhanced when intramuscular glycogen is low, indicating that IL-6 isinvolved in energy metabolism during endurance exercise (17, 33-35, 48).
Concerning immunomodulatory cytokines that are potent functional activators of cellular immunity (29, 30, 36, 37, 38), there are no reports showing significant increases in plasma IFN-γ concentration after exercise, whereas there areseveral reports demonstrating a decrease in the plasma concentration of IL-2 after
exercise (11, 28-30, 39, 53). Interestingly, many studies have shown that the capacity of blood leucocytes to produce IL-2 and IFN-γ decreases after endurance exercise (28-30, 39, 53). The findings that pro-inflammatory and immunomodulatory
EIR 19 2013

Exhaustive exercise, renal damage and cytokine release • 31
cytokines only increase to a small extent, or are even down-regulated after exercise, could well be attributed to the actions of anti-inflammatory cytokines suchas IL-1ra, IL-4 and IL-10. Indeed, we previously observed more than 200-foldincrease of plasma IL-1ra after a marathon race (45) and delayed secretion of IL4 several hours after short-duration maximal-intensity exercise (46). Furthermore,
we also observed renal excretion of several cytokines, which may be one of theunderlying mechanisms that plasma cytokines either remain unchanged or exhibit
relatively small, delayed increments following exhaustive exercise.
Although many researchers have consistently shown that circulating levelsof IL-6, IL-8, IL-1ra and IL-10 increase remarkably following endurance exercise
longer than 2 h such as marathon and triathlon races (4, 14, 24, 33-37, 45-49), lessis known concerning changes in urinary cytokine excretion after endurance exercise and during the recovery period (29, 30, 46, 47, 53). The present study investigated kinetic changes in the urinary levels of cytokines following endurance
exercise lasting several hours to evaluate the magnitude of change in comparison
to the plasma concentrations of the same cytokines. It is known that deteriorationof glomerular filtration rate and oliguria are induced following endurance exercise (50), and that endurance exercise causes rhabdomyolysis (5, 31, 41), produces reactive oxygen species (ROS), and contributes to the onset of acute renal
failure through reduced renal blood flow and damage of tubular cells (15). In theprevious studies, after renal ischaemia-reperfusion, IL-2, IL-8, IL-10, MCP-1 andIFN-γ appeared in the kidney (7, 8, 12, 21, 42, 51). Accordingly, in this study, weevaluate acute kidney injury (AKI) following endurance exercise in relations withthe changes of plasma and urine levels of various cytokines. Our hypothesis wasthat plasma levels of cytokines depend on urinary excretion and the excreted
cytokines into urine might provide important information on renal damage andinflammation after endurance exercise.

METHODS

Subjects
Fourteen male triathletes (age 28.7 ± 7.9 (mean ± SD) yr and body mass 63.2 ±6.0 kg) participating in the 19th Kikunotsuyu duathlon race, held on March 16th
2008, volunteered for this study. The protocol was approved in advance by theinstitutional ethics committee of Waseda University. None of the athletes had
been ill in the month prior to the race, which was confirmed from medical questionnaire.

Race conditions
All participants agreed to avoid the use of vitamin/mineral supplements, herbs
and medications from the day prior to the race until the end of experimental period (3 h post-exercise). They ate the same breakfast containing 574 kcal with 22.1
g protein, 13.7 g fat, and 88.8 g carbohydrate at 08:30. They emptied their bladders before 08:30 and just before the race to eliminate any effect of remaining
urine. 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, with the ambient temperature at
24.6 degrees Celsius (°C). They did not exercise for approximately 18 h before

EIR 19 2013

32 • Exhaustive exercise, renal damage and cytokine release
the pre-race blood and urine sampling. With the subjects resting quietly, the prerace blood and urine samples (Pre) were collected at 10:30. They ate the same
lunch containing 211 kcal with 9.3 g protein, 2.4 g fat, and 38.6 g carbohydrate at
11:00. The post-race blood and urine samples were collected immediately (0 h),
1.5 h (1.5 h) and 3 h (3 h) after the race. All participants drank the same quantityof fluid during exercise. They each drank 600 ml of fluid before the race, 1400 mlof fluid during the race and 1500 ml of fluid until 3 h after the race, respectively.

Blood and urine sampling
Approximately 7 ml of blood was drawn by a standard venipuncture techniquefrom the antecubital vein using vacutainers containing no additive or 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 one 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 ofanalysis. Serum concentrations of creatinine (Cr) and albumin (ALB) were measured using an automated analyzer (Model 747-400, Hitachi, Tokyo), and urine
osmolality was determined using an auto-osmometer (OSMOSTAT, Kyoto Daiichi Kagaku, Kyoto).
Urine samples were collected directly in graduated cylinders and the volume was measured and then approximately 8 ml of urine was stored at 4 °C without centrifuge until analysis of sediments, respectively. Remaining urine samples
were centrifuged immediately at 1000 g for 10 min to remove sediments, and thesupernatants were stored at –80 °C until the day of analysis. Urinary concentrations of Cr, ALB and β-N-acetyl-D-glucosaminidase (NAG) activity were measured using an automated analyzer (Model 747-400, Hitachi, Tokyo). White blood
cells and red blood cells in urinary sediments were counted by flow cytometer(UF-1000i, Sysmex, Kobe), and other cells and casts in urinary sediments wereanalyzed by microscope (x400 high power field: HPF).

Assays for cytokines
Cytokine concentrations were measured in EDTA-plasma and urine samples withenzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers’ instructions. Concentrations of IL-1β, IL-6, IL-12 and TNF-α were measured
with Quantikine high sensitivity (HS) kits (R&D Systems, Inc., Minneapolis,
MN). IL-1ra and MCP-1 were measured with Quantikine kits (R&D Systems,Inc.). IL-2, IL-4, IL-8, IL-10 and IFN-γ were measured with OptEIA kits (Beckton Dickinson Biosciences, San Diego, CA). For all assays, the absorbance wasmeasured spectrophotometrically on a microplate reader (VERSAmax, Molecular
Devices, Sunnyvale, CA), and the concentration of each cytokine was calculated
by comparison with a calculation curve established in the same measurement.
Data analyses
Data are presented as means ± SD. Statistical validation was made using Friedman’s test. If significance was detected, the Scheffe method was used for multiple
comparisons. Statistical significance was evaluated at p < 0.05.

EIR 19 2013

Exhaustive exercise, renal damage and cytokine release • 33

RESULTS

Plasma and urine cytokines
As shown in Table 1, the plasma concentrations of TNF-α did not change significantly following the race. Plasma IL-1β concentration increased significantly at
1.5 h after the race (16-fold) and remained elevated at 3 h after the race (5-fold)compared with the pre-exercise values. Plasma IL-1ra concentration showed
more substantial changes significantly above pre-exercise values at immediately
after the race (100-fold) and at 1.5 h post-exercise (169-fold), and at 3 h postexercise (66-fold). The plasma concentrations of IL-6, IL-8, IL-10 and MCP-1
also increased significantly at immediately after the race, but decreased thereafter. The plasma concentrations of IL-2, IL-4, IL-12 and IFN-γ did not change
significantly following exercise.
Urine volume and urinary creatinine concentration changed markedly following exercise as shown in the bottom of Table 1. Therefore, the urinary concentrations of cytokines are reported as raw data, those corrected for creatinine concentration, the gross amount (raw concentration × urine volume), and the grossamount per minute (urinary excretion rate). Urinary TNF-α did not increase significantly. Urinary IL-1ra, IL-6, IL-12 and MCP-1 were significantly higher after
the race compared with pre-race values, but these differences were no longer significant when corrected for creatinine. Urinary levels of IL-2 (59-fold), IL-4
(348-fold), IL-8 (20-fold), IL-10 (54-fold) and IFN-γ (18-fold) were significantly
elevated at 3 h post-exercise compared with pre-exercise values.

Urinary sediments
As shown in Table 2, renal tubular epithelial cells and renal tubular epithelial
casts were observed in the urinary sediments of 7 subjects, among the fastest 8
subjects for the race time. Granular casts were also observed in the urinary sediments of 6 of these 7 subjects. White blood cells were more dominant than red
blood cells in most subjects, and 13 of the 14 subjects (1~3 counts/ every field)
recovered at 3 hours after the race, but only one subject (No.6) did not recover(11~15 counts/ every field). Based on these results, we analyzed between two
subgroups that were divided according to existence (damaged group, n=7) or nonexistence (minor-damaged group, n=7) of renal tubular epithelial cells in the urinary sediments as follows.

Parameters of renal functions
As shown in Figure 1, in the damaged group, serum Cr concentration increased
significantly at 0 h post-exercise (0.54mg/dL; 1.66-fold) and at 1.5 h after therace (0.36mg/dL; 1.44-fold) compared with the pre-exercise values, whereas
serum Cr concentration increased significantly at 0 h post-exercise (0.32mg/dL;
1.41-fold) as compared to pre-exercise values in the minor-damaged group. In theminor-damaged group, urinary excretion volume per minute (excretion volumerate) exhibited no significant change. Excretion volume rate in the damaged
group and creatinine clearance (Ccr) in both groups decreased after the race compared with the pre-exercise values. Ccr and excretion volume rate in both groups
did increase from 0 h, but still remained below the pre-exercise levels at 3 h after
exercise (damaged group only: Ccr p<0.05). There was a significant decrease in
EIR 19 2013

34 • Exhaustive exercise, renal damage and cytokine release
Table 1. Changes in plasma and urinary cytokines following the duathlon race.
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Values:means ± SD
Statistics:**p < 0.01, *p < 0.05.
5 6
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9(:" 60;<00;
-P: plasma. -U: urine. Cr: creatinine was used for the adjustment of urine concentration. The!6 !6  6     =       gross amount of urine (pg) was calculated by the raw concentration (pg/mL)×urine volume
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Exhaustive exercise, renal damage and cytokine release • 37

Ccr at 0 h following exercise in the damaged group (p<0.05). Urinary excretion
rate of ALB increased significantly at 0 h (30.3-fold) after the race in the minordamaged group, and until 1.5 h (43.4-fold) after exercise in the damaged group,
but were then reduced. The change in urinary excretion rate of NAG activity inthe minor-damaged group was not significant. In the damaged group, urinary
excretion rate of NAG activity was not significantly different from pre-exercise at
0 h, however, levels then increased significantly compared with 0 h post-exercise.
Urinary excretion rate of NAG activity did not return to pre-exercise levels at 3 h

EIR 19 2013

38 • Exhaustive exercise, renal damage and cytokine release
after exercise in the damaged group. Thus, the degree of renal disorder of Ccr,
urinary excretion rates of ALB and urinary excretion rate of NAG activity in thedamaged group were higher than the minor-damaged group.

Plasma and urine cytokines in relation with renal damage
In both groups, plasma concentrations of IL-1ra, IL-6, IL-8 and MCP-1 increased
significantly after the race compared with the pre-exercise values. Plasma IL-10
concentration increased significantly at 0 h after exercise compared with the preexercise value in the damaged group only. However, urinary excretion rates of
IL-8, IL-10 and IFN-γ increased significantly, and urinary excretion rates of IL-2

  

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 
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      '$()* "+* '$()* "#%* '()* "%/* "#%*  /()!0 *     '$()!
4    5 (       /0
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1 )#  ! "#$  '()!
1 )%  ! "% ! "1 )  /()!0 *     '$()!

      '$()* "+* '$()* "#%* '()* "%/* "#%*Modified by Cruz et al. Critical care, 13:211.2009 (6)Abbreviations: Risk, Injury, Failure, Loss, End Stage Kidney Disease (RIFLE), acute kidney
injury network (AKIN), creatinine (Cr), glomerular filtration rate (GFR), acute renal failure(ARF).

and IL-4 tended to increase after the race compared with the pre-exercise values
in the damaged group. There was a trend for urinary excretion rate of IL-8 toincrease following exercise in the minor-damaged group. In both groups, urinary
excretion rates of IL-6 were significantly elevated at 3 h after the race as compared to 0 h-post exercise values. In the damaged group, urinary excretion rates of
IL-2, IL-4 and MCP-1 increased significantly at 3 h after the race compared with
0 h-post exercise. On the other hand, urinary excretion rates of TNF-α, IL-1β, IL1ra and IL-12 did not change significantly after the race in both groups (Figure 1,
Table 4).

EIR 19 2013

Exhaustive exercise, renal damage and cytokine release • 39           
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Values: means ± SD (n=7). Statistics: **p < 0.01, *p < 0.05, †p < 0.1.
-P: Data are plasma concentration. -S: Data are serum concentration.
-U: Data are the gross amount in the volume of urinary excretion per one minute.
pre-exercise (Pre), immediately post-exercise (0 h), 1.5 hour post-exercise (1.5 h) and 3 hourpost-exercise (3 h) are sampling points.
(+): damaged group; renal tubular epithelial cells existed in the urinary sediments.
(-): minor-damaged group; renal tubular epithelial cells did not exist in the urinary sediments.
Abbreviations: tumour necrosis factor (TNF), interleukin (IL), IL-1 receptor antagonist (IL-1ra).
EIR 19 2013

40 • Exhaustive exercise, renal damage and cytokine release
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Figure 1. Changes of cytokines and parameters of renal damage following the duathlon race.
pre-exercise (Pre), immediately post-exercise (0 h), 1.5 hour post-exercise (1.5 h) and 3 hourpost-exercise (3 h) are sampling points.
damaged group: renal tubular epithelial cells existed in the urinary sediments.
minor-damaged group: renal tubular epithelial cells did not exist in the urinary sediments.
Abbreviations: creatinine (Cr), creatinine clearance (Ccr), albumin (ALB),
β-N-acetyl-D-glucosaminidase (NAG) activity, interleukin (IL), interferon (IFN),
monocyte chemotactic protein (MCP)-1.
Values: means ± SD (n=7). Statistics: *p < 0.05, **p < 0.01, †p < 0.1.
Box plot: minimum values means SD means means SD maximum values.
-P: Data are plasma concentration.
-S: Data are serum concentration.
-U: Data are the gross amount in the volume of urinary excretion per one minute (urinary
excretion rate).
EIR 19 201342 • Exhaustive exercise, renal damage and cytokine release

DISCUSSION

Studies investigating changes in the urinary levels of cytokines several hours after
exhaustive endurance exercise have not yet been reported. In the present study,
we present new evidence that changes in the urinary excretion of some cytokines
were much greater than plasma cytokine changes, and might provide sensitive
biomarkers of responses to exercise. IL-8 and IL-10 increased in both plasma andurine after exercise. In contrast, IL-1β, IL-1ra, IL-6 and MCP-1 mainly increasedin plasma, whereas IL-2, IL-4 and IFN-γ only increased in urine after exercise.
These results suggest that following exercise IL-1β, IL-1ra and MCP-1 werecleared slowly from the circulation with the plasma levels reflecting the systemic
release, whereas IL-2, IL-4 and IFN-γ were rapidly excreted from the circulation
into urine, without any increase in the systemic circulation, or the kidney might
be the source of these cytokines as discussed later.
TNF-α has been considered as a primary mediator of the SIRS response, but
the plasma and urine concentrations of TNF-α did not change significantly in thepresent study. On the other hand, the plasma concentration of IL-6 increased significantly by more than 30-fold immediately after the race, which is consistent
with previous findings (4, 9, 14, 28-30, 32-34, 37, 46). However, the urine concentration of IL-6 did not change markedly—especially when corrected for urinary creatinine clearance. Although the plasma concentration of IL-1β increased
significantly 1.5 h after the race, this response occurred later than the IL-6
response, and the concentration of IL-1β was relatively lower than IL-1ra, indicating that the cascade of cytokine production during exercise differs from theclassical pro-inflammatory cytokine cascade that occurs during acute inflammation such as sepsis (1, 10, 19, 33, 37, 46). Chemokines are also considered important in the pro-inflammatory process. IL-8 is a potent neutrophil chemotactic andactivation protein, whereas MCP-1 promotes monocyte activation and extravasation into inflammatory tissues (22). The plasma concentrations of IL-8 and MCP1 increased significantly immediately after the race in the present study, which isalso consistent with previous studies (24-27, 32-35, 44-49). Although exhaustive
exercise enhances the capacity of neutrophils to produce reactive oxygen species(27, 43, 44), which is also one of the main pathogenic mechanisms of multiple
organ failure in SIRS (1), these chemokines may, at least in part, mediate such
exercise-induced pathogenesis as muscle inflammatory damage, exertional rhabdomyolysis and heat-related multiple organ failure (27, 44-46).
In contrast to these pro-inflammatory cytokines, we demonstrated that plasma IL-1ra concentration increased by more than 170 times in the present study.
We have previously shown an increase of IL-1ra by 214-fold after a marathon
race (45). IL-1ra is a natural antagonistic cytokine that competes with IL-1 forreceptor binding without inducing signal transduction. Regardless of whether IL1β is released in a small quantity after exercise in a delayed-onset manner, thehigher concentration of IL-1ra appears to block the IL-1 bioactivity in advance—at least in the circulation. In the present study, IL-10 increased not only in plasma(5-fold) but also in urine by more than 50-fold. IL-10 causes immunosuppression
associated with various forms of trauma by attenuating IL-1β, TNF-α, IFN-γ, IL6 and IL-8 and enhancing IL-1ra production (16, 33, 52). There was no increasein plasma IL-4, but we observed a significant increase in urinary concentration of
EIR 19 2013

Exhaustive exercise, renal damage and cytokine release • 43
IL-4 after exercise in the present study. In support of the present findings, wehave previously shown no changes in plasma IL-4 concentration following amarathon (45). However, we have reported an increase in urinary IL-4 concentrations 2 h after short-duration, maximal exercise (46). The 350-fold increase in urinary IL-4 concentration in the present study is, to our knowledge, the most dramatic change reported among existing studies on exercise and cytokines. IL-4 has
been shown to down-regulate pro-inflammatory cytokines and up-regulate IL-1ra
production (52). In view of the large changes and biological characteristics ofthese anti-inflammatory cytokines, it is possible that they play a role in immunosuppression and increased susceptibility to infection after endurance exercise (3,23, 39). IL-4 is also a strong inducer of type-2 cytokines such as IL-6 and IL-10,
and its involvement in patients with atopic disposition and past history of exercise-induced allergy and anaphylaxis seems worthy of further investigation (13,40).
We did not collect blood samples during exercise, so it is uncertain whether
IL-2 was elevated and active within the bloodstream during exercise. IL-2 maybind to soluble receptors, and/or act locally within lymphoid tissues (36), which
could explain why it did not appear in plasma following exercise (11, 46). We
observed that plasma concentration of IFN-γ was low, whereas urinary excretionof both IL-2 and IFN-γ was large following exhaustive exercise in this study.
Although one study reported the urinary excretion of IFN-γ after 20-km running(38), other groups have not reported any change (11, 29, 53). Several factors
might explain these inconsistencies. Firstly, we used a different manufacturer's
ELISA kit from the previous studies that may capture a different epitope or haveenhanced sensitivity. Secondly, the exercise conditions and time course of sampling points especially for the recovery period in the present study were different
from previous studies. Lastly, other investigators have not standardized fluid
intake or corrected for glomerular filtration by expressing cytokine concentrations relative to urinary creatinine content. Although we observed, somewhat
unexpectedly, that the post-exercise urinary concentrations of IL-2, IL-4, IL-8,
IL-10 and IFN-γ were several orders of magnitude higher compared with plasma,
this might be because we provided fluid replacement during exercise to prevent
dehydration and augment urine output with enhanced renal clearance and reduced
re-absorption, or the kidney in itself might be the main source of the cytokine production. In any case, the present data demonstrate that IL-2, IL-4 and IFN-γ were
produced in the body following exhaustive endurance exercise.
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” (2) and “acute kidney injury network: AKIN” (18, 20) (Table 3),
AKI following endurance exercise showed “Risk” or “Stage1”at 0 h and 1.5 hafter the race in the damaged group and at 0 h after exercise in the minor-damaged group. Moreover, the degree of renal disorder of Ccr, urinary excretion rateof ALB and urinary excretion rate of NAG activity in the damaged group werehigher than the minor-damaged group. In the present study, the relations among
the changes of plasma and urine levels of various cytokines and AKI were investigated. In both groups, plasma concentrations of IL-2, IL-4 and IFN-γ did notchange significantly after the race as compared with the pre-exercise values
whereas urinary excretion of IFN-γ increased significantly and urinary excretion
EIR 19 2013

44 • Exhaustive exercise, renal damage and cytokine releaseof IL-2 and IL-4 tended to increase after the race compared with the pre-exercise
values in the damaged group only. Therefore, it might be possible that IL-2, IL-4
and IFN-γ were produced in the kidney that caused AKI following endurance
exercise. Moreover, urinary excretion of IL-8 and IL-10 also increased significantly after the race as compared with the pre-exercise values in the damaged
group only, but plasma concentrations of IL-8 and IL-10 also increased significantly post-exercise when compared with the pre-exercise in both groups of IL-8
and in the damaged group of IL-10. In the damaged group only, urinary excretion
rates of MCP-1 increased significantly at 3 h after the race as compared with 0 hpost exercise values. However, urinary excretion of MCP-1 was increased until 3
h-post exercise. Furthermore, urinary excretion of IL-8, IL-10 and MCP-1 in thedamaged group was elevated markedly more than the ones in the minor-damaged
group. Accordingly, it might be possible that IL-8, IL-10 and MCP-1 were produced in the circulation and/or kidney, which caused AKI following endurance
exercise.
The present data suggest that although some cytokines may not appear in
the circulation during exercise, they do appear in urine after several hours following endurance exercise, which indicates their production and/or clearance fromthe bloodstream in the kidney. These findings might help to develop reliable biomarkers of immunity and inflammation for the assessment of endurance exercise
workload as well as the pathological mechanisms of strenuous exercise. Further
research is needed to determine the mechanisms of the production and clearanceof the cytokines in the kidney from the circulation following prolonged endurance
exercise. Although the wider physiological and pathological implications are still
not clearly understood, such cytokine kinetics may partly explain the exerciseinduced suppression of cell-mediated immunity (3, 23, 34, 36, 39), organ damage
and inflammation (4, 14, 29, 32-34, 37, 43-49), and increased allergic reactions(13, 40, 46). The relationships among these changes, their clinical significanceand the possibility of intervention must be clarified in future research.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Jonathan Peake and Dr. Cecilia Shing forediting this manuscript. This study was partly supported by grants from the Ministry of Education, Culture, Sports Science and Technology of Japan, the Grantin-Aid for the Scientific Research (A) (No. 23240097) and the Global COE Program “Sport Science for the Promotion of Active Life”.
1.

2.

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