bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute effects of dietary nitrate supplementation on peripheral and central blood pressure in post‐menopausal women
Authors: Kim Danielle Jin‐Kwang , Barrett Megan A , Moore David J , Flanagan Michael P , Roe Carly , Kim‐Shapiro Daniel B , Proctor David N
Published: November 2023

Dietary nitrate supplementation (in the form of beetroot juice) has been shown to have multiple cardiovascular health benefits, including both acute and chronic reductions in resting arterial blood pressure. The purpose of this study was to examine the effects of acute dietary nitrate supplementation on peripheral and central blood pressures in postmenopausal women, a population that exhibits accelerated vascular aging and cardiovascular disease progression. We hypothesized that 1) postmenopausal women would have higher resting central blood pressure compared to premenopausal women, and 2) acute dietary nitrate supplementation would reduce resting peripheral and central blood pressure in postmenopausal women. Eighteen women participated in this study (9 premenopausal women, 20–30 yrs; 9 postmenopausal women, 55–70 yrs). Supine peripheral blood pressure (Omron) and radial pulse wave derived central aortic pressure (SphygmoCor CvM, AtCor Medical) were measured after 10 minutes of quiet supine rest. A subset of the postmenopausal women also participated in a randomized double‐blind crossover study during which they consumed beetroot juice (BRJ; 140 mL Beet‐It Sport, James White Juice Company) or placebo (PLC; 140 mL nitrate‐depleted Beet‐It Sport, James White Juice Company) approximately 1 week apart. Supine peripheral and central blood pressures were measured prior to and 90 minutes following juice consumption.

Results

Postmenopausal women had significantly higher brachial and central blood pressures (systolic, diastolic, and mean), and augmentation index (AI) compared to premenopausal women (all p < 0.05). Acute nitrate supplementation resulted in greater reductions (p<0.05) in brachial mean arterial pressure, central mean arterial pressure, and central systolic blood pressure compared to those observed during the placebo visit. These findings suggest that acute dietary nitrate supplementation favorably modifies resting peripheral and central blood pressures in postmenopausal women.

Support or Funding Information

Penn State Ambulatory Research Fund (JAFFE Endowment)
Knockout of TSP‐1 Prevents Hyperglycemia‐induced Atherosclerosis in STZ‐treated ApoE−/− Mice
Authors: Ganguly Rituparna , Sahu Soumyadip , Raman Priya
Published: November 2023

Atherosclerosis, a macrovascular disease is responsible for ~70–80% long term deaths in diabetic patients. Several clinical studies and trials as well as recent animal studies have identified hyperglycemia, hallmark of Type 1 and Type 2 diabetes, as a critical risk factor for atherosclerotic complications associated with diabetes. Thrombospondin‐1 (TSP‐1) is a proatherogenic and anti‐angiogenic ‘matricellular’ protein genetically associated with development of atherosclerosis, coronary artery disease and myocardial infarction. TSP‐1 level is significantly increased in diabetic patients, diabetic animal models and glucose‐stimulated vascular cells (endothelial cells, vascular smooth muscle cells, fibroblasts) in vitro. Previous reports, including earlier evidence from our laboratory, suggest TSP‐1 as a potential link between hyperglycemia and accelerated atherogenesis in diabetics. We showed earlier that in STZ‐induced hyperglycemic ApoE−/− mice, the micronutrient chromium picolinate in vivo attenuates development of atherosclerotic lesions concomitant to downregulation of TSP1 expression in the aortic vessels. The goal of the current study was to investigate a direct role of TSP‐1 in hyperglycemia‐induced atherosclerosis. We generated TSP1−/−/ApoE−/− double knockout mice by crossing TSP1−/− mice with atherosclerotic ApoE−/− mice, both being in C57BL/6J background. Hyperglycemia (blood glucose>250mg/dl) was induced in six weeks old male ApoE−/− mice and TSP1−/−/ApoE−/− double knockout mice via intraperitoneal injection of 50 mg/kg/day of Streptozotocin (STZ) or sodium citrate buffer (vehicle) for 5 consecutive days. At endpoint, mice (18 wks. of age) were harvested for atherosclerotic lesion analyses. Specifically, Oil‐Red‐O and Hematoxylin‐eosin staining of the aortic root area showed a significant reduction in lipid laden atherosclerotic lesions (~2.4 fold) and intimal thickening (~32%) in STZ‐induced hyperglycemic TSP1−/−/ApoE−/− mice compared to STZ‐treated ApoE−/− mice. Characterization of lesion cellularity by immunostaining for PCNA (proliferating cell nuclear antigen) and alpha‐SMA (alpha smooth muscle actin) revealed reduction in PCNA staining (~33%) and vascular smooth muscle cell abundance (~24%) in hyperglycemic TSP1−/−/ApoE−/− mice compared to hyperglycemic ApoE−/− mice. Further, atherosclerotic lesions in the aortic sinus of STZ‐treated hyperglycemic TSP1−/−/ApoE−/− mice showed a significant reduction in both macrophage content and collagen accumulation (~68%) as compared to the STZ treated hyperglycemic ApoE−/− mice. Together these data clearly demonstrate that knockout of TSP‐1 in ApoE−/− mice protects against hyperglycemia‐induced atherosclerosis, suggesting TSP‐1 as a potential therapeutic target in diabetic macrovascular disease.

Support or Funding Information

Supported by Diabetes Action Research and Education Foundation (DAREF) and NEOMED start‐up funds.
β‐cell Deletion of Nr4a1 and Nr4a3 Nuclear Receptors Impedes Mitochondrial Respiration and Insulin Secretion
Authors: Reynolds Merrick S , Hancock Chad R , Ray Jason D , Kener Kyle B , Hardman Jeremy M , Tessem Jeffery S
Published: November 2023

Insulin secretion from β‐cells is dependent on the stimulation of mitochondrial respiration in response to an increase in glucose supply. Various studies have clearly demonstrated that the Nr4a family of orphan nuclear receptors are essential for fuel utilization and mitochondrial function in liver, muscle and fat. We have previously demonstrated that overexpression of Nr4a1 and Nr4a3 are sufficient to induce proliferation of pancreatic β‐cells. We examined the impact of nuclear receptors Nr4a1 and Nr4a3 on pancreatic β‐cell mitochondrial function and proliferation using lentivirus mediated shRNA knock‐down of Nr4a1 and Nr4a3 in INS‐1 derived 832/13 rat insulinoma cells. β‐cells deficient in Nr4a1 and Nr4a3 have a 37% reduction in proliferation rates (p<0.05). The reduction in β‐cell proliferation with Nr4a1 and Nr4a3 deficiency was accompanied by a significant decrease in glucose stimulated insulin secretion rates (GSIS) (26% decrease, p<0.05). GSIS was determined in cells preincubated in secretion assay buffer (SAB) for 1.5 hours containing 2.5 mM glucose followed by incubation in SAB with 16.7 mM glucose. High resolution respirometry (Oroboros O2k) was used to evaluate mitochondrial respiratory function and capacity with substrate‐uncoupler‐inhibitor titration protocols for both intact and permeabilized β‐cells. Permeabilized cells allow for an in depth assessment of chemiosmotic coupling within the β‐cells while the intact protocol evaluates a more physiologically relevant condition. We have shown that deletions of the Nr4a nuclear receptors clearly impact cellular respiration, lowering the oxygen consumption rate in all respiratory states of intact β‐cells between 40–65% and permeabilized β‐cells by 23–50% compared to GFP (p<0.05). Our results indicate that knockdown of Nr4a1 and Nr4a3 results in decreased expression Idh3a, Idh3g, Sdhb, and Mdh2, which would impede TCA cycle progression, and is consistent with the reduction observed in O2 consumption, ATP production and ultimately glucose stimulated insulin secretion. These data demonstrate for the first time that the Nr4a orphan nuclear receptor family members Nr4a1 and Nr4a3 are critical for basal β‐cell mitochondrial function and insulin secretion.

Support or Funding Information

Brigham Young University
CARBONIC ANHYDRASE MEDIATES HEPATIC STEATOSIS AND VASCULAR STIFFNESS INDUCED BY A WESTERN DIET
Authors: Malcolm Threshia , Aroor Annayya , Bivin William , Sowers James , Woodman Ryan , Lockette Warren
Published: November 2023

The typical “Western diet” (WD) is higher in fat and high fructose corn syrup when compared to a healthful diet (CD) that is lower in fat and does not contain high fructose corn syrup. A WD leads to insulin resistance and the metabolic syndrome that is characterized by fatty steatosis in the liver, a precursor of non‐alcoholic fatty liver disease, and vascular stiffness that presages atherosclerotic vascular disease and hypertension. Bicarbonate is a necessary precursor for fatty acid synthesis and the storage of depot fat in the liver and in perivascular tissue. We hypothesized that acetazolamide (ACT), a carbonic anhydrase inhibitor that blocks bicarbonate‐dependent formation of fatty acids and triglycerides, could prevent the fatty steatosis and vascular stiffness that is induced by a WD. Laboratory mice were fed a WD (46% kcals from fat; 36% kcals from carbohydrate, sucrose [17.5%] and high fructose corn syrup [17.5%]) or CD (10% kcals from fat; 72% kcals from carbohydrate; no high fructose corn syrup) for sixteen weeks with, and without, 2mM ACT in the drinking water ad libitum. Subsequently, rodents were weighed, and fat mass, lean mass, and body water was measured with magnetic resonance imaging (MRI). Hepatic steatosis was graded on a scale of 1–9 arbitrary units (A.U.) on hematoxylin and eosin stained liver tissue. Vascular stiffness was measured by atomic force microscopy (AFM) on aorta explants.

CD
WD
WD + ACT

Total Body Weight (values in g ± SEM)
23.1 ± 0.4
31.3 ± 1.1**
20.1 ± 1.5 (N.S.)

Total Fat Mass (values in g ± SEM)
4.7 ± 11.2
11.2 ± 1 **
5.1 ± 1.2 (N.S.)

Total Body Water/Total Body Weight (in % ± SEM)
65.1 ± 0.8
49.9 ± 2.0**
57.9 ± 2.6 (N.S.)

Hepatic Steatosis (in arbitrary units, AU, ± SEM)
3.75 ± 1.4
7.4 ± 0.5*
5.0 ± 1.1 (N.S.)

Vascular Stiffness (in kPa ± SEM)
4.72 ± 0.6
17.18 ± 3.2*
5.22 ± 1.0 (N.S.)

N = 20,

denotes P < 0.05,

denotes P < 0.01,

N.S. denotes not significantly different from control diet (CD).

Compared to a CD, sixteen weeks of a WD was associated with a gain in total body weight that was completely inhibited by ACT. The increase in total adipocyte fat mass and percentage of body weight from water in WD animals were also inhibited by ACT. Notably, inhibition of carbonic anhydrase resulted in a decrease in hepatic steatosis and aortic vascular stiffness. If excess adiposity is responsible for the untoward sequelae of the metabolic syndrome, chronic inhibition of carbonic anhydrase‐dependent formation of bicarbonate and fatty acid synthesis could result in the normalization of the metabolic profile associated with an unhealthy Western diet

Support or Funding Information

Support for this project was provided by the University of Missouri.
Differential Effects of Toll‐like Receptors 2 and 4 in Endotoxin‐Induced Vascular Dysfunction
Authors: Battson Micah L , Lee Dustin M , Hou Shoufei , Jarrell Dillon K , Santangelo Kelly S , Cox‐York Kimberly A , Gentile Christopher L
Published: November 2023

Bacterial endotoxin (lipopolysaccharide, LPS) may contribute to diabetes‐related inflammation and has been shown to cause generalized vascular dysfunction, including arterial hypocontractility and impaired endothelium‐dependent and –independent vasodilation. These vascular impairments are thought to be mediated in large part via Toll‐like receptor 4 (TLR4). However, contribution of other TLRs, in particular TLR2, has not been well characterized. We therefore examined the relative contribution of TLR2 and TLR4 in mediating the acute effects of LPS on vascular dysfunction. Vascular reactivity was assessed via ex vivo pressure myography in isolated carotid arteries from male wild‐type (WT), TLR2‐KO and TLR4‐KO mice following 1 hour incubation with or without endotoxin (E. coli LPS 50ug/ml). LPS decreased maximal constriction to the thromboxane A2 agonist U46619 in WT (CON: 30 ± 3% vs LPS: 17 ± 3%) but not TLR2‐KO (CON: 29 ± 4% vs LPS: 25 ± 3%) or TLR4‐KO (CON: 33 ± 2% vs LPS: 32 ± 6%). LPS reduced endothelium‐dependent dilation (EDD) to acetylcholine in WT (CON: 95 ± 1% vs LPS: 73 ± 4%) and TLR2‐KO (CON: 84 ± 3% vs LPS: 64 ± 5%) but not TLR4‐KO (CON: 94 ± 2% vs LPS: 89 ± 3%). Finally, LPS reduced maximal endothelium‐independent dilation (EID) to sodium nitroprusside in WT (CON: 94 ± 1% vs LPS: 76 ± 6%) but not TLR2 (CON: 78 ± 5% vs LPS: 74 ± 6%) or TLR4‐KO (CON: 89 ± 3% vs LPS: 80 ± 2%). These findings suggest that lack of TLR2 protects carotid arteries against the LPS‐induced reductions in constriction and EID and that arteries of TLR4‐KO mice display additional protection against impaired EDD. Future studies will investigate the mechanism underlying these differential effects and may provide insight into potential treatments for endotoxin‐related vascular dysfunction.

Support or Funding Information

Funding: Boettcher Foundation's Webb‐Waring Biomedical Research Program (CLG); National Institutes of Health DK087777 (CLG)
Ufm1 E3 ligase Ufl1 is essential for adaptive hypertrophy and ER stress response in the heart
Authors: Li Jie , Yue Guihua , Ma Wenxia , Li Huizhong , Zhang Aizhen , Li Faqian , Li Honglin , Su Huabo
Published: November 2023

Post‐translational modifications of proteins are important regulatory mechanisms of protein function. Protein modifications by Ufm1, a novel ubiquitin‐like protein, require Ufm1 E1 UBA5, E2 Ufc1 and E3 ligase Ufl1 and are emerging as a central theme in protein activity regulation. To date the significance of Ufm1 modification in health and disease is largely unknown. In this study, we reported that Ufl1 was down‐regulated in human failing hearts of ischemic cardiomyopathy and in the ischemic regions of mouse hearts subject to ischemia reperfusion injury. To investigate the effects of decreased Ufmylation in the heart, we generated mice with cardiomyocyte‐restricted deficiency of Ufl1 (Ufl1KO). Loss of Uf11 impaired Ufm1 conjugation and reduced the protein levels of its two known targets, RGDDK1 and C53, in mouse heart. The KO mice were viable and had no morphological abnormality at 2 months after birth. However, during ageing, Ufl1KO mice displayed reduced heart weight to body weight ratio, upregulation of cardiac fetal genes, increased apoptosis and increased fibrosis, which eventually led to left ventricular chamber dilatation, wall thinning and cardiac Huidysfunction at 6 months of age. When challenged by transverse aortic constriction (TAC)‐induced pressure overload, Ufl1KO mice quickly developed dilated cardiomyopathy and heart failure at 2 weeks post procedure without a prior phase of adaptive hypertrophy. RNA sequencing analysis revealed a significant alteration in the expression profile of ER stress‐related genes in Ufl1KO hearts, which was further confirmed by real‐time PCR analysis and western blots of several ER stress response factors. In primary cultured cardiomyocytes, knockdown of Ufl1 also blunted ER stress response and aggravated ER stress‐induced cell injury. Hence, our data demonstrate an essential role of Ufm1 modification in the heart under physiological and pathological conditions and reveal a critical role of Ufl1 in mediating adaptive ER stress response in cardiomyocytes.

Support or Funding Information

This work was supported in part by NIH grant R01HL124248 (to H.S.) and American Heart Association grant 11SDG6960011 (to H.S.).
Repetitive ROS Injury Leads to Inactivation of NRF2‐Induced Antioxidant Defense and Mitochondrial Dysfunction in Ischemic Heart Failure
Authors: Liu Tingting , Knowlton Anne A
Published: November 2023

Ischemic heart failure (IHF) is associated with an imbalance of reactive oxygen species (ROS) and antioxidants. In IHF, the failing heart is repeatedly exposed to increased ROS secondary to multiple factors including mitochondrial dysfunction. Repetitive injury, such as occurs in IHF with increased ROS, can have effects much different from a single treatment. Nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2), is the master regulator of antioxidant genes. Nrf2 binds to Kelch like‐ECH‐associated protein (KEAP) 1 under normal conditions. When stressed by ROS, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it activates the antioxidant response element (ARE) pathway. We found that Nrf2 was paradoxically decreased in left ventricles from rats with IHF, as are antioxidant genes. In isolated adult rat cardiac myocytes subjected to repetitive H2O2 treatment, this repetitive ROS exposure decreased Nrf2 expression, nuclear binding activity, and reduced cytoplasmic Nrf2 translocation to the nucleus. Moreover, mitochondria isolated from the stressed cardiac myocyte showed decreased expression of mitochondrial dynamics related proteins, including OPA1, Parkin, and VCP, consistent with our findings with in vivo heart failure. The Nrf2 knockout heterozygous mice had impaired cardiac function. Overall, our data show that repetitive ROS treatment leads to paradoxical inactivation of the Nrf2‐induced antioxidant defense and mitochondrial dysfunction in heart failure.
The Effect of Monocrotaline‐Induced Heart Failure on Left‐ and Right‐Ventricular Function
Authors: Guild Sarah‐Jane , Han June‐Chiew , Power Amelia , Ward Marie‐Louise , Nisbet Linley A , Loiselle Denis S
Published: November 2023

Monocrotaline is well established to cause pulmonary arterial hypertension and right heart failure. To characterize the development of monocrotaline‐induced heart failure in Wistar rats, Millar telemetry devices were used to follow arterial pressure and heart rate for six weeks following a single subcutaneous injection of either monocrotaline (60 mg/kg) or saline. Four weeks after monocrotaline injection, mean arterial, systolic, diastolic and pulse pressures all began to decrease progressively. This decrease in pressure was accompanied by a progressive increase in heart rate until 5 weeks post‐injection when heart rate began to fall again. Ventricular function was assessed 6 weeks post‐injection by placement of pressure volume catheters in both the left and right ventricles. Reduced ejection fraction and dP/dt max indicated reduced contractile function of both the left and right ventricles in the monocrotaline‐treated rats compared to the saline‐treated controls.

Support or Funding Information

Supported by the National Heart Foundation of New Zealand
Longitudinal Study on Independent and Joint Effect of Brachial‐Ankle Pulse Wave Velocity and Blood Pressure Control on Incident Stroke in Hypertensive Patients
Authors: Song Yun , Xu Benjamin , Xu Richard , Tung Renee , Frank Eric , Tromble Wayne , Yang William , Zhang Yan , Li Jianping , Qin Xianhui , Wang Binyan , Hou Fan Fan , Huo Yong , Wang Hong
Published: November 2023

Background

Pulse wave velocity (PWV) has been shown to affect the effects of anti‐hypertensive treatment in prevention of cardiovascular diseases. Data are very limited regarding whether PWV is an independent predictor of stroke above and beyond hypertension control. In their current guidelines for the management of arterial hypertension, the European Society of Hypertension included PWV as a potential consideration in managing middle‐aged hypertensive patients with increased aortic stiffness. However, none of the current stroke prevention guidelines have considered PWV due to limited data.

Objective

This longitudinal analysis examined the independent and joint effect of brachial‐ankle PWV (baPWV) with hypertension control on the risk of first stroke.

Methods

This report included 3,310 hypertensive adults (age: 59.6 ± 7.5 years), a subset of the China Stroke Primary Prevention Trial (CSPPT) with baseline measurement for baPWV. All the participants had no prior history of stroke or myocardial infarction (MI).

Results

Mean baseline baPWV was 18.76 (SD: 3.82) m/sec. During a median follow‐up of 4.5 years, 111 participants developed first stroke. The risk of stroke was higher among participants with baPWV in the highest quartile than that in the lower range (6.3% vs. 2.4%). Similarly, the participants with inadequate hypertension control had a higher risk of stroke than those with adequate control (5.1% vs. 1.8%). When baPWV and hypertension control were examined jointly, participants in the highest baPWV quartile and with inadequate control had the highest risk of stroke compared to their counterparts as demonstrated by Kaplan‐Meier curves (log rank test p<0.0001) and Cox proportional hazard modeling (hazard ratio (HR): 3.6; 95% confidence interval (CI): 1.9–6.8, p<0.001), after adjusting for demographic and cardiovascular risk factors. Furthermore, there was a significant and independent effect of high baPWV on stroke as shown among participants with adequate hypertension control (HR=2.3, 95%CI: 1.1–4.8, p=0.029).

Conclusion

Among hypertensive patients, baPWV and hypertension control were demonstrated to independently and jointly affect the risk of first stroke. Participants with high baPWV and inadequate hypertension control had the highest risk of stroke compared to other groups. Our findings warrant additional investigation and raise a possibility that baPWV may serve as a simple noninvasive screening tool in clinical or community settings to identify hypertensive adults at high‐risk of stroke. It may also offer a novel therapeutic target to further reduce stroke risk beyond conventional management.
Involvement of Inhibitor Kappa B Kinase 2 in the Elevated Vascular Tone of Spontaneously Hypertensive Rats
Authors: Choi Soo‐Kyoung , Lim Mihwa , Kwon Youngin , Lee Young‐Ho
Published: November 2023

Introduction

Hypertension is one of the leading causes for cardiovascular disease worldwide. A contributing factor to hypertension is elevated vascular tone in small arteries and arterioles. The 20 KDa myosin light chain (MLC20) phosphorylation is determinant of vascular tone. Recently, it has been reported that inhibitor kappa B kinase 2 (IKK2) is involved in the regulation of MLC20 phosphorylation. We tested whether IKK2 is involved augmented vascular tone and increased blood pressure in spontaneously hypertensive rats (SHRs).

Methods and Results

The vascular diameter was measured in mesenteric arteries from 16‐week old male SHRs and their control animals, wistar kyoto rats (WKYs). Western blot analysis was performed to determine changes in expression of IKK2, phosphorylation of inhibitor of kappa B alpha (IκBα), and phosphorylation of MLC20. We found that IC50 values of IKK2 inhibitor, SC‐514 were significantly higher in mesenteric arteries from SHRs compared to those from WKYs. Furthermore, expression of IKK2 is significantly increased in mesenteric arteries from SHRs, which is associated with enhanced phosphorylation of MLC20.

Conclusion

Our results show that IKK2 expression and activation are enhanced in the mesenteric arteries of SHRs compared to WKYs. Thus, we suggest that IKK2 contributes to increased vascular tone in SHRs.
Altered Nitric Oxide Signaling and Oxidative Stress Increase Pulmonary Arteriolar Tone and Cause Lung Fibrosis in a Mouse Model of Alzheimer's Disease
Authors: Roberts Andrew M , Jagadapillai Rekha , Vaishnav Radhika , Friedland Robert P. , Drinovac Robert , Lin Xingyu , Gozal Evelyne
Published: November 2023

Vascular dysfunction and decreased cerebral blood flow in aging have been linked to Alzheimer's Disease (AD). A common feature of aging and cerebrovascular disease is decreased endothelial nitric oxide (NO). Existing evidence suggests that the loss of NO in human cerebrovascular endothelium and increased reactive nitrogen species increase expression of APP and production of Aβ peptides, supporting the concept that a loss of endothelial NO might significantly contribute to the initiation and progression of AD pathology. However, the decrease of systemic NO bioavailability in AD patients is not well characterized and, when occurring in the lung, the resulting endothelial dysfunction may impact pulmonary microcirculation. We hypothesized that systemic NO decrease in AD may induce pulmonary endothelial dysfunction, impairing vascular responses, and result in lung injury. The acute effect of NO synthase (NOS) inhibition on pulmonary arteriolar tone was assessed in anesthetized, male transgenic mice (TgAD; 15–29 months old) expressing the human amyloid beta precursor protein APP gene, 770 isoform with the Swedish, Dutch and Iowa mutations (C57BL/6‐Tg(Thy‐1APP (SwDut Iowa) Bwevn/J mice) and age‐matched wild type controls (C57BL/6J). Pulmonary arteriolar diameters (ranging from 22 to 54 μm) were measured before and after administration of the nitric oxide synthase (NOS) inhibitor, L‐NAME (0.1mg/g body wt). Lung superoxide formation (DHE) and lipid peroxidation (4‐HNE) were assessed as indicators of oxidative stress, and type I and type III collagen deposition (Sirius Red) was used as a marker of pulmonary fibrosis. Inducible NOS (iNOS) expression was determined by Western blotting and immunohistochemistry. In WT mice, administration of L‐NAME caused constriction of pulmonary arterioles (13.6 ± 8.6%, mean ± SD, p<0.05, n = 4). In contrast, L‐NAME administration caused significant dilation of pulmonary arterioles in TgAD mice (14.7 ± 6.2%, p<0.05, n = 6). DHE, 4‐HNE, Sirius Red and iNOS staining increased in TgAD lung tissue samples, compared to WT mice. These data suggest that increased pulmonary arteriolar tone in WT mice may result from a loss of the vasodilator influence of bioavailable NO. However, although bioavailable NO may be decreased in TgAD mice, inhibition of NOS with L‐NAME appears to have a vasodilator effect by decreasing formation of reactive oxygen species. We conclude that increased pulmonary microvascular tone may occur in AD as a result of loss of bioavailable NO and increased oxidative stress.
Salt and Angiotensin II Treatment Induces Fluid Congestion and Heart Failure with Increased Mortality in Balb/C Mice but Not in C57BL/6J
Authors: Joensson Sofia Tora , Becriovic‐Agic Mediha , Hulström Michael
Published: November 2023

Heart and kidney failure causes many hospitalizations, and is associated with high mortality rate. We have previously shown that 3% sodium diet and Angiotensin II (0.5 μg/min/kg) leads to clinical signs of heart failure with decreased blood pressure, edema and mortality in Balb/C while C57BL/6J are protected. The aim of the present investigation was to study heart function using left ventricular catheterization and to formally analyze mortality using meta‐analysis across all our studies.

Mice were treated with a 3% sodium diet and AngII (0.5 μg/min/kg) using Alzet osmotic pumps for four days. Left ventricular catheterization showed lower heart rate and cardiac output in Balb/C (596±9.8 bpm, 7.1±0.74 ml/min) compared to C57BL/6J (517±6.2 bpm, 5.2±0.29 ml/min p<0.05) after Salt+AngII treatment. Stroke volume was lower in Balb/C treated with Salt+AngII compared to control treatment, but no difference was seen in C57BL/6J. The results were reproduced using echocardiography, which also showed a diastolic dysfunction. However, compliance as measured using the pressure volume relationship was not affected. This may be consistent with a fluid congestion and right heart failure as Balb/C show a lower urine output (0.24±0.04 vs. 0.46±0.02 ml/h, p<0.001) and sodium excretion (2.7±0.5 vs.3.5±0.3, p<0.05) with the combination treatment in spite of similar intake.

Meta‐analysis of the mortality of 121 animals from four different experimental series showed a relative risk of 0.16 (95% CI 0.05–0.49) in favor of C57BL/6J after combination treatment with high‐sodium diet and AngII. This provides a rigorous statistical analysis of our observation that Balb/C is much more susceptible to heart failure. Similar analysis of sodium diet (n=96, RR 0.3, 95% CI 0.04–2.54), or AngII treatment alone (n= 83, RR 0.4, 95% CI 0.12–1.36) showed no significant difference.

In conclusion, Balb/C mice have reduced cardiac function compared to C57BL/6J when treated with Salt and AngII, which leads to a higher mortality in Balb/C mice.

Support or Funding Information

Swedish Heart‐Lung foundation, Ake Wiberg foundation, Swedish Society for Medical Research
Renal denervation normalizes blood pressure and improves glucose metabolism in obese genetically hypertensive Schlager mice
Authors: Asirvatham‐Jeyaraj Ninitha , Banek Christopher T. , Han Ruijun , Razzoli Maria , Burbach Brandon J. , Bartolomucci Alessandro , Shimizu Yoji , Osborn John W.
Published: November 2023

Renal nerve ablation has been reported to decrease arterial pressure and improve glucose metabolism in drug‐resistant hypertensive humans, the majority of who are overweight or obese. However, the mechanism mediating these responses is unknown. The Schlager mouse (BPH/2J) is a genetically inbred strain that has chronically elevated sympathetic activity and mean arterial pressure (MAP). We used this model to assess the effect of renal denervation (RNDX) on MAP and glucose metabolism in lean and obese BPH/2J mice. In addition, since both obesity and renal efferent nerves have been shown to drive renal inflammation, we also assessed the effect of RDNX on markers of renal inflammation. Hypertensive BPH/2J and normotensive BPN mice were maintained on either a low fat (LFD; 10 KCal% from fat) or a high fat (HFD; 45 KCal% from fat) diet for 10 weeks. Radio telemeters were then implanted for measurement MAP. MAP was significantly higher in LFD BPH/2J mice (128 ± 2 mmHg) than the LFD BPN controls (107 ± 2 mmHg). Surprisingly, 10 weeks of HFD had no effect on MAP in either strain. MAP was normalized in HFD BPH/2J mice two weeks following RDNX, (~110 mmHg). In contrast, RDNX had no effect on MAP in LFD BPH/2J mice (~134 mmHg). Glucose handling was assessed by glucose tolerance tests three weeks after RDNX/Sham surgery. Glucose tolerance, as quantified by the area under the curve, was impaired to a similar extent in LFD and HFD BPH/2J mice. Moreover, RDNX improved glucose metabolism in both the LFD and HFD groups. Real time PCR was used to determine expression of MCP‐1 (monocyte chemoattractant protein‐1), a chemokine that recruits monocyte and macrophages to the site of injury, and the pro‐inflammatory renal cytokine, tumor necrosis factor alpha (TNFa). In both LF and HF fed groups MCP‐1 (135 ± 37.8 fold) and TNFa expression (56.9 ± 1.9 fold) were elevated in BPH/2J mice compared to normotensive BPN controls (2–4 fold). Results indicate that RDNX may reduce MCP‐1 expression (to 34.1 fold) in BPH/2J mice but have no effect on TNFa. These results suggest that RDNX is effective in the normalization of MAP and glucose metabolism in HFD BPN/2J mice. Future studies will focus on the assessing the mechanisms by which RDNX mediates these responses.

Support or Funding Information

RO1 HL 067357‐15 and RO1 HL 116476‐02
Identification of Dipeptidyl Peptidase III as the Angiotensin‐(1–7)‐Degrading Metallopeptidase in Human Proximal Tubule Cells
Authors: Cruz‐Diaz Nildris , Wilson Bryan A , Marshall Allyson C , Pirro Nancy T , Chappell Mark C
Published: November 2023

We previously identified a soluble metallopeptidase activity present in various compartments of the sheep including the brain medulla, cerebrospinal fluid (CSF) and renal cortex, as well as in the human proximal tubule (HK‐2) cells that efficiently metabolized Angiotensin‐(1–7) [Ang‐(1–7)], an alternative product of the renin‐angiotensin that functionally antagonizes the Ang II‐AT1 receptor axis. Indeed, peptidase activity in the CSF was markedly higher in betamethasone‐exposed sheep, a model of fetal programming‐induced hypertension. Moreover, the peptidase activity was associated with higher blood pressure and reduced CSF content of Ang‐(1–7). Since the peptidase accounted for the sole degrading activity of Ang‐(1–7) in the HK‐2 cells, we purified and sequenced the enzyme from these cells. The 100,000 xg supernatant fraction was subjected to sequential Cibacron‐blue and ion‐exchange chromatography. A single 80kDa band was revealed by both native and SDS polyacrylamide gels. The protein band exhibiting Ang‐(1–7) peptidase activity and sensitivity to the JMV‐390 inhibitor was subsequently excised from the native gel and submitted for protein sequencing by MALDI‐MS. The MS analysis revealed only one peptidase candidate identified as dipeptidyl peptidase III (DPP III, EC 3.4.14.4). Cleavage specificity of Ang‐(1–7) and sensitivity to JMV‐390 were then determined utilizing human recombinant DPP III. DPP III metabolized Ang‐(1–7) to the primary products Ang‐(3–4) and Ang‐(5–7); however, Ang‐(3–7) was a minor product. Comparison of Ang‐(1–7) and Ang‐(3–7) metabolism by recombinant DPP III revealed a ~3‐fold greater rate of hydrolysis for Ang‐(3–7) [14.8 vs. 5.5 nmol/min/ug] that is consistent with the catalytic properties of DPP III and suggests the enzyme catalyzes the sequential cleavage of Ang‐(1–7) to Ang‐(3–7) and then to Ang‐(5–7) and Ang‐(3–4). The recombinant DPP III also exhibited potent inhibition by JMV‐390 (IC50 = 1.4 ± 0.01 nM) that was comparable to the purified peptidase from HK‐2 cells (IC50 = 0.2 ± 0.01 nM). The metabolism of Ang‐(1–7) was then assessed in the HK‐2 cells following chronic treatment with a low (20 nM) and high (200 nM) dose of the JMV‐390 inhibitor over a 72 hour period. The cellular metabolism of exogenous Ang‐(1–7) was reduced at both JMV concentrations (20nM: 54 ± 7% of control, n=2; 200nM: 19 ± 5% of control, n=3, p<0.05). Moreover, the extent of the conversion of Ang I to Ang‐(1–7) was higher in the JMV‐treated HK‐2 cells (JMV 20nM: 135 ± 33% of control, n=2; JMV 200nM: 190 ± 33% of control, n=3). In summary, we identify DPP III as the Ang‐(1–7) degrading activity in human HK‐2 proximal tubule cells. We conclude that that DPP III may constitute a potential pharmacological target to enhance the intracellular levels of Ang‐(1–7) under pathological conditions.

Support or Funding Information

Supported by NIH HD47584, HD‐17644 and K12‐GM102773.
Deficiency of Prorenin Renin Receptor in Adipocyte induces Liver Steatosis and increases Blood Pressure in Lean and Obese Male Mice
Authors: Wu Chia‐Hua , Moradi Shayan , Gong Ming , Nguyen Genevieve , Yiannikouris Frederique
Published: November 2023

Objectives

Adipose tissue is an important source of the prorenin renin receptor (PRR). During the development of obesity, the expression of PRR gene is increased in adipose tissue. However the role of adipocyte PRR during the development of obesity is not clear. The purpose of this study was to characterize a new model of adipocyte PRR deletion and define the metabolic consequences during the development of obesity related hypertension in male mice.

Methods and results

Male PRRfl/Y mice expressing Adiponectin‐driven Cre recombinase were bred to female mice PRRfl/fl to generate littermate controls (PRRfl/Y) and adipocyte‐PRR deficient mice (PRRAdi/Y). Mice were fed a low fat diet (LF, 10% kcal as fat) or a high fat diet (HF, 60% kcal as fat) for 16 weeks (n=5–8/groups). Body weight of LF fed‐PRRAdi/Y mice were decreased significantly compared to LF fed‐PRRfl/Ymice. Adipocyte PRR deficient mice were resistant to diet induced obesity (body weights: PRRfl/Y, 48 ±1 g; PRRAdi/Y, 34 ± 1 g; P<0.05). Fat mass, measured by echo‐MRI, were drastically decreased in PRRAdi/Y mice fed a LF‐or HF‐diet compared to PRRfl/Y mice (fat mass: LF: PRRfl/Y, 9 ± 1 g; PRRAdi/Y, 2 ± 0 g. HF: PRRfl/Y, 21 ± 1 g; PRRfl/Y, 3 ± 0 g. P<0.05). Surprisingly, adipocyte PRR deficiency induced an increase in lean mass in LF− and HF− fed mice. The increase in lean mass was attributed mainly by an increase of liver weights in PRRAdi/Y male mice fed a LF− or a HF‐diet compared to PRRfl/Y mice (LF: PRRfl/Y, 1.5 ± 0.1 g; PRRAdi/Y, 3.0 ± 0.4 g. HF: PRRfl/Y, 2.6 ± 0.2 g; PRRfl/Y, 3.6 ± 0.2 g. P<0.05). Microscopic examination of liver sections revealed an increase of hepatic fat accumulation in male PRRAdi/Y fed a LF‐or HF‐diet compared to control male PRRfl/Y mice. To determine the role of adipocyte PRR deficiency on glucose homeostasis, we performed an intraperitoneal glucose tolerance test (ipGTT). At 12 weeks, LF and HF‐fed PRRAdi/Y mice had an improved glucose tolerance compared to PRRfl/Y mice. At 15 weeks, we implanted radiotelemetry devices into anesthetized mice to measure blood pressure. Surprisingly, LF‐fed PRRAdi/Y mice exhibited a significant increase in systolic blood pressure compared to LF‐fed controls (LF: PRRfl/Y, 129 ± 1 mmHg; LF: PRRAdi/Y, 134 ± 2 mmHg, P< 0.05). Moreover, when challenged with HF‐feedings, adipocyte‐specific deficiency of PRR exaggerated diet induced increased‐blood pressure (HF: PRRfl/Y, 132, ± 2 mmHg ; HF: PRRAdi/Y, 138 ± 1 mmHg, P< 0.05).

Conclusion

Our new PRRAdi/Y mouse model provides evidence for an important link between adipocyte PRR, fat distribution and regulation of blood pressure. The mechanism by which adipocyte PRR induces increase in blood pressure is currently under investigation.

Support or Funding Information

13SDG17230008, COBRE P20 GM103527, pilot grant NIH CTSA UL1TR000117.
The role of Gαi2 proteins in the regulation of PVN neuronal activation in response to isotonic volume expansion in conscious rats
Authors: Carmichael Casey Y , Patel Neha M , Wipfler Rachel E , Wainford Richard D
Published: November 2023

Aim

An acute volume expansion (VE) evokes inhibition of sympathetic outflow and the paraventricular nucleus (PVN) is a key region involved in this response. Extending our prior studies demonstrating a role of CNS Gαi2 proteins in PVN neuronal activation during acute hypertonic saline challenge, we determined the role of brain Gαi2 proteins in PVN neuronal activation during an acute VE.

Methods

24‐h intracerebroventricular scrambled (SCR) or Gαi2 oligodeoxynucleotide (ODN; 25μg/5μl)‐pretreated conscious Sprague‐Dawley rats were IV infused with isotonic saline (0.9% w/v NaCl) equal to 10% of body weight in 10‐min, followed by a maintenance infusion of 0.5 ml/min for 90‐min, and then a physiological infusion of 20 μl/min for 20‐min. Rats were monitored for changes in cardiovascular and urine excretory parameters and sacrificed at control (C) or 120‐min post‐VE for PVN cFos IHC.

Results

In both SCR and Gαi2 ODN pretreated rats, acute isotonic VE produced no significant change in systemic cardiovascular parameters (P>0.05). VE evoked a significant increase in urine flow rate (V) and urinary sodium excretion (UNaV) in SCR ODN pretreated rats, while the magnitude of the diuretic and natriuretic response was attenuated in Gαi2 ODN pretreated rats (peak Δ UNaV [μeq/min] SCR: 101.29±4.20 vs. Gαi2: 63.12±9.78, P<0.05). No difference in the number of PVN Fos+ cells was observed between ODN pretreated groups at control. Significant increases in the number of Fos+ PVN magnocellular and parvocellular neurons were observed post‐VE in both SCR and Gαi2 ODN groups as compared to C (P<0.05). Gαi2 ODN rats exhibited significantly less activated magnocellular (120‐min [Fos+ cells] SCR: 24±3 vs Gαi2: 16±2, P<0.05) and parvocellular neurons (120‐min [Fos+ cells] SCR: 168±6 vs Gαi2: 102±4, P<0.05) as compared to their SCR ODN counterparts following VE (P<0.05).

Conclusion

Endogenous brain Gαi2 proteins contribute to the activation of PVN neurons during isotonic VE to maintain body fluid homeostasis. Owing to the established sympathoinhibitory actions of PVN parvocellular neurons post VE, we speculate Gαi2 proteins are central to the activation of sympathoinhibitory parvocelluar neurons. This hypothesis is supported by our prior data demonstrating 1) CNS Gαi2 proteins are essential to facilitate the suppression of renal sympathetic nerve traffic during acute VE and 2) CNS Gαi2 proteins mediate activation of PVN parvocellular neurons and sympathoinhibition in response to an acute bolus sodium challenge. Collectively, these data highlight a novel role of PVN Gαi2 proteins in the acute neural sympathoinhibitory pathways that regulate fluid and electrolyte homeostasis. Further, impairments in these neural pathways potentially contribute to altered sympathetic outflow and sodium retention in the pathophysiology of hypertension.

Support or Funding Information

NIH HL107330

K02HL112718
A High Fat Diet Increases Blood Pressure and Leads to a Renal Proinflammatory Immune Cell Profile in Female Dahl Salt‐Sensitive Rats
Authors: Taylor Lia , Musall Jacqueline , Baban Babak , de Chantemele Eric J Belin , Sullivan Jennifer
Published: November 2023

Diets high in fat and cholesterol have been linked to weight gain and hypertension, and T cell activation has been suggested to mediate these diet‐induced changes in male animal models. Male Dahl‐salt‐sensitive (DSS) rats on a high‐fat (HF) diet were previously shown to have increased blood pressure (BP), renal injury, renal T cell infiltration. There is little known, however, regarding the impact of a HF diet on T cell activation in females. This is particularly alarming since females represent ~50% of hypertensive cases and are more likely than men to be obese. We hypothesize that a HF‐diet will lead to increased BP and renal T cell infiltration in female DSS rats. Twelve‐week old female DSS rats were maintained on control diet (7.2% fat, n=5) or HF diet (35% fat, n=6) for a 4 week period. Systolic BP was measured by tail‐cuff and renal T lymphocyte infiltration (total CD4+ T cells, T regulatory cells (Tregs), Th17 cells, IL‐10+ cells) was measured by flow cytometric analysis. Metabolic parameters, including food intake, body weight, and plasma leptin levels, were also measured. A 4‐wk HF diet significantly increased systolic BP (mmHg: 136 ± 0.5 control vs 166 ± 0.6 HF; P<0.05). Total fat intake was significantly higher in the HF group after 4 wks (kcal/24hr: 46 ± 3, HF vs 12 ± 1 control; P<0.05), and this likely contributed to the greater increase in body weight in HF‐fed females after 4 weeks (% increase: 15 ± 1 control vs 20 ± 1 HF; P<0.05). Plasma leptin levels were also ~1.7‐fold higher in rats on a HF‐diet (ng/mL: 3.7 ± 1.2 HF, vs. 2.1 ± 0.7 control; P=0.36). Although there was no increase in total renal CD4+ T cells, immunosuppressive FoxP3+ T regulatory cells (% CD3+: 17 ± 3, control vs. 10 ± 4 HF; P=0.17) decrease and IL‐10+ cells (% CD3+: 45 ± 5 control vs 35 ± 6 HF; P=0.25) tended to decrease. There was no change in pro‐inflammatory Th17 cells (%CD3+: 5.2 ± 1.4 control vs 8.0 ± 4.0 HF, P=0.56). We conclude that a 4wk‐HF diet increased BP and decreased renal anti‐inflammatory T cells in female DSS rats. The loss of renal anti‐inflammatory T cells is expected to lead to a more pro‐inflammatory immune cell profile that may sensitize female DSS rats to HF‐diet induced increases in BP.
Both Alpha and Beta Adrenergic Receptor Expression Is Increased in the Renal Medulla of Spontaneously Hypertensive African Green Monkeys
Authors: Rhoads Megan Kathleen , Munhoz Caroline , Osborn Jeffrey
Published: November 2023

The African Green Monkey (Chlorocebus aethiops sabaeus), the Caribbean vervet, is a highly translational model of spontaneous hypertension (HT). The Caribbean vervet has a very close phylogenetic relationship with humans with an estimated divergence time of ~12 million years ago. Within a cohort of adult animals, 36% (124 of 345) of African Green Monkeys exhibit systolic blood pressures (SBP)> 140 mmHg, as measured by forearm cuff plethysmography. Heart rate is elevated in HT vs. normotensive (NT) animals (121±2 bpm vs. 136±2 bpm, p<0.05) suggesting that sympathetic nerve activity is likely elevated in the HT African Green Monkey that behaviorally maintains upright posture throughout the day and night. This study tested the hypothesis that adrenergic receptor subtypes are upregulated in the renal cortex, outer medulla, and inner medulla of adult male HT African Green Monkeys. Primers were custom designed and evaluated for appropriate specificities and efficiencies (95–105%) for α1a, α1d, α2a, α2c, β1 and β2 adrenoceptors. Gene expression was measured with qRT‐PCR and normalized to RPS32 or RPS13A expression and RQs compared with the Mann‐Whitney U Test. The HT cohort of African Green Monkeys (n=18) averaged SBP of 168.24 ± 7.25mmHg compared with NT animals (n=18) that averaged 96.61 ± 3.20mmHg. Expression of each adrenoceptor subtype was similar in the renal cortex between the two groups. Expression of α1a, α1d, α2a, and α2c genes were increased in the renal outer medulla of HT animals (α1a: NT RQ 1 ± 0.43 vs. HT RQ 2.26 ± 0.50, p<0.05; α1d: NT RQ 1 ± 0.34 vs. HT RQ 2.25 ± 0.32, p<0.05; α2a: NT RQ 1 ± 0.24 vs. HT RQ 1.92 ± 0.19, p<0.05; and α2c: NT RQ 1 ± 0.15 vs HT RQ 1.65 ± 0.15, p<0.05). β1 and β2 adrenergic receptor expression in the outer medulla was also upregulated in HT animals (β1: NT RQ 1 ± 0.24 vs. HT RQ 1.88 ± 0.18, p<0.05; β2: NT RQ 1 ± 0.21 vs HT RQ 1.87 ± 0.24; p<0.05). Expression of adrenergic receptors in the inner medulla of HT animals was similar (p>0.05), with the exception of the β2 adrenergic receptor, which was upregulated in the HT group (NT RQ 1 ± 0.25 vs HT RQ 1.62 ± 0.10, p<0.05). These data suggest that the renal outer medulla of HT African Green Monkey may be sensitized to sympathetic nerve activity via elevated adrenergic receptor gene expression. Thus, subtle changes in renal sympathetic outflow may elicit renal medullary vasoconstriction and sodium retention and contribute to the development of the spontaneous hypertensive phenotype in the African Green Monkey. This study further characterizes a unique non‐human primate model of spontaneous hypertension with genetic, behavioral, and social similarities to humans, which may lead to the identification of novel therapies for the treatment of idiopathic hypertension.
ALMS1 (Alstrom Syndrome 1), a new interacting protein of NKCC2, regulates apical NKCC2 trafficking, urinary concentration and blood pressure
Authors: Jaykumar Ankita Bachhawat , Caceres Paulo , Ares Gustavo , Beierwaltes William , Ortiz Pablo
Published: November 2023

NaCl absorption by the Thick Ascending Limb (TAL) is mediated by the apical Na/K/2Cl cotransporter, NKCC2. Increased NKCC2 activity and apical trafficking are associated to hypertension. However, only few proteins are known to bind and regulate NKCC2 trafficking. A 150 amino acid region in the carboxyl terminus of NKCC2 (C‐NKCC2) was shown to be important for apical trafficking of NKCC2. We hypothesized that proteins which bind to this C‐terminus region in NKCC2 play a role in regulating NKCC2 trafficking in the TAL. To identify new TAL proteins that bind C‐NKCC2, we performed a proteomics‐based screening of TAL proteins which interacted with Glutathione‐S‐Transferase‐C‐NKCC2, and identified Alstrom syndrome 1 (ALMS1) as an interacting partner. ALMS1 has been linked to human hypertension and renal function in Genome Wide Association Studies. Thus, we hypothesized that ALMS1 is involved in NKCC2 trafficking in the TAL, sodium reabsorption and blood pressure regulation. First, we confirmed that ALMS1 is expressed in TALs by Western blot and immuno‐labeling of isolated perfused TALs. To study the role of ALMS1 we obtained ALMS1 KO rats in collaboration with the Genome Editing Rat Resource Consortium. To study the effect of ALMS1 deletion on NKCC2 trafficking we isolated outer medullary TALs and measured surface and total NKCC2 expression. In TALs from ALMS1 KO, the percentage of total NKCC2 at the surface was higher compared to WT (13.8 ± 1.2% vs 9.1 ± 1.0%, p<0.05, n=6). Total NKCC2 expression was not different between strains. Urine osmolality was 45% higher in ALMS1 KO rats (2800±37 vs. 1927±167 mOsm/kg H2O, p<0.001, n=5), whereas urinary volume was lower in ALMS1 KO (ALMS1: 10.1±0.5 vs. WT: 14.4± 1.7 ml/day, p< 0.05, n=5). Water intake was also lower in ALMS1 KO rats (18.4±1.1 vs. 28.2±1.5 ml/day, p<0.001, n=5). At three months of age, ALMS1 KO rats had higher mean arterial pressure (ALMS1: 141±5 vs WT: 99 ±6 mmHg, p< 0.001). Combined, these data indicate that ALMS1 binds and regulates NKCC2 trafficking and suggest that higher urine concentration is in part due to increased NKCC2 activity in the TAL of ALMS1 KO rats. We conclude that ALMS1 plays a role in NKCC2 trafficking and regulates blood pressure. The mechanism causing hypertension in ALMS1 KO rats may involve an increase in NKCC2 activity and higher sodium reabsorption by the TAL.