bluuebunny/crossref_metadata_2025 huggingface dataset:
Type 2 diabetes critically redefined for nonhuman primates (854.9)
Authors: Hansen Barbara , Newcomb Jennifer , Linden Ellen
Published: June 2021

Nonhuman primates (NHPs) naturally develop type 2 diabetes (T2DM) with extraordinary concomitance to T2DM in humans. In order to determine valid criteria for the onset of T2DM in NHPs, a colony of 326 adult NHPs (232 male and 94 female; rhesus monkeys, Macaca mulatta) was studied prospectively, including fasting plasma glucose (FPG), glucose tolerance, HbA1c, insulin, lipid profiles, β‐cell function and insulin sensitivity. A subset of 105 metabolically healthy NHPs was used to establish normal/baseline fasting levels, with normal FPG, 57.3±6.6 mg/dl comparable to ~85 mg/dl in humans and normal HbA1c level, 4.2±0.3 comparable to ~5.5 in humans, thus, 37% and 27% lower than for humans. 112 monkeys developed diabetes while under study and showed the necessity of redefining the onset of T2DM for rhesus, specifically at a fasting glucose of 100 mg/dl or 5.6 mmol/l. Monkeys with a fasting glucose >70 mg/dl were in the progression toward diabetes, and 80‐99 mg/dl was redefined as prediabetes, a level that coincided with sharp changes in β‐cell responses to glucose, and in fasting insulin levels. HbA1c was highly correlated with fasting glucose (r=0.852, p<0.001), with a T2DM diagnostic level of > 5.5% for rhesus. NHPs provide the optimal animal model for studies of the in vivo and molecular pathophysiology of T2DM, and these new diagnostic criteria are essential to improved translation to humans.
Monophosphoryl lipid A alters the inflammatory response of endothelial cells challenged with lipopolysaccharide (855.4)
Authors: Stark Ryan , Choi Hyehun , Nguyen Hong , Sherwood Edward , Lamb Fred
Published: June 2021

Monophosphoryl lipid A (MPLA) is a lipid A mimetic that induces a state of endotoxin tolerance when given prior to lipopolysaccharide (LPS) challenge. As endothelial cells regulate inflammation, we hypothesized that MPLA would downregulate the inflammatory response of endothelial cells to LPS in a serum dependent manner. We cultured human umbilical vein endothelial cells (HUVECs) and exposed them to MPLA or a vehicle control for 6 hours. Afterwards, HUVECs were challenged with TNFα or LPS or saline for 24 hours. We assessed activation by quantifying IL‐6 levels in the culture media. In the presence of 2% serum, MPLA alone enhanced IL‐6 production compared to controls. LPS increased IL‐6 production much more potently, but this effect was reduced by 47 ± 5% when pre‐exposed to MPLA (p<0.05). MPLA had no effect on TNFα‐induced IL‐6 production. In the absence of serum, LPS induction of IL‐6 was nearly abolished secondary to lack of serum co‐factors. However, MPLA enhancement of IL‐6 production persisted in a serum free environment (p<0.05). These studies demonstrate that MPLA alters the inflammatory response of endothelial cells after LPS challenge and that the effects of MPLA are serum independent.
The anti‐hypertensive response to renal denervation in aged SHR is independent of changes in body sodium (856.1)
Authors: Phelps Jeremiah , Fink Gregory
Published: June 2021

Since interruption of kidney functions influenced by renal sympathetic nerve activity (RSNA) may cause the fall in blood pressure (BP) after renal denervation (RDX), then the chronic anti‐hypertensive effect of RDX should be larger when RSNA is higher. RSNA is inversely related to body sodium content, so we hypothesized that the BP response to RDX in rats would be agreater during sodium depletion than during sodium loading. In male retired breeder spontaneously hypertensive rats (SHR) on a 0.1% sodium chloride (NaCl) diet, RDX chronically reduced BP by 10.6±2.5 mmHg whereas sham‐operation decreased BP by only 0.2±0.7 mmHg (a net difference (∆) of ‐10.4 mmHg). Additional sodium depletion with chlorthalidone lowered BP modestly in both groups but did not accentuate the effect of RDX (∆ = ‐10.0 mmHg.). Similar findings were noted with more aggressive sodium depletion with furosemide (∆ = ‐7.5 mmHg). Furthermore, the BP response to RDX was not mitigated when rats were taken off drug treatment and placed on a 0.4% NaCl diet (∆ = ‐10.1 mmHg), or when additional sodium loading was achieved by adding 1% NaCl to the drinking water (∆ = ‐13.4 mmHg). Finally, the BP effect of RDX remained when the animals were returned to drinking water only (∆ = ‐15.5 mmHg). These data suggest that the magnitude of the sustained BP response to RDX in this experimental model is not directly related to body sodium content or the level of RSNA.
Angiotensin II and mineralocorticoid receptor antagonism decrease cardiac nitrotyrosine content and recover glutathione peroxidase activity in Angiotensin II‐infused rats (856.10)
Authors: Minas Jacqueline , Thorwald Max , Conte Debra , Vázquez‐Medina Jose‐Pablo , Nishiyama Akira , Ortiz Rudy
Published: June 2021

Angiotensin II (Ang II) and aldosterone (aldo) contribute to cardiovascular damage via oxidative stress, but their respective contributions remain elusive. To independently assess the contributions of Ang II and aldo during Ang II‐dependent hypertension, five groups of Sprague Dawley rats (n =11‐14/group) were studied: 1) Control, 2) Ang II infused (80 ng/min x 28d), 3) Ang II + angiotensin receptor blocker (ARB; 10 mg losartan/kg/d x 21d), 4) Ang II + mineralocorticoid receptor antagonist (Epl; 100 mg eplerenone/d x 21d), and 5) Ang II + ARB + Epl (Combo). ARB and Combo reduced the 65% Ang II‐induced increase in systolic blood pressure (SBP), but Epl had no effect. Epl exacerbated the increase in plasma and heart aldo while ARB reduced both. Ang II increased relative heart mass 19% and heart cardiotrophin‐1 content 57%. All treatments prevented these increases. Ang II reduced mean glutathione peroxidase activity 29%, while ARB and Combo recovered levels beyond Control by 25 and 28%, respectively. Ang II also increased superoxide dismutase (SOD) activity 20% whereas all treatments reduced SOD. Ang II increased mean heart nitrotyrosine (NT) content 3‐fold and mean 4‐hydroxynonenal (HNE) content 26%. All treatments improved NT, but Epl had no effect on HNE levels. Despite the inability to reduce SBP, chronic MR blockade prevented oxidative stress suggesting aldo as a therapeutic target for AT1‐mediated oxidative damage.

Grant Funding Source: Supported in parts by: NHLBI K02HL103787/R01HL091767, NIH NIMHD T37MD001480, NIH
Spontaneous hypertension induced pulmonar and diaphragm disorders in rats (856.11)
Authors: Souza Pamella , Flues Karin , De Moraes Wilson , Santos Fernando , Ferreira Janaina , Medeiros Alessandra , De Angelis Katia , Irigoyen Maria Cláudia
Published: June 2021

Hypertension induces modifications of standard rhythm breathing. However, the interaction of these effects with the cardiopulmonary and diaphragm morphological changes is not well known. We evaluated the influence of high blood pressure (BP) on cardiovascular modulation, chemoreflex sensitivity, right ventricle (RV) function, arterial blood gases and pulmonary morphometric parameters in rats. We also evaluated the diaphragm muscle profile in response to possible ventilator alterations. Hypertension adversely changed arterial blood gases patterns (pH, pO2, HCO3, SpO2) and the autonomic cardiovascular modulation (systolic BP variance, low frequency band and pulse interval variance), as well exacerbated chemoreflex pressor response and increased RV end diastolic pressure. We observed that SHRs present higher lung to body weight index, arterial lumen to wall ratio (lung arteries), and collagen deposition compared to aged matched Wistar rats. The SHR diaphragm muscle showed increase in type I cross‐sectional fiber (CSA) (16%) and reduction in type II CSA area (41%), increased activity of the ubiquitin proteasome system and lipid peroxidation, with no differences between groups in the analysis of ubiquitinated proteins and misfolded proteins. In conclusion, hypertension induced cardiopulmonary morphometric and functional compensatory/adverse alterations associated with diaphragm fiber type changes and protein degradation. These data suggest a possible adaptation in ventilatory pattern as an attempt to improve ventilatory endurance and prevent the development of fatigue and muscle weakness.
Inhibition of the bradykinin B1 receptor attenuates blood pressure and cardiac hypertrophy in DOCA‐salt hypertensive mice (856.12)
Authors: Sriramula Srinivas , Lazartigues Eric
Published: June 2021

DOCA‐salt hypertension is associated with increased activity of the kallikrein‐kinin system. We sought to explore whether bradykinin B1 receptor (B1R)‐mediated inflammation contribute to DOCA‐salt hypertension. While baseline blood pressure (BP; telemetry) was not different, DOCA‐salt treatment (1 mg/g body weight DOCA, 1% NaCl for 28 d) resulted in significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to non‐transgenic (NT) mice (138 ±3 mmHg, n=8). In DOCA‐treated NT mice, B1R expression (Western blot) in the brain was increased by 2 fold (n=4, p<0.01). To dismiss potential developmental alterations in knockout mice, we complemented our study using a pharmacological approach with inhibition of B1R in the brain of NT mice. ICV infusion of R715, a specific B1R antagonist, attenuated the DOCA‐salt‐induced increase in BP in NT mice (125 ±3 mmHg, n=3, p<0.01). Cardiac hypertrophy was also attenuated in NT+R715 mice (HW/BW: 4.1 ±0.1 vs. 5.6 ±0.2; HW/tibia length: 7.9 ±0.3 vs. 9.2 ±0.7; p<0.01 vs. NT+DOCA). NT+DOCA mice had increased inflammation (qRT‐PCR), as shown by elevated gene expression of TNF, IL‐1β and IL‐6 (3‐, 8‐, and 5‐fold, respectively, n=9, p<0.01 vs. NT) in the hypothalamic paraventricular nucleus, which was attenuated in NT+R715 mice. Taken together, these data provide evidence that B1R contributes to neuroinflammation, thereby the development of DOCA‐salt hypertension.

Grant Funding Source: Supported by AHA 12EIA8030004 and 13POST16500025
Role of afferent and efferent renal nerves in the antihypertensive effect of renal denervation in Ang II‐salt hypertension (856.4)
Authors: Foss Jason , Mayerhofer Tim , Wood Laurel , Collister John , Osborn John
Published: June 2021

Renal denervation (RDNX) attenuates drug resistant hypertension (HT) in humans, but it is unclear whether this is due to ablation of afferent or efferent renal nerves. We have previously shown that RDNX attenuates the intravenous (IV) AngII‐salt model of HT in the rat. The present study tested the hypothesis that the antihypertensive effect of RDNX in the IV AngII‐salt model is due to ablation of afferent renal nerves, specifically. Male Sprague Dawley rats underwent SHAM surgery, RDNX or selective ablation of afferent renal nerves by periaxonal capsaicin (renal‐CAP). Rats were instrumented with an IV catheter for delivery of AngII and a radio‐telemeter to measure mean arterial pressure (MAP) and heart rate (HR). Rats were placed on a 4% NaCl diet and, after recovery and a baseline period, AngII was infused IV (10 ng/kg/min) for 16 days. Rats were then sacrificed and kidneys were harvested and assayed for the efferent nerve marker, norepinephrine and the afferent nerve marker, CGRP. Initial results suggest that RDNX and renal‐CAP have similar antihypertensive effects in AngII‐salt rats. On the last day of AngII treatment, MAP was 161 ± 2.8 mmHg in SHAM (n = 4) rats, compared to 133 ± 11.5 mmHg in RDNX (n = 5) and 140 ± 5.2 mmHg in renal‐CAP (n = 4) rats. These preliminary results support the hypothesis that the antihypertensive effect of RDNX in the IV AngII‐salt model is primarily due to the ablation of afferent renal nerves.
Autocrine/paracrine function of angiotensin 1‐7 in tissue repair during hypertension (856.8)
Authors: Meng Weixin , Zhao Wenyuan , Zhao Tieqiang , Liu Chang , Chen Yuanjian , Liu Hongyu , Sun Yao
Published: June 2021

The purpose of this study is to explore 1) whether circulating AngII affects ACE2/MasR expressions in the hypertensive heart and kidney and 2) whether Ang1‐7 via MasR regulates cardiac repair responses during hypertension. In the first portion of the study, rats received either an AngII infusion (400ng/kg/min) for 4 weeks, leading to hypertension with high circulating AngII; or an aldosterone (ALDO, 0.75μg/h) infusion for 4 weeks, leading to hypertension with low circulating AngII. Cardiac and renal ACE2/MasR expressions were examined. We found that cardiac ACE2 was increased and MasR attenuated in both AngII and ALDO groups. However, renal ACE2 and MasR remained unchanged in both AngII and ALDO‐treated animals. In the second portion, rats received AngII infusion with/without MasR antagonist (A779, 1mg/kg/day) for 4 weeks. The roles of MasR blockade in cardiac inflammation, fibrosis, and apoptosis were examined. Chronic AngII infusion caused scattered cardiac injuries; and A779 co‐treatment exacerbated cardiac injury, resulting in aggravated inflammatory, fibrogenic and apoptotic responses compared to AngII group. Thus, ACE2 and MasR are differently expressed in the hypertensive heart and kidney, which are independent of circulating AngII level. Ang1‐7 involves multiple repair responses and protects the hypertensive heart from remodeling.

Grant Funding Source: NIH
Hydrogen sulfide treatment attenuates Ang‐II induced endothelial‐mesenchymal transition in renal fibrosis in aged mice (857.2)
Authors: Pushpakumar Sathnur , Kundu Sourav , Givvimani Srikanth , Metreveli Naira , Sen Utpal
Published: June 2021

Advanced age and hypertension are risk factors for chronic kidney disease. Emerging evidence suggests endothelial‐mesenchymal transition (EndoMT) as a possible source of renal myofibroblasts which play a crucial role in renovascular fibrosis. This complex process is driven by transforming growth factor‐β (TGF‐β) and involves loss of endothelial cell polarity and acquisition of myofibroblast phenotype. Although angiotensin‐II (Ang‐II) activates TGF‐β expression in the kidney, it is not known whether exogenous ang‐II induces endoMT in aged mice. Hydrogen sulfide (H2S), a gasotransmitter has recently been shown to suppress phenotypic transformation in atrial and lung fibroblasts. In the present study, we hypothesized that Ang‐II induced endoMT contributes to renal fibrosis and treatment with H2S attenuates endoMT by inhibition of TGF‐β. Wild type mice were infused with Ang‐II (1000ng/Kg/min) using osmotic pumps for 4 weeks and treated without or with H2S in drinking water (30μM/L). Ang‐II treated animals showed significant renal injury on histology, increased expression of TGF‐β and double labeling of endothelial (CD31) and mesenchymal markers, fibroblast specific protein‐1(FSP‐1) and α‐smooth muscle actin (α‐SMA) revealed significant coexpression suggesting endoMT. Treatment with H2S reduced oxidative stress, TGF‐β activation and inflammation. The expression of FSP‐1 and α‐SMA were decreased including the protein and mRNA levels of collagen. Our findings suggest that endoMT plays an important role in Ang‐II induced renal fibrosis in aged mice and inhibition of TGF‐β pathway by H2S therapy reduces endoMT thus preventing the progression of renovascular fibrosis.

Grant Funding Source: HL104103
Chronic effects of obstructive apnea on renal and lumbar sympathetic nerve activity in conscious rats (857.3)
Authors: Miki Kenju , Sukeguchi Chie , Yoshimoto Misa
Published: June 2021

This study investigated responses of renal (RSNA) and lumbar sympathetic nerve activity (LSNA) of rats to repeated obstructive apnea over 4 days. Wistar male rats were chronically instrumented for measurements of RSNA, LSNA, and oxygen saturation of the brain tissue, and a tracheal balloon for induction of apnea was implanted. At least 3 days after the surgery, the rats were subjected to obstructive apnea by inflating the tracheal balloon for 40 seconds. This process was repeated 6 times/hour for 5 hours/day over a 4‐day period. The basal level of RSNA increased gradually and significantly throughout the 4‐day experimental period. The magnitude of the LSNA increase was less than that of RSNA. The basal levels of arterial pressure and heart rate remained within the normal ranges throughout the experimental period. These data suggest that repeated obstructive apnea activates RSNA in a cumulative manner, which may in part explain the underlying mechanism for development of hypertension in sleep apnea syndrome. JSPS Grant.
Acute behavioral stress‐induced circulating endothelin‐1 is derived from the endothelium (857.9)
Authors: Loria Analia , Johns Robin , Hyndman Kelly X , Yanagisawa Masashi , Pollock Jennifer
Published: June 2021

Acute behavioral stress increases plasma ET‐1 levels. We hypothesize that a source of the stress‐induced ET‐1 is the endothelium. We utilized a unique mouse model of vascular endothelial‐specific ET‐1 knockout (VEETKO) mice. Male VEETKO and flox control mice were exposed to cage switch stress (CSS). No differences in ET receptor expression in lung tissue were found between flox and VEETKO mice, revealing a normal ET‐1 clearance capability in both genotypes. Measurements of plasma ET‐1 levels and tissue prepro‐ET‐1 mRNA were assessed at baseline (BL) or after 30 min of CSS. VEETKO mice had reduced BL plasma ET‐1 levels compared with flox mice (p<0.05), as previously reported. At BL, pre‐proET‐1 mRNA levels were lower in aorta and renal vessels (p<0.05) in VEETKO mice compared to flox control, but no differences were found in renal or adrenal tissues. Flox control mice displayed a CSS‐induced rise in plasma ET‐1 (p<0.05), however CSS did not induce an elevation in plasma ET‐1 in VEETKO mice. Plasma norepinephrine (NE) was not different between flox and VEETKO mice either at BL or CSS. In response to CSS, there was a reduction in adrenal tissue NE content in both VEETKO and flox mice (p<0.05). We conclude that endothelium‐derived ET‐1 is an important mechanism by which acute behavioral stress increases cardiovascular reactivity.

Grant Funding Source: PO1 HL 69999
The role of brain angiotensin AT2 receptors and NO in the renal sympatho‐inhibitory response to acute volume expansion in conscious rats (858.2)
Authors: Abdulla Mohammed , Johns Edward
Published: June 2021

This study investigated the role of AT2 receptor and NO on the renal sympatho‐inhibitory response to volume expansion (VE). Conscious rats were subjected to VE (0.25% bwt/min saline for 10min IV) following intracerebroventricular (I.C.V.) infusion of saline, angiotensin II (Ang II) or a combination of Ang II with either losartan, PD123319 (PD), an AT2 receptor antagonist or L‐NAME, an NO synthase inhibitor. I.C.V. losartan, PD or L‐NAME did not change baseline mean arterial pressure (MAP), heart rate (HR) or renal sympathetic nerve activity (RSNA). However, I.C.V. Ang II increased MAP and decreased both HR and RSNA (113±2 vs. 107±2 mmHg, 365±7 vs. 379±5 beat/min, 1.03±0.13 vs. 1.29±0.15 µV.s, respectively, all P<0.05). VE decreased RSNA in all groups by some 27±2% (P<0.05) at the end of the 10min. Ang II, losartan or PD had no effect on the magnitude of renal sympatho‐inhibitory response to VE. However, L‐NAME decreased the sympatho‐inhibitory response compared to saline (14±3 vs. 30±5%, P<0.05). I.C.V. Ang II in combination with losartan enhanced (41±3 vs. 29±5%) but with PD decreased (15±2 vs. 28±4%, all P<0.05) the renal sympatho‐inhibition compared to Ang II. The sympatho‐inhibitory response to VE following Ang II plus L‐NAME was similar to Ang II alone. These findings suggest that activation of central AT2 receptors enhances the renal sympatho‐inhibitory response to VE but this effect is not dependent on NO.

Grant Funding Source: Funded by Wellcome Trust
Central leptin sensitization of angiotensin II‐induced hypertension (858.3)
Authors: Xue Baojian , Beltz Terry , Guo Fang , Johnson Alan
Published: June 2021

Obesity has been associated with selective leptin resistance whereby the anorexic effect of leptin is attenuated, but its action to increase sympathetic nervous activity (SNA) and blood pressure (BP) remains intact. A recent study showed an interaction between leptin and the renin angiotensin system in regulating SNA. Our previous studies using an Induction‐Delay‐Expression experimental design have demonstrated that a subpressor dose of angiotensin (ANG) II pretreatment sensitizes the brain to produce an enhanced hypertensive response to a subsequent treatment with pressor dose of ANG II. The present study tested the hypothesis that central chronic leptin pretreatment can also sensitize the pressor actions of ANG II. Male rats were implanted for telemetered BP recording and were pretreated during Induction with either peripheral subpressor ANG II (10 ng/kg/min) or central leptin (20 ng/kg/min) for one week. After one week Delay, rats were treated during Expression with subcutaneous ANG II (120 ng/kg/min) for 2 weeks. The animals with the subpressor ANG II pretreatment responded with enhanced hypertension to ANG II (Δ44.5±3.4 mmHg vs.Δ 21.2±5.2 mmHg). Central infusion of leptin during Induction mimicked the sensitization produced by the subpressor ANG II (Δ39.3±3.8 mmHg). The results indicate that central chronic leptin can predispose rats to display an enhanced ANG II‐elicited hypertensive response.
Validation of lower body negative pressure as an experimental model of hemorrhage (859.5)
Authors: Hinojosa‐Laborde Carmen , Shade Robert , Bauer Cassondra , Muniz Gary , Pidcoke Heather , Goei Kathleen , Chung Kevin , Cap Andrew , Convertino Victor
Published: June 2021

Lower body negative pressure (LBNP), a model of hemorrhage (Hem), shifts blood volume to the legs and elicits central hypovolemia. This study compared the effects of LBNP to actual Hem. Baboons (n=14) were instrumented to measure pulse pressure (PP), central venous pressure (CVP), and stroke volume (SV). Blood was removed in four steps: 6.25%,12.5%, 18.75%, and 25% of total blood volume. Four weeks after Hem, the same animals were subjected to four levels of LBNP which changed PP and CVP as seen during Hem. Blood sampled at baseline and maximum Hem or LBNP measured blood gases, hematocrit, hemoglobin, plasma renin activity (PRA), vasopressin (AVP), epinephrine (EPI) and norepinephrine (NE). Hemodynamic responses to Hem and LBNP were identical. Hem decreased hematocrit, hemoglobin, and central venous oxygen saturation (ScvO2). In contrast, LBNP increased hematocrit, and hemoglobin, while ScvO2 remained unchanged. Hem caused greater (p蠄0.01) elevations in AVP and NE than LBNP, while PRA and EPI did not differ between studies. Thus, stepwise Hem elicited hemodynamic changes which could be mimicked with distinct levels of LBNP, but loss of red blood cells during Hem may result in greater neurohumoral activation. We conclude that while LBNP does not elicit the same effect on blood cell loss as Hem, LBNP mimics the integrative cardiovascular response to Hem, and validates the use of LBNP as an experimental model of Hem.

Grant Funding Source: Supported by US Army MRMC
Lower oxidative stress is associated with angiotensin II and salt‐induced acute cardiorenal failure in BalbC mice but not C57Black6 (860.10)
Authors: Jonsson Sofia , Becriovic Agic Mediha , Tveitaras Maria , Skogstrand Trude , Karlsen Tine , Liden Åsa , Leh Sabine , Iversen Bjarne , Reed Rolf , Hultström Michael
Published: June 2021

Combined heart and kidney failure has a high mortality rate and no specific treatment. The aim of the present investigation was to identify genetic and physiological differences between a susceptible and a protected mouse strain.

In preliminary experiments C57Black6 were found to tolerate angiotensin II (1µg/min/kg AngII) and 5% sodium diet for 5 weeks, while 90% of Balb‐C developed edema, hypotension and acute myocardial and tubular necrosis within four days. Experiments with 0.5µg/min/kg AngII, high sodium diet (3%), or a combination were used for urine collection in metabolic cages and microarray gene expression, which identified enrichment of genes involved in oxidative stress.

Water and food intake was similar in the strains. AngII reduced sodium and urine excretion slightly, while salt‐diet alone caused increased excretion in both strains. The combination increased sodium excretion and urine volume more in C57Black6 mice (48%, p<0.05). This was associated with higher oxidative stress in C57Black6 measured as TBARS (125% p<0.01).

In conclusion, BalbC are susceptible to acute cardiorenal failure after combined AngII and salt treatment while C57Black6 are not. Global gene expression indicates a difference in oxidative stress. Interestingly, increased oxidative stress was associated with more efficient sodium and volume excretion and protection from cardiorenal failure.

Grant Funding Source: Supported by Swedish Heart‐Lung Foundation, Åke Wiberg Foundation, Helsevest Research Council
Genomic differences in glutathione metabolism determines susceptibility to cardiorenal failure in mice (860.11)
Authors: Becirovic Agic Mediha , Jonsson Sofia , Tveitaras Maria , Karlsen Tine , Skogstrand Trude , Liden Åsa , Leh Sabine , Iversen(late) Bjarne , Reed Rolf , Hultström Michael
Published: June 2021

The susceptibility to acute cardiorenal failure is very different between patients and between different mouse strains. The aim of the present project was to identify novel regulatory pathways involved in genetic susceptibility to cardiorenal failure in mice. Preliminary results showed that the mouse strain BalbC develop acute cardiorenal syndrome with massive edema when treated with 1 µg/min/kg Angiotensin II (AngII) and 5% sodium diet, while the strain C57Black6 do not. The objective was to identify the genetic cause of this difference using a less extreme protocol with 0.5 µg/min/kg AngII and 3% sodium for four days. This increased urine, and sodium excretion more in C57Black6 than in BalbC (31.2‐fold vs. 22.5‐fold p=0.04).

A comparison of global gene expression using Illumina microarrays identified oxidative stress as an important pathway. Indeed, renal oxidative stress, measured as TBARS, increased in both strains (2.3‐fold p= 0.049 and 5.0‐fold p=1.1e‐6), but more so in C57Black6 (p=1.5e‐3). Further, combining gene‐expression with SNP‐analysis identified glutathione‐transferase‐activity as the most enriched pathway (p=4.8e‐6). N‐Acetylcysteine (NAc,150 mg/kg/24h) was used as a glutathione supplement. NAc reduced urine excretion in C57Black6 but not in BalbC. Interestingly, it markedly increased the mortality of C57Black6 from 10% to 60% while having no effect on BalbC.

In conclusion, the genetic findings and NAc response indicate that the glutathione system is important in determining the response to AngII and high‐salt diet and that increased oxidative stress is essential to protect the C57Black6 strain.

Grant Funding Source: Supported by Swedish Heart and Lung Foundation, Åke Wiberg Foundation, Helsevest Research Council
Normal pregnant T regs inhibit pathophysiology associated with hypertension in response to reduced uterine perfusion pressure in pregnant rats (860.18)
Authors: Cornelius Denise , Wallace Kedra , Harmon Ashlyn , Paige Adrienne , Moseley Janae , LaMarca Babbette
Published: June 2021

The RUPP rat model of preeclampsia (PE) exhibits much of the pathology characterizing this disease such as hypertension, inflammation, suppressed regulatory Tcells (Tregs), oxidative stress (ROS), and AT1‐AA during pregnancy. The objective of this study was to determine if supplementation of normal pregnant (NP) Tregs into RUPP rats would attenuate the pathophysiology associated with preeclampsia. CD4+/CD25+ T cells were isolated from spleens of NP rats and differentiated into Tregs in culture. On day 12 of gestation NP Tregs were injected into pregnant rats undergoing RUPP; day18 carotid catheters were inserted and day19 blood pressure (MAP) was recorded, blood and tissues were analyzed. MAP increased from 100±2 mmHg in NP (n=17) to 125±2 mmHg in RUPP (n=22) but was 117±3 mmHg in RUPP+NPTregs (n=18; p<0.05). Importantly, pup weight was 1.91±0.06 in RUPPs but increased to 2.06 ±0.09 in RUPP + NPTregs (p>0.05). Inflammatory markers in RUPPs decreased with NP Tregs; placental and renal ROS was 339 and 603 RLU in RUPP but was 178 and 171 RLU in RUPP+Tregs (p<0.05); TNF‐α was 20.9 pg/mL in RUPP and 12.59 pg/mL in RUPP+Tregs rats (p>0.5); AT1‐AA was 17.27 bpm in RUPP but was attenuated with NP Tregs to 0.50 bpm (p>.0001). We conclude that suppressed Tregs may play an important role in the inflammation and hypertension associated with PE.

Grant Funding Source: Supported by NIH grants RO1HD067541 and T32HL105324
Lack of an effect of collecting duct‐derived adenylyl cyclase 3 or 4 knockout on renal sodium and water excretion and arterial pressure (860.2)
Authors: Kittikulsuth Wararat , Stuart Deborah , Mironova Elena , Stockand James , Kohan Donald
Published: June 2021

Adenylyl cyclase (AC)‐stimulated cAMP is a key mediator of collecting duct (CD) Na and water transport. AC isoforms 3, 4 and 6 are expressed in the CD; while AC6 is involved in modulating CD Na and water transport, the roles of AC3 and AC4 in this regard are unknown. Therefore, we generated mice with loxP‐flanked critical exons in AC3 and AC4 genes and bred with mice expressing the AQP‐2/Cre transgene to yield CD principal cell (PC)‐specific knockout (KO) of AC3 or AC4. Isolated inner medullary CD (IMCD) showed 100% genomic target gene recombination in both KO lines, while renal papillary AC3 or AC4 mRNA were significantly reduced in CD AC3 or AC4 KO mice, respectively. CD AC3 or AC4 KO had no effect on AVP‐stimulated cAMP generation in isolated IM, although AngII‐stimulated cAMP was reduced in IM from CD AC3 KO mice. CD AC3 or AC4 KO did not alter water intake, urine volume, urine osmolality or plasma AVP during baseline or water restriction as compared to control mice. Na intake, urinary Na excretion, plasma renin concentration and blood pressure on a normal or high Na diet were not affected by CD AC3 or AC4 KO. Further, CD AC3 KO did not alter baseline or AVP‐stimulated ENaC activity as assessed by patch clamp of split‐open isolated cortical CD. In summary, these data suggest that AC3 and AC4 do not play a key role in physiologic regulation of CD Na and water handling.

Grant Funding Source: a Merit Review from the Department of Veterans Affairs and by NIH R01 DK097007
Dehydration induced reduction in body fat content (860.21)
Authors: McKinley Michael , Mathai Michael
Published: June 2021

The aim of this study was to determine the feasibility of body fat being metabolized by dehydrated rats thereby providing an endogenous source of body water to partially ameliorate dehydration. We measured total body fat mass (TBF) and total body water (TBW) using magnetic resonance relaxation analysis with an EchoMRI 100/900 Body Composition Analyser (Echo Medical Instruments, Singapore) before and after depriving rats (n = 6) of water for 24 h. When rats fed a normal pelleted lab diet (Na content of 0.5%) were water deprived, body mass (BM) was reduced from 400 ± 29 to 368 ± 22 g (mean ± SEM) and TBW from 264 ± 15 to 242 ± 15 g, while TBF fell from 29.6 ± 4.3 to 24.7 ± 4.1 g (p < 0.01). Food intake fell from 32.7 ± 1.2 to 20.9 ± 0.7 g/24h. Following rehydration, BM (405 ± 23 g) and TBW (268 ± 14.3 g) had returned to pre‐dehydrational levels by 24 h, whereas, TBF (28.1 ± 4.5 g) was still reduced in 5 of 6 rats at this time, but had recovered (29.1 ± 5.0 g) after 48 h of rehydration. When water replete rats were restricted to the same low intake of food for 24 h, the reduction of TBF (1.1 ± 0.5 g) during 24 h was significantly less than in dehydrated rats (4.9 ± 0.9 g). We also deprived rats (n = 4) of water for 24 h that were fed a diet of low NaCl content (0.02%). No reduction in TBF occurred (37.5 ± 8.2 pre‐dehydration versus 37.6 ± 8.6 g post‐dehydration), whereas TBW fell from 304 ± 8 to 285 ± 8 g. These data show that TBF is metabolized in dehydrated rats, but the effect is dependent on sodium status. They show also that it is feasible that body fat may be metabolized to water in dehydrated rats.

Grant Funding Source: NHMRC of Australia
HDAC1 is a novel regulator of nitric oxide synthase 3 in endothelial cells (860.3)
Authors: Hyndman Kelly , Ho Dao , Sega Martiana , Pollock Jennifer
Published: June 2021

Recent studies have identified that acetylation of lysines on NOS3 regulates NO production; however, there is little information regarding deacetylation of NOS3. Histone deacetylases (HDACs) remove acetyl groups from histone and non‐histone proteins. Recently it was reported that HDAC3 antagonizes aspirin‐induced NOS3 acetylation and reduces NO production in HUVECs. The role of other HDAC isoforms in mediating NOS3 deacetylation and/or NO production has not been determined. We tested the hypothesis that HDAC1 interacts with NOS3 leading to a decrease in lysine acetylation and NO production. Bovine Aortic Endothelial Cells (BAECs) were transfected with empty vector (N = 6) or human HDAC1 (N = 6) and lysine acetylation and NO production determined. In BAECs, we found that HDAC1 and NOS3 interact, and cells over‐expressing (OE) HDAC1 expressed reduced NOS3 acetylation (vector control = 1.0 ± 0.04, OE HDAC1 = 0.6 ± 0.04 A.U.; P < 0.001). We observed no differences in NOS3 phosphorylation at T495, S633, or S1177. Basal nitrite production was also reduced in the OE HDAC1 cells (356 ± 15 pmol/mg prt/h vs 223 ± 35 pmol/mg prt/h; P < 0.05). Stimulation of BAECs by 100 nM endothelin‐1 resulted in blunted nitrite production in OE HDAC1 cells (607± 115 pmol/mg prt/h vs 321 ± 61 pmol/mg prt/h; P < 0.05). In conclusion, HDAC1 is a novel inhibitor of NOS3 acetylation and NO production.

Grant Funding Source: Supported by NIH to JSP
Elucidation of the distal convoluted tubule transcriptome identifies Pcbd1 as new causative gene for hereditary hypomagnesemia (860.4)
Authors: Bindels Rene , de Baaij Jeroen , Ferre Silvia , Hoenderop Joost
Published: June 2021

The distal convoluted tubule (DCT) is instrumental in fine‐tuning of the renal Mg2+ handling. Here, we aim to discover new Mg2+‐related genes in DCT. Transgenic mice expressing eGFP under a DCT‐specific parvalbumin promoter were subjected to Mg2+‐deficient or Mg2+‐enriched diets. Subsequently, the Complex Object Parametric Analyzer and Sorter (COPAS) allowed for the first time isolation of eGFP‐positive DCT cells. RNA extracts thereof were analyzed by DNA microarrays to identify Mg2+ regulatory genes and 46 genes showed differential expression. Several known magnesiotropic genes, such as Trpm6 and Parvalbumin, were upregulated under low dietary Mg2+. Moreover, new genes were identified that are potentially involved in renal Mg2+ handling. Among others, Pcbd1 was identified to be higher expressed in low Mg2+ conditions. Pcbd1 encodes a transcriptional co‐activator of Hnf1b, which was previously implicated in renal Mg2+ handling and maturity onset diabetes of the young (MODY). Interestingly, when examining three independent patients with Pcbd1 mutations, two individuals were diagnosed with hypomagnesemia and renal Mg2+ loss. Moreover, two patients also developed diabetes with characteristics of MODY regardless of the serum Mg2+ levels. By elucidating the Mg2+‐sensitive DCT transcriptome new candidate genes in renal Mg2+ handling have been identified.
Role of the H,K‐ATPase type 2 in the regulation of blood pressure during K+ depletion (860.8)
Authors: Lamouroux Christine , Modyanov Nikolai , Crambert Gilles
Published: June 2021

Alteration of blood pressure (BP) level is one of the side effects associated to K+‐depletion. However, the mechanisms linking K+ balance to BP remain unsettled and debated. Adaptation to K+‐depletion requires ability to retain K+. In kidney and colon, this is handled by the stimulation of H,K‐ATPase type 2 (HKA2) and its absence leads to severe K+‐depletion under low‐K+ diet condition. Our aim is to investigate whether HKA2 may contribute to determination of BP level under low‐K+ diet. Wild‐type mice (WT) displayed a decrease of BP after 10 days of low‐K+ diet (from 113 to 103 mm Hg). In HKA2‐null mice, the decrease of BP (109 to 100 mm Hg) occurred much earlier (day 2‐3). At early stage of K+‐depletion, plasma volume remained constant in WT but decreased by 20% in HKA2‐null mice, indicating that decrease of BP is linked to a problem of volemia. However, this decrease of BP and volemia are not related to a leak of Na+ and fluid by the kidney. On the contrary, this hypovolemic state stimulates urinary Na+ retention through thiazide‐ and amiloride‐sensitive Na+ transport. Measurements of tissue Na+ contents suggest that the origin of the hypovolemia could be a transfer of fluid from the extracellular space toward the intracellular compartment. Presence of HKA2 is therefore required to limit the decrease of plasma K+ value and to protect against development of hypotension.

Grant Funding Source: Supported by the Fondation du Rein
Early post‐natal ventricle resection is accompained by neoformed cardiomyocytes and maintenance of cardiac function under stress in adult rats (862.1)
Authors: Zogbi Camila , de Carvalho Ana Elisa , Nakamuta Juliana , Caceres Viviane , Rochitte Carlos , Krieger Jose
Published: June 2021

One‐day‐old mice hold a brief and robust capacity of heart regeneration after apex resection. We tested whether the rat displays comparable response and its impact in adult cardiac overall performance. Apical resection in rats at postnatal day 1 (P1) and 7 (P7) rendered comparable loss of cardiac tissue (16 vs.18%) estimated by magnetic resonance imaging. P1 was associated with robust cardiac regeneration at 21 and 60 days while in P7 rats there was essentially scar tissue replacement. Cardiac tissue perfusion in the neoformed region decreased in both P1 and P7 groups at 21 and 60 days using SPECT scanning. General cardiac function at 60 days was comparable among the groups, but under hemodynamic stress the degree of change on stroke work decreased significantly in P7 (sham: 18.4±4.0; P1: 47.0±17,8; P7: ‐0.5±7.5; P<0.05). Furthermore, P7 displayed increased chamber stiffness and interstitial collagen (P1: 6.9±0.6; P7: 11.4±0.6; P<0.05). Taken together, we provide evidence that adult P1 rats undergo robust neoformation of cardiomyocytes enabling preservation of overall cardiac function under hemodynamic stress despite decreased tissue perfusion. In contrast, P7 rats display mainly scar tissue replacement and compromised overall cardiac function.

Grant Funding Source: FAPESP/Capes
The transition from fetal to postnatal life: feasibility of telemetric measurements of pressure and flow in the pulmonary and systemic circulations of lambs (862.8)
Authors: Johnson Rebecca , Raff Gary , Johengen Michael , Bennett Stephen , Pitsillides Koullis , Fineman Jeffrey
Published: June 2021

The hemodynamic changes that occur during transition from fetal to postnatal life are dramatic: the pulmonary circulation goes from a high‐pressure, low‐flow system to a low‐pressure, high‐flow system that must accommodate a full cardiac output; native shunts close (ductus arteriosus and foramen ovale); and the left ventricle must fully support the systemic circulation. Continuous hemodynamic measurements during natural birth have never been achieved given the challenging environment. We present a feasibility study demonstrating successful instrumentation of fetal lambs with hemodynamic measurements through postnatal life. A telemetry system (Transonic EndoGear Inc, Davis, CA) was implanted in fetal lambs at approximately 130 days gestation. Non‐occlusive, epivascular Doppler flowprobes and solid‐state blood pressure sensors allowed simultaneous monitoring in both the pulmonary and systemic circulations. Following implantation of the telemetry system, baseline measurements were obtained of blood flow and blood pressure in utero for two weeks, through the birth transition, followed by four weeks of monitoring post‐natal hemodynamics. The novel data acquired of blood flow, blood pressure and temperature will help us further understand the crucial physiologic transition from fetal to postnatal hemodynamics, and physiologic derangements in disease states including congenital heart disease.
Dysfunctional calcium homeostasis in aged mice primary tenocytes ő a potential functional link to tendon disorders (863.10)
Authors: Vallejo Julian , Spence Madoka , Brotto Leticia , Brotto Marco , Abreu Eduardo
Published: June 2021

Musculoskeletal diseases (MSKs) afflict more than 1.7 billion people worldwide. In the US, tendon disorders account for a surprising 30‐50% of all sports‐related injuries and close to half of all occupational illnesses. These diseases are further complicated by aging. Tendon disorders are difficult to treat in part because of our poor understanding of tendon physiology and pathophysiology at the cellular and molecular levels. Historically, more attention has been given to other cells of the musculoskeletal unit (bone‐tendon‐muscle) than to tendon cells (i.e., tenocytes), maybe because we lack good functional tests for tenocytes. Calcium plays an important role in tendon physiology and its response to mechanical load, and as a result calcium studies can be a useful tool to evaluate tenocytes. We compared the resting levels of calcium as well as the responses to Ionomycin in young and aged mouse primary tenocytes and found magnitude and the time to the maximal response were reduced in aged tenocytes. We are now conducting systematic studies to dissect the potential mechanisms for these differences. We postulate that dysfunctional calcium homeostasis might be a critical functional link between tenocytes and the organismal manifestation of tendon and MSKs.

Grant Funding Source: Supported by Dale & Dorothy Thompson Endowment Fund (MB)
Diaphragm muscle sarcopenia and neuromuscular junction morphology (863.6)
Authors: Greising Sarah , Stowe Jessica , Mantilla Carlos , Sieck Gary
Published: June 2021

We recently determined the presence of sarcopenia in the diaphragm muscle (DIAm), specifically an age‐related reduction in force and muscle fiber cross‐sectional area, the latter being more pronounced at type IIx and/or IIb DIAm fibers. In limb muscles, age‐related changes at neuromuscular junctions (NMJs) include retraction of presynaptic terminals, disaggregation and fragmentation of the motor end‐plate. However, it is presently not clear whether aging impacts the morphology of DIAm NMJs. Adult male mice (n=14) at 6 and 24 months of age, representing survival rates of 100 and 75%, respectively, were used to examine DIAm NMJ structure, pre‐ and post‐synaptic morphology and overlap as measured in 3D by confocal microscopy (643 NMJs). No age related differences were evident in 2D planar area (mean: 328 µm2) or complexity (i.e., fragmentation) of motor end‐plates. Pre‐ and post‐synaptic volumes were also similar across age groups. Evidence of partial or full denervation was present in ~6% of NMJs in both age groups. Thus, these results indicate that by 24 months of age, DIAm sarcopenia is not associated with evidence of ongoing denervation. Examination of earlier age groups will be necessary to rule out the contribution of NMJ denervation to DIAm sarcopenia given the loss of type IIx and/or IIb fibers by 24 months of age.

Grant Funding Source: Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.
Muscle specificity of age‐related changes in markers of autophagy (863.9)
Authors: Russ David , McCoy Katherine , Boyd Iva
Published: June 2021

The purpose of this investigation was to determine the degree, if any, of muscle‐specificity regarding the effect of aging on markers of autophagy in skeletal muscle. The idea that impaired basal autophagy may contribute to age‐related declines in tissue and organ function has gained traction in recent years. Our own laboratory has reported that aging phasic hindlimb muscles of aging rats exhibit some signs of impaired autophagy, along with marked declines in force generating capacity, even in the absence of sarcopenia. However, age‐related declines appear to occur to a lesser degree, or lesser rate, in the tonic soleus muscle. Accordingly, here we examined expression, via immunblotting, of several markers of autophagy and stress in the medial gastrocnemius (MG) and soleus (SOL) muscles of adult (6‐8 months) and aged (24‐25 months) male, F344/BN rats (n = 7/grp). Two‐way ANOVA (age X muscle) revealed significant age X muscle interactions in several markers of autophagic signaling and ER stress (Atg3, Beclin, Parkin, Grp78),but not others (Atg7, Atg4b). Where interactions were present, age increased expression in the MG (+25‐50%), but not the soleus. Interestingly, the LC3b‐II/I ratio, a common index of autophagy was reduced in the MG, but not the soleus. This change difference was largely driven by age‐related increases in LC3b‐I, without increases in LC3b‐II. These data are consistent with age‐related failure of autophagic (perhaps at Atg7 or Atg4b) contributing to functional decline in the MG, but not the soleus. These data have important implications for mechanisms of muscular senescence and may direct the development of future intervention strategies to enhance aging muscle function.
Striatin: a novel regulator of cardiomyocyte calcium homeostasis and contraction (864.3)
Authors: Nader Moni , Khalil Bariaa , Kvietys Peter , Bakheet Dana , Dzimiri Nduna
Published: June 2021

Although cardiac arrhythmias are still a major health threat for human, there is paucity of information on the molecular mechanisms involved in this pathology. Striatin (STRN) is a multivalent protein with dynamic domains including caveolin (Cav), Calmodulin (CaM), and Protein Phosphatase 2A (PP2A) binding sites, all of which are important regulatory units of the L‐type Calcium Channels (LCC) in cardiomyocytes. Previous studies indicate that mutations/deletions of the striatin gene correlate with the development of cardiac arrhythmias in animal models. We aimed to investigate whether STRN regulates calcium homeostasis and cardiomyocyte contraction rate. Data indicate that STRN is highly expressed in atrial myocardium when compared to the ventricles. Overexpression of STRN elevated the contraction rate (~ 3 fold) and resulted in sustained levels of KCl‐induced increases in intracellular calcium in neonatal rat cardiomyocytes. Conversely, silencing (>80%) the STRN gene using shRNA was associated with decreased contraction rate (~50%) of cardiomyocytes. Interestingly, pharmacological challenge with isoproterenol (β‐adrenergic agonist) failed to improve the contraction rate of cardiomyocytes with low levels of striatin (shRNA‐STRN). Biochemical analysis (CaM pull down) revealed that the interaction between CaM/PP2A/Cav‐3/LCC was increased in shRNA‐STRN cardiomyocytes and reduced when overexpressing STRN. Collectively our data indicate that striatin is a novel regulator of cardiomyocyte calcium homeostasis and contraction rate by modulating the CaM/PP2A/Cav‐3/LCC complex and influencing the activity of the LCC in the myocardium.

Grant Funding Source: King Faisal Specialist Hospital and Research Center
Activation of Toll‐like receptor 4 mediates bladder hypercontractility in diabetes (865.10)
Authors: Szasz Theodora , Nunes Kenia P. , Webb R. Clinton
Published: June 2021

Diabetic bladder dysfunction (DBD) is a common lower urinary tract complication of diabetes. Manifestations of DBD progress from bladder hypercontractility and urge incontinence in the initial phase to bladder hypocontractility and overflow incontinence in the late phase. Activation of the innate immune system via Toll‐like receptors (TLRs) like TLR4 leads to inflammation and oxidative stress and was implicated in diabetes pathophysiology. We hypothesized that bladder hypercontractility in the initial phase of DBD is mediated by TLR4 activation. Urinary bladder strips without urothelium from male C57bl/6 mice treated with vehicle or streptozotocin (65 mg/kg twice) were used to evaluate contractile function and protein expression of TLR4 and downstream mediator MyD88. Bladder expression of TLR4 and MyD88 was increased in STZ compared to control mice (TLR4=178±26, MyD88=138±12 % control). Carbachol‐mediated contraction was increased in bladder strips from STZ mice. The TLR4 inhibitor CLI095 (10 μM) decreased the maximal CCh‐induced contraction in STZ, but not in control mice (CON= 67.6±2.3, STZ=93.7±11.6, CON+CLI=76.3±6.7, STZ+CLI=66.2±7.2 % KCl‐induced contraction). These data suggest that activation of TLR4 during diabetes mediates bladder hypercontractility and contributes to DBD.

Grant Funding Source: NIH