ß-Caryophyllene Inhibits Monoacylglycerol Lipase Activity and Increases 2-Arachidonoyl Glycerol Levels In Vivo: A New Mechanism of Endocannabinoid-Mediated Analgesia?

The mechanisms of ß-caryophyllene (BCP)-induced analgesia are not well studied. Here, we tested the efficacy of BCP in an acute postsurgical pain model and evaluated its effect on the endocannabinoid system. Rats were treated with vehicle and 10, 25, 50, and 75 mg/kg BCP. Paw withdrawal responses to mechanical stimuli were evaluated using an electronic von Frey anesthesiometer. Endocannabinoids, including 2-arachidonoylglycerol (2-AG), were also evaluated in plasma and tissues using high-performance liquid chromatography-tandem mass spectrometry. Monoacylglycerol lipase (MAGL) activity was evaluated in vitro as well as ex vivo. We observed a dose-dependent and time-dependent alleviation of hyperalgesia in incised paws up to 85% of the baseline value at 30 minutes after administration of BCP. We also observed dose-dependent increases in the 2-AG levels of about threefold after administration of BCP as compared with vehicle controls. Incubations of spinal cord tissue homogenates from BCP-treated rats with isotope-labeled 2-arachidonoylglycerol-d8 revealed a reduced formation of the isotope-labeled MAGL product 2-AG-d8 as compared with vehicle controls, indicating MAGL enzyme inhibition. In vitro MAGL enzyme activity assessment using 2-AG as the substrate revealed an IC50 of 15.8 µM for MAGL inhibition using BCP. These data showed that BCP inhibits MAGL activity in vitro and in vivo, causing 2-AG levels to rise. Since the endocannabinoid 2-AG is a CB1 and CB2 receptor agonist, we propose that 2-AG-mediated cannabinoid receptor activation contributes to BCP's mechanism of analgesia. SIGNIFICANCE STATEMENT: ß-Caryophyllene (BCP) consumption is relatively safe and is approved by the Food and Drug Administration as a flavoring agent, which can be used in cosmetic and food additives. BCP is a potent anti-inflammatory agent that showed substantial antihyperalgesic properties in this study of acute pain suggesting that BCP might be an alternative to opioids. This study shows an additive mechanism (monoacylglycerol lipase inhibition) by which BCP might indirectly alter CB1 and CB2 receptor activity and exhibit its pharmacological properties.

Arginine-NO metabolites are associated with morbidity in single ventricle infants undergoing stage 2 palliation.

BACKGROUND: Infants with single ventricle heart disease (SVHD) suffer morbidity from insufficient pulmonary blood flow, which may be related to impaired arginine metabolism. No prior study has reported quantitative mapping of arginine metabolites to evaluate the relationship between circulating metabolite levels and outcomes. METHODS: Prospective cohort study of 75 SVHD cases peri-Stage 2 and 50 healthy controls. We targeted pre- and post-op absolute serum quantification of 9 key members of the arginine metabolism pathway by tandem mass spectrometry. Primary outcomes were length of stay (LOS) and post-Stage 2 hypoxemia. RESULTS: Pre-op cases showed alteration in 6 metabolites including decreased arginine and increased asymmetric dimethyl arginine (ADMA) levels compared to controls. Post-op cases demonstrated decreased arginine and citrulline levels persisting through 48 h. Adjusting for clinical variables, lower pre-op and 2 h post-op concentrations of multiple metabolites, including arginine and citrulline, were associated with longer post-op LOS (p < 0.01). Increased ADMA at 24 h was associated with greater post-op hypoxemia burden (p < 0.05). CONCLUSION: Arginine metabolism is impaired in interstage SVHD infants and is further deranged following Stage 2 palliation. Patients with greater metabolite alterations experience greater post-op morbidity. Decreased arginine metabolism may be an important driver of pathology in SVHD. IMPACT: Interstage infants with SVHD have significantly altered arginine-nitric oxide metabolism compared to healthy children with deficiency of multiple pathway intermediates persisting through 48 h post-Stage 2 palliation. After controlling for clinical covariates and classic catheterization-derived predictors of Stage 2 readiness, both lower pre-operation and lower post-operation circulating metabolite levels were associated with longer post-Stage 2 LOS while increased post-Stage 2 ADMA concentration was associated with greater post-op hypoxemia. Arginine metabolism mapping offers potential for development using personalized medicine strategies as a biomarker of Stage 2 readiness and therapeutic target to improve pulmonary vascular health in infants with SVHD.

Sex-specific hypnotic effects of the neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile are mediated by peripheral metabolism into an active hypnotic steroid.

BACKGROUND: The novel synthetic neuroactive steroid (3ß,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3ß-OH) blocks T-type calcium channels but does not directly modulate neuronal γ-aminobutyric acid type A (GABAA) currents like other anaesthetic neurosteroids. As 3ß-OH has sex-specific hypnotic effects in adult rats, we studied the mechanism contributing to sex differences in its effects. METHODS: We used a combination of behavioural loss of righting reflex, neuroendocrine, pharmacokinetic, in vitro patch-clamp electrophysiology, and in vivo electrophysiological approaches in wild-type mice and in genetic knockouts of the CaV3.1 T-type calcium channel isoform to study the mechanisms by which 3ß-OH and its metabolite produces sex-specific hypnotic effects. RESULTS: Adult male mice were less sensitive to the hypnotic effects of 3ß-OH compared with female mice, and these differences appeared during development. Adult males had higher 3ß-OH brain concentrations despite being less sensitive to its hypnotic effects. Females metabolised 3ß-OH into the active GABAA receptor positive allosteric modulator (3α,5ß,17ß)-3-hydroxyandrostane-17-carbonitrile (3α-OH) to a greater extent than males. The 3α-OH metabolite has T-channel blocking properties with sex-specific hypnotic and pharmacokinetic effects. Sex-dependent suppression of the cortical electroencephalogram is more pronounced with 3α-OH compared with 3ß-OH. CONCLUSIONS: The sex-specific differences in the hypnotic effect of 3ß-OH in mice are attributable to differences in its peripheral metabolism into the more potent hypnotic metabolite 3α-OH.

Metabolic reprogramming in a slowly developing orthologous model of polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and affects 1 in 1,000 individuals. There is accumulating evidence suggesting that there are shared cellular mechanisms responsible for cystogenesis in human and murine PKD and that reprogramming of metabolism is a key disease feature. In this study, we used a targeted metabolomics approach in an orthologous mouse model of PKD (Pkd1RC/RC) to investigate the metabolic modifications a cystic kidney undergoes during disease progression. Using the Kyoto Encyclopedia of Genes and Genomes pathway database, we identified several biologically relevant metabolic pathways that were altered early in this disease (in 3-mo-old Pkd1RC/RC mice), the most highly represented being arginine biosynthesis and metabolism and tryptophan and phenylalanine metabolism. During the next 6 mo of disease progression, multiple uremic solutes accumulated in the kidney of cystic mice, including several established markers of oxidative stress and endothelial dysfunction (allantoin, asymmetric dimethylarginine, homocysteine, malondialdehyde, methionine sulfoxide, and S-adenosylhomocysteine). Levels of kynurenines and polyamines were also augmented in kidneys of Pkd1RC/RC versus wild-type mice, as were the levels of bacteria-produced indoles, whose increase within PKD kidneys suggests microbial dysbiosis. In summary, we confirmed previously published and identified novel metabolic markers and pathways of PKD progression that may prove helpful for diagnosis and monitoring of cystic kidney disease in patients. Furthermore, they provide targets for novel therapeutic approaches that deserve further study and hint toward currently understudied pathomechanisms.NEW & NOTEWORTHY This report delineates the evolution of metabolic changes occurring during autosomal dominant polycystic kidney disease (ADPKD) progression. Using an orthologous model, we performed kidney metabolomics and confirmed dysregulation of metabolic pathways previously found altered in nonorthologous or rapidly-progressive PKD models. Importantly, we identified novel alterations, including augmentation of kynurenines, polyamines, and indoles, suggesting increased inflammation and microbial dysbiosis that provide insights into PKD pathomechanisms and may prove helpful for diagnosing, monitoring, and treating ADPKD.

15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction.

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.

Metabolomic profiling demonstrates evidence for kidney and urine metabolic dysregulation in a piglet model of cardiac surgery-induced acute kidney injury.

Acute kidney injury (AKI) is a common cause of morbidity after congenital heart disease surgery. Progress on diagnosis and therapy remains limited, however, in part due to poor mechanistic understanding and a lack of relevant translational models. Metabolomic approaches could help identify novel mechanisms of injury and potential therapeutic targets. In the present study, we used a piglet model of cardiopulmonary bypass with deep hypothermic circulatory arrest (CPB/DHCA) and targeted metabolic profiling of kidney tissue, urine, and serum to evaluate metabolic changes specific to animals with histological acute kidney injury. CPB/DHCA animals with acute kidney injury were compared with those without acute kidney injury and mechanically ventilated controls. Acute kidney injury occurred in 10 of 20 CPB/DHCA animals 4 h after CPB/DHCA and 0 of 7 control animals. Injured kidneys showed a distinct tissue metabolic profile compared with uninjured kidneys (R2 = 0.93, Q2 = 0.53), with evidence of dysregulated tryptophan and purine metabolism. Nine urine metabolites differed significantly in animals with acute kidney injury with a pattern suggestive of increased aerobic glycolysis. Dysregulated metabolites in kidney tissue and urine did not overlap. CPB/DHCA strongly affected the serum metabolic profile, with only one metabolite that differed significantly with acute kidney injury (pyroglutamic acid, a marker of oxidative stress). In conclusion, based on these findings, kidney tryptophan and purine metabolism are candidates for further mechanistic and therapeutic investigation. Urine biomarkers of aerobic glycolysis could help diagnose early acute kidney injury after CPB/DHCA and warrant further evaluation. The serum metabolites measured at this early time point did not strongly differentiate based on acute kidney injury.NEW & NOTEWORTHY This project explored the metabolic underpinnings of postoperative acute kidney injury (AKI) following pediatric cardiac surgery in a translationally relevant large animal model of cardiopulmonary bypass with deep hypothermic circulatory arrest. Here, we present novel evidence for dysregulated tryptophan catabolism and purine catabolism in kidney tissue and increased urinary glycolysis intermediates in animals who developed histological AKI. These pathways represent potential diagnostic and therapeutic targets for postoperative AKI in this high-risk population.

Metformin Therapy in Autosomal Dominant Polycystic Kidney Disease: A Feasibility Study.

RATIONALE & OBJECTIVE: Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder that leads to kidney failure and has few treatment options. Metformin is well tolerated and safe in other patient populations. The primary objective of this clinical trial was to determine the safety and tolerability of metformin in patients with ADPKD and without diabetes mellitus. STUDY DESIGN: Prospective randomized controlled double-blind clinical trial. SETTING & PARTICIPANTS: 51 adults aged 30-60 years with ADPKD, without diabetes, and an estimated glomerular filtration rate (eGFR) 50-80 mL/min/1.73 m2. EXPOSURE: Metformin (maximum dose 2,000 mg/d) or placebo for 12 months. OUTCOME: Coprimary end points were the percentage of participants in each group prescribed at the end of the 12-month period: (1) the full randomized dose or (2) at least 50% of the randomized dose. Secondary and exploratory outcomes were the effect of metformin compared with placebo on (1) the percentage change in total kidney volume (TKV) referenced to height (htTKV in mL/m) and (2) the change in eGFR over a 12-month period. RESULTS: The participants' mean age was 48 ± 8 (SD) years, and eGFR was 70 ± 14 mL/min/1.73 m2. The metformin group had no cases of lactic acidosis, and there was 1 episode of mild hypoglycemia in each group. Participants in the metformin group reported more adverse symptoms, mostly related to the gastrointestinal tract. Eleven of 22 metformin-treated participants (50%) completed the treatment phase on the full dose compared with 23 of 23 in the placebo group (100%). In the metformin group, 82% of participants tolerated at least 50% of the dose, compared with 100% in the placebo group. In exploratory analyses, changes in htTKV or eGFR were not significantly different between the groups. LIMITATIONS: Short study duration. CONCLUSIONS: We found that 50% or more of the maximal metformin dose was safe and well tolerated over 12 months in patients with ADPKD. Safety of other preparations of metformin as well as its efficacy should be tested in future clinical trials. FUNDING: Government and philanthropic grants (NIDDK and the Zell Foundation). TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT02903511.

Endocannabinoid System in Polycystic Kidney Disease.

INTRODUCTION: Autosomal dominant polycystic kidney disease (ADPKD) is a commonly inherited disorder characterized by renal cyst formation. A major pathological feature of ADPKD is the development of interstitial inflammation. The endocannabinoid (EC) system is present in the kidney and has recently emerged as an important player in inflammation and the pathogenesis of progressive kidney disease. METHODS: Data on ECs were collected using a validated mass spectrometry assay from a well-characterized cohort of 102 ADPKD patients (at baseline and after 2- and 4 years on standard vs. rigorous blood-pressure control) and compared to 100 healthy subjects. RESULTS: Compared to healthy individuals, we found higher interleukins-6 and -1b as well as reduced plasma levels of anandamide (AEA), 2-arachidonoyl-glycerol (2-AG), and their congeners in ADPKD patients. Baseline AEA concentration negatively associated with the progression of ADPKD as expressed by the yearly percent change in height-corrected total kidney volume and positively with the yearly change in renal function (measured as estimated glomerular filtration rate, ΔeGFR). AEA analog palmitoylethanolamide (PEA) is also associated positively with the yearly change in eGFR. DISCUSSION AND CONCLUSION: The results of the present study suggest that ADPKD patients present with lower levels of ECs and that reestablishing the normality of the renal EC system via augmentation of AEA, PEA, and 2-AG levels, either through the increase of their synthesis or through a reduction of their degradation, could be beneficial and may present a promising therapeutic target in said patients.

Changes in infant porcine pulmonary tissue oxylipins induced by cardiopulmonary bypass.

BACKGROUND: Oxylipins are metabolites derived from fatty acids such as arachidonic acid (AA) and are key mediators in inflammation, host defense, and tissue injury. Serum oxylipins increase in adults after cardiopulmonary bypass (CPB) but tissue-level changes are poorly defined. The objective of this study was to characterize pulmonary tissue oxylipins in an infant porcine model of CPB with deep hypothermic circulatory arrest (DHCA). METHODS: Infant pigs underwent CPB with DHCA. Controls received anesthesia only. Right upper and lower lobes of the lung underwent oxylipin analysis via liquid chromatography-tandem mass spectrometry. One-way ANOVA was utilized to assess differences in oxylipin concentrations across groups, followed by pairwise comparisons. RESULTS: AA and multiple AA metabolites via cytochrome P450 (CYP450), lipoxygenase (LOX), and cyclooxygenase (COX) pathways were significantly increased in the upper and lower lobe of pigs exposed to CPB/DHCA as compared to controls. Multiple prostaglandin metabolites produced via COX were also significantly elevated in the lower lobes of control animals. CONCLUSIONS: CPB/DHCA induces a significant increase in pulmonary tissue AA, with subsequent metabolism via COX, LOX, and CYP450 pathways. Interestingly, prostaglandins were also elevated in the lower lobes of the controls, suggesting a mechanism separate from CPB/DHCA. Future oxylipin studies are needed to better understand CPB-induced acute lung injury. IMPACT: CPB/DHCA and, to a lesser extent, lung region influence pulmonary tissue-level AA metabolite production. Inflammatory mediator AA metabolites have been noted in previous studies to increase following CPB; however, this is the first study to look at pulmonary tissue-level differences following CPB/DHCA. Increases in many AA metabolites, including LOX- and CYP450-derived products, were seen in both upper and lower lobe of piglets following CPB/DHCA. COX-derived prostaglandin metabolites were increased not only in CPB upper and lower lobe but also in mechanically ventilated control lower lobe, suggesting an additional, separate mechanism from CPB/DCHA.

Regional lung metabolic profile in a piglet model of cardiopulmonary bypass with circulatory arrest.

INTRODUCTION: Acute lung injury is common following cardiopulmonary bypass and deep hypothermic circulatory arrest for congenital heart surgery with the most severe injury in the dorsocaudal lung. Metabolomics offers promise in deducing mechanisms of disease states, providing risk stratification, and understanding therapeutic responses in regards to CPB/DHCA related organ injury. OBJECTIVES: Using an infant porcine model, we sought to determine the individual and additive effects of CPB/DHCA and lung region on the metabolic fingerprint, metabolic pathways, and individual metabolites in lung tissue. METHODS: Twenty-seven infant piglets were divided into two groups: mechanical ventilation + CPB/DHCA (n = 20) and mechanical ventilation only (n = 7). Lung tissue was obtained from dorsocaudal and ventral regions. Targeted analysis of 235 metabolites was performed using HPLC/MS-MS. Data was analyzed using Principal Component Analysis (PCA), Partial Least Square Discriminant Analysis (PLS-DA), ANOVA, and pathway analysis. RESULTS: Profound metabolic differences were found in dorsocaudal compared to ventral lung zones by PCA and PLS-DA (R2 = 0.7; Q2 = 0.59; p < 0.0005). While overshadowed by the regional differences, some differences by exposure to CPB/DHCA were seen as well. Seventy-four metabolites differed among groups and pathway analysis revealed 20 differential metabolic pathways. CONCLUSION: Our results demonstrate significant metabolic disturbances between dorsocaudal and ventral lung regions during supine mechanical ventilation with or without CPB/DHCA. CPB/DHCA also leads to metabolic differences and may have additive effects to the regional disturbances. Most pathways driving this pathology are involved in energy metabolism and the metabolism of amino acids, carbohydrates, and reduction-oxidation pathways.

Analysis of 14 endocannabinoids and endocannabinoid congeners in human plasma using column switching high-performance atmospheric pressure chemical ionization liquid chromatography-mass spectrometry.

The endocannabinoid system (ECS) is a complex cell-signaling system. To address the growing need of analytics capturing endocannabinoid levels to investigate the ECS, we developed and validated an assay for the quantitative analysis of 14 endocannabinoids and congeners. A simple extraction using protein precipitation with acetonitrile followed by online-trapping high-performance liquid chromatography-tandem mass spectrometry (LC/LC-MS/MS) was used to monitor the levels of 14 endocannabinoids in plasma. The assay was validated and intra-run and inter-run accuracies and imprecisions as well as matrix effects, recoveries, and sample stabilities were determined. As a proof of concept, a subset of study samples after naturalistic administration of Cannabis flower and concentrate was analyzed. With the exception of N-oleoyl dopamine and oleamide, all endocannabinoids fulfilled the predefined acceptance criteria. Reproducible recoveries and no significant matrix effects were observed. Sample stability was an issue. Analysis of the proof-of-concept study samples revealed a significantly (p = 0.006) higher concentration of docosatetraenoyl ethanolamide in concentrate users (300 ± 13 pg/mL) compared to flower users (252 ± 11 pg/mL). A robust, sensitive high-throughput assay for the quantitation of 14 endocannabinoids and congeners was successfully validated. Our study showed that it is mandatory to (A) appropriately stabilize samples and (B) separate and separately quantify 1-AG and 2-AG; otherwise, study results are unreliable. The analysis of study samples from Cannabis flower users versus Cannabis concentrate users revealed higher levels of docosatetraenoyl ethanolamide and anandamide (n.s.) in high THC concentrate users in accordance with the existing literature, supporting the validity of the assay measurements. Graphical abstract.

Serum metabolic profile of postoperative acute kidney injury following infant cardiac surgery with cardiopulmonary bypass.

BACKGROUND: We sought to determine differences in the circulating metabolic profile of infants with or without acute kidney injury (AKI) following cardiothoracic surgery with cardiopulmonary bypass (CPB). METHODS: We performed a secondary analysis of preoperative and 24-h postoperative serum samples from infants ≤ 120 days old undergoing CPB. Metabolic profiling of the serum samples was performed by targeted analysis of 165 serum metabolites via tandem mass spectrometry. We then compared infants who did or did not develop AKI in the first 72 h postoperatively to determine global differences in the preoperative and 24-h metabolic profiles in addition to specific differences in individual metabolites. RESULTS: A total of 57 infants were included in the study. Six infants (11%) developed KDIGO stage 2/3 AKI and 13 (23%) developed stage 1 AKI. The preoperative metabolic profile did not differentiate between infants with or without AKI. Infants with severe AKI could be moderately distinguished from infants without AKI by their 24-h metabolic profile, while infants with stage 1 AKI segregated into two groups, overlapping with either the no AKI or severe AKI groups. Differences in these 24-h metabolic profiles were driven by 21 metabolites significant at an adjusted false discovery rate of < 0.05. Prominently altered pathways include purine, methionine, and kynurenine/nicotinamide metabolism. CONCLUSION: Moderate-to-severe AKI after infant cardiac surgery is associated with changes in the serum metabolome, including prominent changes to purine, methionine, and kynurenine/nicotinamide metabolism. A portion of infants with mild AKI demonstrated similar metabolic changes, suggesting a potential role for metabolic analysis in the evaluation of lower stage injury.

Activation of kynurenine pathway of tryptophan metabolism after infant cardiac surgery with cardiopulmonary bypass: a prospective cohort study.

BACKGROUND: Serum kynurenic acid is associated with poor outcomes after infant cardiopulmonary bypass (CPB), but comprehensive mapping of the kynurenine pathway (KP) after CPB has yet to be performed. AIMS: To map changes in the KP induced by infant CPB. METHODS: Compared changes in serum KP metabolites through 48hrs post-op with liquid-chromatography-tandem mass spectrometry. RESULTS: Infant CPB results in marked increase in proximal, but not distal metabolites of the KP. CONCLUSIONS: Infant CPB leads to accumulation of circulating KP metabolites, which have important neurologic and immunologic activities. Thus, further exploration of the KP is warranted in these high-risk infants.

Theophylline dosing and pharmacokinetics for renal protection in neonates with hypoxic-ischemic encephalopathy undergoing therapeutic hypothermia.

BACKGROUND: Theophylline, a non-selective adenosine receptor antagonist, improves renal perfusion in the setting of hypoxia-ischemia and may offer therapeutic benefit in neonates with hypoxic-ischemic encephalopathy (HIE) undergoing hypothermia. We evaluated the pharmacokinetics and dose-exposure relationships of theophylline in this population to guide dosing strategies. METHODS: A population pharmacokinetic analysis was performed in 22 neonates with HIE undergoing hypothermia who were part of a prospective study or retrospective chart review. Aminophylline (intravenous salt form of theophylline) was given per institutional standard of care for low urine output and/or rising serum creatinine (5 mg/kg intravenous (i.v.) load then 1.8 mg/kg i.v. q6h). The ability of different dosing regimens to achieve target concentrations (4-10 mg/L) associated with clinical response was examined. RESULTS: Birth weight was a significant predictor of theophylline clearance and volume of distribution (p < 0.05). The median half-life was 39.5 h (range 27.2-50.4). An aminophylline loading dose of 7 mg/kg followed by 1.6 mg/kg q12h was predicted to achieve target concentrations in 84% of simulated neonates. CONCLUSIONS: In neonates with HIE undergoing hypothermia, theophylline clearance was low with a 50% longer half-life compared to full-term normothermic neonates without HIE. Dosing strategies need to consider the unique pharmacokinetic needs of this population. IMPACT: Theophylline is a potential renal-protective therapy in neonates with HIE undergoing therapeutic hypothermia; however, the pharmacokinetics and dose needs in this population are not known. Theophylline clearance was low in neonates with HIE undergoing therapeutic hypothermia with a 50% longer half-life compared to full-term normothermic neonates without HIE. As theophylline is advanced in clinical development, dosing strategies will need to consider the unique pharmacokinetic needs of neonates with HIE undergoing therapeutic hypothermia.

Temsirolimus metabolic pathways revisited.

Temsirolimus, a derivative of sirolimus, exhibits potent antitumor properties. It was the goal of this study to identify yet unknown temsirolimus metabolites generated after incubation with human liver microsomes. Previously, 23-hydroxy-, 24-hydroxy, 12-hydroxy, hydroxy-piperidine and 27-O-desmethyl temsirolimus had been described.Metabolite structures were identified using high-resolution mass spectrometry, MS/iontrap (MSn) and comparison of fragmentation patterns of the metabolites with those of temsirolimus and other known sirolimus derivatives. Moreover, enzyme kinetic parameters of temsirolimus metabolite formation as well as the contribution of individual recombinant cytochrome P450 (CYP) enzymes to temsirolimus metabolism were investigated.Human liver microsomes mainly hydroxylated and/or demethylated temsirolimus. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl and 27-O-desmethyl temsirolimus; hydroxylated metabolites: hydroxy piperidine temsirolimus, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 45/46-hydroxy and 49-hydroxy temsirolimus; demethylated-hydroxylated metabolites: 16-O-desmethyl, 24-hydroxy; 16-O-desmethyl, 23-hydroxy and 16-O-desmethyl 46-hydroxy temsirolimus; didemethylated metabolite: 27,39-O-didesmethyl temsirolimus; and dihydroxylated metabolite: 12,24-dihydroxy temsirolimus. It was confirmed that CYP3A4 represents the predominant enzyme responsible for temsirolimus metabolism. Moreover, CYP3A5 as well as CYP2C8 also showed significant activities especially resulting in the formation of 27-O-desmethyl, 25-hydroxy and hydroxy-piperidine temsirolimus.It is concluded that temsirolimus is metabolized to more than 20 metabolites, not counting metabolism via the sirolimus pathway. Eighteen of these metabolites could be structurally identified using ion trap MSn and high-resolution mass spectrometry. Moreover, the present study showed that, in addition to CYP3A4, metabolism via CYP3A5 and CYP2C8 also represent significant metabolic pathways.

Vitamin D receptor regulates autophagy in the normal mammary gland and in luminal breast cancer cells.

Women in North America have a one in eight lifetime risk of developing breast cancer (BC), and a significant proportion of these individuals will develop recurrent BC and will eventually succumb to the disease. Metastatic, therapy-resistant BC cells are refractory to cell death induced by multiple stresses. Here, we document that the vitamin D receptor (VDR) acts as a master transcriptional regulator of autophagy. Activation of the VDR by vitamin D induces autophagy and an autophagic transcriptional signature in BC cells that correlates with increased survival in patients; strikingly, this signature is present in the normal mammary gland and is progressively lost in patients with metastatic BC. A number of epidemiological studies have shown that sufficient vitamin D serum levels might be protective against BC. We observed that dietary vitamin D supplementation in mice increases basal levels of autophagy in the normal mammary gland, highlighting the potential of vitamin D as a cancer-preventive agent. These findings point to a role of vitamin D and the VDR in modulating autophagy and cell death in both the normal mammary gland and BC cells.

Cyclophilin D knockout protects the mouse kidney against cyclosporin A-induced oxidative stress.

Mitochondrial dysfunction and oxidative stress have been implicated in cyclosporin A (CsA)-induced nephrotoxicity. CsA interacts with cyclophilin D (CypD), an essential component of the mitochondrial permeability transition pore and regulator of cell death processes. Controversial reports have suggested that CypD deletion may or may not protect cells against oxidative stress-induced cell death. In the present study, we treated wild-type (WT) mice and mice lacking CypD [peptidylprolyl isomerase F knockout (Ppif-/-) mice] with CsA to test the role and contribution of CypD to the widely described CsA-induced renal toxicity and oxidative stress. Our results showed an increase in the levels of several known uremic toxins as well as the oxidative stress markers PGF2α and 8-isoprostane in CsA-treated WT animals but not in Ppif-/- animals. Similarly, a decline in S-adenosylmethionine and the resulting methylation potential indicative of DNA hypomethylation were observed only in CsA-treated WT mice. This confirms previous reports of the protective effects of CypD deletion on the mouse kidney mediated through a stronger resistance of these animals to oxidative stress and DNA methylation damage. However, a negative effect of CsA on the glycolysis and overall energy metabolism in Ppif-/- mice also indicated that additional, CypD-parallel pathways are involved in the toxic effects of CsA on the kidney. In summary, CsA-mediated induction of oxidative stress is associated with CypD, with CypD deletion providing a protective effect, whereas the reduction of energy production observed upon CsA exposure did not depend on the animals' CypD status.

Complement factor H protects mice from ischemic acute kidney injury but is not critical for controlling complement activation by glomerular IgM.

Natural IgM binds to glomerular epitopes in several progressive kidney diseases. Previous work has shown that IgM also binds within the glomerulus after ischemia/reperfusion (I/R) but does not fully activate the complement system. Factor H is a circulating complement regulatory protein, and congenital or acquired deficiency of factor H is a strong risk factor for several types of kidney disease. We hypothesized that factor H controls complement activation by IgM in the kidney after I/R, and that heterozygous factor H deficiency would permit IgM-mediated complement activation and injury at this location. We found that mice with targeted heterozygous deletion of the gene for factor H developed more severe kidney injury after I/R than wild-type controls, as expected, but that complement activation within the glomeruli remained well controlled. Furthermore, mice that are unable to generate soluble IgM were not protected from renal I/R, even in the setting of heterozygous factor H deficiency. These results demonstrate that factor H is important for limiting injury in the kidney after I/R, but it is not critical for controlling complement activation by immunoglobulin within the glomerulus in this setting. IgM binds to glomerular epitopes after I/R, but it is not a significant source of injury.

Mineralocorticoid Antagonism and Vascular Function in Early Autosomal Dominant Polycystic Kidney Disease: A Randomized Controlled Trial.

RATIONALE & OBJECTIVE: Vascular dysfunction, characterized by impaired vascular endothelial function and increased large-elastic artery stiffness, is evident early in autosomal dominant polycystic kidney disease (ADPKD) and is an important predictor of cardiovascular events and mortality. Aldosterone excess has been implicated in the development of endothelial dysfunction and arterial stiffness, in part by causing increased oxidative stress and inflammation. We hypothesized that aldosterone antagonism would reduce vascular dysfunction in patients with early-stage ADPKD. STUDY DESIGN: Prospective, randomized, controlled, double-blind, clinical trial. SETTING & PARTICIPANTS: 61 adults aged 20 to 55 years with ADPKD, estimated glomerular filtration rate ≥ 60mL/min/1.73m2, and receiving a renin-angiotensin-aldosterone system inhibitor. INTERVENTION: Spironolactone (maximum dose, 50mg/d) or placebo for 24 weeks. OUTCOMES: Change in brachial artery flow-mediated dilation (FMDBA) was the primary end point and change in carotid-femoral pulse-wave velocity (CFPWV) was the secondary end point. RESULTS: 60 participants completed the trial. Participants had a mean age of 34±10 (SD) years, 54% were women, and 84% were non-Hispanic white. Spironolactone did not change FMDBA (8.0% ± 5.5% and 7.8% ± 4.3% at baseline and 24 weeks, respectively, vs corresponding values in the placebo group of 8.4% ± 6.2% and 8.0% ± 4.6%; P=0.9for comparison of change between groups) or CFPWV (640±127 and 603±101cm/s at baseline and 24 weeks, respectively, vs corresponding values in the placebo group of 659±138 and 658±131cm/s; P=0.1). Brachial systolic blood pressure was reduced with spironolactone (median change, -6 [IQR, -15, 1] vs -2 [IQR, -7, 10] mm Hg in the placebo group; P=0.04). Spironolactone did not change the majority of circulating and/or endothelial cell markers of oxidative stress/inflammation and did not change vascular oxidative stress. LIMITATIONS: Low level of baseline vascular dysfunction; lack of aldosterone measurements. CONCLUSIONS: 24 weeks of aldosterone antagonism reduced systolic blood pressure without changing vascular function in patients with early-stage ADPKD. FUNDING: NIDDK, NIH National Center for Advancing Translational Sciences, and the Zell Family Foundation. TRIAL REGISTRATION: Registered at ClinicalTrials.gov with study number NCT01853553.

A proteomics-metabolomics approach indicates changes in hypothalamic glutamate-GABA metabolism of adult female rats submitted to intrauterine growth restriction.

PURPOSE: Intrauterine growth restriction (IUGR) has been shown to induce the programming of metabolic disturbances and obesity, associated with hypothalamic derangements. The present study aimed at investigating the effects of IUGR on the protein and metabolite profiles of the hypothalamus of adult female rats. METHODS: Wistar rats were mated and either had ad libitum access to food (control group) or received only 50% of the control intake (restricted group) during the whole pregnancy. Both groups ate ad libitum throughout lactation. At 4 months of age, the control and restricted female offspring was euthanized for blood and tissues collection. The hypothalami were processed for data independent acquisition mass spectrometry-based proteomics or targeted mass spectrometry-based metabolomics. RESULTS: The adult females submitted to IUGR showed increased glycemia and body adiposity, with normal body weight and food intake. IUGR modulated significantly 28 hypothalamic proteins and 7 hypothalamic metabolites. The effects of IUGR on hypothalamic proteins and metabolites included downregulation of glutamine synthetase, glutamate decarboxylase, glutamate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate, and up-regulation of NADH dehydrogenase and phosphoenolpyruvate. Integrated pathway analysis indicated that IUGR affected GABAergic synapse, glutamate metabolism, and TCA cycle, highly interconnected pathways whose derangement has potentially multiple consequences. CONCLUSION: The present findings suggested that the effects of IUGR on GABA/glutamate-glutamine cycle may be involved in the programming of obesity and hyperglycemia in female rats.