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Archives of Toxicology

, Volume 89, Issue 12, pp 2273–2289 | Cite as

Glutathione and mitochondria determine acute defense responses and adaptive processes in cadmium-induced oxidative stress and toxicity of the kidney

  • Ambily Ravindran Nair
  • Wing-Kee Lee
  • Karen Smeets
  • Quirine Swennen
  • Amparo Sanchez
  • Frank ThévenodEmail author
  • Ann CuypersEmail author
Molecular Toxicology

Abstract

Cadmium (Cd2+) induces oxidative stress that ultimately defines cell fate and pathology. Mitochondria are the main energy-producing organelles in mammalian cells, but they also have a central role in formation of reactive oxygen species, cell injury, and death signaling. As the kidney is the major target in Cd2+ toxicity, the roles of oxidative signature and mitochondrial function and biogenesis in Cd2+-related stress outcomes were investigated in vitro in cultured rat kidney proximal tubule cells (PTCs) (WKPT-0293 Cl.2) for acute Cd2+ toxicity (1–30 µM, 24 h) and in vivo in Fischer 344 rats for sub-chronic Cd2+ toxicity (1 mg/kg CdCl2 subcutaneously, 13 days). Whereas 30 µM Cd2+ caused ~50 % decrease in cell viability, apoptosis peaked at 10 µM Cd2+ in PTCs. A steep, dose-dependent decline in reduced glutathione (GSH) content occurred after acute exposure and an increase of the oxidized glutathione (GSSG)/GSH ratio. Quantitative PCR analyses evidenced increased antioxidative enzymes (Sod1, Gclc, Gclm), proapoptotic Bax, metallothioneins 1A/2A, and decreased antiapoptotic proteins (Bcl-xL, Bcl-w). The positive regulator of mitochondrial biogenesis Pparγ and mitochondrial DNA was increased, and cellular ATP was unaffected with Cd2+ (1–10 µM). In vivo, active caspase-3, and hence apoptosis, was detected by FLIVO injection in the kidney cortex of Cd2+-treated rats together with an increase in Bax mRNA. However, antiapoptotic genes (Bcl-2, Bcl-xL, Bcl-w) were also upregulated. Both GSSG and GSH increased with chronic Cd2+ exposure with no change in GSSG/GSH ratio and augmented expression of antioxidative enzymes (Gpx4, Prdx2). Mitochondrial DNA, mitofusin 2, and Pparα were increased indicating enhanced mitochondrial biogenesis and fusion. Hence, these results demonstrate a clear involvement of higher mitochondria copy numbers or mass and mitochondrial function in acute defense against oxidative stress induced by Cd2+ in renal PTCs as well as in adaptive processes associated with chronic renal Cd2+ toxicity.

Keywords

Oxidative stress Metallothionein Antioxidative enzymes Mitochondrial DNA content Apoptosis 

Notes

Acknowledgments

The authors would like to thank Rosette Beenaerts, Biomedical Research Institute, Hasselt University for her technical assistance and Dr. Michael D. Garrick at the Department of Biochemistry, SUNY, Buffalo, NY 14214, USA for the Fischer 344 rats. This work was supported by Hasselt University [BOF (Bijzonder onderzoeksfonds) projects; BOF08G01] through a PhD grant for Ambily Ravindran Nair and a grant from the Deutsche Forschungsgemeinschaft (DFG TH345/11-1) to Frank Thévenod. Additional funding came from tUL-impulsfinanciering (project toxicology), and Methusalem project (08M03VGRJ).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

204_2014_1401_MOESM1_ESM.pdf (66 kb)
Supplemental Table 1. Primer details of genes investigated. Column 2: The name of the gene and its official symbol; Column 3: The source of primers- (a) author name and year represents published literature, (b) NCBI/ PrimerBLAST represents primers designed at our lab using the PrimerBLAST software available from www.ncbi.nlm.nih.gov and (c) RTPrimerDB, a database that provides primer sequences and is available from www.rtprimerdb.org; Column 4: The amplicon length of the PCR product; Column 5: The primer efficiency of all genes with two percentages, originating from either pooled control samples or pooled treated samples tested for their efficiency. (PDF 66 kb)

References

  1. Barbier O, Jacquillet G, Tauc M, Cougnon M, Poujeol P (2005) Effect of heavy metals on, and handling by, the kidney. Nephron Physiol 99(4):105–110CrossRefGoogle Scholar
  2. Bhat HK, Epelboym I (2004) Quantitative analysis of total mitochondrial DNA: competitive polymerase chain reaction versus real-time polymerase chain reaction. J Biochem Mol Toxicol 18(4):180–186. doi: 10.1002/jbt.20024 CrossRefPubMedGoogle Scholar
  3. Cadenas E, Davies KJ (2000) Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 29(3–4):222–230CrossRefPubMedGoogle Scholar
  4. Cannino G, Ferruggia E, Luparello C, Rinaldi AM (2009) Cadmium and mitochondria. Mitochondrion 9(6):377–384. doi: 10.1016/j.mito.2009.08.009 CrossRefPubMedGoogle Scholar
  5. Chen Y, Shertzer HG, Schneider SN, Nebert DW, Dalton TP (2005) Glutamate cysteine ligase catalysis: dependence on ATP and modifier subunit for regulation of tissue glutathione levels. J Biol Chem 280(40):33766–33774. doi: 10.1074/jbc.M504604200 CrossRefPubMedGoogle Scholar
  6. Collino M, Patel NS, Lawrence KM et al (2005) The selective PPARgamma antagonist GW9662 reverses the protection of LPS in a model of renal ischemia-reperfusion. Kidney Int 68(2):529–536. doi: 10.1111/j.1523-1755.2005.00430.x CrossRefPubMedGoogle Scholar
  7. Cuypers A, Plusquin M, Remans T et al (2010) Cadmium stress: an oxidative challenge. Biometals 23(5):927–940. doi: 10.1007/s10534-010-9329-x
  8. de Brito OM, Scorrano L (2008) Mitofusin 2 tethers endoplasmic reticulum to mitochondria. Nature 456(7222):605–610. doi: 10.1038/nature07534 CrossRefPubMedGoogle Scholar
  9. Dikalov S (2011) Cross talk between mitochondria and NADPH oxidases. Free Radic Biol Med 51(7):1289–1301. doi: 10.1016/j.freeradbiomed.2011.06.033 PubMedCentralCrossRefPubMedGoogle Scholar
  10. Gobe G, Crane D (2010) Mitochondria, reactive oxygen species and cadmium toxicity in the kidney. Toxicol Lett 198(1):49–55. doi: 10.1016/j.toxlet.2010.04.013 CrossRefPubMedGoogle Scholar
  11. Halliwell B (1994) Free radicals, antioxidants, and human disease: curiosity, cause, or consequence? Lancet 344(8924):721–724CrossRefPubMedGoogle Scholar
  12. Handschin C, Spiegelman BM (2006) Peroxisome proliferator-activated receptor gamma coactivator 1 coactivators, energy homeostasis, and metabolism. Endocr Rev 27(7):728–735. doi: 10.1210/er.2006-0037 CrossRefPubMedGoogle Scholar
  13. Hiratsuka H, Katsuta O, Iwata H, Matsumoto J, Umemura T (1993) Acute toxicity of cadmium in rats with or without cadmium pretreatment. J Toxicol Sci 18(3):197–201CrossRefPubMedGoogle Scholar
  14. Jones DP (2006) Redefining oxidative stress. Antioxid Redox Signal 8(9–10):1865–1879. doi: 10.1089/ars.2006.8.1865 CrossRefPubMedGoogle Scholar
  15. Jones AW, Yao Z, Vicencio JM, Karkucinska-Wieckowska A, Szabadkai G (2012) PGC-1 family coactivators and cell fate: roles in cancer, neurodegeneration, cardiovascular disease and retrograde mitochondria-nucleus signalling. Mitochondrion 12(1):86–99. doi: 10.1016/j.mito.2011.09.009 CrossRefPubMedGoogle Scholar
  16. Klaassen CD, Liu J, Choudhuri S (1999) Metallothionein: an intracellular protein to protect against cadmium toxicity. Annu Rev Pharmacol Toxicol 39:267–294CrossRefPubMedGoogle Scholar
  17. Lee HC, Yin PH, Lu CY, Chi CW, Wei YH (2000) Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. Biochem J 348(Pt 2):425–432PubMedCentralCrossRefPubMedGoogle Scholar
  18. Lee WK, Bork U, Gholamrezaei F, Thévenod F (2005) Cd2+-induced cytochrome c release in apoptotic proximal tubule cells: role of mitochondrial permeability transition pore and Ca2+ uniporter. Am J Physiol Renal Physiol 288(1):F27–F39CrossRefPubMedGoogle Scholar
  19. Lee WK, Torchalski B, Thévenod F (2007) Cadmium-induced ceramide formation triggers calpain-dependent apoptosis in cultured kidney proximal tubule cells. Am J Physiol Cell Physiol 293(3):C839–C847CrossRefPubMedGoogle Scholar
  20. Lenaz G (2012) Mitochondria and reactive oxygen species. which role in physiology and pathology? Adv Exp Med Biol 942:93–136. doi: 10.1007/978-94-007-2869-1_5 CrossRefPubMedGoogle Scholar
  21. L’hoste S, Chargui A, Belfodil R et al (2009) CFTR mediates cadmium-induced apoptosis through modulation of ROS level in mouse proximal tubule cells. Free Radic Biol Med 46:1017–1031CrossRefPubMedGoogle Scholar
  22. Li S, Nagothu KK, Desai V et al (2009) Transgenic expression of proximal tubule peroxisome proliferator-activated receptor-alpha in mice confers protection during acute kidney injury. Kidney Int 76(10):1049–1062. doi: 10.1038/ki.2009.330 PubMedCentralCrossRefPubMedGoogle Scholar
  23. Liesa M, Borda-d’Agua B, Medina-Gomez G et al (2008) Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta. PLoS ONE 3(10):e3613. doi: 10.1371/journal.pone.0003613 PubMedCentralCrossRefPubMedGoogle Scholar
  24. Liu J, Qu W, Kadiiska MB (2009) Role of oxidative stress in cadmium toxicity and carcinogenesis. Toxicol Appl Pharmacol 238(3):209–214. doi: 10.1016/j.taap.2009.01.029 PubMedCentralCrossRefPubMedGoogle Scholar
  25. Lowry O, Rosebrough N, Farr A, Randall RJ (1951) Protein measurements with the Folin phenol reagent. J Biol Chem 194:265–275Google Scholar
  26. Maret W (2011) Redox biochemistry of mammalian metallothioneins. J Biol Inorg Chem 16(7):1079–1086. doi: 10.1007/s00775-011-0800-0 CrossRefPubMedGoogle Scholar
  27. Martindale JL, Holbrook NJ (2002) Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol 192(1):1–15. doi: 10.1002/jcp.10119 CrossRefPubMedGoogle Scholar
  28. Mishra P, Chan DC (2014) Mitochondrial dynamics and inheritance during cell division, development and disease. Nat Rev Mol Cell Biol 15(10):634–646. doi: 10.1038/nrm3877 PubMedCentralCrossRefPubMedGoogle Scholar
  29. Nair AR, Degheselle O, Smeets K, Van Kerkhove E, Cuypers A (2013) Cadmium-induced pathologies: where is the oxidative balance lost (or not)? Int J Mol Sci 14(3):6116–6143. doi: 10.3390/ijms14036116 PubMedCentralCrossRefPubMedGoogle Scholar
  30. Nair AR, Smeets K, Keunen E et al (2014) Renal cells exposed to cadmium in vitro and in vivo: normalising gene expression data. J Appl Toxicol (in press) doi: 10.1002/jat.3047
  31. Orrenius S, Gogvadze V, Zhivotovsky B (2007) Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 47:143–183. doi: 10.1146/annurev.pharmtox.47.120505.105122 CrossRefPubMedGoogle Scholar
  32. Owen JB, Butterfield DA (2010) Measurement of oxidized/reduced glutathione ratio. Methods Mol Biol 648:269–277. doi: 10.1007/978-1-60761-756-3_18 CrossRefPubMedGoogle Scholar
  33. Queval G, Noctor G (2007) A plate reader method for the measurement of NAD, NADP, glutathione, and ascorbate in tissue extracts: application to redox profiling during Arabidopsis rosette development. Anal Biochem 363(1):58–69. doi: 10.1016/j.ab.2007.01.005 CrossRefPubMedGoogle Scholar
  34. Scarpulla RC (2008) Transcriptional paradigms in mammalian mitochondrial biogenesis and function. Physiol Rev 88(2):611–638. doi: 10.1152/physrev.00025.2007 CrossRefPubMedGoogle Scholar
  35. Scarpulla RC (2011) Metabolic control of mitochondrial biogenesis through the PGC-1 family regulatory network. Biochim Biophys Acta 1813(7):1269–1278. doi: 10.1016/j.bbamcr.2010.09.019 PubMedCentralCrossRefPubMedGoogle Scholar
  36. Scarpulla RC (2012) Nucleus-encoded regulators of mitochondrial function: integration of respiratory chain expression, nutrient sensing and metabolic stress. Biochim Biophys Acta 1819(9–10):1088–1097. doi: 10.1016/j.bbagrm.2011.10.011 PubMedCentralCrossRefPubMedGoogle Scholar
  37. Singhal RK, Anderson ME, Meister A (1987) Glutathione, a first line of defense against cadmium toxicity. FASEB J 1(3):220–223PubMedGoogle Scholar
  38. Sinha K, Das J, Pal PB, Sil PC (2013) Oxidative stress: the mitochondria-dependent and mitochondria-independent pathways of apoptosis. Arch Toxicol. doi: 10.1007/s00204-013-1034-4 Google Scholar
  39. Small DM, Morais C, Coombes JS, Bennett NC, Johnson DW, Gobe GC (2014) Oxidative stress-induced alterations in PPARgamma and associated mitochondrial destabilisation contribute to kidney cell apoptosis. Am J Physiol Renal Physiol 307(7):F814–F822. doi: 10.1152/ajprenal.00205.2014 Google Scholar
  40. Taguchi K, Motohashi H, Yamamoto M (2011) Molecular mechanisms of the Keap1-Nrf2 pathway in stress response and cancer evolution. Genes Cells 16(2):123–140. doi: 10.1111/j.1365-2443.2010.01473.x CrossRefPubMedGoogle Scholar
  41. Thévenod F (2010) Catch me if you can! Novel aspects of cadmium transport in mammalian cells. Biometals 23(5):857–875. doi: 10.1007/s10534-010-9309-1
  42. Thévenod F, Lee WK (2013) Toxicology of cadmium and its damage to Mammalian organs. Met Ions Life Sci 11:415–490. doi: 10.1007/978-94-007-5179-8_14 CrossRefPubMedGoogle Scholar
  43. Thijssen S, Cuypers A, Maringwa J et al (2007a) Low cadmium exposure triggers a biphasic oxidative stress response in mice kidneys. Toxicology 236(1–2):29–41. doi: 10.1016/j.tox.2007.03.022 CrossRefPubMedGoogle Scholar
  44. Thijssen S, Maringwa J, Faes C, Lambrichts I, Van Kerkhove E (2007b) Chronic exposure of mice to environmentally relevant, low doses of cadmium leads to early renal damage, not predicted by blood or urine cadmium levels. Toxicology 229(1–2):145–156CrossRefPubMedGoogle Scholar
  45. Trachootham D, Lu W, Ogasawara MA, Nilsa RD, Huang P (2008) Redox regulation of cell survival. Antioxid Redox Signal 10(8):1343–1374. doi: 10.1089/ars.2007.1957 PubMedCentralCrossRefPubMedGoogle Scholar
  46. Wang Y, Fang J, Leonard SS, Rao KM (2004) Cadmium inhibits the electron transfer chain and induces reactive oxygen species. Free Radic Biol Med 36(11):1434–1443CrossRefPubMedGoogle Scholar
  47. Westermann B (2010) Mitochondrial fusion and fission in cell life and death. Nat Rev Mol Cell Biol 11(12):872–884. doi: 10.1038/nrm3013 CrossRefPubMedGoogle Scholar
  48. Woost PG, Orosz DE, Jin W et al (1996) Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats. Kidney Int 50(1):125–134CrossRefPubMedGoogle Scholar
  49. Zhan M, Brooks C, Liu F, Sun L, Dong Z (2013) Mitochondrial dynamics: regulatory mechanisms and emerging role in renal pathophysiology. Kidney Int 83(4):568–581. doi: 10.1038/ki.2012.441 PubMedCentralCrossRefPubMedGoogle Scholar
  50. Zorzano A, Liesa M, Sebastian D, Segales J, Palacin M (2010) Mitochondrial fusion proteins: dual regulators of morphology and metabolism. Semin Cell Dev Biol 21(6):566–574. doi: 10.1016/j.semcdb.2010.01.002 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Ambily Ravindran Nair
    • 1
  • Wing-Kee Lee
    • 2
  • Karen Smeets
    • 1
  • Quirine Swennen
    • 1
  • Amparo Sanchez
    • 1
  • Frank Thévenod
    • 2
    Email author
  • Ann Cuypers
    • 1
    Email author
  1. 1.Environmental Biology, Centre for Environmental SciencesHasselt UniversityDiepenbeekBelgium
  2. 2.Chair of Physiology, Pathophysiology and Toxicology, Centre for Biomedical Education and ResearchWitten/Herdecke UniversityWittenGermany

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