Wild-type Males Research Articles

Methionine Restriction Affects Metabolic Parameters in a Fibroblast Growth Factor 21- and Sex-Dependent Manner

ObjectivesIn rodents, dietary restriction of the amino acid methionine produces profound protection against obesity and obesity-related comorbidities. Fibroblast growth factor 21 (FGF21) is now recognized as an important mediator of methionine restriction's (MR) metabolic benefits. Most preclinical work investigating the impact of diet on weight gain and metabolism has been conducted in males, leaving a gap in our understanding of female responses to dietary changes. This study aimed to examine the impact of high-fat diet (HFD) feeding and MR on body weight and adiposity in male and female mice. Furthermore, we sought to determine the sex-dependent role of endogenous FGF21 in mediating metabolic responses to HFD and MR.MethodsMale and female wild-type (WT) and Fgf21 knockout (Fgf21−/−) mice were fed low-fat diet (LFD) or HFD versions of control (0.86% methionine) or MR (0.17% methionine) diets for 5 weeks. Average body weight (BW) and food intake were recorded weekly. Insulin tolerance tests were performed at week 4. After 5 weeks, mice were euthanized, and adipose depots were collected.ResultsNeither HFD nor MR significantly altered BW of female mice regardless of genotype. However, while HFD did not significantly affect BW, MR significantly reduced BW of both WT and Fgf21−/− males (p < 0.0001). Neither HFD nor MR significantly altered adiposity (gonadal adipose tissue weight/body weight) in female mice regardless of genotype. However, HFD significantly increased adiposity in male WT and Fgf21−/− mice, and MR prevented this increase in both genotypes. MR increased energy intake in LFD- and HFD-fed WT mice. The ability of MR to increase energy intake was abolished in LFD- and HFD-fed Fgf21−/− mice.ConclusionsThe effects of MR on BW and adiposity are sex-dependent, but FGF21-independent. Short-term HFD feeding affects adiposity differently in males and females. The effects of MR on energy intake are dependent upon FGF21. As the field progresses towards developing FGF21 as a therapeutic agent, it will be essential to understand its impact on both sexes.Funding SourcesSources Center for Neuroinflammation and Cardiometabolic Diseases Seed Grant.

1393-P: Cardiac and Skeletal Muscles Exhibit Discordant Flux Adaptations as a Result of Western Diet–Induced Obesity

The metabolic complications of obesity are distributed throughout the body, yet studies assessing its impact on metabolic fluxes have primarily focused on single tissues. Here we use a 13C tracer approach to simultaneously quantify metabolic flux dysregulation in the liver, skeletal muscle, and heart induced by Western diet (WD) feeding in a mouse model of obesity. Male wildtype (WT) and Mc4r-/- mice (KO) were fed chow until 8wks of age, when ∼50% of the KO cohort transitioned to WD (KO+WD) for 8wks. KO and KO+WD had increased adiposity, hepatic steatosis, HOMA-IR, and reduced glucose tolerance relative to WT by 16wks of age; however, only KO+WD had extensive liver fibrosis. Stable- and radio-labeled isotopes were infused and blood was sampled from fasted, conscious, and unrestrained mice (n ≥ 6 per group) through jugular vein and carotid artery catheters, respectively. Metabolic fluxes were estimated by fitting an integrated mathematical model of liver, skeletal muscle, and cardiac metabolism to 13C enrichment measurements of plasma and tissue metabolites taken at the end of the infusion. Endogenous glucose production was elevated in both KO cohorts, with WD further increasing hepatic gluconeogenic and citric acid cycle (CAC) fluxes. Glycolytic, anaplerotic, and CAC fluxes were lower in the skeletal muscle of KO and KO+WD compared to WT, which coincided with a decrease and increase in gene expression of glycolytic and β-oxidation enzymes, respectively. In the heart, however, WD feeding increased glycolytic and CAC fluxes as well as the gene expression of glycolytic and β-oxidation enzymes. Thus, cardiac and skeletal muscles exhibit discordant flux adaptations in obese, WD-fed mice, as WD accelerates aerobic glycolysis and terminal oxidation in the heart but not skeletal muscle. We expect the multi-organ fluxomics platform described here to broadly benefit research to understand systemic metabolism in health and disease. Disclosure C.M.Hasenour: Employee; Eli Lilly and Company. M.Rahim: None. T.Bednarski: None. D.R.Banerjee: None. J.Young: Consultant; Pfizer Inc., Research Support; Merck &amp; Co., Inc., Stock/Shareholder; Metalytics, Inc. Funding National Institutes of Health (R01DK106348, U01CA235508, T32DK101003)

The Effect of DNAJB3 Deficiency on Metabolic Dysfunctions in Diet-induced Obese Mice

Abstract Objectives Obesity and related metabolic dysfunctions are partly mediated by chronic low-grade inflammation and altered expression of numerous proteins including stress responsive heat shock proteins (HSPs). We are specifically interested in HSP40 (Heat Shock Protein-40), subfamily B, member 3 (DNAJB3), a chaperone protein that aids in restoring protein homeostasis. Patients with obesity and type 2 diabetes (T2D) expressed low levels of DNAJB3, which was partly restored by physical activity. Accordingly, we hypothesized that lack of DNAJB3 will increase body weight, inflammation, glucose intolerance and insulin resistance in diet-induced obese mice, compared to B6 wild type (WT) littermates fed the same diets. Methods DNAJB3 knockout (KO) mice were generated using the CRISPR/Cas 9 approach. Male and female KO and wild type (WT) mice were fed high fat (HF: 45 kcal% fat) or low fat (LF: 10 kcal% fat) diets for 12 weeks. Body weight and food intake were measured weekly; body composition measurements and glucose tolerance tests were also conducted during the intervention. Following euthanasia, blood, and tissues were harvested for further analyses. Serum adipokines and hormones were measured using a multiplex immunoassay. Results Compared to WT male and female, KO mice fed HF diets demonstrated higher body weight and fat mass. Similarly, both male and female KO HF groups demonstrated a slower glucose clearance rate as measured by GTT compared to the LF KO and WT groups as well as HF WT groups. Interestingly, these findings were more significant across the board in the female HF-KO group. Female HF group demonstrated a higher leptin, IL-6 and Insulin levels compared to the other groups (p &amp;lt; 0.0001). Additionally, Male Leptin and Resistin levels were higher in the HF-KO group compared to the other groups (p = 0.0001, p = 0.0253). Conclusions Lack of DNAJB3, increases adiposity and glucose intolerance in diet-induced obese male and female mice. Thus, DNAJB3 potentially plays an important role in metabolic functions and glucose homeostasis, warranting further research on DNAJB3 as a potential therapeutic target for obesity and T2D. Funding Sources Funded by Qatar National Research Funds, Hamad Bin Khalifa University &amp; Qatar Biomedical Research Institute, Qatar.

TXNIP knockout improves cardiac function after myocardial infarction by promoting angiogenesis and reducing cardiomyocyte apoptosis.

Myocardial infarction (MI) is a common cause of death. Thioredoxin-interacting protein (TXNIP) expression increases after MI, and it exerts a negative regulatory effect on cardiac function after MI. Our study aimed to investigate the specific regulatory mechanism of TXNIP on angiogenesis and cardiomyocyte apoptosis after MI. The TXNIP gene knock-in (TXNIP-KI) and knock-out (TXNIP-KO) mice were generated, respectively. Eight-week-old male TXNIP-KO, TXNIP-KI, and wild type (WT) mice were subjected to MI by permanent ligation of the left anterior descending artery. Cardiomyocyte apoptosis was detected by TUNEL assay on the 4th post-surgery day. The expressions of TXNIP, hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF), phosphorylated protein kinase B (p-AKT), p-AMP-activated protein kinase (p-AMPK), cleaved caspase-3, and caspase-3 were detected by Western blot. Quantitative real-time PCR was performed to detect the expression of TXNIP, HIF-1α, VEGF, prolyl hydroxylase (PHD) 1, and factor inhibiting HIF (FIH). In addition, the superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in each group were also measured. On day 7 after MI, the hearts of sacrificed animals were analyzed by immunohistochemistry to assess CD31 expression and determine the density of angiogenesis. One month after treatment, the cardiac functional and structural changes were determined by echocardiography and the level of myocardial fibrosis was observed by Masson staining. Compared with WT mice, TXNIP-KO mice had a significantly improved cardiac functional recovery after MI, and the proportion of myocardial fibrosis area was dramatically reduced, cardiomyocyte apoptosis was decreased, and angiogenesis was significantly increased; TXNIP-KI mice reversed in these changes. The expression of HIF-1α, p-AKT, and p-AMPK increased after MI in TXNIP-KO mice, and the mRNA expression of PHD 1 and FIH decreased. TXNIP-KI mice reversed in these changes. After MI, TXNIP down-regulated the level of HIF-1α and VEGF, reduced the number of angiogenesis, increased cardiomyocyte apoptosis, and ultimately led to a poor prognosis of ischemic myocardium. TXNIP was a protein with negative effects after MI and was expected to be a target for the prevention and treatment of MI.

Interleukin-10 deficiency aggravates traumatic brain injury in male but not female mice

The goal of this study was to determine whether deficiency of anti-inflammatory cytokine interleukin-10 (IL-10) affects traumatic brain injury (TBI) outcomes in a sex-dependent manner. Moderate TBI was induced by controlled cortical impact in 8-10 week-old wild-type and IL-10–deficient mice. In wild-type mice, serum IL-10 was significantly increased after TBI in males but not in females. At 4–5 weeks after TBI, sensorimotor function, cognitive function (Y-maze and novel object recognition tests), anxiety-related behavior (light-dark box and open field test), and depression-like behavior (forced swim test) were assessed. IL-10–deficient male mice had larger lesion volumes than did wild-type mice in the early recovery phase and worse performance on sensorimotor tasks, cognitive tests, and anxiety- and depression-related tests in the late recovery phase, whereas female IL-10–deficient mice had lesion volume equivalent to that of wild-type females and worse performance only on sensorimotor tasks. At 3 days after TBI, the number of GFAP- and Iba1-positive cells were augmented in areas in proximity to the injury (cerebral cortex and hippocampus) and in remote functional regions (striatum and amygdala) of IL-10–deficient male, but not female, mice. Moreover, on day 35, significantly fewer NeuN-positive cells were present in cortex, striatum, and amygdala of IL-10–deficient male mice than in wild-type males. This difference was not evident in females. We conclude that IL-10 deficiency aggravates cognitive and emotional recovery from TBI in association with enhanced gliosis and neuronal loss selectively in males, suggesting that recruitment of this cytokine limits damage in a sex-dependent manner.

Further Evidence that Inhibition of Neuronal Voltage-Gated Calcium Channels Contributes to the Hypnotic Effect of Neurosteroid Analogue, 3β-OH.

We recently reported that a neurosteroid analogue with T-channel-blocking properties (3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile (3β-OH), induced hypnosis in rat pups without triggering neuronal apoptosis. Furthermore, we found that the inhibition of the CaV3.1 isoform of T-channels contributes to the hypnotic properties of 3β-OH in adult mice. However, the specific mechanisms underlying the role of other subtypes of voltage-gated calcium channels in thalamocortical excitability and oscillations in vivo during 3β-OH-induced hypnosis are largely unknown. Here, we used patch-clamp recordings from acute brain slices, in vivo electroencephalogram (EEG) recordings, and mouse genetics with wild-type (WT) and CaV2.3 knock-out (KO) mice to further investigate the molecular mechanisms of neurosteroid-induced hypnosis. Our voltage-clamp recordings showed that 3β-OH inhibited recombinant CaV2.3 currents. In subsequent current-clamp recordings in thalamic slices ex vivo, we found that selective CaV2.3 channel blocker (SNX-482) inhibited stimulated tonic firing and increased the threshold for rebound burst firing in WT animals. Additionally, in thalamic slices we found that 3β-OH inhibited spike-firing more profoundly in WT than in mutant mice. Furthermore, 3β-OH reduced bursting frequencies in WT but not mutant animals. In ensuing in vivo experiments, we found that intra-peritoneal injections of 3β-OH were less effective in inducing LORR in the mutant mice than in the WT mice, with expected sex differences. Furthermore, the reduction in total α, β, and low γ EEG power was more profound in WT than in CaV2.3 KO females over time, while at 60 min after injections of 3β-OH, the increase in relative β power was higher in mutant females. In addition, 3β-OH depressed EEG power more strongly in the male WT than in the mutant mice and significantly increased the relative δ power oscillations in WT male mice in comparison to the mutant male animals. Our results demonstrate for the first time the importance of the CaV2.3 subtype of voltage-gated calcium channels in thalamocortical excitability and the oscillations that underlie neurosteroid-induced hypnosis.

Comparative Transcriptome Analyses Characterize Expression Signatures Among Males, Females, Neo-Males, and Gynogenetic Females in the Yellow Drum (Nibea albiflora)

The yellow drum (Nibea albiflora) is one of the most important marine economic fish in China, and its sexually dimorphic growth makes it preferable for mono-sex culture. Although gynogenesis and neo-male induction techniques have been established, the molecular pathways and regulatory mechanisms of sex determination and maintenance in gynogenetic females and neo-males remains far from fully understood. In this study, the gene expression profiles were investigated in the gonads and brains of wild-type male, wild-type female, neo-male, and gynogenetic female yellow drum using comparative transcriptome analyses. Generally, a total of 52,999 novel transcripts were obtained in RNA-seq, of which 45,651 were isoforms of known protein-coding genes, 1,358 novel protein-coding genes, and 5,990 long non-coding RNAs. We found that the differences between wild-type males and neo-males and between wild-type females and gynogenetic females were relatively small at both the histological and transcriptomic levels, indicating that artificial gynogenesis or hormonal sex reversal may have minimal effects on normal female or male life function, respectively. In the brain, pathways such as “Oocyte meiosis”, “Cell cycle”, and “Riboflavin metabolism” were found to be significantly enriched. In the gonads, pathways such as “Prolactin signaling pathway”, “PPAR signaling pathway”, “Cholesterol metabolism”, and “Jak-STAT signaling pathway” were found to play important roles in maintaining the regular proliferation and differentiation of females and males in yellow drum. In particular, we found that zp4 might be an effective molecular marker to differentiate between gynogenetic and normal females owing to its unique expression pattern. The results of this study may help to elucidate the molecular mechanisms involved in sex maintenance in the gonads and brain and provide basic data for genetic breeding of the yellow drum.

Abnormal male reproduction and embryonic development induced by downregulation of a phospholipid fatty acid-introducing enzyme Lpgat1 in zebrafish

Phospholipids in the membrane consist of diverse pairs of fatty acids bound to a glycerol backbone. The biological significance of the diversity, however, remains mostly unclear. Part of this diversity is due to lysophospholipid acyltransferases (LPLATs), which introduce a fatty acid into lysophospholipids. The human genome has 14 LPLATs and most of them are highly conserved in vertebrates. Here, we analyzed the function of one of these enzymes, lysophosphatidylglycerol acyltransferase 1 (Lpgat1), in zebrafish. We found that the reproduction of heterozygous (lpgat1+/−) male mutants was abnormal. Crosses between heterozygous males and wild-type females produced many eggs with no obvious cleavage, whereas eggs produced by crosses between heterozygous females and wild-type males cleaved normally. Consistent with this, spermatozoa from heterozygous males had reduced motility and abnormal morphology. We also found that the occurrence of lpgat1 homozygous (lpgat1−/−) mutants was far lower than expected. In addition, downregulation of lpgat1 by morpholino antisense oligonucleotides resulted in severe developmental defects. Lipidomic analysis revealed that selective phospholipid species with stearic acid and docosahexaenoic acid were reduced in homozygous larvae and spermatozoa from heterozygotes. These results suggest that the specific phospholipid molecular species produced by Lpgat1 have an essential role in sperm fertilization and in embryonic development.

MO612: Relationship of Renal Timp-1, Stat3 and Inflammatory Factors in Diabetes

Abstract BACKGROUND AND AIMS The balance of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMP) is disrupted in diabetic nephropathy and renal TIMP-1 overexpression is observed in both animal experimental models and human biopsies. The STAT3 transcription factor participates in several inflammatory pathways; however, its interaction with TIMP-1 in diabetes remains unknown. We aimed to investigate the progression of nephropathy and its relationship to renal STAT3 activation in type-1 diabetic TIMP-1 knock-out (KO) mice. METHOD Diabetes was induced in 6-week-old male TIMP-1 KO (DM-KO, n = 8) and C57Bl6 wild type (DM, n = 5) mice by intraperitoneal streptozotocin injections (50 mg/kg/day for 5 days). Control mice (CTL, n = 6) received citrate buffer injections. Eight weeks after onset of diabetes blood and urine chemistries, renal histology, renal mRNA and protein expression were assessed. Statistical significance was evaluated using Kruskal–Wallis test and Tukey post-hoc analysis. RESULTS Despite similar hyperglycemia, DM-KO mice developed significantly less proteinuria (P &amp;lt;0.01) and histologically milder glomerulosclerosis and tubular damage as compared with wild type DM mice. Kidneys of DM mice depicted 4-fold STAT3 phosphorylation, but DM-KO kidneys had similar values as CTL nondiabetic controls. DM kidneys also showed 20-fold CCL2, 10-fold C3 and 2.5-fold C4 overexpression, which in DM-KO kidneys remained all near control (CTL) levels. CONCLUSION Based on our results, we conclude that TIMP-1 influences the progression of diabetic kidney disease in mice not solely through the inhibition of MMP-s, but also by influencing the extent of renal inflammation via STAT3 phosphorylation. Therefore, TIMP-1 might emerge as promising future therapeutic target. FUNDING Hungarian Academy of Sciences BO/00 304/20/5, ÚNKP-21–5-SE-1.

Chronic Stress Induces Hippocampal Mitochondrial Damage in APPPS1 Model Mice and Wildtype Littermates.

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia worldwide. Despite decades of investigation, the etiology of AD is not fully understood, although emerging evidence suggest that chronic environmental and psychological stress plays a role in the mechanisms and contributes to the risk of developing AD. Thus, dissecting the impact of stress on the brain could improve our understanding of the pathological mechanisms. We aimed to study the effect of chronic stress on the hippocampal proteome in male APPPS1 transgenic mice and wildtype (WT) littermates. APPPS1 and WT mice were subjected to 4 weeks of chronic stress followed by 3 weeks of continued diurnal disruption. Hippocampal tissue was used for proteomics analysis using label-free quantitative DIA based LC-MS/MS analysis. We identified significantly up- and downregulated proteins in both APPPS1 and WT mice exposed to chronic stress compared to the control groups. Via interaction network mapping, significant proteins could be annotated to specific pathways of mitochondrial function (oxidative phosphorylation and TCA cycle), metabolic pathways, AD pathway and synaptic functions (long term potentiation). In WT mice, chronic stress showed the highest impact on complex I of the oxidative phosphorylation pathway, while in APPPS1 mice this pathway was compromised broadly by chronic stress. Our data shows that chronic stress and amyloidosis additively contribute to mitochondrial damage in hippocampus. Although these results do not explain all effects of chronic stress in AD, they add to the scientific knowledge on the topic.

Sex-specific lipid dysregulation in the Abca7 knockout mouse brain.

Alzheimer’s disease is a devastating neurodegenerative disease that affects more women than men. The pathomechanism underlying the sex disparity, especially in the brain, is unclear. ABCA7 is one of the strongest susceptibility genes for Alzheimer’s disease. It mediates the transport of lipids across membranes and is associated with pathways related to amyloid-β neuropathology. However, the role of ABCA7 in the regulation of brain lipids is largely unknown. Sex-specific differences in the pathological link between brain lipid dysregulation and amyloid-β are also unknown. Here, we undertook quantitative discovery lipidomics of male and female Abca7 knockout (n = 52) and wild type (n = 35) mouse brain using sophisticated liquid chromatography/mass spectrometry. We identified 61 lipid subclasses in the mouse brain and found sex-specific differences in lipids that were altered with Abca7 deletion. The altered lipids belong to cellular pathways that control cell signalling, sterol metabolism, mitochondrial function and neuroprotection. We also investigated the relationship between lipids and amyloid-β levels in the Abca7 knockout mice and found elevated free cholesterol only in female mice that was significantly correlated with amyloid-β42 levels. In male Abca7 knockout mice, the neuroprotective ganglioside GD1a levels were elevated and inversely correlated with amyloid-β42 levels. Collectively, these results demonstrate that Abca7 deletion leads to sex-specific lipid dysregulation in the brain, providing insight into the underlying sex disparity in the aetiology of Alzheimer’s disease.

Metabolic and Cardiac Manifestations in a Mouse Model of Genetic Mitochondrial Hepatopathy Without Obesity: Evidence for a Sexual Dimorphism

ObjectiveIn non‐alcoholic fatty liver disease (NAFLD), liver mitochondrial dysfunction plays an important role in the development of cardiac abnormalities independent of obesity per se. However, the mechanisms underlying the development of these abnormalities, and whether these are affected by sex remain unclear. In the present study, we sought to address this question using a mouse model of hepatic mitochondrial deficiency caused by loss of Lrpprc (H‐Lrpprc‐/‐) as these mice develop microvesicular steatosis and a NAFLD lipidomic‐like profile without obesity.HypothesisOur working hypothesis is that metabolic defects in the liver would trigger metabolic and functional abnormalities in the heart in a sex‐dependent manner.MethodsDetailed phenotyping experiments were performed in male and female H‐Lrpprc‐/‐mice and their controls at 14 weeks of age. Glucose metabolism was monitored by blood glucose as well as insulin, and glucose tolerance tests (ITT, OGTT). Cardiac manifestations were studied using molecular (qPCR), lipidomic (mass spectrometry) and functional (oximetry on isolated mitochondria, intraventricular pressures by Millar probe) analyses.ResultsGenetic inactivation of Lrpprc resulted in an &gt;60% reduction of LRPPRC protein content in both males and females, which resulted in comparable structural liver abnormalities. In contrast, the resulting metabolic and cardiac functional changes displayed a marked sexual dimorphism. Lrpprc deficiency in the liver was associated with fasting hypoglycemia and increased insulin sensitivity in both sexes, but more so in males. Lrpprc deficiency was also associated with a remodeling of specific markers of mitochondrial function exclusively in the female myocardium. This included 1) reduced expression of gene markers for mitochondrial biogenesis (Tfam), fusion (Mfn1‐2), and fatty acid (FA) utilization and oxidation (Pparα, Cd36, Cpt1b, Cpt2, Vlcad, Lcad, Mcad), 2) impairment of FA‐dependent mitochondrial respiration, and 3) increased cardiac triglyceride levels. Paradoxically, cardiac function was preserved in females, while males displayed mild diastolic dysfunction as evidenced by increased early and late diastolic pressures (0.29 and 2.39 fold vs. male wild type respectively) amongst other parameters.ConclusionCollectively, these results indicate that metabolic defects in the liver can result in significant sex‐dependent abnormalities that affect both the mitochondrial/metabolic phenotype and contractile function independent of obesity. This experimental model may prove useful to better understand the mechanisms underlying the sex‐related variability in the progression of lean NAFLD in humans.

Fatty acid amide hydrolase (FAAH) inhibition mitigates TGF‐β1‐induced fibrogenesis via the anandamide‐COX‐2‐dependent pathway

Chronic proximal tubular injury with subsequent fibrosis is the key factor in the transition of acute kidney injury (AKI) to chronic kidney disease (CKD). An increasing body of evidence has shown that endocannabinoids such as anandamide (AEA) and 2‐arachidonoylglycerol (2AG) and their cannabinoid receptors (CBs) play important roles in renal diseases. It remains unclear whether substrates or metabolites of AEA‐metabolizing enzyme FAAH participate in fibrogenesis of kidneys. Though AEA binds the CB1 receptor with high binding affinity, its in vivo pharmacological effects mediated by cannabinoid receptors are of short duration. Likely, active metabolites produced from the rapid catabolism of AEA exert physiological actions. We have previously showed that inhibition of FAAH protects against TGF‐β1‐induced damages via actions independent of CBs in renal tubular cells. Therefore, our aims are to study the effects of downstream metabolites derived from FAAH as well as AEA‐ cyclooxygenase‐2 (COX‐2) pathways on TGF‐β1‐induced renal fibrogenesis. To produce the AKI to CKD transition model, male wild type (WT) C57BL6 and Faah−/− mice were subjected to post ischemia‐reperfusion injury (PIR) by unilateral kidney ischemia‐reperfusion and contralateral nephrectomy 10 days after ischemia‐reperfusion. Our previous studies showed that Faah−/− mice protected against PIR‐induced renal fibrogenesis. Here, LC‐MS/MS assay demonstrated that PIR‐injured WT mice had a significant downregulation of renal AEA level, which was blocked in Faah−/−‐PIR mice (68% reduction in WT‐PIR versus 12% in Faah−/−‐PIR mice compared with WT‐sham group, p&lt;0.001), whereas 2AG showed no difference between WT‐PIR and Faah−/−‐PIR mice (1.2‐fold versus 1.1‐fold increase, p&gt;0.9). In addition, Faah−/− mice did not show difference in the levels of arachidonic acid (AA) (1.13‐fold to WT‐sham, p=0.25), the downstream metabolites of FAAH. Our previous in vitro studies showed that TGF‐β1‐induced profibrotic effects in HK‐2 cells (human kidney proximal tubular epithelial cells) were abolished after the treatment of FAAH inhibitor (PF‐04457845, PF, 1μM) in a CB2‐independent manner. In the current study, blockage of AEA‐COX‐2 derived prostamides pathway by a substrate‐selective COX‐2 inhibitor (LM‐4131, 300nM) reversed the decreased level of a profibrotic marker α‐smooth muscle actin (α‐SMA) caused by PF (1.9‐fold in TGF‐β1, 0.83‐fold in PF+TGF‐β1 and 2.88‐fold in PF+LM‐4131+TGF‐β1 compared to the control group, p&lt;0.0001). Further, supplement of AA did not reverse the protection of FAAH inhibition against TGF‐β1‐induced expression of α‐SMA (2.53‐fold in TGF‐β1, 0.62‐fold in PF+TGF‐β1 versus 0.48‐fold in AA+PF+TGF‐β1 compared to the control group, p&gt;0.99). The AEA‐COX‐2 metabolite, prostamide E2 mimicked the protective effect of FAAH inhibition against TGF‐β1‐induced α‐SMA expression (2.17‐fold in TGF‐β1 vs. 0.9‐fold in prostamide E2 +TGF‐β1 compared to the control group, p&lt;0.0001). Overall, the present study suggests that inactivation of FAAH augments the actions of downstream metabolites of AEA‐COX‐2, which mediates the protection against TGF‐β1‐induced renal fibrogenesis.

Uncovering a sex‐specific role for olfactory receptor 558 (Olfr558) in blood pressure regulation

Olfactory receptors are primarily studied in the nose; but are also expressed and play pivotal roles in non‐olfactory tissues as ecnomotopic receptors. We find that olfactory receptor 558 (Olfr558) is expressed in vascular smooth muscle cells in the kidney, including the renal afferent and efferent arterioles. We developed Olfr558 whole‐body knockout (KO) mice to test the hypothesis that Olfr558 plays a role in blood pressure (BP) regulation. Olfr558 wild‐type (WT) vs. KO (males and females) exhibited no genotypic differences in body weight, kidney weight/body weight, heart weight/body weight, glomerular filtration rate, or blood values (electrolytes, glucose, blood urea nitrogen, creatinine, and hemoglobin). However, Olfr558 KO males had lower diastolic blood pressure (DBP) (81.1±1.6 mmHg, n=9, p=0.009) than WT males (89.1±2.2 mmHg, n=7) during the light cycle (telemetry) without a change in mean arterial (MAP) or systolic blood pressure (SBP). In contrast, Olfr558 KO females had increased MAP (KO: 95.0±1.7 mmHg, WT: 89.0±0.9 mmHg, p=0.003), SBP (KO: 108.0±1.7 mmHg, WT: 102.1±1.4 mmHg, p=0.02), and DBP (KO: 81.6±1.6 mmHg, WT: 75.0±0.9 mmHg, p=0.003) during the light cycle (n=8 for WT and KO). A similar trend was observed during the dark cycle. These data suggest that Olfr558 regulates BP in a sex‐specific manner.To determine the mechanisms underlying the sexually dimorphic effect of Olfr558 on BP regulation, we are examining renin signaling and vascular tone. In males, kidney renin mRNA levels were significantly decreased in Olfr558 KO (0.43±0.04, n=12, p=0.001 vs WT 1±0.14, n=11). Akr1b7 (renin cell marker) mRNA was also reduced in male KO (0.5±0.06, n=12, p=0.002, vs WT 1±0.09, n=10). We then evaluated the % of renin positive to total glomeruli and found that it was reduced in male KO (29.0±0.95, p=0.004, vs WT 43.2±1.88; n=8). We also found that plasma renin activity (PRA) was dramatically decreased in male KO (200.8±20.5, n=11, p=0.001) than WT (401.9±15.2, n=10). These results suggest that Olfr558 regulates BP in males at least in part via renin. In females, there were no differences in renin or Akr1b7 mRNA, nor in the % of renin positive glomeruli, nor PRA. These results suggest that, in females, Olfr558 modulates BP independent of renin. Studies of ex vivo vascular reactivity in males show that aortic rings exhibited less constriction to Phenylephrine (PE) in KO vs WT, which could contribute to the hypotension seen in these mice. In contrast, constriction to KCl and relaxation to Acetylcholine (ACh) and sodium nitroprusside (SNP) showed no genotypic difference. KO mesentery exhibited more relaxation to SNP, but not ACh, and similar constriction to KCl and PE as WT. In females, KO aortic rings exhibited more constriction to KCl, but not PE, and similar relaxation to ACh and SNP as WT. However, mesentery showed no genotypic difference in constriction to KCl and PE, relaxation to ACh and SNP. Thus, our findings suggest that the hypotension seen in Olfr558 KO males is likely contributed to both by decreased renin and by changes in vascular reactivity, whereas the origin of hypertension seen in Olfr558 KO females is still an area of active investigation.

Angiotensin Ii‐induced Hypertension and Glomerular and Tubulointerstitial Injury in Mutant Mice With Kidney Proximal Tubule‐selective Deletion of Mitochondrial Nad+‐dependent Deacetylase Sirtuin 3: Focus on Sex Differences

Sirtuin 3 (SIRT3) is a major mitochondrial NAD‐dependent Deacetylase closely involved in energy production, cell metabolism, oxidative stress responses, and mitochondrial homeostasis during hypertension and kidney diseases. However, it remains unknown whether deletion of SIRT3 in the proximal tubules alters the hypertensive and kidney glomerular and tubulointerstitial fibrotic responses to angiotensin II (Ang II) in sex‐dependent manners. In the present study, adult male and female wild‐type (WT) and mutant mice with proximal tubule‐specific deletion of SIRT3, PT‐Sirt3‐/‐, were infused without (time controls) or with a slow pressor dose of Ang II via an osmotic minipump (0.5 mg/kg/day, i.p.), supplemented with a 2% NaCI diet or losartan, 20 mg/kg/day, for 2 weeks. Systolic (SBP), diastolic (DBP), and mean arterial blood (MAP) pressure were determined using the tail‐cuff method, whereas 24 hr. urinary sodium and potassium excretion were determined using a metabolic cage. Serum and urine creatinine were measured using colorimetric assays, whereas glomerular and tubulointerstitial injury was evaluated by Masson’s Trichrome staining. Basal SBP, DBP, and MAP were significantly lower in PT‐Sirt3‐/‐ mice than WT (WT‐SBP: 112 ± 2 vs. PT‐Sirt3‐/‐‐SBP: 93 ± 2 mmHg, P&lt;0,01). The magnitude of Ang II‐induced hypertension was similar between WT and PT‐Sirt3‐/‐ mice with or without losartan treatment. After the establishment of hypertension, both WT and PT‐Sirt3‐/‐ animals significantly increased urine creatinine levels in both males and females (P&lt;0.05). However, male PT‐Sirt3‐/‐ mice had higher urine creatinine excretion than male WT (WT‐Ang II: 734 ± 59 vs PT‐Sirt3‐/‐‐Ang II: 1155 ± 92, µg/mL, P&lt;0.05), but this sex difference was absent in female WT and PT‐Sirt3‐/‐ mice. In general, females had a lower urinary creatinine excretion (UCr) than males. Losartan treatment decreased urinary creatinine excretion in all groups (P&lt;0.05). Similar responses were observed with plasma creatinine levels (SCr), which were higher in PT‐Sirt3‐/‐mice than in WT mice (P&lt;0.05), but without significant sex differences in response to Ang II infusion or losartan treatment. Losartan significantly increased 24 hr urinary potassium and chloride excretion in Ang II‐infused male and female PT‐Sirt3‐/‐ mice (P&lt;0.01). Finally, Ang II‐infused PT‐Sirt3‐/‐ mice showed significant glomerular and renal cortical tubulointerstitial fibrotic responses, compared with WT (P&lt;0.05), without sex differences. Interestingly, losartan treatment did not show decreased fibrotic responses, instead, showed a tendency to higher collagen deposition than in hypertensive PT‐Sirt3‐/‐ mice. In summary and conclusion, male and female PT‐Sirt3‐/‐ mice have lower basal blood pressure, and Ang II‐induced hypertension and renal fibrotic responses are similar in male and female PT‐Sirt3‐/‐ mice without significant sex differences. Our results suggest that AT1a receptors and SIRT3 play important roles in basal blood pressure homeostasis and Ang II‐induced hypertension and glomerular and tubulointerstitial injury in both male and female mice.

Sex Difference in Renal Artery Contractility in a Novel CRISPR/Cas9‐Generated P2X7 Knockout Rat

Purinergic signaling regulates numerous physiological processes, including renal hemodynamics. The P2X7 receptor (P2X7) is a non‐selective cation channel activated by extra‐cellular adenosine triphosphate (ATP) which plays an important role in immune cell response and inflammation. P2X7 is also expressed in endothelial cells. In the kidney, acute pharmacological blockade of P2X7 increases renal perfusion in healthy rats (Kidney Int. 88(5): 1079–1087, 2015). Here, we generated a novel P2X7 knockout (KO) rat and examined ex vivo contractility of the renal artery in male and female rats.A P2rx7 global KO rat was generated on a F344 IcoCrl background using CRISPR/Cas9 technology. KO was achieved via a two‐adenine insertion predicted to introduce a premature stop codon in exon 2. RNA and protein were extracted from KO and wild‐type (WT) kidneys to detect P2X7 by RTqPCR (n=4‐5/group) and western blotting (n=2/group). Bone marrow‐derived macrophages (BMDM) from KO and WT rats were used to assess P2X7 function by measuring interleukin (IL)‐1β release (ELISA) after stimulation with lipopolysaccharide (LPS) (1μg/mL, 4h) and ATP (3mM, 1h) (n=5‐6/group). RNA from BMDM was extracted after LPS to detect P2rx7, Il1b, Tnf and Il6 mRNA by RTqPCR (n=3/group), and Griess reaction was used to assess BMDM nitric oxide (NO) production (n=5‐6/group). One main renal artery from 2.5‐to‐4.5‐month‐old male and female KO and WT rats (n=8‐11/group) was mounted on a wire myograph to assess the response to 125 mM potassium physiological solution (KPSS) and to cumulative doses (1 nM‐30 μM) of phenylephrine (PE), acetylcholine (ACh) and sodium nitroprusside (SNP).The P2rx7 mutation in exon 2 was confirmed by genome sequencing (Sanger). P2rx7 mRNA abundance was decreased by ~60% in kidney and ~53% in BMDM from KO vs WT. P2X7 protein was detected in kidneys from WT but not from KO rats. P2X7 KO did not alter BMDM polarization after LPS stimulation. The induction of NO production was not different between genotypes (2.14±0.26 vs 2.31±0.43 μM, NS) and mRNA expression of M1 markers Il1b, Tnf and Il6 was similarly increased. In BMDM from male WT rats, ATP stimulation induced IL‐1β release; this response was markedly suppressed in KO BMDM (2577±70 vs 413±19 pg/mL, p&lt;0.0001). In the renal artery, the constriction induced by KPSS depolarization or by PE was not different between genotypes for either sex. In male rats, the maximum vasodilation induced by ACh was significantly decreased in KO compared to WT (IC50=0.25 vs 0.20 μM, NS; %Imax= 24±3 vs 10±2%, p=0.0015); this was not observed in females (IC50=0.11 vs 8.28 μM, NS; %Imax= 19±4 vs 12±4%, NS). The dilation induced by the direct NO donor, SNP was not affected by either genotype or sex.Global genetic deletion of P2X7 impaired endothelial‐dependent vasodilation in the renal artery of male rats that may reflect diminished NO production in response to ACh. Thus, the increased renal perfusion induced by P2X7 inhibitor may arise from off‐target effects. The potential sexual dimorphism in vascular expression and/or function of P2X7 requires further investigation.

Leucine rich repeat containing 8A anion channels modulate vascular reactivity in the pudendal artery

ABSTRACT Introduction Erectile dysfunction (ED) is characterized by an inability to develop or maintain penile erection during sexual activity. The internal pudendal artery (IPA) is the primary supplier of genital blood flow. Reactive oxygen species (ROS) generation is necessary for intracellular signaling, but excessive ROS production can promote ED. NADPH oxidase (Nox) produces extracellular superoxide (O2 •-) in the vasculature and we have demonstrated that Leucine Rich Repeat Containing 8 (LRRC8) Volume Regulated Anion Channels (VRACs) physically associate with and regulate Nox1. LRRC8A downregulation or channel inhibition decreases extracellular O2 •- production by Nox1 and dramatically reduces the inflammatory response to tumor necrosis factor alpha. Objective We hypothesize that smooth muscle-specific LRRC8A knockout (KO) increases vascular relaxation in IPA. Methods The IPAs from male wildtype (WT) and LRRC8A KO (smooth muscle-specific) mice were used in wire myograph studies. Results Following phenylephrine (PE, 2x10-6 M)-induced contraction, cumulative relaxation-response curves to acetylcholine (ACh, 10-9 to 3x10-6 M), sodium nitroprusside (SNP, 10-9 to 10-5 M), and a soluble guanylyl cyclase activator (BAY60-2770, 10-9 to 10-6 M) were measured. Endothelium-dependent relaxation to ACh was increased in LRRC8A KO compared to WT (WT LogEC50 -7.3 ± 0.08; LRRC8A KO LogEC50 -7.7 ± 0.12; *p&amp;lt;0.05, n=5). Relaxation to SNP or BAY60-2770 showed no difference between WT and KO. The LRRC8A-targeted VRAC inhibitor, montelukast (10 μM) reduced PE (10-6 M)-induced contractile responses in the IPA (56 % decrease). Conclusions These data demonstrate that LRRC8A plays an important role in controlling vascular reactivity that may be associated with ED. We speculate that LRRC8 channels are novel pharmacologic targets. Inhibition of the channels may reduce oxidative stress and improve sexual function. Disclosure Work supported by industry: no.

Role of Potassium Channel Function and Long‐term Exercise Training in Soleus Resistance Arterioles

IntroductionNuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) expression has been linked to potassium (K+) channel function. We hypothesized that myogenic responses are enhanced by exercise training and also modulated by K+ channel function but would be impaired in arterioles from skeletal muscle of trained rats in which Nrf2 (Nrf2 KO) is deleted.MethodsExercise training was performed in male wildtype (WT) Sprague‐Dawley rats, and their littermates lacking the Nrf2 gene (Nrf2 KO). Rats were exercise trained using a mixed moderate and high‐intensity interval treadmill program for 10‐12 weeks. A WT cage‐confined control group was also studied to compare the impact of exercise training. After exercise training or sedentary cage‐confinement, soleus resistance arterioles were isolated and cannulated for the assessment of vascular reactivity. Vasodilatory responses to low concentrations of KCl [10 and 20mM} were assessed. Vasoconstrictor responses to higher concentrations of KCl [30–100mM] and myogenic responses to increasing intraluminal pressure (pressure 0cm H2O – 130cm H2O) were also assessed. The contribution of inward rectifying K+ channels (Kir) to vasodilation during low concentrations of KCl was assessed by treatment with barium chloride (BaCl2, 100mM).ResultsSoleus resistance arteriole myogenic responsiveness was reduced in trained Nrf2 KO as compared to WT (p&lt;0.01). Trained WT rats produced a significantly higher myogenic responsiveness than sedentary WT controls (p&lt;0.05). Vasodilation to low concentrations of KCl was reduced in soleus arterioles from trained Nrf2 KO rats as compared to arterioles from trained WT rats (p&lt;0.01). Exercise training did not alter vasodilation to low KCl concentrations in WT rats (p&gt;0.05). Constriction to higher concentrations of KCl was similar in arterioles from trained WT and Nrf2 KO rats. Inhibiting Kir channels with BaCl2 eliminated the genotype‐ and training‐related differences in myogenic responses and vasodilation to KCl.ConclusionOur data indicate that Nrf2 may be a critical regulator of vascular adaptation that occurs in skeletal muscle arterioles in response to long‐term exercise training. Elimination of differences in myogenic responsiveness by BaCl2 suggest that Nrf2‐mediated adaptation of the myogenic responsiveness may involve Kir channels.

Promoting the Resolution of Thromboinflammation

BackgroundThrombosis is viewed as a multi‐step, multi‐cellular process driven by inflammatory stimuli and leukocyte (e.g. neutrophil) activation, and is a major pathophysiological contributor of stroke. Accumulating data supports the hypothesis that anti‐inflammatory therapies may limit thrombosis and anti‐thrombotic therapies may reduce vascular inflammation. One such target is the anti‐inflammatory and pro‐resolving endogenous mediator Annexin A1 (AnxA1). The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of stroke and other thromboinflammatory diseases. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in thrombosis.Material and MethodsWe used a well‐characterized light/dye thrombosis model, coupled with pharmacological and genetic approaches. Male and female adult wild type (WT, C57/BL6) or AnxA1 knock‐out (AnxA1‐/‐) mice were used. Fluorescence intravital microscopy (IVM) was used to monitor cerebral thrombosis, along with flow cytometry. Mice were treated with AnxA1 (1 µg/mouse) or saline vehicle 20 minutes before the onset of cerebral thrombus formation. Thrombogenesis and blood flow cessation times were quantified. (All animal experiments were approved by relevant institutional animal welfare panels and were conducted in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines and complied with ARRIVE [Animal Research: Reporting In Vivo Experiments]).ResultsIVM revealed that AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, via regulation of the key inflammation resolution receptor: FPR2/ALX, as evidenced by the co‐administration of the FPR2/ALX antagonist WRW4. Using AnxA1−/− mice, we found that a deletion of AnxA1 impacts thrombosis as shown by the accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) observed in both arterioles and venules of these mice compared to their WT counterparts (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p&lt;0.05). Furthermore, we found that in an inflammatory backdrop (induced by the administration of lipopolysaccharide [LPS]), AnxA1 treatment was able to suppress both platelet αIIbβ3 and P‐selectin expression.ConclusionHerein, we found a previously unknown phenomenon that the anti‐inflammatory protein AnxA1 can modify a thromboinflammatory environment to promote resolution. These findings have far‐reaching applicability for drug discovery programs focused on resolution biologics targeting thromboinflammatory diseases such as cardiovascular disease and stroke.

Inhibition of Ileal Bile Acid Transport is Protective Against Cholestatic Liver Injury in Cyp2c70‐/‐Mice with Humanized Bile Acid Composition

Cyp2c70 is the key murine liver enzyme responsible for the synthesis of 6‐hydroxylated muricholates. Cyp2c70 knockout (2c70‐/‐) mice present with a more human‐like bile acid (BA) composition devoid of protective, hydrophilic muricholic acids and with features of cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be protective in cholestatic models such as the Mdr2‐/‐ mouse. The aims of this study were to determine if IBAT inhibition is protective in 2c70‐/‐ mice and identify the underlying molecular mechanisms.Methods2c70‐/‐ mice (C57Bl/6J) were generated using CRISPR/Cas9 technology. Male and female wild type (WT) and 2c70‐/‐ mice were fed chow or chow containing 0.006% SC‐435 for 9 weeks following weaning at 3 weeks of age. Standard growth parameters were recorded; organs, gallbladder bile and blood were collected at necropsy for histologic, RNA and biochemical analyses.ResultsMale and female 2c70‐/‐ mice maintained on chow diet exhibited increased levels of serum liver injury markers ALT, AST, and BAs compared to WT mice. Evaluation of 2c70‐/‐ liver histology revealed increased hepatic inflammation, macrophage infiltration and bilary cell proliferation, which were markedly reduced with SC‐435 treatment. IBAT inhibition in 2c70‐/‐ mice improved liver histology, and reduced hepatic inflammation and bilary cell proliferation to levels indistinguishable from WT. The liver BA content was increased by 53% in 2c70‐/‐ mice, and was enriched in more hydrophobic BA species with an overall hydrophobicity similar that of humans (Figure 1). Treating 2c70‐/‐ mice with SC‐435 reduced the hepatic BA burden by 61%, but did not affect the overall hydrophobicity. RNA‐Seq and KEGG pathway analysis performed using liver RNA revealed genes involved in hepatic steroid and lipid metabolism (Sult2a1, Sult1e1, Hsd17b7), and detoxification (Cyp2c39, Cyp2c54, Cyp2c59, Cyp3a41a/b) were down‐regulated in 2c70‐/‐ mice and their expression were rescued with SC‐435 treatment. Conversely, genes in pathways related to immune cell activation and inflammation were significantly up‐regulated in 2c70‐/‐ mice and reduced to WT levels with IBAT inhibition (Figure 2).ConclusionsOur findings indicate that reducing the hepatic BA burden without altering the hydrophobic compostion is sufficient to protect 2c70‐/‐ mice from hepatic injury. The findings suggest that there may be a threshold in liver for pathological accretion of hydrophobic BAs and reducing the hepatic BA pool is sufficient to alleviate liver injury, independent of hydrophobicity. Moreover, these data support the inhibition IBAT activity, and other approaches to reduce hepatic return of BAs in the enterohepatic circulation, as therapeutic targets to treat cholestasis.