bluuebunny/crossref_metadata_2025 huggingface dataset:
Sex Dependent Effects of Prenatal Nicotine Exposure on Cardiovascular Reactivity: Are Males at a Greater Risk for Cardiovascular Disease?
Authors: Hayward Linda F , Watkins Jacqueline
Published: November 2023

Prenatal nicotine exposure (PNE) has been linked to an increased incidence of anxiety‐disorders, risk of nicotine use, and dysregulation of the hypothalamic orexin system. Recent data link dysregulation of the orexin system to cardiovascular disease. The aim of this study was to evaluate cardiovascular reactivity of adult offspring exposed to nicotine in utero and correlate differences with changes in orexin system gene expression in the hypothalamus and amygdala. Pregnant Sprague Dawley rats had an osmotic mini‐pump filled with nicotine implanted on day 6 of pregnancy (exposure dose 3 mg/kg). At 12 weeks of age (n=4–6/group) offspring were instrumented with arterial catheters and tested 2 days later. PNE male and female weighed ~20% more than controls (P<0.05) but resting mean arterial pressure (MAP) was not significantly different between groups. After an acute dose of nicotine (0.37mg/kg), MAP increased ~ 9 mm Hg in females and 6 mm Hg in male within 2 min. of a subcutaneous (sc) injection, independent of prenatal treatment At 18 minutes' post nicotine injection, MAP in both male and female controls and PNE females decreased significantly below baseline (−9 mmHg) while the MAP of PNE males returned to baseline. There was a strong trend (P<0.07) for the pressor response to angiotensin II (10 microg/kg) to be greater in the PNE males compared to control but not for the PNE females when compared to sex‐matched controls. In the hypothalamus orexin‐type 1 receptor gene expression was increased significantly in PNE males compared to controls (P<0.006). Alternatively, in the amygdala a decrease in orexin‐type 2 receptors in PNE females compared to controls (P<0.03) was identified These results suggest that male offspring exposed to nicotine in utero may be more susceptible to the negative cardiovascular consequences of nicotine when compared to females due to sex‐dependent changes in orexin‐receptor gene expression in the brain.

Support or Funding Information

Florida Department of Health‐James and Esther King Biomedical Research Program
Relaxin contributes to the regulation of atrial pressure during pregnancy
Authors: Denton Kate M , Mirabito Collafella Katrina M , Samuel Chrishan
Published: November 2023

Relaxin is a potent vasodilatory and anti‐fibrotic hormone. Recently we demonstrated that these effects of relaxin are mediated via heterodimers formed between the cognate relaxin family receptor 1 (RXFP1) and the angiotensin type 2 receptor (AT2R). We, and others, have demonstrated that adult females are protected from the hypertensive effects of angiotensin II via an enhanced contribution of the AT2R in the regulation of arterial pressure and renal function as compared to age‐matched males and aged females. Moreover, during pregnancy, the refractoriness to angiotensin II is associated with an increase in the AT2R/AT1R ratio. In the present study we hypothesized that relaxin contributes to the regulation of arterial pressure in females at baseline and during pregnancy. Mean arterial pressure (MAP) was measured via radiotelemetry in 14 week‐old male and female wild‐type (WT; C67BL/6xSv129) and relaxin knockout (KO) mice and female relaxin KO mice. Thereafter, female mice were time‐mated with a (non‐telemetered) male of the same genotype and MAP was measured throughout gestation. Basal MAP was ~6 mmHg lower in WT females than males (P<0.05). Relaxin deficiency increased basal MAP in females (P<0.05 versus WT female), but not males. As expected, MAP decreased during gestation in WT mice, returning to near preconception levels during late gestation. Conversely, in relaxin KO mice, arterial pressure increased during mid and late gestation (P<0.05 as compared to WT). Moreover, relaxin deficiency impaired gestational weight gain and reduced litter size. This is the first study i) to demonstrate that relaxin contributes to the sexual dimorphism of arterial pressure in mice and ii) to document the changes in the arterial pressure profile of pregnant relaxin KO mice. Understanding the mechanisms that underlie the regulation of arterial pressure in females of reproductive age may uncover new strategies to treat hypertension in women and men
Time Course of Changes in Calf Venous Compliance during Pregnancy and Postpartum in Humans
Authors: Parker Rosemary , Yoo Jeungki , Jarvis Sara , Okada Yoshiyuki , Best Stuart , Stickford Abigail , Levine Benjamin , Fu Qi
Published: November 2023

Background

Pregnancy is associated with dramatic changes in volume status and vascular function. Previous human studies have focused extensively on endothelial function and arterial stiffness in pregnancy, but time‐course changes in venous compliance throughout gestation have received little attention.

Purpose

To examine the changes in calf venous compliance before, during and after pregnancy.

Methods

17 healthy normotensive women aged 31 ± 5 (mean ± SD) years completed all longitudinal studies including pre‐pregnancy (mid‐luteal phase of the menstrual cycle), early‐pregnancy (4–8 weeks gestation), late‐pregnancy (32–36 weeks gestation), and postpartum testing (6–10 weeks post‐delivery). Subjects were in the supine (rotated about 15° into the left lateral) position, and venous occlusion plethysmography with strain gauges was used to measure calf venous compliance in the right leg. Brachial blood pressure (BP, SunTech) and heart rate (HR, lead II of electrocardiogram) were also measured on each testing day.

Results

The changes in calf venous compliance between pre, early, and late pregnancy (0.05 ± 0.01, 0.06 ± 0.01, and 0.05 ± 0.01 ml/100 ml/min, respectively) appears to be relatively stable. However, there was a significant decrease in calf venous compliance post pregnancy (0.04 ± 0.01 ml/100 ml/min, ANOVA p<0.05) as compared to during pregnancy. Resting systolic BP was significantly lower during early pregnancy as compared to pre and late pregnancy (101±7 vs. 107±8 and 108±7 mmHg, respectively; p<0.05) but not different from post pregnancy (103±7 mmHg). Resting diastolic BP during early pregnancy (59±4 mmHg) had a tendency to differ from pre (63±3 mmHg, p=0.081) and post pregnancy (64±6 mmHg, p=0.072). HR, however, decreased significantly during postpartum (58±9 bpm, ANOVA p<0.001) as compared to pre, early, and late pregnancy (74±13, 69±11, 78±12 bpm respectively).

Conclusion

Despite blood volume expansion and a fall in systemic vascular tone during pregnancy, venous compliance remains relatively stable throughout the entire gestation. The decrease in venous compliance during postpartum may be related to the hormone oxytocin, as approximately 90% of the women were lactating after delivery.

Support or Funding Information

NIH R21 grant (HL088184)

AHA Grant‐in‐Aid (13GRNT16990064)

The Harry S. Moss Heart Foundation
TNFα and IL‐17 downregulates βENaC expression and reduces migration in vascular smooth muscle cells
Authors: Duncan Jeremy W. , Warrington J. Paula , Ryan Michael J. , Granger Joey P. , Drummond Heather A.
Published: November 2023

Studies from our laboratories have shown that beta Epithelial Na+ Channel protein (βENaC) mediates vascular smooth muscle cell (VSMC) and cytotrophoblast cell migration. Inflammatory cytokines have also been shown to play a role in VSMC and trophoblast migration. However, whether cytokines regulate βENaC protein expression has never been determined. Therefore, the purpose of this study was to determine if the cytokines, Tumor Necrosis Factor‐alpha (TNFα) and Interleukin‐17 (IL‐17), alter expression of βENaC and migration in VSMCs. To address this, cultured VSMCs (A10; rat) were treated with recombinant TNFα or IL‐17 (0 – 100 ng/mL). βENaC expression was assessed after 16 hours using Western blotting. Cellular migration responses were assessed at 24 and 48 hours using a scratch wound assay. TNFα inhibited the expression of βENaC in VSMCs by 51% (49.3 ± 9.6 vs 100 ± 0; p= 0.006) and 59% (40.7 ± 12.8 vs 100 ± 0; p= 0.01) at 50 and 100 ng concentrations vs control cells, respectively. TNFα (50 ng) impaired wound healing compared to control cells at 24 hr (89 ± 7.8% vs 58 ± 4.8%) and 48 hr (84 ± 8.2% vs 34 ± 9.7%) following injury, as measured as a percentage of initial wound area (p < 0.05). This effect persisted at 100 ng, where TNFα abolished wound healing at 24 hr (100 ± 12% vs 58 ± 4.8%) and 48 hr (93 ± 17% vs 34 ± 9.7%) vs control cells (p< 0.05). IL‐17 also significantly reduced βENaC by 30% (100ng vs Control; 70.1 ± 10.6 vs 100 ± 0; p= 0.037). Similarly, IL‐17 (100 ng) impaired wound healing at 24 hr (85 ± 7.4% vs 62 ± 2.4%) and 48 hr (83 ± 8.3% vs 45 ± 5.2%) compared to control cells (p< 0.05). These data indicate that the inflammatory cytokines TNFα and IL‐17 reduce βENaC protein expression and cell migration in VSMCs. Whether TNFα and IL‐17 attenuate βENaC expression in cytrotrophoblasts or attenuate migration through βENaC inhibition remains to be determined. These findings suggest VSMC and cytotrophoblast migration may be compromised in conditions of high circulating levels of cytokines, such as pre‐eclampsia. Further studies to determine the molecular mechanisms, as well as potential approaches to rescue βENaC could be beneficial in certain pathophysiological cases where appropriate cell migration is critical.

Support or Funding Information

This study was supported by a National Institutes of Health Grant, HLHL51971, and an NRSA Institutional Training Grant, T32HL105324.
Validation of LTP nanoparticles as a gene delivery system: Maternal and Fetal Outcomes Unchanged by Delivery of Nonsense Plasmid VEGFR2 Nanoparticles
Authors: Novak Jacqueline , Balser Brittany Ann , Yun Yang H , Ramirez Rolando JJ
Published: November 2023

Placental under‐perfusion is a hallmark of preeclampsia. Alterations in the maternal circulation have been well documented in preeclamptic mothers, however treatment options remain limited. An imbalance in angiogenic factors (via the VEGF pathway) favoring a vasoconstrictory phenotype and inadequate vascular remodeling contribute to the maternal hypertension. Delivery of LTP VEGFR2 nanoparticles shows normalization in mean arterial pressure, uterine vessel myogenic reactivity, and fetal growth restriction. We previously demonstrated that LTP nanoparticles seeded with plasmid for VEGFR2 improved MAP and vascular reactivity. The goals of these studies were to validate the maternal and fetal safety of our nanoparticle delivery system. The reduced uterine perfusion pressure rat model (RUPP) is used to investigate preeclampsia pathology with nonsense plasmid for VEGFR2 nanoparticles. In the RUPP Sprague‐Dawley dams, silver clips are placed on the abdominal aorta (0.2mm ID) and the utero‐ovarian arteries (0.1mm ID) on pregnancy day 14. SHAM rats undergo surgery without clip placement. On the same day as RUPP surgery, LTP nanoparticles with nonsense plasmid for VEGFR2 are injected into the uterine wall. On day 21 of gestation an anesthetized blood pressure is measured via carotid catheter then resistance‐sized uterine arteries (< 300μm) are harvested for study in an isobaric arteriograph. Resistance‐sized uterine arteries from nonsense VEGFR2 LTP nanoparticle treated RUPP rats displayed increased myogenic response to intraluminal pressure increases. Animals treated with nonsense plasmid for VEGFR2 were also hypertensive, similar to untreated RUPP (100.4±5.8 Hg). Finally, RUPP rats injected with non‐sense VEGR2 LTP nanoparticles displayed growth restriction comparable to untreated RUPP rats. . In conclusion, uterine injection of LTP nanoparticles with nonsense plasmid for VEGFR2 did not significantly alter resistance‐sized uterine artery myogenic responsiveness, maternal blood pressure or fetal weights in RUPP animals, thus pathology of RUPP remained intact. SHAM animals showed no changes to uterine artery myogenic reactivity, MAP and fetal outcomes. These data suggest LTP nanoparticles themselves did not detrimentally alter pregnancy outcomes. Additionally these results increase support for the efficacy of LTP nanoparticles as a novel gene therapy device to treat preeclampsia.
Effect of Maternal Antioxidant MitoQ Treatment on Offspring Cardiovascular Function in a Rat Model of Intrauterine Growth Restriction (IUGR)
Authors: Aljunaidy Mais M , Morton Jude S , Kirschenman Raven , Case Patrick , Cooke Christy‐Lynn , Davidge Sandra T
Published: November 2023

Introduction

A suboptimal environment in fetal life is linked to cardiovascular disease in adult life. Maternal hypoxia can lead to placental oxidative stress, which is associated with abnormal cardiovascular function in the offspring. We hypothesize that maternal treatment with MitoQ, a mitochondrial antioxidant, may prevent the development of cardiovascular disease in adult offspring.

Methods

Pregnant rats were injected with either MitoQ loaded nanoparticles (nMitoQ; 125 μM‐nanoparticles prevent MitoQ from crossing to the fetus) or saline via tail vein on gestational day (GD) 15. Rats then were subdivided into two groups exposed to either hypoxia (11% O2) or normoxia (21% O2) from GD 15–21 (term; 22 days). At 7 months of age, blood pressure (tail‐cuff plethysmography), in vivo cardiac function (echocardiography) and ex vivo cardiac susceptibility to ischemia‐reperfusion (I/R) injury were assessed in both male and female offspring. A 2‐way ANOVA was used for analyses.

Results

Hypoxia led to diastolic dysfunction in male offspring (P<0.05) and tended to cause diastolic dysfunction in female offspring (P=0.07). nMitoQ treatment improved diastolic function in only female offspring in both normoxic and hypoxic groups [mitral valve E wave velocity/A wave velocity (MV E/A): normoxia + saline: 1.52±0.10 vs. normoxia + nMitoQ: 1.23±0.09 and hypoxia + saline: 2.18±0.40 vs. hypoxia + nMitoQ: 1.29±0.08, P<0.01]. In male offspring only, hypoxia reduced the pulmonary valve peak velocity (PV Peak Vel) and nMitoQ treatment ameliorated this effect (PV Peak Vel: hypoxia + saline 620±28 mmHg vs. hypoxia + nMitoQ 781±53 mmHg, P<0.05). Neither hypoxia nor nMitoQ altered blood pressure and cardiac susceptibility to I/R injury.

Conclusion

nMitoQ treatment in mid gestation restored PV Peak Vel in hypoxic male offspring. In female offspring, maternal treatment with nMitoQ improved diastolic function in both normoxic and hypoxic animals. This study suggests that maternal/placental treatment can alter cardiovascular function of offspring later in life.

Support or Funding Information

CIHR, WCHRI, SCHF and RAHF
The Androgen Receptor Contributes to Hypertension in Ovariectomized IUGR but Not in Ovariectomized Control Rats
Authors: Davis Gwendolyn K. , Newsome Ashley D. , Alexander Barbara T.
Published: November 2023

The prevalence of hypertension (HTN) is two‐fold higher in low birth weight (LBW) women at age 60 relative to normal birth weight (NBW) counterparts. Yet, the mechanisms that contribute to greater cardiovascular risk (CVR) in LBW women are unknown. Previous studies from our laboratory demonstrate that intrauterine growth restriction (IUGR) programs persistent estrus and a significant increase in circulating testosterone (T) and blood pressure (BP) in female Sprague Dawley (SD) IUGR rats at 12 months of age. Androgen receptor (AR) blockade abolishes HTN in female IUGR at 12 months of age indicating a role for the AR in the etiology of age‐dependent HTN in female IUGR. We previously reported that ovariectomy (OVX) at 10 weeks of age has no effect on BP or T levels in NBW (control) SD rats at 4 months of age. However, OVX at 10 months of age induces a significant increase in BP and T in SD controls compared to intact SD controls by 12 months of age. However, the importance of T in OVX‐induced HTN, a model of menopausal HTN, is not known. We tested the hypothesis that OVX‐induced HTN in female SD rats in later life is T dependent. Rats underwent sham (intact) or OVX at 10 months of age followed by vehicle or flutamide, an AR antagonist (8mg/kg/day, SC at 12 months of age for two weeks). Total fat and lean mass were measured via Echo MRI pre‐OVX and after treatment (Paired T‐test). BP was measured in conscious, chronically catheterized rats following treatment (2way ANOVA; Tukey's multiple comparison). BP was increased in vehicle OVX control relative to vehicle intact control (137±3 vs.129±3 mmHg, respectively). However, BP did not differ upon comparison of flutamide OVX control versus vehicle OVX control (140±7 mmHg) suggesting that OVX‐induced increase in BP in control rats does not involve a role for the AR. As observed previously, BP was not further elevated by OVX in IUGR. Furthermore, BP was decreased by 19 mmHg in flutamide intact IUGR relative to vehicle intact IUGR. Yet, BP was decreased by only 10 mmHg in flutamide OVX IUGR versus vehicle OVX IUGR suggesting that the importance of the AR in BP control may differ in ovarian intact IUGR versus ovarian deficient IUGR. OVX induced an increase in total fat mass, but attenuation of the OVX‐induced increase in total fat mass by flutamide was specific to IUGR (Table). Therefore, this study indicates that the AR contributes to enhanced CVR in female IUGR regardless of ovarian status, but not in OVX control SD rats.

Support or Funding Information

NIH: T32HL105324‐07, HL074927, HL51971, P20GM104357; AHA: GRNT19900004.

Total Fat Mass (grams):
Pre‐OVX
Post‐Treatment

Control Vehicle
26±2
62±4*

Control Flutamide
34±7
60±10*

IUGR Vehicle
27±10
53±12*

IUGR Flutamide
22±3
35±8

P<0.05 vs Pre‐OVX.
Chronic Central Leptin Infusion Improves Cardiac Function in Diabetic Rats With Heart Failure
Authors: da Silva Alexandre A. , Gava Fábio N. , Lataro Renata M. , Silva Carlos Alberto A. , Rodrigues Driéli P. , Guida Tamires , do Carmo Jussara M. , Salgado Hélio C.
Published: November 2023

Myocardial infarction (MI) is a major cause of mortality and morbity and the leading cause of heart failure (HF) worldwide. We previously demonstrated that leptin, a peptide hormone produced by adipocytes, exerts powerful antidiabetic effects via activation of its receptors in the central nervous system (CNS). We also showed that chronic central leptin infusion restores intrinsic heart rate, cardiac sympathetic‐vagal balance and baroreflex sensitivity in uncontrolled diabetes. Therefore, in this study we examined if chronic intracerebroventricular infusion of leptin improves cardiac function in rats with HF induced by MI. Wistar Munich rats (275–300 g, n=4–5/group) were submitted to MI by ligation of the left descending coronary artery. Diabetes was induced one week later by a single injection of streptozotocin (STZ, 35 mg/kg, i.p.). Seven days later, rats were instrumented with an intracerebroventricular (ICV) cannula into the lateral ventricle for continuous infusion of leptin (0.62 μg/h, 0.5 μL/h, n=5) or vehicle (0.5 μL/h, n=4), for 4 weeks using osmotic pumps (Alzet, model 2002). Cardiac function was evaluated 6 days after MI surgery and on days 14 and 28 of ICV leptin or vehicle infusion using the echocardiograph VEVO 2100 (VisualSonics, Canada). STZ caused severe hyperglycemia in both groups (ranging from 472±17 to 477±14 mg/dl) which was completely normalized by chronic ICV leptin infusion (120±8 mg/dl). Compared to vehicle group, leptin infusion for 4 weeks also markedly ameliorated cardiac function as evidenced by lower left atrium/aorta ratio (1.73±0.07 vs. 2.63±0.23 mm) and higher cardiac output (113.8±27.6 vs. 69.5±10.0 ml/min) and ejection fraction (36.7±2.3 vs. 30.7±8.7 %). These results suggest that in addition to its powerful CNS‐mediated antidiabetic effects, leptin attenuates the progression of HF after MI in diabetic rats by improving cardiac function.

Support or Funding Information

NHLBI‐PO1HL51971, NIGMS‐P20GM104357, FAPESP, CNPq
A role for Sweet Taste Receptors (T1R2/T1R3) in fructose‐induced increase in NKCC2 Phosphorylation in thick ascending limbs (TALs)
Authors: Kassem Kamal M , Ares Gustavo , Ortiz Pablo
Published: November 2023

High fructose consumption has been implicated in hypertension, renal damage and metabolic syndrome in humans. We previously showed that a fructose‐enriched diet does not increase blood pressure in rats unless it is combined with high salt intake. This effect was accompanied by a large increase in phosphorylation of the renal Na/K/2Cl cotransporter NKCC2 at threonine 96/101. The mechanisms by which fructose induces salt‐sensitive hypertension are not clear. In a proteomics screen, we found that the sweet taste receptor T1R2 was present in TALs. Others reported mRNA expression of T1R2 and T1R3 in whole kidney homogenates. Both T1R2 and T1R3 are required to initiate signaling by sweet agonists such as fructose. Unlike glucose, fructose is not fully reabsorbed in the proximal tubule, thus it is possible that activation of sweet taste receptors induce signaling leading to NKCC2 phosphorylation. We hypothesize that a fructose‐enriched diet (20% fructose in drinking water) stimulates NKCC2 phosphorylation in part by acting on sweet taste receptors (T1R2/T1R3). To test this, we used C57 wild‐type mice (WT) and mice on the C57 background with dual deletion of T1R2 and T1R3 (T1R2/T1R3 KO). Mice were given tap water or 20% fructose in drinking water for 7–8 days. TAL suspensions from WT and T1R2/T1R3 KO mice were obtained to measure total NKCC2 expression, GAPDH (loading control) and NKCC2 phosphorylation at Thre96/101 by Western blot. In WT mice, fructose in drinking water increased phosphorylated NKCC2 by 248 ± 82% (n=5, p<0.01), whereas it decreased total NKCC2 expression by 38 ± 18% (p<0.05). In contrast, in TALs from T1R2/T1R3 KO mice, fructose in drinking water did not affect phosphorylated NKCC2 (PNKCC2/total NKCC2: − 16 ± 28%, p=0.88). We conclude that 1‐week of a fructose‐enriched diet enhances NKCC2 phosphorylation in the mouse TAL, and this effect is in part due to sweet taste receptors T1R2/T1R3. Hence, our data shows for the first time that sweet taste receptors T1R2 and T1R3 play a role in regulating renal transporters. These receptors could play a role in the deleterious effects of fructose intake in the kidney.

Support or Funding Information

NIH‐NIDDKAHA
Central Angiotensin II Modulation of Hypoxia‐Inducible Factor (HIF)‐1 Alpha in the PVN: Possible Mechanism for Sympathetic Activation
Authors: Haibara Andrea , Sharma Neeru , Zheng Hong , Liu Xuefei , Patel Kaushik P
Published: November 2023

Upregulation of Angiotensin II (Ang II) in the paraventricular nucleus (PVN) of the hypothalamus has been associated with the increased sympathetic outflow observed in chronic heart failure (CHF). Recently, we have shown the contribution of HIF‐1α in augmentation of the NMDA‐NR1 mediated sympathoexcitatory responses from the PVN in CHF. HIF‐1α silencing in the PVN normalizes the increased renal sympathetic nerve activity observed in rats with CHF. However, the mechanism of increased expression of HIF‐1α remains unclear. In the present study, we evaluated whether the chronic increase in Ang II levels in the brain could cause augmentation of the HIF‐1α expression in the PVN which in turn may contribute to the sympathoexcitation. Sprague‐Dawley rats (250–300 g) were subjected to 14 days of ICV infusion of Ang II (20 ng/min) or 0.9% sterile saline (0.5μl/h) through osmotic mini‐pumps. Ang II infusion increased baseline mean arterial pressure (84 ± 4 vs. 126 ± 9 mmHg), increased baseline renal sympathetic nerve activity (6.4 ± 1.9 vs. 20.5 ± 2.3 % of Max), increased cardiac sympathetic tone (75 ± 6 vs. 96 ± 7 bpm) and decreased cardiac parasympathetic tone (36 ± 3 vs. 16 ± 2 bpm) compared to saline. The Ang II‐infused group also showed an impairment of baroreflex control of heart rate (1.5 ± 0.3 vs. 0.5 ± 0.1 ms/mmHg), a potentiation of the pressor chemoreflex response (30 ± 7 vs. 53 ± 7 mmHg) and increased sympathoexcitation induced by NMDA (200 pmol) microinjected into the PVN (23 ± 4 vs. 73 ± 16 %). Concomitantly, Ang II infusion increased the expression of HIF‐1α mRNA (2.79 fold) and protein l(1.54 fold) and also the expression of NMDA‐type1 (NR1) receptors mRNA (3.17 fold) and protein (1.52 fold) in the PVN. In contrast, the Ang II treatment failed to induce any significant changes in mRNA expression of HIF‐1α and NR1 receptors in the cerebral cortex. Taken together, our studies suggest that increased central levels of Ang II contribute to the regulation of HIF‐1α expression in the PVN, which may be involved in the potentiation of glutamatergic activation of pre‐autonomic neurons in the PVN, suggesting a relationship between Ang II, HIF‐1α, and sympathoexcitation.

Support or Funding Information

Supported by CNPq‐Brazil 234520/2014‐0 and NIH grants HL124104 & HL62222.
Sodium Chloride Cotransporter Upregulation in Settings of Zinc Deficiency Offers New Insight into Blood Pressure Dysregulation in Chronic Diseases
Authors: Williams Clintoria R. , Mistry Monisha , Mallick Rickta , Mistry Abinash , Ko Ben , Gooch Jennifer L. , Hoover Robert S.
Published: November 2023

Background

Zn2+ deficiency is a common comorbidity with numerous chronic diseases including type II diabetes and chronic kidney disease. Experimental data show that Zn2+ deficiency exacerbates hypertension in these settings. Moreover, Zn2+ deficiency alone may be sufficient to alter blood pressure; however, the mechanisms involved are unknown.

Objective

The renal distal convoluted tubule sodium chloride cotransporter (NCC) plays a critical role in blood pressure regulation and hypertension. The objective of this study was to determine if Zn2+ deficiency alters NCC expression and/or activation in vivo and in vitro.

Experimental Design

To this end, WT mice were pair‐fed a diet with 50 ppm Zn2+ (Zn2+ adequate [ZnA]) or a diet with 1 ppm Zn2+ (Zn2+ deficient [ZnD]) for 6 weeks. To reduce intracellular Zn2+ levels in vitro, WT mouse distal convoluted tubular (mDCT‐15) cells were cultured in 1 nM N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN)‐ or vehicle (DMSO)‐containing medium for 24 hours. To replete intracellular Zn2+, TPEN‐exposed cells were then cultured in 1 nM Zn2+‐supplemented medium for 24 hours. NCC mRNA and protein expression were examined in vivo and in vitro via qRT‐PCR, western blot and immunohistochemistry. NCC activation was assessed by cellular localization (via biotinylation and immunofluorescence) and Na+ uptake activity (via thiazide‐sensitive assays).

Results

In ZnD mice, NCC protein expression is increased in DCT cells compared to mice receiving a ZnA‐diet. In the in vitro model of Zn2+ deficiency, Zn2+ chelation with TPEN stimulates NCC mRNA and protein expression compared to vehicle‐treated cells. Furthermore, TPEN promotes NCC surface localization and Na+ uptake activity. Notably, restoration of intracellular Zn2+ levels by supplementation reverses TPEN effects on NCC.

Conclusions

Our findings indicate that NCC is a Zn2+‐sensitive transporter and is upregulated in settings of Zn2+ deficiency.

Significance

This sodium transporter represents a novel mechanism by which Zn2+ deficiency may alter blood pressure and contribute to hypertension in chronic disease settings. Future experiments are needed to determine what additional factors are involved in Zn2+ deficiency‐induced upregulation of NCC and to test if protecting NCC from Zn2+ regulation has a positive impact on hypertension.
Novel Cardiorenal Effects of the Lactate Receptor GPR81 Activation
Authors: Jones Natalie K , Wallenius Kristina , Menzies Robert I , Stewart Kevin , Mullins John J , Bailey Matthew A
Published: November 2023

G protein‐coupled receptor 81 (GPR81) is highly expressed in adipocytes and activation of the receptor by the endogenous ligand lactate inhibits lipolysis. The receptor is also expressed in non‐adipose tissue but the function of GPR81 at these sites is not known. In the present study we first used end point PCR to screen for GPR81 expression in the kidney and vasculature isolated from male Sprague‐Dawley rats (n=4). GPR81 was expressed in the whole kidney, aorta, mesenteric, renal and interlobar arteries. We next used a specific GPR81 agonist, AZ13415538, to assess the function of the receptor on renal haemodynamics in vivo. Male Sprague Dawley rats (n=14) were anesthetised with Inactin (120mg/kg) and a Doppler ultrasound probe placed round the left renal artery. Mean arterial blood pressure was measured via a cannula inserted into the femoral artery. Data are expressed as mean±sem. The agonist was injected into the jugular vein as a bolus in a 5 point cumulative dose response curve from 0.5μmol to 5μmol. Injection of AZ13415538 caused a rapid, dose‐dependent rise in blood pressure and a reduction in renal blood flow. At the highest dose of 5μmol, the increase in mean arterial blood pressure over the pre‐injection baseline was 20.4±1.6 mmHg; the nadir of renal blood flow was 3.0±0.5 mL/min. Both effects were transient and recovered after 75 seconds. The 5% mannitol vehicle (0.5ml) also caused an increase in blood pressure (11.4±0.8 mmHg) and a reduction of renal blood flow (1.8±0.2 mL/min) but these effects were not sustained. The effect of AZ13415528 was therefore expressed as area over and under the curve for blood pressure and renal blood flow, respectively. A significant dose‐dependent effect for both blood pressure and blood flow was identified by One‐Way Anova (P<0.0001). Post hoc comparisons against the effect of vehicle showed significant increases in blood pressure at 2–4μmol (P<0.05) and 5μmol (P<0.01). Renal blood flow was significantly reduced at 4μmol AZ13415528 (P<0.05) and 5μmol (P<0.01). The increase in renal vascular resistance observed after GPR81 activation shows novel cardiovascular effects of this receptor. The mechanisms underlying these actions have not been investigated.

Support or Funding Information

Supported by The British Heart Foundation (fellowship number ‐ FS/15/63/32033) and AstraZeneca
Sodium Butyrate Protects against Renal Injury in DOCA Salt Hypertensive Rats
Authors: Zhu Qing , Wang Lei , Zhang Linlin , Lu Aihua , Yang Tianxin
Published: November 2023

We previously found that infusion of sodium butyrate (NaBu) into the renal medullary suppressed angiotensin II‐induced hypertension and improved renal injury. The present study was to test whether systemic administration of NaBu protects the kidneys from damage in DOCA‐salt rats. Uninephrectomized male Sprague‐Dawley rats were treated with a subcutaneous DOCA pellet (200mg/rat) and 1% NaCl drinking water for 2 weeks. Sham rats were uninephrectomized and drank tap water. Animals received an oral gavage of NaBu (0.1g/kg) or vehicle once per day. Hypertension induced by DOCA‐salt was significantly attenuated in rats treated with NaBu. Urinary albumin excretion increased from 0.24 ± 0.05 mg/24h in sham rats to 12.01 ± 5.13 in DOCA‐salt rats, which was significantly reduced to 2.32 ± 0.94 in NaBu‐treated DOCA‐salt rats (p<0.05). Morphological examination showed that the glomerular damage indices were 1.04 ± 0.1, 2.87± 0.08 and 1.69 ± 0.12 in sham, DOCA salt‐treated, and NaBu‐treated DOCA‐salt rats, respectively. There was no difference in creatinine clearance rate in sham or NaBu‐treated DOCA‐salt rats compared with only DOCA‐salt rats. These results suggest that sodium butyrate may protect against renal injury in DOCA‐salt hypertension and that NaBu could serve as a novel therapeutic approach for the treatment of chronic renal injury.

Support or Funding Information

National Natural Science Foundation of China Grant No. 91439205, No. 31330037 and No. 81600322; Fundamental Research Funds of Sun Yat‐sen University 16ykpy47; Veterans Affairs Merit Review, and National Institutes of Health Grant DK094956.
Inactivation of Hv1 proton channels is not responsible for bicarbonate‐mediated reductions in tubular cast formation in Dahl salt‐sensitive rats
Authors: Ray Sarah , Ocasio Hiram , Sun Jingping , O'Connor Paul M
Published: November 2023

We have shown that oral bicarbonate promotes a marked reduction in tubular cast formation and fibrosis in the Dahl salt‐sensitive (SS) rat model when fed a high salt diet, independent of arterial blood pressure, glomerular sclerosis, and proteinuria. Rats lacking the voltage‐gated proton channel (Hv1), which is highly expressed in mTAL, denoted a similar phenotype. In the current study, we examined whether bicarbonate intake protected from cast formation in the Dahl SS rat model by inactivation of the voltage‐gated Hv1 proton channel. Wild‐type Dahl SS (WT) and Hv1 knock‐out (KO) Dahl SS rats were treated with either vehicle (0.1M NaCl; n=9) or bicarbonate (0.1M NaHCO3; n=11) in drinking water for the length of the study. Following a 4 day baseline period in which both groups were fed a 0.4% NaCl diet (Dyets AIN76A), rats were placed on an 8% NaCl diet for 14 days. Urine was collected at day 4 of baseline, day 7, and day 14 high salt; telemetry was used to measure blood pressure. On day 14, rats were anesthetized, and the right kidney taken for histology. Mean arterial pressure (mmHg) showed no difference between the WT and KO groups regardless of vehicle or bicarbonate treatment. Tubular casts were significantly decreased (pSTRAIN=0.008) in KO rats when compared to WT. Importantly, bicarbonate treatment had an additive effect, further decreasing tubular cast formation and by a similar magnitude in both KO and WT rats (PTREATMENT=0.02; PTREATMENT*STRAIN=N.S). In conclusion, bicarbonate‐induced inactivation of Hv1 proton channels does not mediate the reduction of cast formation in Dahl SS rats. Further investigation will be required to determine whether a common downstream mechanism is responsible for limiting tubular cast formation in the absence of blood pressure changes or glomerular injury in bicarbonate treated and Hv1−/− Dahl SS rats.

Support or Funding Information

The authors would like to acknowledge funding from the National Institute of Health: NIDDK R01099548
Large conductance potassium (KCa1.1) channel mediated potassium secretion provides the driving force for water secretion in organoids from rat distal colon
Authors: Rottgen Trey , Nickerson Drew , Minor Emily , Rajendran Vazhaikkurichi M
Published: November 2023

Organoid culture has been adapted as a method to recapitulate colonic physiology, especially epithelial electrolyte transport in an in vitro model. We tested the hypothesis that K+ secretion can provide a novel pathway for water secretion. Colonic crypts, containing Lgr5+ cells, were isolated from male Spraque‐Dawley rats (n=4) and plated in an extracellular support (Matrigel) supplemented with crypt culture media containing specific niche factors necessary for proliferation and organoid formation. Utilizing an activator (BMS‐204352, 10μM) of large‐conductance of potassium (KCa1.1) channels, we were able to induce swelling of organoids and increase the luminal to total volume ratio that was significantly different from controls (63.3±4.7 vs. 33.5±5.2, x<0.05). Paxilline (KCa1.1 blocker, 1μM) completely inhibited the BMS‐204352 induced swelling and was not significantly different from unexposed organoids (34.4±3.8 vs. 33.5±5.2, ns). CFTR‐172 inhibitor did not prevent BMS‐204352‐induced swelling, which was not significantly different from BMS‐204352 alone (55.8±5.6 vs. 63.3 ns). We conclude from these data that large conductance of potassium (KCa1.1) channels mediate potassium secretion that contribute to a novel mechanism of water secretion that could be clinically impactful in certain pathophysiological states.

Support or Funding Information

NIH/NIDDK DK‐018777
Regulated Expression of the Na+/K+‐ATPase Pump in Colonic Epithelium by Bile Acids
Authors: Borg Johan Fredrik , Yde Jonathan , Wu Qi , Lajczak Natalia , Keely Stephen , Fenton Robert A , Moeller Hanne
Published: November 2023

Bile acid malabsorption (BAM) is a common cause of chronic diarrhea. The mechanism underlying the development of bile acid induced diarrhea is unclear. We aimed to determine if expression levels of mRNA and protein, as well as localization of Na+/K+‐ATPase (NKA) are influenced by bile acids (BA) in colonic epithelial cells during BAM.

Rats were fed a 1% Na‐cholate containing diet for three days. Colonic epithelium was isolated using Ca2+‐chelation and prepared for protein analysis and RT‐qPCR. Colonic tissue was prepared for immunohistochemistry (IHC). T84 cells were treated with deoxycholic acid (DCA) to examine the direct effect of this bile acid on epithelial cells.

The treated rats developed diarrhea with an increased feces water content compared to controls. In a previous experiment with similar results we used liquid chromatography tandem mass spectrophotometry (LC‐MS/MS) on colonic epithelial tissue from the animal model of BAM to create a protein profile. We found that that the Na+/K+‐ATPase subunit β1 (NKA β1) was significantly down‐regulated in the colon during BAM. In our BAM model, western blotting confirmed the down‐regulation of the NKA β1 in colon. Na+/K+‐ATPase subunit α1 (NKA α1) abundance was also decreased in proximal, but not distal colon. RT‐qPCR analysis showed no regulation of the genes Atp1b1 or Atp1a1 at the mRNA level. IHC showed no morphological differences in cross‐sections from Na‐cholate treated rats, nor any change in NKA β1 or α1 localization.

T84 cells treated with 150 μM DCA for 48 hrs showed a similar down‐regulation of NKA β1 at protein level. mRNA levels were also down‐regulated for both the ATP1B1 and ATP1A1 gene. Transepithelial electrical resistance decreased on average by 65% after 48 hrs of DCA treatment.

In conclusion, we have found the NKA β1 to be down‐regulated at the protein level in the colonic epithelium of rats after 3 days of Na‐cholate treatment. This down‐regulation was also found in T84 cells treated with DCA for 48 hrs. Additionally, mRNA levels of ATP1B1 and ATP1A1 in T84 cells were down‐regulated by DCA. Due to the central role for NKA in the absorption and secretion of water and electrolytes in colon, this regulation could be central in development of diarrhea during BAM.
Dietary K+ and Cl− independently regulate basolateral conductance in the principal and intercalated cells of the collecting duct
Authors: Zaika Oleg , Tomilin Viktor , Mamenko Mykola , Pochynyuk Oleh
Published: November 2023

Kidneys play a central role in maintaining systemic homeostasis by matching dietary variations of electrolyte intake with their excretion in urine. Final adjustments take place in the collecting duct where transport rates are highly sensitive to electrolyte intake. The collecting duct comprises of electrically uncoupled principal and intercalated cells mediating Na+/K+ exchange and Cl− reabsorption, respectively. Basolateral membrane conductance sets up an electrochemical driving force governing electrolyte transport. Previous work established that potassium Kir4.1/5.1 channels underlie basolateral conductance in principal cells, whereas ClC‐K2 channels are essential for basolateral Cl− flux in intercalated cells. However, little is known whether variations in dietary K+ and Cl− affect activity of these channels. Using patch clamp electrophysiology at the single channel and whole cell levels in freshly isolated mouse collecting ducts, we found a marked upregulation of the basolateral K+‐selective current and hyperpolarization of basolateral membrane potential in principal cells of mice subjected to a high (5%) K+ diet for one week compared to the control. This stimulation stemmed from the significantly increased functional levels of Kir4.1/5.1 but not from channel open probability. When high K+ was accompanied with Cl−, this led to drastically decreased basolateral Cl− ‐selective current, the number of active ClC‐K2 and reduced channel open probability in intercalated cells. In contrast, when high K+ was given as citrate, ClC‐K2 macroscopic current and channel activity were significantly increased. Consistently, dietary Cl− restriction with regular K+ augmented ClC‐K2‐dependent conductance but had little effect on Kir4.1/5.1 activity. Overall, our results suggest independent regulation the basolateral Kir4.1/5.1 and ClC‐K2 in principal and intercalated cells by dietary K+ and Cl−. We propose that this mechanism contributes to precise tuning of urinary excretion in response to variable dietary electrolyte intake.

Support or Funding Information

This research was supported by NIH‐NIDDK DK095029 (to O. Pochynyuk), AHA‐15SDG25550150 (to M. Mamenko)
A novel Na+ transport system in the A‐type intercalated cells of the CCD that mediates Na+ secretion through a H/K‐ATPase type 2‐dependent pathway
Authors: Edwards Aurelie , Morla Luciana , Walter Christine , Doucet Alain , Crambert Gilles
Published: November 2023

In the last few years, our understanding of the manner in which the cortical collecting duct (CCD) transports Na+ has significantly evolved. Indeed, in addition to the classical ENaC‐mediated Na+ reabsorption by principal cells, it has been shown that an electroneutral coupled transport system expressed in B‐type intercalated cells involving pendrin and a Na+/Cl−/HCO3− exchanger (NDBCE) also contributes to renal Na+ reabsorption (1). In the present study, we used in vitro microperfusion experiments in the presence of the transepithelial ion concentration gradients that prevail in vivo (asymmetric conditions), combined with the predictions of a mathematical model, to fully characterize the relevant Na+ transport systems in the CCD of Na+‐depleted mice.

Under these asymmetric conditions, we first assessed the contribution of the paracellular route to Na+ transport by inhibiting all known and possible transcellular pathways with amiloride, hydrochlorothiazide (HCTZ), Schering 28080, and bumetanide. Under these conditions, the CCD excreted Na+ (JNa = − 16.8 ± 0.8 pmol/min/mm; n = 5) whereas the Cl− flux was negligible (JCl = − 3.4 ± 2.4 pmol/min/mm), and the transepithelial voltage (DVte) was – 12.8 ± 1.8 mV. To estimate the contribution of each cell type, we then removed one or more inhibitors. Removal of amiloride abolished JNa showing that Na+ reabsorption by principal cells counterbalances Na+ secretion via the paracellular route. Conversely, removal of all inhibitors except amiloride increased Na+ secretion (JNa = −32 ± pmol/min/mm,) in wild‐type mice but not in H/K‐ATPase type 2 (HKA2) knock‐out mice or in the presence of bumetanide.

Altogether our results indicate that type A intercalated cells mediate the secretion of Na+ via basolateral Na+‐K+‐2Cl− (NKCC1) cotransporters in tandem with HKA2 pumps functioning as apical Na+ pumps. We have therefore identified a third CCD transcellular sodium transport pathway, in A‐type intercalated cells, which mediates Na+ secretion in the CCD.

Support or Funding Information

Supported by a Nestlé Waters Grant
Intestine‐specific dual oxidase deficiency lacks ATP‐P2Y‐mediated luminal H2O2 production in mice
Authors: Maruta Koji , Akiba Yasutada , Kaji Izumi , Kaunitz Jonathan D
Published: November 2023

The intestinal epithelium affects luminal microbial composition via anion and mucus secretion, and the secretion of anti‐bacterial peptides or chemorepellents such as H2O2. Dual oxidase‐2 (Duox‐2) an enzyme predominantly expressed in the intestinal epithelium among NADPH oxidase family generates luminal H2O2 via intracellular Ca2+ increase. We found that luminal ATP‐P2Y signals increased luminal H2O2 output in rat duodenum, which we hypothesized was due to the activity of Duox‐2. Lacking specific Duox inhibitors, we created mice selectively deficient for DuoxA1–A2 (accessory proteins essential for membrane trafficking of Duox‐1 and ‐ 2, respectively) by breeding villin‐Cre mice with mice bearing floxed DuoxA1–A2, needed since global Duox knockout (KO) causes hypothyroidism. We examined the effect of luminal ATP on H2O2 production in wild type (WT) and Duox KO mice.

We measured H2O2 output using the fluorogenic substrate Amplex® and HCO3− secretion in an ATP‐perfused duodenal loop in vivo using flow‐through pH and CO2 electrodes. We also measured luminal H2O2 production in response to luminal ATP or ionomycin in Ussing chambered muscle‐stripped duodenum and cecum.

Luminal perfusion of ATP (0.1 or 1 mM) augmented HCO3− secretion with increased luminal H2O2 output in WT duodenum, whereas ATP‐induced HCO3− secretion was reduced and ATP‐induced H2O2 output was abolished in KO duodenum. In Ussing chambered WT duodenum and cecum, basal H2O2 concentrations were higher in the cecum than in the duodenum. Luminal H2O2 production was similarly increased in the duodenum and cecum in response to luminal ATP (1 mM) and ionomycin (1 μM) in WT, whereas no additional H2O2 production was observed in KO intestine. Furthermore, luminal pretreatment with the non‐specific P2Y receptor antagonist suramin (1 mM) abolished ATP‐induced H2O2 production in WT.

These results confirm that epithelial Duox‐2 generates luminal H2O2 via luminal ATP‐P2Y signals. We propose that H2O2 produced by Duox‐2 is a key factor governing the composition of the gut microbial flora.

Support or Funding Information

Supported by VA Merit Review, NIH R01 DK54221
The Epithelial Sodium Channel (ENaC) α‐ and γ‐subunits are cleaved by furin in human kidney
Authors: Langkilde Rikke Zachar , Mikkelsen Maiken Krogsbæk , Skjødt Karsten , Zamani Reza , Marcussen Niels , Jensen Boye , Svenningsen Per
Published: November 2023

The aldosterone‐sensitive Epithelial Sodium Channel (ENaC) plays an essential role in sodium homeostasis and blood pressure regulation. In vitro data suggest that during biosynthesis, the proprotein convertase furin cleaves the alpha and gamma subunit. Full activation takes place at the apical membrane by proteolytical excision of an inhibitory domain from the gamma subunit. The GPI‐anchored serine protease prostasin is a possible candidate to activate ENaC under physiological conditions, while aberrant filtrated plasmin cleaves under pathophysiological conditions. The aim of the study was to investigate proteolytical cleavage of ENaC in human kidney tissue and it was hypothesized that intracellular furin cleavage can be detected in the alpha and gamma subunit in human kidney; that apical plasmin/prostasin‐cleavage is associated with proteinuria and that cleaved products are detectable predominantly in human urinary exosomes.

Monoclonal antibodies directed against αENaC (AA 436–44, constant region at the c‐terminal side of putative furin cleavage) and against epitopes in the cleaved γENaC (AA 138–147 (furin‐cleavage site, C‐terminal) and AA 173–190 (overlapping prostasin‐cleavage site)) were developed in balb/c mice and tested on human kidney tissue from nephrectomized patients and on urinary exosomes from healthy persons. A furin cleavage site‐specific γENaC antibody revealed protein at 75 kDa compatible with furin‐cleavage in a human kidney cortex pool. An antibody overlapping and therefore requiring intact prostasin/plasmin cleavage site showed bands compatible with full‐length (100 kDa) and furin‐cleaved (75 kDa) γENaC. No difference was detected in patients with proteinuria. The furin‐cleaved part was mainly detected within the membrane. The αENaC antibody showed a dominant 50kDa product compatible with predominance of furin‐cleaved α‐subunit in the kidney tissue. All three antibodies revealed labeling associated with collecting ducts in kidney tissue sections when immunostained. In urinary exosomes furin‐cleaved, but not intact γENaC were detected. αENaC was detected in both its intact (≈80kDa) and furin‐cleaved form in exosomes.

The results indicate that proteolytic processing by furin of both alpha and gamma ENaC occurs in human kidney. The presence of prostasin‐cleavage was not clearly present. Cleavage products of both γENaC and αENaC and intact αENaC were detectable in urinary exosomes.

Support or Funding Information

Odense University Hospital PhD Fund, University of Southern Denmark ‐ Faculty of Health, The Danish Diabetes Academy – supported by the Novo Nordisk Foundation, Leo Pharma, A.P. Møller and Hustru Chastine Mc‐Kinney Møllers Foundation.