bluuebunny/crossref_metadata_2025 huggingface dataset:
Suppression of hepatic hepcidin expression in response to acute iron deficiency is correlated with an increase of matripatase‐2 protein in the liver
Authors: Zhang An‐Sheng , Anderson Sheila A , Wang Jiaohong , Yang Fan , DeMaster Kristina , Eisenstein Richard S , Tsukamoto Hidekazu , Enns Caroline A
Published: June 2021

Recent studies demonstrate a pivotal role of bone morphogenic protein‐6 (BMP6) and matriptase‐2, a protein encoded by the TMPRSS6 gene, in the induction and suppression of hepatic hepcidin expression, respectively. We examined their expression profiles in isolated liver cells and found an exclusive expression of TMPRSS6 mRNA in hepatocytes. In rats fed an iron deficient (ID) diet for 24 hrs, the rapid decrease of transferrin saturation from 71% to 24% (control versus ID diet) was associated with a marked decrease of hepatic hepcidin mRNA, by approximately 100‐fold in comparison with the corresponding controls. These results indicated a close correlation of low transferrin saturation with the decrease of hepcidin mRNA. The decrease of phosphorylated Smad1/5/8 detected in the ID rat livers suggests that the suppressed hepcidin expression resulted from the inhibition of BMP signaling. qRT‐PCR analysis revealed no significant change in either BMP6 or TMPRSS6 mRNA in the liver. However, an increase of matriptase‐2 protein in the liver from ID rats was detected, suggesting that suppression of hepcidin expression in response to acute iron deficiency is mediated by the increase of matriptase‐2 protein levels.
Characterization of FasL expression of rat Mesenteric Lymph Node Dendritic Cells during Inflammatory Bowel Disease (IBD)
Authors: Rivera Edelmarie , Rodriguez Freddy , Appleyard Caroline Barbara
Published: June 2021

Strong evidence demonstrates that a breakdown of tolerance against intestinal microflora is a major factor in the pathogenesis of IBD. Dendritic cells (DCs) appear to play a role in modulating the innate and adaptive immune response. During IBD DCs are activated and the toll like receptors upregulated. The DC populations present in inflamed and control mesenteric lymph nodes (MLNs) are unknown.

AIM

To examine the expression of FasL on MLN DC during acute colitis.

METHODS

Acute colitis was induced in Sprague Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS i.c.). At 72 hours the rats were sacrificed and the colons removed and scored for macroscopic damage. The cecum and small intestine were exteriorized and the MLN chain identified. After dissection of overlying mesenteric fat, the MLN were removed. The lymph nodes adjacent to the cecum were also removed. Isolation of DC and phenotypic characterization of FasL expression were performed by flow cytometry.

RESULTS

Animals receiving TNBS had significantly higher damage scores than untreated animals (p<0.0001). Expression of FasL on MLN DC was significantly higher in colitis animals (83%) than in controls (38% p<0.05).

CONCLUSION

Our results suggest that loss of FasL expression on MLN DC may be one of the mechanisms that contribute to the inappropriate apoptosis and subsequent T cell accumulation that occurs in IBD. Supported by 5F31DK077584.
Downregulation of aging‐related Klotho gene in experimental colitis
Authors: Thurston Robert D , Larmonier Claire B , Majewski Pawel M , Ramalingam Rajalakshmy , Midura‐Kiela Monica T , Laubitz Daniel , Vandewalle Alain , Mühlbauer Marcus , Jobin Christian , Kiela Pawel R , Ghishan Fayez K
Published: June 2021

Klotho (Kl) deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis. Since osteopenia/osteoporosis are fequently associated with human IBD, we investigated the changes in Klotho gene expression as a consequence of experimental colitis. We utilized three models: TNBS colitis, microflora‐induced colitis in germ‐free IL‐10−/− mice, and adoptive CD4+CD45RBhigh T‐cell transfer colitis, followed by in vitro approaches using renal epithelial cells and the cloned murine Kl gene promoter. Renal expression of Kl mRNA and protein was suppressed in all three IBD models. The degree of inhibition correlated with colitis severity, and was reversed by anti‐TNF antibody. In vitro, TNF significantly inhibited Kl expression and was further potentiated by IFNγ. TNF/IFNγ combination resulted in increased iNOS expression and elevated NO production. The effect of IFNγ could be reproduced by SNAP (NO donor), and reversed by iNOS inhibition. These effects were transcriptionally mediated since Klotho mRNA stability remained unaffected, while reporter constructs with the mKl gene promoter was downreguled in transiently transfected renal epithelial cells. These novel findings could help explain several extraintestinal complications including abnormalities in bone homeostasis in patients with chronic colitis.

Grant support: R37DK033209 (F.K.G.), 5R01DK067286 (P.R.K.), 2R56DK047700 (C.J.)
Regulation of ghrelin production by gastric mTOR signaling
Authors: Zhang Weizhen , Xu Geyang , Li Yin , An Wenjiao , Mulholland Michael
Published: June 2021

The role of gastric mTOR signaling in ghrelin production and food intake was examined in this study. Here we showed the reciprocal relationship of gastric mTOR signaling and ghrelin during changes in energy status. mTOR activity was down‐regulated, while gastric preproghrelin and circulating total and acyl ghrelin were increased by fasting. In db/db mice, gastric mTOR signaling was enhanced, whereas gastric preproghrelin and circulating total anc acyl ghrelin were decreased. Inhibition of the gastric mTOR signaling by rapamycin stimulated the expression of gastric preproghrelin and ghrelin mRNA, increased the plasma total and acyl ghrelin in both wildtype and db/db mice. Activation of the gastric mTOR signaling by L‐leucine decreased the expression of gastric preproghrelin and the level of plasma total and acyl ghrelin. Ghrelin receptor antagonist D‐Lys3‐growth hormone releasing peptide‐6 abolished the rapamycin‐induced increment in food intake despite that plasma ghrelin remained elevated. mTOR is therefore a gastric fuel sensor whose activity is linked to the regulation of energy intake through ghrelin.
Increased sympathoexcitation to angiotensin II (A II) and AT1 receptor mRNA in the paraventricular nucleus (PVN) of pregnant rats
Authors: Heesch Cheryl M. , Kvochina Lyudmyla I. , Burcks Shannon
Published: June 2021

Previously we reported that pressor and sympathetic nerve activity (SNA) responses due to AT1 receptor activation in the SFO are potentiated in term pregnant (P) compared to nonpregnant (NP) rats. The pathway requires ionotropic glutamate receptors (iGluRs) in the PVN and rostral ventrolateral medulla (RVLM) in both P and NP rats. AT1 receptors in the PVN and RVLM contribute to potentiated responses to SFO activation in P rats. Since the PVN is obligatory to this pathway, and PVN to RVLM projections are almost exclusively glutamatergic, we considered that the enhanced role of A II in the RVLM might be related to an A II dependent increase in excitatory drive from the PVN, which could then stimulate local production of A II in the RVLM. In sinoaortic denervated inactin anesthetized rats, increases in mean arterial pressure (MAP) and lumbar SNA (LSNA) due to microinjected A II (50 nl, 1.6 mM) in the PVN were greater in P (n = 4) compared to NP (n = 7) rats (ΔMAP: P= 25±5 mmHg; NP= 15±2 mmHg; ΔLSNA: P= 13±3%; NP= 6.5±2%). Real‐time RT‐PCR was used to assess relative expression of AT1 receptor mRNA (reference gene = GAPDH) in PVN tissue punches from 5 P and 5 NP rats. Expression of AT1 receptors in the PVN tended (P = 0.1) to be greater in P (2.4±0.82) compared to NP rats (1.0±0.22). These results are consistent with an increased role of PVN AT1 receptors in sympathoexcitation in pregnancy. (NIH HL 091164)
Increased expression of CAPON (Carboxy‐terminal PDZ ligand of nNOS) within the paraventricular nucleus (PVN) of rats with heart failure (HF).
Authors: Sharma Neeru M , Zheng Hong , Li Yi‐Fan , Patel Kaushik P
Published: June 2021

Activation of NMDA receptor initiates a Ca2+ influx that stimulates the production of NO by neuronal nitric oxide synthase (nNOS). A physical coupling of the NMDA receptor and nNOS occurs by an intermediary adaptor protein PSD95 (post synaptic density 95). CAPON, a protein rich in PVN competes with PSD95 for interaction with nNOS. Previously, we showed a decreased expression of nNOS and enhanced expression of NMDA NR1 within the PVN of rats with HF. To further elucidate the underlying mechanism of upregulation of NMDA NR1 with a concomitant downregulation of nNOS, we measured the levels of CAPON in left coronary ligation‐induced HF model of rats. CAPON expression is augmented by 22% in rats with HF. Previously we showed that Angiotensin II receptor (AT1) blocker, losartan (LOS) treatment in rats with HF normalized the changes in expression of NR1 and nNOS protein in the PVN. Consistent with these data, LOS treatment (10 mg/kg/day in the drinking water for three weeks) in rats with HF normalized protein expression of the CAPON in the PVN. Studies in neuronal NG108‐15 hybrid cell line treated with Ang II (6.25–100 μM) for 24h revealed 30% increase in CAPON expression at highest dose as compared to untreated cell samples. Co‐treatment with LOS (1μM) for 24h significantly, ameliorates the increased CAPON expression to Ang II. Immunostaining revealed colocalization of CAPON and nNOS in NG108 cell line as well as in PVN. These data suggest that perhaps the disparate changes in NR1 (upregulation) and nNOS (downregulation) may be due to an over expression of CAPON and this over expression of CAPON may be mediated by increased Ang II influence in rats with HF.
Hypothalamic corticotrophin releasing hormone and norepinephrine contribute to sympathoexcitation in heart failure
Authors: Kang Yu‐Ming , Yu Xiao‐Jing , Guo Yan‐lin , Yan Ning , Qi Jie , Yang Li‐Min , Qin Da‐Nian , Elks Carrie , Francis Joseph
Published: June 2021

Systemic administration of tumour necrosis factor (TNF)‐α induces the release of norepinephrine (NE) in the paraventricular nucleus (PVN) of hypothalamus and an increase in expression of corticotrophin releasing hormone (CRH) and CRH type 1 receptors. NE converges in the PVN to influence its neuronal activity. We explored the hypothesis that CRH and norepinephrine in PVN mediate the sympathetic responses to HF. Sprague‐Dawley rats with HF or SHAM rats were treated for 4‐weeks with a continuous bilateral PVN infusion of the CRH antagonist α‐helical CRH (αh‐CRH) or the α1‐adrenergic antagonist prazosin, or artificial cerebrospinal fluid (aCSF). HF rats had higher levels of norepinephrine (NE) and tyrosine hydroxylase (TH) and lower level of nNOS in the PVN when compared to SHAM rats. Plasma levels of cytokines, NE, epinephrine and angiotensin II, were increased in HF rats; renal sympathetic nerve activity (RSNA) was also increased in these animals. Bilateral PVN infusions of the CRH antagonist αh‐CRH attenuated the increases in PVN NE and TH in HF rats. Bilateral PVN infusions of the CRH antagonist αh‐CRH or the α1‐adrenergic antagonist prazosin attenuated, the decrease in PVN nNOS, the increases in plasma levels of cytokines, NE and angiotensin II and in RSNA in HF rats. This study demonstrates that norepinephrine and CRH in the PVN contribute to sympathoexcitation in heart failure.
A high fat diet attenuates fermentation effects of resistant starches and fructans
Authors: Goldsmith Felicia , Martin Roy J , Raggio Anne M , McCutcheon Kathleen L , Vidrine Kirk , Goita MFamara , Zhou Jun , Keenan Michael J
Published: June 2021

In our previous studies, ingestion of prebiotics in low fat diets resulted in decreased cecal pH from 8 to about 6 indicating robust fermentation. However, in some preliminary studies this effect on fermentation was not seen when rodents were fed a high fat diet. Therefore, this comprehensive high fat diet study was conducted to determine which sources and combinations of prebiotics would enhance fermentation. The effects of prebiotics in a high fat diet (44% energy) on pH, cecal weights, abdominal fat, and body weight were studied in mature male C57Bl/6 mice fed one of 9 isocaloric diets (4 kcal/g) for 12 weeks. A control (C) was compared to 4 prebiotics: Hi‐Maize® RS2 (R), Novelose® RS3 (N), Nutraflora® fructooligosaccharide (F), Orafti HP gel® inulin (I) fed individually and combined (F+I, R+F, R+I, and R+N). Results were significant at p<0.05. Fermentation (lower pH of 7) occurred with F and I diets. All prebiotic diets had larger empty ceca, but only F and I had greater full ceca than the RS groups. It is proposed that dietary fat modulates monogastric fermentation and microbiota similar to the effect it has in ruminants. This agrees with our previous data showing that high fat diets reduce the number of specific bacteria that ferment prebiotics. Beneficial health effects of prebiotic ingestion may be most effective if consumed with a low fat diet. Funding: LSU AgCenter, GTC Nutrition and National Starch.
Effect of melatonin on the vestibulocollic reflex in humans
Authors: Cook Jonathan S , Chin‐Sang Stephanie A , Sauder Charity L , Ray Chester A
Published: June 2021

Melatonin has been reported to decrease nerve activity of the medial vestibular nuclei in rats. The purpose of the present study was to determine if melatonin alters the vestibulocollic reflex (VCR) in humans. Based on our previous findings with melatonin's affect on the vestibulosympathetic reflex (VSR), we hypothesized that exogenous melatonin ingestion would attenuate the VCR. To test this hypothesis, vestibular evoked myogenic potentials (VEMP) were recorded in 10 healthy subjects (26 ± 1 yr; 4 male, 6 female) before and 45 min after ingestion of 3 mg melatonin. Additionally, we recorded VEMP in 8 healthy subjects (25 ± 1 yr; 1 male, 7 female) who underwent the same experiments but without melatonin to serve as time controls. Melatonin did not alter the timing of the first VEMP peak (13 ± 0.4 and 14 ± 0.3 ms, pre and post melatonin, respectively) nor the second VEMP peak (21 ± 0.6 and 21 ± 0.7 ms, pre and post melatonin, respectively). More importantly, VEMP interpeak amplitude was not altered by melatonin (before: 22 ± 5 vs. after: 23 ± 5 a.u.). Comparable results were observed in the control group (before: 26 ± 6 vs. after: 26 ± 7 a.u.) These data indicate that melatonin does not alter the VCR in humans. In conjunction with our previous findings demonstrating an attenuated VSR with exogenous melatonin, the present findings suggest that melatonin's affect on the VCR and VSR are different in humans.

NIH HL077670, NASA Space Fellowship
Prehypertension alters arterial pressure and forearm vascular responses to mental stress in humans
Authors: Schwartz Christopher E , Durocher John J , Carter Jason R
Published: June 2021

Neurovascular responses to mental stress (MS) have been linked to several cardiovascular diseases, including hypertension. Mean arterial pressure (MAP), muscle sympathetic nerve activity (MSNA) and forearm vascular responses to MS are well documented in normotensive (NT) subjects, but responses in prehypertensive (PHT) subjects remain unclear. We tested the hypothesis that PHT would augment MS‐induced increases in MAP through altered MSNA and forearm vascular responses. We examined 11 PHT (systolic 120–139 or diastolic 80–89 mmHg; 21±1 yrs) and 11 NT (systolic <120 and diastolic <80 mmHg; 22±2 yrs) subjects during 5 min of mental arithmetic. MSNA, MAP, heart rate (HR), forearm vascular conductance (FVC) and resistance (FVR) were measured. Resting MSNA (8 ± 2 vs. 14 ± 5 bursts/min; P<0.01) and MAP (80 ± 1 vs. 90 ± 1 mmHg; P<0.001) were significantly higher in PHT subjects. PHT augmented MS‐induced increases in MAP (Δ10 ± 2 vs. Δ14 ± 4 mmHg; P<0.05) and blunted MS‐induced decreases in FVR (Δ−16 ± 2 vs. Δ−8 ± 1 units; P<0.01). PHT tended to blunt MS‐induced increases in FVC (Δ3 ± 1 vs. Δ2 ± 0 units; P=0.10). MS elicited similar increases in MSNA (Δ8 ± 2 vs. Δ8 ± 3 bursts/min) and HR (Δ23 ± 4 vs. Δ20 ± 4 beats/min) in NT and PHT subjects, respectively. We conclude that MS elicits an augmented pressor response in PHT subjects. This augmentation appears to be associated with altered forearm vascular, but not MSNA, responses. Supported by NIH.
Proteomic evaluation of chicken brush‐border membrane during the early post‐hatch period
Authors: Gilbert Elizabeth Ruth , Williams Patricia M , Ray William Keith , Li Huifeng , Emmerson Derek A , Wong Eric A , Webb Kenneth E
Published: June 2021

Many of the proteins responsible for mediating changes in nutrient assimilation in the chicken small intestine are uncharacterized. The objective of this study was to conduct a proteomic analysis of the chicken brush‐border membrane. We isolated brush‐border membrane from broiler small intestine at day of hatch and days 1, 3, 7 and 14 post‐hatch, and performed 2D‐electrophoresis. A total of 1,693 spots were analyzed by matrix‐assisted laser desorption/ionization‐tandem time‐of‐flight mass spectrometry (MALDI‐TOF/TOF). A total of 256 unique proteins were identified and grouped according to biological function. Of these, there were 12 nutrient transporters, 17 digestive enzymes and 47 proteins associated with cytoskeletal structure and microvilli organization. The remaining proteins were classified as tight junction (3), basolateral membrane (3), endosomal/membrane trafficking (10), signaling (34), metabolic (41), degradative (12), stress‐related (10), protein synthesis machinery/mitochondria/nucleus (29), immunologic (6), unknown (14), or other (18). Identification of brush‐border membrane‐associated proteins is an important step in furthering our understanding of digestion and absorption in the chicken. Further analysis of proteins with unknown function may lead to elucidation of novel pathways involving nutrient utilization.
Survival time after hemorrhage (STaH) in inbred rats with known blood volumes
Authors: Klemcke Harold G. , Rose Rajiv , Oh Thomas , Calderon Mariam L.
Published: June 2021

Eight‐fold differences in STaH were detected among 15 inbred rat strains (Klemcke et al., Shock 29:748, 2008) suggesting genetic effects on STaH. In that study, blood volumes (BV) for all strains were assumed to be 5.83 % of body weight and 55% of that BV was removed. We also showed that differences in STaH might reflect strain‐dependent differences in BV (Klemcke et al., FASEB J 23: 801.1, 2009). To address this hypothesis, each rat of 5 inbred rat strains was hemorrhaged based on its own BV measured using Evans Blue dye 24 hr prior to hemorrhage and its STaH determination. BV varied (P <0.01) among Brown Norway (BN/Mcwi; n = 14; 7.43 ± .10 ml/100 g body weight, mean ± SEM), Salt Sensitive (S; n = 10; 7.08 ± .15), Fawn Hooded Hypertensive (FHH; n=10; 6.74 ± .08), Lewis (LEW; n=10; 6.74 ± .08), and DA (n=10; 6.74 ± .08) rat strains. With equal hemorrhages (47.1 ± .05 % of measured BV), STaH did not differ among rat strains (155 ± 12 min; P = 0.95). When considering both studies it is apparent that: 1) Strain‐related differences in BV contributed to differences in STaH in our 1st study; 2) In FHH rats 1.3% more of total BV was removed in the current study, yet they survived 1.5 hr longer; and 3) In LEW rats identical BV were removed in both studies, yet currently LEW rats survived 1 hr longer. Such observations suggest that Evans Blue dye and/or blood sampling for BV determinations might be preconditioning stimuli that improve STaH in some rat strains.
Intermittent hypoxia conditioning reduces DOR‐2 receptors in the canine SA node and improves vagal transmission
Authors: Mallet Robert T , Downey H Fred , Denning Gerene M , Barlow Matthew A , Caffrey James L
Published: June 2021

Intermittent hypoxia conditioning (IHC) decreased infarct size in dogs remarkably from 38% to less than 5% of the area at risk. Since vagal stimulation and vagotonic delta‐1 opioid receptors (DOR‐1) are both cardioprotective, dogs underwent IHC to test the hypothesis that IHC improves vagal transmission and DOR‐1 influences in the canine heart. The IHC protocol was progressively increased from 5 cycles of 5 min of 10% oxygen and 4 min reoxygenation on day one to the maximum of 7 cycles of 10 min hypoxia and 4 min reoxygenation on days 11–20. On day 21 dogs were anesthetized, the right heart exposed and a microdialysis probe was inserted into the SA node. Increasing doses (5 fmol‐1.5 nmol/min) of the DOR agonist methionine‐enkephalin‐arg‐phe (MEAP) were introduced by microdialysis. The right vagus was stimulated at each dose to evaluate the influence and mix of prejunctional DORs. Prior to the dose response, vagally mediated declines in heart rate were exaggerated by ~ 20% in IHC vs. sham and non‐conditioned animals. MEAP generated a clear DOR‐2 mediated vagolytic effect in the control animals that appeared at the midpoint of the dose response curve. This robust vagolytic response was completely absent in the IHC animals. Thus, IHC may exert its cardioprotective influence by increasing vagal transmission and shifting the cardiac DOR receptors to favor the vagotonic DOR‐1 phenotype at the expense of the vagolytic DOR‐2.
Role of Caldesmon in hypoxia‐mediated endothelial cell response
Authors: Desai Ankit A , Moreno‐Vinasco Liliana , Parikh Devang S , Gera Anjali , Garcia Joe GN
Published: June 2021

Pulmonary hypertension (PH) involves stress‐mediated pathways producing endothelial and smooth muscle cell dysfunction. The actin cytoskeleton is important in vascular remodeling and our earlier studies strongly suggested the role of caldesmon (Cald1) in endothelial cytoskeleton remodeling. Cald1 exhibits 2 isoforms: h‐Cald1 found in smooth muscle and l‐Cald1, expressed in non‐muscle cells and involved in cell proliferation and contractility. Both Cald1 isoforms are important in hypoxia‐induced PH and Cald1 surfaced as a candidate gene in 2 rodent PH therapy trials (simvastatin and sorafenib, Sor). We hypothesized that l‐Cald1 is an important regulator of vascular remodeling in PH. We validated Sor array data with increased l‐ and h‐ Cald1 protein levels in lung homgenates from hypoxia‐(1.5‐ & 2.5‐fold) and in hypoxia/SU5416 (H‐SU)‐ challenged rats (2‐ & 4‐fold compared to normoxia at 3.5 wks). Sor treatment reduced l‐ Cald1 levels in H‐SU while h‐ Cald1 remained elevated. Given isoform‐specific changes, we examined l‐Cald1 in vitro in HLMECs exposed to hypoxia (30 min‐4h). Gene and protein l‐Cald1 levels were increased at 1h (2.5‐ & 1.8‐fold) and 4h (1.6‐fold). Diphosphorylated myosin light chain (dpMLC) levels increased (2‐fold) by 30 min and decreased (1.5‐fold) by 2h with no change in total MLC. Thus, l‐Cald is potentially a key regulator in vascular remodeling in PH. NIH support: F32 HL090359‐01
Impact of Hyperthyroidism and Its correlation on regional blood flow in rats
Authors: Gomes Dayane Aparecida , Mostarda Cristiano , Jorge Luciana , De Angelis Katia , Irigoyen Maria Claudia , Barreto‐Chaves Maria Luiza
Published: June 2021

Thyroid hormones (TH) exert multiple effects on the heart and vascular system. In the hyperthyroidism, besides the direct effects of TH on cardiac tissue inducing hypertrophy in rats, TH also lead to an important increase in heat rate and reduction in systemic vascular resistance. In the present study we evaluated the effect of hyperthyroidism (T3, 7ug/100g body wt/2 wk) on regional blood flow (coronary, lung and kidney) and blood pressure parameters in Wistar rats. Hyperthyroidism promoted significant left ventricular (+27.56%) and right ventricular (+33.56%) hypertrophy. Although T3 has promoted a marked increase on blood flow in the myocardium (+98,7%), an important decrease on lung blood flow (−35 %) was observed, which was followed by discrete but significant decrease on cardiac output (−14%). Regard to hemodynamic variables, the hyperthyroidism promoted a significant increase in mean, systolic and diastolic arterial pressure as well as HR compared to control group. These findings show that hyperthyroidism is accompanied by intense changes on the blood flow to lung and myocardium and hypertension. Considering that the reduction in cardiac output and pulmonary blood flow are associated with right ventricular hypertrophy, these findings may have clinical implications by serving as an explanation for the development of pulmonary hypertension and heart failure observed in hyperthyroid patients and point out the importance of adequate control of TH levels.
Bmp2 and Bmp4 exert opposing effects in hypoxic pulmonary hypertension
Authors: de Caestecker Mark Peter , Anderson Lynda , Jones Mark , Lowery Jonathan
Published: June 2021

Patients with Familial Pulmonary Hypertension have mutations in the BMP receptor, BMPR2, but it is unknown how these mutations cause disease. These observations also raise questions as to the role of BMPR2 signaling in other forms of pulmonary hypertension (PH). To address this, we evaluated the regulation and function of Bmpr2 ligands in a mouse model of hypoxic PH. Pulmonary Bmp2 and Bmp4 are up‐regulated by hypoxia: Bmp2 is up‐regulated in the peripheral pulmonary vasculature, while Bmp4 is widely expressed in respiratory epithelia. Loss of hypoxia‐induced Bmp4 in Bmp4+/− mice is associated with reduced PH and decreased pulmonary vascular remodeling. This contrasts with Bmpr2ΔEx2/+ mice which show greater susceptibility to hypoxic PH, and suggests that an alternative ligand regulates Bmpr2‐dependent effects in the vasculature. Unlike Bmp4+/− mice, Bmp2+/− mice develop more severe hypoxic PH than wild type littermates. There is also reduced eNOS expression and activity in the pulmonary vasculature of hypoxic Bmp2+/−, but not Bmp4+/− mice. Bmp2 also up‐regulates eNOS expression and activity in intrapulmonary artery and endothelial cell preparations, indicating that eNOS is a target of Bmp2 in the pulmonary vasculature. These data indicate that Bmp2 and Bmp4 exert opposing roles in hypoxic PH, and suggest that protective effects of Bmp2 are mediated by increasing eNOS activity in the pulmonary vasculature.
Novel role of Pre‐B‐Cell Colony Enhancing Factor (PBEF) in pulmonary arterial hypertension (PAH)
Authors: Machado Roberto F , Moreno‐Vinasco Liliana , Chiang Eddie , Zaidi Syed Rafe , Sammani Saad , Mirzapoiazova Tamara , Toth Peter T. , Ayetkin Meitin , Dweik Raed A , Archer Stephen L. , Garcia Joe G.N
Published: June 2021

The discovery of novel mediators in PAH allows for novel insights into PAH pathobiology and identification of therapeutic targets. PBMC expression profiles from 7 PAH patients (49±7 years, mPAP 55±18 mmHg, PVR 14±11 U) and 10 healthy controls (45±7 years) identified 224 differentially expressed genes including PBEF as an upregulated gene in PAH. We previously identified PBEF as a mediator in lung injury, with extracellular proinflammatory cytokine‐like activity and intracellular enzymatic activity regulating NAD levels, cell redox state, histone deacetylases and inhibiting apoptosis. Plasma PBEF levels were higher in PAH patients (n=10, 46±17 years, mPAP 56±18 mmHg, PVR 10±4 U) than controls (n=9, 33±7 years), (5000±2000 vs. 7500 ± 3000 pg/ml; P=0.04) and trended with BNP levels (R=0.6, P=0.08) and inversely with 6‐minute walk distance (R=−0.57, P=0.08). Immunohistochemistry in explanted PAH lungs (n=3) revealed significant PBEF expression in plexiform lesions and in remodeled PAs, without PBEF staining in PAs of controls. Finally, compared to WT control mice, PBEF heterozygous mice (PBEF+/−) developed less severe PH (FIO2=10% and SU5416 for 3 weeks) with less significant elevations in RVSP (PBEF+/+ 19 ± 2 vs. PBEF+/− 12 ± 4 mmHg, P=0.03) and less RVH (RV/LV+S: PBEF+/+ 0.44 ± 0.05 vs. PBEF+/− 0.34 ± 0.08, P=0.06). Our results implicate PBEF as a novel biomarker and therapeutic target in PAH.
Regulation of apical choline transport in cultured choroid plexus by oxidative stress
Authors: Villalobos Alice R , Young Robin K
Published: June 2021

Modulation of solute transport by choroid plexus may accompany the cellular stress response induced by physicochemical and pathological stressors. This laboratory reported that in cultured choroid plexus cadmium exposure elicited a latent but marked increase in apical choline transport, an effect dependent on induction of oxidative stress. We hypothesize that in absence of heavy metals induction of oxidative stress may regulate apical choline transport in choroid plexus. Thus, apical transport of 3H‐choline was compared in primary cultures of choroid plexus treated without and with inhibitors of the mitochondrial electron transport (ETC) chain to induce oxidative stress. The complex III inhibitor, antimycin‐A depolarized mitochondrial membrane potential and increased cellular accumulation of reactive oxygen species. Antimycin‐A also markedly increased gene expression of stress proteins, metallothionein‐I (MT‐I), heme oxygenase‐1 (HO‐1), and Hsp70, consistent with induction of the cellular stress response. Moreover, 3H‐choline transport was stimulated by 50–80%. Rotenone, a complex I inhibitor, also induced gene expression of MT‐I, HO‐1, and Hsp70; however, apical choline transport was comparable to controls. These preliminary data suggest that in choroid plexus apical choline transport might be regulated in response to oxidative stress elicited by select inhibition of complex III of the ETC.
Vasopressin‐Regulated Phosphorylation of Urea Transporter Proteins, UT‐A1 and UT‐A3, in Rat Inner Medullary Collecting Duct (IMCD)
Authors: Hwang Shelly , Chou C‐L , Yu M‐J , Rinschen M , Gunaratne R , Pisitkun T , Hoffert JD , Knepper MA
Published: June 2021

Vasopressin‐regulated urea transport in the renal IMCD is mediated by two transporters, UT‐1A and UT‐A3, derived from the same gene by alternative splicing. The initial 459 amino acids are the same in both. To study UT‐A1/3 phosphorylation, we made phospho‐specific antibodies to UT‐A sequences targeting phospho‐serines at positions 84 and 486. Both antibodies proved specific, recognizing only the phosphorylated forms of UT‐A1 and ‐A3. Immunoblotting of rat IMCD suspensions or inner medullas from intact rats showed that the V2R‐selective vasopressin analog dDAVP increases phosphorylation at both S84 and S486 by ~8‐fold. The increase in S84 phosphorylation was seen in both UT‐A1 and ‐A3. Time‐course studies in rat IMCD suspensions showed phosphorylation within 1 minute of dDAVP exposure, consistent with the time course of urea transport increases in isolated perfused tubules. Confocal immunofluorescence immunocytochemistry in rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. These studies demonstrate regulated phosphorylation of both UT‐A1 and UT‐A3 in response to vasopressin, potentially contributing to the regulation of urea transport in the IMCD.
ROLE OF DOPAMINE IN MODULATING FLOW‐DEPENDENT SODIUM AND BICARBONATE TRANSPORT IN MOUSE PROXIMAL TUBULE
Authors: Du Zhaopeng , Yan QingShang , Weinbaum Sheldon , Weinstein Alan M , Wang Tong
Published: June 2021

We have previously demonstrated that increases in luminal flow rate produce proportional increases in Na+ reabsorption by mouse proximal tubules. Since dopamine inhibits sodium reabsorption by stimulating NHE3 endocytosis and antagonizes the stimulation of proximal tubule transport by norepinephrine, we investigated whether increase of NHE3 endocytosis by dopamine will block flow‐stimulation of proximal tubule transport. Mouse proximal tubules were microperfused in vitro at perfusion rates of 5 and 20nl/min, and the net reabsorption of fluid (Jv) and HCO3− (JHCO3) were measured. The effect of dopamine and dopamine receptor inhibitors SCH23390 (DA1), sulpiride (DA2), 8‐Bromo‐cAMP and a cAMP‐dependent protein kinase inhibitor H89 on flow‐stimulated proximal tubule transport was examined. Increase in perfusion rate from 5 to 20nl/min significantly increased Jv from 0.88 to 1.5 nl/min/mm and JHCO3 from 61.72 to 128.53 pmole/min/mm. Luminal dopamine did not change Jv and JHCO3 at low flow rate but abolished the increments of Jv and JHCO3 induced by high flow rate. SCH23390 completely blocked, but sulpiride partially blocked the effect of dopamine. 8‐Bromo‐cAMP reduced Jv and JHCO3 at both flow rates and the effect of dopamine was blocked by H89. These results indicate that dopamine activates its receptor and inhibits flow‐stimulated proximal tubule transport by a PKA‐mediated mechanism. Recruitment of NHE3 to the apical membrane is critical for flow‐stimulated Na+ and HCO3− absorption in the kidney proximal tubule.
Generation and Phenotype of a Transgenic Knockout Mouse Lacking the Urea Transporters UT‐A2 and UT‐B
Authors: Yang Baoxue , Lei Tianluo , Bankir Lise , Zhou Hong
Published: June 2021

UT‐A2 and UT‐B are two distinct urea‐selective channels, coded by two different genes on the same chromosome. UT‐A2 is mainly expressed in the thin descending limb of Henle's loop, and UT‐B in descending vasa recta and red blood cells. We generated mice with simultaneous knockout (KO) of these two genes (DbleKO). A targeting vector for homologous recombination was constructed using a mouse UT‐A genomic fragment in which UT‐A2 promoter sequence was deleted. UT‐A gene targeting was performed on UT‐B gene knock‐out embryonic stem cells. The [−/−] mice lacked detectable UT‐A2 and UT‐B proteins by Western blot and immunofluorescence. Survival and growth of DbleKO were not different from that in wild‐type mice (WT). Daily urine output was 2.63 ± 0.13 ml in DbleKO, which was higher than that in WT (2.01 ± 0.17, p < 0.01) and lower than that in UT‐B KO (3.65 ± 0.16, p < 0.01). Urine osmolality (Uosm, in mosm/kg H2O) in DbleKO (2176 ± 181) was between those in UT‐B KO (1697 ± 164, p < 0.01) and WT (2524 ± 193, p < 0.01). After 18 h water deprivation, Uosm was 2976 ± 197 in DbleKO, 2375 ± 228 in UT‐B KO and 4281 ± 257 in WT. These results show that UT‐A2 deletion in UT‐B KO mice attenuates the urine concentrating defect caused by UT‐B deletion. They suggest that UT‐A2 may allow an exit of urea from the thin limbs rather than an entry of urea, as usually assumed, and thus challenge established intrarenal urea recycling mechanism.
Primary cilium is required for vasopressin mediated aquaporin‐2 trafficking
Authors: Saigusa Takamitsu , Bell Phillip Darwin , Kolb Robert J
Published: June 2021

The collecting duct cell lines PCDNA (cilia (−)) and BAP2 (cilia (+)/rescue) derived from a PKD mouse model (orpk mouse) were used to determine the role of the primary cilium in vasopressin (AVP) mediated aquaporin 2 (AQP2) trafficking and fluid absorption. With apical AVP addition, cilia (−) cells grown on impermeable supports demonstrated an increased net fluid absorption as evident by the formation of epithelial domes, which were not seen in cilia (+) cells. Immunofluorescence studies showed AQP2 localization along the ciliary shaft and in subapical membrane compartment in cilia (+). Moreover, after apical AVP treatment, there was an increase in AQP2 expression at the apical membrane in cilia (−) but not in cilia (+) cells. This was confirmed by biotinylation and Western blot analysis, which demonstrated an AVP induced elevated glycosylated form of apically located AQP2 in cilia (−) vs. cilia (+) cells. Interestingly, in cilia (−) cells there was evidence for basolateral membrane insertion of AQP2. In the absence of cilia, there appears to be an AVP mediated targeting of the V2 receptor to the apical membrane and enhanced AQP2 localization at both apical and basolateral membranes. Thus, we propose AQP2 trafficking requires a normal cilium and when absent, the collecting duct cells becomes dedifferentiate leading to enhanced salt and water absorption which may contribute to the hypertension observed in PKD patients.
Time Course of Urinary Concentrating Defect in Vitamin D‐Induced Hypercalcemic Rats
Authors: Khositseth Sookkasem , Sriwong Siriwan , Uawithya Panapat
Published: June 2021

We studied the onset of hypercalcemia‐induced nephrogenic diabetes insipidus and its relationship to aquaporin‐2 (AQP2) and bumetanide‐sensitive Na‐K‐2Cl cotransporter (NKCC2) expression. Rats were treated with or without food supplement of 1mg dihydrotachysterol (DHT)/kg BW/day for 1, 2, 7 days. After treatment for 1 day, serum calcium was significantly increased (control: 9.7±0.3 vs. DHT: 10.8±0.2 mg/dl). Urine volume was also significantly increased (0.27±0.18 vs. 0.83±0.14 ml/gram BW/day). Urine osmolality was decreased after 2 days of DHT ingestion (1.53±0.5 vs. 0.91±0.00 osmol/kgH2O, P < 0.05), suggesting DHT rats lose their ability to concentrate urine. Immunoblotting showed that NKCC2 abundance in kidney outer medulla of DHT‐treated rats was decreased by 59% after 1 day treatment and remained decreased until day 7. The AQP2 abundance in inner medulla was decreased by 44% after 2 days of DHT diet and remained decreased until day 7. In conclusion, the onset of polyuria and hypercalcemia were found to occur within 1 day after DHT treatment. The early development of polyuria in association with hypercalcemia was most likely due to a downregulation of NKCC2 in medullary thick ascending limb, whereas the urinary concentrating defect after 2 days DHT treatment involved downregulation of both NKCC2 and AQP2.

This study was supported by a grant from Thammasat University to SK and PU.
Effect of knocking out receptor protein tyrosine phosphatase γ (RPTPγ) in the CO2‐induced stimulation of HCO3 reabsorption by mouse renal proximal tubules
Authors: Zhou Yuehan , Boron Walter F
Published: June 2021

Previous work on isolated perfused proximal tubules (PTs) shows that: (1) raising basolateral (BL) [CO2] markedly increases HCO3 reabsorption rate (JHCO3) and (2) blocking apical AT1 receptors or knocking out AT1A receptors eliminates the JHCO3 response to [CO2]BL. We hypothesize that the “orphaned” RPTPγ, with an extracellular carbonic‐anhydrase‐like domain, is a CO2 sensor. We perfused PTs from wild‐type vs RPTPγ‐null mice, using out‐of‐equilibrium solutions to vary [CO2]BL at fixed [HCO3]BL and pHBL. In wild‐type PTs, raising [CO2]BL from 0% to 5% (“baseline”) to 20% (fixed [HCO3]BL = 22 mM, pHBL = 7.4) increased JHCO3 from 79 ± 6 (n = 14) to 136 ± 7 (n = 40) to 189 ± 4 pmol mm−1 min−1 (“pmm”, n = 14). In RPTPγ‐null PTs, baseline JHCO3 was reduced by 23% to 105 ± 7 pmm (n = 24, p = 0.002, 1‐way ANOVA), and the responses to 0% [CO2]BL (JHCO3 = 98 ± 7, n = 6) and 20% [CO2]BL (JHCO3 = 105 ± 12, n = 6) were eliminated. Finally, when we added 10−8 M candesartan (non‐peptide AT1 antagonist) to the lumen of RPTPγ‐null PTs, baseline JHCO3 was further fell by 29% to the “pedestal” level (JHCO3 = 75 ± 8 pmm, n = 18, p = 0.012, 1‐way ANOVA). Moreover, and the responses to 0% [CO2]BL (JHCO3 = 58 ± 8, n = 4) and 20% [CO2]BL (JHCO3 = 83 ± 23, n = 5) were eliminated. Thus, knocking out RPTPγ eliminates the PT response to changes in [CO2]BL, consistent with the idea that RPTPγ is a CO2 sensor, as part of a signal‐transduction cascade that also requires the apical AT1 receptor.
Activation of P2Y1 purinergic receptors (P2Y1Rs) induced calcium influx in rat supraoptic neurons (SON) through stretch inactivated cation (SIC) channels
Authors: Song Zhilin
Published: June 2021

Activation of P2Y1Rs contributes to the ATP‐induced increase in intracellular calcium ([Ca++]i) in SON. Although activation of P2Y1Rs induced release of Ca++stores presumably as a result of IP3 production, it also induced a small Ca++ influx, possibly through plasma membrane IP3 receptors or modulation of other ion channels such as osmo‐sensitive SIC channels. Current studies evaluated the role of SIC channels in P2Y1R induced Ca++ influx by directly monitoring [Ca++]i changes using live cell calcium imaging. Hypothalamo‐neurohypophyseal explants were loaded with the Ca++‐sensitive dye, fura 2AM, and the change in [Ca++]i in SON was determined by ratiometric analysis of fluorescence emitted by excitation at 340 and 380 nm. Activation of P2Y1Rs by 2‐methyl‐thio‐ADP (2‐MeSADP, 100 μM) induced a large increase in [Ca++]i. The amplitude of the increase was reduced by ruthenium red (RR, 10 μM) and gadolinium (Gd, 100 μM), both of which block mechano‐sensitive channels including the SIC channels. Furthermore, the decay after peak response was much faster in the presence of RR and Gd. These results indicate that activation of P2Y1Rs may modulate SIC channels, thus inducing a small component of influx of extra‐cellular Ca++, in addition to release of Ca++ from intracellular stores. Further studies are ongoing to clarify the underlying mechanisms and functional importance of the Ca++ influx.

Supported by AHA SDG 0735329N.
Enhanced oxidative and carbonyl stress predicts salt sensitivity in obese young adults
Authors: Mori Takefumi , Kawamata Akihiro , Hosoya Takuma , Yoneki Yoshimi , Mitui Eiko , Hasegawa Yoko , Ohta Michi , Ito Megumi , Sato Hiromi , Kabayama Shigeru , Ito Sadayoshi , Hida Wataru
Published: June 2021

Background

Methylglyoxal (MGO), a carbonyl substance, is increased in CKD patients and can induce salt sensitive hypertension and insulin resistance together with enhanced oxidative stress in a rat model. The present study determined whether carbonyl/oxidative stress is enhanced in obese young adults and would associate with salt sensitivity.

Methods

Young adult students (2335students, male:1794, female:541, age:18to28)were enrolled for the study. Daily Na excretion (UNa, as a indicator of Na intake), albumin (UAE, as a marker of CKD), TBARS (U‐TBARS, as a marker of oxidative stress) and MGO (U‐MGO, as a marker of carbonyl stress) was analyzed. Correlations were determined among the parameters in the health checkup.

Results

Although, SBP, U‐TBARS, U‐MGO and UNa were increased with higher BMI or AC in male students, SBP was increased without increase in U‐TBARS and UNa in high BMI or AC female students. U‐TBARS and U‐MGO had a positive correlation with UNa, and UAE significantly increased with BMI of 30 and above compared to under 30 in either sex. Finally, SBP correlated with BMI of 30 and above in male students while no correlation was observed in those under 30.

Conclusion

These results indicate that obese has a higher risk of salt sensitive hypertension and CKD in young adults which can be predicted by carbonyl/oxidative stress. Supported by the Grants‐in‐Aid from the Min. of ECSST of Japan (No.20590970)
Baroreflex unloading blunts chemoreceptor sensitivity in Postural Tachycardia Syndrome (POTS)
Authors: Taneja Indu , Clarke Debbie A. , Medow Marvin S. , Ocon Anthony J. , Stewart Julian M
Published: June 2021

POTS have increased minute ventilation (VE) and decreased end tidal carbon dioxide (ETCO2) compared to healthy subjects. Chemoreceptor sensitivity was measured in 18 POTS (14F, 4M), 10 healthy controls (6F, 4M) 16–35 years old by exposing them to eucapneic hyperoxia‐30%oxygen, eucapneic hypoxia (10%oxygen) and hypercapnia 30%oxygen+5%carbon dioxide) in supine and HUT70 position. We measured heart Rate (HR), mean arterial pressure (MAP) oxygen saturation, ETCO2, mean cerebral blood flow velocity (mCFV) and VE. Chemoreflex sensitivity was ascertained by comparing slopes determined from eucapneic hypoxia‐hyperoxia to measure peripheral chemoreflex sensitivity, and hyperoxic hypercapnia –eucapnia to measure central chemoreflex sensitivity. POTS had significantly higher resting HR, lower MAP, stroke volume (SV) and mCFV as compared to controls (p<0.05). Hypoxia and hypercapnia increased the HR, MAP, CO, mCFV and VE (p<0.05). POTS had increases in VE during supine hypoxia (POTS vs. controls, 3.6± 0.9 vs. 1.6± 0.2, p=0.003) which reversed in HUT70 (1.96± 0.3 vs. 5.4± 1.2). In both POTS and controls, hypercapnia resulted in similar increases in VE when supine but no further increases when HUT70. POTS have higher peripheral chemoreflex sensitivity than controls which attenuates during unloading of baroreflexes.
Prolonged Maintenance of Cardiopulmonary Stability in Mice: Strain‐Specific Ventilatory Requirements
Authors: Pichard Luis , Kostuk Eric , Pashai Pejmon , Fitzgerald Robert , Shirahata Machiko
Published: June 2021

Traditionally, large mammals have been used to characterize carotid body (CB) function in vivo. Recently, due to our ability to modify the genome, the mouse is often used. However, a challenge in the mouse is to regulate arterial blood gas (ABG). It is known that baseline cardiopulmonary (CP) parameters vary among different strains of mice. Thus, we explored the conditions for mechanical ventilation and fluid balance that support predictable CP parameters in the DBA/2J and A/J mice. Four to six‐week old male mice were anesthetized with urethane and paralyze with pancronium. Experiment I: Tidal volume (Vt), respiratory frequencies and fluid administration were systematically altered while monitoring ABG, electrolytes, blood pressure (BP) and heart rate (HR). We found that high rate of fluid administration (100 μL/gr/hr) was necessary to obtain stable BP and HR without any adverse effects in the lung and heart. DBA/2J mice required higher Vt (15 μL/g) than A/J mice (13 μL/g) to maintain reasonable ABG values. Experiment II: We examined if the parameters obtained from Experiment I were adequate to maintain stable CP conditions for prolonged (4–5 hours) mechanical ventilation. ABG samples were attained every 1.5 hours. ABGs were highly predictable and consistent: pH= 7.40±0.01, PCO2=36.4±1.6 & PO2=307±32 (n=9). Electrolytes BP did not significantly change throughout the experiments. Experiment III: Mice were ventilated as described above. Inspired O2 was reduced and time‐course of ABG changes was examined. A nadir of PaO2 was obtained within 90 seconds of hypoxia. Here, we have established an anesthetized and mechanically ventilated mouse model for addressing the highly sensitive modalities of the CB. Support: F31HL096450, HL081345, AHA 09GRNT2080158, HL50712 and HL61596.
Connexin 26 is responsible for ATP release underlying central CO2 chemosensitivity
Authors: Huckstepp Robert Timothy Richard , Eason Robert C , Bihi Rachid id , Dicke Nikolai , Willecke Klaus , Spyer K. Michael , Gourine Alexander V , Dale Nicholas
Published: June 2021

Although chemosensitive areas of the medulla oblongata have been defined, the cell types and mechanisms involved in CO2 chemosensory transduction there remain unknown. Recently, a causal link between ATP release from the surface of the ventro‐lateral medulla (VLM) and the hypercapnic ventilatory response (HVR) in vivo has been established. By employing horizontal slices of the VLM, we studied CO2‐triggered ATP release in vitro, which was greatly reduced by pharmacological inhibition of connexin hemichannels. The dorso‐ventral distribution of connexin 26 (C×26) ascertained via real time PCR, immunofluorescence and a lacZ knock‐in reporter, combined with the CO2‐senistivity of C×26 transfected HeLa cells makes it the most likely candidate. Furthermore we have documented direct gating of C×26 in response to changes of PCO2 in excised membrane patches. CO2‐ dependent dye loading of tissue slices with carboxyfluorescein stained cells in the pia mater, sub‐pial astrocytes and cells associated with penetrating blood vessels. Application of gap‐junction antagonists greatly reduced ATP release in response to elevated CO2 and the HVR in vivo. Therefore we propose that ATP is released from sub‐pial astrocytes and pial cells through C×26 hemichannels in response to alterations in PCO2, with C×26 acting as the chemosensory transducer and the conduit for ATP release. Research was supported by the BBSRC and MRC