bluuebunny/crossref_metadata_2025 huggingface dataset:
Persistent HIF‐1 Activation by Long‐Term Intermittent Hypoxia
Authors: Nanduri Jayasri , Wang Ning , Khan Shakil A , Semenza Gregg L , Prabhakar Nanduri R
Published: June 2021

Long‐term (30 days) exposure of rats to intermittent hypoxia (LT‐IH) results in persistent elevation of reactive oxygen species levels in central and peripheral nervous system (J. Nanduri et al., J. Physiol.2016,2018). HIF‐ regulates pro‐oxidant enzyme (POE) genes during IH. Present study examined the effect of LT‐IH on HIF‐1α protein and NADPH oxidase (Nox) 4 mRNA abundances in the carotid body (CB) and adrenal medullae (AM) of LT‐IH treated rats. CB and AM of LT‐IH treated rats exhibited robust increase in HIF‐1α protein, Nox4 mRNA and ROS levels. Remarkably, these effects persisted after recovery in room air for 30d. The effects of LT‐IH on HIF‐1α, Nox 4 mRNA, and ROS abundance were absent in rats treated with digoxin, an inhibitor of HIF‐1α during LT‐IH as well as in mice with partial deficiency of HIF‐1a (HIF‐1α heterozygote mice). These findings suggest that HIF‐1 dependent upregulation of POE genes contributes to LT‐IH induced persistent oxidative stress in the CB and AM.

Support or Funding Information

Supported by NIH‐ HL‐90554.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Effects of Continuous versus Intermittent Dietary Fat Intake on Markers of Serotonin Signaling in the Circumvallate Papillae of the Tongue
Authors: Primeaux Stefany , El‐Desoky Dalia
Published: June 2021

Taste is a primary sensory modality that guides organisms in their determination of food preferences. Previous studies suggest that orosensory fat sensing may contribute to the intake and preference of dietary fat leading to the development of obesity. CD36 is the proposed fat taste receptor on the circumvallate papillae (CV) of the tongue and mediates fat sensing. Fat signaling via CD36 on the CV involves the regulation of serotonin (5‐HT) in taste receptor cells and activation of gustatory afferent nerves. The aim of these experiments was to investigate the effects of dietary fat intake on 5‐HT signaling in the CV using two feeding paradigms: continuous access to high fat diet (HFD) vs. limited access to dietary fat. We hypothesized that consumption of dietary fat, in specific patterns, contributes to the decreased sensitivity (i.e. hyposensitivity) of fat sensing through 5‐HT signaling dysregulation. The first experiment investigated the effects of continuous access to either a HFD or a low fat diet (LFD) for 1, 3 or 14 days on 5‐HT signaling in the CV. The second experiment investigated the effects of limited daily and intermittent access to dietary fat on 5‐HT signaling in the CV. Body weight and kilocalorie intake were assessed. In the CV, expression levels of CD36, 5‐HT1A, SERT, TPH1, and TPH2 mRNA were determined by Real Time PCR. Continuous HFD consumption for 14 days increased CD36, 5‐HT1A and SERT mRNA expression on the CV, suggesting reduced lingual fat sensing via decreased gustatory signaling from taste receptor cells. Limited, daily access to dietary fat increased CD36 and TPH1 mRNA expression on the CV suggesting enhanced lingual fat sensing compared to chow fed controls. Limited, intermittent access to dietary led to an escalation of fat intake without significant alterations in lingual expression of 5‐HT signaling. These data suggest that different patterns of fat consumption differentially affect 5‐HT signaling in the CV and that continuous access to HFD may lead to decreased orosensory fat perception by altering 5‐HT signaling in taste receptors cells.

Support or Funding Information

LSUHSC to SDP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Early Postnatal Intermittent Hypoxia Increases Rat Microglial Anti‐inflammatory Gene expression, Effects that Are Sex‐specific and Last into Adulthood
Authors: Kiernan Elizabeth A , Wang Tao A , Watters Jyoti J
Published: June 2021

Chronic intermittent hypoxia (IH), a hallmark of sleep‐disordered breathing (SDB), is prevalent in pediatric and pre‐term infant populations, and is often comorbid with infection. IH increases reactive oxygen species formation, causes neuronal apoptosis, promotes peripheral and CNS inflammation, and results in long‐term neurocognitive deficits in males. However, in the developing brain, little is known about how IH impacts the acute or long‐term inflammatory activities of microglia (CNS resident innate immune cells), or whether these responses differ by sex. Since inflammation impacts cognitive development, we tested the hypothesis that early life IH exposure will sex‐dependently alter microglial inflammatory gene responses and that these effects would last into adulthood. We exposed rat pups to 8 nights of IH beginning on the first postnatal day (P1) and assessed microglial responses to a peripheral lipopolysaccharide (LPS 1mg/kg, 3 hrs) challenge either the day after the last IH exposure (P9), or 6 weeks later (adult). Using RNA sequencing and qRT‐PCR, we first found that IH increased basal expression levels of the anti‐inflammatory cytokines interleukin‐4 (IL‐4) and interferon‐beta (IFNβ) in P9 microglia of both sexes. As expected, LPS‐stimulated inflammatory cytokine expression was decreased after postnatal IH, suggesting that IH may acutely skew neonatal microglia of both sexes towards an anti‐inflammatory phenotype. At P9, there were also sexual dimorphisms in non‐inflammatory microglial genes, both in basal and LPS‐stimulated conditions. For example, multiple genes involved in impaired cognition were upregulated in male microglia, potentially playing a role in the SDB‐induced cognitive impairments that are more prominent in males. Motif analyses to detect the transcription factors that may be driving these male‐specific differences in cognitive genes surprisingly identified the androgen receptor as the top transcriptional driver. Lastly, in adults exposed to neonatal IH, enhanced basal anti‐inflammatory cytokine gene expression was evident only in male microglia, even though their inflammatory gene responses to LPS did not appear to differ at this later time. Ongoing studies are investigating the consequences of persistent increases in anti‐inflammatory cytokines in male microglia on cognitive outcomes since they are known to alter neuroplasticity.

Support or Funding Information

Supported by NIH R01 NS085226 and F31 NS100229

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prenatal Exposure to Bisphenol A and Diethylhexyl Phthalate Induces Hyperprolactinemia in Female offspring
Authors: Al Mansi Maryam H , MohanKumar Sheba M. J. , Mohankumar Puliyur
Published: June 2021

Exposure to endocrine disrupting chemicals (EDC) such as Bisphenol A (BPA) and Diethylhexyl Phthalate (DEHP) are widespread affecting people of all ages. Pregnant women who are exposed to these chemicals through the use of personal care products and plastics invariably expose their fetus to these chemicals as well. Female offspring are then exposed to endogenous estrogens from menarche till menopause and usually, in their prime years, they are also exposed to estrogens through oral contraceptives. This places them at increased risk of developing hyperprolactinemia which causes infertility. In order to mimic real‐life exposures, we developed the “dual exposure paradigm” where we administered saline, 5μg/Kg BW of BPA, 7.5mg/kg BW of DEHP or a combination of BPA+DEHP to pregnant Sprague Dawley rats from day 6–21 of gestation. Female offspring were sham implanted or implanted with slow release estradiol‐17β (E2) pellets for 90 days when they were 3 months old. At the end of E2 treatment, animals were sacrificed, and the brain was removed, frozen and sectioned. We hypothesized that the dual exposure to EDCs and E2 would decrease dopamine (DA) levels in the median eminence of the hypothalamus to increase prolactin levels. The median eminence was microdissected from the brains and DA levels were measured using HPLC‐EC. Serum prolactin levels were assessed by EIA. DA levels in E2‐treated control rats were significantly reduced compared to sham‐implanted rats (p<0.001). Prenatal exposure to EDCs produced a modest reduction in DA levels in sham implanted rats (p<0.05) but decreased further with E2 treatment (p<0.001). Prolactin levels in control animals were significantly higher in E2 implanted rats compared to sham(p<0.05). Prolactin levels in EDC‐exposed rats were variable and were higher than normal in both sham and E2 treated animals. These results indicate that dual exposure to EDC and E2 is able to affect TIDA activity and induce hyperprolactinemia in female offspring. This could have implications in the development of mammary and pituitary tumors.

Support or Funding Information

Supported by UGA research foundation and startup funds.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin‐dependent Decrease of Excitatory Neurotransmission in Preautonomic PVN Neurons Is Reduced in Diet‐induced Obese Mice
Authors: Molinas Adrien Jacques Raymond , Desmoulins Lucie Delphine , Gao Hong , Zsombok Andrea
Published: June 2021

The liver plays a crucial role in the maintenance of glucose homeostasis and evidence supports a governing role for the autonomic nerves in regulating hepatic functions. Activation of hepatic sympathetic nerves increases hepatic glucose production and glycogenolysis; while activation of parasympathetic nerves decreases glucose production and promotes glucose storage. Moreover, excessive hepatic glucose production is a major contributor to hyperglycemia; however, the neural mechanisms underlying the elevated glucose production remain to be determined. In this study, we tested the hypotheses that 1) insulin decreases excitatory neurotransmission in liver‐related neurons in the paraventricular nucleus (PVN), and 2) the insulin‐dependent decrease of excitatory neurotransmission is reduced in pre‐sympathetic PVN neurons in diet‐induced obese (DIO) mice. First, liver‐related PVN neurons were identified with a retrograde, trans‐synaptic viral tracer and whole‐cell patch‐clamp recordings were conducted in male C57BL/6J mice. Bath application of insulin (1μM) decreased the frequency of miniature excitatory post‐synaptic currents (mEPSCs) in liver‐related PVN neurons (1.4±0.3 Hz vs. 1.0±0.2 Hz, n=6, paired t‐test p=0.002). Then, the effect of insulin was determined on excitatory synaptic currents in pre‐sympathetic PVN neurons in male control and DIO mice. In 16‐week‐old control mice, insulin decreased the frequency of mEPSCs (1.6±0.5 Hz vs. 1.3±0.4 Hz, n=10, paired t‐test, p=0.008); whereas, in age‐matched DIO mice insulin failed to decrease mEPSC frequency (1.8±0.4 Hz vs. 1.8±0.5 Hz, n=8). Next, we used channel rhodopsin‐assisted circuit mapping to identify the origin of inputs to liver related PVN neurons. PVN receives inputs from key nuclei implicated in glucose metabolism, including the dorsomedial, ventromedial and the arcuate nucleus; therefore, a viral construct (rAAV5‐hSyn‐hCHR2(H134R)‐mCherry) was injected to the mediobasal hypothalamus (MBH) and light evoked currents were recorded from liver‐related PVN neurons. Light stimulation of ChR2‐expressing fibers in the PVN revealed that one third of the recorded liver‐related PVN neurons received inputs from the MBH. Together, our data demonstrate that excitatory neurotransmission to liver‐related PVN neurons and to pre‐sympathetic PVN neurons is regulated by insulin and this insulin‐dependent suppression is diminished in DIO mice. We also revealed that a subset of liver‐related PVN neurons receives inputs from the MBH. In summary, our findings suggest synaptic plasticity and altered autonomic circuits at the level of the hypothalamus, which likely contributes to dysfunctional autonomic regulation of the liver in diet‐induced obesity.

Support or Funding Information

NIH R01 DK099598

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute neurosteroid treatment with allopregnanolone enhances dorsal vagal motor neuron inhibition
Authors: Littlejohn Erica , Boychuk Carie
Published: June 2021

Neurons in the dorsal motor nucleus of the vagus (DMV) comprise the preganglionic parasympathetic motor output to much of the viscera and critically contribute to autonomic regulation of energy homeostasis. DMV neuron function is tightly regulated by gamma‐aminobutyric (GABA) input to GABAA receptors, which inhibits neuronal excitability. Extrasypnatically localized GABAA receptors, primarily containing δ‐subunits, respond to ambient GABA, setting the tonic inhibitory tone in DMV neurons. Allopregnanolone is a centrally and peripherally produced neuroactive steroid metabolite which readily crosses the blood brain barrier and is a potent allosteric activator of GABAA receptors. Outside of the DMV, allopregnanolone has been shown to increase inhibitory tonic current in a GABAA‐δ dependent manner. DMV neurons have significant tonic inhibition. These currents are largely mediated by the GABAA receptors containing the δ‐subunit. However, no studies to date have investigated the potential for allopregnanolone to induce changes in GABAergic signaling to DMV neurons. Therefore, we hypothesized that acute allopregnanolone exposure increases GABAA receptor mediated inhibition in DMV neurons through a GABAAR δ‐subunit dependent manner. To test this hypothesis, we incubated brainstem slices in allopregnanolone (100nM) or vehicle containing artificial cerebral spinal fluid (ACSF) for 30 minutes. We then analyzed GABAA receptor mediated inhibition in DMV neurons at 30, 60, and 120 minutes following the completion of this incubation. We examined both modalities of GABAA receptor inhibition, phasic and tonic using the specific GABAA antagonist bicuculline (BIC) and wholecell patch‐clamp electrophysiology. Allopregnanolone treatment increased most measures of inhibition in DMV neuron at 30 minutes, including spontaneous inhibitory postsynaptic current (sIPSC) decay time and BIC‐sensitive tonic current. DMV neurons from slices incubated in either allopregnanolone or control ACSF were probed for choline acetyltransferase and GABAA‐δ subunit expression using immunohistochemistry. Brief exposure to allopregnanolone transiently potentiates GABAA receptor response to ambient GABA. Allopregnanolone may play an important role in dynamically modulating vagal inhibition in models of disease.

Support or Funding Information

American Heart Association Grant# 16SDG26590000 and NIH Grant# 5T32HL007446‐37

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Estrogen Act as a Neuroprotective Agent on In Vitro‐Induced β‐Amyloid Plaques in N38 Hypothalamic Neuronal Cells?
Authors: Yakubov Rakhsim , Almathhur Rayan , Suh Catherina , Yoon Jihyung , Cooperstein Deborah , Chakraborty Sanjoy , Chakraborty Tandra R
Published: June 2021

A key characteristic of Alzheimer's disease (AD) is the deposition of beta‐amyloid fibrils in extracellular plaques, as well as the intracellular accumulation of tau in neurofibrillary tangles in the brain. This study is designed to induce plaques on N38 hypothalamic neuronal cells under in vitro conditions using synthetic beta‐amyloid and subsequently to treat with estrogen as a neuroprotective reagent. The cell viability, toxicity and proliferation were determined using different bioassays like cell count, Congo red, MTT and lactate dehydrogenase assay. Results indicate a significant loss in cell death, amyloidosis, mitochondrial dysfunction and increased cytotoxicity in cells treated with beta‐amyloid and neuroprotection when exposed to estrogen. Further, in order to understand the mechanism by which neuronal death by plaque formation and neuroprotection by estrogen western blotting was performed to determine the changes associated with biomarker Tau protein. Taking together the biochemical and molecular data, the neuroprotective effect of estrogen on beta amyloid plaque formation will be analyzed.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Central Circuits Inhibiting Skeletal Muscle Shivering
Authors: Morrison Shaun F. , da Conceição Ellen Paula Santos
Published: June 2021

Background

Skeletal muscle shivering is the most important source of thermogenesis in the human cold‐defensive maintenance of core body temperature (Tcore) and the elevated Tcore during fever. We have described the thermoregulatory reflex pathway through which skin and core cooling, or injection of prostaglandin E2 into the preoptic area of the hypothalamus, leads to activation of skeletal muscle shivering (J Physiol 589.14: 3641–3658, 2011). Here we describe brain regions containing neurons that can provide an inhibitory regulation of shivering.

Results

EMG activities in masseter, neck and gastrocnemius muscles were recorded in Inactin‐anesthetized rats during cold exposure or injection of PGE2 into the preoptic hypothalamus. Cold‐evoked and febrile shivering EMGs were eliminated by activation of neurons in the ventral part of the lateral preoptic nucleus (vLPO), or by increasing the discharge of neurons in the paraventricular hypothalamus, in the nucleus of the tractus solitarii, or in the ventrolateral medulla. Inhibiting neuronal discharge or blocking glutamate receptors in the vLPO elicited a sustained increase in shivering EMGs in warm rats.

Conclusion

These results indicate the strong, tonic inhibitory influence of neurons in the vLPO on skeletal muscle shivering, likely through a GABAergic input to shivering premotor neurons in the rostral raphe pallidus. The discovery of the vLPO and other neuronal populations capable of inhibiting shivering supports the potential for the therapeutic control of shivering in the settings of neurogenic fever, brain injury, or induction of hypothermia.

Support or Funding Information

Supported by NIH grants NS040987 and 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.
Neural Responses of the Cervical Sympathetic Chain to Barorecepter Activation and to Hypoxic Challenge in Sprague‐Dawley Rats
Authors: Muntzel Martin , Lewis Stephen , Hsieh Yee‐Hsee
Published: June 2021

The superior cervical ganglion (SCG) and cervical sympathetic chain (CSC) contain cell bodies that project to the cerebral arteries, and to peripheral structures such as the carotid bodies and upper airways. Despite the importance of these pathways, little is known about how the CSC responds to baroreceptor stimuli and to hypoxic challenges that mimic upper airway obstruction and central apneas. To characterize sensory control of CSC activity, we anesthetized male Sprague‐Dawley rats (275–300g) with isoflurane (2%) and placed bipolar recording electrodes under the CSC at approximately 3 mm caudal to the SCG, and a blood pressure catheter into the left femoral artery (Telemetry Research, model TR56SP). A catheter was placed in the left femoral vein for intravenous (IV) injections. Heart rate, blood pressure (BP), and multi‐fiber CSC neural activity were recorded during 10% oxygen challenge, tracheal occlusion, and during IV drug administration. At the end of the protocol, the rats were euthanized to determine background noise activity. In on‐going studies (n = 4), we found that CSC recordings yielded typical sympathetic responses to phenlyephrine (2.5 μg) and sodium nitroprusside (2.5 μg). For phenyephrine, BP increased by 24 ± 5 mmHg with CSC decreases of 100% baseline to 69 ± 11%. For sodium nitroprusside, BP decreased by 26 ± 3 mmHg with CSC increases of 100% to 111 ± 14%. Ganglionic blockade with hexamethonium (10 mg/kg) decreased BP by 49 ± 2 mmHg and lowered CSC activity by 69 ± 8%. Temporary occlusion of the tracheal airway decreased BP by 16 ± 4 mmHg and increased CSC activity from 100% to 120 ± 17%. Finally, a 1 min 10% oxygen challenge lowered BP by 33 ± 9 mmHg but did not alter CSC activity (100% to 103 ± 5%). These findings indicate that the CSC, just caudal to the SCG, contains approximately 69% post‐ganglionic sympathetic nerve fibers. These nerve fibers respond appropriately to baroreceptor loading and unloading, and show increased activity to temporary upper airway occlusion.

Support or Funding Information

This work was supported in part by NIH award 3OT2OD023680‐01S2 and Galvani Bioelectronics

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cellular Senescence in the Rostral Ventrolateral Medulla (RVLM) – Novel Implications for Obesity‐Induced Sympathoexcitation
Authors: Subramanian Madhan , Csiszar Anna , Ungvari Zoltan , Balasubramanian Priya
Published: June 2021

Oxidative stress in the RVLM, a key brainstem region that regulates sympathetic nerve activity (SNA) and blood pressure has been associated with obesity‐induced hypertension. Chronic oxidative stress is a known stimulus for induction of cellular senescence, a state of irreversible growth arrest in proliferating cells. Senescent cells are also a potential source of neuroinflammation as they secrete cytokines, chemokines and proteases through acquisition of senescence‐associated secretory phenotype (SASP). Whether obesity induces senescence in the brainstem to promote neuroinflammation and increases in SNA is not clear. Hence, we hypothesized that obesity triggers the induction of cellular senescence in the RVLM, which in turn contributes to neuroinflammation and increases in SNA leading to the development of hypertension. To address this hypothesis, 2 months‐old male C57BL/6J mice were fed a high‐fat diet (HFD; 60% fat; n=5) or normal chow diet (Controls;10% fat; n=5) for 16 weeks. At the end of the dietary treatment, brainstem was isolated from the animals for assessment of senescence in the RVLM. Data were analyzed by unpaired students t‐test and expressed as mean ± SE. HFD treatment significantly increased the expression of senescence marker p16ink4a (1.15 ± 0.22 vs 2.17 ± 0.37, fold change, normal diet vs HFD, p<0.05), which is involved in cell cycle arrest. This was accompanied with SASP characterized by increased expression levels of cytokines and chemokines, namely IL‐1β (1.02 ± 0.09 vs 1.35 ± 0.05, p<0.05), MCP1 (1.03 ± 0.13 vs 1.48 ± 0.05, p<0.05) and TNFα (1.04 ± 0.15 vs 1.42 ± 0.09, p<0.05) compared to controls. In addition, the number of cells positive for senescence associated β‐galactosidase activity (SA‐β‐gal) was significantly higher in the RVLM of HFD animals compared to controls. In the next series of experiments, we utilized p16‐senescence reporter mice to investigate obesity‐induced senescence in the RVLM. These mice express red fluorescent protein (RFP) under the control of p16 promoter and hence senescent cells are genetically labeled with RFP expression. HFD treatment in p16‐reporter mice resulted in significant increases in the number of RFP positive cells in the RVLM, further confirming obesity‐induced senescence in the RVLM. Collectively, our results show for the first time, evidence for obesity associated cellular senescence in the RVLM. Future studies will characterize the cellular type and investigate the mechanistic role of senescence 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.
Sympathetic Neurotransmission in Resistance Mesenteric Arteries in Obesity‐Related Hypertension
Authors: Sangsiri Sutheera , Galligan James J
Published: June 2021

Hyperactivity of sympathetic nerves has an important role in the pathogenesis of obesity‐related hypertension. The mechanisms linking obesity to hypertension are unclear. Small mesenteric arteries (MA, <300 mm dia.) are resistance blood vessels that are densely innervated by sympathetic nerves. Nerve varicosities release norepinephrine (NE) and ATP which cause arterial constriction. Impaired control of NE and/or ATP release and clearance would cause hypertension. Alterations in sympathetic nerves supplying MAs contribute to hypertension in several animal models. This study examined whether alterations of sympathetic neurotransmission in the resistance MAs contribute to high blood pressure in high fat‐fed (HF) Dahl salt‐sensitive rats. Sympathetic nerves supplying MAs maintained in vitro were stimulated focally. Electrically‐evoked NE and ATP release were detected by using amperometry with carbon fiber electrodes, and intracellular recording of excitatory junction potentials (EJPs) from arterial smooth muscle cells, respectively. Data were obtained from male and female rats at 10, 17, and 24 weeks of control and HF diet. Peak amplitudes of NE oxidation current and EJPs were assessed. NE oxidation current from all groups were blocked by a Na+ channel blocker, tetrodotoxin (TTX, 300 nM), suggesting that NE release in neurogenic. EJPs from all groups were abolished by TTX, and PPADS (10 μM), a P2X receptor antagonist, indicating that EJPs were neurogenic and purinergic. Frequency (1–30 Hz, 50 pulses) response curves using amperometric measurement of NE release showed similar responses in MAs from control and HF fed male and female rats. EJP frequency (0.5–10Hz, 5 pulses) response curves were also similar in MA from control and HF fed male and female rats. We investigated prejunctional α2‐adrenergic receptor function using the α2 receptor agonist (UK14,304) and antagonist (yohimbine). UK14,304 (0.0001–10 μM) inhibited NE and ATP release equally well and in a concentration‐dependent manner in MAs from control and HF rats. In the presence of 1 μM yohimbine, NE and ATP release was increased equally in MA from all rats. We conclude that adrenergic and purinergic neurotransmission by periarterial sympathetic nerves are not affected in HF Dahl salt sensitive rats. Elevated blood pressure in this animal model is not dependent on altered sympathetic neurotransmission to the mesenteric vasculature.

Support or Funding Information

NIH 2P01HL070687 (For JJG), and Post‐doctoral training scholarship, Faculty of Medicine, Thammasat University, Thailand (For SS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of claudin‐5, ‐2, occludin and PPAR alpha in a model of early diabetic nephropathy
Authors: Rosas Lorena , Rodríguez Rafael , del Carmen Namorado María , Reyes Jose Luis , Cervantes Luz Graciela
Published: June 2021

The WHO estimates that there are more than 422 million people with diabetes in the world, of whom 90% are diagnosed as type 2. Hyperglycemia plays an important role in renal damage, thus diabetic nephropathy is the most common complication of this pathology. Claudins are components of paracellular transport which are determinants to the ion balance in the kidney. Any alteration in the tight junctions carry a pathophysiological renal consequences that have not yet described in diabetic type 2 model. PPARs are nuclear receptors ligand‐dependent and their alpha isoform is activated by fibrates. Pharmacological therapy with fibrates in diabetes had showed to prevent cardiovascular risk, if they have renal protective effects in diabetes and if they are involved in paracellular ion transport, is not well understood. Those actions, could be useful as a target to identify, prevent and treat diabetic nephropathy may contribute to a better understanding of this disease. Type 2 diabetes model that we used was induced to neonatal rats by the intraperitoneal administration of streptozotocin, a chemical that selectively destroys the β cells of the pancreas, diabetics rats showed moderate hyperglycemia since 4th week and at 14‐week, had a decrease in body weight gain, glucose intolerance, lower insulin secretion and an increase in renal mass. We evaluated renal function in this model and we observed that proteinuria was associated with a decrease in claudin‐5 expression in the glomerulus and the increased fractional excretion of Na+ and K+ was related with a decrease of claudin‐2 and occludin in the proximal tubule. In addition, we observed a loss of renal expression of peroxisome proliferator‐activated receptor alpha (PPAR‐α) in both segments of the nephron. The present work provides evidence that type 2 diabetes alters the renal expression of tight junctions proteins such as claudin‐5, claudin‐2 and occludin, as well as PPAR‐α. This is related to the loss of renal function, suggesting that the loss of these proteins is a fundamental part of the early stages of type 2 diabetic nephropathy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Esm‐1 Protects Mice from Glomerular Macrophage Infiltration and Macroalbuminuria in Diabetic Nephropathy
Authors: Zheng Xiaoyi , Higdon Lauren , Sayed Nazish , Liu Chun , Wu Joseph , Maltzman Jonathan , Bhalla Vivek
Published: June 2021

Diabetic nephropathy (DN) is the most common cause of kidney failure in the world, but only a minority of individuals with diabetes develop nephropathy. Extending this observation to mice, different mouse strains are susceptible or resistant to DN. In comparing glomerular transcriptome profiles, we published that DN‐susceptible mice up‐regulate pro‐inflammatory pathways and have diminished expression of endothelial cell‐specific molecule‐1 (Esm‐1) mRNA and protein. Esm‐1 is a secreted glycoprotein that is enriched within kidneys and specifically, in glomeruli. We now test the hypothesis that Esm‐1 over‐expression would inhibit inflammation and rescue DN‐susceptible mice and conversely, Esm‐1 deletion would promote nephropathy in DN‐resistant mice. We first confirmed that Esm‐1 is enriched in glomerular endothelial cells by single cell qPCR. Esm‐1 expression was restricted to Cdh5(+) or CD31(+) cells and absent in cells enriched for podocyte or mesangial cell markers. We demonstrate by flow cytometry that in diabetic mice, endogenous, plasma Esm‐1 inversely correlates with glomerular leukocyte infiltration (R2 = 0.61). Next, by hydrodynamic tail‐vein injection in DN‐susceptible mice, we increased circulating Esm‐1 and demonstrate diminished albumin‐to‐creatinine ratio vs. saline‐injected controls (1.0 ± 0.5 mg/mL vs. 3.6 ± 1.4 mg/mL, p = 0.008). Interestingly, over‐expression of non‐glycosylated Esm‐1 did not decrease albuminuria. In a corollary experiment, genetic deletion of Esm‐1 in DN‐resistant mice, increases urine albumin‐to‐creatinine ratio vs. wild‐type controls (806.5 ± 332.0 ug/mL vs. 1.4 ± 0.29 ug/mL, p < 0.001). In mechanistic studies, we previously published that Esm‐1 decreases leukocyte transmigration across endothelium in vitro. We now demonstrate by flow cytometry that increasing circulating Esm‐1 decreases glomerular macrophage infiltration (0.9 ± 0.5 % vs. 3.1 ± 0.7 % of total single cells, p = 0.002) in diabetic mice in vivo. Neutrophil and lymphocyte infiltration were not significantly different between groups. In conclusion, glycosylated Esm‐1, an endothelial cell‐specific inhibitor of leukocyte infiltration, protects mice from macroalbuminuria, possibly through inhibiting glomerular macrophage infiltration.

Support or Funding Information

Larry L. Hillblom Foundation; Holmgren Family Fund; NIH/NIDDK

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of TLR4 Activation Prevents Renal Autoregulatory Impairment in Ang II Hypertensive Rats
Authors: Freeman Brian , Zhang Shali , Cook Anthony , Inscho Edward W.
Published: June 2021

Autoregulation by afferent arterioles regulates renal blood flow and glomerular filtration rate across widely ranging arterial pressures. Autoregulatory function is impaired in hypertension, perhaps due to release of damage associated molecular patterns that activate renal toll‐like receptor (TLR4). Autoregulatory impairment can promote hypertensive kidney injury. We hypothesize that inhibition of TLR4 activation preserves afferent arteriole autoregulatory function in Angiotensin II (AngII) hypertension.

Three groups (n=4/group) of male Sprague‐Dawley rats were implanted with osmotic minipumps set to deliver: saline (0.9% NaCl) or AngII (60ng/min). Group three received AngII+anti‐TLR4 Ab (3 μg/ml/kg/day, i.p.) for 14 days. Systolic blood pressure (SBP) was assessed (days 0, 3, 7 and 13). Kidneys were harvested on day 14 for juxtamedullary nephron assessment of afferent arteriole autoregulatory function.

SBP increased in the AngII and AngII+Anti‐TLR4 Ab groups compared to saline controls over the 14 days (P<0.05), averaging 198 ± 11, 200 ± 8 and 135 ± 3 mmHg, respectively. Baseline arteriole diameters (100 mmHg) averaged 14 ± 0.8, 15.6 ± 1.7, and 13 ± 0.3 microns for control, AngII and AngII+Anti‐TLR4 Ab, respectively. Perfusion pressure was decreased to 65 mmHg and increased to 170 mmHg in 15 mmHg increments. At 170 mmHg, arteriole diameters decreased by 31 ± 6% and 33 ± 8% of baseline in the control and Ang II+Anti‐TLR4 Ab groups whereas the AngII group had a decline of 11.7 ± 3.7%.

These data establish that autoregulatory function is Impaired in AngII hypertensive rats while it is preserved by anti‐TLR4 Ab‐treatment. These findings implicate chronic TLR4 activation as causative in the hypertensive decline in autoregulatory capability.

Support or Funding Information

Funded by NHLBI; 1R25 HL115473‐01 and DK044628‐20.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute SGLT2 Inhibition and the Pressure Natriuresis Response in Rats with Type 1 Diabetes
Authors: Bailey Matthew A , Costello Hannah M , Stewart Kevin , Binnie David , Culshaw Geoffrey C , Webb David J
Published: June 2021

Increasing blood pressure (BP) induces natriuresis due to downregulation of sodium transport in the proximal tubule. This pressure natriuresis (PN) response influences long‐term BP. Type‐1 diabetic (T1DM) patients show enhanced tubular sodium reabsorption and high BP. We have found that tubular sodium reabsorption is not modulated by BP in T1DM rats. The molecular mechanism of impaired PN is unknown but the sodium‐glucose cotransporter SGLT2 is a plausible candidate and inhibitors acutely induce diuresis in T1DM rats. This study tested the hypothesis that SGLT2 inhibition with dapagliflozin would improve the acute PN response in T1DM rats.

T1DM was induced in male Sprague‐Dawley rats by streptozotocin (30–45mg/kg IP; plasma glucose 13.3–>35mmol). After 3 weeks rats were anaesthetised with Inactin (120mg/kg, IP). The jugular vein was cannulated and isotonic saline (containing FITC‐inulin and p‐amino hippurate for measurement of glomerular filtration rate (GFR) and renal plasma flow (RPF)) was infused. Rats were randomly allocated to receive either dapagliflozin (50mg/kg; n=7) or saline vehicle (n=7) and after an equilibration period and baseline urine collection, BP was increased by sequential arterial ligation of first the coeliac and mesenteric arteries and then the distal aorta and the natriuretic response was measured. Experiments were performed under a single blind and rats were euthanised by an overdose of anaesthetic. Data are mean +SD and comparisons were made by 2‐way ANOVA. GFR and RPF were not different between treatment groups and were unaffected by the experimentally‐induced rise in BP, consistent with autoregulation. In vehicle‐treated rats, incremental rises in BP increased urine flow rate from a baseline of 6 ± 3 to a peak of 51 ± 28μl/min/gkw (P<0.01), sodium excretion from 1.7 ± 0.5 to 16.7 ± 1.4 μmol/min/gkw (P<0.01) and fractional sodium excretion from 0.2 ± 7.6 to 5.4 ± 4.5% (P<0.01). The dapaglifozin‐treated rats responded to increasing pressure with a diuresis and natriuresis but the peak absolute (3.8 ± 4.1μmol/min/gkw; P=0.010) and fractional (15.2 ± 8.0%; P=0.018) sodium excretion were significantly lower than in the vehicle‐ group. Dapagliflozin increased urinary glucose excretion from 0.1 ± 0.2 to 0.4 ± 0.2μmol/min/gkw: glucose excretion was not modified by increasing BP in either group. Although dapagliflozin increased urine flow and glucose excretion at steady‐state BP, it did not enhance the acute PN response in T1DM rats. Thus, impaired PN in T1DM does not reflect increased SGLT2 activity.

Support or Funding Information

British Heart Foundation studentship FS/16/54/32730 & Kidney Research UK Project Grant RP2/2014

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CACNB2 expression is positively associated to the Ras/MAPK pathway in vitro and in an experimental model of hypertension
Authors: Pandian Vishnuprabu Durairaj , Giovannucci David R , Vazquez Guillermo , Sivarajan Kumarasamy
Published: June 2021

Several independent genome‐wide association studies highlighted the strong association of voltage‐dependent L‐type calcium channel subunit beta‐2 (CACNB2) gene with hypertension. One such study showed that hypertensive individuals who carry a promoter polymorphism (rs2357928) in CACNB2 exhibited adverse effects during treatment with calcium channel blocker (CCB) ‐ verapamil but responded well to treatment with the β‐blocker‐ atenolol. However, the exact molecular signature of CACNB2 during the pathophysiology of hypertension is currently remains to be explored. In the clinical setting, CCBs are widely used to treat hypertensive individuals, and the inhibition of L‐type Ca2+ channels with CCBs significantly increases the glomerular filtration rate and renal blood flow. Hence, the goal of this present study is to understand the molecular signaling of CACNB2 during hypertension via in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt‐Sensitive (SS) rats. The over‐expression of CACNB2 in HEK293 cells increased cell proliferation and upregulated the Ras‐MAPK pathway significantly when compared to the controls. These results were consistent with a recent study showing that overexpression of CACNB2, activate Ras/ERK/CREB pathways in vitro. Additionally, the ER stress markers IRE‐1α and PERK were upregulated by CACNB2 over expression compared to the control cells. In contrast, treatment with the L‐type CCB‐ nifidepine in CACNB2 overexpressed cells did not abolish upregulation of Ras‐MAPK and ER stress pathway genes. Similarly, we observed an increase in apoptotic signaling exemplified by the downregulation of Bcl‐2 and upregulation of Caspase 7 and Caspase 9, which were also insensitive to nifidepine treatment. Correspondingly, we investigated whether CACNB2 levels were altered using the SS rats under low salt (0.3% NaCl) and high (2% NaCl) diet fed conditions. We observed higher CACNB2 levels in high salt fed SS rat kidneys compared to SS rats maintained on low salt diet (n=4, p<0.05). In addition, our gene expression studies showed that Mapk1 and Mapk4 levels were upregulated in the kidneys of SS rats under high salt diet (n=4, p<0.05). In summary, our study reveals that CACNB2 significantly associated with the activation of Ras‐MAPK pathway in SS rats. In summary, understanding the regulation of CACNB2 and associated Ras‐MAPK pathway could help in expanding the knowledge of molecular mechanism of hypertension and may help in identifying novel drug targets.

Support or Funding Information

Funding support for this study was met from the American Heart Association grant 16SDG27700030 (to S. Kumarasamy) and funding from UT‐COMLS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α1‐Adrenoceptor Antagonism Attenuates the Development and Progression of Dahl Salt Sensitive Rat Hypertension by Modulating NCC Activity
Authors: Puleo Franco , Frame Alissa A , Moreira Jesse D , Faudoa Elizabeth , Wainford Richard D
Published: June 2021

Background

Dietary sodium intake plays a significant role in blood pressure (BP) regulation, as increased sodium retention is a well‐established precursor of hypertension risk. In particular, increased dietary sodium intake evokes increased BP in individuals that demonstrate the salt sensitivity of BP and promotes the development of salt sensitive hypertension (SSH). Recent studies suggest that excessive Sympathetic Nervous System (SNS) release of norepinephrine (NE) has the ability to stimulate the sodium chloride cotransporter (NCC) via synergistic α1 and β adrenergic receptor pathways to drive SSH.

Hypothesis

Endogenous SNS release of NE activates an α1‐adrenoceptor pathway to mediate increases in NCC activity that drive the development and maintenance of Dahl rat SSH.

Methods

Groups of naïve Dahl Salt Sensitive (DSS) rats, DSS rats receiving a continuous subcutaneous (s.c.) terazosin (10 mg/kg/day), or s.c. propranolol (10 mg/kg/day) infusion were fed a normal salt (NS, 0.6% NaCl) or high salt (HS, 4% NaCl) diet for 21 days respectively. On day 21 basal MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) were assessed. NCC expression and phosphoNCC levels were assayed via immunoblotting (N=6/gp). Additional groups of DSS rats were fed a 42 day HS diet. On day 21, rats were given s.c. saline/DMSO or s.c. terazosin/DMSO (10 mg/kg/day). On day 42, measurements were taken as described previously (N=4–6/gp).

Results

DSS rats develop SSH when fed a 21 day HS diet and show increased NCC activity compared to rats on a NS diet. NCC expression and phospho levels are similar in both the diets. Use of an α1‐adrenoceptor antagonist, terazosin (confirmed pharmacologically) attenuates SSH and reduces NCC activity and expression as well as phosphoNCC levels in rats on a HS diet. Use of a β‐adrenoceptor antagonist, propranolol fails to attenuate SSH or reduce NCC activity. Significantly, α1 antagonism has a trend to reduce BP and NCC activity in DSS rats with established HTN.

Conclusion

Our data reveals that SNS release of NE modulates NCC activity via an α1‐adrenoceptor pathway to evoke dietary evoked SSH. Significantly, α1‐adreoceptor antagonism can attenuate the development of SSH by evoking downregulation of NCC activity and expression. In a model of established HTN, α1‐adreoceptor antagonism reduces NCC activity and BP. Taken together, these findings suggest that SNS release of NE activates an α1‐adreoceptor pathway that stimulates NCC activity to drive the development and maintenance of SSH.

Support or Funding Information

This work was funded by NIH R56 AG057687, R01 HL139867, R01HL141406, AHA17GRNT33670023 to RDW, and F31DK116501 to AAF.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myogenic Constriction is not Dependent on Endothelium in Arcuate and Mesenteric Arteries of Normotensive and Hypertensive Rats
Authors: Nademi Samera , Lu Chao , Dickhout Jeffrey G.
Published: June 2021

Background

Myogenic response (MR) is defined as contraction of small arteries in response to increased intraluminal pressure. Impaired MR is associated with hypertension and Chronic Kidney Disease (CKD). MR has been shown to be regulated by endothelium (ENDO) through production of vasodilators and vasoconstrictors which are differentially produced in different vascular beds. Despite this evidence, there are conflicting literature reports on the dependency of MR on ENDO. This dependency was never investigated in arcuate and mesenteric arteries of normotensive and hypertensive rats. We hypothesized that MR is dependent on ENDO and removing ENDO abolishes MR in arcuate and mesenteric arteries of Wistar Kyoto (WKY, normotensive) and Spontaneously Hypertensive Rats (SHR, hypertensive) rats.

Methods

Arteries were mounted in a pressure myograph chamber (PMC) filled with oxygenated Hank Balance Salt Solution (HBSS) to create a blind sac and allowed 30 min to equilibrate. Intact vascular smooth muscle cell (VSMC) and ENDO layers were evaluated by addition of a pre‐determined median effective dose of phenylephrine (0.2 μM) and carbachol (10 μM) respectively. MR was measured in response to step‐wise increases in intraluminal pressure from 80 to 180 mmHg (20 mmHg increments, 5 min each) (E‐intact MR). The arteries were then transferred into a petri dish containing human hair and moved through the length of the hair 5 times to remove ENDO (Osol et al., 1989). The vessels were re‐mounted, HBSS was flushed through the arteries for 5 mins, and blind sac was re‐created. Phenylephrine and carbachol were added and MR was re‐measured (E‐removed MR). Physical absence of ENDO was further assessed by Scanning Electron Microscopy (SEM).

Results

All arteries contracted to phenylephrine before and after ENDO removal and did not relax significantly to carbachol after ENDO removal. SEM micrographs showed complete physical absence of the ENDO. MC in E‐removed vessels were not significantly different from MC in E‐intact in mesenteric and arcuate arteries of WKY and SHR rats.

Conclusions

Our study validated an ENDO removal method that was described by Osol, et al. in 1989. Additionally, MR was shown to be independent of ENDO in mesenteric and arcuate arteries of WKY and SHR rats.

Support or Funding Information

This work was supported by Canadian Institutes of Health Research grants to Dr. Jeffrey G. Dickhout (PJT148499).

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