bluuebunny/crossref_metadata_2025 huggingface dataset:
Contribution of High Mobility Group Box 1 to Obesity‐Induced Podocyte Dysfunction and Glomerular Injury
Authors: Koka Saisudha , Mohammad Razia S. , Singh Gurinder Bir , Kshirasagar Naresh , Hussain Tahir , Li Ningjun , Li Pin‐Lan , Li Xiang , Boini Krishna M.
Published: June 2021

High mobility group box 1 protein (HMGB1), a nuclear DNA binding protein is released under pathological conditions and locally act as one of potent damage‐associated molecular patterns (DAMPs) to produce tissue injury and chronic inflammation. However, it remains unknown whether HMGB1 is implicated in obesity‐induced podocyte injury. In the present study, we found that obesity‐induced adipokine visfatin dose‐dependently increased the production of HMGB1 in cultured podocytes compared to control cells. However, pretreatment with HMGB1 binder, glycyrrhizin (Gly) completely blocked HMGB1 release which was induced by visfatin. Further, immunofluorescence analysis showed that visfatin treatment significantly decreased the podocin expression (podocyte damage) compared to control cells. However, prior treatment with glycerrhizin attenuated the visfatin‐induced podocin reduction. Furthermore, glycyrrhizin treatment was found to preserve podocyte morphology by maintaining the distinct arrangement of F‐actin fibers normally lost in response to visfatin. It also prevented podocyte dysfunction by restoring visfatin‐induced suppression of VEGF production and secretion. In addition, functional studies showed that dextran flux was significantly increased in visfatin treated podocytes compared to control cells. This visfatin‐induced increase in permeability was markedly reduced in the presence of glycyrrhizin. In in vivo studies, C57BL6 mice were fed with high fat diet or normal chow for 14 weeks to produce obesity. Western blot analysis showed that high fat diet treatment significantly increased the HMGB1, TLR4 and RAGE expression in glomeruli of high fat diet fed mice compared to normal diet fed mice. However, such increase of HMGB1, TLR4 and RAGE expression were attenuated in glomeruli of glycyrrhizin treated mice. Based on these results, it is concluded that HMGB1 is one of important mediators of obesity‐induced podocyte injury and glomerular sclerosis. HMGB1 may be a therapeutic target for treatment or prevention of glomerulosclerosis associated with obesity.

Support or Funding Information

supported by NIH grant, DK104031

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Upregulation of cystathionine‐γ‐lyase/hydrogen sulfide pathway underlies the celecoxib counteraction of the cyclosporine‐induced hypertension and renal insult in rats
Authors: Helmy Mai M. , Helmy Maged Wasfy , El‐Mas Mahmoud M.
Published: June 2021

We recently reported that celecoxib, a selective cyclooxygenase‐2 inhibitor, counteracts the adverse circulatory and renal actions of cyclosporine (CSA). Despite the advantageous nature of this interaction particularly when the two drugs are prescribed together in the management of immune‐related arthritic conditions, the underlying mechanism remains elusive. This prompted us to test the hypothesis that the facilitation of the cystathionine‐γ‐lyase (CES)/hydrogen sulfide (H2S) signaling accounts for the celecoxib‐CSA interaction. The data showed that the 10‐day cotreatment of rats with celecoxib (10 mg/kg/day) ameliorated the hypertensive and biochemical and structural renal damages caused by CSA (20 mg/kg/day). Celecoxib also reversed the CSAevoked (i) reductions in the tubular and glomerular protein expression of CES and levels of H2S, prostaglandin E2, and total antioxidant capacity, and (iii) increases in tumor necrosis factor‐α, transforming growth factor‐β1 and caspase‐3. These favorable effects of celecoxib disappeared when rats were treated concomitantly with the CES inhibitor DL‐propargylglycine (PAG), indicating the importance of the CES‐derived H2S in mediating the celecoxib counteraction of the CSA effects. This view is bolstered by the observation that the beneficial hemodynamic and renal actions of celecoxib were replicated after supplementation of rats with sodium sulfide (H2S donor). Together, the increased abundance of renal CES and H2S generation and subsequent dampening down of inflammatory, fibrotic, oxidant, and apoptotic pathways play pivotal roles in the celecoxib counteraction of the troublesome hypetensive and nephrotoxic insults caused by CSA in rats.

Support or Funding Information

the running research was funded by authors themselves.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Calcium Sulfide Nanoclusters in the Migration of Non‐Small Cell Lung Adenocarcinoma cell line HCC827
Authors: Reyes Amalia Marie Rivera , Forti Kevin Muñoz , Martínez Gerardo Arroyo , Torres Geraline Trossi , Rivera Abigail Arroyo Ruiz , Suarez‐Martínez Edu Arroyo
Published: June 2021

Lung cancer is the most common malignancy worldwide independently of gender. Non‐small cell lung carcinoma (NSCLC) is responsible for 80% of diagnosed cases. Prognosis for patients suffering this cancer is poor, with only 7% of patients surviving five years after the diagnosis. Current treatments include surgery, radiotherapy, and chemotherapy. Although the aforementioned seem successful, they do not address the development of secondary tumors due to cell migration resulting in metastasis. Evidently, there is a need for developing novel therapeutic approaches specifically targeting malignant cells. In our laboratory, we evaluate calcium sulfide (CaS) nanostructures as potential multi‐cancer malignant‐cell targeted specific treatment. The objective of this study was to determine the effect of CaS nanoclusters in the migration of NSCLC HCC827 cells. We hypothesized that CaS will decrease the area of migration compared to the control vehicle (DMSO), will increase the number of dead cells and decrease the live cells in the collected supernatant. We used the Scratch Wound Healing Assay, to determine the area of migration after 24, 48, and 72hrs upon a single exposure at time 0 of CaS treatment and controls (DMSO: vehicle control; etoposide: positive control; and media: negative control). We also collected the supernatant to characterize detached cells in suspension. Preliminary data shows that at 72hrs, cells treated with CaS exhibited a significant decrease in cell migration. We categorized the cells in the supernatant as live or dead. Although there were not statistically significant differences when compared CaS treatment with the controls, we observed a time‐dependent trend. This trend showed a decrease of the number of dead cells and an increase of live cells as the time increases. Based on this preliminary data, we partly accept our hypothesis due to the observed decreasing of migration in CaS treatment. However, the supernatant data was the opposite to what we hypothesized. We are currently optimizing experiments to increase statistical power and correlate the area of migration with the supernatant findings. In the next step of this project, I will be determining the effect of CaS by means of migration and number of live and dead cells in the supernatant in the non‐malignant lung cells MRC5.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G Protein‐Coupled Receptors in Human Lung Fibroblasts: Novel Therapeutic Approaches for Idiopathic Pulmonary Fibrosis?
Authors: Michkov Alexander V. , Sriram Krishna , Mukherjee Subhendu , Janssen Luke J. , Insel Paul A.
Published: June 2021

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, often fatal interstitial lung disease and thus in need of new, effective therapies. A feature of IPF is fibroblast‐to‐myofibroblast transition and resultant increased deposition of extracellular matrix (ECM) proteins, which can lead to loss of functional lung tissue. Previous studies have shown that increases in cyclic AMP (cAMP) can decrease the number and function of myofibroblasts. Cellular synthesis of cAMP is mediated by adenylyl cyclase, the activity of which is regulated by G‐protein signaling and G‐protein coupled receptors (GPCRs). We hypothesized that alteration in GPCR expression in lung fibroblasts from IPF patients may identify new therapeutic targets for lung fibrosis. Using RNA‐seq, we have identified and quantified GPCRs of lung fibroblasts isolated from six IPF patients and six controls grown in low passage primary cultures. RNA‐seq analysis revealed the expression of ~120 GPCRs (of ~380 endoGPCRs [regulated by endogenous ligands]). On average ~20 highly expressed GPCR were detected in all samples, with small differences in biological replicates; these may be “housekeeping” GPCRs. We also identified a group of GPCRs with highly variable expression and found that lung fibroblasts from IPF patients selectively expressed a subset of GPCRs. To assess changes in GPCR expression during fibroblast‐to‐myofibroblast transition, we treated fibroblasts with TGFβ1 (10 ng/ml, 48 hrs or 20 ng/ml, 16 hrs). As expected, TGFβ1 treatment increased the expression of pro‐fibrotic markers (e.g., collagens Iα1, Iα2, III; α‐smooth muscle actin; plasminogen activator inhibitor‐1). In addition, TGFβ1 altered (increased or decreased) the expression of numerous GPCRs. TGFβ1 treatment of control fibroblasts did not fully mimic the GPCR expression changes of fibroblasts from IPF patients. We conclude that GPCR expression (GPCRomic) analysis identifies GPCRs that may contribute to the fibrotic state in the lung and thus, may be novel therapeutic targets for lung fibrosis.

Support or Funding Information

Supported by NIH T32 HL 098062‐04, Bristol Myers Squibb and CIHR 201403MOP‐RS‐323925.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolvin D1 blocks ROS‐mediated inhibitory crosstalk between SHP2 and PP2A and suppresses endothelial‐monocyte interactions
Authors: Singh Nikhlesh , Chattopadhyay Rima N. , Mani Arul M. , Rao Gadiparthi N.
Published: June 2021

In recent years, various studies have demonstrated a role for endogenously derived specialized lipid mediators such as resolvins in the resolution of inflammation. In exploring the signaling mechanisms, in the present study, we show that RvD1 reduces LPS‐induced endothelial cell (EC)‐monocyte interactions via blocking ROS‐mediated PP2A inactivation, NFκB activation and ICAM1 and VCAM1 expression. In addition, we found that ROS‐mediated SHP2 inhibition leads to tyrosine phosphorylation and inactivation of PP2A by LPS, which in turn, accounts for increased NFκB activation and ICAM1 and VCAM1 expression facilitating EC‐monocyte interactions and all these LPS‐mediated responses were reversed by RvD1. Furthermore, the suppression of NFκB activation, ICAM1 and VCAM1 expression and EC and monocyte interactions by RvD1 involved its receptors ALX/FPR2 and GPR32 as inhibition or neutralization of these receptors negated its effects. Together, these observations show that RvD1 via protecting SHP2 from ROS‐mediated inactivation blocks PP2A inactivation and thereby shuts of NFκB activation, ICAM1 and VCAM1 expression and EC‐monocyte interactions, which could be one of the several possible mechanisms underlying its anti‐inflammatory functions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in human neutrophil function by Bisphenol A
Authors: Balistrieri Angela , Meier Angela , Corriden Ross
Published: June 2021

Bisphenol‐A is a synthetic organic compound frequently present in consumer plastics, including plastic‐lined cans, water bottles, toys, and teeth sutures. Previous studies have shown that BPA can produce adverse health effects that include defects in reproductive function and altered prenatal/childhood development. However, little is known regarding the effects of BPA on immune function. In this study, we assessed the effect of BPA on human neutrophils, a critical component of the innate immune system's defense against pathogens. We found that BPA induces a concentration‐dependent increase in reactive oxygen species generation by PMNs that is inhibited by the estrogen receptor β antagonist PHTPP and the NADPH oxidase inhibitor diphenyleneiodonium chloride. Furthermore, incubation with the membrane‐permeable calcium chelator BAPTA‐AM and/or removal of extracellular calcium inhibited BPA‐induced ROS production, indicating that the process is calcium dependent. Transwell chemotaxis assays revealed that BPA exposure reduces the chemotactic capacity of PMNs in a gradient of the bacterial cell wall component f‐Met‐Leu‐Phe, a potent chemoattractant. Exposure to BPA also inhibits the ability of neutrophils to kill methicillin‐resistant Staphylococcus aureus, a leading human pathogen. Our findings reveal that BPA alters the in vitro function of PMNs, including ROS production, chemotaxis and bacterial killing, and raise the possibility of altered innate immunity in vivo, especially in those with compromised immune function and who can be exposed to BPA in a wide variety of products.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nrf2 Deletion is Associated with Impaired BKCa Channel Expression and Function in Rat Cerebral Arterial Muscle Cells
Authors: Gebremedhin Debebe , Harder David Rae , Lombard Julian H.
Published: June 2021

The BKCa channel is ubiquitous in cerebral arterial muscle cells and other brain cell types and regulates cell excitability by sensing and reacting to changes in [Ca2+]. The BKCa channels are formed by tetramers of the pore‐forming α‐subunit and the accessary β1 subunit. The accessary β1 subunit unit is the sensor for changes in Ca2+ and voltage to regulate BKCa channel function. The BKCa channel is one of the downstream targets regulated by Nrf2 antioxidant defenses. We investigated the influence of deletion of Nrf2 on the expression and activities of BKCa single‐channel current in cerebral arterial muscle cells of Nrf2 wild type and knockout rats. In this study we found that the openings of a large conductance BKCa single‐channel current was markedly reduced in cerebral arterial muscle cells of Nrf2 knockout rats compared to those recorded from Nrf2 wild type rat cerebral arterial muscle cells at the same patch potential and symmetrical KCl (145 mM) recording solution. Nrf2 knockout also resulted in reduced expression of the proteins for both the pore forming BKCa channel α‐subunit and the accessary BKCa channel β‐subunit in rat cerebral arteries compared to cerebral arteries of Nrf2 wild type rats. Furthermore, knockout of Nrf2 significantly attenuated the ability of the Nrf2 activator sulforaphane (3 μM) to increase the openings of the BKCa single‐channel currents recorded from cell‐attached patches of Nrf2 knockout rat cerebral arterial muscle cells at a patch potential of 40 mV compared to those measured in Nrf2 wild type cerebral arterial muscle cells. These findings suggest that Nrf2 antioxidant defenses regulate BKCa channel expression and activity in rat cerebral arterial muscle cells and may influence the dynamics of the cerebral circulation under normal or oxidative stress conditions.

Support or Funding Information

NIH #R01‐HL128242 and # R21‐OD018309) and NIH RO1 HL0338833.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative Stress‐Induced Changes in Ca2+ Sensitivity of Cardiomyocytes Do Not Recover
Authors: Piedrahita Tatiana Mejia , Osorio Sara , Han Young Soo , Diaz Rienzi , Sieck Gary
Published: June 2021

Ca2+ sensitivity plays a critical role in regulating cardiac contraction, and pathophysiological changes in Ca2+ sensitivity of cardiac contraction may significantly impact cardiac function. For example, oxidative stress causes myocardial dysfunction, which is due in part to a decrease in Ca2+ sensitivity. Oxidative stress (e.g., elevated H2O2) underlies, at least in part, various conditions of cardiac dysfunction including ischemia‐reperfusion injury, heart failure, hypertension, and atherosclerosis. In cardiac muscle, Ca2+ sensitivity can be dynamically assessed using a phase loop plot of concurrently measured cytosolic Ca2+ concentration ([Ca2+]cyt) and sarcomere length shortening (Spurgeon et al J Physiol 447:83–102, 1992; Han et al Am J Physiol 298:890–897, 2010; Schaible et al Am J Physiol 310:1533–40, 2016). We hypothesized that oxidative stress reversibly reduces Ca2+ sensitivity of cardiomyocytes within minutes. Cardiomyocytes were isolated from adult rat hearts (Sprague Dawley) using a modified Langendorff system. Cardiomyocytes were loaded with Fura‐2 AM (Ca2+ fluorescent indicator; 1 mM) and electrically stimulated every 2 s. [Ca2+]cyt and sarcomere shortening responses to stimulation were simultaneously measured using an IonOptix System. After stabilization for 30 min at 35°C, cardiomyocytes were exposed to PSS for 10 min, then to H2O2 (200 μM) for 10 min and finally to either PSS washout or DTT (1 mM)‐induced reduction for 10 min. H2O2‐induced oxidative stress increased basal [Ca2+]cyt and the amplitude of the evoked [Ca2+]cyt response. H2O2‐induced oxidative stress also reduced the extent of sarcomere shortening and slowed the kinetics of shortening/relaxation. As a result, H2O2‐induced oxidative stress reduced Ca2+ sensitivity reflected by a rightward shift in the phase loop plots. Interestingly, with both PSS washout and DTT‐induced reduction Ca2+ sensitivity was further reduced. These results support the hypothesis that H2O2‐induced oxidative stress reduces Ca2+ sensitivity of cardiomyocytes, but this effect was not immediately reversed, but indeed worsened by PSS washout or by DTT‐induced reduction. Together, these results provide insight into the dynamic effects of oxidative stress on Ca2+ sensitivity of cardiomyocytes that may be reflected under pathological conditions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pannexin 1 channels in renin expressing cells regulate RAAS and blood pressure
Authors: DeLalio Leon J. , Keller TC Stevenson , Page Henry Askew , Masati Ester , Barrett Paula Q. , Gomez Ariel , Le Thu , Isakson Brant E.
Published: June 2021

The homeostatic regulation of blood pressure by renin lineage cells in the renal vasculature and the adrenal zona glomerulosa (ZG), is crucial for controlling tissue renin expression and adrenocortical aldosterone synthesis. In the renal vasculature, renin lineage cells respond to changes in blood pressure by differentiating and re‐expressing their fetal renin gene program to compensate for lowered blood pressure. In the adrenal cortex, ZG cells of the renin lineage centripetally turnover, differentiating from an aldosterone synthetic program to a glucocorticoid synthetic one, thus maintaining steady state aldosterone production. The cellular signals coordinating these transitions is unclear, but evidence from our lab posits a role for purinergic signaling mediated by Pannexin 1 channels. We hypothesized that Pannexin 1 (Panx1) channels regulate purinergic communication and cell differentiation in the renal vasculature and adrenal cortex to regulate RAAS and blood pressure homeostasis. To test this hypothesis, we generated a novel renin cell Panx1 knockout mouse (Ren1‐Px1 KO). At baseline, mice exhibit no differences in kidney size, morphology, or plasma renin concentration. However, Ren1‐Px1 KO mice have increased plasma sodium concentration, reduced urine volume, and a significant elevation in aldosterone levels, which corresponded with aberrant expression of aldosterone synthetic enzyme (CYP11B2) and ZG cell differentiation in the adrenal cortex. Using radiotelemetry, we measured blood pressure in Ren1‐Px1 KO mice, which were hypertensive at baseline. Lastly, we assessed renin dynamics in the kidney when blood pressure is lowered. Ren1‐Px1 KO mice retain the capacity to secrete renin at baseline, but fail to completely recruit tissue renin expression in the renal vasculature. We conclude that loss of Panx1 from renin lineage cells causes activation of RAAS primarily by influencing ZG trans‐differentiation, despite changes in renal renin dynamics. Thus, Panx1 channels may be a novel regulator of long term blood pressure control.

Support or Funding Information

1 F31 HL1376270/NHLBI NIH P01 HL120840/NHLBI NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPA1‐Induced Pulmonary Spinal Sympathetic Afferent Activation is Attenuated in Rats with Chronic Heart Failure
Authors: Adam Ryan J. , Xia Zhiqiu , Shanks Julia A. , Rozanski George J. , Lisco Steven J. , Zucker Irving H. , Wang Han‐Jun
Published: June 2021

In a recent preliminary study (Association of the University Anesthesiologists 64th annual meeting, Abstract #1109, 2017), we reported that activation of pulmonary spinal afferents by topical application of bradykinin (BK) and the TRPV1 agonist capsaisin on to lung surfaces (especially on the ventral surface) evokes a previously undocumented potent sympatho‐excitatory reflex that includes increased bood pressure (BP), heart rate (HR) and renal sympathetic nerve activity (RSNA) in urethane‐α‐chloralose anesthetized, vagotomized rats. These data suggested a new pulmonary sympathetic afferent reflex (PSAR) in rats. However, it remains unclear whether pulmonary sympathetic afferents can also be activated by the transient receptor potential ankyrin 1 (TRPA1) agonist, allyl isothiocyanate (AITC). In the current study, we examined the TRPA1‐induced PSAR in both normal and chronic heart failure (CHF) states. After bilateral vagotomy, we applied filter paper (3 × 3 mm) saturated with 30 mM AITC to the ventral lung surfaces to stimulate the PSAR in urethane‐ α‐chloralose anesthetized sham‐operated and CHF (8 weeks post myocardial infarction) rats. We found that application of AITC to the lungs resulted in a biphasic BP response whereas HR and RSNA remained elevated during the AITC application in sham rats (Table 1). Compared to sham rats, AITC caused a predominantly depressor response in CHF rats. The increases in HR and RSNA in response to AITC were also attenuated in CHF rats. These data suggest that activation of the TRPA1 receptor in pulmonary spinal afferents can cause sympatho‐excitation in normal rats whereas this response was blunted in CHF rats. Ongoing studies are validating TRPA1 receptor protein expression in pulmonary sensory neurons in sham and CHF rats.

Support or Funding Information

This work was, in part, supported by grants from the National Heart, Lung, and Blood Institute (1R01HL121012‐01 and 1R01HL126796‐01).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autonomic Responses during Atrial Fibrillation Ablation
Authors: Cui Jian , Gonzalez Mario , Blaha Cheryl , Hill Ashley , Sinoway Lawrence I.
Published: June 2021

Left atrial radiofrequency catheter ablation (RFCA) is a therapy for atrial fibrillation. RFCA may alter sensory input from left atrium and pulmonary veins. It is speculated that RFCA may alter autonomic afferents and alter indices of sympathetic function. Although prior studies have shown that RFCA may alter autonomic function, which was assessed with heart rate variability (HRV), the effects of left atrial RFCA on sympathetic nerve activity have not been assessed. In this study, we hypothesized that RFCA would raise muscle sympathetic nerve activity (MSNA) during and after the RFCA procedure. Total of 18 patients (16 M, 2 F, 57 ± 3 years, 181 ± 2 cm, 97 ± 5 kg) were studied. In Protocol 1 (n = 10), the ECG, BP and MSNA from the peroneal nerve were recorded through the RFCA procedure in the electrophysiology laboratory. In Protocol 2 (n = 8), ECG, BP and MSNA were recorded before (within one week) and one day after the RFCA in a research laboratory. In Protocol 1, MSNA was obtained prior (n = 9), during (n = 3) and just post (within 15 min, n = 7) the RFCA procedure. Compared to prior RFCA, MSNA decreased during (28.4 ± 3.7 to 16.0 ± 2.6 bursts/min, P < 0.05) and just post (28.6 ± 3.1 to 16.5 ± 4.1 bursts/min, P < 0.05) the RFCA. Cardiac sympathetic tone (HRV) also decreased (prior, during, post LF/HF: 0.64 ± 0.15, 0.49 ± 0.11, 0.16 ± 0.04, P < 0.05, n = 10). Vagal tone (HRV indexes: RMSSD, SDSD, HF) increased during RFCA (all P < 0.05, n = 10). In Protocol 2, MSNA increased one day after RFCA (21.3 ± 3.7 to 35.7 ± 2.6 bursts/min, P < 0.05, n = 6), and blood pressure did not change. Vagal tone (HRV indexes) decreased (all P < 0.05, n = 8), while LF/HF also tended (P = 0.09, n = 8) to decrease one day after RFCA. Despite body motions and electronic noise during the RFCA procedure, we successfully obtained MSNA recordings during the RFCA procedure. The MSNA data suggest that the sympathetic activity is suppressed, and the HRV indexes suggest that the vagal tone is activated during the procedure. We speculate that the radiofrequency energy stimulates the vagal afferents, which in turn induces the observed effects during the procedure. Our data suggest the RFCA induces autonomic denervation in the heart and induces decreases in vagal and cardiac sympathetic tone one day post procedure. The rise in the sympathetic output to muscle after the procedure may represent a compensatory response to maintain blood pressure.

Support or Funding Information

Supported by American Heart Association Grant 15GRNT24480051 (Cui), Penn State Heart and Vascular Institute Penny A. Garban Endowment (Cui), National Institutes of Health Grants P01 HL096570 (Sinoway) and UL1 TR000127 (Sinoway).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Treatment with Streptozotocin (STZ) increases expression of macrophage migration inhibitory factor (MIF) but not metalloproteinase‐13 (MMP‐13) mRNA in the liver of channel catfish
Authors: Nevarez Ericka , Spainhour Rebekah , Kobayashi Yass
Published: June 2021

Tissue repair during liver damage depends on the recruitment and activation of macrophages. In rodents, the production of MIF is essential for the recruitment of scar‐forming macrophages. In addition, production of MMP‐13 appears to be critical for successful remodeling and hepatic tissue repair. In our previous studies with channel catfish, STZ treatment induced hypoglycemia and an abnormal gross morphology of the liver and gall bladder. This suggests that the liver is the target organ for STZ in channel catfish and causes acute liver damage. This results in hypoglycemia, necrosis and eventually organ failure. However, whether liver damage induced by the STZ treatment results in recruitment and activation of macrophage in the liver of channel catfish has never been examined. Liver samples were collected from juvenile channel catfish 7 days after intraperitoneal administration of 0, 3.6, 36, 180, or 360 mg/kg STZ on day 0 (n=4 fish/treatment). Expression of genes (MMP‐13 and MIF) were measured using quantitative real‐time PCR. Expression of MIF and MMP‐13 changed in response to increases in STZ dosage (p<0.01, and p=0.05, respectively.) Expression of MMP‐13 mRNA decreased in response to an increase in STZ dosage. Expression of MIF mRNA was significantly higher in fish treated with 360 mg/kg body weight compared to control (p<0.05). Decreased expression of MMP‐13 suggests STZ treatment inhibits macrophage‐mediated tissue repair. Given that MIF has been associated with multiple liver diseases, STZ treatment may activate multiple mechanisms other than those associated with fibrosis to induce liver damage in channel catfish.

Support or Funding Information

This study is supported by Kansas Idea Network of Biomedical Research Excellence (P20GM103418).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Oleanolic Acid on Inflammatory Cytokines on Rats Fed with High Fructose Diet & Metformin.
Authors: Matumba Mashudu Given , Nyakunda Trevor , Mukwevho Emmanuel
Published: June 2021

Inflammatory cytokines are signaling molecules that play a crucial role in regulation of human immune response. Current research has demonstrated that inflammatory processes also play a significant role in the etiology of type‐2 diabetes (T2D) and obesity. High levels of circulating inflammatory concentration of cytokines have been linked to insulin resistance, the hallmark of T2D. In this study we have assessed the effect of oleanolic acid on some inflammatory cytokine biomarkers (TNF‐α, IL‐6, IL‐10, MCP‐1 and VEGF). Oleanolic acid is a plant derived compound that has shown some promising therapeutic effects on insulin resistance and T2D.

In this study, Sprague Dawley rats were fed with high fructose diet (HFD) and also metformin. Analysis of inflammatory cytokines concentration in blood plasma was done using Bio‐Plex Pro magnetic bead‐based assay. Results showed that all five cytokines were decreased when OA was used and also increased when HFD was used. Similar pattern when gene expression was studied was also observed.

These results clearly indicate that OA influences their concentration and also their gene expression. On the contrary HFD, showed the opposite by increasing both the concentration as well as the gene expression of these cytokines.

In conclusion these study further suggests that OA plays a key role on inflammatory cytokine regulation and it can be used as an effective therapeutic agent to ameliorate insulin resistance and T2D.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Coupling of mDia1‐dependent fast endocytosis to exocytosis in pancreatic beta cells helps to maintain normal glucose tolerance in mice
Authors: Wei Lisi , Du Wen , Zhao Shiqun , Chen Liangyi
Published: June 2021

Previously we have identified a novel Ca2+‐triggered clathrin‐independent but actin‐dependent fast endocytosis (CIE) in pancreatic beta cells. In contrast to well‐defined clathrin‐dependent endocytosis, neither the molecular mechanism nor physiological function of this clathrin‐independent endocytosis is clear. By combining patch clamp capacitance recording, super‐resolution imaging and specific molecular interventions, we have identified that an actin polymerization factor, mDia1, mediates this fast CIE in pancreatic beta‐cells. Using shRNA of mDia1 and knockout mice model, we have shown that removal of CIE leads to impaired exocytosis evoked by sustained stimulation, and disturbed glucose tolerance in mice, which cause ER stress and cellular function degeneration. Interestingly, during the progression of type II diabetes in KKAy mice, we have found out that down‐regulation of mDia1 expression and defects in fast CIE precede the dysregulation of insulin secretion in beta cells, highlighting defects of CIE may cause the beta cell failure. Overall, we conclude that exocytosis‐coupled fast CIE in pancreatic beta cells is essential to sustain insulin secretion during glucose elevation in chronic experiments, and is important for the maintaining of beta cell function and glucose tolerance in a long run.

Support or Funding Information

This study was supported by grants from the National Science and Technology Major 224 Project Program (2016YFA0500400), the Major State Basic Research Program of China 225 (2013CB531200), and the National Natural Science Foundation of China (31327901, 226 31521062, 31570839, 31428004, 61375018, and 61672253).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Mechanism by which PRDM16 Maintains the Adult Small Intestinal Epithelium
Authors: Stine Rachel , Seale Patrick
Published: June 2021

The intestinal epithelium is a well‐organized tissue that undergoes continuous cell turnover, with most cells being replaced every 4–5 days. PRDM16 is a large zinc‐finger containing transcription factor that, in addition to its well‐studied role in brown/beige adipose, regulates maintenance of neuronal and hematopoietic stem cells. However, the role of PRDM16 in other tissues is not well understood. We find that acute global deletion of Prdm16 in adult mice causes death within ten days. The Prdm16‐deleted mice begin to lose weight and appear weak and lethargic, with visibly distended and pale intestines. Histological analysis shows that PRDM16 is selectively and highly expressed in the intestinal epithelium of the duodenum and jejunum. Apoptotic cells are prevalent in the crypt, particularly in the transit amplifying zone by 3 days after Prdm16 deletion. By 5 days after deletion, intestinal tissue appears disorganized, with an abnormal accumulation of secretory cells in the crypt that express markers of both the goblet and Paneth cell lineages, indicating defects in differentiation. Interestingly, we could detect no obvious defects in Notch signaling. We performed RNA‐seq on isolated crypts to identify candidate signaling pathways that change in the intestine shortly after global knockout of Prdm16. A striking upregulation of P53‐activated apoptotic genes occurs immediately following the disappearance of PRDM16 protein, as well as an induction of Type‐I Interferon signaling. Independent of this, we see marked downregulation of genes involved in metabolic pathways. Studying the effects of PRDM16 ex vivo, we find that enteroid culture recapitulates the phenotypes observed in vivo, with Prdm16 deletion causing induction of apoptosis and a failure of intestinal enteroids to form buds. Further, we show that shRNA mediated knockdown of Prdm16 is sufficient to block the formation of enteroids. Our phenotype is ameliorated but not fully rescued by additional deletion of p53 both in vivo and ex vivo. We are currently focusing on defects in mitochondrial function, which appear to be a second major driver of our intestinal phenotype.

Support or Funding Information

NRSA NIDDK F32 DK105743‐03 Pilot Grant NIDDK P30‐DK050306

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Searching for a New Anti‐Cancer Drug: Investigation of KY Hemp‐Induced Apoptosis in Ovarian Cancer Cells
Authors: Sumanasekera Wasana K. , Johnson Christina , Pham Anh‐Thoa , Patel Brijesh , Sivamohan Anjali , Cox Art
Published: June 2021

Marijuana (cannabis sativa) is a schedule 1 drug that has been recently approved by some states in the US for its therapeutic benefit. Although there are a few reports about its anti‐cancer potential, currently it has been used mainly for treatment‐resistant epilepsy and to alleviate pain. However, marijuana is addictive and causes mind‐altering actions. Hemp, which belongs to the same genus and species as marijuana, shows similar therapeutic benefits without addictive potential. Our laboratory is interested in examining for unconventional therapies for ovarian cancer. The main objective of the current study is to investigate hemp‐induced modulation of A2780 ovarian cancer cell apoptosis.

Methods

KY‐Hemp extracts were made via supercritical CO2 extraction, and its cannabidiol content was analyzed using HPLC by KY commonwealth Extracts and MRX laboratories respectively. In our laboratory this hemp extract, which contained cannabidiol (CBD) and minimal tetrahydrocannabinol (THC) was tested for its anti‐cancer effects. A2780 epithelial ovarian cancer cells (OCCs) were seeded in 12‐well plates. Following serum starvation, the OCCs were treated with vehicle (no hemp negative control), Staurosporine (positive control for apoptosis) or different concentrations of hemp extracts for 24 hours. Then cell supernatants were removed and subject to Bradford protein assay followed by caspase‐3 ELISA. Caspase‐3 is one of the major caspases released from apoptotic cells. Caspase‐3 released from each treatment group was detected using caspase‐3 ELISA based on manufacturer's protocol. In brief, caspase‐3 standards and cell supernatants from all treatment groups, including negative and positive controls, were incubated in ELISA plates coated with caspase‐3 antibodies. After a series of steps that included treatment with secondary antibodies and a substrate solution, caspase‐3 levels were detected spectrophotometrically. To ensure the reproducibility, each experiment was repeated a minimum of three times. We also studied pure CBD (analytical standard from Sigma Aldrich) induced apoptosis in A2780 OCCs. Our results indicate that KY hemp extract cause significant increase in caspase‐3 release, which is indicative of apoptosis of OCCs. Caspase‐3 release is significant at hemp extracts that contain 83.5 μM and 167 μM CBD. KY hemp‐induced caspase‐3 secretion is somewhat comparable to pure CBD‐induced caspase‐3 release. Currently we are investigating the anti‐apoptotic effects of hemp at lower concentrations. Based on the data here we conclude that KY hemp has anti‐cancer potential against ovarian cancer

Support or Funding Information

Funding was provided by Sullivan University System via a faculty development grant awarded to Dr. Sumanasekera

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epstein Barr virus and Multiple Sclerosis
Authors: Rodriguez‐Pérez Jose Alejandro , Pérez‐Vallín Vanessa , Mirabal‐Viel Alejandro , Padrón‐González Alexander Ariel , Castillo‐González William , González‐Losada Cristobal , Dorta‐Contreras Alberto Juan
Published: June 2021

Cause of Multiple Sclerosis is not well known yet. There are theories that oint out a viral origen that may initiate the autoimmune response. There are a lot of biological agents that could be causative. Among them is Epstein Barr Virus but there are controversies about this topic. With the evaluation of the specific antibody index developed by Reiber, perhaps it will be possible to find some evidences that sign Epstein Barr virus like the cause of the disease. The aim of this paper is to evaluate the antibody index anti‐Epstein Barr virus in 27 patients with multiple sclerosis and 19 control cases. There were significant differences between Qspecific (Specific antibodies in CSF/Specific antibodies in serum) between the sclerosis multiple group and the control group (p<0.0001). The antibody index( Qspecific/Q IgG or Q lim) between both groups was not significant differences (p=0.0519). Anti‐Epstein Barr Qspecific in Multiple Sclerosis could contribute to clarify the etiology of this disease but it is necessary to increase the total of patients involved in the sample in order to confirm that the antibody index in patients could be definitively a probe of Epstein Barr virus as the causative of the disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Furosemide Reduces BK‐αβ4‐Mediated K+ Secretion in Mice on an Alkaline High K Diet
Authors: Wang Bangchen , Sansom Steven
Published: June 2021

Because of its cardio‐protective benefits, a high K diet is often warranted in conjunction with diuretics such as furosemide for treating hypertension. It is critical to understand how such diets influence the diuretic actions on renal K handling in order to choose the best drug for patients. Studies have shown that furosemide acidifies the urine by increasing acid secretion from thick ascending limb. On the other hand, the Ca2+‐activated large conductance K channel (BK‐αβ4) is regulated by pH. We hypothesized that in mice on an alkaline high K diet (HK), furosemide reduces BK‐αβ4‐mediated K+ secretion in the distal nephron by acidifying the urine. To test our hypothesis, HK‐adapted wild‐type (WT) and BK‐β4 knockout mice (β4KO) were given either control water or furosemide water (0.1 g/L). Urine was collected in metabolic cages on the day before treatment and 1, 4, 7, 11, and 14 days after treatment. Mice were then sacrificed to collect blood and kidneys. The urine pH was reduced after furosemide treatment in both WT and β4KO. Urinary K+ excretion was reduced on day 1 of furosemide treatment in WT (2801 ± 235 vs. 2175 ± 119 μmol/day) but not β4KO (1934 ± 497 vs. 2395 ± 207 μmol/day). After 14 days of treatment, renal K+ clearance was decreased and plasma [K+] increased in WT (ctrl water: 591 ± 45 mL/day; 4.46 ± 0.13 mM vs. furo water: 424 ± 32 mL/day; 5.13 ± 0.10 mM) but not β4KO (ctrl water: 453 ± 42 mL/day; 5.04 ± 0.19 vs. furo water: 516 ± 64 mL/day; 5.03 ± 0.06 mM). Additionally, renal K+ clearance was positively correlated with urine pH in WT but not β4KO. Western blotting and immunofluorescence staining showed that furosemide decreased total protein expression of BK‐β4 and reduced apical localization of BK‐α. These results indicate that in mice on HK, furosemide reduced BK‐αβ4‐mediated K+ secretion by acidifying the urine, making furosemide a K‐sparing diuretic in the setting of an alkaline high K diet.

Support or Funding Information

This project was supported by the National Institute of Diabetes and Digestive and Kidney Diseases grants R01‐DK‐092474, R01‐DK‐071014 and F30‐DK‐108456.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CHIP regulates Aquaporin‐2 Quality Control and Body Water Homeostasis
Authors: Wu Qi , Moeller Hanne B. , Stevens Donté A. , Sanchez‐Hodge Rebekah , Childers Gabrielle , Kortenoeven Marleen L.A. , Cheng Lei , Rosenbaek Lena L. , Rubel Carrie , Patterson Cam , Pisitkun Trairak , Schisler Jonathan C. , Fenton Robert A.
Published: June 2021

The importance of the kidney distal convoluted tubule (DCT) and cortical collecting duct (CCD) are highlighted by various water and electrolyte disorders that arise when the unique transport properties of these segments are disturbed. Despite this critical role, little is known about which proteins play a regulatory role in these cells and how these cells can be regulated by individual physiological stimuli. By combining proteomics, bioinformatics, and cell biology approaches alongside gene targeted animal models, we found an essential role of the E3 ubiquitin ligase CHIP for modulating the abundance of the water channel aquaporin‐2 (AQP2). CHIP is highly expressed throughout the collecting duct, modulated in abundance by vasopressin, interacts with AQP2, Hsp70 and Hsc70 and can directly ubiquitylate AQP2 in vitro. shRNA knockdown of CHIP in CCD cells increased AQP2 protein half‐life and reduced AQP2 ubiquitylation, resulting in greater AQP2 and phosphorylated AQP2 levels. Plasma membrane abundance of AQP2 was increased in CHIP knockdown cells. CHIP knockout mice or novel CRISPR/Cas9 mice without CHIP E3 ligase activity had greater AQP2 abundance and altered renal water handling, with decreased water intake and urine volume, alongside higher urine osmolality. No significant changes in other water or sodium transporting proteins were observed in the gene‐modified mice. In summary, CHIP has a regulatory role for AQP2 and subsequently renal water handling.

Support or Funding Information

Q.W. is supported by European Union Horizon 2020 Marie Skłodowska‐Curie Individual Fellowship project 705682, Danish Medical Research Council grant 6110‐00118B, and Lundbeck Foundation grant R192‐2015‐804. M.L.A.K. is supported by Danish Medical Research Council grant 1333‐00279. Funding is provided by National Institutes of Health grant R37 HL065619 (to C.P. and J.C.S.). Funding is also provided by the Novo Nordisk Foundation (R.A.F.), the Lundbeck Foundation (R.A.F.), and the Danish Medical Research Council (R.A.F.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the Occluding Septate Junction Protein Mesh in Epithelial Integrity and Ion Transport in the Drosophila Renal Tubules
Authors: Jonusaite Sima , Rodan Aylin
Published: June 2021

Background/objective

Epithelial occluding junctions are specialized cell‐cell junctions that establish and maintain cell polarity and control paracellular ion, solute and water transport. Tight junctions are the occluding junctions in the epithelia of vertebrates, whereas the epithelia of invertebrates possess septate junctions (SJs). Molecular and genetic analysis of Drosophila SJs have identified a number of transmembrane as well as cytoplasmic scaffolding proteins expressed in a tissue‐specific manner. However, many questions about the role of these proteins in SJ assembly, maintenance and function remain open. Recently, an integral protein, Mesh, has been identified within the SJs of Drosophila midgut and renal tubule epithelia, and shown to be required for the barrier function of the midgut. Here, we report, for the first time, examination of the role of Mesh in adult fly renal tubules, whose function in the maintenance of internal homeostasis relies on regulated transepithelial ion and water secretion/absorption.

Methods

mesh knockdown in the fly tubules was achieved using the GAL4‐UAS system. Expression and localization of Mesh and a scaffolding SJ protein, Discs large (Dlg), in control and mutant tubules were determined using confocal microscopy and available antibodies. Transepithelial fluid and K+ transport in the main segment of the control and mesh knockdown tubules were measured using the Ramsay assay and a K+‐specific electrode.

Results

Adult fly renal tubules consist of four major morphologically and functionally distinct segments and have two cell types, the larger principal cells and smaller intercalated stellate cells. We found that developmental mesh knockdown in the tubule principal cells was associated with enlarged initial and main segments of the adult fly tubule and reduced septate junctional localization of a scaffolding protein, Dlg, especially in the enlarged initial segment. In addition, the main segment transepithelial fluid and K+ transport in isotonic condition was abolished in mesh knockdown tubules compared to the control groups.

Conclusions and significance

These data suggest that the SJ protein Mesh is required for SJ assembly, epithelial integrity and ion transport in the fly renal tubule epithelium. Since the Mesh mammalian homolog, Susd2, is also highly expressed in the mouse renal epithelium with unknown function, these studies pave the way for further characterization of the Mesh/Susd2 family of proteins in both invertebrates and vertebrates.

Support or Funding Information

This project was supported by NIH/NIDDK to ARR

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.