bluuebunny/crossref_metadata_2025_split huggingface dataset:
Alternative splice variant of the thiazide‐sensitive NaCl cotransporter: the dominant role of the novel phosphorylation site S811
Authors: Tutakhel Omar A. Z. , Hoederop Joost G.J. , Bindels René J.M. 
Published: November 2023

IntroductionThe thiazide‐sensitive sodium‐chloride cotransporter (NCC) is located in the apical membrane of epithelial cells lining the distal convoluted tubule of the kidney, and plays an instrumental role in blood pressure regulation by fine‐tuning the renal sodium excretion. The human SLC12A3 gene, encoding NCC, gives rise to three isoforms, of which only the 3rd isoform (NCC3) has been extensively investigated in patho(physiological) conditions. All three human NCC isoforms contain phosphorylation sites at threonine 55 (T55) and 60 (T60) that are essential mediators of NCC activity. Recently, we identified a new phosphorylation site located at S811 in NCC isoform 1 and 2 (NCC1/2).ObjectiveThe aim of the present study was, therefore, to i) investigate the role of the new S811 phosphorylation site in NCC1/2 function; ii) determine the interaction of NCC1/2 with NCC3; iii) study the effect of NCC1/2 S811 phosphorylation on the function of NCC3.MethodsFirst, with the use of immunoblot analysis the phosphorylation of T55 and T60 was investigated in two NCC1/2 S811 mutants (phosphorylation‐deficient S811A and constitutively phosphorylated S811D) compared to wild type NCC1/2 and NCC3. The NCC wild types and mutants were transiently transfected in human embryonic kidney 293 (HEK‐293) cells and incubated for 30 minutes in hypotonic low Cl− buffer, a well‐known stimulator of NCC T55/T60 phosphorylation. Second, to investigate the dimerization of NCC1/2 with NCC3, co‐immunoprecipitation analysis of NCC was performed. Finally, to determine the effect of NCC1/2 S811 mutants on NCC3 T55/T60 phosphorylation, NCC3 was co‐transfected with either NCC1/2 wild‐type, NCC1/2 S811D or NCC1/2 S811A. In the co‐transfection experiments, transiently transfected HEK293 cells were also incubated for 30 minutes in hypotonic low Cl− buffer.ResultsOur results demonstrate that i) in the presence of hypotonic low Cl− buffer the S811A mutant of NCC1/2, but not S811D, prevents the phosphorylation at T55 and T60 of NCC1/2; ii) NCC1/2 and the two NCC1/2 S811 mutants form dimers with NCC3; iii) S811A mutant, not only abolishes the hypotonic low Cl− induced phosphorylation at T55/60 phosphorylation sites of NCC1/2, but also prevents the phosphorylation at T55 and T60 of NCC3.ConclusionThe present study highlights that the phosphorylation of S811 in NCC1/2 is likely to act as a dominant regulatory site for phosphorylation of T55 and T60. A better understanding of the regulation of NCC function in the kidney is critical to elucidate both the molecular events underlying the pathogenesis of essential hypertension, and to subsequently design and utilize effective therapeutic strategies to combat the dysregulation of blood pressure.Support or Funding InformationOmar A.Z. Tutakhel is funded by the Dutch Kidney Foundation (PHD12.14).
NADPH oxidase 4 mediates ROS‐induced NKCC2 activity in thick ascending limbs
Authors: Saez Fara , Garvin Jeffrey 
Published: November 2023

Luminal flow through the nephron is highly variable, and it is increased in pathological conditions including diabetes and hypertension. Luminal flow increases thick ascending limb superoxide production by NADPH oxidase 4. Exogenously added superoxide increases NaCl reabsorption in this segment. We hypothesize that NADPH oxidase 4‐dependent flow‐induced superoxide stimulates Na/K/2 Cl cotransporter (NKCC2) activity. We measured NKCC2 activity in isolated thick ascending limbs perfused at 20 nl/min in the presence and absence of a superoxide dismutase mimetic and a NADPH oxidase 1/4 inhibitor. During the control period NKCC2 activity was 3.1±0.2 arbitrary fluorescent units (AFU/s). After the superoxide dismutase mimetic MnTBAP (30μM) was added to the bath, NKCC2 activity fell to 2.3±0.2 AFU/sec (n=8, p<0.03), a 30% reduction. Next we tested the effect of a selective NADPH oxidase 1/4 inhibitor. During the control period NKCC2 was 8.4±1.4 AFU/s. After treating thick limbs with GKT137831 (5μM), NKCC2 activity decreased to 3.9±0.7 AFU/sec (n=4, p<0.03), a 54% reduction. Time controls showed no significant change in NKCC2 activity with time (4.1±0.4 vs. 4.3±0.4 AFU/s; n=8). We conclude that flow‐induced superoxide generated by NADPH oxidase 4 stimulates NaCl reabsorption by activating NKCC2 in thick ascending limbs.Support or Funding InformationThis work was supported by a grant from the National Institutes of Health (HL 070985) to J.L. Garvin and a fellowship from the American Heart Association (15POST22650010) to F. Saez.
Segmental and Cellular V1a Receptor Distribution in the Mammalian Kidney
Authors: Giesecke Torsten , Giesecke Yvonne , Kawahara Katsumasa , Koshimizu Taka Aki , Gimber Niclas , Schmoranzer Jan , Smorodchenko Alina , Bachmann Sebastian , Mutig Kerim 
Published: November 2023

Vasopressin (AVP) promotes urinary concentration via its V2 receptors in the distal nephron and collecting duct of the kidney. Parallel activation of V1a receptors (V1aR) has been implicated in renal acid‐base handling, but cell type‐related information on this receptor type is still scarce. We have analyzed segmental and cellular distribution of V1aR in mouse, rat, and human kidneys using new antisera. Localization data were supported by functional studies.An anti‐V1aR was generated and its specificity verified using V1aR‐knockout tissues. Immunofluorescence and super resolution imaging (SIM) were applied for localization studies. AVP‐deficient Brattleboro were analyzed using the V1aR‐specific agonist, AO‐4‐67.The V1aR antibody showed basolateral membrane‐associated and punctate cytoplasmic signal in alpha‐type intercalated cells of connecting tubules and collecting ducts in rodent and human kidneys. In contrast, beta‐type intercalated cells showed only punctate perinuclear and subapical V1aR signal. Double‐labeling of V1aR with markers for flotillin‐1 (lipid rafts), GM130 (trans‐Golgi network protein), Rab9 (endosomal marker), or LAMP1 (lysosomal marker) suggested broad distribution of the receptor in the respective cellular structures in both types of intercalated cells. Administration of AO‐4‐67 to Brattleboro rats for 4h resulted in luminal trafficking of V‐ATPase in alpha‐type intercalated cells, whereas basolateral V‐ATPase or luminal pendrin of beta‐intercalated cells were not affected by the stimulation.In summary, divergent cellular V1aR distribution in alpha‐ vs. beta intercalated cells may explain their distinct responsiveness to the V1aR agonist. These results corroborate and extend data on the role of AVP in renal acid‐based handling.
Flow Stimulation of Endothelin‐1 Production by the Collecting Duct: Role of Nitric Oxide and Reactive Oxygen Species
Authors: Kohan Donald E , Wheatley Will 
Published: November 2023

Collecting duct (CD)‐derived endothelin‐1 (ET‐1) is an important autocrine inhibitor of CD Na+ and water reabsorption; CD‐specific ET‐1 deletion causes marked salt‐sensitive hypertension. Volume expansion increases CD ET‐1 production thereby facilitating elimination of the volume load. We have previously shown that increased luminal flow, as occurs in salt and/or water loading, stimulates CD ET‐1 production via Ca2+‐dependent pathways; however, how this flow effect occurs is incompletely understood. To examine this, a mouse inner medullary CD cell line (IMCD3) was exposed to shear stress in flow chambers and ET‐1/GAPDH mRNA assessed (ET‐1 mRNA very accurately reflects ET‐1 protein and ET‐1 protein levels are too low to measure in this system). A shear stress of 9.8 dyne/cm2 for 2 h was used based on previous studies (N=6–12 for all of the following data). Perfusion with HBSS + 0.25 mM L‐Arg (used in all subsequent studies) increased ET‐1 mRNA by 4‐fold compared to no flow (ET‐1 flow response). Exposure to 1 μM L‐NAME reduced the ET‐1 flow response to 67% compared to vehicle. Inhibition of NOS1 (1 μM SMTC or 200 μM TRIM) or NOS2 (0.1 nM 1400W) did not alter the ET‐1 flow response. NOS3 siRNA (no specific NOS3 antagonists exist) reduced NOS3 levels by 70%, however cells detached on exposure to flow (likely due to lower cell density needed for transfection). Inhibition of NO signaling pathways, including PKG (1 μM KT5823), guanylyl cyclase (10 μM ODQ) or NFkB (50 μg/ml NFkB inhibitory peptide), did not affect the ET‐1 flow response. These findings suggested NO may not be involved, but rather NOS (at least in part)‐generated reactive oxygen species (ROS) may play a role in the ET‐1 flow response. Inhibition of ROS with 5 mM tempol reduced the ET‐1 flow response to 67% vs. vehicle, no further inhibition was seen with tempol + L‐NAME. Superoxide dismutase (SOD) inhibition (50 μM MnTBAP or 100 U/ml PEG‐SOD) reduced the ET‐1 flow response to 65% of vehicle, while 1000 U/ml PEG‐catalase was without effect. Apocynin (100 μM) also reduced the ET‐1 flow response to 60% of vehicle, suggesting NAPDH oxidase (NOX) might be involved. Inhibition of NOX1/4 with 5 μM GKT137831 also reduced the ET‐1 flow response to 70% of vehicle. Taken together, these data suggest that both NOS and NOX contribute to flow stimulated superoxide‐mediated stimulation of CD ET‐1 production. Since ROS have been reported to augment ENaC activity, the current studies suggest that ROS‐stimulated ET‐1 may mitigate their Na+‐retaining effects in the CD. Finally, to our knowledge, this is the first report of flow‐stimulated reactive oxygen species in the CD, as well as the first report of such flow‐stimulated ROS exerting a biologic effect in the CD.Support or Funding InformationSupported by NIH R01 DK107454.
ROMK is Required for the Net K Secretion in the Thick Ascending Limb of Mice on a Low Na High K Diet
Authors: Wang Bangchen , Li Huaqing , Wang‐France Jun , Bales Cayla , Wen Donghai , Sansom Steven 
Published: November 2023

Understanding the effect of a cardio‐ and reno‐protective low Na, high K diet (LNaHK) on renal K handling is crucial to choosing diuretics and anti‐hypertensive agents for patients on such diets. We previously showed that furosemide, a K‐wasting diuretic that inhibits the Na‐K‐Cl‐cotransporter‐2 (NKCC2) in the thick ascending limb (TAL), increased urinary K clearance in mice on a control diet but decreased that in mice on LNaHK. We hypothesized that there is a net K secretion in the TAL of mice on LNaHK via the renal‐outer‐medullary‐K channel (ROMK) located in the apical membrane of TAL. Wild‐type (WT) and ROMK knockout mice (ROMK KO) were given either a control diet or LNaHK (0.01% Na, 5% K) for 4–7 days. Free‐flow micropuncture was performed using K‐selective microelectrodes to measure the K concentration in the early distal tubule (EDT [K]) before and after intravenous administration of vehicle or furosemide (5mg/kg). Urine was collected via a bladder catheter. Na concentration was measured by a flame photometer. Within 5 minutes of administration, furosemide increased the EDT [K] of WT on a control diet (Δ[K] = 2.33 ± 0.46 mM; n = 6) but decreased that of WT on LNaHK (Δ[K] = −3.07 ± 0.61 mM; n = 11). However, furosemide did not affect the EDT [K] of ROMK KO on LNaHK (Δ[K] = −0.09 ± 0.23 mM; n = 3). Vehicle had no effect on EDT [K] of mice on either diet. The furosemide‐sensitive Na excretion was significantly greater in mice on LNaHK (1298 ± 354 nmol/min; n = 5) than those on a control diet (362 ± 132 nmol/min; n = 5). Furthermore, single‐channel patch clamp analysis was used to evaluate the activity of ROMK in the apical membrane of split‐open TALs from mice on a control diet or LNaHK. The 70‐pS ROMK channel exhibited a higher open probability (Po) in mice on LNaHK than those on a control diet (Po = 0.65 ± 0.11 vs 0.35 ± 0.13, p = 0.07). However, the density of the 70 pS ROMK was not different in LNaHK compared with control diet. No significant difference was found in the number or Po of the 30‐pS K channels between the two groups. These results indicate ROMK‐dependent net K secretion in the TAL of mice on LNaHK, and it is associated with increased NKCC2 and ROMK activities.Support or Funding InformationThis project is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grants R01‐DK‐092474, R01‐DK‐071014 and F30‐DK‐108456.
Impact of Exercise on Stress Responses and Memory in an Undergraduate Student Population
Authors: Carranco Ana I. , Monthony Justine M. , Mumtaz Shiraz A. , Morton Lindsay C. , Burns Daniel J. , Cohen Brian D. 
Published: November 2023

When the body perceives a stressor, the the hypothalamic‐pituitary‐adrenal (HPA) axis is activated leading to the synthesis and release of cortisol from the adrenal cortex. Exercise is a physical stressor that increases cortisol levels in the body. Studies have considered exercise as a self‐regulatory strategy to adapt to stressful events. People who exercise regularly have higher baseline cortisol levels and condition their body to higher levels of cortisol but lower self‐reported stress. Many studies focus on professional athletes who exercise vigorously and more frequently than college athletes or college students in general. The aim of this study is to examine the relationship between exercise and physiological responses to stress in college students who exercise compared with students who don't exercise on a regular basis. Similarly, the study also aims to investigate if exercise modulates the impact of stress on memory performance.Stress was induced in the undergraduates (with the approval of the Union College Human Subjects in Research Committee) using the Trier Social Stress Test (TSST). Saliva samples were collected and heart rate and blood pressure were measured four times during the study; before the TSST, immediately after the TSST, and then 5 and 30 minutes after the TSST. Salivary cortisol was measured using an enzyme‐linked immunosorbent assay. Salivary amylase was measured using an enzymatic assay as a proxy for activation of the sympathetic nervous system. In addition to physiological measures, participants completed the brief mood introspection scale, the revised undergraduate hassles scale, and a self‐reported exercise survey describing the amount and kind of exercise they perform. They also reported how stressed they felt before and after the TSST. A negative correlation was observed between salivary cortisol concentrations and salivary amylase activity when comparing the pre and immediately post TSST samples. Surprisingly, most participants had a decrease in amylase levels and an increase in cortisol levels. In contrast, participants who reported a higher number of hours of vigorous activity in the last 7 days had a greater increase in amylase and decrease in cortisol compared to participants who reported a lower number of hours of vigorous activity. Together these data indicate the need for specific stress‐reduction programs for college students that include physical activity as one aspect of the intervention.
Central Angiotensin Type 2 Receptor (AT2R) Stimulation Promotes Enhanced Extinction of Fear Learning Independent of Cardiovascular Measures
Authors: Swiercz Adam P. , Wiaderkiewicz Jan P. , Moshfegh Cassandra , Hopkins Lauren , Marvar Paul J. 
Published: November 2023

Angiotensin II receptor subtypes (AT1R and AT2R) are found in regions of the brain critical to the expression and extinction of conditioned fear, however the neurobiological role(s) remain unknown. We propose that brain angiotensin receptors contribute to modulating inhibitory and excitatory conditioned fear circuits, that maybe important in the pathology of posttraumatic stress disorder (PTSD). The objective of this study was to investigate the role of brain AT2R activation in fear memory. To study learned fear, C57BL/6 J mice (n= 6–10/group) underwent classical Pavlovian fear conditioning, pairing auditory cues with foot shocks. The neural circuitry underlying the fear response is highly conserved across mammalian species, making rodent models a valuable tool in the study of fear learning disorders, such as PTSD. The percentage of time spent freezing in response to a conditioned stimulus is used to quantify the ability to learn and remember fearful associations. Using this model, the expression of learned fear is tested 24 hours after conditioning, and memory retention one day later. Following the acquisition of fear, AT2R mRNA expression was elevated in the central amygdala (CeA) (t(22) = 2.5; p<0.05), a critical region involved in the consolidation of fear memory. To further evaluate the functional role of brain AT2R, we administered (intra‐cerebral ventricle ‐ ICV) the highly selective AT2R agonist Compound 21 (C21). A single ICV injection of C21 at either 0.06ug/ul or 0.1ug/ul was administered prior to fear expression testing. Acute C21 (0.1ug/ul) treated mice showed a significant reduction in both fear expression (% freezing) (saline – 62.5% vs C21 ‐ 41.2%) (t(27) = 2.8; p<0.05) and fear memory retention (saline – 52.9% vs C21 – 22.6%) (t(28) = 3.8; p<0.05) following the acquisition of fear. Similarly, mice receiving C21 ICV for 2 weeks showed a trend for a reduction in fear memory retention (saline – 57.2% vs C21 – 39.6%) (t(14) = 1.0; p=0.06 and this was independent of behavioral measures of anxiety as determined by open field testing. Moreover, C21 did not affect blood pressure or heart rate in telemetry‐implanted mice (Saline‐144 ± 3 SBP mmHg, HR 507 ± 13 bpm, n=5; C21 ‐ 136 ± 3 SBP mmHg; HR 518 bpm ± 26, n=4). These data suggest that activation of central AT2 receptors promote the extinction of learned fear. Further studies are required to determine the neurobiological mechanism(s), which may involve changes in cerebral vascular blood flow and/or modulation of neuronal excitability and plasticity.Support or Funding InformationNIH R00 HL107675‐03 AHA 15CSA24340001
Numerical Determination of Wall Shear Stress in Red Blood Cell Membranes
Authors: Cabrales Pedro , Jani Vivek 
Published: November 2023

Red cells in microvessels deform extensively in order to flow, thus resulting in high observed apparent viscosities and high stress concentrations localized to the red cell membrane. Flow through microvessels is consequently aided by the high compliance in the red cell membrane. However, changes in the mechanical properties of Red Blood Cells (RBC) as a result of storage lesions, can increase stress concentrations in the membrane, impairing oxygen transport and resulting in increased vesiculation and likely cell lysis. The objective of this study is to determine the mechanical properties and thus wall shear stress experienced by a RBC membrane during capillary flow. The RBC membrane is modeled as a two dimensional Hookean Isotropic Solid. Videos of capillary flow of both fresh RBCs and RBCs stored for 2 weeks were taken in the rat cremaster muscle. The mechanical properties, including the elastic modulus, shear stress, and shear strain, of both conditions were compared for both groups. In order to determine strain, each RBC membrane will be parameterized by N (X,Y) points determined by the Moore Neighbor Tracing algorithm of videos of capillary flow obtained by intravital microscopy of the rat cremaster muscle, from which N deformation gradient tensors, [F], can be obtained numerically for each frame of the video. From these deformation gradient tensors, the updated Cauchy Strain Tensor will be determined as a function of position on the Cartesian plane relative to the centroid of the RBC. The strains determined from the updated Langrangian Green Strain are numerically fit to the Navier‐Lame Equations along the boundary of the RBC to experimentally fit the two Lame's Constants during capillary flow. These constants will then be used to determine the Cauchy Stress Tensor, using Hooke's Law, along the boundary of the RBC, allowing for the calculation of the Wall Shear Stress. The average Elastic Modulus of fresh cells was found to be 5.4 ± 0.4 × 106 N/m2, while of that of RBCs stored for two weeks was found to be statistically larger and was calculated to be 6.0 ± 1.0 × 106 N/m2. Additionally, stored RBCs experienced higher shear strains and wall shear strains over larger areas of the membrane as compared with stored cells. Additionally, stress concentrators in the membrane were observed at the points of contact with the capillary wall. However, for stored cells, the high stress acted on a larger segment of the membrane. This algorithm for determining wall shear stress experienced by RBC membranes in vivo during capillary flow was thus able to detect statistically significant differences in the both the mechanical properties and stress concentrations between fresh cells and stored cells.Support or Funding InformationThis work was supported by NIH grants from the Heart Lung and Blood Institute, P01‐HL110900, R56‐HL123015, and R01‐HL126945.
Effect of Smoking on Hearing: Evidence from Auditory Evoked Potentials
Authors: Dixit Abhinav , Singh Yogendra Raj , Sharma Praveen 
Published: November 2023

IntroductionAuditory Brainstem Responses (ABR) are an important clinical tool for assessment of hearing. Acetylcholine is a neurotransmitter present in the auditory pathway and nicotine acts through acetylcholine receptors. Since smoking is widely prevalent, it is essential to know its effect on auditory pathways especially for interpreting the reports of ABR in smokers.MethodologyThe study was done on 15 normal, healthy non‐smoker males and 15 healthy, smoker males in the age group of 18–40 years. The study was approved by Institutional Ethics Committee and written informed consent was taken from all volunteers. The recordings of ABR were done using Biopac MP 150. A total of 1000 responses were averaged. The recordings of ABR were done in a state of abstinence of 12 hours (overnight abstinence) and 10 minutes after smoking in the smoker group. The smokers were allowed to smoke their own brand of cigarette. Only one set of recordings were done for non‐smoker group. The peak latencies and amplitudes of the ABR waves were analyzed.ResultsThe latencies of wave I and II along with Inter‐peak latency I–III and III–V were significantly longer in the smoker group (after abstinence) in comparison to the controls. The amplitudes of waves I, II and V were also significantly reduced. A comparison between the controls and smokers (10 minutes after smoking) revealed significantly prolonged latency of waves I and II in the smoker group accompanied by a significant decrease in the amplitudes of all waves of ABR. There were no significant differences in latencies and amplitudes of the smokers after abstinence and 10 minutes after smoking.ConclusionSmoking brings about a decrease in the amplitudes along with prolongation of latencies which is not affected by abstinence or acute consumption.Support or Funding InformationIntramural Grant of All India Institute of Medical Sciences, Jodhpur