The proposed work is designed to be the first in a series of studies investigating the health benefits and risks related to high intensity training (HIT) exercise. Our specific aims are to determine, 1) if participation in a single bout of HIT induces hematological markers consistent with acute kidney injury (AKI), and 2) if risk is predicted by the pre-exercise concentration of plasma proenkephalin-A.

This investigation is an observational case control study. In year one, data collection procedures will be refined with ~40 participants local to the University of Wyoming and training will occur for collaborators from Wyoming community and tribal colleges. In year two, data collection will expand to some of the 12 CrossFit® gyms in Wyoming with assistance from the community and tribal colleges. Blood and urine samples will be collected before and up to 48 h after a standardized bout of HIT exercise on ~100 participants. Baseline blood samples will be analyzed for proenkephalin-A. All blood samples will be analyzed for markers of muscle damage (e.g., creatine kinase and myoglobin), and markers of kidney function (e.g., serum creatinine and blood urea nitrogen). Urine will be analyzed for markers of filtration function (e.g., albumin, creatinine, neutrophil gelatinase-associated lipocalin [NGAL], and kidney injury molecule 1 [KIM-1]). Lastly, the severity of kidney damage will be compared with the number of risk alleles and proenkephalin-A concentration.

The investigators envision that the bout of HIT exercise will induce markers consistent with skeletal muscle damage in most participants and, based on literature from other styles of intense exercise, that acute kidney injury will be diagnosable in between 50-75% of participants. Secondarily, the investigators predict that the concentration of proenkephalin-A will be inversely related to the change in kidney function from before to after the HIT exercise bout.

The HIT-KH investigation is proposed to take place over the course of two calendar years. During year one collaborations with gyms specializing in High Intensity Resistance and Functional Training style workouts (referred to from this point forward as satellite data collection sites) as well as community and tribal colleges (referred to from this point forward as partner institutions) local to these facilities will be solidified. Also, during the summer of year one data collection will occur on a small subset of individuals (~40) at a data collection site local to the University of Wyoming (Figure 1.). It is our plan to use this first data collection in Laramie, WY as a training session for all students from partner institutions so that the students are prepared when the data collection is expanded to the satellite data collection sites in year 2.

In order to properly power this investigation the investigators have estimated that about 100 participants will be needed. Given that the investigators can expect about 40 participants from the local Larmie data collection gym (CrossFit® 7220)this will require the investigators to partner with other high intensity resistance and functional training gyms around the state of Wyoming in year 2. The investigators have chosen to reach out to CrossFit® gyms specifically because it is common practice for most CrossFit® gyms to program the workout the investigators are interested in observing (Murph), at least once per year. Collection data on 60 additional participants will require partnership with 3 additional gyms (i.e., 20 participants / facility). At this point the investigators have been in contact with 3 gym owners all of whom have offered verbal agreement to assisting with data collection. Among these gyms is CrossFit® Leveler, located on the Wind River Reservation in Fort Washikie, Wyoming. This facility is a Native American owned business and it primarily caters to those Native American's living on the reservation. Because the state of Wyoming is ~2% Native American in population, the inclusion of this facility in our investigation is very important for proper sampling (see attached letter of support from owner Mike Ute). Additional gyms that have been contacted and are in the process of providing written approval are CrossFit® Frontier (Cheyenne), and, Cloud Peak CrossFit® (Sheridan).

Because our data collection will be occurring throughout the state the investigators also plan to partner with local community and tribal colleges local to many of the above satellite data collection sites. Briefly, the investigators plan to incorporate up to three students and one faculty mentor from each of the following educational institutions; Laramie County Community College, Wind River Tribal College, and Sheridan Community College. Regarding the inclusion of Wind River Tribal College and the Wind River Indian Reservation, Investigator Johnson is under the guidance of Dr. Christine Porter who is also in the University of Wyoming Division of Kinesiology and Health. Dr. Porter has successfully implemented several research studies on the Wind River Reservation and is well versed in the intricacies and requirements necessary for the completion of data collection in this partnership. Lastly, the partner institutions are beneficial to the project because the data collection takes place over the course of 5 consecutive days, training and working with local researchers will enable this project to reduce the cost because a large research team from Laramie, Wyoming will not be required for each data collection.

As mentioned above 1-3 students from each of the above institutions will be requested to assist with data collection for HIT-KH. Part of our grant funding is devoted to payment of fees for these student's training. This includes travel fees to travel and stay in Laramie, Wyoming during year 1 for observation and basic level assistance with the initial data collection. During the end of year 1 and/or beginning of year 2 additional funds have been allocated to provide phlebotomy training to interested students. These students will then be utilized during year two data collections to assist with blood draws. Finally, funds have been allocated for travel to and attendance of the Rocky Mountain Regional American College of Sports Medicine conference. It is our goal that at least one student will be able to use the preliminary data from the year 1 data collection to compile a research abstract that will be submitted for presentation. All other students will be offered the ability to attend and participate in the conference during the spring of 2019.

Blood samples will be collected one day before participants take part in the workout, and 24 and 48hrs after completion. Sampling will take place by single venipuncture to an antecubital vein by a trained investigator using sterile technique and universal precautions. Following any needle removal, firm pressure will be applied over the site of the blood draw with gauze to minimize the occurrence of a hematoma. Blood will be drawn into vacutainers specific for each of the analytes to be analyzed later. Tubes will be centrifuged as soon as possible, then the blood plasma, serum, or white blood cells will be removed and aliquoted into separate cryovials. These cryovials will be frozen in the -80C freezer as soon as possible. For satellite data collections, if -80C freezers are not available at the local partner institution, liquid nitrogen will be used to freeze and store samples until the samples can be transported back to the University of Wyoming for more permanent storage.

Urine samples will be collected in 24 hr collection jugs for the period immediately prior to the workout and for two 24hr periods following. Each sample will be weighed for measurement of total volume, evaluated for urine color and urine specific gravity. Dipsticks will be used on fresh samples while three 2mL aliquots of each 24hr sample will be separated and stored at room temperature until the samples are returned to the University of Wyoming where the samples will be frozen at -80C for future analysis.

Fresh 24hr urine samples will be analyzed using traditional dipsticks for the following variables: leukocytes, nitrite, urobilinogen, protein, pH, hemoglobin, ketone, bilirubin, and glucose. Additionally, fresh samples will be evaluated for urine color and specific gravity via handheld refractometer in order to establish hydration state. Further biochemical urinary analysis are reviewed below.

Proenkephalin-A will be analyzed at the Clinical Research Center by the use of a chemiluminometric sandwich immunoassay. The laboratory group in Malmo has previously successfully measured this hormone (Schulz et al., 2017). All remaining blood and urine samples will be analyzed within the Human Integrated Physiology laboratory located at the University of Wyoming using colorimetric enzyme-linked immunosorbent assays (ELISA). These samples will be analyzed for the following variables; serum creatinine, blood urea nitrogen, urinary creatinine, neutrophil gelatinase-associated lipocalin [NGAL], and kidney injury molecule 1 [KIM-1]). All ELISA antibodies will be supplied by "Abcam", (Cambridge, Massachusetts, USA) and microplates will be read according the manufacturer's instructions on spectrophotometer (Epoch 2, Biotek, Winooski, Vermont, USA).

Inclusion Criteria:

1. Healthy individual who has completed this specific workout on at least one prior occasion

Exclusion Criteria:

1. Score of "0" on the Functional Movement Screening (FMS) test as evaluated by two investigators * indicating pain during any of the seven functional movements
2. Self- reported Kidney (chronic kidney disease, polycystic kidney disease, glomerulonephritis, diabetic nephropathy, interstitial nephritis, Goodpasture syndrome) or other medical condition contraindicating participation in HIFRT (Hypertension, dyslipidemia, type II diabetes mellitus, BMI >30)
3. Pregnancy, suspected pregnancy, or breastfeeding
4. Blood donations within the last eight weeks leading up to testing day
5. Any musculoskeletal injuries which have resulted in > 1 week of absence from HIFRT within the last six months
6. Not passing the physical activity readiness questionnaire (PAR-Q)
7. Allergy to ibuprofen or non-steroidal anti-inflammatory medication
8. Surgical operation on digestive tract or kidneys, except appendectomy
9. Inability to participate in the entire study
10. Recurrent urinary tract infections or kidney stones
11. History of protein or blood in urine
12. Moving from a location of low altitude (< 3,000') to Laramie within the past 3 months
13. Inability to understand and write English

During entire protocol enrollment. This was approximately 4 days (96 hours). Participants were able to contact investigators after 96 hours should they have experienced an adverse event following their final visit to the laboratory (i.e., allergy to bandage, increasing muscle soreness, etc.).

No other parties besides the participants will be masked.