Advertisement

Metabolomics

, 15:131 | Cite as

Metabolomic analysis of Shiga toxin 2a-induced injury in conditionally immortalized glomerular endothelial cells

  • Christian Patry
  • Kathrin Plotnicki
  • Christian Betzen
  • Alba Perez Ortiz
  • Kirk L. Pappan
  • Simon C. Satchell
  • Peter W. Mathieson
  • Martina Bielaszewska
  • Helge Karch
  • Burkhard Tönshoff
  • Neysan RafatEmail author
Original Article

Abstract

Introduction

Shiga toxin 2a (Stx2a) induces hemolytic uremic syndrome (STEC HUS) by targeting glomerular endothelial cells (GEC).

Objectives

We investigated in a metabolomic analysis the response of a conditionally immortalized, stable glomerular endothelial cell line (ciGEnC) to Stx2a stimulation as a cell culture model for STEC HUS.

Methods

CiGEnC were treated with tumor necrosis factor-(TNF)α, Stx2a or sequentially with TNFα and Stx2a. We performed a metabolomic high-throughput screening by lipid- or gas chromatography and subsequent mass spectrometry. Metabolite fold changes in stimulated ciGEnC compared to untreated cells were calculated.

Results

320 metabolites were identified and investigated. In response to TNFα + Stx2a, there was a predominant increase in intracellular free fatty acids and amino acids. Furthermore, lipid- and protein derived pro-inflammatory mediators, oxidative stress and an augmented intracellular energy turnover were increased in ciGEnC. Levels of most biochemicals related to carbohydrate metabolism remained unchanged.

Conclusion

Stimulation of ciGEnC with TNFα + Stx2a is associated with profound metabolic changes indicative of increased inflammation, oxidative stress and energy turnover.

Keywords

Hemolytic uremic syndrome Shiga toxin Conditionally immortalized glomerular endothelial cells Metabolomics 

Notes

Acknowledgements

We thank Prof. Peter Nawroth for kindly providing the laboratory facilities and Dr. Thomas Fleming for his valuable input and methodical assistance.

Author contributions

CP designed and performed research, analyzed and interpreted the data and wrote the first draft of the paper; KLP, CB and APO performed research, analyzed and interpreted the data; KLP, SCS, PWM, MB, HK and BT designed research, analyzed and interpreted the data, NR directed, designed, analyzed and interpreted the data and wrote the paper.

Funding

This work was supported by a Physician Scientist fellowship grant from the Medical Faculty of the University of Heidelberg to C.P., C.B. and N.R.

Compliance with ethical standards

Conflict of interest

KL.P. is an employee of Metabolon, Inc. and, as such, has affiliations with or financial involvement with Metabolon, Inc. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Besides that, there are no other financial or non-financial conflicts of interest.

Research involving human and/or animal participants

This article does not contain any studies with human and/or animal participants performed by any of the authors.

Supplementary material

11306_2019_1594_MOESM1_ESM.docx (121 kb)
Supplementary material 1 (DOCX 121 kb)
11306_2019_1594_MOESM2_ESM.docx (17 kb)
Supplementary material 2 (DOCX 17 kb)

References

  1. Bauwens, A., Bielaszewska, M., Kemper, B., Langehanenberg, P., Von Bally, G., Reichelt, R., et al. (2011). Differential cytotoxic actions of Shiga toxin 1 and Shiga toxin 2 on microvascular and macrovascular endothelial cells. Thrombosis and Haemostasis, 105(3), 515–528.CrossRefGoogle Scholar
  2. Betzen, C., Plotnicki, K., Fathalizadeh, F., Pappan, K., Fleming, T., Bielaszewska, M., et al. (2016). Shiga Toxin 2a—Induced endothelial injury in hemolytic uremic syndrome: A metabolomic analysis. Journal of Infectious Diseases, 213(6), 1031–1040.CrossRefGoogle Scholar
  3. Chan, Y. S., & Ng, T. B. (2016). Shiga toxins: From structure and mechanism to applications. Applied Microbiology and Biotechnology, 100(4), 1597–1610.CrossRefGoogle Scholar
  4. Dehaven, C. D., Evans, A. M., Dai, H., & Lawton, K. A. (2010). Organization of GC/MS and LC/MS metabolomics data into chemical libraries. Journal of Cheminformatics, 2(1), 9.CrossRefGoogle Scholar
  5. Ergonul, Z., Hughes, A. K., & Kohan, D. E. (2003). Induction of apoptosis of human brain microvascular endothelial cells by Shiga Toxin 1. The Journal of Infectious Diseases, 187(1), 154–158.CrossRefGoogle Scholar
  6. Evans, A. M., DeHaven, C. D., Barrett, T., Mitchell, M., & Milgram, E. (2009). Integrated, nontargeted ultrahigh performance liquid chromatography/electrospray ionization tandem mass spectrometry platform for the identification and relative quantification of the small-molecule complement of biological systems. Analytical Chemistry, 81(16), 6656–6667.CrossRefGoogle Scholar
  7. Fakhouri, F., Zuber, J., Frémeaux-Bacchi, V., & Loirat, C. (2017). Haemolytic uraemic syndrome. The Lancet, 390(10095), 681–696.CrossRefGoogle Scholar
  8. Ferraris, V., Acquier, A., Ferraris, J. R., Vallejo, G., Paz, C., & Mendez, C. F. (2011). Oxidative stress status during the acute phase of haemolytic uraemic syndrome. Nephrology, dialysis, transplantation: Official publication of the European Dialysis and Transplant Association—European Renal Association, 26(3), 858–864.CrossRefGoogle Scholar
  9. Gomez, S. A., Abrey-Recalde, M. J., Panek, C. A., Ferrarotti, N. F., Repetto, M. G., Mejías, M. P., et al. (2013). The oxidative stress induced in vivo by Shiga toxin-2 contributes to the pathogenicity of haemolytic uraemic syndrome. Clinical and Experimental Immunology, 173(3), 463–472.CrossRefGoogle Scholar
  10. Gvozdjáková, A. (2008). Carnitine. Mitochondrial medicine: Mitochondrial metabolism, diseases, diagnosis and therapy, 5(3), 357–366.CrossRefGoogle Scholar
  11. Hughes, A. K., Stricklett, P. K., Schmid, D., & Kohan, D. E. (2000). Cytotoxic effect of Shiga toxin-1 on human glomerular epithelial cells. Kidney International, 57(6), 2350–2359.CrossRefGoogle Scholar
  12. Johannes, L., & Römer, W. (2010). Shiga toxins—from cell biology to biomedical applications. Nature Reviews Microbiology, 8(2), 105–116.CrossRefGoogle Scholar
  13. Kaur, J., & Debnath, J. (2015). Autophagy at the crossroads of catabolism and anabolism. Nature Reviews Molecular Cell Biology, 16(8), 461–472.CrossRefGoogle Scholar
  14. Komatsu, M., Waguri, S., Ueno, T., Iwata, J., Murata, S., Tanida, I., et al. (2005). Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. Journal of Cell Biology, 169(3), 425–434.CrossRefGoogle Scholar
  15. Lee, M. S., Cherla, R. P., Jenson, M. H., Leyva-Illades, D., Martinez-Moczygemba, M., & Tesh, V. L. (2011). Shiga toxins induce autophagy leading to differential signalling pathways in toxin-sensitive and toxin-resistant human cells. Cellular Microbiology, 13(10), 1479–1496.CrossRefGoogle Scholar
  16. Meisen, I., Rosenbrück, R., Galla, H. J., Hüwel, S., Kouzel, I. U., Mormann, M., et al. (2013). Expression of Shiga toxin 2e glycosphingolipid receptors of primary porcine brain endothelial cells and toxin-mediated breakdown of the blood-brain barrier. Glycobiology, 23(6), 745–759.CrossRefGoogle Scholar
  17. Melton-Celsa, A. R. (2014). Shiga toxin (Stx) classification, structure, and function. Microbiology Spectrum, 2(3), 1–21.Google Scholar
  18. Mizushima, N., & Klionsky, D. J. (2007). Protein turnover via autophagy: Implications for metabolism. Annual Review of Nutrition, 27, 19–40.CrossRefGoogle Scholar
  19. Nakatogawa, H., Suzuki, K., Kamada, Y., & Ohsumi, Y. (2009). Dynamics and diversity in autophagy mechanisms: Lessons from yeast. Nature Reviews Molecular Cell Biology, 10(7), 458–467.CrossRefGoogle Scholar
  20. Nieman, D. C., Shanely, R. A., Gillitt, N. D., Pappan, K. L., & Lila, M. A. (2013). Serum metabolic signatures induced by a three-day intensified exercise period persist after 14 h of recovery in runners. Journal of Proteome Research, 12(10), 4577–4584.CrossRefGoogle Scholar
  21. Noris, M., & Remuzzi, G. (2005). Hemolytic uremic syndrome. Journal of the American Society of Nephrology, 16(4), 1035–1050.CrossRefGoogle Scholar
  22. Obrig, T. G., Louise, C. B., Lingwood, C. A., Boyd, B., Barley-Maloney, L., & Daniel, T. O. (1993). Endothelial heterogeneity in Shiga toxin receptors and responses. Journal of Biological Chemistry, 268(21), 15484–15488.PubMedGoogle Scholar
  23. Obrig, T. G., Seaner, R. M., Bentz, M., Lingwood, C. A., Boyd, B., Smith, A., et al. (2003). Induction by sphingomyelinase of Shiga toxin receptor and Shiga toxin 2 sensitivity in human microvascular endothelial cells. Infection and Immunity, 71(2), 845–849.CrossRefGoogle Scholar
  24. Potter, B. J., Sorrentino, D., & Berk, P. D. (1989). Mechanisms of cellular uptake of free fatty acids. Annual Review of Nutrition, 9(1), 253–270.CrossRefGoogle Scholar
  25. Satchell, S. C., Tasman, C. H., Singh, A., Ni, L., Geelen, J., Von Ruhland, C. J., et al. (2006). Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF. Kidney International, 69(9), 1633–1640.CrossRefGoogle Scholar
  26. Schmid, D. I., & Kohan, D. E. (2001). Effect of shigatoxin-1 on arachidonic acid release by human glomerular epithelial cells. Kidney International, 60(3), 1026–1036.CrossRefGoogle Scholar
  27. Singh, R., Kaushik, S., Wang, Y., Xiang, Y., Novak, I., Komatsu, M., et al. (2009). Autophagy regulates lipid metabolism. Nature, 458(7242), 1131–1135.CrossRefGoogle Scholar
  28. Tang, B., Li, Q., Zhao, X., Wang, H., Li, N., Fang, Y., et al. (2015). Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway. Autophagy, 11(2), 344–354.CrossRefGoogle Scholar
  29. Tesh, V. L. (2012). Activation of cell stress response pathways by Shiga toxins. Cellular Microbiology, 14(1), 1–9.CrossRefGoogle Scholar
  30. Zou, M.-H. (2015). Tryptophan-kynurenine pathway is dysregulated in inflammation and immune activation. Frontiers in Bioscience, 20(7), 4363.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Christian Patry
    • 1
    • 2
  • Kathrin Plotnicki
    • 1
  • Christian Betzen
    • 1
    • 3
  • Alba Perez Ortiz
    • 4
  • Kirk L. Pappan
    • 5
  • Simon C. Satchell
    • 6
  • Peter W. Mathieson
    • 7
  • Martina Bielaszewska
    • 8
    • 10
  • Helge Karch
    • 8
  • Burkhard Tönshoff
    • 1
  • Neysan Rafat
    • 1
    • 4
    • 9
    Email author
  1. 1.Department of Pediatrics IUniversity Children’s Hospital HeidelbergHeidelbergGermany
  2. 2.Division of Cardiovascular Physiology, Institute of Physiology and PathophysiologyUniversity of HeidelbergHeidelbergGermany
  3. 3.Division of Functional Genome AnalysisGerman Cancer Research Center (DKFZ)HeidelbergGermany
  4. 4.Department of Neonatology, University Children’s Hospital MannheimUniversity of HeidelbergMannheimGermany
  5. 5.Metabolon, Inc.DurhamUSA
  6. 6.Learning and Research Southmead Hospital BristolUniversity of BristolBristolUK
  7. 7.The Principal’s OfficeUniversity of EdinburghEdinburghUK
  8. 8.Institute for Hygiene, University of MünsterMünsterGermany
  9. 9.Department of Pharmaceutical SciencesBahá’í Institute of Higher Education (BIHE)TeheranIran
  10. 10.Reference Laboratory for E. coli and ShigellaNational Public Health InstitutePragueCzech Republic

Personalised recommendations