bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of TLR4 Activation Prevents Renal Autoregulatory Impairment in Ang II Hypertensive Rats
Authors: Freeman Brian , Zhang Shali , Cook Anthony , Inscho Edward W. 
Published: June 2021

Autoregulation by afferent arterioles regulates renal blood flow and glomerular filtration rate across widely ranging arterial pressures. Autoregulatory function is impaired in hypertension, perhaps due to release of damage associated molecular patterns that activate renal toll‐like receptor (TLR4). Autoregulatory impairment can promote hypertensive kidney injury. We hypothesize that inhibition of TLR4 activation preserves afferent arteriole autoregulatory function in Angiotensin II (AngII) hypertension.Three groups (n=4/group) of male Sprague‐Dawley rats were implanted with osmotic minipumps set to deliver: saline (0.9% NaCl) or AngII (60ng/min). Group three received AngII+anti‐TLR4 Ab (3 μg/ml/kg/day, i.p.) for 14 days. Systolic blood pressure (SBP) was assessed (days 0, 3, 7 and 13). Kidneys were harvested on day 14 for juxtamedullary nephron assessment of afferent arteriole autoregulatory function.SBP increased in the AngII and AngII+Anti‐TLR4 Ab groups compared to saline controls over the 14 days (P<0.05), averaging 198 ± 11, 200 ± 8 and 135 ± 3 mmHg, respectively. Baseline arteriole diameters (100 mmHg) averaged 14 ± 0.8, 15.6 ± 1.7, and 13 ± 0.3 microns for control, AngII and AngII+Anti‐TLR4 Ab, respectively. Perfusion pressure was decreased to 65 mmHg and increased to 170 mmHg in 15 mmHg increments. At 170 mmHg, arteriole diameters decreased by 31 ± 6% and 33 ± 8% of baseline in the control and Ang II+Anti‐TLR4 Ab groups whereas the AngII group had a decline of 11.7 ± 3.7%.These data establish that autoregulatory function is Impaired in AngII hypertensive rats while it is preserved by anti‐TLR4 Ab‐treatment. These findings implicate chronic TLR4 activation as causative in the hypertensive decline in autoregulatory capability.Support or Funding InformationFunded by NHLBI; 1R25 HL115473‐01 and DK044628‐20.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute SGLT2 Inhibition and the Pressure Natriuresis Response in Rats with Type 1 Diabetes
Authors: Bailey Matthew A , Costello Hannah M , Stewart Kevin , Binnie David , Culshaw Geoffrey C , Webb David J 
Published: June 2021

Increasing blood pressure (BP) induces natriuresis due to downregulation of sodium transport in the proximal tubule. This pressure natriuresis (PN) response influences long‐term BP. Type‐1 diabetic (T1DM) patients show enhanced tubular sodium reabsorption and high BP. We have found that tubular sodium reabsorption is not modulated by BP in T1DM rats. The molecular mechanism of impaired PN is unknown but the sodium‐glucose cotransporter SGLT2 is a plausible candidate and inhibitors acutely induce diuresis in T1DM rats. This study tested the hypothesis that SGLT2 inhibition with dapagliflozin would improve the acute PN response in T1DM rats.T1DM was induced in male Sprague‐Dawley rats by streptozotocin (30–45mg/kg IP; plasma glucose 13.3–>35mmol). After 3 weeks rats were anaesthetised with Inactin (120mg/kg, IP). The jugular vein was cannulated and isotonic saline (containing FITC‐inulin and p‐amino hippurate for measurement of glomerular filtration rate (GFR) and renal plasma flow (RPF)) was infused. Rats were randomly allocated to receive either dapagliflozin (50mg/kg; n=7) or saline vehicle (n=7) and after an equilibration period and baseline urine collection, BP was increased by sequential arterial ligation of first the coeliac and mesenteric arteries and then the distal aorta and the natriuretic response was measured. Experiments were performed under a single blind and rats were euthanised by an overdose of anaesthetic. Data are mean +SD and comparisons were made by 2‐way ANOVA. GFR and RPF were not different between treatment groups and were unaffected by the experimentally‐induced rise in BP, consistent with autoregulation. In vehicle‐treated rats, incremental rises in BP increased urine flow rate from a baseline of 6 ± 3 to a peak of 51 ± 28μl/min/gkw (P<0.01), sodium excretion from 1.7 ± 0.5 to 16.7 ± 1.4 μmol/min/gkw (P<0.01) and fractional sodium excretion from 0.2 ± 7.6 to 5.4 ± 4.5% (P<0.01). The dapaglifozin‐treated rats responded to increasing pressure with a diuresis and natriuresis but the peak absolute (3.8 ± 4.1μmol/min/gkw; P=0.010) and fractional (15.2 ± 8.0%; P=0.018) sodium excretion were significantly lower than in the vehicle‐ group. Dapagliflozin increased urinary glucose excretion from 0.1 ± 0.2 to 0.4 ± 0.2μmol/min/gkw: glucose excretion was not modified by increasing BP in either group. Although dapagliflozin increased urine flow and glucose excretion at steady‐state BP, it did not enhance the acute PN response in T1DM rats. Thus, impaired PN in T1DM does not reflect increased SGLT2 activity.Support or Funding InformationBritish Heart Foundation studentship FS/16/54/32730 & Kidney Research UK Project Grant RP2/2014This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CACNB2 expression is positively associated to the Ras/MAPK pathway in vitro and in an experimental model of hypertension
Authors: Pandian Vishnuprabu Durairaj , Giovannucci David R , Vazquez Guillermo , Sivarajan Kumarasamy 
Published: June 2021

Several independent genome‐wide association studies highlighted the strong association of voltage‐dependent L‐type calcium channel subunit beta‐2 (CACNB2) gene with hypertension. One such study showed that hypertensive individuals who carry a promoter polymorphism (rs2357928) in CACNB2 exhibited adverse effects during treatment with calcium channel blocker (CCB) ‐ verapamil but responded well to treatment with the β‐blocker‐ atenolol. However, the exact molecular signature of CACNB2 during the pathophysiology of hypertension is currently remains to be explored. In the clinical setting, CCBs are widely used to treat hypertensive individuals, and the inhibition of L‐type Ca2+ channels with CCBs significantly increases the glomerular filtration rate and renal blood flow. Hence, the goal of this present study is to understand the molecular signaling of CACNB2 during hypertension via in vitro and in vivo approaches using HEK293 cells and genetically hypertensive, Dahl Salt‐Sensitive (SS) rats. The over‐expression of CACNB2 in HEK293 cells increased cell proliferation and upregulated the Ras‐MAPK pathway significantly when compared to the controls. These results were consistent with a recent study showing that overexpression of CACNB2, activate Ras/ERK/CREB pathways in vitro. Additionally, the ER stress markers IRE‐1α and PERK were upregulated by CACNB2 over expression compared to the control cells. In contrast, treatment with the L‐type CCB‐ nifidepine in CACNB2 overexpressed cells did not abolish upregulation of Ras‐MAPK and ER stress pathway genes. Similarly, we observed an increase in apoptotic signaling exemplified by the downregulation of Bcl‐2 and upregulation of Caspase 7 and Caspase 9, which were also insensitive to nifidepine treatment. Correspondingly, we investigated whether CACNB2 levels were altered using the SS rats under low salt (0.3% NaCl) and high (2% NaCl) diet fed conditions. We observed higher CACNB2 levels in high salt fed SS rat kidneys compared to SS rats maintained on low salt diet (n=4, p<0.05). In addition, our gene expression studies showed that Mapk1 and Mapk4 levels were upregulated in the kidneys of SS rats under high salt diet (n=4, p<0.05). In summary, our study reveals that CACNB2 significantly associated with the activation of Ras‐MAPK pathway in SS rats. In summary, understanding the regulation of CACNB2 and associated Ras‐MAPK pathway could help in expanding the knowledge of molecular mechanism of hypertension and may help in identifying novel drug targets.Support or Funding InformationFunding support for this study was met from the American Heart Association grant 16SDG27700030 (to S. Kumarasamy) and funding from UT‐COMLS.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
α1‐Adrenoceptor Antagonism Attenuates the Development and Progression of Dahl Salt Sensitive Rat Hypertension by Modulating NCC Activity
Authors: Puleo Franco , Frame Alissa A , Moreira Jesse D , Faudoa Elizabeth , Wainford Richard D 
Published: June 2021

BackgroundDietary sodium intake plays a significant role in blood pressure (BP) regulation, as increased sodium retention is a well‐established precursor of hypertension risk. In particular, increased dietary sodium intake evokes increased BP in individuals that demonstrate the salt sensitivity of BP and promotes the development of salt sensitive hypertension (SSH). Recent studies suggest that excessive Sympathetic Nervous System (SNS) release of norepinephrine (NE) has the ability to stimulate the sodium chloride cotransporter (NCC) via synergistic α1 and β adrenergic receptor pathways to drive SSH.HypothesisEndogenous SNS release of NE activates an α1‐adrenoceptor pathway to mediate increases in NCC activity that drive the development and maintenance of Dahl rat SSH.MethodsGroups of naïve Dahl Salt Sensitive (DSS) rats, DSS rats receiving a continuous subcutaneous (s.c.) terazosin (10 mg/kg/day), or s.c. propranolol (10 mg/kg/day) infusion were fed a normal salt (NS, 0.6% NaCl) or high salt (HS, 4% NaCl) diet for 21 days respectively. On day 21 basal MAP and NCC activity (peak natriuresis to iv hydrochlorothiazide (HCTZ; 2mg/kg) infusion) were assessed. NCC expression and phosphoNCC levels were assayed via immunoblotting (N=6/gp). Additional groups of DSS rats were fed a 42 day HS diet. On day 21, rats were given s.c. saline/DMSO or s.c. terazosin/DMSO (10 mg/kg/day). On day 42, measurements were taken as described previously (N=4–6/gp).ResultsDSS rats develop SSH when fed a 21 day HS diet and show increased NCC activity compared to rats on a NS diet. NCC expression and phospho levels are similar in both the diets. Use of an α1‐adrenoceptor antagonist, terazosin (confirmed pharmacologically) attenuates SSH and reduces NCC activity and expression as well as phosphoNCC levels in rats on a HS diet. Use of a β‐adrenoceptor antagonist, propranolol fails to attenuate SSH or reduce NCC activity. Significantly, α1 antagonism has a trend to reduce BP and NCC activity in DSS rats with established HTN.ConclusionOur data reveals that SNS release of NE modulates NCC activity via an α1‐adrenoceptor pathway to evoke dietary evoked SSH. Significantly, α1‐adreoceptor antagonism can attenuate the development of SSH by evoking downregulation of NCC activity and expression. In a model of established HTN, α1‐adreoceptor antagonism reduces NCC activity and BP. Taken together, these findings suggest that SNS release of NE activates an α1‐adreoceptor pathway that stimulates NCC activity to drive the development and maintenance of SSH.Support or Funding InformationThis work was funded by NIH R56 AG057687, R01 HL139867, R01HL141406, AHA17GRNT33670023 to RDW, and F31DK116501 to AAF.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myogenic Constriction is not Dependent on Endothelium in Arcuate and Mesenteric Arteries of Normotensive and Hypertensive Rats
Authors: Nademi Samera , Lu Chao , Dickhout Jeffrey G. 
Published: June 2021

BackgroundMyogenic response (MR) is defined as contraction of small arteries in response to increased intraluminal pressure. Impaired MR is associated with hypertension and Chronic Kidney Disease (CKD). MR has been shown to be regulated by endothelium (ENDO) through production of vasodilators and vasoconstrictors which are differentially produced in different vascular beds. Despite this evidence, there are conflicting literature reports on the dependency of MR on ENDO. This dependency was never investigated in arcuate and mesenteric arteries of normotensive and hypertensive rats. We hypothesized that MR is dependent on ENDO and removing ENDO abolishes MR in arcuate and mesenteric arteries of Wistar Kyoto (WKY, normotensive) and Spontaneously Hypertensive Rats (SHR, hypertensive) rats.MethodsArteries were mounted in a pressure myograph chamber (PMC) filled with oxygenated Hank Balance Salt Solution (HBSS) to create a blind sac and allowed 30 min to equilibrate. Intact vascular smooth muscle cell (VSMC) and ENDO layers were evaluated by addition of a pre‐determined median effective dose of phenylephrine (0.2 μM) and carbachol (10 μM) respectively. MR was measured in response to step‐wise increases in intraluminal pressure from 80 to 180 mmHg (20 mmHg increments, 5 min each) (E‐intact MR). The arteries were then transferred into a petri dish containing human hair and moved through the length of the hair 5 times to remove ENDO (Osol et al., 1989). The vessels were re‐mounted, HBSS was flushed through the arteries for 5 mins, and blind sac was re‐created. Phenylephrine and carbachol were added and MR was re‐measured (E‐removed MR). Physical absence of ENDO was further assessed by Scanning Electron Microscopy (SEM).ResultsAll arteries contracted to phenylephrine before and after ENDO removal and did not relax significantly to carbachol after ENDO removal. SEM micrographs showed complete physical absence of the ENDO. MC in E‐removed vessels were not significantly different from MC in E‐intact in mesenteric and arcuate arteries of WKY and SHR rats.ConclusionsOur study validated an ENDO removal method that was described by Osol, et al. in 1989. Additionally, MR was shown to be independent of ENDO in mesenteric and arcuate arteries of WKY and SHR rats.Support or Funding InformationThis work was supported by Canadian Institutes of Health Research grants to Dr. Jeffrey G. Dickhout (PJT148499).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fumarase Overexpression Abolishes Hypertension Attributable to eNOS Haploinsufficiency in Dahl Salt‐Sensitive Rats
Authors: Xue Hong , Geurts Aron , Usa Kristie , Wang Feng , Phillips Jenifer , Henderson Lisa , Baker Angeles , Tian Zhongmin , Liang Mingyu 
Published: June 2021

BackgroundHuman blood pressure salt sensitivity is associated with changes in urinary metabolites related to fumarase (Fh) and nitric oxide (NO) metabolism, and fumarase promotes NO production through a malate‐aspartate‐arginine pathway.ObjectiveWe examined the role of the fumarase‐NO pathway in the development of hypertension in the genetic background of the Dahl salt‐sensitive (SS) rat, a model of human salt‐sensitive hypertension and renal injury used widely.Methods and ResultsSS rats with heterozygous mutation of endothelial NO synthase (eNOS or Nos3) (SS‐Nos3+/−) were bred with SS rats with a hemizygous Fh transgene. SS‐Nos3+/− rats without the Fh transgene (SS‐Nos3+/−/Fh0/0) developed substantial hypertension with a mean arterial pressure (MAP) of 138.5 ± 3.8 mmHg on a 0.4% NaCl diet, and 183.0 ± 3.7 mmHg after 14 days on a 4% NaCl diet. MAP decreased remarkably to 123.1 ± 1.4 mmHg on 0.4% NaCl, and 143.3 ± 1.5 mmHg on 4% NaCl in SS‐Nos3+/− rats with a Fh transgene (SS‐Nos3+/−/Fh0/1), and proteinuria, renal fibrosis, and tubular casts were attenuated in SS‐Nos3+/−/Fh0/1 rats compared to SS‐Nos3+/−/Fh0/0 rats. eNOS protein abundance decreased in rats with the Nos3 heterozygous mutation, which was not influenced by Fh overexpression. However, the decrease in NO metabolite in the renal outer medulla of SS‐Nos3+/−/Fh0/0 rats was reversed in SS‐Nos3+/−/Fh0/1 rats. Levels of L‐arginine, but not the other 12 amino acids analyzed, were significantly higher in SS‐Nos3+/−/Fh0/1 rats than in SS‐Nos3+/+/Fh0/0 rats.ConclusionsFumarase has potent effects in restoring NO production in renal outer medulla and blunting the development of hypertension attributable to eNOS haploinsufficiency.Support or Funding InformationThis work was supported by US National Institutes of Health grant HL116264‐5244 (M.L.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolic Insults Drive the Development of Glomerular Sclerosis and Proteinuria in Salt‐Sensitive Hypertensive Nephropathy
Authors: Rinschen Markus , Palygin Oleg , Siuzdak Gary , Staruschenko Alexander 
Published: June 2021

Hypertensive nephropathy causes more than one third of end stage renal cases, but its molecular mechanisms are not fully understood. Here, we performed a multi‐layered omic analysis of a well‐established animal model of hypertensive nephropathy, the Dahl salt‐sensitive (SS) rat. Specifically, we performed a time‐course untargeted and targeted metabolomics as well as quantitative phosphoproteomic and proteomic analysis of glomerular and whole kidney cortex. For this experiments three groups of age matched 8 weeks old SS rats were fed either a low salt (0.4% NaCl) or high salt (4% NaCl; 7 and 21 days of HS). Analysis of microalbuminuria, as a marker of kidney injury, revealed that SS rats had a progressive kidney damage following HS treatment (Alb/Cre ratio was 4.6 ± 0.8, 10.7 ± 1.5 and 12.2±2.0, for SS rats fed LS and HS for 7 and 21 days, respectively). Metabolomics, phosphoproteomic and proteomic analyses were performed either in the kidney cortex homogenate or pure fraction of glomeruli. Glomeruli were isolated by consecutive sieving as we previously reported. Metabolomic analysis of the glomeruli revealed a decrease in energy fuels before the onset of proteinuria, including glucose‐6‐phosphate, alpha‐Ketoglutarate, and branched chain amino acids, suggesting a wide‐spread energy depletion. In addition, proinflammatory molecules such as free fatty acids were increased before onset of proteinuria. Notably, these changes did not occur in the kidney cortex where glomeruli fraction was negligeable, suggesting glomerular vulnerability as a key event in hypertensive nephropathy. Phosphoproteomics revealed that podocyte‐specific nutrient‐sensing 5′ AMP‐activated protein kinase (AMPK), as well as regulatory‐associated protein of mTORC1 Raptor, were activated before the onset of structural damage. Structural changes as shown by complement deposition and fibrosis proteins were detectable upon onset of heavy proteinuria. Furthermore, comparative proteomics across glomerular insult models revealed that increased expression of alpha‐Ketoglutarate depleting enzymes is common in glomerular injury. Oxidative protein modifications was also detected in the extracellular matrix of the glomeruli, explaining the energy depletion. In the kidney cortex, reduced uptake of amino acids was observed among other metabolic changes. These data suggest metabolome‐driven insults in the glomeruli of the kidney that proceed the insult in the other parts of nephron. Therefore, this study provides a novel framework for the study of hypertensive nephropathy.Support or Funding InformationSupported by the DFG.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel method for establishing claudin knockout clones using TALENs
Authors: Tokuda Shinsaku , Yu Alan 
Published: June 2021

In the multicellular organisms, epithelia function as a barrier between external and internal environment. Tight junctions (TJs) are one of the cell‐cell junctions between epithelial cells and regulate the permeability of the paracellular pathway. Claudins, a large family of transmembrane proteins, are major component of TJ strands. Most epithelia express multiple different claudins, and the expression pattern of claudins are thought to provide diverse permeability of TJs in each epithelium. However, it is difficult to measure the permeability of each claudin with the current technique, and the permeability property of each claudin is still poorly understood. To clarify the permeability property of each claudin, we attempted to knockout claudin genes using transcription activator‐like effector nucleases (TALENs) in MDCK cells. In the previous study, we succeeded in establishing claudin‐2 knockout clones and claudin‐4 knockout clones in MDCK cells. However, the efficiency of the knockout is low and a lot of effort was required to establish knockout clones. To improve the efficiency of establishing claudin knockout clones, we made a construct in which forward and reverse TALENs were connected with internal ribosome entry site (IRES). Using the constructs, we succeeded in establishing claudin‐2, −3, −4 and −7 knockout clones. In conclusion, we developed the method for establishing claudin knockout clones. The analysis of these claudin knockout clones are thought to provide an important insight in the permeability property of each claudin and TJ physiology.Support or Funding InformationJSPS Overseas Research FellowshipsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High mobility group box‐1 increases epithelial sodium channel activity and inflammation in small airway epithelial cells
Authors: Grant Garett J , Coca Camila , Helms My N 
Published: June 2021

In Cystic Fibrosis (CF) patients, inherited defects in anion transport leads to hyperactive epithelial Na+ channel (ENaC) activity. ENaC dysfunction in CF lung is known to cause airway surface liquid dehydration. Thick and dry mucus, in turn, leads to an inflammatory lung phenotype that often occurs without detection of infection in CF patients. Many investigative groups have reported that airway tissue and fluid from murine models exhibiting the CF lung phenotype (Scnn1b‐Tr mice), as well as CF patients, have increased levels of high mobility group box‐1 (HMGB‐1). HMGB‐1 is a cytokine mediator of inflammation and is a biomarker for severity of CF lung disorder. Herein, we hypothesize that HMGB‐1 plays an important role in the pathogenic regulation of ENaC, inflammation, and fibrosis in sterile airways. To begin testing this hypothesis, we measured transepithelial sodium current (Isc) and single channel ENaC open probability (Po) in human small airway epithelial cells (SAEC) in the presence or absence of 1 mg/mL human HMGB‐1 peptide. Electrophysiological measurements show that HMGB‐1 significantly increased amiloride‐sensitive ENaC Isc from −9.7±1.4 μA/cm2 to −18.4±2.9 μA/cm2 in confluent monolayers of SAEC (n=12; p=0.01) within 30 min. Subsequent amiloride treatment of the same SEAC monolayers decreased Isc 71%. Single channel measurements were conducted in primary SAEC obtained from 4 separate donor lungs in the cell‐attached configuration; HMGB‐1 increased ENaC Po from 0.18±0.02 to 0.35±0.04 (n=17; p<0.01) in single channel analysis. Mean absolute total cell counts were significantly higher in bronchoalveolar lavage fluid (BALF) from mice intraperitoneally (IP) injected with HMBG‐1 (10 mg in 1 mL saline/kg) vs vehicle injected mice. Total immune cell counts were 27.4×103± 8.8×103 (in HMGB‐1) vs. 5.2×103±1.3X103 (in vehicle) injected mice (n=8 counts from 2 independent studies; p<0.05). Masson's Trichrome labeling of IP injected mice show that HMGB‐1 significantly increases pulmonary fibrosis (see Fig 1). Flow cytometric analysis of HMGB‐1 and vehicle injected mice showed significant increase in IL‐1β, IL‐10, IL‐6, IL‐27, IL‐17A, IFN‐β, and GM‐CSF (n=3; p<0.05; see Fig 2). Together, our data suggests that HMGB‐1 plays an important role in the CF lung phenotype; additional studies are underway to strengthen these observations. Impact: Successful completion of our work can lead to novel therapies needed to counteract ENaC dysfunction and inflammation in the CF lung.Support or Funding InformationThis work was supported by NIH 1 R01 HL137033‐01A1 awarded to MH.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.