bluuebunny/crossref_metadata_2025 huggingface dataset:
Glutathione Transferase P1 (GSTP1) is Modified by Palmitate
Authors: Marensi Vanessa , Yap Megan , Ji Yuhuan , Lin Cheng , Berthiaume Luc , Leslie Elaine M
Published: November 2023

Glutathione transferase P1 (GSTP1) protects cells from carcinogens by catalyzing their conjugation with the tripeptide glutathione (γ‐Glu‐Cys‐Gly). GSTP1 is also involved in cell signaling, proliferation and apoptosis. Overexpression of GSTP1 in tumors and single nucleotide polymorphic variants are associated with anti‐cancer drug resistance and poor prognosis. In contrast, inactivation of GSTP1 due to epigenetic promoter silencing increases susceptibility to certain cancer types, with prostate cancer as the best studied example. GSTP1 is classically described as a cytosolic enzyme; however, we have reported that it is strongly associated with the plasma membrane and the strength is comparable to the integral membrane protein Na+/K+‐ATPase. We hypothesize that the addition of a hydrophobic component is required to allow its strong interaction with membranes. Palmitoylation is the reversible post‐translational addition of a 16‐C saturated fatty acid to proteins, most commonly on Cys residues through a thioester bond. We found that GSTP1 is modified by palmitate. However, Cys‐less (Cys to Ser or Cys to Ala) mutants expressed in MCF7 cells surprisingly retained palmitoylation. In addition, treatment of palmitoylated GSTP1 with NaOH, which cleaves thioester bonds, did not remove palmitate. These data suggested that GSTP1 is modified by palmitate on at least one non‐Cys residue. We also demonstrated that GSTP1 can be non‐catalytically palmitoylated (or autoplamitoylated). Peptide sequencing by ESI‐MS/MS of the autopalmitoylated GSTP1 revealed that Cys48 and Cys102 undergo S‐palmitoylation and Lys103 undergoes N‐palmitoylation. N‐palmitoylation of an internal Lys residue is a rare observation and provides an explanation for the resistance of GSTP1 palmitoylation to NaOH treatment. In conclusion, we have identified palmitoylation as a novel post‐translational modification of GSTP1. This research lays the foundation for understanding the fundamental biology of GSTP1 and how palmitoylation may influence its structure, function and membrane association.

Support or Funding Information

Canadian Institute of Health Research (CIHR), Alberta Innovates Health Solutions (AIHS) and Alberta Cancer Foundation (ACF)
Anti‐vascular ageing effects of endothelial SIRT1 ‐ communicating with smooth muscle cells of the arteries
Authors: Wang Yu , Xu Aimin , Vanhoutte Paul M
Published: November 2023

Arterial remodeling is not only a physiological process during the formation of blood vessels, but also plays an important role in the development of cardiovascular diseases. Crosstalks between the endothelium and the surrounding vascular smooth muscle cells control the arterial remodeling process in response to changes in blood flow and nutrient supply. Endothelial disruption and dysfunction facilitate the adverse arterial remodeling and alter the mechanical responses of the blood vessel wall. Despite this information, little is known about the molecular pathways by which arterial remodeling is controlled and regulated. SIRT1 is a longevity regulator possessing potent anti‐cardiometabolic ageing properties. The present study identifies endothelial SIRT1 as a key modulator of arterial remodeling. SIRT1 facilitates the ubiquitination and proteasomal degradation of LKB1, a senescence‐inducing factor in endothelial cells. LKB1 binds to the promoter sequences of a gene encoding transforming growth factor beta (TGFbeta). By promoting the protein complex formation with HERC2, a giant scaffold protein possessing an E3 ligase activity, SIRT1 mediates the selective degradation of acetylated LKB1, thus preventing the trans‐activation of TGFbeta. In mice, overexpression of SIRT1 in endothelial cells abolishes the hypertrophic arterial remodeling caused by eNOS deficiency. In conclusion, SIRT1 prevents endothelial senescence‐induced pathological vascular remodeling and represents a promising anti‐vascular ageing drug target for the prevention of diseases such as restenosis, hypertension and atherosclerosis.

Support or Funding Information

This work was supported by the grants from Hong Kong Research Grant Council [General Research (HKU780613M and 17121714 ) and Collaborative Research Fund (C7055‐14G)].
AMELIORATIVE EFFECT OF METHANOLIC EXTRACT OF AZADIRACHTA INDICA LEAF ON LEAD ACETATE‐INDUCED CARDIOTOXOCITY, NEPHROTOXICITY AND GENOTOXICITY IN MALE WISTAR RATS
Authors: Adeyeoluwa Temitayo Esther , Adejumobi Abiola Olumuyiwa , Ola‐Davies Olufunke Eunice , Adedapo Adeolu Alex , Oyagbemi Ademola Adetokunbo , Omobowale Temidayo Olutayo
Published: November 2023

The persistent existence of lead in the environment is associated with severe health issues due to its cumulative effect and its ability to affect virtually every organ in the body. Recent discoveries have attributed the tissue damaging and inflammatory effects of lead toxicity to oxidative stress. Natural antioxidants are now being employed in the alleviation of lead‐induced toxicity and oxidative stress as they have been observed to be effective with little or no side effects associated.

This study is designed to investigate the ameliorative and antioxidative effects of Azadirachta indica on lead induced cardiotoxicity and nephrotoxicity. Cardiotoxicity and nephrotoxicity was established following oral administration of lead acetate (1mg/ml and 2mg/ml) for six weeks. However, rats were treated with 100mg/kg and 200mg/kg body weight of methanolic extract of Azadiracta indica (MEAI) after oral administration of lead acetate.

Lead acetate administration led to a significant increase in heart rate, blood pressure and markers of oxidative stress in cardiac and renal tissues. Enzymic and non‐enzymic antioxidant defense systems were increased by lead acetate toxicity, there was however a persistent increase in glutathione‐S‐transferase (GST) activity. Renal nitric oxide (NO) content was significant reduced whereas cardiac NO content increased significantly. However, treatment of rats with 100mg/kg and 200mg/kg body weight of methanolic extract of Azadiracta indica (MEAI) after oral administration of lead acetate led to improvement in antioxidant defense systems, reduction in markers of oxidative stress, restoration of blood pressure and electrocardiographic (ECG) changes respectively. Lead acetate also precipitated genotoxicity which was significantly attenuated by MEAI. The pericardial inflammation and necrosis as well as the peritubular inflammation observed in the histology of the heart and kidney tissues. This architectural anarchy was ameliorated by MEAI.

In conclusion, the results of this study show that Azadirachta indica ameliorated lead acetate‐induced cardiotoxicity and nephrotoxicity through free radical scavenging activity and antioxidant defense system. Furthermore, heart rate, systolic and diastolic pressure and other haemodynamic parameters were restored to near normal by MEAI. Hence, Azadirachta indica is a potential drug candidate against environmental pollutant such as lead acetate.
Analysis of the vasodepressor responses induced by NaHS, sodium nitroprusside and acetylcholine in rats treated with high‐fat diet
Authors: Becerril‐Chacon Maria E , Gutierrez‐Lara Erika J , Sanchez‐Lopez Araceli , Centurion David
Published: November 2023

Endothelial and vascular dysfunction is reported in isolated arteries from rats treated with high‐fat diet. However, up to date, it is unknown whether this effect is observed in the complete systemic vasculature in vivo. Thus, the aim of this study was to determine the effect of high‐fat diet on the vasodepressor responses to NaHS, acetylcholine or sodium nitroprusside in pithed rats infused with methoxamine. This experimental model allows evaluating the cardiovascular responses without the influence of the central nervous system. For this purpose, 12 animals were divided into two groups. The first group (n=6) was treated with normal diet and the second group (n=12) was treated with high‐fat diet with lard 30% during 12 weeks. Next, body weight, blood triglyceride levels as well as blood glucose and plasma insulin were determined before postprandial glucose. In both groups the blood glucose and plasma insulin were determined after administration of glucose (1 g Kg‐1, p.o.) at 5–120 min. Then, rats were: (1) anaesthetized with isoflurane; (2) pithed with a stainless steel road; (3) assisted with artificial ventilation; (4) cannulated for i.v. administration of several drugs; and (5) infused with methoxamine (20 μg Kg‐1 min‐1). The vasodepressor effects to i.v. administration of NaHS (1–10 mg Kg‐1), acetylcholine (0.03–10 μg Kg‐1) and sodium nitroprusside (0.56–18 μg Kg‐1) were determined in animals infused with methoxamine. High‐fat diet produced increases in body weight without significant changes in basal blood glucose, basal plasma insulin or HOMA‐index or blood triglycerides. However, high‐fat diet significantly decreased glucose tolerance after administration of 1 g Kg‐1 glucose (p.o.) while plasma insulin levels remained unchanged. Furthermore, i.v. administration of NaHS, acetylcholine and sodium nitroprusside elicited dose‐dependent vasodepressor responses in either animals treated with normal diet or high‐fat diet. These responses were not significantly different in both groups. The above results suggest that high‐fat diet induced obesity but not insulin resistance and produced no significant changes in the vasodepressor responses to the above drugs in systemic vasculature.
Increased Coronary Artery Dissection Rate and Severity in Murine Model of Marfan Syndrome
Authors: Milad Nadia , Sellers Stephanie L , Seidman Michael , Bernatchez Pascal N
Published: November 2023

Purpose

Marfan syndrome (MFS) is an autosomal dominant genetic disorder that leads to a variety of clinical presentations ‐ most seriously, aortic root dissection and aneurysm. Spontaneous coronary artery dissection, although rare in the general population, is associated with high rate of mortality and occurs more frequently in MFS patients due to their characteristic arterial wall weakness. MFS patients are often prescribed anti‐hypertensives such as Losartan, an angiotensin II type 1 receptor (ATR1) antagonist, but recent clinical trials have cast doubt on their effectiveness in preventing clinical endpoints. We investigated the rate and severity of coronary artery dissection in a murine model of MFS compared to wild‐type mice.

Methods

Experimental mice were bred with littermate controls using the common Fbn1C1039G/+ model of MFS giving rise to wild‐type and MFS groups. At 6 or 22 months of age, hearts were fixed, sectioned and stained with hematoxylin and eosin, Movat's pentachrome and iron stains in order to visualize coronary artery dissection. Dissection was scored using a binary system (dissection or no dissection) and a severity‐weighted system (0 represents no dissection, 1 ‐ slight dissection, 2 ‐ clear dissection, 3 ‐ severe dissection).

Results

At 6 months of age, we saw an unexpectedly high rate of mild coronary artery dissection in wild‐type mice compared to MFS alone (100% in wild‐type vs. 50% in MFS) but when weighted for severity there was no difference between groups (1.0 for wild‐type vs. 0.88 in MFS). However at 22 months of age, the MFS group displayed a higher rate of dissection compared to wild‐type (80% vs. 33%) as well as an increase in dissection severity (1.6 vs. 0.33, respectively).

Conclusions

To our knowledge, this is the first genetic mouse model of coronary artery dissection. The unexpectedly high rate of mild dissection in wild‐type mice at 6 months of age (all were scored 1 for slight dissection) may be due to low N values at this time‐point or may be part of normal vessel maturation, unfortunately little study on coronary artery dissection in mice has been conducted. However, the observed increase in both rate and severity in MFS mice at 22 months compared to wild‐type is likely due to vessel wall fragility and elastic fibre fragmentation characteristic of MFS which left coronary arteries susceptible to further damage. Future research into the effects of Losartan treatment on rate and severity of coronary artery dissection should be done.

Support or Funding Information

This research was funded by CIHR grant awarded to Dr. Pascal N Bernatchez.
Decreased Interleukin‐6 Signaling in Female Mice Early Post‐Myocardial Infarction Attenuates Neutrophil Infiltration and Limits Cardiac Dilation and Rupture
Authors: DeLeon‐Pennell Kristine , Iyer Rugmani Padmanabhan , Ma Yonggang , Yabluchanskiy Andriy , Lindsey Merry L
Published: November 2023

Survival after myocardial infarction (MI) is improved in female compared to male mice, yet the mechanisms remain undefined. We hypothesized lower acute inflammation in female mice leads to improved survival and cardiac function post‐MI. We used our Mouse Heart Attack Research Tool (mHART) 1.0 database that has 888,000 data points. We queried the database and found >350 3–7 month C57BL/6J mice that qualified for this study. In addition to improved survival, rupture of the left ventricle (LV; rupture/total deaths) was also reduced in females (11%; 1/9) versus males (46%; 16/35; p<0.05). Out of 52 plasma analytes measured at day (D)0‐D7 post‐MI (n≥10/sex/day), interleukin (IL)‐6 positively correlated with end diastolic dimension (EDD) in males only (R2=0.47; p<0.05). Female IL‐6 levels were significantly lower at D1 and D3 compared to males (all p<0.05). LV dilation was decreased in females (EDD/LV mass index=1318 ± 67 mm/mg) versus males (1487 ± 48 mm/mg; p<0.05; n≥10/sex) at D7 post‐MI. Bioinformatic analysis of 168 genes at D1 post‐MI identified IL‐6ra, the IL‐6 receptor, as the top feature linked to sex differences (p<0.05; n≥8/sex). IL‐6ra was decreased 2‐fold in females versus males. Since IL‐6 regulates neutrophil infiltration, we assessed neutrophil numbers; there was a 2‐fold reduction in females compared to males at D1 and D3 post‐MI (both p<0.05; n=6/sex/day). In conclusion, utilizing mHART 1.0, females had decreased neutrophil infiltration, as well as LV dilation and rupture, possibly due to reduced IL‐6 signaling.

Support or Funding Information

This work was supported by AHA [14POST18770012 to RPI and 15SDG22930009 to YM], and by NIH [HHSN 268201000036C (N01‐HV‐00244) for the San Antonio Cardiovascular Proteomics Center, U54GM114833 for the UCLA Center of Excellence for Big Data, and R01HL075360 to MLL] and from the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award [5I01BX000505 to MLL], and by HL051971 and GM104357.
Non‐Resolving Chemokine Response Dysregulates Cardiosplenic And Cardiorenal Network Following Myocardial Infarction In Aging
Authors: Halade Ganesh V , Kain Vasundhara , Ingle Kevin , Black Laurence M
Published: November 2023

Chemokine signal drives the innate inflammatory response following myocardial infarction (MI), though how it provokes non‐resolving chronic inflammation in aging is unclear. To answer this, we hypothesized that resolution reprograms, specifically proresolving lipid mediators formed in innate immune response, may be dysregulated in aging post‐MI, influencing cardiorenal and cardiosplenic network. Young (6 months) and aging (18 months) mice were fed standard lab chow (LC) and safflower oil (SO)‐enriched diet for 2 months and were then subjected to coronary artery ligation in order to induce MI. Despite similar infarct areas and reduced fractional shortening post‐MI, splenic mass spectroscopy lipid profiling revealed higher levels arachidonic acid (AA) derived pro‐inflammatory metabolites, but minimal formation of eicosanoids, docosanoids D‐ and E‐ series resolvins in SO‐fed group compared with LC group. SO fed aging mice displayed higher levels of pro‐inflammatory thromboxanes and tetranor‐12‐HETEs metabolites (p<.0.5) compared to adult‐SO fed mice. Data suggests that aging decreases the expression levels of 12‐lipoxyegenase (LOX) and 15‐LOX and 5‐LOX (all p<0.05) in infarcted left ventricle compared to young LC fed mice, but increased in kidney and spleen post‐MI. Excess intake of SO decreased formyl peptide receptor 2 (FPR2) compared to young SO‐fed mice, indicating non‐resolving response Increased SO intake also caused higher NGAL expression, suggesting kidney injury with LC group. The decreased expression of Arg‐1 in response to MI suggested the delayed healing in aging mice compared to young mice. In summary, aging magnifies the post‐MI chemokine‐driven innate response and impairs the healing response by stimulating prolonged neutrophil trafficking and proinflammatory lipid mediators.

Support or Funding Information

The authors acknowledge support from NIH‐NCCAM K99/R00 AT006704 and UAB start‐up funds, awarded to GVH.
Pericytes Improve Motor Recovery After Spinal Cord Injury in Both Sexes
Authors: Mayo Jamie N. , Bearden Shawn E.
Published: November 2023

Objective

New capillary growth can improve recovery after spinal cord injury (SCI). Pericytes, microvascular cells, can be stimulated in vitro to induce new capillary growth. The hypothesis was tested that stimulated pericytes would improve motor recovery in both sexes in a pediatric model of SCI.

Methods

Human placental pericytes and pericytes stimulated with cobalt chloride were injected into the site of a thoracic hemi‐section of the spinal cord in rat pups on postnatal day three (P3). Hindlimb motor recovery (assessed using the Basso, Beattie, and Bresnahan motor score scale) and Doppler blood flow perfusion at the site of transection were measured on P10. Immunohistochemistry was used to quantify vessel and neurofilament density on P10.

Results

Pericyte, but not stimulated pericyte, treatment improved motor recovery in both sexes (males, 2.722 ± 0.31 fold score improvement; females, 3.824 ± 0.58 fold score improvement, p < 0.05). Pericyte treatment supported a stable, flow regulated vascular bed in both sexes and greater neurofilament density (males, 1.9 ± 0.09 fold increase; females, 2.031 ± 0.1553 fold increase, p < 0.05). There were significant effects of treatment and interactions between treatment and sex in vessel density after SCI (p < 0.05). In the vehicle control group, females had a higher vessel density seven days after injury than males (p < 0.05). This pattern persisted following pericyte injection (p < 0.05), and the average vessel density was not different than their sex‐matched controls (p > 0.05). With stimulated pericyte injection, males had a greater vessel density then their sex‐matched controls (p < 0.05) and no longer had lower vessel density then females. Sex was a significant source of variation (p < 0.05) for endothelial proliferation. Within the vehicle control group there was less endothelial proliferation in female tissue than there was in male tissue (p < 0.05).

Conclusions

Pericytes and stimulated pericytes interact differently with the host environment after SCI. Injected directly after injury, naïve pericytes as opposed to stimulated pericytes have more potential to improve motor recovery. It appears that either the timing of or capacity for the initiation of an angiogenic program following CNS injury might differ between males and females, even before sexual maturity. Additionally, because female rats had greater vessel density than males but lesser endothelial proliferation it is possible that females may be better able to maintain vessel viability after initial injury. The results from this study highlight the importance of appreciating that even when males and females reach the same gross functional end measurements, they might do so by different structural and molecular mechanisms.

Support or Funding Information

HL106548, P20 GM103408
Regional Differences in Placental and Myoendometrial Vascularization and Placental and Fetal Development in Hypertensive‐Pregnancy
Authors: Dias‐Junior Carlos Alan , Ren Zongli , Zhu Minglin , Li Jun , Khalil Raouf A.
Published: November 2023

Preeclampsia (PE) is a form of hypertensive‐pregnancy (HTN‐Preg) with unclear mechanism. General reduction of uterine perfusion pressure (RUPP) could be an initiating event leading to uteroplacental ischemia, abnormal angiogenic/anti‐angiogenic responses and HTN‐Preg. Additional regional differences in placental blood flow could further affect the pregnancy outcome and may be a factor in the increased risk of PE in twin or multiple pregnancy. To test the hypothesis that regional differences in uterine perfusion could affect uteroplacental vascularization and placental and fetal development, we compared the number and size of pups and placentae, and the number of placental and adjacent myoendometrial arteries in the proximal (prox‐U), middle (mid‐U) and distal (distal‐U) portions of the uterus (in relation to distance from the ovarian arteries) in normal pregnant (NP) and RUPP rats. Maternal blood pressure was higher (127±2 vs. 105±4mmHg), and uterine weight (3.2±0.1 vs. 4.6±0.2g), number of placentae (9.5±0.7 vs. 12.6±0.5), placenta weight (401±5 vs. 456±4mg), litter size (9.5±0.7 vs. 12.6±0.5), and pup weight (1.18±0.05 vs. 1.59±0.04g) were less in RUPP than NP group. Further examination of the RUPP uterus showed a decrease in the number and weight of placentae and pups in distal‐U (0.4±0.3, 210±120mg; 0.4±0.3, 1.16±0.51g) vs. prox‐U (1±0, 400±175mg; 1±0, 1.67±0.49g) and mid‐U (0.6±0.5, 337±184mg; 0.6±0.5, 1.58±0.46g). The number and diameter of placental arteries were smaller in distal‐U (2.6±0.5, 70±8μm) vs. prox‐U (5.0±0.4, 105±4μm) and mid‐U (5.0±0.5, 95±7μm) of RUPP rats. Also, the number of the adjacent myoendometrial arteries was less in distal‐U (1.3±0.04) vs. prox‐U (3.5±0.06) and mid‐U (2.5±0.04) of RUPP rats. No significant differences were observed in placentae and pups number and weight or number of placental and myoendometrial arteries in different uterine regions of NP group. Thus, in addition to the general reduction in placental and fetal development observed during uteroplacental ischemia, there is a greater decrease in placental and myoendometrial vascularization associated with greater placental and fetal growth restriction in distal‐U, suggesting regional differences in blood flow, placental perfusion and angiogenic responses in HTN‐Preg that could be a factor in the incidence of PE in multiple pregnancy.

Support or Funding Information

This work was supported by grants from National Heart, Lung, and Blood Institute (HL‐65998,HL‐111775).
ET‐1 and Nitric Oxide Stimulate Trans‐Epithelial Electrical Resistance (TER) in Inner Medullary Collecting Duct Cells
Authors: Arguello Alexandra M , Hyndman Kelly A , Goodchild Traci T , Pollock Jennifer S
Published: November 2023

In the renal collecting duct, it is the epithelial Na channel (ENaC) that is the rate‐limiting step for Na reabsorption. We recently reported, that collecting duct nitric oxide synthase‐1 (NOS1) is necessary for endothelin‐1 (ET‐1) dependent inhibition of ENaC activity. Loss of either ET‐1 or NOS1 expression specifically in the collecting duct generates a salt‐sensitive blood pressure phenotype. In the kidney, it is the IMCD that expresses the highest NOS activity in the kidney, and produces the most ET‐1 throughout the body. Previous studies demonstrating ENaC regulation in the collecting duct have utilized patch‐clamp electro‐physiology in the cortical collecting duct, however the inner medullary collecting duct (IMCD) is technically difficult to patch clamp. Thus, the purpose of this study was to utilize TER, which is inversely related to ion transport, to determine the effect of ET‐1 and NOS inhibition on IMCD ENaC activity. TER was measured with a volt‐ohmmeter and STX2 electrode. Transwell cell culture inserts were used, allowing mouse or rat IMCD cells to polarize. Both rat primary IMCD and mouse mIMCD‐3 exhibited amiloride‐sensitive ENaC inhibition. Amiloride (1 uM) increased TER (decreased ENaC activity) in rat IMCD cells (+5.27 ± 0.63%) and mIMCD‐3 cells (+9.53 ± 1.33%) compared to vehicle controls (+0.29 ±0.19%). ET‐1 (100 nM) stimulated a significant increase in TER for rat IMCD (+2.66‐‐ ± 0.23%) and mouse mIMCD‐3 cells (+3.25 ± 0.62%−) compared to vehicle, indicating an inhibition of ion transport. NOS inhibition (5 mM L‐NAME) also stimulated a significant decrease in TER in rat IMCD (−3.50 ± 0.5%) and mouse mIMCD‐3 cells (−8.35 ± 1.20%). The decrease in resistance, and therefore increase in ion transport, with NOS inhibition could not be reversed with the addition of ET‐1, indicating that the NOS system contributes to the tonic inhibition of ion transport in the IMCD. In conclusion, these data support the hypothesis that the ET‐1 and NOS systems regulate ion transport, most likely ENaC, in the IMCD of rats and mice.

Support or Funding Information

NIHP01HL95499 and The University of Alabama at Birmingham Preparation for Graduate and Medical Education Program (PARADiGM) R25 HL120883
Impaired renal oxygenation in obese Zucker rats following orthopedic trauma
Authors: Xiang Lusha , Thompson Michael S , Mittwede Peter N
Published: November 2023

Wepreviously found increased acute kidney injury (AKI) following orthopedictrauma in obese (OZ) as compared to lean Zucker rats (LZ), in agreement with results reported in lean and obese trauma patients. Impaired renal oxygenation due to elevated oxygen consumption (VO2) and/or decreased oxygen delivery(DO2) has been shown to be an important mechanism of AKI in in many critical illnesses, but few studies have investigated the mechanisms of AKI following orthopedic trauma, particularly in obesity. We hypothesized that the increased AKI in OZ following orthopedic trauma is associated with impaired renal oxygenation. Soft tissue injury, fibula fracture, and bone component injection were performed in both hind limbs in male LZ and OZ (~12wk old) to induce orthopedictrauma. A day later, food intake, circulating aldosterone, renal blood flow (RBF), GFR, and renal medullary glycogen levels were measured. Renal DO2 and VO2 were calculated from renal arterial and venous blood gases. Sodium (Na+) reabsorption was calculated based on GFR, plasma and urine [Na+], and urine output. Following trauma, RBF, GFR, Na+ filtration, renal DO2, and medullary glycogen levels were decreased in OZ but not in LZ. Food intake was markedly decreased in both LZ and OZ, which was associated with significantly increased Na+ reabsorption and aldosterone levels. However, compared to LZ, OZ exhibited lower Na+ uptake but higher aldosterone levels, renal VO2, and oxygen extraction ratio (VO2/DO2). These results suggest that trauma‐induced AKI in OZ is associated with impairments in renal oxygenation and the efficient oxygen utilization.

Support or Funding Information

Grants: AHA‐12SDG12050525, National Institutes of HealthP20GM104357, HL‐51971, HL‐89581, and T32 HL‐105324.

DoD Disclaimer: The opinions orassertions contained herin are the private views of the author and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.
Development of Renal Dysfunction in an Infant Model of Polymicrobial Sepsis
Authors: Sims Clark R , Gurley Patrick C , Mayeux Philip R
Published: November 2023

Sepsis‐induced acute kidney injury increases mortality from 25% to nearly 45% in infants. We used a clinically relevant cecal ligation and puncture (CLP) sepsis model in rat pups 17–19 days old (developmentally comparable to a 5 to 6‐month‐old human infant) to characterize the development of renal dysfunction. Renal injury was assessed using intravital video microscopy (IVVM), blood urea nitrogen (BUN), and glomerular filtration rate (GFR) at 4, 6, and 18h following CLP. Temperature was measured as an indicator of Systemic Inflammatory Response Syndrome. Temperature was decreased as early as 6h following CLP and remained decreased through 18h. Lactate, an indicator of general organ hypoperfusion and hypoxia, was nearly doubled as early as 4h following CLP and remained elevated through 18h. IVVM analysis of the renal microcirculation showed a significant decrease in the percentage of continuously flowing capillaries (72% to 36%) and an increase in the percentage of capillaries with no flow (2% to 26%) at 6h following CLP that persisted through 18h. Clinically, measurement of BUN is used to assess renal function when GFR is difficult to measure. In this model, BUN was significantly increased by 4h following CLP and was increased even further by 18h. GFR is the gold standard measurement of renal function and it was measured using non‐invasive transcutaneous measurements of sinistrin clearance in conscious rat pups. GFR was decreased 50% as early as 4h following CLP and remained decreased through 18h. These findings show that this model of polymicrobial sepsis produces early and sustained renal dysfunction suggesting it could be used in preclinical studies to evaluate new targeted therapies for sepsis‐induced renal injury in infants.

Support or Funding Information

CRS supported by T32 GM106999, F31DK104533, AHA PRE20450050; PRM supported by AHA 15GRNT2508025 and UAMS Medical Research Foundation; PCG supported by ASPET Institutional SURF Award 2015.
Muc1 Modulates the Beta‐Catenin Protective Pathway During Ischemia‐Reperfusion Injury
Authors: Al‐bataineh Mohammad M , Kinlough Carol L , Pastor‐Soler Nuria M , Sutton Timothy A , Poland Paul A , Mang Henry E , Gendler Sandra J , Madsen Cathy S , Singh Sucha , Bastacky Sheldon I , Monga Satdarshan P , Hughey Rebecca P
Published: November 2023

The HIF‐1 and β‐catenin protective pathways represent the two most significant cellular responses that are activated in response to acute kidney injury. We previously reported that murine Mucin 1 (Muc1) protects kidney function and morphology in a mouse model of ischemia‐reperfusion injury (IRI) by stabilizing HIF‐1α, enhancing HIF‐1 downstream signaling and thereby preventing metabolic stress (Pastor‐Soler et al. 2015 Am. J. Physiol. Renal Physiol. 308, F1452‐F1462). We asked if Muc1 regulates the β‐catenin protective pathway during IRI as published data indicate that (i) β‐catenin is found in co‐immunoprecipitates with human MUC1 in extracts of both cultured cells and tissues, (ii) β‐catenin nuclear targeting is MUC1‐dependent in cultured human cells, and (iii) MUC1 prevents β‐catenin phosphorylation by glycogen synthase kinase 3β (GSK3b) and thereby β‐catenin degradation. Using our same mouse model of IRI, and by using immunohistochemistry and immunoblotting of kidney tissue, we found that levels of active GSK3β were significantly lower in kidneys of control mice compared to Muc1 KO mice. Consequently, β‐catenin was significantly increased at 24 and 72 h recovery and appeared in the nuclear fraction at 72 h in control mouse kidneys. Both β‐catenin induction and nuclear targeting were absent in Muc1 KO mice. In accord with these data, we also found downstream induction of β‐catenin pro‐survival factors (activated Akt, survivin, transcription factor TCF4 and its downstream target cyclin D1) and repression of pro‐apoptotic factors (p53, active Bax and cleaved caspase 3) in control mouse kidneys that were absent or aberrant in kidneys of Muc1 KO mice. Altogether, the data clearly indicate that Muc1 protection during AKI proceeds by enhancing both the HIF‐1 and β‐catenin protective pathways.

Support or Funding Information

This work was supported by National Institutes of Health (NIH) Grants DK‐099345 (to T. A. Sutton), DK‐084184 (to N. M. Pastor‐Soler), CA‐64389 (to S. J. Gendler), DK‐62277, DK‐100287, and DK‐095498 (to S. P. Monga), DK‐097889 (to M. M. Al‐bataineh), DK107632, DK‐079307 (Pilot Project) and Samuel and Emma Winters Foundation grant (to R. P. Hughey), and P30‐DK079307 (to the Microscopy Core N. M. Pastor‐Soler and S. Bastacky).
Role of Nox4 and p67phox in ROS production in response to increased tubular flow in the mTAL of Dahl salt‐sensitive (SS) rats
Authors: Zheleznova Nadezhda N , Yang Chun , Cowley Allen W
Published: November 2023

Reactive Oxygen Species (ROS) in the renal outer medulla play an important role in the development of salt‐sensitive hypertension. NADPH oxidases (Nox) are the major source of ROS production in the kidney. We have examined the contribution of Nox4 and p67phox, related to the most abundant isoforms of NADPH‐oxidase Nox4 and Nox2 in the kidney, to the ROS production in response to sodium delivery and tubular flow in medullary thick ascending limb (mTAL) isolated from Dahl salt‐sensitive (SS) rats. We have shown in our previous studies that SSNox4−/− and SSp67phox−/− mutant rats exhibited a significant reduction of salt‐induced hypertension compared to SS rats after 21 day of 4.0% NaCl and a large reduction of albuminuria, tubular casts, and glomerular injury. In this study we have shown that increase in mTAL luminal flow stimulates cellular hydrogen peroxide (H2O2) and superoxide (O2·−), as well as mitochondrial H2O2 production in SS rats indicated by changes in fluorescence of sensitive dyes for detection of ROS (PF6‐AM, DHE, Mito‐PY1). In the present study we have determined that deletion of Nox4 (SSNox4−/−) resulted in significant reduction of intracellular H2O2 compared to SS in response to increased luminal flow in mTAL isolated from rats fed a 4% NaCl diet for 3 days. Deletion of the p67phox subunit in SS rat (SSp67phox−/−) also decreased H2O2 production compared to SS but to a lesser extent than that observed in mTAL of Nox4 rats (SSNox4−/−). There were no differences between cellular O2·− production in mTAL of SSNox4−/− rats compared to SS. However there was a significant decrease in cellular O2·− production in mTAL of mutant SSp67phox−/− rats. We conclude from this study that Nox4 is a major player in the production of cellular H2O2 in response to increased mTAL tubular flow of the SS rat whereas the p67phox subunit of Nox2 is involved in cellular O2·− production. Neither Nox4 nor the p67phox subunit of Nox2 seems to contribute to the mitochondrial H2O2 production in response to increased mTAL luminal flow.

Support or Funding Information

HL 122662HL 082798
Angiotensin II Type 2 Receptor Agonist C21 Reduces Kidney Damage And Improves Renal Tubular Functions In High Sodium Diet‐Fed Obese Zucker Rats
Authors: Patel Sanket Niranjanbhai , Hussain Tahir
Published: November 2023

Salt‐sensitivity has been implicated in chronic kidney damage (CKD) and impairing renal function, particularly in obesity. Earlier we have reported blood‐pressure and oxidative stress reducing effects of angiotensin AT2 receptor (AT2R) agonist C21 in kidney of high sodium diet (HSD)‐fed obese Zucker rat (OZR). Present study tested the effects of C21 on indices of renal function in HSD‐fed OZR. Obese rats were fed normal sodium diet (NSD) or 4% HSD for 14 days, with/without C21 (1 mg/kg/day) delivered subcutaneously via osmotic pump. Compared to NSD controls, HSD rats exhibited severe glomerulosclerosis, interstitial fibrosis, decline in estimated glomerular filtration rate (GFR), and an increase in urinary leak and activity of N‐acetyl‐β‐D‐glucosaminidase, a lysosomal enzyme and a marker of tubular damage, which were all improved by C21. C21 treatment reduced protein‐to‐creatinine (uPcr), albumin‐to‐creatinine (uAcr) as well as fractional excretion of protein (FEpro) and albumin (FEalb) in HSD‐fed OZR, which is independent of changes in protein recycling receptors – megalin and cubilin. HSD intake has also altered renal excretory and reabsorptive capacity as evident by elevated plasma urea nitrogen‐to‐creatinine (UN‐to‐cr) and fractional excretion of urea nitrogen (FEUN), and reduced urine‐to‐plasma creatinine (UPcr), which were modestly, but insignificantly, improved by C21. Collectively, we conclude that AT2R agonist C21 reduced glomerular protein leak and preserved lysosomal function and thus reduced exposure of nephrotic proteins to tubular lumen suggesting a potential role of AT2R in protecting glomerulus and renal tubules during HSD intake in obesity.

Support or Funding Information

Acknowledgement: This study was supported by NIH R01 grant DK61578 to TH
Renal‐tubular NEDD4‐2 action is fundamental for renal adaptation during potassium deficiency
Authors: Staub Olivier , Basquin Denis , Roy Ankita , Stifanelli Matteo , Rajaram Renuga Devi , Maillard Marc , Subramanya Arohan R , Al‐Qusairi Lama
Published: November 2023

The adaptation to K+ deficiency requires high coordination between organs involved in K+ homeostasis including kidney, muscle and liver. However, how the kidney performs functional and molecular changes to ensure K+ retention is not well understood. Here, we show that the ubiquitin‐protein ligase NEDD4‐2, known as a negative regulator of ENaC, NCC and more recently WNK1, is essential for the adaptation of the kidney to potassium deficiency. Our analyses revealed that the induciblerenal‐tubular NEDD4‐2 (NEDD4‐2Pax8/LC1) mice have normal K+homeostasis when challenged by high K+ (HKD) or low K+ (LKD)diet for one week. Upon longer exposure to LKD (2 weeks), mutant mice exhibited hypokalemia together with increased urinary K+ loss. Under the sehypokalemic conditions, increase of the large, Ca2+‐activated K+channel BK and decrease of ROMK protein levels and membrane localization are observed in NEDD4‐2Pax8/LC1 mice. SPAK‐mediated NCC phosphorylation was enhanced in the knock‐out animals with no change in total NCC. Moreover, the mutant mice showed increased WNK1 signaling in the CNT and the CD, which may explain these deregulations. In addition, ENaC expression and activity were both enhanced in the mutant mice. Interestingly, IP injection of Benzamil, anENaC inhibitor, restored K+ loss in NEDD4‐2Pax8/LC1 mice. Altogether, our data strongly suggest that NEDD4‐2plays an essential role in K+ retention via coordination of K+ channels including BK and ROMK on the one hand and of Na+ transporters including ENaC and NCC on the other hand.

Support or Funding Information

Swiss National Science Foundation grant 310030_159735 (to OS), Swiss Kidney.ch NCCR (to OS), National Institutes of Health grant DK098145 (to ARS). LAQ was supported by a fellowship of the Marie Curie Co‐funding IKPP (International Fellowship Program on Integrative Kidney Physiology and Pathophysiology).
Histamine stimulates mucus secretion through IL‐4 induced TMEM16A in human nasal epithelium
Authors: Cho Hyung‐Ju , Lee Yong Hyuk , Kim Chang‐Hoon , Yoon Joo‐Heon
Published: November 2023

Background

Histamine released from mast cells is an important mediator of allergic reaction, and mucus hypersecretion is a major allergic symptom.

Objective

We studied whether histamine directly induces mucus secretion from epithelial cells or submucosal glands (SMG) in human and pig nasal epithelium.

Methods

The short circuit current (Isc) change in response was determined in cultured primary human nasal epithelial (HNE) cells using Ussing chamber experiment. Freshly excised nasal mucosae were harvested from patients who were classified into AR and non‐AR group to quantify mucus secretion from individual SMGs using bubble method. The expression of H1R, H2R, and H3R in human nasal turbinates was identified using in situ hybridization. Pig nasal and tracheal tissues were also investigated.

Results

Histamine induced a modest alteration of Isc in HNE cells with increasing intracellular calcium but no gland secretion. Pretreatment of IL‐4 augmented histamine response that was suppressed by TMEM16A inhibitor. The expression of H1R, H2R, and H3R was identified in human nasal epithelium by in situ hybridization. However, H1R or H2R was not noted in SMG. In pig airway tissues, histamine was a potent secretagogue that directly induced robust responses, indicating a species‐difference from human.

Conclusion

H1R agonist induces modest anion secretion under normal condition but the response was enhanced under IL‐4 stimulation that might be related to mucus hypersecretion in allergic rhinitis. We first elucidated histamine‐induced mucus secretion in synergy with IL‐4 through TMEM16A in human nasal epithelium.

Support or Funding Information

Supported by grants NRF‐2013R1A1A 1010151 to H. J. Cho from the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education, Science and Technology.
Characterizing TRPA1 and TRPV1 in Isolated Mouse Cardiac Myocytes
Authors: Andrei Spencer , Sinharoy Pritam , Bratz Ian N. , Damron Derek S
Published: November 2023

Rationale

Transient receptor potential channels of the ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) subtypes are extensively coexpressed in dorsal root ganglions and other heterologous systems; however, expression, localization and function of these ion channels within primary isolated adult mouse ventricular cardiomyocytes (MVCs) has not been established.

Objective

Since the localization of TRPA1 and TRPV1 on cardiomyocytes has not been reported, we aimed to determine the expression patterns of the ion channels on MVCs and further studied channel‐specific agonistic effects on intracellular free calcium concentrations.

Methods and Results

Western blot, immunocytochemistry and intracellular free calcium measurements were used to determine localization and function of the ion channels. Immunoblot analysis of MVCs revealed the presence of TRPA1 and TRPV1 at protein levels. Moreover, TRPA1 and TRPV1 were coexpressed at the Z‐discs, costameres and intercalated discs, which was demonstrated with immunocytochemistry using α‐actinin (Z‐disc marker) and vinculin (costamere/intercalated disc marker). Furthermore, both allyl isothiocyanate (AITC), a TRPA1 agonist, and capsaicin, a TRPV1 agonist, dose‐dependently increased intracellular calcium influx. Real‐time measurements of intracellular free calcium concentrations revealed that AITC and capsaicin elicited transient influxes of calcium on cardiomyocytes stained with Fura‐2AM.

Conclusions

In summary, these findings demonstrate functional TRPA1 and TRPV1 ion channels on MVCs.

Support or Funding Information

This work was supported by departmental funds and grants from NIH HL60571 to D.D.
KCa3.1 Inhibition Decreases Integrin/Extracellular Matrix Interaction in Vascular Smooth Muscle
Authors: Tharp Darla L. , Sun Zhe , Meininger Gerald A. , Bowles Doug
Published: November 2023

We and others have shown intermediate‐conductance, Ca2+‐activated K+ channel (KCa3.1) activation contributes to vascular smooth muscle cell (VSMC) migration. VSMC migration necessitates dynamic changes in integrin‐ECM interaction, including focal adhesion turnover, though little is known regarding the mechanical properties of the integrin/ECM interaction in VSMC and how it is regulated when cells are stimulated with a pro‐migratory agonist. We tested the hypothesis that KCa3.1 activation alters matrix‐integrin adhesion. We used atomic force microscopy (AFM) to determine the binding properties of fibronectin (FN)/α5β1‐integrin and collagen I/ α1β1‐integrin in mouse aortic VSMC (MAVSMC). Sub‐confluent MAVSCM were serum‐starved overnight and treated with PDGF‐BB (10 ng/ml) with and without the KCa3.1 inhibitor, TRAM‐34 (100 nM). AFM probes with a FN‐ or collagen I‐coated beads were brought into contact with the cell surface for 2 min and the probes subsequently pulled away from the cell surface in the z‐axis. With FN coated‐beads, PDGF‐BB increased total force required to break adhesion of bead with the MAVSMC compared to control (2539 ± 705 vs. 1093 ± 114 pN) indicative of increased adhesion to FN. This increase was inhibited by TRAM‐34 and reduced to control levels (1068 ± 182 pN). With collagen I coated‐beads, PDGF‐BB had no effect on the force required to break adhesion with the MAVSMC compared to control (1036 ± 263 vs. 1207 ± 237 pN), however TRAM‐34 did reduce adhesion to collagen I compared to control (636 ± 142 pN). Thus, KCa3.1 activation stimulates acute integrin/ECM interaction in an integrin and ECM specific manner in response to PDGF‐BB.
Respiratory Adaptations to High‐Altitude Hypoxia in Deer Mice (Peromyscus maniculatus)
Authors: Ivy Catherine , Scott Graham
Published: November 2023

The unremitting hypoxia at high altitudes challenges small endotherms to extract enough oxygen from the environment to survive, exercise, and generate enough heat for thermoregulation. We compared the control of breathing and pulmonary gas‐exchange between highland and lowland populations of deer mice to determine the physiological specializations important for living in high‐altitude hypoxia. Mice were acclimated to normoxia or hypobaric hypoxia (simulating hypoxia at ~4300 m elevation) for 4–5 months. The hypoxic ventilatory response (HVR) was measured using whole‐body and restraint plethysmography in response to step‐wise decreases in inspired O2 fraction. Hypoxia acclimation enhanced the HVR in lowlanders, such that total ventilation and arterial saturation were higher across a range of inspired O2. Hypoxia acclimation also made breathing pattern more effective in lowlanders, as reflected by higher tidal volumes and lower breathing frequencies at a given total ventilation, and it allowed lowlanders to maintain higher heart rates in deep hypoxia. In contrast, hypoxia acclimation had no effect on these traits in highlanders, who exhibited a fixed HVR and breathing pattern that was similar to hypoxia‐acclimated lowlanders, and highlanders also maintained higher arterial saturations in hypoxia than lowlanders. In contrast, hypoxia acclimation similarly enhanced the hypercapnic ventilatory response (measured in normoxia during step‐wise increases in inspired CO2, from 0% to 6%) in both populations. Therefore, ventilatory acclimatization to hypoxia is absent in highland deer mice, which have a fixed HVR that is effective for gas exchange, through a mechanism that appears independent of evolved changes in the plasticity of CO2 chemosensitivity.

Support or Funding Information

Supported by NSERC of Canada.
Untargeted Metabolomics Reveal State‐Dependent Neurochemical Changes in Frontal Association (FrA) and Motor Cortex (M1) of C57BL/6J (B6) Mouse
Authors: Bourdon Allen K. , Spano Giovanna , Bellesi Michele , Tononi Giulio , Cirelli Chiara , Serra Pier Andrea , Baghdoyan Helen A. , Lydic Ralph , Cammpagna Shawn R.
Published: November 2023

Neurochemical monitoring using targeted and untargeted metabolomic approaches can provide unique insights into changes in brain chemistry that occur normally, in response to pathology, and after therapeutic intervention. This study used an established untargeted metabolomics method (Anal. Chem., 82:3212, 2010) to identify known and unknown brain metabolites by means of ultra‐performance liquid chromatography coupled with high‐resolution mass spectrometry (UPLC‐HRMS) on an Exactive Plus Orbitrap Instrument (Thermo Fisher Scientific). To date, analyses have been performed on microdialysis samples (n=408) obtained from FrA and M1 of intact, behaving B6 mice (n=5). Microdialysis samples were collected in conformance with the FASEB Statement of Principles for the use of animals in research during electroencephalographically confirmed intervals of extended wakefulness, recovery sleep, and subsequent extended wakefulness. Partial least squares discriminant analyses showed reliable and statistically significant state‐dependent clustering of metabolites. In addition to identifying approximately 20 known molecules, analyses revealed consistent associations with the first wakefulness period (acetoacetate and lactate), sleep (valine and leucine/isoleucine), and the second wakefulness period (pyruvate and tyrosine). The results also provided a list of unidentified spectral features in FrA (n=33) and M1 (n=43) that are being subjected to additional analyses using the metabolite databases (https://metlin.scripps.edu/index.php and http://www.hmdb.ca). These spectral features may ultimately lead to the discovery of novel molecules not previously known to be associated with states of sleep and wakefulness. By integrating the dynamic sampling tool of microdialysis with the unparalleled capabilities of UPLC‐HRMS, these studies are identifying and quantifying state‐dependent and region‐specific changes in mouse brain metabolome.

Support or Funding Information

NIH grants HL65252 (RL and HAB); MH099231 and 1P01NS083514 (CC and GT); and support from the UT Joint Institute for Biological Sciences.