Analysis of apoptosis of kidney tissue by the tunel method and histomorphological changes in rabbit kidney model due to unilateral supravesical obstruction

Year 2020, Volume: 4 Issue: 11, 1057 - 1062, 01.11.2020

Ramazan Uçak , Şükrü Oğuzkan Topçu , İbrahim Sarı

https://doi.org/10.28982/josam.811241

Abstract

Aim: There is a need for effective, cheap, and fast method to detect apoptosis. In some studies, we see that newer, more difficult, expensive, or less effective methods are used. We wanted to show that the TUNEL method has serious advantages and can still be used alone. In this study, it was aimed to investigate whether there is a significant difference in the number of apoptotic cells in partial obstruction (PO) and complete obstruction (CO) by using terminal deoxytransferase-mediated bio-dUTP nick and labeling (TUNEL) method in renal tissue. We also evaluated histopathological changes after renal obstruction.
Methods: In this study, 29 rabbits were used. Supravesical obstruction was created in 24 rabbits. Five rabbits were used as the control group. Twelve kidneys were examined after creating unilateral partial obstruction and 12, after the creation of unilateral complete obstruction. Histomorphological changes in kidney tissues in routine Haemetoxylene-Eosine (HE) preparations and apoptosis in preparations obtained by TUNEL method were examined.
Results: Apoptotic cells were observed especially in the tubules by the TUNEL method. The average number of apoptotic cells in CO and PO groups were 190.66 and 40.58, respectively. In the CO group, the number of apoptotic cells was significantly higher than that in the PO group (P<0.001). Interstitial fibrosis, chronic inflammatory infiltration, tubular destruction (vacuolar changes, cystic and atrophic tubules) were observed in both groups. These changes were more limited, and mild, in the PO group, and severe and widespread in the CO group.
Conclusion: TUNEL method is one of the highly effective methods in detecting apoptosis. It was observed that apoptosis and pathological changes developing in the kidney tissue after complete obstruction were more severe and widespread.

Keywords

Renal obstruction, Apoptosis, TUNEL method

Tavşan böbrek modelinde tek taraflı supravezikal tıkanıklığa bağlı böbrek dokusunda oluşan apoptozisin tunel yöntemiyle analizi ve histomorfolojik değişiklikler

Abstract

Amaç: Literatürde, bazı çalışmalarda, daha yeni, ama pratikte değerlendirme güçlükleri olan, pahalı veya etkinliği az yöntemlerin kullanıldığını görüyoruz. Terminal deoxytransferase-mediated bio-dUTP nick and labeling (TUNEL) yönteminin ciddi avantajları olduğunu ve halen tek başına kullanılabileceğini göstermek istedik. Bu nedenle, deneysel olarak tavşan üreterinde kısmi ve tam obstrüksüyon oluşturulmasından sonra böbrek dokusunda gelişen apopitozisin şiddetinin, TUNEL yöntemi kullanılarak belirlenmesi, gruplar arasında anlamlı bir fark olup olmadığı ve yanısıra oluşan histopatolojik değişiklikleri değerlendirme amaçlandı.
Yöntemler: Mesane üstü seviyesinde üreter geçişinin kısmi ve tam olarak engellenmesi yoluyla-12 tam obstrüksiyon, 12 kısmi obstrüksiyon grubu-, 5 taneside kontrol grubu olmak üzere- toplam 29 adet tavşan böbreği kullanıldı. Böbrek dokusunda gelişen histomorfolojik değişiklikler Hematoksilen-Eozin (HE) preperatlarda, apoptozis ise TUNEL yöntemiyle hazırlanan preperatlarda mikroskopik olarak incelendi. Obstrüksiyon sonrası oluşan apopitozisin tam ve kısmi obstrüksiyon grupları arasında istatistiksel olarak anlamlı farklılığı olup olmadığı araştırıldı.
Bulgular: Apopitotik hücreler, TUNEL yöntemiyle net olarak gösterildi. Apopitozisin, özellikle tübül epitelinde yoğunlaştığı görüldü. Tam obstrüksiyon grubunda, ortalama apopitotik hücre sayısı 190,66 ve kısmi obstriksüyon grubunda ise 40,58 idi. Tam obstrüksiyon grubunda apopitotik hücre sayısı kısmi obstrüksiyon grubuna göre anlamlı derecede yüksekti (P<0,001). 2 grupta da interstisyel fibrozis, kronik iltihabi infiltrasyon, tübüler yıkım (vakuoler değişiklikler, kistik ve atrofik tübüller) gözlendi. Bu değişiklikler kısmi tıkanma grubunda daha sınırlı, hafif, tam tıkanma grubunda şiddetli ve yaygın idi.
Sonuç: TUNEL yönteminin apopitozisi belirlemede etkinliği yüksek yöntemlerden biri olduğu gösterildi. Tam tıkanma sonrası böbrek dokusunda gelişen apopitozisin ve patolojik değişikliklerin çok daha şiddetli ve yaygın olduğu görüldü.

Keywords

Renal obstrüksiyon, Apopitozis, TUNEL yöntemi

References

• 1. Kerr JF, Wylie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239-57. PMID:4561027. doi: 10.1038/bjc.1972.33.
• 2. Horvitz HR. Genetic control of programmed cell death in the nematode Caenorhabditis elegans. Cancer Res. 1999;59:1701–6. PMID: 10197583.
• 3. Kumar V, Abbas AK, Aster JC. Cell injury, cell death, and adaptations. Robbins Basic Pathology, 10.th ed. 2018. Chapter 2, 31-56. Elsevier, Philadelphia, Pennsylvania.
• 4. Ucero AC, Gonçalves S, Benito-Martin A, Santamaría B, Ramos AM, Berzal S, et al. Obstructive renal injury: from fluid mechanics to molecular cell biology. Open Access J Urol. 2010;2:41–55. PMID: 24198613 doi: 10.2147/rru.s6597
• 5. Shanley FF. The pathology of Chronic Renal İschemia. Seminars in Nephrology. 1996;16(1);21-32.
• 6. Ucero AC, Benito-Martin A, Izquierdo MC, Sanchez-Niño MD, Sanz AB, Ramos AM, et al. Unilateral ureteral obstruction: beyond obstruction. International Urology and Nephrology. 2014;46(4):765–77. doi: 10.1007/s11255-013-0520-1.
• 7. Truonga L, Petrusevska G, Yang G, Gurpınar T, Shappeli S, Lechago J et all. Cell apoptosis and proliferation in experimental chronic obstructive uropathy. Kidney İnt. 1996;50;200-7. doi: 10.1038/ki.1996.303 PMID: 8807589.
• 8. Truonga L, Gabera L, Eknoyand G. Obstructive Uropathy. Experimental Models for Renal Diseases: Pathogenesis and Diagnosis. Contrib Nephrol. 2011;169:311–26. doi: 10.1159/000314578.
• 9. Yeh CH, Chiang HS, Lai TY, Chien CT. Unilateral Ureteral Obstruction Evokes Renal Tubular Apoptosis via the Enhanced Oxidative Stress and Endoplasmic Reticulum Stress in the Rat Neurourology and Urodynamics. 2011;30:472–9. doi: 10.1002/nau.20855
• 10. Chevalier RL. Prognostic factors and biomarkers of congenital obstructive nephropathy. Pediatric Nephrology. 2016;31(9):1411–20. PMID:26667236.doi: 10.1007/s00467-015-3291-3
• 11. Xu Y, Ruan S, Wu X, Chen H, Zheng K, Fu B. Autophagy and apoptosis in tubular cells following unilateral ureteral obstruction are associated with mitochondrial oxidative stress. International journal of molecular medicine. 2013;(31):628-36. doi: 10.3892/ijmm.2013.1232.
• 12. Hughes J, Gobe G. Identification and quantification of apoptosis in the kidney using morphology, biochemical and molecular markers. Nephrology. 2007;12(5):452-8. doi: 10.1111/j.1440-1797.2007.00854.x.
• 13. Martinez MM, Randall DR, Pappas D. Detection of apoptosis: A review of conventional and novel techniques (Critical Review). Anal Methods. 2010;2:996-1004. doi: 10.1039/c0ay00247j
• 14. Loo DT. In Situ Detection of Apoptosis by the TUNEL Assay: An Overview of Techniques. Methods in Molecular Biology. 2011;682:3-13. doi 10.1007/978-1-60327-409-8_1.
• 15. Lebon C, Rodriguez GV, El Zaoui I, Jaadane I, Behar-Cohen F, Torriglia A. On the use of an appropriate TUNEL assay to identify apoptotic cells, Analytical Biochemistry. 2015; doi: doi: 10.1016/j.ab.2015.04.007.
• 16. Crowley LC, Marfell BJ, Waterhouse NJ. Detection of DNA Fragmentation in Apoptotic Cells by TUNEL. Cold Spring Harb Protoc. 2016;(10)3 doi: 10.1101/pdb.prot 087221.
• 17. Archana M; Bastian, Yogesh TL, Kumaraswamy KL. Various methods available for detection of apoptotic cells--a review. Indian J Cancer. 2013 Jul-Sep;50(3):274-83. doi: 10.4103/0019-509X.118720. PMID: 24061471.
• 18. Bozza WP, Twomey JD, Kim S-R, Zhang B. Detection of Apoptosis: From Bench Side to Clinical Practice.Apoptosis Methods in Toxicology. Methods in Pharmacology and Toxicology, Muganda PM. (ed.), 2016, Chapter 2, Pages 13-29, Springer Science+Business Media New York. doi: 10.1007/978-1-4939-3588-8_2.
• 19. Banfalvi G. Methods to detect apoptotic cell death. Apoptosis. 2017;22(2):306‐23. doi: 10.1007/s10495-016-1333-3.
• 20. Nambiar KS, Hegde V. Apoptosis detection modalities: A brief review. International Dental & Medical Journal of Advanced Research. 2016;2:1–5. doi: 10.15713/ins.idmjar.53.
• 21. Eddy A. Experimental insight into the tubulointertitial disease accompanying primary glomerular lesions. J Am Soc Nephrol. 1994;5(6):12373-87.
• 22. Chevalier RL. The proximal tubule is the primary target of injury and progression of kidney disease: role of the glomerulotubular junction. Am J Physiol Renal Physiol. 2016;311(1):145–61. doi: 10.1152 / ajprenal.00164.2016.
• 23. Docherty NG, O'Sullivan OE , Büyük DA, Fitzpatrick JM, Watson RW. Evidence that inhibition of tubular cell apoptosis protects against renal damage and development of fibrosis following ureteric obstruction. Am J Physiol Renal Physiol. 2006;290:4–13. doi: 10.1152 / ajprenal. 00045.2005.
• 24. Nagalakshmi VK, Li M, Shah S, Gigliotti JC, Klibanov AL, Epstein FH, et al. Changes in cell fate determine the regenerative and functional capacity of the developing kidney before and after release of obstruction. Clinical Science. 2018;132(23):2519-45; doi: 10.1042 /CS2018062.
• 25. Sanz AB, Santamaría B, Ruiz-Ortega M, Egido J, Ortiz A. Mechanisms of renal apoptosis in health and disease. J Am Soc Nephrol. 2008;19(9):1634‐42. doi: 10.1681/ASN.2007121336.
• 26. Adachi T, Sugiyama N, Yagita H, Yokoyama T. Renal atrophy after ischemia–reperfusion injury depends on massive tubular apoptosis induced by TNFα in the later phase. Medical Molecular Morphology. 2014;47(4):213–23. PMID:24407718 doi: 10.1007/s00795-013-0067-3.
• 27. Ophascharoensuk V, Giachelli CM, Gordon K, Hughes J, Pichler R, Brown P, et al. Obstructive uropathy in the mouse: Role of osteopontin in interstitial fibrosis and apoptosis. Kidney International. 1999;56(2):571-80. doi: 10.1046/j.1523-1755.1999.00580.x.
• 28. Misseri R, Meldrum KK. Mediators of Fibrosis and Apoptosis in Obstructive Uropathies. Curr Urol Rep. 2005;6:140–5. doi: 10.1007/s11934-005-0083-5.
• 29. Kim J, Kim DS, Park MJ, Cho H-J, Zervos AS, Bonventre JV, et al. Omi/HtrA2 protease is associated with tubular cell apoptosis and fibrosis induced by unilateral ureteral obstruction. Am J Physiol Renal Physiol. 2010;298:1332–40. doi: 10.1152/ajprenal.00737.2009.
• 30. Jackson L, Woodward M, Coward RJ. The molecular biology of pelvi-ureteric junction obstruction. Pediatr Nephrol. 2018;33(4):553‐71. doi: 10.1007/s00467-017-3629-0.
• 31. Topcu SO, Celik S, Erturhan S, Erbagci A, Yagci F, Ucak R. Verapamil prevents the apoptotic and hemodynamic changes in response to unilateral ureteral obstruction. Int J Urol. 2008;15(4):350‐5. doi: 10.1111/j.1442-2042.2008.01992.x.
• 32. Yang H, Zhang W, Xie T, Wang X, Ning W. Fluorofenidone inhibits apoptosis of renal tubular epithelial cells in rats with renal interstitial fibrosis. Braz J Med Biol Res. 2019;52(11):e8772. doi: 10.1590/1414-431X20198772.
• 33. Cho JH, Choi SY, Ryu HM, et al. Fimasartan attenuates renal ischemia-reperfusion injury by modulating inflammation-related apoptosis. Korean J Physiol Pharmacol. 2018;22(6):661‐70. doi: 10.4196/kjpp.2018.22.6.661.
• 34. Park E, Cox M, Scotland K, Buttyan R, Lange D. Erythropoietin promotes functional recovery via anti-apoptotic mechanisms in mouse unilateral ureteral obstruction. Cell Stress Chaperones. 2020;25(2):245‐51. doi: 10.1007/s12192-020-01067-3
• 35. Chen Y, Mu L, Xing L, Li S and Fu S. Rhein alleviates renal interstitial fibrosis by inhibiting tubular cell apoptosis in rats. Biol Res. 2019;52:50. doi: 10.1186/s40659-019-0257-0.
• 36. Kim S, Jung ES, Lee J, Heo NJ, Na KY, Han JS. Effects of colchicine on renal fibrosis and apoptosis in obstructed kidneys. The Korean Journal of Internal Medicine. 2018;33:568-76. doi: 10.3904/kjim.2016.131.
• 37. Kaeidi A, Taghipour Z, Allahtavakoli M, Fatemi I, Hakimizadeh E, Hassanshahi J. Ameliorating effect of troxerutin in unilateral ureteral obstruction induced renal oxidative stress, inflammation, and apoptosis in male rats. Naunyn Schmiedebergs Arch Pharmacol. 2020;393(5):879‐88. doi: 10.1007/s00210-019-01801-4.
• 38. Zhang QF. Ulinastatin inhibits renal tubular epithelial apoptosis and interstitial fibrosis in rats with unilateral ureteral obstruction. Mol Med Rep. 2017;16(6):8916‐22. doi: 10.3892/mmr.2017.7692.
• 39. Yuan J, Shen Y, Yang X, Xie Y, Lin X, Zeng W, et al. Thymosin β4 alleviates renal fibrosis and tubular cell apoptosis through TGF-β pathway inhibition in UUO rat models. BMC Nephrol. 2017;18:314. doi.org/10.1186/s12882-017-0708-1.
• 40. Hosseinian S, Bideskan AE, Shafei MN, Sadeghnia HR, Soukhtanloo M, Shahraki S, et al. Nigella sativa extract is a potent therapeutic agent for renal inflammation, apoptosis, and oxidative stress in a rat model of unilateral ureteral obstruction. Phytother Res. 2018;32(11):2290‐8. doi: 10.1002/ptr.6169.
• 41. Kartal H, Büyük B. Effects of alpha-lipoic acid on skeletal muscle ischemia-reperfusion injury in mice. J Surg Med. 2020;4(7):567-72. doi: 10.28982/josam.764953.
• 42. Büyük B, Karakoç E. Effects of thiopental in cold ıschemia in liver transplation: An experimental study. J Surg Med. 2019;3(2):143-8. doi: 10.28982/josam.460075