bluuebunny/crossref_metadata_2025 huggingface dataset:
Reactive Oxygen Species activate the NLRP3 Inflammasome in a model of lung transplant
Authors: Chatterjee Shampa , Tao Jian Qin
Published: June 2021

Background

Lung transplant involves a period of storage (ischemia) followed by the transplant (reattachment or reperfusion) event. The resultant ischemia‐reperfusion (I/R) injury, clinically known as primary graft dysfunction (PGD), is the most common cause for post‐transplant failure. Indeed, studies have shown that the poor outcome of lung transplant is primarily due to PGD that drives morbidity and mortality in ~30% of lung transplant recipients. We showed earlier, that one of the pivotal events with lung I/R associated with transplant is the production of reactive oxygen species (ROS). We also showed that this ROS generation is not the result of hypoxia‐deoxygenation as the lung oxygen supply is not compromised with stop or restart of blood flow. ROS produced with lung I/R reflects “flow” sensitive endothelial signaling that triggers NADPH oxidase‐2 (NOX2) activation. We postulated that injury with transplant was caused due to ROS initiated inflammation signaling. Among the inflammatory moieties of interest to us was the NOD like receptor protein 3 (NLRP3 inflammasome), which has been reported to be a major cause of cell death with I/R in systemic organs via pathways that were anoxia/hypoxia dependent. Could NLRP3 be activated by normoxic lung I/R as well? To answer this, we investigated NLRP3 activation and the mechanism of its regulation with lung I/R. Our hypothesis was that NOX2 activated with I/R regulates the NLRP3 inflammasome which in turn is a major driver of graft injury (PGD).

Methods

Mice (wild type and NOX2 null) were subjected lung I/R by hilar clamp and NLRP3 inflammasome activation measured by monitoring the colocalization of NLRP3 with its adaptor molecule ASC. Lung Injury was monitored by indices such as lipid peroxidation, isoprostanes and lung wet to dry ratio. The correlation between NLRP3 and PGD in post‐transplant human subjects was assessed by monitoring plasma NLRP3 and PGD outcome (obtained from Lung Transplant Outcomes Group Database).

Results

We observed a ROS dependent increase in NLRP3 activation post lung I/R. We also found that NLRP3 blockade significantly reduced lung injury. Additionally, post‐transplant (human) recipients with detectable NLRP3 protein in plasma developed PGD.

Conclusion

The NLRP3 inflammasome is regulated via NOX2 induced ROS production and plays a crucial role in injury with I/R. It can thus be considered as a risk factor with PGD.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long Term Hypoxia Reduces Levels of Oxylipins in Pulmonary Arteries and Venous Plasma of Fetal Sheep
Authors: Lopez Vanessa , La Frano Michael , Bosviel Remy , Hillman Tyler C , Newman John , Thorpe Richard , Fiehn Oliver , Zhang Lubo , Wilson Sean M
Published: June 2021

High‐altitude long term hypoxia (LTH) during gestation compromises the development and function of the pulmonary vasculature in the fetus and later in the newborn. This may manifest as fetal growth restriction as well as pulmonary hypertension following birth. While the physiological regulation regarding the effects of hypoxia are well‐known, the dysregulation that occurs at the cellular and molecular level is not yet completely understood. Exploring the effects of LTH using a metabolomic approach is advantageous in deciphering the etiology associated with the development of disease. LTH is well regarded for inducing oxidative stress and causing inflammation. A number of possible regulators associated with LTH stress include oxylipins and endocannabinoids, products of polyunsaturated fatty acid (PUFA) oxidation. We hypothesized that high‐altitude LTH would reduce the levels of oxylipins and endocannabinoids of pulmonary arteries and plasma in fetal sheep. To test the hypothesis, we obtained samples of plasma and pulmonary arteries from fetal normoxic and hypoxic sheep raised at 3,800 m. altitude starting on gestation day 30. Metabolite levels were quantified using ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). Chemical similarity enrichment analyses and visualization by complex pathway analyses was performed on the datasets. Our results support our hypothesis that select oxylipin concentrations were diminished in both venous plasma and pulmonary arteries. Omega‐3 PUFAs alpha linolenic acid (ALA) and eicosapentaenoic acid (EPA) were reduced in both plasma and pulmonary arteries. 15‐HEPE and 12‐HEPE, derivatives of EPA and important anti‐inflammatory mediators, were also significantly reduced by LTH in plasma and pulmonary arterial samples from fetal animals. We believe that our studies provide insight toward identifying key biomarkers of LTH stress that will afford effective and prompt diagnosis of the neonate's physiological response. This is important when it comes to treating diseases associated with gestational hypoxia, which affects numerous mothers and newborns worldwide.

Support or Funding Information

This material is based upon work supported by NIH grants P01HD083132 (LZ) and 1U24DK097154 through a pilot project grant to SMW. VL is a recipient of the American Physiological Society's Short‐Term Research Education Program to Increase Diversity in Health‐Related Research (STRIDE) Fellowship funded by the APS and a grant from the National Heart, Lung and Blood Institute R25HL115473.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
S‐nitroso‐L‐cysteine increases intracellular calcium in prenatal rat carotid body glomus cells
Authors: Getsy Paulina M , Coffee Gregory A , Ghosh Monica , Damron Derek , Lewis Stephen J
Published: June 2021

Carotid body (CB) glomus (Type I) cells detect decreases in arterial oxygen (O2) tension (hypoxia) by depolarizing, opening voltage‐gated calcium channels (VGCCs) and releasing neurotransmitters. These neurotransmitters activate chemoafferent terminals within the carotid sinus nerve (CSN), which signal respiratory regions of the brainstem to increase ventilation and restore blood O2 levels. Low‐molecular mass S‐nitrosothiols (SNOs), such as S‐nitroso‐Lcysteine (LSNC), are recently being used as chemical agents for studying the biochemistry underlying the ventilatory response to hypoxia. Unpublished work from our lab shows that LSNC alters macroscopic outward potassium currents in carotid body glomus cells under both normoxic and hypoxic conditions. However, the effect of LSNC on intracellular calcium concentration ([Ca2+]i) is unknown. Our objective was to determine whether LSNC elicits changes in [Ca2+]i in CB glomus cells. We hypothesized that LSNC increases [Ca2+]i via influx through VGCCs. To test this we isolated CB glomus cells from postnatal (P) P11–P15 Sprague Dawley male rat pups, and used Ca2+ imaging to investigate for the first time the effect of LSNC on CB glomus cell [Ca2+]i. Our preliminary data reveals that 50μM LSNC causes a rise in [Ca2+]i under normoxic conditions (n=2). This data suggests that LSNC increases CB glomus cell [Ca2+]i, and this rise may trigger Ca2+‐dependent release of neurotransmitters and activation of chemoafferent fibers within the CSN.

Support or Funding Information

NIH SPARC 1OT2OD023860‐02 SJLewis

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hypoxia‐induced depolarization of pulmonary arterial smooth muscle cells relies on simultaneous inhibition of K+‐currents and redox state alterations
Authors: Knoepp Fenja , Wahl Joel , Ramser Kerstin , Weissmann Norbert
Published: June 2021

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to localized alveolar hypoxia that is intrinsic to the pulmonary circulation. By hypoxia‐induced contraction of pulmonary arterial smooth muscle cells (PASMCs), pulmonary capillary blood flow is redirected to alveolar areas of high oxygen partial pressure (pO2), thus maintaining the ventilation‐perfusion ratio. Although the principle of HPV was recognized decades ago the underlying pathway still remains elusive.

Voltage gated K+‐channels (Kv‐channels) are known to be redox‐sensitive and crucial for mediating membrane potential in PASMCs ‐ thereby controlling Ca2+‐entry and subsequently vascular tone. Here, we investigated whether acute hypoxia alters the redox state of isolated PASMCs from mice and if this impacts Kv‐channel activity, membrane potential and intracellular Ca2+‐ level.

Kv‐currents and membrane potential in response to hypoxia or the oxidizing agent H2O2 were investigated by whole cell patch clamp experiments (voltage and current clamp, resp.) on freshly isolated murine PASMCs. Simultaneously, pO2 was recorded using an optical oxygen sensor and intracellular Ca2+ was measured by ratiometric FURA‐2 imaging. The redox state was monitored by Raman spectroscopy using an excitation wavelength that was in resonance with the hemeproteins (532 nm).

Raman‐spectra values for reduced myoglobin (1607 cm−1) and cytochrome c (1622 cm−1) both increased upon exposure of PASMCs to hypoxia (4 % O2) and returned to the initial value upon reverting to normoxia (21 % O2), thus indicating a shift in the cells' redox state. In accordance, Kv‐currents were dose‐dependently inhibited by hypoxia and H2O2, resulting in a depolarization from −37,5 ± 2 mV (before) to −28.09 ± 2 mV after exposure to hypoxia (Mean ± SEM; n = 23; p = < 0,001; paired t‐test). The oxidizing agent H2O2 (124 nM) mimicked this effect under normoxic conditions by elevating the membrane potential of the PASMCs from −37.4 ± 3 mV (before) to −25,2 ± 4 mV upon application of 124 nM H2O2 (Mean ± SEM; n = 5, p = 0,005, paired t‐test) ‐ thereby triggering a rise in intracellular Ca2+.

In conclusion, hypoxia induced a shift in redox state in PASMCs, thereby mediating the inhibition of KV‐channels. The resulting depolarization and Ca2+‐entry may account for the contraction of PASMCs – thus initiating HPV.

Support or Funding Information

This study was funded by the German Research Foundation (DFG, CRC 1213) and the Swedish Research Council (Grant 2016‐04220).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Corticospinal and Spinal Excitability to the Biceps and Triceps Brachii is Modulated Differently between Forward and Backward Arm Cycling
Authors: Nippard Anna , Lockyer Evan , Button Duane , Power Kevin
Published: June 2021

The purpose of this study was to evaluate supraspinal and spinal excitability to the biceps and triceps brachii when comparing forward (FWD) and backward (BWD) arm cycling. Supraspinal and spinal excitability were assessed non‐invasively using transcranial magnetic stimulation (TMS) to elicit motor evoked potentials (MEPs) and transmastoid electrical stimulation (TMES) to elicit cervicomedullary evoked potentials (CMEPs), respectively. MEPs and CMEPs were recorded from the biceps and triceps brachii during FWD and BWD arm cycling at two positions, the 6 and 12 o'clock position. The 6 o'clock position corresponded to mid elbow flexion and extension during FWD and BWD cycling, respectively, while the 12 o'clock position corresponded to mid elbow extension and flexion during FWD and BWD cycling, respectively. Participants completed four arm cycling trials, two FWD and two BWD, at 60 rpm and 25 W. During the flexion phase MEP (p = .001) and CMEP (p = .001) amplitudes of the biceps brachii were higher during FWD cycling. However, during the extension phase MEP (p = .006) and CMEP (p = .003) amplitudes were higher during BWD cycling. For the triceps brachii MEP amplitudes were higher during FWD cycling compared to BWD (p = .027) regardless of the functional phase (flexion vs. extension) of the movement cycle. However, spinal excitability to the triceps brachii was dependent on the functional phase of the movement cycle. During the flexion phase CMEPs of the triceps brachii were higher during FWD cycling compared to BWD (p = .032), but during the extension phase CMEPs were higher during BWD cycling compared to FWD (p = .001). This data suggests that the modulation of CSE and spinal excitability to the biceps brachii is dependent on the functional phase of the movement cycle and on the cycling direction. Also, spinal excitability but not CSE to the triceps brachii is dependent on the functional phase of the movement cycle when comparing FWD and BWD cycling.

Support or Funding Information

NSERC Discovery Grant awarded to K.E. Power

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Balancing Neuroprotection with Functional Recovery: The Potential Role of the PNN in Preventing Excitotoxicity after SCI
Authors: Stoltz Daimen , Silverstein Aaron , Huffman Emily , Alilain Warren
Published: June 2021

In spinal cord injury (SCI), initial mechanical trauma causes debilitating primary damage to neural cells and blood vessels. Following this, secondary cascades of downstream events occur, including inflammation, ischemia, and excitotoxicity — an increase in intracellular Ca2+ concentration from overactive glutamate (Glu) receptors leading to cell death. Additionally, there is an upregulation of the perineuronal net (PNN), a lattice‐like structure of the extracellular matrix (ECM) which modulates neural communication and homeostasis. The PNN is partially composed of negatively charged chondroitin sulfate proteoglycans (CSPGs). While CSPGs can stabilize plasticity and neuronal growth during development, these molecules become inhibitory to regeneration, sprouting and plasticity after injury, as well as contribute greatly to the glial scar. However, administration of the bacterial enzyme chondroitinase ABC (ChABC) can digest the PNN and CSPGs, ultimately promoting functional recovery. What remains unknown are the other impacts of removing the PNN at very acute stages of injury. We hypothesize that the PNN and its negatively charged CSPGs are upregulated after SCI as a neuroprotective response that attenuates excitotoxicity by acting as a buffer for Ca2+. To test our hypothesis, we induced excitotoxicity by injecting rats with a threshold dose of NMDA with or without ChABC utilizing the well defined spinal respiratory motor system. 59% of SCI occurs at the cervical level, and mechanical ventilation is the leading cause of death and restriction of independence in these cases. Therefore, we administered the dose instraspinally at the C4 level and paired treatment with intrapleural injection of cholera toxin‐B to retrogradely label the phrenic motor neuron pool, which innervates the diaphragm. Preliminary findings suggest that animals lacking the PNN have exacerbated cell death versus control. This implies that, following SCI, the body's main focus is to survive and not necessarily to preserve function. PNN upregulation after injury could be a conserved mechanism which promotes survival and CNS tissue preservation at the expense of plasticity and functional regeneration. The next steps are to detail the spread of ChABC and how long it takes for the PNN to regenerate. Future directions will investigate the optimal timing of ChABC administration to balance preserving cell survival with promoting functional recovery.

Support or Funding Information

NINDS R01NS101105 (WJA)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dopaminergic Projections to Raphe Pallidus Inhibit Brown Adipose Tissue Thermogenesis
Authors: da Conceição Ellen Paula Santos , Madden Christopher J , Morrison Shaun F.
Published: June 2021

Background

The observations that patients with Parkinson's disease experience weight loss, while dopamine (DA) replacement therapy causes body weight gain, suggest that suppressing the DA system could stimulate energy expenditure. Peripheral administration of dopaminergic drugs decreases heat production by inhibition of brown adipose tissue (BAT) thermogenesis and by an increase in the threshold for muscular shivering. However, the exact neuroregulatory site through which DA modulates the activity of thermogenic thermoeffectors remains undefined.

Results

In urethane/chloralose‐anesthetized, artificially‐ventilated rats, activation of type 2 or 3 dopamine receptors in the rostral raphe pallidus (rRPa) inhibits cold‐evoked BAT sympathetic activity, reduced BAT temperature and expired CO2, and produced hypotension and bradycardia. Anatomic evidence shows the co‐localization of TH and CTb immunoreactivities in neurons in the in the periventricular gray zona following injection of the retrograde tracer in the rRPa. These data indicate that this dopaminergic cell group projects to rRPa and is the likely source of the dopaminergic inhibition of BAT sympathetic activity.

Conclusion

Our data provide the first evidence that a DA input to the rRPa from periventricular gray zona modulates BAT thermogenesis and cardiovascular function. Modulating the dopaminergic input to rRPa could provide a novel therapeutic approach to promote fat oxidation, favor better glycaemic control, and accelerate weight loss.

Support or Funding Information

NIH R01‐NS091066

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Assessment of Central and Peripheral Chemoreceptor Reflex Regulation of Muscle Sympathetic Nerve Activity and Ventilation
Authors: Keir Daniel A , Millar Philip J , Duffin James , Floras John S
Published: June 2021

We tested the hypothesis that central and peripheral chemoreceptor‐mediated sympathetic activation elicited by increases in CO2 tension (PCO2) mirror concurrent ventilatory responses. Twelve healthy young men (mean±SD; age: 24±4 yr; BMI: 24.5±1.3 kg·m−2) performed a modified rebreathing protocol designed to equilibrate central and peripheral chemoreceptor PCO2 tensions with end‐tidal PCO2 (PETCO2) at two isoxic end‐tidal PO2 (PETO2) tensions such that central chemoreceptor reflex responses (hyperoxia) can be isolated from additive peripheral responses (i.e., hypoxia minus hyperoxia). Subject's ventilation (volume turbine) and muscle sympathetic nerve activity (MSNA; microneurography, right fibular nerve) responses to rebreathing at isoxic PETO2 tensions of 150 mmHg (hyperoxia) and 50 mmHg (hypoxia) were recorded continuously. During rebreathing, the PETCO2 (mmHg) at which ventilation (L·min−1) and total MSNA (au) began to rise were identified as the PETCO2 recruitment thresholds (RT). Slopes of these responses above RT also were determined. In hyperoxic rebreathing (i.e., the central chemoreflex response), the mean RT for ventilation (46±3 mmHg) and MSNA (45±4 mmHg) did not differ (p>0.05). Mean slopes were 2.3±0.9 L·min−1·mmHg−1 and 2.1±1.5 au·mmHg−1, respectively. In hypoxic rebreathing (i.e., the summed central and peripheral chemoreflex response), the RT for both ventilation and MSNA were not different (41±3 mmHg vs 41±3 mmHg, p>0.05) but were lower (p<0.05) compared to hyperoxia. Mean slopes for ventilation (4.0±1.8 L·min−1·mmHg−1) and MSNA (5.1±3.4 au·mmHg−1) in hypoxia were greater than in hyperoxia yielding a mean peripheral chemoreflex sensitivity (hypoxic slope minus hyperoxic slope) of 1.7±0.1 L·min−1·mmHg−1 for ventilation and 3.0±2.6 au·mmHg−1 for MSNA. The 95% limits of agreement of RT between ventilation and MSNA for the central chemoreflex were −7 to 6 mmHg (bias=−1 mmHg, p>0.05) and −5 to 5 mmHg (bias=0 mmHg, p>0.05) for the peripheral chemoreflex. There was no relationship (p>0.05) between the ventilatory and MSNA sensitivity for either the central (r2=0.16) or peripheral (r2=0.01) chemoreceptor reflexes. These data highlight two novel findings: 1) ventilatory and sympathetic arms of the central and peripheral chemoreceptor reflexes are initiated at similar PETCO2 recruitment thresholds and 2) sympathetic responsiveness cannot be predicted from the sensitivities of the ventilatory central and peripheral chemoreceptor reflexes.

Support or Funding Information

Experiments were funded by a Project Grant from the Canadian Institutes of Health Research (PJT 159491). Dr. Floras holds the Canada Research Chair in Integrative Cardiovascular Biology.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Selective Reductions in Pulmonary Artery Pressure Lowers Sympathetic Neural Activity in Healthy Humans at High Altitude
Authors: Simpson Lydia L , Steele Andrew , Meah Victoria L , Thapamagar Suman , Gasho Christopher , Drane Aimee , Oliver Samuel J , Tymko Michael M , Ainslie Philip N , Steinback Craig D , Stembridge Mike , Moore Jonathan P
Published: June 2021

Chronically elevated sympathetic neural activity is a characteristic of many disease states. Tonic chemoreflex activation has been implicated in the development of sympathetic overactivity; however, other reflexes may also contribute. For example, in an animal model, stimulation of pulmonary baroreceptors induces a reflex increase in sympathetic neural activity. In humans, pulmonary pressure has been shown to be related to basal sympathetic activity in pulmonary hypertension; however a critical step in determining a causal link is to establish whether changing pulmonary pressure alters sympathetic outflow. In this pilot study, we exposed thirteen healthy humans (5 females, 28 [ ± 7] years) to high altitude hypoxia (HA; 4383m), a setting known to increase both pulmonary pressure and sympathetic activation, before selectively lowering pulmonary pressure via inhalation of the pulmonary vasodilator nitric oxide. Muscle sympathetic nerve activity (MSNA; Microneurography), blood pressure (BP; Photoplethysmography), heart rate (HR; Electrocardiogram), and peripheral oxygen saturation (SPO2; Pulse oximetry) were continually measured during ambient air breathing (Amb) and during a 6‐minute inhalation of nitric oxide (iNO; 40ppm in 21% O2). Pulmonary artery systolic pressure (PASP), pulmonary vascular resistance (PVR) and stroke volume (SV) were determined via cardiac ultrasound. iNO reduced PASP (−23 ± 12%) and PVR (−21 ± 16%) compared to Amb (Table 1). iNO also reduced MSNA burst frequency (−18 ± 18%), burst incidence (−16 ± 17%) and total activity (−21 ± 20%) compared to Amb. MSNA changes during iNO were not related to changes in SPO2 (r = −0.16, P = 0.61) or MAP (r = −0.28, P = 0.35). Our data demonstrate that selectively reducing pulmonary pressure reduces sympathetic nerve activity in healthy humans; therefore, this provides evidence for a causal link in the relationship between pulmonary pressure and basal sympathetic activity. Further investigation is warranted to establish whether elevated pulmonary pressure is a mechanism contributing to chronically elevated sympathetic outflow in disease.

Support or Funding Information

This study was supported by the Natural Sciences and Engineering Research Council of Canada and a Canada Research Chair in Cerebrovascular Physiology grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nrf2 Dysfunction in the RVLM is Associated with Obesity‐Induced Sympathoexcitation
Authors: Hall Delton , Asirvatham‐Jeyaraj Ninitha , Balasubramanian Priya , Subramanian Madhan
Published: June 2021

Sympathetic overactivity contributes to the pathogenesis of obesity‐induced hypertension. The rostral ventrolateral medulla (RVLM) is an important brainstem region that regulates sympathetic nerve activity (SNA) to peripheral tissues. Oxidative stress and neuroinflammation in the RVLM are believed to be important contributors for augmented SNA in obesity, however, the molecular mechanisms that initiate and sustain the pro‐inflammatory milieu in the RVLM during obesity are not clear. Nuclear factor erythroid 2‐related factor (Nrf2) is a master transcriptional factor that regulates the expression of anti‐oxidant and anti‐inflammatory genes. We hypothesized that obesity impairs Nrf2 signaling to increase oxidative stress in the RVLM leading to increases in SNA and hypertension. To test this, 2 months‐old male C57BL/6J mice were fed a high‐fat diet (HFD; 60% fat) or normal chow diet (10% fat for 16 weeks and were instrumented with radiotelemeters in the last 2 weeks for measuring blood pressure and SNA. Heart rate variability and depressor response to ganglionic blockade were used to assess SNA. At the end of 16 weeks, animals were sacrificed, brainstem was isolated and sectioned. Superoxide levels were assessed in the frozen sections by dihydroethidium staining (DHE). RVLM was microdissected for gene expression analysis. Data were analyzed by students t‐test and p<0.05 was considered statistically significant. HFD feeding resulted in a mild but significant increase in mean arterial pressure (103.7 ± 0.9 vs 108.6 ± 0.73 mmHg, normal chow diet vs HFD; P<0.05; n=4) and systolic blood pressure (121.2 ± 1.2 vs 129 ± 0.67 mmHg, p<0.05; n=4) compared to normal chow‐fed animals. Ganglionic blockade using hexamethonium showed greater depressor response (−0.21 ± 2.9 vs 13.13 ± 1.3 mmHg, normal chow diet vs HFD; p<0.05; n=4) to HFD feeding. Furthermore, low‐frequency blood pressure variability and the ratio of low‐frequency to high‐frequency heart rate variability in HFD mice were significantly higher than in the control mice suggesting HFD upregulated SNA. In addition, obesity‐induced sympathoexcitation was associated with increased superoxide levels (DHE staining) and decreased mRNA levels of Nrf2 (1.05 ± 0.16 vs 0.48 ± 0.06 fold, normal diet vs HFD; p<0.05) and its target genes (e.g. NQO1 1.08 ± 0.22 vs 0.51 ± 0.08 fold, p<0.05 and SOD2 1.03 ± 0.13 vs 0.48 ± 0.05 fold; p<0.05) in the RVLM of HFD mice. Collectively, our results show for the first time, evidence for obesity‐associated Nrf2 dysfunction in the RVLM. Future studies will investigate the mechanistic role of Nrf2 signaling in the RVLM in obesity‐induced sympathoexcitation.

Support or Funding Information

RAC fund, CVHS, Oklahoma State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Brain Selective Overexpression of ACE2 Attenuates Blood Pressure Response to Central Ang II: Potential Role of Nrf2
Authors: Ma Anyun , Gao Lie , Zucker Irving
Published: June 2021

Introduction

Essential hypertension is characterized by increased neurohumoral activation. Central Angiotensin II (AngII) increases sympathetic outflow. Our previous studies showed dysfunctional central ACE2/Ang1‐7/Mas receptor axis and increased ACE in heart failure animals with increased sympathetic activation. Mice overexpressing the human isoform of ACE2 (synhACE2) in the brain exhibit reduced sympathetic nerve activity (SNA) and a reduction in central oxidant stress. However, the mechanism by which ACE2 overexpression alters neurohumoral outflow is unknown. Our preliminary experiments showed evidence of increased central Nrf2, a redox sensitive transcription factor, in synhACE2 mice with heart failure. Nrf2 promotes antioxidant enzyme expression, but the role of this pathway in the central regulation of sympathetic outflow in response to central Ang II has not been explored.

Hypothesis

We hypothesized that upregulation of central ACE2 inhibits the pressor response to AngII by a Nrf2–related, antioxidant effect.

Methods

synhACE2+ mice and their synhACE2‐ littermates/wild type controls (n=3–7/group) were used to evaluate blood pressure, metabolic responses, Nrf2, antioxidant enzyme expression and oxidative stress in the brain during and following 2 weeks of intracerebroventricular (icv) AngII (100 ng/kg/min) infusion. In vitro experiments were carried out using Neuro 2A cells to assess Nrf2 translocation to the nucleus in response to AngII (100 nM/24 hrs) stimulation.

Results

icv AngII induced a significant increase in mean arterial blood pressure and polydipsia (Fig.1), as well as 24‐hour norepinephrine (NE) excretion. Brain selective overexpression of synhACE2 attenuated the blood pressure (95.2 ± 4.3 mmHg vs 125.4 ± 13.1 mmHg, p<0.05), polydipsia (11.3 ± 2.5mL/day vs 27.0 ± 2.0mL/day, p<0.05) and NE in response to central Ang II (Fig.1). The Mas receptor agonist Ang1‐7 and blockade (A779) had no effects on blood pressure or drinking responses following central Ang II infusion (Fig.1). In Neuro 2A cells, nuclear Nrf2 increased in response to AngII treatment.

Conclusions

These data indicate that the hypertensive effects of icv AngII are attenuated by brain selective overexpression of synACE2—most likely mediated by the effects of ACE2 on Ang II‐AT1R signaling. These data reinforce our previous finding that brain‐selective overexpression of ACE2 attenuates sympathetic nerve activity in CHF. The cell data show that AngII is capable of translocating Nrf2 to the nucleus, which is counteracted by common antioxidants, reflecting acute Nrf2 response to oxidative stress changes.

Support or Funding Information

Supported by NIH grant P01 HL62222 and a UNMC pre‐doctoral fellowship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
γ‐secretase inhibitor DAPT mitigates cisplatin‐induced acute kidney injury by suppressing Notch1 signaling
Authors: Soni Hitesh M , Matthews Anberitha T. , Pallikkuth Sandeep , Gangaraju Rajashekhar , Adebiyi Adebowale
Published: June 2021

Organ toxicity, including kidney injury, limits the use of cisplatin for the treatment of multiple human cancers. Hence, interventions to alleviate cisplatin‐induced nephropathy are of benefit to cancer patients. Recent studies have demonstrated that pharmacological inhibition of the Notch signaling pathway enhances cisplatin efficacy against several cancer cells. However, whether augmentation of the anti‐cancer effect of cisplatin by Notch inhibition comes at the cost of increased kidney injury is unclear. We show here that treatment of mice with cisplatin resulted in a significant increase in Notch ligand Delta‐like 1 (Dll1) and Notch1 intracellular domain (N1ICD) protein expression levels in the kidneys. N‐ [N‐ (3,5‐difluorophenacetyl) ‐L‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT), a γ‐secretase inhibitor reversed cisplatin‐induced increase in renal N1ICD expression and plasma or urinary levels of predictive biomarkers of acute kidney injury (AKI). DAPT also mitigated cisplatin‐induced tubular injury and reduction in glomerular filtration rate. Real‐time multiphoton microscopy revealed marked necrosis and peritubular vascular dysfunction in the kidneys of cisplatin‐treated mice which were abrogated by DAPT. Cisplatin‐induced Dll1/Notch1 signaling was recapitulated in a human proximal tubule epithelial cell line (HK‐2). siRNA‐mediated Dll1 knockdown and DAPT attenuated cisplatin‐induced Notch1 cleavage and cytotoxicity in HK‐2 cells. These data suggest that Dll1‐mediated Notch1 signaling contributes to cisplatin‐induced AKI. Hence, the Notch signaling pathway could be a potential therapeutic target to alleviate renal complications associated with cisplatin chemotherapy.

Support or Funding Information

We acknowledge a fellowship (to A. Adebiyi) from the O'Brien Center for Advanced Renal Microscopic Analysis (NIH‐NIDDKP30DK079312).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Diallyl trisulfide improves kidney function through alteration of fatty acid receptor expression in diabetic mice
Authors: Weber Gregory , Pushpakumar Sathnur , Sen Utpal
Published: June 2021

Approximately 10% of the United States population suffers from diabetes causing diabetic nephropathy (DN) and eventually leading to renal failure. Fatty acids (FAs) are byproducts of gut microbiota and can regulate physiological process such as metabolism and inflammation as well as influenced through dietary supplements. Certain FA receptors have been shown to exert anti‐inflammatory effects in diabetes and other diseases; however, the role of these receptors in the kidney is not well defined. Diallyl trisulfide (DATS) is a major organosulfur component of garlic and is metabolized into hydrogen sulfide, an important gasotransmitter with noted cardiovascular and renal benefits. Whether DATS regulates FAs and their receptors in the kidney is unclear. In this study, we sought to determine if dietary supplementation with DATS improves kidney function by reducing inflammation through FA receptors in a Type‐II diabetic mouse model. Initial results showed improved kidney function of diabetic mice supplemented with DATS, as evidenced by an increase in glomerular filtration rate (GFR) as well as a lower resistive index (RI) of the renal artery compared to diabetic mice on a normal diet. The expression of short chain fatty acid receptor Gpr43 and long chain fatty acid receptor Gpr120 was altered in kidney of mice given DATS compared to untreated animals. In addition, Ptgs2, an enzyme implicated in the inflammatory response and can be inhibited by some naturally occurring FAs, was reduced in the kidney of diabetic mice supplemented with DATS. 16S sequencing showed DATS supplementation affected host gut microbiome composition, showing an enrichment in Verrucomicrobia and increased alpha diversity in diabetic mice on DATS diet. In conclusion, our data suggests FA receptors may play a role in the diabetic kidney by reducing inflammation with DATS supplementation, influencing the expression of these receptors, and improving kidney function. Further, a shift in the gut microbiome of diabetic mice toward that of non‐diabetic mice suggests a role for gut microbiota in DN and DATS supplementation may help delay or prevent the onset of this disease.

Support or Funding Information

This study was supported in part by U.S. National Institutes of Health (NIH) Grant DK104653 (U.S.) and American Heart Association Scientist Development Grant 15SDG25840013 (S.P.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Saturated Fatty Acids Stimulate Renal Inflammation‐Related Gene and Protein Expression by Activating the ERK Signaling Pathway
Authors: Zhao Xueying , Chen Xiaoming , George Jasmine , Zhang Yuanyuan , Cobbs Alyssa , Wang Guoshen , Li Lingyun , Emmett Nerimiah
Published: June 2021

Diabetic nephropathy is increasingly recognized as a major contributor to renal dysfunction and kidney failure in obesity and type 2 diabetes. To identify the molecular mediators of kidney injury and dysfunction associated with metabolic syndrome with or without hyperglycemia, we compared renal gene expression profiles in Zucker lean (ZL), Zucker obese (ZO) and Zucker diabetic (ZD) rats. Compared to 20‐week‐old ZL control (glucose: 110 ± 8 mg/dL), both prediabetic ZO (glucose: 157 ± 11 mg/dL) and diabetic ZD (glucose: 481 ± 37 mg/dL) rats displayed hyperlipidemia and kidney injury with high‐degree albuminuria/proteinuria. Using cDNA microarray, we identified 25 inflammatory and fibrotic transcriptomes similarly changed in ZO and ZD kidneys, including kidney injury molecule‐1 (KIM‐1), secreted phosphoprotein‐1 (SPP‐1) and CD44. Immunostaining and Western blot analyses confirmed an increase in their protein levels in the kidney of ZO and ZD rats. Moreover, our in vitro cell culture study revealed that administration of saturated fatty acid palmitate resulted in an upregulation of KIM‐1, SPP‐1 and CD44 protein expression in primary tubular epithelial cells. Palmitate‐induced tubular inflammation and injury‐associated proteins were greatly attenuated in the presence of U0126, an inhibitor of extracellular signal‐regulated kinase 1/2 (ERK1/2). Taken together, our results demonstrate a stimulatory role of lipids for inflammation‐related gene and protein expression in the kidney, suggesting that lipotoxicity may play an important part in renal inflammation and nephropathy progression in metabolic syndrome associated with obesity and type 2 diabetes.

Support or Funding Information

This work was supported by the NIH SC1DK112151, NIH/NCRR/RCMI 8G12MD007602 and 8U54MD007588.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Salt Sensing and Salt Response of Renal Neuropeptide FF Receptor 2
Authors: Villar Van Anthony M , Asico Laureano D , Wang Xiaoyan , Konkalmatt Prasad , Yang Jian , Gomes Jerald , Rozyyev Selim , Tiu Andrew C , Cuevas Santiago , Armando Ines , Jose Pedro A
Published: June 2021

The causal role of dietary NaCl intake in the development of hypertension and salt sensitivity has long been recognized. The kidney sits at the fulcrum of maintaining an appropriate sodium balance and blood pressure homeostasis. As such, identifying new regulators of sodium balance in the kidney is crucial to understand the intricate mechanisms involved. Neuropeptide FF (NPFF) is a hormone expressed in the central nervous system that subserves nociception, hormonal modulation, and body temperature control. We now demonstrate for the first time that NPFF and its receptors, NPFF‐R1 and NPFF‐R2, are expressed in the kidney and endowed with pro‐hypertensive properties. The renal‐restricted bolus (1‐hr) or chronic infusion (7‐day) of NPFF increased blood pressure (*P<0.05, n=4/group) and decreased sodium excretion (UNaV; *P<0.05, N=4/group) in C57Bl/6 mice. This tubular effect was due solely to NPFF‐R2 since its silencing resulted in ~3‐fold increase in UNaV, unlike NPFF‐R1 silencing which did not affect UNaV. Therefore, we further explored the changes in NPFF‐R2 expression or activity in response to changes in salt. We now report that we have newly identified a “Sodium Response Element” (SRE), a homolog of “Dehydration‐Responsive Element” (“TACCGACAT”) in Arabidopsis thaliana genome, at the NPFF‐R2 promoter ~ 2.3 Kb upstream of the transcription start point. Exposing human renal proximal tubule cells (hRPTCs) from normal (145 μM NaCl) to low (90 μM NaCl) sodium concentration resulted in increased promoter activity (~2.5‐fold, *P<0.05, vs. DSRE, 3/group; via luciferase activity) and increased mRNA and protein expression of NPFFR2 (*P<0.05, 3–4/group; 0–8 hr). Exposure of hRPTCs from normal to high (175 μM NaCl) sodium concentration was accompanied by a reduction in promoter activity (−0.5‐fold, *P<0.05), and a decrease in mRNA and protein expression of NPFFR2 (*P<0.05, 0–8 hr). Moreover, there was a markedly increased co‐immunoprecipitation between pro‐hypertensive NPFF‐R2 and anti‐hypertensive dopamine D1 receptor, resulting in increased antagonism between the two receptors on cAMP response and sodium transport (*P<0.05, vs. fenoldopam or NPFF treatment [1 mM/30 min], 4/group). Lastly, exposure to high salt concentration resulted in greater co‐immunoprecipitation of NPFF‐R2 with Gαi3 and none with Gαs (“Gα switch”), which may explain its pro‐hypertensive activity. NPFF, via NPFF‐R2, may be a crucial determinant of sodium balance and blood pressure homeostasis and thus, may be an important target for new pharmacological and biological intervention.

Support or Funding Information

R01DK039308, R01HL092196, and P01HL074940

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeted inhibition of mitochondrial ROS maintains TFAM and mitochondrial DNA homeostasis in acute kidney injury
Authors: Zhao Meng , Liu Shunyun , Wang Chengshi , Cui Danli , Li Lan , Yuan Yujia , Chen Younan , Lu Yanrong , Liu Jingping
Published: June 2021

Background

Acute kidney injury (AKI) is a serious worldwide health issue with high mortality and morbidity, and oxidative stress / mitochondrial dysfunction have been recognized as key features of AKI. Mitochondrial transcription factor A (TFAM) is a key regulator of mitochondria DNA (mtDNA) transcription and replication, which maintains the cellular mitochondrial redox and energy homeostasis in various physiological conditions, but its alteration in AKI is not complete clear. In this study, we aim to investigate the effect of mitochondrial ROS (mtROS) on TFAM signaling and mtDNA damage in AKI.

Methods

In vitro, human proximal tubule epithelial cell lines (HK‐2) were incubated under hypoxia/reoxygenation (H/R) condition, and cellular mtROS were specifically eliminated by mitochondria‐targeted antioxidant Mito‐Tempo (MT). The changes of intracellular ROS, TFAM, mitochondrial biogenesis, mtDNA copy number, cell apoptosis and inflammation in HK2 cells were analyzed. In vivo, AKI mice were induced by renal ischemia/reperfusion (I/R) injury and treated with MT, and the changes of renal function, tubular injury, inflammation, mitochondrial function, mtROS, ATP production, and mtDNA level were measured.

Results

In vitro, H/R condition induced mtROS with declined TFAM and mtDNA injury, as well as increased inflammation and apoptosis in HK2 cells. By contrast, targeted inhibition of mtROS restored TFAM, mtDNA copy number and mitochondrial biogenesis (PGC1α), and reduced proinflammatory cytokines (IL‐1β/TNF‐α) and pro‐apoptotic factors (FAS) expression in HK2 cells. However, the cytoprotective role of mtROS inhibition on mtDNA and mitochondrial biogenesis was diminished in HK2 cells when knockdown of TFAM by siRNA, In vivo, targeted inhibition of mtROS also reduced the levels of serum BUN/CREA, necrotic tubules number, tubular apoptosis and renal inflammation in AKI mice. Moreover, targeted inhibition of mtROS rescued the TFAM and mtDNA damage with improved mitochondrial morphology, ATP production and mtDNA copy number in kidney of AKI mice.

Conclusion

This study highlights that TFAM plays an essential role in maintaining mitochondrial redox homeostasis, and mtROS induce renal dysfunction via disturbing TFAM and mtDNA, thus targeted inhibition of mtROS is a promising therapy for AKI.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Treatment with lisinopril slows the early progression of proteinuria in obese Dahl salt‐sensitive rats independent of lowering arterial pressure and GFR
Authors: Pennington Alyssa P. , Poudel Bibek P. , Shields Corbin A. , Thompson Willie , Williams Jan M.
Published: June 2021

Prepubertal childhood obesity (PPO) has emerged as an epidemic and major health problem in the United States. Previous studies have demonstrated that patients who developed obesity prior to puberty are at higher risk of developing progressive proteinuria and chronic kidney disease. Moreover, the early stages of renal injury associated with obesity is characterized with renal hyperfiltration. Recently, we observed the early development of renal injury in the obese Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) strain is associated with elevations in GFR. Furthermore, these renal abnormalities develop prior to puberty (6 weeks of age) and independent of hyperglycemia and elevations in arterial pressure. The standard care of treatment for obese children with hypertension and increased risk of renal disease is an angiotensin converting enzyme inhibitor (ACEi) such as lisinopril. Therefore, the current study examined whether treatment with lisinopril prevents the early development of proteinuria in the SSLepRmutant strain. Experiments were performed on four week‐old SSWT and SSLepRmutant rats that were either treated with vehicle or lisinopril (20 mg/kg/day, drinking water) for four weeks. We did not observe any differences in MAP between SSWT and SSLepRmutant rats (156±6 and 163±7 mmHg, respectively). While treatment with lisinopril markedly reduced MAP in SSWT rats (108±4 mmHg), lisinopril had no effect on MAP in the SSLepRmutant strain (146±7 mmHg). Proteinuria was significantly higher in the SSLepRmutant strain compared to SSWT rats at baseline (105±19 and 17±5 mg/day, respectively) and remained elevated over the course of the study (400±48 and 60±9 mg/day, respectively). Treatment with lisinopril significantly decreased the progression of proteinuria by 68% in SSWT rats (19±3 mg/day) and 40% in SSLepRmutant rats (242±31 mg/day). GFR was elevated in the SSLepRmutant strain compared to the values measured in the SSWT rats (4.80±0.50 vs. 3.87±0.48 mL/min, respectively). Interestingly, lisinopril treatment had no effect on GFR in both strains. These data suggest that obese SSLepRmutant rats develop progressive proteinuria associated with hypertension and elevations in GFR, and treatment with lisinopril blunted the progression of proteinuria independent of lowering GFR and arterial pressure during PPO.

Support or Funding Information

This work was supported by GM104357 and DK109133.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Depletion of macrophages with clodronate partially reduces the progression renal injury in obese Dahl salt‐sensitive rats during prepubertal obesity
Authors: Poudel Bibek , Shields Corbin A. , Pennington Alyssa , Cornelius Denise C. , Williams Jan M.
Published: June 2021

Prepubertal obesity (PPO) is now considered a risk factor for the development of proteinuria and renal injury in children. Recently, we reported that, prior to puberty, Dahl salt‐sensitive leptin receptor mutant (SSLepRmutant) rats display proteinuria and renal injury as early as 6 weeks of age independent of hyperglycemia and elevations in arterial pressure. Moreover, the early development of renal injury was associated with an increase in the renal infiltration of macrophages in the SSLepRmutant strain compared to their lean SSWT counterparts (39±14 vs. 11±3 % gated, respectively; p<0.05 vs. SSWT). Therefore, the current study examined whether depletion of macrophages with clodronate would reduce renal injury in the SSLepRmutant strain during PPO. Four week‐old SSWT and SSLepRmutant rats were separated into four groups (n=4/group): (1) SSWT and (2) SSLepRmutant rats treated with vehicle‐PBS and (3) SSWT and (4) SSLepRmutant rats treated with clodronate (50 mg/kg, i.p., twice/week) for 4 weeks. While administration of clodronate did not have an effect in SSWT rats, clodronate significantly depleted macrophages in the kidneys of SSLepRmutant rats compared to vehicle‐treated rats without altering the monocyte population (8±4 versus 33±10 % gated, respectively; p<0.05 vs. vehicle). At baseline, protein excretion was significantly higher in SSLepRmutant rats compared to SSWT rats (70±14 vs. 10±3 mg/day, respectively; p<0.05 vs. SSWT) that further increased over the course of the study (335±82 vs. 45±14 mg/day, respectively; p<0.05 vs. SSWT). Clodronate had no effect on the progression of proteinuria or renal histopathology in SSWT rats. After 2 weeks of treatment with clodronate, protein excretion was markedly reduced in the SSLepRmutant strain (86±36 vs. 200±35 mg/day; p<0.05 vs. vehicle). However, after 4 weeks of treatment, the effect of clodronate was no longer observed in the SSLepRmutant strain (270±90 vs. 335±82 mg/day). The kidneys from SSLepRmutant rats displayed glomerular injury with increased mesangial expansion and renal fibrosis versus SSWT rats. Interestingly, treatment with clodronate had no effect on glomerular injury but significantly decreased renal fibrosis in the SSLepRmutant strain compared to their vehicle‐treated obese counterparts. Overall, these data indicate that the early development of renal injury in the SSLepRmutant strain is associated with macrophage infiltration, which can be blunted by chronic macrophage depletion.

Support or Funding Information

This study was supported by GM104357 and DK109133.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.