bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of cerebral blood flow during exercise in men and women
Authors: Clausen Mary , Leddy John , Willer Barry , Pendergast David
Published: June 2021

Cerebral autoregulation (CA) maintains cerebral blood flow (CBF) constant over a wide range of perfusion pressures (MAP). However, arterial PCO2 (PETCO2) may modify CBF if brain metabolism and total oxygen consumption (VO2) are modified. During exercise MAP, VO2 and VCO2 are increased and may affect CBF. Female (13) and male (9) subjects completed a CO2 sensitivity test at rest and a graded exercise treadmill test. Heart rate (HR), MAP, ventilation (VE) and CBF velocity (CBFV) were measured in both tests. During rest CBFV (45%) and VE (221%) increased linearly with PETCO2 (58%). During exercise CBFV increased (31%) from 61 ± 16 cm/s to 78 ± 16 cm/s at 80% of VO2max. Once above the ventilatory threshold, CBFV decreased to 65 ± 16 cm/s at 100% VO2max. The changes in CBFV paralleled the changes in PETCO2 (36 ± 5 to 46 ± 6 to 36 ± 6 mmHg), despite a linear increase in MAP with VO2. Multiple linear regression indicated that PETCO2 was contributing more to CBFV than MAP (CBFV = 5.001 + (0.759 * PETCO2) + (0.313 * MAP)). During exercise CA did not maintain CBFV and PETCO2 strongly influenced it, thus ventilatory regulation (PETCO2) in exercise is critical in determining CBF. Resting CO2 sensitivity did not predict exercise PETCO2. Regulation of PETCO2 during exercise may explain the differences in CBF among individuals or conditions. One example may be the dysregulation of CBF in post concussion syndrome patients.
Effects Of Genistein And Exercise On Ovariectomized And Intact Rats
Authors: Djandjoulia Tea , Batia Lyn , Broderick Tom , Al‐Nakkash Layla
Published: June 2021

Genistein and exercise are independently purported to abate cardiovascular disease, thus, in this study we aimed to examine the effects of a 6 week treatment with genistein (300 mg/kg day, G) or placebo control (0G) with (EX) or without exercise (SED) in ovariectomized (OVX) and intact (INT) Sprague‐Dawley rats randomly divided into the following groups; OVX‐EX, OVX‐G/EX, OVX‐SED, OVX‐G/SED, INT‐EX, INT‐G/EX, INT‐SED, INT‐G/SED. Final rat weight, heart weight, and tibia length were all significantly decreased in OVX‐G/EX (n=7) compared to OVX‐EX (n=6). Heart weight and tibia length were significantly increased in OVX‐EX (n=6) compared to OVX‐SED (n=6). Femur weight was significantly increased in INT‐EX (n=6) and INT‐G/EX (n=9) compared to INT‐G/SED (n=5). Tibia length was significantly increased in INT‐EX (n=6) compared to INT‐G/SED (n=5). There were no changes in total liver weight, left liver lobe weight, total liver weight/body weight ratio, femur length, tibia weight, fat pad weight, uterine weight between groups. At the end of the study; systolic BP and mean arterial pressure (MAP) were increased in OVX‐G/EX, MAP was decreased in INT‐G/SED (n=5), pulse pressure was increased in OVX‐EX (n=6) and decreased in INT‐EX (n=6) and cardiac work was decreased in INT‐EX (n=6). Interestingly, there was an EX‐induced increase in aorta luminal diameter of INT‐EX (762.5±9.6 μm, n=4) compared to INT‐SED (723.1±7.5 μm, n=5), without change in aortic wall thickness. Our results show that a 6 week treatment of genistein and/or EX can contribute towards changes in both physical characteristic and blood pressures, and that INT and OVX rats do not respond similarly. Supported by NIH (1R15DK071625‐01A2), MWU intramural & CHS funds.
Endurance Exercise, Mitochondrial Rejuvenescence and Aging: On Your Mark, Get Set, GO!
Authors: Safdar Adeel , Bourgeois Jacqueline M , Mocellin Nicholas , Hettinga Bart P , Kujoth Gregory C , Prolla Tomas A , Tarnopolsky Mark A
Published: June 2021

The role of mitochondrial abnormalities has been extensively characterized in the etiology of systemic decline with aging. A causal role for mitochondrial DNA (mtDNA) mutagenesis in mammalian ageing is supported by recent studies demonstrating that the polymerase gamma (PolG) mutator mouse, harbouring a proofreading‐deficient copy of PolG, exhibits an accelerated aging phenotype, systemic mitochondrial dysfunction, multisystem failure, and reduced lifespan. Longitudinal studies in humans have demonstrated that endurance training reduces the risk of chronic diseases and extends life expectancy. We aimed to delineate if endurance training can prevent the premature aging, and reverse and/or attenuate systemic mitochondrial decline in PolG mice. At 3 months of age, 36 PolG mice (♀ = ♂) were randomly assigned to a sedentary (SED) or forced‐endurance training (END; 15m/min for 45 min, 3x/week for 5 months) group. END induced systemic mitochondrial biogenesis, increased oxidative capacity, restored mitochondrial morphology, and prevented mtDNA depletion in multiple tissues in PolG mice. These adaptations synergistically conferred complete phenotypic protection, rescued multisystem pathology (sarcopenia, cardiac hypertrophy, macrocytic anemia and splenomegaly), and increased lifespan of these mice. We conclude that endurance exercise promotes systemic mitochondrial oxidative capacity, contributing to the complete rejuvenation of PolG mice. However, it seems unlikely that the multi‐organ mitochondrial biogenesis observed is attributable to a single factor which can be mimicked by an “exercise pill.” (Funded by CIHR, and Mr. Warren Lammert and family)
Whole body to gene: Adaptations with high volume bicycle training
Authors: Slivka Dustin Russel , Dumke Charles L , Hailes Walter S , Cuddy John S , Ruby Brent C
Published: June 2021

Metabolic changes occur with exercise and exercise training. However, whole body change is rarely investigated along with enzymatic and mRNA changes. The purpose of this study was to inclusively describe exercise metabolic adaptation. Ten trained males cycled ~100 miles per day for 21 days. Gas was collected on days 0, 10, and 20 during a graded exercise. Muscle biopsies were obtained before and after exercise on days 1, 11, and 21. Fat oxidation increased (p<0.05), carbohydrate oxidation decreased (p<0.05), and gross efficiency increased (p<0.05) by day 10 without further increases by day 20. CS enzyme activity increased from day 11 to day 21 (p<0.05). B‐HAD and COX enzyme activity did not change. CS mRNA did not change acute exercise or training. B‐HAD mRNA was higher post exercise on days 11 (p=0.041) and 21 than (p=0.048) on day 1. COX mRNA increased with exercise (p=0.001) but did not change over the 21 days. PGC‐1 increased as a result of exercise (p=0.000), but did not increase to the same degree on day 11 and 21 as it did on day 1. UCP3 mRNA decreased by day 21 and showed a trend to increase (p=0.073) as a result of exercise. These data indicate that the rapid training adaptations observed in substrate use and gross efficiency cannot be explained by classical metabolic enzymes in these trained individuals.

Supported by Air Force Research Laboratories, FA8650‐06‐1‐679 and the University of Montana Grant Program.
Effect of ischemic heart failure on the muscle mechanosensitive reflex in conscious cats
Authors: Matsukawa Kanji , Wakasugi Rie , Kadowaki Akito , Asahi Rie , Myoi Sachika
Published: June 2021

It has been reported that ischemic heart failure (IHF) augments the muscle mechanoreflex, which may in turn cause a greater sympathetic and pressor response during exercise. However, there are no previous studies analyzing the muscle mechanoreflex before and after IHF in the same animals. We conducted a longitudinal study over 2 months to examine how IHF modified not only the cardiovascular response during mechanical stretch of a hindlimb but also during audiostress with white noise using 4 conscious cats. The left anterior descending coronary artery (LAD) was ligated to induce IHF. In the intact condition, the audiostress increased heart rate (HR) and mean arterial blood pressure (MAP), whereas the hindlimb mechnaical stretch altered neither HR nor MAP. In more than 6 weeks following the LAD ligation, the pressor response during mechanical stretch was augmented, whereas the cardiovascular response during the audiostress was blunted. At the end of the experiments, the cats were anesthetized to examine the reflex response in cardiac sympathetic outflow during mehancial stretch and static contraction of the triceps surae muscle. Cardiac sympathetic nerve activity substantially responded to both interventions. It is likely that ischemic heart failure augments a muscle mechanosentitive reflex, with increased cardiac sympathetic outflow, but blunts the cardiovascular response to stress.
Integrated autonomic control of bronchial blood flow and third generation airway dimensions during exercise
Authors: White Saxon William , McLeod Damian , Quail Anthony , Cottee David , Pitsillides Koullis , Seah Peng , Parsons Gibbe , McIlveen Stephen , Gunther Robert
Published: June 2021

Neural mechanisms controlling 3rd generation airway blood flow and dimensions were studied in 8 sheep trained to run on a treadmill during moderate exercise O2 uptake 20 ml.kg−1.min−1 for 2 min, 10 min recovery. We measured rapid changes in bronchial blood flow and conductance (Qbr, Cbr), circumference (Circbr) and wall thickness (Thbr), using pulsed‐Doppler flowmetry, transit‐time sonomicrometry, and single crystal sonomicrometry, respectively. At exercise onset Pa rose to 111% (P< 0.05) of resting and HR from 85 b/min to 164 b/min (P< 0.001) before returning in recovery at 5 min. Qbr fell immediately to 91% and to 90% at 1.5 min (P< 0.05), and early in recovery to 84% (P< 0.05) before returning to pre‐exercise levels. Cbr followed a similar pattern (P< 0.05). Circbr fell immediately to 96% (P< 0.001) where it remained for the duration of exercise, and Thbr increased and was sustained during exercise before returning to control levels in recovery. Cholinoceptor block by i.v. methscopolamine bromide modified these responses. Pa rose to 109% of resting (P< 0.01), and HR rose from 120 b/min to 168 b/min (P< 0.001). A minor Qbr and Cbr fall was not significant, and only an immediate transient fall in Circbr was evident (P< 0.01). Thus raised vagal activity dominates the exercise‐induced bronchovascular and airway wall constriction in 3rd generation airways during and after exercise.

Funding: Hunter Medical Research Institute
Laser capture microdissection of spinally‐projecting RVLM neurons: Analysis of NMDA receptor subunit gene expression.
Authors: Mischel Nicholas , Holt Avril G. , Mueller Patrick J.
Published: June 2021

Spinally‐projecting neurons in the rostral ventrolateral medulla (RVLM) are important in blood pressure regulation. Functional and anatomical studies suggest that N‐methyl‐D‐aspartate receptors (NMDARs) are located in the RVLM and play important roles in cardiovascular reflexes. Since NMDARs are also associated with neuroplasticity, we determined the feasibility of comparing NMDAR subunit gene expression in the RVLM. To test this, Fluorogold (FG) was injected into the intermediolateral cell column of Sprague Dawley rats (n=4) to label spinally‐projecting neurons. After 1–2 wks, FG‐labeled cells were obtained under RNAase free conditions from fresh, frozen sections (8 μm) using laser capture microdissection (Arcturus PixCell II). Total mRNA was isolated, and NMDAR subunits (NR1 and NR2A) and GAPDH were quantified by real time PCR (qRT‐PCR) using Taqman probes. Preliminary findings suggest that we are able to detect NMDAR subunit gene expression in spinally‐projecting RVLM neurons. Alterations in NMDAR number or subunit composition may influence the excitability of RVLM neurons and contribute to changes in sympathetic outflow in physiological and pathophysiological states. Future studies will compare physical activity‐related changes in gene and protein expression levels of NMDAR subunits. (AHA# 09PRE2050230 and HL‐089364)
Down‐regulation of SK channel function among RVLM projecting PVN neurons (PVN‐RVLM) and sympathetic activation in AngII‐Salt hypertensive (HT) rats
Authors: Chen Qing‐Hui , Dong Ying , Andrade Mary A , Calderon Alfredo S , Toney Glenn M
Published: June 2021

A neural mechanism responsible for sympathetic activation in HT rats was investigated. We hypothesized that reduced SK current in PVN‐RVLM neurons could contribute to increased excitability and sympathoexcitation in HT rats. In voltage‐clamp recordings, the amplitude of SK current was significantly reduced in neurons from HT rats. In current‐clamp recordings, the frequency of current injection‐evoked spikes was greater in neurons from HT rats compared to normotensive (NT) controls. The SK channel blocker apamin caused a greater increase in spikes in NT controls compared to HT group. The amplitude of a medium after‐hyperpolarization potential (mAHP) was significantly reduced in the HT group. An after‐depolarization potential (ADP) was also observed in the HT group, but not in NT controls, though apamin did reveal an ADP in NT controls while failing to alter the ADP already present in the HT group. This suggests that diminished SK current likely underlies the smaller mAHP and the uncovered ADP in the HT group. Microinjection of apamin into the PVN of anesthetized rat induced a greater increase in renal SNA and MAP in NT compared to HT rats. We conclude that increased excitability of PVN‐RVLM neurons may contribute to sympathoexcitation and maintenance of AngII‐salt hypertension and reduced SK current in these neurons may constitute an underlying neural mechanism. Support: AHA0865107F (CQH) & HL076312 (GMT).
Electroacupuncture Improves Left Ventricular Dysfunction and Arrhythmia in a Rabbit Model of Prolonged Ischemia/Reperfusion
Authors: Zhou Wei , Ko Yoshihiro , Patel Sunny , Mahajan Aman
Published: June 2021

Augmentation of cardiac sympathetic tone during myocardial ischemia has been shown to increase myocardial oxygen demand, arrhythmias, and infarct size in animal models. We previously demonstrated that electroacupuncture (EA) inhibits the visceral sympathoexcitatory cardiovascular reflex. The purpose of this study was to determine the effects of EA on left ventricular (LV) mechanics, oxygen demand, arrhythmias, infarct size and in vivo cardiac norepinephrine release in rabbits with myocardial ischemia and reperfusion (MIR). Anesthetized rabbits underwent 60 min of left coronary artery (LCA) occlusion followed by 3 h of reperfusion. We evaluated the LV twist by 2D speckle tracking imaging, elevated ST segments of electrocardiogram (ECG), ventricular arrhythmias, infarct size, and myocardial norepinephrine release by cardiac microdialysis under three experimental conditions: 1) control, 2) EA at P 5–6 acupoints, and 3) EA at nonacupoints. MIR decreased the LV twist and increased ventricular arrhythmias, elevation of ST segments, infarct size, and oxygen demand as well as the concentrations of myocardial norepinephrine. EA at P 5–6 significantly attenuated the deleterious effects of MIR, while EA at nonacupoints had no effects. These data suggest that EA produces cardioprotective effects against MIR through an inhibition of cardiac sympathetic nervous system. Supported by AHA BGIA‐0765001Y.
HIGH FAT DIET ALTERS SPLANCHNIC SYMPATHOINHIBITORY RESPONSES TO GASTRIC HORMONES
Authors: Sartor Daniela Maria , How Jackie May‐Yen , Fam Barbara Christianne , Verberne Anthony Johannes Maria
Published: June 2021

Gastric leptin and cholecystokinin (CCK) act on vagal afferents to induce reflex inhibition of splanchnic sympathetic nerve discharge (SSND), and may act co‐operatively in this response. We sought to determine whether this response is altered in animals exposed to a medium high fat diet (MHFD). Male Sprague‐Dawley rats were placed on a low‐fat diet (LFD; n=8) or a MHFD (n=24) for 13 weeks, after which animals were anesthetised and artificially ventilated. Baseline arterial pressure (AP) was measured and blood was collected for determination of plasma leptin and CCK. SSND responses to leptin (15 μg/kg) and CCK (2 μg/kg) administered close to the celiac artery were evaluated. Animals on the MHFD were grouped as obesity prone (OP; n=8) or obesity resistant (OR; n=8); the effects of diet alone were also examined. OP rats had significantly higher AP, fat pad mass and plasma leptin but lower plasma CCK, compared to LFD rats (P < 0.05 for all). Collectively, MHFD animals had elevated leptin but lower CCK levels than LFD rats (P < 0.05 for both) and this corresponded with attenuated/reversed SSND responses to CCK (LFD: −21 ± 2 Units vs MHFD −10 ± 2 Units; P < 0.05) and leptin (LFD: −6 ± 2 Units vs MHFD: 4 ± 1 Units; P < 0.001). High fat diets may induce leptin resistance that reduces the sensitivity of vagal afferents to gastrointestinal hormones. Increased gastrointestinal vascular resistance may contribute to hypertension in obesity.
Chronic Superoxide Signaling in the Rostral Ventrolateral Medulla (RVLM) is Essential For Goldblatt Hypertension
Authors: Oliveira‐Sales Elizabeth B , Colombari Debora SA , Davisson Robin L , Kasparov Sergey , Campos Ruy R , Paton Julian FR
Published: June 2021

Reactive oxygen species (ROS) in peripheral cardiovascular tissues are implicated in the pathogenesis of Goldblatt (two kidney one clip, 2K1C) hypertension. We identified an imbalance between ROS generation and oxidant scavenging in the RVLM of Goldblatt rats. In 2K1C rats, the RVLM exhibited increased mRNA expression of NAD(P)H oxidase subunits. With a role in sympathetic activity generation, we tested whether chronic superoxide scavenging in RVLM would affect Goldblatt hypertension. In Wistar rats (male, 150–180g) superoxide dismutase was over expressed (Ad‐CMV‐CuZnSOD) chronically using an adenoviral vector 3 weeks after renal artery clipping. In 2K1C rats, the elevated AP and heart rate returned to control values one week after transfection, was maintained for 3 weeks and associated with decreased low frequency spectra of systolic blood pressure variability. We confirmed both reduced ROS levels and the presence of CuZnSOD in RVLM neurons immunohistochemically. No changes in arterial pressure were observed in control rats receiving Ad‐CMV‐eGFP in the RVLM. Thus, chronic superoxide signaling in the RVLM of the Goldblatt model is essential for the hypertension. Supported by CAPES and British Heart Foundation.
Repetitive Electroacupuncture causes Prolonged Enhancement of Preproenkephalin mRNA Expression in Rostral Ventrolateral Medulla of Unanesthetized Rats
Authors: Li Min , Tjen‐A‐Looi Stephanie S.C. , Longhurst John C.
Published: June 2021

The prolonged influence of acupuncture in hypertension has been reported in clinical studies. Analogous to human patient studies, we performed repeated EA on unanesthetized rats. Since the opioid system may play a role in prolonged inhibition of neuronal activity in the rVLM, we hypothesized that repetitive EA increases preproenkephalin mRNA in this brain stem region of unanesthetized rats. Adult male Sprague‐Dawley rats (200 g) were immobilized. They received repetitive EA (1–2 mA, 2 Hz, 0.5 ms) at P5–P6 acupoints (overlying median nerve) or sham (absence of needle stimulation) for 30 min twice over a 72 hr period. Then, 24 hr following repetitive EA or sham, brains were removed quickly, stored in RNA Later solution and punch biopsies of the rVLM were obtained for assay. PPE mRNA copies were analyzed by reverse transcription and absolute quantitative real‐time PCR. Relative ratios of PPE mRNA levels (normalized with the 18s house keeping gene) was increased in EA group (6.62±1.5, n=6) compared with sham (3.05±0.4, n=3), while the baseline value in rats that were restrained but not subjected to EA or sham was 3.84, n=2. Thus, EA increased PPE mRNA expression 24 hr after two applications of EA, suggesting that the prolonged inhibitory effect of EA may be due to an increase opioid mRNA expression in the rVLM (Supported by NIH HL‐63313 and HL‐072125).
Understanding Gene Regulatory Networks and Their Variations
Authors: Koller Daphne
Published: June 2021

A key biological question is to uncover the regulatory networks in a cellular system and to understand how this network varies across individuals, cell types, and environmental conditions. In this talk I will describe work that uses statistical learning techniques to address this goal. Specifically, I will show how gene expression data from a population of genetically diverse individuals (eQTL data) can be used both to uncover regulatory networks and to understand the mechanisms by which genetic variations perturbs those networks. We also show how a prior knowledge and data regarding biological networks and mechanisms can be used to guide the construction of regulatory networks and allow the robust selection of genetic variations that are causal for phenotypic change. I will also show how gene expression from a collection of diverse cell types can be used to understand the role of gene regulation in immune cell differentiation.
Driving backwards the evolution of antibiotic resistance
Authors: Kishony Roy
Published: June 2021

The application of antibiotics is hindered by a well‐known “catch‐22”: The use of a drug promotes the emergence and spread of drug‐resistant mutants that ultimately render it ineffective. But is this problem inevitable? While certain combination therapies are known to be more effective than single drugs, the impact of drug interactions on the evolution of drug resistance is unclear. I will describe a combined theoretical‐experimental approach to understand drug interactions and their effect on the evolution of resistance. Our results demonstrate that certain drug interactions can diminish and even invert the selection pressure for antibiotic resistance.
Diabetes‐induced NOS1 and NOS2 activity blunts oxygen consumption in renal medullary thick ascending limbs
Authors: Foster Jan M. , Carmines Pamela K. , Pollock Jennifer S.
Published: June 2021

We recently reported that diabetes increases superoxide anion and NO production by the rat renal medullary thick ascending limb (mTAL). We hypothesized that endogenously‐produced NO and superoxide anion exert opposing effects on Na transport by the mTAL during diabetes. O2 consumption (QO2) by mTAL suspensions was measured using the BD™ Oxygen Biosensor System, which discloses QO2 as a time‐related increase in relative fluorescence units (ΔRFU/min/mg protein). Ouabain‐sensitive QO2 (a measure of active Na+ transport) averaged 0.08±0.02 in mTALs from sham rats and 0.22±0.04 in mTALs from rats with STZ‐induced diabetes (P<0.05; n=8–9). NOS inhibition (100 μM 1400W) did not alter ouabain‐sensitive QO2 by mTALs from sham or STZ rats. NADPH oxidase inhibition (100 μM apocynin) had no effect on QO2 by sham mTALs, but caused a 60% decrease in ouabain‐sensitive QO2 by STZ mTALs (P<0.05). In the presence of apocynin, NOS inhibition significantly increased ouabain‐sensitive QO2 by mTALs from STZ rats, with similar effects evoked by isoform‐specific inhibitors of NOS1 and NOS2 (1 μM VNIO and 100 nM 1400W, respectively). Thus, while neither NO nor superoxide anion tonically influences Na transport by mTALs from the normal rat kidney, diabetes triggers superoxide anion stimulation of Na transport that is attenuated by NOS1‐ and NOS2‐dependent mechanisms likely reflecting an inhibitory influence of NO. (Supported by the AHA)
Induction of HIF normalizes oxygen utilization and oxygenation in the diabetic kidney
Authors: Palm Fredrik , Fasching Angelica , Nangaku Masaomi , Hansell Peter
Published: June 2021

Hyperglycemia results in increased oxygen consumption (QO2) and decreased oxygen tension (pO2) in the kidney. We hypothesize that activation of hypoxia‐inducible factors (HIF) protect against diabetes‐induced alterations in kidney function and oxygen metabolism.

The experimental groups consisted of control and streptozotocin‐induced diabetic rats with and without chronic cobalt chloride (CoCl2; inducer of HIF; n=9–10/group).

Diabetic rats had increased glomerular filtration rate (2.3±0.2 vs 1.5±0.2 ml/min/kidney; P<0.05) and CoCl2 prevented this increase (1.5±0.1 ml/min/kidney). QO2 was increased in the diabetic kidney (23±2 vs 11±2 μmol/min/kidney; P<0.05) and CoCl2 prevented this alteration (12±2 μmol/min/kidney). Tubular sodium transport (TNa)/QO2 was decreased in the diabetics (14±2 vs 33±8; P<0.05), which was prevented by CoCl2 (25±4). Diabetic kidneys had reduced pO2 in both cortex and medulla compared to control (29±1 vs 45±2 and 22±1 vs 30±1 mmHg, respectively; both P<0.05). CoCl2 prevented these decreases (cortex 42±1; medulla 39±1 mmHg). All investigated parameters were unaltered by CoCl2 in control rats.

Diabetes‐induced alteration in kidney oxygen metabolism is prevented by HIF activation via two different mechanisms. Normalizing GFR, which reduces tubular electrolyte load, and maintaining TNa/QO2 contribute to normal QO2 and pO2 in the diabetic kidney. Early pharmacological activation of the HIF system may prevent the development of diabetic nephropathy. Funding: NIH/NIDDK DK077858 and Swedish Research Council.
POLY(ADP‐RIBOSE) POLYMERASE INHIBITION OR GENE DEFICIENCY ALLEVIATE DIABETIC KIDNEY DISEASE
Authors: Shevalye Hanna , Stavniichuk Roman , Xu Weizheng , Zhang Jie , Lupachyk Sergey , Maksimchyk Yury , Drel Viktor R , Floyd Elizabeth , Slusher Barbara , Obrosova Irina G
Published: June 2021

Evidence for the important role for poly(ADP‐ribose) polymerase activation in diabetic complications is emerging. This study evaluated the role for PARP in diabetic kidney disease using both pharmacological and genetic approaches. In experiment 1, control and streptozotocin (STZ)‐diabetic rats were maintained with or without treatment with a highly specifc and orally active PARP inhibitor GPI‐15,427 (Eisai Inc.), 30 mg kg−1d−1, for 26 weeks after first 2 weeks without treatment. Urinary albumin, isoprostane, and 8‐hydroxy‐2′‐deoxyguanosine excretions, urinary albumin/creatinine ratio, renal poly(ADP‐ribosyl)ated protein expression, and renal transforming growth factor‐β1, vascular endothelial growth factor, and fibronectin concentrations were increased in diabetic rats, and all these changes were at least partially prevented by GPI‐15,427 treatment. In experiment 2, wild‐type and PARP−/− mice were made diabetic with STZ and were maintained for 12 wks. PARP gene deficiency partially prevented diabetes‐induced albuminuria and kidney hypertrophy. Further studies in this animal model are in progress. In conclusion, PARP activation plays an important role in diabetic kidney disease. These findings provide rationale for development of PARP inhibitors, for prevention and treatment of diabetic nephropathy.
Asymmetric Dimethylarginine (ADMA) Regulation in Puromycin Aminonucleoside (PAN) Induced Chronic Kidney Disease (CKD)
Authors: Cunningham Mark W. , Chen Gin‐Fu , Tain You‐Lin , Sasser Jennifer , Baylis Chris
Published: June 2021

Nitric Oxide (NO) is generated from L‐ arginine (L‐Arg) by nitric oxide synthase (NOS). ADMA, a NOS inhibitor, is synthesized by protein arginine methyltransferase (PRMT) and metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Increased plasma ADMA concentrations predict mortality and cardiovascular events in end‐stage renal disease (ESRD). The purpose of this study was to examine the regulation of ADMA in PAN induced CKD. We examined the protein abundance of PRMT‐1 and abundance and activity of DDAH in kidney cortex (KC) in low dose (LD) and high dose (HD) PAN rats. Male Sprague‐Dawley rats were separated into 3 groups: Control (saline vehicle), LD (25mg/kg BW initial, 10mg/kg boosters), and HD (50mg/kg BW initial, 20mg/kg BW boosters). Both LD and HD rats exhibited increased blood pressure (BP) and glomerular injury (GSI) with decreased urine NOx. HD rats had a fall in GFR (Ccr, Table, n=8,*p<0.05 vs. con. +p<0.05 vs. LD). KC PRMT‐1 abundance increased with HD, and KC DDAH activity progressively decreased with LD and HD. The fall in L‐Arg/ADMA ratio likely contributes to reduce NO production in HD PAN induced CKD, by both increased formation (increased PRMT‐1 abundance) and decreased metabolism (decreased DDAH activity).

BP (mmHg)
GSI
Urine Nox (μmol/day)
Ccr (ml/min/100gBW)
L‐Arg/ADMA
KC PRMT‐1 (IOD/Pos/Pons)
KC DDAH Activity (Cit/g protein/min)

Control
100±2
0.11±0.02
2.62 ±0.28
0.73±0.07
283±24
100±16
0.62±0.05

LD PAN
116±5*
1.0±0.3*
0.37 ±0.21*
0.66±0.09
248±15
241.±31
0.39±0.04*

HD PAN
134±8*
2.2±0.3*+
0.38 ±0.21*
0.16±0.07*+
159±30*
326±57*
0.10±0.06*+
Effect of diet and age on expression of renal NOS isoforms and splicing in female Zucker rats
Authors: Slyvka Yuriy , Inman Sharon R , Nowak Felicia V
Published: June 2021

The underlying hypothesis is that changes in renal oxidative status and nitric oxide synthases (NOS) that occur with progression of nephropathy in obesity‐related Type 2 diabetes mellitus (T2bDM) will be altered in response to an antioxidant‐fortified (AO) diet. Female obese Zucker rats (fa/fa), a model of T2bDM, were studied at 6, 13 and 20 weeks of age, on a regular (REG) or AO diet. Glomerular filtration rate (GFR) was measured and NOS isoforms (n‐neuronal, e‐endothelial and i‐inducible) analyzed with Western blots. The AO diet resulted in better preservation of GFR in 20 week animals. In rats on REG diet eNOS, iNOS and nNOS were highest at 13 weeks, while rats on the AO diet showed significantly decreased levels at this time point. Thus levels of all three isoforms were decreased at 13 weeks in animals on the AO diet compared to animals on the REG diet. This may indicate a protective effect of AO diet at earlier stages of nephropathy. Multiple monomeric splice forms, up to 8 for eNOS and iNOS, and 5 for nNOS were detected; 4–5 dimeric forms were also detected for each isoform. Increased prevalence of active monomers and dimers and detection of a greater number of splice variants were observed in the AO diet groups at 6 and 20 weeks.

Conclusion

AO diet has a protective effect on renal function in females that may be due, at least in part, to changes in NOS protein expression and activity. Supported by NIH R15 DK073066 to SRI and FVN.
Expression of the aquaglyceroporin HC3 and morpholino delivery in Hyla chrysoscelis erythrocyte cultures
Authors: Mutyam Venkateshwar , Puccetti Matthew V. , Frisbie James , Goldstein David L. , Krane Carissa M.
Published: June 2021

Cold acclimation in Cope's gray treefrog, Hyla chrysoscelis entails changes in handling and distribution of water and glycerol with concomitant changes in aquaglyceroporin protein expression. We hypothesize that HC3, an ortholog of mammalian aquaporin 3, enhances membrane permeability to glycerol which acts as a natural cryoprotectant to regulate osmotic gradients formed by extracellular ice crystal formation during freezing. In this study, H. chrysoscelis erythrocytes cultured in complete cell media exhibited 85% viability after 72 hrs at 20°C. After 24 hrs, HC3 protein expression was reduced by 92%. HC3 membrane localization was confirmed by immunocytochemistry. HC3 expression increased by 1.7 fold in erythrocytes cultured for 24 hrs in media made hyperosmotic (400 mOsM) by the addition of glycerol as compared to control. Since no specific HC3 channel inhibitors exist, a morpholino‐mediated expression knockdown approach was optimized. Efficient uptake of 10 μM of fluorescein‐labeled control morpholino with 6 uM of the endosomal delivery vehicle Endo‐Porter (GeneTools®) was observed by fluorescent microscopy, with no evidence of compromised cell viability. These results show that HC3 protein expression in erythrocyte cultures from H. chrysoscelis can be induced by hyperosmolarity and potentially modulated by morpholino knockdown. This research was supported by NSF Research Grant IOB‐0517301.
Adherens junction proteins in glomerular podocytes of quail kidney
Authors: Yaoita Eishin , Karasawa Tamaki , Nameta Masaaki , Yamamoto Tadashi , Uchiyama Makoto , Nishimura Hiroko
Published: June 2021

Avian kidney glomerular filtration rate linearly relates to body mass, but glomeruli (diameter, μm) vary in size from small (12–15) to large (25–30). Intercellular junctions of glomerular podocytes are important for filtration function, but their localization and properties, especially those of adherens junctions (AJs), are not well characterized in birds. Quail glomeruli contain podocytes with numerous foot processes and slit diaphragms. We investigated whether glomerular podocytes of Japanese quail, Coturnix japonica (3–12 wks of age), express AJ proteins. We found: 1) podocin, N‐cadherin and alpha‐ and beta‐catenins are prominent in fractions from isolated glomeruli (immunoblot analysis) and are localized along glomerular capillary walls (immunofluorescence microscopy); 2) quail podocytes have at least two types of intercellular junction, slit diaphragms and junctions with close contact (electron microscopy); and 3) in both junction types, significant accumulations of immunogold particles for N‐cadherin and alpha/beta‐catenins are visualized. The results suggest that podocyte intercellular junctions have AJ properties and that they may play a role in supporting the glomerular capillary wall against high intraglomerular pressure. (Grant‐in‐Aid for Scientific Research from the Japanese Ministry for Education, Science, and Culture 21591021 to Yaoita and 21390307 to Nishimura)
Extracellular chloride modulates the desensitization kinetics of acid‐sensing ion channel 1a (ASIC1a)
Authors: KUSAMA NOBUYOSHI , Benson Christopher J
Published: June 2021

Acid‐sensing ion channels (ASICs) have Cl−‐binding sites in the extracellular domain that are highly conserved between ASIC isoforms. In this experiment, we investigated the effect of Cl−‐substitution on heterologously expressed ASIC1a current and ASIC current in hippocampal neurons by whole‐cell patch‐clamp method.

Substitution of Cl− with the impermeable and inert anion, methansulfonate (MeS) did not alter the maximal current amplitude, pH sensitivity or steady‐state desensitization. However, the rate of desensitization was faster in MeS than in Cl−, and the tachyphylaxis of ASIC current was attenuated in MeS. Other anions (thiocyanate, Br− and I−) also modified the kinetics of desensitization and tachyphylaxis.

Next, we studied the effect of mutation of Cl−‐binding sites. The pH sensitivity of mutants (K211A, R309A, E313A) was similar to that of wild type channel. However, these mutants showed a faster desensitization and an attenuated rundown. These mutations abolished the effect of anion substitution.

Finally, we investigated the effect of Cl− substitution in hippocampal neuron. The modulation of the ASIC1a current in hippocampal neuron was similar to that in heterologously expressed ASIC1a.

In addition to sensing pH changes, modulation of ASICs by anion fluxes associated with pathological conditions further positions the channels to sense changes in the extracellular environment.
Voltage‐gated chloride channels in storage cells of the anhydrobiotic eutardigrade Richtersius coronifer
Authors: Willumsen Niels J , Jensen Christel Barker , Ramløv Hans
Published: June 2021

Tardigrades comprise polyextremophilic species that can survive in an anhydrobiotic state for years. Anhydrobiosis may be dependent on properties of the free floating storage cells found in the body cavity of all tardigrades. We studied ion channels in the storage cell membrane with patch clamp technique, using native cells from the eutardigrade Richtersius coronifer.

In cell‐attached configuration the most frequently encountered ion channel had a linear i/V‐relationship with a reversal potential (Vrev) of ~0.4 mV (n ≥6). The single‐channel conductance was 82.4±0.0 pS (mean±SE, n≥6). Binomial analysis of patches with 2–4 active channels showed stochastic behavior. The open‐state probability increased with the pipette potential, indicating voltage‐gating. In whole‐cell configuration the currents showed outward rectification with Vrev = −18.2±1.1 mV (mean±SE, n=24), closest to the equilibrium potential for Cl−. Perfusion with Na‐gluconate Ringer right‐shifted Vrev from −19.5±1.9 to 4.6±1.3 mV, and the current decreased significantly (mean±SE, n=5), confirming that a significant component of the whole‐cell current is attributed to Cl− channels. This was verified by perfusion with KCl Ringer (n=5) and addition of BaCl2 (n=7), neither of which significantly changed Vrev. Future studies are needed to firmly establish the identity and function of the demonstrated ion channel in storage cells.
Evaluation of a mathematical model of the acid‐base chemistry of blood
Authors: Rees Stephen Edward , Klæstrup Elise , Handy Jonathan , Andreassen Steen , Kristensen Søren Risom
Published: June 2021

The objective of this study is to evaluate whether a mathematical model of acid‐base chemistry of blood [1,2] can simulate physiological situations such as the mixing of blood with different acid‐base status. To do so, two samples of venous blood from 21 normal subjects and 6 patients were equilibrated at different O2 and CO2 partial pressures and then mixed. The mathematical model was used to simulate this mixing and model‐simulated and measured values compared. Model simulations compared well with measured values with a small bias and standard deviation, within normal analytical variability, e.g. for normal subjects 0.000±0.006 pH, −0.06±0.11 kPa PCO2, −0.1±0.8 % SO2 and −0.02±0.13 kPa PO2. This model is the first based on a mass‐balance, mass‐action formulation which can perform simulations of this type, including both hemoglobin buffering and the competitive binding of O2, CO2 and H+ on hemoglobin.
In Xenopus oocytes, stimulation of the electrogenic Na/HCO3 transporter NBCe1 by IRBIT can be explained by relief of transporter autoinhibition and is unaffected by endogenous phosphatases.
Authors: Lee Seong‐Ki , Parker Mark , Boron Walter
Published: June 2021

Other have shown in Xenopus oocytes that NBCe1 lacking an amino‐terminal (Nt) autoinhibitory sequence has a ~3‐fold greater transport activity than full‐length NBCe1. In a separate study, it was reported that co‐expression of full‐length NBCe1 with its Nt binding partner IRBIT results in a ~7‐fold stimulation of transporter activity. At face value these data suggest that the stimulatory action of IRBIT cannot be explained solely by relief of autoinhibition. However, these data pertain to different splice variants of NBCe1.

In the present study, we work only with the pancreatic splice form NBCe1‐B. We find that, compared to oocytes expressing full‐length NBCe1‐B alone, oocytes expressing (1) a mutant NBCe1‐B that lacks the autoinhibitory sequence manifest a 7.5‐fold greater transporter activity (2) full‐length NBCe1‐B plus IRBIT manifest a 5.2‐fold greater transporter activity.

Oocytes have endogenous protein phosphatase 1 (PP1) and PP1 is reported to deactivate IRBIT following binding at a non‐consensus PP1‐binding site ‘KQIQF’ in IRBIT. Thus we hypothesized that the full stimulatory effect of IRBIT was masked by the action of endogenous PP1. However, we find that removing the putative PP1 binding site in IRBIT does not result in a substantial increase in NBCe1‐B stimulation. Conversely an IRBIT in which we replaced ‘KQIQF’ with a well characterized PP1‐binding consensus sequence of the type ‘RVxF’ was unable to stimulate NBCe1‐B activity.

In conclusion, our findings are consistent with a model in which the action of IRBIT is explained solely by relief of autoinhibition. The results are not confounded by endogenous oocyte PP1 activity, because the endogenous PP1 has no detectable functional interaction with the ‘KQIQF’ motif in overexpressed IRBIT.
Differential intracellular localization of lysyl oxidase in vitro and in vivo
Authors: Okkelman Irina Alexandrovna , Pestov Nikolay Borisovich , Fedotova Anna Sergeevna
Published: June 2021

Lysyl oxidase is a copper‐dependent amino oxidase that plays an important role in protein cross‐linking. LOX is usually secreted into extracellular space but LOX was also found in the nucleus of many cell types including smooth muscle cells and 3T3 fibroblasts. Using immunocytochemical and immunohistochemical techniques, we compared subcellular localization of LOX in various cells cultured in vitro as well as in different tissues. Endogenous LOX was detected predominantly in nuclear dot‐like structures of cultured cells: NIH 3T3 fibroblasts, Hela, epithelial derived lung cancer cell lines A549 and CALU‐1, and also in primary embryo fibroblasts. In contrast with these results obtained on in vitro cultured cells, such a striking pattern of intracellular distribution was never observed in various histological samples. Interestingly, LOX appears to be abundant in platelets and neutrophils and, in the latter case, LOX staining was also completely extranuclear. In addition, overexpression of recombinant LOX in Hela cells leads to its accumulation outside the nucleus. Therefore, abundant nuclear localization of LOX protein in cultured cells in vitro may be not relevant to its distribution in vivo. Supported by MCB Program of the Russian Academy of Sciences.
Gene expression profiling and immune fluorescence implicate cellular redifferentiation induced by hypertonicity in primary cultured IMCD cells
Authors: Edemir Bayram , Klokkers Jens , Pavenstädt Hermann , Schlatter Eberhard
Published: June 2021

The cells of the renal inner medulla are challenged with an environment consisting of high osmolality mediated mainly by Na+ and urea. High tonicity leads to the activation of the tonicity enhancer binding protein, which induces the expression of several transporters and enzymes. This in turn leads to the cellular accumulation of organic osmolytes thereby protecting the cells from high extracellular tonicity. Although several genes that are induced by tonicity are known, a global gene expression profile and analysis of pathways is missing. We used primary cultured rat renal inner medullary collecting duct cells which were cultivated for 6 days at 300, 600 or 900 mosmol/kg. Total RNA was isolated and prepared for hybridization on Rat Gene 1.0 ST GeneChip. Differentially expressed genes were identified using the BRB Array Tools and functional molecular network were identified with Ingenuity Pathway Analysis.

Comparing cells grown in 300 vs. 600 mosmol/kg, more than 1600 and vs. 900 mosmol/kg more than 3300 genes were differentially expressed. In both cases the ratio between the up and down regulated genes was approximately one. While 1300 were common in both comparisons 290 at 600 and more than 2000 genes at 900 mosmol/kg were unique. Besides known genes the expression of several other genes were identified for which regulation by tonicity had not been described before. Cellular morphology was markedly modified with increases in culture medium tonicity. Tonicity induced actin reorganization and morphological changes at the cell to cell contacts. It also induced a hypertrophic phenotype. The adaptation to a higher tonicity is not only associated with massive changes in gene expression but also with changes in cell morphology. This implicates that tonicity induces cellular redifferentiation.
Mre11 expression in mitochondria of renal cells
Authors: Dmitrieva Natalia I. , Malide Daniela , Burg Maurice B.
Published: June 2021

Mre11 is a key component of the nuclear DNA damage repair network. Nevertheless, thermal stress causes translocation of some of the nuclear Mre11 into the cytoplasm, and we previously found that high NaCl does also. The location of the Mre11 in cytoplasm has been unknown. Noting that Mre11 was identified in yeast mitochondria, we searched for Mre11 in mitochondria of mammalian cells under various conditions. Mre11 is present in the mitochondria isolated from mIMCD3 cells (Western blot), and the amount in the mitochondria increases when NaCl bathing the cells is elevated. In non synchronized primary kidney cells Mre11 forms foci that colocalize with mitochondria (Confocal microscopy). When the primary kidney cells are grown in Matrigel (BD Biosciences), they differentiate and form tubules. In non differentiated cells Mre11 is mostly in the nucleus, but it is mostly in the cytoplasm in cells that have formed tubules. In sections from mouse kidney we find abundant MRE11 colocalized with mitochondria in the cytoplasm of mitochondrial rich proximal tubule and thick ascending limb cells. In collecting ducts, which contain fewer mitochondria, MRE11 is concentrated in nuclei. We conclude that 1) Mre11 localizes in mitochondria as well as in the nuclei of renal cells and 2) its distribution varies depending on the stress to which the cells are exposed and on their differentiation. Supported by the Intramural Program of NHLBI.
Contribution of SHP‐1 protein tyrosine phosphatase to osmotic regulation of the transcription factor TonEBP/OREBP
Authors: Zhou Xiaoming , Gallazzini Morgan , Burg Maurice B. , Ferraris Joan D.
Published: June 2021

Hypertonicity activates the transcription factor TonEBP/OREBP resulting in increased expression of osmoprotective genes, including those responsible for accumulation of organic osmolytes and heat shock proteins. Phosphorylation of TonEBP/OREBP contributes to its activation. Several of the kinases that are involved have been identified, but the phosphatases have not. By screening a genome‐wide human phosphatase siRNA library in HEK293 cells, we found that siRNAs against the protein tyrosine phosphatase SHP‐1 increases tonicity‐dependent TonEBP/OREBP activity, implying that SHP‐1 normally reduces that activity. We further studied in detail SHP‐1. We confirmed that SHP‐1 is inhibitory by over expressing it, which reduces TonEBP/OREBP transcriptional activity at 500 mosmol/kg. SHP‐1 dephosphorylates TonEBP/OREBP at a known regulatory site, Y143, both in vivo and in vitro. It inhibits TonEBP/OREBP by both reducing TonEBP/OREBP nuclear localization, which is TonEBP‐Y143‐dependent, and by lowering high NaCl‐induced TonEBP/OREBP transactivating activity. SHP‐1 coimmunoprecipitates with TonEBP/OREBP and vice versa, suggesting that they are physically associated in the cell. High NaCl inhibits the effect of SHP‐1 on TonEBP/OREBP by increasing phosphorylation of SHP‐1 on Ser591, which reduces its phosphatase activity and localization to the nucleus. Thus, SHP‐1, whose inhibition by high NaCl increases phosphorylation of TonEBP/OREBP at Y143, contributes to the nuclear localization and activation of TonEBP/OREBP.
Phosphorylation May Mediate Normal Trafficking of The Betaine/GABA Transporter (BGT1) During Hypertonic Stress in Renal Medullary Cells
Authors: Anderson Bailey A , Gordon Sashana S , Chaidir Nadia I , Kempson Stephen A
Published: June 2021

BGT1 mediates uptake of the osmolyte betaine in renal medullary cells. During hypertonic (Hyp, 550 mOs) stress BGT1 is synthesized and inserted in the plasma membrane of MDCK cells. The phorbol ester PMA downregulates BGT1 by endocytic removal. To further test if PKC regulates BGT1, mutants of EGFP‐tagged BGT1 were prepared by substituting alanine at consensus sites for PKC phosphorylation and were expressed in MDCK cells. Microscopy showed that mutant T40A remained intracellular during Hyp stress, but substitution of aspartate at T40 did not interfere with the normal trafficking response. Next, native MDCK cells were incubated in Hyp growth medium for 24 h in absence (controls) or presence of PKC inhibitors (staurosporine 0.3 μM or Go6976 10 μM), or PMA (75 nM) for downregulation of PKC. In Hyp controls GABA transport was increased 10‐fold compared to isotonic groups, but the increase was only 3–4 fold when inhibitors or PMA were present (p<0.001, n=3). In contrast, amino acid transport system A was upregulated normally under all Hyp conditions. Westerns of cell lysates showed the abundance of BGT1 protein was not changed by drug treatment. However, consistent with transport data, microscopy revealed a reduction in plasma membrane BGT1 after drug treatment. Data suggest PKC‐dependent phosphorylation at T40 may be required for normal trafficking of BGT1 to the plasma membrane during Hyp stress. AHA 09GRNT2010156.