Differential expression of the Nrf2-linked genes in pediatric septic shock

The Nrf2 pathway has been shown in previous microarray analyses of pediatric sepsis to be in the top 5 signaling and metabolic pathways upregulated in whole blood–derived RNA, with 24 genes represented [23]. The present study delves more deeply into the discovery of Nrf2-linked genes as a critical pathway in pediatric sepsis. We found differential expression of Nrf2-linked genes in children with septic shock compared with controls and that the Nrf2-linked genes showed a greater degree of repression in endotype A subjects, who tend to have the most organ dysfunction and highest mortality.

Nrf2 plays a role in degradation of triglycerides and/or phospholipids and enzymes involved in fatty acid oxidation. In addition, Nrf2 negatively regulates many genes encoding enzymes involved in lipid biosynthesis, fatty acid desaturation, and fatty acid transport through unknown mechanisms [19]. Langley and colleagues performed a metabolomic study of adults with septic shock and demonstrated that fatty acid transport, β-oxidation, gluconeogenesis, and the citric acid cycle pathways are all differentially regulated in non-survivors compared with survivors of septic shock [11]. Specifically, lactate, pyruvate, α-ketoglutarate, oxaloacetate, and acyl-carnitine are all higher in the plasma of sepsis non-survivors than in sepsis survivors [11], purportedly owing to an inability of sepsis non-survivors to harness energetic substrates for aerobic catabolism [11]. Rogers et al also performed metabolomic analysis of critically ill adults using the same cohort as that analyzed by Langley et al and noted that, of the 31 metabolites that differentiated survivors from non-survivors, the six metabolites that were lower in the patients who died were a part of the lipid metabolism pathway [10]. Mickiewicz and colleagues performed nuclear magnetic resonance analysis of metabolites in children with sepsis compared with healthy control children and found an increase in three compounds (2-hydroxybutyrate, 2-hydroxyisovalerate, and lactate) that are associated with enhanced fat breakdown resulting in ketoacid and lactic acid production owing to increased energy demands during septic shock [12]. Our data are consistent with these previous studies and, overall, support the concept that pediatric septic shock is characterized by alterations in the expression of genes essential for oxidative stress responses and lipid metabolism.

Concurrent measures of oxidative stress and metabolite data are not available to determine how the observed Nrf2 pathway gene expression changes might alter the total antioxidant status, fatty acid metabolism, and ATP production in early pediatric sepsis. It is likely that changes in intermediary metabolism and oxidative state affect Nrf2-linked gene expression, and researchers in future studies should measure the effects of oxidative stress, metabolite concentration, and flux through various metabolic pathways influenced by Nrf2, such as glycolysis, the citric acid cycle, the PPP, fatty acid oxidation, and fatty acid synthesis.

The present study is constrained by the fact that we limited the analysis to 267 genes linked to Nrf2, as determined by previous human and murine studies. Linkage was defined as either genes regulated by Nrf2 or genes that regulate Nrf2 function. It is certainly possible that our selection method excluded some relevant genes. Limiting our working gene list in this manner is a potential source of bias. We attempted to mitigate this bias by conducting the initial statistical analyses, with corrections for multiple comparisons, using all gene probes available on the array. We subsequently searched the resulting gene lists for our selected Nrf2 genes and found that many of these genes were differentially regulated when we considered all available gene probes.