bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased local production of homocysteine elicits endothelial dysfunction via a mechanism of angiotensin‐dependent oxidative stress
Authors: Sun Dong , Huang An , Hintze Thomas H
Published: June 2021

To test the hypothesis that vascular production of homocysteine (Hcy), independent of its circulating level, contributes significantly to endothelial dysfunction, isolated mesenteric arteries of rats were incubated with methionine (1mM). Endothelial (EC) and smooth muscle cell (SMC) protein were collected separately for the HPLC/fluorescent detection of Hcy, showing a more than twice level of Hcy in EC compared with SMC. Roles of vascular Hcy in the modulation of vascular function were assessed by incubation of vessels with methionine in the presence or absence of losartan, enalapril or apocynin for 48 hours, followed by testing vascular production of Hcy, nitrite, and superoxide, as well as shear stress‐induced vasodilation (SSID). Vascular production of Hcy was significantly higher in methionine‐treated than control groups (37.3 ± 9.6 vs 1.6 ± 0.2 nm/mm2), which caused a ~40% reduction in SSID and reduced SS‐stimulated nitrite production. Vascular production of superoxide was significantly enhanced in methionine‐treated compared to control vessels (14.0 ± 0.9 vs 6.6 ± 1.0 pmol/μg protein), which however, was prevented by losartan, enalapril and apocynin, respectively. Thus, vascular endothelium de novo synthesizes Hcy to attenuate SSID via an angiotensin‐dependent, superoxide‐mediated impairment of nitric oxide bioavailability (This study was supported by NIH HL‐43023/‐070653).
Estrogen‐dependent methylation of sEH gene in mesenteric arteries of mice
Authors: Huang An , Sun Dong
Published: June 2021

Roles of soluble epoxide hydrolase (sEH) inhibitors in the regulation of blood pressure were assessed by feeding both genders’ eNOS‐KO mice with t‐TUCB (1mg/Kg, sEH inhibitor) via oral gavage for three‐weeks. There was a significant reduction in blood pressure in male t‐TUCB‐treated compared to vehicle‐treated eNOS‐KO mice, revealing an EET‐dependent prevention of hypertension. However, t‐TUCB did not significantly affect blood pressure in female eNOS‐KO mice. To this end, we hypothesized that there is a gender difference in the vascular expression of sEH, in which estrogen downregulates sEH gene via DNA methylation in the promoter region of Ephx2. Thus, mesenteric arteries isolated from male mice were incubated with physiological concentrations of 17β‐ or 17α‐estradiol (E2) for 24 hours. 17β‐E2 dose‐dependently increased DNA methylation that however, was not affected by 17α‐E2, indicating an estrogen‐specific response. As a result, sEH mRNA was attenuated significantly by ~60% compared to controls. Consistently, sEH protein expression was also greatly reduced in vessels of female compared to male mice. These results provide explanation for the lack of hypotensive effect in response to the treatment with sEH inhibitors in females. This is due to the in vivo presence of estrogen‐dependent inhibition of vascular sEH. (This work was supported by NIH HL‐070653)
Treadmill running improves diabetes‐induced capillary regression in skeletal muscle through activation of VEGF and angiopoietin pathways without inhibition of thrombospondin‐1
Authors: Fujino Hidemi , Kondo Hiroyo , Murakami Shinichiro , Tanaka Masayuki , Nagatomo Fumiko , Fujita Naoto , Ishihara Akihiko
Published: June 2021

PURPOSE

Exercise has been shown to be an effective treatment in the prevention of some complications due to type 2 diabetes (T2DM). The purpose of this study was to investigate the effects of treadmill running on the capillary regression and associated angiogenic factors in the soleus muscle of T2DM rats.

METHODS

Goto‐Kakizaki (GK) and Wistar rats were randomly divided into an exercise training (T2DM+Ex, Con+Ex) and a sedentary group (T2DM, Con). The T2DM+Ex and Con+Ex rats were run at a low‐intensity on a treadmill 6 times a week for 3 weeks. The capillary in soleus muscle was visualized using 3‐D method. The expression of pro‐angiogenic factors, and anti‐angiogenic factor, thrombospondin‐1 (TSP‐1), in the soleus muscle were determined by TaqMan probe‐based PCR.

RESULTS

Although the capillary volume of the soleus muscle was lower in T2DM than in Con rats, that in T2DM+Ex and Con+Ex was higher than that in Con. VEGF and Angiopoietin pathways in the T2DM+Ex were significantly increased compared with those in other groups although TSP‐1 level increased.

CONCLUSION

These findings suggest that treadmill running improves the regression of capillary in diabetic muscle, and the pro‐angiogenic pathways in diabetic muscle could be up‐regulated by exercise. Supported by Grants‐in‐Aid for Science Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Contribution of the Actin Cytoskeleton to the Viscoelastic Characteristics of Inwardly Remodeled Arterioles
Authors: Castorena‐Gonzalez Jorge Augusto , Staiculescu Marius Catalin , Zhao Guiling , Martinez‐Lemus Luis A.
Published: June 2021

In hypertension, one of the most important structural changes in the vasculature is the inward remodeling of arterioles, which is characterized by a reduction in passive luminal diameter. In arterioles, prolonged exposure to vasoconstrictor agonists or the reducing agent Dithiothreitol (DTT) induces inward remodeling. Therefore, we hypothesized that DTT induces inward remodeling via prolonged vasoconstriction and remodeling of the actin cytoskeleton. In rat cremaster isolated arterioles, acute exposure to DTT induced a concentration‐dependent constriction. A 4‐hour exposure to 200μM DTT caused prolonged vasoconstriction and induced an 11±4% reduction in passive diameter, with an increase in media‐to‐lumen ratio from 0.14±0.01 to 0.20±0.02. All this was inhibited in the presence of Cytochalasin‐D (500nM). Passive pressure‐diameter curves were obtained for control and remodeled arterioles before and after a 1‐hour incubation with the actin depolymerization agent Mycalolide‐B (2μM). The elasticity of control arterioles was not affected by Mycalolide‐B, but was increased by 21% in remodeled arterioles. These results indicate that DTT induces inward remodeling via prolonged vasoconstriction and that the change in viscoelastic characteristics of inwardly remodeled arterioles, namely increased stiffness, is partially dependent on the F‐actin components of the cytoskeleton. NIH HL‐088105 to LAM
Intrinsic balance of small artery active and passive diameter‐tension relations
Authors: Tuna Bilge Guvenc , Bakker Erik NTP , VanBavel Ed
Published: June 2021

Arterial remodeling is important in the pathogenesis of vascular diseases and target organ damage. In this study we induced matrix remodeling to smaller diameters and studied active diameter‐tension relations to understand the link between active and passive biomechanics.

Diameter‐tension relations were obtained in the passive state and upon full activation (125 mM K+ + 10−6 M NE) by wire myography before and after matrix cross linking by incubation with transglutaminase 2 (1, 5 hours TG2, 50μg/ml, n=6) or after low flow induced remodeling by ligation of rat mesenteric small arteries (1 day LF1, 3days LF3, n=8). The diameter for peak force capacity (Dopt, control 289±6, TG2 258±9*, LF1 258±12*, LF3 247±5*) shifted in the same direction as the passive diameter at 100 mmHg equivalent pressure (D100, control 353±9, TG2 317±17#, LF1 302±13#, LF3 301±7#), normalizing Dopt/D100 balance in vitro and in vivo. Active force at large distention fell simultaneously with increased passive force in all groups. Only LF3 vessels had increased contractile capacity at small diameters.

In conclusion, active tension capacity at high distension is masked by passive tension, challenging the concept that active and passive elements are arranged simply parallel, and suggests that there is an intrinsic mechanism linking biomechanical properties of active and passive components.
Deletion of macrophage focal adhesion kinase signaling impairs neither arteriogenesis nor angiogenesis in the mouse ischemic hindlimb model
Authors: Murrell Kelsey Paige , Meisner Joshua , Bouton Amy , Price Richard
Published: June 2021

We tested whether the myeloid specific deletion of focal adhesion kinase (FAKΔmyeloid), which regulates macrophage differentiation and motility, impairs arteriogenesis and perfusion restoration. Laser doppler perfusion imaging showed a significant increase in perfusion recovery in FAKΔmyeloid as compared to WT mice (n=6/5, p=0.011) after unilateral femoral artery ligation. Gracilis collateral arteries showed no differences in end‐state (day 28) or baseline (non‐ischemic limb) diameter between FAKΔmyeloid [88.4 ± 11.2 μm/42.1 ± 6.5 μm (ischemic/non‐ischemic)] and WT mice [92.4 ± 1.0 μm/40.4 ± 4.7 μm], and there were no differences in angiogenesis in the ischemic calf muscles (capillary:muscle fiber ratio of 2.42 ± 0.20/2.24 ± 0.25 ischemic/non‐ischemic for FAKΔmyeloid vs 2.36 ± 0.32/2.06 ± 0.17 for WT control soleus, 1.57 ± 0.32/1.41 ± 0.18 for FAKΔmyeloid vs. 1.71 ± 0.32/1.46 ± 0.22 for WT oxidative gastrocnemius, and 0.98 ± 0.24/0.77 ± 0.13 for FAKΔmyeloid vs. 0.75 ± 0.03/0.75 ± 0.07 for WT glycolytic gastrocnemius). In conclusion, FAK in myeloid cells does not regulate arteriogenesis or angiogenesis but may affect perfusion recovery through alternative means, such as improved muscle regeneration and/or decreased necrosis. Supported by NIH R21HL098632, AHA 10GRNT3490001, the UVA Cancer Center (CCSG P30 CA44579 and Patients and Friends Fund), and the Arnold and Mabel Beckman Foundation Scholars Program.
Comparing the degree of collateral vessel dilation necessary to compensate for a major arterial occlusion in mouse and man
Authors: Arciero Julia , DiStasi Matthew , Unthank Joseph
Published: June 2021

The dilation of pre‐existing collateral arteries is critical for providing adequate compensation following a major arterial occlusion, as seen in peripheral arterial disease (PAD). The lack of robust techniques for assessing perfusion in mouse has led to challenges in translating experimentally‐supported PAD therapies to the clinic. In this study, we develop a mathematical model to determine the impact of changes in collateral diameter and capillary number on blood flow at rest and during exercise after focal arterial occlusion in mice and humans. The model predicts the ratio of ligated to non‐ligated flow in the mouse hindlimb to be 0.54 if there is a combined 60% increase in collateral diameter and 71% increase in capillary number in the occluded limb. According to the model, the collateral diameter would have to increase in total by 133% to restore resting flow. The degree of enlargement for the mouse gracillis collateral artery is then compared with the enlargement necessary for the geniculate arteries in the human leg to restore resting flow.
Targeting of 11β‐hydroxysteroid dehydrogenase type 2 (11βHSD2) in the nucleus of the solitary tract (NTS) links salt appetite and hypertension
Authors: Evans Louise Christine , Kenyon Christopher , Mullins John , Holmes Megan , Bailey Matthew
Published: June 2021

11βHSD2 catalyses the inactivation of cortisol to cortisone, and thus ensures aldosterone‐specific activation of MR in target tissues. Cre‐lox technology was used to selectively target hsd11b2 expression in the NTS of mice. Telemetry was used to measure blood pressure during which Cre+ and Cre− mice (n=6 per group) had free access to either H2O or H2O and 1.5% NaCl. Cre+ mice were normotensive when given access to water. When offered a choice of water or saline, Cre+ mice had a significant salt preference (68% total fluid intake was NaCl), contrasting with Cre‐mice, which had salt aversion (40% total fluid intake was NaCl). The increased salt intake resulted in significant hypertension, (P=0.0004 active period, P=0.0178 inactive period). In Cre+ mice hypertension occurred over a time period during which balance studies showed that sodium was not retained. When salt‐intake was matched in Cre+ and Cre− mice, only the mice with NTS targeting of hsd11b2 developed a salt induced hypertension.

Here we have defined a role of mineralocorticoid receptors in the NTS that unifies the behavioral drive to consume sodium with the long‐term control of blood pressure. These data indicate a primary central component to the hypertension in the Cre+ mice. Funded by British Heart Foundation.
Autophagy in Neurogenic Hypertension induced by Systemic Inflammation
Authors: Wu Kay LH , Chan Julie , Chan Samuel
Published: June 2021

The reactive oxygen species (ROS) in the central nervous system increase sympathetic outflow contributing to neurogenic hypertension. We reported preciously that ROS in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons located, increases sympathetic outflow and mediates neurogenic hypertension following sustained systemic inflammation. Autophagy serves to balance organelle biogenesis, protein synthesis and their clearance. Cross‐talk with ROS engages autophagy to mediate stress signaling including synaptic plasticity under pathological status. In this study, we examined whether ROS activates the autophagic pathway to increase the sympathetic outflow and arterial pressure. Autophagy markers, including beclin‐1, Atg5, Atg12 and LC3 II, in RVLM were significantly increases under sustained systemic inflammation. Intracisternal infusion of ROS scavenger, tempol or mitoTEMPO, suppressed autophagy marker protein increase and the induced pressor response. Inhibition of autophagy in RVLM by 3‐MA reversed the pressor response. At cellular level, LC3 II immunoreactivity was colocalized with synaptophysin in RVLM and was inhibited by tempol. Together, these data suggest that autophagy in RVLM mediates ROS‐induced neurogenic hypertension under sustained systemic hypertension. This study is supported by CMRPG8A0801 to KLHW from Kaohsiung Chang Gung Memorial Hospital.
Endoplasmic reticulum stress is upstream of angiotensin‐II (Ang‐II)‐induced reactive oxygen species (ROS) formation in the subfornical organ (SFO)
Authors: Young Colin N. , Wang Gang , Iadecola Costantino , Mark Allyn L. , Davisson Robin L.
Published: June 2021

We have demonstrated that oxidative stress and endoplasmic reticulum (ER) stress in the brain SFO mediate Ang‐II‐dependent hypertension (HTN). Whether these two mechanisms are linked remains unknown. Here we tested the hypothesis that ER stress mediates Ang‐II‐induced oxidative stress in the SFO. First, freshly dissociated murine SFO cells were treated with Ang‐II in the presence or absence of the chemical ER stress inhibitor TUDCA. Dihyroethidium (DHE) was used as the ROS indicator. Ang‐II caused a significant increase in ROS production in SFO cells and this response was abrogated by pretreatment with TUDCA (1.28±0.04 vs 1.00±0.01 fold vehicle, Ang‐II vs Ang‐II + TUDCA, p<0.05). Second, C57Bl/6 mice underwent SFO‐targeted microinjections of an adenovirus encoding the ER chaperone GRP78 (AdGRP78, n=4), an approach we have shown to reduce ER stress and prevent Ang‐II HTN. SFO‐targeted AdLacZ (n=4) served as a control. Following recovery, mice were implanted with osmominpumps for 2 wk delivery of low‐dose Ang‐II. Ang‐II caused a significant increase in DHE fluorescence in the SFO at 14 days of Ang‐II infusion. This increase in ROS formation was prevented by SFO‐selective reduction in ER stress using AdGRP78 (1.78±0.21 vs 1.02±0.03 fold untreated, AdLacZ vs AdGRP78, p<0.05). These findings demonstrate that Ang‐II‐induced ER stress is a source of SFO oxidative stress in this model of HTN.

HL63887, HL96571, HL84207
Humoral immune system activation promotes the development of hypertension
Authors: Mathis Keisa W. , Wallace Kedra , Lamarca Babbette , Ryan Michael J.
Published: June 2021

Circulating autoantibodies are elevated in patients with essential hypertension suggesting the possibility of humoral immune system activation. Whether humoral immunity underlies the pathogenesis of hypertension is unclear. We hypothesized that humoral immune suppression (B cell depletion) would prevent the development of hypertension in a mouse model of the autoimmune disease systemic lupus erythematosus (SLE). Female SLE (NZBWF1) and control mice (NZW) were treated weekly with anti‐CD20 (10 mg/kg, Genentech) or vehicle (anti‐IgG) starting at 20, 26, and 30 weeks of age and ending at 34 weeks. Anti‐CD20 treatment reduced spleen weight in all groups regardless of treatment duration. Mean arterial pressure (MAP; mmHg) was higher in SLE mice compared to controls in all groups [(20 weeks; 134±1 vs 115±3, p<0.01); (26 weeks; 139±5 vs 116±2, p<0.001); (30 weeks; 138±2 vs 117±6, p<0.001)]. Anti‐CD20 did not alter MAP in SLE mice treated from 26 (131±3) or 30 (135±4) weeks of age. However, MAP was lower in SLE mice treated with anti‐CD20 starting at 20 weeks of age (120±3; p=0.01). Anti‐CD20 did not affect MAP in any control group. These data suggest that suppression of humoral immunity early in disease progression, but not later, can prevent the development of hypertension and supports the idea of autoimmunity as an underlying mechanism.

AHA4350019/5T32HL105324, HL085907/HL092284/HL085907S1/UMMC‐IRSP, HL051971
Hsd11b2 Targeting in the rat
Authors: Mullins Linda Jane , Peter Audrey , Kenyon Christopher , Sourgia‐Koutraki Yolanda , Bailey Matthew , Mullins John
Published: June 2021

We have used zinc finger nucleases (ZFNs) to target the rat Hsd11b2 gene by microinjection of ZFN mRNAs into single cell rat embryos. We generated multiple targeted alleles predicted to truncate the gene transcript. Having segregated the alleles by breeding, detailed metabolic and telemetric analyses of one line was undertaken.

The Hsd2ZFNΔ120 line carries a 120bp deletion, removing half of exon 2. Animals homozygous for the deletion show symptoms of the syndrome of apparent mineralocorticoid excess (SAME). They are polydypsic and polyuric from an early age, but have increased haematocrit suggesting volume depletion. The increase in Na+/K+ ratio on sodium loading (0.3% Na+ to 3% Na+) is blunted, compared to controls.

Homozygotes are hypertensive on 0.3% Na+ diet with a mean arterial blood pressure of 180mmHg (control MABP −115mmHg), and demonstrate a distinct lack of circadian rhythm (“non‐dippers”), though circadian rhythm for heart rate is normal. Sodium restriction (0.03% Na+) reduces BP to 140mmHg, but enhances its circadian rhythm. Meanwhile the concomitant tachycardia is sustained. We are currently investigating heart rate variability under different salt diet regimes. This rat model will extend experimental possibilities for exploring the mechanism underlying SAME.
A delivery system for protein and peptide‐based therapeutics to treat preeclampsia
Authors: Bidwell Gene Leflore , Liu Huiling , George Eric Matthew
Published: June 2021

Preeclampsia is one of the leading causes of maternal, fetal, and perinatal morbidity and mortality. There is currently no effective intervention for preeclampsia short of induced delivery of the fetus, which is why it is also a leading cause of premature birth. Failure of the maternal vasculature to adequately remodel during placental formation leads to a state of hypoxia/ischemia. In response, the placenta produces pathogenic factors, especially the VEGF antagonist sFlt‐1, inflammatory cytokines, and reactive oxygen species (ROS). Development of new therapies for preeclampsia, including therapies targeted to these pathways, has been hampered by dose‐limiting fetal toxicity. We have developed a drug delivery system capable of stabilizing therapeutic agents in the maternal circulation while excluding them from the fetal circulation. This system utilizes a polypeptide carrier based on Elastin‐like polypeptide (ELP) fused to therapeutic peptides or proteins specifically targeting each of the above mentioned pathways. Here we present in vitro evidence for the restoration of angiogenic activity with ELP‐delivered VEGF, inhibition of the inflammatory response with an NF‐κB inhibitory polypeptide, and inhibition of hypoxia‐induced ROS with a NAD(P)H oxidase inhibitory polypeptide. These data suggest the therapies could be a novel approach for preeclampsia management.
Alterations in placental TGF‐beta signaling pathways in rats with placental ischemia‐induced hypertension
Authors: Bauer Ashley J , Banek Christopher T , Regal Jean F , Gilbert Jeffrey S
Published: June 2021

Preeclampsia is a pregnancy‐specific condition characterized by new‐onset hypertension and decreased signaling by pro‐angiogenic factors such as transforming growth factor beta (TGF‐β). Although previous studies indicate the endogenous TGF‐β inhibitor soluble endoglin (sEng) is increased in preeclampsia and by placental ischemia, little is known about the effects of placental ischemia on other molecules in the TGF‐β signaling cascade such as the TGF‐β receptors, Smad2/3 and heat shock protein (hsp) 90. Therefore, we hypothesized that TGF‐β receptors (TGF‐βR)1, TGF‐βR2, hsp90 would be decreased in the reduced utero‐placental perfusion pressure (RUPP) model of placental ischemia‐induced hypertension in the rat. On day 14 of pregnancy (term=21), silver clips were placed on the inferior abdominal aorta and ovarian arteries to generate RUPP‐hypertension. On day 19, placentas were excised and placental TGF‐βR1, TGF‐βR2, sEng, pSmad2/3 and Hsp90 were measured by Western blot. Placental sEng and HSP 90 were increased (P<0.05) in RUPP compared to NP dams. Placental TGF‐βR2 was decreased (P<0.05) in RUPP compared to NP controls. Placental TGFβ‐R1 and pSmad2/3 were unchanged in RUPP compared to NP controls. These data indicate placental ischemia may decrease TGF‐β signaling via TGFβ‐R2 but not via Smad2 and 3 pathways. Increased HSP 90 expression may be an adaptive response to maintain TGF‐β signaling.
Tumor Necrosis Factor induces cerebral edema and increased cerebrovascular permeability in normal pregnant rats
Authors: Warrington Junie Paula , Ryan Michael J , Drummond Heather A , Spradley Frank T , Granger Joey P
Published: June 2021

Cerebrovascular events contribute to ~40% of preeclampsia/eclampsia deaths and preeclamptic patients commonly present with neurological symptoms. Our laboratory recently showed that placental ischemia, induced by reducing uterine perfusion pressure, closely mimics preeclamptic patients resulting in cerebral edema and increased inflammatory cytokines such as tumor necrosis factor (TNFα). In this study, we examined whether TNFα, infused via intraperitoneal implanted mini‐osmotic pumps (100ng/day from gestational day 14 to 19), contributes to cerebral edema and blood‐brain barrier (BBB) disruption in normal pregnant rats. Mean arterial pressure, measured via carotid catheters, was significantly increased in TNFα infused rats (112 vs. 107 mmHg; p=0.01). Brain water content, calculated as (wet weight ‐ dry weight)/ wet weight × 100, was also significantly increased in TNFα infused rats (78.2 vs. 77.9%; p=0.01). BBB permeability, assayed using the Evans blue extravasation method, was increased in the hippocampus of TNFα‐infused rats (p<0.05) but was unchanged in the striatum, cerebellum, and anterior brain. Our results indicate that TNFα released from ischemic placentas, could mediate cerebral edema through increased BBB permeability. TNFα may be an important clinical target for preventing cerebrovascular complications associated with preeclampsia.

Funding: NIH Grant HL108618
The renal functional response to Ang(1–7) infusion in DOCA‐salt hypertensive rats.
Authors: Barry Elaine , Johns Edward
Published: June 2021

Angiotensin 1–7 (Ang1–7) has natriuretic properties at the kidney that counterbalance the anti‐natriuretic actions of angiotensin II. This study investigated whether the renal actions of Ang1–7 are deranged in Deoxycorticosterone (DOCA) salt hypertensive states where the renin‐angiotensin system (RAS) is suppressed. Groups of anaesthetised male Wistar rats (n=6) were prepared for renal interstitial infusion at 1ml/h of Ang 1–7 (3×10–9 M) into the left kidney and cannulation of its ureter. Data, means±SEM were subject to one‐way ANOVA. Ang 1–7 had no effect on blood pressure, at 118mmHg for control rats and 153mmHg for DOCA Hypertensive rats. In control rats, urine volune (UV), significantly increased from 100.9±6.9 to139.1±4.5μl/min, sodium excretion (UVNa), from 16.0±1.3 to 24.3±1.0μmol/min and fractional sodium excretion (FENa) by 2.3±0.5 to 2.9±0.5% (all P<0.05). Intra‐renal infusion of the same dose of Ang 1–7 in the DOCA model had no effect on the excretory variables. The reasons for the lack of response to the Ang (1–7) are uncertain but one possibility is that the action of Ang(1–7) may correlate with the level of RAS activation.
Natriuresis induced by angiotensin II infusion is attenuated in knockout mice lacking tumor necrosis factor‐α receptors
Authors: Castillo Alexander , Majid Dewan S.A.
Published: June 2021

Angiotensin II (AngII) induces the production of tumor necrosis factor‐α (TNF‐α) which causes reduction in renal blood flow (RBF) as well as natriuresis. To assess the contribution of TNF‐α in the renal actions of AngII, the responses to AngII infusion (1 ng/min/g for 30 min, iv) were evaluated in anesthetized knockout mice lacking TNF‐α receptor type 1 (R1KO, n=6) and type 2 (R2KO, n=6) and compared these responses with those in wild type (WT, n=6) mice. Arterial pressure (AP) was recorded from a cannula placed in the carotid artery. RBF and glomerular filtration rate (GFR) were measured by PAH and inulin clearances respectively. Urine was collected from a catheter placed in the bladder. Basal mean AP was lower in R1KO (81±3 mmHg) but not in R2KO (90±4 mmHg) compared to WT (92±4 mmHg). AngII caused similar increases in AP (WT, 37±5%; R1KO, 38±4%; R2KO, 30±4%) in these mice. The AngII induced reductions in RBF (−39±5%) and GFR (−17±7%) in WT were also not significantly different in R1KO (−28±7% and −17±7%) and R2KO (−31±4% and −12±7%). However, despite similar increases in AP, AngII induced natriuresis was significantly attenuated in RIKO (Δ, 2.5±0.9 μmol/min/g) compared to that in WT (Δ, 6.0±0.9 μmol/min/g) and in R2KO (Δ, 4.1±0.4 μmol/min/g). These data indicate that the natriuresis induced by AngII infusion is, at least partly, mediated by concomitant production of TNF‐α acting mainly via the receptor type 1.
Evaluating the role of interstitial ang‐II and ATP in insulin induced hypoglycemia by chronic renal microdialysis
Authors: Prathipati Priyanka , Quadri Syed , Jackson Debra W , Jackson Keith E
Published: June 2021

Interstitial metabolic changes in organs can be monitored in real time by microdialysis, a minimally invasive method. Various endogenous and exogenous analytes such as ATP and angiotensin II (Ang‐II) can be continuously collected from multiple sites of the same animal without any fluid loss. Previous studies demonstrated the importance of ATP in the tubuloglomerular feedback (TGF) mechanism and few would argue the importance of AngII in the regulation of blood pressure. Emerging evidence has shown the relation between Ang‐II, heme oxygenase‐1(HO‐1) and blood pressure. Since ATP plays a paracrine role in regulating hemodynamic function, our current study was performed to evaluate the hypothesis that renal derived ATP plays a significant role in maintaining elevated AngII levels during hypertension. Microdialysis probes were inserted into the renal cortex of anesthetized male Sprague Dawley rats (200–225g). Post surgery rats were treated with insulin (7U/kg body weight) or vehicle for 2 weeks. Food and water intake were monitored daily and dialysate samples were collected after insulin dosing and analyzed for ATP and Ang‐II by luciferin‐luciferase assay and ELISA respectively. The present study is the first of its kind to adapt microdialysis for chronic collection of renal interstitial samples and to demonstrate the ability of hypoglycemia to promote the development of hypertension.(Support: BOR Comp 2010).
Clinical and Genetic Determinants of Blood Pressure Under Ambulatory Conditions on Days With and Without Acute Exercise
Authors: Ash Garrett Igo , Eicher John D. , Tsongalis Gregory J. , Thompson Paul D. , Pescatello Linda S.
Published: June 2021

OBJECTIVE

To identify clinical and genetic factors accounting for the variability in the BP response to acute bouts of dynamic exercise of varying intensity.

METHODS

48 men (X±SEM 43.9±1.4yr, 29.5±0.7kg•m−2) with hypertension [systolic (SBP)/diastolic (DBP) 145.7±1.5/85.7±1.1mmHg] completed 3 randomly assigned experiments: a cycle exercise bout at LIGHT and VIGOROUS intensity and a CONTROL session of seated rest. Subjects left the laboratory wearing an ambulatory BP monitor. We obtained blood samples for the fasting cardiometabolic profile and genotyping. Multiple variable regression tested correlates of the daytime ambulatory BP response following exercise and CONTROL.

RESULTS

Clinical (30/18%) and genetic (0/6%) factors accounted for 30/24% of the variability in the SBP/DBP response following LIGHT (p=0.01); 27/52% [clinical (13/31%) & genetic (14/21%)] in the SBP/DBP response following VIGOROUS (p=0.01); and 18/29% [clinical (18/29%) & genetic (0/0%)] in the SBP/DBP response following CONTROL (p=0.02).

CONCLUSION

Our findings indicate that for every 10% increase in the intensity of a bout of acute dynamic exercise, the genetic contribution accounting for the variability in the SBP/DBP response increased by 1.2/2.0% (R2=0.779, p=0.08/R2=0.967, p=0.01), respectively. No such trends were found with clinical factors.

Supported by American Heart Association Grant‐in‐Aid # 0150507N.
Angiotensin Receptor Blocker (ARB) prevents albuminuria and cardiac hypertrophy but does not protect the cuffed kidney in murine 2K1C hypertension
Authors: Hartono Stella , Knudsen Bruce E , Grande Joseph P
Published: June 2021

Despite proven efficacy, ARB therapy reduces glomerular filtration in patients with unilateral renal artery stenosis (RAS). The objective of this study was to determine whether ARB exacerbates renal injury in 2K1C hypertension. RAS was established in mice by placement of a cuff on the right renal artery, which reduces blood flow by 70%. Both Hydralazine (H) and Valsartan (ARB) reduced blood pressure to a similar extent. RAS‐mediated cardiac hypertrophy (heart weight 134% sham (S) p<0.001) was prevented by ARB (102% S, p=ns) but not H (116% S, p=0.03); cardiac fibrosis was prevented in both ARB and H. Although ARB did not exacerbate renal atrophy (91.4 ± 2.1% atrophy vehicle (V) vs 75.3 ± 9.5% ARB, p=ns), H reduced renal atrophy (91.4 ± 2.1% V vs 28.4 ± 13.5% H, p<0.01). The contralateral kidney underwent compensatory hyperplasia (123% S; p<0.001), which was blunted with ARB or H treatment. No significant histopathologic alterations were observed in the contralateral kidneys of any group. RAS induced proteinuria in V mice. Urine protein excretion in ARB RAS mice was reduced to levels observed in S mice; H did not significantly alter urine protein excretion. Severe renal atrophy was observed in the cuffed kidneys of V mice, which was not exacerbated by ARB. However, ARB reduced proteinuria to baseline levels, indicating that ARB may protect overall renal function in unilateral RAS.

These studies were supported by NIH grant P01 HL85307.
Using Drosophila to identify inhibitors of oxalate kidney stone formation
Authors: Tanamoto Nana , Hirata Taku , Cabrero Pable , Lieske John C , Ritman Erik L , Dow Julian AT , Romero Michael F
Published: June 2021

Background

Kidney stones are an expensive and painful health problem with calcium oxalate (CaOx) being 70% of cases. We developed a Drosophila model of CaOx stones [Hirata et al., AJP‐Renal Physiol, 2012]. This work showed that dPrestin (Slc26a5/a6) exchanges Cl− for oxalate (Ox). Thiosulfate (S2O3) and tannic acid (TA) are implicated to inhibit CaOx crystals in supersaturated urine. Here, we report transporter, renal tubule and whole body effects of S2O3 and TA on crystal formation.

Methods

S2O3 and TA transport effects were examined by voltage clamping of dPrestin‐expressing Xenopus oocytes. Cis‐inhibition of oxalate transport was also examined in (a) dissected Malpighian tubules (MT) or flies fed ox‐enriched food plus S2O3 or TA. MT CaOx crystals were viewed by DIC, or whole flies by microCT.

Results

dPrestin transports S2O3 but not TA, yet both inhibited Ox transport (Ki(S2O3)~10mM; Ki(TA)~100μM). S2O3 competitively inhibits while TA blocks dPrestin. MT treated with S2O3 or TA ↓ crystal formation compared to MT soaked in Ox only. Flies fed with S2O3 or TA also showed ↓ CaOx in MTs.

Conclusions

S2O3 and TA are both inhibitors of CaOx crystallization. These experiments show that these inhibitors have distinct mechanisms of action. Our Results indicate that (a) this Drosophila model can be used as a chemical screening assay for CaOx stone prevention and (b) TA, due to low Ki, might be a reasonable therapy for certain CaOx stones patients.[DK83007, DK92408]
Src‐family protein tyrosine kinase (SFK) stimulates KCNJ10 K channels in the basolateral membrane of distal convoluted tubules (DCT).
Authors: Wang Wen‐Hui , Zhang Cheng‐Biao , Lin Dao‐Hong , Yue Peng , Wang Lijun , Rinehart Jesse
Published: June 2021

The basolateral K channels in the DCT are composed of KCNJ10 and KCNJ16. Since KCNJ10 have several SFK‐consensus phosphorylation sites (Tyr 8, 9, 51 & 349), we tested the role of tyrosine phosphorylation in regulating KCNJ10. The patch‐clamp experiments were performed in HEK cells transfected with GFP‐tagged KCNJ10 and in the mouse DCT1. Suppression of SFK with PP1 reversibly inhibited the basolateral 40 pS K channels in cell‐attached mode and decreased the whole cell K currents in DCT1 cells. Western blot shows that KCNJ10 is a tyrosine phosphorylated protein. GC‐MASS confirmed that Tyr 8 & 9 were phosphorylated in KCNJ10 protein harvested from HEK293 cells. Mutation of Tyr 9 to phenyalanine decreased the whole cell K currents by 60% while mutations of Tyr 8, 51 and 349 had no significant effect on the whole‐cell K currents. To examine the physiological role of tyrosine phosphorylation of KCNJ10 in DCT1, we measured the whole‐cell K currents in the DCT1 from mice on a high K diet (HK) which is know to decrease SFK activity. The whole cell K current in DCT1 of mice on HK was significantly smaller than those of control animals. We conclude that Tyr 9 is a tyrosine phosphorylation site and that dephosphorylation of Tyr9 inhibited the KCNJ10 activity. We speculate that a decrease in basolateral K channels in DCT1 in mice on HK diminished the basolateral Cl exit thereby decreasing NaCl transport in DCT1.
Flow‐sensitive K+‐coupled ATP secretion modulates ENaC activity in the distal nephron
Authors: Bugay Vladislav , Sansom Steven , Wen Donghai , Hatcher Lori , Stockand James , Mironova Elena
Published: June 2021

The epithelial Na+ channel (ENaC) in the aldosterone‐sensitive distal nephron (ASDN) is under tonic inhibition by paracrine purinergic signaling responding to changes in dietary sodium intake. Normal BK(Ca) channel function is required for ATP secretion in the ASDN. We test here whether ATP secreted through connexin channels in a coupled manner with K+ efflux through BK(Ca) channels is required for inhibitory purinergic regulation of ENaC in response to increases in sodium intake. Inhibition of connexin relieves purinergic inhibition of ENaC. Deletion of the BK‐β4 regulatory subunit, which is required for normal channel function and ATP secretion, mutes sodium intake‐dependent increases in urinary ATP resulting in inappropriately elevated ENaC activity. ENaC in BK‐β4 null mice, though, responds normally to exogenous ATP consistent with lower urinary ATP causing elevated activity. Moreover, disruption of purinergic regulation increases ENaC activity in wild type but not null mice. Consequently, sodium excretion is impaired in null mice. These results demonstrate that ATP secreted into the ASDN in a BK(Ca) channel‐dependent manner is physiologically available for inhibition of ENaC in response to changes in sodium intake. Impaired sodium excretion resulting form the loss of normal inhibitory purinergic control of ENaC likely contributes to the elevated blood pressure of BK‐β4 null mice.
Flow stimulates inner medullary collecting duct endothelin‐1 production
Authors: Pandit Meghana , Gusella Gabriele L , Rohatgi Rajeev , Kohan Donald E
Published: June 2021

Collecting duct (CD)‐derived endothelin‐1 (ET‐1) inhibits Na and water reabsorption; its deficiency causes marked hypertension. CD ET‐1 synthesis is enhanced by extracellular fluid volume expansion. In cultured cortical CD (CCD) cells, increased flow (as would occur in tubule fluid during volume expansion) stimulates ET‐1 production; this is ENaC‐dependent. Since the inner medullary CD (IMCD) is the major site of renal ET‐1 synthesis, we examined the effect of flow on IMCD ET‐1 production. Mouse IMCD3 cells were subjected to static conditions or flow (2 dyne/cm2 for 2 hr), followed by determination of ET‐1 mRNA. Flow increased ET‐1 mRNA by 2.2‐fold. Absence of perfusate Ca prevented the flow response. BAPTA, W‐7, calmidazolium, KN‐93, calphostin C and cyclosporin A reduced the ET‐1 flow response, indicating that PKC and Ca/calmodulin/calmodulin kinase/calcineurin pathways are essential for the flow response. Amiloride or benzamil did not affect the ET‐1 response to flow. Increasing perfusate osmolality to 450 mOsm with NaCl, mannitol or urea elicited a marked flow response (4.4‐fold increase in ET‐1 mRNA). Removal of cilia with chloral hydrate reduced the flow response, while flow failed to stimulate ET‐1 mRNA in mIMCD3 cells deficient in polycystin‐2. These data suggest that IMCD ET‐1 synthesis is stimulated by tubule fluid flow via increased solute delivery and possibly cilia deformation which in turn activates PKC‐ and Ca‐dependent pathways. We propose that Na delivery stimulates CCD ET‐1 which, in turn, inhibits CCD Na reabsorption, while solute and water delivery stimulate IMCD ET‐1 which, in turn, inhibits IMCD water and urea reabsorption.
Feedback inhibition of the epithelial Na+ channel (ENaC) during acute sodium loading in vivo
Authors: Patel Ankit Bharat , Frindt Gustavo , Palmer Lawrence G.
Published: June 2021

The in vivo role of feedback inhibition of ENaC by intracellular [Na+] ([Na+]i) was studied by investigating the effect of amiloride during an acute sodium load to Na+‐deprived rats in the presence of an aldosterone mini‐pump. Amiloride blocks sodium entry into principal cells of the cortical collecting ducts (CCD) and presumably decreases [Na+]i. ENaC activity was evaluated by measuring amiloride‐sensitive currents (INa) in split‐open CCDs using whole‐cell patch clamp. ENaC surface expression was assessed by an in vivo biotinylation protocol. A four‐hour acute sodium load decreased INa by 70%. The presence of amiloride did not significantly change INa after an acute sodium load. Whole‐kidney expression of cleaved α, β, and cleaved γ ENaC decreased with an acute sodium load by 2.6, 3.4, and 6.2 fold, respectively. In addition, cell‐surface expression of β and cleaved γ ENaC decreased with an acute sodium load by 2.5 and 4.7 fold, respectively. Amiloride increased whole‐kidney expression of cleaved α and cleaved γ ENaC after an acute sodium load by 2.1 and 3.0 fold, respectively, and cell‐surface expression of β and cleaved γ ENaC by 2.2 and 5.2 fold, respectively. These results suggest that [Na+]i can regulate ENaC expression in vivo but changes in factors other than [Na+]i and aldosterone are responsible for down regulating ENaC activity in response to an acute sodium load.

NIH RO1‐DK59659 (LGP) and T32‐GM07739 (ABP).
ASK1 and p38 MAP kinase regulate proteolytic processing of ENaC
Authors: Cook David Ian , Lee Il‐Ha , Song Sung‐Hee , Dinudom Anuwat
Published: June 2021

These Ussing chamber studies were undertaken to investigate the mechanism by which hydrogen peroxide (0.5 mM) inhibits mouse epithelial Na+ channels (ENaC) transfected into FRT cell monolayers.

We found the inhibition was blocked by expression of a dominant negative construct of the kinase ASK1, and was replicated by expression of a constitutively active mutant of ASK1. The effects of both hydrogen peroxide and of the constitutively active mutant of ASK1 were blocked by the p38 MAP kinase inhibitor, SB203580, and by expression of a dominant negative mutant of p38 MAP kinase.

Western blotting with an antibody for phosphorylated p38 MAP kinase confirmed that ASK1 modulates the amount of phosphorylated p38 MAP kinase. Epithelial Na+ channels are regulated by the Nedd4–2 system that controls their surface expression. We found, using surface biotinylation methods, however, that neither ASK1 nor p38 MAP kinase affected surface expression of the channels.

Epithelial Na+ channels are normally activated by proteolysis of the α‐ and γ‐subunits during their intracellular processing. When we used ENaC subunit constructs in which the N‐ and C‐terminals are labelled with V5 or FLAG tags to examine the effect of the kinases on proteolytic processing, we found that they inhibit proteolytic cleavage of both α‐ and γ‐ENaC.

In summary, we have found that hydrogen peroxide inhibits epithelial Na+ channels expressed in FRT cell monolayers via the kinases ASK1 and p38 MAP kinase, which reduce the activity of the channels by inhibiting the normal activation of the channels by proteolysis.
Activation of rat and human organic anion transporter 1 and 3 by transcription factor B‐cell CLL/lymphoma 6 (BCL6)
Authors: Henjakovic Maja , Wegner Waja , Burckhardt Birgitta C. , Burckhardt Gerhard
Published: June 2021

In rat kidneys, organic anion transporter 1 (Oat1) and Oat3 are male‐dominant and testosterone‐dependently expressed. Sex‐differences in the secretion of several drugs known to be substrates of human OAT1 and OAT3 suggest a male‐dominant expression of these transporters also in humans. The aim of this study was to identify the regulators of sex‐dependent rat Oat1 and Oat3 transcription and the effect of these regulators on human OAT1 and OAT3 promoters.

RNA was isolated from renal cortical slices of four adult rats per sex. Expression profiling was done using SurePrint G3 rat GE 8×60K microarray. Selected candidate genes were verified using real‐time PCR. Luciferase assays were carried out by transient transfection of opossum kidney (OK) cells with promoter fragments and expression vectors.

Our results suggest that the male‐dominant expression of Oat1 and Oat3 is not directly regulated by the testosterone/androgen receptor‐complex. The human OAT1 and OAT3 promoters were also not affected by testosterone. The transcription factor B‐cell CLL/lymphoma 6 (BCL6) was male‐dominantly expressed and activated the promoters of Oat1/Oat3 and OAT1/OAT3, independent of predicted BCL6‐binding sites, possibly mediated by the interaction with other transcription factors.

BCL6 constitutes a promising candidate gene for the sex‐dependent regulation of rat Oat1/Oat3 and human OAT1/OAT3 transcription.
Signaling pathways regulating renal proximal tubular endocytosis under proteinuric conditions
Authors: Rajapakse Angana , Heim YuYuan , Scolari Brigitte , Matthey Patricia , Uldry Danièle , Theilig Franziska
Published: June 2021

Receptor‐mediated endocytosis is a pivotal function of the renal proximal tubule (PT) to reabsorb proteins from the ultra filtrate. PT dysfunction occurring in nephrotic syndrome results in an impairment of this process. The regulation of endocytosis and signaling pathways involved remain unknown. We aimed to identify whether low (0.1mg/ml) vs. high albumin concentration (10mg/ml) affect endocytosis and to uncover responsible signaling pathways. OK‐cells incubated with 0.1mg/ml albumin significantly augmented receptor‐mediated endocytosis at 1h and 4h. In contrary, incubating OKC with 10mg/ml albumin significantly diminished endocytosis after 30 min till 4h. Analyses of the expression pattern and phosphorylation status of the kinases including PI3K, Akt, TORC1/‐2, WNK1/OSR1 revealed that the WNK1‐induced pathway shows strongest changes upon low vs. high albumin concentrations. Transient transfection of OKC using WNK1, KD‐K233M‐WNK1, OSR1 and KD‐D164A‐OSR1 strongly inhibited endocytosis to 2.8±1.1*; 6.7±1.0*; 2.8±0.5*; and 7.4±2.9*; *P<0.05, respectively, suggesting a regulation by protein‐protein interaction. In rat kidney, IHC of WNK1 and OSR1 demonstrated a strong expression in the BBM of PT, supporting a role in endocytosis. We conclude that WNK1 and OSR1 are the main kinases activated upon high protein concentrations leading to consequent impairment of receptor‐mediated endocytosis.
An interaction between Bcl‐XL and VDAC facilitates mitochondrial Ca2+ uptake and bioenergetics
Authors: Huang Huiya , Hu Xiangxin , Li Chi , White Carl
Published: June 2021

The role of the anti‐apoptotic protein Bcl‐XL in regulating mitochondrial Ca2+ ([Ca2+]mito) handling was examined in wild‐type (WT) and Bcl‐XL knockout (KO) cells. Upon application of InsP3‐ generating agonists to evoke cytosolic Ca2+ ([Ca2+]cyto) transients, [Ca2+]mito uptake was larger in WT cells compared to KO. This was correlated with higher steady‐state ATP levels in intact WT cells compared to KO cells, and chelation of cytosolic Ca2+ or uniporter knockdown reduced [ATP] to the same level in both cells. In permeabilized cells, stepping external [Ca2+] from 0 to 3 μM also produced a larger [Ca2+]mito uptake in the WT, and the [Ca2+]mito uptake capacity of KO cells was restored only by re‐expression of mitochondrially but not ER‐targeted Bcl‐XL. Bcl‐XL enhancement of [Ca2+]mito uptake persisted after dissipation of the mitochondrial membrane potential, but was absent in mitoplasts lacking an outer mitochondrial membrane. The outer membrane‐localized voltage‐dependent anion channel (VDAC) is a known Ca2+ permeability that directly interacts with Bcl‐XL. Application of peptides based on the VDAC1 sequence disrupted Bcl‐XL‐VDAC interactions and reduced [Ca2+]mito uptake and ATP levels in the WT but not in KO cells. Taken together, these data suggest that Bcl‐XL facilitates mitochondrial Ca2+ loading and bioenergetics by regulating VDAC Ca2+ permeability.
Bestrophin‐ and TMEM16A‐associated Cl‐ conductances in rat mesenteric small arteries
Authors: Matchkov Vladimir V. , Dam Vibeke Secher , Bødtkjer Donna , Aalkjaer Christian
Published: June 2021

The presence of Ca2+‐activated Cl− channels in the vascular smooth muscle cells (VSMCs) is well established, but their molecular identity is still controversial. Bestrophins and TMEM16 proteins are the most prominent candidates. We have previously characterized a cGMP‐dependent Ca2+‐activated Cl− current (ICl(Ca)) with unique characteristics which is present in VSMCs simultaneously with a classical ICl(Ca). We aimed to study the role of TMEM16A and bestrophin‐3 in the VSMC and downregulated these proteins in 2nd order branches of rat mesenteric arteries in vivo using siRNA. Knockdown was confirmed at mRNA and protein levels 3 days after transfection. The downregulation of bestrophin‐3 induced secondary decrease in bestrophin‐1 and ‐2. The currents were validated by patch clamp and arteries were tested in isometric myograph. In contrast to bestrophin downregulation, which resulted in suppression of the cGMP dependent ICl(Ca), TMEM16A‐siRNA suppressed both the cGMP‐dependent and the classical ICl(Ca) currents. Downregulation of TMEM16A or bestrophins significantly suppressed the amplitude of agonist‐induced rhythmic arterial contractions – vasomotion. The downregulation of bestrophins was without effect on agonist‐induced tonic contraction. In contrast, arteries downregulated for TMEM16A had suppressed contractility to agonist (noradrenaline and vasopressin) and extracellular K+ depolarization. We conclude that both bestrophins and TMEM16A are essential for vasomotion in mesenteric small arteries but only TMEM16A is involved in agonist‐induced tonic contraction. Our results suggest modulatory interactions between these proteins at the expressional and functional levels.