bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterogeneity of Pulmonary Endothelial Cyclic Nucleotide Response to Pseudomonas aeruginosa Infection
Authors: Morrow Kyle , Seifert Roland , Britain Andrea , Ochoa Cristhiaan , Cioffi Eugene , Frank Dara , Rich Thomas , Stevens Troy
Published: June 2021

Here, we tested the hypothesis that a promiscuous bacterial cyclase generates a diverse cyclic nucleotide response in pulmonary endothelium. Pulmonary endothelial cells were infected with a gram‐negative bacterium Pseudomonas aeruginosa. A strain that introduces only ExoY (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY+) via a type 3 secretion system was used for cellular infections. Pulmonary artery and pulmonary microvascular endothelial cells (PAECs and PMVECs) were infected with the ExoY+ strain at a multiplicity of infection of 20:1. PAECs and PMVECs both possess basal levels of 4 different cyclic nucleotides in the following rank order: cAMP > cUMP > cGMP ∼ cCMP. Endothelial gap formation was induced in a time‐dependent manner following ExoY+ intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h. Increasing levels of cGMP were seen by 1 h post‐infection, with cAMP and cUMP increasing at 3 h followed by cCMP by 4 h. In PMVECs, intercellular gaps did not form until 4 h with increased levels of only cGMP and cUMP at 3 and 6 h post‐infection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased much earlier in response to ExoY+ intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.
Differential Effects of Chronic Hypoxia on Basal and Agonist‐Induced NO‐Dependent Pulmonary Vasodilation in Neonatal Rats
Authors: Sheak Joshua , Jernigan Nikki , Walker Benjimen , Resta Thomas
Published: June 2021

Endothelial dysfunction contributes to chronic hypoxia (CH)‐induced pulmonary hypertension (PH) in neonates. However, whether basal and stimulated endothelium‐derived nitric oxide (EDNO)‐dependent pulmonary vasodilation are similarly impaired in neonatal PH is unclear. We hypothesized that CH attenuates EDNO‐dependent pulmonary vasodilation both basally and in response to receptor activation in a neonatal rat model of PH. To test this hypothesis, we assessed effects of the NO synthase inhibitor Nω‐nitro‐L‐arginine (L‐NNA; 300 μM) on baseline pulmonary vascular resistance (PVR) in salt‐perfused lungs (in situ) from 2‐wk‐old control and CH (2 wk at 0.5 atm) rats. The contribution of active tone to PVR was assessed by examining responses to exogenous NO (spermine NONOate, 100 μM). Lungs from CH rats showed greater baseline PVR compared to controls (p<0.05) in the absence of L‐NNA. This response to CH reflected active tone, whereas no basal tone was observed in control lungs. Consistent with our hypothesis, L‐NNA modestly increased PVR in the control group (p<0.05 vs. vehicle), but had no effect in lungs from CH neonates. We next examined EDNO‐dependent vasodilatory responses to arginine vasopressin (AVP, 25 nM) in U‐46619‐constricted lungs. Surprisingly, lungs from CH rats showed augmented responses to AVP compared to controls (p<0.05). L‐NNA abolished AVP responses in both groups (p<0.05 vs vehicle). We conclude that CH 1) increases basal PVR in part through vasoconstriction, and 2) impairs EDNO‐dependent pulmonary vasodilation under basal conditions, but paradoxically augments AVP‐induced EDNO‐dependent vasodilation in a neonatal rat model of PH.
Aquaporin‐1 retards renal cyst development in polycystic kidney disease by inhibition of Wnt signaling
Authors: WANG Weiling , LI Fei , ZHOU Hong , Verkman Alan , YANG Baoxue
Published: June 2021

Objective

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human monogenic diseases. Aquaporin‐1 (AQP1) is expressed mainly at epithelial cell plasma membranes in renal proximal tubules, as well as in epithelial cells lining the cysts in ADPKD kidneys, postulating that AQP1 may be involved in cytogenesis. Here we investigated the role of AQP1 on ADPKD development.

Methods: We used Aqp1/Pkd1 double knockout mice, embryonic kidney organ cultures, and matrix‐grown MDCK cells in this study to investigate the role of AQP1 in ADPKD. Expression of Wnt signaling‐related proteins were detected in MDCK and AQP1‐MDCK cells. Co‐immunoprecipitation was used to determine the interaction between AQP1 and β‐catenin,GSK3β,LRP6 and Axin1.

Results: Kidney size and cyst number were significantly greater in AQP1‐null PKD mice than in AQP1‐expressing PKD mice. In embryonic kidney cultures, AQP1 deletion increased cyst development. In matrix‐grown MDCK cells, AQP1 inhibited cystogenesis and promoted tubulogenesis. AQP1 decreased β‐catenin and cyclinD1 expression, suggesting down‐regulation of Wnt signaling. Co‐immunoprecipitation showed AQP1 interaction with β‐catenin, GSK3β, LRP6 and Axin1. Biochemical analysis revealed decreased p‐β‐catenin and increased β‐catenin expression in Aqp1/Pkd1 double knockout mice, suggesting enhanced Wnt signaling.

Conclusions

These results suggest that AQP1 may be involved in maintaining the stability of the “destruction complex” to promote β‐catenin phosphorylation and suppress the Wnt signaling. This study reveals a previously unrecognized role of AQP1 in ADPKD and hence may provide new therapeutic targets.
Hypoxia Induced Redistribution of OS‐9 in Kidney Cells
Authors: Harris Regine , Stanley Corshe , Ongeri Elimelda Moige
Published: June 2021

OS‐9 is a component of the endoplasmic reticulum‐associated degradation machinery. OS‐9 has been shown to interact with and promote hydroxylation of the hypoxia‐inducible factor (HIF1α). Meprin metalloproteases are abundantly expressed in the brush border membranes (BBM) of proximal kidney tubules. Redistribution of meprins from the BBM to the cytosol and basolateral membranes occur in ischemia‐reperfusion induced renal injury. Meprin β interacts with and cleaves OS‐9 in an isoform‐specific manner. The present study sought to determine how hypoxia impacts OS‐9 localization in Mardin‐Darby canine kidney (MDCK) cells transfected with meprin β cDNA. The cells were depleted of oxygen by exposure to 125 µM cobalt chloride (CoCl2) for 0, 0.5, 1, 2, and 3 h. Proteins were extracted from the cells and fractionated into cytosolic‐ and nuclear‐enriched fractions. Western blot analysis and optic densitometry were used to quantify the levels of OS‐9 in the cytosolic‐ and nuclear‐enriched protein fractions, and the levels of HIF1α in nuclear‐enriched protein fractions. Additional cells were cultured in slide chambers, fixed in paraformaldehyde, and probed with anti‐OS‐9 and anti‐HIF1α antibodies using immunofluorescent staining. Localization of HIF1α and OS‐9 in the cells was evaluated by confocal microscopy. Exposure to CoCl2 resulted in increased HIFα levels in the nucleus and a time‐dependent redistribution of OS‐9 from the nucleus to the cytosol. In the meprin transfected cells, the cytosolic levels of OS‐9 subsequently decreased. The decrease in cytosolic OS‐9 levels was not observed in non‐transfected control cells, suggesting meprin mediated degradation of OS‐9. These data provide evidence that meprins may impact the response to hypoxia in part via modulation of cytosolic OS‐9 levels.
Analysis of Cardiorenal Physiological Dysfunction in a Mouse Model of Neonatal‐lethal Congenital Obstructive Nephropathy
Authors: Lui Vanessa , Lee Amanda , Polgar Noemi , Tamashiro Kadee‐Kalia , Napoli Josephine Andrea , Walton Chad , Fogelgren Ben
Published: June 2021

Congenital obstructive nephropathy is the most common cause of pediatric chronic kidney disease and end stage renal disease. This disease is caused by obstruction of the urinary tract during fetal development and most commonly occurs at the ureteropelvic junction (UPJ), where the renal pelvis transitions into the ureter. The exocyst is a highly‐conserved 8‐protein trafficking complex and we recently generated a novel conditional knockout mouse for the central exocyst subunit, Sec10 (Sec10‐CKO). Upon Sec10 deletion in the developing kidney and ureter epithelial cells, the mice displayed late‐gestational congenital UPJ obstructions that caused death 10‐14 hours after birth. To further investigate the physiological consequences of these lethal urinary obstructions, cardiorenal and pulmonary parameters were compared in wild‐type and mutant newborn littermates. Upon necropsies, atrial distension with severe hemorrhaging was grossly visible in the mutants compared to littermate controls, which was also confirmed with histological analysis. We performed M‐mode and Doppler echocardiography on the hearts of newborn mice, which indicated signs of diastolic dysfunction. Assessment of newborn lung mass and surface area showed no significance between control and mutant mice. Although our analysis is ongoing, these preliminary results suggest this new mouse model may be a useful tool in studying the cardiovascular consequences of severe urinary obstructions in humans.
Transcriptome Profiling of Human Prostate Cancer Cell Line LNCaP Treated with IndividualSoy Isoflavones or Their Combination
Authors: Jang Chan Ho , Schmidt Carl , Wu Changqing
Published: June 2021

Prostate cancer (PCa) is the second leading cause of cancer death in American men. Increasing epidemiologic evidence and laboratory studies suggest soy isoflavones might reduce PCa risk. However, attention has been drawn to the safety of using high levels of soy isoflavones in human, which is especially the concern about taking soy isoflavone dietary supplements. Our previous work showed that daidzein and genistein had a synergistic effect on inhibiting proliferation and inducing apoptosis of PCa cells, which suggests a combination of isoflavones at lower levels may be more effective for PCa prevention. Based on this work, RNA sequencing (RNA‐seq) was performed in this study to search underlying mechanisms in human prostate cancer cell line LNCaP treated with individual isoflavones (genistein or daidzein, 50 or 200 μM) or their combination (50 μM). Transcriptome analysis showed that several genes were differently expressed in LNCap cells among treatments with individual isoflavones or their combination.Genes such as transcription factor CP2‐like 1, activating transcription factor 3, PRKC apoptosis WT1 regulator, DNA‐damage‐inducible transcript 3 and interferon gamma‐inducible protein 16 are highly expressedin the cells treated with genistein orthecombination of daidzein and genistein, while not highly expressed in the cells treated only with daidzein. To conclude, these findings have indicated that RNA‐seq is valuable to simultaneously search multiple molecular targets involved in the cancer preventive effect of soy isoflavones and a reliable means to quantify absolute transcript levels. The findings might also help explain the synergistic effect of daidzein and genistein.

The project is funded by University of Delaware Research Foundation.
Regulation of Renal TRPV4 Activity by Dietary Potassium Intake is Essential for The Maintenance of K+ Homeostasis
Authors: Boukelmoune Nabila , Mamenko Mykola , Zaika Oleg , O'Neil Roger , Pochynyuk Oleh
Published: June 2021

Flow‐dependent K+ secretion via Ca2+‐activated BK channel in the collecting duct (CD) is an important regulator of K+ homeostasis in the body, specifically during elevated K+ intake. Our recent findings suggest that elevated flow induces [Ca2+]i elevations in the CD cells via Ca2+‐permeable TRPV4 channel. Here we probe the involvement of TRPV4 in regulation of BK‐dependent potassium secretion in the CD and its contribution to K+ balance at the systemic level. We found that TRPV4 expression and m‐RNA levels were significantly increased in the kidneys from mice kept on high potassium diet (5% K+), while dietary K+ restriction (0.003% K+) markedly decreased TRPV4 abundance in the kidney. Regulation of TRPV4 protein expression occurs independently of aldosterone, as saturation of systemic mineralocorticoid status by DOCA does not alter renal TRPV4 levels. Confocal immunofluorescence in split‐opened CDs shows that high K+ intake results in a pronounced redistribution of TRPV4 towards the apical plasma membrane of the CD cells, while K+ restriction in mice causes intracellular retention of TRPV4. Augmented TRPV4 expression and abundance on the apical plasma membrane during high K+ intake were associated with significantly increased flow‐induced [Ca2+]i responses in the CD cells. TRPV4‐/‐ mice kept on high potassium diet exhibit hyperkalemia and have drastically reduced BK channel activity, the major route for the flow‐mediated potassium secretion, in CD cells, when compared to wild‐type controls. Overall, our findings unequivocally demonstrate that TRPV4 regulates K+ secretion in the CD in a BK‐dependent manner, thus, contributing to the maintenance of systemic potassium homeostasis.
Reduced Renal K+ Excretion With Compensatory Hyperaldosteronism In KCa1.1 Channel β2‐subunit KO Mice
Authors: Larsen Casper , Sorensen Mads , Praetorius Helle , Leipziger Jens
Published: June 2021

The kidneys are the primary organs responsible for excreting K+, ensuring whole body K+ homeostasis by precisely matching K+ excretion to dietary K+ intake. K+ is secreted into the urine in the collecting ducts, and two distinct mechanisms for K+ secretion exist; a constitutive mechanism mediated by ROMK (Kir1.1) in principal cells and a flow‐induced mechanism mediated by BK channels (KCa1.1) in intercalated cells. Both mechanisms are up‐regulated by aldosterone. Here we studied renal K+ excretion in KO mice for the β2‐subunit of the BK channel.

The β2 KO mice have increased plasma aldosterone, low renin expression and normal plasma [K+]. The low renin in β2 KO mice indicates that hyperaldosteronism was triggered by a K+ handling deficiency, rather than hypotension and activation of the renin‐angiotensin‐aldosterone system. We hypothesize that β2 KO mice have decreased BK channel‐mediated renal K+ secretion, which is compensated by hyperaldosteronism and up‐regulation of ROMK‐mediated K+ secretion, allowing β2 KO mice to maintain normal plasma [K+]. In fact, when treated with eplerenone (mineralocorticoid receptor antagonist) for 4 days, β2 KO mice develop slight hyperkalemia (4.15 mM ± 0.13 in WT vs. 4.60 mM ± 0.10 in KO, P = 0.013).

Urinary K+ excretion following oral K+ load (20% of normal daily intake) was not different between WT and KO mice under control conditions. However, when treated with eplerenone, β2 KO mice had a significantly lower urinary K+ excretion rate (P=0.044) and significantly higher plasma [K+] 3 hours after oral K+ load (10.0 mM ± 0.4 in WT vs. 11.4 mM ± 0.5 in KO, P = 0.044). Our data support that hyperaldosteronism in β2 KO mice is part of a chronic compensation to a decreased BK channel‐mediated renal K+ secretion.

Supported by the Lundbeck foundation.
Net K Secretion in Thick Ascending Limb in Mice on Low Na High K Diet
Authors: Wang Bangchen , Wen Donghai , Cornelius Ryan , Yuan Yang , Li Huaqing , Wang‐France Jun , Sansom Steven
Published: June 2021

A low Na and high K diet is considered a healthy choice for its cardiovascular benefits. Understanding the effect of such diet on renal handling of K is imperative for choosing effective diuretics and preventing hyperkalemia and hypokalemia. Wild‐type (WT) and Ca‐activated K channel (BK) β4‐subunit knockout mice (KO) were fed either control or a low Na high K diet (LNaHK) for 7‐10 days (all groups: N 蠅 4). Urine samples were collected from mice in metabolic cages 12 hours after intra‐peritoneal injection of vehicle (veh), furosemide (furo), amiloride (amil), or furo + amil. In WT on a control diet, the urinary K excretion (UKV, μmol/day) was higher in furo‐treated mice compared to veh due to increased distal flow (furo: 390 ± 48 vs veh: 297 ± 26). However, in WT on LNaHK, UKV was significantly lower in furo‐treated mice compared to veh despite increased distal flow (furo: 609 ± 113 vs veh: 1057 ± 121). Furo + Amil treatment decreased UKV more than either drug alone in WT on LNaHK (furo + amil: 143 ± 41; furo: 609 ± 113; amil: 413 ± 68). In KO on LNaHK, however, UKV was not different between furo and veh groups (furo: 571 ± 80 vs veh: 464 ± 80). Using HPLC, we found the furosemide concentrations in plasma of both WT and KO mice were 2.3 μM and 1.4 μM 2 hours and 6 hours, respectively, after treatment, which would inhibit the basolateral NKCC1 in the collecting ducts by only 20%. Immunofluorescence staining showed that BK was also expressed in the apical membrane of medullary collecting ducts (MCD) of LNaHK for WT, but not KO. These results suggest that there is a net K secretion in the thick ascending limb of loop of Henle in mice on the LNaHK diet, which is dependent on the BK‐mediated K recycling and high interstitial K concentration in the MCD.
Female Sex Hormones Protect Against Salt‐Induced Increases in Immune System Activation in Dahl Salt‐Sensitive Rats (DSS)
Authors: Ebeledike Chiemelie , Ralph Erica , Musall Jacqueline , Sullivan Jennifer
Published: June 2021

T cells contribute to hypertension and renal injury in response to a high‐salt (HS) diet in male DSS. This study tested the hypotheses that 1) salt increases renal T cell infiltration in female DSS and 2) sex hormones protect against the pro‐inflammatory effects of salt. Female DSS were randomized to the following groups: gonad‐intact + normal‐salt (NS), ovariectomized (OVX) + NS, intact + 4% HS, OVX + HS for 2 weeks (n=3/group). Blood pressure (BP) was measured by telemetry, protein excretion was measured weekly and kidneys were processed for flow cytometric analysis of T cells and high mobility group box 1 protein (HMGB1), HMGB1 is released by damaged cells and stimulates pro‐inflammatory T cells. OVX exacerbated HS‐induced increases in BP and protein excretion (32% increase and 122% increase, respectively) compared to intact DSS (22% increase and 80% increase; p<0.05). OVX and HS increased pro‐inflammatory renal CD4+ T cells (p<0.05) and Th17 cells (p<0.05) and decreased immuno‐suppressive T regulatory cells (Tregs; p<0.001), however, HS induced a greater increase in CD4+ T cells in OVX vs. intact (p<0.001) and a greater decrease in Tregs in intact vs. OVX DSS (p<0.001). Renal HMGB1 increased with HS (p<0.01) and OVX (p<0.001), although the increase was greater in OVX vs. intact DSS (p=0.03). In summary, OVX DSS have the greatest increase in T cells and HMGB1 as well as the highest BP and proteinuria. We propose that female sex hormones protect against the pro‐inflammatory effects of a HS diet by mitigating the release of HMGB1.

CD4+ cells (expressed as %CD3+ cells)
Th17 cells (expressed as %CD3+CD4+ cells)
Tregs (expressed as %CD3+CD4+ cells)
HMGB1 (expressed as % total kidney cells)

Intact NS
25±0.6
8±0.6
9±0.3
2±0.3

Intact HS
29±0.3
10±0.6
5±0.6
4±0.6

OVX NS
29±0.3
14±1.0
2±0.3
5±0.6

OVX HS
40±0.7
17±1.0
1±0.3
9±0.6
Placental Ischemia‐Induced TH17 Cells Mediate the Pathophysiology Associated with Preeclampsia
Authors: Cornelius Denise , Wallace Kedra , Amaral Lorena , Scott Jeremy , Campbell Nathan , Thomas Alexia , Moseley Janae , LaMarca Babbette
Published: June 2021

Preeclampsia (PE) is associated with hypertension, inflammation, intrauterine growth restriction (IUGR), increased CD4+ THelper 17 cells (TH17s) and agonistic auto‐antibodies to the AT1 receptor (AT1‐AA). The objective of this study was to determine a role for TH17s in mediating pathophysiology associated with PE using the reduced uterine perfusion pressure (RUPP) rat model of PE. On gestation day 12 (GD12) RUPP‐induced TH17s were injected into normal pregnant (NP) rats; On GD19 blood pressure (MAP) was recorded, and blood and tissues were collected. One‐way ANOVA was used for statistical analysis. MAP increased from 99±2 in NP (n=8) to 128±4 in RUPP (n=9) and to 111±3 mmHg in NP+TH17 (n=11; p<0.05). Placental and renal ROS increased from 238 and 411 RLU, respectively, in NP to 328 and 603 RLU in RUPP and to 339 and 833 RLU in NP+TH17 (p<0.05). Importantly pup weight (gm) decreased significantly from 2.19 in NP to 1.91 in RUPP, and to 1.9 in NP+TH17 (p<0.05). Adoptive transfer of TH17s also increased inflammation: IL‐6 increased significantly from 27.5 pg/mL in NP to 62.2 pg/mL in RUPP and to 75.9 pg/mL in NP+TH17 (p<0.5); IL‐17 increased from 0.5 pg/mL in NP to 1.29 pg/mL in RUPP and increased to 5.5 pg/mL in NP+TH17 (p>0.05). AT1‐AA significantly increased from 0.1beats/min in NP to 16.82beats/min in RUPP and to 15.6beats/min in NP+TH17. TH17s induced hypertension, ROS, IUGR, inflammation, and AT1‐AA in pregnant rats, demonstrating the importance of TH17s to mediate the pathophysiology associated with PE and their potential as a new therapeutic target for treatment of PE.

NIH grants RO1HD067541 and T32HL105324
N‐Acetyl‐Seryl‐Aspartyl‐Lysyl‐Proline (Ac‐SDKP) Delays the Development of Hypertension and Renal Damage in Systemic Lupus Erythematosus (SLE)
Authors: Nakagawa Pablo , Liao T‐D , Worou M , Basha H , Peterson E , Janic B , Yang X‐P , Rhaleb N‐E , Carretero Oscar
Published: June 2021

SLE is an autoimmune disease with high prevalence of hypertension. Ac‐SDKP is naturally occurring peptide with anti‐inflammatory and immunomodulatory properties. In classical hypertension models, Ac‐SDKP prevents renal damage without affecting systolic blood pressure (SBP). We hypothesize that in autoimmune systemic diseases associated with hypertension, such as SLE, in addition to preventing renal damage, Ac‐SDKP also prevents the development of hypertension. Here we used NZBWF1 mice as SLE hypertension model.
Female NZW (control) or NZBWF1 (SLE) mice were divided in 4 groups: 1) control+vehicle, 2) control+Ac‐SDKP, 3) SLE+vehicle, 4) SLE+Ac‐SDKP. Animals were treated with vehicle or Ac‐SDKP (800 µg/kg/d via minipumps) from 25 to 38 wks of age. Urine albumin and SBP were recorded weekly. Plasma anti‐dsDNA antibodies were measured at 38wks.
At 28 wks of age 40 % of the animals in the SLE+vehicle group developed albuminuria (defined as urine albumin 蠅 30 µg/24hs), while in the SLE+Ac‐SDKP group the incidence of albuminuria was 10 %. At 32 wks of age the incidence of hypertension (defined as SBP 蠅 140 mmHg) in SLE+vehicle was 40 %, while none of the animals in SLE+Ac‐SDKP group developed hypertension. At 32 wks, the survival rate was 60 % in SLE+vehicle group and 90% in SLE+Ac‐SDKP 90 % group. However, at 38 wks of age the incidence of albuminuria, hypertension, mortality and plasma dsDNA antibody levels were similar between SLE+vehicle and SLE+Ac‐SDKP groups.
In SLE, Ac‐SDKP delays the incidence of renal damage, hypertension and mortality without affecting the dsDNA antibody levels.
Supported by NIH HL028982 and AHA 13PRE17100074
Expression of claudin‐2 in the uroepithelium increases urinary bladder activity
Authors: Montalbetti Nicolas , Kullmann F Aura , Rued Anna , Clayton Dennis , Prakasam H Sandeep , Ruiz Wily , Apodaca Gerard , Carattino Marcelo
Published: June 2021

Bladder filling promotes a reorganization of the umbrella cell tight junctions (TJ) ring, which is accompanied by a large drop in junctional resistance. The goal of this study was to assess the impact of increased paracellular permeability on bladder function. Claudins are structural components of the TJ that define the permeability of the paracellular barrier present in epithelia. To increase the paracellular permeability of the rat uroepithelium we expressed claudin‐2 using in situ adenoviral transduction. Fluorescent microscopy showed prominent expression of claudin‐2 in the umbrella cells of the transduced bladders. The electrical resistance and potential difference of the urinary bladders mounted in Ussing chambers were significantly lower in rats transduced with claudin‐2, 0.5 ± 0.1 kΩ.cm2 and ‐1.3 ± 0.2 mV, than in rats transduced with GFP, 4.6 ± 0.3 kΩ.cm2 and ‐12 ± 2 mV. Conversely, the permeability of the uroepithelium to large molecules (MW >350), fluorescein and NHS‐biotin, was similar in rats transduced with GFP and claudin‐2. In vivo studies of bladder function using continuous cystometry revealed higher intravesical basal pressures and shorter intervoiding intervals in rats transduced with claudin‐2 than in rats transduced with GFP. In conclusion, the expression of claudin‐2 in the uroepithelium increases its permeability to ions and augments voiding frequency, consistent with the notion that paracellular ion transport modulates bladder function.

Support: NIH R01 DK099196 and P30 DK079307 Pittsburgh Center for Kidney Research ‐ O'Brien Pilot.
Adipose triglyceride lipase (ATGL) deletion from adipocytes, but not skeletal myocytes, impairs acute exercise performance in mice
Authors: Sitnick Mitchell , Dube John , Schoiswohl Gabriele , Kershaw Erin
Published: June 2021

Fatty acids (FA) and glucose are essential energy substrates that supply exercising muscle. While the selection of substrates is highly influenced by their availability and the intensity and duration of exercise, for prolonged exercise FA are the key substrate. Adipose Triglyceride Lipase (ATGL) is the rate‐limiting enzyme mediating intracellular triacylglycerol hydrolysis and should play a key role in the mobilization of FA during exercise. However, the contribution of adipose versus skeletal muscle fatty acid mobilization to support exercise remains unclear. To gain insight, we generated adipocyte or skeletal myocyte‐specific ATGL knockout mice and determined their response to acute peak and submaximal exercise.

We found that adipocyte ATGL‐mediated lipolysis was crucial to support exercise performance, likely owing to limited FA availability and reliance on a rapidly depleted carbohydrate store. Impaired skeletal muscle ATGL‐mediated lipolysis did not impact exercise tolerance and these mice exhibited improved metabolic flexibility during peak exercise.

This suggests that adipocyte rather than skeletal myocyte ATGL‐mediated lipolysis plays a greater role during acute peak and submaximal endurance exercise due to limits in substrate availability and greater reliance on carbohydrate oxidation. While further investigation is needed, this suggests the role of IMCL is more of a stop‐gap substrate store to bridge carbohydrate use and FA mobilization and delivery from adipocytes to working muscle.

Supported by NIH R01DK090166, HHMI Physician‐Scientist Early Career Award; NIH T32 DK007052
The Profile of Exercise‐induced Neuronal Activation in Autonomic Control Nuclei Changes According to Thermoregulatory Phase
Authors: Lima Paulo , Santiago Henrique , Coimbra Candido
Published: June 2021

The purpose of this study was to examine whether the neuronal recruitment induced by exercise in autonomic nuclei is dependent of thermoregulatory phase and/or physical performance. Rats that were subjected to submaximal running exercise stopped in 3 different times: REST (control group), NADIR (the minimum heat dissipation phase), 20MIN (the stabilization phase of both body temperature ‐ Tb ‐ and tail skin temperature ‐ Tsk), and FATIGUE (Tb and Tsk reach their highest values​).The brains were removed and processed for determination of c‐Fos expression in the medial, median, and ventromedial preoptic areas (mPOA, mnPOA, and vmPOA, respectively), and paraventricular and supraoptic nucleus of hypothalamus (PVN and SON, respectively). Physical exercise induced an increase of c‐Fos expression in all brain areas. The c‐Fos expression in the vmPOA was similar for all exercising groups, while for mPOA, mnPOA, and PVN, it was gradually increased as the exercise and hyperthermic response are prolonged. Only after 20 min of exercise an increase in SON c‐Fos expression was verified. The neuronal recruitment within the mPOA, mnPOA, PVN, and SON was directly correlated to both physical performance and heat storage. The thermoregulatory brain areas are recruited even at small Tb changes, while the SON is recruited only when Tb reaches its higher values, possibly inducing changes on fluid balance. The recruitment rate in these areas is also influenced by duration of stress stimulus, either by increasing running time, as the prolonged hyperthermic response arising of exercise. Cnpq, Capes, and Fapemig.
Differential Response in Arterial Stiffness Measures Between Obese and Normal Weight Individuals
Authors: Baynard Tracy , Ranadive Sushant , Lane Abbi , Yan Huimin , Kappus Rebecca , Fernhall Bo
Published: June 2021

Acute exercise appears to have positive influence on arterial function during the recovery period, yet the effect of obesity is poorly understood in recovery. We examined the effects of an acute bout of exercise on arterial function in obese versus healthy weight individuals. Young (23 yr), lean (n=38, BMI=22.3 kg/m2) and obese (n=20, BMI=34.0 kg/m2) individuals had arterial measurements obtained at rest, and 15 and 30 min following a maximal bout of aerobic exercise. Arterial tonometry and ultrasound were used to obtain central BP, arterial compliance and stiffness (beta stiffness, elastic modulus (Ep), and central pulse wave velocity (PWV)). Despite similar central mean BP, the obese group had higher cPWV following exercise. Beta stiffness and Ep responded similarly to exercise between groups. Arterial compliance was higher in obese but the response in recovery was similar between groups.

Lean
Obese

Pre
15‐min post
30‐min post
Pre
15‐min post
30‐min post

Mean aortic BP
82
83
82
87
86
87

Central PWV
5.67
5.46
5.35
5.84
6.11
6.16

Beta Stiffness
5.89
6.34
5.78
4.88
6.04
5.31

Elastic Modulus
69
75
67
67
78
66

In this young cohort, obesity was associated with increased central arterial stiffness, independent of BP, but higher arterial compliance. This suggests that measures of arterial stiffness provide differential information potentially associated with where in the arterial tree measures were made.
Deep Water Exercise Training is Associated with Decreased Arterial Stiffening in Women
Authors: Moore Corey , Fournier Sara , DeVallance Evan , Lee Kyuwan , Bonner Daniel , Donley David , Sherlock Lori , Chantler Paul
Published: June 2021

Metabolic syndrome (MetS) is associated with an increase in cardiovascular (CV) mortality, partially due to arterial stiffening, which can be measured non‐invasively via pulse wave velocity (PWV). Arterial stiffness is a clinically relevant CV marker of mortality, an increase in PWV by only 1 SD is associated with an increase of CV risk by 15%. We tested the hypothesis that 8 weeks, 3 days per week, of deep water exercise can lower the PWV in individuals with MetS.

10 women (age=58±5) were recruited to participate in the study. Carotid to femoral PWV was measured using applanation tonometry; radial artery waveforms were used for pulse wave analysis and to calculate central pressures. Aquatic exercise significantly lowered PWV by 0.53 m/s (p<0.05). Other significant changes that were observed were BMI (p=0.03) from 33.5±1.5 to 32.9±1.5, blood glucose (p=0.01, from 110±8 to 101±7), insulin (p=0.02, from 20±2 to 14±1), insulin resistance (p=0.01, from 5.39±0.81 to 3.55±0.50), augmentation index (p=0.05, from 25±2 to 22±2), and aerobic capacity (p=0.02, from 15.9±1.3 to 17.8±1.7).

These findings indicate that the introduction of deep‐water exercise positively influences the PWV. This decrease in PWV portends improvements in overall CV health and it may be useful in managing other CV related diseases. Much of the data showed significant improvement in clinical measures and this shows the efficacy of deep‐water exercise. While the benefits of land based exercise have long been well known, these findings show that aquatic based exercise is also a very effective method for improving CV health.
Daily exercise may alter anti‐oxidative responses to heat in mouse brain
Authors: NAGASHIMA KEI , TOKIZAWA KEN , Obata Chisa , Tsunakawa Mizuki , Marui Shuri
Published: June 2021

Purpose

Daily exercise increases heat tolerance. However, a line of evidence seems to indicate that peripheral changes such as blood volume and sweat gland are involved in the mechanism. In the present study, we tested the hypothesis that the brain per se acquires heat tolerance.

Methods

Male B6 mice (8 m.o., 40‐45 g b.w.) were used. Placed a radio telemetry device (F20, DSI), they were individually housed at 25°C and 50% RH for 6 w with or without a running wheel (n=20 each; Ex and C, respectively). After the period, each group of mice had exposure of 39.5°C for 4 h or remained at 25°C. The brain was excised: the left brain was frozen with liquid N2 for mRNA and protein expression of SOD 1 and 2 and 5‐HNE (RT‐PCR and Western blotting, respectively).

Results

Body temperature reached >41°C at the end of 39.5°C exposure in both groups. mRNA of SOD 1 and 2 remained unchanged between 25°C and 39.5°C in both groups. Although SOD 1 was not different between the C and Ex groups, SOD 2in the cortex and basal brain was greater in the Ex group than in the C group. In the C group, SOD2 became after the heat; however, remained unchanged in the Ex group.

Discussion

Hyperthermia may be a stimulus decreasing anti‐oxydative ability in the brain. However, daily exercise mayincreases anti‐oxydative enzyme and lessen influence of heat on the brain.
Reliability and Validity of Ventilatory Threshold and Respiratory Compensation Point Determined by Near‐infrared Spectroscopy
Authors: Fu Tieh‐Cheng , Mao Tso‐Yen , Wang Jong‐Shyan
Published: June 2021

The purpose of this study was to investigate the reliability and validity of ventilatory threshold (VT) and respiratory compensation point (RCP)determined by near‐infrared spectroscopy (NIRS). One hundred healthy sedentary males performed the grade exercise test (GXT) using a bicycle ergometer. An NIRS was employed to assess oxygenation (Δ[O2Hb]VL) in vastus lateralis muscle, whereas VT and RCP were determined by an automatic gas analysis. The results showed that variables VT and RCP determined by ventilatory analysis were significantly associated with changes of exercising Δ[O2Hb]VL measured by NIRS. The first and secondary timing points of declined Δ[O2Hb]VL during GXT reflected high coefficients of determination for VT (R2 = 0.88, P<0.05) and RCP (R2 = 0.89, P<0.05), respectively. Furthermore, the values of VO2 at VT and RCP measured by gas analysis were not significantly different from those variables determined by NIRS method.Additionally,the values of VT and RCP obtained from ventilatory and hemodynamic methods were highly reproducible day to day, and the single‐measure intraclass corrections were 0.93 to 0.95.Hence, we conclude that the analysis of NIRS is reliable and valid to determine VT and RCP during GXT.
Effect of Hyperoxia on Central Nervous System Intercellular Communication Mediated by Tunneling Nanotubes
Authors: Ray Rupa , Kernagis Dawn , de Ridder Gustaaf , Pizzo Salvatore
Published: June 2021

Oxygen toxicity occurs when exposure to elevated partial pressures of oxygen results in hyperoxia. Central nervous system oxygen toxicity (CNS‐OT) can culminate in seizures, and elucidating mechanisms of CNS‐OT will provide approaches for prevention, delay to onset or amelioration of hyperoxic damage. However, little is known of the pathophysiology of hyperoxia. A possible mediator is the tunneling nanotube (TnT). TnTs are thin, membranous connections mediating the transfer of intracellular materials, including proteins and organelles. We recently identified TnTs between normal brain and glioblastoma cells in mouse xenograft brain sections. When neurons or astrocytes are exposed to H2O2, stressed cells form TnTs and unidirectionally transfer materials, indicating that TnTs may be induced by stress as a defense mechanism. Therefore, we are evaluating the effect of oxygen tension on TnT formation and materials transfer and function in neurons and glia. These studies will reveal molecular changes in CNS cell connectivity. Preliminary data indicate that SK‐N‐SH cells grown in 85% oxygen produce more TnTs than cells grown in standard conditions. We also found increased expression of putative TnT markers, ezrin, cxn43, myoX and rhot1, in hyperoxic conditions. The anticonvulsant vigabatrin, which prevents hyperbaric oxygen‐induced seizures in rats, increases the number of TnTs per cell under norm‐ and hyperoxia, suggesting that vigabatrin may upregulate TnT production to facilitate the transfer of materials that protect against hyperoxia. Our studies will elucidate TnT formation and function and identify regulatory mechanisms governing TnT‐mediated behaviors following hyperoxia.
What Can We Learn From Deep‐Diving Elasmobranchs to Help Humans Adapt to Extreme Underwater Environments?
Authors: Ari Csilla , Decker Summer , Ford Jonathan , D'Agostino Dominic
Published: June 2021

Operational Divers are often exposed to high pressure for extended periods while conducting deep dives. To enhance the performance and safety of these divers elevation of ketone bodies in the blood might be considered, as those have proven to serve as alternative fuel for the brain, to enhance physical and cognitive performance and delayed the onset of CNS oxygen toxicity seizures in rats exposed to 5 ATA O2. Some elasmobranchs developed adaptations to preserve and maintain brain function during repeated dives at extreme depths, in environments with low temperature and oxygen. We hypothesized that studying the morphological, physiological and behavioral adaptations of deep‐diving elasmobranchs can lead to the development of new technologies to aid humans underwater.

Brain morphology of shark and ray species were examined on a GE 3T MRI scanner. 3D volumetric reconstructions were used to analyze brain volume and geometric morphology. Histological studies determined astroglia to neuron ratio and biochemical analysis of blood measured ketones and glucose.

Our results demonstrate that deep‐diving (~2000m) elasmobranchs have brains with largest weight, volume and surface area of all fish, high glia to neuron ratio and remarkably high blood ketones (~2.5mM). Considering these observations, we speculate that ketone utilization is an important physiological adaptation to preserve brain metabolism in deep‐diving animals with large brains. These preliminary observations offer insights into physiological countermeasures of elasmobranchs that can be exploited for extending human performance and resilience in extreme environments.
Inhaled CO2 and CO2 Retention during Heavy Exercise Underwater
Authors: Shykoff Barbara , Warkander Dan
Published: June 2021

Underwater breathing apparatus increases resistive work of breathing. That and other loads from breathing underwater may alter responses to inspired CO2 and exercise, and lead to CO2 retention. Minute ventilation (V’) and end tidal CO2 partial pressure (PETCO2), one‐minute averages in late exercise, were measured in 16 men during dry and underwater endurance exercise at nominally 85% of divers' maximum oxygen uptake rates. Divers reported relative perceived exertion (RPE) scores. Underwater, supplied PCO2 (PsCO2) was 0, 1, or 2 kPa, and breathing resistance was symmetrical to give 1 kPa at 62.5 L/min, the minimal resistance of mask and large‐bore hoses, about 1/3 of the maximum acceptable. Oxygen partial pressure underwater was 135 kPa, and dry, 100 kPa. Without added CO2, V’ decreased from dry to wet (89 to 70 L/min). Mean V’ increased linearly with PsCO2 [slope 3.3 (L/min)/kPa] but so did its variance, increasing 6‐fold from PsCO2=0 to 2 kPa. Mean PETCO2 also increased linearly from 5.4 kPa (41 Torr) (slope 0.66 kPa/kPa), indicating that V’ was inadequate for the CO2 load. RPE scores at end exercise with no CO2 showed mean and median 16.5, interquartile distance (IQ) 1.5. With PsCO2=1 kPa, the distribution was irregular, median 16, IQ 3. With PsCO2=2kPa, the distribution was almost uniform from 14 to 20, median 17, IQ 4. Most divers increased V’ somewhat with increasing PsCO2, but mean PETCO2 was 46 and 51 Torr with PsCO2 of 1 and 2 kPa, respectively. For 3 and 8 divers with PsCO2 of 1 and 2 kPa, respectively, PETCO2 was greater than 51 Torr, the value above which Sayers et al (1987) found cognitive decrements. The combination of even minimal resistance, heavy exercise, and inhaled CO2 underwater leads to substantial and potentially hazardous CO2 retention. Support: NAVSEA Deep Submergence Biomedical Program.
Chronic Intermittent Hypoxia (CIH) has state‐dependent effects on rhythmogenesis from the preBötzinger complex
Authors: Garcia Alfredo , Khuu Maggie , Ramirez Jan
Published: June 2021

Exposure to chronic intermittent hypoxia (CIH) experimentally replicates many changes in cardio‐respiratory physiology that result from untreated obstructive sleep apnea (OSA). Thus, CIH has become an established paradigm to understand the potential mechanisms involved with the pathophysiology of OSA. The objective of this study is to understand how CIH affects respiratory rhythmogenesis from the preBötzinger complex (preBötC). Rhythmic preBötC brainstem slices were prepared from neonatal mice (P10‐12; CD1) that were exposed or unexposed to CIH (10days). Electrophysiological recordings were made under control (95% O2; 5% CO2) or hypoxic (0% O2; 5% CO2) conditions from the preBötC. Under control conditions CIH increases the short‐term variability of burst amplitude while blockade of the persistent sodium current (INaP) using, 10µM riluzole, reduced burst amplitude irregularity caused by CIH. Differences in rhythmogenesis were also found between when transitioning between states of oxygenation and during steady‐state hypoxia. During the initial period of hypoxia, the latency to the first occurrence of period doubling was larger in the CIH cohort. In steady state hypoxia, rhythms from the CIH cohort did not show a reduction in the frequency of rhythmogenesis. Moreover, in the CIH group, post‐hypoxic augmentation was more pronounced. These findings indicate that CIH appears to affect the contribution of INaP causing state dependent effects on network physiology. Specifically, CIH conditions the dynamic responsiveness of the preBötC to changes in oxygenation, but causes irregularities in well‐oxygenated states.
Chronic Intermittent Hypoxia (CIH) Impairs Neurogenesis and Alters Paired Pulse Synaptic Plasticity In the Dentate Gyrus of Hippocampus
Authors: Khuu Maggie , Hodge Rebecca , Ramirez Jan‐Marino , Garcia Alfredo
Published: June 2021

CIH is an established model simulating the pattern of oxygenation experienced by individuals suffering from untreated sleep disordered breathing (SDB). In addition to the cardio‐respiratory effects, SDB has been shown to cause deficits in memory and learning. Previous studies conducted in CA1 neurons of the hippocampus indicate that CIH impairs synaptic plasticity in this neuronal population important to executive function. This ongoing study examines how CIH impacts the dentate gyrus (DG), another region of the hippocampus also important to memory and learning and known to exhibit neurogenesis throughout life. Histological analyses and electrophysiological experiments were conducted in the DG of mice (P60‐P80) exposed to CIH for 30 days. Although no difference in structural volume was observed within the hippocampal formation, the number of doublecortin positive cells found in the granular cell layer of the DG were fewer following CIH. Field recordings conducted in the DG of hippocampal brain slices revealed that CIH suppresses paired pulse facilitation at later interpulse intervals (200‐400ms). This significantly alters the paired pulse profile observed in control experiments. While CIH does not cause changes in gross structure of the DG, it results in altered neurogenesis and changed synaptic plasticity indicative of diffuse neuronal injury within an area central to executive function.
Short‐Term Hypoxia Training Improves Anaerobic Performance in Amateur Women Boxers
Authors: Wang Ran , Hu Yang , Fan Mengjiao , Fukuda David , Stout Jeffrey , Hoffman Jay
Published: June 2021

The Live Low‐Exercise High (LoHi) method has shown to effectively improve athletic performance in various sports. However, the examination of this method in female combat sports athletes is limited.

Purpose

To investigate the effect of LoHi on anaerobic performance of amateur women boxers.

Methods

Sixteen amateur women boxers were randomly assigned to hypoxic training (HT) or normal training (NT) groups during a 21‐day period utilizing identical daily training sessions and dietary protocols. The HT group trained under normobaric hypoxic conditions (with a simulated altitude of 2500 m) three times per week on alternating days, while the NT group trained in the standardized environment at sea level. Peak power (PP), mean power (MP), fatigue index (FI) and blood lactate concentrations (BL) from a 30‐s Wingate cycling test, and maximal heart rate (MHR) and one‐min heart rate recovery (HRR) from a simulated sparring test were recorded before and after the training period.

Numbers (N)
Age (Year)
Height (cm)
Weight (kg)
Training Status (Year)

HT Group
8
17.1 ± 0.8
165.4 ± 5.5
56.9 ± 6.6
3.6 ± 0.9

NT Group
8
17.3 ± 1.3
164.8 ± 2.3
55.4 ± 4.4
3.5 ± 0.8

Results

Two‐way analysis of variance with repeated measures exhibited a significant interaction for MP (p = .004), BL (p = .033), MHR (p = .008) and HRR (p = .036). Follow‐up paired sample t‐tests showed significant improvement in MP (p = .005), FI (p = .017), BL (p = .017), MHR (p = .019) and HRR (p = .034) in HT group and no change in these measures for the NT group.

HT‐Pre
HT‐Post
NT‐Pre
NT‐Post

Peak Power (Watts)
8.3 ± 1.0
8.7 ± 0.9
8.2 ± 1.0
8.3 ± 0.8

Mean Power (Watts)
6.5 ± 0.6
6.9 ± 0.7*
6.6 ± 0.8
6.6 ± 0.8

Fatigue Index (%)
49.3 ± 5.7
44.5 ± 4.4*
50.3 ± 5.2
49.7 ± 3.5

Blood Lactate (mmol/L)
9.6 ± 1.5
11.3 ± 1.6*
9.5 ± 1.3
9.9 ± 1.6

Maximal Heart Rate (beat/min)
182.4 ± 4.2
175.5 ± 9.2*
181.3 ± 4.2
181.8 ± 4.2

Heart Rate Recovery (beat/min)
45.9 ± 12.4
55.9 ± 9.9*
46.4 ± 9.4
47.4 ± 9.1

Conclusion

Short‐term LoHi can improve anaerobic performance in amateur women boxers with apparent benefits in sustainable power output, lactate clearance and post‐exercise recovery.
Cox2 Inhibition in Early Pregnancy Improves Adverse Outcomes in the BPH/5 Mouse Model of Preeclampsia (PE)
Authors: Sones Jenny , Butler Scott , Shapiro Stephanie , Cha Jeeyeon , Dey Sudhansu , Davisson Robin
Published: June 2021

PE presents as maternal hypertension and proteinuria during the 2nd half of pregnancy and often results in a preterm or growth restricted baby. Despite being a leading cause of perinatal morbidity/mortality, the underlying mechanism is unknown. BPH/5 mice spontaneously develop the cardinal features of PE and poor fetoplacental outcomes, such as fetal growth restriction (FGR). Prior to placenta formation, BPH/5 show a 2‐fold increase in Cox2 in e7.5 implantation sites vs C57. Since Cox2‐derived prostaglandins (PG) are crucial for placentation, we tested the hypothesis that BPH/5 have dysregulated PG synthesis contributing to adverse pregnancy outcomes. Selective Cox2 inhibition with celecoxib at e6.5 (10mg/kg, oral gavage) normalized Cox2 and reduced PGE2 levels in e7.5 BPH/5 implantation sites (51.6±17.3 vs veh: 135.6±49.7 pg/mg; n=4‐5, p<0.05). Celecoxib abolished resorptions in BPH/5 pregnancies at e12.5 (0% vs veh: 40%), which was associated with improved fetal and placental weight at e12.5 (n=29‐32, p<0.05) and e18.5 (n=49‐51, p<0.05). Late‐gestational proteinuria was decreased in BPH/5 celecoxib‐treated mothers (trace‐1+) vs veh (1‐3+) as measured by urine dipstick. These data indicate that Cox2 and PGE2 play a key role in fetoplacental development in a mouse model of PE, and that celecoxib improves adverse pregnancy outcomes in BPH/5. This highlights the significance of Cox2 signaling in early pregnancy and suggests that aberrations may contribute to the maternal syndrome and FGR seen in PE.