bluuebunny/crossref_metadata_2025 huggingface dataset:
Gene Expression Profiling in Male Mice with Kidney Specific KO of the Circadian Gene Bmal1
Authors: Masten Sarah Howland , Crislip Gene Ryan , Douma Lauren , Matheny Irma Jeanette , Holzworth Meaghan , Cheng Kit‐Yan , Esser Karyn , Wingo Charles , Gumz Michelle L
Published: June 2021

Nearly all living organisms contain an internal circadian clock that regulates physiological functions. This mechanism is regulated by isoforms of four core transcriptional factors: Cryptochrome (CRY), Period (PER), CLOCK, and BMAL. Every clock gene knockout to date affects blood pressure, highlighting the importance of circadian proteins in physiological homeostasis. Recently we found that kidney‐specific BMAL1 KO mice (KS‐BMAL KO) exhibit a lower blood pressure at baseline when compared to their littermate controls. The goal of this study was to explore potential mechanisms that contribute to this phenotype. Because BMAL1 functions as a transcription factor, we focused on changes in mRNA expression.

Our previous data with kidney‐specific PER1 KO mice revealed changes in expression of genes related to renal sodium reabsorption (Douma et. al, 2018). Thus, we hypothesized that KS‐BMAL KO mice may also have differences in the expression of renal sodium handling genes compared to control mice.

Kidneys were collected from male kidney‐specific cadherin Cre+ (KS‐BMAL KO) and Cre‐(control) mice at noon and midnight. RNA was isolated and converted to cDNA using reverse‐transcriptase with random hexamers. Finally, a candidate gene approach was used with quantitative RT‐PCR to compare relative gene expression between control and KS‐BMAL KO mice at noon and midnight.

After measuring gene expression for numerous sodium transporters, we found none were differentially regulated between KS‐BMAL KO and control mice. Some of these genes, such as NCC and aENaC, did exhibit significant changes in expression between noon and midnight (~20% higher expression at noon vs midnight, n=6 at noon, n=4 at midnight, P<0.05). However, in noon samples, KS‐BMAL KO mice exhibited a significant 50% increase in ATP12A, the transcript that codes for the catalytic subunit of the non‐gastric H+/K+ ATPase (p<0.01, n=6). Additionally, other potassium channels were differentially regulated, including ROMK, a voltage gated potassium channel, which was increased by 85% in KS‐BMAL KO mice. (p<0.05, n=4).

Unlike the KS‐PER1 KO mice, KS‐BMAL1 KO mice do not appear to have differences in expression of sodium handling genes. Rather, alterations in the intra‐renal mechanisms of potassium handling may play a critical role to reduce systemic blood pressure.

Support or Funding Information

NIH/NIDDK 1R01DK109570, The Gatorade Trust through the UF Department of Medicine

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intrarenal Metabolite (α‐Ketoglutarate) Signaling Pathway Is Activated by Hnf1α‐Mediated Transcriptional Regulation of OAT10
Authors: Grimm P. Richard , Wellling Paul A
Published: June 2021

We recently discovered an unusual pathway that couples alterations in intermediate metabolism to salt retention. We found that thiazide diuretics or genetic loss of NCC phosphorylation (SPAK KO) or hypokalemia with intravascular volume contraction activates the synthesis and secretion of α‐ketoglutarate (αKG) from the proximal tubule (PT). αKG is then delivered in the tubular fluid to the distal nephron where it activates its cognate G‐protein coupled receptor, OxGR1, and this stimulates a salt reabsorption network (Pendrin/NDCBE) as a part of a homeostatic response that limits urinary sodium and potassium loss. Hypokalemia appears to stimulate αKG synthesis, whereas volume contraction stimulates αKG transport through angiotensin II (AngII). In the present study, we explore the mechanism by which αKG secretion is activated.

Gene profiling studies identified OAT10 (Slc22a13) as an upregulated gene in each of the hypokalemic volume contraction models. As assessed by quantitative immunofluorescence confocal microscopy, OAT10 localized to the PT, and was increased at the apical membrane in SPAK null and thiazide treated WT mice relative to WT control mice. In LLC‐PK1 cells (porcine PT model), we found AngII (100nM) was sufficient to increase OAT10 transcript abundance, and protein localization at the apical membrane. siRNA mediated knockdown of OAT10 resulted in a proportional decrease in αKG secretion. Taken together, the results indicate that OAT10 is regulated to stimulate αKG secretion in response to hypokalemic volume contraction.

Activation of OAT10 gene expression is paralleled by up‐regulation of 7 other PT genes that transport amino‐acid substrates for αKG synthesis. In silico promoter analysis (1500bp from start codon) identified Hnf1α as a candidate transcriptional regulator of all these transport genes. Treating LLC‐PK1 cells with AngII decreased phosphorylated Hnf1α and increased its nuclear localization. Luciferase assays using the porcine and mouse Slc22a13 promoter verified that Hnf1α mediates AngII‐stimulated transcription of OAT10 gene.

Collectively these data support OAT10 as a mediator of regulated PT αKG secretion and demonstrate that AngII stimulates Slc22a13 transcription via Hnf1α.

Support or Funding Information

Support was provided to PAW from NIH (DK63049, DK54231, and DK093501) and to PRG from the National Kidney Foundation of Maryland.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery and characterization of a novel class of phenylsulfonylpiperazine containing compounds as inhibitors of the Aedes aegypti Kir1 (AeKir1) potassium channel
Authors: Mccarthey James Bryson , Aretz Christopher D , Morwitzer Melody D , Sanford Austin G , Hogan Alicia M , Portillo Madeline V , Kharade Sujay V , Kramer Meghan , Rusconi Renata , Piermarini Peter M , Denton Jerod S , Hopkins Corey R
Published: June 2021

Vector‐borne diseases such as Zika virus, dengue fever and chikungunya virus are transmitted by infected Aedes aegypti female mosquitoes and affect a large portion of the world's population. The use of neurotoxic insecticides to reduce mosquito populations has led to the emergence of genetically resistant strains of mosquitoes over the last several decades, prompting us to explore other physiological targets that might be suitable for insecticide development. Inward rectifier potassium (Kir) channels play essential roles in the physiology of the mosquito Malpighian (renal) tubule and represent “druggable” targets that can be exploited to inhibit renal excretory function. We have previously reported on the discovery of various scaffolds that are active against the Aedes aegypti Kir1 (AeKir1) channel. Herein we report the synthesis and biological characterization of a new 2‐nitro‐5‐(4‐(phenylsulfonyl) piperazin‐1‐yl)‐N‐(pyridin‐4‐ylmethyl)anilines scaffold as an inhibitor of the AeKir1 channel. This new scaffold is more potent in vitro compared to the previously reported scaffolds, in addition, the molecules kill mosquito larvae.

Support or Funding Information

This work is funded by National Institutes of Health grant 1R21 AI128418‐01A1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Increased vasopressin secretion during preeclampsia despite normal plasma osmolality
Authors: Zhang Shao Yang , Ray Alyssa T , Lewis Emma , Deng Guorui , Sandgren Jeremy A , Scroggins Sabrina M , Xue Jiarui , Pearson Nicole A , Zepeda‐Orozco Diana , Sigmund Curt D , Santillan Mark K , Pierce Gary L , Santillan Donna A , Grobe Justin L
Published: June 2021

Preeclampsia (PE) is a cardiovascular disorder that affects 4–7% of pregnancies and typically involves new onset hypertension, proteinuria, and edema. Diagnosis of the disorder is formally performed in the second half of gestation based upon clinical signs, but we and others have discovered that maternal plasma levels of copeptin (CPP, the stable C‐terminal fragment of arginine vasopressin, AVP) are elevated already in the first trimester and are highly predictive of the development of PE. Further, chronic low‐dose AVP infusion is sufficient to model almost all the clinical features of PE in pregnant mice. Our objective is therefore to understand the cause of elevated AVP secretion in human PE. Previous studies indicate that blood volume is reduced during PE, but no change in osmolality has been noted. We therefore hypothesized (i) that plasma AVP levels are elevated in early gestation of pregnancies that later develop PE, and (ii) that elevated AVP and CPP secretion during PE is secondary to altered volume regulation. Blood samples (1st and 2nd trimester) from women who later developed PE or represent clinically‐matched case controls were obtained from the Maternal Fetal Tissue Bank (IRB#200910784) at the University of Iowa. Consistent with our first hypothesis, plasma levels of AVP, assessed by ELISA after Sep‐Pak C18 reversed solid phase extraction, were increased in 1st and 2nd trimester from women who later developed PE (25.5±3.6, n=63 vs 52.1±22.2, n=5 pg/mL, mean±SEM, p=0.01) despite no change in osmolality (279.6±0.7, n=46 vs 277.5±1.2, n=4 mOsm/kg H2O). Plasma sodium was increased in 1st and 2nd trimesters before the onset of PE (133.9±0.6, n=91 vs 138.5±0.6, n=6 mmol/L, p<0.05), but potassium was unchanged (4.13±0.05 vs 4.38±0.14 mmol/L). These findings support the conclusions that (i) AVP levels are indeed increased in early pregnancy before the onset of clinical symptoms of PE, as was suspected based upon previous studies of CPP; that (ii) the defended osmolality set‐point is similar between control and PE pregnancies, and therefore chronically elevated osmolality is unlikely to be the major cause of elevated AVP secretion during PE, and that (iii) offsetting changes in concentrations of individual osmolytes may be present during PE.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Luteolin protects human glomerular endothelial cells from TNF‐α‐induced endothelial dysfunction by attenuating ET‐1 and ROS production
Authors: Yang Weiwei , Bradshaw Jessica , Bakrania Bhavisha , Duncan Jeremy , Satchell Simon , Spradley Frank , Granger Joey , Rana Sarosh
Published: June 2021

Preeclampsia (PE) is a pregnancy‐specific syndrome that affects 5~8% pregnancies and is a leading causes of maternal and neonatal morbidity and mortality, especially in low‐income and middle‐income countries. Although the underlying mechanism remains unclear, PE is associated with maternal endothelial dysfunction characterized by elevated endothelin (ET‐1) and reactive oxygen species (ROS). Tumor necrosis factor‐α (TNF‐α) has been reported as one of the regulators of endothelial dysfunction and this inflammatory factor is elevated significantly in the placenta of patients with PE. Recently, luteolin has drawn wide scientific attention due to a number of health benefits and reduced risk of several diseases, including hypertension and cardiovascular disease. However, whether luteolin might have therapeutic potential in PE is unknown. We tested the hypothesis that luteolin protects endothelial cells against TNF‐α by reducing ET‐1 and ROS production. We pretreated immortalized human glomerular endothelial cells (hGEnCs) with luteolin for 1 hour before recombinant human TNF‐α exposure. We found that exposure of hGEnCs to TNF‐α for 24 h significantly increased the release of ET‐1 by 1.9 fold (3.10±0.70 pg/mL vs 1.07±0.20 pg/mL, p<0.05). Pretreatment with luteolin significantly attenuated TNF‐α‐induced ET‐1 release in a dose‐dependent manner, in particular, 20μM luteolin completely attenuated the effect of TNF‐α on ET‐1 production (3.10±0.70 pg/mL vs 0.56±0.08 pg/mL, p<0.01). We further utilized 2',7'‐dichlorodihydrofluorescein diacetate (H2DCFDA) assay to assess whether luteolin has the potential to prevent TNF‐α‐induced ROS in hGEnCs. We found that luteolin significantly ameliorated the effect of TNF‐α on ROS generation. Pretreatment with luteolin at physiologically relevant concentrations (2 μM) significantly decreased TNF‐α‐induced ROS production within cells (fluorescent intensity: 1795±89.26 vs 1237±98.29, p<0.01), while at 20μM luteolin, TNF‐α‐induced ROS was markedly inhibited by 66% (fluorescent intensity: 1795±89.26 vs 618.5±40.81, p<0.01). Taken together, our preliminary data suggest that luteolin significantly attenuates TNF‐α‐induced ET‐1 release and ROS generation.

Support or Funding Information

81601318, P01HL051971, P20GM104357, T32HL105324, 18POST33990293, HL130577

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Medullary Histone Deacetylase Dependent Regulation of Fluid‐Electrolyte Homeostasis During High Salt Feeding
Authors: Hyndman Kelly A , Speed Joshua S , El‐Dahr Samir , Olson Eric , Pollock David M , Pollock Jennifer S
Published: June 2021

Histone deacetylase enzymes (HDACs) regulate gene transcription through epigenetic modification of chromatin structure. HDAC inhibitors (HDACi) are being examined as anti‐inflammatory and anti‐fibrotic therapies in cardiovascular disease. However, adverse events of HDACi use include hyponatremia, hypokalemia, and hypertension suggesting that HDACs are involved in regulation of fluid‐electrolyte balance. Thus, we sought to define the renal physiology and pathophysiology role of HDACs to understand mechanisms of these potentially fatal side effects. We found that class I HDACs are expressed in the human and rat kidney, and that inner medullary HDAC1 abundance is increased 4‐fold with high salt feeding in normotensive male rats. During high salt loading, inhibition of renal medullary class I HDACs with chronic intramedullary interstitial infusion of MS275 (1 mg/kg/day) resulted in a significant increase in polyuria (vehicle = 34.4 ±2.0 ml/day, MS275 = 49.8 ±5.0 ml/day, n = 5, P = 0.012) and decreased plasma potassium (vehicle = 4.9 ±0.2 mmol/L, MS275 = 3.9 ±0.13 mmol/L, n = 5, P = 0.0046). Furthermore, HDACi led to changes in the homeostatic mechanisms of fluid‐electrolyte balance including renal nitric oxide deficiency (vehicle = 9.6±2.7 mmol NOx/day, MS275 = 3.1±0.66 mmol NOX/day, n = 5, P = 0.033). We generated doxycycline‐inducible, collecting duct specific, HDAC1 and HDAC2 knockout mouse (iCDHDAC12KO). In male iCDHDAC12KO mice on a high salt diet, there was significant polyuria (control = 12.9±1.8 ml/day, KO 26.1±5.6 ml/day, n=4–5, P =0.04) and reduced urinary NOX(control = 0.9±0.2 mmol/day, KO = 0.3±.06 mmol/day, n=4–5, P = 0.04). Our findings point to a mechanism whereby HDAC1 promotes NO synthase expression through epigenetic regulation of chromatin structure at the Nos1and Nos3promoters. Given the promising research of HDACi to treat cardiovascular disease, this study provides insight on the mechanisms of the adverse events, which may be fatal to critically ill patients.

Support or Funding Information

NIH NIDDK K01DK105038 and the UAB Pittman Scholarship to KAH, NHLBI HL127178 to JSS, and P01 HL123267 to DMP and JSP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysine Specific Demethylase‐1 Deficiency Accelerates the Development of Hypertension and Renal Damage During Long Term Sodium Intake
Authors: Katayama Isis Akemi , Cullinane Danielle L , Williams Gordon H , Heimann Joel Claudio , Pojoga Luminita H
Published: June 2021

Aims

This study aimed (1) to evaluate the timing of onset for changes in cardiovascular and renal (CVR) health during long term sodium loading in Lysine Specific Demethylase 1 (LSD1) deficiency and (2) to assess whether salt restriction can prevent these effects.

Methods

LSD1‐HET and WT mice were randomized to high salt (HS), moderate salt (NS) or low salt (LS) and followed longitudinally for 12 months. BP, albumin/creatinine ratios (A/C), plasma aldosterone (PA) and renin activity (PRA) were assess monthly. At the end of study the renal arterial resistance index was measure by Doppler ultrasound.

Results

The SBP (mmHg) increased progressively during the study, and reached significance on the 5thmonth for HS‐Het, 6thmonth for HS‐WT, 7thmonth for NS‐Het, 11thmonth for NS‐WT and 8thmonth for LS‐HET (132±3, 128±2, 125±3, 127±3 and 127±1 respectively, p<0.01 vs. baseline) but not for LS‐WT. Similar results were obtained for DBP (p<0.05), suggesting a volume mediated effect. The LS diet prevented the increase in BP only in WT mice.

The A/C (μg/mg) was progressively increased in all groups, interestingly always preceding the BP change. Namely, A/C reached significance on the 3th,5th, 6th, 7th, 7th, 11thmonth for HS‐Het, HS‐WT, NS‐Het, NS‐WT, LS‐Het and LS‐WT (44±3,44±5, 35±2, 35±1, 38±3 and 40±2 respectively, p<0.05vs. baseline).

PA and PRA were appropriately activated by dietary salt restriction and suppressed by aging.

With the exception of LS‐WT (renal resistive index 0.69±0.01)., all groups developed renal resistance

Conclusions

Our novel study showed that long‐term exposure to HS induced a progressive increase in BP in the WT, which was preceded by kidney damage. Interestingly in the LSD1‐Het, similar changes are initiated at an earlier age, suggesting that LSD1 is a critical component of mechanisms involved in CVR health.

Importantly, the observed changes in BP and AC seem to be prevented by long‐term reductions in sodium intake in the WT. However, a stricter control of sodium intake was only able to delay and blunt the development of target‐organ damage and the hypertension in the LSD1‐Het model.

Our study, performed in a clinically relevant animal model, suggest that LSD1 deficiency is a risk factor for cardiovascular and renal health, particularly during long‐term sodium loading, but also during moderately and severely restricted sodium diets.

Support or Funding Information

FAPESP, CAPES and Amercian Heart Association

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Multiple Poloxamers Improve Membrane Repair Capacity in a Model of Duchenne Muscular Dystrophy
Authors: Rose Aubrey , Kwiatkowski Thomas , McElhanon Kevin , Paleo Brian , Capati Ana , Beck Eric X , Weisleder Noah
Published: June 2021

Muscular dystrophies, such as Duchenne Muscular Dystrophy (DMD), are characterized by severe muscle degeneration and weakness. A major contributor to this pathology is sarcolemmal membrane fragility, which leads to increased damage to muscle fibers. A potential therapeutic approach is to improve membrane repair, allowing cells to more effectively resist damage that otherwise leads to necrotic cell death. Previous literature demonstrates that use of Poloxamer 188 (P188) can increase membrane repair capacity due to its affinity for damaged cell membranes. P188 is part of a family of poloxamers, all of which consist of a hydrophobic region of polyoxypropylene flanked by two hydrophilic chains of polyoxyethylene. These chains vary in length between different poloxamers. While P188 has been extensively researched in terms of membrane repair, other poloxamers in the same family have not been tested in this capacity. Based on the similarities between these polymers, we hypothesized that other poloxamers in the P188 family will reseal membrane wounds as effectively as P188. To investigate this hypothesis, we first used a rotation damage assay, in which Human Embryonic Kidney (HEK293) cells were exposed to various poloxamers and injured through impact with small glass beads (106 m). After injury, the lactate dehydrogenase (LDH) level in the cell supernatant was measured and compared to no damage and no poloxamer controls. A laser injury assay was subsequently used to investigate the effects of poloxamers on both HEK293 cells and murine muscle fibers. For these experiments, cells were injured with a multiphoton laser in the presence of a lipophilic dye (FM4‐64), which fluoresces when it comes in contact with the lipids of the cell membrane. Change in fluorescence is measured over time to quantify changes in membrane repair capacity. Muscle fibers for the laser injury assay were taken from mdx mice, a mouse model of DMD. Results indicate that multiple poloxamers improve membrane repair capacity similarly to P188. Our results also suggest that certain poloxamers have this effect in a cell‐type specific manner. This investigation improves our understanding of the effect of poloxamer size and composition on membrane repair. Our results support these polymers as promising therapeutics for DMD and other diseases where necrotic cell death contributes to disease progression.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Imoxin, a PKR inhibitor, ameliorates ER stress induced by palmitate or tunicamycin in C2C12 myotubes
Authors: Eo Hyeyoon , Valentine Rudy J
Published: June 2021

Prevalence of obesity has increased worldwide for decades and has becomes a major public health issue. Obesity is a key inducer of endoplasmic reticulum stress (ER stress), insulin resistance and sarcopenia. ER stress cause accumulation of unfolded or misfolded proteins leading to an unfolded protein response (UPR). Chronically prolonged ER stress consequently can interfere with insulin signaling and cell survival. The double‐stranded RNA (dsRNA)‐activated protein kinase R (PKR) plays a significant role in mediating inflammation, insulin sensitivity and glucose homeostasis. However, pathological roles of PKR in ER stress in muscle cells under obese condition is not fully understood. The current study aimed to investigate the effect of imoxin, a PKR inhibitor, on ER stress induced by palmitate or tunicamycin in C2C12 myotubes. The cells were treated with imoxin at 5 uM with or without bovine serum albumin (BSA)‐conjugated palmitate at 0.5mM or 500 ng/ml tunicamycin for 24 hr. Levels of ER stress signaling proteins including CHOP and BiP were measured by Western blotting. Palmitate significantly augmented the protein levels of both CHOP (288 ± 56%) and BiP (224 ± 17%) compared with BSA control. However, imoxin treatment attenuated palmitate‐induced ER stress signaling (140 ± 30% for CHOP and 89 ± 4% for BiP compared with control, respectively). Similarly, tunicamycin treated cells showed higher protein levels of CHOP (1132 ± 96%) and BiP (794 ± 34%) than control. Imoxin treatment prevented tunicamycin‐induced ER stress signaling (67 ± 40% for CHOP and 245 ± 27% for BiP compared with control, respectively). Thus, these results suggest that palmitate and tunicamycin induce ER stress through PKR in muscle cells.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Roles of Zip8 and Zip14 Transporters in Myogenesis
Authors: Gordon Shellaina Joy Victoria , Vest Katherine E , Padilla‐Benavides Teresita
Published: June 2021

Transition metals like manganese (Mn) are necessary catalytic and structural cofactors in enzymes and as second messengers in cell signaling. Studies on the molecular mechanisms by which Mn contributes to development are focused on its role as co‐factor of the mitochondrial enzyme superoxide dismutase 2 (SOD2). However, Mn has been proposed to participate in protein glycosylation during bone and neural development. Our laboratory is investigating the biological relevance of Mn in the differentiation of skeletal muscle cells. During myogenesis, total mitochondrial content increases and cells increase expression of SOD2, which protects against oxidative stress. Our lab has shown that the gene and protein levels of ZIP8 and ZIP14, two membrane Zn/Mn transporters, increase during differentiation of the C2C12 immortalized mouse myoblasts. Preliminary data shows a significant increase in the cellular Mn quota during the differentiation of primary myoblasts derived from mouse satellite cells, which are the stem cell pool that sustains and regenerates myofibers in adult skeletal muscle. We hypothesized that ZIP8 and ZIP14 might be associated with the Mn influx during myogenesis. To determine the role of ZIP8, ZIP14, and Mn during myogenesis we used shRNA to delete Zip8 and Zip14 transporters in primary myoblasts derived from primary myoblasts. Partial deletion of these transporters resulted in changes in cellular Mn levels. Zip8 knockdown myoblasts are unable to differentiate compared to control and Zip14 knockdown myoblasts, as shown by the expression of muscle‐specific differentiation markers and alterations on the morphology of differentiating cells. Sod2 activity is reduced also in Zip8 knockdown myoblasts. This work will contribute considerably to the very limited literature related to Mn biology in myogenesis and would provide a foundation for future efforts to understand the contribution of Mn in other differentiation and disease models.

Support or Funding Information

This project was funded by the Faculty Diversity Scholars Award from the University of Massachusetts Medical School Awarded to TP‐B. S. Gordon is supported by the NIH Grant R25 HL092610‐11

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Comparison of Ischemia‐Reperfusion Injuries Induced by Tourniquet and Femoral Artery Ligation in Mouse Hindlimb
Authors: Qian Junliang , Tu Huiyin , Zhang Dongze , Wadman Michael C. , Li Yu‐Long
Published: June 2021

The tourniquet and artery ligation are used widely to stop extremity hemorrhage or create a bloodless operating field. However, both of them also induce ischemia‐reperfusion (IR) injuries. To further evaluate the application of the tourniquet and artery ligation, we compared tourniquet and femoral artery ligation‐induced IR injuries in mouse hindlimb.

C57/BL6 mice were anesthetized and 3 hours of unilateral hindlimb ischemia was induced by placing an orthodontic rubber band (ORB) at the hip joint or undergoing a surgery procedure of femoral artery ligation, then the ORB tourniquet or artery ligation was released for 24 hours of reperfusion. Immunohistochemistry data demonstrated that tourniquet‐induced IR caused severe skeletal muscle damage, compared to femoral artery ligation‐induced IR. Triphenyl Tetrazolium Chloride Assay further confirmed that the infarct size in the gastrocnemius muscle was higher in tourniquet‐induced IR mice than that in femoral artery ligation‐induced IR. Sciatic nerve‐stimulated gastrocnemius muscle contraction was weak in tourniquet‐induced IR mice (0 g), compared to femoral artery ligation‐induced IR (46.42±3.74 g). Tourniquet‐induced IR also resulted in more serious damage of lymphatic vessels in mouse hindlimb, compared to femoral artery ligation‐induced IR.

Our present study indicate that tourniquet‐induced IR causes more severe skeletal muscle injuries than that induced by femoral artery ligation‐related IR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
During Ca2+‐dependent gastric epithelial repair, Ca2+ is sourced from both Ca2+ uptake and intracellular Ca2+ release
Authors: Engevik Kristen A , Hanyu Hikaru , Matthis Andrea L , Aihara Eitaro , Montrose Marshall H
Published: June 2021

Background

Calcium (Ca2+) is a known accelerator for gastric repair. However the mechanism by which Ca2+ mobilizes to promote repair remains unclear, and cannot be readily evaluated in vivo. Using gastric organoids derived from transgenic mice expressing a fluorescent Ca2+ reporter (yellow cameleon‐nano15; YC‐Nano), we previously observed intracellular Ca2+ increases in cells directly adjacent to a damaged cell. Using this Ca2+ sensor, we investigate the potential sources of intracellular Ca2+ essential for repair.

Methods

Gastroids generated from YC‐Nano mouse stomach corpus were cultured 4–5 days prior to experiments. Photodamage and resultant cell death was induced to 1–2 gastroid epithelial cells by ~3 sec high intensity 840 nm light. YC‐Nano reports Förster resonance energy transfer (FRET) from CFP to YFP in response to increased intracellular Ca2+. Change in intracellular Ca2+ was measured as FRET/CFP ratio, in cells adjacent to damaged cells. Inhibitors were used to test roles of Ca2+ channels (10 μM verapamil, 20 μM YM58483), intracellular Ca2+ (50 μM BAPTA/AM), Phospholipase C (10 μM U73122), and IP3R (50 μM 2‐APB).

Results

Unperturbed gastroid cells maintain a constant FRET ratio, indicating stable Ca2+ levels. In response to photodamage of the gastroid epithelium, increased levels of Ca2+ were observed specifically within the lateral membranes of cells neighboring the damaged cell. Chelation of intracellular Ca2+ by BAPTA/AM resulted in significant dampening of Ca2+ response, as well as blocking prompt repair. Inhibition of L‐type channels (verapamil) or store operated Ca2+ entry (YM58483) resulted in delaying repair and dampening intracellular Ca2+ response. Furthermore, inhibition of PLC (U73122) or IP3R (2‐APB) resulted in delayed repair and dampened Ca2+ response.

Conclusion

These results suggest both extracellular and intracellular Ca2+ sources are essential for supplying the Ca2+ that stimulates repair. The findings implicate an intracellular Ca2+ raise mediated via Ca2+ uptake via plasma membrane Ca2+ channels and intracellular Ca2+ release from the ER. Collectively this work indicates the usefulness of YC‐Nano to further assess intracellular Ca2+ dynamics and further investigate the signaling cascade behind Ca2+‐mediated repair.

Support or Funding Information

This work was supported by the National Institutes of Health (NIH) R01DK102551 (Montrose; Aihara) and F31DK115126 (Engevik). This project was also supported in part by the NIH P30 DK078392; Live Microscopy Core and DNA Sequencing and Genotyping Core of the Digestive Disease Research Core Center in Cincinnati.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of CD44 Variant Isoforms is an Early Epithelial Response to Helicobacter pylori Infection
Authors: Chakrabarti Jayati , Steele Nina , Holokai Loryn , Broda Taylor , Biesiada Jacek , Pitstick Amy , Mayhew Christopher , Medvedovic Mario , Wells James , Zavros Yana
Published: June 2021

Background

Helicobacter pylori (H. pylori) is the major risk factor for the development of gastric cancer. The progression of H. pylori induced chronic inflammation to gastric cancer may result from the aberrant expression of the cancer stem cell marker, CD44 variant isoform 9 (CD44v9). H. pylori induces reactive oxygen species (ROS) that subsequently leads to cell apoptosis via caspase activation. It is reported that in cancer stem cells, CD44v9 stabilizes the cystine‐glutamate transporter xCT, thus promotes resistance to ROS and subsequently leads to tumor growth. However, it is unknown whether CD44v9 protects the gastric epithelium against H. pylori‐induced apoptosis during early infection. In addition, CD44 variant isoform 6 (CD44v6), is not only a biomarker for diffuse‐type gastric cancer, but also promotes H. pylori‐induced epithelial cell proliferation.

Hypotheses

H. pylori‐induced CD44v9 is an early epithelial response to infection that subsequently promotes resistance to ROS‐induced epithelial cell apoptosis.

Methods

Antral and fundic gastric organoids were generated from induced pluripotent stem cell (IPSC) lines derived from the whole blood of normal patients (HGOs). HGOs were microinjected with the H. pylori G27 or a cagA mutant strain. After 72 hr of infection organoids were immunostained for CD44v9, CD44v6 and H. pylori. The expression of IL‐8 and caspase 3 were measured by qRT‐PCR. In a separate series of experiments, infected organoids were treated with either vehicle or 300 mM sulfasalazine (SSZ) for 72 hrs and changes in phosphorylation (activation) of p38MAPK, a downstream target of ROS was measured by western blot. RNA was isolated from uninfected and infected antral and fundic HGOs and RNA‐sequencing was performed to analyze differential expression of genes among those samples.

Results

H. pylori induced the expression of CD44v9 and CD44v6 in HGOs and this correlated with the suppression of caspase 3 and increased IL‐8 gene expression. Despite the Increased expression of ROS observed in H. pylori Infected HGOs, there was increased epithelial cell proliferation within the infected organoids. Proliferation significantly decreased in H. pylori infected HGOs treated with xCT inhibitor SSZ. H. pylori infected fundic HGOs showed 94% dissimilarity in the differentially expressed genes compared to infected antral HGOs, which fall mostly in cancer and apoptotic pathways.

Conclusion

Induction of CD44v9 and v6 are early epithelial changes in response to H. pylori infection, and may play a role in the protection against H. pylori‐induced apoptosis

Support or Funding Information

This work was supported by NIH 1U19AI116491‐01 (Weis and Wells, YZ Project Leader),

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neonatal necrotizing enterocolitis alters the development of brainstem neurocircuitry controlling gastrointestinal functions
Authors: Meister Alissa L , Doheny Kim K , Browning Kirsteen N , Travagli R. Alberto
Published: June 2021

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal (GI)‐related death in premature infants and is most often characterized by a slow progressive onset of symptoms including feeding intolerance and abdominal distention prior to the development of sepsis and necrosis with potentially life‐long effects on both GI health and neurodevelopmental outcomes. The high‐frequency power spectra of heart rate variability (HF‐HRV) is a non‐invasive measure of vagal tone, our laboratory has previously shown that HF‐HRV is decreased in NEC in both human neonates (Doheny et al., Neurogastroenterol. Motil., 2014) and in rodent pups (Meister et al., Neurogastroenterol. Motil., 2018). The present study examined the hypothesis that NEC induces long term alterations in central and peripheral control of the intestine including i) attenuated developmental maturation of brainstem neurocircuits and ii) decreased transit.

Mild NEC (stage≤2) was induced in newborn Sprague‐Dawley rats through twice daily exposure to cold stress and hypoxia and HRV was measured using bilateral electrodes to collect an electrocardiogram before and after stress exposure on postnatal days (PD) 1, 5, 10 and 28. Whole cell patch clamp recordings examined whether, in young adult rats (4–8 weeks of age), dorsal motor nucleus of the vagus (DMV) neurons innervating the intestine received inhibitory synaptic currents that were entirely GABAergic (indicating normal development, i.e., unaffected by the glycine antagonist, strychnine; 1mM) or were mixed GABA‐glycine (indicating arrested developmental maturation; inhibitory currents prolonged by strychnine). Intestinal transit was measured using the non‐invasive lactose ureide breath test at the same time‐points.

The induction of mild NEC was confirmed by histological grading, these pups showed a significant reduction in HF‐HRV at all time points, except for baseline before stress induction, as compared to controls. Mild NEC rats showed no change in gastric emptying as compared to controls, but a significant increase in gastro‐cecal transit time. In control rats, 0/11 adult DMV neurons showed evidence of inhibitory glycinergic inputs whereas 9/12 DMV neurons from rats in which mild NEC was induced displayed mixed GABA‐glycine currents. Data are summarized in the table below.

Our data suggest that mild NEC is associated with adult comorbidities including arrested development of brainstem neurocircuits innervating the intestine as well as delayed intestinal transit. Neonatal insults, such as NEC, may have long term developmental consequences that impair intestinal functions at both central and peripheral sites.

Support or Funding Information

NIH grant DK99350 to R.A.T. & K.K.D.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Low‐Dose Dihydrotestosterone Lowers Lipogenic Master Regulator in Liver and Adipose Tissue from Female Mice
Authors: Andrisse Stanley , McWhorter Patrick , Seidu Tina , Alamgir Rabita
Published: June 2021

Hyperandrogenemia (HA) and insulin resistance (IR) are hallmarks of polycystic ovary syndrome (PCOS), a common endocrine disorder that affects 1 in 10 women. These hallmarks are also integral elements of non‐alcoholic liver disease (NALFD), a disorder that is common in women with PCOS. In lean female mouse models of dihydrotestosterone (DHT)‐induced PCOS, a low dose of (DHT) promotes IR and hepatic lipogenesis via the androgen receptor (AR), thus resulting in NAFLD. However, the molecular mechanism of HA‐induced NAFLD has not been determined. We hypothesized that a low dose of DHT would interrupt hepatic lipid metabolism leading to NAFLD. In this project, we aimed to investigate how androgen and androgen receptor (AR) impact a key player of lipogenesis, SREBP1. We extracted liver tissue, white adipose tissue (WAT), and skeletal muscle from lean, female wild type mice and mice. Next, we analyzed the effect of low dose dihydrotestosterone (DHT) on lipogenic protein and gene expression. We performed Western blot and real‐time quantitative polymerase chain reaction (qRT‐PCR) analysis of lipogenic intermediates on homogenates of energy storage tissues (liver, WAT, and skeletal muscle). Low‐dose DHT lowered the protein expression of the active form of cytosolic SREBP1 in the liver and WAT. Additionally, low‐dose DHT lowered the protein expression of the inactive SREBP1 in the liver. DHT did not alter the levels of the active and inactive forms of SREBP2 in the liver and WAT. Interestingly, the active form of SREBP2 was lowered in skeletal muscle. Further, phospho acetyl co‐A carboxylase (p‐ACC) protein levels were unaltered in liver and WAT. Fatty acid synthase (FAS) levels were unchanged in WAT and skeletal muscle. Taken together, our findings suggest that hepatic and white adipose cytosolic SREBP1 is being decreased due to its translocation to the nucleus, where it regulates lipogenic protein levels.

Support or Funding Information

Thank you to Howard University College of Medicine, Johns Hopkins Medicine, and Georgetown University Medical Center (GUMC) for supporting this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Integration of Transcriptomics And Proteomics Reveals That miR‐33‐5p Is A Key Player of Lipogenesis Signaling in Fructose‐Induced NAFLD in C57BL/6N Mice
Authors: Redding Mersady , Beane Kaleigh , Tang Jingsi , Lee Jin Hyup , Kong Byungwhi C , Kim Jae Kyeom , Pan Jeong Hoon
Published: June 2021

Fructose is a strong risk factor of non‐alcoholic fatty liver disease (NAFLD), but limited studies have investigated mechanisms by which microRNAs (miRNA) are interlinked in the etiology of NAFLD. Here, we aimed to investigate the significance of miRNAs in fructose‐induced NAFLD conditions through integration of liver transcriptomics and proteomics. C57BL/6N mice (male and female) were fed with either water (n=14) or 34% fructose (n=14) for 6 wk ad libitum. After, liver transcriptomics and proteomics datasets were integrated to narrow down key signaling pathways and to find upstream regulator(s) that are responsible for fructose induced NAFLD in which srebp1 was found to be the most significant upstream regulator. In an attempt to explore regulatory roles of miRNAs in NAFLD, Ingenuity Pathway Analysis software miRNA database was used to list miRNAs that are known to interact with the upstream regulator, srebp1. Out of seven final miRNA candidates, miR‐33a was selected after our qPCR validation in mice liver and AML12 mouse hepatocytes that were treated with fructose. In order to further validate the interaction between miR‐33a and srebp1, AML12 hepatocytes were transfected with the miR‐33a; in this, srebp1, and its downstream proteins, acc1 and scd1 were all decreased by miR‐33a transfection whereas suppression of miR‐33a, achieved by anti‐miR‐33a transfection, restored those protein expression changes. Altogether, based on our unbiased approaches as well as in vitro validation experiments, fructose induces NALFD conditions via suppressing miR‐33a in liver thereby increasing srebp1 and its downstream lipogenic proteins, acc1 and scd1.

Support or Funding Information

This work was supported by the University of Arkansas, VPRED Start‐up fund (JKK, and JHP). Support has been provided in part by the Arkansas Biosciences Institute, a partnership of scientists from Arkansas Children's Hospital, Arkansas State University, the University of Arkansas‐Division of Agriculture, the University of Arkansas, Fayetteville, and the University of Arkansas for Medical Sciences. The Arkansas Biosciences Institute is the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000 (JKK, JHP, KEB, and BCK). This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF‐2015R1C1A1A01053746; JHL) as well as the China Scholarship Council (JT).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prevalence of Thiamine (B1) deficiency in a cohort diagnosed with Postural Orthostatic Tachycardia (POTS)
Authors: Del Pozzi Andrew Thomas , Blitshteyn Svetlana
Published: June 2021

Thiamine (vitamin B1), a water‐soluble vitamin that plays a pivotal role in energy metabolism, growth, development and the function of cells. Has been found to be deficient in conditions such as malnutrition, eating disorders, and bariatric surgery complications as well as alcoholism. Previously vitamin B12, vitamin D and Iron deficiencies have been noted in patients with POTS. Yet the prevalence of thiamine deficiency and response to thiamine supplementation in POTS has yet to be determined.

PURPOSE

to determine the prevalence of Thiamine deficiency in those patients diagnosed with POTS. As well as to indicate if supplementation of thiamine affects outcomes.

METHODS

Sixty‐five consecutive patients (59 women and 6 men) ranging in age from 13 to 54 years old, who met the diagnostic criteria for POTS were evaluated and followed by our clinic. As part of clinic initial diagnostic workup all clinic patients have vitamin panels drawn (Quest Diagnostics Laboratory), thus we reviewed patient medical records noting whole blood vitamin B1. Those patients that were found to be deficient in B1 were given thiamine hydrochloride at 100 mg daily for 4 weeks and then followed up with office exams and self‐report of changes in symptoms.

RESULTS

Six percent (n = 4) of the patients tested positive for thiamine deficiency. Those that showed deficiency were only mildly deficient (72–77 nm/L vs. Normal 78–185 nm/L). Other vitamin and mineral levels including B6, B9, B12, vitamin E, iron, copper, and magnesium as well as homocysteine level were normal in all deficient B1 patients. All patients were supplemented vitamin B1, however, only 25% (n = 1) responded favorably to the supplementation. Although 100% of those supplemented had vitamin B1 levels returned to the normal range.

CONCLUSION

A small subset of POTS patients may have vitamin B1 deficiency. Testing for and correcting the deficiency could prove helpful to those patients. Further research should be done on a larger scale and should include a healthy control group as well as objective measures of improvement such as tilt‐table‐testing as part of the follow‐up.

Support or Funding Information

none

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Apple pomace influences liver‐adipose tissue inflammatory crosstalk in young female rats consuming a Western diet
Authors: Skinner Roy Christopher , Warren Derek C , Agarwal Garima , Naveed Minahal , Benedito Vagner A , Chantler Paul D , Tou Janet C
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease in Western countries and it has been attributed to poor diet. NAFLD is defined by the presence of hepatic steatosis, which can progress to the more severe non‐alcoholic steatohepatitis (NASH) characterized by inflammation. Apple pomace, a waste byproduct of apple processing, is a rich source of anti‐inflammatory/antioxidant polyphenols. The objective of this study was to determine the effects of caloric replacement with apple pomace on hepatic‐adipose tissue inflammatory crosstalk and oxidative stress status. Growing (age 22–29 days) female Sprague‐Dawley rats were randomly assigned (n=8 rats/group) to consume a purified standard rodent diet (AIN‐93G), AIN‐93G/10% g/kg apple pomace (AIN/AP), Western diet, or Western/10% g/kg apple pomace (Western/AP) diets for 8 weeks. Based on histological evaluation, rats consuming Western diet had the highest hepatic fat infiltration and inflammation. Gene expression of nuclear transcription factor kappa B (NFκB), a key transcription factor of inflammation and reactive oxygen species, was upregulated in liver (p=0.009) and the adipose tissue (p=0.0003) of rats fed Western diet compared to the other diets in the experiment. Gene expression of proinflammatory interleukin‐6 (IL‐6) in both tissues as well as the tumor‐necrosis factor‐α (TNF‐α) in the adipose was upregulated in rats fed Western diet, but not Western/AP compared to rats fed standard diets. Replacing calories in Western and AIN diets with apple pomace upregulated (p<0.01) gene expression of the endogenous antioxidant glutathione (GSH). Additionally, serum total antioxidants were highest (p<0.04) and urine total antioxidants were lowest (p<0.04) in rats fed Western/AP. Collectively the results showed caloric replacement with 10% apple pomace in the Western attenuated risk factors associated with progress to NASH in a rat model suggesting the potential of apple pomace as a sustainable functional food.

Support or Funding Information

This research was funded by Hatch WVA 1017641 and the Davis College Dean's discretionary fund.

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