Hydration Status and Acute Kidney Injury Biomarkers in NCAA Female Soccer Athletes During Preseason Conditioning

rcise training in extreme temperatures concurrent with hypohydration status may potentiate the development of acute kidney injury (AKI) in young, healthy persons.Back- ground/Objectives: It is unknown how repeated training bouts in ambient higher tem- peratures and humidity may influence measures of AKI. The purpose of this study was to investigate hydration status and renal biomarkers related to AKI in NCAA Division I female soccer athletes during preseason conditioning.Methods: A convenience sample of n= 21 athletes were recruited (mean±SEM; age: 19.3±0.25 y; height:169.6±1.36 cm; mass: 68.43±2.46 kg; lean body mass: 45.91±1.13 kg; fat mass: 22.51±1.69 kg; body fat %: 32.22±1.32%). The average temperature was 27.43±0.19 ◦ C, and the humidity was71.69±1.82%. Body composition, anthropometric, workload, and 14 urine samples were collected throughout the preseason training period for urine specific gravity (USG), creatinine (uCr), cystatin C (uCyst-C), and neutrophil gelatinase-associated lipocalin (uN- GAL) analyses.Results: Our investigation showed that, when compared to baseline (D0), the athletes maintained a USG-average euhydrated status (1.019±0.001) and

average temperature was 27.43±0.19 ◦ C, and the humidity was71.69±1.82%. Body composition, anthropometric, workload, and 14 urine samples were collected throughout the preseason training period for urine specific gravity (USG), creatinine (uCr), cystatin C (uCyst-C), and neutrophil gelatinase-associated lipocalin (uN- GAL) analyses.Results: Our investigation showed that, when compared to baseline (D0), the athletes maintained a USG-average euhydrated status (1.019±0.001) and were euhy- drated prior to each exhibition game (D5-Pre:p= 0.03; 1.011±0.001; D10-Pre:p= 0.0009; 1.009±0.001 ); uCr was elevated on D8 (p= 0.001; 6.29±0.44 mg·dL −1 · LBM −1 ) and D10- Post (p= 0.02; 6.61±0.44 mg·dL −1 · LBM −1 ); uCyst-C was elevated on D6 through D10 (p= 0.001; ~0.42±0.01 mg·dL −1 ); no differences were found in uNGAL concentration. The highest distance (m) displaced was found during exhibition games (D5:p= <0.0001; ~8.6 km and D10:p= <0.0001; ~9.6 km). During the preseason conditioning, the athletes maintained a euhydrated status (~1.019) via USG, an increase in uCr that averaged within a normal range (208 mg·dL −1 ), and an increase in uCyst-C to near AKI threshold levels (0.42 mg·L −1 ) for several practice sessions, followed by an adaptive decline. No differences were found in uNGAL, which may be explained by athlete variation, chosen time sample collection, and variation in training and hydration status.Conclusions: The athletes main- tained a euhydrated status, and this may help explain why urinary markers did not change or meet the reference threshold for AKI. Keywords:acute kidney injury; exercise training; preseason; renal dysfunction Nutrients2025,17, 2185 https://doi.org/10.3390/nu17132185

Nutrients2025,17, 2185 2 of 23 1. Introduction The physiological demands of soccer athletes incorporate both aerobic and anaero- bic systems for muscular strength, endurance, power, and agility to optimally perform in90–120 minmatches [1,2]. The National Collegiate Athletic Association (NCAA) only allows 21 practice units (including exhibition games) before the first scheduled date of competition [3]. With a relatively short time frame in the preseason training period, coaches are responsible for preparing student-athletes for the competitive season. During preseason, collegiate soccer athletes complete 2–3 daily sessions, including conditioning, strength, technical, and tactical training, and exhibition games. Geographic location can influence environmental factors that worsen exertional heat illnesses (EHIs), with hot, humid condi- tions increasing risk [4]. In the southern U.S., NCAA Division I soccer preseason occurs in August, exposing athletes to high temperatures and humidity, raising dehydration risk from increased water loss and insufficient fluid intake [5]. It has been shown that >2% of body mass loss is often associated with an increased risk of EHIs and a decrease in athletic performance [6]. From the archived National Oceanic and Atmospheric Administration (NOAA) data, South Texas experienced an average temperature of 29.78 ◦ C (24.3–35.0 ◦ C) and a relative humidity of 74% during August. These environments can elevate the risk of dehydration, which may lead to severe health complications caused by heat strain from increased core body temperature [5,7]. This, in turn, may further heighten the risk of acute kidney injury [8]. Acute kidney injury (AKI) is defined as a rapid decline in renal function occurring within hours, encompassing both structural injury and functional impairment of the kid- neys. AKI is classified into three categories: (a) pre-renal AKI, (b) acute post-renal ob- structive nephropathy, and (c) intrinsic AKI. Of these, only intrinsic AKI denotes true parenchymal kidney disease. In contrast, pre-renal and post-renal forms are secondary to extra-renal pathologies that compromise the glomerular filtration rate (GFR). If these conditions persist, they may induce renal parenchymal injury and progress to intrinsic renal pathology [9]. Recent research suggests that acute kidney injury (AKI) can result from strenuous exercise, dehydration, and severe heat illness

intrinsic AKI denotes true parenchymal kidney disease. In contrast, pre-renal and post-renal forms are secondary to extra-renal pathologies that compromise the glomerular filtration rate (GFR). If these conditions persist, they may induce renal parenchymal injury and progress to intrinsic renal pathology [9]. Recent research suggests that acute kidney injury (AKI) can result from strenuous exercise, dehydration, and severe heat illness (e.g., exertional heat stroke) caused by pro- longed heat strain and elevated core body temperature [10,11]. Over the past decade, serum, plasma, and urinary biomarkers have been used for early AKI detection [12]. Tradi- tionally, serum creatinine (sCr), a catabolic byproduct of creatine phosphate metabolism in skeletal muscle, has been the primary marker for AKI assessment [13,14]. Approximately 2% of creatine stores are irreversibly converted to creatinine, with levels influenced by lean body mass, age, protein intake, and muscle function. In healthy individuals, creatinine is freely filtered by the kidneys, with minimal extrarenal metabolism. Creatinine is almost entirely excreted in urine, with about 10–30% secreted by the proximal tubules in those with normal basal kidney function [15]. However, exercise can increase urinary creatinine (uCr) excretion without significantly affecting serum levels [13,14,16]. Urinary creatinine (uCr) levels typically range from ~20 to 400 mg·dL −1 [17], with higher values indicating potential kidney dysfunction. ICU patients with AKI have reported uCr concentrations of ~133 mg·dL −1 , compared to ~82 mg·dL −1 in non-AKI patients. However, creatinine alone may be an unreliable AKI biomarker, as levels can be affected by diet, malnutri- tion, exercise-induced muscle damage, and athletes seeking to acquire muscle mass [18]. While creatinine is used to estimate glomerular filtration rate (GFR), it does not specifically indicate a tubular or glomerular injury and may not reliably identify AKI. The American Society of Nephrology has identified potential biomarkers for AKI detection, including cystatin C (Cyst-C), neutrophil gelatinase-associated lipocalin (NGAL), and creatinine (Cr) [19–21]. Cyst-C, a 13 kDa protein, is filtered by the glomerulus, reab-

of Nephrology has identified potential biomarkers for AKI detection, including cystatin C (Cyst-C), neutrophil gelatinase-associated lipocalin (NGAL), and creatinine (Cr) [19–21]. Cyst-C, a 13 kDa protein, is filtered by the glomerulus, reab-

Nutrients2025,17, 2185 3 of 23 sorbed, and metabolized in the renal tubule (Figure). Even a slight increase in urinary Cyst-C (uCyst-C) signals proximal tubule injury [12]. As a key extracellular inhibitor of cysteine proteases, Cyst-C is typically reabsorbed in a healthy kidney and absent from urine in significant amounts [22]. It has been reported that the normal reference range for urinary Cyst-C is 0.06–0.16 mg·L −1 [23]. However, renal injury reduces reabsorption, leading to an increase in urinary presence. Given that >99% of Cyst-C is filtered and catabolized by the renal tubules, any detectable urinary levels may indicate impairment [19,20]. Previous studies show that in AKI patients, uCyst-C concentrations≥0.45 mg·L −1 suggest tubular dysfunction, while non-AKI levels are≤0.07 mg·L −1 . Increased uCyst-C levels appear to be a valid method of detecting tubular dysfunction [24]. Figure 1.The approximate location of the synthesis of urinary markers of tubular AKI (created using BioRender). Recent studies have explored the effects of exercise on uCyst-C and AKI markers in healthy individuals. Bongers et al. (2018) found uCyst-C levels increased more after prolonged (150 min) than acute (30 min) exercise and rose 1.8-fold after the first day of repeated exercise (0.05–0.09 mg·L −1 ) [25]. However, it was noted that this increase dissipated and returned to near baseline levels after 3 days [26]. Lastly, an increase in uCyst-C was also found in individuals after both 10 and 100 km runs. There was a 2.56-fold increase after 10 km and a 4.96-fold increase after 100 km [27]. In addition to uCyst-C as an AKI measure, neutrophil gelatinase-associated lipocalin (NGAL), also known as human neutrophil lipocalin or lipocalin 2, exists in three molecular forms in blood and urine: a 25 kDa monomer, a 45 kDa disulfide-linked homodimer, and a 135 kDa heterodimer. Synthesized in the bone marrow during myelopoiesis, it is stored in neutrophil granules. NGAL mRNA is also expressed in non-hematopoietic tissues, including the colon, trachea, lung, and kidney epithelium, with synthesis stimulated by Interleukin-1β(IL-1β). In AKI, kidney epithelial cells primarily secrete the 25 kDa monomeric form [28]. Under normal conditions, NGAL is filtered by the

and a 135 kDa heterodimer. Synthesized in the bone marrow during myelopoiesis, it is stored in neutrophil granules. NGAL mRNA is also expressed in non-hematopoietic tissues, including the colon, trachea, lung, and kidney epithelium, with synthesis stimulated by Interleukin-1β(IL-1β). In AKI, kidney epithelial cells primarily secrete the 25 kDa monomeric form [28]. Under normal conditions, NGAL is filtered by the glomerulus, reabsorbed in the proximal tubules (Figure), and minimally excreted in urine. It has been reported that the normal urinary concentration of NGAL in females is≤65.0 ng·mL −1 [29]. It is produced by activated neutrophils in the proximal tubules. Following ischemic, septic, or toxic kidney injury, NGAL transcription and protein levels rise sharply, increasing plasma and urinary NGAL. Elevated NGAL levels indicate early structural renal tubular damage. It is considered a more specific AKI marker, particularly in healthy individuals, where

Nutrients2025,17, 2185 4 of 23 systemic inflammation and multi-organ damage may otherwise elevate its levels in clinical settings [12,28]. Regarding exercise and AKI, NGAL has been investigated as a potential biomarker. Studies on marathon running found urinary NGAL (uNGAL) concentrations increased from ~8–12 ng·mL −1 at baseline to ~33–47 ng·mL −1 immediately post-marathon. However, 24 h post-race, one study reported a return to baseline (~10 ng·mL −1 ), while another observed a further rise to ~59 ng·mL −1 [30,31]. In shorter duration, higher intensity bouts of exercise, uNGAL concentration changes are variable. Previous studies showed an increase in uNGAL immediately after a high-intensity sprint (800 m) from <10 to~12 ng·mL −1 25 min post-exercise [32] and after high-intensity interval resistance training (HIIT) and an increase from ~18 to 33 ng·mL −1 2 h post-HIIT [33]. However, similar to some of the observations in the marathon runners, uNGAL concentrations fell back to baseline levels. This may suggest that these AKI markers may be transient, relative to the duration of the exercise bout, and a potential kidney adaptation to exercise stress over time [12]. The purpose of this study was to investigate the roles of athlete workload and hy- dration status during multiple practice sessions on urinary biomarkers reflective of AKI during preseason training in the summer month of August in NCAA Division I female soccer student-athletes located in South Texas. We hypothesized that the concurrent effect of higher temperatures and humidity, and the assumed preseason training status of the athletes, may negatively affect the markers of hydration and AKI. 2. Materials and Methods 2.1. Sample Population This study was approved by the Institutional Review Board (IRB 50–19) and the Insti- tutional Biosafety Committee (IBC) at Texas A&M University-Corpus Christi. Although all convenience samples have less clear generalizability than probability samples, not all convenience samples are the same. Homogeneous convenience samples have clearer generalizability relative to conventional convenience samples with more constrained so- ciodemographic characteristics [34]. A homogenous convenience sample of 21 female soccer student-athletes competing in NCAA Division I soccer was recruited for partici- pation (age: 19.38±0.25 y; height: 169.6±1.36 cm;

convenience samples have less clear generalizability than probability samples, not all convenience samples are the same. Homogeneous convenience samples have clearer generalizability relative to conventional convenience samples with more constrained so- ciodemographic characteristics [34]. A homogenous convenience sample of 21 female soccer student-athletes competing in NCAA Division I soccer was recruited for partici- pation (age: 19.38±0.25 y; height: 169.6±1.36 cm; mass: 68.43±2.46 kg; lean body mass:45.91±1.13 kg; fat mass: 22.51±1.69 kg; body fat %: 32.22±1.32%). Athletes were medically cleared to participate by the team physician and athletic training staff to participate in this study. Due to the observational nature of this study, all individuals who were medically cleared to participate in preseason were recruited to participate in the study. Athletes designated for urinary AKI biomarker analysis were selected based on the greatest workload performed over the preseason schedule collected by the Polar Team Pro System. 2.2. Anthropometrics and Body Composition Height and mass were assessed via a portable SECA scale and stadiometer (Seca model 769). Whole body composition, regional body composition, and bone mineral density were assessed using the GE Lunar Dual X-Ray Absorptiometry (iDXA) technology (iDXA, Lunar Prodigy; GE Healthcare, Madison, WI, USA). The iDXA is a relatively quick, safe, and noninvasive method to assess body composition measures [35]. 2.3. Hydration Status Hydration status was measured utilizing changes in clothed-body mass pre- and post-practice sessions using the same portable scale and stadiometer (Seca model 769, Seca GmbH & Company, Hamburg, Germany). Urine was collected and analyzed using urine- specific gravity (USG) analysis using a clinical refractometer (Sper Scientific; model 300005;

Nutrients2025,17, 2185 5 of 23 Scottsdale, AZ, USA). The manufacturer reported the accuracy of the clinical refractometer was±0.002 for USG measurements. Additionally, non-nude body mass was recorded for each participant pre- and post-practice session as a sensitive and simple assessment to determine acute changes in body water for all types of dehydration [36]. Athletes were instructed to wear the same clothing for their pre- and post-practice session body mass measures. Perspiration that may have accumulated in clothing post-practice session was not controlled. For this study, hypohydration was defined as USG >1.020 [37] and >2% of body mass loss [36]. The index for the range of hydration status for USG is listed in Table for reference. Additionally, water ingestion during practice sessions was not controlled or monitored due to the difficulty of observing the daily intake of water in multiple athletes (n= 21) over the preseason time period. Table 1.Index for hydration status. Condition USG Value Well hydrated <1.010 Minimal dehydration <1.010–1.020 Significant dehydration 1.021–1.030 Serious dehydration >1.030 Urine specific gravity (USG),≤1.020, is an indication of euhydration status [38]. 2.4. Environmental Analysis All practice and competition events occurred on artificial turf located on the university campus. Since artificial turf can raise surface temperatures by ~10–15 ◦ F, human heat stress can increase, which can adversely affect kidney function [4]. Wet-bulb globe temperature (WBGT) is a measurement of the environment that incorporates radiant heat, humidity, am- bient temperature, and wind [39]. As relative humidity rises, the ability to evaporate sweat becomes challenging and can also lead to excess stored metabolic heat from exercise [40]. Utilizing WBGT as an index of human heat strain is the most appropriate environmental measure in this context. Ambient temperature (Td), dry-bulb temperature (Tg), wet-bulb temperature (Tw), and percent relative humidity (%RH) were assessed via both the Kestrel 5000 environmental meter and a Kestrel DROP 2 device (Nielsen-Kellerman Co., Boothwyn, PA, USA) on the playing surface. Dry-bulb temperature (Tg) measures were not enabled on these devices. All dry-bulb data for the training sessions were acquired from the National Oceanic and Atmospheric Administration (NOAA) National Centers for Environmental Information

percent relative humidity (%RH) were assessed via both the Kestrel 5000 environmental meter and a Kestrel DROP 2 device (Nielsen-Kellerman Co., Boothwyn, PA, USA) on the playing surface. Dry-bulb temperature (Tg) measures were not enabled on these devices. All dry-bulb data for the training sessions were acquired from the National Oceanic and Atmospheric Administration (NOAA) National Centers for Environmental Information (NCEI) [41]. The Corpus Christi Naval Air Station (NAS) served as the closest weather station (4.1 miles) to the practice and competition field. Due to this limitation, WBGT calculations are approximations. After the average height of the athlete population was determined, environmental measures were collected at ~15 cm above the turf with the Kestrel DROP 2 device and at the average one-half height among athletes’ Anterior Superior Iliac Spine (ASIS). Environmental data were continually collected using the Kestrel 5000 unit starting 30 min prior to each outdoor practice and exhibition match. Environmental sampling and storage of data automatically occurred every 15 s. Each practice session was approximately 97 min in duration. Wet-bulb globe temperatures (WBGTs) were calculated using the following equation: 0.7 Tw + 0.2 Tg + 0.1 Td, where Tw = wet-bulb temperature, Tg = globe (dry bulb) temperature, and Td = ambient temperature [42]. Since dry-bulb measurements were not assessed on-site with the environmental data loggers, the dry-bulb data were pooled from the NOAA weather station at Corpus Christi NAS for the preseason period at the times when practices and competitions occurred. All other on-site measures were utilized in the WBGT equation. Due to this limitation, measures are approximations

Nutrients2025,17, 2185 6 of 23 since dry-bulb measures were not enabled on the devices. WBGT approximations were 27.43±0.19 ◦ C and 71.69±1.82 %RH during the intervention. 2.5. Athlete Workload Data Collection The athletes were provided with and fitted for a Team Polar Pro monitor, which utilized a 10 Hz global navigation satellite system (GNSS) device tracked using the Polar Team Pro System (Polar Electro Co., Kempele, Finland). This 10-Hz system has been shown to be an accurate and reliable device for tracking team sports work variables [43]. However, it should be highlighted that this system inherently uses the commonly used “HRMax= 220−Age”, which has been suggested to be inaccurate with a standard deviation of 10–12 bpm and has been shown to underestimate HRMaxin younger and older adults [44]. Workload metrics to highlight the subject workload per session were total distance (m), average velocity (m·min −1 ), age-predicted heart rate average (APHRAvg%), and max (APHRMax%). The Polar Team Pro System was owned and operated by the Athletics Department. 2.6. Data Collection Procedures Athletes were asked to provide 14 urine samples during preseason training, which included two exhibition games. Prior to arriving, athletes were informed of training protocols and were provided consent forms via email. After consenting to participation, athletes were then scheduled between 05:00 and 07:00 on D0 (Table). Athletes were then asked to report to the laboratory for baseline anthropometrics and urine collection assessments. Table 2.Preseason practice schedule. Day Preseason Practice Name Time of Day (h) D0 Pre-Preseason (Baseline) 04:30–07:00 D1 Fitness Testing Day #1 06:00–10:00 and 18:00–21:00 D2 Fitness Testing Day #2 06:00–10:00 and 18:00–21:00 D3 Regular Practice Day #1 06:00–10:00 and 18:00–21:00 D4 Regular Practice Day #2 06:00–10:00 and 18:00–21:00 D5 Exhibition Game #1 17:00–19:00 D6 Scheduled Rest Day #1 05:00–07:00 * D7 Regular Practice Day #3 06:00–10:00 and 18:00–21:00 D8 Midweek #1—Regular Practice Day06:00–10:00 and 18:00–21:00 D9 Regular Practice Day #4 06:00–10:00 and 18:00–21:00 D10 Exhibition Game #2 19:00–21:00 D11 Scheduled Rest Day #2 05:00–07:00 * D12 Midweek #2—Regular Practice Day 18:00–21:00 D13 Regular Practice Day #5 18:00–21:00 D14 Post–Preseason (End) 04:30–07:00 Preseason practice schedule, name, and

This study investigates hydration status and renal biomarkers related to AKI in NCAA Division I female soccer athletes.