bluuebunny/crossref_metadata_2025 huggingface dataset:
Blood volumes in inbred rat strains with different survival times after severe hemorrhage
Authors: Klemcke Harold G. , Calderon Mariam L , OH Taesung
Published: June 2021

We have previously shown 8‐fold differences in survival time among inbred rat strains after a 55% hemorrhage, and found a strong genetic component for this trait (Klemcke et al., Shock 29: 748, 2008). Blood volumes (BV) for all strains were assumed to be 5.83 % of body weight (BW), and BV removed was based on this figure. However, BV might vary in a strain‐dependent manner. If true, this may be one genetic variable affecting survival time after hemorrhage (STaH). To examine this possibility, we measured BV in select strains using an Evans blue procedure. BV varied among Brown Norway, Salt Sensitive, Fawn Hooded Hypertensive, Lewis, and DA rat strains respectively (9.16±.21a ; 7.45±.21b; 7.01±.24b,c ; 7.01±.22b,c ; 6.57±.22c % of BW; means with different letter superscripts are different, P<.01), and showed a heritability (h2) of 0.75. When these BV were used to calculate the % of BV removed from rats in our previous study, those percentages differed (37.0±0.3a; 45.4±0.5b; 50.0±0.4d; 47.8±0.9c; 52.1±.2e %; P<.01) and were highly and inversely correlated with previously determined STaH (r = ‐0.77; P <.01). These data indicate that previously noted differences in STaH among strains might be accounted for in part by removal of a different % of BV. They also suggest that BV −which is amenable to environmental (e.g., exercise) and genetic manipulation− is a genetically‐controlled characteristic that might be used to improve STaH.
Prostaglandin E2 (PGE2) type‐1 receptors (EP1R) mediate the voltage‐gated calcium currents and superoxide production induced by Angiotensin II (AngII) in subfornical organ (SFO) neurons
Authors: Wang Gang , Peterson Jeffrey R , Davisson Robin , Iadecola Costantino
Published: June 2021

The SFO is a critical brain region for the cardiovascular effects of systemic administration of slow pressor doses of AngII. AngII signaling in neurons depends on superoxide (O2−.) and modulation of Ca2+ channels. Cyclooxygenase (COX)‐derived PGE2 and EP1R are involved in AngII‐induced slow pressor hypertension (Peterson et al, Hypertension, 2008, 52:E128), but it is unknown whether AngII signaling in the SFO requires PGE2 or EP1R. We tested the hypothesis that EP1R play a role in AngII‐induced O2−. production and Ca2+ currents in SFO neurons. In whole‐cell patch‐clamp recordings in dissociated mouse SFO neurons, AngII (100nM) increased nifedipine‐sensitive Ca2+ currents (ICa) (+29±5%; p<0.01; n=8). The increase was blocked by the EP1R antagonist SC51089 (10 μM) or the COX1 inhibitor SC560 (10 μM), but not by the COX2 inhibitor NS398 (10 μM; p>0.05; n=4). PGE2 (100nM) also increased ICa (+36±4%; p<0.01; n=6), an effect blocked by SC51089 (n=4). In addition, AngII or PGE2 failed to increase ICa in SFO neurons lacking the CaV1.3 L‐type channel. O2−. production in SFO neurons, assessed using dihydroethidium, was increased (p<0.05) by AngII (+152±55%; n=14) or PGE2 (+126±57%; n=14), an effect blocked by SC51089 (n=15). Our results suggest that AngII signaling in SFO neurons depends on COX1‐derived PGE2 and EP1R, and provide the cellular bases for the prominent role that EP1R play in AngII slow pressor hypertension (supported by HL18974)
KIDNEY FIBROSIS IN HYPERTENSIVE RATS: Role of Oxidative Stress
Authors: Zhao Wenyuan , Chen Sue S , Ahokas Robert A , sun Yao
Published: June 2021

Renal fibrosis occurs in patients with hypertension. Oxidative stress appears in hypertensive kidney disease. Herein, we tested the hypothesis that oxidative stress contributes to the development of renal fibrosis during hypertension. SD rats were treated with angiotensin (Ang)II (9ug/h sc) for 4 wks with/without co‐treatment of antioxidants, apocynin and tempol (120mg/kg/day each). Appearance of renal oxidative stress and its effect on the expression of TGF‐b1, population of myofibroblasts, collagen synthesis/degradation and fibrosis in kidneys were examined. Chronic AngII infusion elevated systemic blood pressure, which was accompanied with extensive renal fibrosis and oxidative stress represented as upregulated NADPH oxidase and suppressed SOD. Antioxidants led to: 1) markedly decreased renal NADPH oxidase; 2) significantly attenuated gene expression of TGF‐b1, type‐I collagen, and TIMP‐I/‐II in the kidney; 3) largely reduced population of myofibroblasts in both the cortex and medulla; 4) significantly reduced renal collagen; 5) and partially suppressed blood pressure. Thus, AngII‐induced hypertension leads to renal oxidative stress by increasing NADPH oxidase and reducing SOD in the kidney. Renal oxidative stress upregulates collagen synthesis, while suppresses collagen degradation, thereby promoting the development of fibrosis in kidneys of hypertensive rats.
High fat (HF) diet during development and throughout life does not increase oxidative stress in female borderline hypertensive rats (BHR)
Authors: Sasser Jennifer M , Mitra Anaya , White Leslie D. , Rowland Neil E. , Baylis Chris
Published: June 2021

HF diet is associated with oxidative stress and cardiovascular disease. Our aim here was to determine if HF stimulates reactive oxygen species in female rats with a genetic predisposition to mild hypertension. Female Wistar rats were fed a HF (60% lard, n=6) or low fat (LF, 10% lard, n=6) diet for 8 wk, then mated with male spontaneously hypertensive rats to yield BHR offspring. Dams were maintained on the same diets though gestation and lactation. At weaning, female offspring from LF dams were fed normal chow while the offspring from HF dams were placed on a HF "junk food" diet for over 10 months. HF diet increased body weight (499±32 vs 412±19 g, p<0.05 vs LF) but had no effect on blood pressure (118±7 vs 128±4, anesthetized direct arterial measurement). Markers of oxidative stress, renal and plasma TBARS and plasma H2O2 levels, were similar between groups. No significant changes were observed in the protein expression of any of the isoforms of superoxide dismutase (SOD) in the renal cortex. In conclusion, female BHR do not exhibit increased oxidative stress in response to prolonged HF diet.
Histone deacetylase blockade improves renal function and attenuates oxidative stress in rats with cisplatin‐induced acute renal failure
Authors: Elks Carrie M. , Haque Masudul , Mariappan Nithya , Francis Joseph
Published: June 2021

Histone deacetylase (HDAC) inhibition has been shown to prevent cytokine induction and to modulate renal disease in rodent models. We tested the hypothesis that HDAC inhibition would improve various renal and oxidative stress parameters in cisplatin (CDDP)‐induced acute renal failure (ARF). 15‐week old male Sprague‐Dawley rats (n=4 per group) were treated with the HDAC inhibitor, valproic acid (VPA; 300 mg/kg, s.c.), or vehicle, for 1 day prior to and 5 days following induction of ARF with a single 6 mg/kg injection of CDDP (or saline vehicle). At the end of 6 days, anesthetized rats underwent acute renal experiments to determine renal blood flow (RBF) and glomerular filtration rate (GFR) using PAH and inulin clearance techniques, respectively. Rats were sacrificed and kidneys removed; renal cortices were isolated for measurement of tissue and mitochondrial free radical levels using electron paramagnetic resonance spectroscopy. Tissue mRNA and protein levels of HDAC and oxidative stress genes were determined using real‐time RT‐PCR and western blot, respectively. No significant changes in mean arterial pressure (MAP) were noted among groups. VPA treatment improved RBF and GFR (p<0.05) and attenuated the higher levels of total tissue and mitochondrial ROS and tissue superoxide in ARF rats. VPA treatment also significantly decreased tissue mRNA and protein expression levels of HDAC and oxidative stress genes, and normalized ATP production and ADP/ATP ratio in rats with ARF. These findings suggest that inhibition of class I HDACs with VPA improves the renal oxidative stress and tissue and mitochondrial injury that accompany CDDP‐induced ARF. Grant support: NIH grant RO1HL080544‐01 for Dr. Joseph Francis.
Nox2 is required for amiloride‐sensitive H+ efflux in a rat medullary thick ascending limb cell line
Authors: O'Connor Paul M , Fang Yi , Feng Di , Liu Yong , Liang Mingyu , Ferreri Nicholas , Cowley Allen W
Published: June 2021

We have recently demonstrated that an amiloride (NMA)‐sensitive outward H+ current is present in mTAL of salt‐sensitive (SS) but not salt‐resistant (SS.13BN) rats and that when activated, this stimulates the production of excess superoxide (O2−). The identity of the H+ transporter remains unclear, however it displays many of the characteristics of a nox2 associated H+ current characterized in macrophages. The aim of this study was to determine which subunits of NAD(P)H oxidase are expressed in a rat mTAL cell line and whether a similar H+ transport pathway exists in these cells. mTAL cells were cultured, transfected with nox2, loaded with BCECF dye, imaged on a inverted microscope and intracellular pH (pHi) recovery determined following an NH4Cl prepulse in 0 Na+ solution with BaCl2. pHi recovery following an NH4Cl prepulse was absent in control transfected mTAL (δpHi/sec ‐1.9±2*10−5) however, in mTAL transfected with nox2, amiloride‐sensitive pHi recovery could be detected (δpHi/sec 7.9±3.2*10−5 vs ‐1.6±2*10−5 with NMA; P<0.03). Cellular mRNA expression of endogenous NAD(P)H oxidase subunits are shown in the table below. We conclude that although nox4 is the primary gp91 homolog expressed in mTAL cells, nox2 over‐expression is required to produce the outward NMA‐sensitive H+ current observed in SS mTAL.

mTAL copy # per 106 18S RNA (n=4)
Augmented afferent arteriole (AA) vasoconstriction induced by chymase‐dependent ANGII formation in diabetic renal disease
Authors: Park S , Bivona B J , Harrison‐Bernard L M
Published: June 2021

The leptin receptor deficient db/db mouse is a genetic model of type II diabetes with progressive renal disease. We hypothesized that chymase is the major enzymatic pathway responsible for intrarenal ANGII formation in diabetes. Renal ACE activity was reduced in diabetic compared to control db/m mice (5±3 vs 37±8 AFU/min/mg), while ACE2 activity was similar. Urinary albumin excretion (822±187 vs 28±4 μg/d) was elevated, while GFR (0.65±0.04 vs 1.00±0.13ml/min/g KW, n=6, 7, P<0.02) was reduced by 35% in diabetic compared to control mice. AA diameters of in vitro blood perfused juxtamedullary nephrons were significantly larger in diabetic compared to control kidneys (14.3±0.4 vs 12.3±0.3 μm). AA responses to ANGI±AT1 receptor blocker (100 μM Candesartan, CV), ±ACE inhibitor (1mM Captopril) ±serine protease inhibitor (1mM PMSF) were measured. AA vasoconstriction to 10, 100, 1000nM ANGI was similar in both groups (‐9±2, ‐29±4, ‐28±3% diabetic, n=11; ‐10±2, ‐24±4, ‐26±3% control, n=12) due to conversion of ANGI→ANGII. AA vasoconstriction produced by ANGI→ANGII was significantly attenuated by CV, Captopril, and Captopril+PMSF (‐4±2, ‐3±5, ‐5±5% at 100nM ANGI), but not by PMSF alone (‐22±4%) in control kidneys. AA vasoconstriction produced by ANGI→ANGII was significantly attenuated by CV, PMSF, and Captopril+PMSF (‐4±3, ‐6±6 ‐6±3 % at 100nM ANGI), but not by Captopril alone (‐20±4%) in diabetic kidneys. AA vasoconstriction to the chymase‐specific [Pro11, D‐Ala12]ANGI was significantly augmented in AA of diabetic compared to control kidneys (‐16±5,‐28±5% vs ‐3±2,‐13±2% at 100, 1000nM). The AA responses to [Pro11, D‐Ala12]ANGI were blocked by CV in both. In summary, we have identified a novel role for renal chymase activity in diabetes. We propose that a switch from ACE‐dependent to chymase‐dependent intrarenal ANGII formation contributes to diabetic renal vascular disease.
Impaired Renal Blood Flow Autoregulation due to Compromised Tubuloglomerular Feedback in Mice Lacking Connexin 40
Authors: Just Armin , Kurtz Lisa , Wagner Charlotte , de Wit Cor , Kurtz Armin , Arendshorst William J.
Published: June 2021

We studied the role of connexin 40 (Cx40) in autoregulation of renal blood flow (RBF). Cx40‐deficient mice (Cx40‐ko) had impaired steady‐state autoregulation (7% of perfect vs. 102% in wild‐type (wt), p<0.01) to a sudden step increase in perfusion pressure. Dynamic analysis markedly reduced tubuloglomerular feedback (TGF, 15 vs. 75%, p<0.01), while myogenic and third mechanisms were not consistently altered. In mice with Cx40 replaced by Cx45 (Cx40‐ki45) steady‐state autoregulation was weaker than in wt (66%). TGF was impaired but tended to be stronger than in Cx40‐ko. L‐NAME augmented the myogenic response similarly in all genotypes. Responses of renovascular resistance and arterial pressure to norepinephrine (NE, 75 μg iv), acetylcholine (ACh, 25 μg), and LNAME (25 mg/kg) were similar among genotypes. Immunhistochemistry showed Cx37, Cx40, and Cx43 in preglomerular endothelial, renin‐producing, and mesangial cells. In Cx40‐ko and Cx40‐ki45, Cx40 was absent and Cx37 and Cx43 reduced.

We conclude that Cx40 contributes to RBF autoregulation and is critical for TGF‐mediated macula densa signaling to the afferent arteriole. This Cx40 function can partly be substituted by Cx45 and is independent of NO. NO modulation of the renal myogenic response does not require Cx40, nor do acute renal vasomotor responses to NE, ACh, or NO. Supported by NIH and German Research Foundation.
Validation of kidney slices as a viable model for studying in situ pericyte regulation of vasa recta capillaries in the renal medulla
Authors: Crawford Carol , Desai Tanmay , Peppiatt‐Wildman Claire M
Published: June 2021

Pericytes have been shown to regulate capillary diameter in situ, in whole retina and the cerebellum [1]. The aim of this study was to identify renal pericytes using similar imaging techniques, with a view to studying the role of pericytes in controlling vasa recta capillary blood flow in the renal medulla.

Kidney slices were obtained from rat kidneys and maintained in a physiological saline solution, bubbled with 95% O2/5% CO2. Confocal microscopy was used to image kidney slices labeled with calcein, propidium iodide and Hoechst to confirm viability of cells in kidney slices. Immunohistochemistry techniques in combination with confocal imaging were used to identify pericytes and vasa recta capillaries in the medulla of fixed kidney slices when labeled with fluorescently conjugated neural‐glial 2 and isolectin B4, respectively. Video‐imaging techniques enabled us to collect DIC images of pericytes residing on vasa recta capillaries, and real‐time vasoconstriction of capillaries at pericyte sites by Angiotensin‐II. Pericytes were identified by their typical 'bump on a log' morphology [1]. The techniques described here have established kidney slices as a viable model which can be used to further investigate the role of pericytes in the regulation of vasa recta capillaries in situ.

[1] Peppiatt CM, Howarth C, Mobbs P, Attwell D. Nature 2006 Oct 12;443(7112):700‐4
Assay limitations of osmotic reflection coefficient of isolated mouse glomeruli ex vivo.
Authors: Sage Leslie May , Bates David O , Harper Stephen J , Salmon Andrew HJ
Published: June 2021

Glomerular reflection coefficient (σ) to albumin (σalb) represents glomerular filtration barrier (GFB) integrity. For molecules perfectly retained (e.g. >100kDa dextran), σ=1. If σalb <1, an albumin solution exerts lower trans‐GFB oncotic pressure (π) than iso‐oncotic dextran solution. We refined and critiqued an assay calculating σalb from the ratio of these effective π, indicated by relative glomerular volumes in each solution.

We created a BSA solution iso‐oncotic to purified >100kDa dextran solution. Glomeruli were sieved from C57bl6 mouse kidney and perifused with BSA in a flow‐controlled chamber, then perifusate was switched to dextran. Volume was calculated from video stills of glomeruli during zero‐flow for each perifusate (dextran: Vdex; BSA: VBSA). Vdex/VBSA was taken to describe σalb. Surprisingly, pronase treatment (0.1mg/ml, 2min) did not change σalb [p=0.97, n=7].

Vdex/VBSA=σalb assumes V represents glomerular capillary volume, but we are limited to calculation of total glomerular volume. Unknown change of non‐capillary glomerular volume fractions confounds calculation of σalb. Vdex/VBSA=σalb also assumes capillary protein retention. Leakage during lowered σ alters effective to an unknown extent, obscuring the relation of π to V. Vdex/VBSA=σalb is invalid in this case. Data must be interpreted cautiously in the case of increased σ.

Kidney Research UK, British Heart Foundation
Water extracts of cigarette smoke elicit smooth muscle dependent relaxation of rat renal arteries
Authors: Halmai Richard , Szijjártó István , Brasnyó Pál , Fésüs Gábor , Tóth Péter , Pusch Gabriella , Koller Akos , Wittmann István
Published: June 2021

The mechanisms responsible for cigarette smoking‐induced increases in glomerular filtration rate and microalbuminuria in healthy persons are not known. We hypothesized that they may be related to changes in renal vascular resistance elicited by the water‐soluble components of cigarette smoke (wCS). The acute effects of wCS on isometric tension of preconstricted isolated rat renal arteries were examined. Increasing doses of wCS (1‐5‐10% of saturated solution) elicited dose dependent relaxations of renal arteries (wCS‐1%: 41±15%, wCS‐5%: 79±9%, wCS‐10% 90±6%). Endothelium removal did not affect relaxations to wCS (wCS‐1%: 15 ± 9%, wCS‐5%: 62±15%, wCS‐10% 90±7%). Similar results were obtained with nicotine free cigarettes (wCS‐1%: 38 ± 3%, wCS‐5%: 77±15%, wCS‐10% 97±0,2%). Relaxation induced by wCS were not affected by inhibition of soluble guanylate cyclase by ODQ (wCS‐1%: 13 ± 4%, wCS‐5%: 58±15%, CS‐10% 94±4%). Thus water soluble components of cigarette smoke elicit relaxations of isolated renal arteries that are independent of endothelium‐, nicotine‐ and cGMP‐related vasomotor mechanisms (such as NO or CO). Although further studies are necessary to elucidate the mechanisms responsible for renal artery relaxation, we propose that cigarette smoking, by modulating directly the smooth muscle tone of renal microvessels, could increases glomerular filtration rate and microalbuminuria observed in healthy smoking men.

(Grants: Hungarian SRF/OTKA T48376, K71591, F68638; HSC/ETT 364/2006, AHA F. Aff. 0855910D, USA)
Expression of endothelin, endothelin type A receptor, isoforms of nitric oxide synthase, and angiotensin receptors in endothelin type B receptor deficient mice
Authors: Rettig‐Zimmermann Juliane , Schildroth Janice , Kalk Philipp , Steege Andreas , Fähling Michael , Persson Pontus Börje , Hocher Berthold , Patzak Andreas
Published: June 2021

Endothelin‐1 (ET‐1) is involved in the control of renal vessel contractions by acting on endothelin type A (ETA) and type B (ETB) receptors. Several studies indicate an interaction of ET‐1 with the angiotensin system; which includes modulation of receptor expression. The aim of this study was to determine the influence of ETB receptor deficiency on the expression of ET‐1, ETA receptors, nitric oxide synthases (NOS) as well as angiotensin receptors (AT) in preglomerular vessels and kidneys of ETB(‐/‐) mice. ETA mRNA expression, determined by real time PCR, was decreased in preglomerular vessels compared to wild type mice (ETB(+/+)). ETA mRNA and protein were also diminished in whole kidney preparations in ETB(‐/‐) mice. The endothelial NOS (eNOS) mRNA was less expressed in preglomerular vessels. No significant differences were found for mRNA expression of eNOS, nNOS, and iNOS in whole kidney preparations. AT1 and AT2 receptor mRNA in the kidney did not differ between ETB(‐/‐) and ETB(+/+). As shown by western blotting analysis the ET‐1 protein was stronger expressed in the kidneys of ETB(‐/‐) The results of ET‐1 and ETA expression confirm earlier findings of a clearance function of the ETB receptor and modulation of ETA expression by ET‐1. Moreover, ET receptor expression/function seems to influence eNOS mRNA expression in renal arterioles.
IL‐6 induced hypertension in pregnant rats is associated with agonistic autoantibodies to the angiotensin II type I receptor
Authors: Speed Joshua , Fournier Lillian , Cockrell Kathy , Dechend Ralf , Granger Joey , Lamarca Babbette
Published: June 2021

Increases in interleukin 6 (IL‐6) and agonistic autoantibodies to the angiotensin II type 1 receptor (AT1‐AA) are proposed to be important links between placental ischemia and hypertension in preeclampsia. We reported that hypertension in response to placental ischemia in pregnant rats is associated with elevated IL‐6 and AT1‐AA. However, whether IL‐6 stimulates AT1‐AA production is unknown. Therefore, the purpose of this study was to determine whether IL‐6 excess in pregnant rats leads to production of AT1‐AA and whether AT1 R activation plays a role in mediating IL‐6 induced hypertension. To achieve this goal, IL‐6 (5ng/day) was infused into NP rats and mean arterial pressure (MAP) and AT1‐AA were analyzed. Serum IL‐6 increased with chronic infusion from 63 ±5 pg/ml to 230 ±54 pg/ml was associated with an increase in MAP from 102 ± 2 to 118 ± 4 mm Hg. Furthermore, AT1‐AA increased markedly with chronic IL‐6 (0.7 ± 0.3 bpm NP vs 14.1 ± 1.4 bpm). To determine the importance of AT1 R activation in mediating hypertension in IL‐6 treated rats, we measured MAP in the presence and absence of an AT1R antagonist, losartan, L. Blood pressure responses to IL‐6 were abolished in losartan pretreated rats (85± 4 in NP+L vs 85±3 mmHg in IL‐6 + L). Collectively, these data indicate that IL‐6 is an important stimulus of AT1‐AA and that activation of the AT1 receptor appears to mediate hypertension associated with IL‐6 during pregnancy.
CHRONIC TESTOSTERONE SUPPLEMENTATION IN ZUCKER RATS WORSEN RENAL FUNCTION IN LEAN, BUT NOT OBESE
Authors: LOPEZ‐RUIZ ARNALDO FABIAN , ILIESCU RADU , ZHANG HUIMIN , RECKELHOFF JANE F.
Published: June 2021

Obesity in men is associated with increases in renal disease. Androgen levels decrease in obese men, and many investigators believe this may contribute to the increased renal disease. The present study was done to test the hypothesis that chronic androgen supplementation protect against renal injury in obese rats. Male lean and obese Zucker rats, aged 24 wks, were untreated (C) or treated with testosterone propionate (5 (5T) or 10 (10T) mg/10 mm Silastic pellets implanted SQ, changed every 3 weeks) for 10 wks. Body weight (BW), GFR, albuminuria, TNF‐α, IL‐6, left ventricular index (LVI) were measured at 34 wks of age. Data shown below.

compared with Lean‐C; #, compared with Obese‐C

The data show that chronic testosterone decreases BW, inflammation, renal injury, LVI in obese rats, but decreases renal function and promotes renal injury in lean rats. Men with obesity may benefit from testosterone replacement.
Estrogen receptor antagonism and ovariectomy exacerbate ventricular remodeling in female rats
Authors: Gardner Jason Davin , Voloshenyuk Tetyana G , Murray David B
Published: June 2021

The ovarian hormone and estrogen receptor dependence of female cardioprotection were investigated using the aortocaval fistula rat model of chronic volume overload. Five groups of female rats were studied: sham‐operated (SHAM), intact fistula (FIST), ovariectomized fistula (FIST‐OX), ovariectomized fistula treated with estrogen (FIST‐OX+EST), and intact fistula rats treated with estrogen receptor blocker ICI 182,780 (FIST+ICI). Cardiac function was assessed temporally for 6 wks using echocardiogram. All rats with fistula had significantly increased cardiac output (119±52 ml/min for FIST‐OX vs. 26±5 ml/min for SHAM, p<0.05). This volume overload induced significant left ventricular (LV) hypertrophy in the fistula groups. All fistula groups developed increased LV internal diastolic dimension (LVIDd), indicating LV dilatation, with the FIST‐OX and FIST+ICI groups having the greatest increases (300% and 290%, respectively, relative to pre‐fistula baselines, p<0.05). This dilatation was associated with significantly reduced ejection fraction. Estrogen treatment attenuated the remodeling developed by ovariectomized females. These data indicate that ovarian hormones are essential for cardioprotection against adverse remodeling in female rats subjected to volume overload, and that this protection is estrogen receptor dependent.

Research supported by the American Heart Association (JG ‐ #0435298N).
Sex differences in cardiovascular and metabolic responses of Sprague‐Dawley rats to chronic subcutaeous leptin infusion
Authors: Kopa Dawn Elizabeth , Knight David , Messina Michelina M. , Seth Rohit , Overton J. Micheal
Published: June 2021

Female rats are reportedly more sensitive to the acute anorectic effects of central leptin (LEP). We tested the hypothesis that female rats would exhibit greater metabolic (indirect calorimetry) and depressor (telemetry) responses to chronic peripheral leptin administration (300 ug/kg/d for 7d via osmotic pump). Surprisingly, there was no significant sex difference in the anorectic actions of LEP, irrespective of estrous cycle phase of the female rats. Compared to male rats, LEP‐treated females exhibited a greater tachycardia, but similar metabolic rate response irrespective of estrous cycle phase. As previously reported in males, LEP infusion decreased mean arterial pressure (MAP), and this response was only evident during diestrus in females. We conclude that there is a complex modulatory role of estrogen on leptin action. While estrogen may augment the central mechanisms associated with leptin‐mediated tachycardia, the results are consistent with the possibility that the hypotensive response to chronic infusion of low dose peripheral leptin is not expressed in the presence of estrogen.
Differences in Hemodynamic Responses to Stress Between Male and Female Rats
Authors: Chen Fangping , Mullenax Heather , Sharma Puneet , Finkel Mitchell S
Published: June 2021

We have previously reported echocardiographic evidence of myocardial dysfunction in male rats with prenatal stress (PS) and restraint stress (R) (PS+R). We now report significant differences in hemodynamic effects of PS+R between male vs female rats. PS + R blunted both systolic and diastolic function in male rats. In contrast, PS+R enhanced systolic function and had no effect on diastolic function in female rats. PS+R blunted both systolic and diastolic responses to isoproterenol (ISO) in male rats, but enhanced the systolic and diastolic responses to ISO in female rats. No differences in baseline hemodynamics or responses to ISO were noted between male and female rats after PS alone or R alone. This cardiomyopathy model may provide insights into sex differences in behavioral and cardiovascular responses to stress in animal models and humans.

Mean± SE; #p<0.05, ##p<0.01, vs Control.
Intrauterine hypoxia inhibits caspase 3 in a NO‐dependent manner in fetal hearts
Authors: Evans LaShauna , Liu Hongshan , Thompson Loren P
Published: June 2021

Nitric oxide (NO) is an important regulator of cardiac function and apoptosis. We have previously shown that hypoxia upregulates iNOS expression in fetal guinea pig hearts. Thus, the aim of this study is to quantify the effect of intrauterine hypoxia on the pro‐apoptotic enzyme, caspase 3, and the role of NO in fetal hearts.

Methods

To test this pregnant guinea pigs were exposed to room air (N=4) or 10.5%O2 (N=5) for 14d prior to term (term=65d) and administered either water or L‐NIL, a selective iNOS inhibitor, at 1‐2mg/kg/d for 7‐10d. At 60d gestation, near term fetuses were removed from anesthetized sows, fetal hearts excised, and left ventricles frozen in liquid N2 and stored in −80C. Caspase 3 protein and activity levels were measured by Western and ELISA kit, respectively.

Results

Chronic HPX decreased (P=.009) active caspase 3 (15kDa) protein levels by 40%, but had no effect on either levels of the pro‐form (32kDa) or active caspase 3 form (22kDa) compared to NMX controls. Hypoxia decreased (P=.029) activity levels by 38% compared to NMX controls. L‐NIL reversed the HPX‐induced decrease and increased activity levels by 72% compared to HPX alone. Thus, chronic hypoxia inhibits apoptosis in near term fetal guinea pig hearts. This may be mediated by an inhibitory effect of NO on caspase 3 activity as a compensatory and cardioprotective response to hypoxic stress. [(NIH HL49999/LT & NIH HL90044/LE)]
Pregnancy outcomes in a Sprague Dawley rat model of periodontal disease
Authors: Palmgren Muriel S. , Corll Conni , Danan Basel , Vastardis Sortirios , Svec David , Svec Frank , Porter Johnny R.
Published: June 2021

Periodontal disease has been linked with preterm, low birth weight babies. However, results of epidemiology studies conflict. An animal model is needed to test this relationship. We propose that periodontal disease in rats would result in preterm birth. Periodontal disease was induced in female Sprague Dawley rats by tying 5/0 silk suture around four molars, one in each quadrant, at least 30 days before the rats became pregnant. In addition to the silk ligatures, the oral cavity of a second group was inoculated with live Porphyromonas gingivalis (ATTC 53978). The control group had neither ligatures nor P. gingivalis. After birth, the jaws of the dams were x‐rayed and bone height measured as percentage of the tooth height. Ligatures reduced alveolar bone height in rats (0.48±0.05, p<0.01) compared to controls (0.055±0.03). The combination of ligatures and P. gingivalis further decreased bone height (0.39±0.08, p<0.0001). In this experiment with Sprague Dawley rats, periodontal disease did not affect birth outcomes, including length of gestation, and number and weight of pups. Supported in part by Research Grant No. 21‐FY1251 from the March of Dimes Birth Defects Foundation.
Differences in spleen mRNA ILs expression in anterior, posterior and total hypophysectomized rats with experimental autoimmune encephalomyelitis (EAE) or adjuvant arthritis (AA); the vasopressin role
Authors: Quintanar‐Stephano Andrés , Villalobos Criseida Egina , Arroyo Karla Isela , Campos Rafael , Reyna Humberto , Kovacs Kalman , Berczi Istvan
Published: June 2021

Neurointermediate pituitary lobectomy (NIL) in Lewis rats causes mild signs of EAE, while hypophysectomy (HYPOX) and anterior pituitary lobectomy (AL) worse the disease, and desmopressin (vasopressin analog) aggravates the EAE in NIL and HYPOX rats. Besides, the AIA severity is low in NIL and no signs of AIA occur in HYPOX animals, while AL induces severe arthritis. Here we compare the splenic expression (RT‐PCR) of mRNA of ILs 1, 2, 6, 10, 12 and INF‐¦Ã between sham operated (SHAM), AL, NIL, and HYPOX groups immunized for EAE (females) or AIA (males). Intact control groups were included for basal IL levels. Rats were operated 3 weeks before immunizations and sacrificed at day 15. Results showed over expression of IL‐2 in AL‐EAE group. Similar augments of IL‐1 and 2 in SHAM, NIL and HYPOX‐EAE groups, and low expression in ILs 6, 10 and INF‐¦Ã in NIL, AL and HYPOX groups. IL‐12 was higher in HYPOX‐EAE rats with no changes in the remaining groups. In AL‐AIA group, significant increases in ILs 1, 2, 6 and INF‐¦Ã occurred, while in NIL‐AIA group ILs 2, 12 and INF‐¦Ã were low respect the remaining groups. HYPOX group did not developed clinical signs of AIA and ILs 2, 6, 10, 12 and INF‐¦Ã levels were similar to the SHAM group. We conclude that neurohypophysial hormones are important in the control of autoimmune diseases and that the ILs involved in the immune responses are different for EAE and AIA.

CONACYT. M̈¦xico
Possible role of the nuclear factor kappa B (NF‐kappa B) (NF) in the development of retained placenta (RP)
Authors: Moustafa Sohair AbdAllah
Published: June 2021

The low NF level seen in the serum of buffalos (B) (P = 0.01) may initiate RP through: a. Myometrial quiescence (MQ) which could be due the inhibited expression of target genes causing increased myometrial contractility e.g. COX‐2 catalyzing prostanoids synthesis. This may account for the abrogated increase in PGE2 level in BFR. The low NF level may cause an inhibition of progesterone withdrawal; decrease in number of progesterone receptors(PR); that accompany parturition. This increases the sensitivity of myometrium to progesterone. A mutual antagonism exists between PR and NF transcriptional activities. A decrease in oxytocin (OX) receptors numbers(OTR) may arise due to the low NF level since NF is involved in the up regulation of OTR. This may cause a low sensitivity of myometrium to OX thus mediating a negative feed back inhibition of the expected decrease in serum OX level accounting for the unchanged OX level in BRP. The low TNF‐alpha (P = 0.02) and NO (P = 0.001) levels in BRP may contribute to MQ since TNF‐alpha activates NF, also the expression of NO‐2 involves the activation of NF. b. Immunosuppression (IM) in which immune response that affects placental rejection at parturition is inhibited in BRP. This hypothesis is based on reduced NF and TNF‐alpha levels in BRP. NF activation induces cell adhesion molecules, cytokines and iNOS expression. And, NF is highly inducible by cytokines including TNF‐alpha.
Endothelium‐independent relaxation induced by estrogen in the chicken ductus arteriosus
Authors: Villamor Eduardo , Flinsenberg Thijs , Zoer Bea , Schuurman Marijn J , Perez‐Vizcaino Francisco , Cogolludo Angel L
Published: June 2021

Estrogen can modulate vascular tone by targeting both endothelial and/or vascular smooth muscle cells. However, The mechanism of estrogen‐induced vascular relaxation appears to be heterogeneous with respect to vessel and/or species. We aimed to characterize the vasoactive effects of estrogen in the chicken ductus arteriosus (DA). Isolated rings of the DA from 19‐d chicken fetuses (total incubation: 21‐d) were mounted in a wire myograph for isometric tension recordings. 17β‐estradiol elicited concentration‐dependent relaxation of KCl‐, phenylephrine‐, and oxygen‐induced active tone in male and female chicken DA. The stereoisomer 17a‐estradiol showed lesser relaxant effects. There were no gender differences in the responses to estrogen. Endothelium removal, or the presence of the soluble guanylate cyclase inhibitor ODQ, the K+ channels blockers glibenclamide and tetraethylammonium, or the estrogen receptor (ER) antagonist ICI 182,780 did not modify 17β‐estradiol‐induced relaxation. Preincubation (in normoxic, Ca2+‐free solution) of DA rings with 17β‐estradiol produced concentration‐dependent inhibition of the subsequent CaCl2‐induced contraction. ER‐α and ER‐β mRNAs (as determined by RT‐PCR) were strongly expressed in the ovary but ER‐α was weakly and ER‐β mRNA was not expressed in the DA. In conclusion, 17β‐estradiol induces endothelium‐independent relaxation of chicken DA, which is not mediated by ER activation. This relaxant effect is, at least partially, due to inhibition of Ca2+ entry from extracellular space.

Supported by "Fundación de Investigación Médica Mutua Madrileña".
Spatial and temporal distribution of nitric oxide synthase (NOS) isoforms in the developing ovine kidney
Authors: Davis Devin W. , Qi Wei , Smith Francine G.
Published: June 2021

Previously we showed that NOS isoforms display an age dependent pattern of expression in the ovine kidney. The present study was designed to further elucidate intrarenal spatial and temporal localization of NOS isoforms in the developing ovine kidney. The in situ hybridization technique was utilized to localize mRNA of all three NOS isoforms (I‐III) in sheep aged: ~1 day, 1, 3, 6, and 12 weeks post natally (N=3). Constitutive isoforms (NOS I and III) were expressed at 1 day to 3 weeks in both the cortex and medulla, with decreased overall expression at 6 and 12 weeks. NOS I showed more intense signal within the cortex than the medulla at all ages. The inducible NOS isoform (NOS II) had less abundant expression than the constitutive isoforms at all ages. NOS I appeared greatest at 1 week, staining the glomeruli intensely, with a decrease after 3 weeks. By demonstrating NOS isoform transcripts to be more abundant in the early post natal period, these findings provide insight into the age dependent role of NO in modulating renal haemodynamics and function during ontogeny.

Supported by the University Research Grant Committee and the Canadian Institutes of Health Research.
Differential effect of nicotine on intracellular calcium concentration ([Ca2+]i) in human umbilical artery and vein endothelial cells
Authors: Undem Clark , Pisarcik Sarah , Nelin Leif D , Shimoda Larissa A
Published: June 2021

Maternal tobacco smoke (TS) exposure is associated with intrauterine growth retardation and low birth weight, both of which increase infant morbidity and mortality. Nicotine, an active component in TS, also decreases birth weight and postnatal weight gain, although the mechanisms involved remain unclear. We hypothesized that in umbilical endothelial cells (ECs), nicotine alters regulation of [Ca2+]i, an important modulator of synthesis and release of endothelial‐derived vasoactive factors and, consequently, umbilical vessel tone and blood flow to and from the fetus. In this study, we used fluorescent microscopy in human umbilical artery ECs (HUAECs) and human umbilical vein ECs (HUVECs) loaded with the Ca2+‐sensitive dye, Fura‐2. In HUAECs, we found that acute (10 min) application of nicotine (100 ng/ml) caused an immediate decrease in [Ca2+]i. Prolonged nicotine treatment (10 or 100 ng/ml; 24 hr) also decreased resting [Ca2+]i. In contrast, both acute (100 ng/ml; 10 min) and prolonged (10 or 100 ng/ml; 24 hr) exposure to nicotine increased [Ca2+]i in HUVECs. Our results indicate that nicotine, at concentrations comparable to plasma levels in smokers and non‐smokers exposed to environmental TS, caused both acute and long‐lasting alterations in [Ca2+]i in HUAECs and HUVECs and suggest that nicotine may differentially regulate vasoactive factor synthesis and vascular tone in umbilical arteries and veins.
Control central ‐ at the intersection of exocytic and endocytic pathways
Authors: Stow Jennifer Lea
Published: June 2021

Recent studies have revealed that the recycling endosome in mammalian cells receives traffic from endocytic and recycling pathways, and from exocytic or secretory pathways. It thus handles a complex mix of membrane and protein cargo, which it then sorts and deploys selectively to the cell surface or other organelles. In epithelial cells this enigmatic compartment helps to control cell polarity, cell adhesion and other functions. In immune cells, recycling endosomes control immune responses through the strategic deployment of cytokines and packets of membrane to enable phagocytosis and migration. This talk will present evidence, including imaging in live cells, to help demonstrate some of the newly discovered and widespread functions of recycling endosomes in trafficking pathways ‐ functions that centrally underpin the physiology of differentiated cells.

Funding from the National Health and Medical Research Council of Australia supports this research.
Cisplatin and carboplatin can cause alteration of protein expression in human renal tubular epithelial cells
Authors: Perez Juliana Dinéia , Sakata Maisa Mayumi , Arita Danielle Yuri , Casarini Dulce Elena
Published: June 2021

Proteomic techniques are a powerful tool in nephrology research and allows the identification of important biomarkers. Using two‐dimensional polyacrilamide gel electrophoresis (2‐DE), we identified an alteration of protein expression in human renal tubular epithelial cells (HK‐2) induced by the treatment with anticancer drugs. HK‐2 were randomized in control (CT‐not treated), cisplatin (CPT‐100 µM) and carboplatin (CB‐50 µM). Sample protein (500 µg) was applied to a IPG strip (pH 3.0‐ 10.0), and electrophoresis ran under denaturing conditions. Gels were stained with coomassie G‐250, scanned and analysed by PdQuest software. Analysis showed a total of 124 spots in CT, 122 in CPT and 127 in CB. Using quantitative analysis (minimum difference of 5x expression between CT and treated groups) we identified a down‐regulation of 8 spots between CT and CPT groups, and also a down‐regulation of 12 spots of CPT and CB, compared with CT group. There was an up‐regulation of spots on CB and CPT groups (14 and 12 spots, respectively) compared with CT, and there was a correpondence on the up‐regulation of 11 spots, between both treatments and CT. Qualitative analysis (100x background) showed that some proteins are only expressed on CT (5 spots), CPT (12 spots) or CB (7 spots) groups. Our results, show that the treatment with anticancer drugs may induce alterations on protein expression in renal cells, which may be related to chemotherapy nephrotoxicity. The identification of these proteins may bring important information for new drug development.

Financial support: Fapesp
Structural studies of Neisseria meningitidis outer membrane protein PorB and the mechanism of recognition by toll‐like receptor 2
Authors: Iverson Tina M. , Tanabe Mikio
Published: June 2021

Neisseria meningitis is one of the causative agents of bacterial meningitis, which is hallmarked by the inflammation of the lining that surrounds the brain and spinal cord. The N. meningitidis outer membrane protein (OMP) PorB has been identified as an attractive candidate for the development of a vaccine against meningitis. In vivo, PorB activates the toll‐like receptor (TLR) pathway to trigger the innate immune system. To provide a framework for the mechanism of PorB recognition by TLRs, each protein is being studied both in isolation and in complex. The x‐ray crystal structure of the N. meningitidis PorB was determined to 2.3 Åresolution. PorB forms a 16‐stranded β‐barrel surrounding a central pore. Solute transport through this pore may be aided by the presence of an internal plug formed exclusively from long, folded inter‐strand loops. Initial studies combining purified PorB and the ectodomain of TLR2 identifies the formation of a 420 kD complex, demonstrating that PorB directly interacts with the ectodomain of TLR2 in vitro. The molecular weight of this complex is consistent with a 6:2 PorB:TLR2 stoichiometric ratio and directly shows complex formation between a TLR and an OMP for the first time.
Attenuation of Acute Doxorubicin‐Induced Cardiotoxicity by the ApoA‐I Mimetic Peptide 4‐F
Authors: Proctor J Bradley , Zhang Zhenghao , White C. Roger
Published: June 2021

Doxorubicin (DOX) is a commonly used chemotherapeutic drug that may induce a dose limiting cardiomyopathy. Cytotoxic effects of DOX are related to its ability to undergo redox cycling, resulting in superoxide anion generation and mitochondrial injury. The apoA‐I mimetic peptide 4F exerts anti‐oxidant effects by improving functional properties of HDL and by upregulating the enzyme superoxide dismutase (SOD). In this study, we tested the hypothesis that 4F treatment attenuates cardiac dysfunction in DOX‐treated rats. Male Sprauge‐Dawley rats were pre‐treated with 4F (5mg/kg/day) or saline by ip injection for 5 days. LV function was measured on day 5 in 4F‐ and saline‐treated rats by echocardiography. DOX (20mg/kg) was then administered by ip injection and echo measurements repeated on day 9. A separate CONTROL group received ip saline over the same time period. Stroke volume (SV) and ejection fraction (EF) were reduced by 25 ± 2% and 44 ± 7% respectively in DOX+saline rats (n=8) on day 9 compared to CONTROL rats (n=10). In contrast, the reduction in SV (8 ± 3%) and EF (7 ± 4%) was significantly attenuated in DOX+4F rats (n=8). It is proposed that a cardioprotective mechanism of 4F is likely due to the induction of SOD resulting in increased scavenging of DOX‐generated superoxide anion. In ongoing experiments, cardiac levels of SOD protein and mRNA expression are being assessed in DOX rats treated with saline or 4F.
Brown Norway chromosome 16 provides protection from cardiac fibrosis with pressure overloading on a Salt‐Sensitive Dahl background
Authors: Kriegel Alison J. , Greene Andrew S.
Published: June 2021

Myocardial fibrosis accumulates with age and often accompanies hypertension induced left ventricle (LV) hypertrophy. Previously we have shown that the normotensive SSBN16 consomic rat (Brown Norway (BN) chromosome 16 in Dahl Salt‐Sensitive (SS) rat genetic background) is better protected from developing LV fibrosis with aging than either the hypertensive SS or normotensive BN. We then hypothesized that the SSBN16 would also be better protected from developing fibrosis with an elevated afterload than the normotensive BN or SSBN13 consomic strains. To test this, 9 week old rats from each strain were subjected to 2 weeks of abdominal aortic occlusion (AAO) or sham operation (SOC). The AAO SSBN13 and BN rats achieved significantly elevated systolic blood pressures (SBP) (162±23 mmHg and 168±24 mmHg, respectively), while the SSBN16 SBP averaged only 141±5 mmHg. Examination of SSBN16 vasculature revealed that collateral blood vessels bypassed the occlusion in the AAO, but not SOC rats. The one‐kidney, one‐clip model was then applied to the SSBN16 producing an average SBP of 163±7 mmHg. With similarly elevated blood pressures the % of LV fibrosis was significantly increased in the SSBN13 and BN rats compared to SOC's, but not in the SSBN16 rats. These results suggest that genes on chromosome 16 protect against cardiac fibrosis associated with pressure overload hypertrophy. Supported by HL‐ U01 HL66579 and HL‐N01‐HV‐28182
Does caveolin‐1 knockout affect matrix metalloproteinase‐2 activity and contractile function in the isolated working mouse heart?
Authors: Chow Ava Kalyca , Daniel Edwin , Schulz Richard
Published: June 2021

Matrix metalloproteinase‐2 (MMP‐2) proteolyzes troponin I (TnI), myosin light chain‐1 (MLC‐1) and α‐actinin within cardiomyocytes in hearts challenged with ischemia and reperfusion (I/R).

MMP‐2 is also co‐localized with caveolin‐1 (Cav‐1) in cardiomyocytes. Cav‐1 inhibits MMP‐2 activity. Cav‐1 null (Cav‐1−/−) hearts have increased MMP‐2 activity. How this affects heart function in response to physiological and pharmacological challenges, and whether this has an impact on known MMP‐2 substrates, is unknown.

Cav‐1−/− or control (Cav‐1+/+) hearts were isolated and perfused as working hearts. Hearts were perfused aerobically for 30 min followed by both physiological (preload varied between 7‐22.5 mmHg) and pharmacological (0.1‐100 nM isoproterenol) challenges.

Cardiac function between the Cav‐1−/− vs Cav‐1+/+ hearts did not differ in either challenge. Gelatin zymography of heart homogenates showed no differences in MMP‐2 activity. Protein levels of MMP‐2, TnI, MLC‐1 and α‐actinin were similar between Cav‐1+/+ and Cav‐1−/− hearts. Hearts briefly perfused for 10 min showed more MMP‐2 activity compared with hearts perfused for 90 min. Prolonged perfusion and the oxidative stress inherent in the isolated working heart procedure may release MMP‐2 from the heart. MMP‐2 proteolysis of intracellular proteins may be dependent upon its activation in I/R.

This work is supported by HSF, CIHR and AHFMR.