Metabolomics assessment reveals oxidative stress and altered energy production in the heart after ischemic acute kidney injury in mice

Acute kidney injury (AKI) is a systemic disease associated with widespread effects on distant organs, including the heart. Normal cardiac function is dependent on constant ATP generation, and the preferred method of energy production is via oxidative phosphorylation. Following direct ischemic cardiac injury, the cardiac metabolome is characterized by inadequate oxidative phosphorylation, increased oxidative stress, and increased alternate energy utilization. We assessed the impact of ischemic AKI on the metabolomics profile in the heart. Ischemic AKI was induced by 22 minutes of renal pedicle clamping, and 124 metabolites were measured in the heart at 4 hours, 24 hours, and 7 days post-procedure. 41% of measured metabolites were affected, with the most prominent changes observed 24 hours post-AKI. The post-AKI cardiac metabolome was characterized by amino acid depletion, increased oxidative stress, and evidence of alternative energy production, including a shift to anaerobic forms of energy production. These metabolomic effects were associated with significant cardiac ATP depletion and with echocardiographic evidence of diastolic dysfunction. In the kidney, metabolomics analysis revealed shifts suggestive of energy depletion and oxidative stress, which were reflected systemically in the plasma. This is the first study to examine the cardiac metabolome after AKI, and demonstrates that effects of ischemic AKI on the heart are akin to the effects of direct ischemic cardiac injury

Antigen Perception in T Cells

Thesis (Ph.D.)--University of Washington, 2023The immune system’s threat detection hinges on T cells’ ability to perceive differences in the quality and quantity of peptide-major histocompatibility complex (pMHC) antigens. Our T cells are tolerant to the vast majority of antigens they sense, as these pMHCs are derived from our own endogenous proteome and represent “self”. In the case of a viral infection, virus-derived pMHC antigens are perceived by T cells as a threat and elicit an effector response to clear the infection. This fundamental perception of “self” vs “foreign” is based on antigen quality. Self pMHCs have a relatively low affinity for the T cell antigen receptor (TCR), while foreign pMHCs have relatively high TCR binding affinities. The current paradigm states that this affinity discrimination is enabled by a signaling network downstream of the TCR that performs kinetic proofreading during antigen sensing, whereby only long-lasting TCR binding events resulting from interactions with high affinity foreign pMHCs pass a series of proofreading reactions and further propagate the signal. However, numerous in vitro and in vivo studies have shown that T cells do not respond uniformly to variations in the quality and quantity of foreign antigens, but rather tailor their response to each unique threat. This suggests an unresolved signal transmission mechanism within the TCR signaling network that regulates T cell responses in a tunable, and not simply all-or-none fashion. The Erk and NFAT signaling pathways connect TCR engagement to gene regulation, and thus their combined signaling dynamics may transmit information about pMHC inputs. We formed this hypothesis from two main bodies of evidence. First, signaling pathway dynamics, including dynamics of the Erk pathway, are known to transmit extracellular signal information in many other contexts to enable input-specific cellular responses. Second, NFAT and the Erk-activated transcription factor AP-1 work cooperatively to regulate gene expression in activated T cells, and disruption of their cooperativity leads to distinct phenotypes. Therefore, it is plausible that signaling activity of these two pathways, in combination, over the course of initial activation can tune gene expression in T cells to generate antigen-specific responses. To test this idea, we developed a dual reporter mouse strain and a quantitative imaging assay that together enable simultaneous monitoring of Erk and NFAT dynamics in live T cells over day-long timescales as they respond to varying pMHC inputs. We found that both pathways initially activate uniformly across various pMHC inputs but diverge over longer (9+ hrs) timescales, enabling independent encoding of pMHC affinity and dose. Next, we combined signaling perturbations and variable pMHC stimulation with RNA sequencing and mathematical modeling to uncover multiple temporal and combinatorial mechanisms for decoding these late signaling dynamics to generate pMHC-specific transcriptional responses. Our results underscore the importance of long timescale signaling dynamics in T cell antigen perception and establish a framework for understanding T cell responses under diverse contexts. Our findings, while only scratching the surface of the complexity and sophistication of T cell signaling and transcriptional regulation, point to a strategy for improving T cell-based therapies by engineering precise temporal control over signaling inputs

Data on how several physiological parameters of stored red blood cells are similar in glucose 6-phosphate dehydrogenase deficient and sufficient donors

This article contains data on the variation in several physiological parameters of red blood cells (RBCs) donated by eligible glucose-6-phosphate dehydrogenase (G6PD) deficient donors during storage in standard blood bank conditions compared to control, G6PD sufficient (G6PD+) cells. Intracellular reactive oxygen species (ROS) generation, cell fragility and membrane exovesiculation were measured in RBCs throughout the storage period, with or without stimulation by oxidants, supplementation of N-acetylcysteine and energy depletion, following incubation of stored cells for 24 h at 37 °C. Apart from cell characteristics, the total or uric acid-dependent antioxidant capacity of the supernatant in addition to extracellular potassium concentration was determined in RBC units. Finally, procoagulant activity and protein carbonylation levels were measured in the microparticles population. Further information can be found in “Glucose 6-phosphate dehydrogenase deficient subjects may be better “storers” than donors of red blood cells” [1]. Keywords: G6PD deficiency, Red blood cell storage lesion, Oxidative stress, Cell fragility, Microparticle

Hydroxylamine Chemical Digestion for Insoluble Extracellular Matrix Characterization

The extracellular matrix (ECM) is readily enriched by decellularizing tissues with nondenaturing detergents to solubilize and deplete the vast majority of cellular components. This approach has been used extensively to generate ECM scaffolds for regenerative medicine technologies and in 3D cell culture to model how the ECM contributes to disease progression. A highly enriched ECM fraction can then be generated using a strong chaotrope buffer that is compatible with downstream bottom-up proteomic analysis or 3D cell culture experiments after extensive dialysis. With most tissues, an insoluble pellet remains after chaotrope extraction that is rich in structural ECM components. Previously, we showed that this understudied fraction represented approximately 80% of total fibrillar collagen from the lung and other ECM fiber components that are known to be covalently cross-linked. Here, we present a hydroxylamine digestion approach for chaotrope-insoluble ECM analysis with comparison to an established CNBr method for matrisome characterization. Because ECM characteristics vary widely among tissues, we chose five tissues that represent unique and diverse ECM abundances, composition, and biomechanical properties. Hydroxylamine digestion is compatible with downstream proteomic workflows, yields high levels of ECM peptides from the insoluble ECM fraction, and reduces analytical variability when compared to CNBr digestion. Data are available via ProteomeXchange with identifier PXD006428

MOESM6 of Trauma/hemorrhagic shock instigates aberrant metabolic flux through glycolytic pathways, as revealed by preliminary 13C-glucose labeling metabolomics

Additional file 6. Blood from trauma/hemorrhagic shock rats (laparotomy with bowel crush, with hemorrhagic shock to MAP <30) was withdrawn before hemorrhagic shock (baseline B). After waiting for 15 min (W15), injection of labeled 13C-glucose was performed and blood was then collected at 5, 10, 15, and 20 min from iLC. Metabolites of glycolysis and Krebs cycles were monitored, as they have been previously shown to increase in plasma after trauma/hemorrhagic shock [14]. In left, the total levels of the metabolite (integrated peak areas—arbitrary units) are indicated through stacked bar graphs, including the unlabeled parent (blue M + 0) and heavy isotopologues (either M + 2, M + 3, M + 4 or M + 6 depending on the expected labeling pattern from catabolism of 13C-glucose). In the right hand panels, only heavy isotopologues (red, yellow, orange, green) are shown. As soon as 5 min after iLC, hemorrhagic shock induced accumulation of lactate and unlabeled glucose (indicative of ongoing gluconeogenesis) and late Krebs cycle intermediates (succinate, fumarate, malate), increased levels of glutamate and totally unlabeled urate, polyamines (spermidine), glutathione (either reduced—GSH and oxidized—GSSG), mannitol and (minimally labeled) citramalate. M + 3 labeling in malate and succinate is suggestive of malate generation from oxaloacetate obtained via pyruvate carboxylase activity and backwards fluxing of complex I and II to generate malate and succinate in the absence of oxygen as a final electron acceptor (following HS top right corner). This figure is an extended version of the in manuscript Fig. 7