Pre-Exercise Protective Effects Against Renal Ischemic Reperfusion Injury in Hsp 70.1 Knockout Mice

Hsp70.1유전자결핍된 마우스에서 허혈 재관류 신장손상에 대한 전처치 운동의 보호효과

  • Lee, Jin (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University) ;
  • Kim, Won-Kyu (Department of Anatomy and Cell Biology, College of Medicine, Hanyang University)
  • 이진 (한양대학교 의과대학 해부세포생물학교실) ;
  • 김원규 (한양대학교 의과대학 해부세포생물학교실)
  • Received : 2010.01.14
  • Accepted : 2010.03.24
  • Published : 2010.04.30


The objective of this study was to investigate levels of serum creatinine, CuSOD and MnSOD protein expression in the kidney after renal ischemic reperfusion with pre-exercise using heat shock protein 70.1 in knock-out mice (KO). The C57/BL6 strain (Wild type: WT) and KO were divided into 4 groups as follows: Sham control group (Sham), pre-exercise group (Ex), pre-exercise +ischemia group (Ex+IR), and ischemia group (IR). CuSOD and MnSOD expression were significantly decreased (p<0.01, p<0.05) and blood creatinine concentration was significantly increased (p<0.01) in the IR group of KO. In contrast, CuSOD and MnSOD expression in the Ex+IR group of KO were higher than the IR group, while creatinine concentration was significantly lower. These results suggest that Hsp70 is directly correlated to renal ischemic reperfusion injury. Pre-exercise in renal ischemia might prevent or inhibit positive oxidative stress inhibitory effects by increasing anti-oxidative enzymes (CuSOD, MnSOD) within the kidney and improve to prevent renal function. Thus, pre-exercise may have a protective role against renal injury after renal ischemia.

이 연구는 Hsp70.1 유전자가 결핍된 생쥐를 이용하여 운동전처치에 따른 신장허혈재관류손상에서 혈청 크레아틴, 신장에서 CuSOD와 MnSOD의 발현변화를 관찰하는데 그 목적을 두고 있다. 실험동물은 c57/BL6 계 수컷(wild type: WT)과 Hsp70.1 knockout (KO) 생쥐를 정상대조군(n=8), 운동군(n=8), 허혈운동군(n=8) 및 허혈군(n=8)의 4군으로 분류하여 이용하였다. 실험종료 후 마취를 한 후 혈청 creatinine을 분석하기 위해서 신장에서 혈액을 추출하였고, 신장을 적출하여 western blot 으로 eCuSOD와 MnSOD 발현변화를 비교하였다. KO 허혈군에서의 CuSOD, MnSOD는 다른 군에 비해 유의하게 낮게(p<0.001, p<0.05) 발현하였으며, creatinine은 높은(p<0.001)농도로 나타났다. 반면 WT에서는 유의한 변화가 나타나지 않았다. 흥미롭게도 KO허혈운동군에서의 CuSOD, MnSOD는 허혈군보다 뚜렷하게 증가하였으며, creatinine은 허혈군에 비해 현저히 감소(p<0.01)하였다. 이상의 결과를 종합하면 Hsp70은 신장허혈재관류손상에 직접적인 관련이 있음을 추정할 수 있다. 따라서 운동전 처치는 허혈성신장기능저하에 예방할 수 있다고 생각된다.



  1. Akita, Y., H. Otani, S. Matsuhisa, S. Kyoi, C. Enoki, R. Hattori, H. Imamura, H. Kamihata, Y. Kimura, and T. Iwasaka. 2007. Exercise-induced activation of cardiac sympathetic nerve triggers cardioprotection via redox-sensitive activation of eNOS and upregulation of iNOS. Am. J. Physiol. Heart Circ. Physiol. 292, H2051-2059.
  2. Alexandra, K., M. Adams, and M. Thomas. 2002. The role of antioxidants in exercise and disease prevention; The physician & sports medicine 1, 30.
  3. Beck, F. X., W. Neuhofer, and E. Muller. 2000. Molecular chaperones in the kidney: distribution, putative roles, and regulation. Am. J. Physiol. Renal. 279, F203-215.
  4. Brady, H. R., M. R. Clarkson, and W. Lieberthal. 2004. Acute renal failure : Brenner & Rector's The kidney. Seventh ed. W. B. Saunders Company.
  5. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72, 248-254.
  6. Chen, S. W., M. Kim, M. Kim, J. H. Song, S. W. Park, D. Wells, K. Brown, J. Belleroche, V. D. D'Agati, and H. T. Lee. 2009. Mice that overexpress human heat shock protein 27 have increased renal injury following ischemia reperfusion. Kidney Int. 75, 499-510.
  7. Chen, S. W., B. Siu, Y. S. Ho, R. Vincent, C. C. Chua, R. C. Hamdy, and B. H. Chua. 1998. Overexpression of MnSOD protects against myocardial ischemia/reperfusion injury in transgenic mice. J. Mol. Cell Cardiol. 30, 2281-2289
  8. Chien, C. T., P. H. Lee, C. F. Chen, M. C. Ma, M. K. Lai, and S. M. Hsu. 2001. De novo demonstration and co-localization of free radical production and apoptosis formation in rat kidney subjected to ischemia/reperfusion. J. Am. Soc. Nephrol. 12, 973-982.
  9. Choi, S., K. A. Park, H. J. Lee, M. S. Park, J. H. Lee, K. C. Park, M. Kim, S. H. Lee, J. S. Seo, and B. W. Yoon. 2005. Expression of Cu/Zn SOD protein is suppressed in hsp 70.1 knockout mice. J. Biochem. Mol. Bio. 38, 111-114.
  10. Ding, Y. H., M. Mrizek, Q. Lai, Y. Wu, R. Jr Reyes, J. Li, W. W. Davis, and Y. Ding. 2006. Exercise preconditioning reduces brain damage and inhibits TNF -alpha receptor expression after hypoxia/ reoxygenation: an in vivo and in vitro study. Curr. Neurovasc. Res. 3, 263-271.
  11. Domenech, R. J. 2006. Precondition. A new concept about the benefit of exercise. Circulation. 113, e1-e3.
  12. Fehrenbach, E. and H. Northoff. 2001. Free radical, exercise, apoptosis, and heat shock proteins. Exerc. Immunol. Rev. 7, 66-89.
  13. Fujimura, M., Y. Morita-Fujimura, K. Murakami, M. Kawase, and P. H. Chan. 1998. Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J. Cereb. blood flow. metab. 18, 1239-1247.
  14. Georgopoulos, C. and W. J. Welch. 1993. Role of the major heat shock proteins as molecular chaperones. Annu. Rev. Cell Biol. 9, 601-634.
  15. Hartmann, S. and P. Bung. 1999. Physical exercise during pregnancy-physiological considerations and recommendations. J. Perinat. Med. 27, 204-215.
  16. Husain, K. and S. R. Hazelrigg. 2002. Oxidative injury due to chronic oxide synthase inhibition in rat: effect of regular exercise on the heart. Biochem. Biophys. Acta. 1587, 75-82.
  17. Hunt, C. and S. Calderwood. 1990. Characterization and sequence of a mouse hsp70 gene and its expression in mouse cell lines. Gene 87, 199-204.
  18. Huot, J., F. Houle, D. R. Spitz, and J. Landry. 1996. HSP27 phosphorylation-mediated resistance against actin fragmentation and cell death induced by oxidative stress. Cancer Res. 56, 273-279.
  19. Ji, L. 1993. Antioxidant enzyme responses to exercise and aging. Med. Sci. Sports Exerc. 25, 225-231.
  20. Jones, D. R. and H. T. Lee. 2007. Protecting the kidney during critical illness. Curr. Opin. Anaesthesiol. 20, 106-112.
  21. Kim, J., I. S. Kil, Y. M. Seok, E. S. Yang, D. K. Kim, D. G. Lim, J. W. Park, J. V. Bonventre, and K. M. Park. 2006. Orchiectomy attenuates post-ischemic oxidative stress and ischemia/reperfusion injury in mice. A role for manganese superoxide dismutase. J. Biol. Chem. 281, 20349-20359.
  22. Lee, J., W. K. Kim, and C. S. Shim. 2008. Effects of exercise on Hsp70 Knock-out mice fetuses exposed to maternal hyperthermia. Kor. J. Spo. Sci. 19, 34-43.
  23. Lee, S. H., M. Kim, B. W. Yoon, Y. J. Kim, S. J. Ma, J. K. Roh, J. S. Lee, and J. S. Seo. 2001. Targeted Hsp 70.1 disruption increases infarction volume after focal cerebral ischemia in mice. Stroke. 32, 2905-2912.
  24. Ling, H., C. Edelstein, P. Gengaro, X. Meng, S. Lucia, M. Knotek, A. Wangsiripaisan, Y. Shi, and R. Schrier. 1999. Attenuation of renal ischemia-reperfusion injury in inducible nitric oxide synthase knockout mice. Am. J. Physiol. 277, F383-390.
  25. Locke, M. and E. G. Noble. 1995. Stress proteins: the exercise response. Canadian. J. Appl. Physiol. 20, 155-167.
  26. Lowe, D. G. and L. A. Moran. 1986. Molecular cloning and analysis of DNA complementary to three mouse Mr=68,000 heat shock protein mRNAs. J. Biol. Chem. 261, 2102-2112.
  27. Marber, M. S., R. Mestril, S. H. Chi, M. R. Sayen, D. M. Yellon, and W. H. Dillmann. 1995. Overexpression of the rat inducible 70-kD heat stress protein in a transgenic mouse increases the resistance of the heart to ischemic injury. J. Clin. Invest. 95, 1446-1456.
  28. Moran, M., J. Delgado, B. Gonzalez, R. Manso, and A. Megias. 2004. Responses of rat myocardial antioxidant defences and heat shock protein HSP72 induced by 12 and 24-week treadmill training. Acta. Physiol. Scand. 180, 157-166.
  29. Pokly, A. G. 2001. Heat shock proteins, anti-heat shock protein reactivity and allograft rejection. Transplantation 71, 1503-1507.
  30. Powers, S. K., D. Criswell, J. Lawler, D. Martin, F. K. Lieu, L. L. Ji, and R. A. Herb. 1993. Rigorous exercise training increase superoxide dismutase activity in ventricular myocardium. Am. J. Physiol. 265, 2094-2098.
  31. Powers, S. K., H. Demirel, J. S. Coombes, H. Naito, K. L. Hamilton, R. H. Shanely, and J. Jessup. 1998. Exercsie train-ing improves myocardial tolerance to in vivo ischemia -reperfusion in the rat. Am. J. Physiol. 275, R1468-1477.
  32. Rajdev, S., K. Hara, Y. Kokubo, R. Mestril, W. Dillmann, P. R. Weinstein, and F. R. Sharp. 2000. Mice overexpressing rat heat shock protein 70 are protected against cerebral infaction. Ann. Neurol. 47, 782-791.<782::AID-ANA11>3.0.CO;2-3
  33. Seok, Y. M., J. Kim, K. C. Choi, C. H. Yoon, Y. C. Boo, Y. Park, and K. M. Park. 2007. Wen-pi-tang-Hab-Wu-ling-san attenuates kidney ischemia/reperfusion injury in mice. A role for antioxidant enzymes and heat-shock proteins. J. Ethnopharmacol. 112, 333-340.
  34. Schefer, V. and M. I. Talan. 1996. Oxygen consumption in adult and aged c57BL/6J mice during acute treadmill exercise of different intensity. Exp. Gerontol. 31, 387-392.
  35. Snoeckx, L. H., R. N. Cornelussen, F. A. Van Nieuwenhoven, R. S. Van Der. Reneman, and G. J. Vusse. 2001. Heat shock proteins and cardiovascular pathophysiology. Physiol. Rev. 81, 1461-1497.
  36. van Ginneken, M. M., E. de Graaf-Roelfsema, H. A. Keizer, K. G. van Dam, I. D. Wijnberg, J. H. van der Kolk, and E. van Breda. 2006. Effect of exercise on activation of the p38 mitogen-activated protein kinase pathway, c-Jun NH2 terminal kinase, and heat shock protein 27 in equine skeletal muscle. Am. J. Vet. Res. 67, 837-844.