bluuebunny/crossref_metadata_2025 huggingface dataset:
Angiotensin II‐induced hypertension results in an oligioclonal T cell receptor expansion in mouse kidney
Authors: Trott Daniel Wayne , Li Chung‐I , Shyr Yu , Harrison David Glenn
Published: June 2021

The purpose of this investigation was to determine if T cells that infiltrate the kidney and promote hypertension exhibit a mono‐ or polyclonal T cell receptor (TCR) phenotype. We produced hypertension in C57Bl/6 mice by infusion of angiotensin II (2 week, 490 ng/kg/min) and used vehicle infused mice as controls. PCR was employed to amplify the 24 TCR Vβ families in CD4 and CD8 cells isolated from the kidney. PCR products were subjected to fragment analysis to determine TCR Vβ transcript length. CD8 TCR length profiles for Vβ 5.1, 8.1, 8.3, and 17 and CD4 Vβ 8.2, 14 and 16 were significantly different (Dirchelet distribution, p ≤ 0.05) with angiotensin II. To determine if mice with an altered TCR phenotype are protected against hypertension, OT1 mice, which have CD8 cells with a transgenic TCR specific for ovaalbumin, were infused with angiotensin II or vehicle. Blood pressure was measured by telemetery. After 2 weeks OT1 mice infused with angiotensin II exhibited increased systolic blood pressure compared to controls (171 ± 10 vs. 117 ± 15 mmHg, mean ± SEM). Analysis of kidney CD8 T cells revealed that OT1 mice possessed a dominant a Vβ 5 TCR specific to ovaalbumin as well as all other Vβ families with the exception of Vβ 17. Overall, these data suggest that angiotensin II infusion results in oligioclonal T cell expansion and that these cells may mediate hypertension.
Metabolic reprogramming and miRNA expression profile in liver of prenatally dexamethasone treated rats
Authors: Pantaleão Lucas Carminatti , Santos Laila Romagueira Bichara , Sakamoto Luciano , Lellis‐Santos Camilo , Gomes Patrícia Rodrigues Lourenço , Rodrigues Sandra Campos , Latorraca Márcia Queiroz , Bordin Silvana
Published: June 2021

We studied the effect of intrauterine exposure to glucocorticoid excess on hepatic miRNA expression and on miRNA target genes expression. Pregnant Wistar rats received dexamethasone (DEX) during the last six days of pregnancy. miRNA profile was evaluated on newborn rat livers. Male offspring was subjected to insulin, glucose and pyruvate tolerance tests (ITT, GTT and PTT) at weaning and at 90th day of life. miRNA potentially involved in insulin signaling or hepatic development were validated by qPCR. Protein content of key target genes were also studied in newborn livers. DEX reduced birth weight by 38 % (P<0.05). Animals born from DEX‐treated dams exhibited glucose intolerance (P<0.05), and increased whole‐body gluconeogenesis at weaning (P<0.05). Dicer protein content was higher in liver of newborn rats from DEX‐treated dams [2.6 fold (P<0.05)], and correlated with upregulation in global miRNA expression [46 miRNA]. Liver of newborn rats from DEX‐treated dams had increased expression of miR‐322 cluster and miR‐29a* (P<0.05). Concomitantly, they showed lower levels of AKT3 and insulin receptor in liver (P<0.05), both putative targets of miR‐322 cluster. Therefore, we propose a link between these miRNA and hepatic glucose metabolism reprogramming through regulation of key insulin signaling proteins. This is the first study relating miRNA and hepatic metabolic reprogramming. Support: FAPESP, CNPq, FAPEMAT
Obesity and insulin resistance in adolescents: impact of renin angiotensin system
Authors: Fernandes Fernanda Barrinha , Fernandes Adriana Barrinha , da Silva Febba Andreia Cristina , de Souza Vitale Maria Sylvia , Jung Flavia Fernandes , Casarini Dulce Elena
Published: June 2021

The aim of this study was to evaluate the relationship between obesity and plasma Angiotensin levels [Ang I, Ang II, Ang (1–7)], Bradykinin (BK) and the presence of insulin resistance (IR). Subjects (n=104) were divided according with HOMA IR and classified as presence of RI (RI+) and absence of insulin resistance (IR−). The Ang I, Ang II, Ang (1–7), BK and Vasopressin (VP) plasmatic levels had been measured by HPLC. The RI+ group had more values of skinfolds, WHR, BMI, leucocytes, uric acid, glucose, insulin, VP, triglycerides, C reactive protein, Des Arg9‐BK, Ang I and lower plasmatic levels of Ang (1–7) and BK. Our results showed a reduction of Ang (1–7) and increased of Ang I associated with weight gain and IR in adolescents suggesting a modulation of Angiotensin Converting Enzyme 2 and Neutral Endopeptidase. Ang (1–7) and BK low levels may contribute to the development of hypertension and inflammatory conditions in obese adolescents with alterations of glucose and insulin metabolism, since these can resulting in cardiovascular target organ damage such as the heart, the vascular wall and the kidney. (Supported by Fapesp and CNPq)
Potential sex differences in the vascular endothelial growth factor (VEGF) signaling pathway in the kidney
Authors: Rouch Al , Fan Liming
Published: June 2021

Sex differences in kidney function and disease are well documented. The VEGF signaling pathway plays important roles in the kidney. The purpose of this study was to test for sex differences in the VEGF pathway in the kidney. Using quantitative real‐time PCR with the RT2 ProfilerTM PCR VEGF Array from SABiosciencesTM, we compared the relative expression of 84 genes in female and male renal tissue. CD‐1 mice, 2–3 months old, consuming normal chow were killed, the left kidney removed, and RNA was extracted from a 10 mg cortical tissue section via the MELTTM nucleotide isolation kit from Ambion®. cDNA was synthesized via the first‐strand cDNA synthesis kit from SABiosciencesTM. Data from four arrays per group were averaged (1 array per mouse). Results demonstrate that expression of most VEGF signaling genes were elevated in the female kidney with 39 having ≥ 4.0 fold‐difference. Notable examples included VEGFa & VEGFb, MAP kinases Mapk1 & Mapk3, and the Akt signals Akt1 & Akt3. These preliminary findings suggest important sex differences in the VEGF pathway. Studies will further test these signals under conditions that stress the kidney, i.e., high protein and high salt diets.
ADP‐ribosyl cyclase (CD38) and superoxide mediate renal vasoconstriction due to thromboxane receptor (TpR) activation
Authors: Moss Nicholas , Kopple Tayler , Arendshorst William
Published: June 2021

The calcium mobilizing second messenger, cyclic ADP ribose (cADPR, the product of ADP‐ribosyl cyclase) is an important component in the renal vasoconstrictor response to G‐protein coupled receptor stimulation. The role of cADPR in TpR‐mediated renal vasoconstriction was tested with the stable thromboxane mimetic U‐46619 in wild type (WT) and CD38 −/− mice. Arterial blood pressure (ABP) and renal blood flow (RBF) were not different between WT and CD38−/− mice during basal conditions. Bolus injections of U‐46619 (10–50ng in 10μl) caused a dose‐dependent decrease in RBF and increase in ABP in both strains but to a lesser extent for both in CD38−/− mice. Compared to control, 50 ng of U‐46619 reduced RBF by 37±4% and 22±3% and increased ABP by 36±6% and 22±6% in WT and CD38−/− mice, respectively (p<0.05 n=7). Intravenous infusion of Tempol to scavenge superoxide reduced renal vasoconstrictor response to U‐46619 by 40–45% in both strains of mice. The data show that the renal vasoconstriction produced by TpR stimulation is mediated by ADP‐ribosyl cyclase activity and depends upon superoxide for full expression. The comparable inhibition of the RBF response by Tempol in both strains of mice indicates that the vasoconstrictor action of superoxide is independent of CD38 and cannot explain the attenuated response to TpR activation in CD38−/− mice. Funded by NIH grant RO1 HL‐02334
Sequential assessment of glomerular filtration rate (GFR) during development of hypertension in freely moving Dahl salt‐sensitive rats
Authors: Cowley Allen W. , Ryan Robert P , Kurth Terry , Schock‐Kusch Daniel , Gretz Norbert
Published: June 2021

A new method for sequential measurement of GFR was applied during the development of hypertension in the conscious Dahl salt‐sensitive (SS) rat. Utilizing a miniaturized device (Kidney Int. 79: 1254, 2011), disappearance curves of fluorescein isothiocyanate (FITC)‐sinistrin were determined by transcutaneous excitation and real time detection of the emitted light through the skin. Two groups of SS rats with implanted femoral venous and carotid catheters were studied before (LS; 0.4% NaCl) and on days 2,5,7,14 and 21 after switching to 4.0% (HS) diet (n=9) or maintaining on LS (n=6). SS rats maintained on LS exhibited no significant changes in mean arterial pressure (MAP; telemetry) or GFR while in the group fed HS, GFR decreased progressively and significantly from 1.52±0.07 (T1/2=21±0.9 min) to 1.1±0.04 ml/min/100g bwgt (28.4±1.1 min.) by d21. MAP increased during this period from 132±2 to 162±7 mmHg in the HS group. In contrast, creatinine clearance (urine and plasma determined by MS), the traditional index of GFR, did not change in either group. In conclusion, this novel non‐invasive method using sinistrin provides a sensitive approach for repetitive tracking of GFR in freely moving rats during disease progression and is more sensitive than standard measurements of creatinine clearance. The data clearly show progressive reduction of GFR in SS rats as salt‐induced hypertension developed.
A Novel Small‐molecule Urea Transporter Inhibitor Acts as a Potential Diuretic
Authors: Yang Baoxue , Li Fei , Lei Tianluo , Zhou Hong
Published: June 2021

Urea transporters (UT) are a family of membrane channel proteins that are specifically permeable to urea. They play an important role in intrarenal urea recycling and in urinary concentrating mechanism. Using an erythrocyte osmotic lysis assay, we screened a small molecule library for inhibitors of UT‐facilitated urea transport. A novel class of UT‐B inhibitors were identified, named as thienoquinolin,one of which, PU‐14, had potent inhibition activity on human, rabbit, rat and mouse UT‐B. IC50 of PU‐14 on rat UT‐B mediated urea transport was about 0.8 μM. PU‐14 did not affect urea transport in mouse erythrocytes lacking UT‐B. PU‐14 also inhibited UT‐A‐type urea transporters, with 36 % inhibition at 4 μM. PU‐14 showed no significant cellular toxicity at concentrations up to its solubility limit of 80 μM. Subcutaneous delivery of PU‐14 (at 12.5, 50 and 100 mg/kg) to rats caused an increase of urine output and a decrease of the urine urea concentration and osmolalities without electrolyte disturbances and liver and renal damages, which suggests that PU‐14 has a diuretic effect by urea‐selective diuresis. These results indicate that PU‐14 or its analogues might be developed as a new diuretic to increase renal fluid clearance in diseases associated with water retention without causing electrolyte imbalance, as well as a tool drug to establish ‘chemical knockout□ animal models to study the physiological functions of UTs.
Outer medullary Na‐K‐ATPase activity and protein expression levels of the highly concentrating kangaroo rat exceed those of Sprague‐Dawley or Munich‐Wistar rats
Authors: Armstrong Tamara M , Shahidullah Mohammad , Pannabecker Thomas L
Published: June 2021

We hypothesize that a greater degree of Na active reabsorption in the thick ascending limb (TAL) of the kangaroo rat relative to that of the common laboratory rat contributes to the ability of the kangaroo rat to produce a more highly concentrated urine. For semiquantitative immunoblotting, outer medulla was dissected free, homogenized, centrifuged at 1100 × g (15 min) and the supernatant centrifuged at 150,000 × g (45 min). The membrane fraction was blotted with an antibody raised against Na‐K‐ATPase alpha subunit (Sigma). For measurement of Na‐K‐ATPase activity, 3 pooled samples, each consisting of tissue from 2–4 outer medullas, were homogenized, centrifuged, and the pellets resuspended in buffer with or without ouabain. ATP hydrolysis was determined by measuring release of inorganic phosphate. Na‐K‐ATPase activity (nmoles ATP hydrolysed/mg protein/30 min) is the difference between ATP hydrolysis in presence and absence of ouabain. Total ATPase activity was 905 ± 122 for kangaroo rat and 721 ± 111 for Munich‐Wistar rat (Mean ± SE). Na‐K‐ATPase activity was greater in the kangaroo rat (204 ± 65) than in Munich‐Wistar rat (79 ± 62). Na‐K‐ATPase protein levels were ~3‐fold higher in the kangaroo rat relative to Sprague‐Dawley rat. We conclude that a greater degree of Na active reabsorption occurs in the TAL of the kangaroo rat relative to that of the Munich‐Wistar or Sprague‐Dawley rats. NIDDK DK083338, NSF IOS095285
RNA‐Seq analysis of glycosylation related gene expression in Streptozotocin‐induced diabetic rat kidney inner medulla
Authors: Chen Guangping , Li Xuechen , Su Hua , Ilori Titilayo O , Klein Janet D , Hughey Rebecca P
Published: June 2021

The UT‐A1 urea transporter plays an important role in the urinary concentration mechanism. Native UT‐A1 from kidney inner medulla (IM) is a heavily glycosylated protein with two glycoforms of 97 and 117 kDa. In diabetes, the protein abundance, particularly of the 117 kD isoform, is significantly increased, corresponding to an increased urea permeability in perfused IMCDs. In this study, by using sugar‐specific binding lectins, we found that the UT‐A1 glycan structure was also dramatically changed under diabetic conditions. UT‐A1 became highly sialyated, fucosylated, and branched. These changes were also accompanied by an alteration of UT‐A1 association with galectin proteins, a group of â‐galactoside glycan binding proteins. To explore the molecular basis of alterations in glycan structures, we then sought to employ the next generation sequencing (NGS) technology Illumina RNA‐Seq to identify genes involved in the process of UT‐A1 glycosylation in the IM tip of streptozotocininduced diabetic rat kidney. We found that the sialylation enzymes, glycosyltransferase Mgat, and galectin were upregulated. In contrast, the fucosylation enzyme Fut8 was downregulated under diabetic conditions. The change of these glycosylation related genes may play an important role in changing UT‐A1 glycan structure, and therefore regulate kidney urea transport activity under diabetic conditions.
Increased UT‐A1 ubiquitination is partially due to decreased deubiquitination activity in Streptozotocin‐induced diabetic rat kidney inner medulla
Authors: Su Hua , Ilori Titilayo O , Klein Janet D , Sands Jeff M , Chen Guangping
Published: June 2021

The urea transporter UT‐A1 protein abundance in kidney inner medulla (IM) is dramatically increased under diabetic conditions to compensate for the osmotic diuresis caused by glucosuria. In this study, we examined UT‐A1 ubiquitination and degradation in a streptozotocin‐induced diabetic animal model. IM tissue lysates were immunoprecipitated with UT‐A1 antibody followed by immunoblotting with ubiquitin antibody. The smeared large size bands indicating ubiquitinated UT‐A1 were significantly increased. We further found that the de‐ubiquitination enzymes, ubiquitin‐specific proteases 18 (USP18) and USP24, were downregulated in diabetic rat IM. In vitro deubiquitination assays were performed and revealed a decrease in deubiquitination activity from diabetic rat IM lysates. We then specifically examined the effect of USP24 on UT‐A1 deubiquitination. USP24 directly interacted with UT‐A1 and promoted UT‐A1 deubiquitination in vitro. Knockdown of USP24 in UT‐A1 HEK293 cells increased both cell surface and total UT‐A1 degradation. By using sucrose gradient ultracentrifugation, we found that USP24 was absent in the cell plasma membrane and mainly present in early endosomes where it is co‐distributed with Rab5. Our study demonstrates that deubiquitination enzymes, like USP24, may play an important role in regulating UT‐A1 expression by affecting protein ubiquitination and degradation
Neither Hypertension nor Sexual Maturation is Responsible for Elevated Mesenteric Arterial Expression of TGF‐β in Female Spontaneously Hypertensive Rats (SHR)
Authors: Tipton Ashlee Joy , Womack Mandy , Sullivan Jennifer
Published: June 2021

TGF‐β has been implicated in vascular damage and hypertension. We have demonstrated that adult female SHR express more vascular TGF‐β than males. Moreover, TGF‐β expression increases as female SHR, but not males, age from 5 to 13 wks. This study tested the hypotheses that 1) increased TGF‐β expression in female SHR is due to hypertension not sexual maturation and 2) greater TGF‐β in females does not equate with greater fibrosis. To test our hypotheses 5 wk old female SHR were randomized to the following groups (N=4): vehicle vs hydralazine‐treated (50 mg/kg/day) or gonad‐intact vs ovariectomized (OVX). Mesenteric arteries were isolated at 13 wks and TGF‐β was measured by immunoblotting. TGF‐β expression was similar in vehicle and hydralazine treated SHR (densitometric units (DU): 27±1 vs 33±5, p=0.3) as well as between intact and OVX (DU: 17±2 vs 14±2, p=0.2). These data suggest that increased TGF‐β with age in female SHR is not dependent on hypertension or sexual maturation. Additional studies measured collagen expression by histological staining in 13 wk male and female SHR. Mesenteric arterial collagen deposition was similar in males and females (area μm2: 6632±898 vs 5507±522, p=0.3; N=3), suggesting that greater TGF‐β in females does not result in greater vascular injury. We propose that the increase in TGF‐β in females might instead be compensatory to offer vascular protection.
Effects of Estrogen on Relaxation Rates in Rat Caudal Artery
Authors: Allawzi Iman , Garcia Ariel , Uzomah Onyx , Pelaez Nancy
Published: June 2021

Menopausal women are more at risk for heart problems such as hypertension, which could arise from altered arterial smooth muscle function. If reproductive hormones impact cardiovascular health by altering vascular muscle contractility, we should note changes in the arteriolar contractility as cyclic hormone levels change. This project studies the effect of varying reproductive hormone levels on relaxation rates in rat caudal arteries. Retired breeding rats experience a peak and decline in natural estrogen and progesterone levels every four days. Experiments were performed on rat caudal artery collected during various phases of the reproductive cycle. Vaginal epithelial cell cytology indicated hormone levels. Isometric tension was recorded after electrical field stimulation of vascular tissue in a muscle bath under physiological conditions. Relaxation curves analyzed using JSIM modeling software matched a double exponential decay curve. The changing hormone levels caused no difference in the relaxation rates for either the slow or the fast portion of the relaxation curve. These results imply that the protective effect of natural hormone replacement therapy may not be directly impacting vascular function as a preventative measure for a healthy heart.
Mechanisms of shear stress mediated nitric oxide production by inner medullary collecting duct cells
Authors: Hyndman Kelly A , Pollock Jennifer S
Published: June 2021

We previously reported that increased shear stress (SS) stimulates nitric oxide (NO) production in mouse inner medullary collecting duct‐3 (mIMCD3) cells. Recent work in mpk‐cortical collecting duct cells determined that SS‐induced endothelin‐1 (ET1) expression was mediated via activation of the epithelial sodium channel (ENaC). We have shown that ET1 via ETB receptor stimulation of NOS1 mediates NO production in mIMCD3 cells. Thus, we hypothesized that SS‐induced NO production in mIMCD3 cells is dependent on an ENaC‐ET1‐ETB‐NOS1 mechanism. mIMCD3 cells were grown to confluency on glass slides, subjected to SS (30 dynes/cm2 for 1 h), and nitrite production measured by HPLC and calculated as fold change from static conditions. Cells were pretreated with ENaC inhibitors, 1 μM amiloride or 0.2 μM benzamil, ETB antagonist 1000 nM BQ‐788, or 100 nM 1400W (NOS2‐selective inhibitor) or 1 μM VNIO (NOS1‐selective inhibitor), and nitrite production determined. Both ENaC inhibitors significantly blunted the SS‐induced increase in nitrite (control = 1.8 ± 0.3 vs 1.3 ± 0.2 and 0.9 ± 0.3 respectively N = 4, P < 0.05). BQ‐788 attenuated the SS mediated increase in NO (control = 3.9 ± 1.0 vs 1.7 ±0.1, N =5, P < 0.05). NOS2 inhibition had no significant effect on SS‐induced nitrite production (p>;0.05), while NOS1 inhibition lead to an ~50% reduction in SS‐mediated nitrite (control = 2.8 ± 0.5 vs 1.5 ± 0.4 N= 5–10, P < 0.05). In conclusion, mIMCD3 SS induced NO production is mediated via activation of ENaC, ETB receptor, and NOS1. Future studies will determine whether ENaC via ET1/ETB/NOS1 directly regulates mIMCD3 NO production. Funding support from AHA to KAH and NIH to JSP.
Protein Carbonylation Regulates Renal Proximal Tubular Na/K‐ATPase signaling and Sodium Transport
Authors: YAN YANLING , Xie Zi‐jian , Shapiro Joseph I. , Liu Jiang
Published: June 2021

We have shown that cardiotonic steroids signaling through the Na/K‐ATPase regulate sodium reabsorption in renal proximal tubule (RPT). Here we report that protein carbonylation is critical in modulation of Na/K‐ATPase signaling and RPT ion transport. In RPT LLC‐PK1 cells, both ouabain (100nM) and glucose oxidase (GO, 1 and 3mU/ml) stimulated protein carbonylation of Na/K‐ATPase α1 subunit and c‐Src, activated c‐Src, stimulated redistribution of Na/K‐ATPase α1 and NHE3, and inhibited active transepithelial 22Na+ flux. Pretreatment with the antioxidant, N‐Acetyl‐L‐Cysteine (NAC), can prevent these effects in a dose‐dependent manner. Furthermore, disruption of the Na/K‐ATPase/c‐ Src signaling complex and c‐Src inhibitor PP2 abolished ouabain‐induced ROS generation and protein carbonylation. Moreover, it appears that there is a self‐regulatory mechanism to reverse carbonylation after removal of ouabain from culture medium, evaluated in the presence of protein biosynthesis inhibitor cycloheximide, proteasome inhibitor MG132, and lysosomotropic weak base agent chloroquine.

In summary, our data indicated that protein carbonylation is involved in a feed‐forward mechanism in regulation of Na/K‐ATPase/ c‐Src signaling and related sodium transport. The data also suggested that the Na/K‐ATPase/c‐Src signaling might be a functional receptor of ROS. (Supported by NIH HL‐109015 to Z.X. and J.I.S.).
What is the physiological relevance of the renal H,K‐ATPase type 2? Example of K+ reabsorption during K+ depletion, gestation and circadian rhythm.
Authors: Crambert Gilles , Lamouroux Christine , Salhi Amel
Published: June 2021

The existence of a renal “nongastric” H,K‐ATPase (HKA2) has been puzzling for decades. Indeed, whereas its stimulation in response to K+ depletion is well established, HKA2 null‐mice do not display a clear renal phenotype under these conditions. This last result had raised doubts about its physiological relevance until now and we started to revisit this problematic. Recently, we identified progesterone as an adrenal mediator that is produced in response to K+ depletion and that activates the renal expression of HKA2. In this situation, acutely preventing progesterone from acting abolishes the stimulation of HKA2 and leads to a higher urinary K+ loss. This finding led us to investigate the role of renal HKA2 during pregnancy. We indeed found that the renal HKA2 is stimulated during gestation and that its absence, in the HKA2‐null mice, leads to gestational problems (decreased fertility, increased maternal mortality) that correlate with an impaired K+ balance. Finally, we identified the renal HKA2 as a component of the renal adaptation to the circadian rhythm, which allows the organism to maintain stable the plasma K+ levels during rest periods by mediating K+ reabsorption when K+ intake is low.

These recent results indicate that the renal HKA2 is not an epiphenomenon and plays important physiological roles.
Usp2–45 represses aldosterone response by decreasing mineralocorticoid receptor availability
Authors: Staub Olivier , Debonneville Anne , Pouly Daniel , Faresse Nourdine
Published: June 2021

Steroid receptors are known to be subject to dynamic ubiquitylation that impacts their stability and activity as transcription factors. We have previously described that the mineralocorticoid receptor (MR) is monoubiquitylated in its basal state and this status is sustained by the tumor suppressor gene 101 (Tsg101) bound to the ubiquitylated receptor. Aldosterone stimulation of MR induced monoubiquitylation removal and disruption of MR/Tsg101 association that prompt polyubiquitin‐dependent destabilization of MR and proteasomal degradation. In this study, we found that the aldosterone induced ubiquitin‐specific protease USP2–45 has a pivotal role in the regulation of MR ubiquitylation state. This enzyme is able to bind to MR and mimic aldosterone effects by inducing MR monoubiquitylation removal, disruption of MR/Tsg101 association and destabilization of MR at protein level. Hence, this study demonstrates a new role for USP2–45 and reveals the existence of a negative feedback loop for limiting aldosterone induced response.
Hyperadrenergic activity and the Postural Tachycardia Syndrome (PoTS)
Authors: Low David A , Akhtar Mohammed Abid , Iodice Valeria , Watson Laura , Mathias Christopher J
Published: June 2021

The aim of this study was to compare cardiovascular autonomic function of Hyperadrenergic PoTS, defined as a plasma norepinephrine >; 600 ng/mol during orthostasis, to PoTS due to other causes. Clinical cardiovascular autonomic function test results were reviewed. 50 PoTS patients were identified; 18 with Hyperadrenergic PoTS. Plasma norepinephrine was higher while upright (383 ± 99 vs 726 ± 112 ng/mol, p<0.05) and supine (233 ± 58 vs 422 ± 128 ng/mol, p<0.05) in Hyperadrenergic PoTS. There were no differences in heart rate or blood pressure during cold pressor challenge, mental stress, isometric exercise, deep breathing and short‐term (9 min) head‐up tilting, between groups (all p>;0.05). Valsalva manoeuvre derived Valsalva ratio, blood pressure recovery time and vagal baroreflex sensitivity were not different (all p>;0.05), whereas adrenergic baroreflex sensitivity was lower in Hyperadrenergic PoTS (p<0.05). Hyperadrenergic PoTS had lower prolonged (60 min or less) head‐up tilt tolerance (p<0.05); termination was predominantly due to patient request not differences in heart rate or blood pressure. These findings suggest that, 1) Hyperadrenergic PoTS have elevated supine and upright, plasma norepinephrine levels and 2) Hyperadrenergic PoTS do not have distinctive cardiovascular autonomic function except for decreased adrenergic baroreflex sensitivity and reduced prolonged orthostatic tolerance.
Water drinking enhances the gain of arterial baroreflex control of muscle sympathetic nerve activity in healthy humans
Authors: Vianna Lauro C , Fernandes Igor A , Martinez Daniel G , Silva Bruno M , Fadel Paul J , Nobrega Antonio C
Published: June 2021

Water drinking increases efferent sympathetic outflow in young healthy subjects in the absence of a pressor response, whereas in autonomic failure patients and healthy older adults water‐induced sympatho‐excitation is accompanied by a large pressor response. The extent to which the arterial baroreflex (ABR) buffers the blood pressure (BP) response to water intake is unclear. We tested the hypothesis that water drinking increases ABR control of muscle sympathetic nerve activity (MSNA) in young healthy subjects. MSNA (microneurography) and arterial BP (Finometer) were measured in 8 men (24 ± 1 yrs) before, and for 60 min after drinking 500 mL of room‐temperature (22°C) tap water or saline. Weighted linear regression analysis between MSNA and diastolic BP was used to assess spontaneous ABR MSNA gain. As expected, MSNA was elevated after water intake (22 ± 4 baseline to 36 ± 5 bursts/100 heart beats at 30 min, P<0.05) and BP was unchanged. ABR MSNA gain for burst incidence (−2.4 ± 0.3 baseline vs. −4.4 ± 0.6 bursts/100 heart beats/mmHg at 30 min; P<0.05) and total MSNA (−3.6 ± 0.8 baseline vs. −5.6 ± 0.8 au/beat/mmHg at 30 min; P<0.05) were increased and tended to return to baseline at 60 min. ABR‐MSNA gain remained unchanged following saline intake. These data indicate that ABR MSNA gain is enhanced following water intake in healthy young subjects, which likely contributes to the maintenance of BP.

Supported by CAPES, CNPq & FAPERJ
Arterial baroreflex control of sympathetic nerve activity during acute hypotension is enhanced in young normotensive black men
Authors: Holwerda Seth W , Keller David M , Credeur Daniel P , Fadel Paul J
Published: June 2021

Although impairments in arterial baroreflex (ABR) control of heart rate have been reported in black compared to white subjects, no studies have examined the potential for racial differences in the ABR control of muscle sympathetic nerve activity (MSNA). Therefore, we tested the hypothesis that ABR control of MSNA is impaired in young black men compared to white men. In 12 non‐hypertensive men (7 black and 5 white, age 18–27 yr) ABR control of MSNA was determined using 100μg i.v. of sodium nitroprusside (NTP) followed 1 min later by 150μg of phenylephrine (PE) during supine rest (3 trials/subject). The slope of the relationship (i.e., gain) between changes in diastolic blood pressure and MSNA was determined. Overall sympathetic baroreflex gain was not different between racial groups (blacks: − 1.9±0.2 vs. whites: −2.4 ± 0.4 AU·beat·mmHg−1, p>;0.05). Similarly, sympathetic baroreflex gain for hypertension (PE phase) was also not different between groups (blacks: −1.9±0.3 vs. whites: −1.7±0.2 AU·beat·mmHg−1, p>;0.05). However, sympathetic baroreflex gain for hypotension (NTP phase) was significantly elevated in blacks (−4.0±0.3) compared to whites (−3.2±0.1 AU·beat·mmHg−1, p<0.05). In contrast to our hypothesis, these preliminary findings suggest that ABR control of MSNA is similar or enhanced in young blacks compared to their white counterparts.

Supported by NIH Grant R15HL096103 (DMK) and RO1 HL093167 (PJF)
Activation of NMDA receptors results in different autonomic and cardiovascular responses along the rostrocaudal axis of the insular cortex
Authors: Marins Fernanda Ribeiro , Limborço‐Filho Marcelo , Vaz Gisele Cristiane , Xavier Carlos Henrique , Fontes Marco Antonio Peliky
Published: June 2021

Unilateral damage of the insular cortex (IC) causes imbalance of the autonomic control. The cardiovascular representation along the IC is unclear. We compared the cardiovascular responses evoked by stimulation of NMDA receptors into left (L‐IC) or right IC (R‐IC) in different levels of the IC rostrocaudal axis (−1.5, 0.0 and +1.5mm). Under urethane (1.4g/kg) anesthesia, Wistar rats were prepared for ECG, MAP, HR and RSNA recording. NMDA was injected unilaterally (0.2mM, 100nL; n=6–8/group). Gene expression of NMDA receptors in the IC was confirmed by RT‐PCR. Nanoinjection of NMDA into IC at −1.5 did not change the cardiovascular parameters. NMDA into IC at 0.0 increased RSNA (Δ: L‐IC 28±4 vs R‐IC31±5%) and HR (Δ: L‐IC 40±8 vs R‐IC 36±7 bpm) with small changes in MAP (Δ: L‐IC 5±1 vs R‐IC 7±2 mmHg). NMDA into IC at +1.5 decreased RSNA (Δ: L‐IC −24±4 vs R‐IC −19±2%), HR (Δ: L‐IC −35±4 vs R‐IC −40±5 bpm) and increased MAP (Δ: L‐IC 17±3 vs R‐IC 15±1 mmHg). NMDA evoked effects were blocked by its antagonist, AP‐5. Arrhythmias were not detected. We found different autonomic and cardiovascular responses in different levels of the IC rostrocaudal axis, but no differences between R‐IC and L‐IC. CNPq, Fapemig, Capes.
The role of α‐adrenergic receptors in mediating beat‐by‐beat sympathetic vascular transduction in resting humans
Authors: Fairfax Seth T , Credeur Daniel P , Holwerda Seth W , Zuidema Mozow Y , Medley John H , Dyke Peter C , Wray D Walter
Published: June 2021

Sympathetic vascular transduction is commonly understood to act as a basic relay, but under basal conditions competing dilatory signals may interact with and alter the ability of sympathetic activity to decrease vascular conductance. Thus, we determined the extent to which spontaneous bursts of muscle sympathetic nerve activity (MSNA) mediate decreases in forearm vascular conductance (FVC). In 7 young men, MSNA (microneurography) and brachial artery blood flow (duplex Doppler ultrasound) were continuously measured during 20 minute infusions of intra‐arterial saline (control), phentolamine (PHEN), and PHEN with angiotensin II (PHEN‐ANG). The latter was used to control for increases in resting blood flow with a‐adrenergic blockade. Signal averaging was used to characterize beat‐by‐beat changes in FVC for 15 cardiac cycles following each individual MSNA burst and a peak response was calculated. During saline, FVC initially increased by +3.3±0.3% following MSNA bursts (P=0.016) and then robustly decreased to a nadir of −5.8±1.5% (P<0.001). PHEN and PHEN+ANG infusion did not alter the initial rise in FVC, but significantly attenuated the ensuing decrease in FVC (−2.1±0.7% and −0.7±0.8%, respectively; P<0.001 vs. saline). These findings indicate that spontaneous MSNA bursts are capable of evoking robust decreases in FVC that are exclusively mediated via α‐adrenergic receptors. Supported by RO1 HL093167
In vitro modeling of inflammation‐induced blood‐brain barrier dysfunction in sickle cell disease
Authors: Parfenova Helena , Pestina Tamara , Persons Derek , Basuroy Shyamali
Published: June 2021

Patients with sickle cell disease (SCD) have a high incidence of cerebral vascular occlusion combined with inflammation. The properties of the blood‐brain barrier (BBB) in SCD are not characterized. We investigated structural and functional properties of SCD BBB using cerebrovascular endothelial cells (CVEC) from Berkeley SCD (BERK) and control C57BL/6 (WT) mice. BERK CVEC in primary cultures had normal growth potency and the ability to form cell contacts visualized by beta‐catenin and VE‐cadherin staining. BBB functions were tested by transendothelial electrical resistance (TEER) and by permeability to Alexa Fluor 488‐dextran in CVEC grown on transwell inserts. The baseline parameters of BBB functions were identical in WT and BERK CVEC. Pro‐inflammatory cytokine TNF‐alpha combined with cycloheximide (CHX) caused a time‐dependent (5–23 h) reduction in TEER and increased dextran permeability in both cell types. TNF/CHX‐induced BBB dysfunction was accompanied by loss of cell‐cell contacts, cell detachment, and by apoptosis. Antioxidant bilirubin conferred protection against TNF/CHX‐increased endothelial permeability, indicating that oxidative stress is a leading factor in inflammation‐induced BBB dysfunction in WT and SCD mice. Overall, CVEC from SCD mice maintain normal functional properties of BBB and are not hypersensitized to inflammation‐related injury. The study is supported by NIH/NINDS.
Intrauterine undernutrition programs the hypothalamic proteome of female rats
Authors: Ribeiro Eliane Beraldi , Pedroso Amanda Paula , de Souza Adriana Pereira , Dornellas Ana Paula Segantine , Estadella Debora , Nascimento Cláudia Maria Oller , Oyama Lila Missae , Rosa José Cesar , Watanabe Regina LH
Published: June 2021

Undernutrition in early life may cause adulthood obesity with disruption of energy homeostasis control. We analyzed the hypothalamic proteome of adult female rats born from dams fed ad libitum or restricted to 50% of ad libitum intake during gestation. Control and restricted progeny were studied at age 4 months. Hypothalamic proteins were analyzed by two‐dimensional gel electrophoresis and MALDI‐TOF/TOF–MS. MASCOT server was used to search Swiss‐Prot protein database. Only proteins with false discovery rate of 0% were considered in the results analysis. The restricted rats had normal body weight and insulin levels with increased fat pads weight and glycemia. Significant density increase of 10 spots and decrease of 9 spots was found. Six overexpressed and 4 under‐expressed proteins were identified. They are enzymes of energy metabolism pathways, and proteins of redox regulation, inflammatory, and cell communication processes. Female rats undernourished intra uterus developed into obese adults and presented alterations of hypothalamic protein profile that may be involved in the disturbances leading to obesity
Correlation and Causation: Systems Level Understanding of Decision‐Making Signals at the Single‐Neuronal Level in the Human Brain
Authors: Patel Shaun R , Sheth Sameer , Mian Matt , Bourne Sarah , Flaherty Alice , Eskandar Emad
Published: June 2021

We explored single neuronal activity from the nucleus accumbens (NAc) and subthalamic nucleus (STN) in patients undergoing deep brain stimulation while actively engaged in a financial decision‐making task. In a subgroup of the subjects, we explored the effects of intermittent electrical stimulation of the STN on financial decision‐making. Subjects are presented with a computerized gambling task modeled as a simplified version of the card game “war”. We recorded 25 NAc and 20 STN from 13 patients. We found single neuronal evidence of a behavioral and prediction error signal in the nucleus accumbens and subthalamic nucleus. The behavioral signal predicted–during a discrete 500 ms interval–their upcoming financial decision well before they physically manifested their choice. The prediction error signal–the difference between expectation and outcome–occurred during a discrete 500 ms interval immediately after receiving feedback on the outcome of the current trial. Furthermore, in a subgroup of subjects, we were able to shift their behavior towards risk‐seeking by applying 1s of electrical stimulation in the STN before the trial began. In conclusion, these findings demonstrate the ubiquitous nature of two reinforcement learning signals during financial‐decision making. In addition, we demonstrate a causal relationship by applying intermittent electrical stimulation to bias the decision signal.
Avoidance of environmental gas irritants mediated by TRPA1
Authors: Yonemitsu Toru , Takahashi Nobuaki , Mori Yasuo , Kanmura Yuichi , Kuwaki Tomoyuki
Published: June 2021

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, is expressed in a subset of sensory neurons such as trigeminal and dorsal root ganglia and implicated as a mediator of nociception evoked by diverse peripheral stimuli including pungent chemicals. We hypothesized that TRPA1 would be involved in detecting environmental chemicals to avoid taking in such a threat. To test our hypothesis, we did place preference test using TRPA1 knockout (KO) mice and age matched wild‐type (WT) mice. The mice were first allowed to freely explore a homemade apparatus that consists of four chambers and the connecting tubes. The number of entry times and the amount of time spent in each chamber were recorded. None of the animals had initial bias for either chamber. Then, each chamber was randomly assigned to a chamber in which a piece of cotton paper soaked with a test solution was placed. We tested a TRPA1 agonist, formaldehyde. During a test period of 20 min, WT mice never tried to enter the chamber with formaldehyde. KO mice entered the chamber without hesitation and even stayed there. This result indicates that WT mice detect and recognize formaldehyde as a dangerous substance via TRPA1.
Behavioral Profile Assessment in Offspring of Swiss Mice Underwent to Neonatal Treatment with D‐fenfluramine
Authors: Melo Roberto Laureano , Silveira Anderson Luis Bezerra , Seara Fernando Azevedo Cruz , da Rocha Fabio Fagundes , Reis Luis Carlos , Cortes Wellington Silva
Published: June 2021

Serotonin (5‐HT) exerts an important role in the central nervous system embryogenesis of mammals, influencing directly several brain systems ontogeny. Therefore, we investigate the effect of neonatal D‐fenfluramine (D‐fen), a specific 5‐HT‐releasing agent, administration on the behavioral expression of adults male Swiss mice. For this purpose, three groups (n=6 each, and ~35g) of pregnant female Swiss mice were mated. The offsprings obtained were treated subcutaneously (s.c) with saline and D‐fenfluramine (1mg/kg or 3mg/kg) from the 5th at 21th post‐natal day. By completing 70 days of life, the animals underwent a behavioral assessment in open field, rotarod, light‐dark box, elevated plus maze and tail suspension test. Statistical analysis was performed by ANOVA one way and the means were considered significantly different when p<0.05. According to our results, the programmed animals had less time of immobility in tail suspension test (D‐fen 3mg/kg: 64.89±6.07; D‐Fen 1mg/kg: 94.15±9.67 vs. control: 130.6±13.26, p<0.001) and less time spent walking on rotarod (D‐fen 3mg/kg: 17.55±7.32; D‐Fen 1mg/kg: 22.75±7.90 vs. control: 189,7±52,33, p<0.001). In the dark light box test, the animals treated with D‐fen 3mg/kg had less time in the light side than the other groups (D‐fen 3mg/kg: 94,56±8.81 vs. D‐Fen 1mg/kg: 121.7±3.43; control: 133.9±8.01, p<0.01). These findings suggest that programming with D‐fenfluramine during the neonatal period alters neural circuits involved with coordination, anxiety and depression‐like behaviors in adults.

Support: FAPERJ & CNPq.
Is myocardial iron content associated with left atrial systolic function in asymptomatic hereditary hemochromatosis?
Authors: Shizukuda Yukitaka , Tripodi Dorothy , Sachdev Vandana , Brenneman Cynthia , Sidenko Stanislav , St. Peter Marilyn , Bolan Charles , Yau Yu Ying , Arai Andrew , Bandettini Patricia , Leitman Susan , Rosing Douglas
Published: June 2021

We previously reported that left atrial (LA) systolic function is augmented in asymptomatic hereditary hemochromatosis subjects (HH) as compared to the age/gender‐matched volunteer control subjects (VC). In this study, we analyzed the relationship between elevated myocardial iron content (MIC) and LA systolic function in these subjects. All HH were NYHA functional class I and homozygous for the HFE gene C282Y mutation under standard therapy. VC did not have HFE mutations. 29 HH (ages; 56±10, 9 females) and 18 VC (ages; 55±8, 5 females) underwent LA function assessment by echocardiography and MRI‐based MIC assessment with T2*. LA ejection force (LAF) was significantly elevated in HH as to VC (14.0±8.3 vs. 9.4±4.7 kdyne, P=0.016) indicating augmented LA systolic function in HH. However, MIC did not significantly differ between the groups (27.6±7.8 msec vs. 28.6±5.5 msec, HH vs. VC, the values were in the normal range) and MIC failed to correlate with LAF or the other parameters of LA systolic function. Our findings suggest that the augmented LA function in asymptomatic HH subjects is independent from MIC and additional factors associated with HH, such as elevated oxidative stress may be responsible for this. However, this study does not address the possibility of elevated MIC to induce abnormal diastolic function in iron overload populations. (This research was supported by the intramural research program of the NHLBI, NIH.)
The role of TNF‐α in cardioprotection induced by adaptation to chronic hypoxia in rats.
Authors: Neckar Jan , Chytilova Anna , Borchert Gudrun H , Kolar Frantisek
Published: June 2021

It has been reported that tumor necrosis factor (TNF‐α) can precondition the heart against acute ischemia/reperfusion injury. This study examined if TNF‐α also plays a role in a long‐lasting cardioprotection induced by chronic hypoxia. Adult male Wistar rats were exposed for 30 days to chronic continuous hypoxia (CCH; inspired O2 fraction 0.1); half of the animals were treated with a TNF‐α inhibitor infliximab (5 mg/kg i.p. once a week). Controls were kept at room air and treated in a corresponding manner. CCH increased the concentration of TNF‐α in particulate fraction of the left ventricle while the concentration of interleukin 10 (IL‐10) and the ratio of IL‐10 to TNF‐α decreased in both cytosolic and particulate fractions. Myocardial infarct size induced by 20‐min coronary artery occlusion and 3‐h reperfusion was reduced in CCH rats by 33% as compared with normoxic controls. This protective effect was attenuated in animals treated with infliximab, which had no effect in normoxic controls. The results suggest that adaptation to CCH results in the myocardial inflammatory response and TNF‐α is involved in the cardioprotective mechanism.

Supported by grants GAAV IAAX01110901, GACR 303/12/1162.
Prohibitin coordinates an anti‐inflammatory/antioxidant feedback loop from mitochondria to nucleus to protect the heart from severe inflammatory stress
Authors: Mattox Taylor Ann , Thayne Kathleen A. , Anderson Ethan J.
Published: June 2021

Cardiovascular and mitochondrial dysfunctions are well‐known complications of sepsis. Prohibitin (PHB), a mitochondrial protein, has been implicated in experimental models involved in coordinating inflammatory signaling; its mechanisms are unknown. We investigated the role of PHB in the heart during sepsis using an in vivo model (LPS) and in vitro model (TNFα/IL1β) in HL1 cardiomyocytes (HL1c). Sepsis caused reductions in mitochondrial function in both models. In rats, LPS reduced PHB expression in whole heart at 4 and 24 hrs post exposure. While the total cardiac PHB decreased, serum levels spiked 3‐fold and the remainder became concentrated in nucleus. In vitro overexpression of PHB (oPHB) in HL1c attenuated the cytokine‐induced lowers O2 consumption, ATP generation, and Ca2+ retention, and prevented raises in H2O2 in response to these cytokines. oPHB protected the HL1c from TNFα/IL1β‐induced cytotoxicity. oPHB and recombinant PHB to HL1c reduced TNFα/IL1β‐induced NFκB activation and transcription of its downstream targets. These findings uncover a novel role for PHB and suggest that PHB can act as a mobile signal transducer which can suppress inflammation and oxidative stress during sepsis. We anticipate that these findings will lead to novel therapies that exploit the pleiotropic functions of PHB, protecting the heart from diseases in which inflammation and oxidative stress are common.
Analysis of mitochondrial MMP‐2 and MMP‐9 in the heart
Authors: Hughes Bryan , Fan Frank (Xiaohu) , Simmen Thomas , Schulz Richard
Published: June 2021

Matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 have been reported to be in cardiac mitochondria. However, these studies relied on non‐specific immunological methods or crude mitochondrial fractions contaminated with endoplasmic reticulum (ER). To determine if MMP‐2 and ‐9 are in the mitochondria we isolated purified mitochondria from adult rat hearts via Percoll density gradient centrifugation. To confirm mitochondrial purity, we showed that the ER marker protein disulfide isomerase, present in crude mitochondria, was absent in the Percoll‐purified mitochondrial fraction. We detected 72 kDa MMP‐2 via gelatin zymography and western blotting in crude mitochondrial fractions; MMP‐9 was only detected by western blotting. Purified mitochondria did not exhibit MMP‐2 or ‐9 gelatinolytic activities, but showed both MMP‐2 and MMP‐9 protein in western blots. In conclusion, MMPs are present in cardiac mitochondria, however, they appear to be in a form that may be resistant to activation. Future research will investigate several putative mitochondrial targets of MMPs identified by previous non‐biased degradomic screens.

Funding: Canadian Institutes of Health Research