bluuebunny/crossref_metadata_2025 huggingface dataset:
NCC regulation by WNK/SPAK pathway is influenced by alkalosis
Authors: Al-Qusairi Lama , Shin Ella , Welling Paul
Published: May 2023

The WNK/SPAK “potassium-switch” pathway provides a homeostatic mechanism that activates the sodium chloride cotransporter, NCC, to limit urinary potassium excretion in dietary potassium deficiency at the expense of increasing salt-sensitivity and blood pressure. Although it is well understood that high dietary potassium turns off the switch, and decreases NCC activity, several reports suggest the counter anion in potassium-rich diets may have an influence on the pathway. The goal of the present study was to determine if the chronic response to a high K diet depends on the counter anion. Wild-type C57BL6J mice were randomized to control (1%K+), potassium chloride (KCl: 5%K+), or potassium bicarbonate (KHCO3: 5%K+) diets, and physiological, molecular, and imaging analyses were performed at 4 and 10 days of diet consumption. In contrast to the classic response to high KCl diet, characterized by reduced NCC phosphorylation and membrane localization, NCC phosphorylation and apical localization increased after 10 days on the high KHCO3 diet. This response was paralleled by increased phosphorylation of the downstream potassium-switch kinase, SPAK, which in turn phosphorylates NCC. Immunofluorescence imaging revealed WNK4 bodies were more numerous and larger in the high KHCO3 group compared to the control, consistent with activation of upstream WNK signaling. Urine excretion studies revealed mice on the high KHCO3 retain more Na+ than mice on control or KCl diets, paralleling enhanced NCC activation. Unlike mice on the high KCl diet, mice on the high KHCO3 diet develop and maintain metabolic alkalosis and do not exhibit profound hyperkalemia, coincident with more robust activation of ENaC and ROMK than with the high KCl diet. In summary, these data identify an unexpected NCC activation mechanism that may limit distal sodium delivery to prevent excessive potassium losses when alkalosis super activates potassium secretion in more distal segments.

NIDDK, LeDucq Foundation

This is the full abstract presented at the American Physiology Summit 2023 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.
Sex-specific differences in the circulatory response to postural stress among young adults with adverse childhood experiences (ACEs)
Authors: Iannarelli Nathan , Wade Terrance , Dempster Kylie , O'Leary Deborah
Published: May 2023

Adverse childhood experiences (ACEs)—a highly prevalent assortment of traumatic childhood events (e.g., abuse)—are strongly linked to adult cardiovascular disease (CVD), though differently among males and females. Biological sex differences in the circulatory response to physiological stress (e.g., postural stress) have been well-characterized; however, whether ACEs affect this response differently among males and females remains unclear. This study tested the hypothesis that ACEs affect the circulatory response to head-up tilt (HUT; postural stress) differently among males and females. Three hundred ( n = 156 female; 22.4 ± 1.8 years) young adults recruited through the Niagara Longitudinal Heart Study (NLHS) were included in the current analyses. ACEs were assessed (Childhood Trust Events Survey 2.0) and participants were dichotomized into high (≥4 ACEs) and low (<4 ACEs) exposure groups. Beat-by-beat mean arterial pressure (MAP; finger photoplethysmography) and heart rate (HR; electrocardiogram) were recorded during 10-minutes of supine rest and 5-minutes of 60° HUT. Sex-stratified, mixed-model regression analyses assessed the effect of ACEs on changes (Δ) in MAP and HR from supine rest to HUT, adjusting for age, body mass index, physical activity, and smoking status. During HUT, MAP increased to a similar extent in males with high and low ACEs exposure (ΔMAP: 11.5 ± 10.8 mmHg vs. 10.1 ± 10.9 mmHg, respectively; p > 0.05). In contrast, MAP increased to a greater extent in females with high ACEs exposure compared to females with low ACEs exposure (ΔMAP: 11.3 ± 10.4 mmHg vs. 5.9 ± 9.8 mmHg, respectively; p = 0.003). HR increased during HUT in males with both high and low ACEs exposure; however, it increased to a lesser extent in males with high ACEs exposure compared to males with low ACEs exposure (ΔHR: 14.7 ± 7.5 bpm vs. 18.7 ± 8.1 bpm, respectively; p = 0.015). In females, HR increased to a similar extent during HUT in those with high and low ACEs exposure (ΔHR: 16.5 ± 8.1 bpm vs. 17.6 ± 8.9 bpm, respectively; p > 0.05). These data suggest that ACEs affect the circulatory response to postural stress differently in males and females. Further, these data imply that sex-specific differences in circulatory regulation can be observed as early as young adulthood in those reporting high ACEs exposure.

This work was supported by the Canadian Institute of Health Research (CIHR) and Brock University

This is the full abstract presented at the American Physiology Summit 2023 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.
Histopathological and Molecular Characteristics of Breast Cancer Patients in Northern Egypt
Authors: Talaat Iman , Soliman Nada , Elnahas Salwa , Elemam Noha
Published: May 2023

Background: In 2020, breast cancer (BC) became the most common cancer worldwide, including Egypt. Despite the extensive research throughout the past years, a detailed analysis of the patients, including the molecular and histopathological characteristics, is needed in order to tailor effective screening and treatment strategies for a particular population of BC patients. In this study, we aimed to explore the various characteristics and survival of a large cohort of BC patients in Northern Egypt. Materials and Methods: The recruited cohort in this study was composed of more than 4,000 BC patients who were managed in different centers across Northern Egypt for 10 years. Various clinical, demographic, and histopathological parameters were retrieved from the records. This included tumor size, grade, and stage, as well as the histological and molecular subtypes. Also, therapeutic modalities and responses, as well as disease-free survival (DFS, no recurrence or metastasis) and overall survival rates (OS, alive/dead), were collected. The SPSS 26.0 software was used for statistical analysis, and p<0.05 was considered statistically significant. Results: Invasive ductal carcinoma was found to be the most common histological type in the recruited cohort (94%). Interestingly, the median age of the patients’ cohort was 50 years, with most of the cohort presenting before the age of 55 years. Around 50% of the patients presented with advanced stage III. Further, the classification of the BC patients according to the Nottingham grading system revealed that the majority were graded as well and moderately differentiated BC (grades 1 and 2). Moreover, 77% of the patients belonged to the hormonal subtypes with positive estrogen (ER) and/or progesterone (PR) receptors. Intriguingly, 45.5% of BC patients were classified as luminal B subtype, 31.8% as luminal A group, 7.6% of BC patients as HER2-enriched subtype, and around 15% were triple-negative subtype. Such stratification by molecular subtyping of BC patients showed a significant difference in both the DFS and OS of the patients. Conclusion: Our findings highlight the different clinical, demographic, and histopathological characteristics of BC patients in Northern Egypt that might be different from other populations. Further studies are needed to identify early detection programs and management strategies for each population in order to improve BC patients’ clinical outcomes.

No funding.

This is the full abstract presented at the American Physiology Summit 2023 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.
Cardiomyocyte beta2AR mediates cardiac fibrosis via maintaining Ca2+ homeostasis in the heart
Authors: Zhu Chaoqun , Zhao Meimi , Xu Bing , Xiang Yang
Published: May 2023

Objective: Cardiac beta2-adrenoceptor (β2AR) has been suggested functions a protective role in defending myocyte apoptosis and cardiac damage induced by various stress. Our recent studies indicate divergent roles of cardiac β2AR in cardiomyocytes and myofibroblasts in regulating high fat diet (HFD) induced cardiac remodeling and dysfunction. While β2AR in cardiomyocytes attenuates HFD-induced cardiac fibrosis, myofibroblast β2AR facilitates cardiac fibrosis. Cell-specific actions of β2AR has been suggested in mediating cardiac fibrosis in the heart. This study is aimed to further determine the role of cardiomyocyte-specific β2AR in cardiac function and cardiac fibrosis. Hypothesis: Cardiomyocyte-specific β2AR regulates cardiac fibrosis via maintaining Ca2+ homeostasis in the heart. Results and Methods: 2-3-month-old mice with cardiomyocyte-specific deletion of β2AR (β2AR-cKO) and β2AR-flox mice were used. While echocardiography showed a comparable cardiac function between cKO and flox mice, the expression of both inflammatory and fibrotic marker proteins, NLR family pyrin domain containing 3 (NLRP3) and connective tissue growth factor (CTGF), was elevated in cKO hearts. As β2AR has been suggested to restrict excessive adrenergic insults, the activities of major molecules in β-adrenergic signaling were examined. Notably, western blot showed the phosphorylation level of Calcium/calmodulin-dependent kinase II (CaMKII) was significantly upregulated in cKO heart which implies a higher level of cytosolic Ca2+. Consequently, the CaMKII phosphorylation site (serine 1700) on L-type Ca2+ channel (LTCC) was also upregulated. To further examine the LTCC activity, whole cell currents (ICa) were recorded from freshly isolated ventricular myocytes, which indicated a higher LTCC activity after isoproterenol stimulation (peak ICa: cKO, -12.87 vs. flox, -10.40, p<0.05). Moreover, cKO and flox were subjected to LTCC blocker (nifedipine,10mg/kg/day) treatment to confirm the effect of Ca2+ activity on cardiac inflammatory and fibrotic response. Two weeks of nifedipine injection completely attenuated over-elevated CaMKII activity and NLRP3 and CTGF expression in cKO hearts. Overall, these results indicate that deletion of β2AR in cardiomyocytes facilitates cardiac fibrosis by altering cytosolic Ca2+ homeostasis via LTCC activity. Conclusion: This study elucidates that cardiomyocyte β2AR is essential to maintain normal cardiac function and plays a protective role against the development of cardiac fibrosis.

National Institutes of Health grants R01-HL147263, Veteran Affair Merit grant 01BX002900, American Heart Association postdoctoral fellowship

This is the full abstract presented at the American Physiology Summit 2023 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.
Small molecule activator of Serca2a improves myocardial bioenergetics and lipid metabolism in diabetic mice
Authors: Majid Abdul , Kang Soojeong , Hammoudi Nadjib , Lebeche Djamel
Published: May 2023

Background: Altered calcium homeostasis, insulin resistance, and dysregulated energy metabolism are major causes of diabetic cardiomyopathy. The expression and activity of Sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) are decreased in the diabetic heart failure. Increasing SERCA2a function reverses both systolic and diastolic dysfunction. However, the mechanisms and consequences underlying the role of SERCA2a in the diabetic failing heart are not clear yet. Objective: To examine the effect of pharmacological activation of SERCA2a on the diabetic heart. Methods and Results: To restore calcium abnormality in leptin-deficient (ob/ob) diabetic mice, we employed and contrasted two different models to elevate the restoration of SERCA2a function: 1) activation of SERCA2 with a novel allosteric agonist, CDN1163 or 2) overexpression of SERCA2a using cardiac-specific AAV9-mediated gene transfer. We demonstrate that SERCA2a activation with CDN1163 or AAV9 repressed the expression of genes involved in lipid synthesis and fatty acid oxidation in obese mice, leading to improved lipid homeostasis. Increased SERCA2a function improved mitochondrial function through elevation of mitochondrial biogenesis, mitochondrial DNA content, oxidative phosphorylation, and ATPase activity in the heart of obese mice. SERCA2a also restored mitochondrial membrane potential to normal levels in cultured H9C2 myocytes in the presence of high glucose and H2O2. SERCA2a ameliorated intracellular lipid toxicity and cell damage, possibly through the upregulation of the gene expression of antioxidant enzymes, and attenuated ROS generation by repression of NADPH oxidase in the obese heart. Furthermore, these molecular changes were paralleled by improvement in diabetes-induced diastolic dysfunction following CDN1163 activation of SERCA2a or AAV9-SERCA2a overexpression in the obese mice. Conclusion: These results suggest that calcium resolving diabetes-induced calcium dyshomeostasis by pharmacological activation of SERCA2a in obese individuals may be a highly effective therapeutic modality for treating obesity-induced diabetes.

This is the full abstract presented at the American Physiology Summit 2023 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.
Colonic butyrate lowers hepatic glucose production via FFAR2-dependant GLP-1 receptor neuronal signaling
Authors: Wachsmuth Hallie , Lane Adelina , Meyer Rachel , Weninger Savanna , Duca Frank
Published: May 2023

Hyperglycemia is a hallmark characteristic of type II diabetes, due in part to increased hepatic glucose production (HGP). The gut microbiota is now recognized as an important contributor to host energy and glucose homeostasis, and our lab has demonstrated that the gut microbiota can impact HGP. One mechanism that the gut microbiome may be impact HGP is through alterations in gut-derived metabolites from breakdown of ingested foodstuff. Specifically, short chain fatty acids (SCFAs) are bacterial metabolites produced primarily in the distal intestine from dietary fibers, and when administered exogenously, SCFAs improve glucose tolerance. However, the mechanisms underlying the effects of SCFAs on glucose tolerance are not fully understood. Given that SCFAs bind to free fatty acid receptors (FFARs) 2 and 3 localized on enteroendocrine cells to induce gut peptide release, and gut peptides regulate HGP via a gut-brain-liver neuronal axis, we propose colonic SCFAs activate a neuronal signaling mechanism to lower HGP mediated via gut peptide signaling. We infused SCFAs into the colon (50min at 0.01mL/min) during a basal insulin euglycemic clamp in rats. Compared with saline infusion, colonic administration of 10mM butyrate increased the glucose infusion rate needed to maintain glycemia due to a reduction in HGP. This was associated with increased portal concentrations of GLP-1 following butyrate infusion compared to saline. We investigated the role of colonic FFAR2 signaling, which is known to induce release of GLP-1, through either co-administration of a FFAR2 antagonist with butyrate or by colon-specific FFAR2 lentiviral knockdown; both abolished the HGP-lowering effect. Next, co-administering a GLP-1 receptor antagonist with 10mM butyrate during a clamp abolished the butyrate-induced reduction of HGP. Additionally, hepatic or celiac branch vagotomy abolished the HGP-lowering effect of colonic butyrate, suggesting a celiac afferent and hepatic efferent neuronal signaling pathway. Together, these studies uncover a FFAR2, GLP-1 receptor, vagal-dependent signaling mechanism by which colonic butyrate lowers HGP.

Research presented was supported under NIH award R01DK121804.

This is the full abstract presented at the American Physiology Summit 2023 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.
High-phosphate diet disrupts ACE/ACE2 balance in mouse kidney
Authors: Elased Dalia , Branch Daniel , Oh Jooho , Pepperney Luke , Elased Khalid , Baskin Kedryn
Published: May 2023

Phosphate binders are a common treatment in dialysis patients and people with chronic kidney disease (CKD) to prevent phosphorous accumulation in the blood. The physiological mechanism behind the relation between excess phosphate and renal damage is largely unknown. The renin-angiotensin-aldosterone system (RAAS) is a hormone regulating system that controls blood pressure and electrolyte balance in cardiovascular organs and dysregulation of RAAS is often an indicator of organ damage. Angiotensin-converting enzyme 2 (ACE2) and Neprilysin (NEP) are both key players in the RAAS due to their role in producing the renoprotective peptide Angiotensin-(1-7). Ang-(1-7) plays a pivotal role in kidney protection due to its anti-inflammatory and vasodilatory effects on various cardiovascular organs including the kidney. Angiotensin-converting enzyme (ACE) is another key enzyme in RAAS which produces opposite effects to those of ACE2. ACE induces the formation of the vasoconstrictor Angiotensin II which causes hypertension and renal fibrosis. To test the hypothesis that a high phosphate diet affects ACE2, NEP, and ACE levels in kidneys, we fed mice high-Pi diet (HP) or normal-Pi diet (NP) for 22 weeks. Serum phosphate levels were increased by the HP diet while body weight and body composition were unaffected. Fluorogenic substrate-based assay and western blot analysis were used to evaluate enzyme activity and protein expression. In mice fed the HP diet, there was a significant downregulation of renal ACE2 activity and expression ( p<0.01), and upregulation of renal ACE expression (p<0.01). NEP activity and protein expression were unchanged. Our data demonstrates that the link between excess dietary Pi and kidney damage could be due to an increased ACE/ACE2 ratio. Future studies will investigate the effect of a high phosphate diet on other key players in the RAAS including Angiotensin II and Angiotensin-(1-7).

This is the full abstract presented at the American Physiology Summit 2023 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.
Meso- and nanoscale structural changes in the hippocampus of control and transgenic Alzheimer's mice exposed to Intermittent Hypoxia
Authors: Sampathkumar Vandana , Polo Ashleigh , Piao Lin , Crowley Lorenzo , Gan Haotian , Dodiya Hemraj , Sisodia Sangram , Sharp Willard , Garcia Alfredo , Kasthuri Narayanan
Published: May 2023

Sleep apnea, a clinical condition characterized by repetitive pauses in breathing that cause intermittent hypoxia (IH), affects a billion people worldwide, increases the risk for mild cognitive impairment, and is estimated to occur in up to 50% of individuals with Alzheimer’s Disease (AD). Previous work in wildtype mice has shown that IH impairs spatial memory that is associated with deficits in NMDAr-dependent synaptic physiology. However, a significant knowledge gap remains in understanding how IH may impact ultrastructural properties in the brain. Using large volume serial electron microscopy (EM), we studied mitochondrial abnormalities and synaptic density changes in the area CA1 of hippocampus of non-transgenic mice and CeAPP/PS1 mice, a mouse model of Aß amyloidosis, exposed to ten days of IH. Immediately after IH, mitochondria became elongated and appeared to undergo fusion (becoming 4x to 8x longer) in pyramidal CA1 neurons. This change in mitochondrial morphology reversed, e.g. mitochondrial length normalized relative to controls, when the IH-exposed mice were allowed to recover in room air for 10 days post-IH. We also found a 20-40% increase in synaptic density in proximal and distal dendrites of IH-exposed pyramidal CA1 neurons, yet the ratio of multi-synaptic boutons to single synaptic boutons in area CA1 did not appear to change. These observations indicate that changes in synaptic density occur without producing significant changes in axonal innervation patterns. Finally, using synchrotron source X-ray microscopy, and automated serial EM in IH-exposed brain tissue from CeAPP/PS1 transgenic mice, we documented an increased amyloid plaque number in both cortex (two-fold increase) and hippocampus (ten-fold increase). Large volume EM showed similarly elongated mitochondrial morphology changes in hippocampal neurons in CeAPP/PS1 mice when exposed to IH. These findings suggest that IH, such as that experienced with untreated sleep apnea, may drive ultrastructural changes that both contribute to mild cognitive impairment and promote the progression of AD and related dementias.

NIH: R01NS107421; R01HL163965; R01DA057767.

This is the full abstract presented at the American Physiology Summit 2023 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.
Characterization of Mitochondria from Primary Isolated Mouse Aortic Smooth Muscle Cells
Authors: Sakamuri Anil , Ogola Benard
Published: May 2023

Sexual dimorphism is demonstrated in cardiovascular disease (CVD). Vascular dysfunction is a critical player in CVD involving mitochondrial respiration in aortic smooth muscle cells. In this study, we characterized contractile and synthetic gene expression, and mitochondrial respiration by sex in aortic smooth muscle cells (ASMC). We hypothesize that female than male ASMC have increased contractile genes and better mitochondrial energetics. Adult male and female C57BL6J mice were euthanized, and thoracic aorta excised. Aortic tissue sections were subject to enzymatic digestion and cultured for subsequent experiments. The 2-delta delta CT method using beta actin as housekeeping gene was used for analysis. For mitochondrial respiration, confluent (70%) cells were trypsinized, enumerated, and seeded in Seahorse mini-culture plates (20,000 cells/well). After overnight incubation mitochondrial stress test was performed to measure basal respiration, maximal respiration, spare capacity, ATP production, proton leak, and non-mitochondrial respiration. All mitochondrial respiratory parameters were expressed as oxygen consumption rate normalized with protein levels (OCR, picomoles/L of O2 per microgram protein, n=8-10 wells/group). Unpaired t-test was used for statistical analysis and P<0.05 was considered significant. To characterize the sex of isolated cells (n=6-8/group), we used sex specific primers that showed significantly higher Sry (P<0.001) and Xist (P<0.001) gene expression of male and female ASMC, respectively. Next, we assessed contractile and synthetic genes including myocardin, alpha actin, and myosin heavy chain 11 that were higher in female than male ASMC (P<0.05). However, myosin heavy chain 10 was higher (P=0.002) in male than female cells. The basal respiration and ATP production were significantly high in female ASMC by 79% and 77%, respectively, when compared with male ASMC (2.48 ± 0.13 vs. 1.38 ± 0.15, P= 0.0001; 2.11 ± 0.11 vs. 1.19 ± 0.11, P= 0.0001). Female cells indicated increased spare respiratory capacity compared to male ASMC (1.67 ± 0.31 vs. 1.1 ± 0.23, p= 0.01). The maximal respiration and proton leak were significantly higher in female than male cells by 69% and 109% (4.21 ± 0.33 vs. 2.49 ± 0.31, P= 0.001: 0.42 ± 0.02 vs. 0.2 ± 0.04, P= 0.001). Female and male cells did not show any significant difference in non-mitochondrial respiration. Based on these results, female than male ASMC exhibit increased contractile gene expression and better mitochondrial energetics at basal and stressed conditions. The sex-specific differences in mitochondrial function may attribute to sex bias in CVD.

R00HL155841 and R25HL105400

This is the full abstract presented at the American Physiology Summit 2023 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.
The clock gene Bmal1 inhibits kidney expression of SGLT2 and glucose reabsorption via the NR1d1/NRF1 pathway
Authors: Pan Xiaoyue , Mowdawalla Cyrus , Mota Samantha , Hussain M. Mahmood , Pessin Jeffrey , Vallon Volker
Published: May 2023

Background & Aims: The nuclear receptor subfamily 1, group D, member 1 (Nr1d1, also called Rev-erbα) is highly expressed in the kidney, and its transcriptional activity is increased by the clock gene Bmal1 (ARNTL or MOP3). Here we studied the roles and functional interactions of Bmal1 and Nr1d1 in mouse kidney, particularly in regulating the expression of the glucose transporter SGLT2 (SLC5A2) and glucose reabsorption. Methods: We performed histologic and biochemical analyses of kidney tissues from male C57BL/6 and Bmal1-knockout mice and performed gene expression analyses to identify genes regulated by Bmal1. The effects of fasting and refeeding on renal expression of Sglt2 was examined in male C57BL/6, Bmal1-knockout, and Nr1d1-knockout mice. Some mice were given hemin to stimulate Nr1d1 gene expression (daily for 1 week) at 12:00 with 100 μg/kg/day; renal glucose reabsorption, and glucose and fatty acid levels were measured. Renal proximal tubule epithelial cells (RPTECs) were generated from C57BL/6 and Bmal1-knockout mice and glucose uptake determined. Luciferase reporter assays were performed using HK2 cell. Results: We found that kidney SGLT2 mRNA and protein expression and glucose absorption were increased in Bmal1-knockout mice compared with C57BL/6 mice. Expression of SGLT2 was affected by fasting and refeeding in C57BL/6 mice, but not in Bmal1-knockout or Nr1d1-knockout mice. Bmal1-knockout mice also presented with higher levels of renal fatty acids compared with C57BL/6 mice. Hemin injection reduced expression of SGLT2, decreased glucose re-absorption in RPTECs, and increased levels of fatty acid in kidney; these changes were not observed in Bmal1-knockout mice. Nuclear respiratory factor 1 (NRF1), which regulates glucose, activated transcription of Sglt2. Fasting caused MAPK signing pathway to phosphorylate Bmal1, which activated NR1d1 and inhibited NRF1 activity and thereby reduced Sglt2 expression. Conclusions: The data indicate that fasting activates Bmal1 and Nr1d1, which inhibits NRF1 and leads to repression of renal SGLT2 expression and glucose reabsorption. This is the first study that mechanistically links food intake to the regulation of kidney glucose transport through a circadian clock gene.

NIH National Heart, Lung, and Blood Institute grant R56 HL137912-01, and American Heart Association grant-in-aid 16GRNT30960027 to PX; NIH R01DK112042 to VV

This is the full abstract presented at the American Physiology Summit 2023 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.
TRPC6 deficiency attenuates renal injury and mitochondrial dysfunction in ischemia/reperfusion-induced acute kidney injury
Authors: Wang Zhen , Fu Yiling , Dai Xuemei , do Carmo Jussara , da Silva Alexandre , Mouton Alan , Li Xuan , Omoto Ana , Hall John
Published: May 2023

Ischemia/reperfusion (I/R)-induced acute kidney injury (AKI) is a major clinical problem without effective therapy and is a significant cause of morbidity and mortality during the perioperative period. The hallmarks of I/R-AKI include renal tubular cell injury and mitochondrial dysfunction. Transient receptor potential channel subfamily C member 6 (TRPC6), a non-selective cation channel that mediates the influx of calcium and other monovalent cations into cells, is widely expressed in the kidney, including proximal tubular cells. TRPC6 deficiency has been reported to be protective in chronic kidney diseases by reducing oxidative stress; however, its role in AKI is still controversial. In this study, we examined the role of TRPC6 during the early phase of renal injury in I/R-induced AKI. To reduce the severity and limit variability in kidney injury after I/R, we performed unilateral I/R surgery with 30 minutes of ischemia in the left kidney of 22-week-old male wild-type (WT) and whole-body TRPC6 knockout (TRPC6 KO) mice. Mitochondrial respiration rate and mitochondria-generated superoxide in the left kidney were examined 18 hours after I/R surgery by OROBOROS Oxygraph Respirometry. Serum creatinine and urinary neutrophil gelatinase-associated lipocalin (NGAL) concentration were determined in addition to histological evaluation by PAS staining in WT and TRPC6 KO mice. Our results show that the mitochondrial oxygen consumption rate was significantly higher in I/R kidneys from TRPC6 KO mice compared to WT (56.5±9.3 vs. 22.9±2.0 pmol/min/mg, n=5-6). Despite higher mitochondrial respiration, mitochondria-generated superoxide production was significantly lower in the kidney of TRPC6 KO mice. TRPC6 KO mice also exhibited significantly lower serum creatinine (0.26±0.02 vs. 0.37±0.08 mg/dL, n=7-10) and urinary NGAL levels (526.6±58.7 vs. 1313.5±192.0 ng/mL, n=6-10) than WT mice. Renal histology showed that I/R kidneys from TRPC6 KO mice have reduced tubular cell death in outer medulla areas and decreased tubular clots and inflammatory cell infiltration. These observations suggest that TRPC6 deficiency may be protective during the early phase of I/R-induced AKI by preserving mitochondrial respiration and attenuating mitochondria reactive oxygen species generation. Targeting TRPC6 and its downstream signaling pathways may provide new avenues for future I/R-induced AKI therapies.

(NIDDK R00DK113280 and R01DK121411, NIGMS P20GM104357 and NIGMS U54GM115428)

This is the full abstract presented at the American Physiology Summit 2023 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.
Role of Nrf2 in myocardial infarction and ischemia-reperfusion injury
Authors: Zuberi Sahar , Rafi Hira , Hussain Azhar , Hashmi Satwat
Published: May 2023

Background: Myocardial infarction (MI) is a leading cause of morbidity and mortality in the world and is characterized by ischemic necrosis of an area of the myocardium permanently devoid of blood supply. With advancements in therapeutics, thrombolytic agents and primary percutaneous coronary intervention (PCI) can now be employed to reperfuse the ischemic area. Reperfusion, in itself generates reactive oxygen species and this causes further insult to the myocardium, thereby resulting in ischemia-reperfusion (IR) injury. Since Nrf2 is a key regulator of redox balance, it essential to determine its contribution in these two disease processes. Conventionally Nrf2 levels have been shown to rise immediately after ischemia and reperfusion but its contribution to disease process in the days that follow remains uncertain. Objective: This study intended to demonstrate the difference in Nrf2 levels one week after myocardial infarction and ischemia-reperfusion injury, respectively. Methods: Mice were divided into MI, IR injury and sham surgery group and were sacrificed 1 week after surgery. In MI model, left anterior descending artery was ligated permanently while in IR model, it was temporarily ligated for 30mins and reperfused. Infarct size was estimated using trichrome staining and expression of Nrf2 was assessed using immunohistochemistry, Western blot and ELISA. Results: Infarct size was the highest in MI group, followed by IR (MI: 31.02 ± 1.45%, IR: 13.03 ± 2.57%; p < 0.01). We observed a significantly higher expression of Nrf2 in MI group compared to IR model using immunohistochemistry, spot densitometry of Western blot (MI: 2.13 ± 0.22, IR: 1.69 ± 0.25; p = 0.039) and ELISA (MI: 80.78 ± 27.08, IR: 31.97 ± 4.35; p < 0.01). Conclusion: There is a significantly higher expression of Nrf2 in MI compared to IR injury group. Modulation of Nrf2 could be a potential target for therapeutics in the future, and its role in cardiprotection can be further investigated.

Aga Khan University University Research Council Funding

This is the full abstract presented at the American Physiology Summit 2023 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.
Therapeutic Potential of Extracellular Vesicles to Restore Alcohol-mediated Impairment of Myoblast Differentiation
Authors: Simon Liz , Bourgeois Brianna , Molina Patricia
Published: May 2023

At-risk alcohol use in people living with HIV (PLWH) and chronic binge alcohol (CBA) administration in simian immunodeficiency virus (SIV)-infected macaques are associated with dysfunctional skeletal muscle mass. Our studies have shown that decreased myomiR-206 expression in skeletal muscle and myoblasts isolated from CBA/SIV macaques contributes to decreased myoblast differentiation. miRs transported in extracellular vesicles (EVs) mediate numerous cellular responses through intercellular communication. This study tested the hypothesis that delivery of miR-206 in EVs would ameliorate CBA-mediated decreased myoblast differentiation. Eight female rhesus macaques received either (CBA, 2.5g/kg/day) or water (VEH) for 14.5 months. Three months following the initiation of CBA/VEH, animals were infected with SIVmac251 and initiated on antiretroviral therapy 2.5 months later. Myoblasts were isolated from the vastus lateralis muscle at study endpoint (blood alcohol concentration= 0 mM) from each animal and used for ex vivo experiments including measuring differentiation, transfecting with a miR-206 mimic, and isolating EVs from myotube culture supernatant via ultracentrifugation. Myoblast differentiation measured by fusion index was lower in CBA myotubes compared to VEH (p<0.05). CBA decreased myotube-derived EV miR-206 expression (p<0.01). Transfection of myoblasts isolated from the CBA group with a mir-206 mimic increased myotube fusion index (p<0.05). Moreover, delivery of exogenous miR-206 in plasma-derived EVs increased myotube expression of miR-206 by over 450-fold (p<0.001) and significantly improved myoblast differentiation as measured by fusion index and myotube density (p<0.05). These results provide evidence that delivering miR-206 through EVs can increase CBA-mediated SKM stem cell differentiation. We propose these findings support the possible use of autologous plasma EVs as therapeutical vehicles devoid of the known adverse effects of synthetic cargo nanocarriers.

Supported by: NIH/NIAAA P60AA009803, F30AA029358, K01AA024494.

This is the full abstract presented at the American Physiology Summit 2023 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.
Gestodene, a Positive Allosteric Modulator of PAR1, Enhances PAR1-Mediated Morphological Changes in MEG-01 Cells
Authors: Park So-Hyeon , Heo Yunkyung , Lee Yechan , Jeon Hyejin , Namkung Wan
Published: May 2023

Protease-activated receptor 1 (PAR1) is a high-affinity thrombin receptor expressed in human platelets and can be activated by low levels of thrombin. Selective inhibition of PAR1 by vorapaxar significantly inhibits thrombin-induced calcium mobilization in human platelets and increases the risk of bleeding, suggesting that positive allosteric modulator (PAM) of PAR1 may increase the risk of thrombosis. In the present study, we performed a cell-based screening to identify novel PAMs of PAR1 and found gestodene, which acts as PAM of PAR1. Gestodene enhanced both thrombin- and PAR1-AP-induced increases in intracellular calcium levels through PAR1 in a dose-dependent manner, but it did not alter the activity of PAR2 and PAR4. Gestodene increased PAR1-AP-induced internalization of PAR1 receptors and phosphorylation of ERK1/2, and the enhancing effects were significantly blocked by vorapaxar. Furthermore, gestodene potently increased PAR1-AP-induced morphological changes in the human megakaryoblastic leukemia cell line MEG-01 cells, a cellular model of PAR1-mediated morphological changes in platelets. Our results reveal that gestodene is a selective PAM of PAR1 and provide a molecular basis for the risk of venous thromboembolism induced by oral contraceptives including gestodene.

This research was supported by a Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2018R1A6A1A03023718).

This is the full abstract presented at the American Physiology Summit 2023 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.
Alveolar macrophages undergo METosis in an inflammatory model of neonatal lung injury
Authors: Higazy Randa , Mohammadi Atefeh , Ivanovska Julijana , Tran Harvard , Gauda Estelle , Palaniyar Nades
Published: May 2023

Background: Activated macrophages can produce macrophage extracellular traps (METs) - DNA strands coated with cytotoxic components that are released to immobilize pathogens and control infection. However, METs can also contribute to the inflammatory process and lead to severe tissue damage. Bronchopulmonary dysplasia (BPD) is a chronic lung disease in premature infants initiated by inflammation and oxidative stress. Gram-negative bacteria and candida increase the risk of developing BPD and are strong inducers of extracellular traps. Macrophages are the most abundant immune cells in the neonatal lung tissue, responsible for controlling the inflammatory process. We aimed to determine whether inflammation triggers METosis in a neonatal model of inflammatory lung injury. Methods: Alveolar macrophages (AMs) from bronchoalveolar lavage fluid (BALF) were isolated from Sprague Dawley rat pups on postnatal day (PND) 8. In vitro - AMs were incubated with METosis agonists (lipopolysaccharide [LPS] and phorbol myristate acetate [PMA]) or control (media) for 4h. Cells were fixed and stained for markers of METosis (myeloperoxidase [MPO] and citrullinated histone H3 [CitH3]) and DNA (DAPI) for immunofluorescence imaging. In vivo - PND6-9 pups were treated with LPS or saline intrapharyngeally and DNase enzyme or saline intraperitoneally. BALF was collected on PND10. BALF was analysed using immunofluoresence imaging and picogreen assay. Results: Immunofluorescence imaging of neonate AMs showed increased expression of METosis markers MPO, CitH3, and DNA strands with in vitro exposure to METosis agonists PMA or LPS compared to control (n =3). In vivo LPS exposure also showed evidence of increased extracellular trap formation in the BALF collected from neonate rat lung compared to control (n=3; p<0.0001); further, rats exposed to both LPS and DNAse enzyme showed decreased extracellular trap formation (p = 0.002) in comparison to the rats exposed to LPS and saline. Conclusions: To the best of our knowledge, this study is first to establish (i) rat AMs undergo METosis, (ii) a plausible role for METosis in lung injury (iii), and the potential relevance of METosis in lung injury during early development. Novel therapies targeting METosis formation may reduce lung inflammation contributing to BPD in premature infants.

This work was supported by the Women's Auxiliary Fund, The Hospital for Sick Children, Toronto, Ontario, Canada

This is the full abstract presented at the American Physiology Summit 2023 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.
Limb bone response to exercise training in High Runner (HR) mice
Authors: Horner Angela , Rawlings Diamond , Guzman Duron Luis , Garland Theodore
Published: May 2023

Physical activity is a primary determinant of musculoskeletal health in vertebrates. The amount and intensity of exercise activity can impact bone morphology and density, muscle fiber type, and the material properties of passive elastic tissues such as tendon and ligaments. To further investigate the role of exercise intensity and duration on bone remodeling, we examined the effects of exercise in a unique model, a line of mice selectively bred for high voluntarily wheel running (high runner, HR) for over 90 generations. We placed HR and control line mice in cages equipped with data logging wheels for a minimum of 9-10 week training periods. Morphometric data collected from humera and femora revealed that exercised mice tended to have shorter, thinner limb bones regardless of genotype. HR mice had longer and thicker femora than control mice, but no significant morphological differences were apparent in humera. Distal femora width correlated with frequency of wheel running, and as HR mice outran control line mice threefold (in average revolutions/day) there was a significant interaction of genotype and exercise intensity. Our data support that the intensity and frequency of running impact limb bone morphology, and that hindlimbs are more responsive than forelimbs both to loading and selective breeding.

This is the full abstract presented at the American Physiology Summit 2023 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.
Intrinsic aerobic capacity and hepatic mitochondrial function influences fecal energy and bile acid excretion
Authors: Kugler Benjamin , Rickabaugh Madi , Hightower Gabrielle , Lysaker Colton , Wilkins Heather , Thyfault John
Published: May 2023

Low aerobic capacity (i.e., capacity to utilize oxygen in metabolic pathways) has proven to be a risk factor for hepatic steatosis. We have previously reported that increased hepatic mitochondrial function caused by exercise and high aerobic capacity is associated with higher fecal bile acid loss. In addition, other labs have shown that bile acid sequestrants (BA-seq) that block bile acid absorption in the gut and drives-up fecal bile acid loss is associated with an upregulation of hepatic mitochondrial function. So far, the connection between bile acid metabolism and mitochondrial function is unknown. Here we utilized rats artificially selected to be higher-capacity runners (HCR) and low-capacity runners (LCR) with distinct intrinsic aerobic capacities and hepatic mitochondrial function. We hypothesized that aerobic capacity would induce distinct differences in fecal bile acid loss and mitochondrial function when on a BA-seq. Male and female HCRs and LCRs (8 months of age) were randomly assigned to either a low-fat diet (LFD, 10% fat), high-fat diet (HFD, 45% fat), or HFD with cholestyramine (i.e., BA-Seq) for 24 weeks. Animals were euthanized, and feces and livers were collected. Fresh mitochondria were then isolated from the liver, and mitochondrial respiration and H2O2 emission was analyzed (O2k high-resolution respirometry). Statistical analysis was performed by a three-way ANOVA within sex with an alpha set at 0.05. BA-Seq increased fecal energy loss and BA content more than control diets (P<0.05 main effect of BA-Seq). However, HCRs with a BA-seq have greater fecal BA content than their LCR counterpart on a BA-Seq (P<0.05). Interestingly, male and female HCR livers displayed elevated hepatic mitochondrial respiration rates (State 2, State 3, FCCP) and mitochondrial coupling efficiency (State 2/State 3) and a reduction in free radical production (H2O2:O2) utilizing a fatty acid fuel source compared to LCRs (P<0.05, main effect of strain). However, diet and BA seq did not alter mitochondrial function in either strain. Overall, we provide evidence that elevated intrinsic aerobic capacity promotes greater sensitivity to BA-seq. However, enhanced fecal energy and BA loss do not alter hepatic mitochondrial function regardless of intrinsic aerobic capacity.

R01DK121497

This is the full abstract presented at the American Physiology Summit 2023 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.
Alterations in the Placental Endothelin-1 System in Response to Hypoxia and Chronic Ischemia
Authors: George Eric , Chapman Heather , Franklin Joshua , Lartigue Elecia , Murphy Hayley
Published: May 2023

Preeclampsia is one of the most common obstetrical complications, commonly characterized by new onset hypertension and proteinuria or other markers of end organ injury. In patients with preeclampsia, the arteries supplying the placenta develop abnormally, resulting in placental ischemia; widely believed to be responsible for the pathophysiology of this disorder. While the placenta is responsible for nutrient and waste exchange, it also releases numerous hormones from its interstitial and syncytialized trophoblasts important in pregnancy. Placental ischemia causes changes in placental hormone function, including endothelin-1 (ET-1). While ET-1 was initially characterized as a vasoconstrictor, it has been shown to be an important regulator of inflammatory and metabolic processes through its two receptors (ETA or ETB), though little is known about the role of the ET-1 system in the placenta. We hypothesized that chronic placental ischemia in vivo, and isolated hypoxia in vitro, would induce significant alteration in the expression of these individual components of the endothelin-1 system. Using the rodent reduced uterine perfusion pressure (RUPP) model to induce placental ischemia, we found that there was a significant (122±32 vs 45±12, p=0.05) decrease in the normalized expression of ETB, with a strong trend (112±26 vs 185±25, p=0.072) to increased ET-1 expression as measured by qRT-PCR. Interestingly, when we syncytialized BeWo choriocarcinoma cells and exposed them for 48 hours to oxygen tensions found in healthy (8%) and ischemic (1%) placentas, we found significant increases in ET-1 (205±79 vs 489±103, p=0.04), ETA (85±31 vs 276±67, p=0.04, and ETB (100±44 vs 388±68, p=0.05) gene expression. This suggests that individual cell types within the placenta are exhibiting differential response in endothelin signaling in response to hypoxia. The effect of endothelin on production of inflammatory cytokines was then determined. Compared to control (106±17) cells, both an ETB antagonist (163±12, p<0.05) and ET-1 itself (179±17, p<0.05) increased normalized TNF-α expression. Likewise, compared to controls (102±9), expression of the anti inflammatory IL-6 was significantly decreased with both ET-1 (69±11, p<0.05) and an ETA antagonist (70±9, p<0.05), suggesting that dysregulation of ET-1 signaling due to hypoxia could be an important regulator of inflammation in preeclampsia. Taken together, these data suggest that the placental ET-1 system could be a potential point of intervention for the management of preeclampsia.

P01HL51971, P20GM104357, and R01HL137791

This is the full abstract presented at the American Physiology Summit 2023 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.