bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of Angiotensin II Type 1 Receptor Agonistic Autoantibody Binding Improves Maternal Outcomes in Rats with Placental Ischemia
Authors: Cunningham Mark Wayne , Castillo Javier , Vaka Venkata Ramana , Amaral Lorena , Cornelius Denise , Spradley Frank , Ibrahim Tarek , Campbell Nathan , Usry Nathan , Williams Jan , LaMarca Babbette 
Published: November 2023

Women with preeclampsia (PE) have hypertension, placental ischemia, reduced glomerular filtration rate (GFR), nitric oxide bioavailability, elevated sFlt, preproendothelin (PPE), agonistic Angiotensin II type 1 receptor autoantibodies (AT1‐AA), oxidative stress, and activation of cytolytic natural killer (NK) cells during pregnancy. The reduce uterine perfusion pressure (RUPP) rat model of placental ischemia displays these same characteristics, indicating that it is a good model to examine factors contributing to the pathophysiology of PE. We hypothesis that administration of a specific 7 amino acid peptide sequence (7AA) to inhibit the AT1‐AA from binding to the AT1receptor, will improve maternal blood pressure and other PE features in RUPP rats.MethodsDay 14 of gestation RUPP rats receive the AT1‐AA inhibitory peptide, 7AA, (2ug/μl saline) via mini‐osmotic pump. On day 19 of gestation, conscious blood pressure (MAP) and GFR were determined. The blood, kidneys, and placentas were collected for molecular analysis and flow cytometry quantification of NK cells.ResultsMAP was elevated in RUPP rats (n=28) vs. normal pregnant (NP) rats (n=10) (121±1 mmHg vs 98±3 mmHg, p<0.05) and this hypertension was attenuated with 7AA (n= 15; 104±3 mmHg, p<0.05). Circulating sFlt1 (176±37 vs. 77±15pg/ml, p<0.05) was elevated in RUPP rats vs. NP rats and was normalized in RUPP + 7AA rats (86±9 pg/ml, p<0.05). There was a 4 fold increase in PPE in RUPP (n=7) vs. NP (n=6) rats which was reduced 2 fold in RUPP + 7AA (n=7) (p<0.05). There was no difference in GFR or plasma creatinine among the groups. Activated cytolytic placental NK cells was increased in RUPP vs. NP (8.29±1.73% vs. 2.05±1.56%, p<0.05), and was normal in RUPP +7AA (2.78±0.95%, p<0.05). Nitrate and nitrite (NOx), was decreased in RUPP vs. NP (14.0± 1.5 vs. 20.0± 1.0 μM NO3, p<0.05) and was improved to 18.3± 1.6 μM NO3 in RUPP + 7AA (ns). Circulating isoprostanes were elevated for RUPP vs. NP (20.1 ± 6.3 vs. 2.8 ± 1.4 ng/mL, p< 0.05) and was normalized with RUPP + 7AA peptide treated rats (4.3 ± 2.2 ng/mL).ConclusionInhibition of AT1‐AA from binding to the AT1R improves many PE characteristics observed in RUPP rats, thus indicating that this may be a potential therapy to further explore for PE patients.Support or Funding InformationResearch Supported by T32HL105324 and RO1HD067541
Obesity‐induced hypertension is associated with increases in adipose tissue‐derived angiotensinogen in female mice exposed to early life stress (ELS).
Authors: Herald Joseph , Murphy Margaret , Cohn Dianne , Loria Analia 
Published: November 2023

ELS is an established independent risk factor for cardiovascular disease. We use Maternal separation and early weaning (MSEW) during postnatal life as a mouse model of ELS. Previously, we have shown that MSEW exacerbates the development of obesity‐induced hypertension (HT) in female mice. Given the well‐known role of renin‐angiotensin system (RAS) during the development of obesity‐induced HT, the goal of this study was to determine the RAS status in female MSEW mice. MSEW was performed in C57BL/6 mice from postnatal days 2–16. Undisturbed litters served as control group (C). Mice were placed on a low‐fat diet (LFD, 10% kcal from fat) or HFD (60% kcal from fat) for 16 weeks upon weaning. Plasma renin concentration (PRC) was reduced in female MSEW compared with C (p<0.05), suggesting increased circulating AngII levels. Angiotensinogen (AGT) protein level, the only known precursor for angiotensin peptides, showed similar levels between groups in plasma (6.2±0.3 vs. 5.5±0.1 ug/ml, p<0.05) and liver (1.6±0.2 vs. 1.7±0.3 ng/ml/g tissue); however, adipose tissue and media explants displayed increased AGT secretion in female MSEW mice compared with C (29±9 vs. 98±8 and 15±1 vs. 22±2 ng/ml/g tissue, respectively, p<0.05). AngII receptor subtypes 1 and 2 (AT1 and AT2) were not different between groups in liver, adipose tissue, brain and kidney. In additional studies, we found increased adipose tissue‐derived AGT in media explants from female MSEW after one week on HFD (27±2 vs. 46±3 ng/ml/g tissue, p<0.05). Finally, AGT mRNA expression in visceral fat was increased in MSEW pups at postnatal day 7 (p<0.05). Taken together, these data indicate that adipose tissue‐derived AGT is particularly susceptible to MSEW and may serve as a major source of circulating angiotensin II peptide in MSEW mice thereby contributing to obesity‐induced HT.Support or Funding InformationHL111354
HNF1B as a New Transcriptional Activator of the Renal KIR4.1/KIR5.1 Potassium Channel
Authors: Kompatscher Andreas , de Baaij Jeroen H.F. , Aboudehen Karam , Hoefnagels Anke P.W.M. , Igarashi Peter , Bindels Rene J.M. , Veenstra Gertjan J.C. , Hoenderop Joost G.J. 
Published: November 2023

BackgroundHepatocyte nuclear factor 1 homeobox B (HNF1B) is an essential transcription factor for the development and functioning of the kidney. Mutations in HNF1b cause autosomal dominant tubulointerstitial kidney disease (ADTKD‐HNF1B), which is characterized by renal cysts and maturity‐onset diabetes of the young (MODY). Moreover, patients suffer from a severe electrolyte phenotype consisting of hypomagnesemia and hypokalemia. Until now, genes that are regulated by HNF1B are only partially known and do not fully explain the phenotype of the patients.MethodsTherefore, a chromatin immunoprecipitation and sequencing (chIP‐seq) was executed in immortalized mouse kidney cells (mpkDCT) to identify HNF1B binding sites at a genome‐wide scale. Luciferase‐promoter assays, siRNA‐mediated knockdown of Hnf1b in DCT cells and RT‐qPCR on HNF1B mutant mouse kidneys were performed to assess the transcriptional regulation of Hnf1b on candidate genes in vitro and in vivo.ResultsIn total 7,421 HNF1B binding sites were identified, including several genes involved in electrolyte transport and diabetes. A highly specific and conserved HNF1B site was identified in the promoter of Kcnj16, encoding the potassium channel Kir5.1. Luciferase‐promoter assays showed a 2.2 fold increase in Kcnj16 expression when HNF1B was present. Expression of the Hnf1b p.Lys156Glu mutant that was previously identified in a ADTKD‐HNF1B patient, did not activate Kcnj16 expression. Knockdown of Hnf1b in mpkDCT cells significantly reduced the expression of Kcnj16 (Kir5.1) and Kcnj10 (Kir4.1) by 38% and 37%, respectively. These results were confirmed in a HNF1B renal knockout mouse, exhibiting downregulation of Kcnj16, Kcnj10 and Slc12a3 transcripts in the kidney by 78%, 83% and 76%, respectively, compared to HNF1B wild‐type mice.ConclusionHNF1B has been identified as a transcriptional activator of Kcnj16. Consequently patients with HNF1B mutations may have a reduced Kir5.1 activity in the kidney, resulting in hypokalemia and hypomagnesemia.
Attenuated cardiovascular‐renal sensitivity of blood pressure and heart rate to infused insulin in obese TallyHo/Jng mice
Authors: Alunan Ashley , Li Lijun , Lee Hwal , Fluitt Maurice , Tiwari Swasti , Ecelbarger Carolyn M. 
Published: November 2023

Obese mice are known to be relatively insulin resistant with regard to cellular glucose uptake; however, it is still uncertain whether the cardiovascular‐renal system develops insulin resistance and what this means in terms of blood pressure (BP) and heart rate (HR). To address this problem, we tested the impact of pre‐existing metabolic syndrome (MetS) on BP, HR (radiotelemetry), and blood chemistry responses to an insulin infusion using a newer inbred polygenic model of MetS. Male TallyHo/Jng (TH) mice were infused with 50 U/kg bw insulin by osmotic minipump for 2‐weeks. C57Bl6 (B6) mice were used as insulin‐sensitive controls (n = 6/strain). All mice were provided with 20% glucose, 0.5% NaCl, 0.5% K2HPO4 water to drink to combat hypoglycemia during the infusion. Mean arterial BP of TH mice was significantly (p < 0.05) higher than B6, e.g., 126±2 versus 112±5 mm Hg prior to infusion. Infusion led to an initial fall in BP in B6 (Day 3‐ 108±2) followed by a slow rise (Day 13‐ 122±8). In contrast BP remained fairly steady in TH (Day 3‐ 123±1; Day 13‐ 125±5). Similarly HR was elevated in TH in the basal period: 649±8 versus 527±20 beats per min, (bpm) in B6, and was reduced by insulin infusion to 614±21 (Day 13). In B6, HR rose to 563±30 (Day 13). Insulin infusion caused a number of changes in blood chemistry including, a reduction in pH and HCO3 levels. Most of these changes were independent of strain; however, insulin infusion caused a reduction in blood glucose levels only in the B6 mice. Overall, we found greater sensitivity of BP and HR to insulin in B6 versus TH. The rise in HR with insulin is likely the result of a rise in sympathetic activity. Insulin infusion produced somewhat “beneficial” changes in TH with a slight fall in HR and BP. In contrast, in B6, after the first few days, BP and HR rose, suggesting primarily detrimental effects likely as mice developed a resistance to the infusion. These findings demonstrate that insulin resistance extends to non‐traditional target tissues in MetS; and that insulin resistance, not insulin action per se, is correlated to rises in both BP and HR during MetS.Support or Funding InformationGeorgetown University internal funding and Georgetown/Howard University CTSA
Forskolin (cAMP) Promotes Insertion of Large Conductance Potassium (KCa1.1) Channels into Apical Membranes of Rat Distal Colon
Authors: Nickerson Andrew , Rottgen Trey , Minor Emily , Rajendran Vazhaikkurichi M 
Published: November 2023

Mammalian colon contributes significantly to overall K+ homeostasis. Under normal conditions, the proximal colon secretes and distal colon absorbs K+. Distal colon also has a secretory capacity, which becomes important to K+ homeostasis under pathological conditions, such as end‐stage renal failure. K+ secretion in the distal colon is primarily mediated through apical large conductance (BK) channels. We hypothesized that the BK opener BMS‐204352 (BMS) and the adenylate cyclase activator forskolin (FSK) induce K+ secretion through separate mechanisms. K+ flux studies were performed in distal colonic mucosal tissues obtained from male Sprague‐Dawley rats (200–225g). Unidirectional 86Rb fluxes (K+ surrogate), and short‐circuit current (ISC) were measured under voltage‐clamped conditions in an Ussing chamber setup. Fluxes were performed in the presence of amiloride (10 mM) to inhibit ENaC‐mediated ISC, as well as VO4 (1mM) to inhibit apical H+/K+ ATPase and unmask BK‐mediated K+ secretion. Results indicate that BMS (10mM) and FSK (10mM) induced a net K+ secretion of 0.083 ± 0.04 and 0.274 ± 0.05 mEq/cm2·hr, respectively (n=4, p<0.05). FSK, but not BMS, increased ISC compared to baseline (220.5 ± 30.5 vs 132.2 ± 18.7 uA/cm2). Immunofluorescence studies performed on the same tissues used in flux experiments showed markedly increased apical BK staining in tissues exposed to FSK, but not in those exposed to only BMS. Taken together, these data suggest that while BMS may only open BK channels already present on apical membranes, FSK may cause further K+ secretion by triggering the insertion of more BK channels into the apical membrane.Support or Funding Information• R01 DK104791/DK/NIDDK NIH HHS/United States
Proximal Tubule Glutamine Synthetase is Critical for the Response to Low Protein Diet
Authors: Weiner I. David , Lee Hyun‐Wook , Osis Gunars , Handlogten Mary E. , Chen Chao , Verlander Jill W. 
Published: November 2023

The mechanisms through which the kidneys increase ammonia generation in response to increased endogenous acid loads have been extensively studied; the mechanisms underlying decreased ammonia metabolism are less well understood. Glutamine synthetase is expressed in the kidney, and mediates reaction of NH4+ with glutamate, generating glutamine and decreasing net ammonia generation. Our previous studies show that dietary protein restriction, which decreases endogenous acid production, increases proximal tubule glutamine synthetase expression. The purpose of the current studies was to determine the functional importance of glutamine synthetase in the response to dietary protein restriction. We used mice with proximal tubule‐specific glutamine synthetase deletion (PT‐GS‐KO), using floxed GS alleles and Cre‐recombinase under control of the PEPCK promoter. Control (C) mice was floxed, Cre‐negative littermates. Mice were studied in metabolic cages, and daily 24‐hour urine samples collected. Changing from normal, 20%, protein to low, 6%, protein diets for one week resulted in a rapid decrease in urinary ammonia excretion that was maximal at day 2 in C mice. In PT‐GS‐KO mice, the ability to decrease ammonia excretion was impaired significantly on day 2–7 as compared to C mice. This impaired ability to alter urinary ammonia excretion occurred despite no significant difference in urine pH on any day between C and PT‐GS‐KO mice. The difference in ammonia excretion was unique to this component of net acid excretion; titratable acid excretion did not differ significantly between C and PT‐GS‐KO mice. Other components of renal ammonia metabolism, specifically PDG, PEPCK, NHE3, NKCC2, Rhbg and Rhcg, were not detectably different between C and PT‐GS‐KO mice. We conclude that glutamine synthetase, whose expression increases in response to dietary protein restriction, is necessary for adaptive changes in renal ammonia excretion, thereby enabling maintenance of acid‐base homeostasis and nitrogen metabolism.Support or Funding InformationThe studies were supported by funds from the NIH (R01‐DK045788 and R01‐DK107798) and from the Department of Veterans Affairs (1I01BX000818).
Identification of a Stress‐Inhibitory Pathway from the Nucleus of the Solitary Tract (NTS) to the Bed Nucleus of the Stria Terminalis (BNST)
Authors: Scheuer Deborah , Hiller Helmut , McCowan Michael , Wang Lei , Krause Eric G 
Published: November 2023

We and others have previously shown that the NTS influences physiological responses to both acute and chronic stress. The NTS sends a robust neuronal projection to the BNST consisting of both catecholaminergic and 11βhydroxysteroid dehydrogenase‐expressing neurons. The BNST contributes importantly to regulation of the stress response, however, the functional significance of the NTS to BNST pathway is not known. We conducted the present experiments to test the hypothesis that activating this pathway would influence stress‐associated physiological endpoints. The Cre‐inducible channelrhodopsin construct, rAAV2/EF1a‐DIO‐hChR2(H134R), was microinjected into the NTS (500 nl bilaterally and at the midline) and the retrograde Cre recombinase expressing virus, CAV2‐Cre, was injected into the BNST (1000 nl bilaterally) in six adult male Wistar‐Kyoto rats. After recovery, rats were implanted with chronic indwelling fiber optics targeting the BNST. At least 12 weeks later, tests were performed in conscious rats to measure the effect of stimulation of NTS neuron terminals in the BNST. Optogenetic activation of the NTS to BNST pathway reduced baseline plasma corticosterone concentration from 5.4±0.9 to 2.1±0.8 μg/dl (P<0.05, within subjects design) and increased the percent of time the rats spent in the open arms of the elevated plus maze from 20±7 to 87±6 % (P<0.05, between subjects design). Rats also underwent tests for sucrose and saline preference. Activation of the NTS to BNST pathway reduced 1.8% saline intake over a 30 min period from 1.40±0.32 to 0.55±0.16 ml (P<0.05, within subjects design). Sucrose (1% solution) intake and water intake were not affected by activation of the pathway. Sucrose intake was 2.01±0.64 ml without stimulation and 1.59±0.24 ml with stimulation. Average water intake was 0.94±0.37 ml without stimulation and 0.89±0.29 ml with stimulation. We conclude that in unstressed normal male rats activation of the neuronal pathway from the NTS to the BNST inhibits the hypothalamic‐pituitary‐adrenal axis, anxiety‐like behavior and saline intake. The pathway may act to restrain the stress response under these conditions. Future studies will determine the function of this pathway under conditions of chronic stress, its role in the control of cardiovascular function, and the role of mineralocorticoid receptors in both catecholaminergic and 11βhydroxysteroid dehydrogenase‐expressing NTS neurons.Support or Funding InformationR01 HL076807, R01 HL‐122494
Differential Role of Nav Isoforms in the Induction vs Conduction of Action Potentials in Vagal Afferent Nerves
Authors: Sun Hui , Meeker Sonya , Herbstsomer Robert A. , Kollarik Marian , Undem Bradley J. 
Published: November 2023

Based on compound action potential studies, it has long been known that tetrodotoxin (TTX) blocks action potential (AP) conduction in the vagus nerve. We recently reported that airways specific nodose and jugular neurons express mainly NaV 1.7, 1.8 and 1.9 channels; and selectively silencing NaV1.7 mRNA expression with an in vivo shRNA strategy mimics the blocking effect of TTX on AP conduction in vagal sensory nerves (J. Physiol, 2011). Here we evaluate the role of TTX‐sensitive Nav 1.7 and TTX‐resistant Nav 1.8 channels in the induction and conduction of APs in airway‐specific vagal afferents derived from nodose and jagular gaglia. Extracellular recordings from nodose or jugular ganglia were used to evaluate the activities of single afferent fibers arising from the isolated, innervated trachea and intrapulmonary airways. The trachea is innervated by jugular C‐fibers and nodose Aδ fibers. TTX 1 μM perfused selectively through the tracheal lumen abolished citric acid‐induced AP discharge in nodose Ad fibers (AP number: 21 ± 6 vs. 0 ± 0, n=7 each group), but did not inhibit citric acid induced AP discharge from jugular C‐fibers (50 ± 8 vs. 50 ± 9, n = 15 each group; the responses in jugular C‐fibers were blocked with lidocaine, n=4). When applied to a segment of the desheathed vagus, however, TTX abolished the conduction of APs induced by vagal electrical stimulation in all nodose Ad fibers and 70% of jugular C‐fibers (n=9). The intrapulmonary airways are innervated by both nodose and jugular C‐fibers. Again, TTX applied via intrapulmonary artery and trachea virtually abolished bradykinin‐induced AP discharge in nodose C‐fibers (AP number: 494 ± 80 vs. 2 ± 2, n=6 each group), but did not affect bradykinin‐induced responses in the jugular C‐fibers (285 ± 95 vs. 318 ± 122, n=6 each group). The NaV1.8 blocker, PF‐01247324, at 30 μM, a concentration we found inhibit the guinea pig NaV1.8 current by about 75%, inhibited the citric acid responses in the jugular C‐fibers inervating the trachea (50 ± 13 vs. 12 ± 6, n=6 each group, P<0.05). Consistent with these findings, patch clamp studies revealed that the Nav1.8‐mediated TTX resistant INa is larger in jugular than nodose neurons (Gmax=6.81±0.81 nS, n=12 vs. 2.38±0.32nS, n=13, p<0.001) while the amplitudes of TTX‐sensitive INa are similar. The voltage dependence of activation and inactivation of Nav1.8 currents was not different between nodose and jugular neurons. Furthermore, TTX increased the amount of current required to evoke an AP to a greater extent in nodose compared to jugular neurons (10‐ vs. 4‐fold increase). In summary, in nodose nociceptors conduction of APs along axons and induction at the nerve endings within the tissue depends on NaV 1.7; whereas in jugular nociceptors, conduction is largely NaV 1.7 dependent, but TTX‐resistant Nav1.8 channels are sufficient to induce APs in the terminals.Support or Funding InformationNIH (5R01HL22228‐04)
Glutamatergic Neurons in the Dorsal Hypothalamic Area Dissociate Thermogenesis from Heat Conserving Pathways During Stress Responses
Authors: Machado Natalia Lima , Abbott Stephen , Fuller Patrick , Fontes Marco , Saper Clifford 
Published: November 2023

Stress elicits a variety of autonomic responses, including thermal changes in humans and animals. In this present study we investigate the circuit basis for stress‐induced hyperthermia with focus on the hypothalamic‐medulary pathway in adaptive thermoregulatory responses.We injected retrograde tracer (CTb) in the raphe pallidus (RPa) of Vgat and Vglut2‐IRES‐cre L10‐GFP reporter mice to identify the phenotype of neurons in the dorsal hypothalamic area (DHA) projecting to RPa.In a separate series of experiment, we bilaterally injected AAV‐Flex‐hGlyR‐mCherry in the DHA of Vglut2‐IRES‐cre mice to promote inhibition of DHAVglut2 neurons. hGlyR is a mutated glycine receptor in which ivermectin (hGlyR ligand) gates a chloride channel. Using AAV8‐hSyn‐DIO‐hM3D(Gq)‐mCherry (DREADD) we induced activation of DHAVglut2 neurons and measured changes in brown adipose tissue (BAT) and tail temperature. An optogenetic approach in freely‐behaving mice was used to promote inhibition of the synaptic terminal of the DHAVglut2 neurons in the RPa.We identified that glutamatergic neurons in the DHA (DHAVglut2) sending direct inputs to RPa are activated during cage switch stress (singly housed male was switched to a cage previously occupied by another male) causing 39 + 6% of Vglut2/CTb neurons to express cFos, whereas only 13 + 2% of Vglut2/CTb neurons expressed cFos in the control animals (mean + SEM, n=4,3 respectively, *p=0.016).The long‐lasting inhibition induced by hGlyR‐IVM reduced cFOS expression in our stress protocol (after IVM 6 + 1% cFOS+/hGlyR Vglut2 labeled neurons vs vehicle 40 + 8% cFOS+/hGlyR Vglut2 cFos doubly labeled neurons in the DHA; n=3 each group, *p=0.013) and also decreased stress‐induced hyperthermia (2.8 + 0.1°C after vehicle vs. 2.0 +0.1°C after IVM, n=11, FTc(interaction) (164,1278)=1.556).During the stress protocol we found a large increase in BAT thermogenesis (rise of 1.2 + 0.2°C), and tail artery vasoconstriction (fall in tail temperature by −2.3 + 0.4°C). Surprisingly, activation of DHAVglut2 neurons using hM3D excitatory receptor caused profound BAT thermogenesis (1.0 + 0.14°C), but it did not cause vasoconstriction, instead resulting in tail artery vasodilation (2.5 + 0.6°C) as a delayed reflex response to central hyperthermia.Finally, optogenetic experiments identified that DHAVglut2—>RPa pathway is a mediator of stress‐induced hyperthermia, as photoinhibition of terminals in the RPa reduced the peak of stress‐induced hyperthermia by 0.4+0.06°C, (comparing same group before vs. during photoinhibitoin). We propose that the DHAVglut2—‐>RPa is a thermogenic pathway regulating BAT thermogenesis but not thermoregulatory vasoconstriction during stress.Support or Funding InformationGrant support: NIH Grant NS085477 (C.B.S.), NS073613 (P.M.F.), CAPES and CNPq