Skip to main content

Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury

Summary

In renal tubular epithelial cells, oxidant injury results in several metabolic alterations including ATP depletion, decreased Na+K+ ATPase activity, and altered intracellular sodium and potassium content. To investigate the recovery of LLC-PK1 cells following oxidant injury and to determine if recovery can be accelerated, we induced oxidant stress in LLC-PK1 cells with 500 µM hydrogen peroxide for 60 min. Identical cohorts of oxidant-stressed cells were incubated in recovery medium without epidermal growth factor (EGF) or recovery medium containing 25 ng EGF per ml. ATP levels, Na+K+ ATPase activity in whole cells, Na+K+ ATPase activity in disrupted cells, and intracellular sodium and potassium ion content were determined at 0, 5, 24, 48, and 72 h following oxidant injury in each cohort of cells. In oxidant-stressed cells recovering in medium without EGF, ATP levels, Na+K+ ATPase activity, and intracellular ion content improved but continued to remain substantially lower than control values at all time points following oxidant stress. In cells recovering in medium with EGF, ATP levels, Na+K+ ATPase activity, and the intracellular potassium-to-sodium ratio were significantly higher at nearly all time points than values in cells recovering in medium alone. In cells recovering with added EGF, Na+K+ ATPase activity had improved to control levels, whereas ATP levels and intracellular ion content approached control values by 72 h following oxidant stress. We conclude that oxidant-mediated ATP depletion, altered Na+K+ ATPase activity, and intracellular ion content remain depressed for several d following oxidant stress and that EGF accelerated recovery of LLC-PK1 cells from oxidant injury.

This is a preview of subscription content, access via your institution.

References

  1. Andreoli, S. P. Reactive oxygen molecules, oxidant injury and renal disease. Pediatr. Nephrol. 5:733–742; 1991.

    PubMed  Article  CAS  Google Scholar 

  2. Andreoli, S. P.; Baehner, R. L.; Bergstein, J. M. In vitro detection of endothelial cell damage utilizing deoxy-D-3H-glucose: comparison with 51chromium, 3H-leucine, 3H-adenine and LDH. J. Lab. Clin. Med. 106:253–261; 1985.

    PubMed  CAS  Google Scholar 

  3. Andreoli, S. P.; McAteer, J. A. Reactive oxygen molecule-mediated injury in endothelial cells and renal tubule epithelial cells in vitro. Kidney Int. 38:785–794; 1990.

    PubMed  CAS  Google Scholar 

  4. Andreoli, S. P.; McAteer, J. A.; Seifert, S. A.; Kempson, S. A. Oxidant induced alterations in glucose and phosphate transport in LLC-PK1 cells: mechanisms of injury. Am. J. Physiol. 265:F377-F384; 1993.

    PubMed  CAS  Google Scholar 

  5. Baliga, R.; Ueda, N.; Walker, P. D.; Shah, S. V. Oxidant mechanisms in toxic acute renal failure. Am. J. Kidney Dis. 29:465–477; 1997.

    PubMed  CAS  Google Scholar 

  6. Baud, L.; Ardaillou, R. Reactive oxygen species: production and role in the kidney. Am. J. Physiol. 251 (Renal Fluid Electrolyte Physiol. 20):F765-F776; 1986.

    PubMed  CAS  Google Scholar 

  7. Bonting, S. L.; Simon, K. A.; Hawkins, N. M. Studies on sodium-potassium-activated adenosine triphosphatase. Quantitative distribution in several tissues of the cat. Arch. Biochem. Biophys. 95:416–423; 1961.

    PubMed  Article  CAS  Google Scholar 

  8. Coimbra, T. M.; Cieslinski, D. A.; Humes, H. D. Epidermal growth factor accelerates renal repair in mercuric chloride nephrotoxicity. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol.):F438-F443; 1990.

    PubMed  CAS  Google Scholar 

  9. Cross, C. E.; Halliwell, B.; Borish, E. T.; Pryor, W. A.; Ames, B. A.; Saul, R. L.; McCord, J. M.; Harmon, D. Oxygen radicals and human disease. Ann. Intern. Med. 107:526–545; 1988.

    Google Scholar 

  10. Ding, H.; Kopple, J. D.; Cohen, A.; Hirschberg, R. Recombinant human insulin-like growth factor-1 accelerates recovery and reduces catabolism in rats with ischemic acute renal failure. J. Clin. Invest. 91:2281–2287; 1993.

    PubMed  CAS  Google Scholar 

  11. Farber, J. L.; Kyle, M. E.; Coleman, J. B. Mechanisms of cell injury by activated oxygen species. Lab. Invest. 62:670–679; 1990.

    PubMed  CAS  Google Scholar 

  12. Fisher, D. A.; Salido, E. C.; Barajas, L. Epidermal growth factor and the kidney. Annu. Rev. Physiol. 51:67–80; 1989.

    PubMed  Article  CAS  Google Scholar 

  13. Gailit, J.; Colflesh, D.; Rabiner, I.; Simone, J.; Goligorsky, M. S. Redistribution and dysfunction of integrins in cultured renal epithelial cells exposed to oxidative stress. Am. J. Physiol. 264:F149-F157; 1993.

    PubMed  CAS  Google Scholar 

  14. Gardner, A. M.; Xu, F. H.; Fady, C.; Jacoby, F. J.; Duffey, D. C.; Tu, Y.; Lichtenstein, A. Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Radical Biol. Med. 22:73–83; 1997.

    Article  CAS  Google Scholar 

  15. Goodyear, P. R.; Kachra, A.; Bell, C.; Rozen, R. Renal tubular cells are potential targets for epidermal growth factor. Am. J. Physiol. 255:F1191-F1196; 1988.

    Google Scholar 

  16. Hannemann, J.; Baumann, K. Cisplatin induced lipid peroxidation and decrease of gluconeogenesis in rat kidney cortex: different effects of antioxidant and radical scavengers. Toxicology 51:119–132; 1988.

    PubMed  Article  CAS  Google Scholar 

  17. Hammerman, M. R.; Miller, S. B. The growth hormone insulin-like growth factor axis in the kidney revisited. Am. J. Physiol. 265:F1-F14; 1993.

    PubMed  CAS  Google Scholar 

  18. Hammerman, M. R.; Miller, S. B. Therapeutic use of growth factors in renal failure. J. Am. Soc. Nephrol. 5:1–11; 1994.

    PubMed  CAS  Google Scholar 

  19. Harris, R. C.; Daniel, T. O. Epidermal growth factor binding, stimulation of phosphorylation, and inhibition of gluconeogenesis in rat proximal tubule. J. Cell. Physiol. 139:383–391; 1989.

    PubMed  Article  CAS  Google Scholar 

  20. Hull, R. N.; Cherry, W. R.; Weaver, G. W. The origin and characteristics of a pig kidney cell strain, LLC-PK1. In Vitro 12:670–677; 1976.

    PubMed  CAS  Google Scholar 

  21. Humes, D. H.; Beals, T. F.; Cieslinski, D. A.; Sanchez, I. O.; Page, T. P. Effects of transforming growth factor-b, transforming growth factor-a, and other growth factors on renal proximal tubule cells. Lab. Invest. 64:538–545; 1991.

    PubMed  CAS  Google Scholar 

  22. Humes, H. D.; Cieslinski, D. A.; Coimbra, T. M.; Messana, J. M.; Galvao, C. Epidermal growth factor enhances renal tubule cell regeneration and repair and accelerates the recovery of renal function in post ischemic acute renal failure. J. Clin. Invest. 84:1757–1761; 1989.

    PubMed  CAS  Google Scholar 

  23. Janssen, Y. M. W.; Houten, B. V.; Borm, P. J. A.; Mossman, B. T. Cell and tissue responses to oxidative damage. Lab. Invest. 69:261–274; 1993.

    PubMed  CAS  Google Scholar 

  24. Johnson, R. J.; Lovett, D.; Lehrer, R. I.; Couser, W. G.; Klebanoff, S. J. Role of oxidants and proteases in glomerular injury. Kidney Int. 45:352–359; 1994.

    PubMed  CAS  Google Scholar 

  25. Kako, K.; Kato, M.; Matsuoka, T.; Mustapha, A. Depression of membranebound Na+K+ ATPase activity induced by free radicals and by ischemia of kidney. Am. J. Physiol. 254:C330-C337; 1988.

    PubMed  CAS  Google Scholar 

  26. Kays, S. E.; Schnellmann, R. G. Regeneration of renal proximal tubule cells in primary culture following toxicant injury: response to growth factors. Toxicol. Appl. Pharmacol. 132:273–280; 1995.

    PubMed  Article  CAS  Google Scholar 

  27. McCord, J. M. Oxygen-derived free radicals in postischemic tissue injury. N. Engl. J. Med. 312:159–163; 1985.

    PubMed  CAS  Article  Google Scholar 

  28. Miller, S. B.; Martin, D. R.; Kissane, J.; Hammerman, M. R. Insulin-like growth factor I accelerates recovery from ischemic acute tubular necrosis in the rat. Proc. Natl. Acad. Sci. 89:11876–11880; 1992.

    PubMed  Article  CAS  Google Scholar 

  29. Molitoris, B. A. Ischemic-induced loss of epithelial polarity: potential role of the actin cytoskeleton. Am. J. Physiol. 260:F769-F778; 1991.

    PubMed  CAS  Google Scholar 

  30. Montrose, M. H. Measurement of intracellular sodium and potassium in cultured epithelial cells. J. Tiss. Cult. Methods 13:211–216; 1991.

    Article  Google Scholar 

  31. Morin, N. L.; Laurent, G.; Nonclercq, D.; Toubeau, G.; Heuson-Stiennon, J. A.; Bergeron, M. G.; Beauchamp, D. Epidermal growth factor accelerates renal tissue repair in a model of gentamycin nephrotoxicity in rats. Am. J. Physiol. 263:F806-F811; 1992.

    PubMed  CAS  Google Scholar 

  32. Nath, K. A.; Croatt, A. J.; Hostetter, T. H. Oxygen consumption and oxidant stress in surviving nephrons. Am. J. Physiol. 258:F1354-F1362; 1990.

    PubMed  CAS  Google Scholar 

  33. Norman, J.; Tsau, Y. K.; Bacay, A.; Fine, L. G. Epidermal growth factor accelerates functional recovery from ischemic acute tubular necrosis in the rat: role of the epidermal growth factor receptor. Clin. Sci. 78:445–450; 1990.

    PubMed  CAS  Google Scholar 

  34. Nowak, G.; Schellmann, R. G. Integrative effects of EGF on metabolism and proliferation in renal proximal tubular cells. Am. J. Physiol. 269:C1317-C1325; 1995.

    PubMed  CAS  Google Scholar 

  35. Paller, M. S.; Hoidal, J. R.; Ferris, T. F. Oxygen free radicals in ischemic acute renal failure in the rat. J. Clin. Invest. 74:1156–1164; 1984.

    PubMed  CAS  Google Scholar 

  36. Paller, M. S.; Neumann, T. V. Reactive oxygen species and rat renal epithelial cells during hypoxia and reoxygenation. Kidney Int. 40:1041–1049; 1991.

    PubMed  CAS  Google Scholar 

  37. Price, P. M.; Megyesi, J.; Saggi, S.; Safirstein, R. Regulation of transcription by the rat EGF gene promoter in normal and ischemic murine kidney cells. Am. J. Physiol. 268:F664-F670; 1995.

    PubMed  CAS  Google Scholar 

  38. Safirstein, R.; Zelent, A. Z.; Price, P. M. Reduced renal prepro-epidermal growth factor mRNA and decreased EGF excretion in ARF. Kidney Int. 36:810–815; 1989.

    PubMed  CAS  Google Scholar 

  39. Schaudies, R. P.; Nonclercq, D.; Nelson, L.; Toubeau, G.; Aznzen, J.; Heuso-Steinnon, J. A.; Laurent, G. Endogenous EGF as a potential renotropic factor in ischemia-induced acute renal failure. Am. J. Physiol. 265:F425-F434; 1993.

    PubMed  CAS  Google Scholar 

  40. Schnellmann, R. G. Mechanism of t-butyl hydroperoxide-induced toxicity to rabbit renal proximal tubules. Am. J. Physiol. 255:C28-C33; 1988.

    PubMed  CAS  Google Scholar 

  41. Sogabe, K.; Roeser, N. F.; Venkatachalam, M. A.; Weinberg, J. M. Differential cytoprotection by glycine against oxidant damage to proximal tubule cells. Kidney Int. 50:845–854; 1996.

    PubMed  Article  CAS  Google Scholar 

  42. Stanton, R. C.; Seifer, J. L. Epidermal growth factor rapidly activates the hexose monophosphate shunt in kidney cells. Am. J. Physiol. (Cell) 253:C267-C271; 1988.

    Google Scholar 

  43. Ueda, N.; Shah, S. V. Endonuclease-induced DNA damage and cell death in oxidant injury to renal tubular epithelial cells. J. Clin. Invest. 90:2593–2597; 1992.

    PubMed  CAS  Google Scholar 

  44. Walker, P. D.; Shah, S. V. Evidence suggesting a role for hydroxyl radical in gentamycin induced acute renal failure in rats. J. Clin. Invest. 81:334–341; 1988.

    PubMed  CAS  Google Scholar 

  45. Weinberg, J. M.; Varani, J.; Johnson, K. J.; Roeser, N. F.; Dame, M. K.; Davis, J. A.; Venkatachalam, M. A. Protection of human umbilical vein endothelial cells by glycine and structurally similar amino acids against calcium and hydrogen peroxide-induced lethal cell injury. Am. J. Pathol. 140:457–471; 1992.

    PubMed  CAS  Google Scholar 

  46. Welsh, M. J.; Shasby, D. M.; Husted, R. M. Oxidants increase paracellular permeability in a cultured epithelial cell line. J. Clin. Invest. 76:1155–1168; 1985.

    PubMed  CAS  Article  Google Scholar 

  47. Yaqoob, M.; Edelstein, C. L.; Wieder, E. D.; Alkhunaizi, A. M.; Gengaro, P. E.; Nemenoff, R. A.; Schrier, R. W. Nitric oxide kinetics during hypoxia in proximal tubules. Kidney Int. 49:1314–1319; 1996.

    PubMed  CAS  Google Scholar 

  48. Yu, L.; Gengaro, P. E.; Niederberger, M.; Burke, T. J.; Schrier, R. W. Nitric oxide: a mediator in rat tubular hypoxia/reoxygenation injury. Proc. Nat. Acad. Sci. 91:1691–1695; 1994.

    PubMed  Article  CAS  Google Scholar 

  49. Zager, R. A.; Burkhart, K. Myoglobin toxicity in proximal human kidney cells: roles of Fe, Ca2+, H2O2, and terminal mitochondrial electron transport. Kidney Int. 51:728–738; 1997.

    PubMed  Article  CAS  Google Scholar 

  50. Zager, R. A.; Burkhart, K. M.; Conrad, D. S.; Gmur, D. J. Iron, heme oxygenase, glutathione: effects on myohemoglobinuric proximal tubular injury. Kidney Int. 48:1624–1634; 1995.

    PubMed  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Andreoli, S.P., Mallett, C.P., McAteer, J.A. et al. Epidermal growth factor accelerates recovery of LLC-PK1 cells following oxidant injury. In Vitro Cell.Dev.Biol.-Animal 34, 824–830 (1998). https://doi.org/10.1007/s11626-998-0037-6

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-998-0037-6

Key words

  • oxidant injury
  • hydrogen peroxide
  • EGF
  • ATP
  • Na+K+ ATPase