bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mitochondrial fragmentation is a novel feature of adherent‐invasive E. coli evoked pathophysiology in enteric epithelia
Authors: Mancini Nicole , Wang Arthur , Shearer Jane , McKay Derek 
Published: November 2023

IntroductionMitochondria are not distinct organelles; they exist as a dynamic network that constantly remodels via the processes of fission and fusion to meet the cells energy demands and allow recycling of damaged mitochondria. Mitochondrial dysfunction has been described in inflammatory bowel disease, where adherent‐invasive E. coli (AIEC) have been described as a pathobiont that can drive epithelial apoptosis. Little is known of microbial regulation of enterocytic mitochondrial dynamics and nothing of the potential of AIEC to perturb the critical metabolic function that mitochondria fulfill for the cell.HypothesisAIEC induce mitochondrial fragmentation in intestinal epithelial cells in an invasion‐dependent manner that requires ROS, and drives the loss of epithelial permeability.AimExplore the relationship between mitochondrial dynamics and epithelial function in vitro and determine if, then how, infection with AIEC affects mitochondrial dynamics and any consequence for epithelial barrier function.MethodsHuman colon‐derived epithelial lines were cultured with AIEC or a non‐invasive E. coli and readouts assessed: (a) visualization of mitochondria morphology and membrane potential with confocal microscopy; (b) immunoblotting of whole cell protein extracts for the mitochondrial fusion protein Optic Atrophy Factor 1 (OPA1); (c) ROS neutralized by the antioxidants; (d) treatment with spent medium from bacterial cultures; and, (e) blockade of fission using specific pharmacological inhibitors and measurement of epithelial permeability in response to AIEC.ResultsAIEC infection resulted in reduced mitochondrial membrane potential and ATP, and dramatic mitochondrial fragmentation accompanied by OPA1 cleavage in gut epithelia in a time‐ and dose‐dependent manner. The mitochondrial fragmentation was not reproduced by exposure to AIEC conditioned medium and was not abrogated by treatment with a general (vitamin C) or a mitochondrial‐specific (mitoTEMPO) anti‐oxidant co‐treatment. Invasion of epithelial cells by AIEC and transepithelial resistance were unaffected by co‐treatment with pharmacological inhibitor of mitochondria fission.ConclusionAs a putative cause or contributor to Crohn's disease, AIEC were found to drive massive mitochondrial fragmentation in model gut epithelia independent of ROS generation or AIEC soluble factors. Neither the invasion of the E. coli nor the increase in paracellular permeability they evoke is sensitive to treatment with inhibitors of mitochondrial fission.Support or Funding InformationAcknowledgements: Supported by: CIHR operating grant to DMM, U. Calgary Eyes High Doctoral Studentship to NLM.
Renal Denervation Prevents Nicotine‐Induced Hypertension and Reduces Renal IL‐17a
Authors: Harwani Sailesh C , Ratcliff Jason , Cicha Michael Z , Chapleau Mark W , Abboud Francois M 
Published: November 2023

Interaction between the nervous and immune systems has become a major focus in the pathophysiology of essential hypertension. Cholinergic activation of nicotinic acetylcholine receptors are known to exert an anti‐inflammatory effect. However, we recently demonstrated that in vivo cholinergic activation with nicotine induces renal inflammation and premature development of hypertension in young Spontaneously Hypertensive Rat (SHR). We hypothesized that renal nerves contribute to nicotine‐induced renal inflammation and premature hypertension in young SHR. Following bilateral renal nerve denervation (RND) (or Sham surgery) in young (3–5 week old SHR), we continuously infused nicotine bitartrate (15mg/kg/day) or saline subcutaneously for 2 weeks. Systolic blood pressure was measured by tail cuff. Norepinephrine (NE) and IL‐17a were measured in kidneys by ELISA and Luminex assay, respectively. RND prevented the development of hypertension in response to nicotine infusion in young SHR (n=6), compared to nicotine‐infused Sham operated young SHR (n=3) (140 ± 4 vs 180 ± 5 mmHg, respectively, p<0.001). RND also significantly decreased IL‐17a in kidneys nicotine‐infused SHR, compared with kidneys from sham operated SHR (12 ± 0.7 pg/mL vs 17 ± 0.9 pg/mL, p<0.01). NE analysis of renal tissues confirmed renal denervation with over an 85% reduction in renal NE levels, compared to sham operation (31±11 ng/mg vs 225±43 ng/mg, P<0.05). We conclude that renal nerves mediate nicotine‐induced renal inflammation and hypertension.Support or Funding InformationUniversity of Iowa, Carver College of MedicineNIH/NHLBI‐HL119588‐01A1 and HL014388‐48
The impaired vasodilatation in rat renal interlobar arteries after hypoxia/re‐oxygenation is endothelium independent
Authors: Braun Diana , Ludwig Marion , Persson Pontus Börje , Dietze Stefanie , Patzak Andreas 
Published: November 2023

Renal vasculature plays an important role in renal ischemia/reperfusion injury. We showed recently that hypoxia/re‐oxygenation combined with low dose norepinephrine treatment increases the angiotensin II responses in interlobar arteries. Since angiotensin II activates constrictor and dilator systems we hypothesized an impaired vasodilatation in this context.To test the hypothesis rat interlobar arteries were studied by using wire myography. The dilatory capacity was investigated by application of acetylcholine (10‐9 to 10‐5 mol/l). Hypoxia (95% N2, 5% CO2 for 60 min) alone followed by re‐oxygenation (95% O2, 5% CO2, 10 min) did not affect vasodilatation. However, norepinephrine in a non‐constrictor dose (10‐9 mol/l) applied during hypoxia resulted in a decreased response to acetylcholine. The contribution of the nitric oxide system, COX‐products, and endothelial derived hyperpolarizing factor to reduced dilatation was tested by pretreatment with L‐NAME (10‐4 mol/l), indomethacin (10‐5 mol/l), TRAM‐34 (10‐6 mol/), and UCL1684 (10‐7 mol/l), respectively. L‐NAME decreased the acetylcholine response after hypoxia/re‐oxygenation stronger compared to control conditions, while indomethacin, TRAM‐34, and UCL1684 did not affect it. Endothelium‐independent effects were analyzed by using the NO‐donor SNP and simultaneous blocking of endothelium‐dependent NO production. SNP significantly reduces vasodilation after hypoxia+norepinephrine/re‐oxygenation (H+NE/R). Passive stiffness of arteries was not altered in our experimental setup.H+NE/R provokes an impairment of the dilatory response to acetylcholine in renal arteries. Our data suggest that the endothelial nitric oxide system, COX products, or TRAM‐34 and UCL1684 inhibitable potassium channels do not contribute to reduced dilatation. The results indicate changed dilatory signaling pathways in vascular smooth muscle cells.Support or Funding InformationThis study was financially supported by a grant of the German Research Council to A.P and P.B.P (FG 1368, DFG PA 479/10‐1 and PA 479/10‐2).
Assessment of Protective Effect of Phikud Navakot Extract against Hypoxia/Reoxygenation Injury on H9c2 Cardiomyoblasts
Authors: Gerdprasert Orapin , Nusuetrong Punnee 
Published: November 2023

Phikud Navakot is composed of nine main medicinal plants in Yahom Navakot, which has been used as traditional medicine for treatment of cardiovascular symptoms such as dizziness and fainting. The present study was to investigate the protective effect of the ethanolic extract of Phikud Navakot (PN) against hypoxia/reoxygenation (H/R)‐injured H9c2 cardiomyoblasts. The cytotoxicity of PN (0.001–0.5 mg/mL) on H9c2 cells was measured by MTT assay, showing no cytotoxicity to H9c2 cells. Cardioprotective ability of PN was investigated during exposure to 6‐h hypoxic buffer in ischemic box followed by 24‐h reoxygenation in complete medium in CO2 incubator. The results showed that PN at a concentration of 0.1 mg/mL was able to increase the percentage of cell survival in H/R‐induced cell death when compared with the H/R control, using 6‐hydroxy‐2,5,7,8‐tetramethylchroman‐2‐carboxylic acid as a positive control. PN at the same concentration was also decreased the intensity of reactive oxygen species (ROS) production measured by 2′,7′‐dichlorofluorescin diacetate, when compared to the H/R control. In conclusion, the present results revealed that PN (0.1 mg/mL) was able to decrease H/R‐induced cell injury in H9c2 cardiomyoblasts, which should be partly through inhibition of the intracellular generation of ROS.Support or Funding InformationThis work was supported by research grant from the National Research Council of Thailand (NRCT 2009‐113). We specially thank Dr. Sanya Hokputsa for giving us the extracts. The authors gratefully thank Mr. Weerasak Ussawawongaraya for preliminary of hypoxic‐simulated H9c2 cells.
Identification of Vesicular Monoamine Transporter 2 (vmat2)‐Containing O2 Chemoreceptors in the Gills of Zebrafish
Authors: Pan Wen , Scott Angela L , Nurse Colin A , Jonz Michael G 
Published: November 2023

All vertebrates “sense” changes in oxygen (O2) via specialized cells, called chemoreceptors, associated with the peripheral nervous system. Aquatic vertebrates are especially prone to fluctuations in environmental O2 levels. Thus it is essential for them to readily detect low O2 and make appropriate ventilatory adjustments. Teleost species, such as zebrafish, have O2 chemoreceptive neuroepithelial cells (NECs) embedded in the gill epithelium. These cells exhibit membrane depolarization and vesicular recycling upon exposure to hypoxia, and express the monoamine neurotransmitter, serotonin (5‐HT). Our research objectives are to identify gill O2 chemoreceptors in live tissue preparations and determine the role of intracellular Ca2+ in O2 sensing. We are currently employing a transgenic zebrafish line, expressing green fluorescent protein (GFP) under the control of vesicular monoamine transporter 2 (vmat2) regulatory elements, in order to identify NECs and characterize their hypoxic responses in vitro and in situ. Immunohistochemistry and confocal microscopy were used to determine the distribution and identity of GFP‐positive cells in gills. Our results show that the population of GFP‐positive cells in our transgenic line significantly overlaps with serotonergic NECs in the gill filaments. Preliminary studies using fura 2‐based imaging indicate that isolated GFP‐positive cells stimulated by hypoxia undergo an increase in intracellular Ca2+ concentration. Identification of NECs both in vitro and in situ using a vital marker will have a great impact on future work addressing the physiological properties of these cells.Support or Funding InformationThis research project is funded by Natural Sciences and Engineering Research Council of Canada (NSERC).
Effects of Different Oxygen Concentrations on Human Differentiated Preadipocytes Lipogenic and Lipolytic functions
Authors: MAHAT BIMIT , Mauger Jean‐François , Imbeault Pascal 
Published: November 2023

ContextFat stored in white adipose tissue is the human body's largest energy reservoir. Adipocytes, the predominant cell population in white adipose tissue, play a central role in the clearance of circulating triglyceride (TG) and the delivery of fatty acids for use by peripheral tissues. Impaired regulation of adipose tissue functions is thought to stimulate lipid deposition in non‐adipose tissues, inducing lipotoxic response and the development of metabolic disorders. During weight gain, adipocytes expand in size, decreasing adipose tissue capillary density and creating areas characterized by excessively low oxygen concentrations (hypoxia). It is not clear how hypoxia modulates adipose tissue TG clearance and fatty acid delivery.ObjectiveThe purpose of this study was to investigate the effects of different hypoxia concentrations on lipogenic (store TG) and lipolytic functions (deliver fatty acids) of human differentiated preadipocytes.MethodsDifferentiated human preadipocytes were exposed to different oxygen concentrations (3%, 10%, and 21%) either during differentiation (14d, continuous exposure) or acutely for 24h after differentiation (acute exposure). Gene expression of lipogenic enzymes and regulating factors will be determined by real‐time PCR. Lipoprotein lipase (LPL) activity was measured using the EnzChek Lipase Substrate and lipolytic rate was determined by measuring glycerol release over 3 hours.ResultsIn differentiated human preadipocytes, a dose‐dependent reduction in LPL activity was observed in both acute and continuous hypoxia (6‐fold reduction under 3% O2 p<0.001, 2‐fold reduction under 10% O2, p<0.05, compared to 21% O2). Basal lipolysis was significantly higher during acute hypoxia (3%) session (p<0.05). No differences in isoproterenol‐ and epinephrine‐stimulated lipolytic responses were observed in subcutaneous abdominal preadipocytes between normoxia and acute hypoxia (3%).ConclusionsThese preliminary results suggest that acute and continuous hypoxia strongly inhibits LPL activity and increases basal lipolysis. These observation supports the hypothesis that hypoxia may adversely affect adipose tissue storage functions, which could in turn contribute to ectopic fat storage, lipotoxicity and a greater prevalence of metabolic disorders in individual with excess adiposity.Support or Funding InformationThis study was supported by the Natural Sciences and Engineering Research Council of Canada as well as the University of Ottawa.
Effect of Carotid Sinus Nerve Transection on Ventilation in Juvenile (P25) Male Sprague‐Dawley Rats
Authors: Getsy Paulina M , Coffee Gregory A , Lewis Stephen J 
Published: November 2023

The carotid body chemoafferents play an essential role in altering ventilation in response to hypoxia, hypercapnia, and acidosis. The retropezoid nucleus in the brainstem also plays a key role in responding to hypercapnia. The effects of exposure to hypoxic (HX), hypercapnic (HC) or hypoxic‐hypercapnic (HH) gas challenges have been studied in young, adult, and elderly subjects. However, little is known about the role of the carotid body in the ventilatory responses elicited by these gas challenges in juvenile rats.Our objective was to determine the role of the carotid body chemoafferents in the ventilatory responses elicited by HX (10% O2, 90% N2), HC (5% CO2, 21% O2, 74% N2) or HH (10% O2, 5% CO2, 85% N2) gas challenges (5 episodes of 5 min in duration separated by 15 min) in freely‐moving juvenile (P25) male Sprague‐Dawley rats.Rats (P21) were subjected to sham or bilateral carotid sinus nerve transection (CSNX) and were allowed 4 days to recover before use in the experiments. On the day of study, the P25 rats were placed in whole‐body plethysmography chambers and after allowing them to acclimatize, they were subjected to one of the above gas exposures. Ventilatory parameters included, frequency of breathing, tidal volume, minute ventilation (= frequency × tidal volume), inspiratory and expiatory times, end inspiratory and expiratory pauses, peak inspiratory and expiratory flows, EF50 (expiratory flow at 50% expired tidal volume) and central inspiratory drive (tidal volume/inspiratory time).The HX challenges elicited robust ventilatory responses in sham‐operated rats. As expected, these responses, such as the increase in minute ventilation, were markedly diminished in CSNX rats. The HC challenges also elicited robust ventilatory responses in sham‐operated rats. Some responses, including the increases in minute ventilation and EF50, were slightly smaller in CSNX rats. The HH challenges elicited very robust ventilatory responses. Unexpectedly, these responses including, (1) increases in frequency of breathing, tidal volume, minute ventilation, peak inspiratory and expiratory flows, EF50, and central inspiratory drive, and (2) the decreases in inspiratory and expiratory times, and end inspiratory and expiratory pauses, were dramatically diminished in CSNX rats.These findings demonstrate that the carotid sinus chemoafferents play a major role in integrating the ventilatory responses to hypoxic‐hypercapnic gas challenges in juvenile rats. Considering that these gas challenges alter arterial blood gas chemistry to values seen during apneic episodes, our findings may be relevant to understanding the role of carotid body chemoafferents in sleep‐disordered breathing.Support or Funding InformationGlaxoSmithKline (GSK) FundingCWRU Internal Number: RES510692
Mesenchymal Stem Cell‐Derived Extracellular Vesicles Improve the Renal Microvasculature in Coexisting Metabolic Syndrome and Renovascular Disease in Swine
Authors: Eirin Alfonso , Zhu Xiang‐Yang , Ferguson Christopher M , van Wijnen Andre J , Lerman Amir , Lerman Lilach O 
Published: November 2023

BackgroundSuperimposition of the metabolic syndrome (MetS) on renovascular disease (RVD) amplifies intrarenal microvascular loss. Extracellular vesicles (EVs) released from adipose tissue‐derived mesenchymal stem/stromal cells (MSCs) may mediate their paracrine effect, but their efficacy to protect the renal microcirculation in RVD complicated by MetS remains unclear. Using a novel swine model, we tested the hypothesis that EVs would attenuate microvascular loss in MetS+RVD.MethodsFour groups of pigs (n=7 each) were studied after 16 weeks of diet‐induced MetS and unilateral RVD (MetS+RVD), MetS+RVD treated 4 weeks earlier with a single intra‐renal delivery of pre‐labeled EVs harvested from autologous MSCs, and Lean and MetS Sham controls. Stenotic‐kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were measured in‐vivo (fast CT), whereas EV characteristics, renal microvascular architecture (micro‐CT), and injury pathways ex‐vivo.ResultsmRNA sequencing of EVs and their parent MSCs revealed that EVs were enriched with pro‐angiogenic genes, including vascular endothelial growth factor (VEGF)‐A, VEGF–C, and Von Willebrand Factor (vWF)‐A Domain Containing‐1 (VWA)‐1 (Figure A). In addition, protein expression of VEGF and vWF was higher in EVs compared to MSCs (Figure B). EVs were detected in the stenotic‐kidney ex‐vivo internalized by tubular and endothelial cells. EVs restored renal expression of the pro‐angiogenic factors VEGF, Notch‐1, and Notch ligand delta‐like‐4 (DLL4) and improved cortical microvascular density (Figure C–D). Tubulointerstitial fibrosis increased and RBF and GFR decreased in MetS+RVD vs. MetS, but were restored in MetS+RVD+EV (Figure E–F).ConclusionsIntra‐renal delivery of MSC‐derived EVs bearing pro‐angiogenic properties in swine MetS+RVD restored stenotic‐kidney microcirculation, and in turn its hemodynamics and function. Our study suggests a novel therapeutic potential for MSC‐derived EVs in restoring renal function in chronic experimental MetS+RVD.Support or Funding InformationThis study was partly supported by NIH grant numbers DK73608, DK104273, HL123160, and DK102325, DK106427, the Mayo Clinic Center for Regenerative Medicine, and the Mayo Clinic Mary Kathryn and Michael B. Panitch Career Development Award.
TRPV4 channels contribute to renal myogenic response in neonatal pigs
Authors: Soni Hitesh , Peixoto‐Neves Dieniffer , Adebiyi Adebowale 
Published: November 2023

Myogenic response, a phenomenon in which resistance size arteries and arterioles swiftly constrict or dilate in response to acute elevation or reduction, respectively in intravascular pressure is a key component of renal blood flow (RBF) autoregulation mechanisms. Although it is well‐established that the renal system is functionally immature in newborns, renal autoregulation mechanisms in neonates remain poorly understood. In this study, we investigated the hypothesis that members of the transient receptor potential vanilloid (TRPV) channels are molecular components of myogenic response in newborns. We show that TRPV4 channels are predominantly expressed in neonatal pig preglomerular vascular smooth muscle cells (SMCs). Stretch‐induced intracellular Ca2+ ([Ca2+]i) elevation in distal interlobular artery SMCs and vasoconstriction were diminished by selective TRPV4 channel blocker HC‐067047. HC‐067047 also inhibited pressure‐induced membrane depolarization, [Ca2+]i elevation, and constriction in the microvessels. In anesthetized and mechanically ventilated neonatal pigs, step increases in arterial pressure up to 120 mmHg induced efficient autoregulation of RBF and cortical perfusion. Furthermore, intrarenal infusion of TRPV4 channel blockers HC‐067047 and RN‐1734 attenuated renal autoregulation in the pigs. These data suggest that renal myogenic autoregulation is functional in neonates. Our findings also indicate that TRPV4 channels are mechanosensors in neonatal pig preglomerular vascular SMCs, and contribute to renal myogenic autoregulation.Support or Funding InformationNIH R01 DK101668
Characterization of pHi Regulation by NBCe1/SLC4A4 Variants with Known Clinical Phenotypes in Human Immortalized Trabecular Meshwork Cells with a Red‐Shifted Genetically‐Encoded pH‐Indicator “pHire”
Authors: Rossano Adam J , Caldas Font Paola N. , Holmes Heather L , Romero Michael F 
Published: November 2023

The electrogenic Na+/HCO3− cotransporter (NBCe1/SLC4A4) maintains systemic acid/base balance by reabsorbing HCO3− in the renal proximal tubule but also contributes to intracellular pH (pHi) regulation in the eye, nervous system, pancreas, and intestine. Human recessive mutations lead to diverse phenotypes (glaucoma, proximal renal tubular acidosis, poor dentition) and mouse models suggest some organ manifestations are partially penetrant. The objective of this study was to develop a full spectrum genetically‐encoded live‐imaging toolkit which could be adapted to assay NBCe1‐mediated pHi regulation across a variety of cell types to further characterize how NBCe1 recessive mutations and polymorphisms functionally interact with each other and wild‐type (WT) NBCe1 to produce alterations in cellular acid/base physiology. WT human NBCe1 (WT‐NBCe1) and a recently characterized human recessive mutation (Mut‐NBCe1) were tagged with GFP. Each construct was co‐expressed via transfection in human immortalized trabecular meshwork cells (TM5) with “pHire”, a red‐shifted (556/630nm ex/em) pH‐sensitive variant of mApple, to assay NBCe1 function through real‐time quantification of pHi via widefield epifluorescent live‐imaging. pHire successfully reported pHi of TM5 cells in response to H+ loading with NH4Cl pulses. Calibration of pHire revealed an in vitro pKa of 7.44, a 5‐fold linear fluorescence response from pH 6.5 to 8.0, and stability following acid loads (pH 5.0) and long imaging protocols (30 min). pHi of TM5 cells fell in response to CO2/HCO3−— buffered solution and displayed a modest Na+‐dependent recovery (dpHi/dt = 0.02 ± 0.01 pH units min−1 in CO2/HCO3−, −0.06 ± 0.02 in Na+‐free), consistent with intrinsic TM5‐cell NBCe1 activity. Transfection of WT‐NBCe1 did not consistently alter HCO3− recovery but accelerated pHi decrease in Na+‐free solution by 10‐fold (1017 ± 3%) relative to neighboring untransfected cells. Transfection with E91R‐NBCe1 failed to similarly potentiate acidification upon Na+ withdrawal (117 ± 81%), and transfection with Mut‐NBCe1 suppressed Na+‐dependent acidification to 72 ± 28% of untransfected cells. These data suggest that although both Mut‐NBCe1 and E91R‐NBCe1 are functionally “transporter‐null” they possess different capacities to impair endogenous WT‐NBCe1, possibly through dimerization and localization deficits. Such differences could manifest discretely in specific cell types and may underlie the partial penetrance of some NBCe1‐associated phenotypes.Support or Funding InformationFunding: T32‐DK007013 to AJR, DK092408, DK100227 and R25‐DK101405 to MFR.