bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of SIRT1 Attenuates Klotho Deficiency‐induced Arterial Stiffness and Hypertension via Enhancing AMPKα and eNOS activity
Authors: Gao Diansa , Zuo Zhong , Ali Quaisar , Tian Jing , Lei Han , Lin Yi , Sun Zhongjie
Published: November 2023

Objective

Haplodeficiency of klotho causes arterial stiffness and hypertension. W found that Klotho, an aging‐suppressor protein, was significantly decreased in serum of patients with arterial stiffness and hypertension. Deficiency of klotho induces arterial stiffness and hypertension. This study was designed to investigate the underlying mechanism.

Methods & Results

Klotho mutant heterozygous (+/−) mice and wild‐type (WT) mice were used. Notably, expression and activity of SIRT1, an important deacetylase, were significantly decreased in aortas of KL (+/−) mice which coincided with elevated pulse wave velocity (PWV) and blood pressure (BP). Treatment with SRT1720 (15mg/kg per day IP), a specific SIRT1 activator, abolished klotho deficiency‐induced arterial stiffness and hypertension in KL (+/−) mice. Interestingly, Klotho deficiency was associated with decreases in the activity of AMP‐activated protein kinase (AMPKa) and endothelial nitric oxide synthase (eNOS) in aortas which can be abolished by SRT1720. Furthermore, klotho deficiency upregulated NADPH oxidase activity and superoxide production, increased collagen expression, and decreased elastin expression in the media of aortas. These klotho deficiency‐related changes were blocked by SRT1720.

Conclusion

This study provided the first evidence that Klotho deficiency‐induced arterial stiffness and hypertension via downregulation of SIRT1. Pharmacological activation of SIRT1 may be an effective therapeutic approach for arterial stiffness and hypertension.

Support or Funding Information

Supported by NIH R01 HL118558, DK093403, HL105302, and HL102074.
Infarct Size Reduction by Remote Ischemic Perconditioning in Pigs Requires Activation of the SAFE but not the RISK Pathway
Authors: Kleinbongard Petra , Gent Sabine , Skyschally Andreas , Heusch Gerd
Published: November 2023

Brief episodes of ischemia/reperfusion in a tissue or organ remote from the heart can reduce myocardial infarct size following sustained severe ischemia/reperfusion. Such remote conditioning can be induced before (pre‐) or during (per‐) myocardial ischemia, and protection by remote conditioning was confirmed in all species tested so far, including humans. In pigs, cardioprotection by remote preconditioning causally involves the activation of the survival activating factor enhancement (SAFE) pathway, whereas the activation of the reperfusion injury salvage kinase (RISK) pathway is not necessary (Circ Res 2015;117:279–88). We have now used a clinically relevant pig model to identify the myocardial signal transduction of remote ischemic perconditioning (RPERC).

Anesthetized open‐chest pigs were subjected to 60 minutes occlusion of the left anterior descending coronary artery (LAD), followed by 180 minutes reperfusion. RPERC (n=10) was induced by 4×5/5 minutes hindlimb ischemia/reperfusion during the last 40 minutes of ischemia. Placebo pigs (PLA; n=10) without hindlimb ischemia/reperfusion served as controls. Regional myocardial blood flow was measured with colored microspheres. The area at risk (AAR) was delineated by atrial injection of 5 ml blue dye after LAD re‐ligation at the end of the experiment. Infarcted tissue was demarcated by TTC staining and calculated as % of the AAR. Myocardial biopsies were taken before LAD occlusion (baseline) and at 10 min reperfusion from the AAR to determine changes in the phosphorylation of protein kinase B and extracellular signal‐regulated kinase 1/2 as the main RISK pathway components and the phosphorylation of signal transducer and activator of transcription 3 as the central SAFE pathway component by Western blot. In additional experiments, RPERC was performed in the presence of either RISK‐blockade (Wortmannin/U0126; n=4) or SAFE‐blockade (AG490; n=4).

Residual myocardial blood flow during ischemia and AAR were not different between groups. RPERC reduced infarct size to 16±10% vs. 42±10% in PLA (mean±SD; p<0.05; t‐test). RPERC increased the phosphorylation of STAT3 at 10 min reperfusion (p<0.05), whereas PLA did not. The SAFE‐blockade abolished both, the increased phosphorylation of STAT3 and the protection by RPERC (infarct size 36±5%; n.s. vs. PLA). In both groups phosphorylation of RISK was increased from baseline to 10 minutes reperfusion. RISK‐blockade abolished this increase, but RPERC still protected the myocardium (infarct size 8±1%; p<0.05 vs. PLA).

The present experiments confirm the causal involvement of STAT3 activation during cardioprotection by RPERC in pigs, whereas an activation of RISK components is not involved. Thus, cardioprotection by RPERC is characterized by the same signal transduction pattern as that of remote ischemic preconditioning and local ischemic postconditioning.
The Cardioprotective Effects of Caffeic Acid Phenethyl Ester (CAPE) on Myocardial Ischemia/Reperfusion (I/R) Injury
Authors: Castellano Andrew J. , Kuhn Tarah , Liu Stephanie , Kucharski Kevin , Venditto Joseph , Kuo Daniel , Barsotti Robert , Young Lindon H. , Chen Qian
Published: November 2023

Oxidative stress plays a major role in causing reperfusion injury following prolonged ischemia. CAPE has been shown to have antioxidant, anti‐inflammatory, and cardioprotective effects following regional myocardial ischemia/reperfusion injury. The effects and mechanisms of CAPE in global myocardial I/R injury are still unclear. In this study, CAPE was tested in isolated perfused rat hearts following global I (30 min)/R (60 min). We recorded left ventricular developed pressure (LVDP), left ventricular end systolic pressure (LVESP), the peak of the first derivative of left ventricular pressure (dP/dtmax), and infarct size. We found that untreated I/R hearts (n=11) recovered LVDP to 45 ± 8% (p<0.05), LVESP to 106 ± 7% (p<0.05), and dP/dtmax to 33 ± 5% (p<0.05) of baseline values, respectively, at the end of 60 minutes reperfusion. By contrast, CAPE (40 μM, n=6) given at reperfusion for 5 minutes significantly restored LVDP to 75 ± 15%, LVESP to 133 ± 13%, and dP/dtmax to 54 ± 12% of baseline values, respectively (all p<0.05). Moreover, CAPE also significantly reduced infarct size to 19 ± 2% (n=6) compared to 30 ± 6% in untreated I/R hearts (n=11, p<0.05). Additionally, a non‐selective nitric oxide synthase inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME, 50 μM, n=5), or a heme oxygenase‐1 inhibitor, SnPP (20 μM, n=5), significantly abolished all the cardioprotective effects of CAPE (all p<0.05). These results suggest that CAPE, when given at reperfusion, improves post‐reperfused contractile function and reduces infarct size. The cardioprotective effects may be mediated by increasing nitric oxide bioavailability and/or heme oxygenase activity.

Support or Funding Information

This study was supported by Division of Research and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.
Cardiovascular Susceptibility to Acute Myocardial Ischemic Injury in Male And Female Rat Offspring Born Growth Restricted
Authors: Shah Amin , Matsumura Nobutoshi , Morton Jude Sandra , Dyck Jason , Davidge Sandra Thomas
Published: November 2023

Background

Ischemic heart disease causes the greatest mortality in the world today. Epidemiological and animal studies have shown that populations who suffer intrauterine growth restriction (IUGR), one of the most common consequences of complicated pregnancies, are more susceptible to develop cardiovascular disorders in later life. We tested the hypothesis that adult male and female IUGR rat offspring are more susceptible to develop cardiac dysfunction following acute myocardial ischemic injury.

Methods

Male and female control or IUGR rat offspring (created by exposing pregnant dams to hypoxia, 11.0±0.5% O2, for the last third of pregnancy) were randomly assigned to undergo sham or myocardial infarction (MI) surgery (by ligation of the left anterior descending coronary artery) at the age of 12 weeks. Thus the experimental groups consisted of: Control‐sham, Control‐MI, IUGR‐sham, and IUGR‐MI. One week after MI surgery, echocardiography was performed to assess cardiac morphometry, systolic function and diastolic function.

Results

Female, but not male, IUGR offspring had lower body weight compared to controls at 12 weeks of age post 1 week of MI. Anterior left ventricular (LV) wall thickness was decreased whereas LV internal diameter during diastole was significantly increased in both control‐MI and IUGR‐MI groups independent of sex or IUGR; indicating wall thinning and LV dilatation due to MI. Ejection fraction, fractional shortening and fractional area change were significantly decreased in both control‐MI and IUGR‐MI groups in male and female offspring. The Tei index was significantly increased in both control‐MI and IUGR‐MI male groups. Interestingly, E/E’ ratio, an index of ventricular filling pressure, was significantly increased in only male IUGR‐MI group compared to IUGR‐Sham (IUGR‐Sham vs IUGR‐MI: 19.84 ± 2.13 vs 35.21 ± 3.66, P<0.05) and control‐MI (Control‐MI vs IUGR‐MI: 22.37 ± 1.73 vs 35.21 ± 3.66, P<0.01).

Conclusion

Ischemic heart disease such as MI was shown to alter LV wall morphometry leading to LV dilatation in both sexes. MI impaired systolic function independent of being born growth‐restricted or sex. However, male IUGR offspring were more susceptible to develop diastolic dysfunction following acute MI. These studies indicate a susceptibility of the male IUGR population to develop diastolic heart failure due to acute myocardial ischemia.

Support or Funding Information

This work is funded by grants from the Canadian Institutes of Health Research (CIHR); and the Women and Children's Health Research Institute (WCHRI) through the generous contributions of the Stollery Children's Hospital Foundation (SCHF) and the Royal Alexandra Hospital Foundation (RAHF). A shah is supported by fellowships from the Heart and Stroke Foundation of Canada.
Membrane Potential Controls Surface Expression of BK Channel Auxiliary β1 Subunits in Arterial Smooth Muscle Cells
Authors: Leo M. Dennis , Bulley Simon , Kuruvilla Korah P. , Jaggar Jonathan H
Published: November 2023

Intravascular pressure stimulates arterial depolarization, leading to smooth muscle cell (myocyte) voltage‐dependent calcium (Ca2+) channel activation and vasoconstriction. Membrane depolarization also activates BK channels, which oppose pressure‐induced depolarization and constriction, but mechanisms involved are unclear. We have previously shown that nitric oxide stimulates rapid surface expression of β1 subunits in arterial myocytes. These β1 subunits activate surface‐resident BK channels, leading to vasodilation. Here, we tested the novel hypothesis that membrane potential controls β1 trafficking to regulate BK channel activity. Using techniques, including surface biotinylation, immuno‐FRET microscopy, western blotting and gene knockdown, we show that membrane depolarization (30–60 mM K+ PSS) rapidly (<10 min) increased the surface abundance of BK channel auxiliary β1 subunits in cerebral artery myocytes. Depolarization‐induced β1 trafficking was inhibited by: 1) brefeldin A, a trafficking inhibitor, 2) knockdown of rab11A, a small GTPase associated with recycling endosomes, 3) rab11A dominant negative mutant overexpression, 4) nimodipine, a CaV1.2 channel blocker, 5) HA‐1100, a rho kinase (ROCK) inhibitor, but not by KN93, a Ca2+/calmodulin‐dependent protein kinase inhibitor, or LY294002, a specific PI3 kinase inhibitor, 6) ROCK2, but not ROCK1, knockdown. HA‐1100 also reduced GTP‐bound active rab11 in arteries. These data indicate that membrane depolarization stimulates surface trafficking of β1 through a mechanism that involves voltage‐dependent Ca2+ channel and ROCK2 activation in arterial myocytes.

Support or Funding Information

RO1 HL67661, HL110347, HL094378 to JHJ and American Heart Assoc.‐SDG to MDL
Leucine Rich Repeat Containing 8A Is Required For NADPH Oxidase 1‐Dependent Superoxide Production and Tumor Necrosis Factor Alpha Receptor Endocytosis
Authors: Choi Hyehun , Rohrbough Jeffrey , Dikalova Anna , Nguyen Hong N , Lamb Fred S
Published: November 2023

Tumor necrosis factor‐α (TNFα) induces superoxide (O2−•) production by NADPH oxidase 1 (Nox1) and activates volume‐regulated anion channel (VRAC) in vascular smooth muscle cells (VSMC). The Leucine Rich Repeat Containing 8A (LRRC8A) protein is part of VRAC, which supports cell proliferation and inflammation. We hypothesized that LRRC8A supports TNFα signaling. Reduction of LRRC8A expression (siLRRC8A) or inhibition of VRAC (DCPIB 30μM) inhibited responses to TNFα including; VRAC and NF‐kB activation (72% decrease), iNOS (52% decrease) and VCAM (36% decrease) expression, and proliferation (siControl 100%, siControl+TNFα 111±1.5%, siLRRC8A+TNFα 101±1.6%). siLRRC8A also reduced extracellular O2−• production (siControl+TNFα 1259±80, siLRRC8A+TNFα 792±86 pmol/mg of protein). Biotinylated TNFα was used to visualize TNFα receptor (TNFR) endocytosis with FITC‐conjugated avidin. TNFR, but not transferrin receptor endocytosis was impaired by siLRRC8A and DCPIB. Extracellular superoxide dismutase (500 U/mL) also inhibited TNFR endocytosis, while catalase (1000 U/mL) did not. LRRC8A co‐immunoprecipitated with the p22phox subunit of Nox1 suggesting a direct functional link to activity of the Nox1 complex. Thus, LRRC8A is required for Nox1‐mediated O2−• production, and extracellular O2−• is required for TNFR endocytosis. These data provide a novel mechanism for the anti‐proliferative and anti‐inflammatory effects of VRAC inhibition.
Leptin, Melanocortin 4 receptor and Renal Nerves Play a Role in High Blood Pressure Programmed by Intrauterine Growth Restriction in Mouse
Authors: Intapad Suttira
Published: November 2023

Intrauterine growth restriction (IUGR) is a risk factor for hypertension and cardiovascular disease in later life. However, the underlying mechanisms remain unclear. We previously showed that male mouse IUGR offspring exhibit high blood pressure and adiposity compared to control offspring. Increased circulating adipocyte‐derived leptin activates the brain melanocortin system, in particular the melanocortin 4 receptor (MC4R); these key factors increase renal sympathetic nerve activity and result in elevated blood pressure. Nonetheless, the effects of the leptin‐MC4R‐renal nerve pathway on blood pressure (BP) in IUGR is unknown. In the present studies, we tested the hypothesis that the leptin‐MC4R‐renal nerve pathway plays a role on the high BP observed in male IUGR mice. To determine the contribution of leptin and renal nerve activity to hypertension in male IUGR offspring, Pregnant C57BL/6J female mice underwent sham or reduced uterine perfusion (RUP) surgery at day 13 of gestation; pregnancy was allowed to come to full term. Offspring from RUP dams were IUGR; progeny from sham surgeries were used as controls. IUGR offspring had lower birth weights than controls (P<0.01). Mean arterial pressure (MAP) was measured via carotid catheter in conscious adult (24 weeks old) male control and IUGR mice with or without bilateral renal denervation (RDV) for 2 weeks. IUGR offspring had a higher BP compared to controls (125.0±3.7 vs 112.1±2.1 mmHg; n=7, P<0.05). RDV did not alter MAP of control mice (110.7±1.8 mmHg; n=3) but significantly decreased MAP of IUGRs (115.0±2.0 mmHg; n=4). Serum leptin levels were significantly (P<0.05) increased in IUGR (28.1±1.9 ng/ml; n=5) compared to controls (12.6±3.1 ng/ml; n=5). We also assessed the role of MC4R on BP of IUGR mice. Male and female MC4R+/−breeders were used to generate MC4R+/+ and MC4R−/− littermates to avoid the undesirable effects of obesity in MC4R−/− dams. RUP induction in pregnant MC4R+/−dams and MAP measurements of their offspring were conducted as aforementioned. IUGR MC4R+/+ mice had a significantly higher (P<0.05) MAP (135.0±4.0 mmHg; n=4) than control MC4R+/+ animals (120.0±5.3 mmHg; n=4). MAP of control MC4R +/− (n=6) and MC4R −/− (n=3) were not significantly different from control MC4R+/+. MAP of IUGR MC4R +/−(125.0±7.0 mmHg; n=5) was slightly lower but not significantly different from IUGR MC4R+/+. However, IUGR MC4R−/− showed a significant decrease (P<0.05) in MAP (117.0±6.7 mmHg; n=3) compared to IUGR MC4R+/+. Together our data suggests that leptin, renal nerves, and MC4R are involved in IUGR programs an increase blood pressure in mice. Thus, the leptin‐MC4R‐renal nerve pathway is a putative mechanism underlying the hypertension of IUGR offspring.

Support or Funding Information

This work was supported by COBRE pilot grant GM104357 from National Institutes of Health
Gene Variants in Sodium Bicarbonate Cotransporter NBCe2 Increase Sodium and Bicarbonate Transport in Human Renal Proximal Tubule
Authors: Gildea John , Carlson Julia M , Tran Hanh T , Xu Peng , Kemp Brandon A , Langouët‐Astrié Christophe J , Carey Robert M , Jose Pedro A , Felder Robin A
Published: November 2023

Rationale

The sodium bicarbonate cotransporter NBCe2 (encoded by SLC4A5) is involved in the regulation of renal tubular sodium bicarbonate transport. We hypothesized that human SLC4A5 single nucleotide polymorphisms (SNPs, rs10177833 and rs7571842) increase NBCe2 expression and renal proximal tubule (RPT) sodium transport and may be a cause of salt sensitivity of blood pressure.

Objective

To characterize the function of wild‐type (WT) and homozygous variants (HV) of SLC4A5.

Methods and Results

The expressions of NBCe2 mRNA and protein were not different between RPT cells (RPTCs) carrying WT (n=4) or HV SLC4A5 (n=6) before or after dopaminergic or angiotensin (II and III) stimulation. However, total transcellular (luminal to basolateral) RPTC sodium transport, NHE3 protein expression, and Cl−/HCO3− exchanger activity were higher in HV than WT SLC4A5. Bicarbonate‐dependent pH recovery after an intracellular acid load, as well as NBCe2 mRNA and protein, was increased when intracellular sodium was elevated (5.2±1.1 mM) in HV SLC4A5 compared with WT SLC4A5 (pH recovery Vmax 0.22±0.02 and Km 6.39±0.81 in HV vs Vmax 0.28±0.02 and Km 4.30±0.53 in WT, P<0.05, P<0.05, n=3). The increased NBCe2‐specific bicarbonate‐dependent pH recovery in HV was verified in stable SLC4A5 shRNA cell lines (n=8/12/group). RPTCs carrying HV SLC4A5 had increased binding of HNF4A to SLC4A5 DNA (1.52±0.10‐fold, N=3, P<0.05, t‐test), using purified HNF4A and ChIP and oligo binding assays. Additionally, the difference between HV and WT in bicarbonate‐dependent pH recovery was abolished by two HNF4A antagonists. RPTCs isolated from human urine of salt‐sensitive subjects who are HV for SLC4A5 had increased bicarbonate dependent pH recovery, relative to salt‐resistant subjects who are WT for SLC4A5 (n=4/group).

Conclusions

NBCe2 activity is stimulated by an increase in intracellular sodium and is hyper‐responsive in RPTCs carrying HV SLC4A5 through an aberrant HNF4A‐mediated mechanism, which may be a key mechanism in the etiology of human salt sensitivity.
Compound 21 Prevents Salt Induced Myocardial Hypertrophy
Authors: Dopona Ellen Priscila Brito , Favoni Veronica , de Oliveira Ivone Braga , Heimann Joel Claudio
Published: November 2023

Objective

To evaluate the role of Compound 21 (C21) in a model of salt overload on left (LV) and right (RV) ventricular hypertrophy and fibrosis.

Methods

Male Wistar rats were fed normal‐ (NS ‐ NaCl = 1,27%, n=8) or high‐ (HS ‐ NaCl = 8%; n=8) salt diet from weaning up to 18 weeks of age. From the 7th week of age, one HS subgroup received Compound 21 (1mg/kg per day by gavage, n=8). Body weight (BW), tail‐cuff blood pressure (BP), cardiac mass to tibia length ratio (CM/TL), cardiomyocyte transverse diameter (TD), percentage of cardiac fibrosis (FI) were measured.

Results

At 7th week of age, BW was higher in HS+C21 and NS than in HS. BW in HS+C21 did not differ from NS from the 7th week to the 18th week of age. BP in HS+C21 was higher than in NS and lower than in HS from the 7th week to the 18th week of age.

Conclusion

In Wistars rats, compound 21 prevents left and right ventricular hypertrophy and left ventricular fibrosis.

Support or Funding Information

This study was supported by FAPESP (2013/08418‐4)

Results: (mean ± standard error of the mean ‐ p<0.05):

Variable
Weeks
NS
HS
HS+C21

Body Weigh (g)
3
65±1.5
65±2.4
64±3.5

18
560±19.4
493±16.4*
566±15.5

BP(mmHg)
6
112±3.0
114±4.2
111±3.4

18
116±2.5
144±1.2*
135±1.8*#

HEART

Total
0.44±0.04
0.47±0.04
0.45±0.06

CM/TL (g/m)
18
RV
0.07±0.007
0.07±0.01
0.07±0.01

LV
0.26±0.02
0.29±0.02
0.29±0.04

TD(μm)
18
RV
9.82±0.21
13.80±0.29*
9.79±0.21

LV
10.17±0.2
15.60±0.9*
10.41±0.09

FI (% área)
18
RV
0.87±0.23
0.92±0.13
0.60±0.05

LV
0.25±0.05
0.82±0.08*
0.33±0.03#

p<0.05 vs. NS;

p<0.05 vs. HS
High Salt Activates Human Monocytes and Promotes their Conversion into Dendritic Cells via Formation of Immunogenic Isoketal‐adducts
Authors: Kirabo Annet , Barbaro Natalia , Foss Jason David , Montaniel Kim Ramil , Chen Wei , Harrison David Glenn
Published: November 2023

High blood pressure is a major modifiable risk factor for cardiovascular disease (CVD) which is the leading cause of death worldwide. Excessive salt intake and inflammation are implicated in the genesis of hypertension. It has recently become evident that sodium accumulates in the interstitial space and can promote inflammation through poorly defined mechanisms. We recently published a new pathway in which increased oxidative stress in dendritic cells (DCs) leads to formation of isoketal‐modified proteins which act as neo‐antigens to activate T cells. We hypothesized that increasing sodium chloride (NaCl) in excess of the physiological interstitial space concentrations activates antigen presenting cells via formation of immunogenic isoketals. We exposed monocytes from human volunteers to normal physiological NaCl (150 mM/L), elevated NaCl concentrations (190 mM/L), or an equiosmoloar concentration of mannitol. We found that exposure of human monocytes to high salt, but not mannitol, caused a 2‐fold increase in formation of isoketal‐modified proteins. This was associated with an increase in activation marker CD86 (466 ± 192 normal salt vs 596 ± 324 high salt) and production of inflammatory cytokines IL‐6, IL‐β and TNF‐α. Interestingly, these cells expressed surface markers indicative of transformation to DCs, as evidenced by their acquisition of surface marker CD83. In additional immunofluorescence studies, we found that monocytes exposed to high salt for 7 days acquire a DC like morphology. Moreover, using flow cytometry, we confirmed that high salt exposure causes these cells to lose the monocyte marker CD14, and gain the DC marker CD209. When co‐cultured with T cells from the same patients, these cells drive T cell proliferation. Scavenging of isoketals during high salt exposure prevented conversion of monocytes into DCs and their ability to drive T cell proliferation. Thus, we have defined a novel pathway whereby high NaCl concentrations lead to transformation of monocytes to DCs due to increased formation of immunogenic isoketals. These observations provide insight into how elevated sodium environments lead to an inflammatory state.

Support or Funding Information

This work is supported by National Institutes of Health grants R01HL039006, P01HL058000, P01HL095070, P01GM015431, R01HL108701, R01HL105294, VITA award HHSN268201400010C and the Strategically Focused Research Network Award from the American Heart Association.
T cell deletion of Serum and Glucocorticoid‐Regulated Kinase 1 (SGK1) Attenuates Hypertension and End‐Organ Inflammation
Authors: Norlander Allison Elizabeth , Saleh Mohamed , Pandey Arvind , Itani Hana , Wu Jing , Dale Bethany , Harrison David Glenn , Madhur Meena
Published: November 2023

We have previously shown that the T cell‐derived pro‐inflammatory cytokine, interleukin 17A (IL17A), is upregulated by and promotes angiotensin II‐induced hypertension and contributes to renal and vascular dysfunction. It was recently demonstrated that an excess of 40 mM of sodium chloride enhances IL17A production from CD4+ T cells in an SGK1 dependent manner. We confirmed the effect of salt on CD4+ T cell differentiation and extended this finding to CD8+ T cells in which 40mM of excess salt increased the expression of IL17A (4.7 fold, p=0.0003), TonEBP (2.3 fold, p <0.002), and the salt‐sensing kinase SGK1 (2.2 fold, p=.001) in naive CD8+ cells cultured under Th17 polarizing conditions. Since dietary salt intake is associated with hypertension, we hypothesized that T cell SGK1 promotes hypertension and contributes to end‐organ dysfunction. To test this hypothesis, we crossed SGK1fl/fl mice with CD4cre mice to delete SGK1 in T lymphocytes. Loss of T cell SGK1 resulted in a blunted blood pressure response to angiotensin II infusion (24.8 mmHg reduction, p=0.01) and DOCA‐salt treatment (15.51 mmHg reduction, p<0.05). Moreover, renal and vascular inflammation in response to angiotensin II infusion and/or DOCA‐salt treatment were abrogated in these mice compared to SGK1fl/fl control mice. Angiotensin II infusion increased total (CD45+) leukocytes in the kidney from 55.4 to 120.4 x103 (p<0.01) in SGK1fl/fl mice while there was no increase in mice with T cell deletion of SGK1 (48.1 to 47.5×103, p=ns). Similarly, angiotensin II increased total (CD45+) leukocytes in the aorta from 5.7 to 52.4×103 (p<0.01) in SGK1fl/fl mice compared to no increase in mice with T cell deletion of SGK1 (16.1 to 10.1×103, p=ns). DOCA‐salt induction increased total (CD45+) leukocytes in the aorta in SGK1fl/fl mice from 7.4 to 20.6×103 (p<0.05) compared to no increase in mice with T cell deletion of SGK1 (8.7 to 10.8×103, p=ns). Furthermore, vascular reactivity studies demonstrated that vessels from mice with T cell deletion of SGK1 were protected from angiotensin II induced endothelial dysfunction. These studies demonstrate that T cell SGK1 may be a novel therapeutic target for hypertension and the associated end‐organ inflammation.
High salt intake alters renal medullary clock genes via ETB receptors
Authors: Speed Joshua S , Hyndman Kelly A , Kasztan Malgorzata , Johnston Jermaine G , Roth Kaehler J , Young Martin E , Pollock Jennifer S , Pollock David M
Published: November 2023

Diurnal rhythms in various physiological functions are driven by the oscillations of several transcription factors including Bmal1, period (Per), cryptochrome (Cry), and clock, collectively termed the molecular clock. The renal inner medullary collecting duct (IMCD) is an important site for Na+ handling and contains several clock factors. The IMCD also produces ET‐1 in response to high salt intake and contains a high density of endothelin B (ETB) receptors that promote the excretion of Na+. Disruption of renal ETB receptors reduces the ability of kidney to excrete a salt load in rodents, especially when salt is given prior to the inactive period vs. the active period. These data suggest an interaction between renal ETB receptors and the molecular clock to promote the excretion of excess Na+. Given that knockout of the circadian clock gene Bmal1 reduces blood pressure during the active period in mice, we hypothesized that a high salt intake suppresses the clock mechanism in the renal inner medulla via ETB receptor activation. Transgenic control (Tg con) or ETB deficient (ETB def) rats were fed normal (NS, 0.49% NaCl) or high (HS, 4% NaCl) salt for two weeks. Rats were euthanized every 4 hours beginning at zeitgeber time 0 (lights on) for tissue collection and subsequent assessment of circadian clock genes. Renal inner medullary Bmal1 oscillation was phase shifted in Tg con rats in response to HS compared to NS fed rats. On the other hand, Bmal1 expression was similar between NS and HS fed ETB def rats indicating that the phase shift in Tg con rats is ET‐dependent. In addition, renal inner medullary Per1 expression was similar between all groups; however expression of Per2, Cry1, and Cry2 were suppressed during normal acrophase in response to HS in both Tg con and ETB def rats suggesting that this is ET‐independent. Further, in cultured mouse IMCD cells, treatment with ET‐1 significantly reduced Bmal1 mRNA. Finally, knockdown of Bmal1 with siRNA significantly reduced mRNA expression of the epithelial Na+ channel alpha subunit (ENaCα) (1.0±0.06 vs. 0.74±0.07, control vs. Bmal siRNA: p<0.05) and increased transepithelial resistance (583.0±43.3 vs. 703.5±32.6 ohms, control vs. Bmal siRNA: p=0.056) in mouse IMCD cells. These data suggest that activation of renal inner medullary ETB receptors during high salt intake functions to regulate the normal circadian pattern of Bmal1 expression in order to suppress ENaCα and promote the excretion of excess Na+.

Support or Funding Information

This work is supported by NIH P01 HL95499 and P01 HL69999 to DMP and American Heart Association grant 15SDG25090194 to JSS.
Intermittent Hypoxia Exacerbates Decreased GFR and Increased Blood Pressure in Rats with Chronic Kidney Disease
Authors: Riggs Jennifer L. , Pace Carolyn , Danielson Leslie , Fischer Edgar , Ward Heather , Kanagy Nancy
Published: November 2023

Kidney injury and sleep apnea (SA) are risk factors for hypertension. This study evaluated interactions between simulated sleep apnea and chronic kidney disease. Rats were exposed to intermittent hypoxia (IH) to simulate SA and were given excess adenine in the diet to cause acute renal injury leading to chronic kidney disease (CKD). We hypothesized that exposing CKD rats to IH would accelerate declines in renal function and increase blood pressure. Male Sprague‐Dawley rats were fed a 0.4% adenine diet or a control diet until plasma urea nitrogen was >140 mg/dL in the adenine group. After two weeks of recovery (normal rat chow for all groups), rats were exposed to IH (20 exposures/hr of 5% O2/5% CO2 7 hr/day) or SHAM for 6 weeks. Arterial pressure (AP) was monitored continuously with indwelling telemeters and plasma and urine samples were collected weekly using metabolism cages to calculate glomerular filtration rate (GFR). AP was increased in rats fed the adenine diet compared to rats on the control diet prior to starting IH. IH treatment further increased AP so that after 6 weeks of Sham or IH treatment AP was elevated in both adenine groups and in the IH only group with pressures in Adenine/IH rats > adenine/Sham rats = Control/IH rats > Control/Sham rats. Histological analysis revealed crystalline deposits and damage to the proximal and distal tubules with interstitial fibrosis in the kidneys of all the adenine fed rats. Fibrosis persisted up to 8 weeks post adenine removal and IH did not cause any apparent additional damage. GFR remained impaired in both the adenine groups throughout the six weeks of IH or SHAM exposures with elevated plasma creatinine levels that were not altered by IH treatment. Therefore simulated SA augments increases in blood pressure in rats with pre‐existing CKD without affecting GFR suggesting that treating apnea should help prevent worsening secondary cardiovascular stresses in CKD patients.

Support or Funding Information

NHLBI 123301, Dialysis Clinic Inc. Reserve Fund Grant
Differential regulation of large conductance K+ (BK) channel mediated K+ secretion in proximal and distal colon by aldosterone
Authors: Nickerson Andrew J , Galbreath Kyla M , Rajendran Vazhaikkurichi M
Published: November 2023

Aldosterone (“aldo”) differentially regulates electrolyte transport processes, as it stimulates Na‐H exchanger isoform‐3 (NHE3) mediated electroneutral Na+ absorption, and down‐regulates NHE3 and induces epithelial Na+ channel (ENaC) mediated electrogenic Na+ absorption in proximal and distal segments of rat colon, respectively. Electrogenic active K+ secretion is present in proximal colon, while H+, K+‐ATPase mediated electroneutral K+ absorption is present in distal colon of normal rat colon. Aldo enhances H+, K+‐ATPase expression and electroneutral K+ absorption in distal colon. Mucosal ortho‐vanadate (VO4; p‐type ATPase inhibitor) unmasked the “aldo” induced BK channel mediated K+ secretion in distal colon. It is not known whether “aldo” also stimulates BK channel mediated active K+ secretion in proximal colon. Studies were, therefore, initiated to identify whether “aldo” also activates K+ secretion in proximal colon. Aldo rats were generated by feeding Na‐free diet for 6 – 7 days. 86Rb (K+ surrogate) fluxes were measured in muscularis stripped proximal colon mounted under voltage clamp condition in Ussing chamber. Mucosal to serosal (m‐s) and serosal to mucosal (s‐m) fluxes were measured, while net fluxes were calculated by subtracting s‐m from m‐s. Net positive and negative fluxes represent active absorption and secretion, respectively. Aldo significantly enhanced K+ secretion (−0.25 ± 0.05 vs −0.47 ± 0.12 μEq/h.cm2; p < 0.05). Forskolin (FSK, adenylate kinase activator) stimulated K+ secretion in both normal (0.25 ± 0.05 vs 0.42 ± 0.09 μEq/h.cm2; p < 0.05) and aldo (0.47 ± 0.12 vs 1.30 ± 0.12 μEq/h.cm2). Mucosal iberiotoxin (IbTX, BK channel inhibitor) inhibited FSK‐stimulated K+ secretion in normal, but it did not affect FSK‐stimulated K+ secretion in aldo proximal colon. In contrast to IbTX, mucosal charybdotoxin [CTX; intermediate K+ (IK) channel blocker) completely inhibited the FSK‐stimulated K+ secretion in aldo proximal. We conclude that FKS stimulates active K+ secretion via: 1) both BK and IK channels in normal proximal colon; and 2) entirely through IK channels in proximal colon.

Support or Funding Information

This work was supported by NIH NIDDK Grant 1006856R to VMR.
Regulation of cell junctions by ClCN2 in cystic fibrosis
Authors: Henry Katherine , Lee Seakwoo , Zeitlin Pamela
Published: November 2023

Cystic Fibrosis (CF) is an autosomal recessive disease, characterized by mutations in the CFTR gene, incorrect protein folding, and impaired chloride secretion. Ultimately, patients with CF exhibit a change in membrane potential regulation and it is possible that some of these changes are the result of impaired cell junctions. Previous data from our lab shows there is a targeted localization of ClCN2 to areas of cell junctions when portions of the c‐terminus are removed (ClCN2‐584). New data shows that this targeted localization is associated with a significant increase in transepithelial resistance in vitro. We hypothesize that this targeted localization and increase in TER suggests a potentially important role for ClCN2 in the regulation of tight junctions during CF, including changes in protein expression, protein interactions and tight junction function.

Support or Funding Information

5T32HL72748‐11: Ruth L Kirschstein National Research Service Award
A Role For 11‐Deoxycortisol In The Regulation Of Tight Junction Permeability In An Extant Agnathan
Authors: Zimina Anna , Kolosov Dennis , Wilkie Michael P , Kelly Scott P
Published: November 2023

Corticosteroid hormones are important regulators of paracellular permeability across vertebrate epithelia. In aquatic vertebrates such as teleost fishes, cortisol reduces the paracellular permeability of the gill epithelium, which in a freshwater (FW) environment has been proposed to contribute to reduced passive ion loss under conditions of environmental or systemic perturbation. However, it is not known whether corticosteroids contribute to the regulated movement of solutes across the paracellular pathway in less derived vertebrates, such as agnathans. This gap in knowledge speaks to the difficulty of pinning down the identity of corticosteroid hormones in agnathans. But recent studies have revealed that 11‐deoxycortisol is the only circulating corticosteroid with high affinity and specificity to the sea lamprey corticosteroid receptor. This knowledge now presents us with an opportunity to examine whether 11‐deoxycortisol contributes to the regulation of paracellular ion transport in sea lamprey. Given the strong link between corticosteroids and paracellular solute movement across epithelia of more derived fishes, as well as other vertebrates, it can be hypothesized that 11‐deoxycortisol will play a similar role in the lamprey. Using the stenohaline FW larval form of the sea lamprey, the objectives of this study were to examine the dose‐dependent effects of 11‐deoxycortisol on: (1) endpoints of salt and water balance and (2) the molecular physiology of the larval lamprey TJ complex in iono/osmoregulatory organs such as the gill and kidney. Results indicated that 11‐deoxycortisol treatment of larval sea lamprey dose‐dependently altered endpoints of salt and water balance as well as the abundance of select TJ proteins. Therefore a third objective of this study was to examine whether changes brought about by 11‐deoxycortisol are beneficial to larval lamprey in terms of coping with ionoregulatory disturbance (i.e. abrupt exposure to an ion deficient environment). While data on the molecular physiology of the lamprey TJ complex suggests that the changes produced by 11‐deoxycortisol are useful for ion retention in a hyposmotic or ion‐deficient environment, net flux of Na+ and Cl‐ upon abrupt exposure to ion‐poor water paradoxically suggest that transepithelial solute permeability is greater following 11‐deoxycortisol treatment. Overall, data from these studies provide evidence to support the hypothesis that 11‐deoxycortisol contributes to the regulation of TJ permeability in an extant FW larval agnathan, but this may differ from the role that corticosteroids such as cortisol play in more derived fishes.

Support or Funding Information

This research was supported by Natural Sciences and Engineering Research Council of Canada.
Claudins of the Thick Ascending Limb of Henle's Loop – From Interaction to Function
Authors: Milatz Susanne , Wulfmeyer Vera Christine , Plain Allein , Drewell Hoora , Mutig Kerim , Breiderhoff Timan , Fromm Michael , Hou Jianghui , Bleich Markus , Günzel Dorothee , Himmerkus Nina
Published: November 2023

The thick ascending limb of Henle's loop (TAL) drives paracellular Na+, Ca2+ and Mg2+ reabsorption via the tight junction (TJ). The TJ is composed of claudins which seal the paracellular cleft or form size‐, charge‐ and water‐specific pores. This is achieved by a concerted interaction of different claudin proteins within the same plasma membrane (cis‐interaction) and of neighboring plasma membranes (trans‐interaction) resulting in the formation of a complex TJ strand meshwork. Claudins consist of four transmembrane segments, a small intracellular loop and two extracellular loops.

Single TAL segments were obtained from cortex or medulla and microperfused. Electrophysiological measurements of single tubules were combined with claudin expression analyses by subsequent immunostaining and confocal laser‐scanning microscopy. In order to gain insight into the molecular composition of TAL TJ strands, claudin cis‐and trans‐interaction was examined by means of live cell imaging and Förster/fluorescence resonance energy transfer (FRET) in an overexpression system. A set of TAL claudin protein chimeras was created to reveal determinants of interaction properties.

Cortical and medullary TAL differed regarding their paracellular permeability properties and their equipment with claudins 3, 10, 11, 16, and 19. A clear correlation between dominance of specific claudins within the TJ and ion selectivity was observed. Although most of these claudins were co‐expressed within the TJ, some of them failed to interact with each other in cis‐or in trans‐configuration. Interaction analyses of claudin chimeras suggested an involvement of the extracellular loops of TAL claudins in cis‐and trans‐interaction.

In conclusion, a specific claudin expression pattern along the TAL corticomedullary axis is revealed. This special composition of claudins can be explained by their particular interaction capabilities. We suggest the existence of at least two spatially distinct types of paracellular pores with different preferences for Na+, Ca2+ and Mg2+ in the TAL.

Support or Funding Information

Supported by DFG FOR 721/2; National Institutes of Health Grant R01DK084059
Hyperglycemia‐induced Elevation of NKCC1 Expression in Blood Brain Barrier Endothelial Cells: Greater Effect of Intermittent Versus Sustained Exposures
Authors: Klug Nicholas R , Raposa Jesse T , Riley Michelle A , O'Donnell Martha E
Published: November 2023

Diabetics with accompanied hyperglycemia (HG) suffer from exacerbated cerebral edema and worsened neurological outcomes following ischemic stroke. We have demonstrated previously that hypoxia and other factors present during cerebral ischemia stimulate cerebral microvascular endothelial cell (CMEC) Na‐K‐Cl (NKCC) cotransport and Na/H exchange (NHE) activities. We also found that inhibition of these blood‐brain barrier (BBB) Na transporters reduces brain Na uptake and edema in the rat middle cerebral artery occlusion (MCAO) model of ischemic stroke. In more recent studies focusing on diabetic stroke, we demonstrated that sustained 6‐hour to 7‐day HG exposures increase expression and activity of CMEC NKCC and NHE, resulting in more pronounced responses to ischemic factor stimulation. Further, we found that inhibition of BBB NKCC and NHE effectively reduces edema and infarct in hyperglycemic rats subjected to MCAO. Diabetics often experience postprandial excursions of blood glucose. Studies have provided evidence that while sustained HG can induce stress/injury responses in endothelial cells, intermittent HG can exaggerate those responses. The present study was conducted to investigate the effects of intermittent HG exposures on BBB Na transporter expression and activity. For this, cultured bovine CMEC were exposed daily over 2 to 5 days to HG medium (DMEM with 30 mM glucose, 320 mOsm) or to osmotic control medium (DMEM with 30 mM mannitol, 320 mOsm) for 6 hours, followed by exposure to normoglycemic medium (DMEM with 5 mM glucose, 295 mOsm) for 18 hours. Quantitative Western blot analysis was then used to assess NKCC1 protein abundance in CMEC lysates. We found that CMEC NKCC1 abundance increased in response to intermittent HG exposures in a graded manner with larger increases observed following greater number of successive exposures. No change in NKCC1 abundance occurred with repeated intermittent exposures to normoglycemic medium. In addition, our results show that 5 days of intermittent HG exposures caused a 124% increase in NKCC1 abundance compared to 40% and 65% increases following sustained 2‐day and 7‐day exposures, respectively. In the present study we also investigated the effects of varying HG levels on CMEC Na transporter abundance. Here, we exposed CMEC for 48 hours to HG media containing 10, 20 and 30 mM glucose, to osmotic control media (with mannitol) or to normoglycemic medium (with 5 mM glucose). We found that NKCC1 and NHE1 protein abundances exhibited a graded response to increasing HG levels. Our findings support the hypothesis that BBB endothelial NKCC1 and NHE1 are highly sensitive to increasing levels of HG and, in particular, that clinically relevant repeated intermittent exposures result in more pronounced BBB Na transporter responses than sustained exposures.

Support or Funding Information

Supported by NIH, ADA and AHA
Molecular Basis of Inhibition of Acid Sensing Ion Channels by Diminazene
Authors: Carattino Marcelo D , Krauson Aram J
Published: November 2023

Acid sensing ion channels (ASICs) are proton‐gated channels that contribute to pain sensation in the peripheral nervous system. We employed site‐directed mutagenesis and electrophysiology to investigate the mechanism of inhibition of ASICs by the aromatic diamidine diminazene. We found that diminazene inhibits mouse ASIC1a and ASIC3 with a half‐maximal inhibitory concentration (IC50) of 2.4 μM and 1.5 μM, respectively. To determine whether diminazene alters ASICs proton sensitivity, we generated dose‐response activation curves for ASIC3 in the absence and presence of diminazene (2 μM). Consistent with a competitive mechanism of action, diminazene shifted the pH of half‐maximal activation of ASIC3 from 6.32 ± 0.06 to 6.00 ± 0.06. To examine whether diminazene binds to a previously identified proton coordination site in the lower palm domain of ASIC1a formed by the residues Glu79 and Glu416, we generated dose‐response inhibition curves with channels bearing Gln substitutions at these positions. E79Q and E416Q ASIC1a channels were inhibited by diminazene with IC50 values of 18 μM (CI 8.2 – 41.8) and 14 μM (CI 5.9 – 32.7), respectively. Likewise, the neutralization of Glu79 (E79Q) on ASIC3 shifted diminazene IC50 from 1.5 μM (CI 1.0 – 2.4) to 5.6 μM (CI 2.6 –12.4). On the contrary, the neutralization of residues in the acidic pocket of ASIC1a, a cluster of negatively charged residues in the extracellular region that serves as binding site for psalmotoxin, did not change apparent diminazene binding affinity. These results strongly indicate that Glu79 and Glu416 in the lower palm domain are part of the diminazene binding site.

Support or Funding Information

R01 DK084060 and P30 DK079307 Pittsburgh Center for Kidney Research
Calreticulin‐dependent VEGF expression promotes neuroblastoma differentiation
Authors: Lin Kuan‐Hung , Weng Wen‐Chin , Ho Ya‐Hsuan , Wu Pei‐Yi , Wang Bo‐Jeng , Liao Yung‐Feng , Hsu Wen‐Ming , Lee Hsinyu
Published: November 2023

Neuroblastoma (NB) is a childhood cancer with a low survival rate and great metastatic potential. Calreticulin (CRT), an endoplasmic reticulum (ER) chaperone protein, has been identified as one of the prognosis factor for NB patients. Our previous study have demonstrated that CRT promotes neuronal differentiation through activating VEGF‐A expression in different NB cell lines. Inhibiting of VEGFR‐1 signaling by blocking antibodies significantly suppressed expressions of neuronal markers, which indicated that VEGF‐VEGFR signaling may play important roles during neuronal differentiation in NB cells. In this study, we further confirmed our hypothesis through xenograft model and clinical data from NB patients. In xenograft experiments, we found that xenograft tumors with induced CRT expression was smaller than control samples. In addition, the expression levels of VEGF‐A and neuron marker GAP‐43 were higher in tumor samples compared to control samples. Consistently, immunofluorescence results indicate that VEGF‐A and GAP43 protein expression were higher in CRT‐induced tumors. However, the expression levels of CD31, an endothelial marker, were decreased under CRT induction. Analyzing Clinical samples from NB patients demonstrated that the mRNA expression levels of CRT and VEGF‐A were positive correlated. Differentiated NB tumor samples have higher CRT and VEGF‐A expression as detected by RT‐qPCR and IHC. Furthermore, patients with higher VEGF‐A expression level have higher survival rate. Our findings suggested that VEGF may promote neuronal differentiation in neuroblastoma patients. This study may provide new therapeutic strategies and prognosis methods for NB patients.
Oxygen consumption in single eggs of a desert pupfish
Authors: Jones Alexander C , Lim David , Hillyard Stanley , van Breukelen Frank
Published: November 2023

Pupfish of the genus Cyprinodon live in environments that may be very warm (>35 °C) and hypoxic. In order to address how these conditions might affect the metabolism of fish eggs, we developed an approach that allowed for the measurement of oxygen consumption of a single egg. Eggs were classified into one of 4 groups depending on the stage of development and reared at either 28 or 33 °C. Expected increases in oxygen consumption as the result of development were found for the first three stages of development. However in well developed embryos, exposure to 33 °C resulted in an unexpected decrease in oxygen consumption. Interestingly, oxygen consumption spiked when eggs hatched during the assay suggesting that eggs are oxygen‐limited by the end of development. Critical partial pressures of oxygen were relatively high (~112 to 125 torr). These data call into question what role oxygen might have in the development of pupfish eggs in the wild.