bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in Cerebral Blood Flow Regulation in Male and Female Rugby Players with Concussions
Authors: Serrador Jorge , Reyes Levy , Tosto Jenna , Falvo Michael
Published: November 2023

Increasing evidence is suggesting that concussions are a possible significant cause of long term cognitive and health problems among athletes. Despite this increasing evidence, there is a lack of data on the physiological effects of a concussion on cerebral blood flow regulation. The goal of this work was to determine if concussion cause impairment of cerebral blood flow regulation in the first few hours after injury and to determine if there is a sex difference in there sponse.

During are creational rugby tournament a total of 91 players were recruited. Of these players, 59 were recruited as controls (12 females) and 34 had suffered a concussion (8 females). All testing was performed on the field to assess cerebral blood flow using Doppler, beat‐by‐beat blood pressure, and end‐tidal CO2. Subjects performed three sit to stand. Data was collected 131±23 min following the head trauma.

We had previously found that concussed players demonstrated greater mean arterial pressure than controls. Both males and females demonstrated greater mean arterial pressures in the concussed players (Males – Ctrl 91±1 vs mTBI 96±2; Females Ctrl 82±3 vs mTBI 90±3 mmHg, P=0.004), with no effect of sex. In contrast cerebral flow velocity while standing tended to be more severely impacted in the females players (Males – Ctrl 95±1 vs mTBI 95±1; Females Ctrl 96±1 vs mTBI 93±1 %), sex interaction (P=0.081). Despite this greater decrease when standing, baseline internal carotid blood flow when seated was not different between sexes with both demonstration significantly lower flow int eh concussed players(Males – Ctrl 479±31 vs mTBI 361±56; Females Ctrl 426±62 vs mTBI 368±76 mL/min).

These data suggest that while both males and females had similar hypertensive responses following a sports related concussion, a tendency towards a greater decrease in cerebral flow when standing was found in the females. Thus further work is necessary to both understand the physiological response following concussion, but specifically with regards to sex differences.

Support or Funding Information

This work was supported by the War Related Illness and Injury Study Center within the Department of Veteran Affairs.
Heterodyning in Tubuloglomerular Feedback Induced Oscillations in Coupled Model Nephrons
Authors: Kesseler Kevin J. , Layton Harold E.
Published: November 2023

In each nephron of the mammalian kidney, a negative feedback mechanism, tubuloglomerular feedback (TGF), participates in the regulation of the glomerular filtration rate. In a previous study, we used a mathematical model to predict the effects of coupling between the TGF systems of two short‐looped rat nephrons (Bull. Math. Biol. 66(6): 1463–1492, 2004). That study predicted parameter regions in which a perturbation of a time‐independent steady state results in a transition to sustained and stable oscillations in the glomerular filtration rate and thus oscillations in nephron tubular flow. These oscillations were predicted to arise for sufficiently large TGF gains and for sufficiently long time delays in TGF response. In the present study, we have used the same two nephron model to investigate how the oscillations are affected by varying time delays in TGF response. We found that when the TGF gains are equal and the TGF delays are sufficiently close, the oscillations in the two model nephrons synchronize: they have identical fundamental frequencies and they oscillate in phase. However, when the delays differ sufficiently, the oscillations in the two model nephrons have differing fundamental frequencies and may exhibit complex behaviors. Power spectra computed from time series generated by the model reveal both harmonic and heterodyne frequencies. These model results may help explain power spectra obtained in experimental studies of rat kidneys: in both normotensive rats and spontaneously hypertensive rats the power spectra frequently exhibit power at non‐fundamental frequencies.

Support or Funding Information

This research was supported by National Institutes of Health grant DK‐42091.
A Newly Developed Technique of in vivo Synchrotron Radiation Renal Micro‐angiography in Spontaneously Hypertensive and Normotensive Rats
Authors: Miya Ken , Matsushita Shonosuke , Hyodo Kazuyuki , Tokunaga Chiho , Sakamoto Hiroaki , Mizutani Taro , Hiramatsu Yuji
Published: November 2023

Introduction

Although real‐time imaging of renal arterioles and glomeruli is desirable, conventional angiography is not capable of such visualization, due to limitations in spatial resolution. We have developed a novel method of visualizing renal arterioles and glomeruli in real‐time in in‐vivo rats, without exteriorizing kidneys, utilizing synchrotron radiation (SR) derived X‐rays. In this study, we aimed to detect morphological differences in the kidney by utilizing SR renal micro‐angiography (SRRM) in normotensive and hypertensive rats.

Method

SRRM was performed at the Photon Factory of the High Energy Accelerator Research Organization (Tsukuba, Japan). The SR derived X‐rays are characterized by high collimation and photon flux, thus resulting in higher spatial resolution compared with conventional X‐ray. Male Wistar rats (WR; n=5) and spontaneously hypertensive rats (SHR; n=5) were anesthetized and iodine contrast agent was administrated via polyethylene tube for angiography inserted into the infra‐renal aorta via laparotomy with its tip just above renal arteries.

Result

In both groups, renal arterioles and glomeruli were clearly visualized (Fig. 1). The average diameter of glomeruli in the WR group was 197±42 micrometer and 193±37 micrometer in the SHR group, respectively. It was also possible to detect local stenosis of interlobular arterioles in SHR rats with diameters of 50 micrometer. Compared with the WR group (Fig. 2), perfusion of contrast agent was apparently decreased in the SHR group (Fig. 3).

Conclusion

The result shows the effectiveness of SRRM for visualizing renal arterioles and glomeruli in in‐vivo rats, in real‐time, without exteriorizing kidneys. The SRRM is a promising method for the investigation of renal physiology as well as pathology.
Meprin β deficiency associated with higher levels of neutrophil gelatinase associated lipocalin (NGAL) and kidney injury molecule (KIM‐1) in mice with streptozotocin induced type 1 diabetes
Authors: Kenley Sharneice Renee , Niyitegeka Jean‐Marie Vianney , Sedighi Rashin , Gooding Jessica , Sumner Susan , Ongeri Elimelda Moige
Published: November 2023

Diabetic nephropathy (DN) is the leading cause of end stage renal disease, and is associated with high morbidity and mortality rates. The pathophysiology of DN includes both glomerular and tubulointerstitial damage. Meprins are metalloproteinases which are most abundantly expressed in the brush border membranes of proximal kidney tubules. Meprins are also expressed in leukocytes (monocytes and macrophages) and podocytes. Meprins have been implicated in the pathology of acute and chronic kidney injury. Single nucleotide polymorphisms (SNPs) in the meprin β gene were associated in human DN in the Pima Indians, suggesting a role for meprins in the pathophysiology of DN. The current study was done to determine the mechanisms by which meprins modulate the progression of DN in mice. Low dose streptozotocin (STZ) was used to induce type 1 diabetes in 8 week old male wild‐type (WT) and meprin β knockout mice (βKO) on a C57BL/6 background. Control mice were injected with sodium citrate buffer. Urine and blood samples were collected at 4 and 8 weeks post STZ injection. The mice were sacrificed at 8 weeks post‐STZ injection and kidney tissue harvested for analysis using metabolomics and proteomic approaches. Enzyme‐linked immunosorbent assays (ELISA) were used to determine the levels of two tubular kidney injury markers, neutrophil gelatinase associated lipocalin (NGAL) and kidney injury molecule 1 (KIM‐1). When compared to non‐diabetic controls, meprin β KO mice with STZ‐induced type 1 diabetes had significantly higher levels of plasma NGAL at 4 weeks but not at 8 weeks post STZ injection. The levels of urinary KIM‐1 were significantly higher in meprin βKO mice at both 4 weeks and 8 weeks when compared to their wild‐type counterparts. Taken together, the data suggest that presence of meprin β partially protects mice from the tubular kidney injury associated with diabetes. This is in contrast to outcomes in ischemia/reperfusion induced acute kidney injury where meprin deficiency was previously shown to be protective. Further analysis will determine the kidney meprin targets and metabolic pathways which play a role in the progression of DN.

Support or Funding Information

NIH/NIGMS # SC3GM102049
Effect of SGLT Inhibition on Medullary Oxygen Consumption: A Multi‐Nephron Model of the Rat Kidney
Authors: Layton Anita , Vallon Volker , Edwards Aurelie
Published: November 2023

The objective of this study was to investigate how changes in sodium reabsorption in the proximal tubule affect oxygen (O2) consumption and the metabolic efficiency of the kidney. To do so, we developed a detailed mathematical model of solute transport in a multi‐nephron representation of the rat kidney. The model represents one superficial nephron and five juxtamedullary nephrons with loops of Henle reaching to differing depths of the inner medulla. The six representative nephrons merge at the entrance of the cortical collecting duct. Glucose is reabsorbed via sodium‐glucose cotransporters (SGLTs) in the proximal tubules, which express SGLT2 in S1–S2 and the SGLT1 in S3. We used the model to investigate the effect of inhibiting SGLT2, a novel treatment for reducing proximal tubule glucose uptake in diabetes, on renal Na+ transport and renal oxygen consumption (QO2). Inhibiting SGLT2 shifts Na+ transport to downstream nephron segments, increasing their QO2. In particular, the S3 segment and medullary thick ascending limb (mTAL) are at risk for hypoxic injury. Dual SGLT1‐SGLT2 inhibition protects the S3 segment, but could further lower mTAL oxygenation. We considered the importance of the GFR‐lowering effect of SGLT2 inhibitors. Additionally, we used the model to determine the optimal combination of SGLT1 and SGLT2 inhibition, in terms of reducing tubular glucose uptake while maintaining sufficiently low S3 and mTAL QO2.

Support or Funding Information

This research was supported in part by NIH grant DK‐89066.
Knockout of Na‐glucose transporter SGLT1 lowers GFR and kidney weight in non‐diabetic and Akita diabetic mice
Authors: Song Panai , Fu Yiling , Onishi Akira , Qiu Hongyu , Koepsell Hermann , Vallon Volker
Published: November 2023

Background

Renal SGLT1 reabsorbs 3% of filtered glucose in euglycemia. Diabetes strongly increases the absolute renal glucose reabsorption by SGLT1. To gain more insights on the functional consequences of SGLT1 inhibition in diabetes, we determined the renal effects of genetic deletion of SGLT1 in Akita mice, a model of type 1 diabetes mellitus.

Methods

SGLT1‐KO mice (C57BL/6J) were backcrossed to DBA2 background for at least 4 generations and crossed with heterozygous Ins2Akita mice, a genetic model of type 1 diabetes, to generate 4 experimental groups; i) WT, ii) SGLT1‐KO, iii) diabetic WT, and iv) diabetic SGLT1‐KO (n=10–21/group). All mice were fed low‐glucose high‐protein diet to prevent diarrhea in SGLT1‐KO. Systolic blood pressure (SBP) was measured by automated tail cuff, and glomerular filtration rate (GFR) by plasma elimination kinetics of FITC‐sinistrin at 20–23 weeks of age, both in conscious mice.

Results

In non‐diabetic mice, SGLT1‐KO increased urine glucose/creatinine ratios vs WT (186±31 vs 9±1 μmol/mg; P<0.01), associated with similar body weight (31±1 vs 30±1 g), blood glucose (114±5 vs 114±5 mg/dl), and SBP (116±1 vs 116±2 mmHg), whereas GFR and kidney weight were modestly reduced (23.6±1.2 vs 28.5±1.5 μl/min/g ; 9.1±0.3 vs 10.2±0.1 mg/g; each P<0.05 ). In SGLT1‐KO and WT, diabetes increased blood and urine glucose and SBP to similar levels (314±21 vs 324±30 mg/dl; 2165±758 vs 2491±872 μmol/mg creatinine; 127±2 vs 128±2 mmHg), increased food and fluid intake (not shown) and decreased body weight (28±1 vs 28±1 g). Diabetes increased GFR and was associated with a small increase in kidney to body weight ratios; this occurred to a similar extent in SGLT1‐KO and WT, such that GFR (28.3±3.7 vs 35.1±2.3 μl/min/g, P<0.05) and kidney weight (10.1±0.2 vs 11.1±0.4 mg/g; P<0.05) remained lower in diabetic SGLT1‐KO vs WT.

Conclusion

1) In the absence of diabetes, SGLT1‐KO increased urine glucose and modestly reduced GFR and KW without affecting blood glucose and SBP. 2) The diabetes‐induced increase in blood glucose levels, SBP, GFR or kidney to body weight ratios was independent of SGLT1. 3) In the diabetic state, SGLT1‐KO mice maintained lower GFR and kidney to body weight ratios compared with WT mice.

Support or Funding Information

National Institutes of Health (R01DK56248, P30DK079337)
Dual Soluble Epoxide Hydrolase Inhibitor/PPAR‐γ Agonist Reduces Kidney Injury In Metabolic Syndrome Rat
Authors: Birschbach Judy , Khan Md. Abdul Hye , Sharma Amit , Hartmann Markus , Blöcher René , Proschak Eugen , Imig John D.
Published: November 2023

Metabolic syndrome (MetS) is a disease that yields a cluster of symptoms including hypertension, hyperglycemia, hypertriglyceridemia and obesity. Due to the complexity of this syndrome, patients are often prescribed numerous medications, leading to an increased risk of negative drug interactions and higher drug cost. We hypothesize that the development of a dual modulator (a drug that combines two pharmacophores) could treat more than one symptom of MetS and prevent renal injury. Soluble epoxide hydrolase (sEH) inhibitors and PPAR‐γ agonists have therapeutic potential in MetS. We synthesized a novel dual sEH inhibitor‐PPAR‐γ agonist (sEHi/PPAR‐γ), RB394 and investigated its efficacy in a rat model of MetS. Three groups of rats were utilized; Group 1: Wister Kyoto (WKY) + Vehicle (n=6); Group 2: Spontaneously Hypertensive Obese (SHROB) + Vehicle (n=3); Group 3: SHROB + RB394 (10mg/kg/d p.o.; n=5). RB394 or vehicle treatments were administered for 56 days, blood pressure measured, and urine and kidney tissues collected at the end of treatment period. The SHROB rats were hypertensive (187±7 mmHg) compared to WKY rats (137±5 mmHg, P<0.05), and RB394 markedly reduced hypertension in SHROB rats (144±4 mmHg, P<0.05). In order to quantify the amount of kidney injury sustained, we assessed albuminuria and utilized Periodic Acid Schiff stain and Picro Sirius Red stains for histological analysis of renal tubular cast formation, collagen formation and glomerular injury. The SHROB rats developed renal damage with marked albuminuria (243±18 mg/d) compared to WKY (1.0±0.1 mg/d, P<0.05), and RB394 decreased the renal damage by reducing albuminuria (59±11 mg/d, P<0.05). The kidney of SHROB rats had markedly elevated protein cast and collagen, and RB394 reduced the cast and collagen formation by 40–50%. Glomerular injury was also prominent in SHROB rats and RB394 reduced it by 45%. The SHROB rats also had marked renal inflammation with infiltrating immune cells in the kidney, and RB394 reduced it by 20%. Overall, our results demonstrate that a dual sEHi/PPAR‐γ, RB394, prevented renal injury in SHROB MetS rats. These results indicate an exciting opportunity for a new way to more effectively treat patients with MetS.
High K+ Intake Attenuates ARPKD Progression by Activation of TRPV4‐mediated Ca2+ Signaling in Cyst Cells
Authors: Mamenko Mykola , Tomilin Viktor , Zaika Oleg , Pochynyuk Oleh
Published: November 2023

Autosomal recessive polycystic kidney disease (ARPKD) is a devastating neonatal pathology, causing development of fluid‐filled cysts in the collecting ducts (CDs) and resulting in kidney failure in the majority of infants. Cyst cells are characterized by increased proliferation rate and cyclic AMP levels, low intracellular Ca2+ concentration ([Ca2+]i) and impaired mechanosensitivity. However, the exact molecular and cellular determinants of the disease remain elusive. We have recently shown that progression of ARPKD is associated with disruption of functional activity of the Ca2+‐permeable TRPV4 channel, which is abundantly expressed in the CD and mediates [Ca2+]i increase in response to elevated tubular flow. Systemic pharmacological activation of TRPV4 retards manifestations of ARPKD. Potassium‐rich diet, elevating tubular fluid flow in the CD, can also serve as a natural stimulant of TRPV4 activity. Here we probed how high K+ intake affects ARPKD progression in PCK 453 rats – a rodent model of the disease. We found that cyst cells isolated from PCK rats supplemented with high K+ diet exhibit a markedly augmented TRPV4 activity (as accessed with a specific activator of the channel GSK1016790A) and increased basal [Ca2+]i levels. Elevated flow elicited a prominent [Ca2+]i response in cystic epithelium of rats placed on high dietary K+, while flow‐induced [Ca2+]i signal was practically abolished in cyst cells from PCK controls on regular K+ regimen. We recently identified a primary contribution of TRPV4 into sustained phase of purinergic [Ca2+]i signal in the CD and demonstrated that cyst cells lack a prolonged ATP‐induced [Ca2+]i response. Here we found that high K+ intake rejuvenates the sustained plateau of ATP‐activated [Ca2+]i signal in cyst cells. Thereby, at the cellular level high dietary K+ at least partially restores TRPV4‐mediated mechanosensitive Ca2+ signaling and [Ca2+]i homeostasis. Systemically, we observed a significant decrease in kidney to body weight ratio in the PCK 453 rats supplemented with high K+ diet. Overall, our results strongly argue high K+ regimen is beneficial for ARPKD patients as it attenuates the morbific manifestations of the disease.
Regulation of 24p3 Receptor (24p3R/SLC22A17) Expression by Tonicity in Rodent Renal Inner Medullary Collecting Duct (IMCD) Cells
Authors: Thevenod Frank , Langelueddecke Christian , Probst Stephanie , Scharner Bettina , Betten Richard , Mese Kemal , Edemir Bayram
Published: November 2023

Background and Aims

The 24p3/NGAL/lipocalin‐2 receptor (24p3R/SLC22A17) is apically expressed in rodent collecting ducts, including IMCD, where it may mediate protein endocytosis (Langelueddecke C. et al. J. Biol. Chem. 287:159–169, 2012) and induce pro‐inflammatory and pro‐fibrotic signalling in response to albuminuria (Dizin E. et al. Am. J. Physiol. Renal Physiol. 305:F1053‐F1063, 2013). Cells of the inner medulla are exposed in vivo to a hyperosmotic environment, which requires an adaptive response for survival. Interestingly, we have identified a putative TonEBP DNA consensus motif in the 24p3R promoter sequence. Here we investigated the expression of 24p3R in cultured IMCD cells that were exposed to increased osmolarity.

Methods

Cultured mouse IMCD cells (mIMCD3) and rat primary IMCD cells were cultured in 300 – 900 mOsm/kg for 1–6 days. Hypertonicity was induced by equimolar addition of NaCl + urea. 24p3R expression and localization were determined by qPCR, immunoblotting, surface biotinylation, flow cytometry and immunofluorescence microscopy.

Results

Hypertonicity decreased cell proliferation and was accompanied by suppression of the cell proliferation genes c‐myc and cyclin‐D1 in mIMCD3 cells, which may reflect cellular stress and damage. Hypertonicity increased 24p3R mRNA and 24p3R protein expression and localization in plasma membranes of mIMCD3 and primary IMCD cells. Furthermore, plasma membrane localization of 24p3R was associated with increased dimerization of the protein.

Conclusions

Hyperosmotic environment decreases proliferation of IMCD cells but increases plasma membrane expression and dimerization of 24p3R, which may be mediated by TonEBP and contribute to adaptation of IMCD cells to osmotic stress.

Support or Funding Information

Funded by DFG TH345/11‐1 and ZBAF
Glutathione (GSH) Absolute Synthesis Rates (ASR) of Multiple Organs in a Pseudomonas aeroginosa (PM) induced Hyperdynamic Sepsis Pig Model
Authors: Ten Have Gabriella A , Engelen Marielle P , Wolfe Robert R , Deutz Nicolaas E
Published: November 2023

Rationale

Sepsis is characterized by a severe redox imbalance. GSH plays a major role in the cellular defense against oxidative and nitrosative stress. It remains unclear whether the GSH defense capacity of tissues is affected differently at an early stage of severe sepsis. Therefore we measured the early response in GSH synthesis of the gut, muscle, liver, and lung in an acute live bacteria (PM) induced hyperdynamic severe sepsis catheterized pig model.

Method

We studied GSH‐ASR in 13 pigs (±25 kg) with intravenous PM induced hyperdynamic sepsis and 9 healthy controls, using primed, constant and continuous infusion of Glycine (1‐13C) stable isotope in a conscious post‐absorptive state. Between 14 and 18 hours after start of the sepsis induction, we collected jejunal mucosa biopsies and erythrocytes, and at eighteen hours we obtained jejunum, jejunal mucosa scraping, ileum, liver, muscle and lung tissue. Enrichments of precursor pools and incorporated glycine in GSH were analyzed by LC‐MS/MS. Data are mean (SE) (in μmol/kg/h); ANOVA and unpaired t‐tests were done by GraphPad Prism.

Results

We found decreased values for GSH‐ASR 14 to18h after sepsis induction in jejunal mucosa biopsies (p=0.032) but not in erythrocytes. At t=18 h, we found lower ASR in sepsis in jejunum (37.3 (5.5) vs 23.7 (2.6), p=0.021) and jejunal mucosa scrapings (45.0 (6.2) vs. 28.9 (3.9), p=0.033). No changes in GSH‐ASR in ileum, liver and muscle were found but GSH‐ASR tended to be higher in the lung (20.2 (3.0) vs 26.9 (2.2), p=0.086).

Conclusion

In acute severe sepsis in the pig, the early defense capacity for oxidative stress is compromised in the mucosa of the jejunum, but not in the muscle, liver, lung, ileum and erythrocytes. We hypothesize that restoring the jejunal GSH defense capacity will attenuate the early sepsis induced gut dysfunction.

Support or Funding Information

This study was supported by NIH R01GM084447 and S10RR027047
Muscle Compression Causes Reduction of Oxidative Phosphorylation (OXPHOS) Subunits and Increase in Autophagy
Authors: Tam Bjorn T. , Yung Benjamin Y. , Wong Cesar S. , Chan Lawrence W. , Tam Eric W. , Siu Parco M.
Published: November 2023

Pressure‐induced injury, skeletal muscle apoptosis and nitrosative stress can be induced in skeletal muscle by moderate mechanical compression in our established mouse model. Nonetheless, the effects of mechanical compression on oxidative phosphorylation (OXPHOS) subunits content and autophagic signalling in compressed skeletal muscle are largely unknown.

PURPOSE

To determine whether mechanical compression decreases the content of OXPHOS subunits and triggers autophagy in skeletal muscle.

METHODS

A single session of 4‐hour compression with static pressure of 150 mmHg was applied over the right lateral gastrocnemius muscle of mice. Tissues were harvested 48 hours after compression for analysis.

RESULTS

Our immunoblot data suggest that mechanical compression decreases OXPHOS subunits content. Furthermore, mechanical compression triggers autophagy in compressed skeletal muscle, resulting in increase in LC3‐II protein abundance and decrease in p62 protein abundance.

CONCLUSION

These results suggest that moderate mechanical compression leads to degradation of mitochondrial complexes which might impair the OXPHOS activity and, hence, the mitochondrial function. The degradation of mitochondrial complexes might possibly be induced by the upregulation of autophagy.

Support or Funding Information

Supported by The Hong Kong Polytechnic University Research Funds (A‐PH69 and G‐U645) and General Research Fund (PolyU 5632/10M).
Coxiella burnetii Infection of Host Cells Involves PKC substrate MARCKS
Authors: Whitlock Sara , Funk Cornelius Joel
Published: November 2023

The human disease Q‐fever is initiated as a respiratory tract infection by the bacterium Coxiella burnetii. Acute infections typically result in flu‐like symptoms and can be treated with antibiotics, but untreated chronic infections can lead to severe conditions including endocarditis. Bacteria enter alveolar macrophages within a phagosome, but instead of being destroyed by cellular enzymes bacterial cells induce the host cell to develop a lysosome‐like organelle called the parasitophorous vacuole (PV) where replication takes place. The PV starts small but grows in size to accommodate the increasing number of replicating cells. Manipulation of the host infection by C. burnetii includes altering Protein Kinase C (PKC) cell signaling events beginning at early times of the infection. PKC activation is initiated through multiple isoforms that sequentially phosphorylate downstream substrates. MARCKS is one of several PKC substrates phosphorylated during infections. The objective of this study was to localize MARCKS using antibodies and examine the role of MARCKS using specific inhibitors. Our results indicate that MARCKS localizes to the PV membrane and surrounding cytoplasm. The level of MARCKS protein was knocked‐down using siRNA treatment of HeLa cells. Infected cells treated with MARCKS siRNA contained C. burnetii, but many PV's were larger in size and contained fewer bacteria than those of infected cells treated with control siRNA. A peptide inhibitor of MARCKS appeared to have a similar effect on infected THP‐1 cells by causing enlarged PV's compared to control peptide treatments. These results indicate that MARCKS may have a role in regulating or controlling the size of C. burnetii‐induced PV's through manipulation of the host cell PKC signaling system.

Support or Funding Information

Supported by a grant from Arkansas INBRE (NIH NIGMS P20 GM103429).
Mechanism of action of Lactobacillus reuteri ATCC PTA 6475 in colon epithelial cells
Authors: Rios‐Arce Naiomy Deliz , Britton Robert A , McCabe Laura , Parameswaran Narayanan
Published: November 2023

Lactobacillus reuteri ATCC PTA 6475 (LR) is a well‐known probiotic that has been shown to be beneficial to the gastrointestinal (GI) system. In addition, LR has been shown to secrete beneficial immunomodulatory factors. In previous studies, we demonstrated that LR can modulate inflammatory genes in the GI tract in mice in vivo. However, the mechanisms of action of LR in the GI system during inflammatory conditions are still poorly understood. In the present study, we investigated the potential role of LR on tumor necrosis factor a (TNFa)‐induced colon epithelial cell biology, including expression of inflammatory genes. For this, we pre‐treated colon epithelial cells (SW480) with live LR, LR conditioned medium (CM), CM fractions (ranging from < 3, 3–10, 10–30, 30–50, 50–100 and >100kD) and heat inactivated CM. Subsequently, we treated the cells with TNFa (10ng/ml) for 6 or 24 hours and examined expression of inflammatory genes. We found that live LR induced IL‐8 mRNA expression in a dose dependent manner. We further identified that the less than 3KD fraction of the CM produced by LR is sufficient to induce IL‐8 at both the mRNA and proteins levels. While we observed similar effects on MIP2a, TNFa gene expression was not regulated by LR. We further found that the factors that induce IL‐8 expression are heat labile. These effects on gene expression were also observed in other intestinal epithelial cell lines including HT29 and CaCo2. The effect on gene expression was not accompanied by cell proliferation. Preliminary examination of signaling pathway activation indicated that LR‐CM small factors do not regulate IL‐8 production by activation of the MAPK or NF‐κB pathways, suggesting other mechanisms. Together, our results confirm that live LR and its secreted factors induce inflammatory genes in epithelial cells that could potentially affect intestinal inflammation without affecting cell growth.
EriC2 Regulates Histamine Production Machinery via an Ion Transport‐Dependent Mechanism in Lactobacillus reuteri
Authors: Hall Anne , Engevik Melinda , Versalovic James
Published: November 2023

Background

Certain strains of Lactobacillus reuteri can reduce inflammation and tissue damage in mouse models of colitis. This anti‐inflammatory capacity stems in part from L. reuteri’s ability to produce and secrete histamine, which can act on specific receptors present on mammalian cells. In the bacteria, histamine is generated by histidine decarboxylase (HdcA), and released from the cell by a histidine/histamine exchanger (HdcP). This process consumes protons, resulting in an increasingly alkaline intracellular pH and an inside‐negative membrane potential. These factors can suppress the activities of HdcA and HdcP. However, hdcA is not constitutively expressed, and it is unclear which signals may trigger its upregulation. Additionally, it is unknown how L. reuteri balances its internal ion environment during histamine production, especially in the presence of a complex external environment like the GI tract. A proton/chloride antiporter, EriC2, was recently identified in L. reuteri as a potential regulator of intracellular pH and membrane potential, and thus histamine production. We hypothesize that EriC2 maintains intracellular pH via proton influx and membrane potential via chloride efflux at levels that allow histamine production by L. reuteri.

Methods & Results

To investigate how EriC2 ion transport might alter expression and function of histamine production machinery in L. reuteri, single‐strand recombineering was used to generate protein knockout, proton transport deficient, and transport null EriC2 strains. These mutations are stable, and do not significantly alter the growth of the resulting strains compared to wild‐type (WT) L. reuteri. Intracellular pH was assessed in each strain with the pH‐sensitive fluorophore, pHrodo Green AM. Loss of ion transport via EriC2 increases intracellular pH, supporting its potential role in maintaining the intracellular proton pool. RT‐qPCR showed that hdcA gene expression is increased in EriC2 transport mutants relative to the WT strain. T84 secretory colonic epithelial cells were co‐cultured with WT and EriC2 mutant L. reuteri strains. Host gene expression was examined via RT‐qPCR, and differential expression of histamine receptors was observed in the presence of mutant L. reuteri strains.

Conclusion

Together these data suggest that EriC2 ion transport mutants differentially regulate histamine production machinery in L. reuteri, and can lead to changes in host physiology. The beneficial effects of L. reuteri‐derived histamine may depend on a regulatory system mediated by ion transport in the GI tract. A mechanistic understanding of histamine production by the gut microbiome may enable the development of diet or probiotic‐based strategies to suppress intestinal inflammation.
EXOCYTOSIS OF ENDOTHELIAL LYSOSOME‐RELATED ORGANELLES HAIR‐TRIGGERS A PATCHY LOSS OF GLYCOCALYX AT THE ONSET OF SEPSIS
Authors: Zullo Joseph A , Fan Jie , Azar Tala , Yen Wanyi , Zeng Min , Chen Jun , Ratliff Brian , Song Jun , Tarbell John M , Goligorsky Michael S , Fu Bingmei
Published: November 2023

Introduction

Endothelial surface glycocalyx (ESG) is a well‐known regulator of leukocyte surveillance, vascular permeability, and a sensor of shear stress. ESG is degraded during sepsis, thus contributing to systemic multiorgan involvement. Our previous demonstration of a very early post‐stress exocytosis of lysosome‐related organelles (LRO) with their lytic enzymes led us to hypothesize that exocytosis of LRO may catapult a synchronized degradation of ESG.

Methods and Results

To test this hypothesis we first analyzed the time‐course of exocytosis of Weibel‐Palade bodies and secretory lysosomes after application of lipopolysaccharides (LPS) to endothelial cells. Time‐lapse video microscopy revealed that LROs undergo agitation and vectorial movement with a time course of a few minutes after LPS. In addition, spectrophotometry measurements illustrated an increase of lysosomal cargo, cathepsin B, within the media after 10min of LPS exposure. Two therapeutic maneuvers, a nitric oxide intermediate, NG‐hydroxy‐L‐arginine (NOHA), and culture media conditioned by endothelial progenitor cells (EPC‐CM) reduced the directionality and motility of LRO. The patchy loss of ESG occurring with the similar fast kinetics was confirmed using confocal fluorescence microscopy and Stochastic Optical Reconstruction Microscopy. The loss of ESG was blunted by pretreatment with NOHA or EPC‐CM. Moreover, these treatments resulted in a significant reduction of mortality of septic mice.

Conclusion

In summary, above studies provide the first demonstration of the very early patchy disintegration of ESG which can be prevented by pretreatment with NOHA and EPC‐CM, maneuvers that reduced exocytosis of LRO. Data support the hypothesis assigning to stress‐induced exocytosis of these organelles the role of a hair‐trigger for local degradation of ESG that in turn initiates leukocyte infiltration, increase in vascular permeability, and partially accounts for the later morbidity and mortality.

Support or Funding Information

NIH grants DK54602, DK052783 and DK45462 (MSG), SC1CA153325 (BMF) and R01HL094889 (JMT and BMF); AHA grant 12SDG9080006 and ASN grant 010973‐101 (BBR); and The New York Community Trust ‐ Renal Clinical Fund (BBR)
Role of brainstem melanocortin‐4 receptor (MC4R) in regulating glucose, energy balance and body fat in female mice
Authors: Fang Taolin , do Carmo Jussara M , Wang Zhen , Aberdein Nicola , de Lara Rodriguez Cecilia Perez , Hall John E
Published: November 2023

The central melanocortin system, particularly the melanocortin‐4 receptor (MC4R), plays an important role in regulation of energy homeostasis. Although the brainstem has a high density of MC4R, their role in regulating metabolic functions, including energy balance, glucose, and body weight has not been fully elucidated. Using Cre‐LoxP technology, we selectively rescued MC4R in preganglionic parasympathetic neurons of the nucleus tractus solitaries/dorsal motor nucleus of the vagus nerve (NTS/DMV) of female mice with whole body MC4R deficiency (Phox2b/LoxTB‐MC4R−/−, n=7). Littermate MC4R‐deficient mice (LoxTB‐MC4R−/−, n=7) were used as controls. Body weight, body composition, and glucose tolerance were determined at 20 weeks of age. At 19–22 weeks of age, the mice were placed in metabolic cages for measurement of daily food intake, oxygen consumption (VO2), heat production, and locomotor activity (MA). Compared to LoxTB‐MC4R−/− mice, Phox2b/LoxT‐MC4R−/− were less obese (38±3 vs. 47±1g) with decreased food intake (2.8±0.5 vs. 4.5±0.2 g), increased VO2 (65.5±2.9 vs. 60.0±0.4 mL·kg−1·min−1) and MA (306±49 vs. 99±6 cm·hr−1), and similar heat production (832±22 vs. 835±54 cal·hr−1). Phox2b/LoxTB‐MC4R−/− mice exhibited similar body fat percentage (45±1 vs. 46±2%) at 20 weeks of age compared to LoxTB‐MC4R−/−. Blood glucose levels and tolerance to a glucose load were similar (140±7 vs.138±13 mg·dL−1; AUC: 25866±2069 vs. 25097±4677 mg·min·dL−1) in Phox2b/LoxT‐MC4R−/− compared to LoxTB‐MC4R−/− mice. Rescuing MC4R in preganglionic parasympathetic neurons in the brainstem did not restore the anorexic (2.8±0.5 vs. 2.9±0.4 g) or the glucose lowering effects of leptin (125 ±32 to 121 ±30.5 mg·dL−1). These results suggest that MC4R activity in the brainstem, specifically in the NTS/DMV, contributes to regulation of appetite, body weight, energy expenditure, and locomotor activity in female mice. We did not observe a major role for MC4R in NTS/DMV in controlling fasting glucose, glucose tolerance, body fat percentage, or rescuing the anorexic and anti‐diabetic effects of leptin.

Support or Funding Information

(Supported by NHLBI‐PO1HL51971, NIGMS P20GM104357, and 15POST22960057).
Acute slow‐paced breathing modulates the pressor and depressor responses following spontaneous oscillations in muscle sympathetic nerve activity
Authors: Vianna Lauro C. , Fadel Paul J. , Joyner Michael J. , Wehrwein Erica A.
Published: November 2023

Previous studies have indicated that acute slow‐paced breathing (6 breaths/min) significantly decreases arterial blood pressure (BP) in both normotensive and hypertensive individuals. More recent work suggested that muscle sympathetic nerve activity (MSNA) was unchanged during acute slow‐paced breathing, however, the pattern of MSNA firing was altered in that more periods of MSNA quiescence were present. Herein, we examined the ability of MSNA to modulate BP on a beat‐to‐beat basis. We hypothesized that the magnitude of the increase in BP following a burst of MSNA is attenuated during acute slow‐paced breathing contributing to the reduction in BP previously reported. BP (brachial catheter) and MSNA (peroneal microneurography) were continuously measured in 10 young (27 ± 1 years), healthy men during 10 minutes of normal spontaneous breathing (baseline) or paced breathing at six breaths per minute. Signal averaging was used to characterize changes in mean BP for 15 cardiac cycles following heartbeats associated with and without MSNA bursts. MSNA bursts were segregated as either single bursts (directly bordered by ≥1 heartbeat lacking MSNA) or multiple bursts (any burst positioned directly adjacent to another MSNA burst). Respiratory rate was 12 ± 1.1 breaths/min during baseline and 6 ± 0.7 breaths/min during slow‐paced breathing. Compared to baseline, mean BP was decreased during slow‐paced breathing (88±2 vs. 85±2 mmHg, P<0.05), whereas MSNA burst frequency was unchanged (13±2.2 vs. 13±2.4 bursts/min, P>0.05). At baseline, mean BP significantly increased following MSNA bursts (+3.2± 0.2 mmHg, P<0.05) and this increase was augmented during slow‐paced breathing (+5.0± 0.5 mmHg, P<0.05). There was a greater increase in mean BP following single as well as multiple MSNA bursts during slow‐paced breathing. In contrast, cardiac cycles without MSNA bursts (non‐bursts) were associated with a significant decrease in mean BP (−0.7± 0.1 mmHg, P<0.05), which was also augmented during slow‐paced breathing (−1.3± 0.3 mmHg, P<0.05). In summary, the pressor and depressor responses coupled to spontaneous oscillations in MSNA are augmented during slow paced breathing with a net effect of reduced BP. Thus, changes in MSNA firing pattern, without changes in burst frequency, can modulate resting BP.

Support or Funding Information

Funding: NIH HL‐083947 (MJJ)
Impaired Arterial Pressure Stability Is Improved by Sodium‐glucose Co‐transporter 2 Inhibitor in Streptozotocin‐induced Diabetic Rat
Authors: Yoshikawa Tomoko , Kishi Takuya , Hirooka Yoshitaka
Published: November 2023

Background

Although all anti‐diabetic agents could not certainly prevent cardiovascular events, arterial pressure (AP) reduction has been determined to have beneficial prevention for cardiovascular mortality in patients with diabetes mellitus (DM). Moreover, patients with DM have impaired AP variability. In these aspects, a new anti‐diabetic agent sodium‐glucose co‐transporter 2 (SGLT2) inhibitor has been focused on its unique AP lowering effect. We examined whether DM model rats would destabilize AP or not, and if so, whether SGLT2 inhibitor could ameliorate the disruptive AP lability.

Methods

We used streptozotocin‐induced diabetic (STZ) rats with telemetric AP monitoring. At 10 days after the injection of streptozotocin, SGLT2 inhibitor (ipragliflozin 1mg/kg/day) was orally administered to established hyperglycemic STZ rats for 15 days. We calculated the average and standard deviation (SD) of AP as the index of lability from AP histograms.

Results

After streptozotocin injection, mean AP did not change both in day (rest)‐time and night (active)‐time. SD of AP increased in rest‐time (3.2 mmHg to 3.7 mmHg, n=2) but not changed in active‐time. SGLT2 inhibitor for 15 days reduced mean AP both in rest‐ (−5.6 mmHg, n=2) and active‐time (−5.9 mmHg, n=2), and ameliorated SD of AP both in rest‐ (3.7 mmHg to 3.0 mmHg, n=2) and active‐time (3.5 mmHg to 2.5 mmHg, n=2).

Conclusions

SGLT2 inhibitor ameliorates the disruptive AP lability in rest‐ and active‐time with depressor response in diabetic model rats.

Support or Funding Information

This study was supported by a Grant‐in‐Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24390198 to Dr. Hirooka and 22790709 to Dr. Kishi).
Analysis of Heart Rate Variability by Multiscale Entropy in Spontaneously Hypertensive Rats
Authors: Silva Luiz Eduardo Virgilio , Silva Carlos Alberto Aguiar , Salgado Helio Cesar , Fazan Rubens
Published: November 2023

Most commonly used approaches to access heart rate variability (HRV) are based on linear models, such as spectral analysis. Recently, much attention has been given to nonlinear methods, because they quantify important additional properties of HRV dynamics. Multiscale entropy (MSE) can quantify the predictability of time series over several time scales, which is demonstrated to be related to neuro‐humoral regulation of cardiac activity. We calculated Sample Entropy over scales 1 to 20 in time series of cardiac interval from spontaneously hypertensive rats (SHR), to identify how hypertension affects the predictability of HR over multiple scales. ECG was continuously recorded during 90 minutes in SHR (N=7) and normotensive Wistar‐Kyoto rats (WKY, N=6) previously (5 days) instrumented with probes that allowed wireless ECG recordings. Beat by beat series of RR were extracted and processed (artifact and missed detections excluded) prior to analysis. Entropy values of SHR are significantly lower compared to WKY for scales four onwards. Total entropy is also significantly lower in SHR (11.6 ± 0.9 vs 16.7 ± 1.2, p=0.008). MSE curve in SHR showed a rapid decrease reaching a plateau after scale 3. In WKY the decrease of MSE was smaller than in SHR, but fully recovered towards the higher scales. The results suggest that mechanisms responsible for long‐term non‐linear dynamics of HRV, such as sympathetic activity and humoral factors, are the most affected in SHR.

Support or Funding Information

FAPESP, CAPES, CNPq
CARDIORESPIRATORY CONTROL IN NEWBORN RATS SUBMITTED TO CHRONIC INTERMITTENT HYPOXIA
Authors: Menezes Miguel F. , Zoccal Daniel
Published: November 2023

Postnatal hypoxia is a major risk factor for the development of cardiorespiratory diseases in adulthood. In this study, we evaluated the effects of postnatal intermittent hypoxia (IH) on baseline cardiorespiratory parameters and on ventilatory responses to hypercapnia. We also verified whether the IH‐induced cardiorespiratory changes were dependent on the DNA hypermethylation. Holtzman newborn rats (P0–1) were subjected to IH (6% O2 every 9 min, 8 hours/day, n= 27) or normoxia (n= 19) during the first 10 days of life and received i.p. injections of decitabine (a hypomethylating agent, 1 mg/kg) or vehicle (control) every 3 days, during 10 days. At 35 day old, mean arterial pressure (MAP), heart rate (HR), respiratory frequency (fR), tidal volume (VT) and minute ventilation (VE) were measured at rest and during hypercapnia (7% CO2, 10 min). Exposure to IH increased baseline MAP (95±1 vs 85±4 mmHg), HR (459±7 vs 414±9 bpm), VT (16.2±0.8 vs 10.7±0.4 mL/kg) and VE (1868±95 ml/kg/min vs 1213±86 ml/kg/min) (P<0.05), but not fR. Hypercapnia elevated fR, VT and VE in both groups, but the magnitude of the responses were significantly greater in IH animals (P<0.05). Decitabine treatment during the period of IH exposure was able to prevent the basal cardiorespiratory changes as well as the augmented hypercapnic‐induced ventilatory responses. The data suggest that postnatal IH promotes persistent changes in basal and chemoreflex mechanisms controlling cardiorespiratory parameters by mechanisms involving DNA hypermethylation.

Support or Funding Information

Support by FAPESP (2013/06077‐5).
Carotid body removal markedly improves survival via sympathoinhibition, depressor response and leftward shifting of the pressure‐natriuresis curve in Dahl salt ‐sensitive rats
Authors: Fujii Kana , Saku Keita , Oga Yasuhiro , Nishizaki Akiko , Akashi Takuya , Takehara Takako , Kishi Takuya , Sunagawa Kenji
Published: November 2023

BACKGROUND

Excessive sympathoexcitation in patients with heart failure is one of the fundamental mechanisms that leads to poor long‐term survival. Carotid body removal (CBR) has shown to reduce sympathetic nerve activity (SNA) and blood pressure (BP) in an animal model of hypertension. The sensitized carotid body is thought to be, in part, responsible for sympathoexcitation in hypertension. We hypothesized that the sensitized carotid body plays a critical role in sympathoexcitation in heart failure. The purpose of this investigation is to examine if CBR reduces SNA, prevents the worsening of heart failure and, in turn, improves survival in a rat model of hypertensive heart failure (Dahl salt‐sensitive rat).

METHODS

We randomly allocated 6 week‐old Dahl salt‐sensitive rats fed with high‐salt diet (8% NaCl) into CBR (N=31) or SHAM (N=55). We removed tissues around the carotid bifurcation and established CBR in 7 week‐old rats. We assessed cardiac function (2D‐echo), 24‐hour urinary norepinephrine (uNE) excretion (an index of SNA), and BP. In 14‐week‐old rats, we estimated the pressure‐natriuresis curve by changing the salt intake. Finally, we compared hemodynamics, cardiac histology and survival rate in 16 week‐old rats.

RESULTS

CBR significantly reduced uNE at 12, 14 and 16 week‐old indicating the sympathoinhibitory effect (Fig. 1), and inhibited the BP elevation from 11 week‐old (Fig. 2). CBR significantly shifted the pressure‐natriuresis curve leftward without changing its slope (Fig. 3). CBR preserved left ventricular (LV) ejection fraction (61±9 vs. 68±8%, p<0.01) and lowered LV end‐diastolic pressure (9±4 vs.5±2 mmHg, p<0.05) suggesting improved cardiac function. In histology, CBR significantly attenuated myocardial fibrosis in both interstitial and perivascular area (p<0.05) (Fig. 4), and reduced the size of cardiomyocyte (804±90 vs. 674±114 μm2, p<0.05). CBR markedly improved the survival rate (Fig. 5).

Conclusion

CBR markedly improves the survival via sympathoinhibition and depressor response with the leftward shift of the pressure‐natriuresis curve in a rat model of hypertensive heart failure.