bluuebunny/crossref_metadata_2025_split huggingface dataset:
Epidermal Growth Factor Reduces Collecting Duct Water Permeability by Inhibiting Aquaporin-2 Phosphorylation at Ser269
Authors: Chou C-L , Limbutara K. , Kao A.R. , Clark J.Z. , Nein E.H. , Viswanathan R. , Knepper M.A. 
Published: May 2024

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism of this response. Using the Burg isolated perfused tubule technique in microdissected rat inner medullary collecting ducts (IMCDs), we found that addition of 0.1 μM EGF to the peritubular bath significantly decreased 0.1 nM 1‐deamino‐8‐D‐arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on dDAVP-stimulated water permeability was associated with a reduction in intracellular cyclic AMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces dDAVP-stimulated aquaporin-2 (AQP2) phosphorylation at S264 and S269. This effect was absent when membrane-permeable 8-cpt-cAMP (0.1 mM) was used to stimulate AQP2 phosphorylation, suggesting that EGF’s effect was on a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for S269 phosphorylation, we used Bayesian analysis to integrate multiple -omic datasets. Thirteen top ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at S256, S264, and S269. None of the other kinases phosphorylated S269. Additionally, PKA inhibitors, H-89 and PKI, strongly inhibited dDAVP-stimulated AQP2 phosphorylation at S269. These results indicate that EGF decreases the water permeability of the collecting duct by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at S269, a site previously shown to regulate AQP2 endocytosis. This work was funded by NHLBI project ZIA-HL001285 and ZIA-HL006129, M.A.K. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Untangling the Impacts of Ethanol on Myoblast Proliferation
Authors: Levitt Danielle , Helmberger Rylea , DiLeo Matthew 
Published: May 2024

Alcohol-mediated myopathy frequently accompanies chronic alcohol misuse. This decreased skeletal muscle mass and function increases risk for frailty and falls, decreases quality of life, and worsens prognosis for other comorbid conditions. After muscle injury or atrophy, satellite cells are activated as myoblasts, which proliferate, differentiate, and fuse with myofibers to aid in repairing and rebuilding skeletal muscle. While decreased myoblast differentiation with ethanol (EtOH) is well-documented, we have also observed fewer myoblasts with in vitro EtOH after the proliferative phase of myogenesis. The extent of this decrease and whether this is due primarily to increased cell death, decreased cell division, or a combination thereof remains unanswered. Therefore, the objective of this study is to quantify effects of EtOH on myoblast cell division and death during the proliferative phase. C2C12 myoblasts were proliferated in the presence or absence of 50 mM EtOH for 24, 48, and 72 hrs. Nuclei were stained with DAPI to quantify differences in cell count at each time point. Lactate dehydrogenase (LDH) activity was measured in cell culture supernatant at 48 and 72 hrs and normalized to cell count to assess overall cytotoxicity. T-tests were used to determine differences between conditions at each time point. There were no significant differences in cell counts between cells grown with 0 and 50 mM EtOH at 24 or 48 hrs. At 72 hrs, EtOH significantly decreased cell count (p=0.03). There were no significant differences for LDH activity at 48 hrs. At 72 hrs, LDH activity was significantly lower in the 50 mM group (p=0.05). These preliminary results suggest that EtOH decreases cell count in a time-dependent manner. Cytotoxicity appeared lower with EtOH, although this is likely due to lower intracellular LDH activity given our previously observed adverse impacts of EtOH on myoblast glycolytic function. Regardless, the present data do not support increased cytotoxicity with 50 mM EtOH. Next steps will be to assess alterations in myoblast apoptosis and cell division with EtOH. Future work based on the results of these experiments will examine possible nutraceutical supplements and exercise-like stimulation as interventions to rescue alcohol-mediated effects on myoblast proliferation, which may improve subsequent differentiation and therefore regenerative capacity. This work was supported by Texas Tech University (TTU) startup funds and a TTU College of Arts & Sciences Undergraduate Research Experiences grant (DL). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Mitochonic Acid 5 Improves Mitochondrial Respiration Following Alcohol-Induced Mitochondrial Dysfunction
Authors: Donovan Meagan , Mobasheran Parnia , Yang Qinglin 
Published: May 2024

Chronic alcohol exposure can lead to the development of Alcohol Induced Cardiomyopathy (ACM). The pathological changes in ACM have been suggested to be largely caused by mitochondrial damage, reduction of ATP, oxidative stress, and apoptosis of the cardiomyocytes. Cardiomyocytes, as cells with high energy demand, rely greatly on mitochondrial homeostasis and ATP production. Thus, therapeutic intervention to specifically target the mitochondria and ATP production can potentially alleviate ACM. Mitochonic acid 5 (MA-5) has been recently identified as a potential therapeutic compound that increases cellular ATP and improves mitochondrial function in several cardiac disorders. Given the potential role of MA-5 in improving mitochondrial function, I speculate that MA-5 could be an effective treatment for alcohol-induced cardiotoxicity. In the present study, I test the hypothesis that MA-5 can mitigate mitochondrial energetics in cultured cardiomyocytes exposed to alcohol. To investigate this, neonatal mouse cardiomyocytes (NMCMs) were isolated from mice at day 1-2 of age and cultured for 6 days with alcohol (50mM). The cells were treated with MA-5 (1μM, 5μM, or 10μM) for 4 hours with serum starvation and then cellular energetic changes were measured using the Mito Stress assay with the Seahorse Bioanalyzer (XF96pro, Agilent). To investigate the impacts of chronic alcohol exposure, rat embryonic myoblasts, H9C2 cells, were treated with 50mM alcohol for 3 weeks. Subsequently, cells were treated with MA-5 (1μM, 5μM, or 10μM) for 24 hours and then attached to the Resipher system (Lucid Lab) for continuous oxygen consumption monitoring and recording. NMCMs treated with 50mM alcohol showed a substantial reduction in basal respiration, maximal respiration, and spare respiratory capacity compared to controls. NMCMs treated with 10μM MA-5 showed a significant improvement in these parameters. Chronically exposed H9C2 cells showed a prolonged decline in OCR. MA-5 treatment improved and maintained oxygen consumption in these cells. In summary, these results demonstrated that alcohol exposed cardiomyocytes experience a decline in cellular energetics that could be corrected by MA-5 treatment. Future in vivo studies on preclinical ACM animal models are warranted to yield further insights into a potential novel therapy for ACM in patients. This research was supported by grant #T32AA007577 from the NIAAA at the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Nr4a3 Loss Impairs Adipose Mitochondrial Respiration Through Downregulaion of Mek1
Authors: Carter Jared , Tessem Jeffery 
Published: May 2024

Type 2 diabetes mellitus (T2D) is a chronic condition affecting nearly half a billion people worldwide. Symptoms of T2D include impaired glucose tolerance, decreased insulin secretion and significant weight gain. While the symptoms of T2D are well-documented, the underlying pathology remains unclear. Recent research has indicated the critical role of the nuclear receptor Nr4a3 in the development of glucose intolerance and weight gain. In individuals with T2D, the Nr4a3 promoter is hypermethylated, leading to decreased Nr4a3 expression. Elucidating the role of Nr4a3 in mitochondrial respiration in adipose will help define the mechanism of T2D onset and treatment. Here we demonstrate the effect of systemic Nr4a3 loss. Full body Nr4a3 KO animals were fed a standard diet. Male, but not female, Nr4a3 KO mice demonstrate impaired glucose and insulin tolerance. Male Nr4a3 KO mice have normal muscle, liver, and kidney respiration rates, while adipose tissue respiration is impaired. The impaired adipose respiration corresponds with increased mass of all adipose depots, increased adipocyte size, and increased lipid droplet size. The analysis of electron transport chain and tricarboxylic acid complex proteins did not reveal significant differences. Instead, the altered respiration is attributed to a decreased Mek1 protein level, and decreased phosphorylation and activation of its downstream target DRP1. Our data demonstrate the critical role of Nr4a3 in adipose tissue through regulating mitochondrial function. NIH R15DK124835-01A1. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Proteomic analysis of central amygdala systems regulated by the glucocorticoid receptor antagonist mifepristone in the context of alcohol dependence
Authors: Lee Sumin , Romero Laura Monteagudo , Klein Jennifer , Edwards Kimberly , Guidry Jessie , Worthylake David , Edwards Scott 
Published: May 2024

Alcohol use disorder (AUD) is a chronic psychiatric disorder characterized by escalated alcohol use and emergence of negative emotional states during withdrawal. Studies show a dysregulation of glucocorticoid signaling in AUD, affecting the hypothalamic-pituitary-adrenal (HPA) axis and the central amygdala (CeA). Excessive activation of glucocorticoid receptors (GRs) is hypothesized to promote cognitive dysfunction, negative emotional states, and escalated drinking. Previous work demonstrates a functional increase in GR activity in the CeA during alcohol withdrawal in alcohol-dependent animal models. Importantly, the GR antagonist mifepristone reduces alcohol-seeking and drinking behaviors in rodents and humans. The aims of this study were to determine differential CeA protein expression in alcohol-dependent rats and examine the effects of mifepristone treatment. We hypothesized that male and female rats will show sex differences in their CeA protein expression and that alcohol-induced reductions in brain anti-stress signaling (e.g., endocannabinoid system) will be normalized by mifepristone treatment. Male and female Wistar rats were exposed to chronic intermittent ethanol vapor (CIEV) or air (control) for 10 weeks. In week 7, half of each group received subcutaneous placebo pellets while the other half received mifepristone pellets (150mg, 21-day slow release). At the end of Week 10, the rats were sacrificed during acute (6-hour) CIEV withdrawal (and identical time in control groups). The CeA was analyzed using proteomics and ingenuity pathway analysis (IPA). In males, a total of 3050 proteins were detected in the CeA. Of these, 142 proteins were altered by alcohol (but not mifepristone), whereas an additional 253 proteins were altered by alcohol and “normalized” by mifepristone. In females, among the 2632 detected proteins, 46 were affected by alcohol (but not mifepristone), and 25 were altered by alcohol and restored by mifepristone. IPA revealed significant changes in canonical pathways, including the endocannabinoid neuronal synapse pathway and oxidative phosphorylation, affected by alcohol but normalized by mifepristone. Future studies will incorporate alcohol self-administration procedures in male and female rats to functionally examine proteins within these pathways as promising “druggable” targets that offer novel therapeutic avenues for AUD. Supported by NIH-NIAAA R01AA025996, P60AA009803, T32AA007577. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Influence of Individual Characteristics on Critical Environmental Limits in Middle-aged and Older Adults (PSU HEAT Project)
Authors: Leach Olivia , Cottle Rachel , Fisher Kat , Kenney W. Larry , Wolf S. Tony 
Published: May 2024

Critical environmental limits represent the temperature/humidity combinations at which heat balance can no longer be maintained for a given metabolic rate. Individual characteristics (e.g., sex, body weight and size, and aerobic fitness) are known to alter environmental heat exchange as well as thermoregulatory effector responses and therefore may modify critical environmental limits. PURPOSE: To determine which individual characteristics, if any, may be associated with higher or lower critical environmental limits [combinations of ambient dry-bulb temperature (Tdb) and relative humidity (rh)] in middle-aged and older adults. Methods: Forty-eight male (n=16) and female (n=32) participants (63±11 yrs; range: 40-92 yrs) were exposed to progressive heat stress at a low metabolic rate designed to reflect activities of daily living. Experiments were conducted in both hot-dry (HD; up to 53°C; ≤25% rh) and warm-humid (WH; ~35°C; ≥50% rh) environments. After determining critical limits for each age group and condition, forward stepwise multiple linear regression analyses were conducted with net metabolic rate (Mnet) and age entered into the model first, followed by sex, body mass, Vȯ2max, body surface area, and LDL cholesterol. Results: After accounting for Mnet and age, both sex and body mass further significantly improved the regression model in the HD environment (R2adj = 0.41, p < 0.001) whereas only sex (R2adj = 0.51, P < 0.001) added significantly to the WH model. Sex explained 11% of the variance in critical environmental limits in HD conditions and 14% of the variance in WH conditions. In both WH and HD environments, females exhibited lower critical combinations of Tdb and rh than their male counterparts. Additionally, body mass was negatively related to critical environmental limits in the HD environments and accounted for 8% of the variance after metabolic rate and age were accounted for. CONCLUSION: These data indicate a modest influence of individual characteristics, specifically sex and body mass, on critical environmental limits in both WH and HD environments in adults between the ages of 40 and 92. SIGNIFICANCE/NOVELTY: These results contrast previously published data in young adults showing no significant contribution of individual characteristics to the upper limits of heat balance at low metabolic rates reflecting activities of daily living. This reflects the heterogeneity of thermal-balance thresholds associated with aging relative to those seen in young adults. NIH R01 AG067471 (WLK) and NIA Grant T32 AG049676 (RMC). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Adenylate cyclase 6 targets in Mucociliary Clearance
Authors: Arora Kavisha , Arora Yashaswini , Sorensen John , Naren Anjaparavanda 
Published: May 2024

Background. Mucociliary clearance (MCC) is a frontline airway defense mechanism that relies on the appropriate interactions between the epithelium, ciliary beat frequency, and the quantity and quality of mucus. MCC is severely impaired in cystic fibrosis (CF) and drives lung function decline and ultimate respiratory failure and death. A great need remains to identify the key molecular players that regulate MCC in CF outside the scope of FDA approved CFTR therapy and to specifically target MCC dysfunction in CF using small molecules. Results. We discover a novel key role of cAMP-synthesizing enzyme Adenylate cyclase 6 (AC6) in MCC via (a) regulation of CFTR chloride channels in the secretory cells that generate airway surface liquid (ASL) component of MCC and (b) AC6 mediated regulation of ciliary beat frequency in the ciliated cells that is required for the mechanical clearance. Specific activators of AC6 (C20: Top candidate) were developed and improved MCC process ex vivo in CF explant tissues and cells. Together these mechanisms and small molecule approach that improve airway fluid balance and cilia function could be beneficial in CF and other respiratory diseases. Experimental Approach. We performed CFTR functional assessment in normal and CF patient derived airway epithelial cells by adapting patch-sequencing approach and using the specific activators of AC6. The effect of AC6 activators on MCC will be evaluated ex vivo in patient derived lung explant tissues , in vivo (micro-CT) in CF rat models and in vitro (ciliary beat frequency and bead flow measurements) in air-liquid interface airway epithelial cultures. Overall, these studies will be mechanistic and translational involving human disease specimens, animal models, drug development and computational analysis. Significance of the results. The proposed research can improve our understanding of epithelial biology and advance activators of AC6 as therapeutic agents to treat mucus clearance defects. These findings can be leveraged to modulate CFTR function and cilia-generate flow, presenting a potential therapeutic avenue for conditions that involve CFTR and cilia dysregulation associated with several respiratory diseases. National Institutes of Health (NIH) DK080834 and P30-DK117467 and HL147351 (to APN). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Kidney Stem Cell Reprogramming Drives Spontaneous Hypertension via Exosomes
Authors: Han Xiaobin , Meyers Rebecca , Kwon Young In , Zhang Bo , Sun Zhongjie 
Published: May 2024

The spontaneously hypertensive rat (SHR) is a commonly used animal model for studying primary hypertension. However, the precise mechanism of these rats developing hypertension remains to be defined. Here, we have first demonstrated that kidney stem cell reprogramming is a driver for the development of hypertension in SHR rats. In this study, we treated 5-week-old SHR rats with WKY-kidney stem cell-derived exosomes (WKY-Exo) and 5-week-old WKY rats with SHR-Exo, respectively, via i.p. injections. Notably, WKY-Exo effectively blocked the development of hypertension and arterial stiffening in SHRs. It is fascinating that WKY rats developed hypertension and arterial stiffening due to treatment of SHR-Exo. We confirmed that both WKY- and SHR-Exo were up taken into aortic endothelial and smooth muscle cells after i.p. delivery of CD63/GFP-labelled exosomes. We demonstrated that WKY-Exo can improve vascular functions and arterial compliance of SHR rats as evidenced by preventing the development of arterial stiffness and restoring renal artery endothelial function. We also demonstrated that the vascular functions were impaired in WKY rats after SHR-Exo treatment. Furthermore, our data show that expression of Elastin, Emilin1, and Myh11 was significantly downregulated in the aorta of SHR rats, which can be repaired by WKY-Exo treatment. SHR-Exo decreased expression of Elastin and Emilin1 without affecting Myh11 in the aorta of WKY rats, suggesting that Elastin and Emilin1 deficiency could contribute to arterial stiffening leading to the development of spontaneous hypertension. Proteomic analysis reveals the enrichment of protein-protein interaction network for elastic fiber assembling, blood vessel development, and regulation of angiogenesis in WKY-Exo vs SHR-Exo. In conclusion, our data first demonstrates that kidney stem cell reprograming is a driver for the development of spontaneous hypertension in SHR rats. WKY-KSCs-derived exosome contains a proteome profile promoting arterial repair, which may provide a therapeutic means to prevent and treat primary hypertension. NIH. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.