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

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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.

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Data availability

All data generated or analysed during this study are included in this published article and its supplementary information files. Further, a list of all analyzed metabolites by name, superordinate metabolite group, biochemical sub-pathway and fold change of the respective intracellular concentration is uploaded as Supplementary Table 1 in the online supplements and is available via figshare.com (https://doi.org/10.6084/m9.figshare.7749521). Original mass spectral data are also uploaded on figshare.com and can be found via this link: https://doi.org/10.6084/m9.figshare.9817115.

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Acknowledgements

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

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.

Author information

Author notes

  1. Martina Bielaszewska

    Present address: Reference Laboratory for E. coli and Shigella, National Public Health Institute, Srobarova 48, 10042, Prague, Czech Republic

Authors and Affiliations

  1. Department of Pediatrics I, University Children’s Hospital Heidelberg, 69120, Heidelberg, Germany

    Christian Patry, Kathrin Plotnicki, Christian Betzen, Burkhard Tönshoff & Neysan Rafat

  2. Division of Cardiovascular Physiology, Institute of Physiology and Pathophysiology, University of Heidelberg, 69120, Heidelberg, Germany

    Christian Patry

  3. Division of Functional Genome Analysis, German Cancer Research Center (DKFZ), 69120, Heidelberg, Germany

    Christian Betzen

  4. Department of Neonatology, University Children’s Hospital Mannheim, University of Heidelberg, 68167, Mannheim, Germany

    Alba Perez Ortiz & Neysan Rafat

  5. Metabolon, Inc., 617 Davis Drive, Suite 400, Durham, NC, 27713, USA

    Kirk L. Pappan

  6. Learning and Research Southmead Hospital Bristol, University of Bristol, Bristol, BS8 1TH, UK

    Simon C. Satchell

  7. The Principal’s Office, University of Edinburgh, Edinburgh, EH8 9YL, UK

    Peter W. Mathieson

  8. Institute for Hygiene, University of Münster, 48149, Münster, Germany

    Martina Bielaszewska & Helge Karch

  9. Department of Pharmaceutical Sciences, Bahá’í Institute of Higher Education (BIHE), Teheran, Iran

    Neysan Rafat

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  1. Christian Patry
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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.

Corresponding author

Correspondence to Neysan Rafat.

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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.

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Patry, C., Plotnicki, K., Betzen, C. et al. Metabolomic analysis of Shiga toxin 2a-induced injury in conditionally immortalized glomerular endothelial cells. Metabolomics 15, 131 (2019). https://doi.org/10.1007/s11306-019-1594-2

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