bluuebunny/crossref_metadata_2025 huggingface dataset:
Paraventricular Nucleus Over Activation Is A Critical Driver In The Development Of Neurogenic Hypertension
Authors: Basting Tyler , Sriramula Srinivas , Epling Joel , Lazartigues Eric
Published: June 2021

Rationale and objectives

In conscious mammals the importance of neural control over sympathetic tone in relation to cardiovascular (CV) function is well established. Neuro‐CV dysregulation leads to increased sympathetic activity and neurogenic hypertension. The paraventricular nucleus (PVN) of the hypothalamus is currently viewed as one of the key hubs for BP control and is implicated in producing or relaying the increased sympathetic tone in hypertension. We hypothesize that it is through chronic over excitation of the glutamatergic PVN neurons that this central sympathetic drive is originating. We test this theory by examining how stimulation or lesioning of the excitatory PVN neurons in conscious mice affects blood pressure and sympathetic activity.

Results & Conclusion

Glutamatergic PVN neurons were unilaterally transduced with channelrhodopsin2 using an adeno‐associated virus (CamKII‐ChR2‐eYFP‐AAV2) in wildtype mice (N = 7). We then measured the effect of acute stimulation of excitatory PVN neurons selectively on resting blood pressure (DSI telemetry) in conscious mice. Stimulation of the PVN glutamatergic population resulted in an immediate frequency dependent (2 Hz, 10Hz, 20Hz) increase in blood pressure from baseline by ~10 mmHg at 20 Hz stimulation (p<0.001). Additionally, in vGlut2‐cre mice glutamatergic neurons of the PVN were bilaterally lesioned utilizing a cre‐dependent caspase (Dio‐Caspase‐AAV). We then recorded resting blood pressure and urinary norepinephrine levels in conscious mice before and after DOCA‐Salt hypertension. Quantified glutamatergic lesions of the PVN (39.3%, p<0.05) resulted in a blunted rise in BP when animals went through the DOCA‐salt protocol (p<0.05 at 7 day time point, N = 8). Finally, we measured nor‐epinephrine levels as a quantification of sympathetic activity between the lesion group and widtype group and observed a significant reduction after DOCA‐salt induced hypertension in the lesioned animals (p<0.05). These experiments demonstrate that stimulation of the PVN is sufficient to cause autonomic dysfunction. Additionally, the dysautonomia observed in neurogenic hypertension is due, in part, to over activation in the PVN.

Support or Funding Information

HL093178‐07, HL135635‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Myo‐inositol in Ischemic Stroke Outcome in a Type 2 Diabetic Mouse Model
Authors: Villalba Heidi , Vaidya Bhuvaneshwar , Abbruscato Thomas
Published: June 2021

Stroke is the leading cause of disability in the United States; ischemic stroke is the common form that occurs in 87% of all strokes. It has been reported that an increase in brain edema after ischemic stroke worsens long‐term clinical outcome. Myo‐inositol (MI), a major organic osmolyte that promotes cell membrane stability and various cellular functions, has been reported to be detected at lower brain levels in stroke patients. Furthermore, glucose can compete with MI transport which could lead to further lowering of brain levels of MI in diabetic patients which is compounded with higher brain edema reported in diabetic stroke patients. Therefore, understanding the relationship between MI and diabetic stroke injury is vital. In this study, we investigated the role of MI transporters during stroke and post‐stroke outcomes especially related to the role of glial cells and post‐stroke reorganization. Our hypothesis is that MI plays a putative role in recovery of motor function after stroke. Restoration of activity in the primary motor cortex has been associated with better motor function in stroke patients. Primary astrocytes were used for in vitro studies and middle cerebral artery occlusion (MCAO) mouse model for in vivo stroke studies. Immunocytochemistry (ICC) studies showed that astrocytes express sodium dependent myo‐inositol transporter 1 (SMIT1) and sodium‐dependent glucose transporter 6 (SGLT6) transporters and level of expression changed in a time‐dependent manner during oxygen‐glucose deprivation (OGD). Further, this was confirmed by time‐dependent uptake of MI during normoxic and OGD conditions. Preliminary animal studies demonstrated that post‐stroke administration of MI attenuated infarction size and edema ratios in a db/db mouse model of type 2 diabetes compared to nondiabetic ischemic animals and also improved motor function in behavioral tests. This preliminary data suggests that myo‐inositol may have a neuroprotective role in ischemic stroke injury and could significantly improve stroke outcome. More studies are required to understand the neuroprotective mechanisms of MI in stroke injury.

Support or Funding Information

RO1 NS070612

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dexmedetomidine Modification of the Chemoreflex Response to Severe Arterial Hypoxia in the Rabbit
Authors: Quail Anthony , Ravindran Bhavi , Cottee David , Johnstone Janice , White Saxon
Published: June 2021

The carotid body contains alpha‐2 adrenoceptors but selective agonists given during hypoxia have shown inhibition or enhancement of carotid sinus nerve activity in vivo (1), and in vitro (2). Dexmedetomidine (DM) is a highly‐selective alpha‐2 adrenergic agonist currently used for clinical sedation and analgesia (3,4) but its effects on integrated chemoreflex control during hypoxia are unknown. We examined the hypothesis that DM would selectively alter components of the cardiorespiratory responses to severe arterial hypoxia (PaO2 <35 mm Hg). An ear artery and vein of 6 New Zealand White female rabbits were cannulated (lidocaine 2%). Mean arterial pressure (AP), arterial blood gases, heart rate (HR), minute ventilation (VE), tidal volume (VT), respiratory frequency (f) and oxygen saturation (ear SpO2) were measured breathing room air, followed by 5 minutes of hypoxia (FiO2 7–8%). After a one‐hour recovery period, the measurements were repeated during intravenous DM infusions (4 or 40 mcg/kg/hr), breathing room air followed by exposure to a matched hypoxic stimulus. One dose of DM was studied on each of two experimental days, one week apart. Behavioural responses were recorded before and during DM infusions. Data were analysed using two‐way repeated measures ANOVA. Significant differences were accepted at the p<0.05 level. In the absence of DM, hypoxia alone evoked a reflex fall in HR and f, with increases in VT, VE and AP. In rabbits breathing room air, DM alone caused dose‐dependent falls in f, VE, HR and AP, while VT was unchanged. With DM plus hypoxia, the reflex bradycardia was absent at the higher dose. The increase in VT remained intact during both doses, while the rise in VE was attenuated due to an unchanged f. The AP increased from a lowered baseline. DM produced dose‐dependent sedation. We conclude reflex vagal control of heart rate during severe hypoxia is modified by DM. There are dose‐dependent effects on AP and components of ventilation. The increase in VT in response to hypoxia during DM infusion indicates that chemotransduction at the carotid body is intact. The data suggest the main sites of action of dexmedetomidine on cardiorespiratory control during hypoxia are within the CNS and not at the carotid body.

Support or Funding Information

John Hunter Hospital Charitable Trust and Hunter Medical Research Institute

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of local Interleukin‐6 on Skeletal Muscle Mitochondrial Physiology
Authors: Abid Hinnah , Hart Corey , Lanza Ian
Published: June 2021

Skeletal muscle is vital to metabolic health and physical function. Skeletal muscle mitochondria have been extensively interrogated as organelles whose function and dysfunction are closely tied with chronic diseases such as diabetes and sarcopenia. There is accumulating evidence suggesting that chronic inflammation that is prevalent with aging and obesity is associated with skeletal muscle abnormalities such as functional impairments in mitochondria. Despite compelling links between inflammation, oxidative stress, and skeletal muscle dysfunction, the local effects of inflammatory cytokines on skeletal muscle mitochondrial physiology are currently not well‐understood. The purpose of this study was to determine how the inflammatory cytokine interleukin‐6 (IL‐6) influences skeletal muscle mitochondrial function. To determine the acute effects of IL‐6, mitochondria were isolated from skeletal muscle of healthy male mice (C57BL6) and were exposed to IL‐6 (IL6+,50 ng/ml) or vehicle (CTRL) for one hour. Multiplexed measurements of mitochondrial respiration (high‐resolution respirometry) and reactive oxygen species production (ROS, fluorometry) revealed that IL‐6 significantly increased ROS production as a percent of oxygen (4.14%±0.40) compared to CTRL (2.74%±0.34). Similarly, IL‐6 increased ROS production in differentiated C2C12 myotubes that were incubated for 18 hrs with 50 ng/mL IL‐6 (4.71%±.57) compared to CTRL (2.21%±.34). To evaluate the possibility that IL‐6 triggers ROS production by stimulating the mitochondrial transition pore (mPTP), isolated mitochondria were ale treated with cyclosporin A (CsA), a mPTP inhibitor in the presence or absence of IL‐6. Inhibition of mPTP attenuated IL‐6 induced ROS production (IL6+ (4.14%±0.40) vs. IL6+/CsA (2.78%±0.58). These results suggest that IL‐6 triggers an increase in mitochondrial ROS production that appears to be mediated by opening of the mPTP. Prolonged opening of the mPTP caused by chronically elevated IL‐6 may contribute to increased oxidative stress that is evident in skeletal muscle with aging and obesity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Urocortin 2 gene transfer reduces fasting glucose, polydipsia, hyperphagia and increases body weight in type 1 diabetes
Authors: Bhargava Raag , Tay May Ling , Gao Meihua , Hammond HK
Published: June 2021

We previously have shown that a single intravenous (IV) injection of Adeno‐associated Virus 8 encoding urocortin 2 (AAV8.UCn2) increases glucose disposal and insulin sensitivity in insulin resistant mice (type‐2 diabetes). The current study tests the hypothesis that UCn2 gene transfer is effective in type‐1 diabetes. We used insulin‐deficient Akita mice and assessed water and food consumption, body weight and fasting glucose before and after UCn2 gene transfer. Akita mice (n=50; 21.3±1.5 g; 2.4±0.2 months of age) were used. Sixteen mice with a mean pre‐treatment 12‐hr fasting glucose of 322±80 mg/dL, received either AAV8.UCn2 (5×1011 genome copies, gc, IV; n=8) or Saline (IV; n=8). Fasting glucose was repeated 8 weeks after gene transfer. Thirty‐four additional mice were used to assess water and food intake and body weight before and 8w after UCn2 gene transfer (5×1011gc, IV; n=17) or Saline (IV; n=17).

Fasting glucose

Before randomization, 12‐hour fasting glucose was similar (Saline: 354±60 mg/dL, n=8; UCn2: 289±88 mg/dL, n=8; p=.11). Eight weeks after treatment, mice that received UCn2 gene transfer showed lower fasting glucose (Saline: 272±69 mg/dL, n=8; UCn2: 106±33 mg/dL, n=8; p<.0001).

Water/food intake and body weight

Water intake over the 8w course of the study was reduced after UCn2 gene transfer (Saline: 1.15±0.09 ml/day/g; UCn2: 0.63±0.05 ml/day/g; p<.0001) (Fig 1). Food intake over 8w was also reduced by UCn2 gene transfer (Saline: 0.32±0.04 g/day/g; UCn2: 0.22±0.02 g/day/g; p<.0001) (Fig 1). Body weight increased 8w after UCn2 gene transfer (Saline: 24.1±1.0 g; UCn2: 27.8±0.7 g; 2‐way ANOVA p<.0001) (Fig 2).

Conclusion

UCn2 gene transfer reduces fasting blood glucose and reduces food and water intake. Despite reduced food intake, Akita mice that received UCn2 gene transfer showed increased body weight. These data suggest a possible non‐insulin therapy for type‐1 diabetes.

Support or Funding Information

I01 BX003774 VA Merit Grant; R42HL122038 NHLBI

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered Body Composition does not Fully Account for Impaired Glucose Tolerance in Women with Spinal Cord Injury
Authors: Li Jia , Hunter Gary R. , Chen Yuying , McLain Amie , Smith Daniel L. , Yarar‐Fisher Ceren
Published: June 2021

Objective

To investigate the differences in insulin sensitivity and glucose tolerance among women with spinal cord injury (SCI) in comparison to their able‐bodied (AB) counterparts after adjusting for differences in body composition.

Design

Cross‐sectional study. Following an overnight fast, each participant consumed a 75‐g glucose solution for oral glucose tolerance test (OGTT). Blood glucose, insulin, and C‐peptide concentrations were analyzed before and 30‐, 60‐, and 120‐minute post‐consumption. Insulin sensitivity index (ISI) was estimated using the Matsuda index. Percentage fat mass (%FM) and relative skeletal muscle index (RSMI) were estimated using data from dual‐energy X‐ray absorptiometry. Visceral fat (VF) was estimated using computerized tomography (CT). Outcome measures were compared among groups using ANCOVA with %FM, VF and RSMI as covariates.

Setting

Research University

Participants

Women with SCI (tetraplegia: n=8; paraplegia: n=14) and their body mass index‐and age‐matched AB counterparts (n=20).

Interventions

Not applicable.

Results

At fasting, there was no difference in glucose homeostasis (glucose, insulin, and C‐peptide concentrations) among 3 groups of women. In contrast, glucose, insulin, and C‐peptide concentrations at OGTT 120 min were higher in women with tetraplegia vs. women with paraplegia and AB women (P < 0.05, adjusted for RMSI and %FM). In addition, women with tetraplegia had lower ISI (P < 0.05, adjusted for RMSI and %FM) vs. AB women. These differences remained after adjusting for VF and RSMI.

Conclusion

Our study confirms that while alterations in body composition may contribute to metabolic deficits in women with SCI, it does not fully account for impaired glucose homeostasis in women with tetraplegia.

Support or Funding Information

This work was supported by the NIH under Grant K12 HD001402/HD/NICHD NIH (Chen), M01 RR000032/RR/NCRR NIH (Chen), NIDLRR – DHHS‐Administration for Community Living Sponsor award 02‐90SI501901 The University of Alabama at Birmingham Spinal Cord injury Model System (McLain).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In Utero Exposure To Cigarette Smoke Promotes Cardiac Dysfunction In High‐fat Fed Mice
Authors: Xia Huijing , Rodrigues Samya Lima , Ronis Martin J. , Noel Alexandra , Penn Arthur , Pullium Casey , Gardner Jason , Varner Kurt J.
Published: June 2021

Maternal smoking during pregnancy is associated with an increased risk for metabolic syndrome, a risk factor for heart disease. In this study, we investigated whether in utero exposure to second hand cigarette smoke would adversely affect cardiac structure/function in the exposed offspring fed a high‐fat diet. Pregnant mice (C57B1/6J) were exposed to cigarette smoke or air (4 hr/d) on gestational days 6–19. The male offspring (age: 35 days) from the smoke and air exposure groups were fed either normal chow, or high‐fat liquid diet (35% of fat; HFD) for 16 weeks (n=12/group). Cardiac function was measured by echocardiography at baseline (30 days) and at the end of pair feeding. Mice were then sacrificed and heart tissue collected for histological analysis. At baseline, left ventricular internal diameter at end systole was significantly greater in the smoke‐exposed compared to the air group (3.2±0.1 vs 2.8±0.1 mm, P<0.05). All other baseline echocardiographic parameters were similar between the groups. 16 weeks of HFD feeding significantly increased the body weight and heart weight/tibial length in both the air and smoke‐exposed mice (P<0.05, smoking+HFD vs. smoking+chow; air+HFD vs. air+chow). However, there was no significant difference in body weight or heart weigh/tibial length between the smoking+HFD and air+HFD groups. Compared to air+HFD mice, smoking+HFD mice had significantly decreased left ventricular internal diameters and volumes during diastole (3.95±0.09 vs. 4.34±0.24 mm; 67.8±1.5 vs. 85.4±4.4 μl, P<0.05) and systole (2.91±0.07 vs. 3.34±0.15 mm; 32.5±1.9 vs. 46.3±5.1 μl, P<0.05), accompanied by increases in left ventricular wall during diastole (0.72±0.03 vs. 0.66±0.03 mm, P<0.01) and septal thickness during systole (1.2±0.06 vs. 0.99±0.05 mm, P<0.05) and increased fractional shortening. There was also increased collagen in the hearts of the smoking+HFD mice. These data indicate the development of concentric cardiac hypertrophy in smoking+HFD mice. We conclude that in utero exposure to second hand cigarette smoke produces differential cardiac responses following challenge with obesogenic high fat diet.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Negative energy balance up‐regulate the levels of hepatokines in dairy cows during the peripartum period
Authors: WANG Jianguo , LIU Lei , ZHAO Baoyu
Published: June 2021

Introduction

The periparturient period is vitally important to the health status and the reproductive performance of dairy cows. Negative energy balance (NEB) is considered as the pathological basis of energy metabolic disorders. Serum non‐esterified fatty acids (NEFA) and β‐hydroxybutyrate (BHBA) concentrations are the indicators of energy balance status. Angiopoietin‐like protein 4 (ANGPTL4) and fibroblast growth factor 21 (FGF21), known as hepatokines, play essential roles in lipid metabolism. However, in vivo the kinetics of ANGPTL4 and FGF21 are not entirely clear of dairy cows, particularly in dairy cows with ketosis and fatty liver. The purposes of this project were to evaluate the variations in serum concentrations of ANGPTL4 and FGF21 in periparturient dairy cows, and to examine the correlations between serum NEFA, BHBA concentrations and ANGPTL4, FGF21 concentrations, respectively, in the serum of experimental cows. Moreover, the serum concentrations of hepatokines from cows with spontaneous ketosis and fatty liver, and healthy cows were compared.

Methods

Experiment I

Blood parameters were measured in healthy periparturient Holstein cows from 4 wk antepartum to 4 wk postpartum (n=219). In this experiment, weekly blood samples were obtained from 4 wk before the expected calving date to 4 wk after calving.

Experiment II

Blood parameters were measured in healthy cows (n=30), cows with ketosis (n=29) and fatty liver (n=25) within the first 4 wk of lactation. In this present study, all blood samples were collected from the coccygeal vein in the early morning before feeding.

Results

Serum concentrations of ANGPTL4 and FGF21 peaked at parturition, and declined rapidly over the following 2 wk. Serum ANGPTL4 and FGF21 concentrations were positively correlated with serum NEFA concentration (ANGPTL4 and NEFA: r=0.856, P=003; FGF21 and NEFA: r=0.848, P=0.004). However, the correlations between serum BHBA concentration and the serum concentrations of ANGPTL4 and FGF21 were not observed in experimental cows. Cows with ketosis and fatty liver had significantly higher serum concentrations of ANGPTL4 and FGF21 when compared with healthy cows (P<0.01).

Conclusion

Serum concentrations of ANGPTL4 and FGF21 were elevated during peripartum period in dairy cows, demonstrating that the energy balance changes that were associated with parturition contributed significantly to these effects. We propose that blood ANGPTL4 and FGF21 concentrations could be used as supplementary biomarkers of “energy balance” status in postpartum dairy cows. Although FGF21 and ANGPTL4 could play important roles in the adaptation of energy metabolism, they may be involved in the pathological processes of energy metabolism disorders of dairy cows in the peripartum period.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of lipogenesis by hematopoietic‐ and neurologic‐expressed sequence1 stimulates hepatocellular carcinoma cell proliferation and metastasis
Authors: Jin Hua , Kim Soo Mi
Published: June 2021

Hepatocellular carcinoma (HCC) is the second most common cause of cancer‐related mortality worldwide and its five‐year survival rate is less than 12%. Since it has been reported that hematopoietic‐ and neurologic‐expressed sequence1 (HN1) gene is upregulated in various cancers, its functional significance in HCC remains unclear. Thus, we investigated the biological role of HN1 in HCC using HepG2 and SNU449 cells. HN1 was significantly overexpressed in HCC tumors compared to normal liver tissues. Silencing of HN1 significantly diminished the viability of HCC cells whereas overexpression of HN1 stimulated the viability of HCC cells. Silencing of HN1 increased apoptotic proteins and increased the number and size of colonies. In addition, silencing of HN1 inhibited the invasion and metastasis of HCC cells whereas overexpression of HN1 promoted the invasion and metastasis of HCC cells. In gene expression profiling, we identified 130 upregulated genes and 379 downregulated genes after HN1 silencing in HCC cells. Putative gene networks revealed suppressed expression of proteins associated with lipogenic signaling pathway. Silencing of HN1 significantly inhibited the expression levels of SREBP1 and SREBP2 of HCC cells whereas overexpression of HN1 increased the expression levels of SREBP1 and SREBP2 of HCC cells. In addition, silencing of SREBP1 also diminished the expression levels of HN1 and suppressed the survival and metastasis of HCC cells. In cholesterol assay and triglyceride assay, silencing of HN1 inhibited the lipid formation of HCC cells whereas overexpression of HN1 promoted the lipid formation of HCC cells. Taken together, HN1 encourages the proliferation, invasion and metastasis of HCC cells in part through the activation of SREBP signaling pathway. Therefore, our results suggest that targeting HN1 may constitute a potential therapeutic strategy for HCC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The ELMO1, a Microbial Sensor Regulates Bacterial Clearance and Endo‐lysosomal Signaling
Authors: Tam Julian , Lau Mitchel , Pranadinata Rama , Ibeawuchi Stella‐Rita , Tindle Courtney , Das Soumita
Published: June 2021

Intestinal antigen presenting cells (APCs) are a major component of the mucosal immune system and maintain the gut homeostasis and the inflammation level. Microbial sensors present on APCs recognize microbes irrespective of their pathogenicity but little is known about their mechanism that leads to host cellular responses unique to bacterial pathogenesis. Previously, we found that BAI1 (Brain Angiogenesis Inhibitor 1) recognizes bacterial LPS from pathogenic and non‐pathogenic bacteria. BAI1 binds cytosolic protein ELMO1 (Engulfment and cell motility protein 1), which facilitates the engulfment of bacteria via Rac1 activation and induces the pro‐inflammatory cytokines and regulates bacterial clearance. Interestingly, ELMO1 interacts with Salmonella effector SifA involved in bacterial survival. Therefore, we hypothesized that ELMO1 is a cytosolic sensor that interacts with bacterial effectors involved in the pathogenesis of enteric infection, controls bacterial clearance and provides differential immune responses between pathogens and commensals. In silico analysis of bacterial database revealed that Salmonella effector SifA shares a WxxxE signature motif present in several effectors from other enteric pathogens, including IpgB1 in Shigella that binds ELMO1. This signature motif is not present in commensal gut microbes. Interestingly, mutation in the WxxxE motif of SifA abolished interaction with ELMO1. SifA is involved in bacterial vacuole integrity and together ELMO1‐SifA is involved in the endosomal‐lysosomal pathway by interacting with the late endosomal protein Rab9. To further elucidate the interaction of SifA and its involvement in interrupting host defenses, we did pulldown of GST‐SifA with murine macrophage cell lysate followed by the identification of host proteins via MALDI‐TOF Spectroscopy. Three major proteins that interact with SifA are Ribophorin 1(RPN1), involved in the transport of newly synthesized proteins across the membrane of the rough endoplasmic reticulum; MOGS, a glycoside hydrolase functions within the N‐glycosylation pathway; Rab14, the endosomal trafficking protein involved in lysosomal fusion. Our functional studies showed the ELMO1‐depleted macrophages had higher acidic and proteolytic lysosomal environment and increased Cathepsin B activity at earlier time points of infection but lower Cathepsin B activity and lysosomal acidification at later time points of infection. Lastly, ELMO1 depleted cells had significant delayed clearance following enteric infection. Our results suggest that ELMO1 is a novel cytosolic sensor that determines bacterial pathogenesis and provides new insight to the degradation of bacteria via mechanisms of the endosomal‐lysosomal system.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of HIV‐1 gp120 and TAT on Endothelial Microparticle Release and Oxidative Stress
Authors: Brewster Lillian M. , Hijmans Jamie G. , Bammert Tyler D. , Stockelman Kelly A. , Levy Ma’ Ayan V. , Greiner Jared J. , DeSouza Christopher A.
Published: June 2021

Human immunodeficiency virus (HIV)‐1 infection is associated with an increased risk and prevalence of atherosclerotic cardiovascular disease (CVD). A major factor underlying the increased CVD burden with HIV‐1‐infection is endothelial damage and dysfunction. Although the mechanisms underlying the HIV‐1‐related vasculopathy are not fully understood, HIV‐1 associated viral proteins are known to have deleterious effects on the endothelium. Endothelial cell release of microparticles and endothelial cell oxidative stress are key characteristics of a proatherogenic endothelial phenotype. The direct effect of HIV‐1 viral proteins on endothelial microparticle release and oxidative stress, however, is currently unknown. The aim of the present study was to determine the effects of X4 and R5 HIV‐1 gp120 and TAT on endothelial cell microparticle release and oxidative stress. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated at a density of 2 × 106 cells/condition. HUVECs were treated with media alone or media containing either lavgp120 (X4; 100 ng/mL), Balgp120 (R5; 100 ng/mL) or TAT (500 ng/mL) for 24 h to generate endothelial microparticles (EMPs). Viral protein stimulated EMPs were pelleted by centrifugation and resuspended in culture media. Characterization and concentration of EMPs was determined by flow cytometry using VE‐Cadherin (CD 144‐PE). To assess oxidative stress, intracellular reactive oxygen species (ROS) production was determined. To do so, 2.5 ×104 HUVECs were plated in 96‐well tissue culture plates and allowed to adhere overnight. Cells were then incubated with 2′,7′ –dichlorofluorescin diacetate (DCFDA; 25 uM), washed and stimulated with media in the presence or absence of viral proteins. All measurements were done in triplicate and calculated as percent (%) of control by fluorescence spectroscopy. Treatment of HUVECs with X4 (117±28 MP/μL), R5 (105 ± 24 MP/μL) and TAT (101±36 MP/μL) induced markedly higher (~105%) EMP release compared with untreated (51±13 MP/μL) cells. Additionally, X4 (135 ±11%), R5 (139±13%) and TAT (142±7%) significantly increased (~40%) ROS production compared with untreated cells. Of note, the magnitude of increase in EMP release and ROS production was not significantly different between viral proteins. These results indicate that HIV‐1 gp120 and TAT induce EMP release and increase oxidative stress in endothelial cells. Viral protein augmentation of cellular ROS may be the impetus for EMP release, and requires further study. EMP generation and increased endothelial oxidative burden likely contribute to atherogenic effects of HIV‐1 viral proteins.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low Sodium Intake Causes Kidney Cortical Hypoxia and Proteinuria in Rats
Authors: Carvalho Carla , Patinha Daniela , Fasching Angelica , Friederich‐Persson Malou , Hansell Peter , Palm Fredrik
Published: June 2021

Reduced sodium intake induces activation of the renin‐angiotensin system to maintain sodium balance and arterial blood pressure. Renin‐angiotensin aldosterone system‐induced oxidative stress is a common pathway to intrarenal hypoxia, which could be detrimental if imposed on an already damaged kidney. We therefore investigated the effect of reduced sodium intake on kidney oxygen homeostasis and function in rats.

Male Sprague Dawley rats were fed standard rat chow containing normal (0.25% sodium) or low sodium content (0.02%) for two weeks and kidney oxygen metabolism and function were studied thereafter. Acute blockade of angiotensin II AT1 receptors by candesartan (1 mg kg−1) was used to estimate the degree of renin‐angiotensin system activation in combination with measurements of intrarenal tissue angiotensin II levels.

Dietary sodium restriction increased intrarenal angiotensin II tissue levels without altered arterial blood pressure. Concomitantly, a pronounced increase in kidney oxygen consumption was observed along with reduced tubular electrolyte transport efficiency and abolished cortico‐medullary oxygen gradient. Urinary protein excretion increased although renal blood flow and glomerular filtration rate were preserved.

In conclusion, reduced sodium intake causing renin‐angiotensin‐aldosterone system activation significantly increases kidney oxygen consumption resulting in cortical tissue hypoxia and proteinuria. This is possibly an undesired effect, especially during pre‐existing kidney disease.

Support or Funding Information

Swedish Research Council, Swedish Diabetes Foundation, Swedish Heart‐Lung Foundation and Family Ernfors Fund.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kir4.1 is involved in Bradykinin‐induced inhibition of NCC and natriuresis
Authors: Wu Peng , Gao Zhong‐Xiuzi , Zhang Dan‐Dan , Vio Carlos P. , Gu Ruimin , Wang Wen‐Hui
Published: June 2021

Stimulation of type II bradykinin (BK) receptor (BK2R) has been shown to increase renal Na+ excretion. However, the mechanism by which BK stimulates natriuresis is not completely understood. Immunohistochemical study demonstrates that BK2R is highly expressed in both apical and basolateral membrane of Kir4.1 positive tubules including thick ascending limb, distal convoluted tubule (DCT) and cortical collecting duct. The aim of the present study is to explore the role of BK in regulating the basolateral K+ channel and apical Na‐Cl cotransporter (NCC) in the DCT. The single channel patch‐clamp experiments show that application of BK (1–10 μM) inhibited the basolateral 40 pS K+ channel (a Kir4.1/5.1 heterotetramer) and this effect was blocked by BK2R antagonist (HOE140) but not by BK1R antagonist. Whole‐cell recordings have also shown that BK decreased the basolateral K+ conductance of the DCT and depolarized the membrane. To examine the role of Kir4.1 in mediating BK‐induced natriuresis, we performed renal clearance experiment in WT and in kidney‐specific conditional Kir4.1 knockout (Ks‐Kir4.1 KO) mice. Acute application of BK increased urinary Na+ and K+ excretion. However, the BK‐induced natriuretic effect was completely abolished in Ks‐Kir4.1 KO mice. The results strongly suggest that Kir4.1 activity is required for BK‐induced natriuresis. Our previous experiments demonstrated that Kir4.1 plays an important role in determining NCC activity such that the deletion of Kir4.1 completely inhibited NCC in the DCT. To test the hypothesis that BK‐induced inhibition of Kir4.1 may cause the inhibition of NCC thereby increasing Na+ and K+ excretion, we examine the basolateral Kir4.1 activity, the expression of NCC and thiazide‐induced natriuresis in the mice with 3 day infusion of BK by an osmotic pump. The infusion of BK for 3 days significantly decreased the basolateral Kir4.1 conductance and the negativity of the DCT membrane. Western blot showed that infusion of BK decreased the expression of total NCC and phosphorylated NCC. Renal clearance experiments demonstrated that thiazide‐induced natriuresis was blunted in the mice receiving BK infusion, suggesting that BK infusion inhibited NCC function in the DCT. Consequently, mice received BK infusion for 3 days were hypokalemic, presumably due to increasing Na and volume delivery to the collecting duct. We conclude that BK2R plays a role in tonic inhibition of the basolateral Kir4.1 channels in the DCT and that BK‐induced inhibition of NCC is, at least in part, responsible for BK‐induced stimulation of Na excretion. We also conclude that BK‐induced inhibition of Kir4.1 in the DCT is an essential step for the inhibition of NCC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Collecting duct principal, but not intercalated, cell (pro)renin receptor modulates sodium and water transport
Authors: Ramkumar Nirupama , Stuart Deborah , Mironova Elena , Lakshmipathi Jayalakshmi , Stockand James D. , Kohan Donald E.
Published: June 2021

The collecting duct (CD) is the predominant site for prorenin receptor (PRR) expression within the nephron. We have previously demonstrated that the CD PRR regulates epithelial Na+ channel (ENaC) activity and water transport; however, which CD cell type is involved is unclear. Hence, we examined the effects of principal cell (PC) or intercalated cell (IC) PRR deletion on renal Na+ and water handling. PC or IC PRR KO mice were obtained by crossing floxed PRR mice with mice harboring Cre recombinase under the control of the AQP2 or B‐1 subunit of the H+ ATPase promoters, respectively. In‐situ hybridization using RNAscope demonstrated abundant PRR RNA in IC with modest staining in PC in control mice with reduced cell‐specific PRR RNA in PC and IC KO mice. Compared to controls, PC KO, but not IC KO mice, had increased urinary volume (Control: 1.2 ± 0.4 vs PC KO: 2.0 ± 0.3, P < 0.05; IC KO: 1.4 ± 0.2 ml/day), reduced urine osmolality (Control: 3160 ± 279 vs PC KO: 2343 ± 247, P < 0.05; IC KO: 2748 ± 356 mosm/Kg H2O) and reduced expression of renal medullary AQP2 (50% of control). Further, PC KO mice had reduced renal medullary ENaC‐α expression with normal and low Na+ diet and lower Na+ balance (24‐hour intake – excretion) following low Na+ intake (controls: 60.5 ± 7.5 vs PC KO: 25.7 ± 10.6 μmol/day, P <0.05). In contrast, no differences in Na+ balance or ENaC expression was observed between control or IC KO mice. In acutely isolated cortical CD, under basal conditions, no significant differences in ENaC activity were observed between control, PC or IC specific PRR KO mice. Treatment with mouse prorenin (10 nM for 30 min) increased ENaC channel number and open probability by 2‐fold in control mice (basal: 0.15 ± 0.04; prorenin treatment: 0.74 ± 0.25). Addition of prorenin did not alter ENaC activity in PC specific PRR KO mice (basal: 0.28 ± 0.06; prorenin treatment: 0.33 ± 0.13) but increased ENaC activity in IC specific PRR KO mice (basal: 0.17 ± 0.08; prorenin treatment 1.29 ± 0.13). However, mean arterial pressure as measured by telemetry (Controls: 104 ± 1, PC KO: 106 ± 2, IC KO: 101 ± 4 mm Hg) was similar between controls and PC or IC KO mice on normal and low Na+ diets. Taken together, these findings indicate that under physiologic conditions, the PC, but not IC, PRR modulates ENaC expression and activity, urinary Na+ excretion and water transport. Further studies are needed to examine the role of PC and IC PRR in pathological states such as Ang‐II induced hypertension.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SPAK (Stk39) is Involved in NKCC2 Phosphorylation and Salt‐sensitive Hypertension in Dahl Salt‐sensitive Rats
Authors: Garcia‐Pedraza Jose Angel , Ortiz Pablo A.
Published: June 2021

In the thick ascending limb (TAL), NaCl reabsorption is mediated by the Na/K/2Cl cotransporter NKCC2, which plays a crucial role in the regulation of blood pressure. NKCC2 activity is partly controlled by phosphorylation via STE20/SPS1‐related proline‐alanine‐rich kinase (SPAK, gene name Stk39) at Thr96 and Thr101 residues in the NH2‐terminus of NKCC2. Previously, we found that NKCC2 phosphorylation was 5‐fold higher in TALs from Dahl salt‐sensitive (DSS) rats, suggesting that this pathway may be related to the salt‐sensitivity in this model. However, the kinase responsible for NKCC2 hyperphosphorylation is unknown. In addition, the role of SPAK in the development of salt‐sensitive hypertension remains unclear. We hypothesized that SPAK is involved in salt‐sensitive hypertension in part by increasing NKCC2 phosphorylation during normal or high salt diet. To test this, we generated SPAK knockout (KO) rats in a DSS genetic background via Crispr/Cas9 targeting of exon 5 in collaboration with the Gene Editing Rat Resource Center (Medical College of Wisconsin). Gene deletion was confirmed by sequencing and RT‐PCR. We used radiotelemetry to continuously measure mean blood pressure (MBP) in these rats on normal Na (0.22% Na chow) or high salt diet (4% Na chow). At baseline, SPAK KO rats had lower MBP than wild‐type DSS (WT) rats (SPAK KO: 117±1 mmHg; WT: 127±2 mmHg; p<0.05, n=7 each). After 4 weeks on high salt diet, MBP rose in all rats reaching 137±3 in SPAK KO and 154±3 mmHg in WT rats (p<0.05). The increase in MBP after switching rats from normal to high salt was lower in SPAK KO than WT rats; at 7 days (8±1 vs 13±1 mmHg, p<0.05), 14 days (12±2 vs 17±2 mmHg, p<0.05), and 28 days (20±3 vs 27±2 mmHg, p<0.05). We then explored total and phosphorylated‐NKCC2 (Thr96 and Thr101) expression in isolated TALs by Western blot. In rats fed normal salt (0.22% Na), phosphorylated‐NKCC2 (corrected over total NKCC2 expression) was lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 39±25%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 103±17%; n=5). After 4 weeks on high‐salt (4% Na), phosphorylated‐NKCC2 was also lower in TALs isolated from SPAK KO (WT: 100; SPAK KO: 38±13%; p<0.05, n=4) whereas total NKCC2 expression (corrected by GAPDH) was not different between both groups (WT: 100; SPAK KO: 113±22%; n=4). We conclude that SPAK KO rats exhibit a lower blood pressure at baseline and decreased salt sensitivity. This may be in part caused by lower NKCC2 phosphorylation in TALs of SPAK KO DSS rats. Our findings support an important role for SPAK in NKCC2 hyperphosphorylation and salt sensitive hypertension in DSS rats.

Support or Funding Information

NIH and NIDDK R01, Grant Number: 5R01DK107263‐02.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bile Acid Membrane Receptor TGR5 Regulates Renal AQP2 and Improves Lithiuminduced NDI
Authors: Li Suchun , Qiu Miaojuan , Kong Yonglun , Liu Qiaojuan , Hu Shan , Zhao Xiaoduo , Han Mengke , Xie Haixia , Levi Moshe , Li Chunling , Wang Weidong
Published: June 2021

The bile acid‐activated receptors, which include the membrane G protein‐coupled receptor (TGR5) and nuclear farnesoid X receptor (FXR), are shown to play roles in kidney diseases. The purpose of current study was to investigate the role of TGR5 receptors in renal water handling and the underlying molecular mechanisms. The activation of TGR5 by lithocholic acid (LCA, a TGR5 endogenous ligand) induced aquaporin‐2 (AQP2) protein expression and intracellular trafficking in rat primary cultured inner medullary collecting duct (IMCD) cells via cAMP‐PKA signaling pathway. A synthetic specific TGR5 agonist INT‐777 has the same effects as LCA in IMCD cells. In mice with lithium‐induced nephrogenic diabetes insipidus (NDI), the activation of TGR5 by LCA markedly decreased urine output and increased urine osmolality, which was associated with significantly up‐regulated AQP2 expression in the inner medulla, whereas chenodeoxycholic acid (CDCA, a FXR endogenous ligand) had no effects. In primary cultured IMCD cells prepared from lithium‐treated rats, INT‐777 or INT‐767 (a FXR and TGR5 dual agonist), but not INT‐747 (a FXR agonist) increased AQP2 protein expression. Compared with wildtype mice, tgr5−/− mice exhibited a mild urine concentration defect after dehydration, which was associated with decreased AQP2 expression in the kidney inner medulla. In lithium‐treated tgr5−/− mice, LCA treatment failed to prevent reduction of AQP2 expression. In conclusion, TGR5 activation increases renal AQP2 expression via cAMP‐PKA signaling pathway and improves impaired urinary concentration in lithium‐induced NDI. TGR5 is thus involved in regulation of water metabolism in the kidney.

Support or Funding Information

This work was supported by the Natural Science Foundation of China (Grants 81370822, 81570635, and 81670646), by the 111 Project, China (No. B13037), and by the Natural Science Foundation of Guangdong Province, China (Grants 2014A020212623, 2014A030313168, and 2016A020215034).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Epithelial Na+ Channel is Regulated by Biliary Components
Authors: Wang Xueping , Im Seohyun Janice , Ray Evan C. , Kashlan Ossama B.
Published: June 2021

Cirrhosis of the liver often leads to edema and increased circulating and urinary levels of bile acids and bilirubin conjugates. We hypothesized that ENaC activation by increased concentrations of biliary metabolites in renal tubular ultrafiltrate could contribute to renal Na+ retention and edema in cirrhosis of the liver. The epithelial Na+ channel (ENaC) plays a key role in regulating Na+ reabsorption in the kidney, and is regulated by an array of factors, including extracellular ligands (e.g. H+, Na+, Cl−) and post‐translational modifications (e.g. cleavage and palmitoylation). To begin testing this, we expressed wild type and mutant ENaCs in Xenopus oocytes and measured effects on channel current by two‐electrode voltage clamp. We found that specific bile acids and conjugated‐bilirubin activate ENaC in a dose‐dependent manner, with deoxycholic acid having the strongest stimulatory effect. Notably, deoxycholic acid stimulated both wild type channels and ‘near silent’ channels in which the furin cleavage sites were mutated. However, fully activating channels by trypsin abolished deoxycholic acid stimulation in both wild type channels and channels lacking furin cleavage sites, consistent with an effect on open probability. We hypothesized that bile acids, conjugated bilirubin and Cys‐palmitoylation (palmitate addition to specific intracellular cysteines on the β and γ subunits) regulate ENaC currents through a common mechanism: by increasing the membrane association of specific intracellular structures, driving conformation changes in the channel's pore. To begin testing our hypothesis, we determined whether mutation of all 4 palmitoylation sites in the β and γ subunits (βC43, βC557, γC33 and γC41) to alanine would affect activation by deoxycholic acid. We found that deoxycholic acid stimulated currents of ENaC lacking of all 4 palmitoylation site cysteines by 7‐fold over wild type ENaC. When we tested the effect of mutating individual sites, we found that removal of the N‐terminal palmitoylation site cysteines resulted in the strongest stimulatory effects. We also found that deoxycholic acid had a stronger stimulatory effect when we removed both sites from the γ subunit as compared to the β subunit. Our results demonstrate that ENaC can be activated by biliary metabolites, including bile acids and bilirubin, and that these metabolites may activate ENaC through a mechanism in common with ENaC activation by palmitoylation.

Support or Funding Information

DK098204 to O.B.K. and DK110332 to E.C.R.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The β‐adrenergic stimulation of the renal NaCl cotransporter is mediated via a cAMP‐dependent activation of protein phosphatase 1 inhibitor 1
Authors: Ribas David Penton , Moser Sandra , Wengi Agnieszka , Czogalla Jan , Rosenbaek Lena Lindtoft , Faresse Nourdine , Fenton Robert , Loffing‐Cueni Dominique , Loffing Johannes
Published: June 2021

Sympathetic β‐adrenergic activation of the thiazide‐sensitive NaCl cotransporter (NCC) in the renal distal convoluted tubule (DCT) was suggested to contribute to the development of salt‐sensitive hypertension. Previous studies demonstrated that β‐adrenergic agonists (e.g. norepinephrine and isoproterenol) increase cytoplasmic cAMP and the phosphorylation and hence the activity of NCC in the mouse DCT. Recently, we identified the endogenous inhibitor 1 (I1) of protein phosphatase 1 (PP1) as a DCT enriched gene product that controls NCC phosphorylation and blood pressure. As I1 is activated by cAMP‐dependent protein kinase A (PKA), we now tested the hypothesis that β‐adrenergic agonists inhibit PP1 via a cAMP/PKA‐dependent activation of I1 ultimately stimulating NCC phosphorylation. To test this hypothesis we performed experiments on MDCK cells stably transfected with NCC and on ex vivo preparations of kidneys from wildtype (WT) and I1‐knockout (I1‐KO) mice. The exposure of isolated DCTs to the cAMP‐elevating drugs forskolin and IBMX rapidly increased the phosphorylation of NCC via a protein kinase A (PKA)‐dependent pathway. The forskolin/IBMX‐induced phosphorylation of NCC was paralleled by I1 phosphorylation at its PKA‐consensus phosphorylation site (T35). Moreover, the forskolin/IBMX‐induced phosphorylation of NCC was diminished in kidney slices of from I1‐KO mice. The transgenic overexpression of a phosphomimetic I1 mutant (T35D) in kidneys of I1‐KO mice restored NCC phosphorylation, but made NCC resistant to forskolin/IBMX stimulation. Using an unbiased yeast‐two‐hybrid screen and co‐immunoprecipitation assays, we demonstrate that NCC and PP1 physically interact in transfected MDCK cells. Finally, the isoproterenol stimulated increase of NCC‐phosphorylation was impaired in I1‐KO mice as assessed in kidney slices and isolated‐perfused mouse kidneys. Remarkably, this occurred independent from any change in the phosphorylation of the kinases SPAK/OSR1. Thus, our data establish a complete signal transduction pathway by which cAMP increases NCC phosphorylation via a PKA‐dependent phosphorylation of I1 and subsequent inhibition of PP1. This pathway likely accounts for beta‐adrenergic NCC activation and hence may contribute to salt‐sensitive hypertension in patients with sympathetic hyperactivity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.