The Effects of Branched Chain Amino Acid Supplement on Kidney Tissue of Exercising Rats

The Effects of Branched Chain Amino Acid Supplement on Kidney Tissue of Exercising Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Effects of Branched Chain Amino Acid Supplement on Kidney Tissue of Exercising Rats Cemre AYDEĞER, Hüseyin Avni EROĞLU This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-326595/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract One of the supplements used in with exercise programs is branched chain amino acids (BCAAs) which preferred because of their effect on novation of muscle protein synthesis. However, BCAAs increase their amount in the blood in a short time due to their properties. In this case, the result can increase the workload of the kidneys. Based on the information, this study investigated the effects of resistance exercise and BCAA supplements on kidney tissue. A total of 24 Wistar Albino male rats were divided equally into 4 groups: Control, BCAA, Exercise and Exercise+BCAA. In six-week study, resistance swimming exercise was applied to exercise groups. BCAA groups were given BCAA supplements at doses of 2.5 mg/kg before exercise. End of the study, histological, immunochemical, and RT-PCR analyses were performed. As a result of the findings obtained, it was found that use of BCAA supplements together with exercise caused tubular necrosis (p=0.002). A significant increase was found in caspase 3 IHC staining findings in BCAA and Exercise+BCAA groups compared to Control group (p=0.011; p=0.02). Also, KIM-1 expression levels were higher in Exercise group compared to all other groups (p=0.004; p=0.003; p=0.008). As a result, BCAA consumption in combination with resistant exercise caused damage to kidney tissue. Molecular Biology Resistance Exercise Kidney Damage Caspase 3 Branched Chain Amino Acids KIM-1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Highlights *Use of BCAA supplements together with exercise caused tubular necrosis. *Exercise and BCAAs were determined to have an antagonistic effect on the mechanism of apoptosis. *KIM-1 expression levels were higher in Exercise group. 1. INTRODUCTION Exercise is defined as the whole of regular, planned, rhythmic and purposeful movements [1]. Exercise can be used for therapeutic purposes and as a preventive against diseases. In addition, exercise can be used to lose weight and achieve aesthetic appearance [2-4]. To achieve these goals, people often prefer for programs that include heavy exercise and diet. In addition, some people use dietary supplements to achieve their goals in less time. Some of the commonly used nutritional supplements have been noted to be protein powder, L-carnitine, whey protein, creatine, and branched chain amino acid (BCAA) supplements [5]. BCAAs composed of leucine, isoleucine, and valine amino acids have anabolic and anti-katabolic effects [6-8]. Therefore, they are often used as supplements in exercise which aimed at bodybuilding or increasing muscle strength [9]. In contrast other amino acids, BCAAs are not metabolized in the liver and are entered directly into the circulation. Therefore, when they are consumed, their amount in the blood increases rapidly [6, 10]. Nephrons, the smallest functional unit of the kidney, are structures that filter the blood and try to retrieve all the useful substances in it. Amino acids, which are main structure matter of the body, are too important molecules to be excreted in the urine that it wants to reclaim all the nephrons. For this reason, amino acids taken into the body with BCAA supplementation and quickly passed into the blood are likely to increase infiltration and reabsorption workloads of nephrons. The main cause of kidney disease pathophysiology is cell death. Because of kidney damage, cell death can occur in different parts of the kidney, and these deaths can be of different types in cells. The processes mediated by cell death occur through apoptosis, necrosis, and autophagy. When the relationship between kidneys and apoptosis is examined, it is stated that although it is common in the developmental stage, it is almost never seen in mature kidneys [11]. Thus, apoptosis is usually a good biomarker for kidney damage in adult kidneys. Another molecule that can be used as a biomarker for kidney damage is glycoprotein-structured Kidney Injury Molecule-1 (KIM-1). KIM-1 molecule, which is normally expressed at low levels in adult individuals, is localized in the proximal tubules. Expression levels increase in possible proximal tubular damages. Because of this property, it can be used as a specific biomarker for proximal tubular damage [12,13]. Based on this information, this experimental study was conducted to investigate the possible effects of resistant exercise and BCAA supplements on kidney tissues in terms of apoptosis and KIM-1. 2. MATERIALS AND METHODS 2.1. Animals Twenty-four male Wistar albino rats (230-250 g), 2 months old, were used in the study. During the experiment, animals were housed in an animal room maintained at a temperature of 22 °C, in 12-hours light periods, and were fed ad-libitum . The animals were fed with commercial diet and allowed to drink water ad libitum. Standard commercial pellet feed was used as feed for the feeding of rats, and normal mains water was used as drinking water. The research was carried out within the organization with animals provided from Çanakkale Onsekiz March University Experimental Research Application and Research Center (ÇOMÜDAM). All animal procedures were approved by the Institutional Animal Care and Use Committee of Çanakkale Onsekiz Mart University (Approved number: 2019/09-02). 2.2. Experimental Design Animals were randomly divided into 4 groups, provided that there were 6 animals in each group: Group I (Control): No application was made to the animals (n=6). Group II (BCAA): The animals were given BCAA supplements every day for 6 weeks (n=6). Group III (Exercise): The animals were underwent resistant swimming exercises 5 days a week for 6 weeks (n=6). Group IV (Exercise+BCAA): The animals were given BCAA supplements every day for 6 weeks and underwent resistant swimming exercises 5 days a week (n=6). 2.3. Preparation and Application of BCAA Supplements BCAA supplementation was prepared by mixing L-leucine, L-isoleucine, and L-valine (Sigma-Aldrich) in a ratio of 2:1:1. No fats or carbohydrates were added to the supplement. Based on research by Lu et al. (2015), prepared supplement was given intragastric by oral gavage at a dose of 2.5 mg/kg, just before exercise every day during the study period [14]. 2.4. Resistant Swimming Exercise Protocol Considering the stress caused by exercises such as stair climbing or treadmills used in experimental studies, swimming exercise was preferred in our study. In this way, animals were prevented from avoiding exercise during exercise and punishment methods that had to be applied were avoided. With weights attached to the tails of the animals, swimming exercises have been made resistant. A swimming model developed by Viera (1988) was used in the swimming exercise (Table 1) [15]. Tail weights for each animal in Exercise and Exercise+BCAA groups were enumerated and stored separately for each rat. The swimming pool is 67.5 cm wide, 94 cm long and 64 cm high, made of rectangular PPC material. The approximate water height was set at 50±2 cm. The water temperature was kept between 32±2 o C. Table 1. Exercise session duration and used lifts [16]. Training Week Session Duration Used Weight 1. Week 20 Minute no weight 2. Week 25 Minute 5% of body weight 3. Week 30 Minute 5% of body weight 4. Week 40 Minute 5% of body weight 5. Week 50 Minute 5% of body weight 6. Week 60 Minute 5% of body weight 2.5. Sample Collection At the end of the study, which lasted a total of 6 weeks, the animals were anesthetized with Xylazine (60 mg/kg) and Ketamine (5 mg/kg) by intramuscular injection. Their lives were ended with cervical dislocation in accordance with ethical rules. Then the kidney tissues were separated and divided into two parts. One parts of kidney tissue were fixed in 10% neutral buffered formaldehyde for histological and immunohistochemical examinations. The other parts immediately placed cryovials and stored −80°C deep freeze for gene expression analysis. 2.6. Histopathological and Immunohistochemical Analysis The kidney tissues taken were embedded in paraffin blocks after routine follow-up. 4 µm thick sections were taken from the prepared tissue blocks. The sections taken were stained with Hematoxylin&Eosin (H&E). In addition, the positively charged slides were taken at 4 µm thickness and immunohistochemically (IHC) stained with Caspase 3 (CPP32) Ab-4 primary antibody (1/200 dilution, Catalogue no: #PA5-77887, Thermo Scientific). Histopathological assessments were performed with camera-attached microscope (Cx43, Olympus, Japan). Damage was assessed using tubular necrosis and vacuolization parameters of H&E staining sections. In H&E staining, a cross-section taken from each animal was graded according to the criteria of tubular necrosis and vacuolization [17]. H&E results were graded, as previously described in the literature, as follows: 0 = No damage 1 = Mild damage 2 = Moderate damage 3 = Severe damage [17,18]. According to their immune positivity in the IHC evaluation, the sections were evaluated as follows: 0= No staining, 1= Mild 2= Moderate 3= Severe [18]. 2.7. Gene Expression Analysis RNA was isolated from the kidney tissues obtained by taking 25-30 mg and using PURE Link RNA Mini Kit (CatNo.121B301BA). Purity and concentration measurements were made using NanoDrop ND-1000 in the obtained RNA samples. As for the purity rate, values between 1.8-2.1 were accepted from the RNAs measured at 260/280 nm. cDNA synthesis was performed from the respective RNAs using the cDNA kit (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems ™). Quantitive Real-Time PCR (StepOnePlus ™ Real-Time PCR System) was applied using the obtained cDNA samples. TaqMan (RealQ Plus 2x Master Mix, Ampliqon, Denmark) was used for analysis of gene expression levels. The PCR condition was 10 minutes at 25 ° C, 120 minutes at 37 ° C, 5 minutes at 85 ° C and 4 ° C infinite [19]. Caspase-3 (Gen-Bank NC_005115.4 and Rn 00563902), Bcl-2 (Gen-Bank NC_005112.4 and Rn99999125_m1) and KIM-1/HAVCR1 (Gen-Bank NC_000005.10 and Rn00597703-mL) genetic codes were evaluated by RT-PCR methods. Results were normalized using the beta-actin (Gen-Bank NC_005111.4 and Rn00667869_m1) housekeeping gene and the 2 −(ΔΔCt) formula. 2.8. Statistical Analysis Statistical evaluation of the data was performed using IBM SPSS Statistics SPSS 18 (SPSS 2009) package program. The results were presented as mean and standard deviation (mean±SD). The one-way analysis of variance (ANOVA) and Mann Whitney U tests were used to compare the groups while results of p<0.05 were considered as significant. 3. RESULTS 3.1. Histopathological Evaluation H&E staining tubular necrosis and vacuolization parameters evaluation results are presented in Table 2. Although minimal tubular necrosis and/or vacuolization were observed in some cases in BCAA group, these changes were not statistically significant (Figure 1B, in order of p=0.07 and p=0.026). As a result of the evaluations, both tubular necrosis and vacuolization were observed in Exercise group and Exercise+BCAA group. As a result of tubular necrosis assessment, a statistically significant increase was found in Exercise group compared to Control group (p=0.007). Also, according to tubular necrosis evaluation results, a significant increase was found in Exercise+BCAA group compared to Control group. (p=0.002). In addition, the increase in Exercise+BCAA group was found to be statistically significant compared to BCAA group (p= 0.007). Table 2. H&E Staining Assessment (*p <0.05 compared to Control group, # p <0.05 compared to BCAA group) Groups (Mean±SD) Histological findings Control BCAA Exercise Exercise+BCAA Tubular Necrosis 0.00±0,00 0.33±0.51 1.17±0.75* 1.67±0.51* , # Vacuolization 0.17±0,40 0.33±0.51 1.00±0.63* 0.83±0.75 3.2. Immunohistochemical Evaluation As a result of the immunohistochemical staining using the rat anti-Caspase 3 primary antibody, there was no difference in the findings of Control and Exercise groups (Figure 3A, C). Caspase 3 concentrations increased in BCAA and Exercise + BCAA groups (Figure 3B, D). According to the evaluation results, a statistically significant increase was found in BCAA group compared to Control group (p=0.011). The increase in the BCAA group was also statistically significant compared to Exercise group (p=0.046). Significant increases were observed in Exercise+BCAA group compared to the control group (p=0.02). 3.3. Gene Expression Levels As a result of the RT-PCR evaluation, a statistically significant decrease was observed in caspase 3 gene expression rates in BCAA and Exercise+BCAA groups compared to Control group (p=0.01). It was also found that Exercise+BCAA group decreased significantly compared to the Exercise group (p=0.036). Another genetic marker in terms of apoptosis assessment, Bcl-2 expression assessment, was not statistically significant between the groups (p>0.05). When the findings of KIM-1's genetic expression rates were examined, statistically significant differences were found between the groups. KIM-1 expression was statistically significantly higher in Exercise group than in Control, BCAA and Exercise+BCAA groups (in order of p=0.004; p=0.003; p=0.008). A statistically significant increase was noted in Exercise+BCAA group compared to BCAA group (p=0.043) (Figure 4). 4. DISCUSSION Exercise physiology is examined; blood flow to the kidneys decreases during exercise, and reperfusion is formed by increasing at the end of exercise. With this mechanism, high frequency and intensity exercise can damage the kidneys [20]. Moreover, it is reported that this situation causes a significant change in urinary markers indicating tubular kidney damage [21]. It is known that high intensity and resistance exercise increases creatinine and some metabolites by causing muscle damage [22-24]. Therefore, it is an expected effect to increase kidney workload. In our study, tubular necrosis parameter was used to evaluate the increase in renal workload and metabolites. Accordingly, we think that the statistically significant increase in Exercise and Exercise+BCAA groups determined in tubular necrosis findings compared to Control group was related to the increased renal workload. In addition, a significant increase was found in Exercise+BCAA group compared to BCAA group. We think that the reason for this increase is the use of supplements just before exercise. The addition of exogenous BCAA use to the increased workload of the kidney can be expected to cause an increase in glomerular filtration and reabsorption workload. Vacuolization is a morphological change in cells, also it is a component of autophagy [25]. Autophagy is an intracellular recycling system that plays a role in clearing damaged cell components. Exercise is also described as one of the stimuli that induces autophagy. The reason why exercise induces autophagy is to limit tissue damage during exercise, maintain tissue continuity, and limit inflammatory responses [26]. As a result, vacuolization can reduce cell activity in kidney cells, as well as lead to cell death because of autophagy. In addition, it is known that there are some changes in the fluid-electrolyte balance after heavy exercise [27]. Among these changes, especially a serious decrease in potassium has been revealed by studies. Although the mechanism of hypokalaemia that occurs after potassium loss leads to kidney disease is not clear, it is histologically associated with vacuolization [28, 29]. In our study, a statistically significant increase was found in Exercise group compared to Control group in the assessment for vacuolization. We think that the significant increase in vacuolization observed in this group was caused by potassium loss after intensity exercise. Caspase 3 is one of the primary mediators of apoptosis. It can be activated both extrinsic and intrinsic pathways [30]. For this reason, caspase 3 was evaluated by both IHC and RT-PCR methods in our study. When the results of the two methods are evaluated together, the two results in these parameters attract attention. The first point is that the number of positively stained apoptotic cells in BCAA supplement groups was higher than in the other groups. In addition, caspase-3 expression rates were decreased within these groups. The PCR method shows the active gene regions, while the IHC method shows the amount of protein in cells regardless of the relevant gene region. If there is enough protein in the cell, the gene regions close by becoming inactive. Therefore, PCR results showed that apoptosis was not inhibited, leading to the deactivation of high levels of caspase-3 in gene regions. Furthermore, caspase 3 is a substance released as an inactive precursor in the form of procaspase 3. Activated by active caspase 8 or 9 in the environment, it becomes caspase 3 [31,32]. As determined by IHC, it is caspase 3 in the environment, but procaspase 3, whose synthesis begins with gene expression. When these findings are evaluated together, it is thought that BCAAs activate the procaspase 3 present in the cell and show the effect of directing the cells to apoptosis. Similarly, Viana et al. in their study, it was stated that in rats fed a rich leucine diet and formed tumours, the increased amount of leucine also increased oxidative stress [33]. It suggests that leucine found in BCAA supplements similarly activates procaspase 3’s by increasing oxidative stress. Another possibility for procaspase 3 activation may be the absence of other amino acids other than BCAA. Damaged cells cannot repair themselves due to insufficient amino acids and therefore go to apoptosis by caspase 3 activation. In short, BCAAs can be the cause of apoptosis as secondary, even if they are not the primary cause. Another point is that although active caspase-3 was not observed in Exercise group, Caspase-3 expression was increased compared to Exercise+BCAA group. This finding suggests that exercise and BCAAs have antagonistic effects on the mechanism of apoptosis. In other words, it can be said that exercise does not directly cause apoptosis but prepares the environment for other stressors such as low glucose levels, oxygen deficiency and increased ADP levels. According to these findings, the use of BCAA in combination with high intensity resistance exercise can cause serious kidney damage in people with a predisposition. Another molecule involved in apoptosis is Bcl-2, which is involved in the intrinsic pathway. Bcl-2, a pro-apoptotic protein, is often examined in apoptosis [34]. Our findings concluded that BCAAs showed no activity in apoptosis mechanisms over Bcl-2 expression. Research involving other molecules is also needed to further examine the effectiveness of these results in relation to this mechanism. KIM-1, which is known to increase kidney damage, can be used as a sensitive marker for the identification of acute tubular damage, especially in proximal tubules [35]. In the results of KIM-1 expression, a statistically significant increase was observed in Exercise group compared to all other groups, and in Exercise+BCAA group compared to only BCAA group. When KIM-1 expression findings and histopathological findings are evaluated together, they show that there is a connection between proximal tubular damage and exercise. It suggests that the damage caused by the consumption of BCAAs with exercise may be in other parts of the kidney, such as the bowman's capsule and glomerular membrane. As a result, the use of BCAA supplements before resistant exercise causes some damage to the kidneys. From a molecular point of view, exercise provides the cellular basis for apoptosis, while BCAAs affect caspase-3 activation, leading to kidney damage. Therefore, it is suggested that it should be investigated whether the use of BCAA supplements causes possible kidney damage in individuals if they are used at rest, not before exercise. Our study is the first study investigating the link between BCAA consumption in kidney tissues in resistant exercises and demonstrating that KIM-1 expression levels increase with exercise as a result of the parameters and findings examined. As a result of these data, we believe that more experimental and clinical studies are needed to understand the mechanisms, clarify, and confirm our evidence. Limitations: Limitations of this study include the experimental design and the lack of some parameters such as creatinine levels, Bax expression levels, glomerular filtration rates and serum potassium levels. On the other hand, both histopathological and genetic evaluations constitute an important strength of our study. Based on this study, extensive studies are needed regarding the effects of exercise and BCAA supplements at different doses and at different times. Abbreviations BCAA, Branched chain amino acid; KIM-1, Kidney injury molecule 1; RT-PCR, Real time polymerase chain reaction; H&E, Hematoxylin&Eosin; IHC, Immunohistochemical; Bcl-2, B-cell lymphoma 2. Declarations Financial Source: This work was supported by Canakkale Onsekiz Mart University The Scientific Research Coordination Unit, Project number: TYL-2019-3141. Conflict of Interest The authors have no financial disclosure or conflict of interest in this study. Availability of data and material: All data and materials of the study are available in contact with the corresponsible author. Author contributions: All authors contributed equally to all parts of the study. Ethics approval: All animal procedures were approved by the Institutional Animal Care and Use Committee of Çanakkale Onsekiz Mart University (Approved number: 2019/09-02) References Booth FW, Roberts CK, Laye MJ. Lack of Exercise is a Major Cause of Chronic Diseases. Comprehensive Physiology. 2012; 2(2): 1143-1211. https://doi.org/10.1002/cphy.c110025. Ardıç F. Egzersiz Reçetesi. Türk Fizyoterapi ve Rehabilitasyon Dergisi. 2014; 60: 1-8. https://doi.org/10.5152/tftrd.2014.25665. Daenen L, Varkey E, Kellmann M, Nijs J. Exercise, Not to Exercise, or How to Exercise in Patients with Chronic Pain? Applying Science to Practice. The Clinical Journal of Pain. 2015; 31(2): 108-114. https://doi.org/10.1097/AJP.0000000000000099. Idorn M, thorStraten P. Exercise and Cancer: From “Healthy” to “Therapeutic”?. Cancer Immunology, Immunotherapy. 2017; 66(5): 667-671. https://doi.org/10.1007/s00262-017-1985-z. Kerksick CM, Wilborn CD, Roberts MD, Smith-Ryan A, Kleiner SM, Jäger R, Greenwood M et al . ISSN exercise & sports nutrition review update: research & recommendations. Journal of the International Society of Sports Nutrition. 2018; 15(1), 38. https://doi.org/10.1186/s12970-018-0242-y. Bifari F, Nisoli E. Branched Chain Amino Acids Differently Modulate Catabolic and Anabolic States in Mammals: A Pharmacological Point of View. British Journal of Pharmacology. 2017; 174(11): 1366-1377. DOI: 10.1111/bph.13624. Goron A, Moinard C. Amino Acids and Sport: A True Love Story?. Amino Acids. 2018; 50(8): 969-980. https://doi.org/10.1007/s00726-018-2591-x. Pitkänen HT, Oja SS, Rusko H, Nummela A, Komi PV, Saransaari P, et al. Leucine Supplementation Does Not Enhance Acute Strength or Running Performance But Affects Serum Amino Acid Concentration. Amino Acids. 2003; 25(1): 85-94. https://doi.org/10.1007/s00726-002-0343-3. Wolfe RR. Branched-Chain Amino Acids and Muscle Protein Synthesis in Humans: Myth or Reality?. Journal of the International Society of Sports Nutrition. 2017; 14(1): 1-7. https://doi.org/10.1186/s12970-017-0184. Holeček M. Branched-Chain Amino Acids in Health and Disease: Metabolism, Alterations in Blood Plasma, and As Supplements. Nutrition&Metabolism. 2018; 15(1): 33. https://doi.org/10.1186/s12986-018-0271-1. Priante G, Gianesello L, Ceol M, Del Prete D, Anglani F. Cell Death in The Kidney. International Journal of Molecular Sciences. 2019; 20(14): 3598. https://doi.org/10.3390/ijms20143598. Ichimura T, Bonventre JV, Bailly V, Wei H, Hession CA, Cate RL, et al. Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. Journal of Biological Chemistry. 1988; 273(7): 4135-4142. https://doi.org/10.1074/jbc.273.7.4135. van Timmeren MM, van den Heuvel MC, Bailly V, Bakker SJ, van Goor H, Stegeman CA. Tubular kidney injury molecule‐1 (KIM‐1) in human renal disease. The Journal of pathology. 2007; 212(2): 209-217. https://doi.org/10.1002/path.2175. Lu M, Zhang X, Zheng D, Jiang X, Chen Q. Branched‐Chain Amino Acids Supplementation Protects Streptozotocin‐Induced Insulin Secretion and The Correlated Mechanism. BioFactors. 2015; 41(2): 127-133. https://doi.org/10.1002/biof.1188. Vieira R, Haebisch H, Kokubun E, Hell NS, Curi R. SistemaDe Natação Para Exercício Físico De Ratos. Swimming System for Physical Exercise of Rats. Arquivos De Biologia E Tecnologia. 1988; 31(3): 387-394. Campos-Ferraz PL, Bozza T, Nicastro H, Lancha JrA H. Distinct effects of leucine or a mixture of the branched-chain amino acids (leucine, isoleucine, and valine) supplementation on resistance to fatigue, and muscle and liver-glycogen degradation, in trained rats. Nutrition. 2013; 29(11-12), 1388-1394. https://doi.org/10.1016/j.nut.2013.05.003 Büyük B, Demirci T, Adalı Y, Eroğlu HA. ¿ Puede el líquido amniótico ser una solución alternativa de conservación de órganos para el almacenamiento renal en frío?. Revista de Nefrología, Diálisis y Trasplante. 2020; 40(1), 14-24. Kandemir FM, Yildirim S, Kucukler S, Caglayan C, Mahamadu A, et al . Therapeutic Efficacy of Zingerone Against Vancomycin-Induced Oxidative Stress, Inflammation, Apoptosis and Aquaporin 1 Permeability in Rat Kidney. Biomedicine & Pharmacotherapy. 2018; 105: 981-991. https://doi.org/10.1016/j.biopha.2018.06.048. Doğanlar ZB, Uzun M, Ovali MA, Dogan A, Ongoren G, Doğanlar O. Melatonin attenuates caspase-dependent apoptosis in the thoracic aorta by regulating element balance and oxidative stress in pinealectomised rats. Applied Physiology, Nutrition, and Metabolism. 2019; 44(2), 153-163. https://doi.org/10.1139/apnm-2018-0205. Lin X, Jiang C, Luo Z, Qu S. Protective Effect of Erythropoietin on Renal Injury Induced in Rats by Four Weeks of Exhaustive Exercise. BMC Nephrology. 2013; 14(1): 130. https://doi.org/10.1186/1471-2369-14-130. Spada TC, Silva JM, Francisco LS, Marcal LJ, Antonangelo L, Zanetta DM, et al . High Intensity Resistance Training Causes Muscle Damage and Increases Biomarkers of Acute Kidney Injury in Healthy Individuals. PloS One. 2018; 13(11). https://doi.org/10.1371/journal.pone.0205791. Baird MF, Graham SM, Baker JS, Bickerstaff GF. Creatine Kinase and Exercise-Related Muscle Damage Implications for Muscle Performance and Recovery. Journal of Nutrition and Metabolism. 2012. https://doi.org/10.1155/2012/960363. Sorichter S, Mair J, Koller A, Calzolari C, Huonker M, Pau B , et al . Release of Muscle Proteins After Downhill Running in Male and Female Subjects. Scandinavian Journal of Medicine&Science in Sports. 2011; 11(1): 28-32. https://doi.org/10.1034/j.1600-0838.2001.011001028.x. Tsatalas, T, Giakas G, Spyropoulos G, Sideris V, Lazaridis S, Kotzamanidis C, Koutedakis Y. The Effects of Eccentric Exercise-Induced Muscle Damage on Running Kinematics at Different Speeds. Journal of Sports Sciences. 2013; 31(3): 288-298. https://doi.org/10.1080/02640414.2012.729135. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell death & differentiation. 2009; 16(1): 3-11. https://doi.org/10.1038/cdd.2008.150. Mooren FC, Krüger K. Exercise, autophagy, and apoptosis. In Progress in molecular biology and translational science. Academic Press 2015; Vol. 135, pp. 407-422. https://doi.org/10.1016/bs.pmbts.2015.07.023. Gladden LB. Lactate Metabolism: A New Paradigm for The Third Millennium. The Journal of Physiology. 2004; 558(1): 5-30. https://doi.org/10.1113/jphysiol.2003.058701. Lindinger MI. Potassium Regulation During Exercise and Recovery in Humans: Implications for Skeletal and Cardiac Muscle. Journal of Molecular and Cellular Cardiology. 1995; 27(4): 1011-1022. https://doi.org/10.1016/0022-2828(95)90070-5. Thomson MN, Schneider W, Mutig K, Ellison DH, Kettritz R, Bachmann S. Patients with Hypokalemia Develop WNK Bodies in The Distal Convoluted Tubule of The Kidney. American Journal of Physiology-Renal Physiology. 2019; 316(2): F292-F300. https://doi.org/10.1152/ajprenal.00464.2018. Jiao C, Chen W, Tan X, Liang H, Li J, Yun H, et al. (2020). Ganoderma lucidum spore oil induces apoptosis of breast cancer cells in vitro and in vivo by activating caspase-3 and caspase-9. Journal of ethnopharmacology. 2020; 247, 112256. https://doi.org/10.1016/j.jep.2019.112256. Bildik A, Bayar İ. Kanserde Apoptotik Yolakların İnhibisyonu. Turkiye Klinikleri Journal of Veterinary Sciences. 2018; 9(2): 42-51. https://doi.org/10.5336/vetsci.2018-62141. Goldar S, Khaniani MS, Derakhshan SM, Baradaran B. Molecular Mechanisms of Apoptosis and Roles in Cancer Development and Treatment. Asian Pacific Journal of Cancer Prevention: APJCP. 2015; 16(6): 2129-2144. https://doi.org/10.7314/apjcp.2015.16.6.2129. Viana LR, Tobar N, Busanello ENB, Marques AC, de Oliveira AG, Lima TI, et al. Leucine-Rich Diet Induces a Shift in Tumour Metabolism from Glycolytic Towards Oxidative Phosphorylation, Reducing Glucose Consumption and Metastasis in Walker-256 Tumour-Bearing Rats. Scientific Reports. 2019; 9(1): 1-11. https://doi.org/10.1038/s41598-019-52112-w. Rautureau GJ, Day CL, Hinds MG. Intrinsically disordered proteins in bcl-2 regulated apoptosis. International journal of molecular sciences. 2010; 11(4), 1808-1824. https://doi.org/10.3390/ijms11041808. Yin W, Kumar T, Lai Z, Zeng X, Kanaan HD, Li W, et al. Kidney Injury Molecule-1, A Sensitive and Specific Marker for Identifying Acute Proximal Tubular Injury, Can Be Used to Predict Renal Functional Recovery in Native Renal Biopsies. International Urology and Nephrology. 2019; 51(12): 2255-2265. https://doi.org/10.1007/s11255-019-02311-1. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-326595","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":17212647,"identity":"66ff0bfc-dbbd-41b2-ba11-a4c8699e9b7c","order_by":0,"name":"Cemre AYDEĞER","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIie3PuwrCMBSA4SOBuES7BlR8hUjBUfFRQsEpdVYcFBwcFGf7FoVAZ4tDl3SvdPEyF+rm4GC94Nh2FMy/HALngxMAne6nC9avQerliVIAu4zg8iQSLwKFxFiuhmkKPb45JP7lKnpNDOh0jnIIVaF0tmBxJx5ZzPes7DBsmiKHMGpLRABxNxZd6nsoIwQ3ckk7kegOM+4e1JPMShBakwhgz92IPMm+mFBly8qaBaajhMlCLyAYFfzFWIYSbuNpaxOoznHiTftGdXG65JHPeTCYfx+ocP1dv+SeTqfT/WMP+lhIddUoWwYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-1654-6406","institution":"Çanakkale Onsekiz Mart Üniversites - Terzioğlu Kampüsü: Canakkale Onsekiz Mart Universitesi","correspondingAuthor":true,"prefix":"","firstName":"Cemre","middleName":"","lastName":"AYDEĞER","suffix":""},{"id":17212648,"identity":"148bb87e-f557-4290-9c6c-f3db13af277b","order_by":1,"name":"Hüseyin Avni EROĞLU","email":"","orcid":"https://orcid.org/0000-0002-1040-3255","institution":"Çanakkale Onsekiz Mart University Faculty of Medicine: Canakkale Onsekiz Mart Universitesi Tip Fakultesi","correspondingAuthor":false,"prefix":"","firstName":"Hüseyin","middleName":"Avni","lastName":"EROĞLU","suffix":""}],"badges":[],"createdAt":"2021-03-13 20:50:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-326595/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-326595/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":7242073,"identity":"bf96b10e-6ef2-4889-9ff8-cbac37dae9ce","added_by":"auto","created_at":"2021-03-22 20:54:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1294717,"visible":true,"origin":"","legend":"Kidney tissues sections of groups (A) Control (B) BCAA (C) Exercise (D) Exercise+BCAA (H\u0026E, 200x). Vacuolization (red arrows) and tubular necrosis (blue arrows) areas were shown.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-326595/v1/b623a004347c24ff86bd831f.png"},{"id":7242484,"identity":"de08a3d0-c02b-4d6a-b4e5-96848fa2b616","added_by":"auto","created_at":"2021-03-22 20:57:33","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":15359,"visible":true,"origin":"","legend":"Caspase 3 immunohistochemical staining assessment (*p \u003c0.05 compared to Control group, + p \u003c0.05 compared to Exercise group).","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-326595/v1/e2db5177d7344ea075b79738.png"},{"id":7241513,"identity":"d8e6c6a4-10fa-4b33-8d4f-8f646e53881f","added_by":"auto","created_at":"2021-03-22 20:51:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1365030,"visible":true,"origin":"","legend":"Kidney tissue sections stained using anti-Caspase-3 primary antibody by IHC method (A) Control (B) BCAA (C) Exercise (D) Exercise+BCAA (200x). The cells indicated by the blue arrow are apoptotic cells that are positively stained, and the cells shown by the green arrow are normal cells that are negatively stained.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-326595/v1/56d37ff5a72d230dffe654ee.png"},{"id":7242071,"identity":"4221153b-ef46-401d-84c1-4fd809034a8d","added_by":"auto","created_at":"2021-03-22 20:54:33","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":16547,"visible":true,"origin":"","legend":"Between group changes in the gene expression levels of Caspase 3 (A), Bcl-2 (B), KIM-1 (C). The data were presented as 2−(ΔΔCt) relative expression after the mRNA levels were normalized with β-actin. All results are presented as mean ± SD for six rats in each group. * compared to Control group, # compared to BCAA group, \u0026 compared to Exercise+BCAA group (p\u003c0.05).","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-326595/v1/bc523094c9d6b38d44cb12a3.png"},{"id":13682069,"identity":"bf7ad2a9-84e4-4aa5-8b9b-5e67d474ca7a","added_by":"auto","created_at":"2021-09-17 11:55:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2761275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-326595/v1/d2366277-1127-4bd7-9201-48a0a5180df7.pdf"}],"financialInterests":"","formattedTitle":"The Effects of Branched Chain Amino Acid Supplement on Kidney Tissue of Exercising Rats","fulltext":[{"header":"Highlights","content":"\u003cp\u003e*Use of BCAA supplements together with exercise caused tubular necrosis.\u003c/p\u003e\n\u003cp\u003e*Exercise and BCAAs were determined to have an antagonistic effect on the mechanism of apoptosis.\u003c/p\u003e\n\u003cp\u003e*KIM-1 expression levels were higher in Exercise group.\u003c/p\u003e"},{"header":"1. INTRODUCTION","content":"\u003cp\u003eExercise is defined as the whole of regular, planned, rhythmic and purposeful movements [1]. Exercise can be used for therapeutic purposes and as a preventive against diseases. In addition, exercise can be used to lose weight and achieve aesthetic appearance [2-4]. To achieve these goals, people often prefer for programs that include heavy exercise and diet. In addition, some people use dietary supplements to achieve their goals in less time. Some of the commonly used nutritional supplements have been noted to be protein powder, L-carnitine, whey protein, creatine, and branched chain amino acid (BCAA) supplements [5].\u003c/p\u003e\n\u003cp\u003eBCAAs composed of leucine, isoleucine, and valine amino acids have anabolic and anti-katabolic effects [6-8]. Therefore, they are often used as supplements in exercise which aimed at bodybuilding or increasing muscle strength [9]. In contrast other amino acids, BCAAs are not metabolized in the liver and are entered directly into the circulation. Therefore, when they are consumed, their amount in the blood increases rapidly [6, 10]. Nephrons, the smallest functional unit of the kidney, are structures that filter the blood and try to retrieve all the useful substances in it. Amino acids, which are main structure matter of the body, are too important molecules to be excreted in the urine that it wants to reclaim all the nephrons. For this reason, amino acids taken into the body with BCAA supplementation and quickly passed into the blood are likely to increase infiltration and reabsorption workloads of nephrons.\u003c/p\u003e\n\u003cp\u003eThe main cause of kidney disease pathophysiology is cell death. Because of kidney damage, cell death can occur in different parts of the kidney, and these deaths can be of different types in cells. The processes mediated by cell death occur through apoptosis, necrosis, and autophagy. When the relationship between kidneys and apoptosis is examined, it is stated that although it is common in the developmental stage, it is almost never seen in mature kidneys [11]. Thus, apoptosis is usually a good biomarker for kidney damage in adult kidneys.\u003c/p\u003e\n\u003cp\u003eAnother molecule that can be used as a biomarker for kidney damage is glycoprotein-structured Kidney Injury Molecule-1 (KIM-1). KIM-1 molecule, which is normally expressed at low levels in adult individuals, is localized in the proximal tubules. Expression levels increase in possible proximal tubular damages. Because of this property, it can be used as a specific biomarker for proximal tubular damage [12,13].\u003c/p\u003e\n\u003cp\u003eBased on this information, this experimental study was conducted to investigate the possible effects of resistant exercise and BCAA supplements on kidney tissues in terms of apoptosis and KIM-1.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003e2.1. Animals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTwenty-four male Wistar albino rats (230-250 g), 2 months old, were used in the study. During the experiment, animals were housed in an animal room maintained at a temperature of 22 \u0026deg;C, in 12-hours light periods, and were fed \u003cem\u003ead-libitum\u003c/em\u003e. The animals were fed with commercial diet and allowed to drink water ad libitum. Standard commercial pellet feed was used as feed for the feeding of rats, and normal mains water was used as drinking water.\u003c/p\u003e\n\u003cp\u003eThe research was carried out within the organization with animals provided from \u0026Ccedil;anakkale Onsekiz March University Experimental Research Application and Research Center (\u0026Ccedil;OM\u0026Uuml;DAM). All animal procedures were approved by the Institutional Animal Care and Use Committee of \u0026Ccedil;anakkale Onsekiz Mart University (Approved number: 2019/09-02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Experimental Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were randomly divided into 4 groups, provided that there were 6 animals in each group:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup I (Control):\u003c/strong\u003e No application was made to the animals (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup II (BCAA): \u003c/strong\u003eThe animals were given BCAA supplements every day for 6 weeks (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup III (Exercise):\u003c/strong\u003e The animals were underwent resistant swimming exercises 5 days a week for 6 weeks (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGroup IV (Exercise+BCAA): \u003c/strong\u003eThe animals were given BCAA supplements every day for 6 weeks and underwent resistant swimming exercises 5 days a week (n=6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Preparation and Application of BCAA Supplements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBCAA supplementation was prepared by mixing L-leucine, L-isoleucine, and L-valine (Sigma-Aldrich) in a ratio of 2:1:1. No fats or carbohydrates were added to the supplement. Based on research by Lu et al. (2015), prepared supplement was given intragastric by oral gavage at a dose of 2.5 mg/kg, just before exercise every day during the study period [14].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Resistant Swimming Exercise Protocol\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsidering the stress caused by exercises such as stair climbing or treadmills used in experimental studies, swimming exercise was preferred in our study. In this way, animals were prevented from avoiding exercise during exercise and punishment methods that had to be applied were avoided. With weights attached to the tails of the animals, swimming exercises have been made resistant. A swimming model developed by Viera (1988) was used in the swimming exercise (Table 1) [15]. Tail weights for each animal in Exercise and Exercise+BCAA groups were enumerated and stored separately for each rat. The swimming pool is 67.5 cm wide, 94 cm long and 64 cm high, made of rectangular PPC material. The approximate water height was set at 50\u0026plusmn;2 cm. The water temperature was kept between 32\u0026plusmn;2\u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1.\u003c/strong\u003e Exercise session duration and used lifts [16].\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003eTraining Week\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e\u003cstrong\u003eSession Duration\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e\u003cstrong\u003eUsed Weight\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003e1. \u003c/strong\u003e\u003cstrong\u003eWeek\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e20 Minute\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003eno weight\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003e2. \u003c/strong\u003e\u003cstrong\u003eWeek\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e25 Minute\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e5% of body weight\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003e3. \u003c/strong\u003e\u003cstrong\u003eWeek\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e30 Minute\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e5% of body weight\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003e4. \u003c/strong\u003e\u003cstrong\u003eWeek\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e40 Minute\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e5% of body weight\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003e5. \u003c/strong\u003e\u003cstrong\u003eWeek\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e50 Minute\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e5% of body weight\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"136\"\u003e\n\u003cp\u003e\u003cstrong\u003e6. \u003c/strong\u003e\u003cstrong\u003eWeek\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"140\"\u003e\n\u003cp\u003e60 Minute\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"156\"\u003e\n\u003cp\u003e5% of body weight\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Sample Collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the study, which lasted a total of 6 weeks, the animals were anesthetized with Xylazine (60 mg/kg) and Ketamine (5 mg/kg) by intramuscular injection. Their lives were ended with cervical dislocation in accordance with ethical rules. Then the kidney tissues were separated and divided into two parts. One parts of kidney tissue were fixed in 10% neutral buffered formaldehyde for histological and immunohistochemical examinations. The other parts immediately placed cryovials and stored \u0026minus;80\u0026deg;C deep freeze for gene expression analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Histopathological and Immunohistochemical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe kidney tissues taken were embedded in paraffin blocks after routine follow-up. 4 \u0026micro;m thick sections were taken from the prepared tissue blocks. The sections taken were stained with Hematoxylin\u0026amp;Eosin (H\u0026amp;E). In addition, the positively charged slides were taken at 4 \u0026micro;m thickness and immunohistochemically (IHC) stained with Caspase 3 (CPP32) Ab-4 primary antibody (1/200 dilution, Catalogue no: #PA5-77887, Thermo Scientific). Histopathological assessments were performed with camera-attached microscope (Cx43, Olympus, Japan). Damage was assessed using tubular necrosis and vacuolization parameters of H\u0026amp;E staining sections. In H\u0026amp;E staining, a cross-section taken from each animal was graded according to the criteria of tubular necrosis and vacuolization [17]. H\u0026amp;E results were graded, as previously described in the literature, as follows:\u003c/p\u003e\n\u003cp\u003e0 = No damage\u003c/p\u003e\n\u003cp\u003e1 = Mild damage\u003c/p\u003e\n\u003cp\u003e2 = Moderate damage\u003c/p\u003e\n\u003cp\u003e3 = Severe damage [17,18].\u003c/p\u003e\n\u003cp\u003eAccording to their immune positivity in the IHC evaluation, the sections were evaluated as follows:\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;0= No staining,\u003c/p\u003e\n\u003cp\u003e1= Mild\u003c/p\u003e\n\u003cp\u003e2= Moderate\u003c/p\u003e\n\u003cp\u003e3= Severe [18].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7. Gene Expression Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA was isolated from the kidney tissues obtained by taking 25-30 mg and using PURE Link RNA Mini Kit (CatNo.121B301BA). Purity and concentration measurements were made using NanoDrop ND-1000 in the obtained RNA samples. As for the purity rate, values between 1.8-2.1 were accepted from the RNAs measured at 260/280 nm.\u003c/p\u003e\n\u003cp\u003ecDNA synthesis was performed from the respective RNAs using the cDNA kit (High-Capacity cDNA Reverse Transcription Kit, Applied Biosystems \u0026trade;). Quantitive Real-Time PCR (StepOnePlus \u0026trade; Real-Time PCR System) was applied using the obtained cDNA samples. TaqMan (RealQ Plus 2x Master Mix, Ampliqon, Denmark) was used for analysis of gene expression levels. The PCR condition was 10 minutes at 25 \u0026deg; C, 120 minutes at 37 \u0026deg; C, 5 minutes at 85 \u0026deg; C and 4 \u0026deg; C infinite [19].\u003c/p\u003e\n\u003cp\u003eCaspase-3 (Gen-Bank NC_005115.4 and Rn 00563902), Bcl-2 (Gen-Bank NC_005112.4 and Rn99999125_m1) and KIM-1/HAVCR1 (Gen-Bank NC_000005.10 and Rn00597703-mL) genetic codes were evaluated by RT-PCR methods. Results were normalized using the beta-actin (Gen-Bank NC_005111.4 and Rn00667869_m1) housekeeping gene and the 2\u003csup\u003e\u0026minus;(\u0026Delta;\u0026Delta;Ct)\u003c/sup\u003e formula.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical evaluation of the data was performed using IBM SPSS Statistics SPSS 18 (SPSS 2009) package program. The results were presented as mean and standard deviation (mean\u0026plusmn;SD). The one-way analysis of variance (ANOVA) and Mann Whitney U tests were used to compare the groups while results of p\u0026lt;0.05 were considered as significant.\u003c/p\u003e"},{"header":"3. RESULTS","content":"\u003cp\u003e\u003cstrong\u003e3.1. Histopathological Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E staining tubular necrosis and vacuolization parameters evaluation results are presented in Table 2. Although minimal tubular necrosis and/or vacuolization were observed in some cases in BCAA group, these changes were not statistically significant (Figure 1B, in order of p=0.07 and p=0.026). As a result of the evaluations, both tubular necrosis and vacuolization were observed in Exercise group and Exercise+BCAA group. As a result of tubular necrosis assessment, a statistically significant increase was found in Exercise group compared to Control group (p=0.007). Also, according to tubular necrosis evaluation results, a significant increase was found in Exercise+BCAA group compared to Control group. (p=0.002). In addition, the increase in Exercise+BCAA group was found to be statistically significant compared to BCAA group (p= 0.007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2.\u003c/strong\u003e H\u0026amp;E Staining Assessment (*p \u0026lt;0.05 compared to Control group, \u003cstrong\u003e#\u003c/strong\u003ep \u0026lt;0.05 compared to BCAA group)\u003c/p\u003e\n\u003ctable border=\"1\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd width=\"168\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd colspan=\"4\" width=\"446\"\u003e\n\u003cp\u003e\u003cstrong\u003eGroups (Mean\u0026plusmn;SD)\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003e\u003cstrong\u003eHistological findings\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"106\"\u003e\n\u003cp\u003e\u003cstrong\u003eBCAA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eExercise\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e\u003cstrong\u003eExercise+BCAA\u003c/strong\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003eTubular Necrosis\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.00\u0026plusmn;0,00\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"106\"\u003e\n\u003cp\u003e0.33\u0026plusmn;0.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e1.17\u0026plusmn;0.75*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e1.67\u0026plusmn;0.51*\u003csup\u003e, #\u003c/sup\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd width=\"168\"\u003e\n\u003cp\u003eVacuolization\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"95\"\u003e\n\u003cp\u003e0.17\u0026plusmn;0,40\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"106\"\u003e\n\u003cp\u003e0.33\u0026plusmn;0.51\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e1.00\u0026plusmn;0.63*\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd width=\"123\"\u003e\n\u003cp\u003e0.83\u0026plusmn;0.75\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Immunohistochemical Evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;As a result of the immunohistochemical staining using the rat anti-Caspase 3 primary antibody, there was no difference in the findings of Control and Exercise groups (Figure 3A, C). Caspase 3 concentrations increased in BCAA and Exercise + BCAA groups (Figure 3B, D). According to the evaluation results, a statistically significant increase was found in BCAA group compared to Control group (p=0.011). The increase in the BCAA group was also statistically significant compared to Exercise group (p=0.046). Significant increases were observed in Exercise+BCAA group compared to the control group (p=0.02).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Gene Expression Levels\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs a result of the RT-PCR evaluation, a statistically significant decrease was observed in caspase 3 gene expression rates in BCAA and Exercise+BCAA groups compared to Control group (p=0.01). It was also found that Exercise+BCAA group decreased significantly compared to the Exercise group (p=0.036).\u003c/p\u003e\n\u003cp\u003eAnother genetic marker in terms of apoptosis assessment, Bcl-2 expression assessment, was not statistically significant between the groups (p\u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003eWhen the findings of KIM-1's genetic expression rates were examined, statistically significant differences were found between the groups. KIM-1 expression was statistically significantly higher in Exercise group than in Control, BCAA and Exercise+BCAA groups (in order of p=0.004; p=0.003; p=0.008). A statistically significant increase was noted in Exercise+BCAA group compared to BCAA group (p=0.043) (Figure 4).\u0026nbsp;\u003c/p\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eExercise physiology is examined; blood flow to the kidneys decreases during exercise, and reperfusion is formed by increasing at the end of exercise. With this mechanism, high frequency and intensity exercise can damage the kidneys [20]. Moreover, it is reported that this situation causes a significant change in urinary markers indicating tubular kidney damage [21]. It is known that high intensity and resistance exercise increases creatinine and some metabolites by causing muscle damage [22-24]. Therefore, it is an expected effect to increase kidney workload. In our study, tubular necrosis parameter was used to evaluate the increase in renal workload and metabolites. Accordingly, we think that the statistically significant increase in Exercise and Exercise+BCAA groups determined in tubular necrosis findings compared to Control group was related to the increased renal workload. In addition, a significant increase was found in Exercise+BCAA group compared to BCAA group. We think that the reason for this increase is the use of supplements just before exercise. The addition of exogenous BCAA use to the increased workload of the kidney can be expected to cause an increase in glomerular filtration and reabsorption workload.\u003c/p\u003e\n\u003cp\u003eVacuolization is a morphological change in cells, also it is a component of autophagy [25]. Autophagy is an intracellular recycling system that plays a role in clearing damaged cell components. Exercise is also described as one of the stimuli that induces autophagy. The reason why exercise induces autophagy is to limit tissue damage during exercise, maintain tissue continuity, and limit inflammatory responses [26]. As a result, vacuolization can reduce cell activity in kidney cells, as well as lead to cell death because of autophagy. In addition, it is known that there are some changes in the fluid-electrolyte balance after heavy exercise [27]. Among these changes, especially a serious decrease in potassium has been revealed by studies. Although the mechanism of hypokalaemia that occurs after potassium loss leads to kidney disease is not clear, it is histologically associated with vacuolization [28, 29]. In our study, a statistically significant increase was found in Exercise group compared to Control group in the assessment for vacuolization. We think that the significant increase in vacuolization observed in this group was caused by potassium loss after intensity exercise.\u003c/p\u003e\n\u003cp\u003eCaspase 3 is one of the primary mediators of apoptosis. It can be activated both extrinsic and intrinsic pathways [30]. For this reason, caspase 3 was evaluated by both IHC and RT-PCR methods in our study. When the results of the two methods are evaluated together, the two results in these parameters attract attention. The first point is that the number of positively stained apoptotic cells in BCAA supplement groups was higher than in the other groups. In addition, caspase-3 expression rates were decreased within these groups. The PCR method shows the active gene regions, while the IHC method shows the amount of protein in cells regardless of the relevant gene region. If there is enough protein in the cell, the gene regions close by becoming inactive. Therefore, PCR results showed that apoptosis was not inhibited, leading to the deactivation of high levels of caspase-3 in gene regions. Furthermore, caspase 3 is a substance released as an inactive precursor in the form of procaspase 3. Activated by active caspase 8 or 9 in the environment, it becomes caspase 3 [31,32]. As determined by IHC, it is caspase 3 in the environment, but procaspase 3, whose synthesis begins with gene expression. When these findings are evaluated together, it is thought that BCAAs activate the procaspase 3 present in the cell and show the effect of directing the cells to apoptosis. Similarly, Viana et al. in their study, it was stated that in rats fed a rich leucine diet and formed tumours, the increased amount of leucine also increased oxidative stress [33]. It suggests that leucine found in BCAA supplements similarly activates procaspase 3\u0026rsquo;s by increasing oxidative stress. Another possibility for procaspase 3 activation may be the absence of other amino acids other than BCAA. Damaged cells cannot repair themselves due to insufficient amino acids and therefore go to apoptosis by caspase 3 activation. In short, BCAAs can be the cause of apoptosis as secondary, even if they are not the primary cause. Another point is that although active caspase-3 was not observed in Exercise group, Caspase-3 expression was increased compared to Exercise+BCAA group. This finding suggests that exercise and BCAAs have antagonistic effects on the mechanism of apoptosis. In other words, it can be said that exercise does not directly cause apoptosis but prepares the environment for other stressors such as low glucose levels, oxygen deficiency and increased ADP levels. According to these findings, the use of BCAA in combination with high intensity resistance exercise can cause serious kidney damage in people with a predisposition.\u003c/p\u003e\n\u003cp\u003eAnother molecule involved in apoptosis is Bcl-2, which is involved in the intrinsic pathway. Bcl-2, a pro-apoptotic protein, is often examined in apoptosis [34]. Our findings concluded that BCAAs showed no activity in apoptosis mechanisms over Bcl-2 expression. Research involving other molecules is also needed to further examine the effectiveness of these results in relation to this mechanism.\u003c/p\u003e\n\u003cp\u003eKIM-1, which is known to increase kidney damage, can be used as a sensitive marker for the identification of acute tubular damage, especially in proximal tubules [35]. In the results of KIM-1 expression, a statistically significant increase was observed in Exercise group compared to all other groups, and in Exercise+BCAA group compared to only BCAA group. When KIM-1 expression findings and histopathological findings are evaluated together, they show that there is a connection between proximal tubular damage and exercise. It suggests that the damage caused by the consumption of BCAAs with exercise may be in other parts of the kidney, such as the bowman's capsule and glomerular membrane.\u003c/p\u003e\n\u003cp\u003eAs a result, the use of BCAA supplements before resistant exercise causes some damage to the kidneys. From a molecular point of view, exercise provides the cellular basis for apoptosis, while BCAAs affect caspase-3 activation, leading to kidney damage. Therefore, it is suggested that it should be investigated whether the use of BCAA supplements causes possible kidney damage in individuals if they are used at rest, not before exercise. Our study is the first study investigating the link between BCAA consumption in kidney tissues in resistant exercises and demonstrating that KIM-1 expression levels increase with exercise as a result of the parameters and findings examined. As a result of these data, we believe that more experimental and clinical studies are needed to understand the mechanisms, clarify, and confirm our evidence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLimitations:\u003c/strong\u003e Limitations of this study include the experimental design and the lack of some parameters such as creatinine levels, Bax expression levels, glomerular filtration rates and serum potassium levels. On the other hand, both histopathological and genetic evaluations constitute an important strength of our study. Based on this study, extensive studies are needed regarding the effects of exercise and BCAA supplements at different doses and at different times.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBCAA, Branched chain amino acid; KIM-1, Kidney injury molecule 1; RT-PCR, Real time polymerase chain reaction; H\u0026amp;E, Hematoxylin\u0026amp;Eosin; IHC, Immunohistochemical; Bcl-2, B-cell lymphoma 2.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFinancial Source:\u003c/strong\u003e This work was supported by Canakkale Onsekiz Mart University The Scientific Research Coordination Unit, Project number: TYL-2019-3141.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e The authors have no financial disclosure or conflict of interest in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material: \u003c/strong\u003eAll data and materials of the study are available in contact with the corresponsible author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: \u003c/strong\u003eAll authors contributed equally to all parts of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval: \u003c/strong\u003eAll animal procedures were approved by the Institutional Animal Care and Use Committee of \u0026Ccedil;anakkale Onsekiz Mart University (Approved number: 2019/09-02)\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBooth FW, Roberts CK, Laye MJ. Lack of Exercise is a Major Cause of Chronic Diseases. Comprehensive Physiology. 2012; 2(2): 1143-1211. https://doi.org/10.1002/cphy.c110025.\u003c/li\u003e\n\u003cli\u003eArdı\u0026ccedil; F. Egzersiz Re\u0026ccedil;etesi. T\u0026uuml;rk Fizyoterapi ve Rehabilitasyon Dergisi. 2014; 60: 1-8. https://doi.org/10.5152/tftrd.2014.25665.\u003c/li\u003e\n\u003cli\u003eDaenen L, Varkey E, Kellmann M, Nijs J. Exercise, Not to Exercise, or How to Exercise in Patients with Chronic Pain? Applying Science to Practice. The Clinical Journal of Pain. 2015; 31(2): 108-114. https://doi.org/10.1097/AJP.0000000000000099.\u003c/li\u003e\n\u003cli\u003eIdorn M, thorStraten P. Exercise and Cancer: From \u0026ldquo;Healthy\u0026rdquo; to \u0026ldquo;Therapeutic\u0026rdquo;?. Cancer Immunology, Immunotherapy. 2017; 66(5): 667-671. https://doi.org/10.1007/s00262-017-1985-z.\u003c/li\u003e\n\u003cli\u003eKerksick CM, Wilborn CD, Roberts MD, Smith-Ryan A, Kleiner SM, J\u0026auml;ger R, Greenwood M \u003cem\u003eet al\u003c/em\u003e. ISSN exercise \u0026amp; sports nutrition review update: research \u0026amp; recommendations. Journal of the International Society of Sports Nutrition. 2018; 15(1), 38. https://doi.org/10.1186/s12970-018-0242-y.\u003c/li\u003e\n\u003cli\u003eBifari F, Nisoli E. Branched Chain Amino Acids Differently Modulate Catabolic and Anabolic States in Mammals: A Pharmacological Point of View. British Journal of Pharmacology. 2017; 174(11): 1366-1377. DOI: 10.1111/bph.13624.\u003c/li\u003e\n\u003cli\u003eGoron A, Moinard C. Amino Acids and Sport: A True Love Story?. Amino Acids. 2018; 50(8): 969-980. https://doi.org/10.1007/s00726-018-2591-x.\u003c/li\u003e\n\u003cli\u003ePitk\u0026auml;nen HT, Oja SS, Rusko H, Nummela A, Komi PV, Saransaari P, \u003cem\u003eet al.\u003c/em\u003e Leucine Supplementation Does Not Enhance Acute Strength or Running Performance But Affects Serum Amino Acid Concentration. Amino Acids. 2003; 25(1): 85-94. https://doi.org/10.1007/s00726-002-0343-3.\u003c/li\u003e\n\u003cli\u003eWolfe RR. Branched-Chain Amino Acids and Muscle Protein Synthesis in Humans: Myth or Reality?. Journal of the International Society of Sports Nutrition. 2017; 14(1): 1-7. https://doi.org/10.1186/s12970-017-0184.\u003c/li\u003e\n\u003cli\u003eHoleček M. Branched-Chain Amino Acids in Health and Disease: Metabolism, Alterations in Blood Plasma, and As Supplements. Nutrition\u0026amp;Metabolism. 2018; 15(1): 33. https://doi.org/10.1186/s12986-018-0271-1.\u003c/li\u003e\n\u003cli\u003ePriante G, Gianesello L, Ceol M, Del Prete D, Anglani F. Cell Death in The Kidney. International Journal of Molecular Sciences. 2019; 20(14): 3598. https://doi.org/10.3390/ijms20143598.\u003c/li\u003e\n\u003cli\u003eIchimura T, Bonventre JV, Bailly V, Wei H, Hession CA, Cate RL, \u003cem\u003eet al.\u003c/em\u003e Kidney injury molecule-1 (KIM-1), a putative epithelial cell adhesion molecule containing a novel immunoglobulin domain, is up-regulated in renal cells after injury. Journal of Biological Chemistry. 1988; 273(7): 4135-4142. https://doi.org/10.1074/jbc.273.7.4135.\u003c/li\u003e\n\u003cli\u003evan Timmeren MM, van den Heuvel MC, Bailly V, Bakker SJ, van Goor H, Stegeman CA. Tubular kidney injury molecule‐1 (KIM‐1) in human renal disease. The Journal of pathology. 2007; 212(2): 209-217. https://doi.org/10.1002/path.2175.\u003c/li\u003e\n\u003cli\u003eLu M, Zhang X, Zheng D, Jiang X, Chen Q. Branched‐Chain Amino Acids Supplementation Protects Streptozotocin‐Induced Insulin Secretion and The Correlated Mechanism. BioFactors. 2015; 41(2): 127-133. https://doi.org/10.1002/biof.1188.\u003c/li\u003e\n\u003cli\u003eVieira R, Haebisch H, Kokubun E, Hell NS, Curi R. SistemaDe Nata\u0026ccedil;\u0026atilde;o Para Exerc\u0026iacute;cio F\u0026iacute;sico De Ratos. Swimming System for Physical Exercise of Rats. Arquivos De Biologia E Tecnologia. 1988; 31(3): 387-394.\u003c/li\u003e\n\u003cli\u003eCampos-Ferraz PL, Bozza T, Nicastro H, Lancha JrA H. Distinct effects of leucine or a mixture of the branched-chain amino acids (leucine, isoleucine, and valine) supplementation on resistance to fatigue, and muscle and liver-glycogen degradation, in trained rats. Nutrition. 2013; 29(11-12), 1388-1394. https://doi.org/10.1016/j.nut.2013.05.003\u003c/li\u003e\n\u003cli\u003eB\u0026uuml;y\u0026uuml;k B, Demirci T, Adalı Y, Eroğlu HA. \u0026iquest; Puede el l\u0026iacute;quido amni\u0026oacute;tico ser una soluci\u0026oacute;n alternativa de conservaci\u0026oacute;n de \u0026oacute;rganos para el almacenamiento renal en fr\u0026iacute;o?. Revista de Nefrolog\u0026iacute;a, Di\u0026aacute;lisis y Trasplante. 2020; 40(1), 14-24.\u003c/li\u003e\n\u003cli\u003eKandemir FM, Yildirim S, Kucukler S, Caglayan C, Mahamadu A, \u003cem\u003eet al\u003c/em\u003e. Therapeutic Efficacy of Zingerone Against Vancomycin-Induced Oxidative Stress, Inflammation, Apoptosis and Aquaporin 1 Permeability in Rat Kidney. Biomedicine \u0026amp; Pharmacotherapy. 2018; 105: 981-991. https://doi.org/10.1016/j.biopha.2018.06.048.\u003c/li\u003e\n\u003cli\u003eDoğanlar ZB, Uzun M, Ovali MA, Dogan A, Ongoren G, Doğanlar O. Melatonin attenuates caspase-dependent apoptosis in the thoracic aorta by regulating element balance and oxidative stress in pinealectomised rats. Applied Physiology, Nutrition, and Metabolism. 2019; 44(2), 153-163. https://doi.org/10.1139/apnm-2018-0205.\u003c/li\u003e\n\u003cli\u003eLin X, Jiang C, Luo Z, Qu S. Protective Effect of Erythropoietin on Renal Injury Induced in Rats by Four Weeks of Exhaustive Exercise. BMC Nephrology. 2013; 14(1): 130. https://doi.org/10.1186/1471-2369-14-130.\u003c/li\u003e\n\u003cli\u003eSpada TC, Silva JM, Francisco LS, Marcal LJ, Antonangelo L, Zanetta DM, \u003cem\u003eet al\u003c/em\u003e. High Intensity Resistance Training Causes Muscle Damage and Increases Biomarkers of Acute Kidney Injury in Healthy Individuals. PloS One. 2018; 13(11). https://doi.org/10.1371/journal.pone.0205791.\u003c/li\u003e\n\u003cli\u003eBaird MF, Graham SM, Baker JS, Bickerstaff GF. Creatine Kinase and Exercise-Related Muscle Damage Implications for Muscle Performance and Recovery. Journal of Nutrition and Metabolism. 2012. https://doi.org/10.1155/2012/960363.\u003c/li\u003e\n\u003cli\u003eSorichter S, Mair J, Koller A, Calzolari C, Huonker M, Pau B\u003cem\u003e, et al\u003c/em\u003e. Release of Muscle Proteins After Downhill Running in Male and Female Subjects. Scandinavian Journal of Medicine\u0026amp;Science in Sports. 2011; 11(1): 28-32. https://doi.org/10.1034/j.1600-0838.2001.011001028.x.\u003c/li\u003e\n\u003cli\u003eTsatalas, T, Giakas G, Spyropoulos G, Sideris V, Lazaridis S, Kotzamanidis C, Koutedakis Y. The Effects of Eccentric Exercise-Induced Muscle Damage on Running Kinematics at Different Speeds. Journal of Sports Sciences. 2013; 31(3): 288-298. https://doi.org/10.1080/02640414.2012.729135.\u003c/li\u003e\n\u003cli\u003eKroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, \u003cem\u003eet al.\u003c/em\u003e Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell death \u0026amp; differentiation. 2009; 16(1): 3-11. https://doi.org/10.1038/cdd.2008.150.\u003c/li\u003e\n\u003cli\u003eMooren FC, Kr\u0026uuml;ger K. Exercise, autophagy, and apoptosis. In Progress in molecular biology and translational science. Academic Press 2015; Vol. 135, pp. 407-422. https://doi.org/10.1016/bs.pmbts.2015.07.023.\u003c/li\u003e\n\u003cli\u003eGladden LB. Lactate Metabolism: A New Paradigm for The Third Millennium. The Journal of Physiology. 2004; 558(1): 5-30. https://doi.org/10.1113/jphysiol.2003.058701.\u003c/li\u003e\n\u003cli\u003eLindinger MI. Potassium Regulation During Exercise and Recovery in Humans: Implications for Skeletal and Cardiac Muscle. Journal of Molecular and Cellular Cardiology. 1995; 27(4): 1011-1022. https://doi.org/10.1016/0022-2828(95)90070-5.\u003c/li\u003e\n\u003cli\u003eThomson MN, Schneider W, Mutig K, Ellison DH, Kettritz R, Bachmann S. Patients with Hypokalemia Develop WNK Bodies in The Distal Convoluted Tubule of The Kidney. American Journal of Physiology-Renal Physiology. 2019; 316(2): F292-F300. https://doi.org/10.1152/ajprenal.00464.2018.\u003c/li\u003e\n\u003cli\u003eJiao C, Chen W, Tan X, Liang H, Li J, Yun H, \u003cem\u003eet al.\u003c/em\u003e (2020). Ganoderma lucidum spore oil induces apoptosis of breast cancer cells in vitro and in vivo by activating caspase-3 and caspase-9. Journal of ethnopharmacology. 2020; 247, 112256. https://doi.org/10.1016/j.jep.2019.112256.\u003c/li\u003e\n\u003cli\u003eBildik A, Bayar İ. Kanserde Apoptotik Yolakların İnhibisyonu. Turkiye Klinikleri Journal of Veterinary Sciences. 2018; 9(2): 42-51. https://doi.org/10.5336/vetsci.2018-62141.\u003c/li\u003e\n\u003cli\u003eGoldar S, Khaniani MS, Derakhshan SM, Baradaran B. Molecular Mechanisms of Apoptosis and Roles in Cancer Development and Treatment. Asian Pacific Journal of Cancer Prevention: APJCP. 2015; 16(6): 2129-2144. https://doi.org/10.7314/apjcp.2015.16.6.2129.\u003c/li\u003e\n\u003cli\u003eViana LR, Tobar N, Busanello ENB, Marques AC, de Oliveira AG, Lima TI, \u003cem\u003eet al.\u003c/em\u003e Leucine-Rich Diet Induces a Shift in Tumour Metabolism from Glycolytic Towards Oxidative Phosphorylation, Reducing Glucose Consumption and Metastasis in Walker-256 Tumour-Bearing Rats. Scientific Reports. 2019; 9(1): 1-11. https://doi.org/10.1038/s41598-019-52112-w.\u003c/li\u003e\n\u003cli\u003eRautureau GJ, Day CL, Hinds MG. Intrinsically disordered proteins in bcl-2 regulated apoptosis. International journal of molecular sciences. 2010; 11(4), 1808-1824. https://doi.org/10.3390/ijms11041808.\u003c/li\u003e\n\u003cli\u003eYin W, Kumar T, Lai Z, Zeng X, Kanaan HD, Li W, \u003cem\u003eet al.\u003c/em\u003e Kidney Injury Molecule-1, A Sensitive and Specific Marker for Identifying Acute Proximal Tubular Injury, Can Be Used to Predict Renal Functional Recovery in Native Renal Biopsies. International Urology and Nephrology. 2019; 51(12): 2255-2265. https://doi.org/10.1007/s11255-019-02311-1.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Resistance Exercise, Kidney Damage, Caspase 3, Branched Chain Amino Acids, KIM-1","lastPublishedDoi":"10.21203/rs.3.rs-326595/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-326595/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOne of the supplements used in with exercise programs is branched chain amino acids (BCAAs) which preferred because of their effect on novation of muscle protein synthesis. However, BCAAs increase their amount in the blood in a short time due to their properties.\u0026nbsp;In this case, the result can increase the workload of the kidneys. Based on the information, this study investigated the effects of resistance exercise and BCAA supplements on kidney tissue. A total of 24 Wistar Albino male rats were divided equally into 4 groups: Control, BCAA, Exercise and Exercise+BCAA. In six-week study, resistance swimming exercise was applied to exercise groups. BCAA groups were given BCAA supplements at doses of 2.5 mg/kg before exercise. End of the study, histological, immunochemical, and RT-PCR analyses were performed. As a result of the findings obtained, it was found that use of BCAA supplements together with exercise caused tubular necrosis (p=0.002). A significant increase was found in caspase 3 IHC staining findings in BCAA and Exercise+BCAA groups compared to Control group (p=0.011; p=0.02). Also, KIM-1 expression levels were higher in Exercise group compared to all other groups (p=0.004; p=0.003; p=0.008). As a result, BCAA consumption in combination with resistant exercise caused damage to kidney tissue.\u003c/p\u003e","manuscriptTitle":"The Effects of Branched Chain Amino Acid Supplement on Kidney Tissue of Exercising Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-03-22 20:51:31","doi":"10.21203/rs.3.rs-326595/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"99563460-f6d4-42aa-92fd-09741305cf0b","owner":[],"postedDate":"March 22nd, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":3124316,"name":"Molecular Biology"}],"tags":[],"updatedAt":"2021-08-06T20:23:59+00:00","versionOfRecord":[],"versionCreatedAt":"2021-03-22 20:51:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-326595","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-326595","identity":"rs-326595","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}