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Ischemic postconditioning inhibits apoptosis of renal cells following reperfusion: a novel in vitro model

  • Urology - Original Paper
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Abstract

Purpose

The majority of renal ischemic/reperfusion (I/R) and ischemic postconditioning (IPO) studies have been based on animal models. To gain mechanistic insights into ischemic postconditioning-induced alterations at the cell level, a novel in vitro model of I/R and IPO is set up by using the rat proximal tubule cell line NRK-52E.

Methods

Cells are incubated in 1 mL ischemic buffer under hypoxia conditions for 3 h to simulate the clinical condition of a cellular microenvironment representative of ischemia, including oxygen deprivation, carbon dioxide elevation, nutrient depletion, and waste accumulation. IPO model is established by exposing the cells to three cycles of ‘mimic reperfusion condition’ for 10 min and ischemic condition for 10 min after placing the cells in ischemic condition for 3 h. Flow cytometry and Hoechst are used to assessing apoptosis. The expression spot and protein levels of PDK, Akt, and ERK are also analyzed.

Results

I/R results in severe injury in NRK-52E cells as evidenced by increased LDH leakage in the culture medium, as well as increased apoptotic index, which may be significantly attenuated by IPO treatment applied before the abrupt reperfusion (P < 0.05 vs. I/R group). Meanwhile, IPO, compared with I/R, increases phosphorylation levels of Akt and ERK (P < 0.05 vs. I/R group), which have been identified to play a vital role in the regulation of cell proliferation, survival, and metabolism.

Conclusion

A new in vitro model of I/R and IPO is established successfully. These results offer evidence that 3 h of simulating ischemic/reperfusion injury may cause cell apoptosis, and IPO is effective to attenuate renal cell apoptosis and potentially mediate via activation of Akt and ERK signal.

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References

  1. Martins PN, Chandraker A, Tullius SG (2006) Modifying graft immunogenicity and immune response prior to transplantation: potential clinical applications of donor and graft treatment. Transpl Int 19:351–359

    Article  PubMed  Google Scholar 

  2. Sehirli O, Sener E, Cetinel S, Yüksel M, Gedik N, Sener G (2008) Alpha-lipoic acid protects against renal ischaemia-reperfusion injury in rats. Clin Exp Pharmacol Physiol 35:249–255

    Article  CAS  PubMed  Google Scholar 

  3. Zager RA, Iwata M, Burkhart KM, Schimpf BA (1994) Post-ischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia. Kidney Int 45:1760–1768

    Article  CAS  PubMed  Google Scholar 

  4. Park KM, Chen A, Bonventre JV (1999) Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J Biol Chem 276:11870–11876

    Article  Google Scholar 

  5. Toosy N, McMorris EL, Grace PA (1999) Ischemic preconditioning protects the rat kidney from reperfusion injury. BJU Int 84:489–494

    Article  CAS  PubMed  Google Scholar 

  6. Jefayri MK, Grace PA, Mathie RT (2000) Attenuation of reperfusion injury by renal ischemic preconditioning: the role of nitric oxide. BJU Int 85:1007–1013

    Article  CAS  PubMed  Google Scholar 

  7. Lee HT, Emala CW (2001) Protein kinase C and Gi/o proteins are involved in adenosine and ischemic preconditioning-mediated renal protection. J Am Soc Nephrol 12:233–240

    CAS  PubMed  Google Scholar 

  8. Torras J, Herrero-Fresneda I, Lloberas N, Riera M, Ma Cruzado J, Ma Grinyó J (2002) Promising effects of ischemic preconditioning in renal transplantation. Kidney In 61:2218–2227

    Article  Google Scholar 

  9. Zhao ZQ, Vinten-Johansen J (2006) Postconditioning: reduction of reperfusion-induced injury. Cardiovasc Res 70:200–211

    Article  CAS  PubMed  Google Scholar 

  10. Liu X, Chen H, Zhan B, Xing B, Zhou J, Zhu H, Chen Z (2007) Attenuation of reperfusion injury by renal ischemic postconditioning: the role of NO. Biochem Biophys Res Commun 359:628–634

    Article  CAS  PubMed  Google Scholar 

  11. Chen H, Xing B, Liu X, Zhan B, Zhou J, Zhu H, Chen Z (2008) Ischemic post- conditioning inhibits apoptosis after renal ischemia/reperfusion injury in rat. Transpl Int 21:364–371

    Article  CAS  PubMed  Google Scholar 

  12. Toback FG (1992) Regeneration after acute tubular necrosis. Kidney Int 41:226–246

    Article  CAS  PubMed  Google Scholar 

  13. Safirstein R, DiMari J, Megyesi J, Price P (1998) Mechanisms of renal repair and survival following acute injury. Semin Nephrol 18:519–522

    CAS  PubMed  Google Scholar 

  14. Aikawa R, Nawano M, Gu Y, Katagiri H, Asano T, Zhu W, Nagai R, Komuro I (2000) Insulin prevents cardiomyocytes from oxidative stress-induced apoptosis through activation of PI3 Kinase/Akt. Circulation 102:2873–2879

    Article  CAS  PubMed  Google Scholar 

  15. Steelman LS, Stadelman KM, Chappell WH, Horn S, Bäsecke J, Cervello M, Nicoletti F, Libra M, Stivala F, Martelli AM, McCubrey JA (2008) Akt as a therapeutic target in cancer. Expert Opin Ther Targets 12:1139–1165

    Article  CAS  PubMed  Google Scholar 

  16. Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, Force TL, Franke TF, Hajjar RJ, Rosenzweig A (2001) Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation 104:330–335

    Article  CAS  PubMed  Google Scholar 

  17. Tsang A, Hausenloy DJ, Mocanu MM, Yellon DM (2004) Postconditioning: a form of ‘‘modified reperfusion’’ protects the myocardium by activating the phosphatidylinositol 3-kinase-akt pathway. Circ Res 95:230–232

    Article  CAS  PubMed  Google Scholar 

  18. Galatou E, Carnická S, Nemčeková M, Ledvényiová V, Adameová A, Kelly T, Barlaka E, Galatou E, Khandelwal VK, Lazou A (2012) PPAR-alpha activation as a preconditioning-like intervention in rats in vivo confers myocardial protection against acute ischaemia-reperfusion injury: involvement of PI3 K-Akt. Can J Physiol Pharmacol 90:1135–1144

    Article  PubMed  Google Scholar 

  19. Shibata M, Yamawaki T, Sasaki T, Hattori H, Hamada J, Fukuuchi Y, Okano H, Miura M (2002) Upregulation of Akt phosphorylation at the early stage of middle cerebral artery occlusion in mice. Brain Res 942:1–10

    Article  CAS  PubMed  Google Scholar 

  20. Harada N, Hatano E, Koizumi N, Nitta T, Yoshida M, Yamamoto N, Brenner DA, Yamaoka Y (2004) Akt activation protects rat liver from ischemia/reperfusion injury. J Surg Res 121:159–170

    Article  CAS  PubMed  Google Scholar 

  21. Toledo-Pereyra LH, Lopez-Neblina F, Reuben JS, Toledo AH, Ward PA (2004) Selectin inhibition modulates Akt/MAPK signaling and chemokine expression after liver ischemia–reperfusion. J Invest Surg 17:303–313

    Article  PubMed  Google Scholar 

  22. Sano T, Izuishi K, Hossain MA, Kakinoki K, Okano K, Masaki T, Suzuki Y (2010) Protective effect of lipopolysaccharide preconditioning in hepatic ischemia reperfusion injury. HPB (Oxford) 12:538–545

    Article  PubMed Central  Google Scholar 

  23. Park KM, Chen A, Bonventre JV (2001) Prevention of kidney ischemia/reperfusion-induced functional injury and JNK, p38, and MAPK kinase activation by remote ischemic pretreatment. J Biol Chem 276:11870–11876

    Article  CAS  PubMed  Google Scholar 

  24. Park KM, Kramers C, Vayssier-Taussat M, Chen A, Bonventre JV (2002) Prevention of kidney ischemia/reperfusion-induced functional injury, MAPK and MAPK kinase activation, and inflammation by remote transient ureteral obstruction. J Biol Chem 277:2040–2049

    Article  CAS  PubMed  Google Scholar 

  25. Sauvant C, Schneider R, Holzinger H, Renker S, Wanner C, Gekle M (2009) Implementation of an in vitro model system for investigation of reperfusion damage after renal ischemia. Cell Physiol Biochem 24:567–576

    Article  CAS  PubMed  Google Scholar 

  26. Meldrum KK, Meldrum DR, Hile KL, Burnett AL, Harken AH (2001) A novel model of ischemia in renal tubular cells which closely parallels in vivo injury. J Surg Res 99:288–293

    Article  CAS  PubMed  Google Scholar 

  27. Pitts KR, Toomb CF (2004) Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro. Am J Physiol Heart Circ Physiol 287:H1801–H1812

    Article  CAS  PubMed  Google Scholar 

  28. Cavdar Z, Oktay G, Egrilmez MY, Genc S, Genc K, Altun Z, Islekel H, Guner G (2010) In vitro reoxygenation following hypoxia increases MMP-2 and TIMP-2 secretion by human umbilical vein endothelial cells. Acta Biochim Pol 57:69–73

    CAS  PubMed  Google Scholar 

  29. Saenz-Morales D, Escribese MM, Stamatakis K, García-Martos M, Alegre L, Conde E, Pérez-Sala D, Mampaso F, García-Bermejo ML (2006) Requirements for proximal tubule epithelial cell detachment in response to ischemia: role of oxidative stress. Exp Cell Res 312:3711–3727

    Article  CAS  PubMed  Google Scholar 

  30. Saenz-Morales D, Conde E, Escribese MM, García-Martos M, Alegre L, Blanco-Sánchez I, García-Bermejo ML (2009) ERK1/2 mediates cytoskeleton and focal adhesion impairment in proximal epithelial cells after renal ischemia. Cell Physiol Biochem 23:285–294

    Article  CAS  PubMed  Google Scholar 

  31. Basnakian AG, Ueda N, Hong X, Galitovsky VE, Yin X, Shah SV (2005) Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation. Am J Physiol Renal Physiol 288:F308–F314

    Article  CAS  PubMed  Google Scholar 

  32. Leichtweiss HP, Lübbers DW, Weiss C, Baumgärtl H, Reschke W (1969) The oxygen supply of the rat kidney: measurements of int4arenal pO2. Pflugers Arch 309:328–349

    Article  CAS  PubMed  Google Scholar 

  33. Holloway JC, Phifer T, Henderson R, Welbourne TC (1986) Renal acid-base metabolism after ischemia. Kidney Int 29:989–994

    Article  CAS  PubMed  Google Scholar 

  34. Bonegio R, Lieberthal W (2002) Role of apoptosis in the pathogenesis of acute renal failure. Curr Opin Nephrol Hypertens 11:301–308

    Article  PubMed  Google Scholar 

  35. Padanilam BJ (2003) Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 284:F608–F627

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (Nos. 30901494 and 30901552), the Hubei Province Natural Science Foundation (No. 2012FFA096), and supported by “the Fundamental Research Funds for the Central Universities (No. 302-274231)”.

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    Authors and Affiliations

    1. Department of Urology, Renmin Hospital of Wuhan University, Wuhan University, Jiefang Road 238, Wuhan, 430060, China

      Xiaodong Weng, Min Wang, Hui Chen, Zhiyuan Chen & Xiuheng Liu

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    1. Xiaodong Weng
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    2. Min Wang
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    3. Hui Chen
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    Correspondence to Xiuheng Liu.

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    Xiaodong Weng and Min Wang have contributed equally to this work and are co-first authors.

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    Weng, X., Wang, M., Chen, H. et al. Ischemic postconditioning inhibits apoptosis of renal cells following reperfusion: a novel in vitro model. Int Urol Nephrol 47, 1067–1074 (2015). https://doi.org/10.1007/s11255-015-0997-x

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    • DOI: https://doi.org/10.1007/s11255-015-0997-x

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