STANOZOLOL UYGULANAN SIÇAN BÖBREĞİNDE PTEN VE PI3K/AKT EKSPRESYONLARININ DEĞERLENDİRİLMESİ

Abstract

Amaç: Stanozolol, gençler ve sporcular tarafından vücut geliştirme, spor ve atletizmde yaygın kullanımı olan bir anabolik androjenik steroiddir (AAS). Stanozololün böbrek fonksiyonlarındaki potansiyel etkileri tanımlanmamıştır. Bu çalışmada, stanozolol ile tedavi edilen sıçan böbreklerinde tümör baskılayıcı protein fosfotaz tensin homolog (Pten), Phosphatidylinositol-3-kinase (Pi3k) ve protein kinaz B (Akt1)’nin mRNA düzeylerinin ekspresyonunu araştırdık. Gereç ve Yöntem: Sıçanlar, kontrol, çözücü kontrol, steroid, çözücü kontrol egzersiz, steroid egzersiz olarak 5 gruba ayrıldı. Yirmi sekiz gün boyunca subkutan stanozolol enjeksiyonu (5 mg/ kg) uygulandı ve egzersiz gruplarındaki hayvanlara 20 dk/gün, 5 gün/hafta yüzme egzersizleri yaptırıldı. PTEN ekspresyonu immünohistokimya ile değerlendirildi. Ayrıca Pten, Pi3k ve Akt1 mRNA ekspresyonları RT-PCR yoluyla analiz edildi. Bulgular: Kontrol, çözücü kontrol ve çözücü kontrol egzersiz gruplarındaki mezanjiyal hücreler ve böbrek tübülleri, steroid grubunda saptanan zayıf PTEN reaktivitesine karşı güçlü (+++) PTEN reaktivitesi gösterdi. Steroid egzersiz grubunun mezanjiyal ve tübül hücrelerinde orta düzeyde PTEN reaktivitesi saptandı. Pten mRNA ekspresyonu, steroid grubunda kontrol ve diğer gruplara göre anlamlı düşüş gösterdi, Pi3k ve Akt1 mRNA ekspresyonu anlamlı olarak arttı (p <0.001). Egzersiz tedavisi ile Pten ekspresyonu artış, Pi3k ve Akt1 ekspresyonu azalma gösterdi (p<0.05). Sonuç: Bulgularımız, AAS kullanımının, artan Pi3k ve Akt1 mRNA seviyeleri ile ilişkili olabilecek böbreklerde PTEN ekspresyonunu inhibe edebileceğini düşündürmektedir. AAS kullanımı ile yapılan egzersiz, PTEN ekspresyon seviyelerinin artması ve Pi3k ve Akt1 seviyelerinin düşmesi nedeniyle stanozolole maruz kalan böbrekler üzerinde koruyucu olabilir.

Keywords

• Stanozolol
• Böbrek
• PTEN
• PI3K
• Akt1

EVALUATION OF PTEN AND PI3K/AKT EXPRESSIONS IN STANOZOLOL-TREATED RAT KIDNEYS

Abstract

Objective: Stanozolol is an anabolic androgenic steroid (AAS) that is widely used by teenagers and athletes in bodybuilding, sports, and athletics. The potential effects of stanozolol in kidney functions have not been defined. In this study we investigated the expression of tumor suppressor protein phosphatase and tensin homolog (Pten) and mRNA levels of phosphatidylinositol 4,5-bisphosphate 3 kinase (Pi3k) and the protein kinase B (Akt1) signaling pathway in rat kidneys treated by stanozolol. Materials and Methods: Rats were randomized to 5 groups as control, solvent control, steroid (stanozolol), solvent-exercise, and steroid-exercise. Subcutaneous injection of stanozolol (5 mg/kg) was applied for 28 days and swimming exercise was performed 20 min/day, 5 days/week in exercise groups. Expression of PTEN was evaluated by immunohistochemistry. Also, Pten, Pi3k, and Akt1 mRNA expressions were analyzed via RT-PCR. Results: Mesangial cells and renal tubules in the control, solvent control, and solvent exercise groups showed strong (+++) PTEN reactivity against weak PTEN reactivity in the steroid group. Moderate PTEN reactivity was detected in cells of the steroid exercise group. Pten mRNA expression was significantly decreased, and Pi3k and Akt1 mRNA expression were significantly increased in the steroid group versus other groups (p<0.001). Pten expression showed increase while Pi3k and Akt1 expression showed decrease with exercise treatment (p<0.05). Conclusion: Our findings suggest that AAS usage may inhibit PTEN expression in kidneys, which can be associated with increased Pi3k and Akt1 mRNA levels. Exercise performed with AAS usage can be protective on stanozolol-exposed kidneys due to increased levels of PTEN expression and decreased levels of Pi3k and Akt1.

Keywords

• Stanozolol
• Kidney
• PTEN
• PI3K
• Akt1

References

1. 1. Bhasin S, Storer TW, Berman N, Yarasheski KE, Clevenger B, Phillips J, et al. Testosterone replacement increases fat-free mass and muscle size in hypogonadal men. J Clin Endocrinol Metab 1997;82(2):407-13. [CrossRef]
2. 2. Yesalis CE, Bahrke MS. Anabolic-androgenic steroids and related substances. Curr Sports Med Rep 2002;1(4):246-52. [CrossRef]
3. 3. Lionikas A, Blizard DA. Diverse effects of stanozolol in C57BL/6J and A/J mouse strains. Eur J Appl Physiol 2008;103(3):333-41. [CrossRef]
4. 4. Dornelles GL, Bueno A, de Oliveira JS, da Silva AS, França RT, da Silva C, et al. Biochemical and oxidative stress markers in the liver and kidneys of rats submitted to different protocols of anabolic steroids. Molecular And Cellular Biochemistry 2017;425(1-2):181-9. [CrossRef]
5. 5. Davani-Davari D, Karimzadeh I, Khalili H. The potential effects of anabolic-androgenic steroids and growth hormone as commonly used sport supplements on the kidney: a systematic review. BMC Nephrology 2019;20(1):198. [CrossRef]
6. 6. Almukhtar SE, Abbas AA, Muhealdeen DN, Hughson MD. Acute kidney injury associated with androgenic steroids and nutritional supplements in bodybuilders. Clin Kidney J 2015;8(4):415-9. [CrossRef]
7. 7. Daher EDF, Fernandes PHPD, Meneses GC, Bezerra GF, Ferreira LDSL, Viana GBD, et al. Novel kidney injury biomarkers among anabolic androgenic steroids usersevidence of subclinical kidney disease. Asian J Sports Med 2018;9(1):e65540. [CrossRef] 8. Juhn M. Popular sports supplements and ergogenic aids. Sports Med 2003;33(12):921-39. [CrossRef]
8. 9. Maravelias C, Dona A, Stefanidou M, Spiliopoulou C. Adverse effects of anabolic steroids in athletes. A constant threat. Toxicol Lett 2005;158(3):167-75. [CrossRef]

9. 10. Yoshida EM, Karim MA, Shaikh JF, Soos JG, Erb SR. At what price, glory? Severe cholestasis and acute renal failure in an athlete abusing stanozolol. CMAJ 1994;151(6):791-3.
10. 11. Habscheid W, Abele U, Dahm HH. Schwere Cholestase mit Nierenversagen durch Anabolika bei einem Bodybuilder [Severe cholestasis with kidney failure from anabolic steroids in a body builder]. Dtsch Med Wochenschr 1999;124(36):1029-32. [CrossRef]
11. 12. Merino García E, Borrego Utiel FJ, Martínez Arcos MÁ, Borrego Hinojosa J, Pérez Del Barrio MP. Kidney damage due to the use of anabolic androgenic steroides and practice of bodybuilding. Nefrologia 2018;38(1):101-3. [CrossRef]
12. 13. Tabatabaee SM, Elahi R, Savaj S. Bile cast nephropathy due to cholestatic jaundice after using stanozolol in 2 amateur bodybuilders. Iran J Kidney Dis 2015;9(4):331-4.
13. 14. Pérez-Ramírez C, Cañadas-Garre M, Molina MÁ, Faus- Dáder MJ, Calleja-Hernández MÁ. PTEN and PI3K/ AKT in non-small-cell lung cancer. Pharmacogenomics 2015;16(16):1843-62. [CrossRef]
14. 15. Zhang J, Li L, Peng Y, Chen Y, Lv X, Li S, et al. Surface chemistry induces mitochondria-mediated apoptosis of breast cancer cells via PTEN/PI3K/AKT signaling pathway. Biochim Biophys Acta Mol Cell Res 2018;1865(1):172-85. [CrossRef]
15. 16. Kim DH, Suh J, Surh YJ, Na HK. Regulation of the tumor suppressor PTEN by natural anticancer compounds. Ann N Y Acad Sci 2017;1401(1):136-49. [CrossRef]
16. 17. Luan X, Tian X, Zhang H, Huang R, Li N, Chen P, et al. Exercise as a prescription for patients with various diseases. J Sport Health Sci 2019;8(5):422-41. [CrossRef]
17. 18. Tahbaz R, Schmid M, Merseburger AS. Prevention of kidney cancer incidence and recurrence: lifestyle, medication and nutrition. Curr Opin Urol 2018;28(1):62-79. [CrossRef]
18. 19. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25(4):402-8. [CrossRef]
19. 20. Vieira TM, Rossi Junior WC, Da Ré Guerra F, Damião B, Marques PP, Esteves A. Effect of testosterone cypionate and stanozolol on the heart of young trained mice: A morphometric study. Steroids 2019;145:19-22. [CrossRef]
20. 21. Tucci P, Morgese MG, Colaianna M, Zotti M, Schiavone S, Cuomo V, et al. Neurochemical consequence of steroid abuse: stanozolol-induced monoaminergic changes. Steroids 2012;77(3):269-75. [CrossRef]
21. 22. Liehr JG. Is estradiol a genotoxic mutagenic carcinogen? Endocr Rev 2000;21(1):40-54. [CrossRef]
22. 23. Giannitrapani L, Soresi M, La Spada E, Cervello M, D’Alessandro N, Montalto G. Sex hormones and risk of liver tumor. Ann N Y Acad Sci 2006;1089:228-36. [CrossRef]
23. 24. Souza LD, da Cruz LA, Cerqueira EM, Meireles J. Micronucleus as biomarkers of cancer risk in anabolic androgenic steroids users. Hum Exp Toxicol 2017;36(3):302- 10. [CrossRef]
24. 25. Salerno M, Cascio O, Bertozzi G, Sessa F, Messina A, Monda V, et al. Anabolic androgenic steroids and carcinogenicity focusing on Leydig cell: a literature review. Oncotarget 2018;9(27):19415-26. [CrossRef]
25. 26. Pak S, Kim W, Kim Y, Song C, Ahn H. Dihydrotestosterone promotes kidney cancer cell proliferation by activating the STAT5 pathway via androgen and glucocorticoid receptors. J Cancer Res Clin Oncol 2019;145(9):2293-301. [CrossRef]
26. 27. Tsitsimpikou C, Vasilaki F, Tsarouhas K, Fragkiadaki P, Tzardi M, Goutzourelas N, et al. Nephrotoxicity in rabbits after long-term nandrolone decanoate administration. Toxicol Lett 2016;259:21-7. [CrossRef]
27. 28. Pozzi R, Fernandes KR, de Moura CF, Ferrari RA, Fernandes KP, Renno AC, et al. Nandrolone decanoate induces genetic damage in multiple organs of rats. Arch Environ Contam Toxicol 2013;64(3):514-8. [CrossRef]
28. 29. Riezzo I, Turillazzi E, Bello S, Cantatore S, Cerretani D, Di Paolo M, et al. Chronic nandrolone administration promotes oxidative stress, induction of pro-inflammatory cytokine and TNF-α mediated apoptosis in the kidneys of CD1 treated mice. Toxicol Appl Pharmacol 2014;280(1):97- 106. [CrossRef]
29. 30. Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis. 2004;9(6):667- 76. [CrossRef]
30. 31. Xu N, Lao Y, Zhang Y, Gillespie DA. Akt: a double-edged sword in cell proliferation and genome stability. J Oncol 2012;2012:951724. doi: 10.1155/2012/951724. [CrossRef]
31. 32. Nassif NT, Lobo GP, Wu X, Henderson CJ, Morrison CD, Eng C, et al. PTEN mutations are common in sporadic microsatellite stable colorectal cancer. Oncogene 2004;23(2):617-28. [CrossRef]
32. 33. Frisk T, Foukakis T, Dwight T, Lundberg J, Höög A, Wallin G, et al. Silencing of the PTEN tumor-suppressor gene in anaplastic thyroid cancer. Genes Chromosomes Cancer 2002;35(1):74-80. [CrossRef]
33. 34. Wang DS, Rieger-Christ K, Latini JM, Moinzadeh A, Stoffel J, Pezza JA, et al. Molecular analysis of PTEN and MXI1 in primary bladder carcinoma. Int J Cancer 2000;88(4):620-5. [CrossRef]
34. 35. Wu H, Goel V, Haluska FG. PTEN signaling pathways in melanoma. Oncogene 2003;22(20):3113-22. [CrossRef]
35. 36. Kanamori Y, Kigawa J, Itamochi H, Shimada M, Takahashi M, Kamazawa S, et al. Correlation between loss of PTEN expression and Akt phosphorylation in endometrial carcinoma. Clin Cancer Res 2001;7(4):892-5.
36. 37. Breuksch I, Welter J, Bauer HK, Enklaar T, Frees S, Thüroff JW, et al. In renal cell carcinoma the PTEN splice variant PTEN-Δ shows similar function as the tumor suppressor PTEN itself. Cell Commun Signal 2018;16(1):35. [CrossRef]
37. 38. Sirianni R, Capparelli C, Chimento A, Panza S, Catalano S, Lanzino M, et al. Nandrolone and stanozolol upregulate aromatase expression and further increase IGF-I-dependent effects on MCF-7 breast cancer cell proliferation. Mol Cell Endocrinol 2012;363(1-2):100-10. [CrossRef]
38. 39. Nourbakhsh M, Golestani A, Zahrai M, Modarressi MH, Malekpour Z, Karami-Tehrani F. Androgens stimulate telomerase expression, activity and phosphorylation in ovarian adenocarcinoma cells. Mol Cell Endocrinol 2010;330(1-2):10-6. [CrossRef]
39. 40. Friedenreich CM, Neilson HK, Lynch BM. State of the epidemiological evidence on physical activity andcancer prevention. Eur J Cancer 2010;46:2593-604. [CrossRef]
40. 41. Winzer BM, Whiteman DC, Reeves MM, Paratz JD. Physical activity and cancer prevention: A system-atic review of clinical trials. Cancer Causes Control 2011;22:811-26. [CrossRef]
41. 42. Yu M, King B, Ewert E, Su X, Mardiyati N, Zhao Z, et al. Exercise activates p53 and negatively regulates IGF-1 pathway in epidermis within a skin cancer model. PLoS ONE 2016;11(8):e0160939. doi: 10.1371/journal.pone.0160939. [CrossRef]
42. 43. Piguet AC, Saran U, Simillion C, Keller I, Terracciano L, Reeves HL, et al. Regular exercise decreases liver tumors development in hepatocyte-specific PTEN-deficient mice independently of steatosis. J Hepatol 2015;62(6):1296-303. [CrossRef]