bluuebunny/crossref_metadata_2025 huggingface dataset:
Presynaptic large‐conductance calcium‐activated potassium channels modulate GABAergic inhibitory synaptic transmission in the superficial dorsal horn of the mouse
Authors: Fukushima Teruyuki , Hori Yuuichi
Published: June 2021

Large‐conductance calcium‐dependent potassium (BK) channels are found in various tissues, including nerve, smooth and skeletal muscles, and other cell types. They are potassium‐selective ion channels that are activated in response to increased intracellular calcium concentrations and transmembrane voltages.

We previously reported that the BK channels that are located in presynaptic terminals control excitatory synaptic transmission in the superficial dorsal horn (SDH), and that functional downregulation of BK channels accompanies the neuropathic pain induced by peripheral nerve injury.

In the present experiments, we attempted to clarify the physiological significance of BK channels in the modulation of GABAergic inhibitory synaptic transmission in the SDH where nociceptive information is processed.

Spontaneously occurring GABAergic miniature inhibitory postsynaptic currents (mIPSCs) were recorded from the GFP‐negative neurons located in the SDH of a mouse spinal cord slice by using slice preparations from adult glutamate decarboxylase 67‐green fluorescent protein (GAD67‐GFP) knock‐in mice, and the effects of Iberiotoxin, a BK channel blocker, on the GABAergic mIPSCs were analyzed.

Iberiotoxin increased the frequency of GABAergic mIPSCs without affecting the amplitude of GABAergic mIPSCs, suggesting that the activation of BK channels attenuates GABAergic synaptic transmission via presynaptic mechanisms.

In order to clarify the role of BK channels in the nociceptive information processing which is modulated by GABAergic transmission, we are currently doing the similar experiments using peripheral nerve‐ligated neuropathic mice.

Those results may provide clues for the underlying mechanisms of the nociceptive actions of BK channels in the SDH.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Leptin Activates Transient Receptor Potential Melastatin 7 (TRPM7) Channels in Mouse Glomus Cells and Leptin‐Receptor Expressing Pheochromocytoma Cells
Authors: Mu Yun‐Ping , Caballero‐Eraso Candelaria , Liu Xiao‐Ru , Shin Mi‐Kyung , Paudel Omkar , Yeung Bonnie , Tang Wan‐Yee , Shirahata Machiko , Polotsky Vsevolod , Sham James
Published: June 2021

Obesity is a leading cause of high cardiovascular morbidity and mortality. It is characterized by an increased leptin level, which has been implicated in increased sympathetic activity and the pathogenesis of hypertension. However, the mechanism by which leptin enhances sympathetic activity is unclear. Leptin receptors (LEPRs) are expressed in the carotid body, the most important arterial chemoreceptor for the cardio‐respiratory chemoreflex. We have previously demonstrated that leptin enhances carotid sinus nerve activity in response to hypoxia; this effect is abolished by non‐selective blockers of transient receptor potential (TRP) channels; and the TRPM7 channel is the most abundant TRP subtype expressed in CB (Shirahata et al., Adv Exp Med Biol. 2015;860:153–9). Since leptin is known to regulate many different ion channels, we hypothesize that leptin may exert its effects through modulation of TRPM7 channel activity. To test this hypothesis, we used glomus cells isolated from the carotid bodies of C57BL/6 mice and the LEPR (LEPRb) expressing pheochromocytoma (PC12LEPRb) cells, which express a high level of TRPM7 channels, as a cell model. Non‐selective cation channels were recorded under amphotericin‐B perforated‐patch techniques with K+ replaced by Cs+ in the presence of voltage‐gated Ca2+ channel and Cl− channel blockers. In the glomus cells, voltage‐ramp from −100 to 100 mV activated a small outwardly rectifying current. Application of leptin 100 ng/ml caused a significant increase in both the outward and inward component of the current, which was completely reversed by the subsequent addition of the TRPM7 antagonist FTY720. In the PC12LEPRb cells, the specific TRPM7 agonist naltriben caused significant increase of a similar outwardly rectifying current, which was abolished by the TRPM7 antagonists NS8593 and FTY720. Leptin at concentration of 10–100 ng/ml caused concentration‐dependent increase in the current, which again was completely blocked by NS8593 and FTY720. These results for the first time demonstrated that stimulation of leptin receptor in both the native glomus cells and the PC12LEPRb cell is capable of activating TRPM7 channels, and suggest that leptin may exert its effect on the carotid body through modulation of TRPM7 activity.

Support or Funding Information

This work is supported by HL133100

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Influence of Sugar and Artificial Sweeteners on Vascular Health during the Onset and Progression of Diabetes
Authors: Hoffmann Brian Robert , Ronan George , Haspula Dhanush
Published: June 2021

As diabetes and obesity become a rising worldwide heath concern there has been an increased awareness of environmental factors, such as diet, that are contributing to the problem. The negative implications of consuming high amounts of dietary sugar on overall health have long been linked to diabetes, obesity, cardiovascular disease, and other systemic health problems. However, it was not until recently that the negative impact of consuming non‐caloric artificial sweeteners in the place of sugar had been increasingly recognized as a potential contributor to the dramatic increase in diabetes and obesity, along with the associated complications. This study tested the response of the vascular endothelium in vitro and the in vivo response of a diabetes susceptible BB‐DR rat model to glucose, aspartame, and acesulfame potassium supplementation through the use of high‐throughput ‘omics' analyses coupled with functional testing. We have demonstrated that an acute in vitro treatment of rat microvascular endothelial cells with high glucose (25 mM) results in impairment of through glycosylation as exhibited by a restored function with PNGaseF enzymatic removal of N‐glycosylations (p<0.05). Conversely, artificial sweetener treatments also exhibited endothelial impairment in an in vitro tube formation assay (p<0.05); however, gene expression analyses suggested the mechanisms of dysfunction were through different mechanisms than the glucose treatment. Furthermore, through a site‐specific proteomic analysis we have identified 87 proteins significantly increased in N‐glycosylation and 143 in O‐glycosylation (p<0.05) on the endothelial cell surface following high glucose treatment as compared to normal glucose (4.5 mM); many targets related to vascular homeostasis. Those experiments have been supplemented with a comprehensive metabolomics analysis of plasma from an acute three week study on diabetes susceptible BB‐DR rats fed a high glucose, aspartame, or acesulfame potassium diet. Through this set of experiments we have identified unique signatures of alterations in lipid metabolism, among others, following artificial sweetener consumption. Overall, results of this study suggests that exposure to high glucose and artificial sweetener administration lead to unique mechanisms of vascular impairment and homeostatic alterations that may be important during the onset and progression of diabetes and obesity.

Support or Funding Information

Support for this project has been provided by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases (K01‐DK105043 to BRH) and the Department of Biomedical Engineering at the Medical College of Wisconsin and Marquette University, Milwaukee, WI, USA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ibuprofen cases Proteasome dysfunction in mice liver
Authors: Gomes Aldrin , Tiwari Shuchita , Sule Rasheed
Published: June 2021

Introduction and objectives

Previous clinical studies have suggested that nonsteroidal anti‐inflammatory drugs (NSAIDs) are associated with increased risk of stroke and cardiovascular disease. Acetaminophen, the active ingredient in Tylenol, is not an NSAID and is associated with increased risk of liver toxicity. The common use of NSAIDs for pain and fever relief is a concern but it is unclear if NSAIDs is also causing adverse effects on other tissues such as the liver, kidney and brain. Previous research in our laboratory has suggested that NSAIDs cause proteasome dysfunction in cardiac tissue. To investigate the possibility that NSAIDs also influence liver function, ibuprofen treated mice were used to determine if this NSAID affects the ubiquitin proteasome system (UPS). The UPS is critical for cell function as it is responsible for degradation of 60–80% of intracellular proteins.

Method/Results

Eight‐week‐old male and female C57BL/6J mice were randomly assigned to one of 4 groups: (A) control (B) Group treated with ibuprofen (C) Group treated with ibuprofen + vitamin C (D) Group treated with vitamin C alone. Mice were treated with water or ibuprofen (100 mg/kg) and vitamin C (150 mg/kg) dissolved in drinking water for 6 weeks. Liver from 6 week treated animals were homogenized and cytosol fractions obtained. The three independent proteolytic proteasome activities, β1 caspase‐like activity, β2 trypsin‐like activity, and β5 chymotrypsin‐like activity were measured in the different cytosolic liver fractions. The proteasome inhibitor bortezomib was utilized to ensure the activity measured was due to the proteasome.

Semi‐quantitative western blotting suggests that the 20S proteasome amount was similar in all 4 groups but the immunoproteasome subunit β5i was increased in both male and female ibuprofen treated and ibuprofen with vitamin C groups. The β1 and β2 proteasome activities were significantly decreased in both male and female mice treated with ibuprofen. Although inclusion of vitamin C with the ibuprofen did not fully restore proteasome activity, it resulted in an improvement in the β1 proteasome activity compared to ibuprofen treated mice. The β5 proteasome activity was also significantly decreased in both male and female mice treated with ibuprofen. However, this decrease was significantly more pronounced in female mice than in male mice suggesting gender‐specific effects of ibuprofen on proteasome function.

Conclusion

These results suggest that ibuprofen significantly disrupts the UPS and causes proteasome dysfunction in liver tissue. The experimental data also suggests that proteasome dysfunction in livers from ibuprofen treated mice differs in female and male mice.

Support or Funding Information

American Heart Association Award #16GRNT31350040

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Throughput Phenotypic Analysis of Circulating Tumor Cells from Pancreatic Adenocarcinoma Patients via Dielectrophoresis and Image Flow Cytometry
Authors: Marra Andre Antonio , Kim Dong , Carpenter Chloe , Varadhachary Gauri , Katz Mathew , Greyscone Peter , Alvarez Hector , Maitra Anirban
Published: June 2021

Effective management of pancreatic ductal adenocarcinoma (PDAC) has remained elusive1. Even with large scale sequencing efforts of cancer genomes providing potential for guidance in informing tumor characterization and therapeutic strategies, the inaccessibility of primary and metastatic tissue precludes the opportunity for further workup following initial tumor sampling2. To circumvent this issue, we utilize circulating tumor cells (CTCs), or those cells shed by primary and metastatic tissue, to noninvasively monitor tumor evolution and changing patterns of drug susceptibility3. As current CTC enrichment methodologies typically involve the use of antibody (anti‐EPCAM) based capture approaches, the ability to detect phenotypic changes as a result of adaptive gene expression patterns such as cells undergoing epithelial to mesenchymal transition (EMT) is lost. We thus aim to isolate CTCs using an antigen independent platform based on a cell's dielectrophoretic (DEP) properties which allow characterization of multiple subpopulations of cells which may have clinical relevance.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ULK2 Regulates p62‐ and NBR1‐Dependent Selective Autophagy In Skeletal Muscle
Authors: Fuqua Jordan , Mere Caleb , Bloome Jay , Melo Vitor , Scudese Estevao , Bae Dam , Turner Kristen , Kronemberger Ana , Lira Vitor
Published: June 2021

Autophagy is an anciently conserved pathway responsible for the degradation of long‐lived proteins, protein aggregates, and organelles, thereby contributing to efficient protein homeostasis. Autophagy is stimulated by nutrient deprivation and is required for certain beneficial adaptations of exercise. Conversely, insufficient autophagy is a common feature of muscle diseases, obesity, type 2 diabetes, and aging; yet the regulation of autophagy is incompletely understood at the molecular level. Here, our goal was to define the role of muscle unc‐51 like autophagy activating kinase 2 (ULK2), and contrast with that of its close homolog ULK1, in regulation of autophagy. To do so, we performed 2 complementary studies. First, DNA plasmids encoding either Ulk1 or Ulk2 pre‐micro RNAs (miR) were electroporated into the tibialis anterior (TA) muscle of one leg, and a control miR plasmid into the contralateral leg of wild type mice. Muscles were harvested 7–8 days afterwards, either at basal conditions or after 24h of starvation. Second, ULK2fl/fl differentiated primary mouse myotubes were transduced with Ad‐Cre‐GFP or Ad‐GFP (control) viruses, and harvested up to 96h afterwards. Our results revealed that ULK2 is highly expressed in skeletal muscle, and that deficiency of ULK2, but not of its close homolog ULK1, leads to accumulation of ubiquitinated proteins. In addition, the autophagy receptors p62 and NBR1, which are degraded by autophagy and serve as receptors for ubiquitinated cargos, accumulate in muscles and primary myotubes deficient only in ULK2, independent of lysosomal fusion and function. Of note, the levels of the autophagy receptor optineurin were unaffected under the same conditions. These results indicate a novel and fundamental role for ULK2 in regulating p62‐ and NBR1‐dependent selective autophagy in skeletal muscle revealing ULK2 as a potential therapeutic target for skeletal muscle contractile and metabolic dysfunction.

Support or Funding Information

Supported by AHA (16SDG30360001) and Dept. of Health & Human Physiology, University of Iowa.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Autophagy Enhances Protective Immunity of Macrophages Against Mycobacterium Tuberculosis
Authors: Lv Jingzhu , Wei Chunfang , Fang Fang , Zhang Yao , Kelly Gabriel , Wang Ting , Qian Zhongqing
Published: June 2021

Autophagy has been considered as an immune mechanism in the last decade, and the involvement of autophagy in individual immune events is being investigated. More recently, autophagy has been linked with innate and adaptive immune responses against Mycobacterium tuberculosis (M. tuberculosis or TB), which can survive within macrophages by blocking the process of lysosome fusion with phagosomes. In addition, previous reports involving M. tuberculosis infection models showed the polarization of Type 1 T helper (Th1) cells in response to IL‐12 production and subsequent induction of IFN‐γ, which are two key players in immunity to TB. We sought to better understand the regulatory role of autophagy in the function of macrophages against M. tuberculosis, and the involvement of Th1 cytokine responses in these events. We found that lipopolysaccharide (LPS), a component of Gramnegative bacteria including M. tuberculosis, and also a pro‐inflammatory mediator, induced autophagy in M. tuberculosis infected THP‐1‐derived macrophages or PMA (phorbol 12‐myristate 13‐acetate) primed THP‐1 macrophages [THP‐1(A)]. This autophagy activation in THP‐1 or THP‐1(A) by LPS is very similar to Rapamycin, a known autophagy inducer. These two autophagy inducers significantly increased Th1 cytokine IL‐12 and IFN‐γ production, as well as M. tuberculosis killing effects in THP‐1 or THP‐1(A) cells. However, the autophagy inhibitor 3‐MA significantly reduced LPS activated IL‐12/IFN‐γ production or M. tuberculosis killing effects in THP‐1 or THP‐1(A) cells. These data demonstrate that autophagy induced by LPS stimulation mediates the protective immune remodeling in macrophages against M. tuberculosis. In parallel, we have shown levels of IFN‐γ presenting peripheral blood mononuclear cells (PBMC) collected from TB patients were significantly reduced compared to healthy volunteers. Similar to results in THP‐1 cells, LPS and Rapamycin restored the levels of IFN‐γ presenting PBMC from TB patients, which could be further raised by 3‐MA treatment. Combined together, our data strongly suggest a critical regulatory role of macrophage autophagy in innate immunity against M. tuberculosis, with involvement of the Th1 cytokine response.

Support or Funding Information

This study was funded by International Science and Technology Cooperation Project‐Key Research and Development Program of Anhui Province 1604b0602026 to ZQ (to support the analysis and interpretation of the data), and National Natural Science Foundation of China 81570011 to TW (to support the analysis and interpretation of the data, and preparation of the manuscript).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Restoration of SERCA ATPase as an Intervention to Muscle Impairment Associated with Oxidative Stress
Authors: Qaisar Rizwan , Bhaskaran Shylesh , Ranjit Rojina , Premkumar Pavithra , Huseman Kendra , Sataranatarajan Kavithalakshmi , Van Remmen Holly
Published: June 2021

Disrupted intracellular calcium homeostasis is associated with various chronic muscle pathologies including sarcopenia. In support of this, the activity of SERCA, the Sarcoplasmic Reticulum (SR) Ca+2‐ATPase, a membrane enzyme that returns calcium to the SR lumen following muscle contraction, is reduced 14% in muscle of old wild type mice. Here we report a 27% decrease in SERCA activity in gastrocnemius muscle from mice lacking the superoxide scavenger CuZnSod (Sod1−/− mice), a mouse model of elevated oxidative stress and accelerated sarcopenia. The mechanism coupling reduced SERCA function with muscle impairment is not known. We hypothesize that restoring SERCA function will attenuate loss of muscle mass and function in the Sod1−/− mice. To test this, 2 month old Sod1−/− mice were treated for 7 weeks with CDN1163, a novel allosteric SERCA activator that acts directly to increase SERCA ATPase activity (50 mg/kg, IP, 3 times per week). Treatment with CDN1163 increased gastrocnemius muscle mass by 8% in CDN1163‐treated versus untreated Sod1−/− mice. In addition, the 22% reduction in specific force measured in untreated Sod1−/− versus age‐matched wild type mice was completely restored by 7 weeks of CDN1163 treatment. CDN1163 also reduced the oxidative damage measured as F2‐isoprostanes in muscle tissues by 50% compared to untreated Sod1−/− mice. Collectively our findings suggest that reduced function of the SERCA pump contributes to muscle atrophy and reduced force generation in the Sod1−/− mice and the pharmacological stabilization of SERCA with CDN1163 may be a powerful tool to counter age‐ and oxidative stress‐associated muscle impairment.

Support or Funding Information

This research was conducted while Dr. Rizwan Qaisar was a Diamond/AFAR award recipient and by a P01 grant (AG051442) to Dr. Holly Van Remmen.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The effect of the KATP channel opener Nicorandil in lipid‐peroxidation in skeletal muscle atrophy
Authors: PADILLA‐MALDONADO PAULINA , SANCHEZ‐DUARTE ELIZABETH , GÓMEZ‐BARROSO MARIANA , SANCHEZ‐PEREZ ALINA , CORTES‐ROJO CHRISTIAN , SAAVEDRA‐MOLINA ALFREDO , MONTOYA‐PEREZ ROCIO
Published: June 2021

Muscle atrophy is defined as a decrease muscle tissue size due to cellular shrinkage caused by loss of organelles, cytoplasm and proteins. The main characteristic of this pathology is the increase in protein degradation and a decrease in protein re‐synthesis. It has been documented that the mitochondrial ATP sensitive potassium channel (mitoKATP), is involved in the resistance of the muscle fatigue and recovery from ischemia‐reperfusion injury and protection during atrophy process; Nicorandil has been described as a selective mitoKATP opener which confers protection to muscle during stressful conditions, the aim of this study was to determine the protective effect of Nicorandil over the damage caused by atrophy.

C57BL/6 male mice 14–16 weeks‐old were used for the experimental basis; these animals were separated into 4 groups: 1) Control, 2) Nicorandil, 3) Atrophy and 4) Atrophy treated with Nicorandil. Atrophy was induced by hindlimb unloading by the tail method. Nicorandil was purveying in 40 mg/kg doses by intramuscular injection during 14 days. Once finished the treatment, the animals were sacrificed by cervical dislocation and the hindlimb were used for mitochondria isolation, once obtained the isoled mitochondria the lipid‐peroxidation was measured by the TBARS method. This reaction was measured in a Spectrophotometer Perkin Elmer Lambda 18 UV/VIS in ʎ of 523 nm. The results were for the group Nicorandil was higher against the Control group 50.5827%, the Atrophy group was 53.03% against the Control group, and the Nicrandil+Atrophy group had an increase in the activity against the Control group.

Lipid‐peroxidation had been used as a marker of oxidative stress, as we know, this has been stablished that atrophy leads to an accumulation of toxic substances as reactive oxygen species, like we observe here the Nicorandil treatment has no effect in healthy mice but the opening of this channel downregulates lipid oxidation in the atrophied that can give us a thought of cytoprotection.

Support or Funding Information

RMP‐CIC2017

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 G Protein‐coupled Estrogen Receptor Promotes Endothelin‐dependent Natriuresis in Female Rats
Authors: Gohar Eman Y. , Obi Ijeoma E. , Daugherty Elizabeth M. , De Miguel Carmen , Kasztan Malgorzata , Pollock Jennifer S. , Pollock David M.
Published: June 2021

The novel estrogen receptor, G protein‐coupled estrogen receptor (GPER), is a membrane‐bound receptor linked to acute signaling pathways. GPER activation elicits cardiovascular and nephroprotective effects against salt‐induced complications. However, its specific role in renal sodium handling is not yet defined. We hypothesized that activation of GPER in the renal medulla facilitates sodium excretion. Isosmotic saline was infused into the renal medullary interstitium (500 μl/h) of anesthetized male and female Sprague‐Dawley rats for an 80 min equilibration period and 20 min baseline urine collection period. Then, G1 (GPER agonist, 50 pmol/kg/min) or vehicle was infused into the renal medulla for an additional 20 min period. Compared with vehicle, G1 significantly increased urinary sodium excretion in females (from 0.6 ± 0.1 to 1.0 ± 0.1 μmol/min n=9, p<0.05). G1 did not change blood pressure or urinary potassium excretion in comparison with vehicle control (106 ± 3 vs. 105 ± 4 mmHg, 0.5 ± 0.1 vs. 0.4 ± 0.1 μmol/min, n=9). Medullary blood flow remained unchanged during these experiments. In males, G1 did not change urinary sodium excretion (from 0.6 ± 0.1 to 0.7 ± 0.1 μmol/min, n=9). No sex‐differences in medullary GPER mRNA expression was observed, suggesting that sex‐differences in G1 effects on sodium excretion could be related to GPER downstream signaling cascade. Given that endothelin‐1 (ET‐1) is a well‐established natriuretic factor that is regulated by sex and sex steroids, we hypothesized that GPER activation promotes natriuresis via an ET‐1‐dependent pathway. To test this idea, we studied the effect of medullary infusion of G1 after dual blockade of ETA and ETB receptors achieved by i.v. bolus injection of ABT‐627 (5 mg/kg) and A‐192621 (10 mg/kg), respectively. We found that the natriuretic response to medullary GPER activation in females was inhibited by dual blockade of ET‐1 receptors (from 0.5 ± 0.2 to 0.5 ± 0.2 μmol/min n=5). These data uncover a novel role for renal medullary GPER in promoting sodium excretion via an ET‐1‐dependent pathway in females, but not in males.

Support or Funding Information

Funded by AHA 15POST25090329 to EYG and P01 HL136267 to DMP and JSP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SPAK disruption increases the glomerular filtration rate
Authors: Yilmaz Duygu Elif , Saritas Turgay , McCormick James , Thomson Martin , Bachmann Sebastian , Mutig Kerim
Published: June 2021

The thick ascending limb (TAL) of the kidney reabsorbs approximately 25% of filtered NaCl via its luminal Na‐K‐Cl cotransporter (NKCC2). In specialized TAL cells constituting the macula densa (MD), NKCC2 serves to sense the luminal NaCl concentration for paracrine adjustments of the glomerular filtration rate (GFR) to the needs of the body. The catalytically active full‐length (FL) SPAK and OSR1 kinases activate NKCC2 by phosphorylation. In addition to the FL‐SPAK, TAL cells express truncated SPAK variants (SPAK2 and KS‐SPAK) arising from alternative splicing or proteolytic cleavage. SPAK2 and KS‐SPAK exert strong dominant‐negative effects on FL‐SPAK/OSR1‐dependent NKCC2 phosphorylation. This study addresses the role of SPAK variants in regulation of NKCC2 in MD cells with respect to the modulation of GFR.

SPAK variants, OSR1, NKCC2 and phospho‐NKCC2 were analyzed in kidneys of wild type (WT) and SPAK‐deficient (SPAK−/−) mice and cultured TAL and MD cells using immunohistochemistry, immunoblotting and quantitative PCR. Key juxtaglomerular components involved in GFR regulation such as cyclooxygenase 2 (COX2), nitric oxide synthase 1 (NOS1) and renin were compared between the two mouse genotypes. Effects of SPAK deficiency on GFR were evaluated by transcutaneous assessment of FITC‐sinistrin clearance in freely moving mice.

Immunohistochemical analysis revealed that NKCC2 phosphorylation levels were significantly higher in TAL cells of SPAK−/− kidneys compared to WT, supporting the presence of the dominant‐negative SPAK2 and KS‐SPAK isoforms in WT TAL cells. In contrast, NKCC2 phosphorylation was not significantly altered in SPAK‐deficient MD cells, suggesting minor effects of truncated SPAK variants on NKCC2 and a redundant role of FL‐SPAK in this cell type. In line with this, immunoblotting of lysates from cultured TAL and MD cells showed predominant abundance of SPAK2 and KS‐SPAK in TAL cells, whereas MD cells mainly expressed FL‐SPAK. Both TAL and MD cells showed substantial OSR1 levels, which may explain the FL‐SPAK redundancy. Juxtaglomerular COX2, NOS1 and renin levels as well as the FITC‐sinistrin clearance were substantially higher in SPAK−/− which might reflect increased NKCC2‐mediated salt reabsorption in preceding TAL and preserved NKCC2‐mediated sensing of luminal NaCl in MD cells.

Our results describe differential expression of SPAK variants in TAL and MD cells and demonstrate that SPAK disruption stimulates GFR likely due increased NKCC2 activity in TAL prior to MD resulting in reduced NaCl concentration at the MD site.

Support or Funding Information

Funding: DFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, R01DK098141 to JAM; DFG Grant MU2924/2‐2

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sorting nexin 27 regulates the lysosomal degradation of AQP2 protein in kidney collecting duct
Authors: Choi Hyo‐Jung , Park Eui‐Jung , Jang Hyo‐Ju , Cho Jeong‐In , Park Hye‐Jeong , Jung Hyun Jun , Kwon Tae‐Hwan
Published: June 2021

We recently demonstrated that vacuolar protein sorting‐associated protein 35 (Vps35), a component of the retromer complex, interacts with the carboxyl terminus of aquaporin‐2, and depletion of Vps35 was associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2. Sorting nexin 27 (SNX27), a PDZ domain‐containing protein, is known to cooperate with the retromer complex in the recycling of early‐endosome to the plasma membrane. Since carboxyl terminus of aquaporin‐2 has the class I PDZ‐interacting motif (X‐Thr/Ser‐X‐Φ), we aimed to examine whether SNX27 could also play a role in the regulation of AQP2 trafficking and protein abundance in the kidney collecting duct cells. Immunohistochemistry revealed that SNX27 was diffusely labeled throughout the cytoplasm in the inner medullary collecting duct cells of rat kidney. Co‐immunoprecipitation assay of rat kidney inner medulla demonstrated that SNX27 interacted with AQP2. The role of SNX27 in the dDAVP‐responsive regulation of AQP2 trafficking and protein abundance was examined in mouse collecting duct mpkCCDc14 cells under siRNA‐mediated knockdown of SNX27. Cell surface biotinylation assay revealed that dDAVP‐induced translocation of AQP2 to the apical plasma membrane was decreased under SNX27 knockdown. Moreover, dDAVP‐induced AQP2 up‐regulation was significantly blunted in the cells with the siRNA‐mediated SNX27 knockdown. During the withdrawal period after dDAVP stimulation, the decrease of AQP2 protein abundance in the cells with SNX27 knockdown was attenuated by lysosomal inhibition with chloroquine co‐treatment. Taken together, SNX27 is an interacting protein with AQP2 and is likely to act as a component of the AQP2 sorting machinery after endocytosis, partly by regulating the lysosomal degradation of AQP2 protein abundance.

Support or Funding Information

This study was supported by the National Research Foundation of Korea Grants 2014R1A5A2009242 and 2016R1A2B4009365 funded by the Ministry of Science, ICT and Future Planning, Korea

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 Medullary Oxygenation Decreases in a Dose‐Dependent Manner with Graded Lower Body Negative Pressure in Healthy Young Adults
Authors: Kim Danielle Jin‐Kwang , Drew Rachel C. , Sica Christopher T. , Yang Qing , Miller Amanda , Cui Jian , Sinoway Lawrence I.
Published: June 2021

Background

Increase in sympathetic nerve activity (SNA) evokes a renal vasoconstrictor response that is powerfully opposed by autoregulatory influences. In the renal medulla, increases in SNA serve to increase Na+/K+‐ATPase activity (an oxygen dependent process) in the distal tubule, a process contributing to salt and water retention. In this report, we engaged SNA with lower body negative pressure (LBNP) as we employed magnetic resonance imaging to examine an index of renal medullary oxygenation, T2*. At a separate time we applied LBNP as we measured changes in renal blood flow velocity. This is an index that largely reflects changes in renal cortical blood flow.

Methods

Thirteen healthy (mean age 28 ± 1 yr) men (n = 7) and women (n = 6) participated in one or both of the experimental visits. Graded negative pressure (−10, −20, and −30 mmHg) was applied during 2 visits; visit 1 was for Doppler and visit 2 for MRI. Blood pressure (BP) and heart rate (HR) were also measured at baseline and at each stage of LBNP and recovery. RBV was obtained in all 13 subjects while T2* was obtained in 8 of 13 subjects.

Results

LBNP −30 mmHg led to a lower T2* (−0.65 ± 0.14 ms; P < 0.001). RBV decreased and renal vascular resistance (RVR) increased at −30 mmHg (ΔRBV −7.44 ± 3.87 cm/s, P = 0.041; ΔRVR arbitrary units 0.28 ± 0.11, P = 0.028). During recovery, T2* remained below baseline for the first and the second minutes of the recovery.

Conclusions

These data suggest that −30 mmHg LBNP leads to renal vasoconstriction and medullary deoxygenation. We suspect that changes in T2* are due to a sympathetically mediated, oxygen requiring, accentuation of Na/K ATP activity in the distal tubule. We postulate that this leads to salt and water retention as the SNS is engaged.

Support or Funding Information

Commonwealth Universal Research Enhancement Program (CURE); P01 HL134609 (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.
Responses to short‐term acid loading in proximal‐tubule‐select insulin/insulin‐like growth factor receptor dual knockout mice
Authors: Ecelbarger Carolyn M. , Li Lijun , Tiwari Swasti , Fluitt Maurice B.
Published: June 2021

Acidosis has been associated with increased renal gluconeogenesis, which in the absence of liver compensation, can lead to hyperglycemia. Acidosis leads to glutamine uptake in the proximal tubule (PT) to liberate bicarbonate and neutralize acids. The remaining carbon skeleton can be used for glucose production at this site. Whether insulin or the related hormone, insulin‐like‐growth‐factor, type 1 (IGF1), modulate these responses to acidosis is unclear. To address this, we bred mice with dual knockout (KO) of the insulin (Insr) and the Igf1r receptors from PT by Cre‐lox recombination using a γ‐glutamyl transferase promoter. KO mice had on average 40% lower expression of both Igf1r and Insr in cortex homogenates. To address the effects of acidosis, male (M) and female (F) wild‐type (WT, Cre negative) and KO (Cre positive) mice were provided ad libitum access to normal drinking water or a water‐based solution containing 1.5% NH4Cl for 7 days (n = 6/treatment/genotype/sex). Fasting (18‐hr) blood glucose was determined in a subset of mice after one week of control then acid treatment. MKO mice showed the greatest mean rise in fasting blood glucose with acid (mg/dl, delta acid‐control): −1.67 (MWT); +15.75 (MKO); −6.40 (FWT); +4.08 (FKO). Urine (24 hr) was collected in metabolic cages on days 5–6. Acid water led to a 30–64% reduction in urine volume in all groups except MWT (ml, delta acid‐control): +0.49 (MWT); −0.54 (MKO); −0.90 (FWT); −1.50 (FKO). Blood chemistry revealed greater sensitivity to acid loading in the male KO. Mean deltas (acid‐control) for blood bicarbonate, base excess, and anion gap, respectively were (mM): −5.73, −7.8, +1.24 (MWT); −8.86, −11.30, +4.67 (MKO); −7.35, −10.36, +4.74 (FWT); −1.67, −1.92, −0.6 (FKO). Western blotting for 3 rate‐limiting enzymes in gluconeogenesis conducted on the cortex from male mice revealed a significant rise in glucose‐6‐phosphatase (G6P), phosphoenolpyruvate carboxykinase (PEPCK), and fructose bisphosphatase (FBP1) in the acid drinking WT males (16–34%). In contrast, MKO mice had a significant reduction in G6P (22%) with acid water; however, FBP1 was increased by acid drinking to a greater extent (60%) in the KO, (as compared to WT, significant interaction). Overall our studies reveal a role for PT‐associated Insr and/or Igf1r signaling in regulating PT responses to an acid load and glucose homeostasis.

Support or Funding Information

Georgetown University Medical CenterGeorgetown/Howard CTSA award

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 farnesoid X receptor stimulates OCT2 function in human renal proximal tubular cells
Authors: Wongwan Teerasak , Chatsudthipong Varanuj , Soodvilai Sunhapas
Published: June 2021

Farnesoid X receptor (FXR) is a member of the nuclear receptor subfamily which highly expresses in liver, intestine, and kidney. It plays an essential role in maintaining of bile acids, glucose and lipid homeostasis. Previous reports showed cholestatic condition‐induced chenodeoxycholic acid (CDCA) inhibited organic cation transporter 1 (OCT1) in human and rat liver. However, there is still lack the information concerning the effects of FXR on expression and transport function of renal OCT2 which plays a major role in the renal elimination of cationic compounds. In this study, we investigated whether FXR activation regulates OCT2 in human renal proximal tubular cells. Treatment human renal proximal tubular cells (RPTEC/TERT1 cell) with FXR agonists, 10–100 μM CDCA and 1 μM GW4064, for 24 hours significantly increased intracellular accumulation of 3H‐MPP+, a prototypic substrate of OCT2. The stimulatory effect of FXR agonists was attenuated by co‐treatment with FXR antagonist, Z‐guggulsterone (10 μM) indicating the effect of FXR agonist was mediated by FXR‐dependent mechanism. Kinetic study revealed that CDCA increased OCT2 transport activity by increasing the maximal transport rate (Jmax) of 3H‐MPP+ with no significant effect on its affinity. CDCA treatment at both physiological level (20 μM) and cholestatic condition (80 μM) significantly increased mRNA expression of OCT2. In conclusion, FXR activation by both endogenous and synthetic ligands stimulates renal OCT2 transport function in the proximal tubules at least at transcriptional regulation. FXR activation might play a role in the regulation of renal OCT2 in both a physiological and pathological conditions.

Support or Funding Information

This study was supported by grant from the Royal Golden Jubilee (RGJ; grant no. PHD/0238/2553).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calibration of intracellular ion probes
Authors: Rana Priyanka S. , Lu Yong , Yurinskaya Valentina E. , Vereninov Alexey A. , Model Michael A.
Published: June 2021

The development of ion‐sensitive fluorescent probes enables the measurement of intracellular concentrations of ions (such as H+, K+, Na+, Ca2+, etc.) in a non‐invasive manner. One major complication with their use arises from their unpredictable behavior inside the cell, which makes it necessary to calibrate them in situ. Calibration implies creating known intracellular ion concentrations Ci (for example, making them equal to extracellular concentrations Ce) and relating them to the measured fluorescent signal. The question is how to ensure Ci = Ce?

Despite the long history of use of intracellular ion probes, the answer to this question is hard to find in the literature. In fact, many of the published calibration protocols clearly fail to achieve the desired goal. We therefore undertook a theoretical and experimental study of this question using K+ as an example. The general recipe for ion equilibration requires two conditions: (1) cell membrane potential should be destroyed; (2) membrane permeability for both K+ and Na+ should be significantly increased. Our results indicate that a combination of gramicidin, valinomycin, nigericin and ouabain is most suitable for achieving equalization of K+ across the membrane. Calibration curves obtained with suboptimal choice of ionophores leads to erroneous potassium measurements.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ANK3 Knockout Reduces ENaC Activity and Slows Adaptation to a Na+ Free Diet
Authors: Berman Jonathan Max , Mironova Elena , Stockand James
Published: June 2021

The Epithelial Na+ Channel, ENaC, is the final arbiter of sodium excretion in the kidneys. As such, discretionary control of ENaC by hormones is critical to the fine‐tuning of electrolyte and water excretion and consequently, blood pressure. We recently showed that phosphorylation of ENaC by CK2 in a putative ANK3 binding domain regulates ENaC activity. Therefore we tested the necessity and sufficiency of ANK3 for normal ENaC function. We produced tissue specific ANK3 knockout mice in the aldosterone sensitive distal nephron. Knockout was verified by immunohistochemistry. Split open tubule recordings from these mice showed significantly decreased baseline ENaC activity. The mice displayed a longer adaptation period to a sodium free diet, and a significantly different response to TBB, a casein‐kinase II inhibitor, compared to control. Together these results suggest that ANK3 modulates ENaC activity by interacting with CK2 phosphorylation sites in Beta and Gamma ENaC. In future work we will examine the mechanism by which this interaction modifies ENaC activity.

Support or Funding Information

This research was supported by American Heart Association grants 15GRNT22930030 & 17GRNT3292002 (to JDS), and 17POST33660468 (to JMB), and National Institutes of Health grants T32HL00744536A1 (to JDS and JMB), K12GM111726 (to JMB), and F32DK104572 (to EM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of ENaC by Casein Kinase II
Authors: Berman Jonathan Max , Mironova Elena , Stockand James
Published: June 2021

The Epithelial Na+ Channel, ENaC, is the final arbiter of sodium excretion in the kidneys. As such, discretionary control of ENaC by hormones is critical to the fine‐tuning of electrolyte and water excretion and consequently, blood pressure. Casein kinase 2 (CK2) phosphorylates ENaC. Phosphorylation by CK2 is necessary for normal ENaC activity. We tested the physiological importance of CK2 regulation of ENaC as the degree to which ENaC activity is dependent on CK2 phosphorylation in the living organism is unknown. This was addressed using patch clamp analysis of ENaC in split‐open collecting ducts in complement with whole animal physiological studies of sodium excretion in mice. We also used ENaC harboring CK2 phosphorylation site mutations to elaborate mechanism. We found that ENaC activity in ex vivo preparations of murine collecting duct had a significant decrease in activity in response to selective antagonism of CK2. In whole animal experiments selective antagonism of CK2 caused a natriuresis similar to benzamil, but not additive to benzamil, suggesting an ENaC dependent mechanism. Regulation of ENaC by CK2 was abolished by mutation of the canonical CK2 phosphorylation sites in beta and gamma ENaC. Together, these results demonstrate that the appropriate regulation of ENaC by CK2 is necessary for the normal physiological role played by this key renal ion channel in the fine‐tuning of sodium excretion.

Support or Funding Information

This research was supported by American Heart Association grants 15GRNT22930030 & 17GRNT3292002 (to JDS), and 17POST33660468 (to JMB), and National Institutes of Health grants T32HL00744536A1 (to JDS and JMB), K12GM111726 (to JMB), and F32DK104572 (to EM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serotonergic immunoreactivity in the brainstem and spinal cord of mdx mice
Authors: Wollman Lila Buls , Sunshine Michael D. , Burns David P. , O'Halloran Ken D. , Fuller David D.
Published: June 2021

The mdx mouse model of Duchenne muscular dystrophy exhibits profound respiratory muscle weakness and muscle fiber remodeling. Initial studies of respiratory control in mdx mice indicate increased respiratory motor output during respiratory challenge, possibly indicating a compensatory neuroplastic response of the respiratory neural control system in the face of muscle pathology (Burns et al., J Physiol, 2017). Serotonin plays a fundamental role in the induction of respiratory neuroplasticity, and in ongoing experiments we are testing the hypothesis that mdx mice have a compensatory increase in serotonergic innervation of the brainstem and spinal cord. The cervical (C4) and thoracic (T4) spinal cord and medulla of three‐month‐old wild type and mdx mice were harvested and incubated with antibodies to recognize serotonin and choline acetyltransferase (ChAT) to identify motoneurons. The intensity of serotonergic immunostaining was quantified in an unbiased manner using a custom Matlab code. In the cervical spinal cord, serotonergic immunostaining was prominent in the ventral horn (lamina 9). The thoracic cord also showed serotonergic immunoreactivity in the aforementioned region, but staining was most prominent in the lateral horn (intermediolateral nucleus). There was no indication of differences in spinal serotonergic staining between control and mdx mice. In contrast, the mdx mice showed a clear trend toward increased serotonin staining in caudal medullary regions important for cardiovascular and respiratory control, including the nucleus ambiguous, hypoglossal motor nucleus, and the dorsal motor nucleus of the vagus. These preliminary findings support the hypothesis that serotonergic neurons undergo neuroplastic changes in mdx mice, and this may have implications for respiratory motor control.

Support or Funding Information

T32‐HL134621‐01A1 (LBW), T32‐HD043730 (MDS), SPARC OT2 OD023854 (DDF), 1 R01 NS080180‐01A1 (DDF)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium Oscillations in Human Pulmonary Artery Smooth Muscle Cells: Developing a Hierarchical Clustering Method for Analysis and the Effects of IL‐1b
Authors: Kim Elizabeth H. , Griffis Khris G. , Sheppard Meredith , Alamro Suliman , Tabari Daniel , Garfinkel Alan , Jones Heather D.
Published: June 2021

Rationale

Acute respiratory distress syndrome (ARDS) is characterized by lung inflammation, edema, and severe hypoxemia. In previous work, we showed that IL‐1b is required for the development of severe hypoxemia in a murine model of acute lung injury, independent of measures of inflammation and edema. We hypothesized that IL‐1b is altering the regulation of pulmonary blood flow to cause severe V/Q mismatching in acute lung injury. To explore possible mechanisms for this, we studied the effects of IL‐1b on calcium flux in human pulmonary artery smooth muscle cells (hPASMCs) in response to the vasoconstrictor endothelin‐1 (ET‐1).

Methods

hPASMCs were grown on glass bottom dishes to confluence and some dishes were treated with IL‐1b (10ng/mL) for 3h. Cells were loaded with 5mM Cal‐520 and stimulated with ET‐1 (10nM) during fluorescent imaging on a Zeiss LSM confocal microscope. All cells from each field were individually selected using Fiji image analysis software. By visual inspection, we noticed that a marked number of cells showed oscillations in calcium flux after ET‐1. To quantify oscillatory from non‐oscillatory responses, we used a mixture of information theoretic and machine learning algorithms and developed a custom classifier tool. We applied this tool to our control and IL‐1b‐treated data (Figure 1) and created a contingency table for the effects of IL‐1b on the induction of oscillatory responses. χ2 confidence intervals and statistical significance were computed by bootstrap simulation with 105 replicates using custom scripts in R(v3.4.1).

Results

In control dishes (n = 6), there were a total of 444 non‐oscillators cells and 379 oscillator cells. In dishes treated with IL‐1b (n = 2), there were 50 non‐oscillator cells and 211 oscillators total. A bootstrap simulation based χ2 test was performed and a strong relationship was found between IL‐1b treatment and the presence of oscillatory responses, χ2 = 96.7 (71.2, 125.5; 95% C.I.), p < 10−5.

Support or Funding Information

Nat'l Heart, Lung & Blood Inst.

K08HL125806

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