Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis.

Literature reports the chemical constituent yields of electronic nicotine delivery systems (ENDS) aerosol collected using a range of aerosol collection strategies. The number of puffs to deplete an ENDS product varies widely, but collections often consist of data from the first 50-100 puffs. However, it is not clear whether these discrete puff blocks are representative of constituent yields over the life of a pod. We aimed to assess the effect of differing aerosol collection strategies on reported yields for select chemical constituents in the aerosol of closed pod-based ENDS products. Constituents analyzed were chosen to reflect important classes of compounds from the Final Premarket Tobacco Product Application Guidance. Yields were normalized to total device mass loss (DML). Collection strategies that consisted of partial pod collection were valid for determining yields of constituents whose DML normalized yields were consistent for the duration of pod life. These included primary aerosol constituents, such as propylene glycol, glycerol, and nicotine, and whole pod yields could be determined from initial puff blocks. However, changes were observed in the yields of some metals, some carbonyl compounds, and glycidol over pod life in a chemical constituent and product dependent manner. These results suggest that collection strategies consisting of initial puff block collections require validation per chemical constituent/product and are not appropriate for chemical constituents with variable yields over pod life. Whole pod collection increased sensitivity and accuracy in determining metal, carbonyl, and glycidol yields compared to puff block-based collection methodologies for all products tested.

IL-6-GP130 signaling protects human hepatocytes against lipid droplet accumulation in humanized liver models.

Liver steatosis is an increasing health issue with few therapeutic options, partly because of a paucity of experimental models. In humanized liver rodent models, abnormal lipid accumulation in transplanted human hepatocytes occurs spontaneously. Here, we demonstrate that this abnormality is associated with compromised interleukin-6 (IL-6)-glycoprotein 130 (GP130) signaling in human hepatocytes because of incompatibility between host rodent IL-6 and human IL-6 receptor (IL-6R) on donor hepatocytes. Restoration of hepatic IL-6-GP130 signaling, through ectopic expression of rodent IL-6R, constitutive activation of GP130 in human hepatocytes, or humanization of an Il6 allele in recipient mice, substantially reduced hepatosteatosis. Notably, providing human Kupffer cells via hematopoietic stem cell engraftment in humanized liver mice also corrected the abnormality. Our observations suggest an important role of IL-6-GP130 pathway in regulating lipid accumulation in hepatocytes and not only provide a method to improve humanized liver models but also suggest therapeutic potential for manipulating GP130 signaling in human liver steatosis.

Efficient engraftment and viral transduction of human hepatocytes in an FRG rat liver humanization model.

Humanized liver rodent models, in which the host liver parenchyma is repopulated by human hepatocytes, have been increasingly used for drug development and disease research. Unlike the leading humanized liver mouse model in which Fumarylacetoacetate Hydrolase (Fah), Recombination Activating Gene (Rag)-2 and Interleukin-2 Receptor Gamma (Il2rg) genes were inactivated simultaneously, generation of similar recipient rats has been challenging. Here, using Velocigene and 1-cell-embryo-targeting technologies, we generated a rat model deficient in Fah, Rag1/2 and Il2rg genes, similar to humanized liver mice. These rats were efficiently engrafted with Fah-expressing hepatocytes from rat, mouse and human. Humanized liver rats expressed human albumin and complement proteins in serum and showed a normal liver zonation pattern. Further, approaches were developed for gene delivery through viral transduction of human hepatocytes either in vivo, or in vitro prior to engraftment, providing a novel platform to study liver disease and hepatocyte-targeted therapies.

Determining the impact of flavored e-liquids on aldehyde production during Vaping.

1,2-Propylene glycol and glycerin, principal components of e-liquids, can thermally degrade to form acetaldehyde, acrolein, and formaldehyde when heated in electronic nicotine delivery systems (ENDS). Recently the flavors in e-liquids were suggested to be the major source of these aldehydes. We used the same 10 ENDS devices to test 5 e-liquid formulations (four flavored & one corresponding non-flavored) and measured device mass loss and levels of acetaldehyde, acrolein, and formaldehyde (30 replicate measurements per formulation). Despite finding reasonable variability in measurements of device mass loss, two out of 10 ENDS devices tested produced outlier values for aerosol levels acetaldehyde, acrolein, and formaldehyde. After removing these devices from further analysis, acceptable variability (≤20% RSD) in aerosol levels of acetaldehyde, and formaldehyde were found. The flavored formulations tested resulted in a consistent and selective increase of 150%-200% in acetaldehyde, no increase or decrease in acrolein and depending on the flavor formulation, an increase, a decrease or no change in formaldehyde levels. Comparison of our results to the literature illustrates the need for development of a standardized ENDS testing protocol. Our results further support that device variability must be fully characterized and considered before assessing the impact of e-liquid formulations.

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.

Aldehyde levels in e-cigarette aerosol: Findings from a replication study and from use of a new-generation device.

PURPOSE: A recent study identified high aldehyde emissions from e-cigarettes (ECs), that when converted to reasonable daily human EC liquid consumption, 5 g/day, gave formaldehyde exposure equivalent to 604-3257 tobacco cigarettes. We replicated this study and also tested a new-generation atomizer under verified realistic (no dry puff) conditions. DESIGN: CE4v2 atomizers were tested at 3.8 V and 4.8 V, and a Nautilus Mini atomizer was tested at 9.0 W and 13.5 W. All measurements were performed in a laboratory ISO-accredited for EC aerosol collection and aldehyde measurements. RESULTS: CE4v2 generated dry puffs at both voltage settings. Formaldehyde levels were >10-fold lower, acetaldehyde 6-9-fold lower and acrolein 16-26-fold lower than reported in the previous study. Nautilus Mini did not generate dry puffs, and minimal aldehydes were emitted despite >100% higher aerosol production per puff compared to CE4v2 (formaldehyde: 16.7 and 16.5 µg/g; acetaldehyde: 9.6 and 10.3 µg/g; acrolein: 8.6 and 11.7 µg/g at 9.0 W and 13.5 W, respectively). EC liquid consumption of 5 g/day reduces aldehyde exposure by 94.4-99.8% compared to smoking 20 tobacco cigarettes. CONCLUSION: Checking for dry puffs is essential for EC emission testing. Under realistic conditions, new-generation ECs emit minimal aldehydes/g liquid at both low and high power. Validated methods should be used when analyzing EC aerosol.

Mitochondrial cyclophilin D ablation is associated with the activation of Akt/p70S6K pathway in the mouse kidney.

The mitochondrial matrix protein cyclophilin D (CypD) is an essential component of the mitochondrial permeability transition pore (MPTP). Here we characterized the effects of CypD ablation on bioenergetics in the kidney. CypD loss triggers a metabolic shift in Ppif-/- male and female mouse kidneys towards glycolysis and Krebs cycle activity. The shift is accompanied by increased glucose consumption and a transcriptional upregulation of effectors of glucose metabolism in the kidney. These included activation of Akt, AMPK (only in males) and p70S6K kinases. Gender specific differences between the Ppif-/- male and female mouse kidneys were observed including activation of pro-surviving ERK1/2 kinase and inhibited expression of pro-apoptotic and pro-fibrotic JNK and TGFß1 proteins in Ppif-/- females. They also showed the highest expression of phosphorylated-ERK1/2 and Akt S473 proteins of all four investigated animal groups. Furthermore, Ppif-/- females showed higher lactate concentrations and ATP/ADP-ratios in the kidney than males. These metabolic and transcriptional modifications could provide an additional level of protection to Ppif-/- females. In summary, loss of mitochondrial CypD results in a shift in bioenergetics and in activation of glucose-metabolism regulating Akt/AMPK/p70S6 kinase pathways that is expected to affect the capability of Ppif-/- mice kidneys to react to stimuli and injury.

Ablation of Cyclophilin D Results in an Activation of FAK, Akt, and ERK Pathways in the Mouse Heart.

Cyclophilin D (CypD) is a mitochondrial chaperone that regulates the mitochondrial permeability transition pore. Metabolically, deletion of Ppif (the gene encoding CypD) in mice is associated with elevated levels of mitochondrial matrix Ca2+ that leads to increased glucose as relative to fatty acid oxidation. Here, we characterized the adaptive mechanisms involved in the regulation of glucose metabolism including the regulation of Akt and ERK kinases that we evaluated by Western blot analysis of Ppif-/- in comparison to wild type (WT) mouse hearts. CypD loss led to adaptive mechanisms in the heart resulting in an upregulation of focal adhesion kinase (phosphorylated at Tyr925) and increased phosphorylation of Akt at S473. The increased activity of this pathway (pAktS473 increased to 170% and 145% in Ppif-/- versus WT males and females, respectively) could be responsible for the observed metabolic switch towards glycolysis. Furthermore, the phosphorylation of ERK1/2 proteins was elevated following CypD ablation. In addition, we observed differences in protein expression and activity in male versus female hearts that were independent of CypD expression. This included an upregulation of pAktS473 (to 273% and 269% in Ppif-/- and WT females as compared to their corresponding males, respectively). Furthermore, decreased levels of endothelial nitric oxide synthase (eNOS) inhibitor asymmetric dimethylarginine were accompanied by an upregulation of eNOS in female mice. The higher extent of kinases phosphorylation may be responsible for the reported lowered tolerance of CypD animals to stress. Moreover, the higher nitric oxide production could be responsible for the cardioprotective properties observed only in female hearts. J. Cell. Biochem. 118: 2933-2940, 2017. © 2017 Wiley Periodicals, Inc.

Pravastatin Therapy and Biomarker Changes in Children and Young Adults with Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND AND OBJECTIVES: Disease-specific treatment options for autosomal dominant polycystic kidney disease are limited. Clinical intervention early in life is likely to have the greatest effect. In a 3-year randomized double-blind placebo-controlled phase 3 clinical trial, the authors recently showed that pravastatin decreased height-corrected total kidney volume (HtTKV) progression of structural kidney disease over a 3-year period. However, the underlying mechanisms have not been elucidated. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Participants were recruited nationally from July 2007 through October 2009. Plasma and urine samples collected at baseline, 18 months, and 36 months from 91 pediatric patients enrolled in the above-mentioned clinical trial were subjected to mass spectrometry-based biomarker analysis. Changes in biomarkers over 3 years were compared between placebo and pravastatin-treated groups. Linear regression was used to evaluate the changes in biomarkers with the percent change in HtTKV over 3 years. RESULTS: Changes in plasma concentrations of proinflammatory and oxidative stress markers (9- hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, and 15-hydroxyeicosatetraenoic acid [HETE]) over 3 years were significantly different between the placebo and pravastatin-treated groups, with the pravastatin group showing a lower rate of biomarker increase. Urinary 8-HETE, 9-HETE, and 11-HETE were positively associated with the changes in HtTKV in the pravastatin group. CONCLUSIONS: Pravastatin therapy diminished the increase of cyclooxygenase- and lipoxygenase-derived plasma lipid mediators. The identified biomarkers and related molecular pathways of inflammation and endothelial dysfunction may present potential targets for monitoring of disease severity and therapeutic intervention of autosomal dominant polycystic kidney disease.

Endothelial dysfunction and oxidative stress in polycystic kidney disease.

Cardiovascular disease (CVD) is the leading cause of premature mortality in ADPKD patients. The aim was to identify potential serum biomarkers associated with the severity of ADPKD. Serum samples from a homogenous group of 61 HALT study A ADPKD patients [early disease group with estimated glomerular filtration rate (eGFR) >60 ml·min(-1)·1.73 m(-2)] were compared with samples from 49 patients from the HALT study B group with moderately advanced disease (eGFR 25-60 ml·min(-1)·1.73 m(-2)). Targeted tandem-mass spectrometry analysis of markers of endothelial dysfunction and oxidative stress was performed and correlated with eGFR and total kidney volume normalized to the body surface area (TKV/BSA). ADPKD patients with eGFR >60 ml·min(-1)·1.73 m(-2) showed higher levels of CVD risk markers asymmetric and symmetric dimethylarginine (ADMA and SDMA), homocysteine, and S-adenosylhomocysteine (SAH) compared with the healthy controls. Upon adjustments for age, sex, systolic blood pressure, and creatinine, SDMA, homocysteine, and SAH remained negatively correlated with eGFR. Resulting cellular methylation power [S-adenosylmethionine (SAM)/SAH ratio] correlated with the reduction of renal function and increase in TKV. Concentrations of prostaglandins (PGs), including oxidative stress marker 8-isoprostane, as well as PGF2α, PGD2, and PGE2, were markedly elevated in patients with ADPKD compared with healthy controls. Upon adjustments for age, sex, systolic blood pressure, and creatinine, increased PGD2 and PGF2α were associated with reduced eGFR, whereas 8-isoprostane and again PGF2α were associated with an increase in TKV/BSA. Endothelial dysfunction and oxidative stress are evident early in ADPKD patients, even in those with preserved kidney function. The identified pathways may provide potential therapeutic targets for slowing down the disease progression.

Bioactive lipid mediators in polycystic kidney disease.

Inflammatory activity is evident in patients with chronic kidney disease with limited data available in autosomal dominant polycystic kidney disease (ADPKD). We hypothesized that inflammation is an upstream event in the pathogenesis of ADPKD and may be a contributing factor in the disease severity and progression. Serum samples from 61 HALT study A group patients were compared with samples from 49 patients from HALT study B group with moderately advanced disease. Targeted MS analysis of bioactive lipid mediators as markers of inflammation was performed and correlated with eGFR and total kidney volume (TKV) normalized to the body surface area (BSAR) to assess if these markers are predictive of ADPKD severity. ADPKD patients with eGFR >60 ml/min/1.73 m(2) showed higher levels of 5- and 12/15-lipoxygenase (LOX) and cyclooxygenase, and generated higher levels of hydroxy-octadecadienoic acids 9-HODE and 13-HODE and HETEs 8-HETE, 11-HETE, 12-HETE, and 15-HETE as compared with healthy subjects. Linear regression of 9-HODE and 13-HODE revealed a significant relationship with eGFR and TKV, while 15-HETE significantly correlated with TKV/BSAR. Production of 20-HETE, a P450-produced metabolite of arachidonic acid, was higher in ADPKD patients as compared with healthy subjects and significantly correlated with eGFR and TKV/BSAR. Perturbation in fatty acid metabolism is evident early in ADPKD patients, even in those with preserved kidney function. The identified LOX pathways may be potential therapeutic targets for slowing down ADPKD progression.

Effects of lovastatin treatment on the metabolic distributions in the Han:SPRD rat model of polycystic kidney disease.

BACKGROUND: We previously demonstrated that lovastatin decreases cyst volume and improves kidney function in the Han:SPRD (Cy/+) rat model of ADPKD. Since endothelial dysfunction and inflammatory activity are evident in patients with ADPKD, we investigated whether lovastatin reduces the inflammation and vascular dysfunction and improves kidney cell energy metabolism of Cy/+ rats. METHODS: Cy/+ and normal littermate control animals (+/+) were treated with either lovastatin (4 mg/kg/day) or vehicle (ethanol) from 3-8 weeks of age. 1H-NMR analysis was performed on water-soluble and lipid kidney fractions following perchloric acid extraction. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess endothelial dysfunction, oxidative stress and inflammation markers in plasma and kidney tissue extracts. RESULTS: Cy/+ rats showed perturbations in fatty acid metabolism and increased synthesis of pro-inflammatory lipoxygenases-produced bioactive lipids was observed. Lovastatin decreased inflammatory markers, specifically 13-HODE, 12-HETE and leukotriene B4. In Cy/+ rats, lovastatin reduced the elevated homocysteine and allantoin plasma levels and increased arginine, that is known to positively affect NO production. CONCLUSION: As previously described, lovastatin was able to decrease kidney weight and cyst volume density in Cy/+ rats. The decrease in cyst volume was accompanied by a reduction in arachidonic acid-mediated inflammation markers, the normalization of metabolism of NO precursors and the improvement of kidney energy cell metabolism.