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.
Subject(s)
Endothelial Cells/drug effects , Endothelial Cells/metabolism , Kidney Glomerulus/cytology , Metabolomics , Shiga Toxin 2/pharmacology , Cell Count , Cell Survival/drug effects , Cells, Cultured , Endothelial Cells/cytology , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/pathology , Lipopolysaccharides , Multivariate Analysis , Shiga Toxin 2/metabolismABSTRACT
BACKGROUND: Endothelial dysfunction plays a pivotal role in the pathogenesis of postenteropathic hemolytic uremic syndrome (HUS), most commonly caused by Shiga toxin (Stx)-producing strains of Escherichia coli. METHODS: To identify new treatment targets, we performed a metabolomic high-throughput screening to analyze the effect of Stx2a, the major Stx type associated with HUS, on human renal glomerular endothelial cells (HRGEC) and umbilical vein endothelial cells (HUVEC). Cells were treated either with sensitizing tumor necrosis factor α (TNF-α) or Stx2a, a sequence of both or remained untreated. RESULTS: We identified 341 metabolites by combined liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry. Both cell lines exhibited distinct metabolic reaction profiles but shared elevated levels of free fatty acids. Stx2a predominantly altered the nicotinamide adenine dinucleotide (NAD) cofactor pathway and the inflammation-modulating eicosanoid pathway, which are associated with lipid metabolism. In HRGEC, Stx2a strongly diminished NAD derivatives, leading to depletion of the energy substrate acetyl coenzyme A and the antioxidant glutathione. HUVEC responded to TNF-α and Stx2a by increasing production of the counteracting eicosanoids prostaglandin I2, E1, E2, and A2, while in HRGEC only more prostaglandin I2 was detected. CONCLUSIONS: We conclude that disruption of energy metabolism and depletion of glutathione contributes to Stx-induced injury of the renal endothelium and that the inflammatory response to Stx is highly cell-type specific.
