bluuebunny/crossref_metadata_2025 huggingface dataset:
Glycoconjugate expression in the extracellular matrix of the mouse lung (87.7)
Authors: Bennett Robert , Ysasi Alexandra , Belle Janeil , Wagner Willi , Ackermann Maximilian , Konerding Moritz , Mentzer Steven
Published: June 2021

Introduction: Glycoconjugates have been implicated in various lung extracellular structures such as the galactose‐binding elastin receptor complex; however, little is known about expression of carbohydrate moieties in lung extracellular matrix (ECM). Methods: To investigate structural implications of glycoconjugate expression in the ECM, we used a panel of 33 biotinylated lectins to label decellularized mouse lungs harvested from 8‐11 week C57/B6 mice. Lungs were decellularized by serial washes through the trachea and pulmonary artery. Standard OCT thin section analysis as well as precision‐cut lung slice (200‐300μm) histology were performed. Lung samples were labeled with lectins (20μg/mL), secondarily labeled with a streptavidin‐fluorescein conjugate and analyzed by fluorescence confocal microscopy. Results: Decellularized mouse lungs showed a lectin‐binding pattern consistent with differential staining of the axial and peripheral connective tissue elements. Some lectins (N=10) demonstrated weak signal interpreted as negative. Others (N=17) stained pleural and subpleural zones suggesting selective staining of the peripheral connective tissue system. Another group (N=7) prominently stained the acinar septae suggesting selective staining of the axial connective tissue system. All lectin labeling was demonstrated at wavelengths that preclude elastin autofluorescence. Conclusions: The differential labeling pattern of the ECM with lectins suggests their utility as monospecific probes of both peripheral and axial connective tissue systems in the decellularized mouse lung.

Grant Funding Source: Supported by NIH grant 04925
microRNA‐98 negatively regulates nicotine‐induced nerve growth factor expression in primary mouse lung fibroblasts (870.4)
Authors: Kang Bum‐Yong , Grooms Kora , Sutliff Roy , Hart C. Michael , Wongtrakool Cherry
Published: June 2021

We previously demonstrated that chronic nicotine exposure 1) increases branching morphogenesis resulting in altered airway geometry associated with increased airway hyper‐responsiveness (AHR) in adulthood, and 2) increases Nerve Growth Factor (NGF) expression levels. To explore post‐transcriptional mechanisms of nicotine‐induced NGF expression, we hypothesized that microRNA (miR‐98) negatively regulates nicotine‐induced NGF expression to promote increased airway remodeling and AHR. Using bioinformatics prediction algorithms, we identified the miR‐98/let‐7 family as potential post‐transcriptional regulators of NGF. Of all of the miRNAs examined, only let‐7c, let‐7g, and miR‐98 were significantly decreased in mouse lung fibroblasts treated with nicotine for 72h compared to control conditions. Nicotine increased NGF, fibronectin, and ET‐1 (a known target of miR‐98) levels and reduced HUWE‐1 (host gene of miR‐98) levels, whereas these effects were reversed by miR‐98 overexpression. Overexpression of miR‐98 decreased branching morphogenesis in lung explants harvested from C57BL/6J mice at embryonic day 11.5 compared to control conditions. These findings suggest that miR‐98 may provide a novel therapeutic target for asthmatics exposed to nicotine in cigarette smoke.

Grant Funding Source: Supported by AHA‐SDG 13SDG14150004, Atlanta VA1I01BX001910,CDA IK2BX001306,NIH HL074518,DK102167
Somatic dysfunction and effect of OMT on pulmonary function in healthy adult male subjects (871.7)
Authors: Kochhar Kanika , Hines Patrick , Heinking Kurt , Henderson Kyle
Published: June 2021

The effects of osteopathic manipulative treatment (OMT) on pulmonary function have been equivocal. Our long‐term goal is to design hypothesis driven research to determine the efficacy of OMT. We hypothesize that treatment of somatic dysfunction with OMT will increase chest compliance and vital capacity. IRB approval was obtained and healthy adult males were randomly divided into a control group (n=17) or OMT group (n=20). Pulmonary function was measured at baseline, ~10min post‐OMT or quiet reading (control), and repeated at 24hrs. Somatic dysfunction was assessed and treated with OMT. There were no statistical differences between the control and OMT groups at baseline, or changes in pulmonary function in controls acutely or 24hrs. Of the OMT group, 75% of the subjects had somatic dysfunction in the C2‐C7, T5‐T9, ribs, and L1‐L5 regions. After OMT, upper thoracic excursion increased from 5.7±0.5 to 6.3±0.5cm (p = 0.03), and forced vital capacity (FVC) increased from 5.13±0.20 to 5.24±0.19L (p = 0.02) at 24hrs. Of note, OMT treatment of subjects with rib dysfunction significantly improved FVC at 24hrs (p = 0.02). Our data demonstrate significant somatic dysfunction in otherwise healthy adult males and suggest OMT can increase upper thoracic excursion and lung volumes. These data support the possibility that OMT can improve pulmonary function in patients with extrinsic restrictive lung disease.

Grant Funding Source: Supported by: AHA 10SDG2640219
Iba1+ microglia/macrophages in the rat hypothalamic paraventricular nucleus are both resident and bone marrow‐derived in Ang II induced hypertension (875.5)
Authors: Santisteban Monica , Zubcevic Jasenka , Kim Seungbum , Marulanda‐Carvajal Jessica , Zingler Michael , Joseph Jessica , Raizada Mohan
Published: June 2021

We have previously demonstrated that Angiotensin II (Ang II)‐induced hypertension increases activated microglial cells in the hypothalamic paraventricular nucleus (PVN). The objective of this study was to determine whether the origin of these cells is, in part, bone marrow (BM). We investigated whether minocycline (Mino), an antibiotic that crosses the BBB, would attenuate the microglial/macrophage responses and thereby the Ang II‐dependent increase in blood pressure (BP). Methods: SD rats underwent BM ablation followed by reconstitution with whole BM from eGFP donors. Following a 3 month recovery, animals were randomized to receive SC saline + H2O; SC Ang II + H2O; or SC Ang II + oral Mino (50 mg/kg). BP was measured weekly by tail‐cuff (n=5 per group) for 7 weeks. Iba1 antibody was used to image and quantify microglia expressing GFP (n=3 per group). Results: The Ang II/H2O group showed increased mean arterial pressure (MAP; 178 ± 9 vs. 104 ± 2 mmHg; p<0.001) and 4.3‐fold increase in GFP+/Iba1+ cells (p<0.05) from Saline/H2O group. Oral Mino attenuated the increased MAP (121 ± 7 mmHg) and caused a 37% decrease in GFP+/Iba1+ cells in the PVN. Conclusion: These data demonstrate that AngII‐induced hypertension is associated with an increase in the BM‐derived microglia/macrophages in the PVN and oral Mino treatment attenuated both the hypertension and extravasation of BM cells into PVN.

Grant Funding Source: This research is supported by NIH grant (HL 33610 to MKR).
Exceptional locomotory performance in Paratarsotomus macropalpis mites (878.1)
Authors: Rubin Samuel , Wright Jonathan , Young Maria , Whitaker Dwight , Ahn Anna
Published: June 2021

The Southern California erythracarid mite species, Paratarsotomus macropalpis, was filmed using a high frame‐rate video camera in the field to analyze speed, stride frequency, acceleration and deceleration. We also recorded higher resolution lab footage of mites starting, stopping and turning to analyze gait and kinematic mechanisms. Mites running in the field on concrete substrates at high temperatures (40°C to 60°C) were shown to travel at mean relative speed of 192.4 bl s−1 (body lengths per second), exceeding the highest currently documented speed for land animals (171 bl s−1). Despite this exceptional value, it conforms broadly with predictions based on interspecific scaling. Stride frequencies were also exceptionally high (as fast as 135 Hz), and increased significantly with substrate temperature. Mites accelerate and decelerate rapidly (mean values of 7.2 ms−2 and −10.1 ms−2, respectively), although the forces involved are simiilar to those found in other running animals. Calculations show that air resistance is a minor contributor to deceleration; the forces are likely borne by a combination of muscle and cuticle ligament strain. During normal running, adjacent and opposite tarsi are 180 degrees out of phase. This gait cycle is preserved during turning, although the two front pairs of tarsi initiate acceleration cycles and duty factors increase for inner tarsi during turns.

Grant Funding Source: Supported by the Howard Hughes Medical Institute
Teaching anatomy in an integrated medical curriculum: getting to the heart of the matter (88.4)
Authors: Nichols Carol
Published: June 2021

In 2009, the Year 1 curriculum at the Medical College of Georgia (MCG) underwent significant curricular reform that resulted in the creation of six integrated systems based courses. All anatomical sciences are now taught as integrated components of these courses with content distributed throughout the academic year. Programatic learning objectives were also revised around this time to align competency based objectives under the six domains of Medical Knowledge, Professionalism, Communication, Practice‐Based Learning, Patient Care, and Systems‐Based Practice. To facilitate practice and assessment of these competencies, a variety of learning activities such as gross anatomy lab handover reviews, peer feedback & self‐reflection exercises, student group presentations, and histology lab quizzes were introduced. Analysis of student performance on these activities indicates that students need and appreciate opportunities to practice these competencies. Indicators of faculty productivity, such as research funding and number of publications, were analyzed as well and did not show a decline. Student perceptions of overall teaching effectiveness also increased.
High levels of the small chaperone HSP25 in naked mole‐rats may be a determinant of rodent longevity (880.5)
Authors: Rodriguez Karl , Buffenstein Rochelle
Published: June 2021

Proteostasis is an integral component of healthy aging. In most metazoans, protein quality declines during aging, resulting in accrual of damaged or self‐aggregating cytotoxic proteins, linked to several age‐associated diseases (e.g, Alzheimer’s Disease, Parkinson’s Disease) and pathology (e.g.,sarcopenia). The mouse‐sized naked mole‐rat [NMRs] lives ~5 times longer than expected based on body size, and despite detected high levels of oxidative damage even at a young age, maintain good health for most of their long lives. Like other long‐lived animal models, both in vivo and in vitro studies reveal that NMRs are resistant to a broad spectrum of environmental stressors. Collectively these findings suggest that NMRs possess efficient mechanisms to maintain protein quality. Our research has previously shown that this is attributed in part to altered proteasome forms and subcellular location. However, changes in proteasome‐related molecular chaperone activity that assists in the transport of damaged proteins into the proteasome may also play a role in this decline. Here we examine key proteasome‐related molecular chaperones [HSPs] and the heat‐shock factor 1 [HSF1] transcription factor in the brain, heart, liver, spleen, kidney, testes, and quadriceps leg muscle in mice and NMRs. HSP70 was higher in NMR for most tissues compared to mice with the exception of the spleen where mouse levels were greater. For HSP110, HSP90, and HSP70 co‐chaperones, such as HSP40, protein levels showed tissue‐specific trends where there was no difference, higher levels in one or the other species. HSF1 and HSP25 both showed higher levels of protein in NMRs compared to mice in all the tissues examined. Hence we measured HSP25 protein content in seven rodents with ages ranging from four to 32 years in both liver and muscle. This comparison resulted in a significant correlation with longevity suggesting that HSP25 may play a key role in age‐related maintenance of protein homeostasis in long‐lived animals.

Grant Funding Source: Supported by NIA Training Grant
Anxiolytic‐like effects of a new 1‐N substituted analogue of melatonin in pinealectomized rats (883.2)
Authors: Picazo Ofir , Espinosa‐Raya Judith , Bustamante‐García Ruth , Gomez‐Martínez Atonatiu , Lira‐Rocha Alfonso , Espejo‐González Ofelia
Published: June 2021

In spite of the wide variety of drugs available for treating anxiety, this disorder continues to represent a worldwide health problem that is classified within the first 10 causes of disability. New antianxiety agents not related to benzodiazepines are being proposed to face this problem. Even though melatonin has been prescribed as an anxiolytic drug, its use is currently reduced due to its short half‐life and photo‐sensitivity, among other disadvantages. The present study explores the antianxiety properties of a new 1‐N substituted melatonin analogue, M3C, in pinealectomized rats submitted to two behavioral tests (the cumulative burying behavior paradigm and the elevated plus‐maze). Results from both tests show that M3C is effective as an anxiolytic agent, at doses lower than any other melatonin analogue previously reported. The blocking of these actions by luzindole, a melatoninergic antagonist showing a high affinity for MT2 receptors, suggests a mechanism independent of MT1 receptors.

Grant Funding Source: Supported by COFAA and SIP‐IPN
Weight loss with or without exercise: effect on classic and novel biomarkers of cardiovascular risk (884.24)
Authors: Mikus Catherine , Huffman Kim , Redman Leanne , Ravussin Eric , Kraus William
Published: June 2021

We previously reported that novel metabolomics‐derived biomarkers (dicarboxyl/hydroxyl acylcarnitines (DC/OH‐ACs) and amino acid‐related metabolites (AAs)) are independent predictors of CAD, CV events, and death and add predictive value to classic CV risk factors in cardiac catheterization patients. Here, we evaluated associations between traditional CV risk factors (FRS; Framingham Risk Score) and DC/OH‐ACs and AAs in sedentary, overweight adults (N=46) randomized to 6 months of: AHA weight maintenance diet (CONTROL), 25% calorie restriction (CR), 12.5% CR + 12.5% increase in exercise energy expenditure (CR+EX), or 15% weight loss via low calorie diet (890 kcals/d; LCD). At baseline, serum DC/OH‐ACs and AAs predicted 97% of FRS. At 6 months, weight loss was greater in LCD (‐11±1 kg) than CR or CR+EX (‐8±1 kg) and in all groups relative to CONTROL (‐1±1 kg). Only in CR+EX did FRS change significantly (‐10±4%) or differ from the change in FRS in CONTROL (+7±4%). Across all groups, changes in serum DC/OH‐ACs and AAs predicted 97% of FRS change. Thus, CR+EX appears to be more effective than CR or LCD in reducing CV risk. Further, DC/OH‐ACs and AAs may have strong predictive value in overweight adults.

Grant Funding Source: Supported by U01 AG20478 (ER) and F32 HL112575 (CRM). and
Adenosine signaling via A2B receptor regulates erythrocyte 2,3‐bisphosphate induction at high altitude (885.6)
Authors: Liu Hong , Zhang Yujin , Sun Kaiqi , Song Anren , Quintana Harry Karmouty , Grenz Almut , Kellems Rodney , Eltzschig Holger , Roach Robert , Blackburn Michael , Xia Yang
Published: June 2021

To function effectively in O2 uptake, and unload, erythrocytes rely on sophisticated regulation of hemoglobin (Hb)‐O2 affinity by allosteric modulators. One of the best known allosteric modulators is 2,3‐bisphophosphoglycerate (2,3‐BPG). Earlier studies demonstrated that erythrocyte 2,3‐BPG levels are elevated at a high altitude. However, what triggers its induction at high altitude is unknown. To address this question, we recruited 24 individuals and placed them at high altitude for different time points. Here we report that 1) Plasma adenosine and erythrocyte 2,3‐BPG levels were significantly elevated in normal individuals at high altitude after 24 hours compared to sea level; 2) their levels are further enhanced at high altitude after 16 days; 3) Elevated circulating adenosine levels significantly correlated with increased erythrocyte 2,3‐DPG levels in normal individuals at high altitude. Next, we provide in vitro evidence that adenosine signaling via A2B receptor (ADORA2B) directly induced 2,3‐BPG production in a protein kinase A‐dependent manner in cultured human erythrocytes. Finally, we provide genetic and pharmacological evidence that that ADORA2B is essential for elevated adenosine induced 2,3‐BPG production and triggers O2 release in mice. Taken together, we reveal ADORA2B signaling cascade regulating erythrocyte 2,3‐BPG induction as a function of altitude and identify new targets to enhance O2 release under hypoxia conditions.

Grant Funding Source: NIH
Renal epithelial electrophysiological response to cobalt chloride induced hypoxia (888.4)
Authors: Nag Subhra , Resnick Andrew
Published: June 2021

Regulation of amiloride‐sensitive sodium current i.e., sodium channel (ENaC) current was found to be dependent on intact cilium in a kidney epithelial cell culture model. We hypothesize that cystic disease, for example Polycystic Kidney Disease (PKD), results in localized hypoxia due to creation of stagnant fluid‐filled cysts, interfering with normal salt and water transport.

In the current study, we used a cortical collecting duct cell line (mCCD 1296 (d)) of an Immortomouse. We simulated hypoxic conditions with 100 µM Cobalt Chloride (CoCl2) and observed sodium transport under static and dynamic cell differentiation conditions. CoCl2 simulates hypoxia conditions by stabilizing the hypoxia inducible transcription factor 1α (HIF1α). We noticed a drop in the sodium current at time points of 24, 48, 72, and 96 hours in the presence of CoCl2. According to our hypothesis, the sodium current is expected to decrease in shorter time points of hypoxia whereas longer hypoxia induces cellular apoptosis totally nullifying the sodium current. We record sodium current in response to hypoxia at shorter time intervals within 48 hours as well as longer time periods upto 8 days. We use MTT cell viability assay to determine the time points at which CoCl2 induced hypoxia or cytotoxic CoCl2 start causing cell death. Correlating the MTT assay results with sodium current results will help us finding the direct correlation between hypoxia and sodium transport. This electrophysiological study will be complimented with immunocytochemistry to image cilium length alterations, and ciliary‐associated proteins, STAT‐6 and Phosphorylated‐STAT6 localizations. Thus, this study will provide better understanding of how hypoxia, sodium ion transport, effect of fluid flow and physiological and molecular cellular responses are interrelated.

Grant Funding Source: Supported by the National Institute of Diabetes and Digestive and Kidney Diseases 1R15DK092716
KLHL3/CUL3 targets the ROMK endocytic protein ARH for degradation directly by ubiquitination (892.14)
Authors: Woodward Owen , Kim Boyoung , Fang Liang , Coleman Richard , Wade James , Welling Paul
Published: June 2021

Mutations in KLHL3 and CUL3, components of an E3 ligase complex, cause a familial disease of hyperkalemia and hypertension (PHAII). Here, we explore how KLHL3/CUL3 normally regulates the ROMK channel for potassium balance. We previously reported that ROMK is targeted for endocytosis by a specific clathrin adaptor molecule, ARH (Fang et al, JCI ’09). Because KLHL3/CUL3 targets ARH for proteasomal degradation (Fang et al, EB ’13), we wondered if ARH is a direct substrate of KLHL3/CUL3 ubiquitination. In vitro protein‐protein interaction studies with purified ARH, KLHL3, CUL3 revealed that ARH directly interacts with KLHL3. We found separate interaction domains allow KLHL3 to simultaneously recruit CUL3 and the E3 ligase, ROC1, into a complex. As measured in ubiquitination assays in vitro with purified components, the direct interaction results in an increase in ARH ubiquitination, indicating that ARH is a direct substrate of KLHL3/CUL3. Importantly, KLHL3 localizes to the late DCT and CNT, overlapping with ROMK and ARH. Thus KLHL3/CUL3 are positioned to limit ARH and ROMK endocytosis to safeguard adequate potassium excretion.

Grant Funding Source: NIDDK
Real time monitoring of NKCC2 endocytosis by total internal reflection fluorescence microscopy (892.19)
Authors: Jaykumar Ankita , Caceres Paulo , Sablaban Ibrahim
Published: June 2021

The apical Na‐K‐2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TALs is determined by dynamic endocytosis from the apical membrane mediated by clathrin and lipid rafts. However, the time resolution of previous methods used to measure endocytosis was low (5 min), and internalization of NKCC2 containing vesicles not measurable. We hypothesize that TIRF microscopy imaging of fluorescently labeled apical NKCC2 would allow us to monitor its endocytosis in real‐time. We transduced polarized MDCK cells (in Transwell filters) with an N‐terminus GFP‐tagged NKCC2 using adenoviruses. Similar to native TALs, GFP‐NKCC2 was located in the apical membrane, at and above tight junctions. Surface biotinylation confirmed targeting of mature GFP‐NKCC2 (≍190 kDa) to the apical membrane. For TIRF imaging, cells were placed in a chamber with the apical membrane facing the TIRF illumination field. To selectively label NKCC2 at the apical surface we generated NKCC2 containing a biotin acceptor domain of 16 aa between one of the extracellular loops. Once expressed in MDCK cells, we specifically biotinylated surface NKCC2 with recombinant biotin ligase. Labeling apical NKCC2 with streptavidin‐Qdot, or streptavidin‐AlexaFluor allowed us to monitor single endocytic events and apical surface NKCC2 distribution in real‐time. In the absence of biotin ligase we did not observe labeling, indicating specificity for NKCC2 labeling. The overall rate of constitutive NKCC2 endocytosis was 1.2±0.1 % per minute (n=6). In MDCK and TAL cells most apical NKCC2 was located within small discrete domains that exhibited little lateral movement. We conclude that TIRF microscopy of labeled NKCC2 allows the imaging of individual endocytic events at the apical membrane.

Grant Funding Source: NIH‐AHA
NKCC2 phosphorylation modulates its clathrin‐mediated internalization (892.20)
Authors: Mutig Kerim , Daigeler Anna‐Lena , Dathe Christin , Wilson Bridget , Bachmann Sebastian
Published: June 2021

The Na‐K‐2Cl cotransporter (NKCC2) of the thick ascending limb of Henle's loop (TAL) is a key player in the urinary concentration and renal salt handling. Activity of the transporter depends on its surface expression and N‐terminal phosphorylation. NKCC2 is integrated into lipid rafts (LR) membrane microdomains during its apical trafficking, whereas endocytic retrieval of the transporter is substantially mediated by clathrin‐coated pits. We hypothesized that the activating N‐terminal NKCC2 phosphorylation interferes with these trafficking events. Evaluation of NKCC2 and phospho‐NKCC2 distribution within the apical membrane showed that the phosphorylated transporter was chiefly distributed in LR, whereas the total NKCC2 signal was also detected in “non‐raft” regions in close co‐localization with clathrin. GST pull down assays of rat kidney lysates using N‐ or C‐terminal tails of NKCC2 as baits revealed substantial binding of clathrin to the N‐terminus of NKCC2. Evaluation of interactions between clathrin and NKCC2 mutants mimicking (de)phosphory[[Unsupported Character ‐ Codename ­]]lation of the transporter at the functionally important residues (T96, T101, T114, and S126) indicated that dephosphorylation of the transporter promotes its N‐terminal association with clathrin coated pits. Calcineurin, a phosphatase involved in NKCC2 dephosphorylation, interacted with clathrin and phospho‐NKCC2 mutants in binding assays suggesting that the phosphatase may dephosphorylate the transporter during its internalization. Finally, stimulation of vasopressin (AVP)/V2 receptor axis in AVP‐deficient Brattleboro rats substantially increased NKCC2 phosphorylation and surface expression and simultaneously attenuated its interaction with clathrin. Our data suggest that NKCC2 phosphorylation attenuates its clathrin‐mediated internalization which may help to maintain the surface expression of the transporter.
NKCC2 activity is inhibited upon the expression of the gain‐of‐function CASR mutant A843E in renal cells (892.23)
Authors: Carmosino Monica , Hendy Geoffrey , Torretta Silvia , Rizzo Federica , Gerbino Andrea , Procino Giuseppe , Svelto Maria
Published: June 2021

The mutation gain‐of‐function A843E of the Calcium Sensing Receptor (CASR) causes a Bartter syndrome type V. Patients carrying this CASR variant showed a remarkable reduced renal NaCl reabsorption in Thick Ascendent Limb (TAL) resulting in renal loss of NaCl in the absence of mutations in renal Na+ and Cl‐ ion transporters. The molecular mechanisms underlying this clinical phenotype are still unknown. Indeed, we investigated, in LLC‐PK1 epithelial renal cells, the possible functional cross‐talk of CASR‐A843E mutant with NKCC2, the main transporter involved in the NaCl reabsorption in the TAL. The steady‐state NKCC2 phosphorylation and activity were inhibited by about 60% in cells transfected with CASR‐A843E mutant compared to CASR WT‐transfected cells, used as control. Of note, the low‐chloride‐dependent NKCC2 activation was also strongly inhibited upon the expression of CASR‐A843E mutant. Interestingly the maximal activation of CASR WT mimicked the effect of CASR‐A843E on NKCC2 regulation. The expression of the CASR mutant did not alter the apical localization of NKCC2 in LLC‐PK1 cells, suggesting that the CASR‐A843E affected intracellular pathway/s modulating NKCC2 activity rather than NKCC2 intracellular trafficking. Our findings opened new avenues not only to gain more insight in the role of CASR in the physiopathology of the kidney, but also to conceive novel therapeutic strategies for the treatment of the Bartter syndrome type V.

Grant Funding Source: Supported by PRIN 2009 n# 2009J53ALK to MS
Novel roles of raft domain in the molecular mechanism of activation of epithelial Na+ channel at the plasma membrane (892.26)
Authors: Yokoyama Noriko , Marunaka Yoshinori
Published: June 2021

ENaC expressed in the apical membrane of epithelial cells plays a critical role in homeostasis of fluid content and blood pressure. Na+ transport via ENaC depends on not only the open probability of the channel, but also the number of channel expressed on the apical membrane. ENaC is cleaved by aldosterone‐induced proteases, resulting in an increase in open probability. The lifetime of ENaC at the apical membrane is regulated by ubiquitin ligase (Nedd4‐2). To elucidate roles of raft domain in the regulation of ENaC activation, we quantified the abundance and localization of ENaC at the plasma membrane with/without aldosterone stimulation in renal epithelial cells. Aldosterone stimulated expression of both full‐length and cleaved ENaCs; i.e., the full‐length ENaC was expressed in both cytoplasm and plasma membrane (raft domain), whereas a large portion of the cleaved ENaC was observed at the non‐raft plasma membrane. Depletion of cholesterol from the raft domain diminished expression of cleaved ENaC not only in raft but also non‐raft domain by aldosterone stimulation. Aldosterone also increased expression of Nedd4‐2 and SGK1 in raft domain. Together, these results suggest that the ubiquitination‐mediated regulation of ENaC would occur in raft domain. Our findings demonstrate novel roles of the lipid raft domain on the regulation of ENaC activity/trafficking.
Cytoskeletal regulation of ENaC: the role of ankyrin G (892.28)
Authors: Klemens Christine , Carananti Maria , Liu Xiaoning , Butterworth Michael
Published: June 2021

The epithelial sodium channel (ENaC) is a major determinant of sodium (Na+) and water balance in the kidney. The hormone aldosterone increases ENaC subunit translation and modifies expression of key regulators that alter ENaC function and membrane density. Using a microarray screen we identified the scaffolding protein, ankyrin G (Ank3), as an aldosterone‐induced protein. We hypothesize that Ank3 regulates Na+ transport by increasing ENaC density at the apical membrane. Ank3 overexpression in a mouse kidney collecting duct epithelial cell line (mCCD‐cl1) increased ENaC‐mediated Na+ transport by 67%±11% (n=12), whereas knockdown reduced ENaC activity by 69%±2% (n=15). Ank3 depletion blunted the ENaC response to aldosterone stimulation in this cell line, from a 70.4±7% increase in control cells to 19.8±5% (n=5) increase in Ank3 knock‐down cells. Membrane permeabilization experiments confirmed the reduction in Na+ transport was due to reduced ENaC, and not Na/K‐ATPase activity. Future studies will identify critical domains governing Ank3’s association with ENaC and the regulation of this interaction. These findings introduce Ank3 as a key regulator in kidney Na+ transport.

Grant Funding Source: Supported by DK078917, DK079307
Rosiglitazone promotes AQP‐2 translocation in renal cells via a Ca2+ dependent/cAMP independent mechanism (892.9)
Authors: Gerbino Andrea , Procino Giuseppe , Milano Serena , Nicoletti Maria , Mastrofrancesco Lisa , Carmosino Monica , Svelto Maria
Published: June 2021

Preliminary results by our group showed that exposure to Rosiglitazone (RGZ) induces phosphorylation and apical translocation of AQP2 in mouse collecting duct clone 4 (MCD4) cells. Here we studied the effect of short‐term exposure to 50 µM RGZ on cAMP‐ and Ca2+‐mediated signaling pathways, both key players for AQP2‐mediated water reabsorption in the collecting duct. Cytosolic Ca2+ and cAMP levels were imaged in real‐time in MCD4 cells loaded with Fura‐2 or transiently transfected with the EPAC‐based fluorescent probe H90, respectively.

Physiologically, AQP2 phosphorylation/translocation depends on cytosolic cAMP levels. Nonetheless, cAMP measurements showed that RGZ did not induce significant changes in cAMP levels. Conversely, 20 minutes RGZ stimulation of Fura‐2 loaded MCD4 cells induced a large, transient cytosolic Ca2+ peak that was not the result of direct blockade of the SERCA pump since the rate of store empting elicited by CPA in the absence of external Ca2+ was not significantly different in the presence of RGZ. Importantly, removal of external Ca2+ and inhibition of Ca2+ channels with ruthenium red prevented the RGZ‐induced increase in cytosolic Ca2+ indicating a prominent role for Ca2+ entry at the plasma membrane.

In conclusion, RGZ‐induced AQP2 phosphorylation/translocation process is likely initiated by a fast, large extracellular Ca2+ influx most likely via Ca2+‐dependent transient receptor potential channels. Further studies to ascertain which cascade of kinases is involved in this scenario are in progress.

Grant Funding Source: Supported by Telethon #GGP1204/AIFA MRAR08P011
Deletion of KCNJ10 eliminates potassium conductance in the basolateral membrane of the early distal convoluted tubule (DCT1). (893.17)
Authors: Wang Wen‐Hui , Zhang Chengbiao , Wang Lijun , Scholl Ute , Zhang Junhui , Lifton Richard , Lin Dao‐Hong
Published: June 2021

The basolateral K channels in the DCT1 are composed of KCNJ10 and KCNJ16. Moreover, a 20 pS K channel was detected in the basolateral membrane of DCT of KCNJ16 knockout mice, suggesting that KCNJ10 alone is able to form homotetramers in the absence of KCNJ16. The aim of the present study is to test whether deletion of KCNJ10 would eliminate the basolateral K channel activity in the DCT1. Global deletion of KCNJ10 significantly impaired the animal growth, and the body weight of KCNJ10‐/‐ mice was significantly lower than that of their +/+ and +/‐ littermates. Moreover, KCNJ10‐/‐ mice did not survive beyond postnatal day 9‐12, while KCNJ10+/‐ mice had a life span similar to that of wt. To study the role of KCNJ10 in constructing the basolateral K channel, we performed patch‐clamp experiments in the isolated DCT1 of 8‐10 days old KCNJ10‐/‐, +/‐ and +/+ mice. The patch‐clamp study detected a 40 pS K channel in the basolateral membrane of the DCT1 of wt and KCNJ10+/‐, but not in KCNJ10 ‐/‐ mice. We also measured the Ba2+‐sensitive whole cell K currents which represent the basolateral K channel activity in the DCT1 since it has no apical K conductance. The mean Ba2+ ‐sensitive K currents at ‐60 mV were 2950 pA (wt), 1400 pA (+/‐) and 250 pA (‐/‐), respectively. Thus, deletion of KCNJ10 largely eliminated the basolateral K conductance in DCT1. We conclude that KCNJ10 is a major type of K channel expressed in the basolateral membrane of the DCT1.

Grant Funding Source: NIH
Characterization of SLC4A11 as a novel ammonia: 2H+ transporter (893.29)
Authors: Zhang Wenlin , Ogando Diego , Bonanno Joseph , Obukhov Alexander
Published: June 2021

SLC4A11 mutations cause corneal endothelial dystrophies. This degenerative condition leads to corneal haze, edema, and vision loss. SLC4A11 is a recently identified membrane transporter, of which multiple substrates have been suggested. Intracellular pH measurements in SLC4A11‐expressing PS120 cells revealed that SLC4A11 is NH3/NH4+ and H+ permeable. To further characterize SLC4A11 substrates and transport stoichiometry, we performed patch‐clamp recordings in SLC4A11‐PS120 cells. We found that extracellular 10mM NH3/NH4+ induced inward current, which was inhibited by bath acidification and potentiated by bath alkalinization. Current density (pA/pF) at pHo 6.5, 7.5 and 8.5 were ‐0.22±0.19, ‐6.18±1.09, ‐18.43±3.43, respectively (mean±SE, p<0.001). Reversal potential shift in response to [NH3]o and pHo was consistent with an NH3/H+ coupled transport model. 10mM methylammonium induced smaller current (p<0.05) with similar pH sensitivity. Removal of K+, Na+, Cl‐ did not affect SLC4A11 NH3/H+ transport (K+ p=0.90, Na+ p=0.06, Cl‐ p=0.65), suggesting neither Na+, K+, nor Cl‐ is substrate. Stoichiometry of SLC4A11 NH3:H+ transport was estimated by measuring Erev at different [NH3]o. A plot of Erev against log[NH3]o was linear, and the slope was consistent with a 1:2 stoichiometry. We here identify SLC4A11 as a new member of ammonia channels/transporters, such as AQP8, Rh, MEP, and Amt.

Grant Funding Source: Supported by 5R01EY008834(JAB); 5R01HL115140(AGO).
Cloning, localization and characterization of a Zn transporter (Zip10) from fly, dog, and human (893.39)
Authors: Williams Paige , Strohmaier Käri , Hirata Taku , Pandey Mukesh , Jiang Huailei , Aditya Bansal , Cabrero Pablo , Dow Julian , DeGrado Timothy , Furrow Eva , Romero Michael
Published: June 2021

Background: Zn is implicated as having a role in the formation of calcium oxalate (CaOx) kidney stones. Our studies indicate that Slc39a10 (Zip10) may be important in this process. Zip10 has identifiable sequences in human, dog and fly (Drosophila). As we are interested in a translational model of kidney stones, we examined Zip10 from all 3 species.

Methods: Zip10 cDNAs were cloned from human & dog (kidney) and fly (whole fly) using gene specific primers and RT‐PCR. Clones were put into a Xenopus oocyte expression vector so that Zip10 function could be measured. Three days post‐injection, oocytes were pre‐incubated in ND96, followed by 30 min in ND96 + 10 μM ZnCl + the PET isotope 63Zn, with varying pHs and ion concentrations. 63Zn uptake was measured in each oocyte using a gamma counter. Zip10 oocytes were also checked using electrophysiology (ion selective electrodes & voltage clamping).

Results: Zip10 mRNA & protein are found in gut and kidney. In mouse kidney, Zip10 antibodies stained the apical membrane of proximal tubules. All clones transported 63Zn (nmol/oocyte/hr): hZip10 = 70±8; cZip10= 44 ±7; dZip10= 64 ±8; water = 0.4 ±0.1. These clones seem to be most active at pH 7.5 as uptakes decreased at 5.5 and 8.5. Replacement of either all Cl‐ (with HCO3舉) or all Na+ (with choline) reduced Zn‐uptake. Electrophysiology revealed no evidence of I or V changes with addition of Zn (electroneutral).

Conclusions: Our results indicated that all three Zip10 clones transport Zn2+. Understanding the detailed mechanisms of these Zn transporters in oocytes presents the opportunity to better use our Drosophila model in the study of CaOx stone formation and absorption.

Grant Funding Source: Supported by DK83007, DK92408
Molecular determinants of the interaction between E3 ubiquitin ligase Nedd4‐2 and ENaC (893.5)
Authors: Labarca Mariana , Goens Donald , Dong Wuxing , Bhalla Vivek
Published: June 2021

Phenotypic characterization of Nedd4‐2 knockout mice demonstrates the critical importance of this E3 ubiquitin ligase to activity of the epithelial sodium channel (ENaC) in several tissues including lung and kidney. However, determinants of the interaction between Nedd4‐2 and ENaC are not well understood and have implications for the treatment of several diseases including cystic fibrosis and hypertension.

We characterized several novel determinants of this interaction using cell‐based biochemical assays to analyze exogenously or endogenously expressed Nedd4‐2 and ENaC.

Nedd4‐2 preferentially binds to proteolytically cleaved ENaC in transfected HEK293T cells (10.9:1 +/‐ 0.009, p < 0.05). The preference for Nedd4‐2 to interact with cleaved channels is independent of Nedd4‐2 species and Nedd4‐2 ligase activity. Additionally, we demonstrate that neither an ENaC channel inhibitor, amiloride, nor ENaC channel mutants with increased or decreased activity alter the preference of Nedd4‐2 for cleaved channels. However, the calcium‐binding C2 domain of Nedd4‐2 decreases the binding of Nedd4‐2 to cleaved and uncleaved ENaC channels by approximately 5‐fold (p < 0.01). Surprisingly, active serum‐and‐glucocorticoid‐induced protein kinase 1 (SGK1) increased interaction between Nedd4‐2 and ENaC. Ongoing studies of the subcellular localization of Nedd4‐2 and cleaved or uncleaved ENaC channels is critical to understanding the determinants of this interaction.

Proteolytic cleavage, but not activity, of ENaC; regulatory domains on Nedd4‐2; and unexpectedly, SGK1 kinase activity enhance Nedd4‐2 and ENaC interaction and can inform our understanding of how to target these pathways for treating lung and kidney disease.

Grant Funding Source: NIH
14‐3‐3 ‐β and ‐ε Contribute to nuclear factor of activated T‐cells 5 (NFAT5) activity by regulating NFAT5 protein expression (895.3)
Authors: Izumi Yuichiro , Burg Maurice , Ferraris Joan
Published: June 2021

NFAT5 is a transcription factor that induces transcription of osmoprotective genes in response to high NaCl. 14‐3‐3s are regulatory proteins that have numerous roles in protein interaction and enzymatic activity. There are multiple 14‐3‐3 isoforms that form homo‐ and hetero‐dimers and play isoform‐specific regulatory roles.

In the present study, the effects of small interfering RNAs against 14‐3‐3 ‐β, ‐ε, ‐γ,‐η,‐θ, and ‐ζ on NFAT5 function were examined in HEK293 cells incubated at 300 and 500 mosmol/kg (NaCl added). We used Luciferase reporters to measure NFAT5 transcriptional (ORE‐X) and transactivating (TAD) activity. We quantified mRNA abundance of the NFAT5 target genes, aldose reductase (AR) and betaine/GABA transporter (BGT1) by real time PCR. We measured nuclear localization of NFAT5 by quantitative Western analysis of nuclear and cytoplasmic proteins. We also measured protein abundance of NFAT5.

Knock down of 14‐3‐3 ‐β and/or ‐ε significantly inhibited high NaCl‐induced ORE‐X and TAD activities, however the effects on TAD activity were minimal. Knockdown of β and/or ε decreased mRNA abundances of AR and BGT1. The effects of knocking down β and ε were not additive. Knock down of β and ε did not affect the nuclear localization of NFAT5 but significantly decreased protein abundance of NFAT5.

We conclude 14‐3‐3 ‐β and ‐ε contribute to NFAT5 transcriptional activity by regulating NFAT5 protein expression.

Grant Funding Source: Supported by The Intramural Research Program of NHLBI, NIH.
Interaction of TM4SF4 with the human thiamine (vitamin B1) transporter‐2 (hTHTR‐2) in human intestinal epithelial cells (896.1)
Authors: Subramanian Veedamali , Nabokina Svetlana , Said Hamid
Published: June 2021

The human thiamine transporter‐2 (hTHTR‐2) is involved in intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients have shown that these transporters have associated proteins that affect different aspects of their physiology and cell biology. Nothing is currently known about protein(s) that may interact with hTHTR‐2 in intestinal cells and influence its physiological function and cell biology.We addressed this issue using the yeast split‐ubiquitin two‐hybrid approach to screen a human intestinal cDNA library with the full‐length hTHTR‐2 as bait. We also performed GST‐pull‐down assay and co‐localization to confirm the existence of protein‐protein interaction. Our screening results have identified the human tetraspanin (transmembrane 4 super family member 4, TM4SF4) protein (which is abundantly expressed in the intestine) as a potential interactor with hTHTR‐2. The interaction between hTHTR‐2 and TM4SF4 were further confirmed by in vitro GST‐pull‐down assay and by live‐cell confocal imaging of HuTu‐80 cells co‐expressing hTHTR‐2‐GFP and Cherry‐TM4SF4. Co‐expression of hTHTR‐2 with TM4SF4 in HuTu‐80 cells led to a significant induction in hTHTR‐2 ‐ mediated thiamine uptake. These results show for the first time the existence of an accessory protein that interacts with hTHTR‐2 and influence its physiological function. (Supported by NIH grant DK 56061‐15).

Grant Funding Source: NIH‐NIDDK
Bacterial immunomodulators in the treatment of patients with nonspecific ulcerative colitis (898.1)
Authors: Omarov T. , Tell L. , Dalenov E.
Published: June 2021

Inflammatory bowel disease to date remains one of the most important and unsolved problems in gastroenterology.

The purpose is to study the immune status of patients with nonspecific ulcerative colitis taking bacterial immunomodulators.

Materials. 20 patients with left‐sided nonspecific ulcerative colitis in the acute stage as an immunomodulator on the background of basic therapy NUC received synbiotics BION 3 per 1 tablet a day for a month.

Results. The study of the immune status before treatment revealed decreasing To‐lymphocytes 42,7±1,2%, Ts 8,3±0,7%, respectively, and indicators of spontaneous NST‐ stimulated 4,5±0,8%, and 6,6±1 6% and increasing in Th‐39, 4±1,5, T act 49,3±3,1% and the ratio of Th/Ts 4,8±0,4 indicating a violation of cellular immunity and phagocyte system depression. Increasing the level of PNA cells 21,0±1,2% and the CIC more than two times 30±2,5 conv unit indicates the severity of the chronic inflammatory process. After treatment, the cell‐mediated immunity returns to normal To 47,4±1,8%, Ts 12,9±0,6%, Tact 37,7±0,8%, Th 35,9±1,0% and the ratio of Th/Ts 2,8±014. Indicators NST test of spontaneous and stimulated significantly normalized and are 5,9±0,5% and 14,9±0,6% and showed activation of phagocytic activity of neutrophils. CIC after treatment is 19,9±2,4 conv units.

Conclusions: 1. Inclusion to the treatment the bacterial immunomodulators normalizes the immune status.
IMP1 loss promotes enhanced recovery from acute colitis (899.3)
Authors: Hamilton Kathryn , Lundsmith Emma , Hahn Christopher , Noubissi Felicite , Spiegelman Vladimir , Rustgi Anil
Published: June 2021

Background: mRNA‐binding proteins, including Mushashi1, Lin28b, and Igf2 mRNA‐binding protein 1 (IMP1), are newly recognized regulators of intestinal epithelial homeostasis via their effects on post‐transcriptional regulation of mRNAs comprising critical growth regulatory networks. Imp1‐null mice exhibit severe morphologic defects in the intestinal epithelium, yet the specific mechanisms of IMP1 in normal intestinal homeostasis and response to injury remain to be elucidated. We hypothesize that Imp1 modulates intestinal epithelial cell proliferation in response to acute inflammation.

Methods: We generated mice with intestinal epithelial‐specific loss of Imp1 (VillinCre;Imp1flox/flox mice), treated with dextran sodium sulfate (DSS) and measured indices during recovery, as well as IL‐6 and IL‐1β, canonical pro‐inflammatory cytokines. Actively proliferating cells were evaluated using Ki67 staining.

Results: Untreated VillinCre;Imp1floxed mice exhibit increased Ki67+ cells in the extra‐proliferative zone compared to controls. Upon DSS treatment followed by 2 days of recovery, VillinCre;Imp1flox/flox mice display decreased histological disease scores and lower cytokine expression compared to controls.

Conclusions: VillinCre;Imp1flox/flox mice have enhanced recovery following DSS and a decreased immune response. Mislocalization of actively proliferating cells may be advantageous in this context, providing a novel link of IMP1 to intestinal epithelial regeneration.

Grant Funding Source: Supported by: F32 DK093207‐01 (KEH), HHMI MRFP (ETL), NIH RO1 DK056645 (KEH, AKR), NIH UO1 DK085551
Importance of scientific social networks (9.1)
Authors: Canady Mary
Published: June 2021

Global scientific social networks have existed for more than 150 years, fueling many great discoveries through the dissemination of published research findings and subsequent scientist communication and collaboration. Indeed, many scientists used email and the world wide web for their research before these internet tools became so commonplace. Online government‐based databases such as PubMed, as well as subject‐specific listservs and forums, played an important part in connecting scientists and advancing many life science ideas and disciplines. Today, scientific social networks go well beyond these early internet tools, allowing scientists to connect, promote their work, and learn on a fast‐paced, global scale. In this introduction to the importance of scientific social networks, we’ll talk about general tools such as blogs, Twitter, and Google+ that researchers as a whole, including anatomists, are using to make important scientific advances.
The role of networking and social media in career advancement (9.2)
Authors: Carpenter Jenna
Published: June 2021

Many people have a misconception about networking and most view this in a very negative vein. We view networking as an important social skill that all professionals should include in his or her toolkit. Networking is sharing your ideas and passion, listening to your colleagues and their current research, finding new collaborators, and introducing people to one another to make ideas and projects grow. There are many resources available about networking online but many researchers feel confused and conflicted towards social media and lack sufficient information or guidance, particularly for those at the beginning of their careers. The importance of networking and issues and strategies used when trying to promote your own work and careers will be the focus of this session. To better familiarize researchers with existing Internet resources, we will discuss prospective benefits that can stem from online science conversations and interactions and explain how engineers and scientists can efficiently and effectively use online resources. Understanding scientific networks and how to use them, how to develop your web presence, or brand, and how to exploit these sites will be the emphasized. The theme will be that successful careers in engineering and science just don’t happen襃. they are crafted!