Signal Response Evaluation Applied to Untargeted Mass Spectrometry Data to Improve Data Interpretability.

Feature finding is a common way to process untargeted mass spectrometry (MS) data to obtain a list of chemicals present in a sample. Most feature finding algorithms naïvely search for patterns of unique descriptors (e.g., m/z, retention time, and mobility) and provide a list of unannotated features. There is a need for solutions in processing untargeted MS data, independent of chemical or origin, to assess features based on measurement quality with the aim of improving interpretation. Here, we report the signal response evaluation as a method by which to assess the individual features observed in untargeted MS data. The basis of this method is the ubiquitous relationship between the amount and response in all MS measurements. Three different metrics with user-defined parameters can be used to assess the monotonic or linear relationship of each feature in a dilution series or multiple injection volumes. We demonstrate this approach in metabolomics data obtained from a uniform biological matrix (NIST SRM 1950) and a variable biological matrix (murine kidney tissue). The code is provided to facilitate implementation of this data processing method.

Extracellular signal-regulated kinase 1/2 regulates NAD metabolism during acute kidney injury through microRNA-34a-mediated NAMPT expression.

Prior studies have established the important role of extracellular signal-regulated kinase 1/2 (ERK1/2) as a mediator of acute kidney injury (AKI). We demonstrated rapid ERK1/2 activation induced renal dysfunction following ischemia/reperfusion (IR)-induced AKI and downregulated the mitochondrial biogenesis (MB) regulator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) in mice. In this study, ERK1/2 regulation of cellular nicotinamide adenine dinucleotide (NAD) and PGC-1α were explored. Inhibition of ERK1/2 activation during AKI in mice using the MEK1/2 inhibitor, trametinib, attenuated renal cortical oxidized NAD (NAD+) depletion. The rate-limiting NAD biosynthesis salvage enzyme, NAMPT, decreased following AKI, and this decrease was prevented by ERK1/2 inhibition. The microRNA miR34a decreased with the inhibition of ERK1/2, leading to increased NAMPT protein. Mice treated with a miR34a mimic prevented increases in NAMPT protein in the renal cortex in the presence of ERK1/2 inhibition. In addition, ERK1/2 activation increased acetylated PGC-1α, the less active form, whereas inhibition of ERK1/2 activation prevented an increase in acetylated PGC-1α after AKI through SIRT1 and NAD+ attenuation. These results implicate IR-induced ERK1/2 activation as an important contributor to the downregulation of both PGC-1α and NAD+ pathways that ultimately decrease cellular metabolism and renal function. Inhibition of ERK1/2 activation prior to the initiation of IR injury attenuated decreases in PGC-1α and NAD+ and prevented kidney dysfunction.

5-HT1F receptor regulates mitochondrial homeostasis and its loss potentiates acute kidney injury and impairs renal recovery.

Our laboratory previously reported that agonists of the 5-hydoxytryptamine 1F (5-HT1F) receptor induce renal mitochondrial biogenesis (MB) and that stimulation of the 5-HT1F receptor following ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) accelerated the recovery of renal function in mice. The goal of this study was to examine the contribution of the 5-HT1F receptor in the regulation of renal mitochondrial homeostasis and renal function in naïve and injured mice. Although 5-HT1F receptor knockout (KO) mice were healthy and fertile, and did not exhibit renal dysfunction, renal mitochondrial DNA copy number and mitochondrial fission gene expression increased at 10 wk of age. The 5-HT1F receptor KO mice exhibited greater proximal tubular injury and diminished renal recovery after I/R-induced AKI compared with wild-type mice. These findings were associated with persistent suppression of renal cortical MB and ATP levels after injury. In summary, the 5-HT1F receptor is a component of physiological MB regulation in the kidney, and its absence potentiates renal injury and impedes recovery.

Extracellular Signal-Regulated Kinase 1/2 Regulates Mouse Kidney Injury Molecule-1 Expression Physiologically and Following Ischemic and Septic Renal Injury.

The upregulation of kidney injury molecule-1 (KIM-1) has been extensively studied in various renal diseases and following acute injury; however, the initial mechanisms controlling KIM-1 expression remain limited. In this study, KIM-1 expression was examined in mouse renal cell cultures and in two different models of acute kidney injury (AKI), ischemia reperfusion (IR)-induced and lipopolysaccharide (LPS)-induced sepsis. KIM-1 mRNA increased in both AKI models, and pharmacological inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling attenuated injury-induced KIM-1 expression in the renal cortex. Toll-like receptor 4 knockout (TLR4KO) mice exhibited reduced ERK1/2 phosphorylation and attenuated KIM-1 mRNA after LPS exposure. TLR4KO mice were not protected from IR-induced ERK1/2 phosphorylation and upregulation of KIM-1 mRNA. Following renal IR injury, phosphorylation of signal transducer and activator of transcription 3 (STAT3) at serine 727 and tyrosine 705 increased downstream from ERK1/2 activation. Because phosphorylated STAT3 is a transcriptional upregulator of KIM-1 and inhibition of ERK1/2 attenuated increases in STAT3 phosphorylation, we suggest an ERK1/2-STAT3-KIM-1 pathway following renal injury. Finally, ERK1/2 inhibition in naive mice decreased KIM-1 mRNA and nuclear STAT3 phosphorylation in the cortex, indicating homeostatic regulation of KIM-1. These findings reveal renal ERK1/2 as an important initial regulator of KIM-1 expression in IR and septic AKI and at a physiologic level.Visual Abstract.Proposed mechanism of IR, LPS, and ROS-induced renal damage that initiates ERK1/2 and STAT3 phosphorylation. STAT3 then binds to the KIM-1 promoter and increases KIM-1 mRNA. By preventing ERK1/2 phosphorylation following renal injury, STAT3 phosphorylation is decreased, leading to less phosphorylated STAT3 within the nucleus, and subsequently less KIM-1 mRNA increases post injury.

Rapid Renal Regulation of Peroxisome Proliferator-activated Receptor γ Coactivator-1α by Extracellular Signal-Regulated Kinase 1/2 in Physiological and Pathological Conditions.

Previous studies have shown that extracellular signal-regulated kinase 1/2 (ERK1/2) directly inhibits mitochondrial function during cellular injury. We evaluated the role of ERK1/2 on the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) gene, a master regulator of mitochondrial function. The potent and specific MEK1/2 inhibitor trametinib rapidly blocked ERK1/2 phosphorylation, decreased cytosolic and nuclear FOXO3a/1 phosphorylation, and increased PGC-1α gene expression and its downstream mitochondrial biogenesis (MB) targets under physiological conditions in the kidney cortex and in primary renal cell cultures. The epidermal growth factor receptor (EGFR) inhibitor erlotinib blocked ERK1/2 phosphorylation and increased PGC-1α gene expression similar to treatment with trametinib, linking EGFR activation and FOXO3a/1 inactivation to the down-regulation of PGC-1α and MB through ERK1/2. Pretreatment with trametinib blocked early ERK1/2 phosphorylation following ischemia/reperfusion kidney injury and attenuated the down-regulation of PGC-1α and downstream target genes. These results demonstrate that ERK1/2 rapidly regulates mitochondrial function through a novel pathway, EGFR/ERK1/2/FOXO3a/1/PGC-1α, under physiological and pathological conditions. As such, ERK1/2 down-regulates mitochondrial function directly by phosphorylation of upstream regulators of PGC-1α and subsequently decreasing MB.

Suppression of mitochondrial biogenesis through toll-like receptor 4-dependent mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling in endotoxin-induced acute kidney injury.

Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1ß) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis.