bluuebunny/crossref_metadata_2025 huggingface dataset:
Netrin-1 increases epithelial cell proliferation and promotes renal cyst formation by activating Rac2 in collecting duct cells
Authors: Mohamed Riyaz , Sullivan Jennifer C
Published: May 2024

Despite recent advances in our understanding of the pathogenesis of polycystic kidney disease (PKD), the underlying molecular mechanisms involved in cystogenesis are not fully understood. Common features of PKD are excessive epithelial cell proliferation and fluid-filled cyst formation in the kidney, leading to loss of kidney function. We recently demonstrated that netrin-1 is upregulated in primary human PKD cystic cells and in the kidneys of rodent models of PKD. Moreover, overexpression of netrin-1 in tubular epithelium increases cell proliferation (p<0.01) and induces renal cyst development (p<0.0001), while decreasing netrin-1 reduces cyst growth (p<0.01) and improves kidney function (p<0.05). However, the underlying mechanism by which netrin-1 induces cystic cell proliferation is unknown. We hypothesize that netrin-1 increases cystic epithelial cell proliferation by activating Ras-Related C3 Botulinum Toxin Substrate 2 (Rac2) in collecting duct (CD). 4-week-old male tubular overexpressed netrin-1 transgenic mice (NTN-1 Tg6) and 8-week old Pkd1RC/RC mice were randomized to receive vehicle or the specific Rac2 inhibitor EHT 1864 (20 mg/kg/48hr i. p; n=4-5/group) for 4 weeks. Blood and kidneys were collected for biochemical, histological, and Western blot analysis. Renal co-localization of Rac2 with CD specific marker Auaphorin-2 in kidney of human and mouse PKD was assessed by immunofluorescence staining and protein expression was measured by Western blot analysis. Renal Rac2 activity was measured using Rac2 activation kit. Cystic index were measured using H&E staining. Cell proliferation was measured using MTT assay and renal function was measured using BUN assay kit. Rac2 is upregulated in the CD of both human PKD and Pkd1RC/RC mice (p<0.01) compared to controls. Moreover, overexpression of netrin-1 activates Rac2 in the cystic kidney (p<0.001) and in human cystic cells (p<0.05). Overexpression of Rac2 increases cell proliferation and phosphorylation of ERK in CD cells (p<0.01). Inhibition of Rac2 reduces netrin-1-induced CD cell proliferation (p<0.01) and cyst growth (p<0.01) in both netrin-1 transgenic (NTN-1 Tg6) and Pkd1RC/RC mice. Our results demonstrate that overexpression of netrin-1 increases cystic epithelial cell proliferation via activating Rac2 in CD cells and contributes to cyst growth. Future studies will be carried out to dissect the downstream signaling mechanism of netrin-1/Rac2 mediated cell proliferation cyst formation in PKD.

This work is supported by Intramural grant award (IGPP00043) from Augusta University to R. Mohamed; National Institute of Health (1P01HL134604-01) to J.C. Sullivan.

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.
β-Hydroxybutyrate Recapitulates the Beneficial Effects of Time-Restricted Feeding and Periodic Fasting in Multiple Rodent Models of Polycystic Kidney Disease
Authors: Torres Jacob A , Holznecht Nickolas , Asplund David , Kroes Bradley , Amarlkhagva Tselmeg , Haeffner Matthias , Sharpe Elizabeth
Published: May 2024

Polycystic kidney disease is a monogenic disease caused predominantly by mutations in the PKD1 or PKD2 genes. These mutations result in the formation of large fluid-filled cysts derived from kidney epithelial cells that slowly replace functional healthy tissue. Affected kidneys rely heavily on glycolysis to meet energy demands, and is accompanied by a reduction in fatty acid oxidation and mitochondrial function. We previously reported that interventions inducing a state of ketosis, including caloric restriction, time-restricted feeding (TRF), ketogenic diet, extended fasting, and ketone supplementation, ameliorate or reverse polycystic kidney disease progression in multiple animal models. To elaborate on the capability of ketosis to alter disease progression, we compared, head-to-head, the effects of a daily 16:8 TRF regimen to periodic 48-hour fasting (PF) in both juvenile and adult Cy/+ rats. We found that alternative fasting interventions prevent juvenile disease progression and partially reverse established kidney disease in adults. Further, to test our hypothesis that an increase in β-hydroxybutyrate (BHB) may mediate these beneficial effects, we administered BHB to adult Cy/+ rats and stereospecific isomers to two orthologous mouse models of PKD ( P kd1RC/RC, Pkd1-Ksp:Cre). BHB recapitulates the effects of fasting in these models independent of stereoisomer, reducing mTORC1 signaling, increasing GSK-3β phosphorylation, expression and translocation of Nuclear factor erythroid 2-related factor 2 (Nrf2) in cystic epithelia, and subsequent downstream targets of Nrf2. These findings extend and expand our previous report on the beneficial effects of ketosis on cystic disease progression and suggest that ketogenic metabolic interventions and BHB supplementation are capable of metabolic reprogramming in polycystic kidney disease.

This work was supported by grants from the NIH (Grants R01DK109563, R01DK124895) and the US Department of Defense (Grant W81XWH2010827) to T.W., and gifts from the Amy P. Goldman Foundation and the Jarrett Family Fund to University of California, Santa Barbara, to support the work of T.W. The MRL Shared Experimental Facilities are supported by the MRSEC Program of the NSF under Award No. DMR 1720256; a member of the NSF-funded Materials Research Facilities Network (www.mrfn.org).

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.
Decoding the role of genetically-distinct sensory nerves in controlling cardiopulmonary reflexes
Authors: Darcey Teresa , Taylor-Clark Thomas , Hooper Shane , Kim Seol-Hee
Published: May 2024

Activation of airway sensory nerves causes respiratory and autonomic reflexes. The majority of the airway sensory afferents are only sensitive to noxious stimuli, such as inflammation, irritants, and pollutants. Activation of these nociceptive afferents evokes protective mechanisms such as apnea, cough, and bradycardia. These reflexes may contribute to disease morbidity when excessively activated. Airway nociceptors, largely projecting from the vagal ganglia (nodose and jugular ganglion), are heterogeneous with respect to gene expression and neuroanatomy, and our objective is to characterize the reflexes evoked by activation of specific afferent subsets using chemogenetics. To selectively activate vagal afferent subpopulations in vivo, mice were exposed to nebulized selective stimuli such as capsaicin (transient receptor potential (TRP) vanilloid 1 (V1) agonist), allyl isothiocyanate (AITC, TRP ankyrin 1 (A1) agonist), and clozapine-N-oxide (CNO, selective agonist for the designer receptor exclusively activated by designer drug (DREADD) stimulatory receptor hM3Dq). hM3Dq expression by ROSA26-loxP-STOP-loxP mice was selectively expressed in sensory subpopulations by breeding with Cre recombinase-expressing mice: TRPV1 (all nociceptors), Tac1-Cre (peptidergic/jugular-originating nerves), and P2X2-Cre (nodose-originating nerves). An intersectional approach using TRPV1-flp mice and the above Cre mice combined with bilateral vagal injections of an AAV delivering dual recombinase-dependent DREADD constructs was used to express hM3Dq in afferent populations defined by two genes (e.g. TRPV1+ and P2X2+). ECGS were recorded via radiotelemetry, and respiration was measured via whole-body plethysmography. CNO administration in TRPV1-hM3Dq mice, P2X2-hM3Dq mice, and Tac1-hM3Dq mice evoked bradypnea and bradycardia. When CNO was administered in the dual recombinase-dependent ConFon (Cre-On/Flp-On) Tac1-Cre/TRPV1-Flp mouse (selectively activating the vagal jugular nociceptors), a significant bradypnea was evoked; however, when CNO was administered in the dual recombinase-dependent ConFon P2X2-Cre/TRPV1-Flp mouse (selectively activating the nodose nociceptors), no bradypnea was observed. Our preliminary data suggest that functionally-distinct nociceptive subpopulations differentially regulate the cardiopulmonary reflexes.

NIH R01: 152219, NIH T32: HL160529.

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.
A preliminary assessment of physical function in aged male mice with constitutive activation of the aryl hydrocarbon receptors in skeletal muscle
Authors: Wimberly Keon , Lackey Jacob , Tice Abigail , Hepple Russell , Ryan Terence
Published: May 2024

BACKGROUND: Aging involves a gradual deterioration of physical and physiological functions, ultimately leading to mortality. Unfortunately, there are no treatments available that successfully mitigate the age-associated decline in physical function. Recent studies have revealed a strong association between elevated levels of L-kynurenine (L-Kyn) and frailty, muscle weakness, and neuromuscular junction degeneration in humans and mice, however a causal relationship has not been tested. L-Kyn is a product of tryptophan catabolism that has been linked to motor neuron death, skeletal muscle atrophy, and mitochondrial dysfunction, all hallmarks of aging muscle. Interestingly, L-Kyn is an endogenous ligand of the aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor widely known for its role in xenobiotic metabolism. Though both pro- and anti-aging effects of the AHR have been reported in the literature, there are several chronic diseases that involve chronic AHR activation and produce strikingly similar phenotypes to exacerbated aging/frailty. OBJECTIVE: The objective of this study was to determine of constitutive activation of the AHR has a causal role in the decline of physical function with aging. METHODS: Aged (16-mo.) C57BL6 male mice (N=48) were obtained from the NIA breeding colony and were randomized to interventions employing muscle-specific adeno-associated virus with the following three treatment groups: 1) a constitutively active AHR mutant was constructed by deleting the ligand binding domain (amino acids 277-418); 2) a transcriptionally deficient AHR mutant (arginine to aspartate mutation at amino acid 39); and an empty vector control. Treadmill endurance, forelimb grip strength, and maximal walking speed were measured at 24-mos. of age. Results: Treadmill endurance capacity in mice with constitutively AHR activation was 5.3% lower than mice treated with the empty vector ( P=0.25) and 12.1% lower than mice treated with the transcriptionally deficient AHR mutant ( P=0.09). Grip strength and walking speed were not significantly different between groups ( P>0.57 and 0.22 respectively) at this 24-mo. age. CONCLUSIONS: These preliminary results in aging male C57BL6 mice may indicate possible role of AHR activation in the decline of physical function. Continued enrollment to increase the sample size and obtaining measures at later ages when physical function declines more severely, as well as examining female mice will provide a more thorough analysis.

Funding Supported by grants from the National Institutes of Health R01-AG076490 (Ryan & Hepple) and T32 AG062728 (Wimberly).

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.
Effect of Sodium Fluoride on Oxidative Stress and Antioxidant System in Male Wistar Rats
Authors: Ayegboyin Olusola A. , Ige Abayomi O. , Adewoye Elsie O.
Published: May 2024

This research aimed to examine the effect of sodium fluoride (NaF) on oxidative stress and antioxidant systems in the serum, aorta, and cardiac tissues of male Wistar rats. The increased attention to sodium fluoride and its detrimental effects on human health in recent years has led to concerns. Excessive ingestion and accumulation of sodium fluoride from water, toothpaste, and pesticides could initiate oxidative stress in the blood, aorta, and heart. This heightened oxidative stress and disturbance in the antioxidant system pose a significant risk and may predispose the body to severe vascular and cardiovascular-related diseases, which are leading causes of global mortality. In this study, ten male Wistar rats weighing 150-180g were grouped into two (n=5). Group I Control received distilled water, and Group 2 received 10mg/kg NaF orally for 21 days. Markers of oxidative stress malondialdehyde (MDA) and reduced glutathione (GSH), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and total antioxidant capacity (TAC) were measured in the serum, aorta, and heart. Data were analyzed using descriptive statistics of student t-test at α0.05. The data were presented as mean ± SEM. The oral administration of sodium fluoride resulted in an elevation of MDA levels in the serum, aorta, and heart of the rats exposed to NaF. Additionally, the oral administration of sodium fluoride led to a reduction in superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) levels in the serum, aorta, and heart of rats exposed to NaF. Furthermore, the oral administration of sodium fluoride resulted in a decrease in the total antioxidant capacity (TAC) in the serum, aorta, and heart of rats exposed to sodium fluoride. This study demonstrated that sodium fluoride exposure diminishes the antioxidant capacity in the serum, aorta, and heart, indicating that sodium fluoride exposure induces oxidative stress, potentially affecting the functions of the aorta and heart. Therefore, the continuous exposure to fluoride in populations residing in regions with endemic fluorosis worldwide should be a topic of considerable concern.

This research work was self-funded.

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.
Effects of age and sex on cardiac function in mice
Authors: Medarev Steven , Evola Vito , Castano Antonio , Gordon Steven , Ogando Anthony , Balenko Jasen , Maggitas Charles
Published: May 2024

It is well established that maximal cardiac output declines with advancing age. Age-related decline in cardiac output could result from changes in either systolic or diastolic function that are also apparent at rest. We investigated the effects of age on systolic and diastolic function in male and female mice. Mice were studied at 4-7months and 17-20 months. Echocardiography was performed with FujiFilm Visual Sonics VevoF2 to obtain ejection fraction, fractional shortening, and E/A ratio. In male mice, fractional shortening decreased by 25% (young: 31.26±1.24, old: 23.31±1.82, P=0.002 young v. old) and ejection fraction decreased by 19.77% (young: 58.23±1.63, old: 46.71±3.05, P=0.002 young v. old) with age. In female mice, fractional shortening and ejection were stable with age. E/A decreased with age in both male (young: 1.53±0.09, old: 1.18±0.07, P=0.02 young v. old) and female (young: 1.64±0.08, old: 1.28±0.07, P=0.002 young v. old) mice. These data indicate that systolic function declines with age in male, but not female mice. In contrast, diastolic function declines with age in both male and female mice. Further analysis is needed to determine how these changes in resting cardiac function correlate with changes in exercise capacity in male and female mice.

NIH R01HL166591.

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.
Impact of Long-Term Ibuprofen Usage on Cardiac Signaling Pathways
Authors: Zheng Emily , Gomes Aldrin , Rivera Gabriela , Sule Rasheed
Published: May 2024

Ibuprofen, a common over-the-counter drug, is taken worldwide to alleviate pain and reduce inflammation. While the recommended dosage is generally regarded as safe for consumption, complications arise when the drug is taken chronically, even in the prescribed quantities. Previous research suggests that ibuprofen alters the productivity of various signal transduction pathways throughout the body and stimulates the production of reactive oxygen species (ROS) in liver tissue. Our research aimed to determine if proteins involved in proteolysis and energy producing pathways were altered in cardiac tissue as a result of ibuprofen usage. The study involved eight nine-week-old female mice divided into two treatment groups: control and ibuprofen. Female control mice received pure water while female ibuprofen mice received 100 mg/kg of ibuprofen in water daily for one week before euthanization. The ibuprofen used corresponds to less than 500 mg ibuprofen/day in humans. Heart samples removed from euthanized mice were homogenized, labeled with Tandem Mass Tags (TMT), and then analyzed by liquid chromatography-tandem Mass Spectrometry (MS). Spectral analysis yielded 120 proteins that were altered in a statistically significant manner (p ≤ 0.0028 according to the Benjamini-Hochberg procedure). Pathway analysis conducted on statistically significantly altered proteins suggests moderate ibuprofen consumption results in changes in aerobic respiration, oxidative stress, the proteasome proteolytic system, the nucleotide metabolic process, and muscle cell development pathways. We conclude that ibuprofen consumption at recommended dosages significantly alters many pathways in cardiac tissue, which may be associated with the clinically observed increased risk of cardiovascular disease for those taking ibuprofen.

This research is funded by the NIEHS/Superfund Research Program (P42 ES004699).

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.
Differential serum inflammatory cytokine elaboration in Nepali brick factory workers
Authors: A Chang , Curtis KL , Fleming AA , Fleming BT , Black AD , Johnston JD , Beard JD
Published: May 2024

Previous studies involving workers at brick kilns in the Kathmandu Valley of Nepal have revealed chronic exposure to hazardous levels of fine particulate matter (PM2.5) common in ambient and occupational environments. Such exposures are known to cause and/or exacerbate chronic respiratory diseases including chronic obstructive pulmonary disease (COPD). However, there is a paucity of data regarding the systemic inflammatory status of exposed workers at brick manufacturing facilities. In the current study, we sought to elucidate systemic inflammatory responses by quantifying the molecular cytokine/chemokine profiles in serum from study participants. A sample of participants (N = 48; 54% female; 31.77±9.76 years-old) were distributed among four different job categories. Blood was procured from participants on-site, allowed to clot at room temperature, and centrifuged to obtain total serum. A human cytokine antibody array was used to screen cytokines/chemokines in serum samples. Comparisons were generated between a control group of administration workers vs. fire master workers, administration vs. green brick hand molders, and administration vs. top loaders. We discovered significantly increased concentrations of eotaxin-1, eotaxin-2, GCSF, GM-CSF, IFN-γ, IL-1γα, IL-1β, IL-6, IL-8, TGF-β1, TNF-α, and TIMP-2 in serum samples from fire master workers vs. administration (p < 0.03). Each of these molecules, with the exception of TIMP-2, were also significantly elevated in the serum from green brick hand molders (p < 0.02). In addition, each molecule was significantly elevated in serum from top loaders compared to administration workers (p < 0.03). With few exceptions, the fire master workers expressed significantly more systemic inflammatory molecular abundance when compared to all other job descriptions. These results reveal an association between pulmonary exposure to PM2.5 and systemic inflammatory responses likely mediated by cytokine/chemokine elaboration. Additional characterization of a broader array of inflammatory molecules may provide valuable insight into susceptibility to lung diseases including COPD.

This work was supported in part by funding from Undergraduate Mentoring and a Widtsoe Research Grant from Brigham Young University.

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.
ER stress CHOP Knockout in Beta Cells protects from type 2 diabetes-induced vascular dysfunction
Authors: Alluri Kiran , Srinivas Balaji , Belmadani Souad , Matrougui Khalid
Published: May 2024

Introduction: Cardiovascular complications are the primary causes of morbidity and mortality in patients with type 2 diabetes (T2D). T2D is a significant risk factor for cardiovascular diseases such as coronary artery disease, stroke, peripheral vascular disease, and heart failure for millions of people in the US. In the present study, we determined the role of the endoplasmic reticulum stress (ER) stress C/EBP homologous protein (CHOP) in beta cells on type 2 diabetes-induced vascular endothelial dysfunction. Methods: male and female CHOPflox/flox and CHOPbeta cells −/− mice were fed a high-fat diet (HFD) for 12 weeks. We measured body weight, running distance, heart/lung/pancreas/kidney weights, glucose tolerance test (GTT), vascular endothelial function, and signaling. Results: Our research indicates that mice with the ER stress CHOPflox/flox genotype, when fed a high-fat diet for 12 weeks, develop obesity and show impaired GTT and running distance. Additionally, they exhibit an increase in the weight of organs such as the heart, lungs, pancreas, and kidneys and experience vascular endothelial dysfunction and a reduction in phosphorylated eNOS levels. However, it's interesting to note that mice that lack the ER stress CHOPbeta cells−/− and were fed the same high-fat diet for 12 weeks displayed a protective effect. Conclusion: The data indicates that beta cell ER stress CHOP plays an essential role in type 2 diabetes. Disrupting the ER stress CHOP, specifically in beta cells, has been shown to protect mice from the vascular endothelial dysfunction caused by type 2 diabetes. Therefore, targeting ER stress CHOP, specifically in beta cells, could be a promising therapeutic approach to safeguard vascular endothelial function.

NIH-HL150014 (Matrougui) and NIH-HL151616 (Matrougui).

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.
Aged HIV-1 Tg26 Transgenic Mice display Vascular Dysfunction with No Alterations in Neurocognitive Function or Cerebral Blood Flow
Authors: Khakina Beryl , Kress Taylor C. , Rosewater Cody , Bosomtwi Asamoah , Kennard Simone , Belin de Chantemèle Eric J.
Published: May 2024

Although combination antiretroviral therapy (cART) has markedly reduced HIV mortality and increased life expectancy, people living with HIV (PLWH) develop premature cognitive and brain aging, classified as HIV-associated neurocognitive disorder (HAND). Vascular dysfunction and expression of HIV-derived proteins have independently been associated with cognitive impairment without causality being established. Thus, we investigated the role of HIV viral proteins in endothelial dysfunction and cognitive impairment in an aged mouse model of HIV. We used 18-month-old Tg26 mice and their wild-type littermate controls. Tg26 mice express 7 out of the 9 HIV viral proteins under the control of the long-term repeat promoter and have previously been shown to exhibit signs of cognitive impairment at eight months of age. Conduction of concentration-response curves to acetylcholine in mesenteric arteries using wire myography revealed impaired endothelium-dependent relaxation in Tg26 mice compared to WT mice. Measurements of brain perfusion with arterial spin labeling magnetic resonance imaging showed no differences in the whole brain and hippocampal cerebral blood flow. In addition, there were no changes in the hippocampus volume, which has been reported to be reduced with cognitive impairments. Two methods were employed to assess the cognitive abilities of the mice: Morris water maze (MWM) and the IntelliCage system. MWM requires human handling while the IntelliCage system is fully automated enabling the testing of mouse cognitive function during their active period and independently of human handling. Tg26 and WT mice underwent ten days of MWM testing and eight days of IntelliCage testing to assess spatial learning and memory. Tg26 mice showed no significant differences in latency to platform, distance swam to platform, or swim velocity when compared to their WT littermates. The IntelliCage results showed no differences in total visits, total visits with nose pokes, or total licks during the reversal learning period. Additionally, there were no significant differences in percent side error or place error indicating that cognitive function is not impaired in Tg26 mice. The Y-maze test further showed no differences in spontaneous alteration, a measure of spatial working memory. Altogether, our data shows that although 18-month-old female Tg26 mice develop endothelial dysfunction, they display no alterations in cerebral blood flow and no cognitive impairment indicating that systemic viral protein expression and vascular dysfunction might minimally contribute to neurocognitive impairment in HIV.

R01HL130301 & R01HL155265 (E.J.B).

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.
Don’t lose your transporters! Optimizing renal transporter recovery after kidney freezing
Authors: Hartman-Houstman Hannah , Ralph Donna , Moronge Desmond , Faulkner Jessica , Sullivan Jennifer , McDonough Alicia
Published: May 2024

Renal ion transporters are critical to maintain fluid and electrolyte homeostasis and are primary targets for (patho)physiology studies and drug discovery. Transporter abundance and covalent modifications are determined by immunoblots. Our standard protocol is quick kidney removal and homogenization in buffer containing 0.2 mM PMSF, 0.9 ug/ml aprotinin, and Sigma P0044 phosphatase inhibitors followed by isolation of a low speed supernatant (the homogenate). Collaborations with other labs requires shipping frozen kidneys. We tested whether flash freezing (FZ) Sprague Dawley (SD) kidneys impacts ion transporter recovery compared to quick removal without freezing (QR) defined as 100% recovery; assessed by immunoblot with specific antibodies. FZ and ~ 3 d storage before homogenization in inhibitors significantly reduced recoveries of proximal tubule (PT) NHE3 and megalin to 20% and Cldn2 to 40%. Medullary NHE3 and NKCC2 remained at 100%, DCT/CD proteins were reduced ~ 10-60% after FZ vs. QR. Renal perfusion pre-FZ further reduced recovery of all transporters vs. non-perfused kidneys. To test whether Fz activates proteases not inhibited in our standard buffer, Roche protease inhibitor (minitabs) were added to homogenization buffer. Adding minitabs to FZ kidneys increased NHE3 recovery to 85% and Cldn2 recovery to 100% of QR, NKCC2 and ENaC-beta to 85% and NCC to100%. Lastly, we tested, in male and female (M,F) SD rats, whether minitab addition to QR renal cortex homogenates followed by Fz prevents proteolysis vs. minitab addition to QR homog without Fz. In F, minitab addition with Fz preserved 100% recovery of transporters: NHE3, Cldn2, AQP1, megalin, NKCC2, NCC, ENaC-beta, Cldn-7 and AQP2. In M, minitab addition with Fz preserved 100% recovery of only AQP1, villin, Cldn7 and AQP2; all other M transporters exhibited ~40% less recovery. In conclusion, flash freezing kidney tissue differentially activates proteolysis of ion transporters - up to 80% in cortex and not at all in medulla - despite inclusion of standard inhibitors in homogenizing buffers. Strong sex differences are evident: addition of minitabs prevents proteolysis of F not M cortical ion transporters. Single cell ‘omics approaches may be useful to identify tubule and sex specific proteases.

NIH: DK083785, DK134695.

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.
Development of CRISPR/Cas9-Based Mouse Models for X-linked Alport Syndrome
Authors: Decker Stephen , Davey Crystal , Cao Guoshen , Al-Rabadi Laith , Funai Katsuhiko
Published: May 2024

Intro: Alport Syndrome (AS) is a rare hereditary disorder characterized by progressive renal impairment and sensorineural hearing loss, resulting from mutations in the genes that encode COL4 synthesis. X-linked Alport Syndrome (XLAS) is the most common form of AS, comprising an estimated 80% of all human AS cases (the others being various forms of autosomal recessive or autosomal dominant AS), and is caused by various mutations on the COL4A5 gene. However, to date, there are no animal models of XLAS. Methods: To facilitate comprehensive preclinical investigations and therapeutic developments, we established two novel mouse models of XLAS using CRISPR/Cas9 technology. Leveraging the robust C57Bl/6J mouse strain, we precisely introduced the L1655R and C1570S mutations, which represent the predominant human variants of XLAS observed in the Western United States. Discussion: Our innovative mouse models not only serve as invaluable tools for elucidating the underlying mechanisms of XLAS pathogenesis but also provide a platform for evaluating the effcacy of potential therapeutic interventions. The establishment of these faithful genetic replicas of human XLAS marks a significant advancement in the field, offering unprecedented opportunities for in-depth mechanistic studies and the development of targeted therapeutic strategies for this debilitating renal and auditory disorder.

This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho.

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.
Tissue dependence of variation in metabolic rates of harvester ants
Authors: Harrison Jon , Perl Craig , Haas James , Johnson Meredith , Johnson Robert
Published: May 2024

Metabolic rates vary tremendously among individuals and across species, and the causes of this variation remain controversial and challenging to identify. We utilized an Agilent Seahorse system to measure the metabolic rates of brains, flight muscles, leg muscles, jaw muscles, digestive tract, fat and reproductive organs of harvester ants of different species, sex and caste, and compared these to whole body metabolic rates. Across Pogonomyrmex species varying an order of magnitude in body size, mass-specific brain metabolic rates declined dramatically in larger species, such that the brain accounted for 43% of whole body metabolism in the smallest species and 14% in the largest. In Veromessor pergandei, males have much higher mass-specific metabolic rates than queens or workers, with queens being slightly higher than workers. The tissue specific bases to metabolic rates varied with sex and caste. Males are "flying gonads", with flight muscle, fat and gonads accounting for 44, 25 and 20%, respectively, of resting metabolic rate, and tissue-specific rates were generally higher in males than females. For queens, which have by far the largest fat content, the fat body contributes the largest fraction of the total tissue metabolic rate (38%), with flight muscle, femoral muscle, brain and gut each contributing 13 – 15% of total cost. For workers, brains are the most important contributors to total tissue metabolic rate (38%), followed by fat body (24%) and gut (19%). Together these results demonstrate that variation in whole body metabolic rate within and across closely related species is strongly affected by size, sex and caste-specific differences in tissue function, likely associated with life history variation.

Supported by NSF IOS-1953419.

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.
IGFBP3 regulates airway basal cell stemness and airway regeneration
Authors: Murthy Shubha , Pathmanathan Aparna , Caceres Adrian , Sease Rosemary , Calvert Ben , Marconett Crystal , Ryan Amy
Published: May 2024

Basal Cells (BCs) are multipotent progenitor cells of the upper airway and critical in airway epithelial regeneration. Impaired self-renewal and differentiation capacity of BCs is a significant cause of chronic respiratory diseases. However, mechanisms mediating these changes remain largely unexplored. We, and others, have observed attenuated growth and differentiation of BCs after repeated cell divisions in vitro. To investigate mechanisms regulating BC stemness, we performed bulk RNA-sequencing to analyze transcriptomes of early and late passage BCs maintained in culture. Our analyses reveal significant differences in the expression of genes involved in IGF- and IGFBP3-dependent pathways that are known to modulate cellular growth and ROS generation. The role of these pathways in regulating BC stemness have not been systematically investigated. We hypothesized that IGFBP3 regulates BC growth and differentiation by modulating cellular redox status and DNA damage repair. Herein, we demonstrate that passage of non-diseased human BCs over 5 passages increases IGFBP3 transcript in 3 independent growth conditions. Interestingly, continued passage decreased IGFBP3 expression and significantly altered its intracellular localization. In contrast to low passage cells where IGFBP3 is largely cytosolic, high passage cells almost exclusively express IGFBP3 in their nuclei where it is known to participate in DNA repair. Consistent with this, we observed increased DNA repair gene expression in high passage cells coupled with significant reduction in thioredoxin levels. Together, these results suggest that serial passage of BCs causes oxidative stress-mediated BC exhaustion and altered IGFBP3 expression and localization. To investigate the role of IGFBP3 in BC growth and differentiation, we knocked down IGFBP3 expression and, as hypothesized, IGFBP3 knock down of low passage cells attenuated growth, BC markers, and DNA damage repair gene expression. In addition, differentiation to secretory, ciliated and club cells at the air-liquid interphase was significantly attenuated. In conclusion, our data support an IGFBP3-dependent regulation of BC stemness by preserving growth and differentiation.

This study was supported by the NIH R01HL139828 (ALR) and Cystic Fibrosis Foundation grants FIRTH17XX0 (ALR), RYAN21XX0, FIRTH21XX0 (ALR, CNM). BCs for this study were provided though the Center for Gene Therapy Cell Core funded by the NIH:NIDDK.

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.
Determining optimal surveillance techniques for the microbial community on human skin using RNAseq data analysis
Authors: Bhargava Disha , Prokop Jeremy
Published: May 2024

Background: The largest organ in the human body, the skin, serves as a physical barrier to the outside world. It also houses millions of bacteria, viruses, and fungi that protect us against invading pathogens or result in altered physiology and disease. These microorganisms can contribute to normal skin health or predispose individuals to pathogenic disorders. Surveillance and observation of the microbiome can give insight into identifying common taxa that compose our skin and indications of new or changing pathogens. However, to do this effciently, it is important to understand what can be detected from skin samples and the differences we may see using various collection methods. Methods: We used publicly available NCBI SRA RNAseq bioprojects that queried “skin,” processing 132 bioprojects with 8,274 samples. Using FastQC, Salmon, KRAKEN2, and MiCXR/Immunarch, we aimed to analyze the quality, human gene transcripts, infections, and B/T- cell responses, respectively. We aimed to identify what skin sampling method provides the best data on the skin microbiome, human gene mapping, and B/T-cell immune repertoire. This project is based on observational methods, not a specific hypothesis. Results: From the KRAKEN2 data, there were 14,383 viruses and 14,691 bacteria assessed through all “skin” BioProjects. From the extensive data pool, we identified unique BioProject signatures for protest (Leishmania), bacteria (Mycoplasma), and viruses (HSV1 dose dependencies, HPV in human warts, Chinese transmitted tick viruses, Human T-cell lymphotropic virus, and the first-ever human case of equine infectious anemia virus). The use of read mapping and coverage is critical to confirming the presence of foreign species, noted through the observation of T4 DNA ligase RNA presence prior to 2018 RNA sequencing. Once pathogens were detected, cellular response mapping was developed, yielding insights into the pathophysiology of multiple viral infections of the skin and sex differences. Conclusion: Detecting a wide variety of microorganisms on the skin can give insight into dermatological pathologies and skin physiology. Using the publicly available skin datasets, we see many unique stories of microorganisms in the skin. This big data mapping strategy highlights the importance of broad observational mapping, possible with cutting-edge sequencing tools.

This work was supported by the MI-SAPPHIRE program of MDHHS and the CDC.

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.
Quantifying Prefrontal-Hippocampal Circuit Engagement Using Scalp EEG and the Paired Associates Learning Task
Authors: Besosa Cristina , Lovett Sarah , Kennedy Jack , Maurer Andrew , Burke Sara
Published: May 2024

The hippocampus (HPC)-prefrontal cortical (PFC) circuit, critical for affective processing and cognition, is vulnerable in mental health disorders. Although disrupted HPC-PFC functional connectivity is a common feature of anxiety, schizophrenia, and autism, how local cellular interactions within this circuit manifest as large-scale temporal coordination remains unknown. The objective of this project is to provide novel mechanistic insights into disrupted neural coordination in mental illness by examining the relationship between invasive local field potential (LFP) recordings from the HPC and PFC, and scalp electroencephalogram (EEG) during the Paired Associates Learning (PAL) task. The PAL task measures visuospatial associative memory by requiring animals to learn the stimulus-location pairing of different images on a touchscreen. We have previously shown that manipulations that disrupt HPC-PFC coordination, such as traumatic brain injury and advanced age, lead to PAL performance impairments. Importantly, EEG recordings during PAL performance in human study participants have shown associations between behavioral deficits and altered neural dynamics in schizophrenia and anxiety. We hypothesize that simultaneous LFP and EEG recordings from rats performing the PAL task will reveal highly correlated theta power across brain regions, as well as significant phase relationships among frequencies when compared to rest periods. Preliminary data show correlated increases in theta power in both the LFP and EEG from HPC as well as PFC, and higher power in the theta frequency band during PAL compared to rest periods. Future analyses will determine phase coherence between frequencies within a signal as well as brain regions. Exploring the relationship between invasive neurophysiological measures of the LFP and non-invasive surface EEG signals in the context of a translatable behavioral paradigm could provide new physiological and behavioral biomarkers that may be capable of predicting HPC-PFC coordination and aid the development of therapies for treating mental health disorders.

The Evelyn F. McKnight Brain Research Foundation and the NIH/NIMH (grant R01MH126236).

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.
Beclin-1-Dependent Autophagy Preserves Perivascular Adipose Tissue Function and Suppresses Hypertension Development in Hyperaldosteronism
Authors: Costa Rafael , Rodrigues Daniel , Gonçalves Tiago , Alves Juliano , Awata Wanessa , Singh Shubhnita , Bruder-Nascimento Ariane
Published: May 2024

Hyperaldosteronism (HA), characterized by the independent overproduction of aldosterone (Aldo), leads to cardiovascular damage and perivascular adipose tissue (PVAT) dysfunction. Beclin-1 (Becn1) is a key protein regulating autophagy, a recycling process of unwanted components. Whether HA disturbs autophagic flux in PVAT via Becn1 is unknown. Thus, we hypothesize that Becn1-dependent autophagic flux will improve PVAT function and inhibit the genesis of hypertension (HTN) in HA. 10-12-weeks-old male C57BL/6J (WT) or Becn1 knock-in mice (Becn1Tg) mice were infused with aldosterone for 14 days (600ug/Kg/day via osmotic mini-pump). Vascular function was studied in PVAT-intact thoracic aortae and blood pressure was analyzed by radiotelemetry. Becn1Tg mice were protected from HA-induced HTN [mean blood pressure (mmHg) − before HA: 101.2±0.5 vs. after HA: 108.2±0.9]. HA disrupted PVAT autophagic flux characterized by accumulation of LC3A/B and p62 and reduction in Becn1 expression and activity. In terms of function, presence of PVAT decreased the contractile response in WT [PVAT (-): 137.2±1.9 vs. PVAT (+): 107.4±2.7, Emax (% KCl)], while HA induced loss of PVAT anti-contractile effect [PVAT (-): 159.9±2.9 vs. PVAT (+): 164.1±3.4]. Becn1Tg mice were protected from this effect. Furthermore, HA induced PVAT oxidative stress and inflammation, characterized by elevated TNF-α, IL-6, IL-1β, IL-17, and IFN-γ mRNA expression, and elevated reactive oxygen species (ROS), measured by lucigenin chemiluminescence. All these effects were prevented in Becn1Tg mice. Pharmacological induction of Becn1 activation, via TB-peptide (16mg/Kg/day for 7 days), improved PVAT function of WT mice with HA. To evaluate the molecular mechanisms, we treated brown adipocyte cells (BAC, WT1) with Aldo (100nM), which triggered a transient autophagic flux, depicted by an initial increase in LC3A/B (12 and 24 h), followed by a decrease at 48 and 96 h. Such effects were prevented by a selective mineralocorticoid receptor antagonist (Eplerenone, 1μM). Finally, Aldo induced BAC inflammation and oxidative stress, which were blunted by TB-peptide. In summary, our findings reveal that autophagy overactivation confers protection against HA-induced PVAT inflammation and dysfunction and HTN. Therefore, activation of Becn1 could be pharmacological tool to prevent HA-associated cardiovascular deleterious effects.

FAPESP (2022/06639-2), NHLBI-R00 (R00HL14013903), AHA (CDA857268 and 23DIVSUP1069230), Vascular Medicine Institute, the Hemophilia Center of Western Pennsylvania Vitalant, and Children's Hospital of Pittsburgh of the UPMC Health System.

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.
Renal Injury and Cross Sex Steroid Therapy in Aging Male Rats
Authors: Mallette Jordan H. , Crudup Breland F. , Nabors Harley S. , Oudomrath Speyrer Adrian , Willis Alex T. , Rawls Adam Z. , Cockrell Kathy L.
Published: May 2024

Significance: Transgender females (TGF) are individuals who were born male but identify as female. Gender affrming hormone therapy (GAHT) is used to align physical characteristics with gender identity. In the TGF, GAHT includes 17β-estradiol (E2) combined with surgical intervention (castration, CTX). The long-term effects of GAHT on cardiorenal health are unclear. Thus, using our model of cross sex steroid therapy whereby E2 is significantly elevated in association with a significant reduction in testosterone in E2+CTX male rats, we hypothesized that renal injury is elevated with GAHT, and that age-related increases in renal injury are enhanced in E2+CTX compared to age-matched control. Methods:Male Sprague-Dawley rats were randomly assigned to: Control or E2+CTX with E2 [5 mg/day, silastic capsule s.c., replaced every 3 weeks] started at 14 (young, Y) or 52-65 (middle-age, MA) weeks of age followed by CTX two weeks later (N=8-10/grp). Experimental protocol: lean and fat mass (EchoMRI), 24-hour metabolic collection for proteinuria, KIM-1, NGAL (ELISA); creatinine (Chemistry Analyzer) up to 14 wks in Y and 20 wks in MA rats post GAHT. Two-way ANOVA with Tukey’s post hoc analysis was performed with P<0.05 considered significant. Results: Body weight and lean mass were significantly decreased in Y and MA E2+CTX vs age-matched Control. Fat mass was significantly reduced in MA E2+CTX vs MA Control. 24-hour urinary protein did not differ in Control vs E2+CTX at either age but was increased with age vs Y groups ( p<0.05). Creatinine was significantly increased in MA groups vs Y but decreased in MA E2+CTX vs MA Control ( p<0.05). NGAL and KIM-1 were significantly increased in MA Control vs all other groups ( p<0.05); NGAL was increased in MA E2+CTX vs its Y counterpart ( p<0.05) but not Y Control. Conclusion: Collectively, these data indicate that markers of renal injury are not increased in Y GAHT males. However, in MA, age-related increases in KIM-1and NGAL are attenuated in GAHT. Future studies will determine if male GAHT are more sensitive to a second hit such as renal ischemia reperfusion and quantitate renal function via a direct method to account for loss of lean mass in E2+CTX males. [Formula: see text]

HL143459, P20-GM-104357, P20-GM121334, AHA CDA 938320, T32-HL105324.

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.