bluuebunny/crossref_metadata_2025 huggingface dataset:
Increases in insulin signaling following electrical pulse stimulation are blunted in myotubes derived from severely obese individuals with or without type 2 diabetes
Authors: LIM SEONGKYUN , CHAVES ALEC , ZHENG DONGHAI , HOUMARD JOSEPH
Published: June 2021

Introduction

Exercise training has been shown to be effective in improving skeletal muscle insulin sensitivity, but individuals with severe obesity and/or type 2 diabetes (T2D) may not respond as robustly as lean individuals. Electrical pulse stimulation (EPS) applied to primary human skeletal muscle cell cultures (myotubes) has been used as an in vitro exercise model to understand responses to contractile activity alone.

Purpose

The purpose of this study was to examine the effect of antecedent EPS on insulin signaling in myotubes from lean and severely obese individuals, with and without type 2 diabetes.

Methods

Human muscle cells were cultured from muscle biopsies obtained from lean (BMI: 21.7± 0.75 kg/m2), obese (BMI: 39.1± 0.99 kg/m2), and diabetic (BMI: 40.7 ± 9.44 kg/m2) individuals. Following differentiation, mature myotubes were stimulated with electrical pulses, set at 11.5 V, 1 Hz and 2 ms for 24 h. Following EPS, myotubes were treated with 100nM insulin for 10 minutes, after which the cell lysate was harvested for western blot analysis to measure the phosphorylation status of AKT (pAKT).

Results

Insulin stimulation increased pAKT levels in all three groups (p = 0.04, Mean ± SEM, 2.86 ± 0.43, 2.1 ± 0.33, 1.85 ± 0.12; fold change over control in lean, obese, and T2D respectively). However, in response to EPS, only the lean group increased insulin‐stimulated pAKT further (p=0.03, 2.86 ± 0.43 vs 4.07 ± 0.11, fold increase over control).

Conclusion

Our data show that EPS increases insulin‐stimulated pAKT levels in myotubes from lean individuals, but this response is absent in myotubes from individuals with severe obesity with or without T2D, indicating that there is an inherent defect in their cellular response to electrical/contractile activity.

Support or Funding Information

National Institutes of Health (DK56112, JAH).
Changes in Hepatic Expression of Hyaluronan Synthases (HAS2 and HAS3) After Treatment with Steptozotocin in Channel Catfish
Authors: Dougherty Megan , Schmidtberger Abigail , Nevarez Ericka , Kobayashi Yasuhiro
Published: June 2021

Streptozotocin (STZ) is commonly used experimentally to induce hyperglycemia in rodents. As an alternative model organism for investigating human obesity, channel catfish show a genetic predisposition toward an accelerated growth rate resulting in a phenotype similar to obese humans. In our previous studies, STZ treatment resulted in hypoglycemia and abnormal liver morphology in channel catfish, indicating that STZ treatment possibly induces liver damage. The mechanisms involved in the STZ‐induced hypoglycemic and tissue damage have never been examined. Another noted marker for liver damage stems from hyaluronan synthases (HAS), which have been associated with tissue damage in both humans and rodents. We hypothesized the expression of HAS mRNA would change in response to liver damage induced by STZ in channel catfish. Juvenile channel catfish were treated with 0, 3.6, 36, 180, or 360 mg STZ/kg body weight (n=4 fish/treatment) on day 0. Liver samples were collected on day 7. Hepatic expression of HAS2 and HAS3 mRNA was examined, using real‐time PCR. Expression of HAS2 and HAS3 mRNA decreased in response to an increased dose of STZ (p < 0.05 and p < 0.10, respectively). These results suggest STZ induces severe liver damage that down regulates the expression of HAS2 and HAS3 mRNA. However, the effects of STZ on gene expression appear to be global, and the tissue damage induced by STZ is not reversible by changes in the amount or activity of HAS.

Support or Funding Information

This research was funded by the APS and a grant from the National Science Foundation Integrative Organismal Systems (IOS) (Grant #IOS‐1238831). It was also supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103418.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Perivascular Adipocytes Store Norepinephrine by Vesicular Transport
Authors: Ahmad Maleeha , Darios Emma S. , Ayala‐Lopez Nadia , Burnett Robert , Watts Stephanie W.
Published: June 2021

Perivascular adipose tissue (PVAT) exerts an anti‐contractile effect on blood vessels in healthy individuals, a mechanism lost in obesity‐related hypertension. To understand this process, we must first investigate the role of the functional adrenergic system residing within PVAT. We have shown that PVAT contains functional norepinephrine (NE), and that PVAT adipocytes have NE transporters, similar to sympathetic neurons. We hypothesize that NE is stored in vesicles within PVAT adipocytes, and that the vesicular monoamine transporter (VMAT) is used to do so. High‐performance liquid chromatography showed the presence of NE in normal male Sprague Dawley rat superior mesenteric artery (SMPVAT; 503.60 ± 34.70 ng/g tissue) and mesenteric (MPVAT; 148.20 ± 15.00 ng/g tissue) PVATs (N=6). Using immunofluorescence, we tested for VMAT1 and VMAT2 in these PVATs (N=6), and both transporters were present surrounding the lipid of the adipocytes. Immunofluorescence of MPVAT (N=4) and SMPVAT (N=4) revealed the co‐localization of NE and VMAT2 surrounding the lipid of PVAT adipocytes, and especially concentrated around the nucleus. We developed a protocol imaging live primary rat MPVAT adipocytes to capture real‐time uptake of Mini202, a fluorescent VMAT probe functioning as a false neurotransmitter (N=4). Cells were stained with 500 μM Mini202 (see data table). Addition of Rose Bengal (100 μM), a potent VMAT inhibitor, significantly reduced Mini202 signal. Addition of plasma membrane transporter inhibitors, nisoxetine (NE transporter, 10 mM) and corticosterone (organic cation 3 transporter, 10 mM) also significantly reduced Mini202 signal. Interestingly, immunofluorescence results support that neither VMAT1 (N=4) nor VMAT2 (N=4) is present in retroperitoneal adipocytes, suggesting that perivascular adipocytes are unique in their ability to store NE. This study supports a new function of perivascular adipocytes in storing amines and provides a basis for understanding NE storage by PVAT in obesity‐related hypertension.

Support or Funding Information

Funding provided by NIH NHLBI PO1HL70687 and the APS UGREF

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inactivation of the Pyruvate Dehydrogenase Complex Results in a Decrease in Butyrate Oxidation and Enhancement of Cellular Proliferation
Authors: Simon Emily , Park Bohye , Spilman Kasey , Donohoe Dallas R.
Published: June 2021

Bacteria in the colon ferment dietary fiber producing the metabolite butyrate, which has been shown to have protective effects against colorectal cancer. Butyrate has two main functions in colonic epithelial cells (colonocytes); metabolically, as the primary energy source, and in gene expression, as a histone deacetylase (HDAC) inhibitor. These two functions are not independent of each other. Cancerous colonocytes oxidize butyrate at a lower capacity than non‐cancerous colonocytes which impacts its role in the inhibition of HDACs. Previously, we found that dichloroacetate, which inhibits pyruvate dehydrogenase kinase 2 (PDK2) resulted in an increased oxidation of butyrate in colorectal cancer (CRC) cells that was associated with diminished pyruvate dehydrogenase complex (PDC) phosphorylation. Increased phosphorylation of this complex inhibits the activity and is a common feature in cancer cells. Therefore, we hypothesized that increased phosphorylation of PDC in CRC cells mediates the metabolic shift away from utilizing butyrate, which is a predominant feature in non‐transformed colonocytes. Here, we report that targeted knockdown of PDK2 by RNA interference showed a significant increase in butyrate oxidation by the CRC cell. This effect was associated with a general decrease in PDH phosphorylation and lactate production, while also having increased levels of acetyl‐CoA. In addition, knockdown of pyruvate dehydrogenase phosphatase (PDPC1) did not significantly alter butyrate oxidation in CRC cells. We next tested whether the oxidation of other short‐chain fatty acids (SCFAs) (acetate and propionate) was also regulated by PDK2 and the phosphorylation of PDH. Interestingly, the oxidation of these two SCFAs was not significantly affected by PDK knockdown. Therefore, this suggests that the phosphorylation of PDH by PDK2 is an important event in regulating the CRC cell's metabolic shift away from the oxidation of butyrate and toward glycolysis.

Support or Funding Information

UTK Start‐up Funds

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of Hippo Signaling by rhBMP‐2 Suppresses the Proliferation of Human Colorectal Cancer Cell
Authors: Liu Yu Chuan , Kim Soo Mi
Published: June 2021

Despite that the use of recombinant human bone morphogenetic protein (rhBMP)‐2 has been debated for a decade due to its oncogenic characteristics. However, the underlying molecular mechanism and safety issues of rhBMP‐2 usage remain poorly understood. In this study, we investigated the effect of rhBMP‐2 and its signaling pathways involved in colorectal cancer cell (CRC) using HT‐29 and HT‐116 cells. RhBMP‐2 significantly inhibited proliferation of CRC cells in dose‐dependent by MTT assay. Cell cycle arrest in G1 phase was induced at 24h after rhBMP‐2 treatment. RhBMP‐2 also stimulated Smad4, p53 and p21 levels, and reduced cyclin D1, cyclin‐dependent kinase (CDK) 4 and CDK6 activities. On the other hand, rhBMP‐2 treatment resulted in reduced protein expression levels of poly (ADP‐ribose) polymerase (PARP) whereas those of cleaved PARP and cleaved caspase‐9 were significantly increased in CRC cells. In addition, rhBMP‐2 increased RASSF1, MST1, MST2, Mob1, LATS1, Sav1, and p‐YAP protein levels. Therefore, our results indicate that rhBMP‐2 suppresses CRC cell proliferation which is mediated via the hippo signaling pathway. Therefore, targeting BMP‐2 may constitute a potential therapeutic strategy for CRC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Live Cell Fluorescence Imaging Reveals Intercellular Calcium Waves and Chloride Channel Activation During Rotavirus Infection
Authors: Chang‐Graham Alexandra , Perry Jacob , Engevik Melinda , Danhof Heather , Hyser Joseph
Published: June 2021

Background

Rotavirus (RV) causes severe diarrheal disease in children worldwide. During infection, RV increases cytosolic calcium in intestinal epithelial cells through release of endoplasmic reticulum calcium and activation of the store‐operated calcium entry (SOCE) pathway. Increased cytosolic calcium is critical for RV replication and activation of secretory pathways in GI epithelium, yet the molecular details of these processes remain poorly defined. Thus, characterizing RV‐induced calcium signaling at the molecular level is needed to understand RV pathogenesis. We conducted live cell fluorescence imaging in cell lines and human intestinal enteroids (HIEs) as a biologically relevant model of the human small intestine to measure calcium signaling dynamics and chloride channel activation during RV infection.

Methods & Results

We first engineered African green monkey kidney MA104 cells and HIEs to stably express GFP‐based genetically encoded calcium indicators to measure calcium dynamics throughout RV infection using live cell epifluorescence microscopy. We found that RV significantly increases the number and magnitude of calcium transients, which were attenuated with SOCE blockers. Furthermore, we observed that single RV‐infected cells triggered long‐distance intercellular calcium waves (ICWs) that encompassed surrounding uninfected cells in both MA104 cells and both duodenum and jejunum HIEs. Treatment with the ectoNTPase apyrase or purinergic receptor inhibitors blocked ICWs and reduced RV replication, mucin release, and serotonin secretion. These results indicate that extracellular ATP/ADP signaling elicits ICWs and is an important mediator of calcium dynamics during RV infection. Finally, we created HEK293 cell lines stably expressing halide‐sensitive YFP, a chloride channel biosensor, and either the calcium‐activated chloride channel anoctamin 1 (Ano1) or cystic fibrosis transmembrane conductance regulator (CFTR). We found that RV activated both mouse Ano1 and CFTR, which were attenuated with channel‐specific blockers, and suggests that multiple chloride channels can be activated during RV infection.

Conclusions

Overall, our results support that RV causes dynamic calcium signaling during infection that is mediated by SOCE and purinergic signaling. Both pathways are potent stimulators of chloride secretion, thus they are potential therapeutic targets for developing life‐saving anti‐diarrheal drugs. Furthermore, we are the first to show that viruses can exploit purinergic signaling to generate ICWs that encompass uninfected cells and thereby potentially amplify the pathophysiological signaling important for diarrhea.

Support or Funding Information

BCM Seed Funding, NIH F30 DK112563‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
WW Domain‐containing Oxidoreductase Regulates Keratinocyte Growth for Maintaining Epidermal Homeostasis
Authors: Chou Ying‐Tsen , Hsu Li‐Jin
Published: June 2021

Human fragile WWOX gene encodes a candidate tumor suppressor WW domain‐containing oxidoreductase. Previous studies have shown that ectopically overexpressed WWOX induces apoptosis in cancer cells and suppresses tumor growth both in vitro and in vivo. Functional suppression of WWOX by antisense mRNA, dominant negatives, and small interfering RNA protects cells from apoptosis by tumor necrosis factor, anticancer drugs, UV light, and ectopic p53 in vitro. Interestingly, substantial evidence revealed that progression of various cancers to a pre‐metastatic stage positively correlates with upregulation of WWOX protein expression, suggesting that WWOX may act more than a tumor suppressor. In normal human epidermis, WWOX is abundant in proliferating epithelial cells of the skin, and its expression is increased in intensity toward the superficial layers. In this study, we determined a reduced number of Ki67‐positive proliferating keratinocytes in the epidermis of our newly developed Wwox gene knockout (Wwox−/−) mice. A marked reduction in the basal (K5‐positive) and suprabasal (K10‐positive) keratinocyte numbers was examined in the keratinized squamous epithelium of the Wwox−/− mouse skin. Apparent decreases in Wwox−/− epidermal and dermal thickness were found. To further verify whether WWOX regulates keratinocyte growth, we detected significantly decreased cell proliferation in both ex vivo Wwox−/− primary keratinocyte cultures and WWOX‐knockdown HaCaT cells as compared to the controls. No significant increase in the number of TUNEL‐positive cells was observed in the Wwox−/− epidermis. Moreover, our recent data showed that keratinocyte differentiation was slightly enhanced in WWOX‐knockdown HaCaT cells. Together, our results clearly demonstrated a crucial role of WWOX in regulating normal keratinocyte proliferation for maintaining skin homeostasis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isoprenoid Depletion Increases Cytokine mRNA Levels in Primary Human Monocytes
Authors: Frey Tiffany Albright
Published: June 2021

Mevalonate Kinase Deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase (MVK) gene, which produces an enzyme responsible for the production of isoprenoids. Previous research in various model systems has demonstrated that depletion of isoprenoids leads to inflammasome activation and increased levels of secreted IL‐1B on a post‐translational level. However, patient data indicates that MKD is a multi‐cytokine disease with increased plasma levels of cytokines such as TNF‐alpha, IL‐6, and IFN‐gamma. In order to study the mechansims responsible for these changes, the mevalonate pathway was inhibited with lovastatin in peripheral blood mononulcear cells (PBMCs) and monocytes isolated from the blood of healthy donors followed by stimulation with lipopolysaccharide (LPS) to induce an inflammatory response. Lovastatin treatment resulted in significantly increased levels of TNF‐alpha, IL‐6, IL‐12p40, and IFN‐gamma mRNA in both PBMCs and monocytes following LPS stimulation as compared to control cells. Lovastatin treatment did not alter IL‐1B, CD14, or IL‐18 mRNA levels either before or after LPS stimulation. Addition of mevalonate to the cells treated with lovastatin prevented the increased cytokine mRNA levels indicating that these changes are due to inhibition of the mevalonate pathway. These results indicate that isoprenoid depletion alters the expression of multiple cytokines on the mRNA level in primary human monocytes, which can be used as a model system ex vivo to study these changes.

Support or Funding Information

Dickinson College Faculty Start‐up and Research & Development Committee Funding

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic alcohol misuse induces alveolar macrophage oxidative stress and mitochondrial dysfunction via upregulation of microRNA‐130a
Authors: Yeligar Samantha M.
Published: June 2021

Objective

Compared to non‐alcoholics, individuals who misuse alcohol are 2–4 times as likely to develop respiratory infections and acute respiratory distress syndrome. Alcohol misuse enhances alveolar macrophage (AM) oxidative stress and causes phagocytic dysfunction via upregulation of microRNA (miR)‐130a. Although studies indicate that alcohol‐induced mitochondrial (MT) redox imbalance in AM may contribute to AM phagocytic dysfunction, the mechanisms by which this occurs is unknown. We hypothesized that alcohol upregulates miR‐130a to destabilize its target peroxisome proliferator‐activated receptor (PPAR)γ, which dysregulates AM MT function and depletes ATP levels necessary for phagocytosis.

Methods

AMs were obtained from the bronchoalveolar lavage fluid of C57BL/6J mice fed ± ethanol (20% w/v) in the drinking water for 12 wks. In parallel, MH‐S cells, a mouse AM cell line, were transfected ± 50 nM miR‐130a inhibitor and treated ± 0.08% ethanol for 3 d. Levels of PPARγ‐related miR‐130a, which binds to the PPARγ 3′untranslated region to decrease PPARγ, and PPARγ mRNA were assessed by qRT‐PCR. PPARγ protein expression was assessed by confocal cytoimmunostaining. MT‐derived oxidative stress was measured using MitoSOX and MitoTracker CMXRos fluorescent molecular probes. MT bioenergetics profile was measured with MT stress testing using a Agilent Seahorse XF96 Extracellular Flux Analyzer. AM function was evaluated by phagocytosis assay (S. aureus internalization).

Results

In vivo and in vitro studies demonstrated that ethanol: 1) increased miR‐130a levels and decreased PPARγ expression; 2) increased MT‐derived oxidative stress; 3) decreased MT oxygen consumption rate, bioenergetics, and ATP; and 4) impaired phagocytic capacity. miR‐130a inhibitor reversed these ethanol‐induced AM derangements.

Conclusions

Chronic alcohol misuse enhances AM oxidative stress and MT dysfunction by upregulating PPARγ‐related miR‐130a. Our studies suggest that therapeutically targeting MT as an intervention will ameliorate alcohol‐induced AM phagocytic dysfunction and may decrease susceptibility of lung infections in patients with a history of alcohol misuse.

Support or Funding Information

Research Funding Source: R00AA021803 (SMY).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Patterns of Suppressed Mitochondrial Respiration in Isolated Muscle Fibers from Type 2 Diabetics
Authors: Turner Kristen D. , Harris Matthew P. , Kronemberger Ana , Ueda Kenichi , Feider Andrew J. , Kenny Helena , Anderson Ethan J. , Abel E. Dale , Casey Darren P. , Lira Vitor A.
Published: June 2021

The quality of skeletal muscle is markedly influenced by mitochondrial function. Aberrant mitochondrial function has been associated with reduced contractile and metabolic dysfunction in skeletal muscle, potentially leading to insulin resistance and type 2 diabetes. However, the direct relationship between mitochondrial dysfunction and type 2 diabetes remains to be fully understood. In the present study, vastus lateralis muscle biopsies were obtained from individuals with type 2 diabetes (N = 7) and non‐diabetic, age, gender and weight‐matched controls (N = 7). Maximal mitochondrial oxygen consumption was assessed in isolated muscle fibers and normalized to wet tissue weight. Preliminary findings illustrate that reductions in carbohydratedependent ADP‐stimulated respiration (~30%) were sustained in muscle fibers from type 2 diabetics, even with the addition of the Krebs Cycle anaplerotic substrates, glutamate (~30%) and succinate (~32%). Interestingly, trends of similar magnitudes persisted for lipid substrate metabolism, as fatty acid‐dependent ADP‐stimulated respiration also tended to be lower (~35%) compared to controls. Furthermore, rotenone‐treated respiration in diabetic skeletal muscle tended to be reduced (~37%). To conclude, these initial findings provide support for a systematic impairment in the oxidative metabolism of both carbohydrates and lipids in skeletal muscle of type 2 diabetic individuals. In addition, findings from rotenone‐treated conditions indicate that investigating functional deficits of Complex I of the Electron Transport Chain (ETC) may provide a more insightful understanding of the relationship between mitochondrial respiratory control and type 2 diabetes.

Support or Funding Information

*This study is supported by funds from the American Diabetic Association, ADA‐1‐16‐ICIS‐015, (D.P.C.) and start‐up funds from the Department of Health and Human Physiology at the University of Iowa (V.A.L.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of Different Mathematical Approaches to Assess Calcium Retention Capacity in Cardiac Isolated Mitochondria following Ischemia Reperfusion Injury
Authors: Lahil Harjeev S. , Salzman Michele M. , Balzer Claudius , Cleveland William J. , Riess Matthias L.
Published: June 2021

A hallmark of myocardial ischemia reperfusion (IR) injury is mitochondrial (m) dysfunction resulting in, e.g., altered electron transport, increased reactive oxygen species formation, decreased adenosine triphosphate synthesis and reduced calcium (Ca2+) retention capacity (CRC). Different cardioprotective strategies aim to attenuate this dysfunction. CRC is typically assessed by adding boluses of extra‐mCa2+ which is taken up by functioning mitochondria to a higher degree, leading to a later opening of the mitochondrial permeability transition pore (mPTP) and release of accumulated intra‐mCa2+ than by damaged mitochondria. We hypothesized that mPTP opening could be predicted by the degree of decrease in the rate of mCa2+ uptake after each exogenous Ca2+ bolus before the mPTP opens. Following IR with and without different cardioprotective stratetgies, mitochondria were isolated from Brown Norway rat (n=38) and Arctic Ground Squirrel (n=19) isolated hearts, and CRC was assessed by measuring changes of extra‐mCa2+ by calcium‐green fluorescence when repeated boluses of exogenous Ca2+ were given in the presence of pyruvate/malate or succinate as complex I or II substrates, respectively. Multiple regression analyses (alpha 0.05) were used to compare the actual number of Ca2+ boluses leading to mPTP opening to the number predicted from absolute mCa2+ uptake and %mCa2+ uptake, respectively. There were no significant differences in regressions between species. Despite acceptable regression coefficients (R2: 0.81/0.81 vs 0.76/0.78), both methods (absolute/%) had a regression slope significantly higher than 1.0 (slope±SEM: 1.28±0.08/1.35±0.09 vs 1.72±0.13/2.00±0.14) for complex I vs II substrates, respectively. This indicates an increasing overestimation of CRC by either method compared to the actual number of boluses before mPTP opening, more so in less dysfunctional compared to more dysfunctional mitochondria. This overestimation was stronger in the presence of complex II vs I substrates. Thus, predictions of eventual mPTP opening from mCa2+ uptake after individual exogenous Ca2+ boluses are more reliable in dysfunctional vs functional mitochondria and when complex I substrate is used compared to complex II substrate.

Support or Funding Information

Merit Review Award (IK2 BX001278 and I01 BX003482) from the United States Department of Veterans Affairs Biomedical Laboratory R&D Service; National Institutes of Health (5R01 HL123227) and institutional funds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ANP antibody attenuates RIHP amplification during acute Saline Volume Expansion in rats
Authors: Briones María Eugenia Sánchez , Lopez‐Rodríguez Juan Francisco , Calvo‐Turrubiartes Miriam Z. , Rodríguez‐Martínez Manuel
Published: June 2021

The role of Atrial Natriuretic Peptide (ANP) on renal interstitial hydrostatic pressure (RIHP), urinary excretion of sodium (UNaV) and water (V) amplification responses during saline volume expansion (SVE, 5% bw, ~30 min, 0.9%), and immediately after (maintained SVE, 10 min) was studied in male adult wistar rats 24 h after antiseptic intra‐arterial infusion of polyglonal IgG anti‐Atrial Natriuretic Factor (ANF) antibody (AbANP, 18 μg, wich theoretically blocked all ANP secretion, n=8) versus control (CTR, gluc5%, n=10). The day of the acute experiment, variables were continously measured in anesthetized animals with hormonal clamp (without ANP), kidney denervation (chemical and mechanical) and renal perfusion pressure control at 100 mmHg during hydropenia (H), SVE, and maintained SVE(mSVE).

The results are shown in table 1. For Ht and plasmatic protein, there were no differences between groups during H period and the hemodilution response was similar between groups. For RIHP, an attenuation in the amplification response was obtained, from 3 times in CTR vs H to 1 time in abANP group (MANOVA test, p<0.05 between groups, SVE and mSVE., t‐student test vs H each group p<0.05). For V and UNaV responses, there were no differences between groups or periods, achieving the amplification response in the same magnitude for both groups. Finally, an increase in UGMPcV and UNOxV during SVE and mSVE were similar in both groups.

Conclusions

in rats, the acute increase in plasmatic ANP is necessary for the complete RIHP amplification during acute SVE, probably involving an independent nitric‐oxide mechanism. Also, a dissociation between RIHP and natriuretic response was observed.

Support or Funding Information

Supported by IPL's financial resources

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal protective effects of the flavonoid (−)‐Epicatechin in a chronic kidney disease (nephrectomy 5/6) model
Authors: Montes Osvaldo , Ramirez‐Sanchez Israel , Villarreal Francisco , Meaney Eduardo , Najera Nayelli , Arellano Monica , Ceballos Guillermo
Published: June 2021

Background

Chronic kidney disease (CKD) is a worldwide public health problem affecting 10% of the adult population. One of the most important factors in the progression of CKD from early stages is oxidative stress. Currently there is limited efficacy to treat this problem so the objective of this study was to test Epicatechin (Epi), effects in a mice model of CKD

Methods

A nephrectomy 5/6 (Nf) in C‐57BL6 mice model was implemented and Epi treatment (14 days doses doses of 0.01, 0.1 and 1 mg/kg) was provided by gavage. Changes in Systolic blood pressure (SBP), serum urea and creatinine, renal cortex morphology, serum tetrahydrobiopterin (BH4), in 3‐nitrotyrosine as oxidative damage marker and of Angiotensin II AT1 receptor (AGTR1) and NOX‐4 protein expression were evaluated

Results

SBP increased in the Nf group and decrease significantly in Nf+ Epi groups. Serum urea and creatinine levels increased in the Nf group Epi treatment induced a significant decrease. Epi treatment induced a dose dependent increase in serum BH4 in Nf animals. Normal renal cortex morphology was conserved at 1 m/kg Epi dose. The expression of AGTR1 and NOX‐4 was increased in the Nf group, Epi treatment in a dose dependent manner decrease the AGTR1 and NOX‐4 expression.

Conclusion

The present findings suggest that Epi could prevent the progression of CKD and may be used as complement for the modulation of blood pressure and f renal dysfunction.

Support or Funding Information

Conacyt 253769

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Renal Expression of Adhesion GPCR Gpr116 (ADGRF5) Plays a Role in Urinary Concentration in Mice
Authors: Zaidman Nathan , Pluznick Jennifer
Published: June 2021

G‐protein coupled receptors (GPCR) are a large and diverse family of integral membrane proteins that recognize a tremendous assortment of extracellular molecules including neurotransmitters, hormones, light and odors. They are a common target of pharmaceutical drug development, and uncovering the function of novel GPCRs in the kidney represents a wealth of untapped therapeutic potential. We previously performed an mRNA screen for novel GPCRs in the kidney to identify promising yet overlooked renal GPCRs. This screen revealed that Gpr116, an adhesion‐class GPCR, is highly expressed in the kidney. To understand the role of Gpr116 in renal physiology, we have taken a multidisciplinary approach. Using transfected HEK293 cells expressing cloned Gpr116, we have validated a monoclonal antibody for Gpr116. In murine kidney, this antibody indicates localization of Gpr116 to intercalated cells in the collecting duct of mouse kidneys. This staining is absent in kidneys from targeted knockout (KO) of Gpr116 in renal tubules (Gpr116flox/flox, ksp‐Cre). Additionally, kidney‐specific KO animals have significantly reduced urine osmolality (1552±71 mOsm/kg, N=11) compared to wild‐type (WT) (2097±76, N=10, p<0.0001) and heterozygous (2423±86, N=6, p<0.0001) littermates. Lastly, preliminary data of urine osmolality following 12‐hour water restriction (dark‐cycle) suggests KO animals are capable of concentrating urine, though only to about 75% that of WT littermates (3492±150 vs. 4361±525; N=3, N=4, respectively). Overall, these results suggest a role for Gpr116 in enhancing the ability to concentrate urine. This study establishes a physiologic role of the previously understudied Gpr116 in the murine kidney and demonstrates the scientific potential of future investigations into novel GPCRs.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kir4.1 activity is essential for dietary Na+ intake induced modulation of Na‐Cl cotransporter (NCC)
Authors: Su Xiao‐Tong , Wu Peng , Gao Zhongxiuzi , Wang Wen‐Hui , Lin Dao‐Hong
Published: June 2021

Dietary Na+ intake has been shown to modulate the thiazide‐sensitive NCC in the distal convoluted tubule (DCT), such that a high Na+ intake (HNa) inhibits whereas a low Na+ diet (LNa) stimulates NCC activity. However, the mechanism by which dietary Na+ intake on NCC is not clear. The aim of the present study is to test the hypothesis Kir4.1 is essential for mediating the effect of dietary Na+ intake on NCC. The patch‐clamp experiments demonstrated that the basolateral 40 pS K+ channel (a Kir4.1/Kir5.1 heterotetramer) activity in the DCT was upregulated in the mice on a LNa diet for 7 days but it was down‐regulated in the mice on HNa for 7 days. The whole‐cell recording further demonstrated that HNa decreased basolateral K+ conductance of the DCT and depolarized the membrane. Conversely, LNa intake augmented the basolateral K+ currents and hyperpolarized the DCT membrane. Western blot and renal clearance studies demonstrated that LNa increased the expression of total NCC (tNCC) and phosphorylated NCC (pNCC) and enhanced hydrochlorothiazide (HCTZ)‐induced natriuresis while HNa intake had opposite effects on the expression of tNCC and pNCC and reduced HCTZ‐induced natriuresis. Thus, an upregulation of Kir4.1 is correlated with a high NCC activity in the mice on LNa while a down‐regulation of Kir4.1 is related with low NCC activity in the mice on HNa. To explore the role of Kir4.1 in mediating the effect of dietary Na+ intake on NCC, we repeated the above experiments in kidney‐specific conditional Kir4.1 knockout mice (KS‐Kir4.1 KO). The deletion of Kir4.1 completely abolished the basolateral K+ conductance and depolarized the DCT membrane. Moreover, the effect of dietary Na+ intake on the basolateral K+ conductance and membrane potential was also absent in KS‐Kir4.1 KO mice. Also, the deletion of Kir4.1 decreased the expression of both tNCC and pNCC and abolished HCTZ‐induced natriuresis. Western blot and renal clearance experiments showed that the effect of dietary Na+ intake on NCC expression and activity was absent in KS‐Kir4.1 KO mice. We conclude that Kir4.1 plays an essential role in mediating the effect of dietary Na+ intake on NCC activity such that LNa‐induced stimulation of Kir4.1 in the DCT is essential for the upregulation of NCC while HNa‐induced inhibition of Kir4.1 is required for the down‐regulation of NCC.

Support or Funding Information

NIH DK54983

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vasopressin V1a Receptor of Renal Collecting Duct Intercalated Cells Mediates Urinary Acidification
Authors: Giesecke Torsten , Himmerkus Nina , Leipziger Jens , Isermann Julian , Ayasse Niklas , Koshimizu Takaaki , Kawahara Katsumasa , Gimber Niclas , Schmoranzer Jan , Bleich Markus , Fähling Michael , Smorodchenko Alina , Bachmann Sebastian , Mutig Kerim
Published: June 2021

Vasopressin (AVP) drives urinary concentration via V2 receptors in the distal nephron and collecting duct of the kidney. Parallel activation of V1a receptors (V1aR) has been implicated in renal acid‐base handling, but cell type‐related information on this receptor is still scarce. We hypothesized that activation of V1aR in intercalated cells (IC) contributes to acid‐base homeostasis by the kidney.

We generated an anti‐V1aR antibody and verified its specificity using V1aR‐knockout tissues. For localization studies in mice, rat and human tissue, we used immunofluorescence and confocal microscopy or 3D‐structured illumination microscopy (3D‐SIM). For functional studies in vivo, V1aR‐specific agonist, AO‐4‐67, was administered in AVP‐deficient Brattleboro rats and C57/BI6J mice (0.2, 2, or 10μg/kg b.w.; 1 to 4h). Urine was collected in metabolic cages or via ureteral catheter and plasma samples were taken. Isolated, microperfused mouse collecting ducts were analyzed for V1aR‐dependent luminal pH changes and cultured inner medullary collecting duct cells for proton‐secreting vH+‐ATPase regulation.

V1aR immunoreactivity of type‐A IC was demonstrated in basolateral membrane and cytoplasmic granules, whereas type B IC revealed perinuclear and subapical granular signal alone. Administration of AO‐4‐67 to Brattleboro rats for 4h resulted in increased urinary proton secretion (+34% after 1h, p<0.05) and decreased bicarbonate excretion (up to −90% after 3h, p<0.001). Similarly, catheterized mice showed rapid urinary acidification in response to AO‐4–67 (pH from 7.18 to 6.78 after 20 min, p<0.05). Basolateral application of V1aR agonist or AVP in isolated perfused collecting ducts resulted in decreased luminal pH values (−5% after 3 min, p<0.05). Cultured collecting duct cells showed significant increases in luminal vH+‐ATPase immunoreactivity upon V1aR stimulation (+90% after 4h, p<0.001).

In summary, our results suggest that activation of V1aR in renal type‐A intercalated cells induces proton secretion into the urine and promote urinary acidification.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interpretation of Whole‐Kidney RNA‐Seq Data
Authors: Clark Jevin , Neijman Kim , Chou Chung‐Lin , Deen Peter , Chen Lihe , Knepper Mark
Published: June 2021

RNA sequencing (RNA‐Seq) is a sensitive means of global identification and quantification of mRNAs. In kidney, we have carried out RNA‐Seq analysis to identify transcriptomes of single microdissected renal tubules and single cells. The resulting datasets provide a community resource for gene expression in kidney structures in the form of a publicly accessible website at https://hpcwebapps.cit.nih.gov/ESBL/Database/Targets/TranscriptomicData.html. We now have added new whole‐kidney RNA‐Seq data for mice (male, age 8 weeks). This dataset reports 13,334 transcripts with mean TPM (“normalized transcripts per million”) greater than 1 for whole kidney (n=3). A large fraction of these are housekeeping genes or genes expressed in multiple cell types. Whole‐kidney RNA‐Seq analysis is of limited use in studying responses in these genes since the responses may be variable among cell types. However, signals were readily quantified for major cell‐type specific transcripts including podocytes (podocin, Nphs2), proximal convoluted tubule (sodium coupled glucose transporter 2, Slc5a2), proximal straight tubule (sodium coupled glucose transporter 1, Slc5a1), thin descending limb of Henle (follistatin, Fst), thin ascending limb (chloride channel ClC‐Ka, Clcnka), thick ascending limb (NKCC2, Slc12a1), distal convoluted tubule (NCC, Slc12a3), principal cell of collecting duct (ENaC gamma subunit, Scnn1g), type A intercalated cell (AE1, Slc4a1), type B intercalated cell (pendrin, Slc26a4), and inner medullary collecting duct cell (urea channel UT‐A1, Slc14a2 long isoform). Beyond this, transcripts were quantifiable for marker genes corresponding to rare cell types in the kidney including granular cells of the afferent arteriole (renin, Ren1), macrophages (macrophage colony‐stimulating factor 1 receptor, Csf1r), dendritic cells (dendritic cell‐specific ICAM‐3‐grabbing non‐integrin, Cd209a), and umbrella cells of the transitional epithelium (uroplakin 1a, Upk1a). In conclusion, numerous cell‐type specific mRNAs are represented in whole‐kidney RNA‐Seq analysis. These markers provide a means of measuring physiological regulatory responses (e.g. renin expression in granular cells) or measuring changes in cell numbers, which can occur with proliferative responses or infiltration of the kidney as part of inflammatory responses (e.g. macrophages and dendritic cells).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Plasiticity in Normoxic Ventilation and Apnea Frequency in Neonatal Rats Exposed to Chronic Hyperoxia
Authors: Kavanagh Julia B. , Pratt Ashley E. , Lewallen Reed M. , Bavis Ryan W.
Published: June 2021

Rats chronically exposed to hyperoxia exhibit hypopnea and lower metabolic rates when returned to room air (e.g., Bavis et al. Respir Physiol Neurobiol 198: 1–12, 2014), but we have not previously measured ventilation and metabolism simultaneously in the same individuals. In this study, we assessed these characteristics in 4–5 and 13–14 day old rats (i.e., P4–5 and P13–14) that were reared in 21% O2 (Control) or 60% O2 (Hyperoxia) from birth; ventilation and O2 consumption rates were measured via head‐body plethysmography paired with continuous monitoring of expired gas concentrations. The ventilation‐to‐O2 consumption ratio was lower in Hyperoxia rats at P4–5 (37.6±1.9 vs. 48.9±3.3 in Control; mean±SEM, P<0.01) and at P13–14 (24.3±1.3 vs. 31.7±2.9 in Control; P=0.04), consistent with hypoventilation. We also quantified the frequency and duration of apneas during the 30 minute recording period. When apnea was defined as a respiratory pause twice the average breath duration (TTot), apnea duration tended to be longer in Hyperoxia rats at P4–5 (1.87±0.15 s vs. 1.48±0.08 s in Control; P=0.03); apnea durations were not different between groups at P13–14. However, this analysis may be confounded by a significantly lower breathing frequency (and thus longer TTot) in Hyperoxia rats. Indeed, apnea durations were no longer different between groups when adjusted for TTot (ANCOVA, P=0.47). To confirm this result, we reanalyzed apneas at P4–5 using the average TTot for all Control rats (rather than for each individual) to identify apneas. This analysis yielded no significant difference in apnea duration between groups (P=0.20), but there was a greater apnea frequency in P4–5 Hyperoxia rats (0.8±0.2 apneas min−1 vs. 0.4±0.1 apneas min−1; P=0.04). We conclude that chronic hyperoxia causes a persistent hypoventilation in neonatal rats that may be accompanied by a modest increase in apnea frequency, but these effects generally diminish with age.

Support or Funding Information

Supported by NIH grant P20 GM‐103423 (Maine INBRE).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prenatal Nicotinic Exposure Prolongs the Apneic Response to Activation of Bronchopulmonary C‐fiber 5‐HT3 Receptor with Upregulation of C‐neural 5‐HT3B Expression in Rat Pups
Authors: Zhao Lei , Gao Xiuping , Zhuang Jianguo , Shi Yuanyuan , Xu Fadi
Published: June 2021

5‐HT3 receptor, an important ionic channel, is heavily expressed in bronchopulmonary C‐fibers (PCFs). Among five 5‐HT3 receptor subunits (5‐HT3A–E), only homomeric 5‐HT3A and heteromeric 5‐HT3AB form functional ion channels. It is reported that the recovery from channel desensitization is much faster in 5‐HT3AB receptor assemblies than that in 5‐HT3A receptor assemblies. Our previous studies have shown that prenatal nicotinic exposure (PNE) significantly prolongs the apneic response to stimulation of PCFs in rat pups. Here, we asked whether PNE would promote 5‐HT secretion in BALF and prolong the 5‐HT3‐mediated apneic response depending on PCFs. We further asked whether this augmented 5‐HT3‐mediated response was associated with alterations of protein and mRNA expressions of 5‐HT3A and/or 5‐HT3B subunits and changes of 5‐HT3 currents in vagal pulmonary C‐neurons. To this end, Ctrl and PNE rat pups were used to detect the differences in: 1) 5‐HT content in BALF; 2) the apneic response to right atrial bolus injection of phenylbiguanide (a selective 5‐HT3 agonist, 10 μg/kg) under PCF intact condition and degenerated condition induced by perivagal capsaicin treatment; 3) 5‐HT3A and 5‐HT3B mRNA (single cell RT‐PCR) and protein expression (immunohistochemistry) in the nodose/jugular ganglionic vagal pulmonary C‐neurons that were retrogradely traced by intratracheal instillation of DiI and marked with TRPV1 expression; and 4) 5‐HT3 currents on these neurons recorded by patch‐clamp in vitro. Our results show that PNE not only upregulates pulmonary 5‐HT concentration but also strengthens the 5‐HT3‐mediated apnea depending on PCF. PNE increases 5‐HT3B but not 5‐HT3A protein expression with little changes in their mRNA level in the vagal pulmonary C‐neurons. Interestingly, PNE significantly accelerates 5‐HT3 channel desensitization and shortens the half‐widths and decay times but has no effect on the rise‐times of these channels. Our results suggest that PNE prolongs PCF 5‐HT3‐mediated apnea likely through altering the formation of 5‐HT subunits (increasing 5‐HT3B) to enhance the sensitivities of 5‐HT3 channels.

Support or Funding Information

HL119683 and HL107462

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Does Shipping Late‐Gestation Rats Alter Respiratory Control in Their Offspring?
Authors: Benevides Ethan S. , Kavanagh Julia B. , Wallace Josefine A. , Bavis Ryan W.
Published: June 2021

Ordering timed‐pregnant rats is convenient for many developmental studies, but the shipping process may be stressful for the animals. Given the sensitivity of the developing respiratory control system to perinatal stress (e.g., Joseph et al., Respir Physiol Neurobiol 185:75–86, 2013), we investigated whether prenatal “shipping stress” alters the respiratory control of neonatal rats. In the first set of experiments, timed‐pregnant rats were shipped to arrive at our animal facility at embryonic age 17 (E17) and their pups were compared to those of rats bred in‐house (IH) at 8–9 days of age (P8–9). Normoxic ventilation (measured by head‐body plethysmography) was 30% greater in E17 pups than in IH pups (138±6 vs. 106±4 ml min−1 100g−1; mean±SEM, P<0.001), and their hypoxic ventilatory responses (HVR) to 12% O2 were lower (45±6 vs. 67±4 % increase from baseline; P=0.02). We then repeated the experiment with timed‐pregnant rats shipped to arrive at E13 or E19. At P4–5, ventilation was approximately 10% greater in E19 pups than in E13 or IH pups (treatment, P=0.02), but the magnitude and time course of the HVR was similar among groups (treatment × time, P=0.53). At P11–12, no differences in normoxic or hypoxic ventilation were detected among the treatment groups (treatment, P=0.24; treatment × time, P=0.08). Similarly, in vitro carotid body responses to hypoxia did not differ among E13, E19, and IH groups at either age (both P>0.05). The different outcomes of these studies could reflect the different gestational ages at which the rats were shipped or different origins/shipping distances of the pregnant rats (Portage, MI vs. Kingston, NY).

Support or Funding Information

Supported by NIH grant P20 GM‐103423 (Maine INBRE).

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