Acute kidney injury during an ultra-distance race.

PURPOSE: Previous studies have noted consequences of ultra-distance trail running on health, but few studies are available regarding the temporal variations of renal biomarker injury during the running. The aim of this study was to assess the of kidney function parameters temporal variation during and on short-term after an ultra-distance race.
METHODS: We performed an observational study with 47 subjects participating in an ultra-distance race (80 km). Urine (47 subjects) and blood (21 subjects) samples were serially collected before (baseline-km 0), during (21 and 53 km), on arrival (80 km), and 9 days after the race (d9).
RESULTS: Mean serum creatinine increased during the race from 90±14 μmol/L (km0) to 136±32 μmol/L (km 80-p<0.0001) corresponding to a 52% increase. Mean creatininuria progressively increased from 4.7±4.5 mmol/L (km 0) to 22.8±12.0 mmol/L (km 80) (p<0.0001). Both urinary biomarkers (Neutrophil Gelatinase Associated Lipocalin and Kidney Injury Molecule-1) of acute kidney injury (AKI) progressively increased during the race (p<0.05 vs baseline). However, after adjustment to urine dilution by urine creatinine, no significant changes remained (p>0.05). On day 9, no significant difference remains in blood and urine biomarkers compared to their respective baseline levels.
CONCLUSIONS: During an ultra-distance race, despite an acute and transient increase in the serum creatinine levels, urinary biomarkers of AKI displayed only limited changes with a complete regression on day 9. These results suggest the absence of the short-term impact of an ultra-distance race kidney function.

Introduction

Practice of high-endurance sports, especially running, has increased in the last two decades.

In 2016, in the United States, more than 64 million runners participated in a marathon [1]. The beneficial effects of regular physical activity, especially on cardiovascular health, are well-established [2]. Physical activity also prevents lifestyle-related metabolic diseases [3] and results in colorectal cancers occurrence [4]. However, very little data exist on the health-related effects of ultra-endurance running, mainly focused on the cardiovascular field [5-8]. Contrary to marathons, trail running is a running race taking place in the wild (forest, plain, mountain) on a long-distance, on a generally marked course with positive and negative denivelation. Most of the time, this is a race in semi or total self-sufficiency requiring the wear a backpack with his food and drink supplies. "Ultra" defines a distance greater than 42 kilometers.

For years, renal function impairment has been defined in two distinct entities, i.e., acute and chronic kidney diseases. This dichotomic classification suggested that acute and chronic renal injuries were, ostensibly, not interconnected. However, during the two past decades, evidence from epidemiological and experimental studies has emerged suggesting that acute kidney injury (AKI) may trigger chronic kidney disease [9]. Independently of its cause, AKI is recognized as an important risk factor for persistent renal function impairment, incident chronic kidney disease, and accelerated progression to end-stage renal disease [9-14]. The ultimate consequence of the acute injury depends on the balance between the results of repair and regenerative pathways. The last one includes cell-cycle arrest, apoptosis, dedifferentiation, inflammatory infiltration, epigenetic factors, and profibrotic changes [9]. Maladaptive repair and disordered regeneration have both been suspected to be contributing mechanisms that may link acute injury to chronic lesions following AKI. Although a number of pathological conditions are well-known risk factors for AKI, more subtle and repeated insults might also trigger acute renal injury, which may worsen long-term renal function [15].

To date, the potential short-term deleterious effect of ultra-distance running on renal injury remains unclear. Previous studies observed an impairment of kidney function by comparing kidney function biomarkers values before and after the race [16-20]. The real temporal variations of the AKI occurrence during an ultra-distance race and the short-term impact of long-distance running are not known.

To help close this knowledge gap, we took advantage of an ultra-distance race in healthy subjects by combining serial blood and urine samples before, during and after an ultra-distance race to assess the temporal variations of acute renal injury during the race and short-term impact.

Methods

Participants

We performed an observational study involving 47 participants of the 80-km Ecotrail of Paris Ile-de-France race on 2014, March 29th. These 47 volunteers were electronically recruited using an announcement on the race’s website (www.traildeparis.com).

Inclusion criteria were adult (age >18 years), male gender, and completion of an ultra-distance race (distance >50 km) during the last 12 months. Subjects with a history of chronic medical disease, e.g. high blood pressure or asthma, were not included.

During and after the race, the participants had free access to food and water, i.e. “ad libitum”, limited to the refreshment points allowed by the race organisation. Participants exclusively drink water (no sport drinks) during the race.

Sports exercise, including running, was permitted between the end of the race and nine days after the end of the race (day 9).

The race’s organizing committee, the French committee on public safety Paris Ile-de-France IV approved the protocol (Reference: 2014/07) as did the National Heart Agency (Number EudraCT: 2014-A00205-42) on 14 March 2014. All participants gave their written informed consent for participation before the start of the race.

Study design and sample processing

Blood and urine samples were collected before the race (<24 hours corresponding to baseline or km 0), during the race at intermediate checkpoints (km 21 and km 53), at arrival (km 80) and on day 9. A nurse collected blood samples from 21 of the 47 subjects and urine samples from all subjects in order to assess serum electrolytes and biomarkers of renal function. The sampling performed during the race required a 5-minute stop at each checkpoint.

Urine and blood specimens were collected and immediately stored on ice and sent to Necker hospital (Paris, France). The urine samples were centrifuged at 1,000 g for 10 minutes. The supernatant was collected after centrifugation and stored with protease inhibitors at -80°C. Frozen aliquots of urine supernatants were tested by ELISA for Kidney Injury Molecule-1 (KIM1, Quantikine ELISA #DKM100, R&D Systems) and for neutrophil gelatinase associated lipocalin (NGAL, NGAL ELISA kit, #036, BIOPORTO Diagnostics) according to the manufacturer’s instructions. Measurement of urine creatinine was performed in the same sample using a Hitachi 917 analyzer (Roche Diagnostics). The results were normalized or not to the urinary creatinine level, and consequently, four biomarkers of AKI were analyzed:KIM1 (pg/mL), KIM1:creatinine ratio (KIM1:Cr, expressed in pg protein/mmoL urine creatinine), NGAL (pg/mL) and NGAL:creatinine ratio (NGAL:Cr, expressed in pg protein/mmoL urine creatinine).

The blood samples were immediately analyzed (analyzer Cobas—Roche Diagnostics) for plasma level of sodium, potassium, chloride, urea, creatinine, protein, C reactive protein (CRP), myoglobin, creatine kinase MB isoenzyme (CK-MB) and lactate.

Statistical analysis

Continuous variables are presented as the mean ± standard deviation (SD). Categorical data are presented as absolute values and percentages. The distribution of each protein biomarker exhibited considerable positive skewness substantially reduced by use of a natural logarithm transformation.

We compared the levels of urinary protein biomarkers across the different time points using the Kruskal–Wallis test followed by Dunn’s post-test. Additional analyses were performed using the Wilcoxon matched-pairs signed rank test to take into account the repeated measure design of the study. A p value <0.05 was considered to be statistically significant.

Statistical analyses were performed with GraphPad Prism (version 6.0f; GraphPad Software, San Diego, CA).

Results

The race took place on 2014, March 29th with a clear weather without rain with a temperature of +14°C.

Volunteer’s parameters and performance

The demographic characteristics of the study population are presented in Table 1.

Table 1

Characteristics of the studied population.

Data are expressed as mean ± standard deviation (SD) excepting time of race which is expressed as mean ± SD (minimal–maximal values).

Parameter	Value
Age (years)	43 ± 7
Body weight (kg)	74 ± 8
Height (cm)	176 ± 7
Weekly covered distance (km)	46 ± 18
Weekly training duration (hours)	5 ± 3
Seniority in running practice (years)	6 ± 4
Number of trails per year	5 ± 3
Time of race (hours)	11.2 ± 0.9 (9.1–12.4)

All 47 subjects reached the race finish line (no follow-up missing) and were examined at all checkpoints and on day 9.

The mean race completion time was 11.2 ± 0.9 hours (9.1–12.4) corresponding to an average race speed of 7.4±0.8 minutes/km. All subjects were trained with a week training mean time of 5 ± 3 hours corresponding to 46 ± 18 km per week.

Blood laboratory data

Blood laboratory data are presented in Table 2.

Table 2

Blood parameters in 47 runners at baseline, during the race (km 21 & 53), on arrival and on day 9.

Data are expressed as mean ± SD.

Parameter	km 0(baseline)	km 21	km 53	km 80(arrival)	Day 9
Na (mmol/L)	143 ± 5	142 ± 2	145 ± 5	141 ± 7	141 ± 5
K (mmol/L)	4.8 ± 0.5	4.9 ± 0.6	4.7 ± 0.5	4.6 ± 0.5	4.8 ± 0.4
Cl (mmol/L)	104 ± 4	102 ± 3	101 ± 3	100 ± 5	101 ± 4
Urea (mmol/L)	5.8 ± 1.6	6.2 ± 1.6	8.3 ± 1.8	9.2 ± 2.6	5.9 ± 0.7
Creatinine (μmol/L)	90 ± 14	111 ± 22	136 ± 32	119 ± 33	90 ± 12
eGFR (ml/min/m2)	89.1 ± 16.1	71.4 ± 15.8	57.7 ± 15.5	68.8 ± 21.4	88.3 ± 12.8
Proteins (g/L)	76 ± 10	78 ± 3	76 ± 6	72 ± 5	72 ± 3
CRP (mg/L)	1.1 ± 0.2	1.4 ± 0.4	2.5 ± 1.5	6.9 ± 4.3	2.9 ± 1.3
Myoglobin (μg/L)	34 ± 16	205 ± 148	1810 ± 2121	2761 ± 1484	2130 ± 1893
CK-MB (ng/mL)	13 ± 5	14 ± 6	32 ± 24	36 ± 34	96 ± 122
Lactate (mmol/L)	2.3 ± 0.8	3.0 ± 1.1	2.9 ± 1.2	3.3 ± 1.1	1.8 ± 0.7

Na = sodium, K = potassium, Cl = chloride, CRP = C reactive protein, CK-MB = creatine kinase MB isoenzyme.

Mean urea plasma levels increased during the race from 5.8 ± 1.6 mmol/L (km 0) to 9.2 ± 2.6 (p = 0.0005) (km 80), corresponding to a 59% increase from baseline. Mean creatinine plasma levels also increased during the race from 90 ± 14 μmol/L (km 0) (p<0.0001) to 136 ± 32 μmol/L (km 80) (p<0.0001). Serum creatinine peaked after 53 km, corresponding to a 52% increase from baseline.

Referring to the Kidney Disease Improving Global Outcome (KDIGO), Acute Kidney Injury (AKI) definition based on serum creatinine increase [16, 17], during the race (comparison with baseline), on km 21: 2 subjects (4%) met the stage 1 (2%) and none met the stages 2 or 3; on km 53: 5 (11%) subjects met the stage 1, 1 (2%) met the stage 2 and none met the stage 3; on km 80: 3 (6%) subjects met the stage 1 and none met the stages 2 or 3 and on day 9: no subject met the stage 1, 2 or 3.

The plasma level of myoglobin and CK-MB increased during the race without reaching significance. No other significant change of blood laboratory parameters was observed during the race.

Urine laboratory data

Despite high interindividual variations, creatininuria progressively increased during the race from 4.7 ± 4.5 mmol/L (km 0) to 22.8 ± 12.0 mmol/L (km 80 p<0.0001) (Fig 1).

Fig 1

Longitudinal evolution of urine creatinine.

Scatter plots of the log (natural)-transformed urine creatinine levels of 47 patients at km 0, km 21, km 55, km 80 and at day 9 after the race. p values are based on the Kruskal-Wallis test. Stars depict pairwise group comparisons by means of Dunn’s post-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001).

Longitudinal evolution of NGAL, NGAL:Cr, KIM1 and KIM1:Cr is summarized in Table 3 and Fig 2.

Table 3

Urinary biomarker levels in 47 runners.

Data are expressed as mean ± standard deviation (SD) and minimal–maximal values (Min–Max).

Parameter	km 0(baseline)	km 21	km 53	km 80(arrival)	Day 9
NGAL, pg/mL
Mean ± SD	15164 ± 19919	39330 ± 34848	75684 ± 65368	38447 ± 41023	24940 ± 28164
Median	7008 (95878)	30090 (142006)	57859 (313305)	24702 (188124)	15547 (132052)
Min–Max	2235–98113	2500–144506	2500–315805	2500–190624	2045–134097
NGAL:Cr, pg/mmol
Mean ± SD	4433 ± 5287	3735 ± 3117	3776 ± 7413	1713 ± 1968	2349 ± 2372
Median (IQR)	3302 (3471)	2771 (3004)	2196 (2345)	1208 (1240.7)	1643 (2178.8)
Min–Max	257.7–31077	130.6–13686	79.47–49999	149.6–12195	143.4–8864
KIM1, pg/mL
Mean ± SD	171.6 ± 177.5	778.2 ± 1156	2204 ± 2222	1875 ± 2212	672.7 ± 551.7
Median (IQR)	80.00 (161)	299.5 (822)	1433 (1927.9)	1261 (2131.4)	475.0 (747.2)
Min–Max	80.00–788.2	55.95–5776	80.00–13947	80.00–13688	80.00–2232
KIM1:Cr, pg/mmol
Mean ± SD	61.07 ± 74.92	47.10 ± 35.66	107.1 ± 176.6	74.07 ± 67.45	51.03 ± 29.27
Median (IQR)	40.56 (51.37)	41.73 (47.87)	53.79 (49.35)	59.76 (55.91)	43.81 (31.84)
Min—Max	8.248–516.1	5.027–184.1	9.660–962.8	2.668–461.0	15.98–147.8

NGAL = neutrophil gelatinase associated lipocalin, NGAL:Cr = NGAL:creatinine ratio (NGAL:Cr, expressed in pg protein/mmoL urine creatinine), KIM1 = Kidney Injury Molecule-1, KIM1:Cr = KIM1:screatinine ratio (KIM1:Cr, expressed in pg protein/mmoL urine creatinine).

Fig 2

Longitudinal evolution of NGAL and KIM1.

Scatter plots of the log (natural)-transformed values of NGAL (A. and B.) and KIM1 (C. and D.) levels of 47 patients at km 0, km 21, km 55, km 80 and at day 9 after the race. Results are normalized (B. and D.) or not (A. and C.) by urine creatinine levels. P values are based on the Kruskal-Wallis test. Stars depict pairwise group comparisons by means of Dunn’s post-test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). NGAL = neutrophil gelatinase associated lipocalin, KIM1 = Kidney Injury Molecule-1.

Urinary concentration of NGAL (Fig 2A) and KIM1 (Fig 2C) followed a similar dynamic pattern and significantly increased during the race.

During the race, NGAL values were, respectively, 15164 ± 19919 pg/mL (km 0), 39330 ± 34848 pg/ml (km 21), 75684 ± 65368 pg/ml (km 53, p<0.0001), and 38447 ± 41023 pg/ml (km 80, p<0.0001) (Table 3).

During the race, KIM1 values were 171.6±177.5 pg/ml (km 0, p<0.0001), 778.2 ± 1156 pg/ml (km 21, p<0.0001), 2204 ± 2222 pg/ml (km53, p<0.0001) and 1875 ± 2212pg/ml (km 80, p<0.0001).

Nine days after the race, NGAL and KIM1 urinary levels decreased but remained significantly higher than baseline levels (15164 ± 19919 pg/mL at baseline versus 24940 ± 28164 pg/mL at day 9, p = 0.007 for NGAL and 171.6 ± 177.5 pg/mL at baseline versus 672.7 ± 551.7 pg/mL at day 9, p<0.0001 for KIM1).

Taking into account the dilution status of the urine samples, the AKI urinary biomarkers displayed only limited variations over time. NGAL:Cr ratio actually decreased over time with similar urine levels at baseline and day 9 (Fig 2B).

KIM1:Cr ratio did not significantly increase at km-80 and returned to baseline levels at Day 9 (Fig 2D).

Considering the repeated measures design and that the samples are not independent, the Wilcoxon matched-pairs signed rank test confirmed the stability of the KIM1:Cr ratio between Km-0 and day 9 (p = 0.66) and even showed the decrease of the NGAL:Cr ratio (p = 0.04) between baseline and day 9 (Fig 3).

Fig 3

Paired analysis of NGAL:Cr and KIM1:Cr ratios between Km 0 and Day 9 after the race.

P values are based on the Wilcoxon matched-pairs signed rank test. NGAL (pg/mL) and NGAL:creatinine ratio (NGAL:Cr, expressed in pg protein/mmoL urine creatinine), KIM1:creatinine ratio (KIM1:Cr, expressed in pg protein/mmoL urine creatinine).

Discussion

In this study, we observed a marked increase in the serum creatinine levels contrasting with limited change of urinary biomarkers of AKI during an ultra-distance race. Nine days after the end of the race, all kidney function biomarkers returned to their respective baseline levels. These results highlight that an ultra-distance race does not impact short-term kidney function.

In the last 10 years, ultra-trail has gained in popularity with a significant increase in races’ length, but little is known regarding its health impact. These last ones were mainly explored in the cardiovascular field [5-7]. To date, ultra-trail is widely practiced by amateur runners. A number of deleterious effects described including acute myocardial infarction, acute cardiac death, rhabdomyolysis, electrolyte disorders and negative impact on kidney function were reported [16-20]. Nevertheless, the temporal variations of kidney injury biomarkers during the race was not previously studied. In this study, results from a few participants evidenced an AKI according to KDIGO AKI score [21, 22]. Nevertheless, after adjustment on urine dilution, no significant dramatic changes remained. Furthermore, all AKI biomarker variations fully disappeared on day 9.

Acute dehydration is a well-known risk factor for prerenal azotemia that can lead to true organ damage by acute tubular necrosis if profound and sustained. The lack of biological parameters of dehydration observed in this study supports the conclusion that an ultra-distance race does not induce a short-term major kidney injury.

Additional factors related to ultra-distance exercise-practice may also trigger AKI, including hypoxemia, production of reactive oxygen species [22], and pro-inflammatory cytokine production [23]. For instance, ultra-distance races have been shown to increase the accumulation of nitrotirosine and protein carbamyl in serum and urine, thereby suggesting the induction of oxidative stress induced by extreme physical exercise [22].

Questioning the temporal variations of AKI occurrence and resolution after an ultra-distance race is not a futile question. For decades, AKI has been considered as a benign pathological condition that usually recovers “ad integrum” with no definitive kidney damage (9). However, epidemiological and experimental studies recently demonstrated a true interconnection of AKI and chronic kidney disease (CKD), and it is now considered that repeated episodes of AKI, including mild cases, may induce CKD over the long term [9, 24]. Observational studies have even demonstrated that AKI leads to new CKD, progression of existing CKD, increasing long-term risk of end-stage renal disease and increased mortality [9, 11, 14, 25]. The question as to whether ultra-distance races might trigger mild and repeated AKI episodes has therefore important implications for CKD prevention.

The high level of urine creatinine concentration, observed during the race, doesn’t necessarily reflect the high level of urine concentration but may simply be reflective of the higher blood creatinine level due to muscle damage [26]. Specifically, in the context of an ultra-distance race, increased serum creatinine may reflect pure prerenal azotemia with no immediate consequence on renal tissue integrity. We cannot know how much of the rise in serum creatinine observed during the race is due to a true creatinine clearance decrease or to an increase in muscle release of creatinine. In this study, we used KIM1 and NGAL urinary levels, two well-known biomarkers of AKI, to assess renal function, because traditional blood (creatinine and blood urea nitrogen concentrations) and urine markers of kidney injury (casts, fractional excretion of sodium, urinary concentration ability) are insensitive and nonspecific [27] contrary to muddy brown urinary casts indicative of tubular injury.

Mc Cullough et al. were the first to study urinary KIM1 and NGAL levels after marathon reporting a transiently increased level of these biomarkers during the race [20]. Lippi et al. [28] confirmed the acute variations of serum creatinine and urinary NGAL occurring after an ultra-distance race. In our study, we didn’t retrieve significant urinary NGAL variations, because we assessed the urinary NGAL value normalized to the urinary creatinine level to take into account the potential confounding effect on acute renal assessment. Due to the highly variable dilution status of the urine samples related to the exercise, urinary AKI biomarker levels need to be cautiously interpreted [29]. Indeed, our findings that urinary creatinine, used as a marker of urine dilution, is highly variable during and after the race should be considered for future urine biomarker interpretation.

Conversely, urine creatinine dramatically increased during the race suggesting extracellular dehydration and antidiuresis. While changes during and after the race of NGAL and KIM1 were quite impressive in our study, the variability over time was profoundly reduced after normalization by urine creatinine. After correction for urine dilution status on urine creatinine level, NGAL and KIM1 urine levels were not significantly different between baseline (km 0) and km 80, suggesting the limited impact of an ultra-distance race on renal tubular cell integrity.

NGAL, a proinflammatory mediator protein of the innate immune system, is a helpful tool to differentiate prerenal and intrinsic AKI [30-34]. Since, in our study, we observed no difference between baseline and other points (during and on day 9 after the running), we can hypothesize that AKI related to exercise practice is prerenal. Thus, the limited impact on kidney function observed herein may partly be explained by the free access to food and water in the study design. Nevertheless, beside extracellular volume depletion, we cannot exclude the contribution from other mechanisms to explain prerenal AKI. Among these, we can cite such the effects of the sympathetic nervous system on renal blood flow, and/or antidiuretic effect of vasopressin released in response to stress and/or pain, and/or cardiac dysfunction.

Beyond the differential diagnosis of AKI, urinary NGAL has a prognostic value to predict adverse outcome in both adult and children [6-11]. Urinary NGAL predicts the severity of AKI after cardiac surgery and long-term renal outcome after ICU stay [35, 36]. Urinary NGAL and urinary KIM-1 have good prognostic value for AKI [35-40]. Conversely, for CKD, they don’t provide robust prognostic information on renal function loss or because both biomarkers are sensitive to detect acute tubular injury but not tubular atrophy [41].

In this study, some limitations deserve consideration. First, this is a single centre study with a small sample size. Second, we did not obtain blood sample for all participants. Third, food and water intakes were not restricted and controlled during the race. Fourth, physical activity and diet in the short term before and after the race were not controlled; thus, we cannot rule out their impact on serum and urinary biomarkers levels. Fifth, we made the assumption, that the runners were healthy, but no comparison with a healthy control population was possible and performed to ascertain it.

Despite of these limitations, this first study with a longitudinal follow-up of blood and urine parameters suggests the restricted impact on short term of an ultra-distance race on the kidney function by combining blood and urinary parameters.

In conclusion, our study is the first to assess the temporal variations of the renal function during an ultra-distance race based on serially collected blood and urine samples with a follow-up 9 days after the end of the race. Half of the subjects experienced acute and transient increase in the serum creatinine levels, but urinary biomarkers of AKI displayed only limited changes over time, thereby suggesting that ultra-trail has no impact on acute kidney function.

23 Jul 2019

PONE-D-19-17069

Acute kidney injury during an ultra-distance race

PLOS ONE

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Reviewer #1: The authors report on the temporal changes in kidney injury biomarkers before, during, and after an 80km ultra-distance race. The study was designed and conducted appropriately. The manuscript has potential and can benefit from some improved clarity with the results and associated conclusions. Additionally, the focus of the introduction and discussion can be expanded. A large focus of the introduction and discussion involves the AKI to CKD transition. Although this is a good point to make, the study had short term follow-up. The authors should expand these sections to include other exercise related AKIs, such as from “regular” marathons and spin classes. Several studies and numerous case reports have been published within the last few years, notably, one published in PLoS ONE. Additionally, Mansour et al. AJKD 2017, is an impactful study and should be discussed. The authors should add a discussion on the strengths and limitations of KIM-1 and NGAL, including the differences between blood and urine measurement.

Also, the novelty the authors make regarding their study of an ultra-distance race rather than a “regular” marathon is probably not as clinically significant as the authors make it out to be. This should be adjusted throughout the manuscript including the last two paragraphs of the discussion.

Minor comments:

The in-text tables and figure legends (pages 8-10) are confusing to follow and should be moved to the proper section according to the journal’s author instructions. Also, the landscape formatting on the later pages needs to be fixed.

Introduction

Page 4, line 3 – clarify what the 480,000 runners refers to specifically. Did 480,000 runners complete a marathon in that one year or in 2001, have 480,000 runners completed a marathon at some point in their life?

Methods

Page 5, line 8 – clarify what medical disease refers to. Is this specifically referring to chronic diseases? What about occasional headache or infections? Only systemic illness?

Page 5, lines 9-10 – The sentence needs to be rewritten. I believe the authors intent to say that the participants had free access to food and water during and after the race. “ad libitum”

The study protocol was approved in 2014, was this the same year the race occurred? The authors should mention the year of the race. At least they mentioned the weather that day.

Results

Page 8 line 12 – Clarify the sentence to specify mean time of the race is referring to “mean race completion time”

Table 1 – what is “Trial experience”?

Page 10, line 18 – elevated urine creatinine does not necessarily reflect a “high level of urine concentration”, which is determined from the measurement of specific gravity or osmolarity. The high urine creatinine may simply be reflective of the higher blood creatinine level. Measurement of urinary osmolarity and electrolytes in stored samples would provide additional insight into kidney function during and after the race.

How much of the rise in serum creatinine during the race is really a true reduction in CrCl vs. and increase in muscle release of creatinine? The authors should comment on this.

It would be interesting to see the breakdown of the kidney injury biomarkers by KDIGO AKI stage. Were the biomarkers more elevated in these participants?

Discussion

Page 14, line 4 – Although the authors comment on AKI to CKD transition in other sections, they should specify here safe for “short term” kidney function as their study followed patients up to 9 days after the race.

Page 14, lines 6-7 – This sentence is not accurate, The impact of long-distance running on kidney function has been studied. As mentioned earlier, the Mansour AJKD paper, and others.

Since the authors talk about acute dehydration leading to ATN in the discussion, they should mention that the clinical parameters in table 2 do reflect any clinical evidence of dehydration and supports their finding of no major kidney injury.

Page 15, line 7 – remove the word “the”

Page 15, line 8 – maybe hyaline and waxy casts are nonspecific but muddy brown urinary casts are not nonspecific and are indicative of tubular injury.

Page 15, lines 14-15 – the assumption that urine was highly diluted before the race is incorrect. There is no evidence to support this claim. Additionally, what does “huge fluid consumption” actually mean?

Reviewer #2: The Authors in their interesting work show that transient increase in creatinine is not accompanied to parallel increase of two urinary biomarkers of acute kidney injury (uNGAL/uCr and uKIM 1/uCr) (ultra-distance race) in trained athletes during intense physical effort.

Major criticisms

-The Authors show that uNGAL/uCr significantly decreases from baseline to day 9 after the physical effort, however in the discussion they state that “…all biomarker variation fully disappeared on day 9...” and “…urine levels of NGAL..were similar at 80 km and at baseline after correction for urine dilution status”. I believe that the decrease of uNGAL/uCr during the physical effort and at day 9 after the race deserves an appropriate discussion. Do the Authors have any data regarding the physical activity/diet in the short term before (ideally 7-10 days) and after the race ? Furthermore it would be interesting to compare baseline athletes’ uNGAL/uCr with healthy control population . Finally it has been previously reported that uNGAL/uCr increases in athletes after 60 kg ultramarathon ( Clin Chem Lab Med. 2012 Feb 14;50(9):1585-9. doi: 10.1515/cclm-2011-0954), please discuss (different population/analysis method?).

Minor criticism

- eGFR should be reported in Table 2

- I would suggest to report the number of subjects with blood test in Table 2 legend (as done by the Authors in Table 3 legend)

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Joshua Rein

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

7 Aug 2019

Dear Editor,

We thank the reviewers for their work and their interesting remarks.

Please consider the revised version of our manuscript.

Please find below the answers to the reviewers’ comments.

All substantial modifications appear in red and bold in the revised manuscript.

Sincerely yours.

Romain Jouffroy

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: The authors report on the temporal changes in kidney injury biomarkers before, during, and after an 80km ultra-distance race. The study was designed and conducted appropriately. The manuscript has potential and can benefit from some improved clarity with the results and associated conclusions. Additionally, the focus of the introduction and discussion can be expanded. A large focus of the introduction and discussion involves the AKI to CKD transition. Although this is a good point to make, the study had short term follow-up. The authors should expand these sections to include other exercise related AKIs, such as from “regular” marathons and spin classes. Several studies and numerous case reports have been published within the last few years, notably, one published in PLoS ONE. Additionally, Mansour et al. AJKD 2017, is an impactful study and should be discussed. The authors should add a discussion on the strengths and limitations of KIM-1 and NGAL, including the differences between blood and urine measurement.

Answer: We thank the reviewer for the remark. In the revised version of the manuscript, the introduction and discussion sections were expended. Nevertheless, we believe that long-distance running is different from regular marathon running because the speed is quite lower, and most of races take place on trails with denivelation contrary to regular marathon on road. Another originality of our study is the kinetic of AKI biomarkers contrary to previous studies with different methodology. In previous studies, the impact of running is evaluated comparing before and after running biomarkers values.

Also, the novelty the authors make regarding their study of an ultra-distance race rather than a “regular” marathon is probably not as clinically significant as the authors make it out to be. This should be adjusted throughout the manuscript including the last two paragraphs of the discussion.

Answer: We thank the reviewer for this interesting suggestion. In the revised version of the manuscript, manuscript and the last two paragraphs of the discussion were adjusted in order to take into account the suggestion.

Minor comments:

The in-text tables and figure legends (pages 8-10) are confusing to follow and should be moved to the proper section according to the journal’s author instructions. Also, the landscape formatting on the later pages needs to be fixed.

Answer: We agree with this remark. In the revised version of the text, the landscape formatting was modified in order to accord with journal’s author instructions.

Introduction

Page 4, line 3 – clarify what the 480,000 runners refers to specifically. Did 480,000 runners complete a marathon in that one year or in 2001, have 480,000 runners completed a marathon at some point in their life?

Answer: We agree with this remark. In the revised version, the sentence was modified in order to avoid confusion.

Methods

Page 5, line 8 – clarify what medical disease refers to. Is this specifically referring to chronic diseases? What about occasional headache or infections? Only systemic illness?

Answer: We fully agree with this comment. The medical disease refers to chronic diseases.

A sentence has been added in the new version of the manuscript to clarify this point.

Page 5, lines 9-10 – The sentence needs to be rewritten. I believe the authors intent to say that the participants had free access to food and water during and after the race. “ad libitum”.

Answer: We thank the reviewer for this remark. In the revised version of the manuscript, the sentence was rewritten.

The study protocol was approved in 2014, was this the same year the race occurred?

The authors should mention the year of the race. At least they mentioned the weather that day.

Answer: We thank the reviewer for the remark and the suggestion. In the revised version of the manuscript, the year of the race and the day race weather were added.

Results

Page 8 line 12 – Clarify the sentence to specify mean time of the race is referring to “mean race completion time”

Answer: We thank the reviewer for this interesting suggestion. In the revised version of the manuscript, the sentence was rewritten.

Table 1 – what is “Trial experience”?

Answer: We thank the reviewer for this remark. In the revised version of the text, the term was changed for “seniority in running practice”.

Page 10, line 18 – elevated urine creatinine does not necessarily reflect a “high level of urine concentration”, which is determined from the measurement of specific gravity or osmolarity. The high urine creatinine may simply be reflective of the higher blood creatinine level. Measurement of urinary osmolarity and electrolytes in stored samples would provide additional insight into kidney function during and after the race.

Answer: We fully agree with this remark and thank the reviewer. In the revised version of the manuscript, the sentence was deleted in the results section and a sentence added in the discussion.

How much of the rise in serum creatinine during the race is really a true reduction in CrCl vs. and increase in muscle release of creatinine? The authors should comment on this.

Answer: We thank the reviewer for this very interesting remark. A sentence was inserted in the discussion to emphasize this very important point.

It would be interesting to see the breakdown of the kidney injury biomarkers by KDIGO AKI stage. Were the biomarkers more elevated in these participants?

Answer: We thank the reviewer for this interesting suggestion. A sentence was added in the results section to describe the kinetics of kidney injury biomarkers by KDIGO AKI stage.

Discussion

Page 14, line 4 – Although the authors comment on AKI to CKD transition in other sections, they should specify here safe for “short term” kidney function as their study followed patients up to 9 days after the race.

Answer: We fully agree with this suggestion and thank the reviewer. In the revised version of the manuscript, the sentence was modified in the abstract and manuscript conclusions.

Page 14, lines 6-7 – This sentence is not accurate, the impact of long-distance running on kidney function has been studied. As mentioned earlier, the Mansour AJKD paper, and others.

Answer: We thank the reviewer for this remark. The sentence was rewritten in order to underline the originality of our study showing the kinetic of kidney injury biomarkers.

Since the authors talk about acute dehydration leading to ATN in the discussion, they should mention that the clinical parameters in table 2 do reflect any clinical evidence of dehydration and supports their finding of no major kidney injury.

Answer: We thank the reviewer for this suggestion. A sentence was added in the results section to describe the kinetics of kidney injury biomarkers by KDIGO AKI stage.

Page 15, line 7 – remove the word “the”

Answer: We thank the reviewer for this remark. In the revised version of the manuscript, the term “the” was deleted.

Page 15, line 8 – maybe hyaline and waxy casts are nonspecific but muddy brown urinary casts are not nonspecific and are indicative of tubular injury.

Answer: We agree and thank the reviewer for this remark. The sentence was rewritten in order to be more accurate.

Page 15, lines 14-15 – the assumption that urine was highly diluted before the race is incorrect. There is no evidence to support this claim. Additionally, what does “huge fluid consumption” actually mean?

Answer: We agree with this remark and thank the reviewer. In the revised version of the manuscript, the sentence was deleted. By “huge fluid consumption”, we wanted to say that a possible explanation was the increase fluid intake before the race by most of runners.

Reviewer #2: The Authors in their interesting work show that transient increase in creatinine is not accompanied to parallel increase of two urinary biomarkers of acute kidney injury (uNGAL/uCr and uKIM 1/uCr) (ultra-distance race) in trained athletes during intense physical effort.

We thank the reviewer for his comment.

Major criticisms

-The Authors show that uNGAL/uCr significantly decreases from baseline to day 9 after the physical effort, however in the discussion they state that “…all biomarker variation fully disappeared on day 9...” and “…urine levels of NGAL were similar at 80 km and at baseline after correction for urine dilution status”.

Answer: We thank the reviewer for the remark. Figure 2B shows no difference between uNGAL/uCr between baseline (km 0) and day 9 whereas a significant difference was observed between baseline and the end of the race (km 80). The sentence, in the discussion, was rewritten in order to avoid confusion; we fully agree that “similar” and “not different” are not, statistically, synonymous.

I believe that the decrease of uNGAL/uCr during the physical effort and at day 9 after the race deserves an appropriate discussion. Do the Authors have any data regarding the physical activity/diet in the short term before (ideally 7-10 days) and after the race?

Answer: We thank the reviewer and fully agree with this comment.

We don’t have any data regarding the physical activity/diet in the short term before and after the race. An add was inserted in the revised version of the manuscript to emphasize the important impact of this limitation.

Furthermore, it would be interesting to compare baseline athletes’ uNGAL/uCr with healthy control population.

Answer: We thank the reviewer for this very interesting suggestion. Unfortunately, we don’t have data to compare baseline athletes’ uNGAL/uCr and healthy control population. A limitation was inserted in the revised version of the manuscript to underline this point.

Finally it has been previously reported that uNGAL/uCr increases in athletes after 60 kg ultramarathon (Clin Chem Lab Med. 2012 Feb 14;50(9):1585-9. doi: 10.1515/cclm-2011-0954), please discuss (different population/analysis method?).

Answer: We thank the reviewer for this reference. The reference has been added and potential explanations for the differences in studies results inserted in the discussion.

Minor criticism

- eGFR should be reported in Table 2

Answer: We thank the reviewer for this suggestion. In the revised version of the manuscript, eGFR (mean +/- SD) was added in Table 2.

- I would suggest to report the number of subjects with blood test in Table 2 legend (as done by the Authors in Table 3 legend)

Answer: We thank the reviewer for this suggestion. In the revised version of the manuscript, the number of subjects with blood test was added in Table 2 legend.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

20 Aug 2019

PONE-D-19-17069R1

Acute kidney injury during an ultra-distance race

PLOS ONE

Dear Dr Jouffroy,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the final minor points raised during by Reviewer 1.

================================

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Kind regards,

Paolo Cravedi

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: I thank the authors for their revisions that adequately addressed my major concerns. I have several additional comments/suggestions to further improve the manuscript for publication.

I appreciate the authors’ clarification regarding the novelty of their study in comparison to prior studies. The authors should add a sentence or two introducing what trail running is as explained in their response to my first comment. They can include aspects such as terrain, running surface, flat vs. hills, carry extra weight, and make a comparison with marathons to help clarify and explain differences in the races.

Throughout the manuscript, the authors should emphasize “ultra-distance” when referring to the race rather than “long-distance” to distinguish from marathons.

Authors use the words running where in some sentences, race would be the more appropriate word. The sentences can be reworded so that running also fits if the authors prefer to use that word. Page 3 lines 4 and 5 – “biomarker injury while running” or “biomarker injury during the race”

Temporal seems to be a more appropriate word than kinetic when describing changes in biomarkers over time. Kinetic refers more to the changes in the rate of something. The authors can consider calculating rates of change at each time point but I don’t think that would be of any clinical significance.

As some of the authors have previously published on changes in cardiac function during ultradistance racing, do the authors think the reduction in eGFR can be partially explained by cardiac strain? Also, the authors state in the discussion on page 18 line 22 that AKI may be prerenal. Besides extracellular volume depletion, it may be worth mentioning other potential mechanisms such effects on renal blood flow by the sympathetic nervous system, and antidiuresis from vasopressin release in response to stress/pain. May be worth commenting on to build the discussion but not critical.

Page 5 line 3 - As the authors discuss recent trends in increasing race distance in several parts of the manuscript, they should consider using a more recent reference than the one used from 2001.

Page 5 line 2 and line 10 – fix the use of two vs. 2

Page 5 line 24 – clarify that “before and after” is referring to the race

Page 6 line 3 – blood and urine samples were also collected before the race.

Did participants exclusively drink water or could some have consumed sports drinks? Might be worth discussing the association with kidney injury and rehydration with sugar sweetened beverages if participants did in fact have access to it.

It is more appropriate to use the word “chloride” rather than “chlorine” in the text and tables.

Page 12 lines 9-10 – Myoglobin increased from 34 to 2761, if not statistically significant, this increase may possibly be of some clinical significance. Although not massively elevated, do the authors believe this elevation could have contributed to any reduction in eGFR?

Page 14 lines 12-16 – Awkward sentence structure. These sentences would benefit from English editing.

Page 18 line 23 - If the hypothesis is that free access to food and water limited the kidney injury, it is safe to say on page 18 lines 12-13 that suggesting dehydration and antidiuresis?

Page 19 line 21 – ultraendurance races do in fact have considerable effects to kidney function such as, as the authors state, antidiuresis. However, it would be more accurate to state that ultra-endurance races specifically have no impact on acute kidney injury.

Reviewer #2: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step.

29 Aug 2019

Dear Editor,

We thank the reviewers for their work and their interesting remarks.

Please consider the revised version of our manuscript.

Please find below the answers to the reviewers’ comments.

In the revised manuscript, all substantial modifications appear in red and bold whereas previous modifications remain in black and bold.

Sincerely yours.

Romain Jouffroy

Responses to Reviewers' comments:

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

6. Review Comments to the Author

Reviewer #1: I thank the authors for their revisions that adequately addressed my major concerns. I have several additional comments/suggestions to further improve the manuscript for publication.

Answer: We thank the reviewer for his work and new comments/suggestions to further improve our manuscript.

I appreciate the authors’ clarification regarding the novelty of their study in comparison to prior studies. The authors should add a sentence or two introducing what trail running is as explained in their response to my first comment. They can include aspects such as terrain, running surface, flat vs. hills, carry extra weight, and make a comparison with marathons to help clarify and explain differences in the races.

Answer: We thank you for this suggestion. In the revised manuscript, three sentences have been added to better explain to the readers what trail running is in comparison with marathons.

Throughout the manuscript, the authors should emphasize “ultra-distance” when referring to the race rather than “long-distance” to distinguish from marathons.

Answer: We thank the reviewer for this suggestion. In the revised manuscript, the term “long-distance” was replaced by “ultra-distance”.

Authors use the words running where in some sentences, race would be the more appropriate word. The sentences can be reworded so that running also fits if the authors prefer to use that word. Page 3 lines 4 and 5 – “biomarker injury while running” or “biomarker injury during the race”.

Answer: We thank the reviewer for this suggestion. In the revised manuscript, the term running was replaced by race.

Temporal seems to be a more appropriate word than kinetic when describing changes in biomarkers over time. Kinetic refers more to the changes in the rate of something.

Answer: We thank the reviewer for this interesting remark. In the revised manuscript, the word “kinetic” running was replaced by “temporal”.

The authors can consider calculating rates of change at each time point but I don’t think that would be of any clinical significance.

Answer: We fully agree with the reviewer. As underlined, the rate of change does not provide more clinical information in this study.

As some of the authors have previously published on changes in cardiac function during ultradistance racing, do the authors think the reduction in eGFR can be partially explained by cardiac strain?

Also, the authors state in the discussion on page 18 line 22 that AKI may be prerenal. Besides extracellular volume depletion, it may be worth mentioning other potential mechanisms such effects on renal blood flow by the sympathetic nervous system, and antidiuresis from vasopressin release in response to stress/pain. May be worth commenting on to build the discussion but not critical.

Answer: We fully agree with this very interesting remark. A sentence has been inserted in the discussion in order to take into account the remark.

Page 5 line 3 - As the authors discuss recent trends in increasing race distance in several parts of the manuscript, they should consider using a more recent reference than the one used from 2001.

Answer: We thank the reviewer for this suggestion. In the revised manuscript, a more recent reference replaced the one of 2001. Consequently, the sentence was modified.

Page 5 line 2 and line 10 – fix the use of two vs. 2

Answer: We thank the reviewer. The modification appears in the revised version of the manuscript.

Page 5 line 24 – clarify that “before and after” is referring to the race

Answer: We thank the reviewer. The modification appears in the revised version of the manuscript.

Page 6 line 3 – blood and urine samples were also collected before the race.

Answer: We fully agree with the reviewer. The term “before” was added in the sentence.

Did participants exclusively drink water or could some have consumed sports drinks? Might be worth discussing the association with kidney injury and rehydration with sugar sweetened beverages if participants did in fact have access to it.

Answer: We thank the reviewer for this suggestion. The participants drink only water during the race. A sentence was inserted in the revised version of the manuscript to clarify this point.

It is more appropriate to use the word “chloride” rather than “chlorine” in the text and tables.

Answer: We thank the reviewer. The modification appears in the revised version of the manuscript.

Page 12 lines 9-10 – Myoglobin increased from 34 to 2761, if not statistically significant, this increase may possibly be of some clinical significance. Although not massively elevated, do the authors believe this elevation could have contributed to any reduction in eGFR?

Answer: We fully agree with the author that the myoglobin elevation at the end of the race vs start could partly explain the reduction in eGFR.

Page 14 lines 12-16 – Awkward sentence structure. These sentences would benefit from English editing.

Answer: We thank the reviewer. The sentences were edited in the new version of the manuscript.

Page 18 line 23 - If the hypothesis is that free access to food and water limited the kidney injury, it is safe to say on page 18 lines 12-13 that suggesting dehydration and antidiuresis?

Answer: We fully agree with the reviewer for this very interesting suggestion. During the race, free access to food and water was limited to the refreshment points allowed by the race organisation. Thus, we cannot exclude that some participants experienced dehydration and antidiuresis during the race (all samples were collected before the participant refreshed). A sentence was inserted in the revised version of the manuscript to clarify this point.

Page 19 line 21 – ultra endurance races do in fact have considerable effects to kidney function such as, as the authors state, antidiuresis. However, it would be more accurate to state that ultra-endurance races specifically have no impact on acute kidney injury.

Answer: We fully agree with this remark and thank the reviewer. The sentence was modified in order to take into account the remark.

Reviewer #2: (No Response)

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

3 Sep 2019

Acute kidney injury during an ultra-distance race

PONE-D-19-17069R2

Dear Dr. Jouffroy,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.

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With kind regards,

Paolo Cravedi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

"Temporal" is an adjective. As a noun, it refers to the temples of the skull.

In the present paper, "temporal" should be used in expressions such as, for instance, "temporal changes" or "temporal variations".

Reviewers' comments:

6 Sep 2019

PONE-D-19-17069R2

Acute kidney injury during an ultra-distance race

Dear Dr. Jouffroy:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

For any other questions or concerns, please email plosone@plos.org.

Thank you for submitting your work to PLOS ONE.

With kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr Paolo Cravedi

Academic Editor

PLOS ONE